WO2015041514A1

WO2015041514A1 - Averrhoa carambola extract and a method for producing the extract

Info

Publication number: WO2015041514A1
Application number: PCT/MY2014/000235
Authority: WO
Inventors: Zunoliza ABDULLAH; Abdul Rashid LI; Norsuhaina ZAKARIA; Khairul Kamilah ABDUL KADIR; Mohd. Radzi AHMAD; Ahmad Shukri ABDULLAH; Muhamad Khair MOHD. AYOB
Original assignee: Forest Research Institute Malaysia
Priority date: 2013-09-19
Filing date: 2014-09-17
Publication date: 2015-03-26

Abstract

An extract containing an effective amount of a bioactive compound derived from Averrhoa carambola comprising soluble dietary fiber, soluble phenolics, soluble protein, acid-soluble lignin and sugar. The bioactive compound has galactoglucan residues as a major component and has an average molecular weight of about 11992 Da. The bioactive compound enables the extract to act as a cholesterol lowering agent suitable for use in the manufacture of a composition for reducing blood lipid levels. The extract is also suitable to be made into a health supplement or into food and beverage products. A method of producing a therapeutically effective extract derived from Averrhoa carambola of the present invention is also provided.

Description

AVERRHOA CARAMBOLA EXTRACT AND A

METHOD FOR PRODUCING THE EXTRACT

The invention relates to an extract derived from Averrhoa carambola and a method for producing the extract. More particularly, this invention relates to an extract derived from Averrhoa carambola comprising a bioactive compound.

DESCRIPTION OF THE PRIOR ART Naturally derived dietary fibers such as those from fruits, vegetables and grains, have been reported to be effective in lowering risk of cardiovascular disease. The effectiveness of such fibers lies in the prevention of cholesterol accumulation from consumed food in a person's bloodstream by the transport of undigested food out of the body, thereby, reducing the risk of elevated blood lipid levels.

Averrhoa carambola is a common fruit found in the South East Asian region. Within communities where traditional knowledge is well preserved, particularly within the Asian community, it has been said that consuming Averrhoa carambola reduces blood lipid levels, but it is not known which compound in the Averrhoa carambola is responsible for this beneficial effect. The compound responsible for this effect is likely to be present in small amounts in the fruit, hence, leading to the common misconception that it is necessary to consume as much Averrhoa carambola as often as possible. This is not always desirable or practicable. It is hard to determine the effective amount of Averrhoa carambola one would need to consume to lower blood lipid levels. Further, the compound responsible for lowering blood lipid levels is likely to vary in concentration and quality in various harvests, sources and even from fruit to fruit from the same harvest and source. Traces of pesticides found in and on the fruit poses a risk to human health, e.g. pesticide poisoning. Traces of pesticide found on raw fruits and vegetables are significantly higher than in the corresponding processed commodities. Although it is beneficial to consume Averrhoa carambola, it has been reported that Averrhoa carambola causes adverse effects in renal patients. Averrhoa carambola contains a powerful neurotoxin that accumulates in the blood of renal patients and can cause intoxication which may lead to death. Symptoms of Averrhoa carambola intoxication can become apparent even after consuming as little as one half of a fruit.

Narain etal. ¹ reported the chemical composition of fresh Averrhoa carambola ruit where 100 g of an edible portion shows the presence of 0.98 g, 2.1 x 10^"4 g, 0.41 g, 4.4g and 90.31 g of crude fiber, tannin, protein, total sugar and moisture content, respectively.

Chau, C. F. etal. ^{2, 3} conducted studies which revealed that the insoluble fiber-rich fraction isolated from pomace collected after extraction of juice from Averrhoa carambola reduces serum triglyceride and total cholesterol levels. It was suggested that the insoluble fiber- rich fraction be used as a cholesterol-lowering ingredient in human diets. Insoluble fiber prevents or eases constipation and reduces the risk of colon-related problems. The moisture retention property of insoluble dietary fiber helps in softening stools. Chau, C. F. et al. reported that insoluble dietary fiber may enhance the excretion of total lipids and cholesterol via stools. The study conducted by Chau, C. F. et al. appears to support the traditional use of Averrhoa carambola in lowering cholesterol levels. However, this study was confined to insoluble dietary fiber and a suggestion that it could be used as a cholesterol-lowering ingredient as it promotes excretion of wastes. One possible drawback of relying on the insoluble fiber-rich fraction of Averrhoa carambola as a cholesterol-lowering agent, is the possibility that the cholesterol could be reabsorbed into the bloodstream prior to being excreted as waste.

