Gayathiri M et al. Int. Res. J. Pharm. 2019, 10 (9) INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407 Research Article ANALYSIS OF BIOACTIVE COMPOUNDS OF HEMIONITIS ARIFOLIA (BURM.) MOORE. AN ANTI-DIABETIC FERN USING GAS CHROMATOGRAPHY AND MASS SPECTROSCOPY Gayathiri M 1, Catharin Sara S 2*, Sujatha S 1, Ramya Roselin I 1 and Gnana Deepa Ruby R 2 1 Ph. D Scholars, PG and Research Department of Botany, Holy Cross College (Autonomous), Tiruchirappalli, Tamil Nadu, India 2 Assistant Professor, PG and Research Department of Botany, Holy Cross College (Autonomous), Tiruchirappalli, Tamil Nadu, India *Corresponding Author Email: sara.kameelasivaraman.catharin@gmail.com Article Received on: 05/08/19 Approved for publication: 21/08/19 DOI: 10.7897/2230-8407.1009287 ABSTRACT Hemionitis arifolia (Burm.) Moore. is an important medicinal plant belongs to the family Hemionitidaceae. In Asian folklore Hemionitis arifolia is used to treat diabetes. The fern has been medically evaluated for its anti-diabetic properties and for intestinal worms. The present work is an important exploration and aims at analysing ethanolic extract of the plant using Gas Chromatography-Mass Spectrometry. The mass spectra of the compounds found in the extract was matched with the National Institute of Standards and Technologies (NIST) library. Gas chromatography mass spectrometry (GC-MS) analysis revealed the presence of nine compounds and the phyto constituents screened were 1. 4-(chloromethyl)-1-azabicydo-(2.2.2)octane (18.26%), 2. 4-Nitrobenzoic acid, 2,4,6-trichlorophenyl ester (18.70), 3. Decanoic acid, 2-hexyl-,methyl ester(19.91), 4. N-(4-(Chlorophenyl)isothiazol-5-yl)-2-methylpiperidin-2-imine (21.73), 5. Pregnane-3,20-dione, 17,21-{(methylborylene)bis(oxy)}-,5a- (23.76), 6. Retinoic acid, 5,8-epoxy-5,8-dihydro (24.80), 7. Androst-5-en-3-one, 19-acetoxy-4, 4-dimethyl-, oxine (26.14), 8. Choestan-26-oic acid, 3,7,12-trihyroxy-, (3a,5a,7a, 12a) (28.07), 9. Benzamide, N-(4-chlorotetrahydro-3-thinyl)-3, 5- dimethoxy, s-dioxide (31.52). The nine compounds identified provide positive light upon anti-diabetic works and drug could be formulated in future for diabetic condition. Hence Hemionitis arifolia is an important medicinal material for further research leading to possible drug development. Keywords: Gas Chromatography-Mass spectrum, Hemionitis arifolia (Burm.) Moore. Bioactive compounds. Decanoic acid, Retinoic acid INTRODUCTION Medicinal plants have been used by human being since ages in traditional medicine due to their therapeutic potential and the search on medicinal plants led to the discovery of novel drug candidates used against diverse diseases. According to the World Health Organisation (WHO) in 2008, more than 80% of the world’s population relies on traditional medicine for their primary healthcare needs. The Pteridophytes from Western Ghats and Eastern Ghats were used by tribal’s for the treatment of various ailments like stomach disorders, poisonous bites, rheumatics, cough, asthma, fever, diabetes, etc1., Moreover the dominant tribes involved in using Pteridophyte as medicines in the District are Santhal, Kol, Bhumija, Bhuyan, Mahalis, Sounti and Saharas. The plant parts like fronds and rhizomes were used as raw or cooked forms for the treatment of malaria, gonorrhea, leprosy and rheumatism, the mostly used genera are Adiantum, Asplenium, Lygodium and Pteris2,3. Bioactive compounds continue to play a dominant role in the maintenance of human health. Reports available on green plants represent a reservoir of effective chemo therapeutants, these are non phytotoxic, more systematic and easily biodegradable4-6. Medicinal value of Pteridophytes lists against bacteria, fungi, virus, cancer, rheumatism, diabetes, inflammation, fertility, diuretic, pesticides, hepatoprotective