NZ550120A - Extracts of passion fruit and uses thereof - Google Patents

Abstract

Disclosed herein is a method for preparing a passion fruit extract which is useful as a medicament for lowering blood pressure, lowering serum nitric oxide levels, and preventing liver damage in a mammal (human and non-human). Also disclosed is the passion fruit extract comprising of a compound of the structure as shown in the disclosure which is prepared by said method.

Description

550120 WO 2005/097153 PCT/US2005/011244 EXTRACTS OF PASSION FRUIT AND USES THEREOF Background of the Invention The invention relates generally to botanical extracts and, more specifically, to extracts of passion fruit (Passiflora sp.), including particularly extracts of the skin of 5 passion fruit, and the use of the extracts for food, nutraceutical, and medical applications.

Hypertension, or a blood pressure higher than 140/90 mm Hg, is the most common risk factor for cardiovascular and cerebrovascular morbidity and mortality. In the United States, high blood pressure is responsible for 40,000 deaths annually, while being the most modifiable risk factor for stroke. Hypertension affects about one in four 10 adults, or almost 50 million people in the United States.

A Framingham study showed that as people aged from 30 to 65 years, their blood pressure increased an average 20 mm Hg systolic and 10 mm Hg diastolic pressure, with systolic blood pressure continuing to rise up to age 90.

While higher blood pressure increases the likelihood of a cardiovascular event, 15 hypertension is not often well controlled and too few patients are adequately treated.

Epidemiologic studies predict that reduction of the systemic blood pressure by the amount usually achieved in major clinical trials could reduce cerebrovascular events by 42% and cardiac events by 24% Hypertension is frequently treated non-specifically, resulting in a large number of 20 minor side-effects, and a relatively high rate of non- or inadequate treatment. Thus, the search for new treatments for hypertension remains ongoing.

Therapies derived from natural products are well known. It has been established that certain flavonoids have a beneficial effect on hypertension. For example, a bark extract from the French maritime pine (Pinus pinaster), which contains a mixture of 25 flavonoids, decreases systolic blood pressure when taken orally by mildly hypertensive patients.

Nitric oxide is an important molecular regulator of blood pressure. Nitric oxide is a potent vasodilator. It inhibits platelet activation, limits leukocyte adhesion to the endothelium, and regulates myocardiocontractility. Synthesis of nitric oxide catalyzed by 30 nitric oxide synthase (NOS) occurs in the vascular endothelium while the production of nitric oxide involving inducible nitric oxide synthase (iNOS) is associated with immune function. However, small amounts of nitric oxide produced by another NOS, epithelial nitric oxide synthase (eNOS), have a cytoprotective effect and vasodilation action on the cardiovascular system. 1 550120 WO 2005/097153 PCT/US2005/011244 Peroxynitrite is a potentially damaging oxidant, formed from nitric oxide (NO+O2 ONOO"). Peroxynitrite can give rise to lipid peroxidation, protein nitration, DNA single-strand breakage, and guanidine nitration.

It has been shown that the flavonoids quercetin and kaempferol inhibit NOLA-5 dependent spontaneous aortic ring contraction in spontaneously hypertensive rat (SHR) cells in vitro. NOLA is a nitric oxide synthase inhibitor. Large dose acetylcholine-induced vascular contraction can also be inhibited by antioxidative flavonoids such as quercetin, kaempferol, rutin, and esculetin. Inhibition of vascular smooth muscle contraction should lead to lower blood pressure.

In addition, the effects of flavonoids on immune function are controversial.

Catechin enhances proliferation of lymphocytes and antibody production, while it exerts an inhibitory effect at high concentration. Some studies show that flavonoids enhance NK cell activity, while other studies show that flavonoids have no effect. Quercetin seems to inhibit non-specific immunological responses and exerts an anti-inflammatory action. 15 Passion fruit (Passiflora edulis) is a subtropical or tropical plant with a vigorous climbing character, growing to 20 fit. The purple passion fruit is native from southern Brazil through Paraguay to northern Argentina. Its fruit is nearly round or ovoid, 1.5 to 3 inches wide, with a tough, smooth and waxy rind.

In a search for bioactive constituents of passion fruit, it has now surprisingly been 20 found that a passion fruit extract lowers systolic blood pressure in spontaneously hypertensive rats (SHR), and decreases nitric oxide production from iNOS, thus improving endothelial dysfunction in SHR. It is therefore also envisaged that the passion fruit extract will exhibit antioxidant properties.