Naturally derived medicines and dietary supplements such as those of plant origin are preferable over synthesized compositions due to the reduced side effects when consumed. However, plant-derived bioactive ingredients are generally unstable and difficult to sustain at elevated temperatures for prolonged periods of time and, hence, are at present unsuitable for incorporation into food and beverages as medicine or supplements.

This invention thus aims to alleviate some or all of the problems of the prior art. Narain, N., Bora, P. S., Holschuh, H. J., Vasconcelos M. A. Da S. (2001). Physical and Chemical Composition of Carambola Fruit (Averrhoa carambola L.) at Three Stages of Maturity. Ciencia y Tecnologia Alimentaria, 3(3), 144-148. Chau, C. F., Chen, C. H. and Wang, Y. T. (2004). Effects of a Novel Pomace Fiber on Lipid and Cholesterol Metabolism in the Hamster. Nutrition Research, 24(5), 337-345. Chau, C. F., Chen, C. H. and Lin, C. Y. (2004). Insoluble Fiber-rich Fractions Derived from Averrhoa carambola Hypoglycemic Effects Determined by in Vitro Methods. Lebenson Wiss Technology, 37(3), 331-335.

SUMMARY OF INVENTION

In an aspect of the invention, there is provided an extract containing an affective amount of a bioactive compound derived from Averrhoa carambola comprising soluble dietary fiber, soluble phenolics, soluble protein, acid-soluble lignin and sugar. The bioactive compound has galactoglucan residues as a major component and has an average molecular weight of about 11992 Da. The bioactive compound enables the extract to act as a cholesterol lowering agent suitable for use in the manufacture of a composition for reducing blood lipid levels.

In an embodiment, the bioactive compound may comprise about 19.0% soluble polysaccharide, 14.6% acid-soluble lignin, 0.4% soluble protein and 3.6% phenolic compounds. In a further embodiment, the bioactive compound may be derived from the pericarp layer of Averrhoa carambola and is stable at temperatures ranging from about 30°C to about 100°C.

A health supplement may be made containing an effective amount of the extract which is stable at temperatures ranging from about 30°C to about 100°C. The health supplement may be in the form of pellets, tablets, liquid, granules or capsules.

An edible product such as yoghurt drinks, protein shakes, nutritional bars and cereals may also be made containing an effective amount of the extract which is stable at temperatures ranging from about 30°C to about 100°C.

In a second aspect of the present invention, there is provided a method for producing a therapeutically effective extract derived from Averrhoa carambola of the present invention. The method comprises the following steps :

I) preparing dried ground Averrhoa carambola;

II) extracting a bioactive compound from the dried ground Averrhoa carambola to produce a crude extract, the extraction performed using water as a solvent; III) adding a polar organic solvent to the crude extract to form a precipitate and separating the supernatant from the precipitate; and

IV) discarding the supernatant and collecting the precipitate containing the bioactive compound, which forms the extract.

In an embodiment, suitably sized pieces o Averrhoa carambola of about 2.4 to about 3.3 cm³ may be provided from the pericarp layer of the fruit, prior to step I.

In an embodiment, the Averrhoa carambola may be ground to a particulate size of about 2000 to about 4000 pm in step I.

In an embodiment, the dried ground Averrhoa carambola may be refluxed in boiling water for about two hours in step II. This may be performed five times. The solid residues formed during the reflux step may be filtered.

In an embodiment, the crude extract may be concentrated under reduced pressure, prior to step III.

In an embodiment, the mixture may be centrifuged at 6500 rpm for about 10 minutes to separate the precipitate in the mixture in step III.

