PHARMA SCIENCE MONITOR

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1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES PHARMACOGNOSTICAL AND PHYTOCHEMICAL STUDY OF LEAVES OF AEGLE MARMELOS (CORR.) H. Y. Hasni Sayyed 1 *, M. R. Patel 2, J. K. Patil 1, R. A. Ahirrao 1, H. P. Suryawanshi 1 and D. M. Pokal 2 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 Aegle marmelos (Rutaceae) leaves. The plant is sacred in Hindu scripture and reported to possess several claims like hypoglycaemic, antidyslipidemic, immunomodulatory, antiproliferative, wound healing, anti-fertility, and insecticidal. The present study is been designed to investigate detailed morphological and microscopical features of the leaves of the plant. In the present work, other identification parameters are also studied which includes powder characterization and fluorescence analysis of the leaves. In the investigation the leaves are subjected to extraction with different solvent and resulted extracts were evaluated for phytochemical profile and the preliminary phytochemical investigation has revealed presence of resins, lipids, alkaloids, phenolic compounds, flavonoids in different extracts. Keywords: Aegle marmelos, Pharmacognosy, Phytochemistry, Morphology, Microscopy. INTRODUCTION Aegle marmelos (Rutaceae) a sacred plant of Hindu scripture [1], commonly known as Bael, growing wildly throughout deciduous forest of India, ascending to an altitude of 1,200 m in western Himalayas and also occurring in Andaman Islands. The fruits and leaves of the plant are valued in indigenous medicine [2]. The plant is small to medium-sized aromatic tree with deciduous; stem and branches with light brown to green in color. Strong axillary spines are present on the branches. The plant bears characteristic trifoliate Leaves which are offered to lord Shiva. [1] The plant has been employed for long time in folk therapy in Sat-Puda region of Maharashtra by Aadivasi people as anthelmentic [3]. Several pharmacological effects are attached to plants like in management of dyspepsia, ulcers, dysentery or gastro-enterocolitis [4], as antifungal [5], IC Value

2 antibacterial [6], antioxidant [7], antidiarrhoetic [8], pesticidal, antidote, anti-inflammatory [9], anti-spermatogenic [10]. The present investigation is aimed to study pharmacognostic features of Aegle marmelos leaves using macroscopy, microscopic characters, powder microscopy, fluorescence analysis, and Phsico-chemical parameters. Present study also covers extraction of phytoconstituents by successive Soxhlet extraction and identification of primary phytochemical content of the drug using various chemical tests. MATERIAL AND METHODS Collection of Crude drug Aegle marmelos leaves were collected from medicinal garden of campus of P.S.G.V.P Mandal s College of Pharmacy, Shahada, Dist. Nandurbar, Maharashtra, India, 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 of macroscopic and microscopical characters and remaining collected drug was air dried. Finally dried leaves 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, and loss on drying, fluorescence analysis and phytochemical constituents. PHARMACOGNOSTIC EVALUATION: [11, 12] Morphological studies: In morphological studies organoleptic characters were studied like shape, size, colour, odour, taste, etc. of the A. marmelos leaves and details are reported. 11, 12] Microscopic studies-[ Transverse section: The transverse section of leaf through midrib was prepared by standard method. The results of the study were reported along with microscopic photo. Powder analysis: Slides of powdered leaves were prepared by boiling pulverized powder of crude drug with chloral hydrate solution in small quantity. To a little quantity of powder taken IC Value

3 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 magnification. The characteristic structures observed for the powdered leaf was studied and reported FLUORESCENCE ANALYSIS: [13] The fluorescence nature of powder drug with different chemicals was analyzed using shorter wavelength and longer wavelength lights and the observations were reported in table no. 1. PHYSICOCHEMICAL STANDARDIZATION The various Physico-chemical values of leaves such as ash values, extractive values, loss on drying, were determined according to the Pharmacopoeial method. 1. DETERMINATION OF ASH VALUES [14] A) Determination Total Ash Value: Accurately weighed (2 gm) of air-dried leaves powder of Aegle marmelos 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 leaves powder was boiled for 5 minutes with 25 ml of dilute hydrochloric acid and the insoluble matter was collected on an 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 leaves 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 [15] IC Value