It is therefore an object of the invention to provide an extract of passion fruit 25 which exhibits therapeutic effects against hypertension, and diseases associated with hypertension, and which is hepatoprotective, or at least to provide a useful choice.

Summary of the Invention In a first aspect, the invention provides a method of lowering blood pressure in a 30 mammal comprising administering an effective amount of a passion fruit extract to the mammal.

In another aspect, the invention provides a method of preventing or treating a disease or disorder in a mammal where it is desirable to lower blood pressure comprising administering an effective amount of a passion fruit extract to the mammal. 2 550120 There is also provided a method of lowering serum nitric oxide levels in a mammal comprising administering an effective amount of a passion fruit extract to the mammal.

The invention further provides a method of treating a disease or disorder related to 5 liver function in a mammal comprising administering an effective amount of a passion fruit extract to the mammal.

The invention therefore provides a method of treating hypertension as well as any other disease or disorder associated with elevated blood pressure. The invention further provides-a method of hepatoprotection in a mammal, as well as a method of treating any 10 disease or disorder related to liver function.

The invention further provides the use of a passion fruit extract as an antioxidant to inhibit damage from free radicals, to reduce serum lipid peroxidation and to preserve healthy tissue antioxidant vitamin levels.

The passion fruit extract of the invention includes one or more of the group 15 selected from quercetin, cyanidin glycoside, catechin, epicatechin, luteolin, phenylpyruvic acid, the novel edulilic acid isolated and described herein, and any glycoside thereof.

Preferably, the passion fruit extract is prepared by a process including the following steps: (i) preferably, cutting the passion fruit into pieces to increase the surface area; (ii) contacting the pieces of passion fruit with water to give an aqueous extract and a solid residue; (iii) separating the aqueous extract from the solid residue; (iv) contacting the aqueous extract with a polymeric matrix to adsorb one or 25 more components of the extract onto the matrix; (v) washing the matrix with water; and/ (vi) eluting the one or more components from the matrix with an organic solvent or mixture of organic solvents.

Optionally, the skin of the passion fruit is removed from the flesh and used in the 30 extraction process.

It is preferred that the organic solvent is methanol, ethanol, isopropyl alcohol, 1-propanol, or acetone.

The invention also provides a composition containing the passion fruit extract.

The composition may be a food or food product. The composition may also be a 3 550120 dietary supplement, such as a nutraceutical or other nutritional composition.

Alternatively, the composition may be a pharmaceutical composition comprising the extract described above, admixed with one or more pharmaceutically acceptable excipients.

In a further aspect, the invention provides the use of a passion fruit skin extract as a nutraceutical, such-as a dietary supplement, or as an active ingredient in the preparation of medical or functional foods and beverages.

Brief Description of the Figures Figs, la-c are HPLC traces of the extract of passion fruit made according to the present invention.

Fig. 2 is a diagrammatical representation of data showing the reduction in blood pressure in a group of spontaneously hypertensive rats administered the extract of passion fruit.

Fig. 3 is a diagrammatical representation of data showing the reduction in serum nitric oxide levels in a group of rats administered the extract of passion fruit.

Figs. 4a and 4b are diagrammatical representations of data showing the in vivo increase in activity of Na-K ATPase and Ca ATPase, respectively, by the extract of passion fruit.

Figs. 5a and 5b are diagrammatical representation of data showing the reduction in systolic blood pressure and diastolic blood pressure, respectively, in a group of humans administered the extract of passion fruit.

Figs. 6a and 6b are drawings showing H-l methylene proton interactions with H-1' and H-2' of the sugar moiety.

Figs. 7a and 7b are HPLC-ELSD chromatograms of the extracts of passion fruit skin and pulp, respectively, obtained under identical conditions Detailed Description As described herein, "passion fruit" means generally the fruit including both the skin and the edible pulp. The term "passion fruit skin" is used to mean the remaining part of the fruit after the edible pulp inside has been removed. HPLC analysis indicates that the passion fruit extract of the invention contains a number of flavonoids, including quercetin, quercetin galactoside, quercetin glucoside, luteolin, luteolin glucoside, cyanidin-3-glucosides, catechin and epicatechin. (Fig. 1). 4 550120 WO 2005/097153 PCT/US2005/011244 The flavonoid and cyanidin components of the extract inhibit superoxide formation and nitric oxide production from iNOS, thus improving endothelial dysfunction and lowering blood pressure. Quercetin has been shown to inhibit iNOS mRNA and the production of nitric oxide.

