nutritional evaluation, antimicrobial … the value moisture, crude protein, crude fat crude fiber...

Chandra Subhash et al. IRJP 2011, 2 (12), 217-221

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY, 2(12), 2011

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY ISSN 2230 – 8407 Available online www.irjponline.com Research Article

NUTRITIONAL EVALUATION, ANTIMICROBIAL ACTIVITY AND PHYTOCHEMICAL SCREENING

OF WILD EDIBLE FRUIT (CARISSA OPACA) Saklani Sarla1, Chandra Subhash1*, Kandari Singh Alok2

1Department of pharmaceutical Sciences, H.N.B. Garhwal University (A Central University) Srinagar Garhwal, 246174, Uttarakhand, India 2Department of university Science instrumentation centre (USIC), H.N.B. Garhwal University (A Central University) Srinagar Garhwal,

246174, Uttarakhand, India

Article Received on: 17/10/11 Revised on: 21/11/11 Approved for publication: 18/12/11 *E-mail: [email protected] ABSTRACT The aim of the present study was to investigate Nutritional profile, Antimicrobial activity and Phytochemical Screening of Wild edible fruit of Garhwal Himalaya, Carissa opaca. The fruits have been found to rich in nutrients such as crude protein1.3%, carbohydrates17.39%, crude fiber3.4%, ash content1.25% and minerals as calcium, magnesium, potassium and phosphorus (1.0, 8.4, 1.98 and 0.24 mg/100g) respectively. The ethanolic fruit extracts of Carissa opaca showed significant activity 15±1mm, 14±1mm and 13±1mm against Streptococcus pyogenes, Streptococcus aureus and Bacillus cereus against food poisoning bacteria, and phytochemical screening for the presence of glycosides, flavonoids, phenols, resin and tannins. However, alkaloids were present in leaf. This analysis revealed that, the fruits contained higher value of fat, protein, fiber and minerals as compared to the cultivated fruits with apple and 200 gm fruits contain sufficient amount of nutrients, required per day by a person. Consumption of fruits may promote general health and well-being as well as reduce the risk of chronic diseases. Key words: Antibacterial, Antifungal, Nutritional value and Phytochemical screening. INTRODUCTION The medicinal plants are important therapeutic aids for alleviating various ailments of humankind. Herbal medicines have a strong traditional or conceptual base and the potential to be useful as drugs in terms of safety and effectiveness leads for treating different diseases. A number of Indian medicinal plants are used as rejuvenators as well as for treating various disease conditions. They may be tonics, antimalarials, antipyretics, aphrodisiacs, expectorants, hepatoprotectives, diuretics etc. Medicinal plants are a major source of drugs for the treatment of various health disorders especially in India1, 2. The ripe fruits having a sweet-sour taste are eaten, and are also used to make pickles, and twigs are browsed by sheep and goats3. In ethno medicine, Carissa opaca is used against a number diseases and conditions. It is used to cure fever, and eye disorders, and the fruit of the plant mixed with roots of Mimosa pudica is taken as aphrodisiac4, 5. It is used to cure jaundice and hepatitis. A paste of the plant root is used by local people for healing small cuts and wounds. The plant is commonly used as a medicine to kill worm infesting cattle wounds, as fly repellent, as stimulant, and to cure asthma, and its leaves are also used for tanning6, 7. The plant is known to have cardio tonic action while roots can be used as purgative. The plant is also used for the treatment of horn injuries and maggot wounds in animals and the root paste of the plant is applied locally8. Carissa opaca Stapf ex Haines is wild plant having a number of ethno medicinal applications. In the present study, the leaves, and fruits of the plant were subjected to antimicrobial and nutritional investigations. Antimicrobial activity of the ethanolic extracts of the fruits of the plant, determined against 13 strains of gram-positive and gram negative bacteria and fungus, exhibits a considerable broad spectrum efficacy. Analysis of mineral elements present in fruits shows that the plant has good quantities of potassium, magnesium, iron, zinc, copper and chromium. The nutritional value of the fruits was calculated by gm/100gm, and they are a good source of fiber, lipids, protein and carbohydrates . MATERIAL AND METHODS Plant Material The fresh parts of fruit of Carissa opaca was collected from adjoining area of Langasu city (Dist- Chamoli Uttarakhand) in the

