ABSTRACTObjective: Ruellia patula (Jacq.) a medicinally important plant belongs to the family Acanthaceae. Traditionally, the leaves are used in the treatment ofmany diseases such as insect bites, itches, eye diseases, and skin problems. In the present study, the qualitative and quantitative analysis of bioactivecompounds by gas chromatography (GC) and mass spectroscopy (MS) techniques.Methods: The chemical compositions of the ethanol extract of R. patula leaves were investigated using Perkinâ€“Elmer GCâ€‘MS.Result: In the GCâ€‘MS analysis of ethanol extract of R. patula L. reported the presence of 15 compounds. The major compound constituents are 3, 7, 11,15â€‘tetramethylâ€‘2â€‘hexadecenâ€‘1â€‘ol (28.75%), and Î±â€‘sitosterol (14.35%).Conclusion: Identification of these compounds can help in the development of new drugs.Keywords: Ruellia patula, Ethanol extract, Phytochemical compounds, gas chromatographyâ€‘mass spectroscopy analysis, Biological activity ofphytoâ€‘components

C, Karthika

G, Yogeshwari

K, MURUGANANTHAM

S, MANIVANNAN

English

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Vol 9, Issue 2, 2016Online - 2455-3891 Print - 0974-2441PHYTOCHEMICAL ANALYSIS OF RUELLIA PATULA USING GAS CHROMATOGRAPHY‑MASS SPECTROMETRYKARTHIKA C, YOGESHWARI G, MURUGANANTHAM K, MANIVANNAN S*Post Graduate and Research Department of Biotechnology, Bharath College of Science and Management, Thanjavur ‑ 613 005,  Tamil Nadu, India. Email: mani3677@gmail.comReceived: 21 November 2015, Revised and Accepted: 03 December 2015ABSTRACTObjective: Ruellia patula (Jacq.) a medicinally important plant belongs to the family Acanthaceae. Traditionally, the leaves are used in the treatment of many diseases such as insect bites, itches, eye diseases, and skin problems. In the present study, the qualitative and quantitative analysis of bioactive compounds by gas chromatography (GC) and mass spectroscopy (MS) techniques.Methods: The chemical compositions of the ethanol extract of R. patula leaves were investigated using Perkin–Elmer GC‑MS.Result: In the GC‑MS analysis of ethanol extract of R. patula L. reported the presence of 15 compounds. The major compound constituents are 3, 7, 11, 15‑tetramethyl‑2‑hexadecen‑1‑ol (28.75%), and α‑sitosterol (14.35%).Conclusion: Identification of these compounds can help in the development of new drugs.Keywords: Ruellia patula, Ethanol extract, Phytochemical compounds, gas chromatography‑mass spectroscopy analysis, Biological activity of phyto‑components.INTRODUCTIONPlant‑derived substances have traditionally played an important role in the components of health care system. They continue to provide primary health care to more than three‑quarters of the world’s population. Ancient traditional medicinal practices, such as Indian Ayurveda, Arabic Unani, and Chinese systems, are mainly based on a large diversity of medicinal plants. Besides medicinal uses, plant‑derived products are also employed in the preparation of personal care products, phyto‑cosmetics, natural health products, and phytopharmaceuticals [1]. During the last few decades, there has been an increasing focus in the study of medicinal plants and their traditional uses in different parts of the world [2].Three main groups of secondary metabolites (phytochemicals)‑alkaloids, phenolic compounds, and terpenoids are present in plants. These biocompounds are the most important plant materials for the development and production of drugs [3,4]. Indeed, the dazzling variety of amazingly diverse chemicals produced by plants may well yield a significant number of novels, more efficacious drugs against human diseases. According to conservative estimates, there are as many as 400,000 