journal of pharmacy research 7 (2013) 20e23

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Original Article Evaluation of a parasitic flowering trigona (Wt. & Arn.) Danser for its phytochemical and antioxidant activities

G.K. Puneetha a, M.C. Thriveni a, M. Murali a, G.R. Shivamurthy a, S.R. Niranjana b, H.S. Prakash b, M.P. Sadashiva c, K.N. Amruthesh a,* a Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, b Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India c Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India article info abstract

Article history: Background: Dendrophthoe trigona (Wt. and Arn.) Danser, a hemi-parasitic flowering plant, Received 9 November 2012 growing on Ficus benghalensis was collected from Western Ghats of Karnataka and inves- Accepted 10 January 2013 tigated for its phytochemical constituents in different solvent extracts. Available online 26 February 2013 Methods and results: The preliminary phytochemical investigations in D. trigona revealed the presence of secondary metabolites like reducing compounds, alkaloids, flavonoids, sapo- Keywords: nins, tannins, sterols, tri-terpenes and anthraquinones. A maximum total phenolic content of about 37 mg of GAE in 100 mg in methanolic extract was recorded. The antioxidant and Secondary metabolites reducing power activities showed a concentration dependent increase in methanolic DPPH extract. GAE Conclusion: It is evident in the present study that though D. trigona is known for its dele- Radical scavenging activity terious effects it could be exploited for its antioxidant properties. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

1. Introduction and it is estimated that about 25,000 effective plant- based formulations are being used in traditional treatment Natural products from plants have been the basis of treatment methods. The commercial market value for ayurvedic medi- of various diseases in plants and animals. Since time imme- cines is estimated to be expanding at 20% annually.3 morial, man has been using plant parts in the treatment of The medicinal value of plants lies in naturally occurring various ailments.1 Herbal products have been used to treat phytochemical constituents that produce a definite physio- a wide range of human diseases because of their richness in logical action on the human body.4 The secondary metabolites bioactive compounds.2 These bioactive compounds are cur- like alkaloids, tannins, saponins, flavonoids, phenolic com- rently in demand and their recognition in medicine is pounds, glycosides, tri-terpenes etc. present in plants are of increasing day by day due to toxicity and side effects of allo- great pharmaceutical interest. Though the secondary metab- pathic medicines. India has a vast repository of medicinal olites have significant biological role including antioxidant,

* Corresponding author. Tel.: þ91 (0) 8212419760; fax: þ91 (0) 8212419759. E-mail addresses: [email protected], [email protected] (K.N. Amruthesh). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.01.020 journal of pharmacy research 7 (2013) 20e23 21

anti-inflammatory and anti-cholinesterase effects,5 but their flavonoids and saponins using the method described by Har- definite active constituents of many crude drugs are still un- borne19 and Trease and Evans.20 known. Thus, it is anticipated that phytochemicals with adequate biological activities will be used for the treatment of 2.4. Determination of total phenolics microbial infections.6 Antioxidants derived from plants are 7 important in controlling the effects of oxidative damage, Total phenolics in methanol extract were determined by the 8 prevention of inflammatory conditions, ageing and neuro- method of Singleton et al.21 20 mL of extract (5 mg/mL) was 9 degenerative diseases. Phenolic components such as flavo- mixed with 0.75 mL of 20% sodium carbonate solution and 10 11 noids and phenolic acids are responsible for antioxidative 0.25 mL of FolineCiocalteau reagent and incubated. After in- effect. There is a great scientific interest in secondary me- cubation, the absorbance was measured at 765 nm using tabolites produced from plants, due to the increasing devel- UVeVisible spectrophotometer. Total phenolics were quanti- opment of resistance against commonly used antibiotics fied by calibration curve (obtained from known concentra- which has led to a major medical problem and challenge tions of Gallic acid standard) and the concentrations were 12 worldwide, leading to a big threat to human community. expressed as mg of Gallic Acid Equivalents (GAE) per mL and all Dendrophthoe sp. is an important medicinal plant belonging to the determinations were performed in triplicates. thefamilyLoranthaceae.Itisan evergreen,shrubby,partial stem parasite mainly found in tropical and sub-tropical regions of the 2.5. Antioxidant activity by DPPH method world. There are about thirty species of Dendrophthoe and seven 13 species are found in India. It has been used in traditional The free radical scavenging capacity of the methanolic extract medicine and found to have , antidiabetic, anti- of the plant was determined by DPPH (2, 2-diphenyl-1-pic- oxidant, anticancer, antilithiatic, hypertensive and antiviral rylhydrazyl) method.22 The reaction mixture contained 5 mLof 14 properties. Among different species, D. falcata is largely studied plant extract and 95 mL of DPPH (300 mM) in methanol. Differ- and is used to control a wide variety of diseases such as skin ent concentrations (100e1000 mg/mL) of test sample and disorder, pulmonary tuberculosis, psychic disorders, asthma, ascorbic acid (control) were prepared and the reaction mix- 15 16 paralysis, ulcers, menstrual disorders and wounds. They are tures were incubated at 37 C for 30 min and absorbance was used as health food for enhancing immunity and used as pain measured at 517 nm. The experiment was repeated thrice and 17 reliever, aphrodisiac, narcotic and diuretic. per cent RSA was calculated using the formula: Hence, the present study has been undertaken to deter- mine the preliminary phytochemical constituents of the leaf Absorbanceof controlAbsorbanceof sample RSA% ¼ 100 extracts, antioxidant and reducing power ability of D. trigona. Absorbanceof control

