INFOKARA RESEARCH ISSN NO: 1021-9056

PHYTOCHEMICAL SCREENING AND GC-MS PROFILING OF ETHANOLIC FRUITS EXTRACT OF TORVUM

Authors R. SATHYA1, S. NIRMALA DEVI2 & V. RAMAMURTHY3 P.G & Research Department of Biochemistry1,2&3, Marudupandiyar College, Thanjavur, 613 403, Tamil Nadu, India Corresponding Author E-mail: [email protected]

ABSTRACT Solanum torvum Swartz (family: ) is commonly known as turkey berry. This is found in tropical Africa, Asia and South America. S. torvum, is an important medicinal plant in tropical and subtropical countries is widely used like food and in folk medicine around the world. To quantify the major secondary metabolites and the antioxidant potential of ethanolic fruits extract of S. torvum. In the present study ethanolic fruits extract of S. torvum was analyzed using Gas Chromatography–Mass Spectrometry (GC-MS) and the compound structures were identified with help of National Institute of Standards and Technology (NIST) library. GC-MS analysis of test plant revealed the presence of 28 bioactive compounds. The prevailing compounds were 9-octadecenoic acid(Z)- (36.9%), Hexadeconoic acid (25.2%), Cholest-5-en-3-ol(3a)- (4.46%), Oleyl alcohol, trifluroacetate (4.94%), D-Neooleana-12,14-diene,(3.xi.,5a)- (2.98%), Stigmast-5-en-3-ol,-(2.92%), (3a,24S)-Lupan-3-ol,acetate (2.72%), 1- Docosanol (2.53%), Fucoxanthin (2.38%), Choloroacetic acid, tetradecyl ester (1.91%), Phytol (1.39%), 4-chlorophenyl tert-pentyl sulphate (1.34%), Hentriacontane (1.11%) are important bioactive compounds which act as essential drugs for dangerous diseases and disorders and other compounds are used in pharmacological activities. Keywords: Solanum torvum, GC-MS analysis, phytochemicals, bioactive compounds.

INTRODUCTION are useful and valuable to us. Human life cycle largely depends on plants. The fruits bark and leaves of a large number of plants are valuable as drugs. They are powdered and used as important ingredients in medicine. However, a very little amount is known about the chemical composition of these plant materials. Therefore, studies on isolation and characterization of the medicinally important compounds from them are very important for the well being of the human society. It became the task of the medicine man to maintain this knowledge and pass it on to his successor. The medicine men were often also priests and thus the actual knowledge became enmeshed in a veil of myth and magic. This process can still be observed in the developing countries; consequently the study of drugs used by traditional healers is an important object of pharmacognostical research. Over 50% of all modern clinical drugs are of natural product origin and natural products play an important role in drug development programs in the pharmaceutical industry (Ramamurthy and Naveen, 2017).

Medicinal plants are the source of many potent and powerful drugs. The plant derived drugs are healthier and safer alternate to the synthetic drugs (Dineshkumar and Rajakumar, 2015). Different parts of medicinal plants like root, stem, flower, fruit, seed etc. are used to obtain pharmacologically active constituents. Medicinal activities of plants can be attributed to the secondary metabolites such as alkaloids, flavonoids, glycosides, tannins, terpenoids

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and essential amino acids present in these plants. These active principles are isolated for direct use as drugs, lead compounds and or pharmacological agents (Kumaradevan et al., 2015). Even today compounds from plants continue to play a major role in primary health care as therapeutic remedies in many developing countries (Smolinske and Susan, 1992). Standardization of plant materials is the need of the day. Several pharmacopoeia containing monographs of the plant materials describe only the physicochemical parameters. Hence the modern methods describing the identification and quantification of active constituents in the plant material can be useful for proper standardization of herbals and its formulations. Also the WHO has emphasized the need to ensure the quality of medicinal plants products using modern controlled technique and applying suitable standards (Sharma et al., 2010). Nowadays there were a number of dramatic advances in analytical techniques including TLC, UV, NMR and GC-MS that were powerful tools for separation identification and structure determination of Phytochemicals. In GC-MS used to identify the bioactive constituents of long chain hydrocarbons, alcohols, acids, esters, alkaloids, steroids, amino and nitro compounds etc.,

