WO2008066370A1

WO2008066370A1 - Nephelium lappaceum extracts for cosmeceutical and nutraceutical applications

Info

Publication number: WO2008066370A1
Application number: PCT/MY2007/000081
Authority: WO
Inventors: Uma Devi Palanisamy; Hwee Ming Cheng; Theanmalar Masilamani; Thavamanithevi Subramaniam; Say Moi Yap; Ammu Radhakrishnan
Original assignee: Sirim Berhad; Universiti Malaya; International Medical University
Priority date: 2006-11-27
Filing date: 2007-11-20
Publication date: 2008-06-05
Also published as: MY174362A

Abstract

There is disclosed a method of extraction of Nephelium lappaceum extracts, a composition of phenolic compounds, with high free radical scavenging activity. The extract is characterised by its radical scavenging activity with IC50 (effective concentration for 50 % inhibition) of 3.8 μg/ml (DPPH), 1.7 μg/ml (ABTS), 1.7 μg/ml (Galvinoxyl), no prooxidant capabilities and a phenolic content of 762-822 mg GAE/g extract. The extract is not cytotoxic to normal mouse fibroblasts cells or splenocytes. The use of this extract alone or in combination with other active principals, is of interest to the nutraceutical, cosmeceutical and pharmaceutical industry.

Description

NEPHELIUM LAPPACEUM EXTRACTS FOR COSMECEUTICAL AND NUTRACEUTICAL APPLICATIONS

Technical Field of the Invention

The present invention relates generally to plant -based extracts and more particularly to Nephelium lappaceum extracts with high free radical scavenging activity.

Background of the Invention

Anti-oxidants are of interest to cosmetic, food and health professionals. Antioxidants represent our front line of defense against major health conditions such as heart disease, cancer and muscular degeneration. At the molecular and cellular levels, anti-oxidants serve to deactivate free radicals, which usually come in the form of O₂, the oxygen molecule, Free radicals are the natural byproducts of many processes within and among cell. They can also be created by exposure to various environmental factors and can cause damage to cell walls, certain cell structures and genetic material within the cells. Such damage can become irreversible and lead to disease.

Some fast facts about free radicals and oxidative stress (Ames, 1993).:

• Free radicals are unstable molecules that often contain oxygen. They are unstable because they have only one electron and they desperately want two to make them stable. To get another electron they rob one from anything they can. Healthy cells in our bodies are a good source for these electrons.

• Free radicals go about their business in our bodies relentlessly and very quickly, and can cause a lot of damage. • it is estimated that our DNA receives about 10,000 "hits" from free radicals per cell per day

• An imbalance of free radicals in the body is called oxidative stress. Our cells can cope with small amounts of oxidative stress as they are quite ingenious and can repair minor free radical damage. However, not all damage can be repaired and the unrepaired damage accumulates in the body.

• Major oxidative stress can cause grave disturbances in cell metabolism and contribute to human disease. Tissue damage and injury can also lead to oxidative stress. Oxidative stress can cause DNA damage, lipid peroxidation, protein damage and ischaemic (lack of oxygen) injury.

• The human body constantly produces free radicals and other oxygen derived ^"molecules (such as hydrogen peroxide). It has a relatively small reserve of antioxidant defence capacity.

Today, people continuously subject themselves to many substances and activities that increase the level of free radicals in their bodies, so the body can easily become overpopulated with oxygen-derived species that upset cell biochemistry. Antioxidants are vital in combating the free radicals that are constantly forming in our bodies due to oxidation. By curbing the activity of free radicals in your body, you can slow down the processes that cause disease and aging, and live a longer, healthier life.

Our bodies gain antioxidants from two sources:

1. Our body's in-house antioxidants, such as superoxide dismutase, catalase and glutathione. 2. Dietary antioxidants, from sources such as fruit, vegetables, nuts and grains.

As we grow older our body's defence system loses its effectiveness and we hold fewer antioxidants. Fewer free radicals are neutralised and there is a slow buildup of damaged molecules in the body.Therefore, to reduce the damage to our bodies caused by free radicals, we need to add antioxidants to our diets.

Natural Antioxidants in Nutraceuticals and Pharmaceuticals

The powerful antioxidant properties of vitamins C, and E, and beta carotene are well known. But there's another group of naturally occurring inhibitors of oxidation receiving a lot of attention lately, namely the phenolics and polyphenolics which are secondary plant metabolites Studies have shown that polyphenols are better scavengers of free radicals than vitamins C and E. For instance, green tea is thought to be particularly effective at protecting against free radical damage in the gastrointestinal tract Orner et α/.,2004) . Other antioxidant herbs include bilberry (Valentova et al., 2006), grape seed extract (Hwang et α/.,2004), pomegranate (Rosenblat et α/.,2006) and milk thistle. Additionally, the use of exotic juices and extracts has been widespread (USP 6858235).

Recent interest in food phenolics has increased greatly, owing to their antioxidant capacity and their possible beneficial implications in human health, such as in the treatment and prevention of cancer, cardiovascular disease, and other pathologies. Much of the interest is in the group of plant phenolics called the flavonoids. Flavonoids are antioxidant compounds found in plants, and are natural dietary disease-preventing, health-promoting, anti-ageing substances.

