商品描述

橄欖多酚(萃取液)

Abstract:

The Mediterranean diet is rich in vegetables, cereals, fruit, fish, milk, wine and olive oil and has salutary biological functions. Epidemiological studies have shown a lower incidence of atherosclerosis, cardiovascular diseases and certain kinds of cancer in the Mediterranean area. Olive oil is the main source of fat, and the Mediterranean diet's healthy effects can in particular be attributed not only to the high relationship between unsaturated and saturated fatty acids in olive oil but also to the antioxidant property of its phenolic compounds. The main phenolic compounds, hydroxytyrosol and oleuropein, which give extra-virgin olive oil its bitter, pungent taste, have powerful antioxidant activity both in vivo and in vitro. The present review focuses on recent works analysing the relationship between the structure of olive oil polyphenolic compounds and their antioxidant activity. These compounds' possible beneficial effects are due to their antioxidant activity, which is related to the development of atherosclerosis and cancer, and to anti-inflammatory and antimicrobial activity.

Leaves of the privet tree, Ligustrum obtusifolium, contain a large amount of oleuropein, a phenolic secoiridoid glycoside, which is stably kept in a compartment separate from activating enzymes. When the leaf tissue is destroyed by herbivores, enzymes localized in organelles start to activate oleuropein into a very strong protein denaturant that has protein-crosslinking and lysine-decreasing activities. These activities are stronger than ever reported from plant systems and have adverse effects against herbivores by decreasing the nutritive value of dietary protein completely. We report here that strong oleuropein-specific β-glucosidase in organelles activates oleuropein by converting the secoiridoid glucoside moiety of oleuropein into a glutaraldehyde-like structure, which is also an α,β-unsaturated aldehyde. Oleuropein activated by β-glucosidase had very strong protein-denaturing, protein-crosslinking, and lysine-alkylating activities that are very similar to, but stronger than, those of glutaraldehyde. Aucubin, another iridoid glycoside, had similar activities after β-glucosidase treatment. We also detected polyphenol oxidase activity in organelles that activate the dihydroxyphenolic moiety to have protein-crosslinking activities. These data suggest that the privet tree has developed an effective defense mechanism with oleuropein, a unique multivalent alkylator ideal as a protein-crosslinker. Our results that iridoid glycosides are precursors of alkylators may elucidate the chemical bases that underlie various bioactivities and ecological roles of iridoid glycosides.

Recently, the importance of alkylating agents in plant–herbivore interactions has been recognized. Alkylating agents, which include quinones, epoxides, aldehydes, sesquiterpene lactone, pyrrolizidine alkaloids, etc., are diverse in their chemical structure, but they all have electrophilic atoms. These structures readily bind to various biological nucleophiles, including nucleophilic side chains of proteins (e.g., -NH2 of Lys and -SH of Cys), and therefore exert adverse effects on herbivores (e.g., loss of nutritive value caused by loss of Lys and inactivation of enzymes), providing plants a chemical defense against herbivores.

The privet tree, Ligustrum obtusifolium (Oleaceae), is a small tree or a shrub that is widespread throughout East Asia and has been naturalized recently in the northeastern parts of the United States. Previously, we reported that the leaves of the privet tree have very strong protein-denaturing, protein-crosslinking, and lysine-decreasing activities that could be explained in terms of alkylating activities. When protein is mixed with the leaf extract, protein denatures and forms a high-molecular-mass complex. At the same time, the lysine content of the protein decreases to one-third to one-fifth of the original, although other amino acids were not affected. As a result, the protein loses its nutritive value. Interestingly, addition of free glycine can inhibit all these activities, and privet-specialist herbivores secrete free glycine in the digestive juice as an adaptation. Purification study established that the compound responsible for the denaturing activity is oleuropein (Fig. 1), a phenolic secoiridoid glycoside found in several Oleaceae species such as the olive tree. Oleuropein makes up 3% of the wet weight of privet leaves. However, this compound itself is stable, does not have any of the activities, and is kept in the vacuoles or cytosol of the leaf cell. When the leaves are mechanically damaged by herbivory and cell compartments are broken, enzymatic activity localized in organelles separate from oleuropein starts to activate oleuropein into a very strong protein denaturant. Although the alkylating activities of privet leaves are stronger than those of other plants, the chemical mechanism of activation was not clear.

