Ameriden Research

Antiviral Res. 2005 Jun;66(2-3):129-36. Epub 2005 Apr 18.

The olive leaf extract exhibits antiviral activity against viral haemorrhagic septicaemia rhabdovirus (VHSV).

Micol V, Caturla N, Pérez-Fons L, Más V, Pérez L, Estepa A.

Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, E-03202-Elche, Alicante, Spain.

Abstract

A commercial plant extract derived from olive tree leaf (Olea europaea) (LExt) and its major compound, oleuropein (Ole), inhibited the in vitro infectivity of the viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus. Incubation of virus with LExt or Ole before infection reduced the viral infectivity to 10 and 30%, respectively. Furthermore, LExt drastically decreased VHSV titers and viral protein accumulation (virucidal effect) in a dose dependent manner when added to cell monolayers 36 h post-infection. On the other hand, both the LExt and Ole were able to inhibit cell-to-cell membrane fusion induced by VHSV in uninfected cells, suggesting interactions with viral envelope. Therefore, we propose that O. europaea could be used as a potential source of promising natural antivirals, which have demonstrated to lack impact on health and environment. In addition, Ole could be used to design other related antiviral agents.

PMID: 15869811 [PubMed - indexed for MEDLINE]

United States Patent	6,117,844
Fredrickson	September 12, 2000

Method and composition for antiviral therapy

Abstract

A method of treatment of diseases of viral origin is disclosed. The method comprises oral or parenteral administration of an antiviral amount of a naturally occurring secoiridoid from plants of the family Oleaceae or derivatives thereof. Preferred oral dosage forms include the secoiridoid oleuropein in pure form or as a component of dried plant material of Olea europaea or a dried extract thereof and a pharmaceutically acceptable carrier.

Inventors:	Fredrickson; William R. (Indianapolis, IN)
Assignee:	F&S Group, Inc. (Pacific Palisades, CA)
Appl. No.:	08/668,324
Filed:	June 26, 1996

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Related U.S. Patent Documents

	Application Number	Filing Date	Patent Number	Issue Date
	335138	Nov., 1994

Current U.S. Class:	514/27 ; 514/460
Current International Class:	A61K 31/351 (20060101); A61K 031/70 (); A61K 031/35 ()
Field of Search:	514/460,27

References Cited [Referenced By]

U.S. Patent Documents

3033877	May 1962	Veer et al.
3737550	June 1973	Nook et al.
3786068	January 1974	Kelly
3793346	February 1974	Kelly

Foreign Patent Documents

	683957		Feb., 1969		ZA

Other References Renis, Antimicro Agent Chemotherapy, 1969 pp. 167-172, 970. . Walter et al Appl Micro vol 26(5) pp 773-76, 1973. . Hirschman, Noter vol 238 Aug. 20, 1972, pp. 277-79, 1972. . “The Bitter Glucoside of the Olive”, W.V. Cruess and C. L. Alsberg, J. Am. Chem. Soc. 56:2115-2117 (1934). . Isolation of a Bacterial Inhibitor from Green Olives, Fleming, et al., Applied Microbiology, vol. 18, No. 5, pp856-860 (1969). . “Preparation of Antimicrobial Compounds by Hydrolysis of Oleuropein from Green Olives”, Walter et al., Applied Microbiology, vol. 26, No. 5, pp773-776 (1973). . “Antimicrobial Properties of Oleuropein and Products of Its Hydrolysis from Green Olives”, Fleming et al., Applied Microbiology, vol. 26, No. 5, pp777-782 (1973). . “In Vitro Antiviral Activity of Calcium Elenolate”, Harold Renis, Antimicrobial Agents and Chemotherapy, 1969 (printed 1970) pp. 167-172. . “Inactivation of DNA Polymerases of Murine Leukaemia Viruses by Calcium Elenolate” S. Z. Hirschman, Nature (New Biology), 238: 277-279 (1972). . “Antiviral Activity of Calcium Elenolate on Parainfluenza Infection of Hamsters”, M. G. Soret, Antimicrobial Agents and Chemotherapy, 1969 (printed 1970) pp. 160-166. . “Preliminary Safety Studies with Calcium Elenolate, an Antiviral Agent”, Elliot et al., Antimicrobial Agents and Chemotherapy, 1969 (printed 1970) pp. 173-176. . “Binding of Eugenol and O-ethoxybenzoic Acid to Bovine Serum Albumin”, S. Fujisawa and E. Masuhara, J. Dent. Res., vol. 60, No. 4, pp. 860-864 (1981). . “Pharmacological Analysis of the Iridoid Oleuropein,” V. Petkov and P. Manolov, Drug Res., 22(9) pp. 1476-1486 (1972). . “A Multichemical Defense Mechanism of Bitter Olive Olea Europaea (Oleaceae)”, Kubo et al. J. of Chemical Ecology, vol. 11, No. 2, pp. 251-263 (1985). . “Secoiridoids from Olea Europaea”, Gariboldi, et al., Phytochem. 25(4) pp. 865-869 (1986). . “Inactivation of Myxoviruses by Calcium Elenolate”, Harold Renis, Antimicrobial Agents and Chemotherapy, vol. 8, No. 2 pp. 194-199 (1975). . “Conversion of Secologanin into Elenolic Acid and 18-Oxayohimban Alkaloids”, R. T. Brown et al, J. of Chem. Soc. Perkin Trans. vol. 1, pp160-162 (1976). . “Mulluscicides from Olive Olea europaea and Their Efficient Isolation by Countercurrent Chromatographies”, I. Kubo and A. Matsumoto, J. Agric. Food Chem., vol. 32, No. 3 pp 687-688 (1984). . “HPLC Analysis of Oleuropein and Some Flavonoids in Leaf and Bud of Olea Europaea L.”, P. Ficarra and R. Ficarra, IL Farmaco, vol. 46, No. 6, pp. 803-815 (1991). . “A New Secoiridoid from Olive Wastewaters”, R. L. Scalzo and M. L. Scarpati, Journal of Natural Products, vol. 56, No. 4, pp. 621-623 (1993). . “Leaf Extract of Olea eropea Rich in Oleuropeine, Products from It, Their Application as Medicines and Compositions Containing Them”, G. Combes and A. Escaut, Chem Abstracts, vol. 98: 221797r (1983). . “(–)-Elenolic Acid Esters”, Takano et al., Chem Abstracts, vol. 106: 67534c (1987). . “Preparation of Dihydropyran Derivative as Antiviral Agents and Precursors for Alkyl (–)-Elenolates”, Takano et al., Chem Abstracts, vol. 113: 58936c (1990). . “Structure and Stereochemistry of Elenolic Acid”, MacKellar et al., J. Am. Chem. Soc. vol. 95 (21) pp. 7155-7156 (1973).

Primary Examiner: Travers; Russell
Attorney, Agent or Firm: Barnes & Thornburg

Parent Case Text

This Application is a continuation of application Ser. No. 08/335/138 filed Nov. 7, 1994 now abandoned.

Claims

I claim:

1. A method of treating a warm-blooded vertebrate suffering from a disease of viral origin, said method comprising the step of administering orally or parenterally to said vertebrate a therapeutically effective amount of an antiviral composition comprising a compound selected from the group consisting of oleuropein which is a compound of the formula ##STR8## wherein R.sub.1 is 2-(3,4-dihydroxyphenyl)ethyl and R is glucosyl, deesterified oleuropein of Formula I above wherein R.sub.1 is hydrogen, pharmaceutically acceptable salts of deesterified oleuropein, and other esters of formula I wherein R.sub.1 is derived from substantially non-toxic physiologically compatible alcohols, and a pharmaceutically

acceptable carrier therefor.

2. The method of claim 1, wherein the compound is oleuropein.

3. The method of claim 1, wherein the disease of viral origin is selected from the group consisting of herpes mononucleosis, hepatitis, and diseases deriving from infection by rotovirus, bovine rhinovirus, canine parvovirus and feline leukemia virus.

Description

FIELD OF THE INVENTION

This invention relates to treatment of disease of viral origin in warm-blooded vertebrates. More particularly, this invention is directed to the use of secoiridoid compounds naturally occurring in plants of the family Oleaceae and derivatives thereof.

BACKGROUND AND SUMMARY OF THE INVENTION

The olive tree and other members of the Family Oleaceae have been documented as a source of medicinal substances since biblical times. Needless to say, many researchers have studied the cocktail of phytogenic substances produced by the olive and other members of the Family Oleaceae. One compound that has received attention from the research community is the secoiridoid glucoside oleuropein, a compound of the formula ##STR1## wherein R is glycosyl and R.sub.1 is 2-(3,4-dihydroxyphenyl) ethyl. Related secoiridoids wherein R.sub.1 is H, CH.sub.3 or 2-(4-hydroxyphenyl ethyl) are also known to be endogenous to many plant species of the family Oleaceae, although in lesser concentrations and in fewer identified species than the ubiquitous oleuropein.

Animal studies have revealed that oleuropein itself or as a component of extracts of plant tissues containing that compound exhibit both hypoglycemic and cardiovascular effects. It is also known that oleuropein can be acid hydrolyzed to produce (-)-elenolic acid, a compound which has been reported to have antiviral properties in vitro, but little, if any, activity in vivo.

The present invention is based on the discovery that secoiridoid glucosides of the formula ##STR2## wherein R is glycosyl and R.sub.1, is hydrogen or an ester-forming group are metabolized in vivo to the dextrorotatory form of elenolic acid [(+)-elenolic acid], a compound of the formula ##STR3## wherein R.sub.1, is hydrogen, a compound which is believed to be more available in vivo than the corresponding diasteromer(-)-elenolic acid of the formula ##STR4## The enhanced in vivo efficacy of the oleuropein metabolite, (+)-elenolic acid relative to the corresponding levorotatory compound is thought to be due, at least in part, to its reduced affinity for serum proteins and thus its greater availability for uptake by virus infected tissues. Antivirally effective blood levels of (+)-elenolic acid can also be achieved by administration of (+)-elenolic acid and its esters of the formula ##STR5## wherein R.sub.1 is hydrogen or a pharmaceutically acceptable ester-forming group and salts thereof, which can be prepared from secoiridoid glucosides naturally occurring in plant material of the family Oleaceae via extraction and controlled enzyme (glucosidase) hydrolysis and/or deesterification/transesterification reactions.

Thus, it is one object of the present invention to provide a method of treatment of disease of viral origin in warm-blooded vertebrates by administering antiviral compositions containing secoiridoid glucosides native to the plant family Oleaceae and derivatives thereof.

Another object of the invention is to provide oral dosage forms of secoiridoid glucosides of Olea europaea and derivatives thereof.

In another more particular aspect of this invention plant material of the family Oleaceae and extracts thereof containing naturally occurring oleuropein glucosides and enzyme hydrolysates thereof are administered in treatment of diseases of viral origin in warm-blooded vertebrates suffering from such diseases.

One further object of this invention is a method for establishing antivirally effective blood levels of (+)-elenolic acid by the administration of oleuropein glucosides native to the family Oleaceae or their derivatives via transesterification, diesterification and/or glucolysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of treatment of disease of viral origin in warm-blooded vertebrates and to pharmaceutical formulations for use in such treatment methods. The method comprises the step of administering to a vertebrate suffering from a disease of viral origin an antivirally effective amount of an antiviral composition comprising a compound of the formula ##STR6## and a pharmaceutically acceptable carrier therefor. In the above formula, the group R is glucosyl and R.sub.1, is hydrogen or a pharmaceutically acceptable ester-forming group. When R.sub.1 is hydrogen, the acid compound represented can be utilized in the form of one of its pharmaceutically acceptable salts.

There are many diseases of viral etiology that afflict man and animal. In man, diseases such as hepatitis, mononucleosis, shingles, herpes, influenza, the common cold and even certain types of leukemia are known to be of viral etiology. Viral infections are also common in many animal species, both in meat producing and in companion animals. Rotovirus infections plague swine. Cattle develop bovine rhinovirus infections (shipping fever) when subjected to conditions of stress. Canine parvovirus and feline leukemia virus are common viral infections in those companion animals species. Such diseases of viral origin can be treated with resultant reduction in clinical symptomology by therapeutic administration of antiviral compositions in accordance with this invention.

The antiviral compositions administered in accordance with this invention comprise a compound of Formula I or II above in combination with a pharmaceutically acceptable carrier. The compounds of Formula I wherein R.sub.1 is 2-(4-hydroxyphenyl)ethyl or 2-(3,4-dihydroxyphenyl)ethyl are

naturally occurring compounds in many plants of the Family Oleaceae, including members of the genus Fraxinus, Syringa and the genus Ligustrum. Preferred plant sources of the naturally occurring secoiridoids of Formula I wherein R.sub.1 is 2-(4-hydroxyphenyl)ethyl and 2-(3,4-dihydroxyphenyl)ethyl are varieties of Olea europaea (the olive). Preferred varieties of Olea europaea as a source of secoiridoid glycosides for use in accordance with this invention are the varieties Manzanillo and Mission.

The most prevalent of the secoiridoid compounds in such varieties is the compound of Formula I wherein R.sub.1, is 2-(3,4-dihydroxyphenyl)ethyl and R is glucosyl, a compound given the common name oleuropein. That compound can be readily isolated from plant material, preferably ground leaves of the olive by aqueous or aqueous-alcoholic extraction at room temperature or above, preferably at elevated temperature of about 40 to about 100.degree. C.

Oleuropein can then be purified, for example, by chromatographic separation procedures. That compound can then be used to formulate antiviral compositions in accordance with this invention or to prepare other antivirally effective compounds represented by Formulas I or II. Thus, for example, oleuropein can be subjected, to base catalyzed transesterification wherein the R.sub.1, group 2-(3,4-dihydroxyphenyl)ethyl is exchanged with another pharmaceutically acceptable ester-forming group. The term “pharmaceutically acceptable ester-forming group” as used in defining the present invention, refers to those ester-forming groups which when cleaved via esterase reactions in vivo produce substantially non-toxic, physiologically compatible alcohols. Suitable pharmaceutically acceptable ester-forming groups include C.sub.1 -C.sub.8 lower alkyl, and substituted C.sub.1 -C.sub.8 alkyl, benzyl, substituted benzyl wherein the substituents are halo, C.sub.1 -C.sub.4 alkoxy, C.sub.1 -C.sub.4 acyloxy, and the like. The compound of Formula I wherein R.sub.1, is hydrogen can be produced by esterase-mediated deesterification of oleuropein, typically in an aqueous medium at a pH between about 6 and about 8.5.

The compounds of Formula II are prepared from the corresponding compounds of Formula I by treatment with glucosidase, preferably that from the olive at a pH of about 4 to about 5. The compound of Formula II wherein R.sub.1 is hydrogen is (+)-elenolic acid.

The compounds of Formula I or II wherein R.sub.1 is hydrogen represent carboxylic acids and such acids can be used in accordance with this invention in the acid form or in the form of their pharmaceutically acceptable salts formed with organic bases or inorganic bases, such as ammonium, alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, and potassium carbonate. Of the salt forms, the potassium and sodium salts are particularly preferred.

The antiviral compositions of the present invention can be administered orally or parenterally in an antivirally effective amount to treat, i.e., reduce the symptoms, of diseases of viral origin. Oral dosage forms can be in a solid or liquid form and comprise an antivirally effective amount of a compound of Formula I or Formula II above and a pharmaceutically acceptable carrier. Such dosage forms can be formulated from pure compounds of Formula I or Formula II, or they can be formulated from ground plant materials of the family Oleaceae, preferably leaves of Olea europaea, or aqueous or aqueous alcoholic extracts thereof. Thus, for example, extraction of dried olive leaves with two volumes of a 12-15% ethanol/water solution for 10 days at room temperature provides an extract containing about 70 to about 250 mg of oleuropein per two ounces of the liquid extract. The extract itself can be administered orally as an antiviral composition in accordance with the method of treatment of the present invention, or aqueous or aqueous alcoholic (preferably methanol or ethanol) extracts can be spray-dried to provide a dry powder which can be formulated into oral dosage forms with other pharmaceutically acceptable carriers.

The solid oral dosage form compositions in accordance with this invention are prepared in a manner well known in the pharmaceutical art, and comprise at least one compound of Formula I or Formula II associated with at least one pharmaceutically acceptable carrier. In making such compositions, the compound of Formula I or Formula II, either in pure form or as a component of ground plant material or extracts thereof, are usually mixed, diluted or enclosed within a carrier. The carrier can be in a solid form, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient. Alternatively, the carrier can be in the form of a capsule or other container to facilitate oral administration. Thus the solid oral dosage forms for administration in accordance with the method of this invention can be in the form of tablets, pills, powders or soft or hard gelatin capsules. Alternatively, the antiviral compositions in accordance with this invention for oral administration can be in liquid form wherein the pharmaceutically acceptable carrier is water or an aqueous alcoholic medium. The compositions for administration in the present method can also be formulated with other common pharmaceutically acceptable excipients, including lactose, dextrose, sucrose, sorbitol, mannitol, starches, gums, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, methylcellulose, water, alcohol and the like. The formulations can additionally include lubricating agents such as talc, magnesium stearate and mineral oil, wetting agents, emulsifying and suspending agents, preserving agents such as methyl- and propylhydroxybenzoates, sweetening agents or flavoring agents. Further the compositions of the present invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.

Orally administered compositions are preferably formulated in unit dosage form with each dosage normally containing from about 30 to about 500 mg of a compound of the Formula I or Formula II, more typically about 100 to about 500 mg of such active compounds. The term “unit dosage form” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical carrier/excipient. The preferred dosage levels of the compounds of Formula I/Formula II for treatment of viral infections in accordance with this invention depend on the route of administration, the nature of the active compound or combination of active compounds, and patient condition. When administered orally for treatment of viral disease, the compounds of Formula I or Formula II are administered at a dose of about 0.1 to about 15 mg/kg, more preferably about 0.2 to about 10 mg/kg of patient/animal body weight. In treatment of viral infections oral dosage forms in accordance with this invention can be administered 1 to 4 times a day, again depending on patient condition and the nature of the disease being treated.

Similar considerations bear on the dosage range for parenteral administration of compounds of Formula I or Formula II in treatment of viral infections in accordance with this invention. Parenteral doses are, however, typically lower than those required for antiviral efficacy via the oral route of administration. Thus, antiviral treatment can be achieved by parenteral administration of about 0.05 to about 3 mg/kg of patient/animal body weight. Parenteral formulations for use in accordance with the present invention are prepared using standard techniques in the art. They are commonly prepared as sterile injectable solutions, using a parenterally acceptable carrier such as isotonic saline solution or as a sterile packaged powder prepared for reconstitution with sterile buffer or isotonic saline prior to administration to a patient. The injectable formulation can contain from about 1 to 50 mg of a compound of Formula I or II per ml of formulation.

Administration of an antivirally effective amount of a composition comprising a compound of Formula I or Formula II and a pharmaceutically acceptable carrier in accordance with this invention produce antivirally effective blood levels of (+)-elenolic acid, a compound of the formula ##STR7## via a heretofore unappreciated in vivo hydrolysis, and in the case of the compounds of Formula I, stereoselective hydrolysis/rearrangement. Administration of antiviral compositions of a compound of Formula I as part of ground/dried native plant material, preferably olive leaves, or as an aqueous or alcoholic extract of olive leaves is a particularly preferred embodiment in accordance with this invention. While not wishing to be bound by theory, the level of antiviral activity associated with such antiviral compositions may derive from a synergistic antiviral effect with other natural components of the olive leaf, such as the flavonoids rutin, hesperidin, and luteolin-7-glucoside.

The following non-limiting examples are illustrative of the method and compositions of the present invention. It is understood, however, that such examples are but representative of various embodiments of the invention and it is not intended that the invention be limited to the scope of the examples.

EXAMPLE 1

(A) A volume of dried leaves of Olea europaea is suspended in 2 volumes of red wine and held at room temperature for about 7 to about 10 days with periodic stirring. Filtration of the mixture provides a tincture containing about 88 mg of oleuropein per ounce of fluid.

(B) A volume of dried leaves of Olea europaea is suspended in two volumes of water and the resulting suspension is then subjected to conditions of high shear in a Waring blender to produce a dispersion of finely divided plant material which was held at a temperature of about 40 to about 65.degree. for two days. Filtration of the mixture provides an aqueous extract containing about 72 mg of oleuropein per ounce of fluid. The aqueous extract is optionally blended with effective amounts of sweetening and/or flavoring agents to provide a palatable liquid oral dosage form of oleuropein.

(C) One volume of leaves or buds of Olea europaea is combined with about two volumes of a 3:2 mixture of methanol and water. The resulting aqueous alcoholic suspension of plant material is heated for 16 hours at about 75.degree. C., cooled and filtered to provide an aqueous alcoholic extract. The extract is spray-dried to produce a powder comprising oleuropein. The powder is filled into gelatin capsules in an amount sufficient to provide 30 to about 500 mg of oleuropein per capsule.

(D) The spray-dried extract of (C) above is subjected to high pressure liquid chromatography to produce oleuropein [Formula I; R=glycosyl; R.sub.1 =2-(3,4-dihydroxyphenyl)ethyl] and ligstroside [Formula I; R=glycosyl; R.sub.1 =2-(4-hydroxyphenyl)ethyl] in substantially pure form. The purified oleuropein is formulated alone or in combination with ligstroside with tabletting starch and a tabletting lubricant, magnesium stearate, to form a tabletting mixture. The tabletting mixture is pressed into compressed tablets containing about 30 to about 500 mg of oleuropein per tablet.

EXAMPLE 2

Oleuropein (1 g) is dispersed in 50 ml of methanol. The solution is cooled to about 10.degree. C. and treated with stirring with about 2 g of potassium hydroxide pellets. The mixture is allowed to warm to room temperature and after about 6 hours, the reaction mixture is diluted with about 60 ml of 6 N HCl (pH about 7.5) and evaporated to dryness. The chromatographic purification of the product mixture provides oleoside [Formula I; R=glycosyl, R.sub.1 =methyl]. The purified oleoside is formulated into a solid oral dosage form containing about 250 mg of oleoside. Alternatively, oleoside is dissolved in sterile isotonic saline at a concentration of about 5 mg per ml to provide a parenteral dosage form for use in accordance with the method of this invention.

EXAMPLE 3

Five grams of oleuropein is dissolved in 500 ml of water buffered at pH 5.0 and treated with glucosidase until analysis of the reaction mixture by thin layer chromatography indicates completion of the reaction. Standard workup of the reaction mixture followed by chromatographic purification of the product mixture provides a compound of Formula II wherein R.sub.1 =2-(3,4-dihydroxyphenyl)ethyl. The purified product is formulated into oral or parenteral dosage forms and administered for treatment of viral diseases in accordance with the method of the present invention.

EXAMPLE 4

A solution of 500 mg of the product of Example 3 in 50 ml of water buffered at about pH 7.5 to about 8.5 is treated with commercially available esterase at a temperature of about 30.degree. C. until thin layer chromatographic analysis of the esterase reaction mixture indicates completion of the reaction due to disappearance of the starting compound. After reaction completion, the pH of the mixture is readjusted to about 8.5 and after washing it twice with ethyl acetate, the pH of the solution is adjusted to about 4.5 in the presence of 30 ml of ethyl acetate. The ethyl acetate acid extract of the reaction mixture is separated, washed with distilled water and brine, and dried over anhydrous sodium sulfate. Evaporation to dryness provides (+)-elenolic acid [Formula II; R.sub.1 =hydrogen] in substantially pure form. The product is further purified by chromatography and/or crystallization as its sodium or potassium salt. It is formulated into oral or parenteral dosage forms in accordance with this invention for treatment of viral infections in warm-blooded vertebrates.

