In-vitro antioxidant and GC-MS analysis ethanolic extract of poly herbal drug

1*&2 Department of Siddha Medicine, Faculty of Science, Tamil University, Thanjavur613010. Professor, Department of Siddha Medicine, Faculty of Science, Tamil University, Thanjavur613010. Author to whom correspondence should be addressed/E-Mail: kumaran.bio14@gmail.com Received: Feb 2017 / Accepted: Feb 2017/ Published: Mar 2017 ABSTRACT: Antioxidants play an important role in inhibiting and scavenging free radicals, thus providing protection to human against infections and degenerative diseases. Current research is now directed towards natural antioxidants originated from plants due to safe therapeutics. Poly herbal drugs is used in Indian traditional medicine for a wide range of various ailments. To understand the mechanism of pharmacological actions, antioxidant properties of the Poly herbal drugs extract were tested using standard in vitro models. The ethanolic extract of Poly herbal drugs exhibited strong scavenging effect on superoxide, nitric oxide radical and reducing power radical scavenging assay. The free radical scavenging effect of Poly herbal drugs extract was comparable with that of the reference antioxidants. The data obtained in the present study suggests that the extract of Poly herbal drugs have potent Invitro antioxidant and Anti Diabetic activity against free radicals, prevent oxidative damage to major biomolecules and afford significant protection against oxidative damage.


INTRODUCTION
Indian medicinal plants and many herbal formulations belonging to the traditional systems of medicine like Ayurveda have been investigated on various aspects of disease curing drugs. Side effects and expenses associated with allopathic drugs have provoked the need for research into drugs which are without the side effects, especially those belonging to the traditional systems of medicine (Jose and Kuttan, 2000) Research emphasis has been directed towards herbal drugs either in single or in combination having specific diagnostic and therapeutic principles. Antioxidant agents of natural origin have attracted special interest because they can protect human body from free radicals (Kunchandy & Rado, 1990) currently, polyhedral formulations are employed for the treatment of various types of diseases, such as respiratory diseases, cancer, acquired immunodeficiency syndrome (AIDS), diabetics and ulcer in order to achieve enhanced therapeutic effects In view of above information the present study has been undertaken to assess the invitro antioxidant and anti-diabetic activity of polyherbal formulation containing drugs from different herbs viz Cassia auriculata , Mimosa pudica , Mangifera indica , Cyperus rotundus and Asparagus racemosus.
1ml of methanol and 0.05 ml of herbal extract. The absorbance of the mixture was measured at 517 nm exactly 30 sec after adding extracts. The experiment was performed in triplicate and percentage of scavenging activity was calculated using the formula 100-[100/blank absorbance X sample absorbance]. The blank was also carried out in similar manner, using distilled water in the place of extracts. The activity was compared with ascorbic acid, which was used as a standard antioxidant.

ABTS radical cation decolourisation Assay
ABTS (54.8 mg) was dissolved in 50 ml of distilled water to 2 mM concentration and potassium persulphate (17 mM, 0.3 ml) was added. The reaction mixture was left to stand at room temperature overnight in dark before use (Re and Pellegrini, 1999). To 0.2 ml of various concentrations of the extracts or standards, 1.0 ml of distilled DMSO and 0.16 ml of ABTS solution was added to make a final volume of 1.36 ml. Absorbance was measured spectrophotometrically, after 20 min at 734 nm. The assay was performed in triplicate.

Scavenging of Nitric Oxide
Sodium nitroprusside (5 μM) in standard phosphate buffer solution (Sreejayan Rao, 1997) was incubated with different concentration of the methanol and aqueous extracts dissolved in standard 0.025 M phosphate buffer (pH-7.4) and the tubes were incubated at 250 C for 5 hours. After 5hours, 0.5 ml of incubation solution was removed and diluted with0.5 ml of Griess reagent (prepared by mixing equal volume of 1%sulphanilamide in 2% phosphoric acid and 0.1% aphthylethylenediamine dihydrochloride in water). The absorbance of chromophore formed was read at 546 nm.

Metal chelating activity
The chelation of ferrous ions by extracts was estimated by method of Dinis et al. (Dinis et al., 1994). Briefly, 50 µl of 2 mM FeCl2 was added to 1 ml of different concentrations of the extract (0.2, 0.4, 0.8, 1.6 and 3.2 mg/ml). The reaction was initiated by the addition of 0.2 ml of 5 mM ferrozine solution. The mixture was vigorously shaken and left to stand at room temperature for 10 min. The absorbance of the solution was thereafter measured at 562 nm. The percentage inhibition of ferrozine-Fe 2+ complex formation was calculated as [(A0-As)/ As] × 100, where A0 was the absorbance of the control, and As was the absorbance of the extract/ standard. Na2EDTA was used as positive control.

