Antioxidant, Antimicrobial and GC-MS Analysis of Pterolobium hexapetalum Seed Extracts

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1 Applied Science Reports E-ISSN: / P-ISSN: DOI: /PSCP.ASR App. Sci. Report. 16 (3), 2016: PSCI Publications Antioxidant, Antimicrobial and GC-MS Analysis of Pterolobium hexapetalum Seed Extracts K. Kokila, N. Elavarasan, V. Sujatha * Department of Chemistry, Periyar University, Periyar Palkalai Nagar, Salem , Tamil Nadu, India. *Corresponding Author chemsujatha888@gmail.com Paper Information A B S T R A C T Many secondary metabolites of the plant are commercially important and Received: 13 September, 2016 find use in a number of pharmaceutical compounds. Seeds of Pterolobium hexapetalum [F: Fabaceae] were chosen to investigate the contents of Accepted: 29 November, 2016 phenol, flavonoid, carbohydrate, protein, in vitro antioxidant assays like superoxide, hydroxyl radical scavenging activity along with antimicrobial Published: 20 December, 2016 properties, in various solvent extracts [hexane, ethyl acetate, acetone, and methanol]. The presence of numerous phytoconstituents was ensured from the preliminary qualitative screening which presented a platform to assess their individual contribution to the antioxidant activity in almost all the extracts. The results of the antioxidant assay evidently outlined the richest source of flavonoids in acetone extract (65.29%) and the amount of phenol content in acetone extract as (21.91%) present in their whole respective extracts. The acetone extract exhibited highest total carbohydrate and total protein content above almost all the extracts. The acetone extract has higher antioxidant potential (lowest IC 50 value), when compared to the other extracts. When the extract has lower IC 50 value, it indicates that extract is highly efficient in comparison to the standard (ascorbic acid). An overall significant activity was established by acetone extract of Pterolobium hexapetalum seed for all the assayed parameters, in particular, carbohydrate (91.25 mg) and superoxide (31.74 µg). Significant zone of inhibition were also recorded against Lacto bacillus (19 mm), Bacillus subtillus (19 mm), Aspergillus niger (23 mm) against control. Moreover, a variety of compounds were identified through GC-MS analysis. Hence, this present study would endow the scientific validation for their medicinal, therapeutic and dietary applications PSCI Publisher All rights reserved. Key words: Pterolobium hexapetalum, phenol, flavonoid, antioxidants, GC-MS Introduction Medicinal plants have been widely deliberated for their antioxidant activity in recent years. It is trusted that an increased intake of food prospers in natural antioxidants is associated with lower risks of degenerative diseases, particularly cardiovascular diseases and cancer [1]. The health promoting benefits of antioxidants of plants to be thought have resulted from their potential effects against the reactive oxygen species. Hence, the development of antioxidants from the natural source has attracted considerable attention and is thought to be a desirable development. Moreover, several investigations have indicated that medicinal plants contain a wide variety of natural antioxidants such as phenols, tannins and flavonoids, which possess antioxidant activity [2, 3]. Pterolobium hexapetalum (F: Fabaceae) is a spiny straggler of dry deciduous forests having an extensive range of medicinal uses. The stem bark is used against fever, cold, cough, tooth ache, chest pain, nausea, dog bite, heat boils and wound healing. The present study is designed to investigate in such a method to assess the efficiency of Pterolobium hexapetalum seed, using in vitro antioxidant assays accompanied through estimation of different contents and antibacterial activities against micro organisms, along with GC-MS analysis to discover the presence of phytoconstituents. Pterolobium hexapetalum seed can be used as a therapeutic agent and as a protective shield against numerous free radicals mediated diseases. Materials And Methods Authentification and extraction Pterolobium hexapetalum seed was collected from the forest range of Nilgiri hills, Tamil Nadu, India and authenticated by a taxonomist [BSI/SRC/5/23/ /Tech-83] of TNAU. Samples were treated with soxhelation using each