secondary plant metabolites on the earth, of which only about 10,000 have been chemically characterized [5,6]. Thus, one can easily imagine the amount of naturally available chemical wealth that lies awaiting in the vegetal pharmacies of our globe.In this context, a systematic evaluation of medicinal plants is of utmost importance and also a major concern for not only a better understanding of plant resources but also in developing safe, better, and novel therapeutic strategies. Moreover, nowadays, due to the increasing number of adverse effects and other potential limitations of synthetic drugs, natural therapeutic strategies such as plant‑based medicines, which are comparatively safe with limited or no adverse effects, are becoming increasingly useful today. So, people are returning to the natural products with the hope of safety and security [7].Ruellia patula (Jacq.) is a member of the family Acanthaceae, commonly known as Puni chedi in Tamil. It is a hairy small under‑shrub, found in Arabia, Africa, India (especially in Southern India), Sri Lanka, and Myanmar. The plant is commonly distributed on the wastelands in Tamil Nadu, India. Its leaves are used for treating syphilis, insect bites, eye diseases, skin diseases, gonorrhea, tumors, rheumatic complaints renal infection, cough, wounds, scalds, toothache, stomachache, and kidney stones problems [8,9]. R. patula and another species, R. brittoniana, were found to possess good cardiotonic properties [10]. Dipteracanthus patulus (Jacq.) Nees. (Syn. R. patula Jacq) has significant antimicrobial and anti‑inflammatory activities [11]. D. patulus uses as a single drug remedy for against the deadly poison of Kaduva chilanthi (Tiger Spider) by Kani tribe of Agasthiarmalai, India [12]. Ruellia tuberose has saponins, tannins, and flavonoids, which are responsible for antiulcer activity [13]. Based on these beneficial effects of R. patula, the objective of the present study was to determine the phytochemical constituents, and potent bioactive compounds from the ethanol extract of R. patula leaves with the aid of GC‑MS analysis.METHODSPlant materialsFresh leaves of R. patula were collected from Thanjavur (Tamil Nadu, India) in January 2011 and authenticated by a botanist at the Botanical Survey of India, Coimbatore. A voucher specimen has been deposited at the museum of our college.Preparation of leaf extractThe fresh, undamaged, and disease‑free leaves were selected and washed thoroughly with sterile double distilled water, shade‑dried and then coarsely powdered in a blender. The coarse powder was successively solvent extracted in a soxhlet extractor using ethanol solvent. The extracts so obtained were further dried in vacuum desiccators. The residue obtained from the extract was used for further studies by preserving it in a refrigerator.Identification of compounds using gas chromatography and mass spectrometry (GC‑MS) analysisGC and MS make an effective combination for chemical analysis [14]. Research ArticleAsian J Pharm Clin Res, Vol 9, Issue 2, 2016, 111-113 Karthika et al. 112GC‑MS analysis of these extracts was performed with GC Clarus 500 Perkin Elmer system and GC interfaced to an MS equipped with an Elite‑1 fused silica capillary column (30 mm × 0.25 mm 1D × 1 µm of the capillary column, composed of 100% Dimethyl poly siloxane). For GC‑MS detection, an electron ionization system with ionizing energy of 70 eV was used. Helium gas (99.999%) was used as the carrier gas at constant flow rate 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 programed from 110°C (isothermal for 2 minutes) with an increase of 10°C/minutes to 200°C, then 5°C/minutes to 280°C, ending with 9 minutes isothermal at 280°C. Mass spectra were taken at 70 eV, a scan interval of 0.5 seconds and fragments from 45 to 450 kDa. Total GC running time was 36 minutes. The plant extract was dissolved in ethanol and filtered with polymeric solid phase extraction column and analyzed in GC‑MS for different components. 