Int.J.Curr.Microbiol.App.Sci (2016) 5(3): 300-308

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 3(2016) pp. 300-308 Journal homepage: http://www.ijcmas.com

Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.503.036

Biological Activities of Different Parts of asoca an Endangered Valuable Medicinal

Ch. Mohan1*, S. Kistamma1, P. Vani2 and A. Narshimha Reddy1

1Department of Botany, Osmania University, Hyderabad-500007, Telangana, 2Department of Botany, Raja Bahadur Venkata Rama Reddy Women's College, Hyderabad, Telangana, India *Corresponding author

ABSTRACT

K eywo rd s This report describes the phytochemical relation of the antibacterial, antioxidant

activity of different explants extract of (Roxb.) De Wilde. In the Saraca asoca, present study preliminary phytochemical analysis of different extracts (bark, Phytochemical and leaves) was conducted, which revealed the presence of Alkaloids, Flavonoids, screening, Glycosides, Saponins, Phenols, Steroids, Tannins and Triterpenoids. Quatitative

Antibacterial estimation of Flavanoids and Phenols was conducted using methanolic, ethanolic activity, and aqueous extracts. Screening for antibacterial activity of extracts of different S. Antioxidant asoca explants were performed against gram positive and gram negative bacteria activity, DPPH. by the disk diffusion method. The activities of the compounds were compared with standard strain for antibacterial properties of the imine base and its solvent extract Article Info evaluated and presenting in indicate that the compounds are active in exhibiting antibacterial role. Methanol bark extract showed maximum DPPH, radical Accepted: scavenging activity which is the measure of antioxidant property at the

15 February 2016 concentration of above compounds at 200 µg/µL-1 follows the order Ascorbic acid

Available Online: > Bark extract of S. asoca while at higher concentration the same order is followed 10 , March 2016 by bark extraction exchanged their position. There present study confirms of photochemical, antibacterial and antioxidant activity of all plant extract of S. asoca.

Introduction

Undoubtingly are a good source of Saraca asoca is a medicinally important and biologically active natural products. While globally vulnerable plant found in investigating the bioactive natural the evergreen forests of India (Thakur et al., compounds, it is essential to have access to 1989). India has often been referred to as the simple biological tests in order to locate medicinal garden of the world and the required activities (Sener et al., 1994). This medicinal plant Saraca asoca has been species is currently listed as a ‘globally regarded as one of the foremost plants vulnerable’ species by the IUCN 2013. utilized from antiquity till date. Saraca (http://www.iucnredlist.org/apps/redlist/deta asoca (Roxb.) De Wilde, is a small ils/34623/0). evergreen , belongs to the 300

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Caesalpiniaceae is commonly known as purified from seed Asoka, Asoka and Haempushpam. It is integument, has been found to agglutinate an evergreen tree which is 9 m in height. human lymphocytes and erythrocytes The are orange yellow in colour and irrespective of the blood group; it causes arranged in dense corymbs. It occurs agglutination of Ehrlich ascites carcinoma throughout India up to an altitude of 750m (EAC)3 cells as well as animal erythrocytes in central and eastern . Leaves are (Cibin et al., 2010).Moreover, chemo- parpinnate 15-20 cm long and the leaflets 6- preventive activity of flavonoid fraction of 12, oblong and rigidly sub-coriaceous. Saraca asoca flower was reported in skin Leaves are narrowly lanceolate, cork like at carcinogenesis (Ghosh et al., 1999). the base and with a shot pestistipules are Larvicidal activity has also been recorded by intra-petiolar and completely united. The using Saraca bark and leaves (Methew et bark is dark brown or grey or almost black al., 2008). Biochemical analyses have with warty surface. Stem bark are rough and shown that leaves of S. asoca contain uneven due to the presence of rounded or carbohydrates, proteins, glycosides, projecting lenticles. Bark channeled, smooth flavonoids, tannins and saponins (Pradhan et with circular lenticles and traversely ridged, al., 2010). Different plant parts of S. asoca sometimes cracked. Fracture splinting provide antibacterial (Nayak et al., 2011) exposing striated surface, a thin whitish and CNS depressant, anti-pyretic, anthelmintic continuous layer is seen beneath the cork and analgesic activities (Nayak et al., 2010 leaver. Flowers are fragrant. Flowers are and Varma et al., 2010). Polygamous apetalous, yellowish orange turning to scarlet, in short laterally placed All the plant parts are considered to contain corymbose, auxillary panicles, bract small, medicinal properties. Leaves of Saraca deciduous and calyx petaloid. Seeds are 4-8, asoca are known to contain carbohydrates, ellipsoid-oblong and compressed (Ali et al., proteins, tannins and saponins and shows 2008 and Jain et al., 1968). antibacterial activity (Pradhan et al., 2010). Barks and Flowers contain glycosides, Useful parts of the plant are barks, leaves, steroids, saponins, carbohydrates and flowers and seed. The earliest chronicled tannins (Pal et al., 1985). The flowers are mention of this tree is in the ayurvedic also regarded as medicinally important plant treatise and later in Charka Samhita (100 part and used as therapeutic agent in A.D.) in which has been treatment of diabetes, cancer and recommended in formulations for the hemorrhagic dysentery, uterine infections as management of gynecological disorders as menorrhagia and other types of uterine anodynes. Ashokarista is a very famous disorders. It is also used in bleeding piles, formulation from the bark of this plant bacillary dysentery. which is available commercially from various reputed companies and used to treat Dried flower buds are reported to have menstrual disorders. Different parts of antibacterial activity. Aqueous suspension of Saraca asoca plant have been attributed Saraca indica flower has antiulcer activity with high medicinal value. Saraca asoca in albino rats (Maruthappan et al., 2010). bark extracts are often used in Leucorrhea Saraca asoca bark and flowers exhibit (Shukla et al., 2008). Flowers have shown antitumour activity against DLA, S-180 and encouraging anti-ulcer activity in albino rats Ehrlich ascites carcinoma tumour cell lines, (Maruthappan et al., 2010). Saracin, a lectin Larvicidal activity has also been recorded

