Cashew Bhavan, Mundakkal P. O, Kollam , Kerala 3 Dept of Zoology, Fatima Mata National College, Kollam , Kerala

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1 Study of Total phenol, Flavonoids, Tannin contents and phytochemical screening of various crude extracts of Terminalia catappa leaf, stem bark and fruit Rajesh B.R 1, Potty V.P 2 and Sreelekshmy S.G 3 1,2 Dept of Biotechnology, The Cashew Export Promotion Council of India Laboratory & Technical Division, Cashew Bhavan, Mundakkal P. O, Kollam , Kerala 3 Dept of Zoology, Fatima Mata National College, Kollam , Kerala Corresponding Author: Rajesh B.R Abstract In the present study, phytochemical screening, total phenolic content, flavonoid content and tannin content of various extracts of Terminalia catappa L. leaf, stem bark and fruit were determined using spectrophotometric methods. T. catappa L. leaf, stem bark and fruit were extracted using petroleum ether, chloroform, methanol and water. Phytochemical screening of the extracts was evaluated using standard methods. Leaf extracts showed the presence of alkaloids, cardiac glycosides, steroids, terpenoids, saponins, flavonoids, phenols and tannin. The stem bark and fruit extracts showed the presence of cardiac glycosides, flavonoids, phenol, saponins and tannin. Alkaloids are absent in stem bark and fruit extracts. The phytochemical constituents were different in different solvents. Methanolic extract of T. catappa leaf showed the highest tannin and flavonoid content. The total tannin content of leaf extracts ranged from 11.8±0.14 to 50.46±1.1 mg/g of dry weight of extract, expressed as tannic acid equivalents. The total flavonoid concentrations of leaf extracts varied from 17.13±0.78 to 98.86±2.32 mg/g, expressed as Quercetin equivalents. The result indicates that Terminalia catappa contains pharmaceutically important phytochemicals. Key words: Phytochemical screening, Terminalia catappa L, Alkaloids, Total phenolic content, Flavonoids. I. INTRODUCTION Plant-derived natural phytochemicals are the potential source for the treatment and prevention of a number of diseases. Herbal extracts act as a potential pipeline for novel bioactive molecules. The rich biodiversity of medicinal plants make them a treasure house for obtaining novel compounds either themselves as drugs or pilot molecules for invention of new drugs with different mechanism of action. Herbal extracts contain numerous phytochemicals or secondary metabolites that may act individually or in synergy against various diseases [1]. The different plant extracts have different mechanism of action for curing diseases. India is highly rich in all the three levels of biodiversity, namely species diversity, habitat diversity and genetic diversity. India has a wealthy culture of medicinal herbs and spices, which includes more than 2000 species and has an immense geographical area with high promising abilities for Ayurvedic, Siddha, Unani and traditional medicines. But only a very few have been studied pharmacologically and chemically for their potential medicinal importance [2, 3]. Ancient Indian literature incorporates a remarkably expansive definition of medicinal plants to the potential sources of medicinal substances. Diseases that remains most challenging for today s health care system is likely to be more difficult than could be treated by current permutation therapies. Nature has bestowed on us a very rich botanical wealth and a large number of diverse types of plants grow in different parts of the country. Since ancient times, in India, thousands of species are known to have medicinal properties and the use of different organs of medicinal plants to cure specific ailments All rights Reserved Page 291

2 been in vogue. Herbal medicine still occupy about 75-80% of the whole population, mainly in developing countries. Indian almond (Terminalia catappa) also known as Tropical Almond, is among one of the most common trees seen throughout India, Sri Lanka, Malaysia and many other parts of South East Asia. T. catappa is cultivating in some places for its striking features as well as its tasty nut. It is a large tropical tree belonging to leadwood family, combretaceae that grows mainly in the tropical regions of the world. It is commonly used as a shady tree in India and Sri Lanka due to its shape with long horizontal branches and large leaves. The leaves of this tree have been used in folk medicine for treating dermatitis and hepatitis in Asian countries [4]. The extract of the leaves shows antioxidative, anti inflammatory and hepataprotective properties [5]. In recent years there has been an interest in T. catappa due to its medicinal properties, especially in cancer [6] and in prevention of diabetes [7]. Apart from the western medicines available for many of these diseases, many herbal preparations are also used. The traditional herbal medicines are becoming popular throughout the world for many of these diseases. Therefore, the valuable properties of T. catappa deserve more research attention than before to exploit its potential in herbal medicinal preparations. II. MATERIALS AND METHODS 2.1 Phytochemical screening of Terminalia catappa plant parts The leaves, unripe fruits and stem bark of Terminalia catappa was collected locally. They were washed thoroughly with double distilled water to remove impurities and air dried for four days at room temperature. They were then coarsely powdered with the help of a blender. 60 g of the dried powder was extracted with petroleum ether, chloroform, methanol and water using Soxhlet apparatus for 8 hours. After extraction, the petroleum ether, chloroform and methanol extracts were separately condensed by using a rotary evaporator. Aqueous extracts were concentrated using distillation method and dried in a hot air oven. The extracts obtained were stored at 4 o C for further analysis. The petroleum ether, chloroform, methanol and water extracts (1g) was completely dissolved in 100 ml of its own mother solvents and was used for the identification of phytochemicals using the methodology of [8, 9]. 2.2 Total phenol determination The total phenolic content was determined using the method of [10]. To 1 ml of extract added 3ml of distilled water and mixed with 0.5ml of Folin Ciocalteau reagent. After 3 min, added 2ml of 20% sodium carbonate to it and mixed thoroughly. The mixture was kept at boiling water bath for one minute and cooled. The blue colour developed was read at 650 nm using UV/visible spectrophotometer (Perkin Elmer, USA). The total phenolic content was calculated from the standard graph of cathechol and the results were expressed as cathecol equivalent (mg/g). 2.3 Total Flavonoid determination The total flavonoid content was determined using the method of [11]. 0.5 ml of the extract was mixed with 4.5 ml of methanol. To the mixture, 0.1 ml of 10% aluminium chloride and 0.1 ml of 1 mol/l sodium acetate was added. The mixture was kept at room temperature for 30 min and the absorbance was measured at 415 nm using UV/visible spectrophotometer (Perkin Elmer, USA). The total flavonoid content of the extracts was calculated using standard graph of quercetin and the results were expressed as quercetin equivalent (mg/g). 2.4 Total Tannin determination The total tannin content was determined using the method of [12]. 1 ml of the extract was transferred to a 100mL volumetric flask containing 75mL of distilled water. To the mixture, 5mL of Folin-Denis reagent followed by 10mL of sodium carbonate solution was added and diluted to 100mL with distilled water. The mixture was shaken well and kept for 30 min at room temperature. The blue colour developed was read at 700nm using UV/visible spectrophotometer (Perkin All rights Reserved Page 292

