PRELIMINARY PHYTOCHEMICAL SCREENING AND GC-MS ANALYSIS FOR IDENTIFICATION OF BIOACTIVE COMPOUNDS FROM ABUTILON FRUTICOSUM GUILL AND PERR. A RARE AND ENDEMIC PLANT OF INDIAN THAR DESERT

PRELIMINARY PHYTOCHEMICAL SCREENING AND GC-MS ANALYSIS FOR IDENTIFICATION OF BIOACTIVE COMPOUNDS FROM ABUTILON FRUTICOSUM GUILL AND PERR. A RARE AND ENDEMIC PLANT OF INDIAN THAR DESERT

Ilham Bano ¹ and G. S. Deora ^{* 2}

Taxonomy and Plant Diversity Laboratory ¹, Center of Advanced Study, Department of Botany, Jai Narain Vyas University, Jodhpur - 342005, Rajasthan, India.

Department of Botany ², Mohanlal Sukhadia University, Udaipur - 313001, Rajasthan, India.

ABSTRACT: Present work was aimed to determine, identify, and characterize the bioactive chemical compounds from methanolic leaves extract of Abutilon fruticosum by GC-MS analysis. Fresh disease-free leaves were collected shade dried and powdered for extraction with HPLC grade methanol. Preliminary phytochemical screening of methanolic leaves extract was performed using standard methods to determine the presence of different chemical compounds; the crude extract was subjected to GC- MS analysis for the identification of bioactive compounds. Phytochemical screening of methanolic leaves extracts revealed the presence of carbohydrates, proteins, alkaloids, phenols, flavonoids, terpenes, phytosterols, etc. Furthermore, GC MS analysis of the extract revealed the presence of 65 bioactive compounds. Some major biologically active compounds identified were Azulene (24.91%), Hexadecanoic acid (13.27%), Phytol (9.51%), Beta-sitosterol (3.19%), Lupeol (1.21%), Campesterol (0.33%) etc. These chemical compounds are biologically active and pharmacologically important. The study provides detailed information about the identification and chemical characterization of various medicinally important phytocompounds from methanolic leaves extract of this plant. Although the plant is rare and endemic to Indian Thar Desert and previously not explored very much, such kind of study about this plant could provide valuable information to be used in pharmacological research.

Abutilon fruticosum, Beta-sitosterol, Endemic, GC-MS, Phytosterols, Terpenes

INTRODUCTION: The ancient medicinal system played an important role in meeting the demand at the global level. Approximately 80-90 percent of the world’s population mainly depends on traditional medicine for primary healthcare; most of them involve the use of plant extracts ¹. Medicinal plants are rich in various bioactive compounds such as alkaloids, steroids, flavonoids, glycosides, terpenoids, phenols, gum, and mucilage, etc.

These components are mainly responsible for the therapeutic activity of plants. To understand the bioactivity of plants, whether it is medicinal, poisonous, or nutritive knowledge of its phytoconstituents, is necessary. Thus, phyto-chemical research is very important in the development and discovery of the drug. With the increasing advancement of technology, GC-MS analysis emerged as a powerful technique for the identification and quantification of bioactive compounds from medicinal plant extract even in very minute quantity. One such an important medicinal plant is Abutilon fruticosum Guill. and Perr. They are commonly known as ‘Imarti’. The plant is a rare and endemic medicinal plant of Indian Thar Desert region ².

It is branched perennial undershrub with a dense slender and thin stem, acute to sub obtuse ovate to chordate leaf, velvety on both the surfaces, light yellow colored solitary flowers, and schizocarpic cylindrical fruit with 8-10 awnless mericarp ³. It is xeriscaping plant, commonly found at small hillocks and rocky plains of the arid region of Rajasthan Fig. 1.

