Extraction, Phytochemical Screening and Antioxidant Potential of Ethanolic Extract of Drypetes roxburghii

Humans are affected by free radicals in over a hundred different ways. Synthetic antioxidants have been linked to harmful health consequences, prompting substantial investigation into naturally occurring antioxidants, particularly those found in plants. The goal of this study was to determine the phytochemical components and in vitro antioxidant activity of Drypetes roxburghii (D. roxburghii, Euphorbiaceae) leaves collected in the Bhopal district of Madhya Pradesh. The 1, 1-diphenyl-2picrylhydrazyl radical (DPPH) test was used to measure antioxidant activity. The presence of cardiac glycosides, flavonoids, phenol, proteins, carbohydrate, and saponins in the ethanolic extracts of D. roxburghii leaves was discovered using phytochemical screening. The ethanolic and aqueous extracts of D. roxburghii leaves yielded 1.9 and 2.6 percent w/w, respectively. The ethanolic extract of D. roxburghii leaves contained 1.48 and 0.70 mg/100 mg of total phenolic and flavonoids, respectively. When comparing the DPPH scavenging activity of leaf ethanolic extract to Ascorbic acid (standard), the IC50 value was 87.80. It suggests that the plant has the ability to scavenge free radicals, and it might lead to new discoveries in the continuing hunt for natural antioxidants in medicinal plants that can be utilised to treat illnesses caused by free radical reactions. Original Research Article Arvind et al.; JPRI, 33(64A): 358-363, 2021; Article no.JPRI.82642 359


INTRODUCTION
Plants have long been used as therapeutic ingredients, and humans continues to rely on them for healthcare [1]. Plant-based remedies are still used by over 80% of the population in poor and underdeveloped nations to treat their problems [2]. Compounds generated from nature have played an important role in the development of novel chemical entities. The process of discovering drugs from nature is multidisciplinary and incorporates numerous fields such as ethnobotany, phytochemistry, biology, and different chemical separation methods, as well as combinatorial synthesis approaches. Approximately 87 percent of medications are considered to be obtained directly or indirectly from nature. In nature, there are around 420 000 plant species [3]. Antioxidants fight oxidation and help to preserve lipids from radical peroxidation. The human body is equipped with a sophisticated antioxidant defence system [4]. The capacity of an antioxidant to trap free radicals is its most important feature. Free radicals and oxygen species with high reactivity can be found in biological systems from a variety of sources. These free radicals can cause degenerative illness by oxidising nucleic acids, proteins, lipids, or DNA. Antioxidant substances such as phenolic acids, polyphenols, and flavonoids scavenge free radicals including peroxide, hydroperoxide, and lipid peroxyl, inhibiting the oxidative pathways that contribute to degenerative illnesses. A number of clinical investigations show that the antioxidants found in fruits, vegetables, tea, and red wine are the major reason for these foods' reported efficacy in lowering the prevalence of chronic illnesses like heart disease. The plant D. roxburghii Wall is a member of the Euphorbiaceae family. Putranjiva roxburghii Wall is its synonym. It grows wild or farmed in practically every corner of India, up to a height of 750 metres. It is a dioecious, evergreen tree with grey bark that grows to a height of up to 18 metres and a girth of up to 2 metres. Long, elliptic oblong to ovate-lanceolate leaves with uneven sides at the base, dark green in colour and gleaming. Saponins, mannitol, arachidic acid, linoleic acid, palmitic acid, glucoputranjivin, putranjivoside, putranoside,sitosterol, carboxylic acid, putric acid, putranjivic acid, glucosides, fatty oil, alkaloids, and gallotannins are among the phytochemicals found in D. roxburghii. For colds and fevers, the leaves and stones are decocted; they are also used for rheumatism, elephantiasis, burning sensations, azoospermia, constipation, and infertility. It is also used as an antidiabetic [5,6]. The aim of this work was to determine the quality (types), quantity (amount) of bioactive compounds and in vitro antioxidant activity of leaves of D. roxburghii in Bhopal region of Madhya Pradesh.

Plant Materials
Fresh D. roxburghii leaves were taken from Vindhya Herbals' Botanical Garden in Bhopal. The leaves were properly cleansed with regular tap water and then sterile distil water. The leaves were then dried at room temperature in a shady environment. Using a grinding machine, the leaves were crushed into powder. The powder was kept at 4°C in an airtight storage bottle. The substances employed in this experiment were all analytical grade.