3 USA). The total tannin content was calculated using standard graph of tannic acid (0-100µg) and the results were expressed as tannic acid equivalent (mg/g). III. RESULTS 3.1 Phytochemical screening of T. catappa plant parts The results of phytochemical screening of petroleum ether, chloroform, methanol and aqueous extracts of leaves, stem bark and fruit were showed in Table 1. The phytochemicals constituents were different in different solvents. The leaves of T. catappa showed the presence of alkaloids, flavonoids, saponins, terpenoids, steroids, glycosides, phenols and tannins, but phlobatannin was not present in the leaf. Saponins were present only in methanol extract. Alkaloids were present in all the extracts of leaf. The stem bark and fruit extracts showed the presence of flavonoids, saponins, glycosides, terpenoids, steroids, phenols and tannins. Alkaloids were absent in stem bark and fruit extracts. Flavonoids are present in methanol and water extracts of all the plant parts while saponins are present only in methanol extracts. Table 1: Phytochemical screening of leaf, stem bark and fruit extracts of T. catappa Phytochemicals Leaf Stem bark Fruit Petro leum ether Chlor ofor m Meth anol Wat er Petrol eum ether Chlor ofor m Meth anol Wa ter Petrol eum ether Chlor oform Metha nol Alkaloids Saponins Terpenoids Phlobatannins Glycosides Steroids Flavonoids Tannin Phenol Water 3.2 Determination of Total phenol The total phenol contents of different extracts of leaf, stem bark and fruit of T. catappa were determined by using Folin-Ciocalteu method and were expressed in terms of gallic acid equivalents. In leaf, the methanol extract contains the highest amount of phenolic compounds (65.88±0.86mg/g) followed by chloroform (48.62±1.2mg/g), water (38.21±2.1mg/g), and petroleum ether extract (19.44± 0.34mg/g) (Table 2). In stem bark, the phenolic content varied from 28.64±1.03mg/g to 71.41±2.05mg/g equivalent of gallic acid (Table 3). In fruit extracts, methanol extract showed highest amount of phenol (73.77±2.1mg/g) (Table 4). 3.3 Determination of Total flavonoid content Total flavonoid content of T. catappa plant extracts was determined in comparison with standard quercetin and the results were expressed in terms of mg quercetin/g. In leaf, the methanol extract contains the highest amount of flavonoid (98.86±2.32mg/g) followed by water (41.23±0.98mg/g), chloroform (27.99±1.23mg/g) and petroleum ether extract (17.13±0.78mg/g). In stem bark, the highest amount of flavonoid was present in methanol extract (83.03±1.76mg/g) All rights Reserved Page 293