In another embodiment, the resultant precipitate of step IV may be washed with the solvent and the mixture may later be centrifuged at 6500 rpm for about 10 minutes. This may be repeated three times.

In a further embodiment, the precipitate of step IV may be further dissolved in distilled water and concentrated under reduced pressure. The dissolved precipitate may be freeze- dried.

It is an object of the present invention to develop an extract derived from Averrhoa carambola containing a bioactive compound capable of lowering blood lipid levels.

The present invention seeks to provide a solution for reducing blood lipid levels by identifying a bioactive compound in an extract of Averrhoa carambola which enables the extract to act as a cholesterol lowering agent. The identified bioactive compound comprises soluble dietary fiber, sugar, soluble phenolics, soluble protein and acid-soluble lignin. The extract produced contains concentrated amounts of the bioactive compound, thus enhancing the ability of the extract to lower blood lipid levels.

The extract is suitable to be consumed by anyone, for example children, pregnant ladies and even patients under medical treatment who are particularly sensitive toward traces of pesticide found on the raw fruit. It is unlikely that the extract will cause pesticide poisoning as any traces of pesticide would have been eliminated during the extraction process. Further, the extract is also suitable to be consumed by renal patients as the naturally occurring neurotoxin would have been eliminated during production of the extract.

Advantageously, the inventors noted that soluble dietary fiber is significantly more effective in lowering blood lipid levels in comparison with insoluble dietary fiber. Besides facilitating excretion of cholesterol from the body, soluble dietary fiber also binds to bile acids, which are made from cholesterol, and prevents the reabsorption of these bile acids into the body.

The extract of the present invention provides a natural and effective prevention method for lowering blood lipid levels, is safe to be included in our daily diet with no side effects and is potentially nutritional as the extract is naturally derived.

The extract is stable and sustainable at elevated temperatures and enables the bioactive compound present to be preserved and functional even after a long period of time. It is suitable for incorporation into food and beverages as medicines or supplements. The extract is also suitable for use as a daily meal enhancer or as an additive in hot food and beverages.

The above advantages will be further elaborated in the detailed description of the embodiments in the following pages of this document. BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated, although not limited, by the following description of embodiments made with reference to the accompanying drawings in which: Figure 1 shows a method of producing an extract of Averrhoa carambola according to this invention.

Figure 2 shows a preferred embodiment of the method of Figure 1. Figure 3a is a high-performance liquid chromatography chromatogram of the extract. Figure 3b is a high-performance liquid chromatography chromatogram of the first eluent. Figure 4a is a gel-permeation chromatography chromatogram of galactoglucan.

Figure 4b is a gel-permeation chromatography chromatogram of water (mobile phase).

Figure 5 is a graph of mean and standard deviation of bile acid present in the faeces of rabbits collected 3 days before the termination of phase 2 of the Example.

Figure 6 is a graph of mean and standard deviation of bile acid present in the faeces of rabbits collected 2 days before the termination of phase 2 of the Example.

Figure 7 is a graph of mean and standard deviation of bile acid present in the faeces of rabbits collected 1 day before the termination of phase 2 of the Example.

Figure 8 shows photographs of the aorta of rabbits stained with Sudan IV at the end of phase 3 of the Example. Figure 9 shows photographs of the aorta of rabbits stained with Hematoxylin and Eosin at the end of phase 3 of the Example. DETAILED DESCRIPTION OF THE EMBODIMENTS

The invention relates to an extract containing a bioactive compound derived from Averrhoa carambola and its method of production. The Averrhoa carambola is commonly known as the "belimbing besi" in South East Asia, particularly in Malaysia and Indonesia, to distinguish it from the Averrhoa blllmblknown as the "belimbing asam".

Averrhoa carambola is green to greenish yellow in colour and becomes orange-yellow in colour when ripe. The edible flesh of the Averrhoa carambola is from the pericarp layer, which tastes sour to mildly sweet. It has five longitudinal, sharp and angular lobes with a star-like cross section. The seeds of the Averrhoa carambola can be found about the centre of the fruit.