Flavonoids exert these antioxidant effects by neutralising all types of oxidising radicals (Bors, et α/., 1998) including the superoxide (Robak, et α/., 1998) and hydroxyl radicals (Husain, et α/., 1987) and by chelation. A chelator binds to metal ions in our bodies to prevent them being available for oxidation. Flavonoids can also act as powerful chain-breaking antioxidants due to the electron-donating capacity of their phenolic groups. In addition, many biological effects countering inflammatory, bacterial, viral, microbial, hormonal, carcinogenic, neoplastic and allergic disorders (Middleton, 1996) have been reported for flavonoids in both in vitro and in vivo systems.

However, studies have proven that our normal intake of flavonoids through eating a range of fruits and vegetables is not enough to keep modern levels of free radicals at bay. Flavonoids are some of the most powerful and effective antioxidant compounds available to humans - and since we are unable to produce flavonoids ourselves, we must get them from the food we eat and from supplements. Food companies are increasingly promoting the health benefits of anti-oxidants contained in fruits and vegetables. The trend has been to investigate a variety of plants as new potential sources of anti-oxidants (Miliauskas et al.,2004; Maisuthisakul et al, 2006). Besides vitamins, a defined range of herbal extracts represents the most potential in the anti-oxidant market. There are plenty of opportunities in the market to place these extracts because they are natural ingredients that have a positive reputation due to the use of herbals as traditional products (Peschel, 2006).

Also important on the antioxidant front are xanthones-polyphenols with chemical configurations that make them very strong antioxidants. Besides being antioxidants, evidence is growing indicating they react at a cellar level with almost all tissues in the human body. Laboratory tests have shown that extracts containing xanthones have anti-microbial, antiproliferative,^' apoptic inductive and cytotoxic actions. Other studies show them to be anti-inflammatory, and to be COX-2 inhibitors. Over 40 types of xanthones are found in the fruit, seeds and peel of the Garcinia mangostana fruit. Pure Fruit Technologies, USA has extracted these xanthones for use in its product Mango^«xan. To mask the bitter flavor of xanthones, Mango»xan also contains pure fruit juices.

Manufacturers appear to be moving toward antioxidant blends that incorporate both traditional vitamins and herbal extracts, rather than stand alone single ingredient formulas, while food manufacturers continue to incorporate natural antioxidants into traditional food formulations. As a result, companies must be careful to have substantial documentation of antioxidant evaluation of their natural ingredients. The antioxidant market is set to grow over the foreseeable future, however what is often lacking is good science that shows the anti-oxidant claims of these compounds.

Antioxidants in cosmetics

There is also growing research showing that topically applied antioxidants can help protect from sun damage (Black, 2004; White, 1984; Hanson, 2003). Although antioxidants don't replace the need to use sunscreens, when used in combination with them, they are highly effective and a wonderful addition to the protective value of a well-formulated sunscreen.

Antioxidants also serve as topical anti-inflammatory agents and that's a good thing, because keeping skin inflammation to a minimum is critical to its healthy functioning and outward appearance. Some cosmetics companies are utilizing antioxidants for their rejuvenating effects in makeup.

Cosmetic chemists often use antioxidants to slow the oxidation of fats and oils in cosmetics, inhibiting rancidity and extending product shelf life. Antioxidants are effective in a vast array of personal care products-from skin creams, facial cleansers and body oils, to shampoos, lipsticks, fragrances and sunscreens (Klingman, 2000). There are both synthetic antioxidants and natural antioxidants on the market today. Tocopherols and citric acid are two well-known natural antioxidants, and technologists continue to conduct studies to show the efficacy of other potential natural antioxidants.

Summary of the Invention

Accordingly, it is the primary object of the present invention to provide a method of preparation of Nephelium lappaceum extracts with high free radical scavenging activity.

It is another object of the present invention to provide a use for Nephelium lappaceum extract prepared according to the method of Claim 1.

It is yet another object of the present invention to provide a use for powdered pulp, seed, rind or leaf of Nephelium lappaceum prepared according to the method of Claim 2.

These and other objects of the present invention are accomplished by providing, A method of preparation of Nephelium lappaceum extracts with high free radical scavenging activity comprising the steps of: a) Adding deionised water or solvent at 1:10 (w/v) concentration to powderised pulp, seed, rind or leaf of Nephelium lappaceum; b) Extracting the powderised pulp, seed, rind or leaf with deionised water at

4O⁰C, or extracting with solvent at room temperature for 24 hours in an orbital shaker; c) Filtering the resulting suspension from (b) using No. 114 Whatman filter paper; and d) Collecting the aqueous or solvent filtrate and concentrating the aqueous filtrate using a freeze dryer or concentrating the solvent filtrate using a rotary evaporator.

Detailed Description of the Invention

The plant, Nephelium lappaceum (rambutan) Native to Southeast Asia, rambutan (Nephelium lappaceum L.) belongs in the same family (Sapindaceae) as the subtropical fruits lychee and longan and is often described as being less aromatic than the lychee. This fruit is an important commercial crop in Asia, where it is consumed fresh, canned, or processed and appreciated for its refreshing flavor and exotic appearance (Almeyada et fl/.,1979, Ong e/ αl.,1998).

Rambutans are most commonly eaten out-of-hand after merely tearing the rind open, or cutting it around the middle and pulling it off. It does not cling to the flesh. In Malayasia the dried fruit rind has been employed in local medicine.