EXAMPLE 5

Dried leaves of Olea europaea are ground to a fine powder and loaded into gelatin capsules such that each capsule contains a volume of ground plant material containing between about 30 and about 500 mg of oleuropein per capsule. Dried olive leaves typically contain about 60 to about 90 mg of oleuropein per gram of dried leaf. The capsules are administered orally for treatment of diseases of viral origin in accordance with this invention.

EXAMPLE 6

The antiviral efficacy of a composition of Example 1(A) above was evaluated in treatment of six subjects afflicted with herpes virus infections. Each of the six subjects ingested two ounces of the formulation every six hours. All subjects reported reduction of herpetic lesions. Three subjects reported disappearance of lesions in 36-48 hours after initiating treatment. One subject, a 34 year old Caucasian female, had several months earlier discontinued use of birth control pills and it was thought that her estrogen surges were causing immunosuppression which complicated her infection. After three days of doubling the dosage of the tincture (four ounces every six hours) most of her lesions were resolved. Two other subjects, a male and a female, more recently infected were also given the higher doses (four ounces every six hours orally) and each reported improvement measured by reduction in severity of their herpetic lesions, and in at least one patient, a 22.8% decrease in antibody titers (IgG) three weeks after initiating therapy with the oleuropein-containing olive leaf extract.

The Olive tree and its by-products have been an important part of everyday life for millions of people from generation to generation, making its way into legend & lore from country to country since the dawn of recorded history.  While olive oil and other components of the olive tree are not only are nourishing, tasteful and even medicinal to a certain degree, it wasn’t until the discovery of the powerful healing nutrients, hidden in the leaves of this tree, that serious attention from the medical community was raised and the tree’s ultimate potential was recognized, resulting in Olive Leaf Extract.

In the medical arena, many persons have recorded Olive Leaf Extracts since the early 1800’s.  In 1811 Mr. M. Cazals tells of intermittent fever giving way quickly by using olive leaf extract.  In addition, Dr. Pallas tells of the doctors of a war of Spain in 1808-1813 using olive leaf extract to stop fever and the spread of disease.  In 1828 Dr. Pallas also observed the remarkable effects from Olive Leaf Extract in the military hospitals at Pampeluna.  We finally read in 1843 where Mr. Sydney H. Maltass of Smyrna observed the Island of Mytelene, gripped by deadly malarial fever, recover completely within a few days by using Olive Leaf Extract.

Since the mid to late 1800’s very little research was conducted on this remarkable tree.  It wasn’t until around the 1960’s that interest in Olive Leaf Extracts began again.  The Upjohn Co. tried to make a synthetic substance out of one of the molecules that comes from olive leaf extract called Oleuropein.  They failed however, in their attempts to get it to work in the body with out blood serum protein binding and gave up on the project.

Then in 1989 William Fredrickson, a lay-chemist from Indiana, was searching for a thesis subject at Purdue University and so began the discovery of an active Olive Leaf Extract here in the United States.  He was particularly interested in why the Oleuropein molecule didn’t work in the body for The Upjohn Co.  That investigation lead to what we now know as one of the most important antimicrobial discoveries ever known to mankind.  His discovery encompassed many long tedious hours of producing liquid extracts, and then finally dried extracts, that would have the efficacy necessary to eradicate virus, bacteria, fungus and yeast.

Sources of funding came in slowly at first, then the Strecker Group and a private company called The N.F.N. Company, of which William was a partner, and finally Bio-Genesis Inc., which succeeded in getting the only U. S. patent for antiviral therapy on a natural substance in the world, (United States Patent #6,117,844).  Sadly, William Fredrickson passed away just a few months before the patent was numbered, stamped and approved for use.  A second patent subsequently has passed (United States Patent #6,455,580) in regards to Elenolic acid.

His book entitled, “The Tree of Life” resides in the Library of Congress and contains the full contents of what Olive Leaf Extract is all about, how to make it and why leaving it in its natural form, a natural extract as opposed to a synthetic, is so important.  His proprietary knowledge and company patent has now been licensed to AMERIDEN® International, Inc. a Nutraceutical Company, based in San Diego County, California, and “The Original Olive Leaf Extract®” continues to serve as a remedy to those suffering from chronic conditions around the world.

Cairo University, EgyptTable of Contents:

• ANTIBACTERIAL
• ANTIFUNGAL
• ANTI-INFLAMMATORY
• ANTIOXIDANT & FREE RADICAL SCAVENGER
• ANTIVIRAL
• BLOOD PRESSURE (HYPERTENSION)
• CANCER
• CHOLESTEROL, CORONARY & HEART
• COSMETIC
• DIABETES (HYPOGLYCEMIA)
• FEVER
• GENERAL
• HIV/AIDS
• IMMUNE SYSTEM
• RADIO-PROTECTIVE
• RESPIRATORY (INFLUENZA, PNEUMONIA ETC)
• ULCERS

NB. Where the word TITLE: is underlined this signifies that a full copy of the scientific paper is held in hard copy at Medifood Pty Ltd.

The therapeutic benefits of Olive Leaf Extract have been widely researched and documented in more than 30 medical and scientific publications including:

• American Journal of Chinese Medicine
• Antimicrobial Agents and Chemotherapy
• Applied and Environmental Microbiology
• Arzneim-Forsch
• Belgian Pharmacology Journal
• European Journal of Cancer Farmacevitsk Revy
• Journal of the American Chemists Society
• Journal of Applied Bacteriology
• Journal of Chemical Ecology
• Journal of Ethnopharmacology
• Journal Universel des Sciences Medicales
• Lancet
• Microbiology
• Nature’s New Biology
• Nutrition Research
• Phytochemistry
• Planta
• Medicine
• Positive Health News

Selection of institutes with published reports on the health benefits of olive leaf extract and oleuropein:

• Cairo University, Egypt
• Harvard Medical School, USA
• King Saud University, Saudi Arabia
• Kobe University, Japan
• New York University School of Medicine, USA
• Rene Descartes University, France
• University of Antwerp, Belgium
• University of Durban, South Africa
• University of Granada, Spain
• University of Milan, Italy
• University of Nebraska, USA
• Wageningen University, Netherlands

Additional publications and institutes can be found inside this document.

ANTIBACTERIAL

TITLE:In vitro antimicrobial activity of olive leaves

AUTHOR:Markin-D; Duek-L; Berdicevsky-I

INSTITUTE:Department of Microbiology, Rappaport Faculty of Medicine, Technion – Institute of Technology, Haifa

SOURCE:Mycoses. 2003; 46(3-4): 132-136

YEAR: 2003

ABSTRACT: We investigated the antimicrobial effect of olive leaves against bacteria and fungi. The microorganisms tested were inoculated in various concentrations of olive leaf water extract. Olive leaf 0.6% (w/v) water extract killed almost all bacteria tested, within 3 h. Dermatophytes were inhibited by 1.25% (w/v) plant extract following a 3-day exposure whereas Candida albicans was killed following a 24 h incubation in the presence of 15% (w/v) plant extract. Olive leaf extract fractions, obtained by dialysis, showed antimicrobial activity consisted of particles smaller than 1000 molecular rate cutoffs. Scanning electron microscopic observations of C. albicans, exposed to 40% (w/v) olive leaf extract, showed invaginated and amorphous cells. Escherichia coli cells, subjected to a similar treatment but exposed to only 0.6% (w/v) olive leaf extract showed complete destruction. These findings suggest an antimicrobial potential for olive leaves.

TITLE:  Antimicrobial Properties of Oleuropein and Products of Its Hydrolysis from Green Olives.

AUTHOR: Fleming-HP; Walter-WM Jr; Etchells-JL

YEAR:1973

SOURCE:Applied-Microbiology; 26 (5) 777-782, 14 ref.

ABSTRACT: Antibacterial action of oleuropein and its hydrolysis products against certain species of lactic acid bacteria involved in the brine fermentation of olives was investigated. Oleuropein was not inhibitory, but 2 of its hydrolysis products, the aglycone and elenolic acid, inhibited growth of the 4 species of lactic acid bacteria tested [Lactobacillus plantarum, L. brevis, Leuconostoc mesenteroides and Pediococcus cerevisiae]. Another hydrolysis product, beta-3,4­dihydroxyphenylethyl alcohol, was not inhibitory. The aglycone of oleuropein and elenolic acid were much more inhibitory when the broth medium contained 5% NaCI; 150 mug of either compound/ml prevented growth of L. plantarum. A crude extract of oleuropein, tested by paper disk bioassay, was inhibitory to 3 [Staphylococcus aureus, Bacillus subtilus and Pseudomonas solanacearum] of 17 species of bacteria screened. The acid hydrolysate of the extract was inhibitory to 11 of the bacteria, which included 4 species of lactic acid bacteria and other Gram-positive and Gram-negative species. Neither crude preparation was inhibitory to growth of the 7 species of yeasts tested. A possible explanation is given for the previously reported observation that heating (3 min, 74 degree C) olives prior to brining render them more fermentable by lactic acid bacteria. Results of a brining experiment indicated that oleuropein is degraded to antibacterial compounds when unheated olives are brined. [See preceding abstract]

TITLE:  Studies on the mechanism of the antimicrobial action of oleuropein.

AUTHOR:Juven-B; Henis-Y; Jacoby-B

INSTITUTE: Div. of Food Tech., Volcani Inst. of Agric. Res., Rehovot, Israel

YEAR: 1972

SOURCE: Journal-of-Applied-Bacteriology; 35 (4) 559-567, 25 ref.

ABSTRACT: This work was undertaken to investigate the mechanism of the antimicrobial action of the phenolic glycoside, oleuropein, the bitter principle of green olives. It was shown to be surface active, and this, in conjunction with its damaging effect on the permeability of cell membranes, seemed to be the basis of its antimicrobial activity. Oleuropein affected a significant leakage of glutamate, K and inorganic phosphate from Lactobacillus plantarum. Oleuropein had no effect on the rate of glycolysis when added to resting cells of L. plantarum, but it caused a decrease in the ATP content of the cells.

TITLE: Inhibition of Salmonella enteritidis by oleuropein in broth and in a model food system.

AUTHOR:Tassou,-C-C; Nychas,-GJ

INSTITUTE:National Agricultural Research Foundation, Institute of Technology of Agricultural Products, Lycovrysi, Athens, Greece.

SOURCE: Lett-Appl-Microbiol. 1995 Feb; 20(2): 120-4

YEAR: 1995

ABSTRACT: The inhibitory effect of commercial ‘pure’ oleuropein was tested against Salmonella enteritidis in a coliform broth and in reconstituted milk (model food system). It was found that the inhibition of this organism in the broth was influenced by the initial inoculum size, the pH of the medium and the concentration of additive. The inhibition was more pronounced in samples with low pH and low inoculum size. No such inhibition was evident in the model food system.

TITLE:Modeling the effectiveness of a natural antimicrobial on Salmonella enteritidis as a function of concentration, temperature and pH,using conductance measurements.

AUTHOR:Koutsoumanis,-K; Tassou,-C-C; Taoukis,-P-S; Nychas,-G-J
                 
INSTITUTE: Agricultural University of Athens, Department of Food Science and Technology, Hellas, Greece.

SOURCE: J-Appl-Microbiol. 1998 Jun; 84(6): 981-7
                                                                  
YEAR: 1998

ABSTRACT: The growth of Salmonella enteritidis in a brain heart infusion medium was monitored using the traditional viable count method and by conductance measurements using a Rabit impedance instrument. Growth curves (Iog10 cfu ml-1 vs time) at three different concentrations of oleuropein (0, 0.2 and 0.8%), pH values in the range of 5-8 and incubation temperatures from 22 to 42 degrees C were modeled using the Gompertz equation. A good correlation between the maximum growth rate from the viable count method and the maximum slope of the conductance curve from the impedance instrument was established. Based on this correlation, the maximum specific growth rate of Salm. enteritidis was modeled as a function of the oleuropein concentration, initial pH values and the incubation temperature with a quadratic equation, using a new, large dataset of growth measurements by conductance. The developed model was validated by statistical comparison of predicted growth rates with growth rates determined by the viable count method, within the limits of the antimicrobial, pH and temperature domain.

TITLE:Characterization of an oleuropein degrading strain of Lactobacillus plantarum. Combined effects of compounds present in olive fermenting brines (phenols, glucose and NaCI) on bacterial activity.

AUTHOR:Marsilio,-V; Lanza,-B. 

SOURCE: J-sci-food-agric. Sussex : John Wiley & Sons Limited. Apr 1998. v. 76 (4) p. 520­-524

YEAR:1998

ABSTRACT:This study aims to examine the effects of glucose and NaCI on the ability of an oleuropein degrading strain of Lactobacillus plantarum, strain B21, to grow in the presence of oleuropein, its degradation product, hydroxytyrosol, and p-coumaric acid. Oleuropein (10 g litre­1) and 2 g litre-1 hydroxytyrosol combined with NaCI did not inhibit bacterial growth, whereas 1 g litre-1 p-coumaric acid showed low inhibitory activity. This study also presents that bacterial beta-glucosidase and esterase are involved in the breakdown of oleuropein. Oleuropein (10 g litre-1) incorporated in the cultivation medium without glucose was completely degraded to derivative products within 20 days, whereas in the presence of glucose, at concentrations up to 50 g litre-1 beta-glucosidase activity was partially inhibited, and 30-70% of oleuropein residual content remained in the cultivation medium. Esterase activity involved in the second step of biodegradation process, was not influenced by glucose. Incorporation of glucose in the growth medium adversely affected the ability of L plantarum to break-down oleuropein. The findings of this study are significant since it could lead to the development of a new biotechnology for removing the bitter principle, oleuropein, from olives replacing the traditional alkali treatment used for debittering olives.

TITLE:Inhibition of Staphylococcus aureus by olive phenolics in broth and in a model food system.

AUTHOR: Tassou,-C.C.; Nychas,-G.J.E.

SOURCE: J-food-prot. Des Moines, Iowa : International Association of Milk, Food and Environmental Sanitarians. Feb 1994. v. 57 (2) p. 120-124.

YEAR:1994

ABSTRACT:The commercial ‘pure’ oleuropein and phenolics extracted from olives inhibited the growth and enterotoxin production by Staphylococcus aureus S-6 in broth as well as in reconstituted milk (model food system). It was found that the inhibition of this organism in N-Z amine A broth was influenced by the initial inoculum size, the pH of the media, and the concentration of additive. In particular, growth and enterotoxin B production by S. aureus were inhibited in broth with a high concentration of oleuropein (0.6%). The inhibition was more pronounced in samples with low pH and low inoculum size. In the case of milk, enterotoxin B production was also influenced by the initial concentration of extract. [Extracts from olives have been shown to inhibit or delay the rate of growth of a range of bacteria and micro fungi such as Pseudomonas fluorescens, Lactobacillus plantarum, Bacillus megaterium, Staphylococcus aureus, Eneterobacteriaceae, Aspergillus spp., Saccharomyces, Pichia, Torulopsis, Candida.]

TITLE:The effect of the olive phenolic compound, oleuropein, on growth and enterotoxin B production by Staphylococcus aureus.

AUTHOR:Tranter,-H.S.;Tassou,-S.C.;Nychas,-G.J

SOURCE: J-Appl-Bacteriol. Oxford : Blackwell Scientific Publications. Mar 1993. v. 74 (3) p. 253-259.

YEAR:1993
 
ABSTRACT:The presence of low concentrations (0.1% w/v) of oleuropein, a phenolic compound extracted from olives, delayed the growth of Staphylococcus aureus in NZ amine A and brain heart infusion media modified by the addition of growth factors and glucose (NZA+ and BHI+), as indicated by changes in conductance, whilst higher concentrations (0.4-0.6% w/v) inhibited growth completely. Intermediate concentrations of oleuropein (0.2%) prevented growth in BHI+ but allowed growth to occur in NZA+ despite an extended lag phase (30 h). Concentrations of oleuropein > 0.2% inhibited growth and production of enterotoxin B in both types of media. Lower levels (0.1 %) did not affect the final viable count and production of toxin in BHI+ but decreased the number of viable organisms and reduced the toxin production in NZA+ by eightfold. An increase in the concentration of oleuropein resulted in a decrease in the amount of glucose assimilated and consequently the amount of lactate produced. In addition, oleuropein prevented the secretion of a number of exoproteins. Addition of oleuropein during the exponential phase appeared to have no effect on the growth of Staph. aureus in NZA+.

TITLE:Bactericidal action of oleuropein extracted from green olives against Lactobacillus plantarum.

AUTHOR:Ruiz-Barba,-J-L; Garrido-Fernandez,-A; Jimenez-Diaz,-R
 
INSTITUTE:Correspondence (Reprint) address, R. Jimenez-Diaz, Unidad Estructural de Biotecnologia de Alimentos, Inst. de la Grasa y sus Derivados (CSIC), Avda. Padre Garcia Tejero 4, Apdo. 1078, 41012 Seville, Spain

SOURCE: Letters-in-Applied-Microbiology. 1991; 12(2): 65-68 ; 15 ref.

YEAR:1991

ABSTRACT:The phenolic compound oleuropein extracted from green olives was shown to be bactericidal against 9 strains of Lactobacillus plantarum isolated from green olive fermentation brines. Heat-treated oleuropein also demonstrated a strong bactericidal effect but not alkali­treated oleuropein, which allowed survival of most of the strains tested. The bactericidal effect was accompanied by changes in the typical bacillary structure and Gram-positive strain of L. plantarum.

TITLE:In vitro antimycoplasmal activity of oleuropein.

AUTHOR:Furneri,-P-M; Marino,-A; Saija,-A; Uccella,-N; Bisignano,-G

INSTITUTE: Department of Microbiological Sciences and Gynecological Sciences, University of Catania, via Androne 81, 95124, Catania, Italy. furneri@mbox.unit. it

SOURCE: Int-J-Antimicrob-Agents. 2002 Oct; 20(4): 293-6

YEAR: 2002

ABSTRACT: The activity of oleuropein, a phenolic glycoside contained in olive oil, was investigated in vitro against Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma pneumoniae and Mycoplasma pirum. Oleuropein inhibited mycoplasmas at concentrations from 20 to 320 mg/I. The MICs of oleuropein to M. pneumoniae, M. pirum, M. hominis and M. fermentans were 160, 320, 20 and 20 mg/I, respectively.

TITLE: On the in-vitro antimicrobial activity of oleuropein and hydroxytyrosol.

AUTHOR:Bisignano,-G; Tomaino,-A; Lo-Cascio,-R; Crisafi,-G; Uccella,-N; Saija,-A

INSTITUTE: Department Farmaco-Biologico, University of Messina, Italy.

SOURCE:J-Pharm-Pharmacol. 1999 Aug; 51(8): 971-4

YEAR: 1999

ABSTRACT: Secoiridoides (oleuropein and derivatives), one of the major classes of polyphenol contained in olives and olive oil, have recently been shown to inhibit or delay the rate of growth of a range of bacteria and microfungi but there are no data in the literature concerning the possible employment of these secoiridoides as antimicrobial agents against pathogenic bacteria in man. In this study five ATCC standard bacterial strains (Haemophilus influenzae ATCC 9006, Moraxella catarrhalis ATCC 8176, Salmonella typhi ATCC 6539, Vibrio parahaemolyticus ATCC 17802 and Staphylococcus aureus ATCC 25923) and 44 fresh clinical isolates (Haemophilus influenzae, eight strains, Moraxella catarrhalis, six strains, Salmonella species, 15 strains, Vibrio cholerae, one strain, Vibrio alginolyticus, two strains, Vibrio parahaemolyticus, one strain, Staphylococcus aureus, five penicillin-susceptible strains and six penicillin-resistant strains), causal agents of intestinal or respiratory tract infections in man, were tested for in-vitro susceptibility to two olive (Olea europaea) secoiridoides, oleuropein (the bitter principle of olives) and hydroxytyrosol (derived from oleuropein by enzymatic hydrolysis and responsible for the high stability of olive oil). The minimum inhibitory concentrations (MICs) calculated in our study are evidence of the broad antimicrobial activity of hydroxytyrosol against these bacterial strains (MIC values between 0.24 and 7.85 microg mL(-1) for ATCC strains and between 0.97 and 31.25 microg mL(-1) for clinically isolated strains). Furthermore oleuropein also inhibited (although to a much lesser extent) the growth of several bacterial strains (MIC values between 62.5 and 500 microg mL(-1) for ATCC strains and between 31.25 and 250 microg mL(-1) for clinical isolates); oleuropein was ineffective against Haemophilus influenzae and Moraxella catarrhalis. These data indicate that in addition to the potential employment of its active principles as food additives or in integrated pest-management programs, Olea europaea can be considered a potential source of promising antimicrobial agents for treatment of intestinal or respiratory tract infections in man.

TITLE: Comparative antibacterial and antifungal effects of some phenolic compounds.

AUTHOR:Aziz,-N-H; Farag,-S-E; Mousa,-L-A; Abo-Zaid,-M-A

INSTITUTE: National Centre for Radiation Research and Technology, Nasr City, Cairo, Egypt.

SOURCE:Microbios. 1998; 93(374):43-54

YEAR: 1998

ABSTRACT:The antimicrobial potential of eight phenolic compounds isolated from olive cake was tested against the growth of Escherichia coli, Klebsiella pneumoniae, Bacillus cereus, Aspergillus flavus and Aspergillus parasiticus. The phenolic compounds included p-hydroxy benzoic, vanillic, caffeic, protocatechuic, syringic, and p-coumaric acids, oleuropein and quercetin. Caffeic and protocatechuic acids (0.3 mg/ml) inhibited the growth of E. coli and K. pneumoniae. The same compounds apart from syringic acid (0.5 mg/ml) completely inhibited the growth of B. cereus. Oleuropein, and p-hydroxy benzoic, vanillic and p-coumaric acids (0.4 mg/ml) completely inhibited the growth of E. coli, K. pneumoniae and B. cereus. Vanillic and caffeic acids (0.2 mg/ml) completely inhibited the growth and aflatoxin production by both A. flavus and A. parasiticus, whereas the complete inhibition of the moulds was attained with 0.3 mg/ml p-hydroxy benzoic, protocatechuic, syringic, and p-coumaric acids and quercetin.

TITLE: Effect of Phenolic Compounds and Oleuropein on the Germination of Bacillus-cereus T Spores.

AUTHOR:TASSOU-C-C {a}; NYCHAS-G-J-E; BOARD-R-G

INSTITUTE: {a} INST FOOD TECHNOL, MINISTRY AGRIC, S VENIZELOU 1, LYCOVRISI 14123, ATHENS, GREECE

SOURCE:Biotechnology-and-Applied-Biochemistry. 1991; 13 (2): 231-237.

YEAR:1991

ABSTRACT: The phenolic compounds extracted from olives with ethyl acetate inhibited germination and outgrowth of Bacillus cereus T spores. Purified oleuropein, a well-characterized component of olive extract, inhibited these processes also. The addition of oleuropein and olive extracts 3 or 5 min after germination began, immediately decreased the rate of change of phase bright to phase dark spores and delayed significantly outgrowth.

ANTIFUNGAL

TITLE:In vitro antimicrobial activity of olive leaves

AUTHOR: Markin-D; Duek-L; Berdicevsky-I

INSTITUTE: Department of Microbiology, Rappaport Faculty of Medicine, Technion – Institute of Technology, Haifa

SOURCE:Mycoses-. 2003; 46(3-4):132-136,

YEAR: 2003

ABSTRACT: We investigated the antimicrobial effect of olive leaves against bacteria and fungi. The microorganisms tested were inoculated in various concentrations of olive leaf water extract. Olive leaf 0.6% (w/v) water extract killed almost all bacteria tested, within 3 h. Dermatophytes were inhibited by 1.25% (w/v) plant extract following a 3-day exposure whereas Candida albicans was killed following a 24 h incubation in the presence of 15% (w/v) plant extract. Olive leaf extract fractions, obtained by dialysis, showed antimicrobial activity consisted of particles smaller than 1000 molecular rate cutoffs. Scanning electron microscopic observations of C. albicans, exposed to 40% (w/v) olive leaf extract, showed invaginated and amorphous cells. Escherichia coli cells, subjected to a similar treatment but exposed to only 0.6% (w/v) olive leaf extract showed complete destruction. These findings suggest an antimicrobial potential for olive leaves.

