Phytochemical evaluation, HPTLC analysis and in-vitro antioxidant activity of hydroalcoholic leaf extract of Grewia hirsuta collected fromWestern Ghats forest

In-vitro antioxidant action of hydroalcoholic leaf extract of Grewia hirsuta (HAEGH) has been examined using one, “1-diphenyl-2-picryl-hydrazil (DPPH) free from radical scavenging” actions. The plant collected from the forest of the Western Ghats region of Karnataka province. The motive for plant collection from a speci ic location is the plant of forests exhibits the variation in growth, quantity, and quality of their active ingredients and secondary metabolites due to in luence ecological factors like effect changes in location, soil, climate, etc. The work corresponds to preliminary phytochemical investigation for diverse phytoconstituents and quantitative phytochemical analysis of total phenolic, lavonoids & alkaloids content (TPC, TFC & TAC respectively) was evaluated with advanced methods. “HPTLC (High performance thin-layer” chromatography) ingerprint investigation was achieved for qualitative determination of the likely number of elements present in the hydroalcoholic extract. In-vitro antioxidant activity of HAEGH has been determined through hydroxyl radical scavenging assay that exhibited strong dosedependent antioxidant activity as comparedwith standards compound, ascorbic acid. The IC50 value of HAEGH found, 25.90 % inhibition and for ascorbic acid, it was 17.68%. The Preliminary phytochemical estimation found presence of lavonoids, alkaloids, glycosides, phenol, proteins, diterpins and quantitative phytochemical analysis estimation of TPC, TFC & TAC found to be 3.627%, 4.059% & 5.671% respectively. HPTLC analysis of HAEGH at 354nm reveals the presence of a compound with Rf value 0.44 compare with Rf value 0.46 of quercetin. These outcomes indicated that the hydroalcoholic leaf extract of Grewia hirsuta plant contains phytoconstituents that exhibit antioxidant activity possible because of the existence of bioactive compounds.


INTRODUCTION
The diseases are caused due to damage of tissues through active nitrogen, active oxygen species, as well as active chlorine, are byproducts of metabolism or entered the body from external sources (Cochrane, 1991). These "free radicals play a" main "role in the" procedure of aging as well as serious diseases like diabetes mellitus, cardiovascular diseases, cataract development, respiratory diseases, neurodegenerative disorders, rheumatoid arthritis, and various cancers. The three basic units of cells nucleic acids, lipids as well as proteins molecules damage owing to generation of nitrosative stress and oxidative stress (Phaniendra et al., 2015).
Antioxidants are naturally occurring compounds that protect the body from oxidative free radicals and obtained from plants mainly are phenolic lavonoids, alkaloids, tannins, and phenolic compounds include phenolic acids, tocopherols, cinnamon acids carotenoids as well as benzoic acid derivatives (Zargoosh et al., 2019). The plant origin chemical compounds have the potential to be replaced by synthetic drugs and reduce side effects than synthetic drugs (Berahou et al., 2007). Hence above 80% of developing or under rising populations primarily using herbal extracts or their active ingredients for health care purposes (Hasan et al., 2016).

Plant Material
Grewia hirsuta's Fresh leaves have been collected from the forest near to Rani Channama University, Belgavi, (Latitude: 16.0073130, Longitude: 74.4968770) of Karnataka, India in November 2018. The fruits have been washed to take away foreign matters, and then shed dried.
Plant identi ication and authenti ication was done as Grewia hirsuta Vahl by an expert taxonomist from "ICMR-National Institute of Traditional Medicine, Belagavi, Karnataka, India".

Total phenol content estimation"(TPC)
The TPC was calculated through the modi ied folinciocalteu technique. Gallic acid 10mg and Methanol 10ml used for the preparation of a standard sample. The test sample of HAEGH was prepared by adding 10mg of dried extract powder into 10ml methanol as where, +: present (mild "amount), ++: present (moderate amount), +++: present" (large"amount), −: absent, based on the" power of generated "color reaction".

