Keywords

Phytoconstituents; Antioxidant activity; Enzymic; Non-enzymic and total antioxidants; Correlation; Gnaphalium polycaulon

Introduction

India is extremely rich in medicinal plants comprising about 8000 species and around 70% are found in the tropical areas mostly in the various forest types spread across the Western Ghats, Eastern Ghats, etc. [1]. Herbal medicines have continued to be a part of the medical field from the early Chinese Empires to modern physicians [2]. Medicinal and aromatic plants are used widely in the later years of the 21st century. It constitute the backbone of traditional medicine practices. The importance of traditional medicine (TM) as a source of primary healthcare was first officially recognized by the World Health Organization by Traditional Medicine Program. Researchers are increasingly turning their attention to herbal products, looking for new leads to investigate phytochemical investigation and to develop better drugs against Oxidative damage of cells. A recent ethno-botanical survey of traditional and folk medicine in India has revealed that most of these plants are still in use by the local tribal people, from ancient time. India is the largest producer of medicinal herbs and is appropriately called the botanical garden of the world. Ethnobotany is the scientific study of relationship that exists between people and plants. It interface between indigenous people and their wild exploit of plants around them, which is a significant aspect of biological diversity conservation [3] Plants have always been the principal source of drugs for the prevention and treatment of disease and also for the production of some drugs currently used in modern medicine. Many reports stated that Asteraceae family is considered as abundant flowering plants with many therapeutic properties and reported as folkloric medicinal plants [4]. The genus, Gnaphalium was reported to

have antioxidant, antibacterial, antifungal, anti-complement, antitussive and expectorant, insect anti-feedant, cytotoxic, antiinflammatory, anti-diabetic and anti-hypouricemic properties [5] due to the presence of flavonoids, phenolic glycosides, diterpenes, triterpenes, phytosterols and other major phytocompounds. The genus Gnaphalium, an herb distributed worldwide, comprises approximately 200 species of the Compositae (Asteraceae) family that belongs to the tribe Gnaphalieae. G. polycaulon is an annual widespread weed in tropical and subtropical Africa, Asia, Australia, and America [6]. The rich availability of G. polycaulon in the Western Ghats particularly in and around Kothagiri areas is due to the humid climatic conditions in hill stations. Based on these reports, G. polycaulon is a unique habitat of Western Ghats, The Nilgiris so this places were selected as the study area to carry out the present investigation Researchers on Biodiversity studies revealed that the plant kingdom has not been exhausted based on the species of medicinal plants which are yet to be discovered. The investigations of biological activity and chemical composition of medicinal plants as a potential source of natural antioxidants are numerous. Most of the modern researches on herbal medicine have hinged around traditional folklore medicine. The modern medicine has brought with it an array of drugs, none of which is non-toxic and quite safer for human consumption [7]. There are hundreds of medicinal plants that have a long history of curative properties against various diseases and ailments. However, screening of plants for their activity is very essential and needs urgent attention in order to know the value of the plant. So, this medicinal plant was validated with main objectives to screening and analyses antioxidant activity.

Materials and Methods

Collection and identification of plant samples

Following the collection, the plant samples were submitted to Botanical Survey of India (BSI), Coimbatore. They have been identified and confirmed as Gnaphalium polycaulon Pers. (Gnaphalium indicum Hook. f.) - Asteraceae family. Herbarium specimen has been authenticated and incorporated by the Madras Herbarium.

Chemicals required

All chemicals used for this study were high quality analytical grade reagents. The solvents such as methanol, acetone, ethanol, and hexane were purchased from S.D. Fine Chemicals Pvt. Ltd, Sigma chemicals, Lobe chemicals, Merck Chemical Supplies, Nice Chemicals and Himedia. All other drugs and chemicals used in the study were obtained commercially and were of analytical grade.

Preparation and extraction of plant extracts

Following the confirmation, the standard protocols were followed to extract phytoconstituents from dried plant parts (leaves, stems and flowers), parallely with methanol as solvent. Resulting extracts were individually concentrated by applying vacuum to get dry powder using rotary evaporator and subjected to lyophilizaion. The obtained crude extracts were packed in airtight plastic containers and stored in refrigerator at 4°C for further applications. The extracts were used for analysis the antioxidant activity in the present study.

Evaluation of in vitro antioxidant activity of Gnaphalium polycaulon extracts

Based on the phytoconstituents analysis, antioxidant activity was analysed, since there is no report available on G. polycaulon on these aspects in further studies.

Enzymic antioxidants

The enzymic antioxidants methods were followed to determine the antioxidant activity of dried G. polycaulon extracts. According to the previously reported standard protocols [7], all the assay methods such as Catalase (CAT), Peroxidase (POD), Glutathione S-Transferase (GST), Polyphenol Oxidase (PPO), Superoxide Dismutase (SOD), Glucose-6-phosphatedehydrogenase (G6PD) have been carried out in all extracts.

Non-enzymic antioxidants

According to the previously reported standard protocols [8], all non-enzymic antioxidants assays are Ascorbic acid, Total carotenoids and lycopene, Reduced glutathione, α-tocopherol, Total phenols, Total flavonols, Flavonoids have been carried out in all dried Gnaphalium polycaulon extracts.

