Vol-3, Issue-4, Suppl-3, Dec 2012 ISSN: Sayyed et al PHARMA SCIENCE MONITOR

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PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES PHARMACOGNOSTICAL AND PHYTOCHEMICAL STUDY OF FRUIT PEEL OF PUNICA GRANATUM LINN H. Y. Hasni Sayyed 1 *, M. R. Patel 2, J. K.

Shah College of Pharmaceutical Education & Research, Modasa, Gujarat, India.

The plant is sacred in religious scripture and reported to possess several claims like hypoglycaemic, antilipidemic, immunomodulatory, antiproliferative, wound healing, anti-fertility, insecticidal

1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES PHARMACOGNOSTICAL AND PHYTOCHEMICAL STUDY OF FRUIT PEEL OF PUNICA GRANATUM LINN H. Y. Hasni Sayyed 1 *, M. R. Patel 2, J. K. Patil 1 1 Research scholar, Shri Jagdishprasad Jhabarmal Tibrewala University, Dist. Jhunjhunu, Rajasthan , India. 2 Department of Pharmaceutics, Shree B. M. Shah College of Pharmaceutical Education & Research, Modasa, Gujarat, India. ABSTRACT The present work is focused to evaluate the Pharmacognostic features and phytochemical profile of the fruit peels of Punica granatum (Punicaceae). The plant is sacred in religious scripture and reported to possess several claims like hypoglycaemic, antilipidemic, immunomodulatory, antiproliferative, wound healing, anti-fertility, insecticidal and many more. The present study is been designed to investigate detailed pharmacognostic feature viz. morphological, microscopical features and fluorescence analysis of fruit peels of the plant. The Punica fruit peels are subjected to extraction with different solvent and resulted extracts were also evaluated for phytochemical profile. The preliminary phytochemical investigation has revealed presence of proteins, fats, carbohydrates, alkaloids, Phenolic compounds, tannins, terpenes, saponins, flavonoids, and cardenolides. Key words: Punica granatum, Pharmacognosy, Phytochemistry, Morphology, Microscopy INTRODUCTION The pomegranate (Punica granatum) is one of the oldest known edible fruits. This fruit is one of the species mentioned in the Bible and Koran and is often associated with fertility. It is native to Persia and cultivated in ancient Egypt, Greece, Italy, Asia (Turkmenistan, Afghanistan, India, China, etc.), North Africa and Mediterranean Europe including Turkey. [1] The plant is deciduous small tree, up to 6 m tall and sometimes spiny. Leaves are opposite, oblong - lanceolate, 7 cm long, entire, glabrous and glossy. Flowers are bisexual, to 4 cm across, solitary or clustered at ends of branchlets. The fruit is technically a berry, it is globose and shiny reddish or yellowish green when mature. It has a persistent calyx opposite the stem end that looks like a little crown. It is filled with IC Value

2 crunchy seeds each of which is encased in a juicy, somewhat acidic pulp that is itself enclosed in a membranous skin. The seeds, juice and pulp are eaten, but the yellowish membrane is too astringent. [2] Punica was reported to possess several health claims as Antioxidant, Anticarcinogenic, Anti-inflammatory, anti-cancer (Prostate), Anti-lipidemic, Anti-hypertensive. Plant also usedin Carotid Artery Stenosis, Dental Conditions, Bacterial Infections and many more [3]. In the Satpuda regions, where present study was carried out, the powder of shade dried peels of mature fruits is used as anthelmentic by local people; the powder is to be taken two times a day, for 3 days. [4] MATERIAL AND METHODS Collection of Crude drug: Punica granatum fruits were collected from medicinal garden of campus of P.S.G.V.P Mandal s College of Pharmacy, Shahada, Dist. Nandurbar, Maharashtra, India, and peels from the fruits were removed manually, which was identified by Taxonomist Dr. S.K. Tayade, Department of Botany, P.S.G.V.P. Mandal s Arts, Commerce and Science College, Shahada, Dist. Nandurbar, Maharashtra. The voucher specimen was deposited in Department of Pharmacognosy and Phytochemistry of the institute for future reference. The collected plant material in fresh condition was used to study the macroscopic and microscopical characters and remaining collected drug was air dried under shade. Finally dried fruit peels were subjected to size reduction to get coarse powder and then passed through sieve no. 40 to get uniform powder. Then uniform powder was subjected for the determination of ash values, extractive values, loss on drying, fluorescence analysis and phytochemical constituents. PHARMACOGNOSTIC EVALUATION: Morphological studies: [5, 6] In morphological studies, Organoleptic characters of Punica granatum fruit peels were studied like shape, size, colour, odour, taste, etc. and details are reported. IC Value