Iridoids (iridoid glycosides) are a group of terpene-derived compounds that have a common structure. At present, almost 600 iridoids have been described from plants of 57 families and are divided into three groups: (i) nonglycosidic iridoids, which have no sugar moiety, such as genipin (Fig. 1); (ii) iridoid glycosides, which typically have a single glucose molecule and a closed cyclopentane ring, such as aucubin and geniposide (Fig. 1); and (iii) secoiridoid glycosides, which also have a glucose molecule but no cyclopentane ring, such as oleuropein. Iridoids are known to have a variety of biological effects and have been implicated to play roles in plant–herbivore and prey–predator interactions. As iridoids generally are toxic or deterrent to generalist herbivores, generalists usually experience a reduced growth rate or are deterred from feeding on iridoid-containing plants. However, iridoids have no negative effects on the feeding and growth of herbivores that specialize in feeding on iridoid-containing plants. Some of these specialists sequester a large amount of iridoid and have conspicuous warning coloration. These insects are toxic and unpalatable to predators such as birds and ants. Iridoids are also reported to have antimicrobial , antitumor , hepatotoxic , bitter , and emetic features . Nevertheless, the chemical bases that lie under these interesting characteristics of iridoids have not yet been well explained.

In this study, we show that the activation of the secoiridoid glycoside moiety of oleuropein by substrate-specific β-glucosidase in organelles is crucial for oleuropein to exert the strong protein-denaturing/protein-crosslinking/lysine-alkylating activities in privet leaves. We also show that other iridoid glycosides could be similarly activated into alkylating agents by β-glucosidase and discuss the chemical bases for the biological activities of iridoids.

MATERIALS AND METHODS:

Materials. Oleuropein was purified from the leaves of the privet tree (Ligustrum obtusifolium) as described . Aucubin was purchased from Nacalai Tesque (Kyoto). Geniposide, genipin, tropolone (polyphenol oxidase inhibitor), catechol, 20% (vol/vol) glutaraldehyde solution, ovalbumin, and p-nitrophenol were from Wako Pure Chemical Industries (Osaka). p-Nitrophenyl-β-glucopyranoside was from Senn Chemicals (Dielsdorf, Switzerland). β-Glucosidase (sweet almond) was purchased from Oriental Yeast (Osaka).

Preparation of Crude Enzyme Fraction (Organelle Fraction) from Privet Leaves. The enzyme fraction of the privet tree, which contains oleuropein-activating enzymes but not oleuropein, was prepared as described . In short, 16 g of fresh privet leaves were homogenized in 100 ml of high-osmotic-pressure sucrose solution (0.4 M sucrose/0.1 M sodium phosphate, pH 7) to collect intact organelles. After differential centrifugation and subsequent washing in the same buffer, collected organelles were resuspended in 20 ml of the same buffer. This suspension of intact organelles, where oleuropein-activating enzymes are localized, was used as the enzyme fraction. Breaking the organelles, a necessary step in assaying the enzyme activities inside them, was accomplished automatically when the organelles were burst in reaction solutions with low osmotic pressure.

Assays of Protein-Denaturing/Protein-Crosslinking/Lysine-Decreasing Activity. The activities were examined by testing whether ovalbumin would be denatured. All the assays were performed in 375 μl of 0.1 M sodium phosphate buffer (pH 7.0), containing 1% ovalbumin and 250 μl of 0 or 11 mM solutions of chemicals whose activities were to be measured (i.e., oleuropein, catechol, glutaraldehyde, geniposide, genipin, and aucubin). In trials designed to examine the effects of enzymes, 375-μl volumes of the reaction solutions also included 50 μl of the enzyme fraction of privet leaves or 50 μl of β-glucosidase solution, which contained 5 units of β-glucosidase (from sweet almond). Assays were performed in triplicate at 25°C for 2 h in 1.5-ml microfuge tubes with vigorous shaking. The effect of tropolone (polyphenol oxidase inhibitor; chelator of copper ion; refs. 21 and 22) on the denaturing activity was assayed by preincubating the 50-μl volumes of enzyme solutions with 90 μg of tropolone for 10 min before the initiation of the reaction; 5 μl of the reaction solution was applied to SDS/PAGE to determine the protein-denaturing/protein-crosslinking activities. The degree of denaturation and crosslinking was determined by the band pattern. For amino acid analysis, 1 ml of 6 M HCl and 700 μl of water were added to 300 μl of the reaction solution and hydrolyzed for 22 h at 110°C. After removal of HCl, lysine contents were analyzed with an auto amino acid analyzer (model L5000, Hitachi, Tokyo).