Evaluation of free radical scavenging activity of Gnaphalium polycaulon extracts

The dried extracts of Gnaphalium polycaulon were analysed for their radical scavenging ability against a free radicals and oxidants using standard methods. The absorbance values were measured in a UVVis spectrophotometer and the percentage inhibition of free radical scavenging activity was calculated with standard formula as follows:

%inhibition of radical scavenging activity=A₅₁₈(control- A₅₁₈(sample)/A₅₁₈ × 100

Where, A₅₁₈ (control) is the absorbance of Control; A₅₁₈ (sample) is the absorbance of free radical+sample extract/ standard. The graph against percentage inhibition versus concentration was plotted using Graph Pad Prism 5.0 program and its IC₅₀ values (Concentration required for 50% inhibition of radicals) were calculated for all antioxidants assays in triplicates.

Dot-plot rapid screening assay

The rapid screening assay was performed by the method proposed by Soler-Rivas [9]. Aliquots of plant extracts were spotted carefully on TLC plates and dried for 3 min. The sheets bearing the dry spots were placed upside down for 10 secs in a 0.4 mM DPPH solution in methanol and the layer was dried. The stained silica layer revealed a purple background with yellow spots, which showed radical scavenging capacity. The ability of the plant extracts to scavenge the DPPH radical was tested in a rapid dot-plot screening and quantified using a spectrophotometer.

DPPH radical scavenging activity

The scavenging ability of the natural antioxidants of the plant material towards the stable free radical DPPH assay was measured by the method of Mensor [10]. About 20 μl of various concentration of each plant extract was added to 0.5 ml of methanolic solution of DPPH and 0.48 ml of methanol. The mixture was allowed to react at room temperature for 30 min. Methanol served as the blank and DPPH in methanol, without the plant extracts, served as the positive control. Following incubation, the dis colouration of the purple colour and the absorbance values was measured at 518 nm using a UV-Vis spectrophotometer.

FRAP radical scavenging activity

The FRAP assay was carried out with a slight modified method, proposed by Benzie [11]. The stock solutions included 300 mM acetate buffer, pH 3.6, 10 mM 2, 4, 6- tripyridyl-s-triazine (TPTZ) solution in 40 mM HCl, and 20 mM FeCl₃.6H₂O solution. The fresh working solution was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ solution, and 2.5 ml FeCl₃.6H₂O solution and then warmed at 37°C before using. Different concentration (100-500 μg/ml) of plant extracts were allowed to react with 2850 ml of the FRAP solution for 30 min in the dark condition. Readings of the colored product were then taken at 593 nm using UV-Vis spectrophotometer. Results are expressed in mM ascorbic acid/g dry mass. Results are expressed in mM TE/g fresh mass. The final result was expressed as the concentration of antioxidant having a ferric reducing ability.

ABTS·+radical scavenging activity

ABTS⁺ or 2, 2’-azinobis (3-ethylbenzthiazoline)-6-sulphonic acid free radical cation decolourization assay of the antioxidant effect in the plant extracts was done according to the method of Shiwaiker [12]. The pre-formed radical monocation of ABTS+ was generated by reacting ABTS⁺ solution (7mM) with 2.45 mM potassium persulfate (K₂S₂O₈). The mixture was allowed to stand for 15 h in dark at room temperature before use. The aliquot of 0.5 ml of each sample was added to 0.3 ml of ABTS⁺ free radical cation solution and the final volume was made upto 1 ml with ethanol. The absorbance, monitored for 5 min was measured spectrophotometrically at 734 nm using UV-Vis spectrophotometer. Appropriate solvent blanks were run in each assay. The absorbance was read at 745 nm in a spectrophotometer.

ORAC radical scavenging activity

The ORAC (Oxygen radical absorbance capacity) free radical cation decolourization assay of the antioxidant effect in the plant extracts was performed by using the method of Prior [13]. In ORAC procedure, an automated plate reader with 96-well plates was used. Analyses were conducted in phosphate buffer pH 7.4 at 37°C. Peroxyl radical was generated using 2,2’-azobis (2-amidinopropane) dihydrochloride which was prepared fresh for each run. Fluorescein was used as the substrate. Fluorescence conditions were excitation at 485 nm and emission at 520 nm. Results are expressed as mM Ascorbic acid/g dry weight.

Reducing power activity

The reducing power was quantified by the Fe³⁺-Fe²⁺ transformation in the presence of the fractions according to the method of Oyaizu [14] Different concentrations of the plant extracts in 1 ml of distilled water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferric cyanide (2.5 ml, 1%). The mixture was incubated at 50oC for 20 min. Trichloroacetic acid (10%, 2.5 ml) was added to the mixture. A portion of the resulting mixture was mixed with FeCl₃ (0.1%, 0.5 ml) and the absorbance was measured at 700 nm using UV-Vis spectrophotometer. The reducing power of the extract was linearly proportional to the concentration of the sample. Higher absorbance of the reaction mixture indicated higher reductive potential.

