PHARMACOGNOSTICAL AND PRELIMINARY PHYTOCHEMICAL PROPERTIES OF AEGLE MARMELOS L. CORREA FRUIT PULP

PHARMACOGNOSTICAL AND PRELIMINARY PHYTOCHEMICAL PROPERTIES OF AEGLE MARMELOS L. CORREA FRUIT PULP

1. K. Sujatha ², S. Rajan*¹, M. Gokila ², P. Jency ² and P. Brindha ³

Department of Microbiology, M. R. Government Arts College ¹, Mannargudy, Thiruvarur District, Tamil Nadu, India

Department of Microbiology, Srimad Andavan Arts and Science College ², Thiruvanaikovil, Thiruchirapalli, Tamil Nadu, India

CARISM, SASTRA University ³, Tanjore, Tamil Nadu, India

ABSTRACT

The present study deals with the Pharmacognostical and preliminary phytochemical studies of fruit pulp of Aegle marmelos. Macroscopy, organoleptic characters, microscopic features, physiochemical standardization parameters such as total ash, acid insoluble ash, water soluble ash, alcohol, water and other organic solvent extractives, preliminary phytochemical assay, fluorescence analysis and microbial limit assay were performed using standard textual methods. The Pharmacognostical results showed the following observations that are total ash (3.8%), acid insoluble ash (2.05%), water soluble ash (2.9%), water extractives (12%), alcoholic extractives (10.4%), foaming index (333.3U), and swelling index (3.3%). Fruit pulp reveals the presence of steroids, terpenoids, flavonoids, Saponins, phenolic compounds, lignin, fat and oil, proteins, carbohydrates, amino acids and reducing sugars. These observations would be of immense value in the botanical identification and standardization of drugs in a crude form

Phytochemical Characters, Pharmacognostic Characters

INTRODUCTION: India has a rich heritage of traditional knowledge and is a birth place to several important time-honored systems of health care like Ayurveda, Siddha and Unani. It has been estimated that the proportion of medicinal plants in India (7, 500 of the 17, 000 higher plant species are medicinal plants) is higher than any other country of the world ^{1, 2}. Aegle marmelos L. Correa commonly known as Bael in English and Vilvam in Tamil belonging to the family Rutaceae has been widely used in Indian medicine due to its various medicinal properties.

Although this plant is native to northern India it is also widely found throughout the Indian peninsula and in Ceylon, Burma, Thailand and Indo-China. All parts of this tree such as root, leaf, trunk, fruit and seed are useful in several ailments. The unripe fruit is said to be an excellent remedy for diarrhoea and is especially useful in chronic diarrhoea ^{3, 4, 5}.

The effectiveness of   Aegle marmelos fruit in diarrhoea and dysentery has resulted in its recognition into the British Pharmacopoeia. The present work therefore attempts to report various necessary phytochemical and pharmacognostical standards of Aegle marmelos fruit pulp.

MATERIALS AND METHODS:

Collection and Authentication of Plant Material: The fruit pulp of Aegle marmelos were collected from the herbal garden of Thiuvanai koil temple, Tiruchirappalli and authenticated by Professor Dr. John Britto, Taxonomist, Department of Botany, St. Joseph’s College, Thiruchirapalli, India. After authentication, the fruit pulp were collected by scooping the pulp using sterile scoop and blade, shade dried and then milled into coarse powder by a mechanical grinder.

Organoleptic Evaluation: Organoleptic evaluation refers to evaluation of the formulation by color, odor, taste, texture, etc. The organoleptic characters of the samples were evaluated based on the textual methods ⁶.

Microscopy: Free hand section of the materials were taken using a sharp blade, suitably stained and subjected to microscopic observations. Photomicrographs were taken using Nikon compound microscope attached with digital camera. Photomicrographs were taken at different magnifications depending upon the anatomical details ⁷.

Physicochemical Parameters: The determination of various physicochemical parameters such as total ash, acid insoluble ash, water soluble ash, water soluble extractive value, alcohol soluble extractive value, swelling index, foaming index, foreign matter were analyzed by the methods given in the ayurvedic Pharmacopoeia of India ^{8, 9}.

Fluorescence Analysis: Fruit pulp powder were subjected to analyze fluorescence features under ultra violet light and day light after giving treatment for 48 hours with various chemical and organic solvents like ethanol, 50% sulphuric acid, 10% sodium hydroxide and dilute hydrochloric acid ^10,11.

