The traditional knowledge forms of codified systems of medicine are exists in the forms of Ayurveda, Unani and Siddha (Sen & Chakraborty, 2016). India is the oldest, richest diverse cultural traditions associated with the use of medicinal plants. This knowledge is accessible from thousands of medical test and manuscript (Kumar et al., 2017). Since ancient time, mankind was using herbal plants for treatment of certain diseases. The study of traditional medicine based on bioactive compounds in the plants is called as ethnomedicine study (Adhikari et al., 2019).

Among medicinal plants, the substances having medicinal value have been extensively used for treating various disease conditions (Sofowora et al., 2013). Herbs being easily available to human beings have been explored to the maximum for their medicinal properties (Ekor, 2013). Phytoconstituents are the natural bioactive compounds found in plants. This phytoconstituents work with nutrients and fibers to form an integrated part of defense system against various forms of diseases and stress conditions (Altemimi et al., 2017).

Gomphrena serrata, the plants belong to the family of amaranthaceae are very rich source of bioactive constituents like carbohydrate, alkaloids, steroids, glycosides, and triterpenoids. In general, the family of amaranthaceae contains nearly 60-70 exotic species (Nandini et al., 2018). The genus Gomphrena, contain about 138 species, some of the important species include G. boliviana, G. celosioides, G. globose, G. haenkeana, G. macrocephala, G. martiana, G. meyeniana, G. perennis, and G. pulchella. The various parts of this plant are used in India for treatment of various ailments need for the traditional healers, including treatment of asthma, diarrhea, indigestion, dermatitis, hay fever, and others (Rahman & Gulshana, 2014).

Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) are products of normal cellular metabolism. These free radicals are the fundamental to any biochemical process and represent as an essential part of aerobic life and metabolism (Di Meo et al., 2016). Antioxidant are the molecules which have capability to prevent the oxidation of other molecules (Kurutas, 2016).

The objective of the study was to analyses phytochemical constituent and antioxidant potential of G. serrata. The plant extracts determined by hydrogen peroxides, hydroxyl radical and DPPH assays which are designed by the EC50 and compared with the standard ascorbic acid. However, there are no reports on phytochemical analysis of whole plant of ethanol extract of G. serrata have been reported.

Plant collection

The fresh whole plants of G. serrata were collected from the local area of Bharathinagara, Mandya, Karnataka. The plants were identified and authenticated by Botanist Dr. Gurukar Mathews, Head of the Department Bharathi College of Post-Graduation and Research Centre, Bharathinagara, Maddur, Mandya, Karnataka, India.

Extraction

After the collection of whole plant of the G. serrata was wash thoroughly with running tap water, cut in to small pieces, and shade dried. The dried whole plant then pulverized separately into coarse powder by a mechanical grinder. As much as 100 g of powdered G. serrata was carried out by hot extraction process using Soxhlet apparatus with ethanol as solvent for 72 hours at 50°C. The distillates were collected and distilled separately to yield the extracts. These extracts concentrated using vacuum rotary evaporator to obtain crude extract. It turned into a greenish black color with yield of 10.8%. The extract was kept in a desiccator over anhydrous calcium chloride until used.

Phytochemical screening

Small quantity of freshly prepared extract of G. serrata were subjected to quantitative chemical tests for identification of various phytoconstituents. Phytochemical investigations were carried out as per the standard methods set by WHO (Khandelwal, 2006; Khadabadi et al., 2013).

Carbohydrates test

1. Molisch test

As much as 1 ml of extract was treated with the compounds of β-naphthol and added with concentrated sulphuric acid along the sides of the test tube. Purple or reddish violet color was formed at the junction between two liquids, which indicated the presence of carbohydrates.

Alkaloids test

1. Dragendorff test

As much as 1 ml of extract was treated with 1 ml of Dragendorff reagent. Orange red precipitate was formed which indicates the presence of alkaloids.

2. Wagner test

As much as 1 ml of extract was treated with 1 ml of Wagner’s reagent. Reddish brown precipitate was formed, which indicates the presence of alkaloids.

3. Mayer test

As much as 1 ml of extract was treated with 1-2 drops of Mayer’s reagent. Cream colored precipitate was formed, which indicates the presence of alkaloids.

4. Hager test

As much as 1 ml of extract was treated with 3 ml of Hager’s reagent. Yellow precipitate was formed, which indicates the presence of alkaloids.

Glycosides test

1. Keller-Killiani test

As much as 2 ml of extract was dissolved in acetic acid containing trace of ferric chloride and transferred to the surface of concentrated sulphuric acid. At the junction of two liquids reddish brown color was formed, which gradually blue color due to the presence of glycosides.