2 of the subsequent solvents in increasing polarity: n-hexane, ethyl acetate, acetone and methanol and crudes stored at 4 5 C until further use. Phytochemical screening The qualitative preliminary screening was analyzed to identify the presence of the secondary metabolites according to the standard methods [4]. Estimation of phenol content Contents of total phenol in the seed extracts were estimated using standard procedures [5] employing Folin and Ciocalteu s phenol reagent, at an absorbance of 725 nm (Analytik Jena spectrophotometer). Gallic acid was used for standard curve ( mm; Y = x ; R 2 = ) and the results were showed as µg of gallic acid equivalents (GAEs)/ mg of extract. Estimation of flavonoid content Flavonoid contents in the extracts were determined [5] using AlCl 3, at an absorbance of 510 nm. (+)-Catechin was used to calculate the standard curve ( mm, Y = , R 2 = ) and the consequences were expressed as µg of (+)-catechin equivalents (CEs)/ mg of extract. Total carbohydrate content The carbohydrate contents were estimated by anthrone method [6]. Glucose was used to calculate the standard curve ( µg/ml, Y= X , R 2 = ) and the results were expressed as µg of glucose equivalents (GEs)/ mg of extract. Total protein content The proteins were estimated by Lowry s method [7]. Bovine serum albumin was used to calculate the standard curve ( µg/ml, Y = x , R 2 = ) and the results were expressed as µg of bovine serum albumin equivalents (BSAEs)/ mg of extract. DPPH radical scavenging activity At the various concentrations of Pterolobium hexapetalum seed extracts were assayed using methanol solution containing DPPH radicals (6 x 10-5 mol/l) at an absorbance of 517 nm. The radical scavenging activity (RSA) was calculated as a % of DPPH discoloration, using the equation: % RSA= [(A DPPH -A S)/ A DPPH] x 100, where A S - the absorbance of the solution, when the sample extract is added at a particular level and A DPPH - the absorbance of the DPPH solution [5]. Ascorbic acid was used as a standard. Reducing power activity Various concentrations of Pterolobium hexapetalum seed extracts were assayed by measuring the reduction system of Fe 3+ to Fe 2+ using the method of Bauer et al., [8]. The absorbance was measured spectrophotometrically at 700 nm. Ascorbic acid was used as a standard solution. Superoxide radical scavenging activity The superoxide anion scavenging assay of Pterolobium hexapetalum ethanol extract was based on the method described by Liu et al., [9]. Superoxide radicals are generated in PMS-NADH systems with oxidation of NADH and assayed by the reduction of nitro blue tetra zolium (NBT) methods. Hydroxyl radical scavenging activity The mixture contain 0.2 ml of sample in various concentration to which 0.1 ml EDTA (1 mm) FeCl 3 (10 mm), 0.36 ml deoxy ribose (10 mm), mixture, 0.1 ml H 2O 2 (10 Mm ), 0.36 ml deoxy ribose (10 mm), 0.33 ml phosphate buffer (50 mm; ph 7.4) and 0.1 ml of ascorbic acid (1 mm) was added in sequence. The mixture was incubated at 37 o C for 1 hour to this was added 1.0 ml each of TCA (10%) and TBA (0.67) in boiling water bath at 20 minutes. The colour developed in the reaction was measured at 532 nm [10]. The control tube contains phosphate buffer instead of sample. Antimicrobial activity Antibacterial activity: [Lacto bacillus (G+ve), Bacillus subtillus (G-ve)] Media Used (Nutrient broth): NaCl-10 g, Peptone-10 g, Yeast extract 5 g and Agar 20 g in 1000 ml of deionised water by using agar diffusion method [11]. 142

