Chemical Profile and Antioxidant Properties of Andrographis producta (C. B. Clarke) Gamble

Andrographis Wall. ex Nees (Family Acanthaceae) consisting 45 species globally according to International Plant Name Index (IPNI)1 and 28 species in India where most of them have distributional range in South Indian states namely Karnataka, Tamil Nadu, Kerala and Andhra Pradesh.2,3 Andrographis paniculata Nees (Kalmegh) is therapeutically well known species in the genus distributed throughout India and other South East Asian countries. Kalmegh has been used to treat fever, cough, sore throat, gastric infections and other acute diseases and is as prime ingredient in several Ayurvedic formulations.4 The plant possesses wide range of pharmacological effects including antimalarial, anticancer, antioxidative, anti-inflammatory, antidiabetic, immunomodulatory and antiretroviral activities. Andrographolide is major diterpene lactone in the plant and neoandrographolide, 14-deoxy11,12-didehydroandrographolide, andrographon, 14-deoxyandrographolide are other bioactive compounds contributing to pharmacological activities together with some flavonoids.4,5


INTRODUCTION
Andrographis Wall. ex Nees (Family Acanthaceae) consisting 45 species globally according to International Plant Name Index (IPNI) 1 and 28 species in India where most of them have distributional range in South Indian states namely Karnataka, Tamil Nadu, Kerala and Andhra Pradesh. 2,3 Andrographis paniculata Nees (Kalmegh) is therapeutically well known species in the genus distributed throughout India and other South East Asian countries. Kalmegh has been used to treat fever, cough, sore throat, gastric infections and other acute diseases and is as prime ingredient in several Ayurvedic formulations. 4 The plant possesses wide range of pharmacological effects including antimalarial, anticancer, antioxidative, anti-inflammatory, antidiabetic, immunomodulatory and antiretroviral activities. Andrographolide is major diterpene lactone in the plant and neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, andrographon, 14-deoxyandrographolide are other bioactive compounds contributing to pharmacological activities together with some flavonoids. 4,5 Andrographis producta (C. B. Clarke) Gamble is an undershrub reaching 150 cm in height. It is indigenous to South Indian states namely Karnataka, Kerala and Tamil Nadu 2 and grows frequently in the edges of shola forests and often high elevated hill forests. The leaves are lanceolate-elliptic, acute-acuminate, sub-membranous, strigose on adaxial surface, 10-20 cm in long. The inflorescence is branched, rather terminal panicle with purplish white flowers. Corolla decurved, with a prominent ventricose protuberance.
Modern technologies and scientific advancements have been facilitating the discovery of synthetic drugs which helped in the treatment and control of even dreadful diseases but prolonged medication of synthetic drugs may lead to toxic side effects in human. The demand for the herbal based medicines, pharmaceuticals, nutraceuticals, food supplements and herbal cosmetics is growing worldwide due to the numerous benefits including non-toxicity, less/ no side effects, better compatibility with human physiology and affordable costs over synthetic drugs. 6 Hence, identification of ethnopharmacologically important plants and extraction, isolation and characterization of chemical compounds using sophisticated chromatography techniques such as GC-MS, LC-MS and HPLC methods can help to find out biologically active compounds.
Andrographis producta is lesser known medicinally important plant of the genus used by tribal/native people in the Western Ghats region where it has been called with regional name 'Chevidantu' . The plant has been used to treat skin diseases. 7 During the present study, we found that the leaf decoction is used to relieve the constipation, to control the intestinal worms in children and also used to eliminate phlegm in women during postpartum. Though species belongs to the medicinally important genus, the investigation on the chemical composition, antioxidant properties and pharmacological activities have not been carried out till date. Hence, in the present work, an attempt has been made to explore the chemical compounds present in the different parts of the plant i.e. root, stem and leaves using spectrophotometric and GC-MS technique. Further, antioxidant potential of root, stem and leaves has been assessed by DPPH free radicals scavenging and FRAP assays to gather comprehensive information. Therefore, the present study aimed to investigate the available chemical compounds in root, stem and leaves of A. producta and their antioxidant properties.