TITLE: Comparative antibacterial and antifungal effects of some phenolic compounds.

AUTHOR: Aziz,-N-H; Farag,-S-E; Mousa,-L-A; Abo-Zaid,-M-A

INSTITUTE:National Centre for Radiation Research and Technology, Nasr City, Cairo, Egypt.

SOURCE: Microbios. 1998; 93(374): 43-54

YEAR:1998

ABSTRACT:The antimicrobial potential of eight phenolic compounds isolated from olive cake was tested against the growth of Escherichia coli, Klebsiella pneumoniae, Bacillus cereus, Aspergillus flavus and Aspergillus parasiticus. The phenolic compounds included p-hydroxy benzoic, vanillic, caffeic, protocatechuic, syringic, and p-coumaric acids, oleuropein and quercetin. Caffeic and protocatechuic acids (0.3 mg/ml) inhibited the growth of E. coli and K. pneumoniae. The same compounds apart from syringic acid (0.5 mg/ml) completely inhibited the growth of B. cereus. Oleuropein, and p-hydroxy benzoic, vanillic and p-coumaric acids (0.4 mg/ml) completely inhibited the growth of E. coli, K. pneumoniae and 8. cereus. Vanillic and caffeic acids (0.2 mg/ml) completely inhibited the growth and aflatoxin production by both A. flavus and A. parasiticus, whereas the complete inhibition of the moulds was attained with 0.3 mg/ml p-hydroxy benzoic, protocatechuic, syringic, and p-coumaric acids and quercetin.

ANTI-INFLAMMATORY

TITLE: Olea europaea L.: stimulant, anti-ulcer and anti-inflammatory effects

AUTHOR:Fehri-B; Aiache-JM; Mrad-S; Korbi-S; Lamaison-JL

INSTITUTE: Dept. of Pharmacol. and Toxicol., Soc. of Pharm. Industries of Tunisia Fondouk Choucha, Rades 2040, Tunisia

SOURCE: Boll-Chim-Farm (Bollettino-Chimico-Farmaceutico); 1996; 135(Jan); 42-49,

YEAR:1996

ABSTRACT: The dried aqueous extract of the leaf of Olea europaea containing 3.25% oleuropein was studied for pharmacological effects using the hole board, open field, rota rod, and automatic reflex conditioner tests. At low doses, the preparation stimulated the CNS, but at high doses it caused CNS depression. The extract induced dose dependent and significant anti-inflammatory effects on carrageenin induced edema in rats. It also provided pronounced protection against aspirin induced gastric ulcers.

TITLE:Oleuropein, the bitter principle of olives, enhances nitric oxide production by mouse macrophages.

AUTHOR: Visioli,-F; Bellosta,-S; Galli,-C

INSTITUTE: Institute of Pharmacological Sciences, Milan, Italy. Francesco.Visioli@unimi.it

SOURCE:Life-Sci. 1998; 62(6): 541-6

YEAR:1998

ABSTRACT: The Mediterranean diet, rich in fresh fruits and vegetables, has been associated with a lower incidence of cardiovascular disease and cancer, partly because of its high proportion of bioactive compounds such as vitamins, flavonoids and polyphenols. The major lipid component of such diet is the drupe-derived olive oil, which can be distinguished from other seed oils for the peculiar composition of its non-triglyceride fraction. In fact, several minor components, including polyphenols, grant the oil its particular taste and aroma. Oleuropein, the most abundant among these components, has been shown to be a potent antioxidant endowed with anti-inflammatory properties. We investigated the effects of oleuropein on NO release in cell culture and its activity toward nitric oxide synthase (iNOS) expression. The results show that oleuropein dose-dependently enhance nitrite production in LPS-challenged mouse macrophages. This effect was blocked by the iNOS inhibitor L-NAME, indicating increased iNOS activity. Also, Western blot analysis of cell homogenates show that oleuropein increases iNOS expression in such cells. Taken together, our data suggest that, during endotoxin challenge, oleuropein potentiates the macrophage-mediated response, resulting in higher NO production, currently believed to be beneficial for cellular and organismal protection.

TITLE: Effect of minor components of virgin olive oil on topical anti­inflammatory assays

AUTHOR:de-la-Puerta-Rocio {a}; Martinez-Dominguez-Eugenia; Ruiz-Gutierrez-Valentina

INSTITUTE: {a} Department of Pharmacology, Faculty of Pharmacy, University of Seville, 41012, Seville: puerta@fafar.us.es, Spain

SOURCE: Zeitschrift-fuer-Naturforschung-Section-C-Journal-of-Biosciences. [print] September-October, 2000; 55 (9-10): 814-819.

YEAR: 2000

ABSTRACT:Interest in the health-promoting effects of virgin olive oil, an important part of the “Mediterranean diet”, prompted us to determine the anti-inflammatory effects of erythrodiol, beta-sitosterol and squalene, identified as major components of the so-called “unsaponifiable fraction” of virgin olive oil, as well as of the phenolic compounds from the “polar fraction”: oleuropein, tyrosol, hydroxytyrosol and caffeic acid. Their activities were compared to those of both, total unsaponifiable and polar fractions. This study was designed to analyse the anti­inflammatory effect of these specific compounds from virgin olive oil on edema in mice induced by either arachidonic acid (AA) or 12-O-tetradecanoylphorbol acetate (TPA). The inhibition of the myeloperoxidase (MPO), marker enzyme of the accumulation of neutrophils in the inflamed tissue, was also investigated by the TPA model. The topical application of the olive oil compounds (0.5 mg/ear) produced a variable degree of anti-inflammatory effect with both assays. In the auricular edema induced by TPA, beta-sitosterol and erythrodiol from the unsaponifiable fraction of the oil showed a potent anti-edematous effect with a 61.4% and 82.1% of inhibition respectively, values not very different to that of the reference indomethacin (85.6%) at 0.5 mg/ear. The four phenolics exerted a similar range of inhibition (33-45%). All compounds strongly inhibited the enzyme myeloperoxidase, indicating a reduction of the neutrophil influx in the inflamed tissues. The strongest inhibitor of AA edema was the total unsaponifiable fraction which inhibition was 34%, similar to that obtained by the reference drug dexamethasone at 0.05 mg/ear. Among the phenolics, oleuropein also produced an inhibition of about 30% with the same dose, but all the other components were found less active in this assay. The anti-inflammatory effects exerted by both unsaponifiable and polar compounds might contribute to the potential biological properties reported for virgin olive oil against different pathological processes.

ANTIOXIDANT & FREE RADICAL SCAVENGER

TITLE: Antioxidant activity of phenolics extracted from Olea europaea L. leaves

AUTHOR:Benavente-Garcia-O; Castillo-J; Lorente-J; Ortuno-A; Del-Rio-JA

INSTITUTE: Research/Development Department, Furfural Espanol S.A., Camino Viejo de Pliego s/n, 80320 Alcantarilla, Murcia

SOURCE: FOOD-CHEM. Food-Chemistry. 2000^-₁68(4):457-462

YEAR: 2000

ABSTRACT: The purpose of this study was to identify the main phenolic compounds present in an olive leaf extract (OL) in order to delineate the differential antioxidant activities of these compounds through the extent of their abilities to scavenge the ABTS(^.+) radical cation and to clarify the structural elements conferring antioxidant capacity in aqueous systems. The results show that the relative abilities of the flavonoids from olive leaf to scavenge the ABTS(^.+) radical cation are influenced by the presence of functional groups in their structure, mainly the B-ring catechol, the 3-hydroxyl group and the 2,3-double bond conjugated with the 4-oxo function. For the other phenolic compounds present in OL, their relative abilities to scavenge the ABTS(^.+) radical cation are mainly influenced by the number and position of free hydroxyl groups in their structure. Also, both groups of compounds show synergic behaviour when mixed, as occurs in the OL. Copyright (C) 2000 Elsevier Science Ltd.

TITLE: In vitro evaluation of the antioxidant activity and biomembrane interaction of the plant phenols oleuropein and hydroxytyrosol

AUTHOR: Saija-A; Trombetta-D; Tomaino-A; Lo-Cascio-R; Castelli-F; et-al

INSTITUTE: Dept. Farmaco-Biol., Sch. of Pharm., Univ. of Messina, 98168 Messina, Italy SOURCE: Int-J-Pharm (International-Journal-of-Pharmaceutics); 1998; 166(May 18); 123-133

YEAR: 1998

ABSTRACT: The scavenging activity against the stable 1,1-diphenyl-2-picrylhydrazyl free radical, the antioxidant activity in a model system consisting of dipalmitoyllecithin (dipalmitoylphosphatidylcholine)/linoleic acid unilamellar vesicles and a water soluble azo compound as a free radical generator, and the biomembrane interaction with dimyristoyllecithin (dimyristoylphosphatidylcholine) vesicles of the plant phenols oleuropein and 3,4­dihydroxyphenethyl alcohol (hydroxytyrosol) were studied in vitro. The results showed that both oleuropein and hydro.

TITLE:Olea europaea L. leaf extract and derivatives: antioxidant properties.

AUTHOR: Briante,-R; Patumi,-M; Terenziani,-S; Bismuto,-E; Febbraio,-F; Nucci,-R Correspondence (Reprint) address, R. Nucci, Istituto di Biochimica delle Proteine ed

INSTITUTE: Enzimologia del CNR, Via Marconi 10, 80125 Naples, Italy.     Tel./Fax +39-081­7257300.      E-mail nucci@dafne.ibpe.na.cnr.it

SOURCE: Journal-of-Agricultural-and-Food-Chemistry. 2002; 50(17): 4934-4940 ; 44 ref,

YEAR: 2002

ABSTRACT: A simple and fast method to collect eluates containing high amounts of hydroxytyrosol from Olea europaea (olive cv. Moraiolo) leaf extract is described. Biotransformation of O. europaea leaf extract was carried out using a thermophilic beta­glycosidase immobilized on chitosan, and some of the phenols in the leaf tissue and in the eluates were identified.Antioxidative activity of the eluates was compared with that of the leaf extract from which they originated.The eluates possessed a higher concentration of simple phenols (characterized by stronger antioxidative activity) than those in extra virgin olive oils and in many commercial olive leaf extract tablets.

TITLE: Anti hypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies africana leaves.

AUTHOR:Somova,-LI; Shode,-FO; Ramnanan,-P; Nadar,-A

SOURCE: J-Ethnopharmacol. 2003 Feb; 84(2-3): 299-305,

YEAR: 2003

ABSTRACT: For the first time a biossay-directed study of triterpenoids isolated from the leaves of Olea europaea from Greece, from wild African olive and from a cultivar of O. europaea grown in Cape Town was reported. The experiment was undertaken since our preliminary analyses showed that the African wild olive leave is rich in triterpenoids and contain only traces of the glycoside oleuropein, which is typical for the European olive leaves.The isolate of the African wild olive leaves (AO) used in the experiments was found to contain 0.27% 1:1 mixture of oleanolic acid and ursolic acid, named oleuafricein.The isolate of Greek olive leaves (GO) was found to contain 0.71% oleanolic acid, and the Cape Town cultivar (CT) contained 2.47% oleanolic acid.No ursolic acid was found in either GO or CT.The antihypertensive, diuretic, antiatherosclerotic, antioxidant and hypoglycemic effects of authentic oleanolic and ursolic acid and the three isolates (GO, AO and CT) were studied on Dahl salt-sensitive (DSS), insulin­resistant rat genetic model of hypertension. All three isolates, in a dose 60 mg/kg b.w. for 6 weeks treatment, prevented the development of severe hypertension and atherosclerosis and improved the insulin resistance of the experimental animals.  GO, OA and CT isolates could provide an effective and cheap treatment of this particular, most common type of salt-sensitive hypertension in the African population. xytyroso.

TITLE: Bioactive derivatives from oleuropein by a biotransformation on Olea europaea leaf extracts.

AUTHOR:Briante,-R; La-Cara,-F; Febbraio,-F; Patumi,-M; Nucci,-R

INSTITUTE: Istituto di Biochimica delle Proteine ed Enzimologia del Consiglio Nazionale delle Ricerche, Via Marconi 10, 80125 Napoli, Italy.

SOURCE: J-Biotechnol. 2002 Feb 14; 93(2): 109-19

YEAR:2002

ABSTRACT: A very simple method is proposed to produce, using non-homogeneous hyperthermophilic beta-glycosidase immobilised on chitosan, 3,4-dihydroxy-phenylethanol (hydroxytyrosol), a commercially unavailable compound with well known biological properties which justify a potential commercial application. Leaf extracts from Olea europaea with high oleuropein content are selected as substrate for biotransformation. Under the biotransformation conditions, high amounts of hydroxytyrosol are collected within a short space of time after being preliminarily purified by a non-treated chitosan column. This is possible due to the capacity of amino groups on the chitosan to bind aldehydic groups of molecules present at the end of the reaction. We have produced a natural and non-toxic product from vegetal source, as opposed to the molecule obtainable through chemical synthesis, as a candidate to test in vivo its biological properties. The proposed process may prove useful for a further application for recycling Olea europaea leaves. The radical-scavenging properties of the bioreactor eluates and their capacity to inhibit fatty acid peroxidation rates are characterized in order to make them candidates as substitutes for synthetic antioxidants commonly used to increase the shelf-life of food products as well as for their possible protective effect in human cells.

TITLE: Isolation and characterization of the antioxidant component 3,4­dihydroxyphenylethyl 4-formyl-3-formylmethyl-4-hexenoate from olive (Olea europaea) leaves.

AUTHOR:Paiva-Martins,-F; Gordon,-M-H

INSTITUTE: Centro de Investigacao em Quimica, Departamento de Quimica, Faculdade de Ciencias, Universidade do Porto, Rua do Campo Alegre, number 687, 4169-007 Porto,Portugal

SOURCE: J-Agric-Food-Chem. 2001 Sep; 49(9): 4214-9,

YEAR: 2001

ABSTRACT:Storage of olive (Olea europaea) leaves for 22 h at 37 degrees C in closed plastic bags caused the content of a nonglycosidic secoiridoid, 3,4-dihydroxyphenylethyl 4-formyl-3­formylmethyl-4-hexenoate (3,4-DHPEA-EDA) to rise from 15% to 50% of the phenolic extract with corresponding falls in the content of oleuropein and two oleuropeindials, which were identified as precursors of 3,4-DHPEA-EDA. Pure product was isolated from one set of stored olive leaves in a 0.16% yield. Storage of olive leaves under various conditions showed that the moisture present in closed plastic bags was important for the formation of 3,4-DHPEA-EDA. The time taken to reach the maximum concentration of the product varied widely for different.
l showed good free scavenging activity and antioxidant effects. samples of olive leaves, with a shorter time for the sample with lower initial oleuropein content. The oleuropeindial precursors of the product were readily hydrolyzed to carboxylic acid derivatives, which have been identified by NMR. The antiradical activity of 3,4-DHPEA-EDA, evaluated by scavenging of 2,2-diphenyl-1-picrylhydrazyl radicals, was comparable to that of alpha-tocopherol.

TITLE: Protective effect of oleuropein, an olive oil biophenol, on low density lipoprotein oxidizability in rabbits.

AUTHOR: Coni,-E; Di-Benedetto,-R; Di-Pasquale,-M; Masella,-R; Modesti,-D; Mattei,-R; Carlini,-E-A

INSTITUTE: Food Department, Istituto Superiore di Sanita, Rome, Italy. e.coni@iss.it

YEAR:2000

ABSTRACT:On the basis of the results obtained with pilot studies conducted in vitro on human low density lipoprotein (LDL) and on cell cultures (Caco-2), which had indicated the ability of certain molecules present in olive oil to inhibit pro-oxidative processes, an in vivo study was made of laboratory rabbits fed special diets. Three different diets were prepared: a standard diet for rabbits (diet A), a standard diet for rabbits modified by the addition of 10% (w/w) extra virgin olive oil (diet B), a modified standard diet for rabbits (diet C) differing from diet B only in the addition of 7 mg kg(-1) of oleuropein. A series of biochemical parameters was therefore identified, both in the rabbit plasma and the related isolated LDL, before and after Cu-induced oxidation. The following, in particular, were selected: (i) biophenols, vitamins E and C, uric acid, and total, free, and ester cholesterol in the plasma; (ii) proteins, triglycerides, phospholipids, and total, free, and ester cholesterol in the native LDL (for the latter, the dimensions were also measured); (iii) lipid hydroperoxides, aldehydes, conjugated dienes, and relative electrophoretic mobility (REM) in the oxidized LDL (ox-LDL). In an attempt to summarize the results obtained, it can be said that this investigation has not only verified the antioxidant efficacy of extra virgin olive oil biophenols and, in particular, of oleuropein, but has also revealed a series of thus far unknown effects of the latter on the plasmatic lipid situation. In fact, the addition of oleuropein in diet C increased the ability of LDL to resist oxidation (less conjugated diene formation) and, at the same time, reduced the plasmatic levels of total, free, and ester cholesterol (-15, -12, and – 17%, respectively), giving rise to a redistribution of the lipidic components of LDL (greater phospholipid and cholesterol amounts) with an indirect effect on their dimensions (bigger by about 12%).

TITLE:Oleuropein evaluated in vitro and in vivo as an antioxidant

AUTHOR:Speroni-E; Guerra-MC; Minghetti-A; Crespi-Perellino-N; Pasini-P; Piazza-F; Roda-A

INSTITUTE:Department of Pharmacology, Bologna University, Via Irnerio, 48, 40126 Bologna

SOURCE:Phytotherapy-Research. 1998; 12(SUPPL. 1): S98-S100,

YEAR: 1998

ABSTRACT: Oleuropein is a phenolic compound extracted from the leaves of Olea europaea. The antioxidant activity of this natural product has been evaluated in vitro and in vivo by means of a chemiluminescent assay, based on a luminol-horseradish peroxidase p-iodophenol O<inf>2</inf> mediated light emission system. In vitro, oleuropein had a remarkable antioxidant activity. Serum samples obtained from treated animals exhibited an antioxidant activity comparable to controls. Bile samples obtained from the same treated animals showed a significant inhibition (90%) of the light emission in the chemiluminescent assay, when compared with the control sample.

TITLE: Effect of phenolic compounds of virgin olive oil on LDL oxidation resistance.

AUTHOR: Moreno-JA; Lopez-Miranda-J; Gomez-P; Fatiha-Benkhalti; EI-Boustani-E; Perez­Jimenez-F

INSTITUTE: Unidad de Lipidos y Arteriosclerosis, Hospital Universitario Reina Sofia, Avda. Menendez Pidal, s/n. 14004 Cordoba, Spain.

SOURCE:Medicina-Clinica-Barcelona. 2003, 120: 4, 128-131; 41 ref.

YEAR:2003

ABSTRACT: Background and objective: Several epidemiological and experimental studies have associated the intake of antioxidants, which are abundant in the Mediterranean diet, with a low incidence of cardiovascular disease. One possible mechanism of this action is the oxidative protection in low density lipoproteins (LDL). The aim of our study was to compare the antioxidative activity of diverse phenolic compounds present in virgin olive oil on these lipoproteins. Subjects and method: LDL was isolated from blood plasma of healthy volunteers by sequential ultracentrifugation. This was followed by oxidation with CuCl2 in the presence of different concentrations of phenolic compounds and virgin olive oil extract. Production of conjugated dienes was determined by the continuous monitoring of increased absorbency at 234 nm as an indicator of LDL oxidation. Results: Virgin olive oil extract prolonged the latency phase and significantly lowered the progression rate (p < 0.05) at low concentrations (2 Ng/ml). This antioxidative effect was also observed with low concentrations (2 NM) of caffeic acid and oleuropein (p < 0.05). However, it was necessary to increase the concentration of flavone up to 50 times to observe a similar effect (p < 0.05). Conclusion: Both virgin olive oil extract enriched in phenolic compounds and phenolic compounds present in olive oil (caffeic acid and oleuropein) are potent antioxidants at very low concentrations. Thus, the beneficial effects of a Mediterranean diet may be partly due to the protective action of these compounds.

TITLE: Antioxidant and other biological activities of phenols from olives and olive oil.

AUTHOR: Visioli-F; Poli-A; Galli-C

INSTITUTE: Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan,Italy.

SOURCE:Medicinal-Research-Reviews. 2002, 22: 1, 65-75; 68 ref.

YEAR: 2002

ABSTRACT:Olive oil is the principal source of fat in the Mediterranean diet, which has been associated with a lower incidence of coronary heart disease and certain cancers. Phenolic compounds, e.g., hydroxytyrosol and oleuropein, in extra-virgin olive oil are responsible for its peculiar pungent taste and for its high stability. Recent findings demonstrate that olive oil phenolics are powerful antioxidants, both in vitro and in vivo, and possess other potent biological activities that could partially account for the observed healthful effects of the Mediterranean diet.

TITLE: Antioxidative activities of Olea europaea leaves and related phenolic compounds.

AUTHOR:Le-Tutour,-B.; Guedon,D.

SOURCE: Phytochemistry. Oxford : Pergamon Press. Apr 1992. v. 31 (4) p. 1173-1178.

YEAR:1992

ABSTRACT: Olea europaea leaves, oleuropein, hydroxytyrosol and tyrosol were compared with vitamin E and BHT, with regard to their antioxidative activities, by kinetic studies in model systems. The thermal initiated oxidation of methyl linoleate was performed in homogeneous solution at 60 degrees or 40 degrees with or without antioxidants. Olea europaea extracts, oleuropein and hydroxytyrosol were much more effective than BHT or vitamin E in extending the induction period. Assuming a stoichiometric factor f=2 for BHT, this coefficient of inhibition reached 5 for hydroxytyrosol, 7 for oleuropein and 10 for 0. europaea extracts. These extracts owe their antioxidative properties to their high oleuropein content (19% w/w) and also to a lesser amount of flavonoids (1.8% w/w with 0.8% of luteolin 7-glucoside). Tyrosol showed neither antioxidant nor pro-oxidant activity. No synergistic effect on the preservation of methyl linoleate was found when vitamin E was used together with tyrosol or O. europaea extracts.

TITLE:GC-MS evaluation of phenolic compounds in virgin olive oil.

AUTHOR:Angerosa,-F.; d’Alessandro,-N.; Konstantinou,-P.; Di-Giacinto,-L.

SOURCE: J-agric-food-chem. Washington, D.C. : American Chemical Society. July 1995. v. 43 (7) p. 1802-1807

YEAR:1995

ABSTRACT: A new gas chromatographic method for detection of phenolic compounds in virgin olive oils was developed. Identification of chromatographic peaks was made by mass selective detection. The presence of a main peak at m/z 192 or at m/z 280, related only to tyrosol and hydroxytyrosol, evident in the mass spectra of linked phenols, was very profitable for assigning the phenolic nature to minor polar compounds extracted by methanol from virgin olive oil. Twelve structures are possible, and some of them are deemed more likely on the basis of the chemical behavior of the compounds studied. The presence of a ligstrosid aglycon containing no carbomethoxy group and of oleuropein aglycon derivatives was evidenced. The levels of these substances is a very important parameter in the evaluation of virgin olive oil quality since phenols are strictly related both to the oil’s resistance to oxidation because of their antioxidative properties (Montedoro, 192; Papadopoulos and Boskou, 1991) and to the typical bitter taste of olive oil (Angerosa and Solinas, 1990; Olias, 1992). Furthermore, some studies showed that the amount of phenolic substances present, just as the fatty acid composition, is related to the health beneficial effects that make virgin oil a very valuable and appreciated commodity (Panizzi et al., 1960; Galli et al., 1992).