Supercritical Fluid Extraction (McHugh and Krukonis,1994) Super critical extraction (SFE) is an extraction process using a supercritical fluid as a solvent. When a fluid is taken above its critical temperature (Tc) and critical pressure (Pc), it exists in a condition called the supercritical fluid state. The supercritical fluid has characteristics of both gases and liquids. By manipulating the pressure and temperature of the fluid, the fluid can solubilize the material of interest and selectively extract it.

SFE is widely used in a variety of industries, including natural products, foods and flavors, pharmaceuticals, nutraceutical, polymers, chemicals, and cleaning.

Unlike traditional solvent based processing, no solvent is used in SFE. This translates into lower operating costs because of the reduction in post-processing steps, clean up and safety and assurance measurements. Most importantly, SFE provides extracts with no residual solvent, keeping your product natural.

Methods

Plant collection

Fresh fruit pulp, rind, seed and leaves of Nephelium lappaceum L were obtained from Kuala Lumpur. The plants were authenticated by Kepong Herbarium,

FRIM.

Preparation of plant extracts

The fruit pulp, seed, rind and leaves were washed with copious amounts of water followed by distilled water and then allowed to air dry at room temperature. It is then placed in an oven at 40⁰C until completely dry, after which is powderised using a Waring blender or milled using the Fritsch dry miller. Extraction of the powderised pulp, seed, rind and leaves was carried out with deionised water and a variety of solvents such as absolute ethanol, methanol, petroleum ether, ethyl acetate, chloroform and polyethylene glycol:deionised water (80:20 and 50:50). The method of supercritical extraction (SFE) was used only on the rind of Nephelium lappaceum.

Water and solvent extraction; deionised water or the respective solvents

(analytical grade) at 1 : 10(w/v) concentrations was added to the powderised pulp, seed, rind and leaves. Water extraction was carried out at 40⁰C while solvent extraction at root temperature for 24 hours in an orbital shaker. The suspension thus obtained was filtered using a 114 Whatman filter paper and filtrate collected. Aqueous filtrate was concentrated using a freeze drier while solvent filtrate was concentrated using a rotary evaporator.

Supercritical fluid extraction Extraction was carried out using the supercritical fluid extraction system Thar

SFE 500 from Thar Technologies (McHugh and Krukonis,1994). 80 g of powderised rind or leaves of Nephelium lappaceum L was charged to the extraction vessel and CO₂ at a pressure of 300 bar and temperature of 50⁰C was passed through the vessel for a period of 2 hours. The gas flow rate was set at 30 g/min. The components (oils and actives) soluble in liquid CO₂ precipitated at the end of the run were collected. The product thus obtained is a completely pure extract with no liquid organic solvents. Ethanol however, is used to wash down the lines and precipitate is further concentrated on a rotary evaporator. The residue thus remaining after the SFE extraction was re-extracted using ethanol as solvent. A ratio of 1 :20 that is 100 g of powderised rind to 2000 ml of ethanol was used for further extraction and processed as described above for ethanol extraction.

Anti-oxidant Assays

Five different anti-oxidant and free radical scavenging assays were performed.

Scavenging activity onto DPPH radicals

Scavenging activity on DPPH (l,l-diphenyl-2-picrylhydrazyl) free radicals by the extracts was assessed according to the modified method reported by Chang et al, (2002). A total of 950 μl of 0.004 % DPPH ethanolic solution was added to 50 μl of an extract of different concentrations to make a final volume of ImI and the mixture was allowed to react at 37 ⁰C. All determinations were performed in triplicates. After 10 minutes, the absorbance value was measured at 515 nm with Cary 50 Bio UV-visible spectrophotometer. DPPH in ethanol (950 μl) plus 50 μl of different samples was used in the assay, while DPPH solution (950 μl) with 50 μl of ethanol/distilled water was used as negative control. The positive controls (standards) consisted of 950μl of DPPH reagent and 50 μl of L-ascorbic acid (Sigma chemicals) and grape seed (Vitis vital ^M).

Scavenging activity in this assay was expressed as ICs₀, which represents the final concentration of the extract (μg/ml) in the reaction mixture required to inhibit 50 % of the free radical scavenging activity. The optical density obtained was converted into of free radical scavenging activity by using the formula:

% free radical scavenging activity = [(A negative control - A gxtrnct)] x 100%

A negative control

IC₅₀ values were calculated by linear regression of plots where x-axis represented the various concentrations of tested plant extracts and the y-axis represented the average percentage of free radical scavenging activities from three replicates. IC_5O of samples were compared against the standards, L-ascorbic acid and grape seed extract, because the lower the IC₅₀ of a plant extract, the better it is as an antioxidant.

Scavenging activity onto Galvinoxyl radicals

Galinoxyl, another stable phenoxyl radical can be reduced by hydrogen-donating free radical scavengers. Concentration of extracts and standards required to achieve 50 % phenoxyl radical scavenging activity was determined according to the method of Shi et al, 2001. A total of 900 μl of Galvinoxyl methanol working solution (5 niM) was added to 90 μl of a sample of different concentrations to make a final volume of 990 ml and the mixture was allowed to react at 37°C. All determinations were performed in triplicates. After 20 minutes, the absorbance value was measured at 428nm. Ethanol or distilled water was used as negative control while L-ascorbic acid and grape seed served as the positive controls. The phenoxyl radical scavenging activity and IC₅₀ of the extracts and positive controls were calculated as described for the DPPH assay. Scavenging activity onto ABTS radicals

ABTS (2,2-azinobis 3-ethylbenzothiazoline 6-sulfonate) is oxidized to the colored nitrogen centered radical cation ABTS⁺ in a persulfate system. The radical scavenging capacity of extracts and positive controls were assessed according to the modified method reported by Roberta et a\, 1999. 200 μl of the working solution (5mM, pH7.4) was mixed with 20 μl of extract in a microtiter plate to a final volume of 220 ml and the mixture was allowed to react for 6 mins before taking its absorbance at 690 run on a AsYS Microplate Reader UVM-340. All determinations were performed in triplicates. Ethanol or distilled water was used as negative control while L-ascorbic acid and grape seed served as the positive controls. The ABTS scavenging activity and IC₅₀ of the extracts and positive controls were calculated as described for the DPPH assay.