2. Borntrager test

As much as 1 ml of diluted H2SO4 was added with 2 ml of extract. The mixture was boiled, filtered, and extracted with ether or chloroform. Organic layer was separated to which ammonia was added. Pink, red, or violet color was produced in organic layer, which indicated the presence of glycosides.

Phytosterols and triterpenes test

1. Liebermann–Burchard test

As much as 1 ml of extract was treated with 2 ml of chloroform in a dry test tube. Then 10 drops of acetic anhydride and 2 drops of concentrated sulphuric acid were added. The solution was turned into red, then blue, and finally green in color, which indicates the presence of phytosterols.

2. Salkowski test

As much as 1 ml of extract was treated with 1 ml of chloroform and added 2 ml of concentrated H2SO4. Bluish red and purple color was formed in chloroform layer, which indicate the presence of triterpenes.

Tannins and flavonoids test

1. Gelatin test

As much as 1 ml of extract was treated with 1% gelatin solution containing sodium chloride. Formation of white precipitate indicates the presence of tannins.

2. Lead-acetate test

As much as 1 ml of extract was treated with 10% lead acetate solution. Formation of yellow precipitate indicates the presence of flavonoids.

3. Shinoda test

As much as 1 ml of extract was treated with a few fragments of magnesium and concentrated HCl were added. Appearance of magenta color after few minutes indicates presence of flavonoids.

Proteins and amino acids test

1. Biuret test

As much as 1 ml of extract was treated with 1 ml of 40% NaOH and 2 drops of 1% copper sulphate. Appearance of violet color indicates the presence of proteins.

2. Xanthoproteic test

As much as 1 ml of extract was treated with 1 ml of 20% of sodium hydroxide or ammonia. Appearance of orange color indicates the presence of aromatic amino acid.

Fixed oils and fats test

1. Spot test

As much as 1 ml of extract was applied as a spot in filter paper. Appearance of a clear-transparent spot indicates the presence of fixed oils.

Saponins test

1. Foam test

As much as 1 ml of extract was treated in hot water sufficiently, and after cooled until room temperature then shake vigorously for 10 seconds. It was produced the foam then 1% HCl was added. Foam that lasts for not less than 10 minutes indicates the presence of saponins.

In vitro antioxidant test

Each sample was dissolved in distilled methanol to make a concentration of 20-100 µg/ml and then diluted to prepare the series concentrations for antioxidant assays. Reference Ascorbic acid was used for standard comparison in all assays.

Hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity of G. serrata was measured according to a method described previously with some modification (Smirnoff & Cumbes, 1989). Briefly, the different concentration of ethanol extract of G. serrata was mixed with 1 ml of 9 mM of Salicylic acid, 1 ml of 9 mM of Ferrous sulphate, and 1 ml of 9 mM Hydrogen peroxide, respectively. The mixture was then incubated at 37°C for 60 min in a water bath. After incubation period, the absorbance of the mixtures was measured at 510 nm. The activity of hydroxyl radical scavenging (%) was calculated as follows:

% inhibition = absorbance of control - absorbance of sample x 100

absorbance of control

Hydroxyl peroxide scavenging activity

Hydrogen peroxide solution (4 mM) was prepared in 50 mM phosphate buffer pH 7.4. As much as 0.1 ml of aliquots from different concentration sample solution was transferred into the test tubes and their volumes were made up to 0.4 ml with 50 mM phosphate buffer. After addition of 0.6 ml hydrogen peroxide solution, mixed solution and absorbance of the hydrogen peroxide at 230 nm was determined after 10 minutes, against a blank (Ruch et al., 1989). The abilities to scavenge the hydrogen peroxide was calculated using the following equation:

% inhibition = absorbance of control - absorbance of sample x 100

absorbance of control

DPPH free radical scavenging activity

As much as 2.36 g of the DPPH was dissolved in 100 ml of methanol to get 6 x 10-5 M methanolic solution of DPPH. A series concentration of standard ascorbic acid and G. serrata extract that is 20, 40, 60, 80, and 100 μg/ml were prepared by diluting with methanol (Pavithra & Mani, 2019). As much as 1 ml of each diluted standard and test solution were mixed with 3 ml of DPPH solution in each test tube. Control solution was prepared by adding 1 ml of methanol and 3 ml of DPPH. The test tubes were covered with aluminum foil to protect from light and kept in dark place for 15 minutes. Methanol was used as blank. Absorbance of standard, control, and test extract was measured at 517 nm using UV-Visible spectrophotometer. The % inhibition was calculated by using following formula and compared with the values of standard ascorbic acid:

% inhibition = absorbance of control - absorbance of sample x 100

absorbance of control

Statistical analysis

All the experiment was carried out in triplicate and data reported are mean ± standard deviation. Then EC50 was calculated from the graph obtained by percentage of inhibition was plotted against concentration.