Preparation of extracts
The plants were separated into different parts viz. root, stem and leaves were shade dried and ground to a coarse powder in blender followed by drying at 35 °C in hot air over 24 h. 10 g powder of each material was extracted with 100 ml methanol individually with Soxhlet extractor at 55-60 °C for 8 h. The extracts of root, stem and leaf were dried to evaporate the excess solvent using rotary evaporator under reduced pressure at 40 °C and used in experiments.

Determination of total phenolics
The amount of total phenolics in the methanolic extracts of root, stem and leaf was analyzed spectrophotometrically, as described by Folin and Ciocalteau 8 with slight modifications. The methanolic extracts (0.5 ml) were taken in test tubes containing 2.5 ml of deionized distilled water and mixed with 0.1 ml (2N) of Folin-Ciocalteau (FC) reagent. The mixture was allowed to stand for 6 min before adding 0.5 ml of 20 % sodium carbonate solution. The absorbance of the developed color after 30 min of incubation at room temperature was detected at 760 nm on UV visible spectrophotometer (UV-1601, Shimadzu). The amount of total phenolic content was calculated by comparing to the standard curve of gallic acid and expressed as the mg gallic acid equivalent (GAE) per g of dry samples.

Determination of flavonoids
The flavonoid content in the extracts of root, stem and leaf was determined spectrophotometrically 9 with some modifications. 0.5 ml of methanolic extracts of each samples were mixed with 2.5 ml of distilled water and solution of 0.15 ml sodium nitrite (5%) was added to take place reaction for 6 min. Then, 0.3 ml of 10 % aluminum chloride was added to take up the reaction for further 5 min. Finally, 2 ml of 1M sodium hydroxide was mixed and absorbance was read immediately on a spectrophotometer (UV-1601, Shimadzu) at 510 nm. The standard calibration curve of quercetin was used to determine the amount of flavonoid contents and expressed as mg of quercetin equivalent (QE) per g of dry samples.

Determination of tannins
The methanolic extracts were used in determination of tannins present in the samples by spectrophotometrically according to the method developed by Schenderi. 10 Each extracts (0.5 ml) were mixed with 2.5 ml of distilled water and 0.25 ml of Folin-Denis reagent, followed by 0.5 ml of 30 % sodium carbonate. Then all the reagents were mixed well to complete reaction and incubated for 30 min at room temperature. The absorbance of developed color was measured at 700 nm using spectrophotometer (UV-1601, Shimadzu). The known amount of tannic acid was used to draw the calibration curve and amount of tannin in samples determined and expressed as mg of tannic acid equivalent (TAE) per gram of dry samples.

Gas chromatography and mass spectrometry (GC-MS) analysis
Gas chromatography and mass spectrometry (Model: QP2010S; Shimadzu Corporation, Japan), a sophisticated analytical instrument equipped with Rxi-5Sil MS capillary column (length 30 m × 0.25 mm ID, 0.25 µm film thickness) was used to separate the chemical compounds present in the methanolic extracts of the samples. Helium (99.9995 %) was the carrier gas with a constant flow rate of 1 ml/min. the column oven temperature was held at 60 °C initially and increased to 260 °C by 5 °C/min held for 5 min. The diluted samples of1 µl were injected in the split injection mode and the solvent delay was 4 min. The total run time was of 30 min. The ion source and interface line temperatures were set at 200 °C and 280 °C, respectively. 11 The separated components were identified by comparing the retention times of authentic compounds and mass spectra from NIST 11 & WILEY 8 mass spectral libraries.

DPPH radicals scavenging assay
The free radical scavenging activity of the extracts was measured in vitro by 2, 2-diphenyl-1-picryl hydrazyl (DPPH) assay. 12 The stock solution was prepared with 24 mg DPPH in 100 ml methanol. The working solution was obtained by diluting DPPH stock solution with methanol to attain an absorbance of about 0.99 ± 0.02 at 515 nm on spectrophotometer (UV-1601, Shimadzu). Aliquots (3 ml) of this working solution (DPPH) were mixed with 0.1 ml sample of various concentrations (0.2-1 mg/ml). The reaction mixture was shaken well and incubated in the dark for 30 min at room temperature. Then the absorbance was recorded at 515 nm. Simultaneously, the control was prepared without any sample. ascorbic acid and butylated hydroxyl anisole (BHA) were used as standards. The percentage of DPPH scavenging activity was calculated using the following equation.