Human essential hypertension is characterized by impaired endothelium- dependent vasodilation, caused by oxidative stress. The extract of the invention has antihypertensive effects. In addition, the flavonoid components of the extract lower blood pressure, inhibit oxidation of LDL, and inhibit platelet aggregation, thereby exerting a cardiovascular protecting action. It is noted that the components of the extract include 10 quercetin, a compound know to have antihypertensive activity, but the amount contained in the extract is less than between about 1 and 5% and is not sufficient to substantially account for the antihypertensive activities of the extract. Indeed, none of the previously known constituents of the extract are present in quantities sufficient to provide the observed effects alone.

It is also envisaged, based on the in vitro data, that the extract will have antioxidant properties.

In studies with spontaneously hypertensive rats (SHR), the applicants found that diets supplemented with 50 mg/kg of the extract lowered blood pressure in SHR, retarding their normal increase in blood pressure due to aging. Systolic blood pressure 20 was 12.3 mm Hg lower in rats fed 50 mg/kg of the extract, compared with a control group (P<0.01) (Fig. 2).

Furthermore, nitric oxide concentration was 18.82 p.mol/L in rats fed with 10 mg/kg of the extract, 40% lower than that in rats fed no extract. The nitric oxide concentration was 11.07 jxmol/L in rats fed with 50 mg/kg of the extract, 65% lower than 25 that in rats fed no extract (Fig. 3). This will prevent the overproduction of nitric oxide and its subsequent reaction to form peroxynitrite which is detrimental to the cardiovascular system.

The applicants have also found, in rat liver studies, that the passion fruit extract is hepatoprotective. Precision-cut rat liver slices were incubated with 20 |ag/ml of passion 30 fruit extract. At 9 and 24 hours incubation, potassium levels in the slices, an index of viability, were not significantly different from control slices.

The extract was then incubated with precision-cut rat liver slices in the presence or absence of ImM chloroform, a hepatotoxicant. At 6 hours incubation and at 9 hours incubation the passion fruit extract showed significant hepatoprotection against 550120 chloroform injury. No toxicity of the extract was found in this study.

In vitro studies have also shown that the extract increases human red blood cell membrane-bound Na-K ATPase and Ca ATPase activity. RBC membrane-bound Na-K pump ATPase had much higher activity when cultured with the extract at either 0.25 5 mg/mL (increased by 102%), 1 mg/mL (increased by 107%), or 25 mg/mL (increased by 170%) than the control group (Fig. 4). The extract at concentrations of 1 mg/mL and 2.5 mg/mL increased membrane-bound Ca ATPase activity by 78% and 41% on average.

The applicants have also carried out human studies (Fig. 5). Figure 5 shows the change in SBP (systolic blood pressure) and DBP (diastolic blood pressure) in 10 hypertensive patients who received placebo or passion fruit extract (2 mg/lb/day, maximum 400 mg /day) for four weeks. Passion fruit extract or placebo pills were given in a randomized, double-blind, parallel group fashion to these patients. They had an average systolic blood pressure of 176.60 ± 4.90 mm Hg (mean ± SEM). Passion fruit treatment decreased systolic blood pressure significantly (p <0.001) to 145.67 ± 4.44 mm 15 Hg (mean ± SEM) as compared to the placebo group. The data also demonstrated that passion fruit extract supplementation decreases diastolic blood pressure significantly (p < 0.001) in hypertensive patients with an average diastolic blood pressure of 103.27 ± 2.30 mm Hg (mean ± SEM), to 78.67 ± 2.78 mm Hg (mean ± SEM). No patient in the study showed electrocardiographic changes after four weeks of therapy.

Because of the above-described activities, it is also expected that the passion fruit extract will benefit patients with inflammatory-related diseases, such as arthritis, asthma and allergies, as well as heart disease and hypertension. In addition, even though very large intakes or amounts of the extract were used in the studies described in this application, no toxicity of the extract was found in humans, rats, mice or cells in culture. 25 It will be appreciated by those skilled in the art that the extract may be administered to a patient by a variety of routes, including oral administration, or injection. The amount of extract to be administered will vary widely depending upon the patient and the nature and extent of the disorder to be treated. Typically, the extract is formulated as a composition which may be administered intravenously or by oral ingestion. The 30 composition may be ingested or intravenously administered in any dosage levels and dosage frequencies suitable for lowering blood pressure and/or increasing immune function.