3 Microscopic studies: Transverse section: [5, 6] The transverse section of fruit peel was prepared by standard method. The results of the study were reported along with microscopic photographs 2. Powder analysis: Slides of powdered fruit peel were prepared by boiling pulverized powder of crude drug with chloral hydrate solution in small quantity. To a little quantity of powder taken onto a microscopic slide, 1 2 drops of 0.1% phloroglucinol solution and a drop of concentrated hydrochloric acid were added, mounted in dilute glycerin, covered with a cover slip and observed under microscope with and magnifications. The characteristic structures observed for the powdered peels were studied and reported.(photograph 3) FLUORESCENCE ANALYSIS: [7] The fluorescence nature of powder drug with different chemicals was analyzed using shorter wavelength (254 nm) and longer wavelength lights (365 nm) and the observations were reported in table no. 1. PHYSICAL STANDARDIZATION The various Physical values of peels such as ash values, extractive values, loss on drying, were determined according to the Pharmacopoeial methods and results are reported in table no 2. DETERMINATION OF ASH VALUES [8] A) Determination Total Ash Value: Accurately weighed (2 gm) of air-dried fruit peel powder of Punica granatum was taken in a silica dish and incinerated at temperature not exceeding 450 C until free from carbon. The resultant ash was cooled and weighed. The percentage of ash was calculated with reference to the air-dried drug. B) Determination of Acid Insoluble Ash: The total ash obtained from 2 gm of fruit peel powder was boiled for 5 minutes with 25 ml of dilute hydrochloric acid and the insoluble matter was collected on an IC Value

4 ashless filter paper. It was washed with hot water, ignited and weighed. The percentage of acid insoluble ash was calculated with reference to the air-dried drug. C) Determination of Water Soluble Ash: The total ash obtained from another 2 gm of fruit peel powder was boiled for 5 minutes with 25 ml of water; the insoluble matter was collected on an ashless filter paper, washed with hot water, and ignited for 15 min at a temperature not exceeding 450ºC. The weight of insoluble matter was subtracted from the weight of the ash, the difference in weight represent the water-soluble ash. The percentage of water soluble ash was calculated with reference to the air-dried drug. The results of ash values were given in Table no. 2. EXTRACTIVE VALUES [9] A) Water-soluble extractive value: Accurately weighed (5 gm) of fruit peel powder of Punica granatum was added to 50ml of boiled water at 80 C in a conical flask. It was then shaken well and allowed to stand for 10 minutes so as to cool it and filtered. 5 ml of filtrate was transferred to an evaporating dish, which was 7.5 cm in diameter, the solvent was evaporated on water bath, allowed to dry for 30 minutes, finally dried in an oven for 2 hours at 100 C and residue was weighed. Percentage of water-soluble extractive was calculated with reference to the air-dried drug. B) Alcohol soluble extractive value: Accurately weighed (5 gm) fruit peel powder of Punica granatum was macerated with 100 ml of alcohol in a closed flask, shaking frequently during the first 6 hours and allowed to stand for 18 hours. Thereafter, it was filtered rapidly taking precaution against loss of alcohol. Evaporated 25ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105 C and weighed. Percentage alcohol soluble extractive was calculated with reference to the air-dried drug. C) Chloroform soluble extractive value: IC Value

