ISSN Original Article

Transcription

1 Available online at International Journal of Research in Biochemistry and Biophysics Universal Research Publications. All rights reserved ISSN Original Article ANALYSIS OF BIOACTIVE COMPOUNDS IN ANTIINFERTILITY FORMULATION USING GC MS AND FTIR TECHNIQUES S. A. MEENAKSHI 1 and S. KALAVATHY 2 1 Department of Biochemistry, S.R.College, Trichirapalli. Tamil Nadu, South India 2 Department of Botany, Bishop Heber College, Trichirapalli. Tamil Nadu, South India 1 Corresponding author: Received 20 August 2015; accepted 12 September 2015 Abstract Traditional medicine is of immense value in the health care systems of developing countries. Hence, the present study has been made to investigate the phytochemical screening of formulated (Mucuna pruriens seed, Murraya koenigii leaf, Saraca asoca bark, Lanthiram bark,) anti-infertility plants. GC/MS results signified the presence of twenty five phytochemical constituents. The prevailing compounds were 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-, Tetradecanoic acid Dibutyl phthalate, 1-(+)-Ascorbic acid 2,6-dihexadecanoate, Phytol isomer, Octadecanoic acid, 9,12- ctadecadienoic acid (Z,Z)-, Hexadecanoic acid, 3,7,11,15-Tetramethyl-2-hexadecen-1-ol and Stigmast-5-en-3-ol, (3.beta.,24S)- (CAS) Clionasterol. The results of FTIR analysis confirmed the presence of amines, alkanes, aldehyde, aliphatic, aromatic amines and ketone compounds. The results of this study offer a platform of using formulation as herbal alternative for infertility Universal Research Publications. All rights reserved Key words: Formulation, Antiinfertility, GC MS, Phytochemicals, FTIR INTRODUCTION In developing countries, 80% of the population continues to use medicinal plants and plant products in handling primary medical problems due to their accessibility, availability and affordability. In these countries, a variety of plants are claimed to have fertility regulating properties and a few have been tested for such effect ( Ganguly et al, 2007; Cherdshewasart et al, 2007). Traditional medicine is of immense value in the health care systems of developing countries. The World Health Organization (WHO) estimates that more than 80% of health care needs in these countries are met through traditional health care practices (World Health Organization, 2002). Research has shown that some medicinal and highly nutritive plants when taken in diet for their known properties have accidental effects on the sex hormones of the individual ingesting it (Williamson et al., 1996). This may possibly be because its affordability and accessibility is more than that of the orthodox medicine. A lot of herbs have been used traditionally to treat infertility for example, Lepidium meyenii (maca), Trifolium pretense (red clover), Dioscorea villosa (wild yam) etc. In the Northern part of Nigeria (Sokoto), Fiscus platyphylla (gamji) has been used for this purpose (Sofowora, 1993; Luoga et al., 2000). Ever increasing world's population has severely depleted the natural resources and has forced mankind to develop new fertility regulation methods. Though considerable progress has been made in the development of effective methods of fertility control but most the methods developed include chemical formulation being non-herbal have several side effects. It has, therefore, become necessary to screened and use biologically active botanical substances as fertility-regulating agents which are safe and interfere with the natural patterns of reproduction (Aladakatti et al., 2010). Some preliminary tests to be screened before preparation of drug. If it works then the drug may enter in the market. Hence, the present study has been made to investigate the phytochemical screening of formulated (Mucuna pruriens seed, Murraya koenigii leaf, Saraca asoca bark, Lanthiram bark,) anti-infertility plants. MATERIALS AND METHODS Phytochemical screening Plant materials: The fully mature Mucuna pruriens seed, Murraya koenigii leaf, Saraca asoca bark, Lanthiram bark were collected in January 2015 from Tiruchirrappali, Tamil Nadu, India. The Mucuna pruriens seed, Murraya koenigii 20