Assay of β-Glucosidase Activity and Assessment of the Degree of Deglucosidation. β-Glucosidase activity of the enzyme fraction (organelle fraction) on oleuropein, aucubin, and geniposide was assayed by measuring the amounts of glucose produced in the reaction solutions. To assay β-glucosidase activity of the enzyme fraction on oleuropein, an enzyme reaction was performed in triplicate in 375 μl of 0.1 M sodium phosphate buffer (pH 7.0) containing 250 ml of 11 mM solution of oleuropein, 10 μl of the enzyme fraction, and 90 μg of tropolone for 15 min at 25°C. Tropolone was preincubated with the enzyme fraction to inhibit the interference of polyphenol oxidase activity as described above. To assay β-glucosidase activity of the enzyme fraction on aucubin and geniposide, enzyme reactions were performed in 375 μl of 0.1 M sodium phosphate buffer (pH 7.0) containing 50 μl of the enzyme fraction and 250 ml of either 11 mM aucubin or geniposide at 25°C for 2 h. These conditions were within the linear range. Reactions were stopped by heat inactivation (96°C for 5 min). After centrifugation and filtration, 100-μl volumes of the supernatants from the reaction solutions were injected into an STR-ODS-H column (150 × 4.6-mm i.d., 5 μm; Shimadzu) to separate the produced glucose from chemicals that interfere with the detection of glucose. The column temperature was 40°C, and the flow rate was 1.0 ml/min. The gradient was as follows: 0–10 min, 0–70% (vol/vol) ethanol in water (linear gradient) and then 10–15 min, 70% ethanol (isocratic). A 1-ml volume of glucose-containing fraction of each sample was collected between 1.0 and 2.0 min, and the glucose concentration was analyzed by using GOD-PODLK (Nagase Biochemical, Kyoto), a glucose assaying kit based on a glucose oxidase-peroxidase-chromogen system, which uses phenol as a substrate of peroxidase and 4-aminoantipyrine as a chromogen. Absorbance at 505 nm was measured. Glucose solutions of known concentrations in 0.1 M sodium phosphate buffer (pH 7.0) were also analyzed as standards by using HPLC and GOD-PODLK.

To assess the degree of deglucosidation of oleuropein and other iridoid glycosides by the enzyme fraction (organelles) or β-glucosidase, assays were performed in 375 μl of 0.1 M sodium phosphate buffer (pH 7.0) containing 11 mM solutions of an iridoid glycoside (oleuropein, geniposide, or aucubin) and 50 μl of the enzyme fraction of privet leaves or 50 μl of β-glucosidase solution, which contained 5 units of β-glucosidase (from sweet almond). In the reaction of oleuropein and the enzyme fraction of the leaves, the enzyme fraction was preincubated with tropolone as described above. Assays were performed in triplicate at 25°C for 2 h in 1.5-ml microfuge tubes with vigorous shaking. The glucose produced was analyzed as described above.

To assay the β-glucosidase activity of the enzyme fraction on p-nitrophenyl-β-glucopyranoside, a reaction was performed in 375 μl of 0.1 M sodium phosphate buffer (pH 7.0) containing 250 μl of 11 mM p-nitrophenyl-β-glucopyranoside, 90 μg of tropolone, and 50 μl of the enzyme fraction at 25°C for 2 h. After the reaction, 100 μl of the reaction solution was mixed with 2 ml of 1 M Na2CO3, and then absorbance at 450 nm was measured. The degree of deglucosidation was determined by comparing this absorbance with that of p-nitrophenol at 450 nm .