month of June - July. The plant was authenticated by botanist Dr. R. D. Guar, Department of Botany; H. N. B. G. U. Srinagar Garhwal. Preparation of plant Extract The plant material was separated into its selected parts (leaf and fruit) air dried ground to moderately fine powder and Soxhlet extracted with increasing polarity solvent (Petroleum ether, chloroform, ethyl acetate, acetone, methanolic, ethanolic and water)9. Each extract was evaporated to dryness under reduce pressure using rotary evaporator. The coarse powder of fruit and leaf was subjected to successive hot continuous extraction with various solvent each time before extracting with next solvent the powdered material will be air dried (weight of crude extract 100gm). The various concentrated extracts were stored in air tight container for further studies. Media Nutrient broth, Nutrient agar, Muller Hinton agar, Malt extract broth and Sabouraud dextrose agar, Alcohol, Hydrochloric acid, alcohol, and sulphuric acid, Distilled water etc all product of Himedia Laboratories Mumbai (India) were used in this study. Bacterial Strains Ten bacterial strains were used namely Escherichia coli, Klebsiella pneumoniae, Enterobacter gergoviae, salmonella entericatyphim, shigella flexneri, Staphyloccus aureus, staphyloccus epidermidis, streptococcus pyogenes, and Bacillus cereus, The bacterial strains were supplied by the Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India (Customer no. 3921). Fungal Strains Three fungal strains were used namely Candida albicans, Aspergillus flavus and Aspergillus parasiticus, The fungal strains were supplied by the Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India. Antibacterial assay The disc diffusion assay methods were used to determine the growth inhibition of bacteria by plant extracts10, 11. Diluted bacterial culture (100μl) was spread over nutrient agar plates with a sterile glass L-rod. 10mg/ml and 50mg/ml of the each extracts were applied to each filter paper disc (Whatman No. 1, 5 mm diam.) and allowed to dry before being placed on the agar plate. Each extract was tested in



triplicate (3 discs/ plate) and the plates were inoculated at 37°C for 24 h. After incubation, the diameter of inhibition zones was measured with a caliper. Antifungal assay The antifungal activity was tested by disc diffusion method12, 13. The Sabouraud dextrose agar plates were each similarly seeded with each fungal strain The 24 hrs. both culture of each bacterium and 7 days inoculated fungus culture were used to seed sterile Sabouraud dextrose agar at 45°C respectively, and fungal plates were incubated at 25-28°C for 7 days after which diameter of zones of inhibition were measured. Each disc filled with extract . Nutritional & Mineral assay The edible portion of fruits was analyzed for moisture, ash, fat14. Fiber as per method reported in AOAC. Total nitrogen was analyzed by microkjeldhal method15. And for crude protein the value was multiplied by 6.25. Total carbohydrates were obtained by subtracting the value moisture, crude protein, crude fat crude fiber and ash from 100%16. The total energy value equal to addition of fat, protein and sugars calorie, each gram of fat give 9 kcal, protein and sugar give 4 kcal energy. The minerals analyzed were Potassium using atomic absorption spectrophotometer, Calcium and Phosphorus by flame photometer. Ascorbic acid in fruits was estimated17. Moisture (%): 76.60 ± 0.25 Ash (%): 1.25 ± 0.05 Total protein (%): 1.30 ± 0.04 Crude fat (%): 0.02 ± 0.15 Crude fibre (%): 3.40 ± 0.01 Soluble carbohydrates (%): 17.39± 0.08 Organic Matter (%): 98.75± 0.12 Ascorbic acid: 3.2± 0.12 N mg/100gm: 0.20 ± 0.07 Ca mg/100gm: 1.0 ± 0.05 Mg mg/100gm: 8.4 ± 0.10 K mg/100gm: 1.98± 0.70 P mg/100gm: 0.24 ± 0.05 Fe mg/100gm: 0.56± 0.03 Phytochemical analysis The qualitative phytochemical properties of the dried powdered sample were determined using standard methods18. RESULT AND DISCUSSION Plants are important source of potentially bioactive constituents for the development of new chemotherapeutic agents. The first step towards this goal is the in vitro antimicrobial activity assay. The results of antibacterial, antifungal, nutritional value and phytochemical screening activity, table 1, 2, 3, and 4, reveals that antibacterial, antifungal, nutritional, and phytochemical screening activity of fruit of Carissa opaca was evaluated against ten bacterial and three fungal pathogenic strains. Antibacterial and antifungal activity Carissa opaca ethanolic fruit extract significant activity 15±1mm, 14±1mm and 13±1mm against Streptococcus pyogenes, Streptococcus aureus and Bacillus cereus against food poisoning bacteria and fungus, the order of the species based on total antibacterial activity is as follows: Streptococcus pyogenes ˃Streptococcus aureus ˃ Bacillus cereus. Nutritional value The level of nutrients such as crude protein1.3%, carbohydrates17.39%, crude fiber3.4%, ash content1.25% and minerals as calcium, magnesium, potassium and phosphorus (1.0, 8.4 & 1.98 and 0.24mg/100g) respectively.