2.6. Reducing power assay

2. Materials and methods Reducing power assay was carried out as described by Nagu- lendran et al.23 with slight modifications. 0.75 mL of meth- 2.1. Collection of plant material anolic extract (1 mg/mL) was mixed with 0.75 mL of 0.2 M phosphate buffer (pH 6.6) and 0.75 mL of 1% potassium ferri- D. trigona Ficus The fresh plant material (leaves) of growing on cyanide and incubated at 50 C for 20 min. After incubation, benghalensis (Moraceae) was collected from Western Ghats of 0.75 mL of 10% trichloroacetic acid was added to the mixture Karnataka, India. The plant was identified with the help of Flora and centrifuged for 10 min at 3000 rpm. To the supernatant of Presidency of Madras18 and a voucher specimen is deposited (1.5 mL), 1.5 mL of distilled water and 0.5 mL of 0.1% FeCl3 was in the Herbarium, Department of Studies in Botany, University added and the absorbance was measured at 700 nm using of Mysore, Manasagangotri, Mysore, Karnataka, India. phosphate buffer as blank and butylated hydroxyl toluene (BHT) as standard. 2.2. Preparation of the extracts 2.7. Statistics The leaves of D. trigona were washed under running tap water; shade dried and powdered using wearing blender. 50 g of dried The values are mean SD of triplicate determinations. The leaf powder was filled in the thimble and successfully data were analysed by ANOVA followed by Tukey’s HSD test extracted with petroleum ether, chloroform, methanol, etha- for significant differences using SPSS 11.0 computer software. nol and distilled water using Soxhlet extractor. All the extracts IC50 values were calculated by Boltzmann’s dose response collected were concentrated using rotary flash evaporator and analysis using Origin 6.1 computer software. stored at 4 C in air tight vials and used for further studies.19

2.3. Phytochemical screening 3. Results

The collected plant extracts were subjected to qualitative 3.1. Phytochemical screening phytochemical screening for identification of various classes of active chemical constituents like reducing compounds, The sequential extraction methods followed for phytochem- sterols, tri-terpenes, tannins, anthraquinones, alkaloids, ical screening in D. trigona revealed the presence of reducing 22 journal of pharmacy research 7 (2013) 20e23

compounds in all the solvent extracts tested. Saponins, tan- 3.0 nins, sterols and flavonoids were present in methanol, etha- BHT D. trigona extract nol and aqueous extracts but absent in petroleum ether and 2.5 chloroform extracts. Alkaloids and anthraquinones were present in methanol extract and tri-terpenes in petroleum ether and chloroform. 2.0

3.2. Determination of total phenolics 1.5 R2= 0.99664 The total phenolic content in methanol extract of D. trigona 1.0

was determined as Gallic Acid Equivalent (GAE). The extract Absorbance at 700 nm showed concentration dependent increase in phenolic con- tent. Tested methanol extract showed significant phenolic 0.5 content of 37 mg of GAE in 100 mg of plant extract.

100 200 300 400 500 3.3. Antioxidant activity by DPPH assay Concentration in µg/mL

Antioxidant activity of the methanol extract measured as free Fig. 2 e Reducing power of D. trigona methanolic extract at radical scavenging activity (RSA) is presented in Fig. 1. The different concentrations as compared with BHT. extract showed a significant dose dependent increase in RSA similar to that of standard. Ascorbic acid used as reference standard showed 75% of inhibition at 50 mg/mL. countries because of the unavailability of medicines and the emergence of widespread drug resistance, the disease impact 3.4. Reducing power assay is more.24 Hence, the production of phytomedicines of plant origin play an important role in herbal drug technology. The reducing power assay of methanolic extract was com- The present study on preliminary phytochemical analysis pared with standard BHT which showed an increase in of D. trigona showed the presence of secondary metabolites in absorbance at 700 nm. The extract of the plant showed different solvent extracts. There are also reports on the phy- promising amount of reducing power ability which reflected tochemical constituents of a few species of .17 its antioxidant potential and increased with increase in con- Plants which contain tannins are used as astringent and in centration (Fig. 2). treating diarrhoea and dysentery25 and also reported to have anticancerous activity.26 Just et al.27 have reported the effect of saponins in managing inflammation of cells. Sterols are 4. Discussion important due to their relationship with various anabolic hormones including sex hormones and its antiviral property Pathogens such as bacteria, fungi and viruses cause many has been confirmed.28 Flavonoids exhibit a wide range of infectious diseases which are major threat to public health biological activities like antimicrobial, anti-inflammatory, despite of advancement in human medicine. In developing analgesic, anti-allergic and antioxidant properties.29 Alka- loids are widely used in the development of pain killer medi- cines.30 These compounds are also found toxic against cells of foreign organisms and used in the elimination of human cancer cells.31 The phenolic and reducing compounds are the major bioactive substances involved in antioxidant activity by eliminating free radicals, stimulation of the immune sys- tem, regulation of gene expression and antibacterial ef- fects.32 The experiments revealed that total phenolics and antioxidant activities of D. trigona were dose dependent. Meyers et al.33 demonstrated that antioxidant activity of the plant extracts were stronger than the synthetic ascorbic acid. The DPPH assay has been largely used as a quick and reliable procedure to estimate antioxidant activity of plant extracts.34 The reducing power assay was dose dependent with increase in the concentration of plant extract and revealed promising amount of compounds with reducing power. This may be due to the biologically active com- pounds present in the plant extract indicating that they are electron donors and can reduce the oxidized intermediates Fig. 1 e DPPH radical scavenging activity of D. trigona of lipid peroxidation process which act as primary and methanolic leaf extract. secondary antioxidants.35 journal of pharmacy research 7 (2013) 20e23 23

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