FIG. 1: ABUTILON FRUTICOSUM FIELD VIEW

Systemic Position: ⁴

The plant belongs to genus Abutilon and its sister species such as Abutilon indicum reported having great medicinal utility. All parts of this plant are useful in the treatment of various diseases and ailments such as leprosy, rheumatism, piles, ulcer, jaundice, bronchitis, inflammation of bladder etc. ^{5, 6, 7}. From the critical literature survey, it was revealed that there is no previous report on phytochemical characterization of this plant. The identification of phytoconstituents through GC-MS analysis from crude methanolic leaf extract is also missing from this plant. As it is an endemic plant of the Indian Thar Desert region and due to lacking knowledge about its phytoconstituents, chemical characterization is necessary to explore the potential of the plant to be used for medicinal purposes. Increasing urbanization and overgrazing lead to habitat destruction of this endemic plant, and it has become rare in occurrence. So new cultivation techniques should be practiced to save the medicinal plant species of this region.

The present study was conducted for the identification of bioactive compounds in the leaves of A. fruticosum by preliminary phytochemical screening and GC-MS analysis, which could provide useful information about this plant for further studies.

MATERIALS AND METHODS:

Plant Material Collection: Fresh and disease-free leaves of the plant A. fruticosum were collected from rocky areas of Kailana and Mandore of Jodhpur, Rajasthan, India, during August 2018. The plant sample was identified and authenticated by BSI, Arid Zone Regional Centre (Plant authentication number- BSI/AZRC/1.12012), and the voucher specimen was deposited in the Herbarium of Department of Botany, Jai Narain Vyas University, Jodhpur (Rajasthan).

Preparation of Plant Extract: Thoroughly washed and shade dried leaves of plant material were coarsely powdered and kept in an airtight container till further use.

10 g of leaf powder was extracted with 100 ml of HPLC grade Methanol and kept in the dark for 48 h with occasional stirring. The extract was then filtered with Whatman filter paper no.1 solvent was evaporated from the filtrate till a semi-solid mass is obtained.

Phytochemical Screening: Preliminary phyto-chemical screening of leaf methanolic extract was performed to test the presence or absence of various primary and secondary metabolites such as carbohydrates, proteins, alkaloids, steroids, terpenoids, phenols, flavonoids, glycosides, etc. using standard methods ^{8, 9}.

GC-MS Analysis: GC-MS analysis of crude extract was performed with GC-MS equipment QP 2010 Shimadzu, Japan. Experimental conditions for GC-MS were as follow: Helium gas as the carrier gas at a constant flow rate of 16.3 ml/min and column flow rate 1.21ml/min. Injector and mass transfer line temp were 200 and 280 °C for 10 min. The total running time of GC-MS was 50 min. The injection volume was 1µl.

As individual compounds eluted from the GC column where these compounds were bombarded with a stream of electrons, causing them to break into fragments. Samples were run fully at a range of 50/650 m/z and mass spectrum graphs obtained, which was a fingerprint of a molecule. The identified compounds were compared with the NIST library and Willey spectral library search programme.

RESULTS: Preliminary phytochemical screening of methanolic extract of Abutilon fruticosum showed the presence of bioactive compounds such as carbohydrate, amino acids, phenols, terpenoids, phytosterol, tannins, glycosides, saponins, Gums and mucilage Table 1.

GC-MS chromatogram of methanolic extract of Abutilon fruticosum shows 68 peaks pertaining to presence of 65 bioactive compounds, as shown in Fig. 2. Major compounds were identified through mass spectrometry attached with GC as listed in Table 2 along with their retention time, molecular formula, molecular weight, and chemical nature. Some major compounds identified with high peak area were Azulene (24.91%), n-Hexadecanoic acid (13.27%), Phytol (9.51%), Neophytadiene (2.17%),9,12-Octadecadienoic Acid, Methyl Ester (3.54%), Hexadecanoic acid methyl ester (5.05%), 9,12,15-Octadecatrienoic Acid, (Z,Z,Z) –or alpha linolenic acid (3.54%), Squalene (3.46%), Beta.-Sitosterol (3.19%), Stigmasta-5, 22- Dien- 3-Ol (1.71%), 8,11,14-Docosatrienoic acid, Methyl Ester (1.65%), Lupeol (1.21%) and other important chemical constituents with less than 1% peak area were 2-Methoxy-4vinylphenol(0.99%), Linoelaidic acid (0.85%), Alpha.-Tocospiro A (0.8%), Lup-20 (29)-En-3-One (0.64%), 1Eicosanol (0.38%), Campesterol (0.33%), Gamma. Tocopherol (0.3%).