RESULTS:

Protein-Denaturing/Protein-Crosslinking/Lysine-Decreasing Activities of Oleuropein: Activation of Phenolic Moiety by Polyphenol Oxidase. Oleuropein itself did not have any protein-denaturing and lysine-decreasing activities. There was no difference in SDS/PAGE pattern and lysine content between untreated ovalbumin and ovalbumin treated only with 11 mM oleuropein (Fig. 2 A and B, lanes 1 and 2). When organelles (the enzyme fraction), which have no protein-denaturing and lysine-decreasing activities (7), were added together in the mixture of ovalbumin and oleuropein, ovalbumin was denatured and high-molecular-mass crosslink products were formed, which are visible in the disappearance of the main band and in the appearance of fuzzy bands in the upper part of the stacking and separation gels (Fig. 2A, lane 3). At the same time, the lysine content of the treated protein decreased to approximately one-third of the original content (Fig. 2B, lane 3), but other amino acids were not affected (data not shown, but see refs. 6 and 7). These results suggest that activation by enzymes retained in organelles is necessary for oleuropein to exert its protein-denaturing/protein-crosslinking/lysine-decreasing activities.

To elucidate the chemical process of enzymatic activation of oleuropein, we first examined whether activation of the phenolic moiety is involved. The rapid browning that we observed when we mixed oleuropein and the enzyme fraction together, a hallmark of polyphenol oxidation and polymerization, supported the idea that the phenolic moiety is involved. Catechol (11 mM), a dehydroxyphenolic moiety of oleuropein itself, did not have any of the activities on ovalbumin without enzymatic activation (Fig. 2 A and B, lane 4). When mixed with the enzyme fraction (organelles) of privet leaves, 11 mM catechol had certain protein-denaturing/protein-crosslinking/lysine-decreasing activities (Fig. 2 A and B, lane 5) that were weaker than those observed when 11 mM of oleuropein was mixed with the enzyme fraction (Fig. 2, lane 3). The color of the reaction solution changed to dark brown. However, when we added 90 μg of tropolone (final concentration of 2 mM), a polyphenol oxidase inhibitor (21, 22), to the reaction between catechol and the enzyme fraction, all the activities and the browning of the reaction solution were inhibited completely (Fig. 2, lane 6). These data suggest that the enzyme fraction of privet leaves has a certain polyphenol oxidase activity that activates the phenolic moiety of oleuropein to some extent. However, contrary to our expectations, tropolone did not inhibit the protein-denaturing/protein-alkylating/lysine-decreasing activities of oleuropein activated by the enzyme fraction of privet leaves (Fig. 2, lane 7), although browning was inhibited completely. This result strongly suggested that some activation mechanism other than oxidation of the phenolic moiety exists and plays a greater role.

Protein-Denaturing/Protein-Crosslinking/Lysine-Decreasing Activities of Oleuropein: Activation of Iridoid Glycoside Moiety by β-Glucosidase. Next, we investigated the iridoid glycoside moiety and hypothesized that, if glucose is removed, after the ring-opening reaction in the hemiacetal-like structure and subsequent keto-enol conversion, the iridoid glycoside moiety may form a glutaraldehyde-like structure (Fig. 1). This conversion had been suggested to occur by several researchers (18, 20, 26), but its chemical, physiological and ecological consequences had not been studied in detail. As glutaraldehyde is well known as an potent alkylator, crosslinker, and denaturant of protein and is used to fix protein in histochemical studies (27–31), we hypothesized that the same reaction might occur in our system. To examine this possibility, we mixed 11 mM oleuropein with 5 units of β-glucosidase from sweet almond. Oleuropein incubated with β-glucosidase had protein-denaturing/protein-crosslinking/lysine-decreasing activities (Fig. 2 A and B, lane 8) just as strong as those observed when oleuropein was activated by the enzyme fraction of privet leaves in the presence of tropolone (Fig. 2, lane 7). Browning of the reaction solution did not occur at all. We next examined whether glutaraldehyde has similar activities. Glutaraldehyde had activities (Fig. 2, lane 9) that are stronger than those observed in catechol activated by the enzyme fraction of privet leaves (Fig. 2, lane 5) but weaker than those observed in oleuropein activated by β-glucosidase (Fig. 2, lane 7). We further examined whether other iridoid glycosides are activated by β-glucosidase and by the enzyme fraction of privet leaves as well. Aucubin, which does not have any of the activities itself (Fig. 2 A and B, lane 14), had considerably strong activities when activated by β-glucosidase from sweet almond (Fig. 2, lane 15). The lysine-decreasing activity was as strong as that of glutaraldehyde (Fig. 2B, lanes 9 and 15), and the protein-denaturing/protein-crosslinking activities were stronger than those of glutaraldehyde and equaled those of oleuropein, as determined by the SDS/PAGE pattern (Fig. 2A, lanes 7–9 and 15). However, aucubin was not activated by the enzyme fraction of privet leaves (Fig. 2, lane 16). Geniposide, another iridoid glycoside that also has no activities itself, had weak activities after activation by β-glucosidase or by the enzyme fraction (Fig. 2 A and B, lanes 10–12). Geniposide is different from other iridoid glycosides in that its aglycone is relatively stable and is available as genipin. In agreement with this result, genipin had weak activities (Fig. 2, lane 13). All of these data indicate that oleuropein and other iridoid glycosides could be activated by β-glucosidase to exert activities very similar to those of privet leaves and glutaraldehyde.