3 Antifungal activity: [Aspergillus niger] The agar well diffusion method was modified. Sabouraud s dextrose agar (SDA) was used for fungal cultures. The diameters of zone of inhibition (mm) observed were measured. GC-MS analysis GC-MS was performed [12] with GC Clarus 500 Perkin Elmer equipment. Compounds were separated on a 30 m x 0.25 mm capillary column coated with a 0.2 µm film of HP-5-MS. Samples were injected with a hole ratio of 50:1; helium was used as carrier gas at 1 ml/min. The column temperature was maintained at 100 o C for 1 min after injection then increased at 10 o C/min to 275 o C which was sustained for 20 min. The time required for chromatography of one sample was 36 min. Statistical analysis All the experiments were carried out in triplicates and the results are expressed as mean value ± S.D using ANOVA analysis (SPSS software version). Results And Discussion The phytochemical qualitative screening analysis was carried out on Pterolobium hexapetalum seed revealed the presence of medicinal active constituents. Phytoconstituents like alkaloids, phenolics, flavonoid, tannins, carbohydrates, glycosides, terpenoids etc, were found to be present in almost all the extracts (Table 1). In addition, the knowledge of these plant constituents would further be able to discover the actual value of folkloric remedies [13]. Table 1: Preliminary qualitative screening of various extracts of Pterolobium hexapetalum seed Constituents Name of the test Pterolobium hexapetalum Seed Hexane Ethyl acetate Acetone Methanol Alkaloids Wagner s Hager s Meyer s Dragendroff s Flavonoids Shinoda s Fecl Alkaline Con.H 2SO Phenolics FeCl Dichromate Lead acetate Tannins Gelatin KOH Saponins Foam Carbohydrate Molisch s Fehling s Barfoed s Borntrager s Proteins Biuret Ninhydrin Steroids Liberman s Salkowski s Terpenoids Hager s Glycosides Keller-Killiani Fats/Oils Biuret s slightly present; moderately present; copiously present Some non-enzymatic assays were estimated. The results of the antioxidant assay clearly outlined the richest source of the phenol, flavonoid, carbohydrate and protein contents. These contents are highly focused factors for a plant to be considered as an effective antioxidant. The results of the antioxidant assay evidently outlined the richest source of flavonoids in acetone extract (65.29%) and the amount of phenol content in acetone extract as (21.91%) present in their whole respective extracts. From Table 2, it can be noted that the acetone extract possess significant antioxidant content, evidencing its ability to have extracted a considerable amount of phenols and flavonoids with specific distribution in the particular representatives. Rest three extracts cannot be considered to be insignificant because satisfactory contents were obtained enough to show their antioxidant activity. Especially, if the acetone extract is non-toxic after the toxicological evaluation, further isolation and purification of antioxidant components is not essential because health benefits of the extract might be from additive and synergistic effects of phytochemicals in the extract [14]. 143

4 Table 2: Estimation of phenolic, flavonoid, carbohydrate and protein content in various extracts of Pterolobium hexapetalum seed Contents (µg/ml) Hexane Ethyl acetate Acetone Methanol Total Phenol 36.60±0.23 a 42.65±0.07 b 65.29±0.71 c 50.49±0.12 b Total flavonoid 13.95±0.25 c 15.13±0.49 a 16.69±0.10 a 5.37±0.12 b Total carbohydrate 7.90±0.03 a 9.07±0.30 b 91.25±0.39 c 16.88±0.08 a Total protein 10.44±0.03 a 80.49±0.10 a 53.81±0.27 c 83.35±0.23 b The statistical significance (p*<0.05), n=3, for all the extracts is calculated from gallic acid, catechin, glucose and bovine serum albumin equivalence. The results of the various contents are expressed as mean±s.d, significantly (a = 0.001> b = 0.01> c = 0.05) through ANOVA using SPSS software (16.0 versions). Intense carbohydrate contents were measured in the acetone extract (91.25 μg). The next potent content was recorded by methanol extract (16.88 μg) in gallic acid equivalents. In agreement to the carbohydrate content, methanol extract showed richer protein content of about 83.35μg. Likewise, the next significance was produced by acetone extract of about 53.81μg. Thus, protein being less polar constituents would have got extracted in such less polar solvents comparatively. Figure 1 DPPH radical scavenging activity of Pterolobium hexapetalum seed extracts In the DPPH assay of the current examination, Fig. 1 shows the amount of each extract required for 50% inhibition/scavenging (IC 50) of the DPPH radicals. The highest significant antioxidant activity in DPPH assay was presented by acetone with IC 50 = mg which is higher than that of methanol and ethyl acetate, against the standard ascorbic acid. Other extracts exhibited appreciable scavenging activity ranging from 50.92% to 95.80% capable of quenching DPPH radicals (Table 3). The advantage of this method is that DPPH was allowed to react with the whole sample and enough time given in the method allows DPPH to react slowly even with weak antioxidants [15]. Table 3: In vitro antioxidant properties of various extracts of Pterolobium hexapetalum seed In vitro antioxidant properties Hexane Ethyl acetate Acetone Methanol Standard (Ascorbic acid) DPPH radical scavenging activity (IC 50 in mg/ml) Reducing power activity (EC 50 in mg/ml) Superoxide radical scavenging activity (IC 50 in µg/ml) Hydroxyl radical scavenging activity (IC 50 in µg/ml) HX- hexane; EA- ethyl acetate; AC- acetone; ME- methanol; STD-ascorbic acid was used as standard. Standards were used for a comparative assessment to determine the 50% inhibition of the extracts against the radicals formed at the reaction system. 144