Scavenging activity onto superoxide anions

An enzyme system (xanthine/xanthine oxidase) forms the superoxide anions which react with NBT(Tetrazolium salt) to form a red formazan. An anti-oxidant substance absorbs or destroys superoxide anions, thereby reducing the formation of formazon. The results are presented as percent inhibition and compared to the positive control superoxide dismutase using the modified method of Chang et al., (1996).

Stock solutions of NBT (0.2 M Tris-HCl, 5 mM MgCl₂, 0.75 mM NBT), xanthine (66 mM xanthine in 0.5 M NaOH) and 27 mM EDTA were prepared. An NBT working solution was prepared by adding stock solutions of the above to give a final concentration of 0.15 mM NBT, 3 mM xanthine, 0.108 mM EDTA, 0.05 M Na₂CO₃. The final pH of the working solution is pH 10 to 10.2. The assay was conducted in 96 wells plates where 25 ul of extracts were added to 200 ul of NBT working solution and 25 ul of xanthine oxidase (0.3 Units/ml). The reaction was allowed to proceed for 5 minutes after which its absorbance at 560 nm was recorded on a AsYS Microplate Reader UVM-340. All determinations were performed in triplicates. Ethanol or distilled water was used as negative control while superoxide dismutase, L-ascorbic acid and grape seed served as the positive controls. Antioxidants present in the extract will inhibit the production of superoxide free radicals and thus a reduction in absorbance will be observed. The percentage inhibition was calculated as follows:

(ΔNegative Control____AE\tract) X 100 -".Negative Control

Inhibition of induced lipid autooxidation

Linoleic acid undergoes auto-oxidation at a specified condition. Autoxidative activity is determined by the capacity to inhibit the peroxidation of linoleic acid. The results are presented as percent inhibition and compared to Tert-Butylphenol (BHT) and α-tocopherol (vitamin E). The modified method of Lee and Lim

(2000) was followed, where a mixture containing extract, 5 % linoelic acid in 0.05M phosphate buffer (pH 7.0 containing 0.5 % Tween 80) was incubated for 16 hours at 40 ⁰C in the dark. lOOμl of this incubated mixture was taken out and remixed with 3 ml of 70 % ethanol, 100 μl of 0.3 g/ml ammonium thiocyanate (Sigma Chemicals) and 100 μl of 2.45 mg/ml ferrous chloride (Merck

Chemicals) in 3.5 % hydrochloric acid. The addition of acid-alcohol solution stopped the autoxidation reaction and hydroperoxides formed complexed with the ferric thiocyanate to produce an absorbance at 500 nm, which was measured after 3 minutes.

All determinations were performed in triplicates. Ethanol or distilled water was used as negative control while butylated hydroxytoluene (1% BHT) (Sigma Chemicals) and α-tocopherol (Fluka Chemicals) served as the positive controls. Extract concentrations used were; rind at O.land 0.4 mg/ml and leaves at 0.3 and 0.7 mg/ml.

Antioxidant activity was determined by the capacity to inhibit the peroxidation of linoleic acid. The percentage inhibition was calculated as follows:

% of inhibition = [(A _nιτat,ve mntmi - A e_Xlrac)l x 100%

A negative control Pro-oxidant assay

Reducing power of iron ion was measured according to the method of Bing et al, 2005 where equal volumes the extract and 1% potassium ferricyanate [K₃Fe(CN₆)] was incubated at 50 ⁰C for 20 minutes. An equal volume of 10% trichloroacetic acid was added and mixture centrifuged at 3000 g for 10 minutes.

The upper layer of the solution (1.0 ml) was mixed with ImI of distilled water and 0.2 ml of 0.1% ferric chloride (FeCl₃) and its absorbance recorded at 700 nm. Ethanol or distilled water was used as negative control while Vitamin C and Emblica™ (a commercial antioxidant with very low pro-oxidant activity) was used as positive controls. Reducing power/Pro-oxidant activity was calculated as below:

Reducing Power = A extract - A negative control

Results are expressed in comparison with positive controls at concentration ranging from 0.1 mg/ml to 0.5 mg/ml.

Heavy metal content determination

Lead, arsenic and mercury content of the powderised rind of Nephelium lappaceum was determined against international standards that is the ICP-OES for the detremination of arsenic and lead and AAS for the determination of mercury.

Phenolic content determination

Total phenolic was determined using the Folin-Ciocalteu method modified according to G. Miliauskas et al., (2002) and based on a colorimetric oxidation and reduction reaction.