Qualitative Phytochemical Screening: Freshly prepared Aegle marmelos fruit pulp extracts were tested for the presence of phytochemical constituents using standard methods ^{12, 13}.

Microbial Limit Assay: Dissolved 1gm of powdered plant material in 10mL of distilled water. It was serially diluted using phosphate buffer as diluent. The sample was inoculated in Nutrient agar by pour plate, Rose Bengal agar and SS agar by spread plate techniques for Bacteria, Fungi and Salmonella respectively. For bacteria, the plates were incubated at 37°C for 48 hrs and for fungi; the plates were incubated 25°C for 96 hrs ¹³.

RESULT AND DISCUSSION: Medicinal plants are the backbone of Traditional system of medicines like Ayurveda and Siddha. Good raw material provides better therapeutic values. Pharmacognostical study will contribute in determining characteristic features of standard and raw materials. Standardization of herbal drug is a topic of great concern recently. They are subjected to variability due to its origin from heterogeneous sources. Hence, in the present investigation efforts were made to provide scientific data to determine standards for the plant materials.  The present study on microscopical features and other physicochemical, phytochemical and fluorescence parameters on the Aegle marmelos fruit pulp will help in identifying the correct species of the plant.

Microscopy: Presence of characteristic stone cells, compact parenchyma cells and oil globules. Phloem cells were characteristically dark pinkish color, radially flattened cork cells. Presence of brownish granules, laticiferous ducts were unique. Medullary ray were mostly uniseriate (Figure 1-10).

Macroscopy: Fruits occur as piece about up to 3.95±0.43 cm long and up to 0.21±0.6cm width, cylindrical thick and hard. The fruit of Aegle marmelos were directly plugged from the tree and the fruit was cut into two halves and transverse sections were prepared (Figure 11-14).

FIG. 11: UNRIPE FRUIT; FIG. 12: FRUIT EXPOSED; FIG. 13: FRUIT PULP; FIG. 14:  FRUIT PULP POWDER

Organoleptic characters: The following organoleptic characters were indicated when observing powdered fruit pulp. Good powder of A. marmelos fruit pulp was yellowish orange in color, bitter taste and latex cum aromatic odor and smooth texture (Table 1).

TABLE 1: ORGANOLEPTIC CHARACTERS OF AEGLE MARMELOS FRUIT

Physicochemical characters: The fruit pulp was dried, powdered and analyzed for its physicochemical characters, which were presented in table 2. Total ash content of the powdered fruit pulp was found to be 3.8%, acid insoluble ash 2.05% and water soluble ash 2.9%. Water extractive value was higher (12%) than alcohol extractive value (10.4%).

TABLE 2: PHYSICOCHEMICAL PARAMETERS OF AEGLE MARMELOS FRUIT PULP POWDER

Phytochemical Analysis: The results of preliminary quantitative phytochemical screening of aqueous and alcoholic extracts of Aegle marmelos fruit pulp revealed the presence of multiple polar and non-polar chemical constituents (Table 3). Steroids, terpenoids, flavonoids, phenolic compounds, lignin, fat, inulin, proteins, carbohydrates were present in both extracts. Alkaloids and tannins were present only in alcoholic extracts whereas saponins and cardiac glycosides were present only in aqueous extract. Terpenoids therapeutically exert wide spectrum of biological activities such as antiseptic and antihelmentic ¹⁴. Phenolic compounds are used in the treatment of burns as they precipitate the proteins of exposed tissue to form a protective covering ¹⁵. They are also used as healing agents in inflammation, leucorrhoea, gonorrhoea, burns, piles and as antidote ¹⁶.

TABLE 3: QUALITATIVE PHYTOCHEMICAL ANALYSIS AEGLE MARMELOS FRUIT PULP EXTRACTS

Fluorescence analysis: Magenta to red colored chromophore was produced when the drug powder is treated with Aqueous and alcoholic NaOH, HCl, Benzene. Black coloration was noted after 48 hrs of H₂SO₄ treatment under visible light. These chromatophoric colors under UV and visible light illustrated the nature of raw materials (Table 4). Fruit pulp exhibited characteristic colors when treated with different chemicals that may be due to chromatophores present in the powder. This may help to assess the purity of the drug ¹⁷.

