Gas Chromatography-Mass Spectrometry Analysis and In vitro Antioxidant Activity of the Ethanolic Extract of the Leaves of Tabernaemontana divaricata

Objective: To identify phytoconstituents present in an ethanolic extract of the leaves of Tabernaemontana divaricata and to evaluate its in-vitro antioxidant potential. Methods: The extract was subjected to gas chromatography-mass spectrometry analysis to identify phytoconstituents, and screened for hydroxyl, superoxide and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power and metal-chelating activity as a measure of potential antioxidant activity. Results: GC-MS analysis of the extract revealed the presence of 96 phytoconstituents, of which 17 are reported to be bioactive and 11 of these to possess antioxidant potential. When tested in-vitro, the extract exhibited the most potent radical-scavenging activity at a maximum concentration of 10 mg/ml, scavenging effects of 64%, 67% and 69% and corresponding half maximal inhibitory concentration (IC50) values of 6.7 mg/ml, 6.8 mg/ml and 6.2 mg/ml on hydroxyl, superoxide and DPPH radicals, respectively. Ascorbic acid used as a standard (10 mg/ml) showed scavenging effects of 73%, 73% and 75% and corresponding IC50 values of 5.3 mg/ml, 5.8 mg/ml and 5.2 mg/ml, respectively, on hydroxyl, superoxide and DPPH radicals. At 10 mg/ml, the extract and an ethylenediaminetetraacetic acid standard exhibited 68% and 78%, respectively, chelation of ferrous ions; at the same concentration, the reducing power of the extract and that of a butylated hydroxytoluene standard was found to be 3.855 and 4.308, respectively. Conclusion: These observations strongly suggest that the ethanolic extract of T. divaricata leaves has potent in-vitro antioxidant activity and thereby could act as a possible therapeutic agent for oxidative stressinduced pathological states.


INTRODUCTION
An antioxidant is a compound that inhibits or retards the oxidation of substrates even if it is present in a significantly lower concentration than that of the oxidised substrate. 1A possible mechanism of antioxidant activity is scavenging of reactive oxygen species (ROS); others include the prevention of ROS formation by binding of metal or inhibition of enzyme.ROS may initiate several human degenerative diseases, and antioxidants may exert defensive and therapeutic effects on many metabolic disorders. 2Interestingly, clinical and epidemiologic studies in some cases have pointed out that antioxidant nutrients may be effective in disease prevention. 3] The developed world is witnessing increasing interest in complementary and alternative medicine (CAM), particularly herbal remedies.Herbal medicines include herbs, herbal materials, herbal preparations, and finished herbal products that contain parts of plants or other plant materials as active ingredients. 124] So also, due to synergistic effects, medicinal plants are reported to be more biologically active than their purified constituents. 15abernaemontana divaricata (T.divaricata) is one such medicinallyimportant plant belonging to the family Apocynaceae. 168] Jain et al. 19 also reported that aqueous and ethanolic extracts of T. divaricata leaves were able to scavenge superoxide radicals in-vitro.Hence, in the present investigation, an attempt was made to identify bioactive constituents of the leaves of T. divaricata and to elucidate various antioxidant characteristics such as potential radicalscavenging and metal-chelating activities and reducing power of the leaf extract.

Preparation of an ethanolic extract of T. divaricata leaves
The leaves of T. divaricata plant were collected from within the Bharathidasan University campus, Tiruchirappalli, Tamilnadu, India.The plant was identified by the Rapinat Herbarium and Centre for Molecular Systematics, St.Joseph's College, Tiruchirappalli, India.The voucher specimen (No: KV 001) has been preserved in the Department of Animal Science, Bharathidasan University, Tiruchirappalli, India.The leaves (fresh and disease-free) were shade-dried and finely powdered.Thirty grams of the powder were extracted with 300 ml of Pharmacognosy Journal, Vol 8, Issue 5, Sep-Oct, 2016 95% ethanol using a Soxhlet apparatus.The solvent was evaporated under reduced pressure at 55-60°C and dried in a vacuum.The residue was filtered and concentrated to a dry mass by vacuum distillation.This dried ethanolic extract of T. divaricata was used for further analyses.

