International Journal of Current Biotechnology

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1 Chandhini Rajendran, Femina Carolin Christopher, Nithyakalyani Ramu, Vivek Pazhamalai, Nithyananthi Mohankumar and Sumathi Ethiraj*, Studies on Phytochemical Screening, Antioxidant Activity and Extraction Active Compound (2-Hydroxy 4-Methoxy Benzaldehyde) From Rhizome Extract of Decalepis Hamiltonii Wight and Arn, Int.J.Curr.Biotechnol., 2014, 2(4): International Journal of Current Biotechnology ISSN: Journal Homepage : Studies on Phytochemical Screening, Antioxidant Activity and Extraction Active Compound (2-Hydroxy 4-Methoxy Benzaldehyde) From Rhizome Extract of Decalepis Hamiltonii Wight and Arn Chandhini Rajendran 2, Femina Carolin Christopher 2, Nithyakalyani Ramu 2, Vivek Pazhamalai 2, Nithyananthi Mohankumar 2 and Sumathi Ethiraj 1 * 1 Poonga Biotech Research Centre, Plant Biotechnology Division, Choolaimedu, Chennai Tamil Nadu, India. 2 Vel Tech High Tech Dr. Rangarajan Dr. Sakunthala Engineering College Avadi, Chennai A R T I C L E I N F O Article History: Received 31 March 2014 Received in revised form 15 April 2014 Accepted 20 April 2014 Available online 30 April 2014 Key words: Decalepis hamiltonii, Antioxidant activity, Phytochemical screening, HPLC analysis, Antibacterial activity. A B S T R A C T The present study was performed to investigate the phytochemical screening, total flavonoid, antioxidant activity, HPLC analysis of 2-hydroxy 4-methoxy benzaldehyde (2H4MB) and antibacterial activity from the rhizome of Decalepis hamiltonii. The phytochemical analysis revealed the presence of active ingredients such as steroids, saponins, phenols, flavonoids, terpenoids, alkaloids and tannins in the rhizome extract of D. hamiltonii followed by others. Total flavonoid content was quantitatively estimated which recorded maximum in Kanchipuram accession (5.25 mg Quercetin Equivalents (QE) /g). The rhizome extracts were evaluated for antioxidant activities by DPPH (1, 1 Diphenyl -2- picryl - hydrazyl) radical scavenging assay. Among the three accessions with different solvents extractions, maximum antioxidant activity was found in the ethanolic rhizome extract (90.9%) of D. hamiltonii followed by others. HPLC analysis of these extracts showed that the main components of the active principles namely 2H4MB were present in the rhizome extract of D. hamiltonii. Different concentrations of ethanolic rhizome extract were tested using the agar disc diffusion technique for the activity against Bacillus cereus, Pseudomonas aeruginosa, and Bacillus subtilis. It was found to be inactive against Staphylococcus aureus and Escherichia coli. Introduction Medicinal plants have been used in traditional treatments for numerous human diseases for thousands of years and they continue to be an important therapeutic aid for alleviating the ailments of human kind (Momin and Kadam, 2011). The therapeutic benefits are generally traced to specific plant compounds; but are specifically due to the active constituents of the plants (Mary et al., 2012). Phytochemical screening of various plants has been reported by many workers (Mojab et al., 2003; Parekh and Chanda, 2008). These studies have revealed the presence of numerous chemicals including alkaloids, flavonoids, steroids, phenols, glycosides and saponins. The phenolic compounds are one of the largest and most ubiquitous groups of plant metabolites (Hagerman et al., 2008). A number of studies have focused on the biological activities of phenolic compounds which are antioxidants and free radical scavengers (Evans et al., 1995; Cespedes et al., 2008; Reddy et al., 2008). *Corresponding author. address: sumathiethiraj@yahoo.com The flavonoids are a category of natural substances belonging to the family of polyphenols. The flavonoids are also widely distributed in plants which have been reported to exert multiple biological benefits, including antioxidant, free radical scavenging abilities, antiinflammatory and anti-carcinogenic (Miller, 1996). The crude extracts of herbs, spices and other plant materials, rich in phenolics and flavonoids are of increasing interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food (Chu et al., 2000). Free radicals (superoxide, hydroxyl radicals and nitric oxide) and other reactive species (hydrogen peroxide, hypochloric acid and peroxynitrite) produced during aerobic metabolism in the body, can cause oxidative damage of amino acids, lipids, proteins and DNA (Gutteridge, 1995; Halliwell, 1995). It has been established that oxidative stress is