The composition of the invention may also be a food product, including, but not limited to, a nutritional supplement. 6 550120 WO 2005/097153 PCT/US2005/011244 In the case of a pharmaceutical composition, the extract may be formulated into solid or liquid preparations, for example tablets, capsules, powders, solutions, suspensions and dispersions. Liquid forms include carriers such as water and ethanol, with or without other agents such as pharmaceutically acceptable surfactants or 5 suspending agents.

The invention is further described with reference to the following examples. However, it is to be appreciated that the invention is not limited to these examples.

EXAMPLES Example 1: Preparation of Passion fruit Extract Passion fruits (Passiflora edulis) were cut into halves and the juicy pulp removed to give empty shells of passion fruit skin. The shells were chopped into small pieces less than 10 mm in length and placed in a container. Hot water (65-75 °C) was added to the 15 container to immerse the chopped shells completely. The mixture was stirred occasionally during the first hour and then left to soak overnight. The mixture was filtered and the filtrate passed through a column of non-ionic polymeric resin to absorb phenolic and other organic compounds. Distilled water was passed through the column to wash out sugars and other polar components. The absorbed compounds were then eluted from the 20 column with methanol and the eluant concentrated under reduced pressure to give a dark concentrate. The concentrate was freeze-dried to give the passion fruit extract as a dark red powder. While methanol was used as the eluting agent, ethanol, isopropyl alcohol, 1-propanol or acetone could also have been used.

Example 2: Determination of Components of Passion fruit Extract The components of the extract were determined by HPLC. Experiments were carried out on a Hewlett Packard 1100 instrument equipped with a DAD detector and a LiChrospher 100 RP-18 (um) column (125 x 4) held at 30 °C. The solvent program started from 3.6% B (2% HO Ac in acetonitrile) in solvent A (2% HO Ac in water) up to 30 12% B in 20 min, to 20% in 30 min, and to 50% B in 45 min. Flow rate was set at mL/min and compounds were monitored by UV absorption set at 280 nm for phenolic acids, 350 nm for flavonoids and 520 nm for anthocyanins. The identity of the compounds was confirmed by comparison of retention times and UV/visible spectra with authentic materials. 7 550120 Example 3: Red Blood Cell Membrane Preparation and ATPase Assay Erythrocyte membranes were prepared as previously described (Farrance, ML., & Vincenzi, FF. (1977) Biochim Biophvs Acta 471:49-58). Briefly, blood was taken from 5 healthy humans. Red blood cells were washed with saline and lysed in a hypotonic imidazole buffer (pH 7.4, 20 mM, Sigma) with EGTA (100 mM, Sigma) and PMSF (10 mM, Sigma). Membranes were washed with imidazole buffer (20 mM) containing EGTA and PMSF, imidazole buffer containing EGTA, and only imidazole buffer each one time in sequence. The final wash was in 40 mM histidine-imidazole buffer (pH 7,4), and the 10 membranes were stored in a refrigerator (4-8°C) under nitrogen. Prior to assay, RBC membrane (0.75 mg/mL) was incubated for 30 minutes at 37°C with 0, 0.25, 1, 2.5 mg/mL of passion fruit skin extract and enough saline to achieve a final volume of 1 mL. Following incubation and centrifugation, the supernatants were removed and the membrane was resuspended in saline up to 1 mL. Thereafter, membrane ATPase 15 activities were measured simultaneously in multi-well plates. The typical assay mixture contained RBC membrane (75 pg/mL), 18 mM histidine-imidazole (pH 7.1, Sigma), 3 mM MgCI2, 80 mM NaCI, 15 mM KCl, 0.2 mM CaCI2, 0.1 mM EGTA, 0.1 mM Ouabain (Sigma) and 30 nM CaM (only for CaM-activated Ca2+ pump, Sigma). After a 15 minute preincubation at 37°C, 5 % SDS (Sigma) was added to the control groups. The enzymatic 20 reaction was started with 3 mM ATP. After 60 minutes at 37°C, the-reaction was stopped with 5% SDS; and the inorganic phosphate released was measured with an ammonium molybdate/ascorbic acid mixture and absorption was measured at wave length 820 nm by Microplate Autoreader (Bio-Tek Instruments, EL31 1. USA). For additional accuracy, a BCA assay was performed to determine the final concentration of protein within the tubes 25 at the end of each assay. Membrane (25 jj.1) from the above experiment, 25 jil ddfLO and 1 mL color reagent were added in a tube and then incubated for 30 mm at 37°C. Standard protein (Albumin, Sigma) at different concentrations was incubated in the meantime. After incubation, each tube was cooled to room temperature. Light absorbance was measured by spectrophotometer (Beckman Coulter, DU640) at X=562 nm. Protein 30 concentration of the membrane was read from the standard curve.