Ferric reducing ability of plasma (FRAP) assay
The FRAP assay was carried out according to the method developed by Benzie and Strain. 13 The different concentration (0.2-1.0 mg/ml) of 0.1 ml plant extracts were added to 3 ml of freshly prepared FRAP reagent (300 mM acetate buffer at pH 3.6, 10 mM 2, 4, 6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl and 20 mM FeCl 3 in the ratio 10:1:1) and heated to 37 °C in an hot water bath for 10 min. Final volume was adjusted to 4 ml using distilled water and incubated in the dark at room temperature for 10 min. Optical density was measured at 593 nm. Ascorbic acid and butylated hydroxyl anisole were used as standards. The increase in the optical density (OD) indicated greater the antioxidant power.
All experiments were conducted in triplicates using three different lots. Mean, percentage, EC 50 values and standard deviations were calculated using Microsoft Office Excel 2007.

Phytochemical analysis
The phytochemicals such as total phenolics, flavonoids and tannins were quantified in the root, stem and leaves of A. producta and expressed as gallic acid equivalent (GAE), quercetin equivalent (QE) and tannic acid equivalent (TAE), respectively. Amount of total phenolics, flavonoids and tannins in the root extract were 94.60 ± 0.51 mg/g DW, 33.50 ± 0.51 mg/g DW and 70.11 ± 0.12 mg/g DW respectively. The stem extract comprised of 163.61 ± 0.45 mg/g DW of phenolics, 35.11 ± 0.53/g DW of flavonoids and 84.52 ± 0.07 mg/g DW of tannins. The spectrophotometric quantifications affirmed that 109.75 ± 0.10 mg GAE/g DW of phenolics in higher proportion followed by 74.78 ± 0.27 mg/g DW of tannins and 32.26 ± 0.96 mg/g DW of flavonoids in the leaves of A. producta.

Gas chromatography and mass spectrometry (GC-MS) analysis
GC-MS analysis was performed to get insight into chemical profiles of root, stem and leaves of A. producta. Chromatograms of root, stem and leaf extracts (Figure 1) revealed that 30 (Table 1), 28 (Table 2) and 64 (Table 3) spectral peaks, respectively.

In vitro antioxidant capacities
DPPH radicals scavenging assay DPPH free radicals were deep purple colored due to their lone pair of electrons and the antioxidants present in the samples scavenge the lone pair of electrons that decolorize the DPPH containing solution. Thus, the decreasing in the purple color is directly linked to the antioxidant potential of methanolic extracts of the samples. The concentration dependent scavenging activity against DPPH radicals was expressed in mg/ml EC 50 value (Figure 2A). The lower the EC 50 value indicated higher the radical scavenging activity and vice versa. The methanolic extracts of root, stem and leaf exhibited DPPH radical scavenging capacity with EC 50 value of 5.02 mg/ml, 3.58 mg/ml and 7.02 mg/ml respectively, which were less than ascorbic acid (2.85 mg/ml) and BHA (3.22 mg/ml) (Figure 2A).

Ferric reducing ability of plasma (FRAP) assay
FRAP assay was rapid and sensitive to assess antioxidant power with a range of concentration. Under low pH, the colorless ferrictripyridyltriazine (Fe 3+ -TPTZ) complex reduced to an intense blue complex, ferrous-tripyridyltriazine (Fe 2+ -TPTZ) by the antioxidants present in the sample on dose-response relationship. The higher in the color development indicated more amount of antioxidants. Antioxidant power of standard ascorbic acid and BHA reached maximum efficacy at low concentration i.e. 1.986 ± 0.02 OD. at 0.2 mg/ml and 2.116 ± 0.04 OD at 0.4 mg/ml respectively, but dose-response of each samples tested was linear ( Figure 2B) with change in concentration. The methanolic extracts of stem showed good antioxidant power i.e. 0.63 ± 0.015 OD and 1.742 ± 0.02 OD at 0.2 mg/ml and 1mg/ml of concentration respectively. The order of antioxidant power of samples was stem > root > leaf ( Figure 2B).