It is traditional knowledge in Asian cultures that consuming Averrhoa carambola aids in combating cholesterol, although the bioactive compound responsible for this phenomenon has never been previously extracted and converted into a therapeutically-effective consumable composition.

The flesh of fresh Averrhoa carambola has a high content of soluble dietary fiber. When dietary fiber is consumed, it forms a bulky gel in the intestine which aids in regulating the flow of waste materials through the digestive tract and facilitates excretion from the body. Soluble dietary fiber prevents the reabsorption of bile acids, which are made from cholesterol, by binding to them and directing them out of the body. The inventors have successfully identified, extracted and converted the bioactive compound having the soluble dietary fiber of Averrhoa carambola, into a therapeutically-effective composition.

Method of Extraction

In order to obtain the bioactive extract, firstly, the pericarp of Averrhoa carambola is cut to suitably sized pieces of about 2.4 to 3.3 cm³. The seeds were not removed in this process. The small pieces of fruit are dried to remove moisture. Any suitable method of drying may be used such as oven-drying or using a drying cabinet. The fruit should preferably be dried to a moisture content of about 8% relative to the fresh fruit. Subsequently, the dried pieces of fruit are ground into a coarse powder of about 2000 to 4000 pm particulate size to increase its surface area to allow the bioactive compound in the Averrhoa carambo/a to be extracted more efficiently. Any suitable method of grinding may be used such as mechanical grinding using a grinder or mortar and pestle. Water is used as a solvent to produce the crude extract from the ground powder of Averrhoa carambola. The volume of water added is about 15 times the volume of the dry mass of the ground powder. The ground powder is then refluxed for two hours in boiling water. This is preferably repeated five times. The solid residues formed are removed by mechanical filtration, for example using a sieve of about 4.2 pm pore size. Any suitable mode of filtering the solid residues from the liquid (filtrate) may be used.

The filtrate, which is the crude extract containing the water soluble bioactive compound, is then concentrated. Any suitable mode of concentration may be used, such as a rotary evaporator, to remove as much water as possible from the crude extract. The concentrated crude extract should preferably have a moisture content of about 8% relative to the crude extract. Any suitable polar organic solvent such as 95% ethanol, acetone or acetonitrile, is then added to the concentrated crude extract. Such solvents are less polar than water and its addition causes a change in the polarity of the original solvent, water. Based on the theory where "like dissolves like", molecules readily dissolve in solvents of similar polarity. Typically, polar solutes will only dissolve in polar solvents, and non-polar solutes will only dissolve in non-polar solvents. Due to the change in polarity of the solution, precipitation occurs for certain compounds which are unable to dissolve in the mixed solution (supernatant). In this case, the inventors observed that precipitation occurred instantaneously once the polar organic solvent is added to the crude extract. The supernatant formed is discarded and finally, the precipitate which forms the extract of the present invention is collected.

To effectively separate the precipitate from the supernatant, the mixture is centrifuged at 6500 rpm for about 10 minutes prior to discarding the supernatant.

After discarding the supernatant, the precipitate is washed with the same polar organic solvent used previously and re-centrifuged at 6500 rpm for about 10 minutes, preferably for about three times. This is to remove any remaining compound in the precipitate which is not precipitated in the organic solvent.

Finally, the precipitate is dissolved in distilled water of about 5 times the volume of the precipitate. The mixture is subsequently concentrated to remove as much water as possible using a rotary evaporator. The concentrated dissolved precipitate should preferably have a moisture content of about 70% relative to the precipitate.

The concentrated dissolved precipitate may be freeze-dried at -46°C to preserve the composition of the extract. Further, freeze drying may be applied to the concentrated dissolved precipitate to produce a powder form of the extract. Other suitable downstream processes may also be applied to the concentrated dissolved precipitate for production of the different consumable forms of the extract.

Analysis of the Extract

A proximate composition analysis of the extract is performed in accordance with the methods and procedures of the Official Methods of Analysis (16^th Edition)⁴ and the results are tabulated in table 1.

