Formulation Development of a Nutraceutical product comprising Manilkara  zapota Fruit Pulp

 

Rebello Norma^1,2*, Bhalerao Suhasini²

¹Department of Pharmaceutics, St. John Institute of Pharmacy and Research, St. John Technical Campus,

Vevoor Village, Manor Road, Palghar (E)-401404, District: Palghar, Maharashtra, India.

²Shri Jagdish Prasad Jhabarmal Tibrewala University, Rajasthan.

 

 

ABSTRACT:

The fresh fruit pulp of the plant Manilkara zapota belonging to family Sapotaceae is nutritious. However the pulp if stored at room temperature for 24 hours or more is easily perishable and succumbs degradation and hence in order to stabilize it, an attempt was made to develop a solid nutraceutical product. The ripe fruits of the plant Manilkara zapota were collected from the local garden from the coastal regions of Maharashtra, India, further processed and freeze dried. The product so obtained was subjected to evaluation for its nutritional value such as antioxidant value, elemental analysis etc. as well as for safe consumption using Animal studies. The results revealed that the fruit pulp on freeze drying increased the stability of the nutritional components. The product was found to be safe for oral consumption even upto a dose of 5000 mg/KG of the animal weight. The research thereby concluded that the nutritional fruits of the various regions which if stored at room temperature could be perishable or may not even be stable at refrigeration temperature for longer times can be made available in natural form to the consumers if appropriate stabilization techniques are used rather than using irrelevant and huge concentrations of preservatives. This shall also provide the local gardeners a huge monitory returns and aid in organic farming thereby maintaining the health of the consumers naturally.

 

 

INTRODUCTION:

Centuries back, Hippocrates had put forth a health key, stating, “Let thy food be thy medicines and thy medicines be thy food”. The key holds so truth that for the centuries earlier and further years to come it’s very much applicable. Indian, Egyptian, Sumerian, and Chinese civilization has provided the evidences about the health key put forth by Hippocrates. Ayurveda, the 5000 years old, ancient health science have reported the benefits of fruits for therapeutic purposes. Chyavanprasha, Brahma Rasayana, Phala Ghrita, Arjuna Ksheerapaka, Shatavari Ghrita, Rasona Ksheerapaka are some of the Ayurvedic nutraceuticals.

 

The broad classes of the nutraceutical components include the antioxidants, dietary fibers, polyunsaturated fatty acids,  probiotics and prebiotics. These components have been reported to provide cardioprotective, cancer protective, hepatoprotective, wound healing etc. beneficial effects for years. Modern nutraceutical industries realized the tremendous potential of fruits to heal the ailments, the deficiencies of consumers eating habits and thus began to bloom as the processed fruits were made available ready to the consumers. Later the nutraceutical foods were synonymously called as designer foods, health foods, fortified foods, pharma foods, functional foods and dietary supplements. Nutraceutical market is growing at the rate of 21% per year. Globally this market is blooming and it supplies products which are incorporated in every consumer’s diet in one or the other form. The important reason been that consumers in this fast and paceful life are unable to find time to eat nutritionally and adequate balanced diet, they dwell on fast foods and hence encounter various lifestyle diseases like diabetes, cardiac hypertension, obesity etc. In fact, as per the euromonitor scale, in developing nations, malnutrition related mortality factor is nearly 40 percent. Thus there is a tremendous need for nutraceutical products, to balance the nutritional intake of the individuals.

 

Consumers are aware of the link between eating healthily and staying fit, many struggle to eat the recommended amounts of fruits, vegetables and dietary supplements needed to achieve this. Nutraceutical products readily available in the market thus fill this gap and provide healthy solutions. Nutraceuticals are thus a growing segment of the global consumer market. Traditional Indian fruits like pomegranate, grapes, chickoo as well as exotic superfruits known for their antioxidant properties and concentrated vitamin content including mangosteen, goji, acai and cupuacu packed with high impact health benefits, tropical and ethnic influences, sustainability and niche appeal have interested the consumers at large. Manilkara  zapota L. Royen belonging to the Sapotaceae family is a tropical fruit plant. commonly known as Baramasi (Bengal and Bihar, India); sapodilla plum (India); sapota, tree potato, chikoo (India); chiku (Malaya, India)¹. It is cultivated throughout India (most extensive in coastal India such as Maharashtra, Gujarat, Andhra Pradesh, Madras and Bengal States), though it is native to Mexico and Central America. Manilkara zapota (L.) P. Royen is extensively found to be planted in Virar-Vapi belt, the western region of Maharashtra-Gujarat border, north of Mumbai as a source of its fruits.