The G. serrata extract was subjected for qualitative chemical analysis for the identification of various phytoconstituents, revealed the presence of carbohydrates, alkaloids, glycosides, phytosterols and triterpenes, tannins and flavonoids, proteins and amino acids, fixed oils and fats, and saponins. Since all these compounds were found to be present in the extracts, it might be responsible for the potent antioxidant capacity of G. serrata. The preliminary phytochemical screenings are helpful in finding phytoconstituents in the plant material that may lead to their quantitative estimation and also in locating the source of pharmacologically active chemical compound (Shrestha et al., 2015). Detail results of each phytochemical screening tests was presented in the Table I.

Table I. Preliminary phytochemical analysis of ethanolic extract of G. serrata

In vitro antioxidant test

Hydroxyl radical scavenging activity

The scavenging activity of ethanol extract of G. serrata on hydrogen peroxide scavenging activity is presented in Table II. The percentage inhibitions were increased with increasing concentrations of the extracts as presented in Figure 1. Free radicals are known to play very important role in a wide variety of pathological manifestations. Antioxidants fight against free radicals and protect us from various ailments. They exert their action either by scavenging the ROS or protecting the antioxidant defense mechanisms (Umamaheswari & Chatterjee, 2007). In biological systems, hydroxyl radical are the most powerful radicals evolved from hydrogen peroxide and superoxide anions in metal ions presence. Hydroxyl radical can damage any cells in the body and responsible for many pathological conditions in DNA, lipids, as well as proteins and can cause mutagenesis, cancer, and cytotoxicity (Phaniendra et al., 2015).

Table II. Hydroxyl radical scavenging activity of G. serrata

Figure 1. Hydroxyl radical scavenging activity of G. serrata

Hydroxyl peroxide scavenging activity

The hydrogen peroxide scavenging activity of ethanol extract of G. serrata is presented in Table III. The plant extract exhibited antioxidant activity at all the concentration of test solutions, with the increase in concentration of the plant extract also increasing the percentage of antioxidant activity. Among all concentration, maximum antioxidant activity was observed at 100 µl/ml as presented in Figure 2. Hydrogen peroxide occurs naturally at low concentration levels in the air, water, human body, plants, microorganisms and food (Gülçin et al., 2005). Hydrogen peroxide is quickly break down into water and oxygen. This will produce hydroxyl radicals (•OH) that can initiate lipid peroxidation and cause DNA damage. Ethanolic extract of G. serrata efficiently scavenging hydrogen peroxide which may be attributed to the presence of phenolic groups that could donate electrons to hydrogen peroxide, thereby neutralizing it into water (Pizzino et al., 2017).

Table III. Hydroxyl peroxide scavenging activity of G. serrata

Figure 2. Hydroxyl peroxide scavenging activity of G. serrata

DPPH free radical scavenging activity

The DPPH free radical scavenging activity of ethanol extract of G. serrata is presented in Table IV. The stable radical DPPH had been used widely for the determination of primary antioxidant activity. The DPPH antioxidant assay is based on the ability of a stable free radical to decolorize in the presence of antioxidants (Kedare & Singh, 2011). Among successive solvent of extracts, the highest percentage inhibition by DPPH radical scavenging assay exhibited in ethanolic extract 81.66 ± 0.11 at 100 µg/ml and the lowest percentage inhibition by DPPH radical scavenging assay exhibited in ethanolic extract 42.67 ± 0.15 at 20 µg/ml. The mean EC50 value of extract is 33.23 µg/ml and for standard ascorbic acid is 25.43 µg/ml. The percentage inhibitions were increased with increasing concentrations of the extracts as presented in Figure 3. That showed the scavenging effect on the DPPH radical increase sharply with increasing concentration of the sample and standards.

Table IV. DPPH free radical scavenging activity of G. serrata

Figure 3. DPPH free radical scavenging activity of G. serrata

The phytochemical screening showed that the whole plant of G. serrata extract contain a mixture of phytochemicals as carbohydrates, alkaloids, glycosides, phytosterols and triterpenes, tannins and flavonoids, proteins, fixed oils, as well as saponins. It is also known that whole plant extract of G. serrata also exhibit good scavenging effects on hydroxyl radical, hydrogen peroxide, and DPPH method. In conclusion, the high antioxidant activity exhibited by G. serrata extract provided justification for the therapeutic use of this plant in folkloric medicine. Further research is needed for G. serrata to identify compounds that have pharmacological properties using an appropriate assay model.

The authors are thankful to the Principal and the Management of Bharathi College of Pharmacy, Bharathinagara, Karnataka, for providing the platform to perform the research work.

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