β-Glucosidase Activity in the Enzyme Fraction Is Highly Specific to Oleuropein. To examine whether β-glucosidase really exists and plays a crucial role in privet leaves, we assayed β-glucosidase activity in the enzyme fraction (organelles) of privet leaves on oleuropein and other related β-glucosides (Table 1). The enzyme fraction (organelles) derived from 1 g of fresh privet leaves had 5.22 ± 1.01 μmol/min β-glucosidase activity on oleuropein. As 1 g of fresh leaves contains 55.5 μmol of oleuropein, the data show, theoretically, that the β-glucosidase activity is strong enough to deglucosidate and activate all the oleuropein in privet leaves in about 10 min. With geniposide as a substrate, the enzyme fraction from 1 g of fresh leaves had 0.142 ± 0.004 μmol/min β-glucosidase activity. With aucubin as a substrate, the same enzyme fraction had practically no activity (0.000 ± 0.000 μmol/min). With p-nitrophenyl-β-glucopyranoside, an artificial substrate often used in assaying β-glucosidase activity, as a substrate, this enzyme fraction had 0.029 ± 0.002 μmol/min activity. These data indicate that the enzyme fraction (organelles) of privet leaves has a strong and substrate-specific β-glucosidase activity on oleuropein and suggest that this β-glucosidase activity is the major factor that activates oleuropein in privet leaves.

Degree of Deglucosidation in Assaying Conditions. To interpret the results shown in Fig. 2 in greater detail, we examined the degree of deglucosidation in the assaying conditions by measuring the amounts of glucose produced after the reactions (Table 2). The enzyme fraction of privet leaves deglucosidated 92.2 ± 2.9% of oleuropein in the presence of tropolone (corresponding to lane 7 of Fig. 2). β-Glucosidase from bitter almond deglucosidated 40.9 ± 3.6% of oleuropein (Fig. 2, lane 8). The enzyme fraction of privet leaves deglucosidated 24.8 ± 0.7% of geniposide (Fig. 2, lane 11), and β-glucosidase deglucosidated 100.3 ± 12.9% of geniposide (Fig. 2, lane 12). Although β-glucosidase deglucosidated 34.4 ± 5.0% of aucubin (Fig. 2, lane 15), the enzyme fraction did not deglucosidate aucubin in assaying conditions (0.0 ± 0.0%; Fig. 2, lane 16). These enzyme assays based on the appearance of glucose showed good agreement with those based on the decrease of iridoid glycosides in the reaction solutions (as estimated by peak areas in HPLC) and also were in good agreement with those based on the appearance of genipin when geniposide was used (data not shown). In the case of oleuropein and aucubin, however, the corresponding aglycones were not observed in the reaction solutions as HPLC peaks, probably because of the instability of these aglycones. These data suggest that the inability of the enzyme fraction of privet leaves to activate aucubin is caused by its inability to deglucosidate aucubin and that the inability of the enzyme fraction to activate geniposide is caused by the inactivity of the corresponding aglycone. For an iridoid glycoside to exert strong activities, both the activeness of aglycone and the existence of efficient β-glucosidase are necessary. The oleuropein system of the privet tree seems to fulfill both criteria.