TITLE: Antioxidant activity of virgin olive oil phenolic compounds in a micellar system.

AUTHOR: Fogliano,-V; Ritieni,-A.; Monti,-S.M.; Gallo,-M.; Della-Medaglia,-D.; Ambrosino,­M.L.; Sacchi,-R

SOURCE: J-sci-food-agric. West Sussex: John Wiley & Sons Limited. Oct 1999. v. 79 (13) p. 1803-1808.

YEAR:1999

ABSTRACT: The antioxidant activity of two virgin olive oils, obtained from the same olive (Olea europaea sativa) batch but processed with different hammer crushing conditions, was evaluated by measuring their protective action towards linoleic acid peroxidation in a micellar system. The antioxidant efficiency (AE) of the oil phenolic fraction was higher when the olives were processed with a higher hammer crusher rotation rate. HPLC analysis demonstrated that one of the main derivatives of oleuropein, indicated as 3,4-DHPEA-EDA (the dialdehydic form of elenolic acid linked with 3,4-dihydroxyphenylethanol), is present only in the oil with higher AE. 3,4-DHPEA-EDA showed the greatest antioxidant ability among virgin olive oil phenols. Its greater efficiency in the micellar system in comparison with 3,4-dihydroxyphenylethanol (3,4­DHPEA) is related to its greater lipophilicity. It is suggested that the behaviour in the water­micellar environment is representative of that in a real system such as tomato-based sauce with added virgin olive oil.

TITLE:Effect of virgin olive oil phenolic compounds on in vitro oxidation of human low density lipoproteins.

AUTHOR:Caruso,-D; Berra,-B; Giavarini,-F; Cortesi,-N; Fedeli,-E; Galli,-G

INSTITUTE:Institute of Pharmacological Sciences, University of Milan, Italy.

SOURCE:Nutr-Metab-Cardiovasc-Dis. 1999 Jun; 9(3): 102-7

YEAR:1999

ABSTRACT:BACKGROUND AND AIM: Substantial evidence suggests that oxidative modifications of low density lipoproteins (LDL) critically contribute to the pathogenesis and progression of human atherosclerosis. Oxidized LDL (oxLDL) are present in atherosclerotic plaques and contain oxysterols that exhibit a variety of adverse biological activities. Antioxidants have also been shown to prevent LDL modification. We have therefore assessed the efficacy of virgin olive oil phenolic compounds in preventing oxidative modifications of human LDL oxidized by UV light.
METHODS AND RESULTS: Cholesterol oxides formed during LDL photo-oxidation were determined  by UV HPLC in the presence of different concentrations of phenolic compounds and their pure components (tyrosol and oleuropein), and probucol, a widely used synthetic antioxidant. Electrophoretic mobility was also assayed. The results demonstrate that phenolic compounds are much more potent in preventing cholesterol oxide formation and apoproteic moiety modification than their pure components and probucol.
CONCLUSIONS: The beneficial effects of a Mediterranean diet may be ascribable not only to the high unsaturated/saturated fatty acid ratio characteristic of olive oil, but also to the unique antioxidant properties of its phenolic compounds.

TITLE:Oleuropein, the bitter principle of olives, enhances nitric oxide production by mouse macrophages.

AUTHOR: Visioli,-F; Bellosta,-S; Galli,-C

INSTITUT: Institute of Pharmacological Sciences, Milan, Italy. Francesco.Visioli@unimi.it

SOURCE : Life-Sci. 1998; 62(6): 541-6

YEAR:1998

ABSTRACT: The Mediterranean diet, rich in fresh fruits and vegetables, has been associated with a lower incidence of cardiovascular disease and cancer, partly because of its high proportion of bioactive compounds such as vitamins, flavonoids and polyphenols. The major lipid component of such diet is the drupe-derived olive oil, that can be distinguished from other seed oils for the peculiar composition of its non-trig lyceride fraction. In fact, several minor components, including polyphenols, grant the oil its particular taste and aroma. Oleuropein, the most abundant among these components, has been shown to be a potent antioxidant endowed with anti-inflammatory properties. We investigated the effects of oleuropein on NO release in cell culture and its activity toward nitric oxide synthase (iNOS) expression. The results show that oleuropein dose-dependently enhance nitrite production in LPS-challenged mouse macrophages. This effect was blocked by the iNOS inhibitor L-NAME, indicating increased iNOS activity. Also, Western blot analysis of cell homogenates show that oleuropein increases iNOS expression in such cells. Taken together, our data suggest that, during endotoxin challenge; oleuropein potentiates the macrophage-mediated response, resulting in higher NO production, currently believed to be beneficial for cellular and organismal protection.

TITLE: Olive-oil consumption and health: the possible role of antioxidants.

AUTHOR:Owen,-R-W; Giacosa,-A; Hull,-W-E; Haubner,-R; Wurtele,-G; Spiegelhalder,-B; Bartsch,-H

INSTITUTE:Division of Toxicology and Cancer Risk Factors, German Cancer Research Center, Heidelberg. R.Owen@DKFZ-Heidelberg. DE

SOURCE: Lancet-Oncol. 2000 Oct; 1: 107-12

YEAR: 2000

ABSTRACT: In the Mediterranean basin, olive oil, along with fruits, vegetables, and fish, is an important constituent of the diet, and is considered a major factor in preserving a healthy and relatively disease-free population. Epidemiological data show that the Mediterranean diet has significant protective effects against cancer and coronary heart disease. We present evidence that it is the unique profile of the phenolic fraction, along with high intakes of squalene and the monounsaturated fatty acid, oleic acid, which confer its health-promoting properties. The major phenolic compounds identified and quantified in olive oil belong to three different classes: simple phenols (hydroxytyrosol, tyrosol); secoiridoids (oleuropein, the aglycone of ligstroside, and their respective decarboxylated dialdehyde derivatives); and the lignans [(+)-1­acetoxypinoresinol and pinoresinol]. All three classes have potent antioxidant properties. High consumption of extra-virgin olive oils, which are particularly rich in these phenolic antioxidants (as well as squalene and oleic acid), should afford considerable protection against cancer (colon, breast, skin), coronary heart disease, and ageing by inhibiting oxidative stress.

TITLE:The antioxidantlanticancer potential of phenolic compounds isolated from olive oil

AUTHOR:Owen-RW; Giacosa-A; Hull-WE; Haubner-R; Spiegelhalder-B; Bartsch-H

INSTITUTE:Division of Toxicology, Cancer Research Centre, Im Neuenheimer Feld 280, D­69120 Heidelberg

SOURCE:EUR-J-CANCER. European-Journal-of-Cancer. 2000; 36(10): 1235-1247

YEAR: 2000

ABSTRACT:In our ongoing studies on the chemoprevention of cancer we have a particular interest in the health benefits of the Mediterranean diet, of which olive oil is a major component. Recent studies have shown that extra virgin olive oil contains an abundance of phenolic antioxidants including simple phenols (hydroxytyrosol, tyrosol), aldehydic secoiridoids, flavonoids and lignans (acetoxypinoresinol, pinoresinol). All of these phenolic substances are potent inhibitors of reactive oxygen species attack on, e.g. salicylic acid, 2-deoxyguanosine. Currently there is growing evidence that reactive oxygen species are involved in the aetiology of fat-related neoplasms such as cancer of the breast and colorectum. A plausible mechanism is a high intake of < omega >-6 polyunsaturated fatty acids which are especially prone to lipid peroxidation initiated and propagated by reactive oxygen species, leading to the formation (via < alpha >,< beta >-unsaturated aldehydes such as trans-4-hydroxy-2-nonenal) of highly pro­ mutagenic exocyclic DNA adducts. Previous studies have shown that the colonic mucosa of cancer patients and those suffering from predisposing inflammatory conditions such as ulcerative colitis and Crohn’s disease generates appreciably higher quantities of reactive oxygen species compared with normal tissue. We have extended these studies by developing accurate high performance liquid chromatography (HPLC) methods for the quantitation of reactive oxygen species generated by the faecal matrix. The data shows that the faecal matrix supports the generation of reactive oxygen species in abundance. As yet, there is a dearth of evidence linking this capacity to actual components of the diet which may influence the colorectal milieu. However, using the newly developed methodology we can demonstrate that the antioxidant phenolic compounds present in olive oil are potent inhibitors of free radical generation by the faecal matrix. This indicates that the study of the inter-relation between reactive oxygen species and dietary antioxidants is an area of great promise for elucidating mechanisms of colorectal carcinogenesis and possible future chemopreventive strategies. Copyright (C) 2000 Elsevier Science Ltd.

TITLE: Free radical-scavenging properties of olive oil polyphenols

AUTHOR:Visioli-F; Bellomo-G; Galli-C

INSTITUTE:Department of Biomedical Sciences, University of Torino, Novara

SOURCE: BIOCHEM-BIOPHYS-RES-COMMUN. Biochemical-and-Biophysical-Research­Communications. 1998; 247(1): 60-64

YEAR:1998

ABSTRACT:Plants in the Mediterranean basin, such as vine and olive trees, have developed an array of antioxidant defences to protect themselves from environmental stress. Accordingly, the incidence of coronary heart disease and certain cancers is lower in the Mediterranean area, where olive oil is the dietary fat of choice. As opposed to other vegetable oils, extra virgin olive oil, which is obtained by physical pressure from a whole fruit, is rich in phenolic components that are responsible for the particular stability of the oil. We have investigated the scavenging actions of some olive oil phenolics, namely hydroxytyrosol and oleuropein, with respect to superoxide anion generation, neutrophils respiratory burst, and hypochlorous acid. The low EC<inf>50</inf>s indicate that both compounds are potent scavengers of superoxide radicals and inhibitors of neutrophils respiratory burst: whenever demonstrated in vivo, these properties may partially explain the observed lower incidence of CHD and cancer associated with the Mediterranean diet.

TITLE: Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: Antiatherogenic properties of Mediterranean diet phytochemicals

AUTHOR:Carluccio-MA; Siculella-L; Ancora-MA; Massaro-M; Scoditti-E; Storelli-C; Visioli-F; Distante-A; De-Caterina-R

INSTITUTE:C.N.R. Inst. of Clinical Physiology, Lecce

SOURCE:ARTERIOSCLER-THROMB-VASC-BIOL. Arteriosclerosis,-Thrombosis,-and­Vascular-Biology. 2003; 23(4): 622-629

YEAR:2003

ABSTRACT: Objective – Epidemiology suggests that Mediterranean diets are associated with reduced risk of cardiovascular disease. Because monocyte adhesion to the endothelium is crucial in early atherogenesis, we evaluated whether typical olive oil and red wine polyphenols affect endothelial-leukocyte adhesion molecule expression and monocyte adhesion. Methods and Results – Phytochemicals in olive oil and red wine, including oleuropein, hydroxytyrosol, tyrosol, elenolic acid, and resveratrol, with or without antioxidant activity, were incubated with human umbilical vein endothelial cells for 30 minutes, followed by co-incubation with bacterial lipopolysaccharide or cytokines to trigger adhesion molecule expression. At nutritionally relevant concentrations, only oleuropein, hydroxytyrosol, and resveratrol, possessing a marked antioxidant activity, reduced monocytoid cell adhesion to stimulated endothelium, as well as vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein by Northern analysis and cell surface enzyme immunoassay. Reporter gene assays with deletional VCAM-1 promoter constructs indicated the relevance of nuclear factor-< kappa >B, activator protein- 1, and possibly GATA binding sites in mediating VCAM- 1 transcriptional inhibition. The involvement of nuclear factor-< kappa >B and activator protein-1 was finally demonstrated at electrophoretic mobility shift assays. Conclusions – Olive oil and red wine antioxidant polyphenols at nutritionally relevant concentrations transcriptionally inhibit endothelial adhesion molecule expression, thus partially explaining atheroprotection from Mediterranean diets.

ANTIVIRAL

TITLE: New applications of herbal extracts for functional food and pharmaceuticals. Part 2.

AUTHOR: Micol,-V; Estepa,-A; Caturla,-N; Perez-Fons,-L; Saura,-D; Ferrer-Montiel,-A; Cartagena,-V

INSTITUTE:Inst. de Biol. Molecular y Cellular, Univ. Miguel Hernandez, E-03202 Elche (Alicante), Spain

SOURCE: Agro-Food-Industry-hi-tech. 2003; 14(6): 14-16 ; 15 ref.

YEAR: 2003

ABSTRACT: In this 2nd part of a 2-part article on use of plant extracts in functional foods and pharmaceuticals, application of natural antiviral supplements (especially olive leaf extract) in foods for animals and the role of polyphenols from herbal sources as neuroprotective agents in functional foods for humans are discussed.

TITLE: In vitro evaluation of secoiridoid glucosides from the fruits of Ligustrum lucidum as antiviral agents.

AUTHOR: Ma-Shuang-Cheng; He-Zhen-Dan; Deng-Xue-Long; But-Paul-Pui-Hay {a}; Ooi­Vincent-Eng-Choon; Xu-Hong-Xi; Lee-Spencer-Hon-Sun; Lee-Song-Fong

INSTITUTE: {a} Department of Biology and Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong; E-Mail: paulbut@cuhk.edu.hk, China

SOURCE: Chemical-and-Pharmaceutical-Bulletin-Tokyo. [print] November, 2001; 49 (11): 1471-1473.

YEAR: 2001

ABSTRACT: Six secoiridoid glucosides, lucidumoside C (1), oleoside dimethylester (2), neonuezhenide (3), oleuropein (4), ligustroside (5) and lucidumoside A (6), isolated from the fruits of Ligustrum lucidum (Oleaceae), were examined in vitro for their activities against four strains of pathogenic viruses, namely herpes simplex type 1 virus (HSV 1), influenza type A virus (Flu A), respiratory syncytial virus (RSV) and parainfluenza type 3 virus (Para 3). Antiviral activities were evaluated by the cytopathic effect (CPE) inhibitory assay. The purpose was to check if the anti-oxidative potency of these glucosides correlated with their antiviral potency. Results showed that none of the glucosides had any significant activity against HSV 1 and Flu A. Oleuropein, however, showed significant antiviral activities against RSV and Para 3 with IC50 value of 23.4 and 11.7 mug/ml, respectively. Lucidumoside C, oleoside dimethylester and ligustroside showed potent or moderate antiviral activities against Para 3 with IC50 values of 15.6-20.8 mug/ml. These results also documented that the anti-oxidative potency of these secoiriodoid glucosides was not directly related to their antiviral effects.

TITLE: In Vitro Antiviral Activity of Calcium Elenolate

AUTHOR: Renis, H.

INSTITUTE: Department of Virology Research, The Upjohn Co., Kalamazoo, Michigan 49001

ABSTRACT:Calcium Elenolate [(C_llH_l30₆)_ZCaI] is a multifunctional monoterpene which can be isolated from acid-hydrolyzed aqueous extracts of various parts of olive plants. This compound has been shown to be virucidal for all viruses against which it has been tested No change in infectivity was noted when infectious ribonucleic acid (prepared from coxsackievirus A21) was incubated with calcium Elenolate.Incubation of calcium Elenolate with influenza A (PR8) virus resulted in loss of infectivity at concentrations which caused no observable change in hemagglutination titer or neuraminidase activity. The virucidal activity of this compound was shown go be greatest under alkaline conditions )_pH 7.5)  The quantity of virus inactivated was dependent on the calcium Elenolate concentration and the time of incubation. Incubation of calcium Elenolate with several nucleic acid constituents prior to the addition of virus caused no alteration in virucidal activity However, when the incubation was carried out with amino acids prior to incubation with virus, losses in virucidal activity were detected with glycine, lysine, cysteine, and histidine, nd to a lesser extend with phenylalanine, tryptophan, serine, and threonine Pretreating cells with calcium eleolate did not alter coxsackievirus A21 attachment.

TITLE: Antiviral Activity of Calcium Elenolate of Parainfluenza Infection of Hamsters

AUTHOR:Soret, M. G.

SOURCE: Antimicrobial Agents and Chemotherapy – 1969, Copyright ©1970 American Society for Microbiology

INSTITUTE: Department of Virology Research, The Upjohn Co., Kalamazoo, Michigan 49001

ABSTRACT: The in vivo antiviral activity of calcium Elenolate was demonstrated in hamsters infected with parainfluenza 3 virus. The drug was virucidal when given within minutes of the viral inoculation. Calcium Elenolate also showed a “therapeutic” effect when given 8 hr after infection. This antiviral activity, after establishment of the infection, reduced the severity of the infectious process.The minimal effective dose of calcium Elenolate produced only minimal histological changes in the sensitive olfactory epithelium of the hamster while exerting significant therapeutic antiviral activity. The effects obtained suggest that calcium Elenolate may affect viral components of cellular origin in both free and cell-associated virus.

TITLE: Specificity of the Antiviral Agent Calcium Elenolate

AUTHOR:Heinze, J. E., Hale, &A., Carl, P.

ABSTRACT: Calcium elenolate, an antiviral agent which inhibits reverse transcriptases, inhibits the growth of chicken embryo fibroblast cells, as well as Echerichia coli and Bacillus Subtilis strains.The drug in vitro inhibits E. coli deoxyribonucleic acid (DNA) polymerase 111 holoenzyme, as well as several unrelated enzymes. The usual DNA polymerase assay components, with the exception of spermidine, have no effect on the observed inhibition. Inhibition of DNA polymerase 11 by the drug appears to be due to a direct and irreversible effect on the enzyme. However, DNA synthesis in E. coli is no more susceptible to the drug than is the increase in cell mass. These results suggest that calcium Elenolate is an inhibitor of rather low specificity.

BLOOD PRESSURE (HYPERTENSION)

TITLE: Anti hypertensive,antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies africana leaves

AUTHOR: Somova,-LI; Shode,-FO; Ramnanan,-P; Nadar,-A 

SOURCE: J-Ethnopharmacol. 2003 Feb; 84(2-3): 299-305,

YEAR: 2003

ABSTRACT: For the first time a biossay-directed study of triterpenoids isolated from the leaves of Olea europaea from Greece, from wild African olive and from a cultivar of O. europaea grown in Cape Town was reported.  The experiment was undertaken since our preliminary analyses showed that the African wild olive leave is rich in triterpenoids and contain only traces of the glycoside oleuropein, which is typical for the European olive leaves. The isolate of the African wild olive leaves (AO) used in the experiments was found to contain 0.27% 1:1 mixture of oleanolic acid and ursolic acid, named oleuafricein. The isolate of Greek olive leaves (GO) was found to contain 0.71% oleanolic acid, and the Cape Town cultivar (CT) contained 2.47% oleanolic acid.No ursolic acid was found in either GO or CT.The anti hypertensive, diuretic, antiatherosclerotic, antioxidant and hypoglycemic effects of authentic oleanolic and ursolic acid and the three isolates (GO, AO and CT) were studied on Dahl salt-sensitive (DSS), insulin­resistant rat genetic model of hypertension. All three isolates, in a dose 60 mg/kg b.w. for 6 weeks treatment, prevented the development of severe hypertension and atherosclerosis and improved the insulin resistance of the experimental animals. GO, OA and CT isolates could provide an effective and cheap treatment of this particular, most common type of salt-sensitive hypertension in the African population.

TITLE: Blood pressure lowering effect of an olive leaf extract (Olea europaea) in L-NAME induced hypertension in rats.

AUTHOR: Khayyal,-M-T; el-Ghazaly,-M-A; Abdallah,-D-M; Nassar,-N-N; Okpanyi,-S-N; Kreuter,-M-H

INSTITUTE:  Department of Pharmacology, Faculty of Pharmacy, Cairo University, Cairo, Egypt. mtkhayyal@hotmail.com

SOURCE: Arzneimittelforschung. 2002; 52(11): 797-802,
  
YEAR: 2002

ABSTRACT: A specially prepared olive leaf extract (EFLA 943) has been tested for its blood pressure lowering activity in rats rendered hypertensive by daily oral doses of L-NAME (NG­nitro-L-arginine methyl ester, 50 mg/kg) for at least 4 weeks. Oral administration of the extract at different dose levels at the same time as L-NAME for a period of 8 weeks showed a dose dependent prophylactic effect against the rise in blood pressure induced by L-NAME, best effects being induced by a dose of 100 mg/kg of the extract. In rats previously rendered hypertensive by L-NAME for 6 weeks and then treated with that dose of the extract for a further 6 weeks without discontinuation of L-NAME, normalisation of the blood pressure was observed. The findings confirm previous reports on the hypotensive effects of olive leaf. The special extract, EFLA 943, was shown to give consistent results with little individual variability. The anti hypertensive effect of the extract may be related to a variety of factors involving reversal of vascular changes involved in the L-NAME induced hypertension.

TITLE: Vasodilator effect of olive leaf.

AUTHOR:Zarzuelo A, Duarte J, Jimenez J, Gonzalez M, Utrilla MP.

INSTITUTE:Department of Pharmacology, School of Pharmacy, University of Granada, Spain.

ABSTRACT: We studied the importance of the smooth vascular muscle endothelium in the vasodilator action of the decoction of olive (Olea europaea) leaf. The decoction caused relaxation of isolated rat aorta preparations both in the presence (IC50 1.12 +/- 0.33 mg/ml) and in the absence (IC50 1.67 +/- 0.16 mg/ml) of endothelium. The results indicate that the relaxant activity of the lyophilized decoction is independent of the integrity of the vascular endothelium. We also showed that oleuropeoside is a component responsible for vasodilator activity but, from the results, it seems likely that at least one other principle is to be found in the olive leaf which is either a vasodilator itself or else potentiates the relaxant effect of oleuropeoside.

TITLE: Pharmaco-chemical studies of Olea in the Azerbaijan Soviet Socialist Republic

AUTHOR:Movsumov-IS; Aliev-AM; Tagieva-ZD

SOURCE:Farmatsiya-Moscow (Farmatsiya); 1987; 36(2); 32-34

YEAR:1987

ABSTRACT:The hypotensive effects of flavonoids extracted from the leaves and fruits of varieties of Olea europaea found in the Azerbaijan Soviet Socialist Republic were studied. The pharmacological effects of the extracts were largely ascribed to oleuropein Leaves and fruits of Olea varieties “Azerbaijan” and “Baku” have been found to contain p-mannitol, phenylcarboxylic acid, apigenin, lulcolin and quercetin derivatives flavonoids. Studies of separate fractions suggested that hypotensive effect of the extracts produced from leaves and fruits of the Olea varieties mentioned is mainly related to oleuropein.

TITLE: Investigation on the extraction and concentration of oleuropein and flavonoids in Olea europaea L. based products.

AUTHOR:De-Laurentis-N {a}; Crescenzo-G; Lai-O-R; Milillo-M-A

INSTITUTE:{a} Dep. Med. Chem., Fac. Pharm., Univ. Bari, Via Orabona 4, 70126 Bari, Italy

SOURCE: Pharmaceutical-and-Pharmacological-Letters. 1997; 7 (1) 27-30.

YEAR: 1997

ABSTRACT: HPLC reversed phase (RP) is the technique used for the identification and dosage of the secoiridoid oleuropein, of flavonoids (apigenin, quercetin, kaempferol), of glycosyl flavonoids (hesperidin, rutin, the leaves and buds of Olea europaea L. This luteolin-7-0­glucoside, apigenin-7-0-glucoside), present in chromatographic method, which is simple and easy to use, presents a high reproducibility. Quantitative results and pharmacological considerations are reported here.Several studies have been carried out on the isolation and therapeutic activities of the flavonoids and secoiridoids contained in Olea europaea L.. These works report data related to the influence of oleuropein on heart activity and on arterial pressure (Messrli, F.H. Ed. 1996)), to the antispasmodic action and the calcium-antagonism of quercetin on smooth muscles of ileum in guinea pig (Ganten D. & Mulrow P. J., 1990) and to the hypocholesterolemic activity of hesperidin in rats kept on a hyperlipidic diet (Timmermans P.B.M.W.M, et al. 1993).