DISCUSSION
Herbal products of Andrographis paniculata are in higher demands in herbal and phamceutical market and it amounts to 2197.3 tons per year (in the year 2005-2006). 14 It has been highly traded and prioritized medicinal plant in the Asian countries that has lead to decline in the availability of natural populations. 14,15 The over-exploitations of A. paniculata have created the necessity to search for alternatives to relieve the burden. In present study, we quantified the total phenolics, flavonoids and tannins in root, stem and leaf from A. producta. The total phenolics (163.61 mg GAE/g), flavonoids (35.11 ± 0.53 mg QE/g) and tannins (84.52 ± 0.07 mg TAE/g) were highest in stem compared   Insecticidal, trypanocidal, oxidative stress, cytotoxic and genotoxic effects 24 25,26 5 Apigenin 7,4'-dimethyl ether Leaf Antifungal activity 27 6 Asaraldehyde Root and stem Antiobesity effects 28 7 Azulene Root, stem and leaf Anti-inflammatory activity 29 8 Cinnamic acid Root, stem and leaf Antibactearial, antioxidant, anti-inflammatory, and antidiabetic activity  Table 4: List of biologically important chemical compounds indentified in Andrographis producta by GC-MS.

10
Methyl cinnamate Root Larvicidal activity 35 11 Neophytadiene Leaf Anti-inflammatory effects 36 12 Phytol Root, stem and leaf Anti-inflammatory and immune-modulating properties 37 38 13 Pinostrobin chalcone Leaf Anti-obesity effects 39 14 Salvigenin Root, stem and leaf Neuroprotective effects 40 15 Solanesol Root Neuroprotective effects and antioxidant activity to leaf and root. The total phenolic and tannins were relatively less in roots (i.e. 94.60 ± 0.51 mg GAE/g and 70.11 ± 0.12 mg TAE/g, respectively) than the leaf (i.e. 109 ± 0.10 mg GAE/g and 74.78 ± 0.27 mg TAE/g, respectively) extract. Similarly, Rafat et al. 16 reported the phenolic content variation in ethanolic extract of stem, leaf and fruits of A. paniculata which were 55.02 ± 0.35 mg of GAE/g, 75.86 ± 0.82 mg of GAE/g and 181.00 ± 1.48 mg of GAE/g respectively. However, amount of phenolic content was superior in stem (163.61 mg GAE/g) and leaf (109 ± 0.10 mg GAE/g) of A. producta compared to that of A. paniculata.
The polyphenols such as total phenolics and flavonoids derived from the plants have been more effective antioxidants. 17 The DPPH radical scavenging activity was highest in the stem samples with 3.58 mg/ ml EC 50 value ( Figure 2A) which was comparable to EC 50 values of standard ascorbic acid (3.22 mg/ml) and BHA (2.85 mg/ml). The high amount of total phenolics (163.61 mg GAE/g) and flavonoids (35.11 ± 0.53 mg QE/g) were found in stem might have contributed to antioxidant effects. Root and leaf extracts were exhibited less radical scavenging activity than stem with EC 50 values of 5.02 mg/ml and 7.02 mg/ml of concentration respectively (Figure 2A). Further, FRAP assay affirmed the antioxidant effects of each sample as displayed by DPPH assay. The reduction of ferric-tripyridyltriazine (Fe 3+ -TPTZ) to ferroustripyridyltriazine (Fe 2+ -TPTZ) found highest in the solution of stem extract with 1.742 ± 0.02 OD ( Figure 2B), whereas root (1.139 ± 0.03) and leaf (0.866 ± 0.016) showed relatively less reduction of Fe 3+ -TPTZ ( Figure 2B). The DPPH and FRAP assays have demonstrated that stem was rich reservoir of antioxidant compounds followed by root and leaf, and antioxidant capacities were attributed to total phenolics and flavonoids that are capable of donating hydrogen to free radicals to reduce oxidative stress ( Figure 2).