2 leaf, Saraca asoca bark and Lanthiram bark were first washed well and dust was removed. Formulation of antiinfertility drug Formulation was prepared by equal concentrations of Mucuna pruriens seed, Murraya koenigii leaf, Saraca asoca bark and Lanthiram bark powder. The plants were chosen based on our earlier reports of phytochemical screening (Meenakshi and Kalavathy, 2015). Preparation of alcoholic extract: The leaves, seeds, and bark were dried at room temperature and coarsely powdered using pestle and mortar. Formulation was extracted with 70% methanol for 48 hours. A semi solid extract was obtained after complete elimination of alcohol under reduced pressure. The extract was stored in refrigerator until used. Chemical tests were carried out on the alcoholic extract and on the powdered specimens using standard procedures to identify the constituents as described by Sofowara (1993), Trease and Evans (1989) and Harborne (1973, 1984). GC MS Analysis GC MS analysis was carried out on Shimadzu 2010 plus comprising a AOC-20i auto sampler and gas chromatograph interfaced to a mass spectrometer instrument employing the following conditions: column RTX 5Ms (Column diameter is 0..32mm, column length is 30m, column thickness 0.50µm), operating in electron impact mode at 70eV; Helium gas (99.999%) was used as carrier gas at a constant flow of 1.73 ml /min and an injection volume of 0.5 µi was employed (split ratio of 10:1) injector temperature 270 ºC; ion-source temperature 200 ºC. The oven temperature was programmed from 40 ºC (isothermal for 2 min), with an increase of 8 ºC/min, to 150ºC, then 8ºC/min to 250ºC, ending with a 20min isothermal at 280ºC. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds and fragments from 40 to 450 Da. Total GC running time is 51.25min. 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 Ver (Srinivasan et al., 2013). Identification of components Interpretation on GCMS 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 (Dr. Dukes, 2013). Fourier Transforms - Infrared Spectroscopy (FTIR) Spectroscopic analysis FTIR analysis was performed using Perkin Elmer Spectrophotometer system, which was used to detect the characteristic peaks in ranging from cm -1 and their functional groups. The peak values of the FTIR were recorded. Each and every analysis was repeated twice for the spectrum confirmation. RESULTS AND DISCUSSION The pharmacological activities of any plant sample are due to the presence of metabolites, secondary metabolites and secretary products in it. These usually consist of the phenolic compounds, alkaloids, tannins, saponins, carbohydrates, glycosides, flavonoids, steroids, etc. Most phenolic compounds such as flavonoids, glycosides, triperinoids, flavonons, carbohydrates and anthraquinones are found distributed throughout the plant kingdom (Harbone, 1973). Similarly, the polyphenolic compounds most commonly found in plant extracts are the phenolic acids, flavonoids and tannins (Naik et al., 2006). These compounds together with other phenolic structures of plant origin have been reported as scavengers of Reactive Oxygen Species (ROS) and are seen as promising therapeutic drugs for free radical mediated pathologies including diabetic, cardiovascular diseases (Velavan, 2011). The work of Gaware et al. (2009) who reported the use of thirty one medicinal plants commonly used by Indian people in the management of female infertility. The phytochemical characters of formulation (Mucuna pruriens seed, Murraya koenigii leaf, Saraca asoca bark, Lanthiram bark) were investigated and summarized in Table-1. The phytochemical screening of formulation showed that the presence of steroids, flavonoids, polyphenol, tannin, saponins, glycosides, terpenoids, amino acids and anthroquinones. GC-MS ANALYSIS Twenty five compounds were identified in formulation by GC-MS analysis. The active principles with their retention time (RT), molecular formula and concentration (%) are presented in (Table 1 and Fig 1). The prevailing compounds were 4H-Pyran-4-one, 2,3-dihydro- 3,5-dihydroxy-6-methyl-, Tetradecanoic acid Dibutyl phthalate, 1-(+)-Ascorbic acid 2,6-dihexadecanoate, Phytol isomer, Octadecanoic acid, 9,12- ctadecadienoic acid (Z,Z)-, Hexadecanoic acid, 3,7,11,15-Tetramethyl-2- hexadecen-1-ol and Stigmast-5-en-3-ol, (3.beta.,24S)- (CAS) Clionasterol. In the present study thirty one chemical constituents have been identified from extract of the plant of formulation by Gas Chromatogram- Mass spectrometry (GC-MS) analysis. The presence of various bioactive compounds justifies the use of the whole plant for various ailments by traditional practitioners. However isolation of individual phytochemical constituents and subjecting it to biological activity will definitely give fruitful results. The biological activity of formulation represented in the table 2. Fig 1. GC MS analysis of formulation extract 21