Nitric oxide radical scavenging activity

The extent of inhibition of nitric oxide radical generation in vitro was followed as per the method reported by Makchuchit [15]. The reaction was initiated by adding 2.0 ml of 100 mM sodium nitroprusside, 0.5 ml of phosphate buffered saline (pH 7.4), 0.5 ml of plant extracts (100-500 μg/ml) and incubated at 25oC for 30 min. About 0.5 ml of Griess reagent was added and incubated for another 30 min. Control tubes were prepared without plant extracts. The absorbance of the chromophore formed during diazotization of nitrite with sulphanilamide and its subsequent coupling with napthyl ethylene diamine was read at 546 nm. The experiment was repeated in triplicate manner. The absorbance was read at 546 nm against the reagent blank, in a spectrophotometer.

Total Antioxidant Capacity (TAC) by phosphomolybdenum method

The total antioxidant capacity of the plant extracts was evaluated by the phosphomolybdenum method according to the procedure described by Prieto [16]. To 0.3 ml of plant extracts (100-500 μg/ ml), 3 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) was added and mixed. The absorbance of the reaction mixture was measured at 695 nm using a spectrophotometer against blank after cooling to room temperature.

Hydrogen peroxide radical scavenging activity

The ability of the plant extracts to scavenge hydrogen peroxide was assessed according to Ruch [17]. Plant extracts were added to 0.6 ml H₂O₂ solution (40 mM H₂O₂ was prepared with phosphate buffer, 0.1 M, pH 7.4) and the total volume was made up to 3 ml and mixed properly. Then the absorbance was measured at 230 nm using a spectrophotometer (Hach DR 5000 UV Spectrophotometer). Phosphate buffer, without H₂O₂ was considered as blank solution.

Superoxide radical scavenging activity

The superoxide scavenging activity of the plant extracts was assessed according to Suprava [18]. Superoxide anions were generated in samples that contained in 3.0 ml, 0.02 ml of the plant extracts (100-500 μg/ml), 0.2 ml of EDTA (0.1 M containing 1.5 mg of NaCN), 0.1 ml of 1.5 mM Nitroblue tetrazolium [NBT]), 0.05 ml of 0.12 mM riboflavin and 2.64 ml of phosphate buffer (0.067 M, pH 7.6). DMSO was used as control. All the tubes were vortexed and the initial optical density was measured at 560 nm in a spectrophotometer. Then the tubes were illuminated using a fluorescent lamp for 30 min and absorbance was measured again at 560 nm. The difference in absorbance before and after illumination was indicative of superoxide anion scavenging activity.

Hydroxyl radical scavenging activity

The extent of hydroxyl radical scavenging was assayed according to Elizabeth [19]. The reaction mixture contained 0.1 ml of 2.8 mM deoxyribose, 0.1 ml of 0.1 mM FeCl₃, 0.1 ml of 0.1 mM EDTA, 0.1 ml of 1 mM H₂O₂, 0.1 ml of 0.1 mM ascorbate, 0.1 ml of KH₂PO₄-KOH buffer (20 mM, pH 7.4) and 20 μl of plant extracts (100-500 μg/ml) in a final volume of 1.0 ml. The mixture was incubated at 37°C for 1 h. Following incubation, 1.0 ml of thiobarbituric acid (TBA) was added and heated at 95°C for 20 min to observe colour development from yellow to orange. Then the content was cooled and TBARS formation was measured spectrophotometrically at 532 nm. Ascorbic acid was used as a positive control. Deoxyribose degradation was measured as thio barbituric acid-reactive species (TBARS).

Ferric Thiocyanate (FTC) method

The antioxidant potential of G. polycaulon extracts was determined according to the FTC method with slight modification of Lee [20]. The reaction mixtures contained 400 μl of different concentration of plant extracts, 200 μl of diluted linoleic acid (25 mg/ml in 99% ethanol) and 400 μl of 50 mM phosphate buffer (pH 7.4) was incubated for 15 min at 4oC. A 100 μl aliquot of the above was mixed with a reaction mixture containing 3 ml of 70% ethanol, 100 μl of ammonium thiocyanate (300 mg/ml in distilled water) and 100 μl of ferrous sulphate, which results in the development of red colour, which spectrophotometrically measured at 535 nm.

Assay of inhibition of free radicals using Thiobarbituric Acid (TBA) method

Assaying of inhibition of free radicals was done according to Kikuzaki and Ohkawa [20,21]. The sample preparation for this assay was similar to FTC method. To 2.0 ml of the sample solution, 1.0 ml of 20% aqueous trichloroacetic acid (TCA) and 2.0 ml of aqueous thiobarbituric acid (TBA) solution were added. The final sample concentration was 0.02% w/v. The mixture was placed in a boiling water bath for 10 min. Following cooling, centrifuged at 3000 rpm for 20 min and the absorbance was measured. On fifth day, prepared sample was spectrophotometrically analysed at 532 nm and recorded.

Metal chelation activity

The ability of chelating Fe²⁺ by the extracts was determined according to Dinis [22,23]. One ml of each extracts were individually incubated with 50 μl of 2 mM ferrous chloride and incubated at room temperature for 2 h. Following incubation, 200 μlferrozine (5 mM) was added and after 10 min, the green colour developed was recorded and measured spectrophotometrically at 562 nm and calculated using standard formula. Na₂EDTA served as standard. The percentage inhibition and IC₅₀ values were calculated and graph was plotted.