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(Methew et al., 2008). Chemo preventive Preparation of Extracts activity of flavonoid fraction of S. asoca is reported in skin carcinogenesis. During Plant samples (bark, flowers and leaves) ‘ashoka-sasthi’ the flower buds are taken were washed with distilled water and air- orally by women. Though phytoconstituents dried at room temperature for 7-10 days, have been reported earlier in case of leaves then oven-dried at 40 0C to remove the and bark of the plant (Pradhan et al., 2010), residual moisture. The dried plant parts were no detail qualitative phytochemical analysis pulverized and stored in air-tight containers are found for flowers. at 4 0C for future use. 50 g of powdered samples of bark, flowers and leaves were The antimicrobial activity of the stem and extracted with methanol by soxhlation bark of Saraca indica have been evaluated method at 60 to 80 0C. The three filtrates against standard strain of Staphylococcus were separately concentrated in water bath aureus, Escherichia coli, Salmonella at 40 0C and evaporated under reduced typhimurium (Sainath et al., 2009). The pressure. leaves of Saraca indica also evaluated for anthelmintic activity (Nayak et al., 2011), Phytochemical Analysis analgesic and antipyretic activities (Pradhan et al., 2010), CNS depressant activity The four extracts obtained from the (Yadav et al., 2008). The reports of powdered flowers of Saraca asoca were quantitative estimation of different subjected to phytochemical tests to antioxidants of the bark of Saraca asoca are determine the presence of active secondary hardly available. The antioxidant property is metabolites using standard procedures also related to the condition of soil and (Mohan et al., 2014). environment where the plant is grown. So in this investigation we are quantitatively Antibacterial Activity estimate different phytochemicals such as total polyphenols, flavonoids, ascorbic acid The disc diffusion method was used to and tannins and free radical scavenging evaluate the antibacterial activity of the activities of DPPH to evaluate antioxidants synthesized compounds against four properties of the bark of Saraca asoca. In bacterial strains viz; E. coli, P. aeruginosa, the Present study was phytochemical K. pnemone and S. aureus. Each organism analysis, antibacterial activity and was cultured in nutrient broth at 37 0C for 24 antioxidant study of the different parts of h. Then 1 % broth culture containing methanol extracts of Saraca asoca bark, approximately 106 colony forming units flower and leaves was carried out. (CFU/mL) of test strain was added to nutrient agar medium at 45 0C and poured Materials and Methods into sterile petri plates. The medium was allowed to solidify. 5 μL of the test Plant material used various plant parts (bark, compound (40 mg/mL in DMSO) was flowers and leaves) of S. asoca were poured on 4 mm sterile paper discs and collected from the Botanical garden, placed on nutrient agar plates. In each plate Osmania University, Hyderabad. The standard antibacterial drug (ampicillin) and collected plant materials were identified and metal complexes were added. The plates voucher specimens were kept at the were incubated at 370Cfor 24 h and the medicinal plant museum (Herbarium) of the antibacterial activity was determined by Department of Botany. 302