among the major causative factors in the induction of many chronic and degenerative diseases including atherosclerosis, ischemic heart disease, ageing, diabetes mellitus, cancer, immunosuppression, neurodegenerative diseases and others (Gulcin et al., 2002 ; Devasagayam et al., 2004). The screening of plant products for antibacterial activity has shown that the higher plants represent a potential source of novel antibiotic prototypes (Afolayan, 2003). There has been an increasing incidence of multiple resistances in human pathogenic microorganism in recent years (Okunji et al., 1999). Decalepis hamiltonii Wight & Arn belonging to the family Asclepiadaceae is commonly known as Magali Kizhangu in Tamil. It is a monogeneric climbing shrub native of the Deccan Peninsula and forest areas of 31 Int.J.Curr.Biotechnol. Volume 2; Issue 4; Apr, 2014

2 Western Ghats of India and also found in the dry and moist deciduous forests of Karnataka, Andhra Pradesh and Tamil Nadu. D. hamiltonii rhizome has been used as an appetizer, blood purifier and for the treatment of various physiological disorders (Nagarajan et al., 2007). Hence, the present study was performed to investigate the phytochemical screening, total flavonoid content, antioxidant activity, HPLC analysis of 2-hydroxy 4- methoxy benzaldehyde (2H4MB) and antibacterial activity from the rhizome of Decalepis hamiltonii. Material and Methods Collection of plant material The healthy rhizome of Decalepis hamiltonii were collected from three different regions of Tamil Nadu namely Kanchipuram, Salem and Trichy district. The collected rhizome were brought to the laboratory and maintained at Poonga Biotech Research Centre, Plant biotechnology division, Chennai , Tamil Nadu, India. Preparation of the plant extract Preparation of the extracts was done according to a combination of the methods used by Pizzale et al. (2002) and Lu and Foo, (2001). About 15g of dried rhizome fine powder of Decalepis hamiltonii plant materials were extracted with 150 ml acetone, ethanol (75%), chloroform, petroleum ether and aqueous extract for 1 min using an Ultra Turax mixer (13,000 rpm) and soaked overnight at room temperature. The sample was then filtered through Whatman No.1 paper in a Buchner funnel. The filtered solution was evaporated under vacuum in a rota-evator at 40 C to a constant weight and then dissolved in respective solvents. The concentrated extracts were stored in airtight container in refrigerator below 10ºC. Phytochemical Screening from rhizome extracts of Decalepis hamiltonii: The phytochemical screening of rhizome extracts were assessed by standard method as described by Brinda et al., (1981); Siddiqui and Ali (1997) and Savithramma et al., (2011). Phytochemical screening was carried out on the rhizome extracts using different solvents to identify the major natural chemical groups such as tannins, saponins, flavonoids, phenols, terpenoids, alkaloids, glycosides, cardiac glycosides, coumarins and steroids. General reactions in these analyses revealed the presence or absence of these compounds in the rhizome extracts tested. Estimation of Total Flavonoid Content from rhizome extracts Decalepis hamiltonii Total flavonoids content in the ethanolic rhizome extracts was determined by the aluminium chloride colorimetric method (Mervat et al., 2009). 0.5ml of rhizome extracts of Decalepis hamiltonii at a concentration of 1mg/ ml were taken and the volume was made up to 3ml with ethanol. Then 0.1ml AlCl 3 (10%), 0.1ml of potassium acetate and 2.8 ml distilled water were added sequentially. The test solution was vigorously shaken. Absorbance was recorded at 415 nm after 30 minutes of incubation. A standard calibration plot was generated at 415nm using known concentrations of quercetin. The concentrations of flavonoid in the test samples were calculated from the calibration plot and expressed as mg quercetin equivalent /g of sample. Qualitative analysis of Antioxidant activity of Decalepis hamiltonii The antioxidant activity of rhizome extracts of Decalepis hamiltonii was determined by following the method as described by George et al. (1996). 50ìl of rhizome extracts of Decalepis hamiltonii were taken in the microtiter plate. 