Table 1 : A proximate composition analysis of extract of Averrhoa carambola

Further chemical analysis of the extract for soluble dietary fiber is performed in accordance with the methods and procedures in Dubois et a/.^s and Krishnaveni et a/.⁶, soluble phenolic in accordance with the methods and procedures in Singleton and Rossi⁷, and soluble lignin in accordance with the methods and procedures of The Technical Association of the Pulp and Paper Industry UM250⁸. Analysis of soluble protein in the extract is performed using the Bicinchoninic Acid protein Assay Kit SIGMA, BCAl and B9643. The results of the chemical analysis are shown in table 2.

Table 2 : Chemical analysis of the extract of Averrhoa carambola

The extract is fractionated by column chromatography for purification and identification. The first eluent which contains the bioactive compound is collected from a DEAE-cellulose column with a gradient elution buffer of increasing salt concentration as its mobile phase. The buffer used is a sodium chloride solution of increasing concentration. The molecules in the extract which are retained in the stationary phase (DEAE-cellulose column) are displaced by the sodium ion in the mobile phase. Molecules which bind less strongly to the DEAE-cellulose column will move more readily and is eluted earlier by a lower concentration of sodium chloride solution. The first eluent containing the bioactive compound is chemically characterized.

The column used may be of any suitable anion exchanger and suitable buffer.

Table 3 shows the analysis of the content of the first eluent.

Table 3 : Analysis of the first eluent

Further analysis by high-performance liquid chromatography (HPLC) of the first eluent from ion-exchange chromatography identified galactose and glucose (galactoglucan) as the building units of the compounds in the first eluent.

The first eluent from the ion-exchange chromatography is also analysed using gel- permeation chromatography with a series of dextran solution. The average molecular weight of the bioactive compound is 11992 Da as shown in Figure 4a.

The extract containing the bioactive compound derived from the pericarp layer of the Averrhoa carambola was observed to be stable and sustainable at temperatures ranging from about 30°C to about 100°C for a prolonged period of time.

This enables the extract to be particularly suitable for use as an edible health supplement in the form of pellets, tablets, capsules, granules, or liquid. Once the extract is processed into a health supplement, it can be kept in long term storage as it has high resistance to microbes since it does not contain any sugar or water, and thus, negating the need for use of preservatives. Further, the stability and sustainability of the extract at elevated temperatures also renders the health supplement suitable for addition into food and beverages as a medicine or supplement, for example, added into hot food and/or beverages.

The health supplement comprising the extract may be incorporated into consumer products. The extract which is odourless and tasteless does not affect the original taste of the consumer product. Some examples of commercial products include instant hot beverages, yoghurt or protein shakes, nutritional bars and cereals.

The amount of the extract to be used in consumable end products e.g. health supplement, food or beverage, is largely dependent on the form of the product and its desired efficacy (prophylactic or treatment).

4 Official Methods of Analysis (16^th Edition) (1996). Association of Official Analytical Chemists. Washington, DC.

5 Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956).

Colorimetric Method for Determination of Sugars and Related Substances. Analytical Chemistry, 28(3), 350-356.

6 Krishnaveni, S., Theymoli Balasubramaniam and Sadasivam, S. (1984). Sugar

Distribution in Sweet Stalk Sorghum. Food Chemistry, 15, 229-232.

7 Singleton, V.L. and Rossi, J. A (1965). Colorimetry of Total Phenolics with

Phosphomolybdic-phosphotungstic Acid Reagents. American Journal of Enology and

Viticulture, 16, 144-58.

8 The Technical Association of the Pulp and Paper Industry UM250 (1991). Acid- soluble lignin in wood and pulp, 1991 TAPPI Useful Methods. Tappi, Atlanta, GA, USA. EXAMPLE

The following Example illustrates different aspects of this invention. This Example does not limit the invention, the scope of which is set out in the appended claims. Hvpercholesterolemic assay

In a study, 28 male New Zealand white rabbits, each weighing about 1.5-2.0 kg, from A Sapphire Enterprise, Selangor, Malaysia were used as test subjects to determine the effectiveness of the extract of the present invention in reducing blood lipid levels. Prior to the study period, the rabbits were acclimatized for one week.