Lipid Peroxidation (LPO) activity

The method of Dudonne [24] was carried out to measure the lipid peroxide formed in the plant samples. Egg homogenate (10% in 0.2 M PBS, 0.5 ml), 0.1 ml of plant extracts (100-500 μg/ml) and deionised water (0.85 ml) were mixed in a test tube. To this, FeSO₄ (0.07 M, 0.05 ml) was added and incubated at 37oC temperature for 30 min to induce lipid peroxidation. Thereafter, acetic acid (20%, 1.5 ml), thiobarbituric acid (TBA) (0.8% prepared in 1.1% sodium dodecyl sulphate, 1.5 ml) and TCA (20%, 0.05 ml) were added, vortexed and then heated in a boiling water bath for 60 min. Following cooling, butan-1-ol (5 ml) was added to each tube and centrifuged for 10 min at 3000 rpm. The absorbance of the organic upper layer was measured at 532 nm spectrophotometerically. The percentage inhibition and IC₅₀ values were calculated and graph was plotted. The coefficient significance of phytoconstituents were correlated between all antioxidant assays of G. polycaulon using Graph Pad Prism 5.0 program. Based on the significant results of antioxidant activities, methanolic extract of dry leaf in G. polycaulon plant was observed to be more significant equal activities than other extracts.

Results

Enzymic antioxidants

The enzymic antioxidants of G. polycaulon reported that the methanolic extract of dried leaf exhibited higher amount of catalase of 0.74 U/mg, peroxidase of 0.827 and 0.938 U/mg, glutathione s-transferase of 0.71 and 0.70 U/mg, polyphenol oxidase of 1.73 U/mg, superoxide of 0.87 U/mg and glucose- 6-phosphate dehydrogenase of 0.76 U/mg than other extracts (Table 1).

Table 1: In vitro enzymic antioxidant activity of methanolic extracts of dried G. polycaulon.

Non-enzymic antioxidants

The non-enzymic antioxidants were estimated in G. polycaulon extracts for the estimation of ascorbic acid (0.623), total carotenoids (0.642), lycopene (0.591), reduced glutathione (0.629), tocopherol (0.019), total phenols (0.78), total flavonols (0.79) and flavonoids (0.81 mg/g) was found that the methanolic extract of dried leaf exhibited maximum yield than other extracts (Table 2).

Table 2: In vitro non-enzymic antioxidant activity of methanolic extracts of dried G. polycaulon.