GC-MS analysis of the T. divaricata extract
The ethanolic extract of T. divaricata underwent gas chromatography-mass spectrometry (GC-MS) analysis (GC-MS -QP-2010 Plus, Shimadzu, Tokyo, Japan) with the thermal desorption (TD) system 20.Experimental conditions of the GC-MS system were as follows: Trace-5 mass spectrometry capillary standard non-polar column, dimension: 30 meters; internal diameter: 0.25 mm; film thickness: 0.25 μm.The flow rate of the mobile phase (carrier gas: helium) was set at 1.2 ml/min.In the gas chromatography phase, the temperature programme (oven temperature) was 80°C, which was raised to 250°C at 10°C/min, and the injection volume was 1 μl.Samples dissolved in chloroform were run fully at a range of 50-650 mass-to-charge ratio (m/z) and the results were compared by using the Wiley Spectral Library Search Programme (http:// www.sisweb.com/software/ms/wiley-search.html).

Determination of putative in vitro antioxidant activities of the ethanolic extract of T. divaricata leaves Determination of possible hydroxyl radicalscavenging activity in the T. divaricata extract
The hydroxyl radical (OH -)-scavenging capacity of the T. divaricata extract was measured according to the modified method of Halliwell et al. 20 Stock solutions of ethylenediamine tetraacetic acid (EDTA, 1 mM), ferric chloride (FeCl 3, 10 mM), ascorbic acid (1 mM), hydrogen peroxide (H 2 O 2, 10 mM) and deoxyribose (10 mM) were prepared in distilled and deionized water.The assay was performed by adding 0.1 ml of EDTA, 0.01 ml of FeCl 3, 0.1 ml of H 2 O 2, 0.36 ml of deoxyribose, 1.0 ml of the extract (2 to 10 mg/ ml) dissolved in distilled water, 0.33 ml of phosphate buffer (50 mM, pH 7.4) and 0.1 ml of ascorbic acid, in sequence.This mixture was then incubated at 37°C for 1 hour.A 1.0 ml portion of the incubated mixture was mixed with 1.0 ml of 10% trichloroacetic acid (TCA) and 1.0 ml of 0.5% thiobarbituric acid (TBA) [in 0.025M sodium hydroxide (NaOH) containing 0.025% butylated hydroxyl aniline (BHA)] to develop the pink chromogen which was measured at 532 nm.L-ascorbic acid was used as a standard.The OH --scavenging activity of the extract was reported as % inhibition of deoxyribose degradation and was calculated as where A cont is the absorbance of the control reaction and A test is the absorbance of the mixture containing the extract or the absorbance of a standard solution.

Determination of putative superoxide radicalscavenging activity in the T. divaricata extract
The superoxide anion radical-scavenging capacity of the T. divaricata extract was measured essentially as described by Liu et al. 21using a minor modification proposed by Rajeshwar et al. 22 The principle of this method is that superoxide radicals are generated in phenazine methosulphate (PMS) -nicotinamide adenine dinucleotide (NADH) systems by oxidation of NADH and assayed by the reduction of nitroblue tetrazolium (NBT).In this experiment, the superoxide radicals were generated in 3.0 ml of Tris-HCl buffer (16 mM, pH 8.0) containing 1.0 ml of NBT (50 μM) solution, 1.0 ml of NADH (78 μM) solution and samples of the extracts (2 to 10 mg/ml) in water.The reaction was initiated by adding 1.0 ml of PMS (10 μM) solution to the mixture.The reaction mixture was incubated at 100°C for 5 minutes and the absorbance was measured at 560 nm against a blank.L-ascorbic acid was used as a standard.Decreased absorbance of the reaction mixture indicated increased superoxide anion-scavenging activity.The percentage (%) inhibition of superoxide anion generation was calculated using the same formula that was used to calculate the OH --scavenging activity.
Determination of possible 1, 1-diphenyl-2picrylhydrazyl (DPPH) radical-scavenging activity in the T. divaricata extract The potential of the T. divaricata extract to scavenge the stable radical DPPH (1, 1-diphenyl-2-picrylhydrazyl) was measured by the method described by Kikuzaki and Nakatani, 23 with slight modifications.In this experiment, 1.0 ml of 0.1 mM DPPH prepared in methanol was mixed with 1.0 ml of the extract (ranging from 2 to 10 mg/ml).The reaction mixture was shaken vigorously and left in the dark at room temperature for 30 minutes.The absorbance was then measured at 517 nm against a blank; L-ascorbic acid was used as a standard.Decreased absorbance of the reaction mixture indicated higher free radical-scavenging activity.DPPH radical-scavenging activity was calculated using the same formula as that used to calculate the OH --scavenging activity.