4 A) Water-soluble extractive value: Accurately weighed (5 gm) of stem leaves powder of Aegle marmelos 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. 5ml 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) Acetone-Water (70:30) soluble extractive value: Accurately weighed (5 gm) of stem leaves powder of Aegle marmelos was macerated with 100 ml of Acetone-Water (70:30) 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 Acetone-Water (70:30). Evaporated 25ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105 C and weighed. Percentage Acetone-Water (70:30) soluble extractive was calculated with reference to the air-dried drug. C) Alcohol soluble extractive value: Accurately weighed (5 gm) of leaves powder of Aegle marmelos was macerated with 100 ml of methanol 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) Chloroform soluble extractive value: Accurately weighed (5 gm) of leaves powder of Aegle marmelos 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. E) Petroleum ether (40-60 C) soluble extractive value: IC Value

5 Accurately weighed (5 gm) of leaves powder of Aegle marmelos 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 [16] Accurately weighed (2 gm) quantity of leaf 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 in table no. 2. Phytochemical screening: [17] The preliminary phytochemical evaluation of leaves was carried on extract prepared by successive extraction method in Soxhlet. The previously dried powdered leaves (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. alkaloids, carbohydrates, phenolics, flavonoids, proteins, amino acids, saponins, mucilage and resins etc. and the results are in table no. 3. RESULT AND DISCUSSION The macroscopical examination revealed that leaves are green in color, attenuate, trifoliate very rarely five foliate. The shape of the leaves is lenceolate. The size is varying ranging from 7 cm to 13 cm in length and 3 to 6 cm in width. The margin is crenate with acuminate apex and symmetric base with petiole around 2.3 to 6.5 cm long. Surface of leaves is glabrous and shiny showing parallel venation with brittle texture. Leaves bear aromatic odour and characteristic taste. The lamina portion of the T.S of A. marmelos leaf showed the presence of upper and lower epidermis both comprised of round to oval shaped cells. The Epidermis is single layered occasionally interrupted with sunken stomata on both surfaces and over- IC Value

6 lined by a thick layer of cuticle. Both upper and lower epidermal layers bear stomata. Each stoma has two guard cells and two subsidiary cells and they correspond to rubiaceous type. Numbers of stomata are high in upper epidermis compared to lower epidermis and both epidermis shown presence of covering trichomes. Interior to the epidermis is a many layered palisade tissue, which consists of closely, packed oval cell without much intercellular space. The palisade layer is continues in midrib in upper portion while it is discontinued in lower portion. Interior to the palisade spongy parenchyma layer is present which makes the bulk of lamina. Spongy parenchyma also shows presence of cluster crystals of calcium oxalate. The chloroplasts are more abundant in the palisade cells and less in the spongy tissue. Midrib portion shows continuation of the epidermal layer of lamina over midrib. Below the upper epidermis and above the lower epidermis are seen strips of the collenchymas (3-4 layered). Midrib shown the presence of xylem and phloem arranged in an arc. The microscopic photograph of transverse section (Fig-no. 1) had shown the characters. The microscopic examination of leaf material was on dried powder. Character observed was covering trichomes and stomata were present in the sample. The covering trichomes were multicelluler, uniseriate and the stomata were paracytic type. It was found that the powdered leaf showed groups of fibres with calcium oxalate crystals. Calcium oxalate crystals were numerous and mainly of cluster crystal type. Some xylem vessels (pitted vessels) were also visible which were lignified and cells of palisade and spongy parenchyma were also observed. 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 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 IC Value

Upper Epidermis Collenchyma Palisade layer Spongy parenchyma Xylem Lower epidermis Figure 1 Transverse section demonstrating microscopic features of leaves of Aegle marmelos Table No.