Evaluation of total free radical scavenging activity of G. polycaulon

Because of the complex nature of phytochemicals, the antioxidant activities of plant extracts must be evaluated by combining two or more different in vitro assays based on different features of the antioxidant effects, such as the ability to scavenge free radicals, or the metal ion chelation. The rapid screening of antioxidant was assayed by dot-blot antioxidant rapid Screening assay to visualize free radicals quantitatively in the tested samples and measured spectrophotometrically. Then it was detected in the TLC plate by the DPPH staining method. Among all plant extracts, stronger yellow spots with purple background effect were observed in methanolic extract of dried leaf of Gnaphalium polycaulon (Figure 1) than other extracts. However, initially faint spots appeared and several hours later, weak spots could be observed in loaded area, similar to the result of DPPH spectrophotometric assay. The results of DPPH radical scavenging activity of G. polycaulon exhibited very high percentage inhibition antioxidant activity of 73.2% at 100 μg/ml in methanolic extract of dried leaf extract than other extracts. Standard ascorbic acid extract was significantly different (p<0.005) but all extracts were significantly different from standard (p>0.005). All fresh and dried extracts showed steady increase in percentage inhibition of the DPPH radicals with concentration. The percentage inhibition of antioxidant values was determined in all extracts (Table 3a). Among all the extracts, methanolic extract of dried leaf exhibited more potential ABTS⁺antioxidants. The percentage inhibition of antioxidant activity was 66.9% at 100 μg/ml concentration with IC₅₀ of 0.131 ± 0.002 mg/ml in methanolic extract of dried leaf than other extracts (Table 3a). The FRAP radical scavenging activity exhibited very high antioxidant percentage activity of 71.2% at 100 μg/ml in methanolic extract of dried leaf than other extracts with an IC₅₀, 71.2 ± 0.009 mg/ml. Standard ascorbic acid extract were significantly different (p<0.005) All dried extracts showed steady increase in percentage inhibition of the DPPH radicals with concentration and dependent. The percentage inhibition of antioxidant values was tabulated in Table 3a. The methanolic extract of dried leaf exhibited very high significant ORAC activity than other extracts and found to be 41%. The IC₅₀ was calculated and found to be high antioxidant potential when compared to the standard and other extracts. It showed more significant than standard values (Table 3b). Reducing power of methanolic extract of dried leaf was significantly higher than that other extracts. All dried extracts were found to be significantly different from ascorbic acid (p<0.005) (Table 3b). However, methanolic leaf extracts showed better activity than the ascorbic acid at 100 μg/mL. The methanolic extract of dried leaf exhibited a maximum nitric oxide radical scavenging activity of 45.9% at 100 μg/ml with IC₅₀ of 200 μg/ml (Table 3b) in methanolic extract of dried leaf had higher scavenging activity than standard. All plant extracts showed consistent increase in scavenging nitric oxide radicals with increase in concentration. The different plant extracts were significantly different with each other as well as from standard ascorbic acid (p>0.005). The results of phosphomolybdenum assay were measured and found to be 45.7% at 100 μg/ml in the methanolic extract of dried leaf than other extracts (Table 3c). The percentage scavenging of hydrogen peroxide in all extracts were determined and found to be 36% at 100 μg/ml in the methanolic extract of dried leaf than other extracts with IC₅₀ of 200 μg/ml (Table 3c). The maximum superoxide radical scavenging activity was found to be 61.6% at 100 μg/ml with IC₅₀ of 100 μg/ml in methanolic dried leaf extract (Table 3c). There was no significant difference in superoxide radical scavenging activity of extracts and standard ascorbic acid (p>0.005) by statistical analysis. The Hydroxyl radical scavenging activity exhibited more activity in methanolic extract of dried leaf than other extracts and found to be 39.8% with IC₅₀ of 300 μg/ml (Table 3d). All plant extracts had been oxidized at 40-45°C for seven days. Initially, the absorbance of all extracts were measured and exhibited as the lowest values from 0.026. After seven days storage, all samples exhibited good effect in inhibiting linoleic acid oxidation compared to standard (Table 3e). Antioxidant activity of four different extracts were measured to inhibit lipid peroxidation by FTC and TBA methods. In general, the antioxidant capacity of all extracts by TBA method is higher than that of FTC method. The methanolic extract of dried leaf revealed high antioxidant capacity than others extracts. The TBA analysis of the methanolic extract of dried leaf at seventh day storage exhibited high antioxidant capacity than other extracts and found to be 0.57% in methanolic extract of dried leaf (Figure 2). The methanolic extract of dried leaf at 100 μg/ml exhibited higher metal chelating effects of 33.8% than other extracts with IC₅₀ of 300 μg/ml. The good chelating effects of G. polycaulon extracts on ferrous ions increased with increasing concentrations (Table 3d). The Lipid peroxidation assay exhibited high inhibiting activity in the methanolic extract of dried leaf at 100 μg/ml (46.2%) with IC₅₀ of 300 μg/ml than other extracts (Table 3d). Methanolic extract of driedleaf of G. polycaulon was found to have maximum antioxidant activity with significant IC₅₀ values, followed by other extracts. The correlation of coefficient significance among major phytoconstituents with antioxidant assays of fresh and dried G. polycaulon plant was analysed (Tables 4a-4c). The correlation results clearly stated that methanolic extract of driedleaf of G. polycaulon statistically exhibited more significant results in total phenol, tannin, saponin, alkaloids and flavonoids contents with maximum antioxidant potential than other extracts. Based on the significant results in phytoconstituents analysis, antimicrobial and antioxidant activities, methanolic extract of dried leaf and aqueous extract of fresh leaf in G. polycaulon plant was found to be more significant equal activities than other extracts. So, both extracts were considered and parallely compared in further analysis.

Figure 1: Dot-plot rapid Screening assay.

Figure 2: Estimation of Free radicals present in methanolic extracts of dried G. polycaulon Thiobarbituric acid (TBA) method.

Table 3a: Total radical Scavenging activity of different extracts of G. polycaulon.

Table 3b: Total Radical Scavenging activity of different extracts of G. polycaulon.

Table 3c: Total Radical Scavenging activity of different extracts of G. polycaulon.

Table 3d: Total radical Scavenging activity of different extracts of G. polycaulon.

Table 3e: Estimation ofFree radicalspresent in methanolic extracts of dried G. polycaulonby Ferric thiocyanate (FTC) method.

Table 4a: Coefficient of correlation between antioxidant assays of methanolic extracts of G. polycaulon.

Table 4b: Coefficient of correlation between antioxidant assays of methanolic extracts of G. polycaulon.

Table 4c: Coefficient of correlation between antioxidant assays of methanolic extracts of G. polycaulon.

Discussion

During the last two decades, there has been in search for new plant derived drugs containing medically useful alkaloids, polyphenolics, steroids and terpenoids derivatives, which contributes to the antioxidant property. Many research studies have demonstrated that medicinal plants contain various phytocomponents with antioxidant activities that are responsible for their beneficial health effects. Antioxidants played an effective defense role in inhibiting and scavenging free radicals with synthetic antioxidants, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) [25]. It providing supported to defense mechanism of the body against risk of free radicals mediated deadly diseases [26]. Therefore, the discovery of natural antioxidants is currently a foremost theme of intensive investigation. It is believed that folk remedies are major sources of new phytoconstituents for antimicrobial and antioxidant treatments. Antioxidant assay was analysed and evaluated as high antioxidant activity in G. polycaulon extracts.