5 Accurately weighed (5 gm) fruit peel powder of Punica granatum was macerated with 100 ml of chloroform in a closed flask, shaking frequently during the first 6 hours and allowed to stand for 18 hours. Thereafter, it was filtered rapidly taking precaution against loss of Chloroform. Evaporated 25ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105 C and weighed. Percentage chloroform soluble extractive was calculated with reference to the air-dried drug. D) Petroleum ether (40-60 C) soluble extractive value: Accurately weighed (5 gm) fruit peel powder of Punica granatum was macerated with 100 ml of Petroleum ether in a closed flask, shaking frequently during the first 6 hours and allowed to stand for 18 hours. Thereafter, it was filtered rapidly taking precaution against loss of Petroleum ether. Evaporated 25ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105 C and weighed. Percentage Petroleum ether soluble extractive was calculated with reference to the bark. The results of extractive values are given in table no. 2. LOSS ON DRYING [10] Accurately weighed (2 gm) quantity of air dried fruit peel powder was taken in a tarred glass bottle and initial weight was taken. The sample was heated at 105 C in an oven and weighed. This procedure was repeated until a constant weight was obtained. The moisture content of the sample was calculated with reference to air-dried drug and the results are reported in Table no. 2. Similar procedure was also performed on fresh peels and the results are reported in Table No. 2 Phytochemical Screening: [11] The preliminary phytochemical evaluation of fruit peel was carried on extract prepared by successive extraction method in Soxhlet. The previously dried powdered peels (50 gm) were extracted in a Soxhlet apparatus with petroleum ether, chloroform, methanol and water successively. The resultant extracts were evaporated to dryness under vacuum. These extract were subjected to chemical test for different phytoconstituents viz. IC Value

6 alkaloids, carbohydrates, phenolics, flavonoids, proteins, amino acids, saponins, mucilage and resins etc. and the results are in table no. 3. RESULT AND DISCUSSION The pomegranate fruit is of sub-globular shaped and brownish yellow to dull crimson red coloured. The fruit was deemed to be a large berry around 2.5 to 5 inches in diameter crowned with a large, deeply five-toothed, tubular, coriaceous calyx, within which are the remains of the stamens and style. Fruit itself gives rise to three parts: the seeds, having weight 4% without juice. the juice, about 34% of the fruit weight, and the peels contributing 56% fruit weight (weights reported are average value ) The peel of fruit was composed of tough and glossy leathery skin that is variously referred to as the rind, husk, or pericarp and the interior membranous walls and white spongy pith referred as carpels or arils of the fruit which forms locules to hold seeds. As depicted in photograph 1, the peel of the fruit, separated from the seeds, occurs in thin curved fragments, granular and brownish yellow or reddish on the outer surface, uniformly yellowish within, and there exhibiting distinct, shallow, angular depressions left by the seeds; to some fragments the remains of the coriaceous calyx are attached. Thickness of the peel was around 2.5 to 3.0 mm with granular, thick, hard, and coriaceous outer surface, while inner surface was with large cavities divided by thin, membranous dissepiments into a number of cells, each of which is completely filled with numerous seeds. peel breaks with a short granular fracture, has little odour, but a powerfully astringent taste.the intensity of acrid- astringent taste was more promising in arils compared to pericarp. The microscopical transverse section of the peels showed presence of a cuticle layer on the outer surface (pericarps) of pomegranate fruits followed by epidermal cells organized by one layer immediately underneath the cuticle. The epidermis forms the outer most cell layer on the structure of pomegranate fruit peel. Epidermis has showed evenly distributed lenticels which function as stomata were observed on the peel of the pomegranate fruit. The observed lenticels were mostly in the shape of a lens and were IC Value

characterized as extending from loosely lined complementary cell surface. In addition to this transverse section has shown presence of pigment layer followed to epidermis.

7 characterized as extending from loosely lined complementary cell surface. In addition to this transverse section has shown presence of pigment layer followed to epidermis. Below pigment layer transverse section had shown presence of several cell thick spongy parenchyma layers. These Parenchyma cells are in the isodiametric shape. Vascular bundles and sclerenchyma cells were located between parenchyma cells. It was found that protective sclerenchyma cells were densely distributed on the peel of fruit. (photograph 2). The microscopic examination of powdered fruit peel material was performed to detect and established various identifying microscopic characters which will be help full in differentiation of the substitute of the drug supplied in the form of dried powder. The photomicrographs of the identifying features of the plant material are shown in (photograph 3) The air dried plant materials were subjected to florescence analysis using different chemicals and lights and results are depicted in table no. 1. The physicochemical parameters like ash value, extractive value and loss on drying were performed on the powdered drug and values found are depicted in table no. 2. Phytochemical investigation was performed on the extract developed by the successive extraction technique and the presence of chemical in individual extract were reported in the table no. 3. Figure 1 Showing fresh peels of Punica granatum fruits IC Value

