PRELIMINARY PHYTOCHEMICAL SCREENING OF VARIOUS EXTRACTS OF WHOLE OF CALYCOPTERIS FLORIBUNDA LAM. IMIRJ, 1(2) ISSN: 2456-4613

Volume No: 1 Issue 2 June 2017

International Multidisciplinary Innovative Research Journal -An International refereed e-journal - Science Issue ISSN: 2456-4613 IMIRJ, 1(2), 2017 [1-13]

Preliminary phytochemical screening of various extracts of

whole plant of Calycopteris floribunda Lam.

BHUVANESWARI SANTHARAM1, SUBBURAYALU. S1 GANESH.P2 SORNAM.R3AND MURUGALAKSHMIKUMARI.R4

1Department of Biochemistry, KR College of Arts and Science, Kovilpatti, 2Department of Microbiology, Annamalai University, Annamalai Nagar 3Department of Environmental Biotechnology, M.S.University, Alwarkurichi 4Department of Botany, V.V.Vanniaperumal College for Women, Virudhunagar TamilNadu, India.

ABSTRACT Medicinal are a rich source of bioactive phytochemicals or bionutrients. Herbal medicines as the major remedy in traditional system of medicine have been used in medical practices since entiquity.The present study was designed to investigate the presence of various phytochemicals in the three different extracts of the whole plant of Calycopteris floribunda which evokes various therapeutic effect. The whole plant of Calycopteris floribunda were extracted with the appropriate three solvents (Harborne, 1984) and these were evaluated for the qualitative phytochemical analysis and these can be further studied for the novel compounds and their biological activities. The petroleum ether and methanol extracts have much lesser compounds when compared with ethyl acetate extracts. When compared to the three extracts, ethyl acetate extracts have high affinity towards the biological activities. The qualitative phytochemical analysis mainly focused on different chemical compounds which can be useful for the drug discovery and effective medicine improvement from the natural resources. The ethyl acetate extract has more phyto constituents than the remaining solvents ,contains phenols, flavonoids, steroids, tannins, saponins and reducing sugars.Our findings provided evidence that ethyl aceate extracts of the tested plant Calycopteris floribunda contain 1

PRELIMINARY PHYTOCHEMICAL SCREENING OF VARIOUS EXTRACTS OF WHOLE PLANT OF CALYCOPTERIS FLORIBUNDA LAM. IMIRJ, 1(2) ISSN: 2456-4613

medicinally important bioactive compounds and it justifies their use in the traditional medicine for treatment of different diseases.

Key words: Calycopteris floribunda, Phytoconstuients, Whole plant, Phytochemical Qualitative Tests.

Introduction replaced by synthetic drugs (Gibson 1998) The importance of plants is known to us well. The plant kingdom Phytochemicals are not essential treasure house of potential drugs and in nutrients and are required by the human the recent years there has been an body for sustaining life, but have increasing awareness about the important properties to prevent or to importance of medicinal plants. fight some common diseases.They Medicinal plants contain some organic protect plants from disease and damage compounds which provide definite and contribute to the plants color, aroma physiological action on the human body and flavor. In general, the plant and these bioactive substance include chemicals from environmental hazards tannin alkaloids, carbohydrate, such as pollution, stress, drought, UV terpenoids, steroids and flavonoids exposure and pathogenic attack are (Mathai 2000 and Mann 1978) These called as phytochemicals. Natural compounds are known as secondary products are the source of synthetic and plant metabolites and have biological traditional herbal medicine.The properties such as antioxidant activity, medicinal importance of plant due to the antimicrobial effect, modulation of presence of some special substances like detoxificationen enzymes, stimulation of alkaloids, glycosides, tannins, the immune system, decrease of platelet flavonoids, saponins etc., Soxhlet aggregation and modulation of hormone apparatus was used for the organic metabolism and anticancer property. extraction. Solvents used were Herbal medicines have been used for so petroleum ether , ethyl acetate and many years that have lesser side effects methanol. and less priced than the synthetic medicine. The Active compounds of Calycopteris floribunda Lam. many drugs found in plants are (Combertaceae) commonly known as secondary metabolites .(Dobelis 1993 Kokkarai in Hindi, Minnarakoti in and Edoga 2005) For a long period of Tamil, a scadent woody and climbing time medicinal plants or their secondary which is 5-10cm long with slender metabolites have been directly or brown streaked branches with indirectly playing an important role in storing water abundantly. So it is the human society to combat diseases. referred as a life-saver by the forest In the early century of mankind, plant dwellers during summer when streams derived secondary metabolites have dry up, people quench their thirst by been used by humans to treat acute using this plant( Chopra 1956,WHO infections, health disorders and chronic 2001 Orient Longman 1995) The illness. Only during the last 100 years have reported to posses anti-diabetic natural products have been largely activity(Sreenu et al .,2012 The hepato protective activity of various stem and 2