Enzymic antioxidants

The dried extracts of G. polycaulon have exhibited interesting phytoconstituents that leads to good antioxidant by enzymic, non-enzymic and total free scavenging assays. The enzymic antioxidants were analyzed and reported as methanolic extract of dried leaf have significant antioxidant activity than other solvents. Hydrogen peroxide is generated by the dismutation of superoxide radical by superoxide dismutase. SOD generates H₂O₂ as a more toxic product to the cells and required catalase or peroxidases to scavenge free radicals. An increase in catalase or peroxidase is essential for beneficial antioxidant effect from increase superoxide dismutase activity. Glucose-6-phospatedehydrogenase enzyme is to maintain GSH in reduced state [27]. In addition to vitamin C, vitamin E and carotenoids, polyphenols exhibited strong antioxidant capacity [28].

Non-enzymic antioxidants

The non-enzymic antioxidants were estimated in different dried plant parts of G. polycaulon extracts and reported for ascorbic acid, total carotenoids, lycopene, reduced glutathione, tocopherol, total phenols, total flavonols and flavonoids in methanolic extract of dried leaf as high antioxidant activity than other extracts. Medicinal plants exhibited free radical scavenging molecules such as vitamins, reduced glutathione, tocopherol, lycopene, phenolic acids, flavonoids, and alkaloids which are rich in antioxidant activity. Specifically, non-enzymic antioxidants are essential for redox buffering [29]. Ascorbic acid is a natural water soluble antioxidant defense that protects cells against lipid peroxidation [30] and acts as strongest reductants and primary defense against radicals due to increasing utilization in the trapping of free radicals. The previous studies reported that lycopene acts as effective dietary antioxidant and free radicals. Reduced Glutathione is a major endogenous antioxidant defense substances and also acts as essential factor for glutathione peroxidase. Carotenoids are particularly efficient scavengers of singlet oxygen but it can also trap peroxyl radicals at low oxygen pressure with efficiency and also played a role in preventing in vivo lipid peroxidation [31]. Total phenolic act as reducing agents, hydrogen donors, singlet oxygen quenchers, and a metal chelating potential. Flavonoids exhibited structural diversity, biological and ecological significance, and health-promoting, anticancer properties [32] and responsible for antioxidant production.

Total free radical scavenging assays

Antioxidant activities (Dot-plot, DPPH, FRAP, ABTS, ORAC, reducing power, nitric oxide radical, phosphomolybdenum method, hydrogen peroxide, superoxide radical, hydroxyl radical, ferric thiocyanate method, thiobarbituric acid method, metal chelation and lipid peroxidation assays) were measured in all dried G. polycaulon extracts. The Dot-plot rapid screening assay is based on the inhibition of the accumulation of oxidized products by scavenging the free radical. When flavonoids were analyzed, fast-reacted and strong intensities of white-yellow spots were appeared When a DPPH solution is mixed with plant extract that can donate a hydrogen atom to the reduced form, diphenylpicryl hydrazine with the loss of its violet color The degree of reduction in absorbance measurement is indicative of free radical scavenging of extract. The scavenging effect elicited by the ethanolic extract of leaves of G. uniflorum was concentration-dependent in DPPH test the amount of antioxidant was necessary to decrease 50% of initial concentration. The reduction of the ferric-tripyridyltriazine to the ferrous complex forms an intense blue colour is related to the amount of antioxidant reductants in the samples. The reductive ability of plant samples suggested that the extracts were able to donate electron to free radicals in biological systems, for stable and unreactiveradicals. A decrease of the ABTS.+ concentration is linearly dependent on the antioxidant concentration Higher concentrations of the extracts were more effective in quenching free radicals in the system. Trolox, BHT, rutin, ascorbic acid and gallic acid can be used as a positive control. The essential oil from G. affine was observed to possess strong radical scavenging activity against 2,2'-azinobis-3-ethylbenzthiazoline-6-sulfonate with the IC₅₀ being 0.32 ± 0.89 μg/mL. The ORAC assay is based on the scavenging of peroxyl radicals generated that prevent degradation of the fluorescein probe and prevent the loss of fluorescence of the probeby spontaneous decomposition of 2, 2’- azo-bis, 2- amidinopropanedihydrochloride (AAPH), was estimated and Similar results was reported. Increasing absorbance indicated an increase in the reductive ability. The reducing capacity of plant extract served as a significant indicator of its potential electron donating and antioxidant activity that inhibition of lipid oxidation and peroxidation