Figure 1 Structures of oleuropein and its related compounds.

Figure 2.Protein-denaturing/protein-crosslinking/lysine-decreasing activities of oleuropein, catechol, glutaraldehyde, and iridoid glycosides in the presence of the enzyme fraction of privet leaves (organelles), β-glucosidase, or tropolone (polyphenol oxidase inhibitor). (A) Protein-denaturing/protein-crosslinking activities on ovalbumin as assayed by SDS/PAGE. The degree of denaturation and crosslinking is indicated by the disappearance of the main band of ovalbumin and the appearance of fuzzy bands in the upper parts of stacking and separation gel. (B) Lysine-decreasing activity. After hydrolysis, the lysine content of ovalbumin in reaction solution was analyzed with an auto amino acid analyzer (Hitachi). Error bars represents SD (n = 3).

Figure 3.Proposed chemical model of oleuropein activation in the privet tree, Ligustrum obtusifolium.

表1

‑

SD (n = 3) 。

Table 2.Percentage of deglucosidation of iridoid glycosides in conditions corresponding to those indicated in Fig.

*Mean ± SD (n = 3); The numbers in parentheses correspond to the lane numbers in Fig.

†The enzyme fraction (organelle) of privet leaves.

‡β-Glucosidase from sweet almond.

Antimicrobial properties – manufacturing problems:

The second historical source indicating that components of the olive tree had biologically important properties came from the European olive fermentation industry. Up until the 1970s, the industry had suffered problems in the fermentation of olives, a process involving lactic acid pickling, because of strong resistance of the fresh fruits to the action of lactic acid bacteria.5,6,7,8

In 1960, Panizzi et al9 had isolated a bitter glucoside, oleuropein, from olive leaves, with the empirical formula C25H32O13. The substance, later determined to be a phenolic compound belonging to the iridoid group,10 was also present in the olive itself. Oleuropein, as with Pallas’ “Vauqueline”, was considered to be the source of the olive tree’s powerful disease-resistant properties. It was subsequently found that removal of oleuropein from olives enabled fermentation to take place successfully.

The olive oil manufacturing industry had also long been well aware of the rich antibacterial properties of the olive tree. The manufacturing process involves milling of olive paste and continuous washing with water, known as malaxation. The waste waters from this process were generally discarded; however, it was found that if the waters found their way into the soil, they displaced beneficial bacterial flora and adversely affected the natural biodegradation process.

The active ingredient in olive leaf extract is called oleuropein. Oleuropein is a polyphenolic fraction derived from the fruit, leaves, bark and roots of the olive tree, which help make it strongly resistant to damage from insects and other factors. Oleuropein is known as an iridoid, a type of plant chemical found throughout the olive tree and in olive oil.

Within Oleuropein is a chemical agent called elenolic acid, which has been shown to assist the body's immune defense. Research studies have found that elenolic acid helps the body to balance levels of friendly bacteria and support the immune system.

The energy-boosting benefits of olive leaf extract are believed to be the result of its ability to help fend off fungi, which overtax the immune system, and yeast overgrowth (such as Candida albicans), which cause fatigue.

Olive leaf extract provides nutritional support for detoxification at the cellular level, when the body is under stress. It has been shown to protect RNA structure.

The chemical components:

Over a period of more than 30 years since Panizzi et al’s9 isolation of oleuropein, extracts from various parts of the olive tree have been extensively investigated. Oleuropein appears to be present throughout the olive tree, including leaves, buds, fruit, wood, bark and roots.16,3,17,18 Olive leaves contain around 60—90 mg per gram (dry weight) oleuropein,19 plus

significant levels of a glucosidic ester of elenolic acid and hydroxytyrosol (3,4-dihydrophenylethanol). However, it turns out that oleuropein and the products of its hydrolysis, oleuropein aglycone, elenolic acid, beta-3,4-dihydroxyphenyethyl alcohol and methyl-o-methyl elenolate,20 are the major molecules of interest biologically.