Extraction by Maceration Methods
By maceration, 175 gramme of powdered D. roxburghii leaves were extracted using various solvents such as chloroform, ethyl acetate, ethanol, and aqueous. The extract was evaporated at temperatures higher than their boiling points. Finally, the dried extracts' percent yield was calculated. After that, the extracts were reduced in a rotary evaporator before being kept in airtight containers at 4°C for later use.

Evaluation of Organoleptic Properties
Color, odour, taste, texture, and other organoleptic characteristics of dried extracts of Drypetes roxburghii were assessed. The sample's organoleptic characteristics were assessed using the approach outlined by Siddiqui and Hakim [7].

Estimation of Total Phenolic Content
The modified Folin-Ciocalteu technique [10] was used to determine the total phenolic content of the extract. In methanol, 10 mg Gallic acid was dissolved in 10 ml methanol, and different aliquots of 5-25g/ml were produced. We dissolved 10 mg of dried extracts in 10 ml methanol and filtered it. For the phenol determination, two millilitres (1 mg/ml) of this solution were utilised. 1 ml Folin-Ciocalteu reagent (previously diluted with distilled water 1:10 v/v) and 1 ml (7.5g/l) sodium carbonate were combined with 2 ml of each extract or standard. The mixture was vortexed for 15 seconds before being let to sit for 15 minutes to develop colour. A spectrophotometer was used to measure the absorbance at 765 nm.

Estimation of Total Flavonoids Content
The total flavonoids content was determined using the aluminium chloride technique [11]. In 10 ml methanol, 10 mg quercetin was dissolved, and different aliquots of 5-25g/ml were made in methanol. We dissolved 10 mg of dried extracts in 10 ml methanol and filtered it. The flavonoid concentration was determined using three millilitres (1 mg/ml) of this solution. 1 ml of a 2 percent AlCl3 methanolic solution was added to 3 ml of extract or standard and allowed to remain at room temperature for 15 minutes before measuring absorbance at 420 nm.

DPPH methods
Total free radical scavenging capacity of the ethanolic extract obtained from Drypetes roxburghii was estimated according to the previously reported method with slight modification [12]. Solution of DPPH (6 mg in 100ml methanol) was prepared and stored in dark place. Different concentration of standard and test (10-100 µg/ml) was prepared. 1.5 ml of DPPH and 1.5 ml of each standard and test was taken in separate test tube; absorbance of this solution was taken immediately at 517nm. 1.5 ml of DPPH and 1.5 ml of the methanol was taken as control absorbance at 517nm. The percentage inhibition of free radical DPPH was calculated from the following equation: % inhibition = [(absorbance of controlabsorbance of sample)/absorbance of control] × 100%.

RESULTS AND DISCUSSIONS
To achieve the real yield of extraction, the crude extracts produced after each consecutive maceration extraction step were concentrated on a water bath by fully evaporating the solvents. Table 1 shows the yield of extracts collected from the plants' leaves using chloroform, ethyl acetate, ethanol, and water as solvents. Table 2 shows the findings of a qualitative phytochemical study of D. roxburghii leaf crude powder. D. roxburghii ethanolic and aqueous extracts included cardiac glycosides, flavonoids, phenol, proteins, carbohydrate, and saponins. The total phenolic content of a sample is measured in mg gallic acid equivalents per 100 mg dry weight of the sample. The extracts' total flavonoids content was calculated as a percentage of quercetin equivalent per 100 mg dry weight of sample. TPC of D. roxburghii ethanolic and aqueous extracts revealed concentration values of 1.48 and 1.08, respectively. Table 3 shows that the total flavonoid concentration of D. roxburghii ethanolic and aqueous extracts was 0.70 and 0.32, respectively. The DPPH test has been widely utilised to determine the in vitro general antioxidant activity of pure chemicals and plant extracts [13][14][15]. The reduction in DPPH radical absorbance with increasing extract concentration, as seen by the fast discoloration of the purple DPPH, suggests that the D. roxburghii hydro alcoholic extracts have antioxidant activity due to its proton donating capacity. The extracts were shown to have dosedependent action, implying that DPPH scavenging activity increased according to the rise in extract concentration. Table 4 also included the IC 50 values for scavenging DPPH radicals for the AA and extract. When compared to AA, the extract's IC50 value for DPPH radical activity was determined to be 87.80.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

NOTE
The study highlights the efficacy of "Herbals" which is an ancient tradition, used in some parts of India. This ancient concept should be carefully evaluated in the light of modern medical science and can be utilized partially if found suitable CONSENT It is not applicable.

ETHICAL APPROVAL
It is not applicable.