Int.J.Curr.Microbiol.App.Sci (2016) 5(3): 300-308 measuring the diameter of zones showing has all the phytochemicals like flavonoids, complete inhibition (mm) (Mohan et al., glycosides, saponins, phenols, steroids, 2015). tannins and triterpenoids, except bark extract it has all phytochemical constituents except Radical Scavenging Activity alkaloids, ethanol extract of flavoinoids and also glycosides and tannins are absent in The percentage of free radical scavenging aqueous extract of the bark. In flower extract activity is shown in Fig-2. This assay is it has all phytochemical constituents except based on decrease in absorbance value of alkaloids are absent in all extract, DPPH at 517 nm on addition of complex. flavoinoids, glycosids and tannins are absent The experiment involves diluting the in aqueous extract and ethanol extract of the working solution of the plant extracts and flower. In leaves extract it has all the ascorbic acid standard (700, 600, 500, phytochemical constituents except alkaloids 400, 300 and 200 μg/μL−1) in methanol. are absent in all extract, steroids are absent DPPH concentration was kept constant (2 in methanolic extract, triterpinoids are mL, 0.004 %). To this varying concentration absent in ethanolic extract and glycosides, of plant extracts and standard were added. tannins and phenols are absent in aqueous The mixture was shaken vigorously and kept extract (Table-1). Which is similar to the in dark for 30 min at room temperature. reports of (Saha et al., 2012; Nayak et al., Then the absorbance was measured at 517 2011). The plant bark, flower and leaves nm in a spectrophotometer. The whole extracts were quantitatively analyzed for experiment was carried out using Flavonoids and Phenol (Table-2). Whereas, spectroscopic grade methanol solvent at 298 present our study reports of Steroids (Nayak K. The radical scavenging activity has been et al., 2011). Indicated that Steroids were measured by using the following Eq. 1. absent in S. asoca. Several medicinal properties have been attributed to Steroids Suppression ratio (%)=[(A0-Ai)/A0)]X 100% (Ghatak et al., 2014 and Gayathri et al., (1) 2013) but surprisingly, Steroids were found in the present study. Flavonoids and Phenol Where Ai=the absorbance in the presence of are however reported in the present study the ligand or its complexes, A0=the which agrees with the findings of (Ghatak et absorbance in the absence of the ligand or its al., 2015). plant extracts. Anti-Bacterial Studies Results and Discussion The antibacterial screening of the S. asoca Phytochemical Analysis different extracts (bark, flower and leaf) were performed against gram positive (S. The present study contributes valuable aureus) and gram negative bacteria (E.coli, information of bioactive compounds in S. P. aeruginosa and K. pnemone) by the disk asoca. Qualitative analysis of plant different diffusion method. The activities of the extract (bark, flower and leaves) was carried compounds were compared with standard out for Alkaloids, Flavonoids, Glycosides, Ampicillin for antibacterial activity. The Saponins, Phenols, Steroids, Tannins and antibacterial properties of the imine base and Triterpenoids. Methanol, ethanol and its solvent extract evaluated and presenting aqueous extract of bark, flower and leaves in Fig-1, Table-3 and Graph-1,indicted that

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the compounds are active in exhibiting bacteria is flower> bark> leaf and for gram antibacterial role like bark 0.4, 0.2, 0.5; positive bacteria the order is bark> flower> flower 0.2, 0.4, 0.6 and leaf 0.6, 0.2, 0.2 in leaf. However reported in the present study gram negative bacteria and bark 0.6, flower which agrees with the findings of (Nayak et 0.4, leaf 0.3 in gram positive bacteria. The al., 2011; Sainth et al., 2009 and Sujatha et order of activity towards gram negative al., 2013).

Table.1 Table Shows Preliminary Phytochemical Screening of Methanol, Ethanol and Aqueous Extract Saraca asoca

S. Phytoconstituents Plant parts No Bark Flower Leaf Met- Et- Aqueous Met- Et- Aqueous Met- Et- Aqueous OH OH OH OH OH OH 1. Alkaloids -ve -ve -ve -ve -ve -ve -ve -ve -ve 2. Flavonoids +ve -ve +ve +ve -ve +ve +ve +ve +ve 3. Glycosides +ve +ve -ve +ve +ve -ve +ve +ve -ve 4. Saponins +ve +ve +ve +ve +ve +ve +ve +ve +ve 5. Phenols +ve +ve +ve +ve +ve +ve +ve +ve +ve 6. Steroids +ve +ve +ve +ve +ve +ve +ve +ve +ve 7. Tannins +ve +ve -ve +ve +ve -ve +ve +ve -ve 8. Triterpenoids +ve +ve +ve +ve +ve +ve +ve -ve +ve Note:-Et-OH- Ethanol, Met-OH- Methanol and –ve-Absent, +ve-Present

Table.2 Quantitative Analysis of the Methonol Extracts of Saraca Asoca for Estimation of Flavonoids and Phenols