8 Epidermis Pigment layer Sclerenchyma cells Spongy paranchyma Figure 2 Showing Transverse section of fresh peels of Punica granatum fruits a. Lignified fibers b. sclerenchyma cells c.epidermis d. spongy paranchyma Figure 3 Showing powder characteristics of peels of Punica granatum. IC Value

9 TABLE 1: EFFECT OF DIFFERENT CHEMICAL REAGENTS ON THE FLUORESCENCE BEHAVIOR OF POWDER OF DRIED FRUIT PEELS OF PUNICA GRANATUM. Sr. No Treatment Day light UV light 254 nm UV light 366 nm 1. Powder as such Brown Dark Brown Green 2. Powder treated with Light brown Dark Brown Dark green distilled water 3. Powder treated with Brown Green Brown Petroleum ether 4. Powder treated with Brown Green Black Chloroform 5. Powder treated with Brown Green Black conc. HCl 6. Powder treated with HNO 3 Orange red Green Black 7. H 2 SO 4 brown Powder treated with Blackish Greenish brown Black 8. Powder treated with Brown Green Black Glacial acetic acid 9. Powder treated with Reddish Brown Greenish black Black 1N NaOH in water 10. Powder treated with black black Black Ammonia 11. Powder treated with Blue Greenish Black Ferric chloride 12. Powder treated with Blackish Brown Black Iodine Brown IC Value

10 TABLE 2: PHYSICO-CHEMICAL PARAMETERS OF DRIED FRUIT PEEL POWDER OF PUNICA GRANATUM Sr. No. Parameter 1. Ash value: A. Total Ash value B. Water soluble value C. Acid insoluble value Extractive value: A. Pet. Ether soluble extractive value 2. B. Chloroform soluble extractive value C. Alcohol soluble extractive value D. Water soluble extractive value Loss On Drying Fresh peels 2. Dried peels Values (%w/w) TABLE 3: PHYTOCHEMICAL PROFILE OF CRUDE EXTRACT OF POWDERED LEAVES OF PUNICA GRANATUM Constituents Punica granatum fruit peel Extracts tested Petroleum ether Chloroform Alcoholic Aqueous Alkaloids Carbohydrates Phenolic compounds Flavonoids Proteins and amino- acids Saponins Mucilage Resins Lipids / Fats Sterol (- : Absent, + : Present) IC Value

11 REFERENCES 1. Salaheddin, ME. and Kader AA: Post-harvest physiology and storage behavior of pomegranate fruits Scientia Horticulturae 1984:24: http: // www. Floridata.com/plant profile/ punica. 3. Julie J: Therapeutic Applications of Pomegranate (Punica granatum L.):A Review Alternative Medicine Review 2008:13 (2): Maria D-souza: Tribal Medicines. Social center, Ahmednagar (India); First edition, 1998: Wallis TE: Text Book of Pharmacognosy. CSB Publishers and Distributors; Fifth Edition 1985: Khandelwal KR: Practical Pharmacognosy, Technique and Experiments. Nirali Prakashan, Ninth Edition, 2002: Woltering EJ. and Van Doon WG: Role of Ethylene in Senescence of Petals- Morphological and Taxonomical Relationships Journal of Experimental Botany 1988: 39: Indian Pharmacopoeia. Controller of Publication, Delhi, Vol. II, 1996: A3: Indian Pharmacopoeia. Controller of Publication, Delhi, Vol. I, 1996: Indian Pharmacopoeia. Controller of Publication, Delhi, Vol. I, 1996: Khandelwal KR: Practical Pharmacognosy, Technique and Experiments. Nirali Prakashan, Ninth Edition, 2002: For Correspondence: Mr. Sayyed Hamid Y. IC Value

CHAPTER Introduction

CHAPTER Introduction 27 CHAPTER 2 2.1 Introduction Pharmacognosy as a subject of pharmaceutical curriculum focused on those natural products employed in the allopathic system of medicine. Coincident with the increasing attractiveness