Determination of putative reducing power in the T. divaricata extract
The putative reducing power of the T. divaricata extract was determined by the method of Oyaizu. 24Various concentrations of the extract (2 to 10 mg/ml) in deionized water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 1% potassium ferricyanide (2.5 ml).The mixture was incubated at 50°C for 20 minutes.Aliquots of TCA (2.5 ml, 10%) were added to the mixture, which was then centrifuged at 1036xg for 10 minutes.The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and a freshly-prepared FeCl 3 solution (0.5 ml, 0.1%).The absorbance was measured at 700 nm.Increased absorbance of the reaction mixture was interpreted as an increase in reducing power.The standard, BHT, was also processed by the same procedures.

Determination of possible metal-chelating activity in the T. divaricata extract
The potential of the extract of T. divaricata to chelate ferrous ions was measured by the method of Dinis et al. 25 Briefly, the extract (2 to 10 mg/ml) was added to a solution of 2 mM FeCl 2 (0.05 ml).The reaction was initiated by the addition of 5 mM ferrozine (0.2 ml), and the mixture was then shaken vigorously and left to stand at room temperature for 10 minutes.The absorbance of the solution was measured spectrophotometrically at 562 nm.EDTA was used as a standard.The percentage (%) inhibition of ferrozine-Fe 2+ complex formation was calculated as [(A 0-A 1 )/A 0 ] × 100 where A 0 was the absorbance of the control, and A 1 was the absorbance of the mixture containing the extract or the absorbance of a standard solution.

Statistical Analysis
Results are presented as mean ± standard deviation of three measurements.Statistical analysis of the data was performed by Student't' test using SPSS (Statistical Package for Social Sciences) software package for Windows (version 16.0;IBM Corp., Armonk, NY, USA).P values <0.05 were regarded as significant.

Scavenging effect of the T. divaricata extract on hydroxyl radicals
In the present study, the T. divaricata leaf extract and the ascorbic acid standard were found to inhibit OH --mediated deoxyribose degradation in the reaction mixture.The OH --scavenging effect of the T. divaricata extract (10 mg/ml) was 64% while that of ascorbic acid (10 mg/ml) was 73%; this difference was statistically significant (p<0.05)(Figure 2).The IC 50 values were found to be 6.7 mg/ml and 5.3 mg/ml, respectively, for the extract of T. divaricata and for ascorbic acid.

Scavenging effect of the T. divaricata extract on superoxide anion radicals
The % inhibition of superoxide radical generation by the ethanolic extract of T. divaricata leaves (10 mg/ml) and that of the standard ascorbic acid solution (10 mg/ml) were found to be 67% and 73%, respectively (Figure 1-6), as measured by the riboflavin-light system in-vitro.The IC 50 values were found to be 6.8 mg/ml and 5.8 mg/ml, respectively, for the extract of T. divaricata and for ascorbic acid.

Scavenging effect of the T. divaricata extract on DPPH radicals
The T. divaricata leaf extract exhibited significant DPPH radicalscavenging effects with increasing concentrations (2-10 mg/ml), when compared with that of ascorbic acid (Figure 4).The % scavenging of DPPH by the T. divaricata extract (10 mg/ml) and that of ascorbic acid (10 mg/ml) was found to be 69% and 75%, respectively, while the IC 50 values were found to be 6.2 and 5.2 mg/ml, respectively.

Metal-chelating activity of the T. divaricata extract
The iron-binding capacity of the T. divaricata leaf extract was compared to that of a known metal chelator, namely EDTA.The chelating effect of the extract and that of EDTA on ferrous ions was found to increase with increasing concentrations (Figure 6).The extract (10 mg/ml) and EDTA (10 mg/ml) exhibited chelating effects of 68% and 78%, respectively (Figure 6).50% chelation of ferrous ions was achieved by the extract and by EDTA in concentrations of 5.4 mg/ml and 4.6 mg/ml, respectively.