Nature's Antimicrobial Agent:

Oleuropein, the bitter glucoside lodged in leaves of the green olive tree (Olea europaea), and products of its hydrolysis, contain components valuable for treating both infectious and degenerative diseases. The empirical formula of oleuropein (C25H32O13) makes it a member of the iridoid group, a uniquely structured chemical class that contains a carbohydrate component appearing as D-glucose.

The first iridoid found in nature, verbenalin, was isolated circa 1835. No investigation into the group's structure began until 1963 because iridoids are extremely unstable--one member of the iridoid group has the capability to transfer into another group. This biogenetic characteristic gives the iridoid oleuropein its therapeutic antimicrobial power.11

In a document describing how oleuropein works, James R. Privitera, M.D., of Covina, Calif., says olive leaf extract brings about:

* A critical interference with certain amino acid production processes necessary for the vitality of a specific virus, bacterium or microbe;

* Interference with viral infection and/or spread by inactivating viruses or by preventing virus shedding, budding or assembly at the cell membrane;

* Direct penetration into infected host cells and irreversible inhibition of microbial replication;

* Neutralization of the retrovirus' production of reverse transcriptase and protease. These enzymes are essential for a retrovirus such as human immunodeficiency virus (HIV) to alter the ribonucleic acid (RNA) of a healthy cell;

* Direct stimulation of phagocytosis as an immune system response to germs of all types.

Oleuropein is a stable chemical except in nature, where certain environmental alterations cause molecular properties to change. For example, during the several thousand years olive leaves have been antimicrobial, pathogenic microorganisms have continued to be adversely affected by the olive leaves' oleuropein. This may indicate that the chemical structure of oleuropein tends to alter with the microbes' mutations, allowing it to continue inhibiting their growth, spread or survival. In contrast, synthetic antibiotics do not change to maintain resistance to bacteria.

The Post Antibiotic Era:

According to well-documented scientific evidence, the myriad of pharmaceutical antibiotics developed over the past fifty years are no longer as effective against bacterial infections as they once were. Bacteria have developed antibiotic resistant mechanisms, which negate the protective effect of many antibiotics commonly used today. Proof of this is that over 2 million people are infected in hospitals every year even when given antibiotics to prevent infection.

Bacteria replicate at very accelerated rates inside an infected animal and if the bacterial numbers exceed the capabilities of the immune defense, dire consequences follow. Bacteria bring about illness by churning out micro-toxins or by digesting and breaking down tissues.

Antibiotics work by killing the growing bacterial colony in one of three methods:

l By interfering with the microbe ability to build its cell wall (penicillin works this way).

l By interfering with the mechanism used by bacteria to assemble vital proteins (as shown by erythromycin and tetracycline).

l By binding to the bacterial chromosomes and shutting down reproduction. Pain Reduction

In order to work, antibiotics must penetrate the bacterium or bind to the outer membrane of the cell wall. With the excessive use of antibiotics over the past 20 years, bacteria have been able to develop mutation strategies, which no longer allow the antibiotic to penetrate the bacteria or bind to the cell. Microbes also have been able to neutralize the antibiotics by using immunological genes called plasmids (small circular strands of DNA). These plasmids help the bacteria to generate enzymes, which can actually destroy the antibiotic and render it harmless. When "Super bugs" come on the scene, it requires a whole new class of more sophisticated antibiotics to overcome them. The problem is that the pharmaceutical industry is not keeping up.

For instance, the super new strain of staphylococcus, although once killed by Penicillin, now has adapted to inactivate every antibiotic used against it except Vancomycin, a potent antibiotic that unfortunately also has major adverse side effects.

Antibiotic resistant bacteria have led to a big increase in ear, lung and sinus infections in children and adults. Drug resistant food-born salmonella cases have drastically increased since 1979. Pharmaceutical companies are having difficulty in developing new super antibiotics to treat these drug-resistant bacteria. They have failed in many cases to produce anti-viral medicines that are effective against many of the super bugs now evolving.

Olive Leaf Extract has now shown itself to be an answer and possible approach to overcome and destroy these super antibiotic resistant microorganisms. Clinical and laboratory studies have clearly shown that the active ingredient in Olive Leaf Extract will kill most viruses, all types of resistant bacteria, Anthrax, yeast, parasites as well as influenza virus, Ebola, HIV and other emerging viruses. This discovery may well provide the safety, protection and healing power in those cases where medical science and drug research have not provided an answer.