7 Upper Epidermis Collenchyma Palisade layer Spongy parenchyma Xylem Lower epidermis Figure 1 Transverse section demonstrating microscopic features of leaves of Aegle marmelos Table No.1 Effect of different chemical reagents on the fluorescence behavior of powder of dried Aegle marmelos leaves Sr. No Treatment Day light UV light 254 nm UV light 366 nm 1. Powder as such Green Dark green Green 2. Powder treated with Light green Dark green Black distilled water 3. Powder treated with Dark green Green Dark green Petroleum ether 4. Powder treated with Dark green Dark green Dark green Chloroform 5. Powder treated with Dark green Radish brown Greenish black conc. HCl 6. Powder treated with HNO 3 Light brown Dark green Dark violet 7. H 2 SO 4 Powder treated with Green Black Blue 8. Powder treated with Yellow Dark yellow Yellow Glacial acetic acid 9. Powder treated with Greenish Greenish black Brown 1N NaOH in water brown 10. Powder treated with Light green Dark green Greenish brown Ammonia 11. Powder treated with Green Radish black Greenish brown Ferric chloride 12. Powder treated with Dark brown Brown Brown Iodine IC Value

8 Table No. 2: Physico-chemical parameters of dried leaves powder of Aegle marmelos Sr. No. Parameter 1. Ash value: A. Total ash value B. Water soluble value C. Acid insoluble value Values (%w/w) Extractive value: A. Pet. Ether soluble extractive value B. Chloroform soluble extractive value C. Alcohol soluble extractive value D. Acetone water (70:30) soluble extractive value E. Water soluble extractive value Loss On Drying 0.63 Table no. 3: Phytochemical profile of crude extract of powdered leaves of Aegle marmelos Constituents Aegle marmelos leaves Extracts tested Petroleum ether Chloroform Alcoholic Aqueous Alkaloids Carbohydrates Phenolic compounds Flavonoids Proteins and amino- acids Saponins Mucilage Resins Lipids / Fats Sterol (- : Absent, + : Present) REFERENCES: 1. Parmar C and Kaushal MK: Aegle marmelos. Wild Fruits 1982; 1: IC Value

9 2. Charakbraty B., Malik C. And Bhatthacharya S: Studies on the effect of green leaves of Aegle marmelos and Piper nigrum on the glucose and cholesterol levels of blood in diabetes mellitus. Indian Med Forum 1960; 9: Maria D-souza: Tribal Medicines. Social center, Ahmednagar; First edition, 1998: Anonymous: The Ayurvedic Pharmacopoeia of India, Govt. of India, Ministry of health and family welfare, Deptt. of AYUSH, New Delhi. First Edition, Vol- I, Part- I: Renu A: Fungitoxicity of leaf extracts of some higher plants against Rhizoctonia solani. Kuehn. Natl. Acd. Sci. Lett 1983; 6: Venkatesan D, Karrunakarn CM, Selva Kumar S and Palani Swamy PT: Identification of Phytochemical Constituents of Aegle marmelos Responsible for Antimicrobial Activity against Selected Pathogenic Organisms, Ethnobotanical Leaflets 2009;13: , 7. Dhalwal K, Shinde VM, Namdeo AG and Mahadik KR: Antioxidant Profile and HPTLCDensitometric Analysis of Umbelliferone and Psoralen in Aegle marmelos. Journal of Pharmaceutical Biology 2008; 46(4): Mazumder R, Bhattacharya S, Mazumder A, Pattnaik AK, Tiwary PM and Chaudhary S: Antidiarrhoeal evaluation of Aegle marmelos (Correa) Linn. root extract. Phytother Res. 2006; 20 (1): Misra KK Chatterjee A and Bose SJ: New Crop Fact Sheet India. Indian Chem. Soc. 1999; 29: Sur TK,. Pandit S and Pramanik T: Antispermatogenic activity of leaves of Aegle marmelos, corr. In albino rats: a preliminary report. Biomed 1990; 19: 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: IC Value

10 15. 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. Hasni Sayyed Hamid Y. IC Value

Pharmacognostical and Phytochemical Studies on Spondia pinnata Leaf Mathew George* and Lincy Joseph

Annals of Plant Sciences ISSN: 2287-688X Original Research Article Pharmacognostical and Phytochemical Studies on Spondia pinnata Leaf Mathew George* and Lincy Joseph Pushpagiri College of Pharmacy, Tiruvalla,