DISCUSSION
T. divaricata, which is used in Chinese, ayurvedic and Thai traditional medicine, has been reported to exhibit diverse medicinal properties. 18,26n the current investigation, an ethanolic extract of T. divaricata leaves was screened to detect phytochemical constituents and putative antioxidant components.GC-MS analysis of the extract (Table 2) revealed the presence of 11 key compounds, which have previously been reported to possess antioxidant properties (Table 1).The major constituent detected in the extract of T. divaricata, namely n-hexadecanoic acid (area percentage 11.18%)(Table 2), has also been reported in an ethanolic extract of Eugenia flocossa leaves 27 and in a methanolic extract of Vitex negundo leaves 28 to possess antioxidant, anti-androgenic and hypocholesterolemic properties.Phytol (2.97%), yet another major constituent (Table 2), has been reported to possess antioxidant properties and to reduce free-radical generation in an in-vitro experimental system. 29umar et al. 28 have also reported that a methanolic extract of Vitex negundo, which was found to have phytol as one of the major constituents, possessed antimicrobial, anti-inflammatory and anti-cancerous properties.
The third major constituent detected in the T. divaricata extract, namely 9, 12, 15-octadecatrienoic acid (Z, Z, Z)-(area percentage 1.47%) (Table 2), is a linolenic acid; it has been reported to exhibit antioxidant activity in the methanolic extract of Vitex negundo leaves. 28The phytoconstituent, hexadecanoic acid ethyl ester (1.28%) (Table 2), has been reported to possess antioxidant potential in an ethanolic extract of Mussaenda frondosa. 30Stigmast 5-en-3-ol (0.99%) and tetradecanoic acid (0.66%), detected in the extract of T. divaricata in the current study (Table 2), are also reported to occur in a methanolic extract of Aegle marmelos and to exhibit antimicrobial, anti-inflammatory, antiarthritic, anti-fungal and anti-cancerous properties. 31Minor components identified in the ethanolic extract of T. divaricata leaves in the present study (Table 2) have also been reported to exhibit antioxidant potential.These include: 9, 12-octadecadienoic acid (Z,Z)-(0.83%),a conjugated linoleic acid reported to occur in an ethanolic extract of Phragmytes vallatoria leaves; 32 2-pyrrolidinone (0.38%), reported in Brassica oleraceae var.capitata; 33 Vitamin-E (0.32%), a potential antioxidant that protects the cell against free radicals; 28,31 hexadecanoic acid methyl ester (0.23%), that has been detected in the methanolic fraction of Aegle marmelos 31 and a well-reported antioxidant compound, retinol (0.17%), that inhibits lipid peroxidation. 34Thus, 11% of the phytoconstituents identified in the ethanolic extract of T. divaricata leaves in the current study have previously been reported to exhibit antioxidant activity.Hence, we hypothesize that these phytoconstituents may contribute to potential antioxidant activity of the extract of T. divaricata leaves.To validate this hypothesis, we tested the T. divaricata leaf extract for hydroxyl, superoxide, and DPPH radical-scavenging activity, reducing power and metal-chelating activity.Hydroxyl radicals can be formed by the Fenton reaction in the presence of reduced transition metals (such as Fe 2+ ) and H 2 O 2 , which is known to be the most reactive of all the reduced forms of dioxygen and is thought to initiate cell damage in-vivo. 35The potential of an ethanolic extract of T. divaricata leaves to inhibit OH --mediated deoxyribose damage was assessed by means of an iron (II)-dependent DNA damage assay in the current study.The extract of T. divaricata was found to exhibit maximum OH -radical-scavenging activity at a concentration of 10 mg/ml; moreover, the capacity to scavenge OH -radicals increased with increasing concentrations of the extract and of ascorbic acid (Figure 2).Similar OH --scavenging activity has been reported in several other plant extracts, including Stevia rebaudiana, 36 Agaricus bisporus, 37 and Greek oregano. 38he superoxide radical (O 2 - ) is known to be very harmful to cellular components since it is a precursor of ROS. 39Photochemical reduction of flavin generates O 2 -, which can reduce NBT, resulting in the formation of blue formazan. 40In the present study, the extract of T. divaricata was found to be a notable scavenger of O 2 -radicals generated in the riboflavin-NBT light system.In a concentration of 10 mg/ml, the extract inhibited the formation of blue formazan at a percentage inhibition of 67%; moreover, the percentage of scavenging O 2 -(O 2 -being scavenged leading to inhibition of blue formazan formation) increased with increasing concentrations of the extract and of ascorbic acid (Figure 3).Ascorbic acid, a natural antioxidant, was used as a positive control for comparison.Interestingly, the O 2 --scavenging potential of the extract of T. divaricata has also been reported earlier by Jain et al. 19 The ethanolic extract of Caesalpinia bonducella seeds has been reported to possess similar O 2 --scavenging effects. 41he DPPH radical is considered to be a model for a lipophilic radical.A chain of lipophilic radicals is initiated by lipid auto oxidation.DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. 