High Technology Letters ISSN NO : 1006-6748

Preliminary Phytochemical Screening o nRoot Part of arborea Roxb.

Nand Kumar Kashyap , Milan Hait * Department of Chemistry, Dr. C.V. Raman University, Kota, BilaspurBilaspur,, C.G. -495113, India. *Corresponding author: [email protected] Abstract Preliminary phytochemical screening is an important step in detecting biologically active compounds present in medicinal and subsequently leading to drug discovery and development. Use standard methods to perform a preliminary phytochemical screenin g of the root extracts and solvent fractions of Roxb . This study involved the phytochemical characteristics of the roots of Careya arborea Roxb . Qualitative chemical examination showed that there are various phytochemicals in root extracts, such as alkaloids, terpenoids, flavonoids, steroids, saponins, sterols, phenol, carbohydrates, tannins, and glycosides. The presence of various biologica lly active ingredients helps traditional healers to apply Careya arborea Roxb. to various diseases. The investigation revealed the specific characteristics of specific raw drugs, which will be suitable for identification and control to reduce the value of raw drugs.

Keywords: Careya arborea , root, extraction yield, phytochemical analysis . 1. INTRODUCTION Phytochemicals are compounds produced by plants. They exist in leaves, stems, flowers, fruits, seeds, and various parts of plants. These phytochemicals are be lieved to protect against various diseases. Plants are the oldest and most important source of medicine. Information from various records reveals the use of plants as medicines about 60,000 years ago. Since the birth of humans on this planet, plants have b een used to diagnose, trea t, and prevent various diseases [1-2] . Conventional medications that are commonly used to treat many diseases are not always safe or effective and sometimes exceed the affordability or accessibility of the majority of people in the world [3] . The traditional medicine system, which is based on the use of herbal medicine as medicine, is one of the most famous alternatives for these patients. Although there are several treatments available from synthetic sources, there is still a need t o evaluate new, inexpensive, and cost -effective drugs to meet future disease-related challenges. According to the World Health Organization (WHO), the use of herbal medicines in the healthcare system is increasing over time [4-6] . Medicinal plants and plant extracts have become an important source of new drugs and rewarding results have been obtained in the treatment of various diseases. Careya arborea Roxb is the generic name for wild guava and is a member of the family. It is a medium -sized d eciduous tree with a height of 20m. Careya arborea Roxb is commonly known as "Padmaka" in Ayurveda [7-8] , Ka Li Yu Rui in Chinese medicine [9] , Katabhi in Sanskrit [10] , and Kumhi in Chhattisgarh. It is widely used in India, the Malay Peninsula, Bangladesh, Sri Lanka, China, and Myanmar. In India, it is found in Jammu and Kashmir, West Bengal, Madhya Pradesh, Chhattisgarh, Tamil Nadu, and forests and grasslands. In 1819, a genus of flowering plants in the Lecythidacea family was described as Careya [11] . The f amily Lecythidaceae is included in the order and is considered a single line [12] . The term Lecythidaceae means large tropical trees that produce large frui ts with woody skin [7] . Lecythidaceae is a tropical tree family consisting of about 20 differ ent

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genera and 450 different species. This family usually lives in tropical humid regions of South America, and there are some genera in Africa and Asia [13] . Careya arborea Roxb is a very attractive tree with a spreading crown. The leaves are simple, glabrous, alternate, widely obovate or oblong, clustered at the ends of branches, strong and marginal, and the leaves turn red in winter. The flowers are large, sessile, yellowish-white [10] , pink, and the persistent calyxes have crowns. The flowering period is from March to April. The fruit is a drupe, berry-shaped, large, green, fleshy, crowned with calyx members, globose and round [7,14] . The bark is dark gray [10] and usually peels off in thin strips [2,13,15-16] . The wood is medium in texture, hard in texture, heavy, and strong. The sapwood is white, while the heartwood is reddish [17] . The seeds are dark brown, not cracked, hard and wrinkled [7] , and nest in large cotyledons of fleshy and fleshy embryos [18] . The root is reddish-brown, spicy, odorless, and wrinkled on the surface. The root of Careya arborea Roxb is used in Ayurveda [19] for tuberculosis and bone fractures and it is also used in Vata and Kapha [20] . It has many pharmacological activities, such as anti-fertility, antibacterial, hyperglycemic, antidiarrheal, and antifungal [21-23] . In this study, preliminary phytochemical screening and their qualitative analysis were done using standard methods to identify the dry form of the drug and monitor for adulteration. 2. MATERIAL AND METHODS 2.1 Collection of plant materials The root of Careya arborea Roxb was collected from the Pamgarh area (Pamgarh, Loharsi, Kesala, and Madanpur) District Janjgir-Champa (Chhattisgarh) in Feb’ 2020. The plant materials were taxonomically identified and authenticated by BSI Allahabad (UP). 2.2 Processing of Plant materials: The plant materials were cleaned, washed with fresh water, and shade dried until all the water molecules evaporated, and the shed dried plant material (root) was taken and grind into a coarse powder. The powder samples were stored in a clean and air-tight container with proper labeling for further analysis. 2.3 Preparation of plant extracts: The root extract of the plant was prepared by the Soxhlet extraction method. About 50 gram coarse powder of plant material ( Careya arborea Roxb. roots) was packed into a thimble and extracted with 250 ml of various solvents separately. The solvent used was petroleum ether, chloroform, ethyl acetate, acetone, ethanol, methanol, and water (according to their polarity nature). The process of extraction continues for 24 hours or till the solvent in the siphon tube of an extractor becomes colorless. After that, the extract was taken in a beaker and kept in a water bath, and heated at 30-40 oC till all the solvent got evaporated. The dried extract was kept in the refrigerator at 4 oC for further use in phytochemical analysis. 2.4 Percentage of Extraction Yield: The coarse powder of the root of Careya arborea Roxb. (5gm) each was weighed into two beakers and was extracted with a various solvent. The percentage extractive yield is calculated according to the following formula, and the values are shown in Table: 1