supported the present study and are significant to superoxide dismutase and catalase activities. Excess production of NO is associated with several diseases. It reacts with O₂ to produce stable product nitrate and nitrite through intermediates NO₂, N₂O₄ and N₃O₄ using Griess reagent. The methanolic extract of dried leaf of G. polycaulon exhibits significant antioxidant activity which compete with oxygen to react with nitric oxide and inhibited the generation of nitrite. The antioxidant capacity measured is based on the reduction of (IV) to (V) by the sample analyte and subsequent formation of green formation / Mo (V) compounds. It might be attributed to the presence of phytochemicals such as flavonoids and biflavones. Hydrogen peroxide is decomposing to yield highly reactive hydroxyl radical. Hydrogen peroxide is not very reactive, but it can sometimes be toxic to cell because it can give rise to hydroxyl radical in the cells. Thus, the removal of H₂O₂ is very important for antioxidant defense in cell systems. Superoxide anions served as precursors of singlet oxygen and hydroxyl radicals. The plant extracts decreased the mean rate of absorption by inhibiting NBT reduction by the superoxide anion radicals through metabolic processes. SOD enzymes present in aerobic and anaerobic organisms catalyzed the breakdown of superoxide radical. The hydroxyl radical scavenging activity is measured as percentage of inhibition of hydroxyl radicals generated in the Fentons reaction mixture by studying the composition between deoxyribose and the plant extract for hydroxyl radical generated. Plant extracts and the standard inhibited the production of hydroxyl radicals. The highly reactive hydroxyl radicals can cause oxidative damage to DNA, lipids and proteins. The Ferric thiocyanate method measured the amount of peroxide value in the beginning of the lipid peroxidation, where ferric ion was formed upon reaction of peroxide with ferrous chloride. The ferric ion reacted with ammonium thiocyanate to formed ferric thiocyanate, a redcoloured substance. The darker colour will gives the absorbance higher The same samples as prepared for the FTC method were used in TBA test. FTC is used to measure the production of peroxide compound at the initial stage of oxidation while TBA test is used to measure the secondary product of oxidation such as aldehyde and ketone. Furthermore, the secondary product is much more stable for a period of time. It turned into alcohol and acid, which cannot be detected by a spectrophotometer. According to the results, the methanolic extract of dried leaf of G. polycaulon reported high ferrous ion-chelating activity through scavenging or chelating processes. Ferrozine combines with ferrous ions forming a red coloured complex. It was reported that the chelating agents which form σ bond with a metal, are effective as secondary antioxidants, because they reduce the redox potential thereby stabilizing the oxidized form of the metal ion. In addition, metal chelating ability of the plant extract demonstrated that they reduce concentration of the catalyzing transition metal involved in the peroxidation of lipids. The high lipid peroxidation scavenging effects was measured in methanolic extract of dried leaf due to high contents of phenolic compounds and other radical scavengers present in the plant extracts which can terminate the peroxidation chain reaction easily and quench reactive oxygen species or nitrogen species, thereby inhibiting the oxidation of lipid and other biological molecules. In addition, it was reported that phenolic substances were associated with antioxidant activity played important role in stabilizing lipid peroxidation. Plant parts having better protection against free radical induced lipid peroxidation that can be used as anti-lipid peroxidation and anti-mutagenic substances. There is growing interest in correlating the phytochemical constituents of a medicinal plant with its pharmacological activity. The correlation results stated that the total phenol content is more significant to antioxidant activity also. A positive correlation was found between phenolics, alkaloids, tannins, saponins, and flavonoid contents and antioxidant properties of the G. polycaulon extracts. These findings indicated that polyphenolic components may have antioxidant potential, as reported in other medicinal plants among major phytoconstituents. Based on the findings of the present investigation, standardization of antioxidant activity clearly reported that the methanolic extract of dried leaf of G. polycaulon exhibited highly significant values than other extracts. Most of the ethnobotanical studies confirmed that leaves are the major portion of the plant used for the treatment of various diseases due to their easily accessible, active in photosynthesis and production of various valuable metabolites, which has been reflected in the present investigation too.