TITLE: Pharmacological analysis of the iridoid oleuropein.

AUTHOR:Petkov,-V; Manolov,-P

SOURCE:Arzneimittelforschung. 1972 Sep; 22(9): 1476-86

YEAR:1972

ABSTRACT: Empirical clinical data about a healing effect of the olive leaves in the case of hypertensive disease stimulated searching for the active component. In 1960 Panizzi et al. (20) succeeded in isolating a bitter glucoside which was given the name of oleuropein and its empirical formula was found to be C₂₅H_g2O1_g. This glucoside belongs to the iridoid group. Recently (1970) Inouye et al, (9) cast light upon the structure of eleuropein.Iridoids represent a new structural-chemical class. The majority of the compounds included in this group are glucosides in which carbohydrate component appears most frequently as D-glucose (occasionally as disaccharide, too). Though the first iridoid found in nature, verbenalin, was isolated as early as 1835 (24), successful investigations into the chemical structure of this group were only commenced in the recent fifteen years. The cause of this delay lies in the extremely great instability of the majority of compounds in this group. Characteristic feature of the aglycons of iridoids is the presence of cyclopentane-pyrane system. In some of them, the so called secoiridoids, the cyclopentane ring is open.This group of compounds was given the name of iridoids by Briggs et al. (2) in 1963. Very interesting data were recently obtained to show the presence of biogenetic correlations between iridoids and alkaloids as well as their ability of principle to transform one into another. Thus it has been established by the radioisotope studies of Popov (22) that the alkaloid gentioflavine isolated from some species of Gentiana might be obtained through a particular transformation of the molecule of the iridoid gentiopicroside. Further the alkaloids gentianidine and gentianadine are obtained by transforming the molecule of gentioflavine. The latter appears to be a precursor also of the alkaloids of the gentiane-structure type (genianine, etc.) through a would-be intermediate formation of a compound similar in structure to the iridoid gentioflavoside.It has been shown by way of isotopic studies, too, tha the new-isolated iridoid gentioside represents an ingermediate phase of the iridoid step in the biosynthesis pathway that leads to the formation of alkaloids in the gentiane plants. Oleuropein is water soluble and unstable against acids. When subjected to tentative studies (personal communication of Dr. Kloss [12]) oleuropein displays the following effects: it provokes an increase of coronary blood stream (experiments with heart after Langendorff), produces an elevation of blood-stream in the guinea pig limb, exerts a spasmolytic effect in case of histamine spasm. A 2% solution of oleuropein applied in a dose of 0.1 ml decreases by 22% the blood pressure of the rat, while administered in a dose of 1.0 ml by 31%. These scarce data about the pharmacodynamics of oleuropein gave us grounds to study in more details its pharmacology.Our investigations into the iridoid of the olive leaves were largely stimulated by the encouraging results obtained from studies of iridoids isolated from valerian (Valerina officinalis L). For a long time the importance of valerian as a sedative drug has been discussed and even contested. However this problem was considered differently after Thies [25-29], Mannetstatter et al. [13], Wagner et al. [30] and some others succeeded in isolating from the valerian root a new group of active substances, reckoned amount the iridoid group These new substances were determined quantitatively and studies under experimental and clinical conditions.Iridoids called valepotriaes (the most important of which are valtratum, didrovaltratum and acevaltratum) contained in a relatively high concentration (0.5 to 2%) in the valerian root displayed a clear-cut activity as sedative drugs [5, 69].

TITLE: Essai Clinique D’Un Extrait Titre de Feuilles D’Olivier Dans Le Traitement De L’Hypertension Arterielle Essentielle [Clinical Assay of Olea europaea Aqueous Extract in Hypertension Arteria Treatment.]

AUTHOR: Cherif, S., Rahal, N., Haouala, M., Hizaoui, B., Dargouth, F., Gueddiche, M., Kallel, Z., Balansard, G., & Boukef, K..

SOURCE:J. Pharm. Belg., 1996, 51, 2, 69-71

YEAR: 1996

ABSTRACT: A clinical assay of Olea europaea L. aqueous extract was carried on two groups of patients suffering with essential hypertension. 12 patients consulting for the first time, 18
patients with anti hypertensive treatment. Treatment based on Olea europaea L.leaf aqueous extract was given (400mg x 4/24h) during 3 months, after 15 days treatment based on placebo. We note for all patients a statistically significant decrease of blood pressure (p<_0,001), we didn’t find any modification of biological parameters, excepted a significant little decrease of glycemia and calcemia p<_0,01 and p<_0,001 respectively.  We didn’t find any side effect in the two groups.

CANCER

TITLE:The inhibitory effects of compounds from olive leaf on tumor necrosis factor production and on beta-hexosaminidase release.

AUTHOR: Nishibe-Sansei {a}; Han-Yingmei {a}; Noguchi-Yukari {a}; Ueda-Hiroshi; Yamazaki­Masatoshi; Mizutani-Kenji; Kambara-Toshimitsu; Kishida-Naoko

INSTITUTE: {a} Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, 061-0293, Japan

SOURCE: Natural-Medicines. [print] August, 2001; 55 (4): 205-208.

YEAR: 2001

ABSTRACT: The extraction and isolation of olive leaf gave luteolin 7-O-glucoside, luteolin 4′-O­glucoside and oleuropein as the major components. The inhibitory effects of these compounds on tumor necrosis factor (TNF-alpha) production and on beta-hexosaminidase release from rat basophilic leukemia (RBL-2H3) cells, which were both recently found to be linked to allergic reaction, were examined. Oleuropein showed a potent inhibitory effect on TNF-alpha production. Luteolin 4′-O-glucoside showed a strong inhibitory effect on beta-hexosaminidase release (IC50: 17.1 mug/ml).

TITLE:The antioxidant/anticancer potential of phenolic compounds isolated from olive oil

AUTHOR: Owen-RW; Giacosa-A; Hull-WE; Haubner-R; Spiegelhalder-B; Bartsch-H

INSTITUTE:Division of Toxicology, Cancer Research Centre, Im Neuenheimer Feld 280, D­69120 Heidelberg

SOURCE: EUR-J-CANCER. European-Journal-of-Cancer. 2000; 36(10): 1235-1247

YEAR:2000

ABSTRACT: In our ongoing studies on the chemoprevention of cancer we have a particular interest in the health benefits of the Mediterranean diet, of which olive oil is a major component. Recent studies have shown that extravirgin olive oil contains an abundance of phenolic antioxidants including simple phenols (hydroxytyrosol, tyrosol), aldehydic secoiridoids, flavonoids and lignans (acetoxypinoresinol, pinoresinol). All of these phenolic substances are potent inhibitors of reactive oxygen species attack on, e.g. salicylic acid, 2-deoxyguanosine. Currently there is growing evidence that reactive oxygen species are involved in the aetiology of fat-related neoplasms such as cancer of the breast and colorectum. A plausible mechanism is a high intake of < omega >-6 polyunsaturated fatty acids which are especially prone to lipid peroxidation initiated and propagated by reactive oxygen species, leading to the formation (via < alpha >,< beta >-unsaturated aldehydes such as trans-4-hydroxy-2-nonenal) of highly pro­mutagenic exocyclic DNA adducts. Previous studies have shown that the colonic mucosa of cancer patients and those suffering from predisposing inflammatory conditions such as ulcerative colitis and Crohn’s disease generates appreciably higher quantities of reactive oxygen species compared with normal tissue. We have extended these studies by developing accurate high performance liquid chromatography (HPLC) methods for the quantitation of reactive oxygen species generated by the faecal matrix. The data shows that the faecal matrix supports the generation of reactive oxygen species in abundance. As yet, there is a dearth of evidence linking this capacity to actual components of the diet which may influence the colorectal milieu. However, using the newly developed methodology we can demonstrate that the antioxidant phenolic compounds present in olive oil are potent inhibitors of free radical generation by the faecal matrix. This indicates that the study of the inter-relation between reactive oxygen species and dietary antioxidants is an area of great promise for elucidating mechanisms of colorectal carcinogenesis and possible future chemopreventive strategies. Copyr

TITLE: Antiatherogenic components of olive oil.

AUTHOR:Visioli,-F; Galli,-C

INSTITUTE:nstitute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, Milan 20133, Italy. francesco.visioli@unimi.it

SOURCE:Curr-Atheroscler-Rep. 2001 Jan; 3(1): 64-7

YEAR: 2001

ABSTRAC:Olive oil is the principal source of fat in the Mediterranean diet, which has been associated with a lower incidence of coronary heart disease and certain cancers. Olive oil is characterized by a high proportion of monounsaturated oleic acid, but the main peculiarity of extra-virgin oil is the presence of remarkable quantities of phenolic compounds, notably hydroxytyrosol and oleuropein, that provide high stability and strong taste. Recently, several studies have demonstrated that olive oil phenolics are powerful antioxidants, both in vitro and in vivo, and exert additional potent biologic activities that could partially account for the observed card ioprotective effects of the Mediterranean diet.

CHOLESTEROL, CORONARY & HEART PROBLEMS

TITLE: Anti hypertensive,antiatherosclerotic and antioxidant  activity of triterpenoids isolated from Olea europaea, subspecies africana leaves.
 
AUTHOR: Somova,-LI; Shode,-FO; Ramnanan,-P; Nadar,-A

SOURCE: J-Ethnopharmacol. 2003 Feb; 84(2-3): 299-305,

YEAR:2003

ABSTRACT: For the first time a biossay-directed study of triterpenoids isolated from the leaves of Olea europaea from Greece, from wild African olive and from a cultivar of O. europaea grown in Cape Town was reported. The experiment was undertaken since our preliminary analyses showed that the African wild olive leave is rich in triterpenoids and contain only traces of the glycoside oleuropein, which is typical for the European olive leaves. The isolate of the African wild olive leaves (AO) used in the experiments was found to contain 0.27% 1:1 mixture of oleanolic acid and ursolic acid, named oleuafricein. The isolate of Greek olive leaves (GO) was found to contain 0.71% oleanolic acid, and the Cape Town cultivar (CT) contained 2.47% oleanolic acid. No ursolic acid was found in either GO or CT. The antihypertensive, diuretic, antiatherosclerotic, antioxidant and hypoglycemic effects of authentic oleanolic and ursolic acid and the three isolates (GO, AO and CT) were studied on Dahl salt-sensitive (DSS), insulin­resistant rat genetic model of hypertension. All three isolates, in a dose 60 mg/kg b.w. for 6 weeks treatment, prevented the development of severe hypertension and atherosclerosis and improved the insulin resistance of the experimental animals. GO, OA and CT isolates could provide an effective and cheap treatment of this particular, most common type of salt-sensitive hypertension in the African po

TITLE: HPLC analysis of oleuropein and some flavonoids in leaf and bud of Olea europaea L.

AUTHOR:Ficarra-P; Ficarra-R; De-Pasquale-A; Monforte-MT; Calabro-ML 

INSTITUTE: Dipartimento Farmaco Chimico, Univ. di Messina, Viale Annunziata, 98168 Messina, Italy

SOURCE:Farmaco-Ed-Sci; 1991; 46(Jun); 803-815

YEAR:1991

ABSTRACT: A rapid, precise and reproducible reversed phase HPLC method is described for the quantitative determination of oleuropein and some flavonoids from the leaves and buds of Olea europaea in order to determine which constituents are responsible for the antilipemic effects of the plant.

TITLE: Protective effect of oleuropein, an olive oil biophenol, on low density lipoprotein oxidizability in rabbits.

AUTHOR:Coni,-E; Di-Benedetto,-R; Di-Pasquale,-M; Masella,-R; Modesti,-D; Mattei,-R; Carlini,-E-A

INSTITUTE: Food Department, Istituto Superiore di Sanita, Rome, Italy. e.coni@iss.it

SOURCE: Lipids. 2000 Jan; 35(1): 45-54

YEAR:2000

ABSTRACT:On the basis of the results obtained with pilot studies conducted in vitro on human low density lipoprotein (LDL) and on cell cultures (Caco-2), which had indicated the ability of certain molecules present in olive oil to inhibit pro-oxidative processes, an in vivo study was made of laboratory rabbits fed special diets. Three different diets were prepared: a standard diet for rabbits (diet A), a standard diet for rabbits modified by the addition of 10% (w/w) extra virgin olive oil (diet B), a modified standard diet for rabbits (diet C) differing from diet B only in the addition of 7 mg kg(-1) of oleuropein. A series of biochemical parameters was therefore identified, both in the rabbit plasma and the related isolated LDL, before and after Cu-induced oxidation. The following, in particular, were selected: (i) biophenols, vitamins E and C, uric acid, and total, free, and ester cholesterol in the plasma; (ii) proteins, triglycerides, phospholipids, and total, free, and ester cholesterol in the native LDL (for the latter, the dimensions were also measured); (iii) lipid hydroperoxides, aldehydes, conjugated dienes, and relative electrophoretic mobility (REM) in the oxidized LDL (ox-LDL). In an attempt to summarize the results obtained, it can be said that this investigation has not only verified the antioxidant efficacy of extra virgin olive oil biophenols and, in particular, of oleuropein, but has also revealed a series of thus far unknown effects of the latter on the plasmatic lipid situation. In fact, the addition of oleuropein in diet C increased the ability of LDL to resist oxidation (less conjugated diene formation) and, at the same time, reduced the plasmatic levels of total, free, and ester cholesterol (-15, -12, and – 17%, respectively), giving rise to a redistribution of the lipidic components of LDL (greaterpulationight (C) 2 phospholipid and cholesterol amounts) with an indirect effect on their dimensions (bigger by about 12%).

TITLE: Simultaneous determination of oleuropein and its metabolites in plasma by high-performance liquid chromatography.

AUTHOR:Tsarbopoulos,-A; Gikas,-E; Papadopoulos,-N; Aligiannis,-N; Kafatos,

INSTITUTE:GAIA Research Center, Bioanalytical Department, The Goulandris Natural History Museum, 13 Levidou street, Kifissia, GR-145 62, Greece. atsarbop@gnhm.gr

SOURCE:J-Chromatogr-B-Analyt-Technol-Biomed-Life-Sci. 2003 Feb 25; 785(1): 157-64,

YEAR: 2003

ABSTRACT: A method based on high-performance liquid chromatography (HPLC) with a diode array detection system was developed and validated aiming at the simultaneous determination of oleuropein (OE) and its metabolites, hydroxytyrosol (HT) and tyrosol (T), in human plasma. These phenolic components are believed to play a vital role in the prevention of coronary artery disease and atherosclerosis. The proposed method includes a clean-up solid-phase extraction procedure (using a C(18) column) with high recovery efficiency (85-100%). The statistical evaluation of the method reveals good linearity, accuracy and reproducibility for all the compounds analyzed with RSD values less than 6.5%, while the detection limit is 50 ng/ml for both OE and T and 75 ng/ml for HT This assay can be employed in bioavailability studies of olive oil phenolic compounds, thus assisting the evaluation of their pharmacological role.

TITLE: Effect of phenolic compounds of virgin olive oil on LDL oxidation resistance.

AUTHOR: Moreno-JA; Lopez-Miranda-J; Gomez-P; Fatiha-Benkhalti; EI-Boustani-E; Perez­Jimenez-F

INSTITUTE: Unidad de Lipidos y Arteriosclerosis, Hospital Universitario Reina Sofia, Avda. Menendez Pidal, s/n. 14004 Cordoba, Spain.

SOURCE:  Medicina-Clinica-Barcelona. 2003, 120: 4, 128-131; 41 ref.

YEAR: 2003

ABSTRACT: Background and objective: Several epidemiological and experimental studies have associated the intake of antioxidants, which are abundant in the Mediterranean diet, with a low incidence of cardiovascular disease. One possible mechanism of this action is the oxidative protection in low density lipoproteins (LDL). The aim of our study was to compare the antioxidative activity of diverse phenolic compounds present in virgin olive oil on these lipoproteins. Subjects and method: LDL was isolated from blood plasma of healthy volunteers by sequential ultracentrifugation. This was followed by oxidation with CuCl2 in the presence of different concentrations of phenolic compounds and virgin olive oil extract. Production of conjugated dienes was determined by the continuous monitoring of increased absorbency at 234 nm as an indicator of LDL oxidation. Results: Virgin olive oil extract prolonged the latency 000 Elsevier Science Ltd  phase and significantly lowered the progression rate (p < 0.05) at low concentrations (2 pg/ml). This antioxidative effect was also observed with low concentrations (2 NM) of caffeic acid and oleuropein (p < 0.05). However, it was necessary to increase the concentration of flavone up to 50 times to observe a similar effect (p < 0.05). Conclusion: Both virgin olive oil extract enriched in phenolic compounds and phenolic compounds present in olive oil (caffeic acid and oleuropein) are potent antioxidants at very low concentrations. Thus, the beneficial effects of a Mediterranean diet may be partly due to the protective action of these compounds.

TITLE: Investigation on the extraction and concentration of oleuropein and flavonoids in Olea europaea L. based products.

AUTHOR: De-Laurentis-N {a}; Crescenzo-G; Lai-O-R; Milillo-M-A

INSTITUTE:{a} Dep. Med. Chem., Fac. Pharm., Univ. Bari, Via Orabona 4, 70126 Bari, Italy

SOURCE: Pharmaceutical-and-Pharmacological-Letters. 1997; 7 (1) 27-30.

YEAR:1997

ABSTRACT: HPLC reversed phase (RP) is the technique used for the identification and dosage of the secoiridoid oleuropein, of flavonoids (apigenin, quercetin, kaempferol), of glycosyl flavonoids (hesperidin, rutin, the leaves and buds of Olea europaea L. This luteolin-7-0­glucoside, apigenin-7-0-glucoside), present in chromatographic method, which is simple and easy to use, presents a high reproducibility. Quantitative results and pharmacological considerations are reported here.Several studies have been carried out on the isolation and therapeutic activities of the flavonoids and secoiridoids contained in Olea europaea L. These works report data related to the influence of oleuropein on heart activity and on arterial pressure (Messrli, F.H. Ed. 1996)), to the antispasmodic action and the calcium-antagonism of quercetin on smooth muscles of ileum in guinea pig (Ganten D. & Mulrow P. J., 1990) and to the hypocholesterolemic activity of hesperidin in rats kept on a hyperlipidic diet (Timmermans P.B.M.W.M, et al. 1993).

TITLE:Effect of virgin olive oil phenolic compounds on in vitro oxidation of human low density lipoproteins.

AUTHOR: Caruso,-D; Berra,-B; Giavarini,-F; Cortesi,-N; Fedeli,-E; Galli,-G

INSTITUTE:Institute of Pharmacological Sciences, University of Milan, Italy.

SOURCE:Nutr-Metab-Cardiovasc-Dis. 1999 Jun; 9(3): 102-7

YEAR:1999

ABSTRACT:BACKGROUND AND AIM: Substantial evidence suggests that oxidative modifications of low density lipoproteins (LDL) critically contribute to the pathogenesis and progression of human atherosclerosis. Oxidized LDL (oxLDL) are present in atherosclerotic plaques and contain oxysterols that exhibit a variety of adverse biological activities. Antioxidants have also been shown to prevent LDL modification. We have therefore assessed the efficacy of virgin olive oil phenolic compounds in preventing oxidative modifications of human LDL oxidized. by UV light.
METHODS AND RESULTS: Cholesterol oxides formed during LDL photo-oxidation were determined by UV-HPLC in the presence of different concentrations of phenolic compounds and their pure components (tyrosol and oleuropein), and probucol, a widely used synthetic antioxidant. Electrophoretic mobility was also assayed. The results demonstrate that phenolic compounds are much more potent in preventing cholesterol oxide formation and apoproteic moiety modification than their pure components and probucol.
CONCLUSIONS: The beneficial effects of a Mediterranean diet may be ascribable not only to the high unsaturated/saturated fatty acid ratio characteristic of olive oil, but also to the unique antioxidant properties of its phenolic compounds.

TITLE: Oleuropein, the bitter principle of olives, enhances nitric oxide production by mouse macrophages.

AUTHOR: Visioli,-F; Bellosta,-S; Galli,-C 

INSTITUTE:Institute of Pharmacological Sciences, Milan, Italy. Francesco.Visioli@unimi.it

SOURCE: Life-Sci. 1998; 62(6): 541-6

YEAR:1998

ABSTRACT:The Mediterranean diet, rich in fresh fruits and vegetables, has been associated with a lower incidence of cardiovascular disease and cancer, partly because of its high proportion of bioactive compounds such as vitamins, flavonoids and polyphenols. The major lipid component of such diet is the drupe-derived olive oil, which can be distinguished from other seed oils for the peculiar composition of its non-triglyceride fraction. In fact, several minor components, including polyphenols, grant the oil its particular taste and aroma. Oleuropein, the most abundant among these components, has been shown to be a potent antioxidant endowed with anti-inflammatory properties. We investigated the effects of oleuropein on NO release in cell culture and its activity toward nitric oxide synthase (iNOS) expression. The results show that oleuropein dose-dependently enhance nitrite production in LPS-challenged mouse macrophages. This effect was blocked by the iNOS inhibitor L-NAME, indicating increased iNOS activity. Also, Western blot analysis of cell homogenates show that oleuropein increases iNOS expression in such cells. Taken together, our data suggest that, during endotoxin challenge, oleuropein potentiates the macrophage-mediated response, resulting in higher NO production, currently believed to be beneficial for cellular and organismal protection.

TITLE: Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: Antiatherogenic properties of Mediterranean diet phytochemicals

AUTHOR:Carluccio-MA; Siculella-L; Ancora-MA; Massaro-M; Scoditti-E; Storelli-C; Visioli-F; Distante-A;De-Caterina-R

INSTITUTE: C.N.R. Inst. of Clinical Physiology, Lecce

SOURCE: Arteriosclerosis,-Thrombosis,-and-Vascular-Biology. 2003; 23(4): 622-629

YEAR:2003

ABSTRACT:Objective – Epidemiology suggests that Mediterranean diets are associated with reduced risk of cardiovascular disease. Because monocyte adhesion to the endothelium is crucial in early atherogenesis, we evaluated whether typical olive oil and red wine polyphenols affect endothelial-leukocyte adhesion molecule expression and monocyte adhesion. Methods and Results – Phytochemicals in olive oil and red wine, including oleuropein, hydroxytyrosol, tyrosol, elenolic acid, and resveratrol, with or without antioxidant activity, were incubated with human umbilical vein endothelial cells for 30 minutes, followed by co-incubation with bacterial lipopolysaccharide or cytokines to trigger adhesion molecule expression. At nutritionally relevant concentrations, only oleuropein, hydroxytyrosol, and resveratrol, possessing a marked antioxidant activity, reduced monocytoid cell adhesion to stimulated endothelium, as well as vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein by Northern analysis and cell surface enzyme immunoassay. Reporter gene assays with deletional VCAM-1 promoter constructs indicated the relevance of nuclear factor-< kappa >B, activator protein- 1, and possibly GATA binding sites in mediating VCAM- 1 transcriptional inhibition. The involvement of nuclear factor-< kappa >B and activator protein-1 was finally demonstrated at electrophoretic mobility shift assays. Conclusions – Olive oil and red wine antioxidant polyphenols at nutritionally relevant concentrations transcriptionally inhibit endothelial adhesion molecule expression, thus partially explaining atheroprotection from Mediterranean diets.

TITLE:Antiatherogenic components of olive oil.