 

Literature review reports sapota fruit, commonly called as chikoo fruit to be consumed mainly due to its sweet taste, and nutritive purpose. Analysis of the chemical composition of Manilkara zapota (L.) fruit, sapota juice reveals that it is one of the rich sources of sugars, proteins, ascorbic acid, phenolics, carotenoids and minerals like iron, copper, zinc, calcium and potassium². Fruits are also identified as rich sources of antioxidants and used to overcome tremendous oxidative stress³. Catechin, epicatechin, leucocyanidin, leucodelphinidin, leucopelarginidin, chlorogenic acid, and gallic acid like polyphenols are present in fruits^2,4.  Thus due to its high contents of the nutrient, sapota fruits could be consumed thereby combating the deficiency of the nutrients. However high activity of many oxidative enzymes makes the fruit highly perishable⁵, thus the stability of the fruit pulp if stored at ambient or refrigeration temperature is a challenge. Hence the present research work aims to formulate a stable nutraceutical product using M. zapota fruit pulp. The Nutraceutical formulation of the Manilkara zapota (L.) P. Royen fruits will help to stabilize the product from deterioration and will provide multiple essential nutrients in a single source. The research in this area is beneficial not only to the consumers but also the nutraceutical market in general. This may explain the research objectives of formulating a stable nutraceutical product containing the fruit pulp..

 

MATERIALS AND METHODS:

Procurement of Plant Material:

Unripe and ripe fruits of Manilkara zapota (L). P. Royen plant were obtained from the local garden of St. John Technical Campus, Palghar, Maharashtra, India and further authenticated by a botanist.

 

Preparation and Standardization of M. Zapota Fruit Juice:

Ripe fruits were washed, wiped with cloth. The outer skin was scraped and the seeds were separated. Pulp was grinded using mixer grinder and the finely ground pulp was used for the further process. Standardization procedures determined the pH of the pulp juice, the solid content as well as the ascorbic acid content using standard pharmacopoeial procedures⁶.

 

Formulation and Characterization of the Nutraceutical Product:

The finely ground fruit pulp was freeze dried to convert into a dry powder form. The diluted samples were pre-frozen for 16-18 hours at -55 to -60°C. The frozen samples were lyophilized under vacuum (10-20 milli Torr) for further 16-18 hrs. at ambient temperature (25°C) using Virtis Bench top Lyophilizer, at Bombay College of Pharmacy, Mumbai. The vials were stoppered under vacuum and stored till further use. The freeze dried samples were further subjected to characterization tests like taste, and flow properties⁷ and moisture content using standard procedures.

 

Screening for Nutraceutical Potential:

The nutraceutical potential of the fruit was determined by estimating the elemental analysis, antioxidant value, ascorbic acid content etc.

 

Elemental Analysis :

The freeze dried sample was dispersed in the distilled water to get 5%w/v solution and then filtered through a Whatman filter paper. The filtrate thus obtained was processed as per the requirement of the instrument and directly aspirated into the Inductively Coupled Plasma-Atomic Emission Spectrophotometer and subjected to determine the composition of elements specifically copper, zinc, iron, calcium, potassium and trace metals like lead and arsenic at Indian Institute of Technology, Bombay (IIT Bombay), Powai, Mumbai.

 

 

Antioxidant Value:

The DPPH free radical scavenging activity of the freeze dried product was determined in comparison to the standard ascorbic acid. Briefly the procedure followed is described as follows. The freeze dried product (0.5 gm) was dispersed in tris-HCL buffer (pH 7.4) to get the concentrations in the range of 10-100 µg/ml. The reactant mixture consisted of 1 ml volume of the freeze dried sample and 1 ml volume of DPPH (500 µM in ethanol). The mixture was vigorously shaken and allowed to stand for 30 mins. Absorbance of the resultant solution was measure at 517 nm Shimadzu UV 1800 double beam, UV visible spectrophotometer. Similarly a blank was run using 1 ml volume of tris-HCL buffer. The concentrations of the standard ascorbic acid used to plot the standard curve were in the range of 1-20 µg/ml².

Radical Scavenging Capacity (RSC) in percent was calculated by following equation.

 

                                                            A_blank- A_sample

% Radical Scavenging Capacity = -------------------X100

                                                                 A_blank

Where,

A_{blank =} Absorbance of blank and A_sample= Absorbance of sample.