Conclusion

According to the knowledge of tribals, G. polycaulon is having variety of medicinal properties. The results also clearly revealed the presence of medicinally important phytoconstituents in the plants studied and showed that G. polycaulon plant is good source of antioxidant due to its phytochemicals. Only very few reports available on this plant and this report is the first report from India. This plant is said to be location specific and present throughout the year. These findings stated that G. polycaulon extracts has a potential application as natural medicine and to treat diseases as well as the microbiological safety of the human health. Further studies are ongoing to isolate, identity, characterize and elucidate the structure of the bioactive components. Such kind of plants should be given with lot of importance and their bioactive properties should be studied in an elaborative manner and should be documented. Specifically, preservation of medicinal plants is urgent need to protect biological diversity for the betterment of future generation.

Acknowledgements

The authors are grateful to Management, School of Biotechnology, Dr. GR Damodaran College of Science, Coimbatore, Tamil Nadu, India for providing all facilities and for their encouragement.

References

1. Shanmugapriya K, Thayumanavan T (2015) Screening for antimicrobial activity and quantitative phytochemicals analysis of aqueous extract of Gnaphalium polycaulon used in Indian folkloric medicine. J Microbiol Biotechnol Res 5:31-37.
2. Sadasivam S, Manickam A (1996) Biochemical methods: A New Age International (P) Limited,New Delhi.
3. Mowobi GG, Abubakar S, Osuji C, Etim VN, Ogechi N, et al. (2016) Ethnobotanical survey of medicinal plants used for the treatment of skin disease in Keffi, Nigeria. Am J Phytomed Clin Therapeut 4: 73-90.
4. Borkataky M, Kakoty BB, Saikia LR (2013) Antimicrobial activity and phytochemical screening of some common weeds of asteraceae family. Int J Pharm Sci Rev Res 23: 116-120.
5. Winterbourn C, Hawkins R, Brian M, Carrell R (1975) The Estimation of red cell superoxide dismutase activity. J Lab ClinMed 85: 337.
6. Ibarra VJR, Sanchez M, Espejo O, Estrada AZ, Valencia JMT, et al. (2001) Antimicrobial activity of three Mexican Gnaphalium species. Fitoterapia 72: 692-694.
7. Pushpa RV, Moorthy BBP, Priya KS, Nancy AA, Kumari GM, et al. (2016) Phytochemical screening and GC-MS analysis in wild variety of Cocciniaindica –A Future Promising Therapeutic Source. Am J Phytomed Clin Therapeut4: 98-105.
8. Sreejith M, Kannappan N, Santhiagu A, Ajith PM (2013) Phytochemical, Anti-oxidant and Anthelmintic activities of various leaf extracts of Flacourtia sepiaria Roxb. Asian Pac J Trop Biomed 3: 947-953.
9. Soler-Rivas SC, Espin JC, Wichers HJ (2000) An easy and fast test to compare total free radical scavenger capacity of foodstuffs. Phytochem Anal 11: 330-338.
10. Mensor LL, Menezes FS, Leitao GG, Reis AS, Santos TC, et al. (2001) Screening of Brazilian plant extracts for antioxidant activity by theuse of DPPH free radical method. Phytoth Res 15:127-130.
11. Benzie IEF, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power the FRAP assay. Anal Biochem 239: 70-76.
12. Shiwaiker A, Rajendran K, Kumar CD (2006) in vitro antioxidant studies of Annona squamosa Linn leaves. Ind J Exp Biol 42:803-807.
13. Prior RL, Hoang H, GuL W, Wu X, Bacchiocca M, et al.(2003) Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORAC (FL)) of plasma and other biological and food samples. J Agric Food Chem 51: 3273-3279.
14. Oyaizu M (1986) Studies on products of browning reaction prepared from glucoseamine. Jpn J Nutr 44:307-314.
15. Makchuchit S, tharat AI, Tewtrakul S (2010) Antioxidant and nitric oxide inhibition activities of Thai Medicinal Plants. J Med Assoc Thai 93: 227-235.
16. Prieto P, Pineda M, Aguilar M (1999) Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem 269: 337-341.
17. Ruch KJ, Cheng SJ, Klauning JE (1998) Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechin isolated from Chinese green tea. Carcinogenesis 10: 1003-1008.
18. Suprava S, Goutam G, Debajyoti D, Sanghamitra N (2013) Phytochemical investigation and in vitro antioxidant activity of an indigenous medicinal plant Alpinia nigra BL BurttAsian Pac J Trop Biomed 3:871-876.
19. Elizabeth K, Rao MNA(1990) Oxygen radical scavenging activity of curcumin.Int J Pharmaceutics 58: 237-240.
20. Lee JC, Kim HR, Kim J, Yang YS (2002) Antioxidant property of an ethanol extract of Optuniaficus-indica Saboten. J Agri Food Chem 50: 6490-6496.
21. Kikuzaki H, Nakatani N (1993) Antioxidant effects of some ginger constituents. J Food Sci 58:1407-1410.
22. Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95: 351-358.
23. Dinis TCP, Madeira VMC, Almeida LM (1994) Action of phenolic derivates (acetoaminophen, salycilate and 5-aminosalycilate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Archives Biochem Biophys 315: 161-169.
24. Dudonne S, Vitrac X, Coutiere P, Woillez M, Merillon JM, et al.(2009) Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J Agric Food Chem 57: 1768.
25. Sharmila S, Kalaichelvi K, Rajeswari M, Anjanadevi N (2014) Studies on the folklore medicinal uses of some indigenous plants among the tribes of Thiashola, Manjoor, Nilgiris South Division, Western Ghats. Int J Plant Animal Envir Sci 4:14-22.
26. Farid MM, Hussein SR, Ibrahim LF, Desouky MAE (2015) Cytotoxic activity and phytochemical analysis of Arum palaestinum Boiss. Asian Pac J Trop Biomed 5: 944-947.
27. Saiba A, Wahab N, Kumaraswamy M (2013) in vitro analysis on bactericidal screening and antioxidant potentiality of leaf and root extracts of Thottea siliquosa (Lam.) Ding Hou. An ethnobotanical plant. Asian Pac J Trop Biomed 3: 859-865.
28. Prasad GJ, Sonal RB, Shailesh LP, Priti ST (2013) Plant profile, phytochemistry and pharmacology of Cordia dichotoma (Indian cherry): A review. Asian Pac J Trop Biomed 3: 1009-1012.
29. Shanmugapriya K, Jinu U, Thayumanavan Tha (2014) Investigation of antioxidant activity in different solvents of Gnaphalium polycaulon. Int J Pharm Pharmaceut Sci 6: 870-873.
30. Li W, Sun H, Zhou J, Zhang Y, Liu L, et al. (2015) Antibacterial activities, antioxidant contents and antioxidant properties of three traditional Chinese medicinal extracts. Bangl J Pharmacol 10: 131-137.
31. Zheng X, Wang W, Piao H, Xu W, Shi H, et al. (2013) The Genus Gnaphalium L. (Compositae): Phytochemical and Pharmacological Characteristics. Molecules 18: 8298-8318.
32. Zeng WC, Zhu RX, Jia LR, Gao H, Zheng Y, et al.(2011)Chemical composition, antimicrobial and antioxidant activities of essential oil from Gnaphalium affine. Food Chem Toxicol49: 1322-1321.