The Solanaceae family comprises about 90 genera and 3000 which are widely distributed in the world. They are a rich source of active secondary metabolites (Coletto da Silva et al., 2004). Within this family, the genus Solanum is the largest and most complex with more than 1500 species (Chowdhury et al., 2007) which yield a great variety of steroidal saponins and glycoalkaloids of interest from ecological and human health viewpoints (Roddick et al., 2001). Solanum torvum Sw. (Solanaceae), commonly known as Turkey berry is native and cultivated in Africa and West Indies (Adjanohoun et al., 1996). The fruits and leaves are widely used in Camerooninan folk medicine. It also occurs commonly in the moist farms of India. The fruits of S. torvum are edible and commonly available in the markets. They are utilized as a vegetable and regarded as an essential ingredient in the South Indian population’s diet. A decoction of fruits is given for cough ailments and is considered useful in cases of liver and spleen enlargement (Siemonsma and Piluek, 1994). The plant is sedative and diuretic and the leaves are used as a haemostatic.

The fruits are berries that are yellow when fully ripe. They are thin-fleshed and contain numerous flat, round, brown seeds. Fruits globose, with numerous small seeds are surrounded by persistent calyx at the base. The plant is sedative and diuretic and the leaves are used as a haemostatic. The ripened fruits are used in the preparation of tonic and haemopoitic agent and also for the treatment for pain (Kala, 2005). It has antioxidant properties (Sivapriya and Srinivas, 2007). Keeping this in view, the present study has been undertaken to investigate the phytoconstituents present in ethanolic extract of S. torvum. Hence the present study focused on Phytochemical profiling of ethanolic fruits extract of S. torvum using Gas chromatography and mass spectrometry.

MATERIALS AND METHODS Plant collection and authentication: Seed of Solanum torvum were procured from local market and were grown organically in the garden area of Marudupandiyar College. Fruits were collected during dawn and were shade dried, powdered and stored in air tight containers for further use. The plant parts were identified taxonomically and authenticated according to various literatures, Flora of Madras Presidency and Wealth of India including other pertinent taxonomic literature.

Plant preparation and extraction: 20g of shade dried plant sample powder was extracted with ethanol in soxhlet apparatus followed by evaporation of solvent by rotary evaporator to

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procure ethanolic extract. 20g of shade dried plant sample powder was extracted using water by cold maceration method, lyophilized and was used. 100mg of extract was dissolved in 100ml of their ethanol solvents and was used as stock solution for further studies.

Phytochemical Screening: The Phytochemical screening for the presence of alkaloids, saponins, flavonoids, phlobatanins, reducing sugars, anthranoids, cardiac glycosides, anthraquinones and polyphenols were carried out according to the method adopted by harborne (Harborne, 1973; Sofowora, 1993; Trease and Evans, 1989).

Gas Chromatography-Mass spectrometry (GC-MS) analysis: The GC-MS analysis was carried out using a Clarus 500 Perkin- Elmer (Auto System XL) Gas Chromatograph equipped and coupled to a mass detector Turbo mass gold – Perking Elmer Turbomas 5.2 spectrometer with an Elite-1 (100% Dimethyl ply siloxane), 300 m x 0.25 mm x 1 μmdf capillary column. The instrument was set to an initial temperature of 110°C, and maintained at this temperature for 2 min. At the end of this period, the oven temperature was raised upto 280°C, at the rate of an increase of 5°C/min, and maintained for 9 min. Injection port temperature was ensured as 250°C and Helium flow rate as 1 ml/min. The ionization voltage was 70 eV. The samples were injected in split mode as 10:1. Mass Spectral scan range was set at 45-450 (mhz). The chemical constituents were identified by GC-MS. The fragmentation patterns of mass spectra were compared with those stored in the spectrometer database using National Institute of Standards and Technology Mass Spectral database (NIST-MS). The percentage of each component was calculated from relative peak area of each component in the chromatogram. Identification of Compounds: The identity of the components in the extract was assigned by the comparison of their retention indices and mass spectra fragmentation patterns with those stored on the computer library and also with published literatures. National Institute of Standards and Technology library sources were also used for matching the identified components from the plant material.