5 In reducing power assay, there was observed a neat uniform and racing increase in the absorbance along the increasing concentration, due to the electron donating capacity of plant extract. To the above assays, hexane extract exhibited its IC 50 value of about mg, which was much convincing (Fig. 2). On the other hand, it was acetone extract being significant with IC 50 value of mg, which was the outstanding evidence for the antioxidant property of Pterolobium hexapetalum seed. However, the activities of antioxidants have been attributed to various mechanisms such as prevention of chain initiation, reducing capacity and radical scavenging [16]. Figure 2. Reducing power activity of Pterolobium hexapetalum seed extracts The versatile propensities of Pterolobium hexapetalum seed extracts illustrated via in vitro assays like superoxide and hydroxyl radical scavenging activities concerned with antioxidant studies have been of interest. Figure 3. Superoxide radical scavenging activity of Pterolobium hexapetalum seed extracts 145

6 In superoxide anion radical scavenging assay, acetone extract exhibited an excellent scavenging activity (31.55µg/ml). The abilities of the Pterolobium hexapetalum seed extracts and ascorbic acid to quench superoxide radicals from the extracts reflected the decrease of the absorbance (Fig. 3). Further, superoxide is also known to indirectly initiate lipid peroxidation as a result of H 2O 2 formation, creating precursors of hydroxyl radicals. Figure 4. Hydroxyl radical scavenging activity of Pterolobium hexapetalum seed extracts For instance, Fig. 4 shows that 250μg/ml, % inhibition recorded were: ascorbic acid (97.38%) > acetone (81.49%) > methanol (72.33%) > ethyl acetate (68.41%) > hexane (64.67%). Altogether, there was observed a neat concentrationdependant manner of scavenging activity. The hydroxyl radical is a very reactive species, but it has very low diffusion ability [17]. Figure 5 Antimicrobial activity of Pterolobium hexapetalum seed extracts against Lactobacillus [A], Bacillus subtillus [B], Aspergillus niger [C] Antimicrobial studies for the seed extracts of Pterolobium hexapetalum have been measured with Lacto bacillus (G+ve), Bacillus subtillus (G-ve) and Aspergillus niger for different micro organisms in Fig. 5. Among the extracts, acetone extract was found to be significant in inhibiting the bacterial growth which is measured in mm i.e. zone of inhibition almost all 146

7 the micro organism were efficiently scavenged by plant extracts, in particular excellent inhibition was measured by acetone extract Lacto bacillus (19 mm), Bacillus subtillus (19 mm) and Aspergillus niger (23 mm) respectively (Table 4). Table 4 Antimicrobial properties of Pterolobium hexapetalum seed against various pathogens Micro-organisms Zone of inhibition (mm) Control Hexane Ethyl acetate Acetone Methanol Lactobasillus Bacillus subtillus ф Aspergillus niger Gram +ve bacteria, ф- Gram ve bacteria, - fungus. The results presented above indicate the efficiency of the extracts being active against the tested organisms. The search for antimicrobials from natural sources has received much attention and efforts have been put in to identify compounds that are able to act as suitable antimicrobials agent to replace synthetic ones. Phytochemicals derived from Pterolobium hexapetalum seed products serve as a prototype to develop less toxic and more effective medicines in controlling the growth of micro organism [18, 19]. Figure 6 GC-MS chromatogram of Pterolobium hexapetalum seed methanol extract GC-MS analysis identified many useful constituents inferring the pharmacological properties of the methanol extract. The compounds present in Pterolobium hexapetalum seed were identified by GC-MS analysis and chromatogram was obtained in Fig. 6. The major components present in the seed extract like Methyl 11,14-octadecadienoate (RT ), hentriacontane (RT ), Decane,1,10-diiodo- (RT ), formyl-2,2,6-trimethyl-3-trans-(3-methyl-but-2-enyl)-5-cyclohexene (RT ) (Table 5). These phytoconstituents are used as anti-cancer, anti-inflammatory, antimicrobial, as well as safe and preventive the free radical mediated diseases [20]. The presence of various bioactive compounds in the Pterolobium hexapetalum seed justifies the use of whole plant for various ailments by traditional medicines. However, isolation of individual phytoconstituents and subjecting to the biological activity will definitely give fruitful results. From the results, it could be concluded that Pterolobium hexapetalum contains various bioactive compounds with immense applications. 147