Phytochemical screening The phytochemical screening for the presence of glycosides, flavonoids, phenols, resin and tannins. However, alkaloids were absent. This analysis revealed that, the fruits contained higher value of fat, protein, fiber and minerals as compared to the cultivated fruits with apple and 200 gm fruits contain sufficient amount of nutrients, required per day by a person. CONCLUSION The in vitro antimicrobial studies present Carissa opaca to have considerable efficacy against various pathogenic bacteria. The study provides a scientific basis for the use of the plant as folk medicine. The fruit of the plant is a good source of essential nutrients including minerals, carbohydrates, proteins and lipids. However, more advanced pharmacological and clinical studies would be required to investigate in vivo mechanism of nutraceuticals effects of this important wild plant. ACKNOWLEDGMENT We sincerely acknowledge the financial support granted by UCS&T/R&D/CHEM-16/09/10/6539/1, 06/01/2010 (UCOST) Dehradun to work on a project. The present research paper is the outcome of the same. REFERENCES 1. Gaur R. D. Flora of the District Garhwal North West Himalaya. Transindia: Srinagar, Uttaranchal, 1999. 2. Rai, S. K., Mallavarapu, G. R., Pandey-Rai, S., Srivastava,S., Singh, D., Mishra, R. and Kumar, S. Constituents of the flower oil of Carissa opaca growing in the Aravalli mountain range at New Delhi, Flavour Fragr. J. 21: 304–305, 2006. 3. Joshi, D. Plants of Kumaon, http://www.uttaranchal.org.uk/articles.php, 2010. 4. Ahmad, S. S., Mahmood, F., Dogar, Z., Khan, Z. I., Ahmad, K., Sher, M., Mustafa, I. and Valeem, E. E. Prioritization Of Medicinal Plants Of Margala Hills National Park, Islamabad On The Basis Of Available Information Pak. J. Bot., 2009, 41(5): 2105-2114. 5. Acharya, D. and Rai, M. K. Traditional knowledge for curing various ailments among Gonds and Bharias of Patalkot valley, M.P., India, 2009. 6. Jabeen, A., Khan, M. A., Ahmad, M., Zafar, M. and Ahmad, F. Indigenous uses of economically important flora of Margallah Hills National Park, Islamabad, Pakistan, African Journal of Biotechnology, 2009, 8(5), p. 777. 7. Ravishankar, T., Vedavalli, L., Nambi. A. A., Selvam V. Role of Tribal People in the Conservation and Utilization of Plant Genetic Resources. MS Swaminathan Research Foundation: Chennai, 1991. 8. Dinesh, K., ripathi, H. C., Tandon, S. K, Lal, J., Malik, J. K. Ethnoveterinary phytomedicines used in India and Nepal in the treatment of fractures, wounds and allied disorders: An update. Ind. J. Vet. Surgery, 1997, 18:2, p 65-72. 9. Lin J, Opak War, and Geheeb-Keller M. 1999. Preliminary screening of some traditional Zulu medicinal plants for anti-inflammatory and antimicrobial activities. Journal of Ethnopharmacology, 68: 267–274. 10. Iennette E.H. 1985. Manual of clinical microbiology, 978–987. 4th edition. American Association for Microbiology, Washington. 11. Rosoanaivo and Ratsimanaga Urverg 1993. Biological evaluation of plants with reference to the Malagasy flora. Monograph for the IFs. NAPRECA Workshop on Bioassays. 12. Taylor, R.S.L., N.P. Manandhar, J.B. Hudson and G.H.N. Towers, 1995. Screening of selected medicinal plants of Nepal for antimicrobial activities. J. Ethnopharmacol., 546: 153-159. 13. Espinel Ingroff, A., A. Fothergill, J. Peter, M .G. Rinaldi, and T.J. Walsh. 2002. Testing conditions for determination of minimum fungicidal concentrations of new and established antifungal agents for Aspergillus spp.: NCCLS Collaborative Study. Journal of Clinical Microbiology 40:3204-3208. 14. Iswaran,V., A Laboratory Handbook for Agreeculural Analysis. New Dehli Today and Tomorrow’s Prienters and Publisher, 209-222, 1980. 15. Ward G.M., Chemical Methods of plant Analysis; Canada: Department of Agriculture Publication 1064, 9-20 1962. 16. Negi, Y.S., Rawat,M.S.M.,. Pant-Joshi G., and Badoni, S., Biochemical Investigation of Fruits of Some Common Ficus Species J. Food Science and Technology 25; 582-584, 1992. 17. Jayaraman, J. Laboratory Manual in Biochemistry. New Dehli, India: Wiley Estern Ltd, 56. 18. Kokate .C. K. , Purohit A. P. and Gokhale S. B, Pharmacognosy, Nirali prakashan 33 edition Nov. 2005, P. No. 108-109,



Table 1, Antibacterial activity of ten bacterial strains against Carissa opaca plant fruit extract. Disc size, 5 Mm, Inhibitory zone size ±1 Mm, Mm means (millimetres) and – indicate (NIZ) No inhibitory zone

Figure 1 and 2 Antibacterial and antifungal activity of ten bacterial strains & three fungal strains against Carissa opaca plant fruit extract.