The activity of ATPase was calculated by: . . . „ N(PI) xO.2778 x protein concentration Activity of ATPase = — Initial protein concentration (0.75mg / mL) 8 550120 WO 2005/097153 PCT/US2005/011244 Example 4: Animals and Diets - SHR Studies Spontaneously hypertensive rats (SHR), 6 weeks old, were kept at 22 to 20 °C and 50% humidity during the experiment. 24 SHRs were divided into 3 groups with 8 rats in 5 each group. They were fed the following diets: basic diet, basic diet supplemented with the passion fruit extract at 50 mg/kg, or basic diet supplemented with the passion fruit extract at lOmg/kg (Table 1). The amount of food intake, body weight and systolic blood pressure were recorded once a week. Systolic blood pressure was measured by tail cuff method (Softron, Co. Ltd, Tokyo, Japan). After 8 weeks of feeding, all the rats were 10 sacrificed under anesthesia with Nembutal (O.lmg/lOOg body weight, Wako, Co. Ltd., Japan). Thymus, spleen, liver and heart were isolated and weighed. No toxicity was observed.

Example 5: Nitric Oxide Measurement 15 Measurement of nitric oxide was carried out as previously described (Rockett, KA., Awburn, MM., Cowden, WB. & Clark, JA. (1991) Infect, lmmun. 59:3280-3).

Nitric oxide is easily converted to nitrite. Nitrate was measured for nitric oxide. Dilutions of NaN02 (BDH; Wako, Co. Ltd., Japan) and test compounds were made in distilled water in 96-well, flat-bottom plates to a final volume of 50ul. 20 microliters of NH4CI 20 borate buffer was added to all wells requiring analysis for nitric oxide/nitrite. 50 microliters of Griess reagent [1 % sulfanilamide plus 0.1% N- (1-napthyl) ethylenenediamine dihydrochloride (Wako, Co. Ltd., Japan) in 2 M H2SO4] was then added to wells to be analyzed for nitric oxide/nitrite. The plate was read at 540nm (test) and nitric oxide and nitrite concentrations were read directly from a nitrite standard curve.

Example 6: Human Study People with hypertension, 14 men aged 57.0 ± 14.48 y (mean ± SD) and 16 women aged 57.56 ± 12.75 y (mean ± SD), were included in the study. Patients had hypertension of stage 1 or 2 according to the guidelines of the Joint National Committee 30 on Detection, Evaluation, and Treatment of High Blood Pressure. In repeated blood pressure measurements, they had systolic blood pressure (SBP) between 144 and 210 mm Hg and diastolic blood pressure (DBP) between 80 and 120 mm Hg. The exclusion criteria included those with renal or cardiac disease, taking oral contraceptives, use of tobacco and alcohol, or taking any vitamin supplements other than a single, daily 9 550120 multivitamin tablet. All of subjects included were taking antihypertensive combination therapy including diuretic, beta-blocker and ACE-inhibitor. More than one hundred were screened to select the thirty participants, none of which dropped out during the study.

At the beginning of the study, the passion fruit and placebo groups did not differ 5 in mean blood pressure (SBP, 176.60 ± 4.90 vs. 179.67 ± 3.79 mm Hg and DBP, 103.27 ± 2.30 vs. 104.33 ± 2.06 mm Hg), age, sex, height, weight, heart rate, and pretreatment medication against hypertension or pattern of ECG.