TABLE 1: PHYTOCHEMICAL SCREENING IN METHANOLIC EXTRACT OF ABUTILON FRUTICOSUM

+ present; - absent

FIG. 2: GC-MS CHROMATOGRAM OF METHANOLIC EXTRACT OF ABUTILON FRUTICOSUM

All the major biologically active compounds identified through GC-MS analysis were listed in Table 3 along with their peak area, molecular structure, chemical nature, and bioactivities.

TABLE 2: PHYTOCHEMICAL COMPOUNDS IDENTIFIED IN THE METHANOLIC LEAVES EXTRACT OF ABUTILON FRUTICOSUM BY GC-MS ANALYSIS

TABLE 3: MAJOR COMPOUNDS IDENTIFIED IN ABUTILON FRUTICOSUM METHANOLIC LEAVES EXTRACT   WITH THEIR BIOACTIVITIES

S. no.	Name of compound	Peak area %	Compound nature	Molecular structure	Biological activity of compound
1	Azulene	24.91	Aromatic hydrocarbon		Anti-microbial and anti-inflammatory, antipyretic activity10
2	n-Hexadecanoic acid /Palmitic acid	13.27	Fatty acid		Antibacterial 11, anti-inflammatory, anti-oxidant, hypocholestrolemic, nematicide, pesticide, anti-androgenic, hemolytic, mosquito larvicidal activity 12
3	Phytol	9.51	Diterpene		Antimicrobial, anti-cancerous, anti-inflammatory and diuretic properties 13
4	Hexadecanoic Acid,2-Hydroxy-1(Hydroxymethyl) Ethyl Ester	5.55	Fatty acid ester		Pesticide, hemolytic, flavoring agent, Antioxidant 20, 21
5	Hexadecanoic Acid, Methyl Ester	5.05	Fatty acid ester		Antioxidant, antimicrobial hypocholestrolemic, nematicide hemolytic20, 21
6	9,12,15-Octadecatrienoic Acid, Z,Z,Z)-/alpha linolenic acid	3.54	Omega  3- Fatty acid		Anti-inflammatory, antibacterial, anticancerous, Vasodilator 22
7	Squalene	3.46	Triterpene		Anti-bacterial, antitumour,anti-inflammatory,antioxidant,anti-atherosclerotic23
8	Beta.-Sitosterol	3.19	Phytosterol		Anticancerous, androgenic, angiogenic, antibacterial, antifertility, anti-inflammatory 24, 25, 26
9	Neophytadiene	2.17	Sesquiterpenoid		Antibacterial, analgesic, anti-inflammatory, antipyretic, antioxidant 27
10	Stigmasta-5,22-Dien-3-ol	1.71	Phytosterol		Anti-inflammatory, antihepatoxic, antiviral, estrogenic, hypocholestrolemic, sedative 28
11	8,11,14-Docosatrienoic Acid, Methyl Ester	1.65	Omega 3 fatty acid		Nutrient, energy source, emulsifier, surfactant, cardioprotective 22
12	Methyl Stearate	1.38	Fatty acid ester		Antifoaming agent, fermentation nutrient, flavoring agent 20, 29, 30
13	Lupeol	1.21	Diterpene		Antimicrobial, anti-inflammatory, anticancerous properties 31
14	Octadecanoic acid	1.04	Fatty acid		Octadecanoic acid; Antimicrobial activity 20
15	Tetradecanoic acid/Myristic acid	1.01	Fatty acid		Antioxidant, antimicrobial, Lubricant, anticancerous, cosmetics 32
16	2-Methoxy-4-Vinylphenol	0.99	Phenol		Anti-tumour, antimicrobial, anti-inflammatory properties 33
17	Linoelaidic acid	0.85	Omega  6 fatty acid		Reduces obesity, melasma treatment, immune function modulation 34
18	Lup-20(29)-En-3-One	0.64	Triterpenoid		Anti-cancerous, antidiabetic, antiviral activity 35
19	alpha tocospiro A	0.47	Tocopherols		Anti-tumour, anti- Inflammatory 36
20	1-Eicosanol	0.38	Fatty alcohol		Emollients, cosmetic Antimalarial, antifungal, antioxidant 34
21	gamma Tocopherol	0.36	Tocopherols		Anti-tumour, anti-inflammatory 37, anti-aging, analgesic, vasodilator 38
22	Campesterol	0.33	Phytosterol		Anti-cancerous, anti-tumour properties 19
23	2-Cyclohexen  -  1-Ol, 3-Methyl-6-(1-Methylethyl)-, Cis-2/Piperitol	0.23	Monoterpenoid		Surfactant, emulsifier, Flavoring agent 34
24	Cyclohexen-1-Ol, 2-Methyl-5-(1-Methylethenyl)-, Trans-/Carveol	0.14	unsaturated, monocyclic monoterpenoid alcohol		Prevent breast cancer 39