PRELIMINARY PHYTOCHEMICAL SCREENING OF VARIOUS EXTRACTS OF WHOLE PLANT OF CALYCOPTERIS FLORIBUNDA LAM. IMIRJ, 1(2) ISSN: 2456-4613

extracts have been reported( Chinna (76-78ºC) for 24 hrs, then marc was et al .,2010, Thalla et al., 2011) and dried and then it was subjected to even claimed to treat jaundice. methanol extraction (80ºC) for 24 hrs. Calycopterone, Isocalycopterone and 4- The solvent from the extracts was dimethyl-calycopterone showed a wide recovered under reduced pressure using range activity against solid cell rotary evaporator and subjected to lines(Wall et al., 1994) freeze drying in a lyophilizer until dry powder was obtained. As far as our literature survey could Calculation of percentage ascertain, no reports concerning the The percentage yield was phytochemical investigation of the calculated for the extracts and major whole plant of C.floribunda given here. compounds with reference to the crude Therefore we undertook the present material taken using the formula given work to screen qualitatively the various below. extracts of the whole plant of C.floribunda through various methods. The Main objective of the present study was to investigate the active phytoconstituents present in various

extracts Calycopteris floribunda (Lam.) The extract was subjected to

preliminary phytochemical screening for Materials and methods the detection of various plant Collection and identification of constituents present. The term Calycopteris floribunda (Lam.) qualitative analysis refers to the The whole plant of Calycopteris establishing and providing the identity floribunda (Lam.), was collected from, of a substance. The pharmacological Pulliyankudi, Nellai District of Tamil action of crude drugs was determined by nadu, India. Taxonomic identification the nature of their constituents. The was made from Botanical Survey of phytoconstituents are responsible for the Medicinal Plants Unit Siddha, desired therapeutic properties. To Government of India, Palayamkottai. obtain these pharmacological effects, The whole plant of Calycopteris the plant materials itself or extract in a floribunda (Lam.) was dried under suitable solvent or isolated active shade, segregated, pulverized by a constituent may be used. The various mechanical grinder and passed through extracts of Calycopteris floribunda were a 40 mesh sieve. The powdered plant subjected to the following chemical tests materials were stored in an airtight separately for the identification of container. various active constituents (Evans, 1997). Preparation of Various extracts from Calycopteris floribunda (Lam.) Tests for Alkaloids (Evans, 1997) The powdered plant materials a) Dragondroff's test were successfully extracted with A fraction of the extract was treated petroleum ether (40-60 ºC) by hot with Dragondroff's reagent and continuous percolation method in observed for the formation of yellow Soxhlet apparatus (Harborne, 1984). for colored precipitate. 24 hrs. Then the marc was dried and then subjected to ethyl acetate extraction 3

PRELIMINARY PHYTOCHEMICAL SCREENING OF VARIOUS EXTRACTS OF WHOLE PLANT OF CALYCOPTERIS FLORIBUNDA LAM. IMIRJ, 1(2) ISSN: 2456-4613