RESULT AND DISCUSSION Qualitative phytochemical analyses for alkaloids, carbohydrates, tannins, phenols, gums and mucilage, fixed oils and fats, saponins, proteins, volatile oils, flavonoids and steroids were screened in ethanolic extracts of Solanum torvum fruits. Table 1 shows the results of phytochemical screening of S. torvum fruits. The results obtained revealed the presence of alkaloids, saponins, flavonoids, polyphenols and reducing sugars in the aqueous extracts while cardiac glycosides, saponins, flavonoids, polyphenols and reducing sugars were present in the aqueous extracts. The results of Phytochemical screening of S. torvum fruits revealed the presence of saponins in alcoholic extract. Saponins are heterogeneous groups of natural products with a marked hormonal activity, strong expectorant and aid in the absorption of nutrients (Rahman, 2010). The results of the phytochemical screening of S. torvum fruits revealed the presence of flavonoids in alcoholic extracts. Flavonoids possess antioxidant properties and ensure healthy circulation of blood. It helps to strengthen capillaries wall. The compound is sometimes referred to as phytoestrogens. Phytoestrogens are associated with relief of menopausal systems, reduction of osteoporosis, improvement of blood cholesterol levels, and lowering the risk of certain hormone-related cancers and coronary heart disease (Rahman, 2010). The phytochemical screening of S. torvum fruits also revealed the presence of polyphenols and reducing sugars this results compared favourable well with the one reported by Osabor et al. (2015) for cola lepidota seeds. Polyphenols have been implicated in medical circle to protect person against ageing and can inhibit cancer growth (Rahman, 2010). The drugs derived from herbs may have the possibility of use in

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medicine because of their biological activity. With onset of scientific research in Ayurvedic system of medicine, it is becoming clearer that the medicinal herbs have a potential in today’s synthetic era, as numbers of medicines are becoming resistant. According to one estimate only 20% of the plant flora has been studied and 60% of synthetic medicines owe their origin to plants. Ancient knowledge coupled with scientific principles can come to the forefront and provide us with powerful remedies to eradicate the diseases.

Table 1. Qualitative phytochemical analysis of Solanum torvum fruits

S. No Name of Test Test applied / Reagent used Alcoholic extract 1 Alkaloids Mayer’s + Wagner’s ++ Hagner’s ++ Dragndorff’s test ++ 2 Flavanoids Shinoda test +++ Alkaline reagent test ++ 3 Cardiac Keller-kilianni test ++ glycosides 4 Phlobotannins HCl Test + 5 Test for Steroids Libermann-Burchard’s Test + 6 Tannins FeCl3 test ++ 7 Triterpenes H2SO4 test +++ 8 Saponins Frothing test + 9 Fixed oils & fats Spot test - 10 Gum & Alcoholic Precipitation - mucilage 11 Protein and Millons test +++ Amino acids Ninhydrin test +++ 12 Carbohydrate Molisch`s test +++ (-): Absence, (+): Less presence, (++): Moderate presence, (+++): High presence

Use of GC/MS enabled identification of the most components in Solanum torvum fruits were analyzed by phytoconstituents. The compounds identified are listed in Table 2. The natural compounds have been a source of medicinal agents for hepatoprotective, antimicrobial, anti-inflammatory properties. The components of the infusion differ from those found in tincture except organic acids derivatives. Twenty eight compounds were identified in ethanolic fraction of S. torvum extract by GC-MS analysis. The active principle, area of the peak, Concentration (%), Retention Time (RT), Molecular formula and Molecular weight were presented. The prevailing compounds were 9-octadecenoic acid(Z)- (36.9%), Hexadeconoic acid (25.2%), Cholest-5-en-3-ol(3a)- (4.46%), Oleyl alcohol, trifluroacetate (4.94%), D-Neooleana-12,14-diene,(3.xi.,5a)- (2.98%), Stigmast-5-en-3-ol,-(2.92%), (3a,24S)-Lupan-3-ol,acetate (2.72%), 1- Docosanol (2.53%), Fucoxanthin (2.38%), Choloroacetic acid, tetradecyl ester (1.91%), Phytol (1.39%), 4-chlorophenyl tert-pentyl sulphate (1.34%), Hentriacontane (1.11%) etc. Figure 1 shows the chromatogram with retention time, molecular weight, height, area% of the standards.