3 Table 1. GC MS analysis of formulation extract Formula S.No R.Time Area% C10H22O5S i Methyl 2,2-dimethyl-3,6,9-trioxa-2-silaundecan-11-oate C7H14O2 1-Butanol, 3-methyl-, 3 Name C6H8O4 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl C15H24 Bicyclo[7.2.0] Undec-4-Ene, 4,11,11-Trimethyl-8-Methylene C8H8O3 Methylparaben C15H24O (-)-5-Oxatricyclo[ (4,6)]Dodecane,,12-Trimethyl-9- Methylene C14H28O2 Tetradecanoic acid C20H40O 2-Hexadecen-1-OL, 3,7,11,15-Tetramethyl-, [R-[R*,R*-(E)]] C18H34O2 9-Octadecenoic Acid (Z) C20H40O 3,7,11,15-Tetramethyl-2-hexadecen-1-ol C17H34O2 Hexadecanoic acid, methyl ester C16H30O2 cis-9-hexadecenoic acid (+)-Ascorbic acid 2,6-dihexadecanoate C16H22O4 Dibutyl phthalate C19H34O2 Methyl 10-trans,12-cis-octadecadienoate C19H36O2 9-Octadecenoic acid, methyl ester, (E) C20H40O Phytol C19H38O2 Methyl stearate C18H32O2 9,12-Octadecadienoic acid (Z,Z) C18H36O2 Octadecanoic acid Stearic acid C18H32O2 13-Hexyloxacyclotridec-10-en-2-one C16H28O cis,cis-7,10,-hexadecadienal C20H40O2 Eicosanoic acid C18H35NO 9-Octadecenamide C29H50O Stigmast-5-EN-3-OL, (3.BETA.)- Table 2. Biological activity of formulation compounds identified by GCMS S.no Compound name Biological activity 1. 4H-Pyran-4-one, 2,3- dihydro-3,5-dihydroxy-6- methyl- 2. Tetradecanoic acid Myristic acid Antimicrobial, Anti inflammatory Anti oxidant, cancer preventive, hypercholesterolemic, nematicide, lubricant, cosmetic. 3. Dibutyl phthalate Vitamin C, antioxidant, immunomodulator (+)-Ascorbic acid 2,6- dihexadecanoate Ester Anti oxidant, anti scorbutic, anti inflammatory, anti nociceptive, anti mutagenic, wound healing property. Phytol isomer Diterpene Anti inflammatory, anti cancer, anti microbial, diuretic. 6. Octadecanoic acid Stearic acid 7. 9,12-Octadecadienoic acid (Z,Z)- Linoleic acid 8. Hexadecanoic acid Palmittic acid ,7,11,15-Tetramethyl-2- hexadecen-1-ol Stigmast-5-en-3-ol, (3.beta.,24S)- (CAS) Clionasterol Terpene alcohol Steroids 5-α reductase inhibitor, hypo cholesterolemic, suppository, cosmetic, lubricant, surfactant & softening agent, perfumery, propecic, flavour. Hypocholesterolemic, 5-Alpha reductase inhibitor, Antihistaminic, Insectifuge, Antieczemic, Antiacne Anti oxidant, hypocholesterolemic, nematicide, pesticide, lubricant, anti androgenic, flavour, hemolytic-5-α reductase inhibitor. Antimicrobial Antihepatotoxic, Antiviral, Antioxidant, Cancer preventive, Hypocholesterolemic 22

4 Functional groups identification The FTIR spectrum was used to identify the functional groups of the active components present in extract based on the peaks values in the region of IR radiation. When the extract was passed into the FTIR, the functional groups of the components were separated based on its peaks ratio. The results of FTIR analysis confirmed the presence of amines, alkanes, aldehyde, aliphatic, aromatic amines and ketone compounds (Fig-2, and Table- 3). Table 3: FTIR Peak Values of formulation extract S.No Range Compound Name Amines, Amide Unknown Alkanes Carboxylic acid Alkanes Organo Phosphorus Compounds Unknown Silicon and boron compounds Alkenes Aromatics Ketones Aliphatic amines Aliphatic amines Aliphatic amines The use of different plant parts in recipes is of common occurrence in ethnobotanical studies. Sometimes leaves may be used as in Acanthospermum hispidum DC Fig 2. FTIR analysis of formulation extract (Lemonica and Alvarenga, 2004), Nicotiana tabacum L. (Emmanuel and Claidette, 2007), and Senna alata (L.) Roxb (Babalola, 2009), other times, bark and roots may be more important as in Vernonia amygdalina Del. (Kumar, 2012; Lawal, 2008) and Baphia nitida Lodd (Babalola, 2009). In others, recipe fruits are common as in Citrullus colocynthis (L.) Schrad (Chaturredi and Dixit, 1997). In