GAS CHROMATOGRAPHY AND MASS SPECTROMETRY ANALYSIS OF BIOACTIVE COMPOUNDS OF DRYOPTERIS HIRTIPES (BLUMZE) KUNTZE

Objective: The objective of this study was to analyze GC–MS analysis of whole plant methanolic extract of Dryopteris hirtipes from Dryopteridaceae family. Methods: Gas chromatography and mass spectrometry analysis of whole plant extract was carried out with instrument GC–MS. Results: The methanolic extract of D. hirtipes reveals to identify more known and unknown bioactive compounds. In this study, seven major bioactive compounds were identified such as Stigmast-5-en-3-ol(56.65%), Phytol (5.39%), Lanost -8-en-3-ol-(3 β )(3.18%), Neophytadiene(2.68%), Tri-o-trimethylsilyl N-heptaflurobutryl derivative of terbutaline(2.19%), 1H-Imidazole 2-methanol(1.28%), and 8A-(2,4-Dimethyl-1-nitrilo-pent-2-yl) dioxy)tocopherone(1.0%) and low concentrations of compounds like hexadecanoic acid(0.6%). Conclusion: These identified compounds are having active pharmacological properties such as antimicrobial property, hypotension, anti-inflammatory, anti-tumor, anti-cancer, anti-hepatitis, analgesic, and antipyretic properties. However, D. hirtipes is a rare pteridophyte and used to cure many diseases, and so there need further studies to isolate and identify the specific active compounds present in it.


INTRODUCTION
Medicinal plants are the major natural resources found in the world, in which it contains more active phytoconstituents. Among these groups, pteridophytes were found abundant to have more than 13,000 species of fern and fern-related groups distributed in all over the world [1]. Most of the pteridophytes grow vigorously in tropical, temperate regions, and eco-geographically threatened regions, and sometimes, they have adapted with varied climatic conditions [2]. More than 400 species of pteridophytes occur all over southern India [3]. Moreover, researchers analyzed that pteridophyte families are not easily infected by pathogenic organisms due to the rich content of phytoconstituents and its evolutionary aspects to survive more than 350 million years [4]. In past days, lower groups of pteridophytes screening were not explored properly and not well documented, and these present days, the priority in the usage of natural drugs has been increased in the research community, and analysis of bioactive compounds may also take in this family of pteridophytes and showed more pharmacological activities [5]. However, plants are the major reservoir of secondary metabolites with pharmacological activities to treat various ailments [6] with less side effects. Many species of pteridophytes are yet to be explored for their bioactive compounds and to isolate specific chemical compounds to treat various disorders.
Dryopteris hirtipes (Blumze) kuntze belongs to the family Dryopteridaceae which occurs in temperate and mountain areas in tropical regions [7], as shown in Fig. 1. In Dryopteris species, the rhizome is used for rheumatism, epilepsy, and leprosy [8], leaf juice used to cure epilepsy, act as an antibiotic [9], and paste of the plant used to cure a bacterial infection, act as antifungal properties, and insecticide [10,11].
Metabolic profiling of various plant extracts provides information on their phytochemical composition by qualitatively, which allows the detection of chemically active known and unknown bioactive compounds by hyphenate technique. GC-MS studies proved to be a reliable method for identifying the volatile and semi-volatile compounds [12,13] fatty acids, lipids, and hydrocarbons [14]. The aim of the present study is to identify the active compounds present in D. hirtipes using GC-MS.

Collection of the plant
The leaves of D. hirtipes were collected from Yercaud hills, Salem District, Tamil Nadu. The plant identification was confirmed with the help of herbarium specimens in the Indian Botanical Survey of India, Coimbatore. The leaves were carefully separated, washed with 2-3 times in tap water, and then dried under shade for more than two weeks. The shade dried fronds with spores of D. hirtipes were powdered by electric blender. The powdered plant materials were stored in a closed glass container until their use in extract preparation. About 100 g of powdered leaves were extracted with various organic solvents such as hexane, ethyl acetate, and methanol using Soxhlet apparatus and then concentrated using rotary evaporator.

GC-MS analysis
GC-MS analysis of the sample was carried out using Shimadzu QP2020.

Identification of compounds
Gas chromatography can separate volatile components in a given sample and mass spectroscopy helps to fragment the components and identify them on the basis of their mass. The identification of compounds was based on the molecular structure, molecular mass, and calculated fragments. Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST) and WILEY Library having more than 62,000 patterns. The relative percentage of each compound was calculated by comparing its average peak area to the total areas. The spectrum of the unknown compound was compared with the spectrum of the known compound stored in the NIST and WILEY library.

Valarmathi and Natarajan
The diverse active components have been considered to have a wide range of activities which may be used to cure various diseases.
Stigmast-5-en-3-ol(56.65%) is a derivative of phytosterol and it has the tendency to reduces blood level of glucose, acts as antiinflammatory, anti-pyretic, anti-arthritic, and anti-ulcer activity were reported in their findings [15][16][17][18][19]. Phytol (5.39%) is a diterpene with antimicrobial properties against many bacterial strains and also possesses antioxidant, anti-inflammatory properties in the methanolic extract of A. capillus-veneris [20,21] reported in ethanolic extract of Jatropha gossypifolia and also reported the bioactive compound in ethanolic extracts of christella dentata [22]. Lanost-8-en-3-ol-3β, a steroid family, possess that anti-inflammatory, antitumor, anticancer, and anti-hepatitis activity were reported [23,24]. The presence of bioactive compound neophytadiene (2.68%) a hydrocarbons possess anti-inflammatory, antioxidant, and disinfectant activity which were reported [22,25,26] in their research findings. 1H-imidazole-2-methanol contains a special pharmacological properties such as antidepressants and antiinflammatory properties which were identified and suggested their findings performed in benzene/ethanol extracts in oil tea cake [27]. 8A-(2,4-Dimethyl -1-nitilo-pent-2-yl)-dioxy)-tocopherone components that it possess antioxidant property reported [28]. The phytocomponents present in D. hirtipes may contribute the major proportion and trace amounts of compounds like hexadecanoic acid that possesses such as an analgesic, anti-inflammatory, antioxidant, anticancer, and antidepressants. These natural components should be explored for the beneficiary in the fields of medical and pharmacy.

CONCLUSION
In the present study, seven major known chemical constituents and some unknown chemical constituents have been identified from the whole plant in the methanolic extract of D. hirtipes Linn. In those, seven bioactive compounds show the medicinal properties to treat various ailments. Further investigation is required for the analysis of specified compounds in D. hirtipes which may lead to explore the development of the new drug to the society.

ACKNOWLEDGMENTS
The authors must be thankful to Dr. D. Natarajan, Assistant Professor, Department of Biotechnology, Periyar University, Salem, for his support and encouragement and for providing laboratory facilities.