The study was approved by Mashhad University's Human Subjects Committee. After providing informed consent and the one week withdrawal of any previous 10 antihypertensive treatment except Triamterene-H in two subjects, eligible patients entered a four week, double-blind, placebo controlled, parallel group study. Patients were requested to attend clinic for follow-up every week during the study to assess side-effects. In addition, blood pressure and heart rate were measured. At the first visit, a complete medical history and a physical examination, including electrocardiogram, were carried 15 out. Blood pressure was measured by a registered nurse, after the subject had been sitting for 10 min rest. Korotkoff phases I and V were taken as the systolic and diastolic blood pressures, respectively. Repeated readings were taken at 2 minute intervals for a total of 3 sitting measurements at each visit. Averages of repeated measurements at a given visit were recorded. At the second visit, blood pressure and heart rate were assessed again and 20 patients were randomized to receive twice daily dosing of 2 mg/lb/day (maximum 400 mg/day) of a statistical formula of passion fruit pill or a similarly appearing placebo for 4 weeks. The data for week 1 and week 0 were.combined as baseline values. In the last visit, for each patient an ECG was performed and the study drug was collected. All changes in concomitant medications and clinical adverse events, either volunteered or 25 elicited by questioning, at baseline and follow-up visits were recorded, with none reported.

Compliance was evaluated by tablet counting. During the four-week period of treatment, all the subjects took 100% percent of the pills provided in a blinded fashion. All tests were two to four hours after the last consumption of pills.

Example 7: Isolation of Edulilic Acid from Passion Fruit Peel Extract Passion fruit peel extract prepared according to Example 1 was dissolved in 50% aqueous ethanol and was treated on a Sephadex LH20 column and eluted with 50% aqueous ethanol. The chromatographic fractions were collected in 20 ml tubes with the 550120 aid of a fraction collector. Fractions were monitored by thin layer chromatography using cellulose TLC developed with 6% aqueous acetic acid and visualized under UV. Under this condition, the novel compound (Rf 0.8) co-eluted with the colored anthocyanins fraction (Rf 0.4-0.5). This fraction was collected and concentrated and re-5 chromatographed on a column of MCI GEL CHP 20P purchased from Mitsubishi Chemical Industries Ltd. Using 15% aqueous methanol as the eluating solvent. Fractions were collected and monitored by cellulose TLC developed with tertiary BuOH-AcOH-H2O (3/1/1 v/v). Fractions containing the novel compound (Rf 0.9 with this solvent) were collected and the solvent evaporated and the residue was freeze dried.

A high resolution electrospray ionization mass spectrum of edulilic acid was made on a MARINER Biospectrometry Workstation at the Victoria University of Wellington, New Zealand operating on the negative ion mode to give (M-H)-1 peak at 301.09691 which corresponded to the molecular formulae of C13H18O8. Various NMR studies (1H, 13C, COSY, HMQC, HMBC and NOSEY) and mass spectrometry were conducted on the edulilic 15 acid. Table 1 contains data from the NMR studies.

Table 1. *H and 13C NMR Spectral Assignment for Edulilic acid Recorded in D?0 C/H position 13C 51H (HMQC) HMBC 1 29.1 2.70 (m) CI, C2, C3, C4, 2 31.8 2.37 (bs) C3,C4 3 130.1 . 6.84 (d) CI, C2, C4, C5 4 150.5 6.52 (d) CI, C2, C3, C5 156.3 - 6 132.7 - 7 167.6 - glucose CI' 103.5 4.57 (d) C2',C3', C6 C2' 76.4 3.30 (m) C2',C2', C3' C3' 75.9 3.35 (m) C4' C4' 73.7 3.32 (m) C3',C4', C5' 69.5 3.30(m) C3',C4',C6' C6' 60.6 3.53 (dd), 3.67 (dd) C5' C7 (CO) 167.6 - On this basis the chemical structure of edulilic acid is as given below. The beta-linkage of the glucose residue is assigned due the large J-coupling magnitude (J= 7.3Hz) of the anomeric proton. 40 11 550120 OH The drawing of the molecule was prepared using ACD/ChemSketch Software, which provided the IUPAC name (2£)-cyclopent-2-en-l-ylidene((3,4,5-trihydroxy-6-5 (hydroxymethyl)tetrahydro-2#-pyran-2-yl)oxy)acetic acid. The compound has been identified throughout this specification as "edulilic acid", as having originally been discovered in the fruit of Passiflora edulis. The structure of edulilic acid depicted above is the E or trans isomer. Edulilic acid is expected to undergo cis/trans enolization to the Z or cis form and back. The electron shift can go from the C-2 protons through the acetic 10 acid moiety, or from the acetic acid moiety and travel in the opposite direction to afford the same cis/trans result.