b) Wagner's test solution and the yellow to orange color A fraction of the extract was treated revealed the presence of glycosides. with Wagner's reagent and observed for c) Borntrager's test the formation of yellow colored A few ml of dilute HC1 was added precipitate. to 1ml of the extract solution. It was c) Mayer's test then boiled, filtered and the filtrate was A fraction of the extract was treated extracted with chloroform. The with Mayer's reagent and observed for chloroform layer was they treated with 1 the formation of yellow colored ml of ammonia. The formation of red precipitate. color showed the presence of d) Hager's test anthraquinone glycosides A fraction of the extract was treated with Hager's reagent and observed for the formation of yellow colored d) Keller Killiani test precipitate. The extract was dissolved in acetic Test for Carbohydrates (Evans, 1997) acid containing traces of ferric chloride a) Molisch test and it was then transferred to a test tube To 2ml of the extract, 1 ml of α- containing sulphuric acid. At the naphthol solution was added, and junction, formation of a reddish brown concentrated sulphuric acid through the color, gradually became blue, confirmed sides of test tube. Purple or reddish the presence of glycosides. violet color at the junction of the two Tests for Phytosterol (Finar, 1986) liquids revealed the presence of a) Libermann Burchard test carbohydrates. 3 ml of test solution was mixed b) Fehling’s test with 3 ml of acetic acid anhydride was To 1ml of the extract, equal heated and then cooled. Few drops of quantities of Fehling's solution A and B concentrated sulphuric acid were added. were added, upon heating formation of a A blue color appeared. brick red precipitate indicated the b) Salkowski test presence of carbohydrates. The extract was dissolved in c) Benedict's test chloroform and equal volume of To 5 ml of Benedict's reagent, concentrate sulphuric acid was added. 1ml of extract solution was added and Formation of bluish red to cherry red boiled for 2 minutes and cooled. color in chloroform layer and green Formation of a red precipitate showed fluorescence in the acid layer the presence of carbohydrates represented the steroid components in Tests of Glycosides (Evans, 1997) the tested extract. a) Legal test Test for Flavonoids (Dey and The extract was dissolved in Harborne, 1987 Evans, 1989 pyridine and sodium nitroprusside a) Shinoda test solution was added to make it alkaline. The dried powder or extract The formation of pink red to red color was treated with 5 ml of 95% ethanol. showed the presence of glycosides. Few drops of concentrated hydrochloric b) Baljet test acid and 0.5 g of magnesium turnings To one ml of the test extract was were added. Development of pink color added with one ml sodium picrate indicates the presence of flavonoids.

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Test for Tannins and phenolic Test for Fixed Oils compounds (Mace, 1963) a) Spot test a) Ferric chloride test A small quantity of extract was One ml of the extract was pressed between two filter papers. Oil added with ferric chloride and observed stains on the filter paper indicated the the formation of a dark blue or greenish presence of fixed oils. black color. b) Saponification test b) Gelatin test (Evans, 1997) One ml of extract was added with A fraction of the extract was few drops of 0.5 N alcoholic potassium treated with 1% gelatin containing 10% hydroxide along with a drop of NaCl and observed for the precipitation. phenolpthalein. The mixture was heated on a water bath for 1-2 hours. The formation of soap or partial Test for proteins and Amino acids neutralization indicated the presence of (Evans, 1997) fixed oils. a) Biuret test One ml of the extract was treated with 1ml of 40% sodium hydroxide RESULTS AND DISCUSSION solution followed by 2 drops of 1% copper sulphate solution. Formation of a Preparation of Extracts and the violet color showed the presence of Calculation of Percentage Yield of proteins. Calycopteris floribunda b) Xanthoprotein test The various extracts of whole One ml of the extract was plant of Calycopteris floribunda treated with 1ml of concentrated nitric screened for the presence of various acid. A white precipitate formed was bioactive phytochemical compounds. boiled and cooled. Then 20% of sodium Specific qualitative tests were hydroxide or ammonia was performed to identify bioactive subsequently added. Formation of compounds of pharmacological orange color indicated the presence of importance through standard methods. aromatic amino acids. The powdered plant materials were c) Lead Acetate test successively extracted with petroleum A fraction of extract was treated ether (40-60°C) by hot continuous with 1 ml of lead acetate. Formation of percolation method in soxhlet apparatus white precipitate indicated the presence for 24hrs. The extracts were stored in of proteins. screw cap vials until further use. The Test for Saponins (Evans, 1997) percentage yields of the whole plant of About 1 ml of methanol Calycopteris floribunda are shown in extract was diluted separately with Table1. Continuous hot percolation distilled water to 20 ml, and shaken in a method of extraction of the whole plant graduated cylinder for 15 minutes. One of Calycopteris floribunda yields5.16% cm layer of foam indicated the presence w/w, 14.52%w/w and 9.70%w/w in of saponins. petroleum ether, ethyl acetate and methanol extracts respectively. The highest yield was obtained in ethyl acetate extract followed by methanol extract.