The rabbits were randomly grouped into four groups, with each group having 7 rabbits. The rabbits in each of the four groups were fed a pre-determined diet as detailed below :

NC - negative control; fed with normal diet

PC - positive control; fed with high cholesterol diet

CA - fed with high cholesterol diet and the extract of the present invention

SG - fed with high cholesterol diet and oats

For the CA group, each rabbit was fed a mixture of high cholesterol diet and a prescribed amount of the extract calculated at 3% of their body weight.

Phase 1

Comparison of lipid profiles and atherogenic index (AI) of the four groups of male New Zealand white rabbits Blood sampling was performed on the male New Zealand white rabbits at weeks 0 and 10 of the study. The blood samples taken were centrifuged to separate out the plasma (blood serum), which was analysed to ascertain lipid profiles as total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-c) and low density lipoprotein- cholesterol (LDL-c), and the atherogenic index (AI) was calculated using the formula (TC - HDL)/HDL as per in the study conducted by Lee eta/. ⁹.

Tables 4 and 5 show the lipid profiles and atherogenic index (AI) at weeks 0 and 10.

Table 4 : Lipid profiles and atherogenic index (AI) at week 0

Note : Values are expressed as mean ± SD; n=7. Means not sharing a common letter in the same column were significantly different; P<0.05. At the end of the study in week 10, the positive control (PC) group having a high cholesterol diet was observed to have increased total cholesterol and low density lipoprotein-cholesterol. A prescribed dosage of the extract of the present invention was fed to the rabbits of the CA group and oats to the rabbits of the SG group. Decreased levels of total cholesterol, low density lipoprotein-cholesterol and atherogenic index were observed with an increase in the levels of high density lipoprotein-cholesterol. Comparing the CA group with PC group, the total cholesterol, low density lipoprotein- cholesterol and atherogenic index had decreased by 8%, 20% and 59%, respectively, and high density lipoprotein-cholesterol increased by 95%.

Comparing the SG group with PC group, the total cholesterol, low density lipoprotein- cholesterol and atherogenic index had decreased by 57%, 69% and 82%, respectively, and high density lipoprotein-cholesterol increased by 103%.

Phase 2

Faeces assessment of the four groups

of male New Zealand white rabbits The faeces of the four groups of male New Zealand white rabbits were assessed to confirm that the extract of the present invention promotes excretion of cholesterol via faeces as bile acids.

Faeces were collected from the rabbits, each day, beginning 3 days before the termination of the study period. Bile acid extraction was performed on the faeces collected according to the method reported by Uchida et a/.¹⁰ The amount of samples tested and test parameters (concentrations, temperature and time) were identical to Uchida eta/, 's method. The ether and bile acid fraction obtained from the extraction was analysed according to the method reported in Chau et a/.^{2, 3} The volume of the ether and bile acid fraction used and other chemicals were one fifth of those used in Chau et al. 's method, except for hydrazine hydrate where 25pL was used. Other parameters which include concentrations, temperature and time were identical to Chau eta/, 's method.

The results of the faeces assessment are shown in Tables 6 to 8.

Table 6 : Day 3 before termination of study period

Faecal bild acids (mg/day) NC group PC group CA group SG group

Average 28.22701 22.57184 37.42241 26.39368

Standard deviation 6.342047 3.815659 8.031498 7.158987

Table 7 : Day 2 before termination of study period

Faecal bild acids (mg/day) NC group PC group CA group SG group

Average 25.69828 28.06897 31.43391 30.36494

Standard deviation 7.183071 3.702082 7.359647 8.048299

Table 8 : Day 1 before termination of study period

Faecal bild acids (mg/day) NC group PC group CA group SG group

Average 20.73276 18.54885 48.68678 23.68678

Standard deviation 5.529781 3.695022 11.86880 5.209560 Graphs showing the mean and standard deviation of bile acid present in the faeces tested, based on Tables 6 to 8 are shown in Figures 5 to 7, respectively. The PC group did not show an increase in faecal bile acids in day 3 and day 1 before the termination of the study period. Overall, the bile acids content of the faeces of CA group was the highest, followed by SG group, in all 3 days before the termination of the study.