From the obtained RSC values the IC₅₀ were calculated, which represents the   

Concentration of the scavenging compound that caused 50 % scavenging.

 

Total Reducing Power:

The procedure determines the capacity of an antioxidant to be able to donate an electron and thereby act as a reducing agent. Thus it is also called as total antioxidant power. Briefly 1 ml volume of the sample, 2.5 ml volume of potassium phosphate buffer (200 mM, pH 6.6) and 2.5 ml volume of potassium ferricyanide (30 mM) were mixed and incubated at 50ºC for 20 mins. Further 2.5 ml volume of trichloroacetic acid (600 mM) was added to the reaction mixture and centrifuged at 3000 rpm for 10 mins. The supernatant layer was allowed to mix with 0.5 ml volume of ferric chloride (6 mM) and the absorbance of the resultant mixture was thus measured at 700 nm using Shimadzu UV 1800 double beam, UV visible spectrophotometer. Ascorbic acid a known reducing agent was employed as a reference standard. The assay was performed in triplicates and the mean values with ± SD are reported².

 

Ascorbic Acid Content:

The concentration of the ascorbic acid in the freeze dried product was estimated using a titration method mentioned as per the monograph of Ascorbic Acid IP in Indian Pharmacopoeia 2010. 

 

 

Toxicity evaluation:

Acute oral toxicity:

Acute Oral Toxicity studies were conducted at Bombay College of Pharmacy, Committee for the Purpose of Control and Supervision of Experiment on Animals, CPCSEA registration Details 242/PO/c/2000/CPCSEA registered on 1/08/2000. Ethical clearance for the study was achieved from Institutional Animal ethics Committee of Bombay College of Pharmacy, Kalina, Mumbai for carrying out Animal Experiments prior to the start of the study.  Acute oral toxicity was determined in rats by administering a dose of 5000 mg/Kg. Purified water was used as a control vehicle. The protocol used in the study was as per the as per the guidelines provided by Organization for economic Co-operation and Development⁸. Statistical significance was evaluated by subjecting the data to student’s t - test.  P< 0.05 as statistical significant interpretation was considered.

 

Stability Testing of The Nutraceutical Product :

The freeze dried product was packed in an air tight container and stored at room temperature for 3 months. The Moisture content of the sample, pH of the solution and ascorbic acid content was determined at periodic intervals like 0 day, 1 months and three months respectively and evaluated for its moisture content, pH and Ascorbic acid value.

 

RESULT AND DISCUSSION :

An extensive search on the literature reviews about the plant was conducted. Manilkara zapota (L). P. Royen has been reported to possess various therapeutic utilities as antioxidant, anti-inflammatory, anthelmintic, anti-bacterial against various gram positive and gram negative organisms etc. As the earlier reports have already evaluated M. zapota fruit pulp for antioxidant activity, presence of polyphenols, carotenoids, elements like Iron, copper, calcium potassium etc. and claimed that the fruit pulp is nutritious². Also Mahattanatawee et al.⁹ have reported the total dietary fiber content, antioxidant ascorbic acid content, total soluble phenol content, pectin content, galactouronic acid content etc. for the sapodilla fruit. However it ripens and decays very fast, within 7-8 days at ambient temperature. Hence the nutritive value is lost. Thus attempts were made to delay the ripening process by application of inhibitor 1-methyl cyclopropene (1-MCP). It has been noted that the consumers are nowadays aware of such artificial treatments of the fruits and hence raised the safety issues. Moreover the consumer acceptance of such fruits which are stored for long time in an unnatural method is unhealthy. The nutraceutical potential of the fruit though proved was not reported to be used in formulation development. Hence the present research aimed to overcome the lacunae in this area. 

The ripe fruit pulp was found to exhibit pH 3.9 on dilution with water during its measurement. This indicates that the fruit pulp was acidic hence could comprise the acidic components like tannins to a greater extent. Solid content of the ripe fruit pulp was determined to be 15 % w/w. Considering the overall fruit size, and comparatively less solid content it could be noted that the fruits comprises more of water content.  Ascorbic acid content of fresh pulp was found to be 0.176 % w/w on titration with 0.05 M Iodine solution using starch as an indicator. Ascorbic acid is a very good free radical scavenger hence M. zapota fruits could be presumed to possess antioxidant activity.