4 lowest amount was present in petroleum ether extract (15.05±0.55mg/g). In fruit, the flavonoid content varied from 15.16±0.76mg/g to 79.58±2.11 mg/g equivalent of quercetin. 3.4 Determination of Total Tannin content Total tannin content of T. catappa plant extracts was determined by its ability to reduce phosphotungstomolybdic acid in alkaline solution. The results were compared with standard tannic acid and were expressed in terms of mg tannic acid/g. Total tannin content was high in methanol extract of leaf (50.46±1.1mg/g). In stem bark, the total tannin content varied from 25.22±0.62 mg/g to 48.05±1.83mg/g equivalent of tannic acid. In T. catappa fruit, the methanol extract contains the highest amount of tannin (48.53±1.87mg/g) followed by water (31.25±1.90mg/g), chloroform (29.90±1.44mg/g) and petroleum ether extract (24.23±0.45mg/g). Table 2. Quantitative Determination of Total phenol, Tannins and Flavonoids in leaf of T. catappa. Leaf Petroleum ether Chloroform Methanol Water Total Phenol (mg/gm Catechol equivalent) Tannin (mg/gm Tannic acid equivalent) Flavonoid (mg/gm Quercetin equivalent) 19.44± ± ± ± ± ± ± ± ± ± ± ±0.98 Table 3. Quantitative Determination of Total phenol, Tannins and Flavonoids in stem bark of T. catappa. Stem bark Petroleum ether Chloroform Methanol Water Total Phenol (mg/gm Catechol equivalent) Tannin (mg/gm Tannic acid equivalent) Flavonoid (mg/gm Quercetin equivalent) 28.64± ± ± ± ± ± ± ± ± ± ± ±1.96 Table 4. Quantitative Determination of Total phenol, Tannins and Flavonoids in fruit of T. catappa. Fruit Petroleum ether Chloroform Methanol Water Total Phenol (mg/gm Catechol equivalent) Tannin (mg/gm Tannic acid equivalent) Flavonoid (mg/gm Quercetin equivalent) 25.74± ± ± ± ± ± ± ± ± ± ±2.11 All rights Reserved Page 294

5 IV. DISCUSSION In the present study, a qualitative phytochemical screening of the various extracts of Terminalia catappa leaf, stem bark and fruit extracts using petroleum ether, chloroform, methanol and water was conducted to detect the presence of various secondary metabolites which may be responsible for the medicinal properties of the plant. The study revealed the presence of various bioactive components such as phenols, tannins, flavonoids, glycosides, saponins and alkaloids in the plant extracts among which phenols, tannins and flavonoids were most prominent. These secondary metabolites were reported to possess biological activities such as antidiabetic, antimicrobial, antioxidant, antiinflammatory, anticarcinogenic, hypoglycemic, activities etc [13]. A study on the phytochemical screening and antibacterial activity of T. catappa leaves by [14] revealed the presence of flavonoids, tannins, saponins and alkaloids. Polyphenols are natural occurred secondary metabolites present in plants that are generally used in defense mechanism against pathogens [15]. They are naturally occurred in fruits, leaves, vegetables and cereals of the plant. Polyphenols are responsible for the bitterness, color, flavor, astringency, odor and oxidative stability in food. Several epidemiological studies strongly recommended the long term consumption of diets rich in plant polyphenols which will offer prevention against the development of various cardiovascular diseases, neurodegenerative diseases, cancer, diabetes and osteoporosis [16, 17]. Flavonoids are water soluble polyphenolic compounds containing 15 carbon atoms. The chemical structure of flavonoids contains two aromatic rings bound together by three carbon atoms to form an oxygenated heterocycle. Based on the difference in the type of heterocycle involved, flavonoids may be classified into six subclasses such as flavonols, flavanones, flavones, anthocyanins, flavanols and isoflavones [18]. According to [9], among the 8000 known phenolic compounds, around 4000 were flavonoids. The previous studies have reported that flavonoids revealed a wide range of biological activities such as anti-inflammatory, antioxidant, antiallergic, and antimicrobial [19, 20]. Studies have shown that flavonoids prevent the oxidation of low-density lipoprotein and thereby prevent the development of atherosclerosis. Tannins are polyphenolic compounds that are present in various parts of plants [21]. They are present mostly in the stem bark of trees which help to prevent the plant cell from bacteria and fungi. Several studies confirmed that the tannins exhibit anti-oxidant, anti-microbial, and antiinflammatory properties [22]. The consumption of tannins rich food will offer a lot of remedial and beneficial effects to human being. The tannin can also be used as drugs to heal the burning injury and on cuts to stop bleeding [23]. Tannins have the ability to form a leather resistance on the exposed tissues and helps in protecting the wounds from being affected further. V. CONCLUSION Phytochemicals found present in the leaf, stem bark and fruit extracts of T. catappa indicates their potential as a source of principles that may supply novel medicines. The results obtained in this study thus suggest that the identified phytochemical compounds may be the bioactive constituents responsible for the efficacy of the plant studied. Hence it could be inferred that the plant extracts could be a source for the industrial manufacture of drugs useful in the chemotherapy of some microbial infection. Further studies are therefore suggested to ascertain their antimicrobial, antioxidant and anti-tumour activities. Furthermore, isolation purification and characterization of the phytochemicals found present will make interesting studies. BIBLIOGRAPHY [1] Srinivasahan, V. and Durairaj, B Antioxidant and free radical scavenging effect of Morinda citrifolia fruit extract. Int J Pharma Pharmaceut Sci., 6(4): [2] Gupta, M. P.; Solis, P. N.; Calderon, A. J.; Guinneau Sinclair, F.; Correa, M. Gladames, C.; Guerra, C.; Espinosa, A.; Alvenda, G.L.; Robles, G. and Olampo, R Medical ethnobotany of the tribes of Bocas del Toro, Panama. Journal of Ethnopharmacology, 96: All rights Reserved Page 295

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