1 ml aliquot of the extracts at defined concentrations was added to 5 ml of Folin- Ciocalteu reagent, 4 ml of 7.5 % Na₂CO₃ solution was added to the mixture after

3 to 5 minutes and was thoroughly mixed. The absorbance at 765 nm was taken after an hour. All determinations was performed in triplicates. Blank consisted of Folin-Ciocalteu reagent (5 ml), ethanol/distilled water (1 ml) and 7.5 % Na₂CO₃ solution (4 ml).

A linear dose response regression was generated using absorbance reading of gallic acid at the wavelength of 765nm. The calibration curve using gallic acid was obtained in the same manner as above except that the absorbance was read after 30 minutes.

Total content of phenolic compounds in the plant extracts was calculated using this formula: C = A/B

C = Total content of phenolic compounds, mg/g extract A = The equivalent concentration of gallic acid established from calibration

Curve (mg) B = The concentration of extract (g)

Total phenolic content of sample was calculated and the content of phenolic compounds in a respective sample was expressed in mg/g of extract, gallic acid equivalent (GAE).

Measurement of cell proliferation of cultured cells by XTT assay 96-well culture plates were removed from the CO₂ incubator at the end of the culture periods (3T3 mouse fibroblasts cells and 4Tl mouse mammary gland tumor cells). Cell proliferation was determined using the XTT cell proliferation kit as recommended by the manufacturers (Roche Applied Science, Germany).

The XTT labelling and electron-coupling reagents was thawed using a water- bath set at 37 °C. Five ml of labelling and electron-coupling reagents was mixed with 0.1 ml of the coupling reagent and the concoction mixed thoroughly to obtain a clear solution. 50 μl of the XTT labelling mixture was pipetted into each of the 96-wells (50 μl/well) of the microtitre plate. Following this, the culture plates were returned to the humidified CO₂ incubator and incubated for six hours. The absorbance of the test samples was read at a wavelength of 450 nm using an ELISA plate reader. The reference wavelength used was 650 nm. The XTT labelling reagent alone was used as blank.

Measurement of cell death by apoptosis in cells cultured in extracts of the rind of N lappaceum

Confluent cells (3T3 or 4Tl cells) were harvested and counted before being plated onto 96- well plates at 5x10³ cells/well. Following this, known concentrations of aqueous and ethanol extracts of the rind of Nephelium lappaceum was added into each of the 96-wells. The extracts were appropriately diluted in complete RPMI 1640 medium to obtain final concentrations of 50 μg/ml, 100 μg/ml and 500 μg/ml. One-hundred μl of the diluted extracts was added into the wells containing the cultured cells.

Wells containing cells in medium without any added extracts were used as negative control. Grape seed extract used in the same concentrations was used as the positive control. The plates were then incubated for an hour at 37°C in the humidified incubator. Cell death due to apoptosis in cells cultured in the presence and absence of the extracts was determined using the cell death detection kit (Roche Applied Science, Germany) as recommended by the manufacturers.

Measurement of oxidant-induced cell death by apoptosis in cells cultured in the extracts of the rind of N lappaceum

Confluent cells (3T3 or 4Tl cells) were harvested and counted before being plated out in 96-well plates at 5x10³ cells/well. Following this, 100 μl of known concentrations (50 μg/ml, 100 μg/ml and 500 μg/ml) of aqueous and ethanol extracts of the rind of Nephelium lappaceum was added into each of the 96- wells. Grape seed extract used in the same concentrations was used as the positive control while cells in medium without any added extracts was used as negative control. The plates were then incubated for an hour at 37°C in the humidified incubator. Following this, 100 μl of known concentration of DPPH was added to all the wells. The culture plates were incubated for 24 hours in a humidified 5% CO₂ incubator. Wells containing cells and oxidants alone served as positive control i.e. where maximum cell death will be expected while wells containing cells, oxidants and grape seed extract were used as negative control where only low levels of cell death is expected. Oxidant-induced cell death by apoptosis in cells cultured in the presence and absence of the extracts was determined using the cell death detection kit (Roche Applied Science, Germany) as recommended by the manufacturers. ';

Effect of culturing murine splenocytes in the presence of extracts of the rind of

N. lappaceum

Seven-week old male BALB/c mice were euthanized with ether. Once the mice were dead, they were dipped in 70 % alcohol to sterilise the animal prior to taking them into the clean area for culture purposes. The spleen from each of the sacrificed animals was removed aseptically. The spleens were placed in a sterile petri dish containing complete RPMI 1640 media. The splenocytes were then gently teased out from the splenic capsule aseptically. The splenocyte suspension was then transferred into sterile 15ml tubes and centrifuged at 500 g for five minutes at room temperature.

After centrifugation, the supernatant was discarded and five ml of complete

RPMI 1640 medium was added to the tube. The pellet was resuspended in the medium through vortexing. The mixture was left on ice while the number of white blood cells were determined using a haemocytometer.

The splenocyte suspension was appropriately diluted in complete RPMI 1640 medium to obtain a final cell concentration of IXlO⁵ cells/ml. One-hundred μl of the diluted splenoyctes was then pipetted into the wells of sterile 96-well tissue culture plates (IXlO⁴ cells/well). Following this, known concentrations (10, 50, 100 and 125 μg/ml) of the aqueous and ethanol extracts of the rind of Nephehum lappaceum was added into the wells containing the splenocytes and cultured for 24, 48 and 72 hour in a humidified 5 % CO₂ incubator

Wells containing cells in culture medium alone with no added extracts was taken as the negative control while wells that contained 25 μg/ml Concanavalin A (Con A) served as positive control. At the end of the specified culture periods (24, 48 and 72 hours), the plates were removed from the incubator and cell proliferation was determined using a cell proliferation determination kit.