 

Drying is the most commonly used unit operation in Pharmaceutical manufacturing having varying applications as aid in preparing of granules, processing of materials, and preparation of powdered extracts. The unit operation can also be used to reduce the bulk and weight of material which would reduce the overall cost of transportation and storage. Other uses include as the aid in preservation of animal and vegetable drugs, where it minimizes the moisture laden material from mold and bacterial growth. Thus drying phenomena offers more stability to the animal and vegetable drugs⁷. Thus in the present research in order to obtain a stable product using M. zapota fruit pulp, drying was considered as a suitable option. This would help to prepare a solid product and would provide greater stability then the liquid moist pulp.

 

Many pharmaceutical products lose their viability in the liquid state and could deteriorate readily if dried in air at normal atmospheric pressure. These materials may be heat sensitive or may readily react with oxygen, hence to render them stable they need to be dehydrated to a solid state. Thus the sample was freeze dried. The dried powder of the M. zapota fruit pulp was tasted by human volunteers and described to be sweet to taste and retains the chiku odor. The freeze dried powder of the M. zapota fruit pulp exhibited a Carr’s index 28.6, a Hausner’s ratio 1.41 and an Angle of repose 42 hence as per the standard scale the flow property was described as poor.

Thus it can be concluded that the freeze dried powder did not possess very free flowing property. This could be due to the presence of mucilaginous mass in the powder. However the flow properties were improved further by addition of 0.5%w/w microcrystalline cellulose. It provided a glidant like effect slightly increased the flow parameters. Flow parameters thus obtained were Carr’s Index (24.2 - Passable, Hausner’s ratio (1.32 - Passable) and Angle of repose (38 - Fair).  Further the moisture content of the freeze dried powder of M. zapota fruit pulp was found to be less than 2%w/w. Thus the final product contains very less moisture, thereby reducing the reactive potential and render stability.

 

Elemental analysis of the freeze dried powder of the fruit pulp juice revealed the composition of elements specifically copper, zinc, iron, calcium, potassium. The copper content in the human body helps in hemoglobin and collagen production, functions of the heart, energy production and absorption of iron. The zinc content in the body is essential in cell reproduction, wound healing, production of sperm and testosterone. The iron content in the body is essential for hemoglobin production, oxygen transport and energy production. The calcium content in the body is essential for maintaining bone and teeth strength, nerve, muscle and glandular function and blood clotting. While the phosphorus in the body helps in phosphorylation process, energy production, bone and teeth strength and formation of genetic material. Considering the diverse and important functions performed by the essential elements it is thus necessary that their supplementation in regular diet is must. A Chiku fruit provides all these essential elements adequately and thus should be consumed regularly. It is very important to note that the trace metals like lead and arsenic were not detected in the sample of the freeze dried pulp provided as their concentration was well below the detection limit i.e less than 0.01 ppm (Table 1).

 

Antioxidant potential of the freeze dried powder of M. zapota fruit pulp was determined using DPPH radical scavenging assay and total reducing power assay. Freeze dried powder of M. zapota fruit pulp exhibited a fairly good DPPH scavenging activity with an IC₅₀ value as 65.54 ± 0.004 µg/ml compared to the standard ascorbic acid with an IC₅₀ value: 12.5 ± 0.004 µg/ml. Further the total reducing power of the freeze dried powder of M. zapota fruit pulp was found to be comparable to the standard ascorbic acid used exhibiting a similar optical density. Thus the test sample also possesses reducing ability (Table 2). The results thus obtained could be due to the constituents like polyphenols, carotenoids etc. as reported by Aradhya et al².

 

 

Table 1. Elemental analysis of the Freeze dried powder

*ND means less than 0.01 ppm, hence not detected.

Table 2. DPPH radical scavenging potential and total reducing power of freeze dried powder of M. zapota fruit pulp.

DPPH Radical Scavenging Potential				Total Reducing Power
Standard Ascorbic acid		M. zapota fruit pulp		Standard Ascorbic acid		M. zapota fruit pulp
Conc. (µg/ml)	% Inhibition	Conc. (µg/ml)	% Inhibition	Conc. (µg/ml)	Optical density*	Conc. (µg/ml)	Optical density*
4	39.90±0.004	10	12.42±0.008	10	0.1893±0.003	10	0.1367±0.001
8	43.79±0.005	20	18.28±0.004	20	0.3581±0.001	20	0.3421±0.002
12	47.87±0.004	30	29.01±0.005	30	0.5244±0.002	30	0.4758±0.002
16	56.36±0.007	40	43.53±0.002	40	0.6421±0.004	40	0.5962±0.005
20	60.92±0.008	60	74.71±0.006	60	0.7521±0.004	60	0.7362±0.003
IC50 value: 12.5±0.004 µg/ml		IC50 value: 65.54±0.004 µg/ml

   * Values are expressed as mean ± SD of three replicate analyses.