Where, W1 = weight of dried extract and W2 = weight of dried root

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2.5 Qualitative phytochemical analysis: The extracts were tested for the presence of various phytochemicals (secondary metabolites) by using the following standard methods [24- 33] as described in Table 2: 2.6 Preliminary phytochemical screening: Phytochemical screening is very important to identify new sources of therapeutic importance for phytochemistry. These tests revealed the existence of several biologically active secondary metabolites, which may be the reason for their medicinal properties. The observations and inferences made in the phytochemical test are described below: I. Test for Alkaloids: Dragendorff test: The extract was treated with few drops of Dragendorff reagent. Extract become orange-red (Reddish brown) colour, which indicates alkaloids are present in extracts. Wagener test: A small amount of extracts was treated with 4-5 drops of Wagner reagent (1.27gram I2 and 2 grams of KI in 100 ml of water). The extract gives the Reddish-brown colour. It means alkaloids are present in these extracts. II. Test for Flavonoids: Sinoda test: Take a small amount of extract then add Mg powder with a few drops of concentrated hydrochloric acid, pinkish type red colour is obtained which indicates the presence of flavonoid. Lead acetate test: Take a small amount of extract then add few drop 10% lead acetates (alcoholic solution) yellowish precipitate observed which indicate the presence of flavonoid . III. Test for Saponins (Foam test) : Take a small amount of sample and added 6 ml water to a test tube. The mixture was shaken well and observed for the formation of persistent foam which indicates the presence of saponins. IV. Test for Phenol (Ferric chloride test) Take a small amount of extract and treated it with an aqueous 5% FeCl 3. The deep blue or black colour is obtained, which means phenol is available in extracts. V. Test for Carbohydrates (Molisch test) Add few drops of Molisch reagent (1% naphthol in 80% ethanol + concentrated sulphuric acid) were added to the sample, without mixing, to form an upper phase. Formation of red or purple colour at the interface of the two layers, which indicates the presence of carbohydrates. VI. Test for Cardiac glycosides (Killer Killiani test) Take a small amount of extract was treated with 2 ml of glacial acetic acid in a test tube and a drop of FeCl 3 solution was added to it. This was deliberately underplayed with 1 ml conc. H2SO 4. An earthy-coloured (brown) ring at the interface showed the presence of deoxy sugar. A violet or purple ring will show up beneath the ring while in the acetic acid layer, a greenish color ring appear indicates carbohydrates are present. VII. Test for Sterols (Leibermann-Burchard test) Take a small amount of sample and treated with 1 ml anhydrous acetic acid, 1 ml chloroform and 2-3 drop concentrate sulphuric acid and observed for the formation of blue or greenish- red colour, which indicates the presence of sterols. VIII. Test for Tannins (Braymer test) 2 ml of extract was treated with 10% alcoholic FeCl3 solution and observed for the formation of a blue or greenish colour solution.