this study, the leaves, seeds and bark are the most used parts, which agree with previous ethnobotanical studies that have been carried out on plants used in the management of fertility (Kumar et al., 2012). Observable biological activities of plant extracts are due to one or more phytochemical components. CONCLUSION GC/MS results signified the presence of twenty five phytochemical constituents. The prevailing compounds were 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6- methyl-, Tetradecanoic acid Dibutyl phthalate, 1-(+)- Ascorbic acid 2,6-dihexadecanoate, Phytol isomer, Octadecanoic acid, 9,12- ctadecadienoic acid (Z,Z)-, Hexadecanoic acid, 3,7,11,15-Tetramethyl-2-hexadecen-1- ol and Stigmast-5-en-3-ol, (3.beta.,24S)- (CAS) Clionasterol. The results of FTIR analysis confirmed the presence of amines, alkanes, aldehyde, aliphatic, aromatic amines and ketone compounds. The results of this study offer a platform of using formulation as herbal alternative for infertility etc. Scientific studies and experimental screening of these plants are on the increase. Hence, forest reserves, farms and medicinal gardens should exist to protect these plants from extinction. Acknowledgement The authors are grateful to Dr. S. Velavan, Director, Harman Institute of Science Education and 23

5 Research ( Thanjavur, Tamil Nadu for his support. References 1. Aladakatti RH, Mukhtar A, Nazeer AR, Ghodesawar MG: Effect of benzene leaf extract of Ocimum sanctum on testis and spermatogenic pattern in albino rats. International Journal of Current Research 2010, 5: Babalola KA (2009). An examination of the usage of herbal contraceptives and Abortifacients in Lagos state Nigeria. Ethnobotanical leaflets 13: Chaturredi M, Dixit VP (1997). Antiferility effect of CitrulluscolocynthisSchrad.In male albino rats. Ind. J. Environ. Sci. 4. Cherdshewasart W, Kitsamai Y, Malaivijitnond S. Evaluation of the estrogenic activity of the Wild Pueraria mirifica by vaginal cornification assay. J. Reprod. Dev., 2007; 53: Dr.Dukes Phytochemical and Ethnobotanical Databases, Phytochemical and Ethnobotanical Databases Emmanuel N, Claidette D (2007). Abortifacient plants of the Buea region their participation in the sexuality of aldolescent girls, Cameroon. Indian J. Trad. Knowledge 6: Ganguly M, Borthakur M, Devi N, Mahanta R. Antifertility activity of the methanolic leaf extract of Cissampelos pareira in female albino mice. J. Ethnopharmacol., 2007; 111: Gaware. V. M. Parjane. S. K. Merekar Abhijit N., Pattan S. R., Dighe N. S.Kuchekar.B. S Godge Rahul K. Female infertility and its treatment by alternative medicine: A review. Journal of Chemical and Pharmaceutical Research, 2009, 1(1): Harborne J B. Phytochemical Methods, A Guide to modern Techniques of Plant Analysis. Chapman and Hall, London, 1973; Kumar D, Kumar A, Prakash O (2012). Potential fertility regulating agents from plants. A comprehensive review. J. Ethnopharmacol. 140: Luoga EJ, Witkowski ETF, Balkwill K: Differential utilization and ethnobotany of trees in Kitulanghalo forest reserve and surrounding communal lands, eastern Tanzania. Econ Bot 2000, 54(suppl 3): Meenakshi SA and Kalavathy S. Phytochemical Screening of Selected Anti-Infertility Indian Medicinal Plants. Indian Journal of Applied Research, 5(8): Naik G H, Priyadarsini K I, Hari M. Free radical scavenging reactions and phytochemical analysis of Triphala, an ayurvedic formulation. Curr. sci. 2006; 90: Sofowora A: Medicinal Plants and Traditional Medicine in Africa. 2 edition. Ibadan: Spectrum Books Ltd; 1993, Srinivasan K, Sivasubramanian S and Kumaravel S,.Phytochemical profiling and GC-MS study of Adhatoda vasica leaves.int.j.pharm.bio.sci, 2013; 5(1): Trease GE, Evans WC (1989). Pharmacognsy. 11th edn. Brailliar Tiridel Can. Macmillian publishers. 17. Velavan S. (2011) Free radicals in health and diseases A mini review Pharmacologyonline 1: (2011) Newsletter 18. Williamson EM, Evans FG, Okpako DT (1996). Selection preparation and pharmacological evaluation of plant material. John Wiley & Sons Baffin Lane Chichester West Sussex PO19 IUD England 11: World Health Organization (WHO): WHO Traditional Medicine Strategy World Health Organisation Geneva; Source of support: Nil; Conflict of interest: None declared 24