 

Ascorbic acid content however was reduced to 26.68 ± 4.5 mg/100 gm of freeze dried sample compared to the fruit pulp. The fruit pulp was obtained from the ripe M. zapota fruit and sent for freeze drying. The sample was freeze dried a week later due to the time required for freezing as part of the drying procedure and subjected to the further procedure of drying. The time duration for which the product was kept in the liquid state before initiation of the drying process thus led to degradation of the ascorbic acid hence the value was found to be reduced post freeze drying. This also proved the instability and degradation produced in the fresh pulp on storage in liquid state hence the research work undertaken to convert the liquid into solid form is apt and useful.

 

Test substance, freeze dried M. zapota fruit powder was appropriately diluted with water and administered 5000 milligrams per kilograms as a single oral dose. None of the rats exhibited abnormalities in gait, fur, eyes, mucous membranes or abnormal behavior. The Body weight is one of the important factors to be considered in Acute Oral toxicity studies as a sign of healthy animal. If the body weight changes significantly, it is considered as a first indicator of the adverse effect exhibited by the test substance frequently. Also at a dose whereby 10% or more reduction in body weight is observed in the animal, is considered toxic. At a particular dose level such reduction in body weight can be also considered as the minimum toxic dose whether it is accompanied by any other toxic effects on the body or as single observation. The food and water consumption was also normal. Changes in body weight and food consumption in both the groups were thus found to be non-significant at the 14^th day compared to the 0 day. Various organs excised after sacrificing the animals were weighed; their organ to body weight ratios were determined and the statistical analysis using unpaired t-test was found to be non-significant of test group compared to the control group.  Statistical analyses using student t test and 95% confidence limit revealed that there was no significant difference between the observed values (Table 3). Hence it was concluded that the test product did not exhibit any gross toxicological signs in Acute Oral Toxicity study as per OECD guidelines, thus can be considered as safe to use in humans even at a high dose of 5000 mg/Kg.

 

Table 3. Comparison of Organ to body weight ratios for Group I and Group II in Acute Oral Toxicity study.

Values are expressed as mean ± SD.

*P<0.05 is considered as statistical significant analyzed by student t- test.

 

In order to assign a quality standard to the product after manufacturing, the stability determination of the product is an important pre-requisite. The freeze dried M. zapota fruit powder was found to be stable when packed in air tight  container during its three months evaluation. The powder did not reveal any significant change in moisture content and pH also the ascorbic acid content was found to be 25.25 ± 2.7 mg/100 gm at the end of three months. Thus the conversion of solid powder from the liquid slurry on freeze drying has offered stability to the M. zapota fruit pulp. This also indicated that the method of freeze drying is a suitable method for drying the fruit pulp. Although the overall cost of freeze drying was bit higher, the bulk of the formulation when subjected to freeze drying technique may reduce the cost of drying step. Thus the product could be used as a nutritional supplement providing very good amounts of antioxidants, vitamin C and at the same time essential elemental load instantly on dispersing the powder in the glass of water or even fluids like milk thus fortifying the milk.

 

CONCLUSIONS:

Dietary phytochemicals are widely used as pharmaceuticals beneficial for human health and other commercial products. Epidemiologic evidence has suggested that certain plant polyphenols, flavonoids, phytoestrogens, terpenoids, phytosterols, fibres, promote good health and help prevent the occurrence of chronic and fatal diseases, like cardiovascular diseases, cancer, diabetes etc¹⁰. The phytoconstituents have been reported to act through various mechanisms like antioxidant, promoting blood flow, maintaining the cholesterol level etc. However the exact mechanism of action for each of the nutritional database needs further research.