Results and Discussion

Extraction Yields Water and solvent extraction of the fruit pulp, seed, rind and leaves of the

Nephehum lappaceum was carried out as described earlier. The extraction yields of aqueous and ethanol are given in Table 1.

Table 1: Aqueous and Ethanolic extraction yields of the fruit pulp, seed, rind and leaves oiNephelium lappaceum

Extraction yields with ethanol was the highest among all the solvents used. Fruit pulp followed by the seed, rind and finally the leaves displayed the highest solids extracted in the ethanolic extraction. Aqueous extraction however, saw fruit pulp followed by rind, leaves and finally the seed. Supercritical fluid extraction (SFE) was carried out on the rind only and the yield of dry matter was 0.42 %. The residue after SFE when re-extracted with ethanol gave a yield of 16.3% of dry matter, very similar to the yield when direct extraction with ethanol was performed (Table 1). Therefore, SFE prior to ethanolic extraction does not increase yield any further as such the simple and direct extraction with ethanol is the recommended way to process the extracts.

Anti-oxidant assays

Scavenging activity onto DPPH radicals

All extracts were evaluated for its DPPH scavenging ability and its activity is as shown in Table 2.

Table 2: DPPH scavenging activity of the fruit pulp, seed, rind and leaves oϊNephelium lappaceum

Figure 1: DPPH scavenging activity, expressed as I/EC₅₀, of the aqueous and ethanolic extracts of fruit pulp, seed, rind and leaves oiNephelium lappaceum

As observed in Table 2 and Figure 1 above, the rind and the leaves of Nephehum lappaceum displayed the highest DPPH radical scavenging ability, with the ethanolic rind extracts having the lowest IC₅₀ value The activity exhibited by the ethanolic rind extracts are comparable to Vitamin C and far better than grape seed extracts. SFE extracts of the rind of N lappaceum did not exhibit any

DPPH scavenging activity, however the residue when re-extracted with ethanol exhibited activity similar to ethanolic extracts. Further studies on the anti-oxidant ability of Nephehum lappaceum was carried out using the rind and leaves only.

Scavenging activity onto Galvinoxyl and ABTS radicals

Rind and leave extracts were evaluated for its Galvinoxyl and ABTS scavenging ability and its activity is as shown in Table 2. Table 2: Galvinoxyl and ABTS scavenging activity of the rind and leave of Nephelium lappaceum

Galvinoxyl ABTS

Figure 2 (a) and (b): The Galvinoxyl and ABTS scavenging assays of the aqueous and ethanolic extracts of the rind and leaf of Nephelium lappaceum

Both Galvinoxyl and ABTS assays show that the ethanolic extracts of the rind of

Nephelium lappaceum have far better radical scavenging activity than Vitamin C and grape seed.

Scavenging activity onto superoxide anions and Inhibition of Induced Lipid Autooxidation Rind and leave extracts were evaluated for its scavenging activity onto superoxide anions and inhibition of induced lipid autooxidation and its activity is as shown in Tables 3 and 4. Table 3: Superoxide scavenging ability of the aqueous and athanolic extracts of the rind and leaf of Nephelium lappaceum

Concentration of extracts in reaction mixture was 50 μg/ml

It was observed that at the concentrations tested (50 μg/ml), the aqueous and ethanolic extracts of the rind of Nephelium lappaceum exhibited similar scavenging ability for superoxides as superoxide dismutase (5 μg/ml) and aqueous grape seed extracts.

Table 4: Inhibition of induced lipid autooxidation of the aqueous and ethanolic extracts of rind and leaf of Nephelium lappaceum

* Concentrations of rind and leaf of Nephelium lappaceum and grape seed tested; aqueous rind- 0.4 mg/ml; ethanolic rind- 0.1 mg/ml; aqueous leaf - 0.7 mg/ml; ethanolic leaf - 0.3 mg/ml; aqueous grape seed - 0.7 mg/ml; ethanolic grape seed - 0.3 mg/ml)

The inhibition of induced lipid autooxidation of the aqueous and ethanolic extracts of rind and leaf of Nephelium lappaceum at its respective concentrations is shown in Table 4. Aqueous extracts of both the rind and leaf managed to protect lipid autooxidation by about 50 % at the very low concentrations of 0.4 and 0.7 mg/ml respectively. The concentration of the ethanolic extracts of rind tested was 0.1 mg/ml which may be too low for effective protection against lipid autooxidation.

Pro-oxidant assay

A pro-oxidant is defined as a substance that can produce oxygen byproducts of metabolism that can cause damage to cells. It is known that Vitamin C at higher concentrations tends to behave like a pro-oxidant. The interaction of vitamin C with 'free', catalytically active metal ions could contribute to oxidative damage through the production of hydroxyl and alkoxyl radicals; whether these mechanisms occur in vivo, however, is uncertain. We have evaluated the rind and leaf of Nephelium lappaceum for its pro-oxidant capability and compared it against vitamin C, grape seed and Emblica™, a commercially available plant extract used in cosmetics and as a nutraceutical, known for its very low pro- oxidant capacity.

i of N lappaceum (Aqueous) i

Concentratιon(mg/ml)

Figure 3: Pro-oxidant capacity of the rind and leaf of Neplielium lappaceum compared with Vitamin C, α-tocopherol, grape seed and Emblica ΓM over a range of concentrations

As expected Vitamin C showed the highest pro-oxidant activity, induced by transition metals. Among the positive controls Emblica™, followed by tocotrienol, green tea (results not shown) and finally α-tocopherol showed lower pro-oxidant capacity. Aqueous rind of N lappaceum exhibited the lowest pro- oxidant capacity, even lower than Emblica™ while the ethanolic rind extract had lower pro-oxidant capacity than vitamin C, tocotrienol and ethanolic grape seed extract. The ethanolic rind extract was seen to have comparable pro- oxidant profiles as aqueous grape seed extract, green tea and tocotrienol.

