GC-MS Analysis and Antioxidant Activity of Bauhinia nakhonphanomensis Leaf Ethanolic Extract

Aims: Leaves of B. nakhonphanomensis were extracted and the extract was used in gas chromatography-mass spectrometry (GC-MS) analysis to evaluate the total phenols, total flavonoids and antioxidant activity. Methods: The extract of B. nakhonphanomensis was analyzed by GC-MS. Quantitative analysis for total phenols was done by the Folin-Ciocatteu method and for total flavonoids by the aluminium chloride method. The antioxidant activity of the ethanolic extract was evaluated by the DPPH method. Results: GC-MS analysis revealed the presence of 19 phytochemical constituents. These compounds were identified by comparing their retention times and peak areas with those from the literature and by interpretation of the mass spectra. The major chemical constituents were inositol (48.55 %), alpha-tocopherol (12.21 %) and phenol (6.61 %). Total phenolic content was 48.69±0.56 mg/100 of Gallic acid equivalent (GE). The total flavonoid contentwas 10539± 6.14 mg/100 of quercetin equivalent (QE). Antioxidant activity was 17.07±0.24 μg/100 of ascorbic acid equivalent antioxidant capacity (AEAC).Conclusion: These findings are the first report and suggest that the rich phytochemical content of B. nakhonphanomensis has good antioxidant activity.


INTRODUCTION
Plant secondary metabolites have been referred to as phytochemicals that are naturally occurring and have potential disease inhibiting capabilities. 1Phyto chemicalsare excellent sources of many bioactive compounds, such as volatile oils, steroids, alkaloids and natural antioxidants,i.e., flavonoids and other phenolic compounds, with beneficial effects on human health.Hence, the screening of active compounds and anti oxidant activity determination from plants have led to the discovery and development of novel drugs to be used against diverse diseases.Drugs from plants are easily available, less expensive, safe, and efficient and rarely have side effects. 2 The modern methods describing the identification and quantification of active constituents in plant material may be useful for proper standardization of herbal drug formulations.Mass spectrometry coupled with chromatographic separations, such as gas chromatography (GC/MS), is normally used for the direct analysis of the com ponents that exist in both traditional medicines and medicinal plants.In recent years, GCMS studies have been increasingly applied for the analysis of medicinal plants as this technique has proved to be a valuable method for the analysis of nonpolar components and volatile essential oils, fatty acids, lipids and alkaloids. 3auhinia is a large genus belonging to the subfamily Bauhinia nakhonphanomensis Leaf Ethanolic Extract.Pharmacog J. 2017;9(5):663-67.
Caesalpinioideae (Leguminosae).It consists of about 300 species and is distributed in pantropical regions of the world. 4,5Plants in the genus Bauhinia have char acteristic butterfly shaped leaves.Most Bauhinia spp.have applications in traditional medicine 6 , such as the bark of B. tomentosa that is used for the treat ment of inflammation, dysentery and skin diseases. 7he leaf ethanolic extracts of B. purpurea exhibited hypoglycemic and antioxidant activity. 8,9Bauhinia is well known for the therapeutic efficacies of its different species.In the last revision of the Flora of Thailand, there were 37 species reported. 4In the year 2013, B. nakhonphanomensis Chatan was collected from the Phulangkha National Park, Nakhon Phanom Province and reported as a new and endemic species to Thailand (Figure 1).This liana species is easy to recognize by having tendrils, acuminate or caudate leaf apices, entire leaf margin, oblong or elliptic floral buds, floral bud 2535 mm long raceme or panical inflorescence, 1013 mm long hypanthium and anther opening by longitudi nal sits. 10However, there are no scientific reports on the phytochemicals and antioxidants of B. nakhonphanomensis leaves.Therefore, the pres ent study aims to investigate the presence of phy tochemicals in an ethanolic extract of the leaves Determination of total flavonoid content Total flavonoid contents were measured using an aluminum chloride colorimetric assay. 12The extract (concentration 1 mg/ml) was mixed with 200 µl of distilled water and 100 µl of 5 % NaNO 2 solution.After 6 min, 200 µl of 10 % AlCl 3 solution was added.After 5 min, 500 µl of 1M NaOH and 275 µl of distilled water were added to prepare the mixture.The absorbance at 510 nm was recorded after 15 min of incubation.The total flavonoid content of the extracts was expressed as a percentage of quercetin equivalent per 100 g dry weight of sample.

Determination of antioxidant activity
The ability of the extract to scavenge DPPH radicals was determined according to the method described by Braca et al. (2001). 13The plant extract (0.1 mL) was added to 3 mL of a 0.004% methanol solution of DPPH.Absorbance at 517 nm was determined after 30 min.The percentage DPPH radical scavenging activity of each extract was determined using the formula % DPPH radical scavenging =[(A 0 -A 1 ) / A 0 ] × 100, where A 0 is the absorbance of the control and A 1 is the absor bance of the extract/standard.The inhibition curves were prepared and the IC 50 values were calculated. 14

GC-MS analysis
The leaf extract of B. nakhonphanomensis was analyzed for its chemical constituents by GCMS (GC 7890A Agilent Technology).The column (DB5) was fused silica 30 m x 0.25 mm ID × 0.25 µm film thickness.The oven temperature was programmed from 80°C @10°C/min to 200 °C @12°C/min to 260°C (30 min).Helium gas (99.999 %) was used as the carrier gas at a constant flow rate of 1ml/min and an injection volume of 1 µl was employed (split ratio of 10:1) at an injector tempera ture of 250°C; the ionsource temperature was set at 280°C.The com pounds were detected in the range 50550 amu.The molecular weight and structure of the compounds of the test materials were ascertained by interpretation of the mass spectrum of the GCMS using the database of the National Institute of Standards and Technology (NIST).