The GC-MS analysis of A. producta revealed that the large number of chemical compounds present in the methanolic extracts of different parts. Total of 89 compounds were identified by GC-MS analysis, in which 14 compounds were common in all the three extracts, 2 compounds in root and stem, 2 compounds in stem and leaf and 1 compound in root and leaf extracts. However, 13, 10 and 47 compounds were specific to root, stem, and leaf extracts, respectively ( Cinnamic acid is belongs to class of aromatic organic compounds and it was present in all three parts of A. producta i.e. root (8.34 %), stem (0.71 %) and leaf (5.74%). The cinnamic acid was reported to exhibit in vivo anti-diabetic activity in Wistar rats. 32 The administration of cinnamic acid at 10 mg/ kg dose to non-obese type 2 diabetic rats had decreased the blood glucose level significantly on time and dose dependent mode and also, at 100 µM concentration, triggered the insulin secretion efficiently (6.06 ± 0.83 ng/islet/hour) which was comparable to insulin secretion by standard drug tolbutamide (6.56 ± 0.81 ng/islet/hour). 32 Alpha-Terpinene was detected in leaf (4.58 %) of A. producta belongs to group of monoterpenoids ( Table 3). The investigation on the toxic effects of the alpha-terpinene in liver tissues of rats was conducted and showed that the hepatic damage caused by alpha-terpinene at a dose of 1.0 mL per kg was reported via quantitative elevation of serum ALT and AST activities. 26 Therefore, alpha-terpinene could induce oxidative stress, cytotoxic and genotoxic damages in the hepatic tissue involving the caspases activation. 26 2-Methoxy-4-vinylphenol is used as flavoring agent found in root (0.70 %) and leaves (0.19 %) of A. producta. 2-methoxy-4-vinylphenol was proved to be potent anticancer agent and suppressed migratory activity of pancreatic cancer cells, Panc-1 and SNU-213 and also reduced the viability of Panc-1 cells by inhibiting the cell nuclear antigen expression. 21 2,4-Di-tert-butylphenol belongs to phenol group was isolated from roots of Humboldtia unijuga showed anticancer effects through the activation of p53 gene in breast cancer cell line i.e. MCF-7 and also, anti-inflammatory effects of 2, 4-di-tert-butylphenol were significantly superior in pro-inflammatory cytokines TNFa, IL-6 and IL-1b. 19 Nair et al. 19 suggested for the development of 2, 4-di-tert-butylphenol as a novel anti-inflammatory and anticancer agent. Further, significant amount of 2, 4-di-tert-butylphenol was detected in root (9.74 %), stem (8.79 %) and leaf (7.43 %) of A. producta. Accordingly, the antioxidant activity of A. producta have attributed to these compounds such as 2-methoxy-4vinylphenol and 2,4-di-tert-butylphenol due to the their capabilities of prevention of free radical mediated oxidation ( Figure 2).
Solanesol is a terpene alcohol, which has absorbed the ultraviolet radiation effectively and inhibited the tyrosinae, a key enzyme in melanin synthesis and pigmentation disorder in human. 42 In the neuroprotective assessment, salvigenin at the concentration of 25 µM decreased the oxidative stress induced apoptosis by activating antioxidant factors in neuroblastoma SH-SY5Y cells. 40 Gamma-Sitosterol belongs to class of organic compound had exhibited potential anticancer activity through the growth inhibition, cell cycle arrest at G2/M phase and the apoptosis on cancer cells. 34

CONCLUSION
This is the first report on the quantification of total phenolic, flavonoid and tannin contents, identification of phytochemicals by GC-MS method and assessment of antioxidant capacities from Andrographis producta. The findings in the present investigation indicate that root, stem and leaf of A. producta can be explored as a good source of potent antioxidants due to the availability of phenolic and flavonoid contents. GC-MS studies showed the presence of important bioactive compounds such as cinnamic acid, 2,4-di-tert-butylphenol, solanesol, phytol, alphaterpinene, 5-hydroxy-7,8-dimethoxyflavone, salvigenin, stigmasterol and gamma-sitosterol and it provides immense opportunities to isolate and validate the phytochemicals for their pharmacological activities. Further, the meticulous assessment of such bioactive compounds from the A. producta would be great contribution in the field of medicine.