AUTHOR: Visioli,-F; Galli,-C

INSTITUTE: Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, Milan 20133, Italy. francesco.visioli@unimi.it

SOURCE:Curr-Atheroscler-Rep. 2001 Jan; 3(1): 64-7
 

YEAR:2001

ABSTRACT: Olive oil is the principal source of fat in the Mediterranean diet, which has been associated with a lower incidence of coronary heart disease and certain cancers. Olive oil is characterized by a high proportion of monounsaturated oleic acid, but the main peculiarity of extra-virgin oil is the presence of remarkable quantities of phenolic compounds, notably hydroxytyrosol and oleuropein, that provide high stability and strong taste. Recently, several studies have demonstrated that olive oil phenolics are powerful antioxidants, both in vitro and in vivo, and exert additional potent biologic activities that could partially account for the observed card ioprotective effects of the Mediterranean diet.

TITLE: Effects of leaves and shoots of Olea europaea L. and oleuropein on experimental hypercholesteolemia in rat

AUTHOR:De-Pasquale-R; Monforte-MT; Trozzi-A; Raccuia-A; Tommasini-S; Ragusa-S

INSTITUTE:Pharmaco-Biological Department, School of Pharmacy, Vill. ss. Annunziata, 98168 Messina

SOURCE:PLANT-MED-PHYTOTHER. Plantes-Medicinales-et-Phytotherapie. 1991; 25(2-3): 134-140

YEAR:1991

ABSTRACT:The effects of glycero-alcoholic extracts of shoots and leaves of Olea europaeea L. and oleuropein on diet and triton hype rcholesterolemia were studied in Wistar rats. It was found that the hypocholesterolemic effects of the Olea extracts can be ascribed to a synergic action of oleuropein and polyunsaturated fatty acids contained in the drugs.

TITLE:Oleuropein Protects Low Density Lipoprotein from Oxidation

AUTHOR:Visoli, F. & Galli, C.

SOURCE:Life Sciences, Vol. 55, No. 24, pp. 1965-1971

YEAR:1994

ABSTRACT:The Mediterranean diet, rich in fruit, vegetables, grain, and vegetable oil (mainly olive oil) is correlated with a lower incidence of coronary heart disease (CHID). Natural antioxidants contained in the Mediterranean diet might also play a role in the prevention of cardiovascular diseases, through inhibition of LDL oxidation. We tested this hypothesis “in vitro” by inducing LDL oxidation with copper sulphate and preincubating the samples with oleuropein, the bitter principle of olives, that is one of the major components of the polyphenolic fraction of olive oil. Oleuropein 10^-5 M effectively inhibited CuS0₄_induced LDL oxidation, as assessed by various parameters. We demonstrate in this investigation that polyphenolic components of the Mediterranean diet interfere with biochemical events that are implicated in atherogenetic disease, thus proposing a new link between the Mediterranean diet and prevention of CHID.

TITLE: Pharmacological analysis of the iridoid oleuropein.

AUTHOR:Petkov,-V; Manolov,-P

SOURCE:Arzneimittelforschung. 1972 Sep; 22(9): 1476-86

YEAR:1972

ABSTRACT:Empirical clinical data about a healing effect of the olive leaves in the case of hypertensive disease stimulated searching for the active component. In 1960 Panizzi et al. (20) succeeded in isolating a bitter glucoside which was given the name of oleuropein and its empirical formula was found to be C_ZSH_gzO,₃. This glucoside belongs to the iridoid group. Recently (1970) Inouye et al, (9) cast light upon the structure of eleuropein.       Iridoids represent a new structural-chemical class. The majority of the compounds included in this group are glucosides in which carbohydrate component appears most frequently as D-glucose (occasionally as disaccharide, too). Though the first iridoid found in nature, verbenalin, was isolated as early as 1835 (24), successful investigations into the chemical structure of this group were only commenced in the recent fifteen years.     The cause of this delay lies in the extremely great instability of the majority of compounds in this group. Characteristic feature of the aglycons of iridoids is the presence of cyclopentane-pyrane system. In some of them, the so called secoiridoids, the cyclopentane ring is open. This group of compounds was given the name of iridoids by Briggs et al. (2) in 1963. Very interesting data were recently obtained to show the presence of biogenetic correlations between iridoids and alkaloids as well as their ability of principle to transform one into another. Thus it has been established by the radioisotope studies of Popov (22) that the alkaloid gentioflavine isolated from some species of Gentiana might be obtained through a particular transformation of the molecule of the iridoid gentiopicroside. Further the alkaloids gentianidine and gentianadine are obtained by transforming the molecule of gentioflavine. The latter appears to be a precursor also of the alkaloids of the gentiane-structure type (genianine, etc.) through a would-be intermediate formation of a compound similar in structure to the iridoid gentioflavoside.It has been shown by way of isotopic studies, too, tha the new-isolated iridoid gentioside represents an ingermediate phase of the iridoid step in the biosynthesis pathway that leads to the formation of alkaloids in the gentiane plants. Oleuropein is water soluble and unstable against acids. When subjected to tentative studies (personal communication of Dr. Kloss [12]) oleuropein displays the following effects: it provokes an increase of coronary blood stream (experiments with heart after Langendorf), produces an elevation of blood-stream in the guinea pig limb, exerts a spasmolytic effect in case of histamine spasm. A 2% solution of oleuropein applied in a dose of 0.1 ml decreases by 22% the blood pressure of the rat, while administered in a dose of 1.0 ml by 31%. These scarce data about the pharmacodynamics of oleuropein gave us grounds to study in more details its pharmacology.      Our investigations into the iridoid of the olive leaves were largely stimulated by the encouraging results obtained from studies of iridoids isolated from valerian (Valerina officinalis L). For a long time the importance of valerian as a sedative drug has been discussed and even contested. However this problem was considered differently after Thies [25-29], Mannetstatter et al. [13], Wagner et al. [30] and some others succeeded in isolating from the valerian root a new group of active substances, reckoned amount the iridoid group. These new substances were determined quantitatively and studies under experimental and clinical conditions. Iridoids called valepotriaes (the most important of which are valtratum, didrovaltratum and acevaltratum) contained in a relatively high concentration (0.5 to 2%) in the valerian root displayed a clear-cut activity as sedative drugs [5, 69].

COSMETIC

TITLE: Natural actives for cosmetics

AUTHOR:Ziolkowsky-B

SOURCE:Seifen-Oele-Fette-Wachse (Seifen-Oele-Fette-Wachse); 2002; 128(1-2); 19-23,

YEAR:2002

ABSTRACT:Today many raw materials and actives are won from natural products, to be used in cosmetic preparations, which support the body’s natural process. These natural actives can effectively be used in preventing negative external and internal processes such as anti-aging, anti-irritants, antioxidants or as moisturizers, thighteners and cell activators.

TITLE: Dietetic and/or cosmetic preparation comprising a mixture of lycopene and olive leaf extract

AUTHOR:Coll,-D; Mathonnet,-J-P; Zannini,-G

YEAR:2000

ABSTRACT:A dietetic or cosmetic preparation comprises an active mixture of lycopene with an olive leaf extract; the olive leaf extract aids solubilization of the lycopene. It is claimed that the lipid regulation action of the olive leaf extract associates with the cell preservation action of the lycopene.

DIABETES (HYPOGLYCEMIA)

TITLE: Hypoglycemic Activity of Olive Leaf

AUTHOR:Gonzalez, M., Zarzuelo, A., Gomez, M. J., Utrilla, M. P., Jimenez, J., and Osuna I.

INSTITUTE:Department of Farmacologia, Facultad de Farmacia, Universidad de Granada, Granada,Spain.

SOURCE:Planta Med. 58 (1992) 513-515

YEAR:1992

ABSTRACT:The hypoglycemic activity of olive leaf was studied. Maximum hypoglycemic activity was obtained from samples collected in the winter months, especially in February.One of the compounds responsible for this activity was oleuropeoside, which showed activity at a dose of 16mg/kg. The compound also demonstrated antidiabetic activity in animals with alloxan-induced diabetes. The hypoglycemic activity of this compound may result from two mechanisms (a) potentiation of glucose-induced insulin release, and (b) increased peripheral uptake of glucose.

TITLE: The Evaluation of Long-term Effects of Cinnamon Bark and Olive Leaf on Toxicity Induced by Streptozotocin Administration to Rats.

AUTHOR:Onderoglu, S., Sozer, S., Mine Erbil, K., Ortac, R. & Lermioglu, F.

SOURCE:J. Pharm. Pharmacol. 1999, 51: 1305-1312

YEAR:1999

ABSTRACT:The effects of cinnamon bark and olive leaf have been investigated on streptozotocin-induced tissue injury, and some biochemical and haematological changes in rats. The effects on glycaemia were also evaluated Long term administration of olive leaf caused significant improvement in tissue injury induced by streptozotocin treatment; the effect of cinnamon bark was less extent.No effects on blood glucose levels were detected. However, significant decreases in some increased biochemical and haematological parameters of streptozotocin-treated rats were observed. Aspartate aminotransferase, urea and cholesterol levels were significantly decreased by treatment with both plant materials, and alanine aminotransferase by treatment with olive leaf. Cinnamon bark also caused a significant decrease in platelet counts In addition, any visible toxicity, except decrease in body weight gain, attributable to the long term use of plant materials was not established in normal rats. The data indicate that long term use of olive leaf and cinnamon bark may provide benefit against diabetic conditions. Determination of underlying mechanism(s) of beneficial effects, toxicity to other systems and clinical assessments of related plant materials are major topics requiring further studies.

TITLE:Essai Clinique D’Un Extrait Titre de Feuilles D’Olivier Dans Le Traitement De L’Hypertension Arterielle Essentielle [Clinical Assay of Olea europaea Aqueous Extract in Hypertension Arteria Treatment.]

AUTHOR: Cherif, S., Rahal, N., Haouala, M., Hizaoui, B., Dargouth, F., Gueddiche, M., Kallel, Z., Balansard, G., & Boukef, K..

SOURCE:J. Pharm. Belg., 1996, 51, 2, 69-71 

YEAR: 1996

ABSTRACT:A clinical assay of Olea europaea L. aqueous extract was carried on two groups of patients suffering with essential hypertension. 12 patients consulting for the first time, 18 patients with anti hypertensive treatment. Treatment based on Olea europaea L.leaf aqueous extract was given (400mg x 4/24h) during 3 months, after 15 days treatment based on placebo. We note for all patients a statistically significant decrease of blood pressure (p<_0,001), we didn’t find any modification of biological parameters, excepted a significant little decrease of glycemia and calcemia ps0,01 and p<_0,0

FEVER

TITLE:A Modern Herbal

AUTHOR:Grieve, M.

SOURCE:Jonathan Cape Ltd, London

YEAR:1931, Reprinted 1974, 1975, 1977, 1979.

EXTRACT:Medicinal Action & Uses: The olive leaves are astringent and antiseptic.Internally a decoction of 2 handsful boiled in a quart of water until reduced to half a pint has been used in Levant in obstinate fevers. Both leaves and bark have valuable febrifugal qualities.

GENERAL

TITLE: Olive oil phenols are absorbed in humans.

AUTHOR:Vissers,-M-N; Zock,-P-L; Roodenburg,-A-J; Leenen,-R; Katan,-M-B

INSTITUTE:Division of Human Nutrition and Epidemiology, Wageningen University, Wageningen, The Netherlands

SOURCE:J-Nutr. 2002 Mar; 132(3): 409-17,

YEAR:2002

ABSTRACT:Animal and in vitro studies suggest that olive oil phenols are effective antioxidants. The most abundant phenols in olive oil are the nonpolar oleuropein- and ligstroside-aglycones and the polar hydroxytyrosol and tyrosol. The aim of this study was to gain more insight into the metabolism of those phenols in humans. We measured their absorption in eight healthy ileostomy subjects. We also measured urinary excretion in the ileostomy subjects and in 12 volunteers with a colon. Subjects consumed three different supplements containing 100 mg of olive oil phenols on separate days in random order. Ileostomy subjects consumed a supplement with mainly nonpolar phenols, one with mainly polar phenols and one with the parent compound oleurope in-glycoside. Subjects with a colon consumed a supplement without phenols (placebo) instead of the supplement with oleuropein-glycoside. Ileostomy effluent and urine were collected for 24 h after supplement intake. Tyrosol and hydroxytyrosol concentrations were low (< 4 mol/100 mol of intake) in the ileostomy effluent, and no aglycones were detected. We estimated that the apparent absorption of phenols was at least 55-66% of the ingested dose. Absorption was confirmed by the excretion of tyrosol and hydroxytyrosol in urine. In ileostomy subjects, 12 mol/100 mol and in subjects with a colon, 6 mol/100 mol of the phenols from the nonpolar supplement were recovered in urine as tyrosol or hydroxytyrosol. In both subject groups, 5–6 mol/100 mol of the phenols was recovered from the polar supplement. When ileostomy subjects were given oleuropein-glycoside, 16 mol/100 mol was recovered in 24-h urine, mainly in the form of hydroxytyrosol. Thus, humans absorb a large part of ingested olive oil phenols and absorbed olive oil phenols are extensively modified in the body.

TITLE: The blessing from the mount of olives

AUTHOR: Hardelius-M

SOURCE: FIP-World-Congress (International-Pharmaceutical-Federation-World-Congress); 2002;62,152,

YEAR:2002

ABSTRACT:The olive-tree appears in many fundamental myths of our culture and the Mount of Olives is the theatre of several essential scenes from the life of Jesus.”Olive oil frightens away all suffering” according to the old proverb and you get the following recommendations on the web: “Research from Europe shows how olive leaf extract can help fight 137 different diseases, including ear infections; hepatitus and pneumonia” The antiquity praised the medical qualities of the olive leaf but even before the 5th century B.C. Hippocrates did advise them in case of ulcer or cholera. Pliny the Elder reminded of the Anecdote “There are two liquids delightful for the human body for internal use the wine and for external use olive oil ” Olive oil is the only vegetable oil to be extracted by cold pressure and it is an ingredient in several preparations such as ointments and plasters in the ancient Pharmacopeias.The olive-tree is since the Neolithic period considered present all around the Mediterranean Sea. Two thirds of the trees are concentrated in the European part of the Mediterranean areas and only 3% in the New World. The consumption is highest in Greece with 20 kg per inhabitant and year. In an International Consensus Statement from EU this year you can read “There is increasing scientific evidence that there are positive health effects from the Traditional Mediterranean Diet where olive oil is the principal source of fat.”

TITLE: In vitro anti-complementary activity of flavonoids from olive (Olea europaea L.) leaves

AUTHOR: Pieroni-A; Heimler-D; Pieters-L; Van-Poel-B; Vlietinck-AJ

INSTITUTE: Pharmazeutisches Institut, Rheinische-Friedrich-Wilhelms-Univ., Kreuzbergweg 26, D-53115 Bonn

SOURCE:PHARMAZIE. Pharmazie-. 1996; 51(10):765-768

YEAR:1996

ABSTRACT:From extracts of olive (Olea europaea L., Oleaceae) leaves showing anti­complementary activity, the flavonoids apigenin, apigenin-4′-O-rhamnosylglucoside, apigenin-7­O-glucoside, luteolin, luteolin-4′-O-glucoside, luteolin-7-O-glucoside, chrysoeriol, chrysoeriol-7­O-glucoside and quercetin-3-O-rhamnoside were isolated. Major isolated constituents strongly inhibited the classical pathway of the compl

TITLE: Natural substance based agent

AUTHOR:Stueckler,-F,

YEAR:1999

ABSTRACT: A food supplement with advantageous physiological effects contains lecithin, red wine extract and D-alpha-tocopherol at concn. of 0.05-10.00 parts by wt. in a support material (e.g. dairy products, margarine, fruit juice, vegetable juice, yeast, pectin or olive oil). One or more other ingredients may optionally be incorporated at concn. of 0.01-1.00 parts by wt., including: squalene, beta-carotene, lycopene, green tea extract, garlic extract, genistein, D­limonene, bisabolol (levomenol), leucocianidol (pynkogenol), Ginkgo biloba leaf extract, hawthorn extract, artichoke extract, our lady’s thistle fruit extract, ascorbic acid, B group vitamins, olive leaf extract, diosmin, escin, troxerutin, anguarate extract, pectin, salmon oil concentrate, shiitake (Lentinus edodes) extract, incense (Boswellia) extract and Uncaria tomentosa root extract.

HIVI/AIDS

TITLE: Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV -1 infection and OLE treatment

AUTHOR:Lee-Huang-S; Zhang-L; Huang-PL; Chang-Y T; Huang-PL

INSTITUTE:department of Biochemistry, NY University School of Medicine, New York, NY 10016

SOURCE:Biochemical-and-Biophysical-Research-Communications. 2003; 307(4): 1029-1037, 

YEAR:2003

ABSTRACT:We investigated the antiviral activity of olive leaf extract (OLE) preparations standardized by liquid chromatography-coupled mass spectrometry (LC-MS) against HIV-1 infection and replication. We find that OLE inhibits acute infection and cell-to-cell transmission of HIV -1 as assayed by syncytia formation using uninfected MT2 cells co-cultured with HIV-1­infected H9 T lymphocytes. OLE also inhibits HIV-1 replication as assayed by p24 expression in infected H9 cells. These anti-HIV effects of OLE are dose dependent, with EC<inf>50</inf>s of around 0.2< mu >g/ml. In the effective dose range, no cytotoxicity on uninfected target cells was detected. The therapeutic index of OLE is above 5000. To identify viral and host targets for OLE, we characterized gene expression profiles associated with HIV-1 infection and OLE treatment using cDNA microarrays. HIV-1 infection modulates the expression patterns of cellular genes involved in apoptosis, stress, cytokine, protein kinase C, and hedgehog signaling. HIV-1 infection up-regulates the expression of the heat-shock proteins hsp27 and hsp90, the DNA damage inducible transcript 1 gadd45, the p53-binding protein mdm2, and the hedgehog signal protein patched 1, while it down-regulates the expression of the anti-apoptotic BCL2-associated X protein Bax. Treatment with OLE reverses many of these HIV A infection-associated changes. Treatment of HIV-1-infected cells with OLE also up-regulates the expression of the apoptosis inhibitor proteins IAP1 and 2, as well as the calcium and protein kinase C pathway signaling molecules IL-2, IL-2R< alpha >, and ornithine decarboxylase ODC1.

TITLE:A new triple combination therapy

AUTHOR: Konlee,-M

SOURCE:Posit-Health-News. 1998 Fall; (No 17): 12-4,

YEAR:1998

ABSTRACT: Elderberry, chondroitin, and glucosamine sulfate have been found to block HIV replication at three distinct points in the replication cycle. For quadruple therapy, a reverse transcriptase inhibitor such as olive leaf extract or Epivir (3TC) could be added. In one case, a female, taking no HIV drugs, used an elderberry extract, called Sambucol, with olive leaf extract and experienced a viral load drop from 17,000 to 4,000. Instructions are given for making both alcohol-free and alcohol-based elderberry extracts. In 1993, researchers at Jerusalem’s Hebrew University Medical School found in a placebo-controlled double-blind study that Sambucol led to a rapid recovery from influenza and inhibited replication of nine other strains of the flu virus. A theory is that elderberry renders viruses nonfunctional by staining and coating them. Another promising treatment is soil based organisms, which improved Natural Killer cell function in a person with CFIDS.

IMMUNE SYSTEM

TITLE: Replenishing your immune system with nature’s antibiotic apothecary

AUTHOR:Wellman-T

SOURCE:Total Health Magazine (TOTAL HEALTH -MAG) 2001 Nov-Dec; 23(6): 76-8,

YEAR: 2001

ABSTRACT:In a world of superbugs that are resistant to antibiotics and the coming plague” of new viruses, scientific research shows that olive leaf extract kills germs and infections of all kinds and that power comes from its active agent, elenolic acid with its salt compound, calcium d-elenolate.

TITLE: Transfer factor.

AUTHOR:Anonymous 

SOURCE:Posit-Health-News. 1998 Fall; (No 17): 21,

YEAR:1998

ABSTRACT:
Transfer factor, a natural substance of the immune system, was discovered in 1949. More than 3,000 scientific articles have established it as an effective treatment for many diseases, usually those related to the immune system. In China, more than six million people have used transfer factor as a prophylaxis for hepatitis. Information on ordering articles on transfer factor, olive leaf extract, and coconut oil is included.

RADIO-PROTECTIVE

TITLE: Radioprotective effects in vivo of phenolics extracted from Olea europaea L. leaves against X-ray-induced chromosomal damage: comparative study versus several flavonoids and sulfur-containing compounds.

AUTHOR:Benavente-Garcia O, Castillo J, Lorente J, Alcaraz M.

INSTITUTE:Research and Development Department, Furfural Espanol S.A., Camino Viejo de Pliego s/n, 80320 Alcantarilla, Murcia, Spain. laboratorio@furesa.es

ABSTRACT:The radioprotective effects of a polyphenolic extract of Olea europaea L. leaves (OL); the flavonoids diosmin and rutin, which are widely used as pharmaceuticals; and the sulfur-containing compounds dimethylsulfoxide (DMSO) and 6-n-propyl-2-thiouracil (PTU) were determined by using the micronucleus test for anticlastogenic activity, evaluating the reduction of the frequency of micronucleated polychromatic erythrocytes (MnPCEs) in bone marrow of mouse before and after X-ray irradiation. With treatment before X-irradiation, the most effective compounds were, in order, rutin > DMSO > OL > PTU > diosmin. These results showed, for the polyphenols studied, a linear correlation (r(2) = 0.965) between anticlastogenic activity and antioxidant capacity. The magnitude of protection with treatment after X-irradiation were lower, and the most effective compounds were, in order, OL > diosmin > rutin; DMSO and PTU lacked radioprotective activity. Therefore, OL is the only substance that showed a significant anticlastogenic activity both before and after X-ray irradiation treatments. Structurally, the free oxygen radicals and lipoperoxyradicals scavenging capacity and, consequently, the anticlastogenic activity of these polyphenolic compounds are based principally on the presence of specific functional groups, mainly catechol groups (rutin, oleuropein, hydroxytyrosol, verbascoside, luteolin), that also increase the stability of the aroxyl-polyphenol radical generated in the above processes.

RESPIRATORY (INFLUENZA, PNEUMONIA ETC)

TITLE: In vitro evaluation of secoiridoid glucosides from the fruits of Ligustrum lucidum as antiviral agents.

AUTHOR:Ma-Shuang-Cheng; He-Zhen-Dan; Deng-Xue-Long; But-Paul-Pui-Hay {a}; Ooi­Vincent-Eng-Choon; Xu-Hong-Xi; Lee-Spencer-Hon-Sun; Lee-Song-Fong

INSTITUTE: {a} Department of Biology and Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong; E-Mail: paulbut@cuhk.edu.hk, China

SOURCE:Chemical-and-Pharmaceutical-Bulletin-Tokyo. [print] November, 2001; 49 (11): 1471-1473.

YEAR:2001

ABSTRACT:Six secoiridoid glucosides, lucidumoside C (1), oleoside dimethylester (2), neonuezhenide (3), oleuropein (4), ligustroside (5) and lucidumoside A (6), isolated from the fruits of Ligustrum lucidum (Oleaceae), were examined in vitro for their activities against four strains of pathogenic viruses, namely herpes simplex type 1 virus (HSV-1), influenza type A virus (Flu A), respiratory syncytial virus (RSV) and parainfluenza type 3 virus (Para 3). Antiviral activities were evaluated by the cytopathic effect (CPE) inhibitory assay. The purpose was to check if the anti-oxidative potency of these glucosides correlated with their antiviral potency. Results showed that none of the glucosides had any significant activity against HSV-1 and Flu A. Oleuropein, however, showed significant antiviral activities against RSV and Para 3 with IC50 value of 23.4 and 11.7 mug/ml, respectively. Lucidumoside C, oleoside dimethylester and ligustroside showed potent or moderate antiviral activities against Para 3 with IC50 values of 15.6-20.8 mug/ml. These results also documented that the anti-oxidative potency of these secoiriodoid glucosides was not directly related to their antiviral effects.