S.No Plant extract Phytochemicals Average Estimated value (mg/gm) (Mean±S.E) 1. Flavonoids Bark 4.72±0.74 Flower 5.22±0.15 Leaf 5.86±0.54 Phenols Bark 115.98±0.67 2. Flower 125.32±0.63 Leaf 146.37±0.64 * Phenols are expressed as Gallic acid equivalent (GAE) and Flavonoids are expressed as Quercetin equivalents (QE) in mg/100 gm

Table.3 Minimum Inhibition Zone (mm) Complexes (µg/ml)

Compound Bacterial inhibition zone (mm) Gram (+) Bacterial inhibition zone (mm) Gram (-) E. coli P. aeruginosa K. pnemone S. aureus 1. Bark 0.4 0.2 0.5 0.6 2. Flower 0.2 0.4 0.6 0.4 3. Leaf 0.6 0.2 0.2 0.3

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Fig.1 Antimicrobial Activity of Bark, Flower and Leaves Extract of S. asoca (a) E.coli, (b) P. aeruginosa, (c) K. pneumoniae (Gram Negative) and (d) S. aureus (Gram Positive) Ampicillin as Positive Control

A B

C D

Fig.2 Radical-Scavenging Activity of the Bark Extract of S. asoca on DPPH Radicals (%)

Graph.1 Minimum Inhibition Zone (mm) Complexes (µg/ml)

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Antioxident Properties quantitatively (Flavonoids and Phenols), methanol extraction is proved to be the best The model of scavenging the stable DPPH solvent system for the all parts S. asoca. radical is a widely used technique to screen Study confirms the antibacterial activity of antioxidant properties by spectrophotometer bark, flower and leaf extract of S. asoca the in a very short time period. When the extract found effective bacterial strain, the reaction between antioxidant molecule and order of activity towards flower antibacterial DPPH radical occurs, it results in decrease activity higher then bark and leaf in gram in absorbance at 517 nm. This is because the negative bacteria, where as bark activity is radical is scavenged by antioxidants through more when compare to flower and leaf in donation of hydrogen to form the reduced gram positive bacteria. The antioxidant form (DPPH-H), and this property is also activity evaluated for the methanolic bark visually noticeable as the color changes extraction of S. asoca has excellent from purple to yellow. The more rapidly the compound activity to eliminate the hydroxyl absorbance decreases, the more potent is the radicals. These observations can lead to the antioxidant compound. In the present study development of new potent antioxidants. the antioxidant activity of bark extract of S. This is valuable information for preparation asoca was evaluated by scavenging stable of drugs in pharmaceutical industry and DPPH radical (Fig:-2). The DPPH radical stress the need for more intensive research scavenging activities were found to be 28.16 since they play a great role in healthcare. % for ascorbic acid, 16.86 % for bark extract (B), at concentration of the 200 µg/µL-1. Acknowledgment Ascorbic acid exhibited higher DPPH scavenging activity than the compound at all We are thankful to Department of Botany, concentrations. At the concentration of 700 Osmania University, Hyderabad for lab µg/µL-1 scavenging activities were found to facilities and valuable suggestions and be 95.08 and 78.12 % for Ascorbic acid and financial assistance is gratefully bark extract of S.asoca respectively. The acknowledged. metal scavenging activity which is the measure of antioxidant property at the References concentration of above compounds at 200 µg/µL-1 follows the order: Ascorbic acid > Ali, M. 2008. Pharmacognosy. CBS Bark extract of S. asoca while at higher Publishers & Distributors, New concentration the same order is followed by Delhi. 668–669. bark extraction exchanged their Arindam, Ghatak, Sidhesh Nair, Ambrish position.However reported in the present Vajpayee, Palak Chaturvedi, study which agrees are similar with their Shardool Samant, Ketki Soley, antioxidative potential (Ghatak et al., 2015; Subhash Kudale, Neetin Desai. 2015. Panchawat et al., 2010). Evaluation of antioxidant activity, total phenolic content, total In conclusion, the Present study involving flavonoids, and LC–MS preliminary phytochemical studies of characterization of Saraca asoca different extract (bark, flower and leaves) (Roxb.) De. Wilde. Int. J. Adv. Res., revealed the presence of all secondary Volume 3, Issue 5: 318–327. metabolites different extract and some Cibin, T.R., Gayathri, D.D, Abraham, A. compounds were also estimated 2010. Chemoprevention of skin

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How to cite this article:

Mohan, Ch., Kistamma, S., Vani, P., Narshimha Reddy, A.2016. Biological Activities of Different Parts of Saraca asoca an Endangered Valuable Medicinal Plant. Int.J.Curr.Microbiol.App.Sci. 5(3): 300-308. doi: http://dx.doi.org/10.20546/ijcmas.2016.503.036

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