GC-MS analysis
The components present in the ethanol extract of the leaf of B. nakhonphanomensis identified by the GCMS chromatogram are shown in Figure 2. The active principles with their retention time (RT), molecular formula, molecular weight (MW) and peak area as a percentage are presented in Table 1.The GCMS analysis showed the presence of mainly three compounds at retention times of 8.12 (6.61%), 13.70 (48.55%) and 46.29 (12.21%), which were phenol, inositol and alpha-tocopherol, respectively.The major phytocompounds and their biological activities obtained through the GCMS analysis of the leaf of B. nakhonphanomensis have been tabulated Table 2.

Total phenolics and flavonoid content
The total phenolic contents of B. nakhonphanomensis determined by the FolinCiocalteu method of the ethanolic extract showed 48.69 ± 0.56.mg/100 of Gallic acid equivalent (GE), while the total flavonoid contents determined by the aluminium chloride method of the extract showed 10539 ± 6.14 mg/100 of quercetin equivalent (QE).The results of the phenolic contents and flavonoids contents are in Table 4.

Antioxidant activity
The extract showed potent DPPH radical scavenging activity.The ethanolic extract of the leaves was found to have an IC 50 value of 17.07 ± 0.24 µg/ml.The IC 50 value of the standard ascorbic acid was 7.88 ± 0.1 µg/ml (Table 3). of B. nakhonphanomensis and to study the antioxidant activity for the first time.This work will help to identify the compounds and antioxi dants that might have therapeutic value.

Plant collection and preparation of extract
Leaves of B. nakhonphanomensis were collected from Phulangkha Nation Park, Nakhon Phanom Province, Thailand during April to May, 2013.The voucher specimen (Chatan 1337) was identified and confirmed by the second author and deposited in Natural Medicinal Mushroom Museum (MSUT), Mahasarakham University, Thailandfor future reference.The fresh leaves were manually isolated.They were cleaned, air dried, powered and subjected to macerate extraction with ethanol.The extract was filtered through filter paper, an evaporator and dried by a lyophilizer.The crude extracts were analyzed by GCMS.

Determination of total phenolic content
The phenolic contents were determined by the FolinCiocalteau method.Briefly, 200 µl of the B. nakhonphanomensis extract at appropriated dilutions was mixed with 1 ml of 0.2 M FolinCiocalteu reagent.After leaving the solution in the dark at room temperature for 30 min, 800 µl of 7% sodium carbonate was added to it.The absorbance of the resulting blue color was measured at 756 nm.Phenolic contents were expressed as mg of Gallic acid equivalent (GAE)/g dry weight of extract. 11

DISCUSSION
The phytochemical analysis conducted on the extract of B. nakhonphanomensis revealed the presence of constituents that are known to exhibit medicinal as well as physiological activities.In the last few years, GC/MS has been the best technique used for screening, identification and quan tification of many susceptible compound in plant extracts. 1517GC/MS data revealed that the ethanoic extract of B. nakhonphanomensis contained three major chemical constituents, i.e., inositol, phenol and alpha tocopherol.Inositol is used for the treatment of Polycystic Ovary Syndrome (PCOS), as an antidiabetic and metabolic syndrome in postmenopausal women. 1820Phenol and alpha.tocopherolhaveanti oxidant uses in humans. 21Phenolic compounds are one of the largest and most ubiquitous groups of plant metabolites. 22Several studies have described the antioxidant properties of medicinal plants that had rich phenolic compounds. 23,24Natural antioxidants, which mainly come from plants, are in the form of phenolic compounds, such as flavonoids, phenolic acids and tocopherols. 25In this study, we demonstrated that the antioxidant activity of B. nakhonphanomensis was a very efficient free radical scavenger due to the lowest IC 50 value.The activity of the reference antioxidant (vitaminc) was much higher when compare with the ethanolic extract.The GCMS analysis of the ethanolic extract of B. nakhonphanomensis revealed the presence of phenol and alpha tocopherol.Therefore, this study can conclude that the leaf ethanoic extract of this plantis a good source of antioxidants.

CONCLUSION
The presence of antioxidant activity and GCMS analysis were the first steps towards understanding the nature of the active principles in this medicinal plant and phytochemically will be helpful for further detailed study.The importance of the study was to identify some of the biological activities of these compounds.The present study, which revealed the presence of components in B. nakhonphanomensis leaves, suggests a contribution from these compounds to pharmacological activity in the future.

ACKNOWLEDGEMENT
We gratefully acknowledge the Faculty of Science, Mahasarakham Uni versity 2015, Thailand, for financial support.Thanks for linguistic advice from Dr. Jolyon Dodgson, Faculty of Science, Mahasarakham Univer sity..

CONFLICT OF INTEREST
None.

Table 4 : Total phenol and flavonoid contents of B. nakhonphanomensis.
The values are means of three replicates.* Gallic acid equivalent, ** quercetin equivalent