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IX. Test for Terpenoids (Salkowski test) 1 ml chloroform was added to 2 ml of extract followed by a few drops of concentrated sulphuric acid. A reddish-brown precipitate formed immediately indicates the presence of terpenoids. X. Test for Amino acid and protein (1% Ninhydrin solution in acetone) 1 ml chloroform was added to 2 ml of extract followed by a few drops of concentrated sulfuric acid. A reddish-brown precipitate formed immediately indicates the presence of amino acid (protein). (Xanthoprotien test): Take 1ml extract and added a few drops of conc. H 2SO 4 makes whit precipitate, which indicates the presence of protein in the extract. XI. Test for Steroids: Liebermann Buchard test: Take a small amount of sample treated with 3-5 drops of chloroform and add a few drops of acetic anhydride followed by concentrated sulphuric acid from the sidewall of the test tube to the chloroform extract. The formation of a purple to blue coloured ring at the junction of the two liquids indicates the presence of steroids. Salkowski test Take a small quantity of sample and 1 ml chloroform was added followed by a few drops of concentrated sulphuric acid and observed form a yellow colour ring at the junction of two liquids which turn into a red colour after 2 minutes, which indicate the presence of steroids. XII. Test for Oxalate Take a small amount of sample and treated it with a few drops of glacial acetic acid. A greenish-black colour was obtained which indicates the presence of oxalates. XIII. Test for Quinones Take little amount of sample and treated with concentrated hydrochloric acid and observed for the formation of a yellow precipitate, which indicates the presence of Quinones. XIV. Test for Phlobatannins (Precipitate test) Take a small amount of sample and boiled with 1ml aqueous hydrochloric acid. The deposition of a red precipitate indicates the presence of phlorotannins.

3. RESULTS Phytochemical analysis refers to the extraction, screening, and identification of medicinally active substances found in plants. Results obtained fromtheir extraction yield andthe qualitative phytochemical screening of the root of Careya arborea Roxb. are shown in Table 1, 2 and 3respectively. A total of 14 phytochemicals were screened in which 11 were available in petroleum ether, chloroform, acetone, ethyl acetate, ethanol, methanol, and aqueous extracts. They are alkaloids, flavonoids, terpenoids, steroids, tannins, saponins, sterols, phenol,carbohydrates, cardiac glycosides, amino acid (protein).Remarkably Alkaloids,flavonoids, terpenoids, saponins, sterols cardiac glycoside, tannins, phenol, carbo-

Table 1:Extraction Yield (%) of root of Careya arborea Roxb.with various solvents S. N. Solvent Colour % Extraction yeild 1 Petroleum Ether Light Yellow 8.18% 2 Chloroform Yellowish Brown 12.10% 3 Ethyl Acetate Rusty Brown 9.11% 4 Ethyl Alcohol Light Orange 9.17% 5 Methyl Alcohol Red Brown 13.03% 6 Acetone Rusty Red 14.59%

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7 Water Orange Brown 18.93% Table 2: Preliminary Phytochemical test for root extract of Careya arborea Roxb. S. N. Phytochemicals Test Observation 1 Flavonoids (Sinoda test) 2ml extract + Mg + conc. HCl Red coloration 2 Alkaloids (Wagener test) 2ml extract + few drops of Wagener reagent Reddish brown coloration

3 Terpenoids (Salkowski Test) 2ml extract + 2ml CHCl 3 + 2-3 drops conc. Reddish brown precipitate H2SO 4 4 Steroids (Salkowski Test) 2ml extract + 2ml CHCl 3 + 2ml H 2SO 4 (conc.) Yellow color ring at the junction 5 Carbohydrates (Molisch’s 2ml extract + 10ml H 2O + 2 drops Ethanolic α- Reddish violet ring at the Test) naphthol (20%) +2ml H 2SO 4 (conc.) junction 6 Glycosides (Kiler Kiliani 2ml extract + 2ml CH 3COOH + FeCl 3 Red or violet coloration Test) 7 Proteins (Xanthoproteic Test) 1ml extract + 1ml H 2SO 4(conc.) White precipitate

8 Tannins (Braymer’s Test) 2ml extract + 2ml H 2O + 2-3 drops FeCl 3 (5%) Green precipitate 9 Saponins (Foam Test) (a) 5ml extract + 5ml H 2O + heat Froth appears (b) 5ml extract + Olive oil (few drops Emulsion forms

10 Phenol (Feric chloride test) 2ml extract +5% FeCl 3 Deep blue or Black coloration 11 Phlobatannins (Precipitate 2ml extract + 2ml HCl (1%) + heat Red precipitate Test) 12 Sterol (Leibermann-Buchard 2ml extract +CH 3COOH + CHCl 3 + conc. Blue or greenish coloration test) H2SO 4 13 Quinone 2ml extract + 2ml HCl Yellow Precipitate 14 Oxalate 2ml extract + 1ml glacial acetic acid Greenish black coloration