As expected, comparing with the PC group, the inclusion of the extract of the present invention and oats in a high cholesterol diet increased the content of faecal bile acids in all 3 days before the termination of the study period.

As presented in Table 6 and Figure 5, in day 3 before the termination of the study period, comparing with the PC group, the inclusion of the extract of the present invention (CA group) and oats (SG group) in a high cholesterol diet increased the content of faecal bile acids at 65.8% and 16.9%, respectively.

As shown in Table 7 and Figure 6, in day 2 before the termination of the study, comparing with the PC group, the content of faecal bile acid in the CA group and SG group increased to 12.0% and 8.2%.

In Table 8 and Figure 7, it is observed that the content of faecal bile acids in the CA group was the highest during the 3 days before the termination of the study. Comparing with the PC group, the content of faecal bile acid in the CA group and SG group increased to 162.5% and 27.7%.

2 Chau, C. F., Chen, C. H. and Wang, Y. T. (2004). Effects of a Novel Pomace Fiber on Lipid and Cholesterol Metabolism in the Hamster. Nutrition Research, 24(5), 337-345. 3 Chau, C. F., Chen, C. H. and Lin, C. Y. (2004). Insoluble Fiber-rich Fractions Derived from Averrhoa carambola: Hypoglycemic Effects Determined by in Vitro Methods. Lebenson Wiss Technology, 37(3), 331-335. Lee, Y. R., Kin, C. E., Kang, M. Y., Nam, S. H. (2007). Cholesterol Lowering and Antioxidant Status-improving Efficacy of Germinated Giant Embryonic Rice (Oryza sativa L.) in High Cholesterol-fed Rats. Annals of Nutrition and Metabolism, 51, 519- 526. Uchida, K., Nomura, Y., Kadowaki, M., Takeuchi, N., Yamamura Y. (1977). Effect of Dietary Cholesterol and Bile Acid Metabolism in Rats. Japan Journal of Pharmacology, 27, 193-204.

Phase 3

Comparison of aorta atherosclerotic plaques assessment of the four groups of male New Zealand white rabbits At the end of the study period, the rabbits were sacrificed by way of exsanguination and the aorta of each rabbit was removed for assessment on the presence of atherosclerotic plaques.

The aorta of each rabbit was stained with Sudan IV and Hematoxylin and eosin. Photographs of the stained aortas are as shown in figures 8 and 9.

Among the aorta of the rabbits stained with Sudan IV, it was observed that the CA group had significant inhibition of atherosclerotic plaque compared to the PC group. The observations were consistent with the atherosclerotic index in Tables 4, 5 and 6.

Microscopic observation with Hematoxylin and eosin stain after 10 weeks indicated thick foam cell formation in the PC group, followed by CA and SG group.

Foam cells are lipid loaded macrophages that are found in the lining of blood vessels. Accumulation of foam cells lead to fatty streaks of plaque atheroma or also known as atherosclerosis. Foam cell formation is induced by low density lipoprotein (LDL), including oxidized LDL or minimally modified LDL. Modified LDL and oxidized LDL are avidly taken up by macrophages which leads to the generation of foam cells. By comparing the aorta atherosclerotic plaques assessment of the four groups of rabbits, it can be concluded that the Averrhoa carambola extract has significant anti-atherogenic activity and that this activity can be attributed primarily to the soluble dietary fiber content of the extract. Conclusion

From Phase 1 of this Example, it is shown that the extract of the present invention has the potential to serve as a cholesterol-lowering agent and has significant anti-atherogenic activity. Although the extract seemed less effective than oats in decreasing the total cholesterol, low density lipoprotein-cholesterol and atherogenic index, an increment of dosage will very likely intensify its beneficial effects.