GC -MS Analysis Preparation of extract
Rhizome powder of Curcuma caesia Roxb were shade dried. 20 g of the powdered tubers were soaked in 95% ethanol for 12 h. The extracts were then filtered through Whatmann filter paper No.41 along with 2 gm sodium sulfate to remove the sediments and traces of water in the filtrate. Before filtering, the filter paper along with sodium sulphate was wetted with 95% ethanol. The filtrate was then concentrated by bubbling nitrogen gas into the solution. The extract contained both polar and non-polar phytocomponents of the plant material used. 2 μl of these solutions was employed for GC/MS analysis (Merlin et al., 2009) GC-MS analysis was carried out on a GC clarus 500 Perkin Elmer system comprising a AOC-20i auto sampler and gas chromatograph interfaced to a mass spectrometer (GC-MS) instrument employing the following conditions: column Elite-1 fused silica capillary column (30 × 0.25 mm ID ×1EM df, composed of 100% Dimethyl poly siloxane), operating in electron impact mode at 70 eV; helium (99.999%) was used as carrier gas at a constant flow of 1ml/min and an injection volume of 0.5 EI was employed (split ratio of10:1) injector temperature 250°C; ion-source temperature 280°C. The oven temperature was programmed from 110°C (isothermal for 2 min), with an increase of 10°C/min, to 200°C, then 5°C/min to 280°C, ending with a 9 min isothermal at 280°C. Mass spectra were taken at 70 eV; a scan interval of 0.5 s and fragments from 40 to 550 Da.

Identification of Components
Identification was based on the molecular structure, molecular mass and calculated fragments. Interpretation on mass spectrum GC MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The name, molecular weight and structure of the components of the test materials were ascertained. The relative percentage amount of each component was calculated by comparing its average peak area to the total area. The spectrum of unknown components was compared with the version, 2005, software, Turbo mass 5.2. This is done in order to determine whether this plant species contains any individual compound or group of compounds, which may substantiate its current commercial and traditional use as an herbal medicine.
Further it helps to determine the most appropriate methods of extraction these compounds (Karthikeyan Ravichandran et al., 2015).

RESULTS AND DISCUSSION
DPPH assay is being used widely as a preliminary test which provides information on the reactivity of test compound. This method is based on the reduction of alcoholic DPPH solution in the presence of hydrogen donating antioxidant (AH) due to the formation of non-radical form DPPH-H. Antioxidant reacts with DPPH which is a stable free radical and is reduced to the DPPH-H and as a consequence the absorbance decreased from the DPPH˙ radical to the DPPH-H form. The degree of discoloration indicates the scavenging potential of the antioxidant compounds present in the poly herbal extract in turns of hydrogen donating ability. (Burits, and Bucar, 2000) DPPH assay is a simple and acceptable method to evaluate the anti-oxidative activity of compounds (Chen and Ho, 1995). DPPH is a stable free radical, which when encounters proton donors such as antioxidants, the radicals get quenched, absorbance gets reduced and thus used to measure the antioxidant activity of specific compound or plant extract (Koleva et al., 2002;Qureshi et al., 2010).
The DPPH radical has been widely used to test the potential of compounds as free radical scavengers of hydrogen donors and to investigate the antioxidant activity of plant extracts. The DPPH free radical scavenging activity of plant extract may be due to neutralization of DPPH radical either by transfer of hydrogen or of an electron (Shimada et al., 1992).
The Poly herbal mixture exhibited a significant dose dependent inhibition of DPPH activity. In this study, at 100 μg /ml, the extract showed highest inhibition of DPPH activity shown in (Table1; Figure 1).The results of DPPH-free radical scavenging assay suggest that the Poly herbal mixture extract is more capable of scavenging free radicals.

ABTS + Activity
ABTS + is a blue chromophore produced by the reaction between ABTS and potassium per sulfate. Previous studies have suggested that the total polyphenolic content of the plant extracts are positively correlated to the scavenging activities and reducing potential of an extract. Our result shows that scavenges pre-generated ABTS+ radicals Addition of polyherbal extracts to this pre-formed