Heavy Metal Content Lead, arsenic and mercury content of powderised N lappaceum was determined against international standards using the ICP-OES (inductively coupled plasma-optical emission spectroscopy) for the determination of arsenic and lead and AAS (atomic absorption spectroscopy) for the determination of mercury. Table 5: Lead, Arsenic and Mercury determination of the rind and leaf of powderised N. lappaceum

The levels of the above heavy metals in the rind and leaf of the powderised N. lappaceum were far below the permissible levels for nutraceuticals.

Total Phenolic Content

The total phenolic content of the extracts were determined following the Folin-

Ciocalteu method.

Table 6: Total Phenolic Content of the aqueous and ethanolic extracts of the rind and leaf of Nephelium lappaceum and compared with grape seed extracts

Figure 4: Total Phenolic Content of the aqueous and ethanolic extracts of the rind and leaf of Nephelium lappaceum and compared with grape seed extracts

Although the total phenolic content of the ethanolic extracts of the rind of Nephelium lappaceum is comparable to grape seed ethanol extracts, its DPPH scavenging activity is far higher than that of grape seed. It has been shown that phenolic content tends to correspond with scavenging activity (Auger et al., 2004; Bagchi et al., 2006; Chen et al, 2004), this however does not hold true when comparing with the commercial grape seed extract. On the other hand, the extracts having lower total phenolic content are seen to have correspondingly lower scavenging activity.

Measurement of Cell Death by Apoptosis in Cells Cultured in the Presence of Nephelium lappaceum Extracts

Confluent 3T3 and 4Tl cells were incubated in the presence and absence of aqueous and ethanolic extracts of the rind of Nephelium lappaceum. The enrichment factor, which refers to the amount of mono- and oligonucleosomes in the cytoplasm of the apoptotic cells is presented in Figures 5 and 6 below. 4Tl cells '

Figure 5 and 6: The effect of aqueous and ethanolic extracts (100 μg/ml) of the rind of Nephelium lappaceum on 3T3 cells (Figure 5) and 4Tl cells (Figure 6) without the addition of oxidants

* designates a significant difference from cell (PO.05)

# designates a significant difference from grape seed (P<0.05)

Apoptosis levels in cells treated with Nephelium lappaceum and grape seed extracts were lower compared to the cells cultured in the absence of plant extracts in both 3T3 and 4Tl cells, at the concentration tested (100 μg/ml). The aqueous and ethanolic extracts of the rind of Nephelium lappaceum appear to protect 3T3 cells from cell death due to apoptosis by about 60 %.

A similar protection from cell death due to apoptosis was observed in 4Tl cells.

However, the level of protection of 4Tl cells from apoptotic death by Nephelium lappaceum extracts appear to be lower than that observed for normal 3T3 cells. The lower levels of apoptosis in 4Tl cells may be due to the defective apoptotic pathways in tumour cells where there is an over-expression of the inhibitor of apoptotic proteins, which suppresses the activity of apoptotic activators (Yang et al., 2003). Therefore, owing to such low levels of apoptosis in untreated cancer cells, it may not be possible to find a significant difference in the apoptotic rate between treated and untreated cancer cells. Apoptosis Levels in Cells Exposed to Oxidants in the Presence of Nephehum lappaceum Extracts

This study suggests that reactive oxygen species (ROS) such as DPPH can induce significant apoptotic cell death. The rate of apoptotic cell due to the free radical DPPH was observed to increase significantly in both normal 3T3 mouse fibroblast and murine mammary cancer cells (4Tl). The rate of apoptotic death in 3T3 cell lines was markedly reduced following the addition of 100 μg/ml Nephehum lappaceum extracts when 5 and 50 μM of DPPH was used (Figures 7 and 8)

Figures 7 and 8: The effect of aqueous and ethanolic extracts of the rind of

Nephelium lappaceum on 3T3 cells with the addition of 5 μM (Figure 7) and 50 μM (Figure 8) DPPH

* designates a significant difference from cell alone (P<0 05)

# designates a significant difference from grape seed (P<0.05)

The extracts may work in two ways, firstly as a direct free radical scavenger of the oxidant DPPH, which can reduce the activation of the apoptotic pathway (Miller et ai, 2001), or secondly the extracts can increase the levels of physiological antioxidants in these cells.

When using 5 μM DPPH, Nephehum lappaceum extracts at the concentration tested were able to limit oxidant-induce cell death by apoptosis to a similar capacity as grape seed extract. However, at 50 μM DPPH grape seed extract was seen to provide a more significant protection against apoptosis. The grape seed extract used in this study is from a commercial source and may contain higher concentration of the active compounds compared to the Nephehum lappaceum extracts used here.