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The various extracts of Calycopteris et al., 2011) Phytochemicals give plants floribunda screened for the presence of their color, flavour, smell and are part of various bioactive photochemical a plant`s natural defense system and compounds. Specific qualitative tests protect them against herbivorous insects were performed to identify bioactive and vertebrates, fungi, pathogens, and compounds of pharmacologically parasites(Ibrahim et al., 2010 Sherman importance through standard methods. and Billing 1999 These tests were helpful in finding Phenols are very important chemical constituents in the plant plant constituents because of their material that may lead to their scavenging ability on free radicals due quantitative estimation and also in to their hydroxyl groups (Sandhyarani locating the source of pharmacologically et al., 2011)Therefore, the phenolic active chemical compound. The curative content of plants may contribute directly properties of medicinal plants are to their antioxidant action. Literature perhaps due to the presence of various survey has revealed a direct relationship secondary metabolites such as alkaloids, between antioxidant activity and total flavonoids, glycosides, phenols, phenolic content. This study has saponins, phytosterols etc. supported that phenols present Phytosterols, fixed oil and fats were contribute to antioxidant activity. present in the petroleum ether extracts Phenolics are secondary metabolites that of Calycopteris floribunda . The ethyl play a role in the maintenance of the acetate extractrevealed the presence of human body. This study reveals alkaloids, flavonoids, carbohydrates presence of phytoconstituents, such as ,glycosides, phenols, saponins, phenols, flavonoids and tannin in plants, phytosterols, protein, amino acid and indicates the possibility of antioxidant fixed oils.. Alkaloids, carbohydrates activity and this activity will helps in and glycosides, saponins, phenolic preventing a number of diseases through compounds and tannins, protein and free radical scavenging activity. amino acid and flavonoids were found Phenolics compound the principal in the methanol extract of whole plant of antioxidant constituents of natural plant Calycopteris floribunda. From this products are composed of phenolic acid analysis, ethyl acetate extract of and flavonoids (Gulcin et al., 2004) In C.floribunda was found to have more this respect, polyphenolic compounds constituents compared to other two commonly found in plants have been extracts. So, the ethyl acetate extract reported to have multiple biological was ascertained as suitable solvent for effects like anticancer, antiproliferative, the extraction of most of the antimicrobial, wound healing and phytochemicals in this experimental antibacterial activities including plant (Table 1.2). antioxidant activity (Sreeram et al., 2005 Supayarg et al., 2004) The plant The preliminary phytochemical undertaken for this study also has all the screening tests may be useful in the above mentioned activities. detection of the bioactive principles and subsequently may lead to the drug The polyphenolic compound discovery and development. The active like flavonoids commonly found in principles usually remain concentrated plants have been reported to have in the storage organs of the plants (Sony multiple biological effects, including

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antioxidant activity (Brown et al.1998, The various phytochemical Gil et al., 1999, Kahkonen et al., 1999,, compounds detected are known to have Vinson et al., 1995). It is reported that beneficial importance in medicinal polyphenols the secondary plant sciences. The steroids are known to be metabolites play a role in the protection important for their cardiotonic activities of plants against ultraviolet radiation, and also insecticidal and antimicrobial pathogens and herbivores (Harborne and properties. They are also used in Williams, 2000). The flavonoids nutrition, herbal medicine and cosmetics commonly found in this plants (Callow, 1936). Calycopteris floribunda to have several Tannins and flavonoids are biological activities including compounds that act as primary antioxidant properties.The flavonoids antioxidants or free radical scavengers. have been reported to as nature’s Since these compounds were present in biological response modifiers, because the extract it might be responsible for of their inherent ability to modify the the potent antioxidant capacity of body’s reaction to allergies and virus Calycopteris floribunda. The Tannins and their anti-allergic, anti- were reported to exhibit antiviral, inflammatory, anti-microbial and anti- antibacterial and anti-tumour activities. cancer activities (Aiyelaagbe and It was also reported that certain tannins Osamudiamen, 2009) were able to inhibit HIV replication selectively and was also used as diuretic Rajasekaran and Periasamy (Callow,1936).The plant selected for 2012) reported the presence of terpenes, this may have the mentioned activity steroids, tannins and flavonoids in due to its presence of tannins and alcoholic leaf extract of Calycopteris flavonoids. floribunda. In this present study, The secondary metabolites alkaloids, saponins and carbohydrates (phytochemicals) and other chemical were also identified along with the constituents of medicinal plants account above mentioned plant constituents. for their medicinal value.For example, Five bioflavonoids were identified by Saponins have hypotensive and Mayer (2004) from the green leaves cardiodepressant properties ( Olaleye, have been isolated from Calycopteris 2007) In medicine, it is used in floribunda (Mayer 1999; Wall et al., hypercholestrolaemia, hyperglycaemia, 1994, Dey et al., (2003) isolated steroid antioxidant, anticancer, and terpene from the leaves of antiinflammatory, weight loss, etc. It is Calycopteris floribunda. Thalla and also known to have antifungal Pentela (2011) revealed the presence of properties(Haslam,1989). Glycosides alkaloids, carbohydrates, phytosterols, are naturally cardioactive drugs used in glycosides, saponins, tannins and the treatment of congestive heart failure phenolic compounds in hydroalcoholic and cardiac arrhythnica ( Brian et al., leaf extracts of C.floribunda.Secondary 1985)The presence of saponins and metabolites may be used for the glycosides in the extracts might play a preparation of drug in a systematic way role in the cardioprotective potential of which may lead to the cure of many Caycopteris floribunda. ailments in the future (Shanthi and It has been evaluated that Amudha, 2010). phytosterols can reduce cancer risk, by various trials.Various studies suggests