Table : Results from the Use of Olive Leaf Extract Against Pathological Organisms - Viruses, Bacteria, and Fungi

Disease Entities	No of Patients	Fully Recovered	Improved	Unchanged	Deteriorated
Respiratory diseases (tonsillitis, pharyngitis, tracheitis, etc.)	119	115	4	none	none
Lung conditions (pneumonia, bronchitis, etc.)	45	42	3	none	none
Dental problems (pulpitis, leukoplakia, stomatitis)	67	60	5	2	none
Skin conditions (herpes and other viral skin problems)	172	120	52	none	none
Bacterial skin infections (pyoderma, injuries)	37	30	7	none	none
Ulcer disease (while experiencing Helicobacter pylori infection)	17	none	17	none	none
Strengthened immunity	43	n/a	40	3	none

Antibacterial actions – in vitro studies:

A variety of antibacterial actions of oleuropein and its associated compounds have been demonstrated in vitro. Fleming et al8 isolated six major phenolic compounds from green olives; one particular compound, possibly a hydrolysis product of oleuropein, was much more inhibitory than oleuropein itself to the lactic acid bacterium Leuconostoc mesenteroides FBB 42. Later on, the oleuropein aglycone and elenolic acid were found to strongly inhibit the growth of three further species of lactic acid bacteria – Lactobacillus plantarum, Pediococcus cerevisiae, and Lactobacillus brevis.20 Since the aglycone is composed of elenolic acid bound to b-3,4-dihydroxyphenylethyl alcohol, and the latter compound was not inhibitory, the investigators concluded that elenolic acid was the inhibitory part of the aglycone molecule. Oleuropein itself was not inhibitory to these bacteria, but did inhibit three species of non-lactic acid bacteria – Staphylococcus aureus, Bacillis subtilis and Pseudomonas solanecearum. In addition, an acid hydrolysate of an extract of oleuropein (containing hydrolysis products of oleuropein not specifically identified) inhibited the growth of a further eight species of bacteria.

Some more recent in vitro studies have shown that oleuropein and/or its hydrolysis products also inhibit the germination and sporulation of Bacillus megaterium15  and inhibit outgrowth of germinating spores of Bacillus cereus T.

Oleuropein: A Potent Antioxidant\

Oleuropein, an active constituent of olive oil and olive leaf, was investigated by Coni and coworkers1. The researchers conducted an in vivo study that evaluated oleuropein’s effects on the serum low density lipoprotein (LDL) levels in rabbits. The study was carried out on the basis of the positive results obtained with in vitro, pilot studies on human LDL. The results of these pilot studies indicated that certain constituents in olive oil inhibited prooxidative processes in human LDL.

The rabbits were fed special diets. Diet A consisted of a standard diet for rabbits. Diet B consisted of the standard diet plus 10% (w/w) extra virgin olive oil, and Diet C consisted of the standard diet plus 10% (w/w) extra virgin olive oil and 7 mg/kg oleuropein. In order to evaluate oleuropein’s effect, biochemical parameters identified in the rabbits’ blood plasma and LDL were measured before and after copper-induced oxidation.

The results verified the antioxidant efficacy of extra virgin olive oil’s biophenols, particularly oleuropein. In measuring the presence of conjugated dienes in the rabbits’ LDL, it was determined that rabbits fed Diet C had a lesser amount of conjugated dienes and therefore of lipid radicals than either rabbits fed Diets A or B. The amounts of conjugated dienes present in the LDL were 51.0 ± 9.3 µM, 25.8 ± 4.1 µM, and 19.8 ± 3.9 µM for rabbits fed Diet A, Diet B, and Diet C, respectively. Similarly, evaluation of other ox-LDL (oxidized LDL) parameters followed the same trend. These results indicate that oleuropein increased the ability of LDL to resist oxidation.

In addition to oleuropein’s antioxidant properties, it was determined that Diet C reduced the rabbits’ plasma levels of total, free, and ester cholesterol by 15%, 12%, and 17%, respectively, compared to rabbits fed Diet B. This reduction caused a redistribution of the lipid components of LDL with an indirect effect on the dimensions.