8 Table 5: Constituents identified by GC-MS analysis of Pterolobium hexapetalum seed S. RT Name of the compound Molecular Formula MW Peak Area Biological Applications No % H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6- C 6H 8O Antifungal Activities methyl ,2,5-Oxadiazol-3 carboxamide, 4,4 -azobis-, 2,2 - dioxide C 6H 4N 8O Antifungal activities Bicyclo[2.2.1] heptan-2-one, 4,7,7-trimethyl-, C 11H 19N 3O Antimicrobial activities semicarbazone ,2,3-Benzenetriol C 6H 6O Anticryptococal, anti-fungal activities Methyl-4,5-tetramethylene-5-ethyl-2-oxazoline C 10H 17NO Antimicrobial activities a-d-glucopyranoside, a-d-glucopyranosyl C 12H 22O Antioxidant activities Hexadecanoic acid, methyl ester C 11H 22O Anti-bacterial, antifungal activities agent Octadecanoic acid, ethyl ester C 20H 40O Anti-inflammatory Octadecynoic acid C 18H 32O Analgesic, cardio-tonic, Anti-inflammatory, antibacterial activities ,12,15-Octadecatrienoic acid, (Z,Z,Z)- C 18H 30O Antioxidant, Antimicrobial, Antiinflammatory activities Pyridine-3-carboxamide, 1,2-dihydro-4,6-dimethyl- C 8H 10N 2OS Antimicrobial activities, Antifungal properties Acetohydrazide, 2-(3-hydroxy-2-pentylcyclopentyl)- C 12H 24N 2O Anticancer property Methyl-Z-tetradecan-1-ol acetate C 17H 32O Anti-viral, antifungal, Anticancer activities ,12,15-Octadecatrienoic acid, methyl ester (Z,Z,Z)- C 19H 32O Anti-inflammatory, Antioxidant property E-11-Hexadecenoic acid, ethyl ester C 18H 34O Antioxidant property Vitamin E C 29H 50O Antioxidant, Anti-aging, Cancer protecting Z,Z,Z 1,4,6,9-Nonadecatetraene C 19H Antibacterial activity (7-heptadecynyloxy)tetra hydro-2h Pyran C 22H 40O Anti-microbial activity. *Source: Dr.Duke s phytochemical and ethnobotanical databases [Online database] Conclusion Pterolobium hexapetalum seed has therefore been actively selected to demonstrate its promising potential against various ailments. Hence to sum up, a clear understanding about the relationship between the phenolic, flavonoid, carbohydrates, protein contents with that of antioxidant, antibacterial, antifungal studies have provided the potency of the Pterolobium hexapetalum seed extracts, acetone in particular. Isolation of new constituents responsible for such antioxidant and antimicrobial activities are interest. This may ultimately lead to the application of Pterolobium hexapetalum seed in various pharmaceutical formulations by the active and potent properties. Acknowledgements The Department of Science and Technology and University Grants Commission is gratefully acknowledged for the award of DST-INSPIRE [IF ] fellowship to K.K. References Ahmad I, Beg AZ, Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multiple drug resistant human pathogens. J. Ethanopharmacol. 74: Aiyegoro OA, Okoh AI, Preliminary phytochemical screening and in vitro antioxidant activities of aqueous extract of Helichrysum longifolium DC. BMC Comp Alternative Med, 10: Alagammal M, Tresina PS, Mohan VR GC-MS Determination of bioactive components of Polygala javana Dc. Int. J. Cur. Pharm. Res. 4(2): Barreira CM, Ferreira CFR, Beatriz M, Olliveira PP, Jose Alberto Pereira, Antioxidant activities of the extracts from chestnut flower, leaf, skins and fruit. Food Chem., 107: Bauer AW, Kirby MM, Sherris JC, Truck M, Antibiotic susceptibility testing by a standardized single disc method. American J. Clin. Pathol. 45: Farhat Al, Khan, Iqbal Hussain, Shahid Farooq, Majed Ahmad, Muhammad Arif, Inayat Ur Rehman, Phytochemical screening of some Pakistanian medicinal plants. Middle-East Journal of Scientific Research, 8 (3): Frankel EN, Neff WE, Selke E, Analysis of autoxidized fats by gas chromatography mass spectrometry: VII. Volatile thermal decomposition products of pure hydroperoxides from autoxidized and photosensitized oxidized methyl oleate, linoleate and linolenate. Lipids, 16: Halliwell B, Cross CE, Gutteridge JMC, Free radicals, antioxidants, and human disease: where are we now? Journal of Laboratory and Clinical Medicine, 119:

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