TITLE: On the in-vitro antimicrobial activity of oleuropein and hydroxytyrosol.

AUTHOR:Bisignano,-G; Tomaino,-A; Lo-Cascio,-R; Crisafi,-G; Uccella,-N; Saija,-A

INSTITUTE:Department Farmaco-Biologico, University of Messina, Italy.

SOURCE:J-Pharm-Pharmacol. 1999 Aug; 51(8): 971-4

YEAR:1999

ABSTRACT:Secoiridoides (oleuropein and derivatives), one of the major classes of polyphenol contained in olives and olive oil, have recently been shown to inhibit or delay the rate of growth of a range of bacteria and microfungi but there are no data in the literature concerning the possible employment of these secoiridoides as antimicrobial agents against pathogenic bacteria in man. In this study five ATCC standard bacterial strains (Haemophilus influenzae ATCC 9006, Moraxella catarrhalis ATCC 8176, Salmonella typhi ATCC 6539, Vibrio parahaemolyticus ATCC 17802 and Staphylococcus aureus ATCC 25923) and 44 fresh clinical isolates (Haemophilus influenzae, eight strains, Moraxella catarrhalis, six strains, Salmonella species, 15 strains, Vibrio cholerae, one strain, Vibrio alginolyticus, two strains, Vibrio parahaemolyticus, one strain, Staphylococcus aureus, five penicillin-susceptible strains and six penicillin-resistant strains), causal agents of intestinal or respiratory tract infections in man, were tested for in-vitro susceptibility to two olive (Olea europaea) secoiridoides, oleuropein (the bitter principle of olives) and hydroxytyrosol (derived from oleuropein by enzymatic hydrolysis and responsible for the high stability of olive oil). The minimum inhibitory concentrations (MICs) calculated in our study are evidence of the broad antimicrobial activity of hydroxytyrosol against these bacterial strains (MIC values between 0.24 and 7.85 microg mL(-1) for ATCC strains and between 0.97 and 31.25 microg mL(-1) for clinically isolated strains). Furthermore oleuropein also inhibited (although to a much lesser extent) the growth of several bacterial strains (MIC values between 62.5 and 500 microg mL(-1) for ATCC strains and between 31.25 and 250 microg mL(-1) for clinical isolates); oleuropein was ineffective against Haemophilus influenzae and Moraxella catarrhalis. These data indicate that in addition to the potential employment of its active principles as food additives or in integrated pest-management programs, Olea europaea can be considered a potential source of promising antimicrobial agents for treatment of intestinal or respiratory tract infections in man.

TITLE:In vitro evaluation of secoiridoid glucosides from the fruits of Ligustrum lucidum as antiviral agents.

AUTHOR:Ma-Shuang-Cheng; He-Zhen-Dan; Deng-Xue-Long; But-Paul-Pui-Hay {a}; Ooi­Vincent-Eng-Choon; Xu-Hong-Xi; Lee-Spencer-Hon-Sun; Lee-Song-Fong

INSTITUTE:{a} Department of Biology and Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong; E-Mail: paulbut@cuhk.edu.hk, China

SOURCE: Chemical-and-Pharmaceutical-Bulletin-Tokyo. [print] November, 2001; 49 (11): 1471-1473.

YEAR: 2001

ABSTRACT:Six secoiridoid glucosides, lucidumoside C (1), oleoside dimethylester (2), neonuezhenide (3), oleuropein (4), ligustroside (5) and lucidumoside A (6), isolated from the fruits of Ligustrum lucidum (Oleaceae), were examined in vitro for their activities against four strains of pathogenic viruses, namely herpes simplex type 1 virus (HSV 1), influenza type A virus (Flu A), respiratory syncytial virus (RSV) and parainfluenza type 3 virus (Para 3). Antiviral activities were evaluated by the cytopathic effect (CPE) inhibitory assay. The purpose was to check if the anti-oxidative potency of these glucosides correlated with their antiviral potency. Results showed that none of the glucosides had any significant activity against HSV 1 and Flu A. Oleuropein, however, showed significant antiviral activities against RSV and Para 3 with IC50 value of 23.4 and 11.7 mug/ml, respectively. Lucidumoside C, oleoside dimethylester and ligustroside showed potent or moderate antiviral activities against Para 3 with IC50 values of 15.6-20.8 mug/ml. These results also documented that the anti-oxidative potency of these secoiriodoid glucosides was not directly related to their antiviral effects.

ULCERS

TITLE:Olea europaea L.: stimulant, anti-ulcer and anti-inflammatory effects

AUTHOR: Fehri-B; Aiache-JM; Mrad-S; Korbi-S; Lamaison-JL 

INSTITUTE: Dept. of Pharmacol. and Toxicol., Soc. of Pharm. Industries of Tunisia Fondouk Choucha, Rades 2040, Tunisia

SOURCE: Boll-Chim-Farm (Bollettino-Chimico-Farmaceutico); 1996; 135(Jan); 42-49,

YEAR: 1996

ABSTRACT:The dried aqueous extract of the leaf of Olea europaea containing 3.25% oleuropein was studied for pharmacological effects using the hole board, open field, rota rod, and automatic reflex conditioner tests. At low doses, the preparation stimulated the CNS, but at high doses it caused CNS depression. The extract induced dose dependent and significant anti-inflammatory effects on carrageenin induced edema in rats. It also provided pronounced protection against aspirin induced gastric ulcers.

TITLE: The blessing from the mount of olives (HP-S-004)

AUTHOR:Hardelius-M

SOURCE:FIP-World-Congress (International-Pharmaceutical-Federation-World-Congress); 2002;62,152,

YEAR: 2002

ABSTRACT: The olive-tree appears in many fundamental myths of our culture and the Mount of Olives is the theatre of several essential scenes from the life of Jesus. “Olive oil frightens away all suffering” according to the old proverb and you get the following recommendations on the web: “Research from Europe shows how olive leaf extract can help fight 137 different diseases, including ear infections; hepatitus and pneumonia” The antiquity praised the medical qualities of the olive leaf but even before the 5th century B.C. Hippocrates did advise them in case of ulcer or cholera. Pliny the Elder reminded of the Anecdote “There are two liquids delightful for the human body for internal use the wine and for external use olive oil ” Olive oil is the only vegetable oil to be extracted by cold pressure and it is an ingredient in several preparations such as ointments and plasters in the ancient Pharmacopeias.The olive-tree is since the Neolithic period considered present all around the Mediterranean Sea. Two thirds of the trees are concentrated in the European part of the Mediterranean areas and only 3% in the New World. The consumption is highest in Greece with 20 kg per inhabitant and year.In an International Consensus Statement from EU this year you can read “There is increasing scientific evidence that there are positive health effects from the Traditional Mediterranean Diet.”

United States Patent	6,455,580
Fredrickson	September 24, 2002

Method and composition for antiviral therapy

Abstract

A method of treatment of diseases of viral origin is disclosed. The method comprises oral or parenteral administration of an antiviral amount of a naturally occurring secoiridoid from plants of the family Oleaceae or derivatives thereof. Preferred oral dosage forms include the secoiridoid oleuropein in pure form or as a component of dried plant material of Olea europaea or a dried extract thereof and a pharmaceutically acceptable carrier.

Inventors:	Fredrickson; William R. (Indianapolis, IN)
Assignee:	F&S Group, Inc. (Pacific Palisades, CA)
Appl. No.:	659204
Filed:	September 11, 2000

Current U.S. Class:	514/460; 514/27
Intern’l Class:	A61K 031/35; A61K 031/70
Field of Search:	514/27,460

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References Cited [Referenced By]

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U.S. Patent Documents

3033877	May., 1962	Veer	260/345.
3737550	Jun., 1973	Nook et al.	424/279.
3786068	Jan., 1974	Kelly	260/340.
3793346	Feb., 1974	Kelly	260/340.
Foreign Patent Documents
68/3957	May., 1968	ZA.
68/3957	Feb., 1969	ZA.

Other References

• Renis, Antimicrobial Agents and Chemotherapy, vol. 8(2), pp194-199, 1975.*
• Fleming et al., “Antimicrobial Properties of Oleuropein and Products of Its Hydrolysis from Green Olives,” Applied Microbiology, vol. 26, No. 5, pp. 777-782 (1973).
• Scarpati et al., “A New Secoiridoid From Olive Wastewaters,” Journal of Natural Products, vol. 56, No. 4, pp. 621-623 (1993).
• Ficarra et al., “HPLC Analysis of Oleuropein and Some Flavonoids in Leaf and Bud of Olea Europaea L., ” IL Farmaco, vol. 46(6), pp. 803-815, (1991).
• Brown et al., “Conversion of Secologanin into Elenolic Acid and 18-Oxayohimban Alkaloids,” J.C.S. Perkin, pp 160-162.
• Kubo et al., “A Multichemical Defense Mechanism of Bitter Olive Olea europae (Oleanceae) Is Oleuropein a Phytoalexin Precursor?,” Journal of Chemical Ecology, Col. 11, No. 2 (1985).
• Cruess et al., “The Bitter Glucoside of the Olive,” Journal of the American Chemical Society, vol. 56, pp. 2115-2117 (1934).
• Fleming et al., “Isolation of a Bacterial Inhibitor from Green Olives,” Applied Microbiology, vol. 18, pp. 826-860 (1969).
• Petkov et al., “Pharmacological Analysis of the Iridoid Oleuropein,” Arzneim-Forsch. (Drug Res.) 22, Nr. 9 (1972).
• Soret, “Antiviral Activity of Calcium Elenolate on Parainfluenza Infection of Hamsters,” Antimicrobial Agents and Chemotherapy, pp. 160-166 (1969).
• Elliott et al., “Preliminary Safety Studies with Calcium Elenolate, an Antiviral Agent,” Antimicrobial Agents and Chemotherapy, pp. 173-176 (1969).
• Kubo et al., “Molluscicides from Olive Olea europaea and Their Efficient Isolation by Countercurrent Chromatographies”, J. Agric. Food Chem., vol. 32, No. 3, pp 687-688 (1984).
• Fujisawa et al., “Binding of Eugenol and O-ethoxybenzoic Acid to Bovine Serum Albumin”, J. Dent. Res., vol. 60, No. 4, pp. 860-864 (1981).
• Renis, Harold, “In Vitro Antiviral Activity of Calcium Elenolate”, Antimicrobial Agents and Chemotherapy, pp. 167-172 (1969).
• Walter et al., “Preparation of Antimicrobial Compounds by Hydrolysis of Oleuropein from Green Olives”, Applied Microbiology, vol. 26, No. 5, pp 773-776 (1973).
• Hirschman, S.Z., “Inactivation of DNA Polymerases of Murine Leukaemia Viruses by Calcium Elenolate”, Nature (New Biology), 238: pp. 277-279 (1972).
• Combes, G. et al., Leaf Extract of Olea eropea Rich in Oleuropeine, Products from It, Their Application as Medicines and Compositions Containing Them, Chem Abstracts, vol. 98: 221797r (1983).
• “(—)-Elenolic Acid Esters”, Chem Abstracts, vol. 106: 67534c (1987).
• Takano et al., “Preparation of Dihydropyran Derivative as Antiviral Agents and Precursors for Alkyl (–)-Elenolates”, Chem Abstracts, vol. 113:58936c (1990).
• MacKellar et al., “Structure and Stereochemistry of Elenolic Acid”, J. Am. Chem. Soc. vol. 95(21), pp. 7175-7156 (1973).
• Renis, Harold, “Inactivation of Myxoviruses by Calcium Elenolate”, Antimicrobial Agents and Chemotherapy, vol. 8, No. 2 pp. 194-199 (1975).
• Gariboldi et al., “Secoiridoids from Olea Europaea”, Phyochem. 25(4), pp. 865-869 (1986).

Primary Examiner: Travers; Russell
Attorney, Agent or Firm: Barnes & Thornburg

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 08/668,324, filed Jun. 26, 1996 now U.S. Pat. No. 6,117,844 (now allowed), as a continuation of U.S. application Ser. No. 08/335,138, filed Nov. 7, 1994, now abandoned.

Claims

I claim:

1. A method of treatment of disease of vital origin in a warm-blooded vertebrate suffering from such disease, said method comprising the step of administering to said vertebrate an antivirally effective amount of an antiviral composition comprising a compound of the formula ##STR8##

and a pharmaceutically acceptable carrier therefor wherein in the above formulas R is glucosyl and R.sub.1 is hydrogen or a pharmaceutically acceptable ester-forming group and when R.sub.1 is hydrogen, the pharmaceutically acceptable salts of the acids represented thereby.

2. The method of claim 1 wherein said composition is administered orally.

3. The method of claim 1 wherein said composition is administered parenterally.

4. The method of claim 1 wherein R.sub.1 is 2-(4-hydroxyphenyl)ethyl or 2-(3,4-dihydroxyphenyl)ethyl.

5. The method of claim 4 wherein the composition is administered orally.

6. The method of claim 1 wherein the compound is extracted from a plant of the Family Oleaceae.

7. The method of claim 6 wherein the antiviral composition is administered orally.

8. The method of claim 1 wherein the compound is extracted from Olea europaea.

9. The method of claim 1 wherein the antiviral composition includes leaves of Olea europaea dried, ground and formulated in a solid oral dosage form.

10. A solid oral dosage form for treatment of viral infections in warm-blooded vertebrates, said solid dosage form comprising about 30 to about 500 mg of a compound of the formula ##STR9##

and a pharmaceutically acceptable carrier therefor wherein in the above formulas R is glucosyl and R.sub.1 is hydrogen or a pharmaceutically acceptable ester-forming group and when R.sub.1 is hydrogen, the pharmaceutically acceptable salts of the acids represented thereby.

11. The solid oral dosage form of claim 10 wherein the compound is extracted from plant material of the Family Oleaceae.

12. The solid oral dosage form of claim 10 wherein R.sub.1 is 2-(4-hydroxyphenyl)ethyl or 2-(3,4-dihydroxyphenyl) ethyl.

13. The solid oral dosage form of claim 12 wherein the antiviral composition includes dried plant material of Olea europaea or a dried extract thereof.

14. A method for establishing antivirally effective blood levels of a compound of the formula ##STR10##

in a warm-blooded vertebrate suffering from a disease of viral origin, said method comprising the step of administering an antiviral composition orally to said vertebrate said composition comprising about 0.1 to about 15 mg/kg of vertebrate body weight of a compound of the formula ##STR11##

and a pharmaceutically acceptable carrier therefor wherein R is glucosyl and R.sub.1 is hydrogen or a pharmaceutically acceptable ester-forming group and when R.sub.1 is hydrogen, pharmaceutically acceptable salts of the acids represented thereby.

15. The method of claim 14 wherein the antiviral composition includes plant material of the Family Oleaceae or a dried extract thereof containing about 0.2 to about 5 mg of the compound/kg of vertebrate body weight.

16. The method of claim 14 wherein the antiviral composition includes an aqueous or aqueous alcoholic extract of Olea europaea.

17. The method of claim 14 wherein R.sub.1 is 2-(3,4-dihydroxyphenyl)ethyl.

18. A method for producing antivirally effective blood levels of a compound of the formula ##STR12##

in a warm-blooded vertebrate suffering from a disease of viral origin, said method comprising the step of administering parenterally to said vertebrate an antiviral composition comprising about 0.05 to about 3 mg/kg of vertebrate weight of a compound of the formula ##STR13##

and a pharmaceutically acceptable carrier therefor wherein in the above formulas R is glycosyl and R.sub.1 is a pharmaceutically acceptable ester-forming group.

19. The method of claim 17 wherein R.sub.1 is 2-(3,4-dihydroxyphenyl)ethyl.

20. The method of claim 18 wherein the antiviral composition consists essentially of oleuropein and a parenterally acceptable carrier therefor.

Description

FIELD OF THE INVENTION

This invention relates to treatment of disease of viral origin in warm-blooded vertebrates. More particularly, this invention is directed to the use of secoiridoid compounds naturally occurring in plants of the family Oleaceae and derivatives thereof.

BACKGROUND AND SUMMARY OF THE INVENTION

The olive tree and other members of the Family Oleaceae have been documented as a source of medicinal substances since biblical times. Needless to say, many researchers have studied the cocktail of phytogenic substances produced by the olive and other members of the Family Oleaceae. One compound that has received attention from the research community is the secoiridoid glucoside oleuropein, a compound of the formula ##STR1##

wherein R is glycosyl and R.sub.1 is 2-(3,4-dihydroxyphenyl) ethyl. Related secoiridoids wherein R.sub.1 is H, CH.sub.3 or 2-(4-hydroxyphenyl ethyl) are also known to be endogenous to many plant species of the family Oleaceae, although in lesser concentrations and in fewer identified species than the ubiquitous oleuropein.

Animal studies have revealed that oleuropein itself or as a component of extracts of plant tissues containing that compound exhibit both hypoglycemic and cardiovascular effects. It is also known that oleuropein can be acid hydrolyzed to produce (-)-elenolic acid, a compound which has been reported to have antiviral properties in vitro, but little, if any, activity in vivo.

The present invention is based on the discovery that secoiridoid glucosides of the formula ##STR2##

wherein R is glycosyl and R.sub.1 is hydrogen or an ester-forming group are metabolized in vivo to the dextrorotatory form of elenolic acid [(+)-elenolic acid], a compound of the formula ##STR3##

wherein R.sub.1 is hydrogen, a compound which is believed to be more available in vivo than the corresponding diasteromer(-)-elenolic acid of the formula ##STR4##

The enhanced in vivo efficacy of the oleuropein metabolite, (+)-elenolic acid relative to the corresponding levorotatory compound is thought to be due, at least in part, to its reduced affinity for serum proteins and thus its greater availability for uptake by virus infected tissues. Antivirally effective blood levels of (+)-elenolic acid can also be achieved by administration of (+)-elenolic acid and its esters of the formula ##STR5##

wherein R.sub.1 is hydrogen or a pharmaceutically acceptable ester-forming group and salts thereof, which can be prepared from secoiridoid glucosides naturally occurring in plant material of the family Oleaceae via extraction and controlled enzyme (glucosidase) hydrolysis and/or deesterification/transesterification reactions.

Thus, it is one object of the present invention to provide a method of treatment of disease of viral origin in warm-blooded vertebrates by administering antiviral compositions containing secoiridoid glucosides native to the plant family Oleaceae and derivatives thereof.

Another object of the invention is to provide oral dosage forms of secoiridoid glucosides of Olea europaea and derivatives thereof.

In another more particular aspect of this invention plant material of the family Oleaceae and extracts thereof containing naturally occurring oleuropein glucosides and enzyme hydrolysates thereof are administered in treatment of diseases of viral origin in warm-blooded vertebrates suffering from such diseases.

One further object of this invention is a method for establishing antivirally effective blood levels of (+)-elenolic acid by the administration of oleuropein glucosides native to the family Oleaceae or their derivatives via transesterification, diesterification and/or glucolysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of treatment of disease of viral origin in warm-blooded vertebrates and to pharmaceutical formulations for use in such treatment methods. The method comprises the step of administering to a vertebrate suffering from a disease of viral origin an antivirally effective amount of an antiviral composition comprising a compound of the formula ##STR6##

and a pharmaceutically acceptable carrier therefor. In the above formula, the group R is glucosyl and R.sub.1 is hydrogen or a pharmaceutically acceptable ester-forming group. When R.sub.1 is hydrogen, the acid compound represented can be utilized in the form of one of its pharmaceutically acceptable salts.

There are many diseases of viral etiology that afflict man and animal. In man, diseases such as hepatitis, mononucleosis, shingles, herpes, influenza, the common cold and even certain types of leukemia are known to be of viral etiology. Viral infections are also common in many animal species, both in meat producing and in companion animals. Rotovirus infections plague swine. Cattle develop bovine rhinovirus infections (shipping fever) when subjected to conditions of stress. Canine parvovirus and feline leukemia virus are common viral infections in those companion animals species. Such diseases of viral origin can be treated with resultant reduction in clinical symptomology by therapeutic administration of antiviral compositions in accordance with this invention.

The antiviral compositions administered in accordance with this invention comprise a compound of Formula I or II above in combination with a pharmaceutically acceptable carrier. The compounds of Formula I wherein R.sub.1 is 2-(4-hydroxyphenyl)ethyl or 2-(3,4-dihydroxyphenyl)ethyl are naturally occurring compounds in many plants of the Family Oleaceae, including members of the genus Fraxinus, Syringa and the genus Ligustrum. Preferred plant sources of the naturally occurring secoiridoids of Formula I wherein R.sub.1 is 2-(4-hydroxyphenyl)ethyl and 2-(3,4-dihydroxyphenyl)ethyl are varieties of Olea europaea (the olive). Preferred varieties of Olea europaea as a source of secoiridoid glycosides for use in accordance with this invention are the varieties Manzanillo and Mission.

The most prevalent of the secoiridoid compounds in such varieties is the compound of Formula I wherein R.sub.1 is 2-(3,4-dihydroxyphenyl)ethyl and R is glucosyl, a compound given the common name oleuropein. That compound can be readily isolated from plant material, preferably ground leaves of the olive by aqueous or aqueous-alcoholic extraction at room temperature or above, preferably at elevated temperature of about 40 to about 100.degree. C.

Oleuropein can then be purified, for example, by chromatographic separation procedures. That compound can then be used to formulate antiviral compositions in accordance with this invention or to prepare other antivirally effective compounds represented by Formulas I or II. Thus, for example, oleuropein can be subjected, to base catalyzed transesterification wherein the R.sub.1 group 2-(3,4-dihydroxyphenyl)ethyl is exchanged with another pharmaceutically acceptable ester-forming group. The term “pharmaceutically acceptable ester-forming group” as used in defining the present invention, refers to those ester-forming groups which when cleaved via esterase reactions in vivo produce substantially non-toxic, physiologically compatible alcohols. Suitable pharmaceutically acceptable ester-forming groups include C.sub.1 -C.sub.8 lower alkyl, and substituted C.sub.1 -C.sub.8 alkyl, benzyl, substituted benzyl wherein the substituents are halo, C.sub.1 -C.sub.4 alkoxy, C.sub.1 -C.sub.4 acyloxy, and the like. The compound of Formula I wherein R.sub.1 is hydrogen can be produced by esterase-mediated deesterification of oleuropein, typically in an aqueous medium at a pH between about 6 and about 8.5.

The compounds of Formula II are prepared from the corresponding compounds of Formula I by treatment with glucosidase, preferably that from the olive at a pH of about 4 to about 5. The compound of Formula II wherein R.sub.1 is hydrogen is (+)-elenolic acid.

The compounds of Formula I or II wherein R.sub.1 is hydrogen represent carboxylic acids and such acids can be used in accordance with this invention in the acid form or in the form of their pharmaceutically acceptable salts formed with organic bases or inorganic bases, such as ammonium, alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, and potassium carbonate. Of the salt forms,.the potassium and sodium salts are particularly preferred.

The antiviral compositions of the present invention can be administered orally or parenterally in an antivirally effective amount to treat, i.e., reduce the symptoms, of diseases of viral origin. Oral dosage forms can be in a solid or liquid form and comprise an antivirally effective amount of a compound of Formula I or Formula II above and a pharmaceutically acceptable carrier. Such dosage forms can be formulated from pure compounds of Formula I or Formula II, or they can be formulated from ground plant materials of the family Oleaceae, preferably leaves of Olea europaea, or aqueous or aqueous alcoholic extracts thereof. Thus, for example, extraction of dried olive leaves with two volumes of a 12-15% ethanol/water solution for 10 days at room temperature provides an extract containing about 70 to about 250 mg of oleuropein per two ounces of the liquid extract. The extract itself can be administered orally as an antiviral composition in accordance with the method of treatment of the present invention, or aqueous or aqueous alcoholic (preferably methanol or ethanol) extracts can be spray-dried to provide a dry powder which can be formulated into oral dosage forms with other pharmaceutically acceptable carriers.