DISCUSSION: Abutilon fruticosum is a rare and endemic plant of the Indian Thar desert. As the plant belongs to the genus Abutilon which has been used since ancient times to treat various diseases and ailments. Most of the compounds identified from the plant mainly belong to phenols, phytosterols, terpenes, fatty acids, and esters. These compounds were reported to contain various medicinal properties such as anti-inflammatory, antimicrobial, anticancerous, mosquito larvicidal, hepatoprotective activity, etc. Azulene is an aromatic hydrocarbon identified with highest peak area reported to have anti-microbial and anti-inflammatory, antipyretic, and soothing properties ¹⁰. Polyunsaturated fatty acids and their esters such as 8,11,14 Docosatrienoic acid, methyl ester, alpha linolenic acid, etc. were identified from the plant extract known to contain anti-inflammatory, anticancerous, vasodilator and antimicrobial properties ¹¹. These are important components for the production and movement of energy throughout the body, for the regulation of transportation of oxygen and for maintaining the integrity of cell structure and to control the cholesterol level of blood. Various fatty acids and their esters were identified from the extract that is known to possess anti-microbial, antifungal, anti-inflammatory properties ¹². Phytol is acyclic diterpene alcohol with known antimicrobial, anticancer, anti-inflammatory, and diuretic properties ¹³. It is used as a precursor of vitamin E ¹⁴ and vitamin K1 ¹⁵. It is used in cosmetics, shampoos, detergents ¹⁶. Phytosterols are plant-based sterols with potential to inhibit lung, stomach, ovarian, breast, colon as well as prostate cancer ^{17, 18, 19}. Some medicinally important phytosterols identified in the plant with less than 1 percent area were Beta-sitosterol, stigmasterol, and gamma sitosterol, respectively.

CONCLUSION: Preliminary phytochemical screening and GC-MS analysis of a methanolic extract of leaves of Abutilon fruticosum reveal the presence of various medicinally valued phytoconstituents such as alkaloids, terpenoids, phenols, phytosterols etc. This is the first report of the identification of active constituents from the leaf of this plant. The biological properties of compounds present in leaf extract of Abutilon fruticosum supports its medicinal utility. Although, other species of this genera have been explored very well and of great medicinal value. The present study could provide a valuable knowledge about this plant to be used in pharmacological research for human welfare after its toxicology test.

ACKNOWLEDGEMENT: Authors are thankful to the Centre of Advanced Study, Department of Botany, Jai Narain Vyas University, for providing infrastructure and technical support. Authors thank to Arid Zone Regional Centre BSI, Jodhpur for plant identification and authentication and AIRF, New Delhi for GC-MS analysis.

FINANCIAL SUPPORT: The research was carried out using fellowship under the scheme of UGC funded NF-OBC provided to one of the authors.

CONFLICTS OF INTEREST: Authors declares no conflict of interest

REFERENCES:

1. Sandhya B, Thomas S, Isabel W and Shenbagarathai R: Ethnomedicinal plants used by the valaiyan community of Piranmalai Hills (Reserved Forest), Tamil Nadu, India.-A Pilot Study. African Journal of Traditional, Complementary and Alternative Medicines 2006; 3(1): 101-14.
2. Pandey RP, Shetty BV and Malhotra SK: A preliminary census of rare and threatened plants of India. In: Jain SK, Rao RR, (eds) An assessment of threatened plants of India: BSI Howarh 1983; 55-62.
3. Bhandari MM: Flora of Indian Desert. MPS Repros, Jodhpur, India 1990.
4. Chase MW, Christenhusz MJ, Fay MF, Byng JW, Judd WS, Soltis DE, Mabberley DJ, Sennikov AN, Soltis PS and Stevens PF: An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Botanical Journal of the Linnean Society 2016; 181(1): 1-20.
5. Baquar SR: Medicinal and poisonous plants of Pakistan, Printas, Karachi, Pakistan 1989.
6. Khadabadi SS and Bhajipale NS: A review on some important medicinal plants of Abutilon spp. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2010; 1(4): 718-29.
7. Ramar K and Ayyadurai V: The present investigation deals with in-vitro Callus induction and plant regeneration of Abutilon indicum (L.). Journal of Pharmacognosy and Phytochemistry 2015; 3(6): 248-51.
8. Harbourne JB: Phytochemical methods of analysis. Jackmann and Hall, London 1973; 64-90.
9. Evans WC: Trease and Evans: Pharmacognosy.14^th London. W.B. Saunders Company Ltd 2000; 19-20.
10. Kulkarni MG and Sathe PS: Phytochemical and GC-MS analysis of Hamiltonia suaveolens (ROXB). International Journal of Chem Tech Research CODEN 2013; 5(1): 212-9.
11. Johannes, Eva, and Litaay M: The Bioactivity of Hexadecanoic acid compound isolated from hydroid Aglaophenia cupressina Lamoureoux as antibacterial agent against Salmonella typhi. International Journal of Biological and Medical Research 2016; 7(2): 5469-72.
12. Ravi L and Krishnan K: Cytotoxic potential of n-Hexadecanoic acid extracted from Kigelia pinnata Asian Journal of Cell Biology 2017; 12(1): 20-27.
13. Islam MT, Ali, ES, Uddin SJ, Shaw S, Islam, MA, Ahmed MI and Billah MM: Phytol: A review of biomedical activities. Food and Chemical Toxicology 2018; 121: 82-94.
14. Netscher T: Synthesis of vitamin E. Vitamins and Hormones 2007; 76: 155-202.
15. Daines AM, Payne RJ, Humphries ME and Abell AD: The synthesis of naturally occurring vitamin K and vitamin K analogues. Current Organic Chemistry 2003; 7(16): 1625-34.
16. Ko GA and Cho SK: Phytol suppresses melanogenesis through proteasomal degradation of MITF via the ROS-ERK signaling pathway. Chemico-Biological Interactions 2018; 286: 132-40.
17. Ramprakash VR and Awad AB: Role of Phytosterols in cancer prevention and treatment. Journal of AOAC International 2015; 98(3): 735-38.
18. Shahzad N, Khan W, Shadab MD, Ali A, Saluja SS, Sharma S and Afify MA: Phytosterols as a natural anticancer agent: Current status and future perspective. Biomedicine and Pharmacotherapy 2017; 88: 786-94.
19. López‐García G, Alegría A, Barberá R and Cilla A: Antiproliferative effects and mechanism of action of phytosterols derived from bioactive plant extracts. Nutraceuticals and Natural Product Derivatives: Disease Prevention & Drug Discovery 2019; 145-65.
20. Anonymous: Dr. Duke's phytochemical and ethanobotanical databases. USDA, Agricultural Research Service 1992-2016.
21. Aleryani SL, Cluette-Brown JE, Khan ZA, Hasaba H, de Heredia LL and Laposata M: Fatty acid methyl esters are detectable in the plasma and their presence correlates with liver dysfunction. Clinica Chimica Acta 2005; 359(1-2): 141-9.
22. Huang CB and Ebersole JL: A novel bioactivity of omega‐3 polyunsaturated fatty acids and their ester derivatives. Molecular Oral Microbiology 2010; 25(1): 75-80.