TABLE 4: FLUORESCENCE ANALYSIS OF AEGLE MARMELOS FRUIT PULP AT 24 HOURS

Microbial Limit assay: Total bacterial load available in the fruit powder was within the limits of ayurvedic pharmacopeia of India. Only 7X10² CFU of bacteria and 3X10¹ funguses were isolated per gram of plant powder. Plant powder was free from enteric bacteria like Escherichia coli, Salmonella sp., and Shigella sp.

CONCLUSION: The following Pharmacognostical standards were determined in the present work. Fruits occur as piece about up to 3.95±0.43 cm long and up to 0.21±0.6 cm width, cylindrical thick and hard, presence of characteristic stone cells, compact parenchyma cells and oil globules. Phloem cells were characteristically dark pinkish color, radially flattened cork cells, presence of brownish granules. Laticiferous ducts were unique. Medullary ray were mostly uniseriate. Color of the fruit pulp power was found to be yellowish orange, mildly aromatic odor and bitter taste. The Pharmacognostical results showed the following observations that are total ash (3.8%), acid insoluble ash (2.05%), water soluble ash (2.9%), water extractives (12%), alcoholic extractives (10.4%), foaming index (333.3U), and swelling index (3.3%). Foreign matter was found to be <0.1%. Flavonoids, phenolic compounds and tannins were the major secondary metabolites present in both the extracts. Microorganisms were present within the limits of ayurvedic pharmacopoeia of India.  These observations would be of immense value in the botanical identification and standardization of drugs in a crude form.

REFERENCE:

1. Shiva MP: Inventory of Forestry Resources for Sustainable Management and Biodiversity Conservation. New Delhi; Indus Publishing Company; 1996.
2. Kala CP, Dhyani PP and Sajwan BS: Developing the medicinal plants sector in northern India: challenges and opportunities. Journal of Ethnobiology Ethnomedicine 2006; 2:
3. Chopra R: Indigenous drugs of India. Calcutta; Academic Publishers; 1982.
4. Nadkarni AK: Indian Materia Medica. Volume 1. 3rd edition. Mumbai; Popular Prakashan; 1954.
5. Satyavati GV, Gupta AK and Tandon N: Medicinal Plants of India. Volume 1. New Delhi: Indian Council of Medical Research; 1976.
6. Siddiqui A and Hakim MA: Format for the pharmacopoeial analytical standards of compound formulation, workshop on standardization of Unani drugs, (appendix), 24-25 January. New Delhi: Central Council for Research in Unani Medicine (CCRUM); 1995.
7. Johansen DA: Plant Microtechnique, Mc Graw Hill Book Co, New York; 1940.
8. Wallis TE: Textbook of Pharmacognosy, 5th ed, CBS Publishers and Distributors, New Delhi, India; 2005.
9. Quality Control of Medicinal Plant Materials. (An authorized publication of World Health Organization, Geneva) New Delhi: A.I.T.B.S. Publishers & Distributors India, 2002.
10. Kokate CK: Practical Pharmacognosy, 1st ed, Vallabh Prakashan, New Delhi 1986.
11. Chase CR and Pratt RS: Fluorescence of Powdered Vegetable drugs with particular reference to Development of a System of Identification. Journal of American. Pharmacology Association 1949; 38: 32.
12. Harborne JB. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. Champman and Hall, 3rd ed. New York, USA: 1998.
13. Anonymous: Indian Pharmacopoeia, Vol. I & II. Government of India, Ministry of Health and Family Welfare, the Controller of Publications, Civil Lines, Delhi– 110054, 1996.
14. Riyanto SS and Mustafa AL: Isolation of some chemical compounds from Aegle marmelos. Majalah Farmasi Indonesia 2000; 11(1):17-32.
15. Handa SS, Sharma A and Chakraborti KK: Natural products and plants as liver protecting drugs. Fitoterapia 1986; 57:307-45.
16. Gopiesh Khanna V and Kannabiran K: Larvicidal effect of Aegle marmelos, Gymnema sylvestrre and Eclipta prostrate against   Culex qinquifaciatus mosquito larvae. African Journal of Biotechnology, 2007; 6(3): 307-311.
17. Kokate C K: Practical Pharmacognosy, Vallabh Prakashan, New Delhi, India. 1994.
    

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