Bacterial Name

Petroleum ether Extract

Chloroform Extract

Ethyl acetate Extract

Acetone Extract

Ethanol Extract

Water Extract

Genus /Species/Subspe.

MTCC (Code)

10 Mg/ ml

50 Mg/ Ml

10 Mg /ml

50 Mg/ml

10 Mg/ml

50 Mg/ ml

10 Mg/ ml

50 Mg/ml

10 Mg/ ml

50 Mg/ml

10 Mg/ml

50 Mg/ml

Bacillus cereus 1272 - - - - 8 9 10 12 10 13 9 11

Escherichia coli 729 - 6 - 8 7 9 - 9 11 12 - 9

Enterobacter gergoviae

621 - - - - 7 9 9 9 9 11 - 8

Klebsiella pneumonia

432 - - - 9 - 9 - 12 9 12 8 10

Salmonella entericatyphim

98 - - - - - 8 - 9 7 10 - 7

Shigella flexneri 1457 - 7 - - 8 10 - 10 9 11 - 8

Staphyloccus aureus

902 - 8 - 8 7 11 11 12 10 14 9 11

Staphyloccus epidermidis

435 - - - - 7 9 - 10 8 12 - 8

Streptococcus pyogenes

1925 - - - 7 - 8 - 10 11 15 9 10

Escherichia coli 443 - 8 -

9 8 9 - 10 9 11 - 9



Table 2, Fungal activity of three fungal strains against Carissa opaca plant fruit extract. Disc size, 5 Mm, Inhibitory zone size ±1 Mm, Mm means (millimetres) and – indicate (NIZ) No inhibitory zone.

Fungal Name

Petroleum ether Extract

Chloroform Extract

Ethyl acetate Extract

Acetone Extract

Ethanol Extract

Water Extract

Genus /Species/Subspe.

MTCC (Code)

10 Mg/ml

50 Mg /ml

10 Mg /ml

50 Mg /ml

10 Mg /ml

50 Mg /ml

10 Mg/m

l

50 Mg/m

l

10 Mg/m

l

50 Mg/m

l

10 Mg/m

l

50 Mg/m

l Candida albicans 3017 - - - - - 6 - 7 - 9 - 7

Aspergillus flavus 2798 - 6 - - - - - - - 9 - 6 Aspergillus parasiticus

2796 - 7 - - - - - 6 - 8 - 8

Table 3, Qualitative estimation of Carissa opaca fruit and leaf phytochemical screening.

Test Carissa opaca fruit Carissa opaca leaf

Carbohydrates/ glycosides (1)Molish test (2)Fehling test

(3)Benedict test

(+) (+) (+)

(-) (-) (+)

Alkaloid (1)Mayer’s test

(2) Dragondroff test

(+) (-)

(+) (+)

Flavonoid

(+) (+)

Saponins

(+) (+)

Tannins (1)Pyrogoll & catechol

(2)Gallic acid

(+) (-)

(+) (-)

Unsaturated sterol/triterpenes (1)Liebermann Burchard test

(2)Salkowiskis test

(+) (+)

(+) (+)

Resin

(+) (-)

Table 5 - Qualitative estimation of Carissa opaca fruit amino acid screening.

Amino acid test Carissa opaca fruit L- Hydroxy proline (+)

DL Serine (-) DL Iso-leucine (+)

DL Valine (+) DL-2-Aminobutyric acid (+)

L-Ornithin (-) L-Cystein hydroxyl (+)

DL-Nor-leucine (-) DL-Tryptopham (+)

DL-Alanine (+) L-Glutamic acid (+)

Glycine (-) L –Proline (+) L- Arginine (+)

DL – Aspartic acid (+) L –Cystein hydroxychloride (+)

L- Histidine (-) L – Leucine (+)

L –Lysine monochloride (+) DL – Methionine (-)

DL – β-Phenyl alanine (+) DL – Threonine (+)

L – Tyrosine (+) 3-C-3-4Dihydroxy phenyl (-)



Figure 3.1; Comparison of per day intake of nutrients by Adults with the nutrients present in the fruits of Carissa opaca

0

2

4

6

8

10

Calcium (gm/day) Magnesium (gm/day)

Pottasium (gm/day) Phosphorus (gm/day)

Adult in take of mineral

Carissa opaca fruit

Figure 3.2, Comparison of per day intake of minerals by Adults with the mineral present in the fruits of Carissa opaca fruit.

Source of support: Nil, Conflict of interest: None Declared

nutritional evaluation, antimicrobial … the value moisture, crude protein, crude fat crude fiber...

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