and sedative. Besides sugar, starch, proteins and amino acids, ferns contain a variety of alkaloids, glycosides, flavonoids, terpenoids, sterols, phenols etc. as potential components used in various industries7. Hemionitis arifolia is an attractive fern belonging to the family Hemionitidaceae. It is both an epiphyte as well as a terrestrial plant. In Asian folklore Hemionitis arifolia is used to treat diabetes. According to references the fern has been medically evaluated for its hypoglycemic properties and for intestinal worms. It is commonly called ‘Heart or Rabbit ear’ fern. This attractive fern has dark green cordate (heart shaped) fronds which grow on blackish stems with fronds that reach 5 to 7.5 cm (2" to 3") in length. Since the experimental plant is used by tribals, people may explore the plants more within short time for its medicinal value. This leads to insufficiency of the plant as well as extinction in future so that the medicinal value should be exploited immediately. References points out anti-diabetic properties in this plant and diabetes is a chronic diseases that kills major population of the World nowadays and hence there is a dire need for drug related to diabetes. Since there is not much work on this plant, the present work aims at screening of bioactive compounds present in this plant by performing GC-MS analysis where the components of long chain hydrocarbons, alcohols, acids and volatile compounds present in the plant could be revealed. 231 Gayathiri M et al. Int. Res. J. Pharm. 2019, 10 (9) MATERIALS AND METHODS Collection of plants materials The whole plant of Hemionitis arifolia was collected from Eastern Ghats (Kolli hills) of Tamil Nadu, India. This fern is both an epiphyte and grows on trees as well as a terrestrial plant. It is an attractive and dwarf fern. The plant sample were identified by Dr. S. John Britto, The Director, the Rapinat Herbarium and Center for Molecular Systematics, St. Joseph’s College, Tiruchirappalli, Tamil Nadu, India (Voucher No.001). The voucher specimens (Voucher No. 001) were deposited in the Department of Botany, Holy Cross College (Autonomous), Tiruchirappalli District, Tamil Nadu for future references. Processing, Preparation and Extraction of sample for GC-MS analysis The plant material of Hemionitis arifolia was collected and the fronds were initially separated from the main plant parts (Frond and Rhizome) and rinsed with distilled water and dried under shade on paper towel in laboratory then homogenized into fine particles and stored in airtight bottles. 10 gm of the powdered whole plant sample was soaked with 20 ml Ethanol for 3 days. The extract was then filtered through Whatman filter paper. From these extract 1ml of sample was extracted with ethanol and analyzed in GC-MS for identification of different components. Methodology GC-MS analysis was carried out on a GC Clarus 500 Perkin Elmer system and gas chromatograph interfaced to a mass spectrometer (GC-MS) instrument employing the following conditions: Column Elite-5MS fused silica capillary column (30 mm × 0.25 mm ID × 1 μmdf, composed of 5% Diphenyl/95% Dimethyl poly siloxane), operating in electron impact mode at 70 eV; Helium (99.999%) was used as carrier gas at a constant flow of 1 ml/min and an injection volume of 2 μl was employed (split ratio of 10:1); Injector temperature 250°C; Ion-source temperature 280°C. The oven temperature was programmed from 110°C (isothermal for 2 min.), with an increase of 10°C/min, to 200°C, then 5°C/min to 280°C, ending with a 9 min. isothermal at 280°C. Mass spectra were taken at 70 eV; a scan interval of 0.5 seconds and fragments from 45 to 450 Da. Total GC running time was 36 min. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was a Turbo Mass Ver 5.2.0. Identification of Components Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The Name, Molecular weight and Structure of the components of the test materials were ascertained. RESULTS The studies on the bioactive components in the ethanolic extract of whole plant of H. arifolia by GC-MS analysis clearly showed the presence of nine bioactive compounds. The active principles with their retention time (RT), molecular formula (MF), molecular weight (MW) and concentration (peak area %) were presented and biological activity of nine peaks were presented in Table 1. The GC-MS chromatogram of the nine peaks of bioactive compounds detected were shown in Figure 1. The total number of compound identified in ethanolic extracts were 1. 4-(Chloromethyl)-1-azabicydo-(2.2.2)octane, 4-(Chloromethyl)quinuclidine (18.26%), 2. 4-Nitrobenzoic acid, 2, 4, 6-trichlorophenyl ester (18.70), 3. Decanoic acid, 2-hexyl-, methyl ester, Methyl 2hexyldecanoate (19.91), 4. N-(4-(-Chlorophenyl) isothiazol-5-yl)-2-methylpiperidin-2amine, 4-(4-Chlorophenyl)-N-{(2E)-1methylpiperidinylidene}-5-isothiazolamine (21.73), 5. Pregnane-3, 20-dione, 17, 21-{(methylborylene)bis(oxy)},5a-dihyroxy-, cyclic methaneboronate (23.76), 6. Retinoic acid, 5, 8-epoxy-5, 8-dihydro, 5, 6-Epoxyretinoic acid (24.80), 7. Androst-5-en-3-one, 19-acetoxy-4, 4-dimethyl-, oxine, 3(Hydroxyimino)-4, 4- dimethylandrost-5-en-19-yl acetate (26.14), 8. Choestan-26-oic acid, 3, 7, 12-trihyroxy-, (3a, 5a, 7a, 12a) – 5a-cholestan-26-oic acid, 3a, 7a-trihydroxy- (28.07), 9. Benzamide, N-(4-chlorotetrahydro-3-thinyl)-3, 5- dimethoxy, s-dioxide (31.52). The listed out major phytocompounds and its biological activities obtained through the GC-MS study of the whole plant of Hemionitis arifolia showed the mass spectrum of seven bioactive constituents. The bioactive compounds find to act as anti-fungal, anti-bacterial, anti-cancerous agents, fluorinating agent, pharmaceuticals, dye, nematicides, pesticides, chemotherapeutic agents, compounds curing fibrosis, obesity and diabetes. There is no bioactivity found for two compounds and these compounds are considered as novel compounds to be explored in future. DISCUSSION Phytochemicals identified from medicinal plants present an exciting opportunity for the development of new types of therapeutics. Phytomedicine has been used since ancient times in various parts of the World where access to modern medicine is limited. In recent years, considerable attention has been directed towards identification of plants with anti-diabetic ability that may be used for human consumption8. According to an estimation of the International Diabetes Federation, approximately 366 million people are suffering from diabetes and this may double by 2030, In India is 40.9 million now which is expected to grow to 60.9 million by 20259. GC-MS analysis of Hemionitis arifolia yielded nine bioactive compounds respectively. All the compounds and its bioactivity are listed in Table 1. Out of nine, seven compounds showed their activity during reference whereas two compounds were found to possess no activity and hence to be possibly new compounds. Studies revealed that the plant can be used as antibacterial, antifungal, anti-diabetic and anti-cancerous. Various authors have worked GC-MS in plants like Nephrolepis cordifolia and Adiantum capillus-veneris and the activities related to the compounds found out coincide with our results10,11. The antidiabetic property of the experimental plant reveals that the plant could be an important source for the investigation related to diabetics. Moreover efforts should be taken to find out the drug present in the plant and also to elucidate and characterize the structure and clinical properties in coming days. 232 Gayathiri M. et al. Int. Res. J. Pharm. 