42In the DPPH assay, antioxidants are able to reduce the stable DPPH radical to the non-radical form DPPH-H.When the odd electrons are paired off in the presence of a free radical-scavenger, the absorption is reduced and the color of the DPPH solution changes from purple to yellow.Measurement of the degree of reduction in absorbance at 517 nm suggests the radical-scavenging potential of a compound. 43In the present study, the DPPH radical-scavenging capacity of the extract of T. divaricata leaves was found to increase in a dose-dependent manner, as did that of ascorbic acid, although it was significantly less (p<0.05).Antioxidants present in the extract possibly effected the increased scavenging of DPPH radical.Such activity has also been reported in several other plant extracts, including Aframomum corrorima (Braun), 44 Stevia rebaudiana, 36 Chromolaena odorata 45 and Agaricus bisporus. 37emonstration of reducing power is based on the principle of increase in the absorbance of reaction mixtures.In this method, an antioxidant compound forms a colored complex with potassium ferricyanide, TCA and FeCl 3 , which is measured at 700 nm.Increase in the absorbance of the reaction mixture provides a measure of the reducing power of the sample, suggesting its antioxidant potential. 46In the present study, the method of Oyaizu 24 was adopted to measure Fe 3+ -Fe 2+ transformation in the presence of T. divaricata leaf extract.We observed that the reducing power of the extract of T. divaricata and that of the standard, BHT, steadily increased with increase in concentrations (Figure 5); the reducing power of the extract was also found to approximate the reducing capacity of the standard BHT.Duh et al. 47 reported that the reducing properties of antioxidants are generally associated with the presence of reductones, such as ascorbic acid.Such reductones exert antioxidant activity by breaking the free radical chain by donating hydrogen atoms; 48 reductones have also been reported to react with certain precursors of peroxide, thus preventing peroxide formation. 49Such a mechanism was possibly effected by the antioxidants present in the extract of T. divaricata, therein increasing its reducing power.Extracts of other plants, such as those of Aloe vera, 50 Barleria prionitis, 51 Caesalpinia crista, 52 and Agaricus bisporus, 53 have also been reported to exhibit reducing power, suggesting their antioxidant potential.Transition metals are believed to catalyze the initial formation of radicals.Chelating agents may stabilize transition metals in living systems and inhibit generation of radicals, consequently reducing free radicalinduced damage.The principle of this method is that ferrozine can quantitatively form red-colored complexes with Fe 2+ .In the presence of chelating agents, the formation of such complexes is disrupted, leading to a decrease in the rate of formation of red-colored complexes.Therefore, measurement of the rate of color reduction allows an estimation of chelating activity of the co-existing chelator. 54To better understand how the T. divaricata extract inhibited generation of radicals, its chelating activity was evaluated against that of Fe 2+ .In the current investigation, at a concentration of 10 mg/ml, the extract, as well as the standard EDTA, were found to chelate 68% and 78% of ferrous ions, respectively; the chelating effect was found to gradually increase with increasing concentrations (Figure 6).Chua et al. 55 reported that an ethanolic extract of Cinnamomum osmophloeum chelated 66.4% of ferrous ions at a concentration of 2.5 mg/ml.A methanolic extract of Black stinkhom, at a concentration of 24 mg/ml, was found to chelate 46.4% -52.0% of ferrous ions 56 and an ethanolic extract of Pleurotus citrinopileatus chelated 22.30% of ferrous ions at 20 mg/ml. 57The results of the present study suggest that an ethanolic extract of T. divaricata exhibits good chelating activity on ferrous ions, thereby preventing the generation of free radicals.The observed hydroxyl, superoxide and DPPH-radical scavenging potential, reducing power and metal-chelating activity of the T. divaricata extract were possibly due to the presence of perceptible quantities of antioxidant constituents.

CONCLUSION
The results of the present study suggest that an ethanolic extract of T. divaricata leaves possesses phytoconstituents with antioxidant potential, which is manifested by concentration-dependent scavenging of hydroxyl, superoxide and DPPH free radicals, reducing power and chelating of ferrous ions.The relevance of these results in the context of medicinal use of the extract of T. divaricata leaves requires further study.

Figure 2 :
Figure 2: Scavenging effect of an ethanolic extract of Tabernaemontana divaricata leaves on hydroxyl radicals compared to that of an ascorbic acid standard.

Figure 3 :
Figure 3: Scavenging effect of an ethanolic extract of Tabernaemontana divaricata leaves on superoxide radicals compared to that of an ascorbic acid standard.

Figure 5 :
Figure 5: Reducing power of an ethanolic extract of Tabernaemontana divaricata leaves compared to that of a butylated hydroxytoluene (BHT) standard.

Figure 6 :
Figure 6: Chelating effect of an ethanolic extract of Tabernaemontana divaricata leaves on ferrous ions compared to that of an ethylenediaminetetraacetic acid (EDTA) standard.