The differences between the compounds that we have found in the roots, stems and leaves of Aristolochia clematitis were studied by GC-FID. This study was performed on the alcoholic extracts of the three parts of the plant. From this study we have concluded that the compounds found in the root and steam are very similar. The aristolochic acid derivatives are

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present in both extracts, but in the leaves these derivatives are in very low concentration (Podea et al., 2001). Ramamurthy et al. (2014) reported that the roots and leaves of H. indicum were studied by GC-MS. This study was performed on the alcoholic extracts of the two parts of the plant. From this study we have concluded that the compounds found in the root and leaves are very dissimilar. The organic acid derivatives are present in both extracts, but in the leaves these derivatives are in very high concentration.

Plants are integral part of human civilization. Medicinal plants are also been relied upon by over 80% of the world population for their basic health care needs. Drugs based on the Plants are of prime importance for several remedies in traditional and conventional medicine throughout the world and serves as a substitute for drug supply in modern medicine (Dineshkumar and Rajakumar, 2016). Medicinal plants with therapeutic properties are used for the treatment of many infectious diseases of humans as they contain many bioactive phytochemical constituents which are of curative effects. By consuming medicinal plants, can boost the immune system and increase antioxidant activity in humans. The higher level use of medicinal plant due to easily available, cheap and relatively no side effects were reported by Chandra et al. (2004).

The analytical methods used GC/MS is suitable for medicinal herbs organic compounds determination. The sample preparation method is rapid and precise. There is a difference between the compounds extracted from herb by infusion and tincture but the important thing is that the organic acid and fatty acids derivatives are present in both of them. On the other side the study shows that their concentration is higher in the roots and steams. The present study focused on identification of several constituents present in the ethanolic extract of Solanum torvum fruits. This type of GC-MS analysis is the first step towards understanding nature of active compounds in this medicinal plant and helpful for the further detailed study. In this plant contains various bioactive compounds justifies the use of the whole plant for various ailments by traditional practitioners.

Fig 1. GC-MS profile of the phytocompound of Solanum torvum fruits

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Table 2. GC-MS profile of the phytocompound of Solanum torvum fruits

Molecular S.No RT Name of the Compound Area% Formula 1 3.26 Diethoxymethyl silanol C5H14O3Si 0.99% 2 4.49 Tetraethyl silicate C8H20O4Si 0.31% 3 4.82 4-chlorophenyl tert-pentyl sulphate C11H15C10 1.34% 4 6.41 Nonanal C9H18O 0.83% 5 9.18 Janoxepin C19H23N3O3 0.30%

6 16.14 Stigmast-5-en-3-ol,(3a,24S)- C29H50O 2.92%

7 16.91 Methanone, (1-hydroxyly clohexyl)phenyl C13H16O2 0.66% 4,7-Methano-1H-indene,3a,4,5,6,7,7a- 8 19.13 C H O 0.75% hexahydro-5-(2-propenyloxy)- 13 18

9 19.87 Neophytadiene C20H38 0.67% Phthalic acid,5-methylhex-2-yl isobuty 10 20.32 C H O 0.30% lester 19 28 4

11 21.269 Oleyl alcohol, trifluroacetate C20H35O2F3 4.94%

12 22.30 Hexadeconoic acid C16H32O2 25.2%

13 22.156 Choloroacetic acid, tetradecyl ester C16H31O2Cl 1.91%

14 23.64 2-Amino-5-chlorobiphenyl C12H10ClN 0.33% 15 25.13 Phytol C20H40O 1.39%

16 25.58 9,12,15-Octadecatrienole acid,(Z,Z,Z) C18H30O2 0.41%

17 26.326 Hentriacontane C33H64O 1.11% 18 27.873 Hentriacontane C33H64O 0.50%

19 28.33 O- isopropylanisole C10H14O 0.56%

20 30.80 Cholest-5-en-3-ol(3a)- C27H46O 4.46% 21 32.01 9-octadecenoic acid(Z)- C18H34O2 36.9% 22 33.15 Rhodopin C40H58O 0.35% 23 35.51 3-methyl-2-(2-oxopropyl)feran C8H10O2 0.77% 24 35.65 Lupan-3-ol,acetate C32H54O2 2.72% 25 35.98 Squalene C50H50 0.36% 26 37.02 Fucoxanthin C42H58O6 2.38% 27 38.61 1- Docosanol C22H46O 2.53% 28 40.43 D-Neooleana-12,14-diene,(3.xi.,5a)- C30H4 2.98% Source: - Dr. Duke’s Phytochemical and Ethno botanical Databases

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