2019, 10 (9) Figure 1: Chromatogram of frond ethanolic extract of GC-MS with the whole plant of H. Arifolia (Burm.) Moore Table 1: Components detected in whole plant ethanolic extract of Hemionitis arifolia (Burm.) Moore. through GC-MS Analysis Name of the compound Molecular formula C8H14CIN Molecular weight 159 Peak area 18.26 Retention time 18.22-18.31 4-Nitrobenzoic acid, 2,4,6-trichlorophenyl ester C13H6C13NO4 345 18.70 18.67-18.74 Decanoic acid, 2-hexyl-,methyl ester, Methyl 2-hexyldecanoate N-(4-(-Chlorophenyl)isothiazol-5-yl)-2methylpiperidin-2-amine, 4-(4-Chlorophenyl)N-{(2E)-1-methylpiperidinylidene}-5isothiazolamine Pregnane-3,20-dione, 17,21{(methylborylene)bis(oxy)}-,5a-dihyroxy-, cyclic methaneboronate Retinoic acid, 5,8-epoxy-5,8-dihydro, 5,6Epoxyretinoic acid Androst-5-en-3-one, 19-acetoxy-4, 4-dimethyl, oxine, 3-(Hydroxyimino)-4, 4dimethylandrost-5-en-19-yl acetate Choestan-26-oic acid, 3,7,12-trihyroxy-, (3a,5a,7a, 12a) – 5a-cholestan-26-oic acid, 3a,7a-trihydroxyBenzamide, N-(4-chlorotetrahydro-3-thinyl)-3, 5- dimethoxy, s-dioxide C17H34O2 270 19.91 19.88-19.98 C15H16CIN38 305 21.73 21.69-21.82 C22H33BO4 372 23.76 23.67-23.87 Anti-bacterial, anti-fungal and anti-cancer activity C20H28NO3 316 24.80 24.76-24.83 C23H35O3 373 26.14 26.10-26.19 chemopreventive and chemotherapeutic agents Activity not found C27H46O5 450 28.07 28.03-28.14 C27H46CINO55 333 31.52 31.49-31.56 4-(Chloromethyl)-1-azabicydo-(2.2.2)octane , 4-(Chloromethyl)quinuclidine Figure 2: Mass spectrum of 4-(Chloromethyl)-1azabicyclo[2.2.2]octane, 4-(Chloromethyl) quinuclidine Bioactivity Used as Fluorinating agents and therapeutic and diagnostic purpose Production of pharmaceuticals and dyes Nematicide and Pesticide Activity not found used to treat inflammation,fibrosis,obesity and diabetes Anti-oxidant and antidiabetic Figure 3: Mass spectrum of 4-Nitrobenzoic acid, 2,4,6trichlorophenyl ester 233 Gayathiri M. et al. Int. Res. J. Pharm. 2019, 10 (9) Figure 4: Mass spectrum of Decanoic acid, 2-hexyl-, methyl ester, Methyl 2-hexyldecanoate Figure 5: Mass spectrum of Retinoic acid, 5,8-epoxy-5,8-dihydro-5,6Epoxyretinoic acid Figure 6: Mass spectrum of Cholestan-26-oic acid, 3,7,12-trihydroxy-,(3ẚ,5ẚ,7ẚ12ẚ)-5ẚ-Cholestan-26-oic acid,3ẚ,7ẚ,12ẚ-trihydroxy CONCLUSION The establishment of natural plant product industries increase in domestic employment, the creation of relevant technological capability, an increase in export earnings and the betterment of the rural population have improved in terms of health care provision. The present experimental plant proves to be a promising plant for the exploration of drug towards diabetics and its implications. ACKNOWLEDGEMENT The authors express their acknowledgements to the Management, Principal Dr. (Sr.) A. Christina Bridget, Head of the Department Dr. P. Shanthi for the support rendered during this work in a successful manner. REFERENCES 1. 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International Journal of Pharmacy and Pharmaceutical Sciences 2014; 6: 60-63. Cite this article as: Gayathiri M. et al. Analysis of Bioactive Compounds of Hemionitis arifolia (burm.) Moore. An anti-diabetic fern using gas chromatography and mass spectroscopy. Int. Res. J. Pharm. 2019; 10(9):231-235 http://dx.doi.org/10.7897/22308407.1009287 Source of support: Nil, Conflict of interest: None Declared Disclaimer: IRJP is solely owned by Moksha Publishing House - A non-profit publishing house, dedicated to publish quality research, while every effort has been taken to verify the accuracy of the content published in our Journal. IRJP cannot accept any responsibility or liability for the site content and articles published. The views expressed in articles by our contributing authors are not necessarily those of IRJP editor or editorial board members. 235