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 turbomass.Interpretation on mass spectrum of GC‑MS was done using the database of the National Institute of Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of known components using computer searches on an NIST database (NIST version 2.1). The name, molecular weight (MW), and structure of the components of the test materials were ascertained.RESULTS AND DISCUSSIONGC‑MS analysis of the ethanol extract of leaves of R. patula identified 15 compounds. The active principal components with their RT, molecular formula, MW, and percentages were also determined. The chemical compounds are shown in Table 1, biological activities of the compounds are tabulated in Table 2, and the corresponding chemical shift peaks of the spectrum are shown in Fig. 1.The qualitative analysis identified the various phytochemicals such as alkaloids, phenols, steroids, glycosides, vitamins, and terpenoids. Interpretation on mass spectrum GC‑MS was conducted using the database of NIST data library [8,10]. 3,7,11,15‑tetramethyl‑2‑hexadecen‑1‑ol (28.75%) was found as the major component followed by α‑sitosterol (14.35%), 9,12‑octadecodienoic acid [z, z], phenylmethyl ester (10.27%), Table 1: Phyto‑components detected in the ethanol extract of the leaves of R. patula by GC‑MSSerial number RT Name of the compound Molecular formula MW Peak area (%)1 2.85 Propane, 1,1,3‑triethoxy C9H20O3 176 8.212 11.71 3,7,11,15‑tetramethyl‑2hexadecen‑l‑ol C20H40O 296 28.753 12.20 3,7,11,15‑tetramethyl‑2hexadecen‑l‑ol C20H40O 296 7.584 13.17 1,2‑Benzenedicarboxylic acid diheptyl ester C22H34O4 362 1.715 13.56 Dodecanoic acid ethyl ester C14H28O2 228 1.716 15.06 Phytol C20H40O 296 8.157 15.84 9,12,15‑octadecatrienal C18H30O 262 2.398 17.45 Pentanal, 2‑methyl C6H12O 100 0.749 19.96 Pentanal, 2,4‑dimethyl C7H14O 114 0.6310 20.99 Didodecyl phthalate C32H54O4 502 0.9711 24.84 Squalene C30H50 410 5.5512 28.01 γ‑tocopherol C28H48O2 416 2.8713 31.35 9,12‑octadecodienoic acid [z, z], phenylmethyl ester C25H38O2 370 10.2714 32.64 α‑sitosterol C29H50O 414 14.3515 33.46 α‑amyrin C30H50O 426 6.11RT: Retention time, MW: Molecular weight, R. patula: Ruellia patula, GC‑MS: Gas chromatography‑mass spectroscopyTable 2: Biological activity of phyto‑components identified in the ethanol extracts of the leaves of R. patulaSerial number Molecular formula Name of the compound Active biological activity1 C20H40O 3,7,11,15‑tetra methyl 1‑2‑hexadecen‑1‑01 Cancer preventive, antioxidant2 C22H34O4 1,2‑benzenedi‑carboxylic acid, diheptyl ester Used as softeners, perfumes, textiles as dyestuffs, cosmetic and glass making3 C14H28O2 Dodecanoic acid, ethyl ester Anti‑inflammatory and anticoronary activity4 C20H40O Phytol Cancer preventive5 C18H30O 9,12,15‑octadecaterienal Cancer preventive, hypo‑cholesterolemic, anticoronary activity6 C30H50 Squalene Anticancer, used in cosmetic, immunologic adjuvant in vaccines7 C28H48O2 C‑tocopherol Antioxidant, fertility factor8 C25H38O2 9,12, octadecadienoic acid (z, z)‑phenylmethyl esterAnti‑inflammatory, Hypo‑cholesterolemic cancer preventive, hepato‑protective, namaticide, insectifuge, antihistaminic, antieczemic, antiacne, 5‑alpha reductase inhibitor, antiandrogenic, antiarthritic, anticoronary9 C29H50O α‑sitosterol Antirachitic, help to prevent cholesterol uptake in GI tract10 C30H50O α‑amyrin Antihyperglycemic activityGI: Gastrointestinal, R. patula: Ruellia