The NOESY spectrum of edulilic acid in D2O shows that there is some interaction of the sugar anomeric proton with the methylene protons on C-l as numbered in the structure shown earlier (Figs. 6a and 6b). This is only possible if the carboxylic acid moiety is on the 15 double bond side of the cyclopentene ring. Further studies using a Dreiding model for edulilic acid show that the orientation of the sugar moiety that offer the least crowding to the cyclopentene ring indeed placed the anomeric proton in close proximity to the methylene protons on C-l.

Example 8: Detection of Edulilic acid in the Pulp of Passion Fruit An aqueous extract of passion fruit pulp was prepared in a similar manner as described in Example 1 with respect to the skin, and its chemical profile was examined using HPLC. A different HPLC solvent programming using methanol instead of acetonitrile was used successfully to resolve the edulilic acid and prunasin peaks. Also, 25 instead of UV detection Evaporative Light Scattering Detection (ELSD) was used for better detection of weak UV absorbing compounds such as prunasin. Figs 7a and 7b show 12 550120 the HPLC chromatograms of the extracts of the skin and pulp respectively obtained under such conditions. While the skin and pulp extracts were clearly distinguishable by their HPLC profile, it was also apparent that both edulilic acid and prunasin were present in both extracts.

The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the 10 order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto, except insofar as the claims are so limited. Those skilled in the art that have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention. 13 550120

Claims (22)

We claim:

1. A method of preparing a passion fruit extract, comprising the steps of: (a) contacting passion fruit skin with water to provide an aqueous extract and a solid residue; (b) contacting the aqueous extract with a polymeric matrix to adsorb at least one component from the aqueous extract; and (c) eluting the component from the matrix.

2. A method as claimed in claim 1, wherein the elution step uses an organic solvent.

3. A method as claimed in claim 2, wherein the organic solvent is selected from the group consisting of an ethanol, isopropyl alcohol, methanol, 1-propanol, and acetone.

4. A method as claimed in claim 1, wherein the polymeric matrix is a non-ionic polymeric matrix resin.

5. A method as claimed in claim 1, further comprising the step of washing the polymeric matrix with water after contact with the aqueous extract and prior to the elution step.

6. An extract of passion fruit prepared according to the method of claim 1.

7. A method of lowering blood pressure in a non-human mammal, comprising administering to a non-human mammal an effective amount of the extract of claim 6.

8. A method of lowering serum nitric oxide levels in a non-human mammal, comprising administering to a non- human mammal an effective amount of the extract of claim 6.

9. A method of preventing liver damage in a non-human mammal, comprising administering to a non-human mammal an effective amount of the extract of claim 6. 14 550120

10. The use of an effective amount of the extract of claim 6 in the manufacture of a medicament for lowering blood pressure in a mammal. 5

11. The use of an effective amount of the extract of claim 6 in the manufacture of a medicament for lowering serum nitric oxide levels in a mammal.

12. The use of an effective amount of the extract of claim 6 in the manufacture of a medicament for preventing liver damage in a mammal. 10

13. The use as claimed in any one of claims 10-12 wherein the mammal is human.

14. An extract of passion fruit skin as claimed in claim 6, which comprises at least one of the compounds of the group consisting of catechin, cyanidine, edulilic acid, epicatechin, 15 luteolin, phenylpyruvic acid, quercetin, and any glycoside of such compounds.

15. An extract of passion fruit as claimed in claim 6, which comprises a compound having the structure 20 and the cis isomer thereof.

16. A method according to claim 1, substantially as herein described with reference to Example 1, 2, 7 or 8. HO OH 15 550120

17. A method as claimed in claim 7, substantially as herein described with reference to Example 4 or 7 or Figure 2. 5

18. A method as claimed in claim 8, substantially as herein described with reference to Example 5 or Figure 3.

19. A method as claimed in claim 9, substantially as herein described with reference to any of the Examples. 10

20. A use as claimed in claim 10, substantially as herein described with reference to Example 4 or 7 or Figure 2.

21. A use as claimed in claim 11, substantially as herein described with reference to 15 Example 5 or Figure 3.

22. A use as claimed in claim 12, substantially as herein described with reference to any of the Examples. 16