100ìl of 0.1% methanolic DPPH was added over the samples and incubated for 30 minutes in dark condition. The samples were then observed for discoloration; from purple to yellow and pale pink were considered as strong and weak positive respectively. The antioxidant positive samples were subjected for further quantitative analysis. Quantitative analysis of Free radical scavenging activity of Decalepis hamiltonii The antioxidant activities were determined using DPPH, (Sigma-Aldrich) as a free radical. Rhizome extract of 100ìl were mixed with 2.7ml of methanol and then 200ìl of 0.1 % methanolic DPPH was added. The suspension was incubated for 30 minutes in dark condition. Initially, absorption of blank sample containing the same amount of methanol and DPPH solution was prepared and measured as a control (Lee et al., 2003). Subsequently, at every 5 min interval, the absorption maxima of the solution were measured using a UV double beam spectra scan (Chemito, India) at 517nm. The antioxidant activity of the sample was compared with known synthetic standard of (0.16%) of Butylated Hydroxy Toluene (BHT). The experiment was carried out in triplicate. Free radical scavenging activity was calculated by the following formula: Inhibition = [(Absorbance of control (Ac 517) Absorbance of sample (As517) X 100 (Absorbance control (Ac517)] High performance liquid chromatography analysis of 2- hydroxy 4-methoxy benzaldehyde (2H4MB) The fine powder of the rhizome biomass was extracted with 75% of ethanol and then the extract was evaporated. The residue of extract was mixed with n-butanol and water (2:1) and both the upper layer of n-butanol and lower layer of water were separated and evaporated under vacuum. The residues were washed with petroleum ether to remove fatty components and then extracted with methanol. The concentrated extract in methanol was separated and analyzed using high performance liquid chromatography as per standard method (Shimizu et al., 1997). The extracts were filtered through sartorius RC-membrane syringe filter (0.20 m) and 20 µl of filtrate was injected in to the HPLC. Chromatography was performed using Shimadzu HPLC (Model SPD-10A UV-VIS Detector) and supelcosil LC-18 column (25 cm x 4.6 mm, 5 m) with mobile phase consisting of acetonitrile, water and acetic acid (50:50:0.1). Flow rate was maintained at 1.0ml/minute with a back pressure of 250 psi and the compounds were read at 210 nm using a UV detector. The total run time was 20 min but preferably it was extended up to 40min (Shimizu et al., 1997). The results were compared with standard. Antibacterial activity from rhizome extract of Decalepis hamiltonii The ethanol rhizome extracts from mother plant of Decalepis hamiltonii plant were used for antibacterial study (ozkan et al., 2004; Janarthanam and Sumathi, 2010). Different concentration (10, 20 and 30 mg/ml) of the concentrated ethanol rhizome extracts was tested for its antimicrobial strain such as Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. The bacterial cultures were grown in Mueller Hinton Agar and Mueller Hinton broth (Himedia) (Lopez et al., 2001). Volume 2; Issue 4; Apr, 2014 Int.J.Curr.Biotechnol. 32

3 Figure - 1: Mother plant of Decalepis hamiltonii a. Mother plant of Decalepis hamiltonii collected from Kanchipuram area, b. Rhizome of Decalepis hamiltonii Figure - 2: Antioxidant activity from rhizome extract of Decalepis hamiltonii (Kanchipuram) 120 Antioxidant activity (%) BHT Aqueous Ethanol Acetone Petroleum ether Different extractions of Decalepis hamiltonii Chloroform Figure 3a: HPLC analysis of 2-hydroxy 4-methoxy benzaldehyde (2H4MB) content in rhizome extract of Decalepis hamiltonii (Kanchipuram) (a) 2H4MB standard (1 mg/ 1ml) (Sigma aldrich) 33 Int.J.Curr.Biotechnol. Volume 2; Issue 4; Apr, 2014

4 Figure 3b: HPLC analysis of 2-hydroxy 4-methoxy benzaldehyde (2H4MB) content in rhizome extract of Decalepis hamiltonii (Kanchipuram) (b) Rhizome extract of D. hamiltonii Figure - 4: Antibacterial activity from ethanol rhizome extract of Decalepis hamiltonii (Kanchipuram) Antibacterial activity of rhizome extract of D. hamiltonii against Bacillus subtilis (a), Bacillus cereus (b), Pseudomonas aeruginosa (c) Staphylococcus aureus (d) and Escherichia coli (e) Volume 2; Issue 4; Apr, 2014 Int.J.Curr.Biotechnol. 34