 

The Indian nutraceuticals sector is growing at a high pace among other sectors of Indian Food and Pharmaceuticals market and is also assumed and predicted to sustain its high growth in the future years. The burgeoning affluent middle class consumers in the country is becoming health conscious tremendously. Also the awareness about the positive impact of consuming nutraceuticals and dietary supplements is the generation next step among the young Indians. It is a preordained fact that affluence been one of the important causes of lifestyle diseases prevailing, and which nutraceuticals and dietary supplements should definitely address. However, India represents merely 2% of the global nutraceutical market and is way behind in terms of per capita spend of nutraceuticals with just USD 2.5 compared to the global average of USD 21. The nutraceutical market is poised to double in the next five years and could rise five-fold by 2020, as per a recent statistics. It is therefore fitting that the current research focuses on development of a nutraceutical product. The major reason for the global demand for such nutraceutical products is that the consumer desires a nutrient rich product readily made available to them.

 

The nutraceutical formulation so prepared by freeze drying the pulp obtained from the ripe fruits of Manilkara zapota exhibited good nutritional value in terms of elemental and antioxidant ascorbic acid content. The presence of whole fiber in the powder is an added benefit compared to the plain juices of other ready juices available in the market. The freeze drying of the pulp thus led to stabilization of the fruit pulp as revealed from stability studies. The Acute Oral Toxicity of the freeze dried powder also showed no abnormal reactions thus concluding it to be safe for oral consumption. The stability of the fruit pulp was thus enhanced which could retain the nutritional value of the product. Also the product was prepared without any preservative and color. Formulation of the freeze dried powder comprising Manilkara zapota (L). P. Royen fruits as nutraceutical product required very few processing steps hence the large scale manufacturing of such products can be considered to be feasible. Also the products were formulated using very few ingredients. The product possess multifunctional activity with pulp of a single fruit in nutraceutical powder. The product was cost effective and thus has a great commercial potential. The society at large will definitely benefit by this product.

 

ACKNOWLEDGEMENTS :

The authors would like to acknowledge the Chairman, Mr. Albert W. D’souza and Management of Aldel Education Trust, Principal, Dr. Savita Tauro, St. John Institute of Pharmacy and Research, for the financial aid to conduct the research work and their constant unwavering support. We would also like to thank all the faculty members and the non-teaching staff of the Institute for the valuable help rendered.

 

REFERENCES :

1.       Morton J. "Sapodilla". In Julia F. Morton. Fruits of Warm Climates. Florida Flair Books, Miami, FL. 1987:393-398.

2.       Aaradhya S, Anand P, Kulkarni R and Policegoudra S. Chemical composition and anti-oxidant activity of Sapota (Achras sapota Linn.) Fruit. Journal of Food Biochemistry 31; 2007:399-414.

3.       Prasanna T, Lavanya M, Prasanthi G and Kumar P. In vitro antioxidant activities of few seasonal fruits. International Journal of Pharma Research and Review 2 (2); 2012:38-43.

4.       Kennelly E, Ma J, Luo X, Yang H, Ma C, Basile M and Weinstein B. Bioactive Novel polyphenols from the fruit of Manilkara zapota (Sapodilla). Asian Pacific Journal of Tropical Biomedicine 1(6) ;2011: 448-451.

5.       Wangdup B, Pal RK, Sangita S and Jha S. Response of different maturity stages of sapota (Manilkara achras Mill.) cv. Kallipatti to in-package ethylene absorbent. Journal of Food Science and Technology. 48(6); 2011:763-768.

6.       Indian Pharmacopoeia. (2010) Volume II, III. Government of India, Ministry of Health and Family Welfare. Published by The Indian Pharmacopoeia Commission, Ghaziabad.

7.       Khar R, Vyas S, Ahmad F and Jain G. Drying. In Lachman-Lieberman’s The Theory and Practice of Industrial Pharmacy. 4^th Edition, CBS Publishers and Distributors Pvt. Ltd. New Delhi, India, 4;2013:89, 98,104.

8.         OECD. Test No. 429: Acute Oral Toxicity - Fixed Dose Procedure. OECD Guidelines for the testing of Chemicals. OECD Publishing, Paris. 2004:1-13.

9.         Mahattanatawee K, Manthey J, Luzio G, Talcott S, Goodner K and Baldwin E. Total Antioxidant Activity and Fiber Content of Select Florida-Grown Tropical Fruits. Journal of Agricultural and Food Chemistry 4;2006: 7355-7363

10.       Rai-Pandey S, Meena R and Rai S. Medicinal plants derived nutraceuticals: A Re-emerging health aid. International Journal of Pharma and Bio Sciences 2(4); 2011:419-44.

 

 

Received on 30.06.2016       Modified on 17.07.2016

Accepted on 28.07.2016     ©A&V Publications All right reserved