patulaAsian J Pharm Clin Res, Vol 9, Issue 2, 2016, 111-113 Karthika et al. 113Fig. 1: Gas chromatography‑mass spectroscopy chromatogram of the ethanol extracts of leaves of the Ruellia patulaPropane, 1,1,3‑triethoxy (8.21%), phytol (8.15%), α‑amyrin (6.11%), squalene (5.55%), γ‑tocopherol (2.87%), 9,12,15‑octadecatrienal (2.39%), 1,2‑benzenedicarboxylic acid diheptyl ester (1.71%), dodecanoic acid ethyl ester (1.71%), didodecyl phthalate (0.97%), pentanal, 2‑methyl (0.74%), pentanal, 2,4‑dimethyl (0.63%).SUMMARY AND CONCLUSIONSThe present study carried out using the ethanol extract of R. patula leaves revealed the presence of active medicinal constituents. This study also helped to identify the formulae and structures of biomolecules, which can be used as drugs. Thus, this type of GC‑MS analysis is a first step toward understanding the nature of active principal components in these medicinal plants. Hence, this type of study will be helpful for further investigations that may lead to the development of new therapeutic drugs.ACKNOWLEDGMENTThe authors express their gratitude to Secretary and Principal, Bharath College of Science and Management, Thanjavur, for providing the laboratory facilities and encouragement.REFERENCES1. Mans DR. From forest to pharmacy: Plant‑based traditional medicines as sources for novel therapeutic compounds. Acad J Med Plants 2013;1:101‑10.2. Lev E. Ethno‑diversity within current ethno‑pharmacology as part of Israeli traditional medicine – A review. J Ethnobiol Ethnomed 2006;2:4.3. Facchini PJ. Alkaloid biosynthesis in plants: Biochemistry, cell biology, molecular regulation, and metabolic engineering applications. Annu Rev Plant Physiol Plant Mol Biol 2001;52:29‑66.4. Goldman P. Herbal medicines today and the roots of modern pharmacology. Ann Intern Med 2001;135:594‑600.5. Firn RD, Jones CG. Natural products – A simple model to explain chemical diversity. Nat Prod Rep 2003;20(4):382‑91.6. Wink M. Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry 2003;64(1):3‑19.7. Bhuvaneswari S, Manivannan S. Anti‑diabetic and anti‑inflammatory activity of Carlluma adscendens var. Adscendens. Int J Pharm Bio Sci 2014;5:42‑9.8. Gopalakrishnan S, Saroja K, Elizabeth JD. GC‑MS analysis of the methanolic extract of the leaves of Dipteracanthus patulus (Jacq.) Nees. Res J Chem Pharm 2011;3:477‑80.9. Samy MN, Khalil HE, Sugimoto S, Matsunami K, Otsuka H, Kamel MS. Three new flavonoid glycosides, byzantionoside B 6’‑O‑sulfate and xyloglucoside of (Z)‑hex‑3‑en‑1‑ol from Ruellia patula. Chem Pharm Bull (Tokyo) 2011;59(6):725‑9.10. Akhtar MF, Rashid S, Ahmad M, Usmanghani K. Cardiovascular evaluation of Ruellia patula and Ruellia brittoniana. J Islam Acad Sci 1992;5:67‑71.11. Yadav S, Arya V, Kumar S, Yadav JP. Anti‑inflammatory activity of root, leaves and stem of Dipteracanthus patulus (Jacq.) Nees (Acanthaceae). Asian Pac J Trop Biomed 2012;2:187‑91.12. Kannikaparameswari N, Chinnaswamy P. Identification and quantification of Lupeol in Dipteracanthus patulus (Jacq.) Nees by high performance liquid chromatography‑photo diode array method. Int J Pharm Biol Sci 2013;4:660‑6.13. Srikumar PP, Pardhasaradhi P. Preliminary phytochemical investigation and anti‑ulcer activity of aerial parts of Ruellia tuberosa L. (Acanthaceae) in male Wistat rats. Int J Pharm Biomed Res 2013;4:145‑8.14. Kumari TK, Muthukumarasamy S, Mohan VR. 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PHYTOCHEMICAL ANALYSIS OF RUELLIA PATULA USING GAS CHROMATOGRAPHYâ€‘MASS SPECTROMETRY

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PHYTOCHEMICAL ANALYSIS OF RUELLIA PATULA USING GAS CHROMATOGRAPHYâ€‘MASS SPECTROMETRY

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