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that phytosterols can play an important improve serum lipid (cholesterol) role in the prevention of several types of profiles and reduce the risk of cancer such as lung, stomach, prostate, cardiovascular disease (Kendall and ovarian and breast cancer (Santans et Jenkins, 2004). Cholesterol in human al., 2013). It is reported that many blood and tissues is derived from the mechanisms by which phytosterols diet as well as endogenous cholesterol show protective effect against cancer synthesis. In contrast, all phytosterols in such as inhibition of the production of human blood and tissues are derived carcinogens, cancer cell growth, from the diet because humans invasion and metastasis and promote cannot synthesize phytosterols(Sudhop apoptosis of cancerous cells (Woyengo et al., 2005) et al., 2009) This plant Calycopteris floribunda might have cancer protective Our results were in accordance effects due to the presence of with the findings who reported ethyl phytosterol in its extracts. Research acetate to be the best solvent for the work done on cholesterol lowering extraction of antioxidant compounds ability of phytosterols at the intestinal (Shah et al., 2015). It would thus mean level (Santans et al., 2013). So this that in this study, the ethyl acetate had plant might possess antihyperlipidemia the highest number of bioactive activity and can be recommended for its compounds. Since the yield of bioactive usage as lipid reducing medicine. The metabolites in a plant extract also varies research article explained in detail the considerably with the method/solvent of potential concern and benefits of extraction (Clark et al., 1997 and phytosterols in prevention of various Marston et al., 1993). It is plausible that diseases (Dhande et al.,2015) In ethyl acetate were generally more potent addition to being rich in fiber and plant than other two extracts probably protein, the diets of our ancestors were because the active principles in the plant also rich in phytosterols—plant-derived dissolved more readily in better sterols that are similar in structure and extracted by the solvent. function to cholesterol. There is increasing evidence that the reintroduction of plant foods providing phytosterols into the modern diet can

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Table 1.1. Percentage yield of the whole plants of Calycopteris floribunda by continuous hot percolation method

Percentage yield Solvent system (%w/w)

Petroleum ether 5.16 Ethyl acetate 14.52 Methanol 9.70

Table 1.2.Phytochemical analysis of various extracts of Calycopteris floribunda Petroleum S.No. Test Ethyl acetate Methanol ether 1. Alkaloids - + +

2. Carbohydrates and glycosides - + + 3. Phytosterols + + -

4. Fixed oil and fats + + - 5. Saponins - + + 6. Phenolic compounds and tannins - + + 7. Protein and Amino Acid - + +

8. Gum and Mucilage - - - 9. Test for flavonoids - + +

+ Present - Absent

Conclusion warranted for isolation and identification of individual phenolic compounds and in vivo In the present study, we have found studies are needed for understanding their that most of the biologically active mechanism of action as an antioxidant prior phytochemicals were present in ethyl acetate to clinical use. In Conclusion , the results extract of the whole plant of Calycopteris obtained herein have been discussed in floribunda .Since the ethyl acetate extract of details to document merits of ethyl acetate of the whole plant of Calycopteris floribunda Calycopteris floribunda as a therapeutic contains more constituents it can be agent which can be used in preventing the considered beneficial for further progress of oxidative stresses may be due to investigation.With the advances in synthetic the presence of large amount of phenolic methodology and the development of more compounds. sophisticated isolation and analytical techniques, all the above mentioned ACKNOWLEDGMENTS phytochemicals present in the extract could be isolated. So it is concluded that the The authors are grateful to the authorities of presence of phytoconstituents make this KR College of Arts & Science, Kovilpatti plant useful for treating different ailments and SB College of Pharmacy Sivakasi, and have a potential of providing useful Tamilnadu, India for providing required drugs of human use. Further studies are facilities. 9

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