The solid oral dosage form compositions in accordance with this invention are prepared in a manner well known in the pharmaceutical art, and comprise at least one compound of Formula I or Formula II associated with at least one pharmaceutically acceptable carrier. In making such compositions, the compound of Formula I or Formula II, either in pure form or as a component of ground plant material or extracts thereof, are usually mixed, diluted or enclosed within a carrier. The carrier can be in a solid form, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient. Alternatively, the carrier can be in the form of a capsule or other container to facilitate oral administration. Thus the solid oral dosage forms for administration in accordance with the method of this invention can be in the form of tablets, pills, powders or soft or hard gelatin capsules. Alternatively, the antiviral compositions in accordance with this invention for oral administration can be in liquid form wherein the pharmaceutically acceptable carrier is water or an aqueous alcoholic medium. The compositions for administration in the present method can also be formulated with other common pharmaceutically acceptable excipients, including lactose, dextrose, sucrose, sorbitol, mannitol, starches, gums, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, methylcellulose, water, alcohol and the like. The formulations can additionally include lubricating agents such as talc, magnesium stearate and mineral oil, wetting agents, emulsifying and suspending agents, preserving agents such as methyl- and propylhydroxybenzoates, sweetening agents or flavoring agents. Further the compositions of the present invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.

Orally administered compositions are preferably formulated in unit dosage form with each dosage normally containing from about 30 to about 500 mg of a compound of the Formula I or Formula II, more typically about 100 to about 500 mg of such active compounds. The term “unit dosage form” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical carrier/excipient. The preferred dosage levels of the compounds of Formula I/Formula II for treatment of viral infections in accordance with this invention depend on the route of administration, the nature of the active compound or combination of active compounds, and patient condition. When administered orally for treatment of viral disease, the compounds of Formula I or Formula II are administered at a dose of about 0.1 to about 15 mg/kg, more preferably about 0.2 to about 10 mg/kg of patient/animal body weight. In treatment of viral infections oral dosage forms in accordance with this invention can be administered 1 to 4 times a day, again depending on patient condition and the nature of the disease being treated.

Similar considerations bear on the dosage range for parenteral administration of compounds of Formula I or Formula II in treatment of viral infections in accordance with this invention. Parenteral doses are, however, typically lower than those required for antiviral efficacy via the oral route of administration. Thus, antiviral treatment can be achieved by parenteral administration of about 0.05 to about 3 mg/kg of patient/animal body weight. Parenteral formulations for use in accordance with the present invention are prepared using standard techniques in the art. They are commonly prepared as sterile injectable solutions, using a parenterally acceptable carrier such as isotonic saline solution or as a sterile packaged powder prepared for reconstitution with sterile buffer or isotonic saline prior to administration to a patient. The injectable formulation can contain from about 1 to 50 mg of a compound of Formula I or II per ml of formulation.

Administration of an antivirally effective amount of a composition comprising a compound of Formula I or Formula II and a pharmaceutically acceptable carrier in accordance with this invention produce antivirally effective blood levels of (+)-elenolic acid, a compound of the formula ##STR7##

via a heretofore unappreciated in vivo hydrolysis, and in the case of the compounds of Formula I, stereoselective hydrolysis/rearrangement. Administration of antiviral compositions of a compound of Formula I as part of ground/dried native plant material, preferably olive leaves, or as an aqueous or alcoholic extract of olive leaves is a particularly preferred embodiment in accordance with this invention. While not wishing to be bound by theory, the level of antiviral activity associated with such antiviral compositions may derive from a synergistic antiviral effect with other natural components of the olive leaf, such as the flavonoids rutin, hesperidin, and luteolin-7-glucoside.

The following non-limiting examples are illustrative of the method and compositions of the present invention. It is understood, however, that such examples are but representative of various embodiments of the invention and it is not intended that the invention be limited to the scope of the examples.

EXAMPLE 1

(A) A volume of dried leaves of Olea europaea is suspended in 2 volumes of red wine and held at room temperature for about 7 to about 10 days with periodic stirring. Filtration of the mixture provides a tincture containing about 88 mg of oleuropein per ounce of fluid.

(B) A volume of dried leaves of Olea europaea is suspended in two volumes of water and the resulting suspension is then subjected to conditions of high shear in a Waring blender to produce a dispersion of finely divided plant material which was held at a temperature of about 40 to about 65.degree. for two days. Filtration of the mixture provides an aqueous extract containing about 72 mg of oleuropein per ounce of fluid. The aqueous extract is optionally blended with effective amounts of sweetening and/or flavoring agents to provide a palatable liquid oral dosage form of oleuropein.

(C) One volume of leaves or buds of Olea europaea is combined with about two volumes of a 3:2 mixture of methanol and water. The resulting aqueous alcoholic suspension of plant material is heated for 16 hours at about 75.degree. C., cooled and filtered to provide an aqueous alcoholic extract. The extract is spray-dried to produce a powder comprising oleuropein. The powder is filled into gelatin capsules in an amount sufficient to provide 30 to about 500 mg of oleuropein per capsule.

(D) The spray-dried extract of (C) above is subjected to high pressure liquid chromatography to produce oleuropein [Formula I; R=glycosyl; R.sub.1 =2-(3,4-dihydroxyphenyl)ethyl] and ligstroside [Formula I; R=glycosyl; R.sub.1 =2-(4-hydroxyphenyl)ethyl] in substantially pure form. The purified oleuropein is formulated alone or in combination with ligstroside with tabletting starch and a tabletting lubricant, magnesium stearate, to form a tabletting mixture. The tabletting mixture is pressed into compressed tablets containing about 30 to about 500 mg of oleuropein per tablet.

EXAMPLE 2

Oleuropein (1 g) is dispersed in 50 ml of methanol. The solution is cooled to about 10.degree. C. and treated with stirring with about 2 g of potassium hydroxide pellets. The mixture is allowed to warm to room temperature and after about 6 hours, the reaction mixture is diluted with about 60 ml of 6 N HCl (pH about 7.5) and evaporated to dryness. The chromatographic purification of the product mixture provides oleoside [Formula I; R=glycosyl, R.sub.1 =methyl]. The purified oleoside is formulated into a solid oral dosage form containing about 250 mg of oleoside. Alternatively, oleoside is dissolved in sterile isotonic saline at a concentration of about 5 mg per ml to provide a parenteral dosage form for use in accordance with the method of this invention.

EXAMPLE 3

Five grams of oleuropein is dissolved in 500 ml of water buffered at pH 5.0 and treated with glucosidase until analysis of the reaction mixture by thin layer chromatography indicates completion of the reaction. Standard workup of the reaction mixture followed by chromatographic purification of the product mixture provides a compound of Formula II wherein R.sub.1 =2-(3,4-dihydroxyphenyl)ethyl. The purified product is formulated into oral or parenteral dosage forms and administered for treatment of viral diseases in accordance with the method of the present invention.

EXAMPLE 4

A solution of 500 mg of the product of Example 3 in 50 ml of water buffered at about pH 7.5 to about 8.5 is treated with commercially available esterase at a temperature of about 30.degree. C. until thin layer chromatographic analysis of the esterase reaction mixture indicates completion of the reaction due to disappearance of the starting compound. After reaction completion, the pH of the mixture is readjusted to about 8.5 and after washing it twice with ethyl acetate, the pH of the solution is adjusted to about 4.5 in the presence of 30 ml of ethyl acetate. The ethyl acetate acid extract of the reaction mixture is separated, washed with distilled water and brine, and dried over anhydrous sodium sulfate. Evaporation to dryness provides (+)-elenolic acid [Formula II; R.sub.1 =hydrogen] in substantially pure form. The product is further purified by chromatography and/or crystallization as its sodium or potassium salt. It is formulated into oral or parenteral dosage forms in accordance with this invention for treatment of viral infections in warm-blooded vertebrates.

EXAMPLE 5

Dried leaves of Olea europaea are ground to a fine powder and loaded into gelatin capsules such that each capsule contains a volume of ground plant material containing between about 30 and about 500 mg of oleuropein per capsule. Dried olive leaves typically contain about 60 to about 90 mg of oleuropein per gram of dried leaf. The capsules are administered orally for treatment of diseases of viral origin in accordance with this invention.

EXAMPLE 6

The antiviral efficacy of a composition of Example 1(A) above was evaluated in treatment of six subjects afflicted with herpes virus infections. Each of the six subjects ingested two ounces of the formulation every six hours. All subjects reported reduction of herpetic lesions. Three subjects reported disappearance of lesions in 36-48 hours after initiating treatment. One subject, a 34 year old Caucasian female, had several months earlier discontinued use of birth control pills and it was thought that her estrogen surges were causing immunosuppression which complicated her infection. After three days of doubling the dosage of the tincture (four ounces every six hours) most of her lesions were resolved. Two other subjects, a male and a female, more recently infected were also given the higher doses (four ounces every six hours orally) and each reported improvement measured by reduction in severity of their herpetic lesions, and in at least one patient, a 22.8% decrease in antibody titers (IgG) three weeks after initiating therapy with the oleuropein-containing olive leaf extract.

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H. Esterbauer, G. Striegl, H. Puhl and M. Rotheneder, “Continuous Monitoring of In Vitro Oxidation of Human Low Density Lipoprotein,” Free Rad. Res. Commms., vol. 6, No. 1, pp. 67-75, 1989.

Katerina Pattichis, Louis L. Louca, Joan Jarman, Merton Sandler, Vivette Glover, “5-Hydroxytryptamine Release from Platelets by Different Red Wines: Implications for Migraine,” European Journal of Pharmacology, Environmental Toxicology and Pharmacology Section 292, Jan. 13, 1995, pp. 173-177.

R. Aeschbach, J. Loliger, B.C. Scott, A. Murcia, J. Butler, B. Halliwell and O.I. Aruoma, “Antioxidant Actions of Thymiol, Carvocrol, 6-Gingerol, Zingerone and Hydroxytyrosol,” Food Chem. Toxic., vol. 32, No. 1, pp. 31-36, Jan., 1994.

Michel de Lorgeril, M.D., Patricia Salen, BSc., Jean-Louis Martin, PhD., Isabelle Monjaud, BSc., Philippe Boucher, PhD., Nicolle Mamelle, PhD., “Mediterranean Dietary Pattern in a Randomized Trial: Prolonged Survival and Possible Reduced Cancer Rate,” Arch. Intern. Med., vol. 158, Jun. 8, 1998, pp. 1181-1187.

A. Ghiselli, A. D’Amicis and A. Glacosa, “The Antioxidant Potential of the Mediterranean Diet,” European Journal of Cancer Prevention, vol. 6 (Supp.1), pp. S15-S19, Mar. 1997.

V.L. Singleton and Joseph A. Rossi, Jr., “Colorimetry of Total Phenolics With Phosphomolybdic-Phosphotungstic Acid Reagents,” American Journal of Enology and Viticulture, vol. 16, No. 3, pp. 144-158, 1965.

Hanbury, On the febrifuge properties of the olive (Olea europaea L.), unknown publication information. Shasha, On the oleuropein, the bitter principle of olives, vol. 26:1948-1954, 1960. .
Redman, In vivo antiviral chemotherapy…, Antimicrobial Agents & Chemotherapy 1966, 497-502.

Benavente-Garcia, Antioxidant activity of phenolics extracted from Olea europaea L. leaves, Food Chem. 68:457-462, 2000.

Alcohol extracts show much higher phenolic compounds than water extracts

Bioresour Technol. 2009 Dec;100(23):6107-13. Epub 2009 Jul 15.

Lee OH, Lee BY, Lee J, Lee HB, Son JY, Park CS, Shetty K, Kim YC.

Department of Nutrition, University of Massachusetts, Amherst, MA 01003, USA.

Recent studies suggest that olive leaf is a significant source of bioactive phenolic compounds comparable to olive oil and fruits. Identifying appropriate extraction methods is thus an important step to increase the yield of such bioactive components from olive leaf, which is otherwise agricultural waste. The present study evaluates phenolic contents and compositions of olive leaf extracted by several solvent methods and to further establish their antioxidant activities using various radical scavenging systems. Total flavonoid and phenolic contents were significantly higher in the 80% ethanol extract, butanol, and ethylacetate fractions than hexane, chloroform and water fractions (p<0.05). Oleuropein was identified as a major phenolic compound with considerable contents in these major three fractions and the extract that correlated with their higher antioxidant and radical scavenging. These results indicate that olive leaf contains significant amounts of oleuropein and phenolics, important factors for antioxidant capacity, which can be substantially modified by different extraction methods.

PMID: 19608415 [PubMed - in process]

Anal Bioanal Chem. 2008 Nov;392(5):977-85. Epub 2008 Sep 2

Optimisation of the extraction of olive (Olea europaea) leaf phenolics using water/ethanol-based solvent systems and response surface methodology.

Mylonaki S, Kiassos E, Makris DP, Kefalas P.

Department of Chemistry, University of Crete, P.O. Box 2208, 71003, Voutes, Heraklion, Greece.

An experimental setup based on a 2(3) full-factorial, central-composite design was implemented with the aim of optimising the recovery of polyphenols from olive leaves by employing reusable and nontoxic solutions composed of water/ethanol/citric acid as extracting media. The factors considered were (i) the pH of the medium, (ii) the extraction time and (iii) the ethanol concentration. The model obtained produced a satisfactory fit to the data with regard to total polyphenol extraction (R(2) = 0.91, p = 0.0139), but not for the antiradical activity of the extracts (R(2) = 0.67, p = 0.3734). The second-order polynomial equation obtained after analysing the experimental data indicated that ethanol concentration and time mostly affected the extraction yield, but that increased pH values were unfavourable in this regard. The maximum theoretical yield was calculated to be 250.2 +/- 76.8 mg gallic acid equivalent per g of dry, chlorophyll-free tissue under optimal conditions (60% EtOH, pH 2 and 5 h). Liquid chromatography-electrospray ionisation mass spectrometry of the optimally obtained extract revealed that the principal phytochemicals recovered were luteolin 7-O-glucoside, apigenin 7-O-rutinoside and oleuropein, accompanied by smaller amounts of luteolin 3′,7-O-diglucoside, quercetin 3-O-rutinoside (rutin), luteolin 7-O-rutinoside and luteolin 3′-O-glucoside. Simple linear regression analysis between the total polyphenol and antiradical activity values gave a low and statistically insignificant correlation (R(2) = 0.273, p > 0.05), suggesting that it is not the sheer amount of polyphenols that provides high antioxidant potency; instead, this potency is probably achieved through interactions among the various phenolic constituents.

PMID: 18762919 [PubMed - indexed for MEDLINE]

J Chromatogr A. 2006 Mar 3;1108(1):76-82. Epub 2006 Jan 26

Dynamic ultrasound-assisted extraction of oleuropein and related biophenols from olive leaves.

Japón-Luján R, Luque-Rodríguez JM, Luque de Castro MD.

Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Córdoba, E-14071 Córdoba, Spain.

A continuous approach for the ultrasound-assisted extraction of olive biophenols (OBPs) from olive leaves is proposed. Multivariate methodology was used to carry out a detailed optimisation of extraction. Under the optimal working conditions, complete extraction of the target analytes (namely, oleuropein, verbacoside, apigenin-7-glucoside and luteolin-7-glucoside with LODs 11.04, 2.68, 1.49 and 3.91 mg/kg, respectively) was achieved in 25 min. The extract was injected into a chromatograph-photodiode array detector assembly (HPLC-DAD) for individual separation-quantification. No clean-up or preconcentration steps were required. Gas chromatography-mass spectrometry (without derivatization of the analytes) was used to identify OBPs at concentrations below the LODs obtained by HPLC-DAD. The efficacy of ethanol-water mixtures to extract OBPs from olive leaves has been demonstrated and compared with that of a conventional method which requires 24h for complete extraction; so these mixtures can substitute toxic extractants used to date.

PMID: 16442552 [PubMed - indexed for MEDLINE]

J Nutr. 2009 Sep 23. [Epub ahead of print]

Olive Leaf Extract and Its Main Component Oleuropein Prevent Chronic Ultraviolet B Radiation-Induced Skin Damage and Carcinogenesis in Hairless Mice.

Kimura Y, Sumiyoshi M.

Division of Biochemical Pharmacology, Department Basic Medical Research, Ehime University Graduate School of Medicine, Toon City Ehime 791-0295, Japan.

Chronic exposure to solar UV radiation damages skin, increasing its thickness and reducing its elasticity, and causes skin cancer. Our aim in this study was to examine the effects of an olive leaf extract and its component oleuropein on skin damage and the incidence of skin tumors caused by long-term UVB irradiation in hairless mice. Male hairless mice (5 wk old) were divided into 6 groups, including a non-UVB group, a vehicle-treated UVB group (control), 2 olive leaf extract-treated UVB groups, and 2 oleuropein-treated UVB groups. Five groups were UVB irradiated (36-180 mJ/cm(2)) 3 times each week for 30 wk and skin thickness and elasticity after UVB irradiation were measured every week. Olive leaf extract (300 and 1000 mg/kg) and oleuropein (10 and 25 mg/kg) were administered orally twice daily every day for 30 wk. The extract and oleuropein significantly inhibited increases in skin thickness and reductions in skin elasticity, and skin carcinogenesis and tumor growth. Furthermore, they prevented increases in the expression of matrix metalloproteinase (MMP)-2, MMP-9, and MMP-13 as well as in levels of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) in the skin. Based on histological evaluation, they prevented increases in the expression of Ki-67 and CD31-positive cells induced by the irradiation. These results suggest that the preventative effects of the olive leaf extract and oleuropein on chronic UVB-induced skin damage and carcinogenesis and tumor growth may be due to inhibition of the expression of VEGF, MMP-2, MMP-9, and MMP-13 through a reduction in COX-2 levels.

PMID: 19776181 [PubMed - as supplied by publisher]

WATER EXTRACT?

Int J Antimicrob Agents. 2009 May;33(5):461-3. Epub 2009 Jan 9

Antimicrobial activity of commercial Olea europaea (olive) leaf extract.

Sudjana AN, D’Orazio C, Ryan V, Rasool N, Ng J, Islam N, Riley TV, Hammer KA.

Discipline of Microbiology and Immunology, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia.

The aim of this research was to investigate the activity of a commercial extract derived from the leaves of Olea europaea (olive) against a wide range of microorganisms (n=122). Using agar dilution and broth microdilution techniques, olive leaf extract was found to be most active against Campylobacter jejuni, Helicobacter pylori and Staphylococcus aureus [including meticillin-resistant S. aureus (MRSA)], with minimum inhibitory concentrations (MICs) as low as 0.31-0.78% (v/v). In contrast, the extract showed little activity against all other test organisms (n=79), with MICs for most ranging from 6.25% to 50% (v/v). In conclusion, olive leaf extract was not broad-spectrum in action, showing appreciable activity only against H. pylori, C. jejuni, S. aureus and MRSA. Given this specific activity, olive leaf extract may have a role in regulating the composition of the gastric flora by selectively reducing levels of H. pylori and C. jejuni.

PMID: 19135874 [PubMed - indexed for MEDLINE]

Phytother Res. 2009 Mar;23(3):347-

Antidiabetic effect of Olea europaea L. in normal and diabetic rats.

Eidi A, Eidi M, Darzi R.

Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. akram_eidi@yahoo.com

The antidiabetic effect of an alcohol extract of olive (Olea europaea L.) leaves was investigated in normal and streptozotocin-induced diabetic rats. The oral administration of the olive leaves extract (0.1, 0.25 and 0.5 g/kg body wt) for 14 days significantly decreased the serum glucose, total cholesterol, triglycerides, urea, uric acid, creatinine, aspartate amino transferase (AST) and alanine amino transferase (ALT) while it increased the serum insulin in diabetic rats but not in normal rats (p < 0.05). A comparison was made between the action of olive leaves extract and glibenclamide (600 microg/kg), a known antidiabetic drug. The antidiabetic effect of the extract was more effective than that observed with glibenclamide. (c) 2008 John Wiley & Sons, Ltd.

PMID: 18844257 [PubMed - indexed for MEDLINE]

J Ethnopharmacol. 2008 Nov 20;120(2):233-40. Epub 2008 Aug 23.

Olea europaea leaf extract exerts L-type Ca(2+) channel antagonistic effects.

Scheffler A, Rauwald HW, Kampa B, Mann U, Mohr FW, Dhein S.

Klinik für Herzchirurgie, Herzzentrum Leipzig, Strümpellstr. 39, 04289 Leipzig, Germany. anja.scheffler@medizin.uni-leipzig.de

ETHNOPHARMACOLOGICAL RELEVANCE: In Southern Europe Olea europaea leafs are known as a folk remedy for hypertension. Cardiovascular diseases are still the leading causes of morbidity and mortality in industrialized countries with hypertension being one of the main risk factors. AIM OF THE STUDY: We investigated effects of a commercial Olea europaea leaf extract (OLE) on isolated hearts and cultured cardiomyocytes. MATERIALS AND METHODS: Isolated rabbit hearts were perfused according to the Langendorff technique and connected to a 256-channel epicardial mapping system. Voltage clamp experiments were performed in cultured neonatal rat cardiomyocytes using a perforated-patch technique. RESULTS: OLE caused a concentration-depended decrease in systolic left ventricular pressure and heart rate as well as an increase in relative coronary flow and a slight, but not significant prolongation of PQ-time. There were no significant changes between the groups in the activation-recovery interval and its dispersion, total activation time, peak-to-peak amplitude, percentage of identical breakthrough-points and similar vectors of local activation. Voltage clamp experiments in cultured neonatal rat cardiomyocytes showed a significant decrease in maximum I(Ca,L) by OLE which was reversible upon wash-out. CONCLUSIONS: OLE suppresses the L-type calcium channel directly and reversibly. Our findings might help to understand the traditional use of OLE in the treatment of cardiovascular disease.

PMID: 18790040 [PubMed - indexed for MEDLINE]

Dear Dr. Hall,

We used the commercial extract of folia olivae eur. of the company Ysat (brand name: Olivysat), which is an ethonal/water extract (80% ethanol). For experimental use it may be necessary to add 0.1-1.o% DMSO, depending on the buffer solution which is used. We hope that we could help you with this information and we remain

Prof. Dr. Stefan Dhein & Anja Hagen
Herzzentrum Leipzig
Klinik f. Herzchirurgie
Struempellstr. 39
04289 Leipzig Germany

A double-blind, randomized, placebo-controlled study recruited 90 people (age range 55 to 75), received an olive leaf extract per day or placebo for eight weeks.

Blood samples were taken in order to measure a variety of biochemical markers. The RA subjects receiving the olive extract had significantly lower homocysteine levels than the placebo group. “High homocysteine levels in patients with RA have been associated with higher rates of mortality from cardiovascular events,” stated the researchers.

C-reactive protein (CRP) levels decreased by about 50 per cent as a result of olive extract supplementation, while CRP levels in the placebo group increased. Supplementation with olive leaf & extract also decreased pain and inflammation of people suffering from osteoarthritis the author wrote:

Thus, introducing this natural and safe food extract into the diet may have the same or better beneficial effects as an intervention diet.

Source: Nutrition Research (Elsevier) August 2007, Volume 27, Issue 8

• Athletes Foot (Tinea Pedis)
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There are many other situations for which The Original Olive Leaf Extract® may act as an antimicrobial agent that are not listed on this page.

The statements set forth have not been evaluated by the Food & Drug Administration and are only intended for information purposes.  Always consult your healthcare provider before using any health or dietary supplement.

Olive Leaf Extract Qualifies for the GRAS List under 21 CFR 170.30 (c) and 170.3 (f)