Total lavonoids content estimation (TFC)
TFC was estimation based on aluminum chloride method. The standard solution is made by 10mg quercetin dissolved in 10ml methanol. The test solution prepared by 10mg of dried HAEGH was dissolved in 10ml methanol and ilters it. From iltrate, 3ml was utilized for the evaluation of TFC (Moteriya et al., 2014).

"Total" alkaloids "content" estimation (TAC)
The principle of TAC is standing on response of alkaloid among bromocresol green solution that forms a yellow-colored solution. The dried 1mg plant extract mixed with "1ml of 2 N HCl" as well as " iltered. This solution" of pH adjusted by 0.1 N NaOH and poured into separating funnel. After that "5 ml of phosphate buffer and 5 ml of bromocresol green solution" had been included. The combi-  nation was shaken vigorously as well as extracted with chloroform. Then extracts have been transferred into a 10ml volumetric lask along with diluted through chloroform. Standard sample prepared by various aliquots "of atropine (40, 60, 80, 100 and 120 µg/ml)" have been organized similarly as described previously. The experiment, as well as standard sample absorbance, was calculated through a UV/Visible spectrophotometer at 470nm wavelength against the reagent blank (Fadhil et al.,  The scavenging activity has been assessed through the spectrophotometer process. In this assay reference, standard and control were used butylated hydroxytoluene and methanol respectively. The principle involved evaluation of antioxidant capacity by the reduction in DPPH radicals that analyzed by transformation of colors of solution from deep violet to light yellow, measured at 517 nm wavelength. A stock solution for test prepared with 6 mg DPPH in 100 ml methanol and 1.5 ml of this solution combined by various concentrations of HAEGH. The absorbance in the presence of test extract at various "concentrations (10-100 µg/ml) was noted after 15 minutes. The"control reading has been noted through the adding up 1.5 ml of DPPH solution with makeup volume 3 ml with methanol and absorbance has been used directly at 517 nm. All tests were carried out thrice and mean taken for calculation. Recording of reduction in absorbance has been mentioned of DPPH through the sample at various concentration after 15 minutes at 517 nm (Olajuyigbe and Afolayan, 2011).

Estimation of Phytochemical Constituents"
The phytochemical investigation of HAEGH exhibited the existence of important biologically active secondary metabolites, like saponins, phenols, alkaloids, ditrepins, lavonoids, glycosides, carbohydrates, and proteins which were established that demonstrated in Table 1. That based on the intensity of the color reaction, the greatest amount of alkaloids, lavonoids, as well as glycosides, were contained by the HAEGH extract

Total phenol content estimation (TPC)
Gallic acid equivalent of TPC was expressed as mg/100mg of dry extract sample utilizing the equation acquired from the calibration curve: Y = 0.011X+0.011, R 2 = 0.998, in which Y stand for absorbance and X referred as gallic acid equivalent (GAE). The TPC was observed to be 3.627mg/100mg of HAEGH, which was shown in Table 2 and Figure 1.

Total alkaloid content estimation (TAC)
TAC was shown "as atropine equivalent mg/100mg"utilizing the equation stand on the calibration curve: Y=0.007X+ 0.024, R 2 =0.995, in which Y measured as absorbance and X represented the Atropine equivalent (AE). The TAC was found 5.671mg/100mg of HAEGH, shown in Table 2 and Figure 3.

HPTLC ingerprint analysis
The HPTLC ingerprinting validates a total of ifteen peaks, peak one to seven represents HAEGH and peaks onwards seven revealed standard drug Quercetin. The sharp and compact peaks of Quercetin was obtained at Rf = 0.46 with absorbance mode at 254nm shown in Figures 4 and 5.
The results at wavelength 254nm for HAEGH revealed the presence of polyvalent phytoconstituents. The component with Rf value 0.44 was found to be superior with the percentage area is 89.41% exposed in Figure 6. The visualization of chromatogram obtained from HAEGH shown in Figure 7.