Effect of Culturing Murine Splenocytes in the Presence of Nephehum lappaceum extracts

Splenocytes, obtained from BALB/c mice, were cultured in the presence of aqueous and ethanol extracts from the rind of Nephehum lappaceum for either

24, 48 or 72 hours in a humidified 5% CO₂ incubator. At the end of each of the incubation period, cell proliferation was determined and its findings are depicted in Figures 9a and 9b

Figures 9(a) and (b): Proliferation curves for murine splenocytes cells cultured in 125 μg/ml 9(a) aqueous and 9 (b) ethanol extracts of the rind of Nephelium lappaceum

Proliferation observed when the splenocytes were cultured in the presence of concanavalin A was used as positive control while splenocytes cultured in medium without addition of plant extracts served as the negative control. At the concentrations (10, 50, 100 and 125 μg/ml) studied, the extracts did not affect the splenocytes proliferation over a period of 72 hours. Conclusion

The ethanolic extract of the rind of Nephelium lappaceum has a significantly high free radical scavenging activity, comparable to that of Vitamin C and much higher than that of Vitamin E. In addition, it has been shown to have superoxide scavenging ability of 30 % while SOD (superoxide dismutase) itself has a 50 % scavenging ability. The aqueous extract also helps prevent auto-oxidation of linoleic acid by 50%. It is an ideal anti-oxidant as it has no prooxidant activity induced by transition metals, unlike Vitamin C at higher concentrations. The extract with the said radical scavenging activity has a phenolic content in the range of 762-822 mg/g GAE. In addition, when using cultured cells the extract was seen to possess a significant amount of anti-oxidative property and limits cell death by apoptosis. More importantly, the extracts did not show any inhibitory effects on cultured normal mouse fibroblast cells or splenocytes, suggesting that the extract does not contain compounds that are cytotoxic to normal cells.

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Claims

Claims:

1. A method of preparation of Nephelium lappaceum extracts with high free radical scavenging activity comprising the steps of: a) Adding deionised water or solvent at 1 :10 (w/v) concentration to powderised pulp, seed, rind or leaf of Nephelium lappaceum; b) Extracting the powderised pulp, seed, rind or leaf with deionised water at 4O⁰C, or extracting with solvent at room temperature for 24 hours in an orbital shaker; c) Filtering the resulting suspension from (b) using No. 114 Whatman filter paper; and d) Collecting the aqueous or solvent filtrate and concentrating the aqueous filtrate using a freeze dryer or concentrating the solvent filtrate using a rotary evaporator.

2. The method of Claim 1 , wherein the solvent is ethanol.

3. The method of Claim 1, wherein the powderised pulp, seed, rind or leaf of

Nephelium lappaceum are prepared comprising the steps of: a) Washing the pulp, seed, rind or leaf with copious amount of water; b) Repeat the washing of the pulp, seed, rind or leaf with distilled water; c) Drying the washed pulp, seed, rind or leaf from (b) at room temperature; d) Further drying the pulp, seed, rind or leaf from (c) in an oven at 4O⁰C until completely dry; and e) Preparing the powderised pulp, seed, rind or leaf using a Waring blender or milling the pulp, seed rind or leaf using a Fritsch dry miller.

4. Ethanolic extracts prepared according to the method of Claim 1, wherein the extraction yield ranged from 15 - 22%.

5. The rind and leaf ethanolic extracts prepared according to the method of Claim 1, wherein the said extracts have highest scavenging activity on DPPH (l,l-diphenyl-2-picrylhydrazyl) free radicals.

6. Rind ethanolic extracts prepared according to the method of Claim 1, wherein the said extract showed comparable DPPH radical scavenging activity to Vitamin C and far better than α-tocotrienol, tocopherol and ethanolic grape seed extracts.

7. Rind ethanolic extract prepared according to the method of Claim 1, wherein the said extract showed 54% higher galvinoxyl scavenging activity and 78% higher ABTS (2,2-azinobis 3-ethylbenzothiazoline) scavenging activity, compared to Vitamin C.

8. Rind ethanolic extract prepared according to the method of Claim 1, wherein the said extract showed lower pro-oxidant profiles compared to Emblica ' ^M, aqueous grape seed, green tea, α-tocotrienol and the positive control.

9. Rind ethanolic extract prepared according to the method of Claim 1, wherein the said extract showed total phenolic content similar to ethanolic grape seed, i.e. 765 mg GAE/g extract.

10. Rind aqueous and ethanolic extracts prepared according to the method of Claim 1, wherein the said extracts when used at concentration of 100 μg/ml protects mouse fibroblast cells 3T3 from cell death due to apoptosis by 60 - 65%.

11. Rind aqueous and ethanolic extracts prepared according to the method of Claim 1, wherein the said extracts when used at a concentration ranging from 10-125 μg/ml exhibited non-toxicity.

12. Powdered rind prepared according to the method of Claim 2, wherein the said powdered rind showed far lower heavy metal content than permissible levels for nutraceutical or cosmeceutical preparations.

13. The use of powdered pulp, seed, rind or leaf of Nephelium lappaceum prepared according to the method of Claim 2 for preparation of Nephelium lappaceum extracts.

14. The use of Nephelium lappaceum extract prepared according to the method of Claim 1 for nutraceutical and cosmeceutical preparations.

15. The extract of Claim 13, wherein the said extract is prepared from powderised pulp, seed, rind or leaf of Nephelium lappaceum.