No Activity
Nitric oxide, a gaseous free radical and is relatively less reactive. But its metabolic product peroxynitrite, formed after reacting with oxygen is extremely reactive and directly induce toxic reactions such as SH group oxidation, protein -tyrosine nitration, lipid peroxidation and DNA modification (Moncada et al., 1991;Yermilov et al., 1995). Nitric oxide has been demonstrated to participate in the beta cell damage during STZ-induced diabetes (Duran Reges et al., 2004). Nitric oxide plays an important role in various inflammatory processes. Sustained level of production of this radical is directly toxic to tissues and contribute to vascular collapse associated with septic shock, whereas chronic exposure of nitric oxide radical is associated with various carcinomas and inflammatory conditions including juvenile diabetes, multiple sclerosis, arthritis and ulcerative colitis (Tylor et al., 1997).
The toxicity of NO increases greatly when it reacts with superoxide radical, producing the highly reactive peroxynitrite (ONOO-) anion (Huie and Padmaja, 1993). Nitric oxide is an important chemical mediator generated by endothelial cells, macrophages, neurons and involved in the regulation of various physiological processes. Excess concentration of nitric oxide is implicated in the cytotoxic effects observed in various disorders like AIDS, cancer, alzheimer's and arthritis. Oxygen reacts with the excess NO to generate nitrite and peroxy nitrite anions, which act as free radicals. (Hashem et al., 2007).

Iron chelating activity
Iron can stimulate lipid peroxidation by the Fenton reaction and also accelerates peroxidation by decomposing lipid hydro peroxides into peroxyl and alkoxyl radicals that can themselves abstract hydrogen and perpetuate the chain reaction of lipid peroxidation. In the presence of chelating agents, the complex formation is disrupted with the result that the red colour of the complex is decreased. Thus, the chelating effect of the coexisting chelators can be determined by measuring the rate of color reduction. The formation of the ferrozine-Fe2+ complex is interrupted in the presence of aqueous extract of poly herbal extract indicating that have chelating activity (Table 4; Figure 4).
Ferrous iron can initiate lipid peroxidation by the Fenton reaction as well as accelerating peroxidation by decomposing lipid hydro peroxides into peroxyl and alkoxy radicals (Hashem et al., 2007). Metal chelating activity can contribute in reducing the concentration of the catalyzing transition metal in lipid peroxidation. Furthermore, chelating agents that forms bonds with a metal are effective as secondary antioxidants because they reduce the redox potential, and thereby stabilize the oxidized form of the metal ion (Cao G et al.,1996).Thus, poly herbal extract demonstrate a marked capacity for iron binding, suggesting their ability as a peroxidation protector that relates to the iron binding capacity. The phytochemical compounds present in the selected poly herbal combined extract were identified by GC-MS analysis. GC-MS chromatogram of the ethanolic extract of combined poly herbal showed the presence of 20 phytochemical constituents. The major phytochemical compounds identified were 3-o-methyl-d glucose, ergosterol, Ercosta-7,22-dien-3ol,(3a,22e)-, resorcinol, n-hexadecannoic acid.
Phenolic compounds have also been known as antioxidant agents, which act as free radical terminators and have shown medicinal activity as well as exhibiting physiological functions. It was reported that compounds such as flavonoids, which contain hydroxyls, are responsible for the radical scavenging effects of this extract.
This study highlights the presence of many secondary metabolites in the poly herbal combined extract provide an overview of the different classes of molecules present that have led to their pharmacological activities. This study confirmed that the plant extract could be used for the treatment of various diseases. The plants sample on subjecting to GC-MS provides the result of different peaks determining the presence of seven different compounds. The molecular weight of these compounds is also known. By interpreting these compounds, it is found that this plant possess various therapeutically uses. This typical gas chromatogram shows the relative concentrations of various compounds getting eluted as a function of retention time. The heights of the peak indicate the relative concentrations of the components present in the plant. The numbers at various peaks are the retention time in minutes. The mass spectrometer analyzes the compounds eluted at different times to identify the nature and structure of the compounds. The large compound fragments into small compounds giving rise to appearance of peaks at different m/z ratios (Kalimuthu et al., 2013).
These observations may be due to the nature of biological active components and the stronger extraction capacity of hydro ethanol could have been produced number of active constituents responsible for medicinal activity (Komal Kumar et al., 2011). The biological activities based on Dr. Duke's Phytochemical and Ethnobotanical Databases.
Our investigation through the present study revealed that the poly herbal combined extract is a reliable source of bioactive compounds like fatty acid esters, alcohols, hydrocarbons, alkanes, amines, terpenes, and sugars that justify the traditional usage of this plants As GC-MS is the first step towards understanding the nature of active principles (Ghorbani, et al., 2011), further investigation in this species is suggested for the development of novel drugs.
The investigation concluded that the stronger extraction capacity of ethanol could have been produced number of active constituents responsible for many biological activities. So that those might be utilized for the development of traditional medicines and