5 Antibacterial activity was measured using the standard method of diffusion disc plates on agar (Erturk et al., 2003). Then 0.1ml of each culture of bacteria was spread on agar plate surfaces. For antibacterial assay, all bacterial strains were grown in Mueller Hinton Broth Medium (Hi media) for 24 hours at 37 C and plated on Mueller Hinton Agar (Hi media) for agar diffusion experiments. Paper disc (6mm in diameter) were placed on the agar medium to load 20µl of different concentrations of ethanol rhizome extracts of Decalepis hamiltonii were tested. Inhibition diameters were measured after incubation for hours at 37 C. Results and Discussion In the present study, phytochemical screening was performed with ethanol, chloroform, petroleum ether, acetone and aqueous rhizome extracts of Decalepis hamiltonii. The ethanolic rhizome extract of Decalepis hamiltonii were rich in terpenoids, quinones, Cardiac glycosides, flavonoids, steroids, phenols, tannins and saponins followed by other extracts (Table 1,2 and 3). Phytochemical constituents such as tannins, flavonoids, alkaloids and several other aromatic compounds or secondary metabolites of plants serve as defense mechanism against predation by many micro-organisms, insects and herbivores. The curative properties of medicinal plants are perhaps due to the presence of various secondary metabolites such as alkaloids, flavonoids, glycosides, phenols, saponins, steroids, etc (Britto and Sebastian, 2011). Thus the preliminary screening test may be useful in the detection of the bioactive principles and subsequently may lead to the drug discovery and development (Doss, 2009). The presence of alkaloids and saponins in the rhizome extract, the biological function of alkaloids and their derivatives are very important and are used in analgesic, antispasmodic and bactericidal activities (Stary, 1998). Saponins have properties of precipitating and coagulating red blood cells and they also have cholesterol binding properties, formation of foams in aqueous solutions and haemolytic activity (Sodipo et al., 2000) and traditionally saponins have been extensively used as detergents and molluscicides, in addition to their industrial applications as foaming and surface active agents and also have beneficial health effects (Shi et al., 2004). Plant steroids are known important for their cardiotonic activities and also used in nutrition, herbal medicine and cosmetics. Thus the preliminary screening studies may be useful in the detection of the bioactive principles, which leads to drug discovery and development. Flavonoids are regarded as one of the most widespread groups of natural constituents found in plants. The values of flavonoid content varied among plants. It has been recognized that flavonoids show antioxidant activity and their effects on human nutrition and health are considerable. The mechanisms of action of flavonoids are through scavenging or chelating process (Kessler et al., 2003; Cook and Samman, 1996). The results of the present study showed that the flavonoid contents of the ethanolic rhizome extracts in terms of quercetin equivalent (QE) were found to be maximum in Decalepis hamiltonii (Kanchipuram) 5.25 mg QE/g followed by Salem ( 3.9 mg QE /g) and Trichy ( 2.3 mg QE/g) (Table 4). Scavenging activity for free radicals of DPPH has widely used to evaluate the antioxidant activity of natural products from plant and natural sources. Free radicals have broad range of effects in biological systems. It has been proved that these mechanisms may be important in the pathogenesis of certain diseases and ageing. Many synthetic antioxidant components have shown toxic and/ or mutagenic effects, which have shifted the attention towards the naturally occurring antioxidants (Galvez et al., 2005; Tepe et al., 2005; Mammadov, 2011). Wild accessions of Decalepis hamiltonii rhizome samples were used for antioxidant studies. Analysis on different extraction of acetone, ethanol (75%), petroleum ether, chloroform and aqueous extract showed the presence of antioxidants. 100ìl of rhizome extracts were estimated for free radical scavenging activity using DPPH assay. The samples were observed for the colour change from purple to yellow and pale pink were considered as strong positive and weak positive respectively (Table 5). Among the three wild accessions and five different solvent extracts of D. hamiltonii, the ethanolic rhizome extract collected from Kanchipuram district recorded the most effective DPPH radical scavenging activity (90.98%) followed by Salem (67.3%) and Trichy district (40.98%) (Fig. 2). Decalepis hamiltonii (Kanchipuram) value being very close to synthetic antioxidant (BHT) as positive control (98.36%). In all accessions, ethanolic rhizome extracts recorded higher percentage of free radical scavenging activity followed by acetone, aqueous, chloroform and petroleum ether. HPLC analysis of 2H4MB compound 2-hydroxy 4-methoxy benzaldehyde (2H4MB) compound from D. hamiltonii eluted through HPLC analysis and based on standard retention time min. Sample Retention time (min) 2H4MB compound Standard (Decalepis hamiltonii rhizome extract (Kanchipuram) The Decalepis hamiltonii rhizome extract