Table 3:Results of Phytochemical analyses of the root of Careya arborea Roxb. Phytochemicals/ Pet. Ethyl S.N. Chloroform Acetone Ethanol Methanol Water Solvent extracts Ether acetate 1 Alkaloids - + + + + + + 2 Cardiac Glycosides - + + + + + - 3 Steroids - - - + + + + 4 Flavonoids + + + + + + + 5 Phenols - + + - - + + 6 Phlobatannins ------7 Carbohydrates - - + - + + + 8 Saponins + - + + + + + 9 Sterols + + + + + + + 10 Tannins - - + - - + - 11 Protien - - - - - + + 12 Terpenoids + + + + + + + 13 Quinones ------14 Oxalates ------(+) = Present, (-) = Absent

hydrates, amino acid (protein), and steroids were present in root parts of the plant Careya arborea Roxb. This indicates that the root of this plant has a large potential for bioactive secondary metabolites. In the phytochemical screening process, all available bioactive secondary metabolites displayed various types of results in various solvents.

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Sixphytochemical alkaloids, flavonoids, terpenoids, saponin, sterol, and cardiac glycoside are wide, five phytochemical namely phenol, tannins, steroids, amino acid (protein) and carbohydrates are less available in the extracts. Whereas oxalate, quinones, and phlobatannins phytochemicals are absent in the root extract of the Careya arborea Roxb. The data in Table 2 shows the phytochemical screening of various extracts of root parts of Careya arborea Roxb. based on phytochemical tests. The percentage of extraction yield w/w of various extracts was also analyzed wherein the highest extraction yield (18.93%) was obtained in aqueous extract and least (8.81%) in petroleum ether extract.

4. DISCUSSION The medicinal significance of plants described certain physiological actions on the human organs due to certain chemical substances. Various bioactive secondary metabolites (Phytochemical) have been found to possess a wide range of activities, which may help in protection against various diseases. Flavonoids, terpenoids, and sterol are obtained in every extract of the root of Careya arborea Roxb. Alkaloids absent in the petroleum ether, steroids absent in the petroleum ether, chloroform and ethyl acetate, glycosides absent in the petroleum ether and aqueous, phenol absent in the petroleum ether, acetone, and ethanol, carbohydrates absent in the petroleum ether, chloroform, and acetone, saponins absent only in chloroform extract. Tannins presence in ethyl acetate and methyl alcohol extract, protein (amino acid) presence in only methyl alcohol and aqueous extract, absent in another extract. Oxalate, phlobatannins, and quinones are not obtained in the root extract of the Careya arborea Roxb . Flavonoids, sterols, and terpenoids obtained in chloroform extract, glycosides, and flavonoids present in ethyl acetate extract are responsible for the antimicrobial action [21] . Methanol extract of the root of Careya arborea Roxb. possesses antifertility activity [22-23, 34] . Alkaloids protect against chronic diseases. Saponin protects against hypercholesterolemia and antibiotic properties. Steroids and triterpenoids display the analgesic for CNS actions [32] . The chloroform extract of the root of Careya arborea Roxb displays antifungal action [21] . 5. CONCLUSION Phytochemical exists naturally in plants and protects itself from various pathogenic microorganisms by playing a very important role in plants, thus showing biological significance. Phytochemical screening must include the identification of active substances, extraction with suitable solvents, purification, and characterization of active principles in medicines. Medicinal plants have been a natural and valuable source of bioactive secondary metabolites for keeping good human health, for a long time. It would be the best source for obtaining a variety of newer drugs as phytochemicals are more specific, biodegradable, and are supposed to have fewer side effects. The screening of plant material samples is essential for biochemical modification in the drug, degradation due to handling, storage, and contamination. Preliminary phytochemical screening is a part of the chemical evaluation. The qualitative chemical examination is useful in the exposure to contamination. Bioactive secondary metabolites obtained in the root extract of Careya arborea Roxb. indicate their prospects as a resource herbal remedy. The phytochemical characterization of the root extract, the isolation of important bioactive compounds, and their biological action are compulsory for future studies. Standardization of bioactive extracts obtained from the medicinal plant will be conceded based on the phytochemical components exist in that plant,

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which is a vital step in recognizing novel and potent source of medically and commercially significant constituents.

Acknowledgment: The authors are grateful to HOD, Dept. of Chemistry, Dr. C. V. Raman University, Kota, Bilaspur (C. G.) for providing research facilities. Authors are also thanks to Principal, Dr. C. V. Raman University, Kota, Bilaspur (C. G.) for his timely help and co-operation.

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