23. Lozano-Grande MA, Gorinstein S, Espitia-Rangel E, Dávila-Ortiz G and Martínez-Ayala AL: Plant sources, extraction methods, and uses of Squalene. International Journal of Agronomy 2018; 1-13.
24. Prieto JM, Recio MC and Giner RM: Anti-inflammatory activity of β-sitosterol in a model of oxazolone induced contact-delayed-type hypersensitivity. Boletín Latin-American y del Caribe de Plantas Medicinales y Aromáticas 2006; 5(3).
25. Kangsamaksin T, Chaithongyot S, Wootthichairangsan, C and Hanchaina R, Tangshewinsirikul C and Svasti J: Lupeol and stigmasterol suppress tumor angiogenesis and inhibit cholangio carcinoma growth in mice via downregulation of tumor necrosis factor-α. PloS one 2017; 12(12): e0189628.
26. Sharmila R and Sindhu G: Modulation of angiogenesis, proliferative response and apoptosis by β-sitosterol in rat model of renal carcinogenesis. Indian Journal of Clinical Biochemistry 2017; 32(2): 142-52.
27. Swamy MK, Arumugam G, Kaur R, Ghasemzadeh A, Yusoff MM and Sinniah UR: GC-MS based metabolite profiling, antioxidant and antimicrobial properties of different solvent extracts of Malaysian Plectranthus amboinicus Evidence-Based Complementary and Alternative Medicine 2017: 1-10.
28. Achika J, Ndukwe G and Ayo R: Isolation, Characterization and Antimicrobial Activity of 3 [beta], 22E-Stigmasta-5, 22-dien-3-ol from the Aerial Part of Aeschynomene uniflora Mey. British Journal of Pharmaceutical Research 2016; 11(5).
29. Mulyono N, Lay BW, Ocktreya L and Rahayu S: Antidiarrheal activity of Apus bamboo (Gigantochloa apus) leaf extract and its bioactive compounds. American Journal of Microbiology 2013; 4(1): 1.
30. Nascimento AM, Conti R, Turatti IC, Cavalcanti BC, Costa-Lotufo LV, Pessoa C, Moraes MO, Manfrim V, Toledo JS, Cruz AK and Pupo MT: Bioactive extracts and chemical constituents of two endophytic strains of Fusarium oxysporum. Revista Brasileira de Farmacognosia 2012; 22(6): 1276-81.
31. Saleem M: Lupeol, a novel anti-inflammatory and anti-cancer dietary triterpenes. Cancer Letters 2009; 285(2): 109-15.
32. McGaw LJ, Jäger AK and Van Staden J: Isolation of antibacterial fatty acids from Schotia brachypetala. Fitoterapia 2002; 73(5): 431-3.
33. Jeong JB, Hong SC, Jeong HJ and Koo JS: Anti-inflammatory effect of 2-methoxy-4-vinylphenol via the suppression of NF-κB and MAPK activation, and acetylation of histone H3. Archives of Pharma Research 2011; 34(12): 2109-16.
34. Wishart DS, Feunang YD, Marcu A, Guo AC, Liang K, Vázquez-Fresno R, Sajed T, Johnson D, Li C, Karu N and Sayeeda Z: HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Research 2017; 46(D1): D608-17.
35. Xu F, Huang X, Wu H and Wang X: Beneficial health effects of lupenone triterpene: A review. Biomedicine and Pharmacotherapy 2018; 103: 198-203.
36. Chen CR, Chao LH, Liao YW, Chang CI and Pan MH: Tocopherols and Triterpenoids from Sida acuta. Journal of the Chinese Chemical Society 2007; 54(1): 41-45.
37. Shin J, Yang SJ and Lim Y: Gamma-tocopherol supplementation ameliorated hyper-inflammatory response during the early cutaneous wound healing in alloxan-induced diabetic mice. Experimental Biology and Medicine 2017; 242(5): 505-15.
38. Munné-Bosch S and Alegre L: The function of tocopherols and tocotrienols in plants. Critical Reviews in Plant Sciences 2002; 21(1): 31-57.
39. Crowell PL: Prevention and therapy of cancer by dietary Monoterpene. The Journal of Nutrition1999; 129(3): 775S-8S.
    

    ---

    How to cite this article:

    Bano I and Deora G: Preliminary phytochemical screening and GC-MS analysis for identification of bioactive compounds from Abutilon fruticosum Guill and Perr. A rare and endemic plant of Indian Thar Desert. Int J Pharm Sci & Res 2020; 11(6): 2671-79. doi: 10.13040/ IJPSR.0975-8232.11(6).2671-79.

    ---

    All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---