DPPH scavenging activity:
The study explored, HAEGH for their antioxidant potential with DPPH scavenging assay. Assay followed criteria IC 50 for interpretation of antioxidant activity. It was based on the linear regression equation which says the correlation between the concentration of the sample (X-axis) with the percent inhibition (Y-axis) shown 50% decreased activity of DPPH concentration. Percentage inhibition was estimated from the reduction of arithmetic DPPH absorbance with the absorbance of the sample.
Outcomes of the antioxidant activity of Grewia hirsuta hydroalcoholic extract compared to the ascorbic acid were revealed in Table 3. The hydroalcoholic extract established a high antioxidant activity compared against ascorbic acid.
Medicinal plants are" bene icial sources of antioxidant compounds as well as reported "an important role in the treatment of diseases" worldwide (Fouche et al., 2015). Various plants have been shown antioxidant pro iles like the reduction of DPPH radicals reducing power because of the existence of bioactive secondary metabolites" that are rich in antioxidant along with "free radical scavenging" qualities (Kipkore et al., 2014). The plant Grewia hirsuta Vahl has conventionally been utilized through people in many region of India and various parts of a plant traditionally used in the treatment of headache, vision problems, sore throat, and cholera (Goyal, 2012). It was reported that the whole plant contains phytoconstituents like steroids, lavonoids, alkaloids, tannins (Yamazaki and Kawano, 2011).
The current study was based on conventional use along with preliminary phytochemical constituents to estimate the antioxidant pro ile of the hydroalcoholic extract of Grewia hirsuta Vahl. The plant leaves collected from the Western Ghats region of Karnataka province and the reasons for the collection of plants from a speci ic location that affects ecological factors. The ecological factors apart from species are climate, soil; along with location lead to changes in the development of medicinal plants moreover the quality and quantity of their active ingredients, like steroids, glycosides, alkaloids, as well as essential oils (Omidbeigi, 2012). A study published in Nature Scienti ic Report showed that an ecological factor-like elevation, climate, and soil features, etc produces considerable consequence on the potency of the extract, antioxidant actions as well as total phenol content of Scrophularia striata Boiss plant (Zargoosh et al., 2019).
One study reported the free radical scavenging activity of Grewia hirsuta whole plant, which was collected from the Nanmangalam forest, Chennai, India state that 200 µg methanol extract of plant exhibited 95 % free radical scavenging inhibition activity (Ema et al., 2013). But in the current study, it has been proved that the hydroalcoholic extract of plant leaves collected from the Western Ghats region of Karnataka province exhibited the better antioxidant activity at dose merely 100 µg with 88.23 % inhibition.
It was envisaged that the entire polyphenols content of hydroalcoholic extract of Grewia hirsuta leaf extract had been the foremost accountable parts for the antioxidant potentials, additionally, it has been con irmed that various ROS (reactive oxygen species) speci ically, as superoxide anion, hydroxyl radicals and hydrogen peroxide" produced from the system had been inhibited through the different antioxidant phytoconstituents present in medicinal plant and preventing further cell damages (Yamunadevi et al., 2011). In DPPH assay, reduction of ferric ion into ferrous ion through antioxidants phytoconstituents present in the hydroalcoholic extract, which develops a change in color, depending upon their concentration of phytoconstituents and reducing power capacity (Sravani and Paarakh, 2012). HPTLC analysis reveals the presence of active constituents in the hydroalcoholic extract of Grewia hirsuta leaf was con irmed quercetin by peak but even though further study is required to con irm the identity of compounds.

CONCLUSIONS
The present study con irmed the hydroalcoholic extract obtained from the leaf of Grewia hirsuta (family: Tiliaceae) plant collected from the Western Ghats forest, possesses signi icant antioxidant activity. The HPTLC ingerprint analysis showed the presence of quercetin compound as compared to the standard quercetin chromatograph. The plant extracts could be invoked as a renewable source of natural safe antioxidants in nutraceutical preparations or food supplements. Further study is under process to con irm the identity of the actual component present in hydroalcoholic extract responsible for the antioxidant property.