was used for HPLC analysis, showed a similar Retention time (Rt) in both rhizome extract (11.14 min) and 2H4MB compound Standard (11.03min). Thus confirming the presence of 2H4MB compound in rhizome extract of Decalepis hamiltonii (Fig. 3). The data presented in Table 6, indicate that the rhizome extracts of Decalepis hamiltonii inhibit the growth of some microorganism to various concentration. The concentrations of 10mg/ml - 30mg/ml ethanolic extract showed antimicrobial activity against Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Pseudomonas aeruginosa and inactivity against Escherichia coli (Fig. 4). The maximum clear zone of inhibition was found at 30mg/ml of 75% ethanolic rhizome extract of Decalepis hamiltonii. In rhizome extract, there is no zone of inhibition was found in lower concentration 10mg/ml. Similar results were obtained on ethanol extracts from rhizome of Sida acuta and Acalypha wilkesiana which exhibited antibacterial activity (Oboh et al., 2007; Gotep et al., 2010). The antimicrobial activities of ethanol extract may be due to the presence of tannins, triterpenoids and flavonoids (Mamtha et al., 2004). Thus from our findings, it is concluded that the 75% ethanolic extracts from dry powdered rhizome of Decalepis hamiltonii had superior level of antimicrobial activity. 35 Int.J.Curr.Biotechnol. Volume 2; Issue 4; Apr, 2014

6 In conclusion, phytochemical composition, total flavonoid, antioxidant activity and antibacterial activity of medicinal plants are very important in identifying new sources of therapeutically and industrially important compounds. It is imperative to initiate an urgent step for screening of plants for secondary metabolites. The present communication attempts to assess the status of phytochemicals, total flavonoid, antioxidant activity, extraction of compound and antibacterial activity, in the rhizome extract of Decalepis hamiltonii to improve the health status of people and also to use it in the nutraceuticals products of commercial importance. The results indicate that the plant material may become an important source of natural drug compounds with health protective potential and natural antioxidants and antibacterial of significant impact on the status of human health and disease prevention. 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7 Table - 1: Phytochemical screening from rhizome extracts of Decalepis hamiltonii (Kanchipuram) Rhizome extract of Decalepis hamiltonii Phytochemicals Petroleum Aqueous Ethanol Chloroform Acetone ether Tannins Saponins Flavonoids Quinones Glycosides Cardiac glycosides Terpenoids Phenol Coumarins Steroids Alkaloids = strong positive; + = positive; - = negative Table - 2: Phytochemical screening from rhizome extracts of Decalepis hamiltonii (Salem) Rhizome extract of Decalepis hamiltonii Phytochemicals Petroleum Aqueous Ethanol Chloroform Acetone ether Tannins Saponins Flavonoids Quinones Glycosides Cardiac glycosides Terpenoids Phenol Coumarins Steroids Alkaloids = strong positive; + = positive; - = negative 37 Int.J.Curr.Biotechnol. Volume 2; Issue 4; Apr, 2014

8 Table - 3: Phytochemical screening from rhizome extracts of Decalepis hamiltonii (Trichy) Phytochemicals Rhizome extract of Decalepis hamiltonii Aqueous Ethanol Chloroform Acetone Petroleum Tannins Saponins Flavonoids Quinones Glycosides Cardiac glycosides Terpenoids Phenol Coumarins Steroids Alkaloids = strong positive; + = positive; - = negative Table - 4: Estimation of Flavonoid content from ethanolic rhizome extract of different wild accessions of Decalepis hamiltonii S. No Plant sample S1 D. hamiltonii - Kanchipuram S2 D. hamiltonii Salem S3 D. hamiltonii Trichy Total flavonoid content (mg QE/g) 1563) * - Includes diameter of disc (6mm); Average three replicates Volume 2; Issue 4; Apr, 2014 Int.J.Curr.Biotechnol. 38 S. No Extractions ether Table - 5: Qualitative analysis of antioxidant activity from rhizome extract of Decalepis hamiltonii D. hamiltonii (Kanchipuram ) BHT (standard) +++ S1 Aqueous + S2 Ethanol +++ S3 Acetone + S4 Chloroform - S5 Petroleum ether - Table - 6: Antibacterial activity from ethanol rhizome extracts of Decalepis hamiltonii (Kanchipuram) Inhibition Zone in diameter (mm)* Micro-organisms Ethanolic extract (rhizome) Bacillus subtilis ( MTCC No ) Bacillus cereus ( MTCC No ) 5.25mg 3.96 mg 2.31mg Pseudomonas aeruginosa ( MTCC No ) Staphylococcus aureus ( MTCC No. 9542) Escherichia coli ( MTCC No. Concentrations of extract 10mg/ml 20mg/ml 30mg/ml - 9mm 13mm mm - - 9mm - - 8mm - - -

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