Based on the faeces assessment of the rabbits in Phase 2 of this Example, it can be concluded that the Averrhoa carambola extract absorbs bile acids from the bloodstream and promotes their excretion via faeces. Soluble dietary fiber interferes with cholesterol absorption by binding to bile acids which are made from cholesterol and directing them out of the body. In Phase 3 of this Example, based on the photographs of the aorta of the rabbits, it is observed that the Averrhoa carambola extract significantly reduces the presence of atherosclerotic plaques on aorta. This observation is consistent with the calculated atherosclerotic index in Phase 2 of this Example. Although the extract seemed less effective than oats in reducing the presence of atherosclerotic plaques, an increment of dosage will likely intensify its anti-atherogenic activity.

Overall, the presence of the Averrhoa carambola extract in the diet of the rabbits is very effective in reducing blood lipid levels, increasing excretion of bile acids and has significant anti-atherogenic activity.

As will be readily apparent to those skilled in the art, the present invention may easily be produced in other specific forms without departing from its scope or essential characteristics. The present embodiments are, therefore, to be considered as merely illustrative and not restrictive, the scope of the invention being indicated by the claims rather than the foregoing description, and all changes which come within therefore intended to be embraced therein.

Claims

1. An extract containing an effective amount of a bioactive compound derived from Averrhoa carambola,

said extract comprising soluble dietary fiber, soluble phenolics, soluble protein, acid-soluble lignin and sugar;

said bioactive compound having galactoglucan residues of average molecular weight about 11992 Da as a major component;

wherein said bioactive compound enables the extract to act as a cholesterol lowering agent suitable for use in the manufacture of a composition for reducing blood lipid levels.

2. The extract according to any one of the preceding claims, wherein said bioactive compound comprises about 19.0% soluble polysaccharide, 14.6% soluble lignin, 0.4% soluble protein and 3.6% phenolic compounds.

3. The extract according to any one of the preceding claims, wherein said bioactive compound is derived from the pericarp layer of Averrhoa carambola and is stable at temperatures ranging from about 30°C to about 100°C.

4. A health supplement comprising an effective amount of the extract of claim 1, said extract being stable at temperatures ranging from about 30°C to about 100°C.

5. A health supplement according to claim 4, wherein said supplement is in the form of pellets, tablets, granules, capsules, or liquid.

6. An edible product comprising an effective amount of the extract of claim 1, said extract being stable at temperatures ranging from about 30°C to about 100°C.

7. An edible product according to claim 6, wherein said edible product is in the form of yoghurt drinks, protein shakes, nutritional bars and cereals.

8. A method of producing a therapeutically effective extract of claim 1, said method comprising the following steps:

I) preparing dried ground Averrhoa carambo/a;

II) extracting a bioactive compound from dried ground Averrhoa carambo/a to produce a crude extract, said extraction performed using water as a solvent;

III) adding a polar organic solvent to said crude extract of step II to form a precipitate and separating the supernatant from the precipitate; and

IV) discarding said supernatant and collecting said precipitate containing said bioactive compound, of the extract.

9. The method according to claim 8, further comprising providing suitably sized cut pieces of Averrhoa carambo/a from the pericarp layer of the fruit of about 2.4 to 3.3 cm³, prior to step I.

10. The method according to any one of claims 8 to 9, further comprising grinding the Averrhoa carambo/a to a particulate size of about 2000 to 4000 pm, in step I.

11. The method according to any one of claims 8 to 10, wherein said dried ground Averrhoa carambo/a is refluxed in boiling water for about two hours in step II.

12. The method according to claim 11, wherein said reflux step is performed five times.

13. The method according to claim 12, wherein said solid residues formed during said reflux step is filtered from the filtrate.

14. The method according to any one of claims 8 to 13, wherein said crude extract is concentrated under reduced pressure, prior to step III.

15. The method according to any one of claims 8 to 14, wherein in step III, said mixture is centrifuged at 6500 rpm for about 10 minutes after addition of said solvent.

16. The method according to any one of claims 8 to 15, further comprising the following steps :

washing the resultant precipitate of step IV with said solvent; and

centrifuging the mixture at 6500 rpm for about 10 minutes,

for three times.

17. The method according to any one of claims 8 to 16, further comprising dissolving said precipitate of step IV in distilled water and concentrating the mixture under reduced pressure.

18. The method according to claim 17, further comprising freeze-drying the dissolved precipitate.