Int J Curr Pharm Res, Vol 10, Issue 6, 16-20 Original Article


PHYTOCHEMICAL PROFILING OF MEDICALLY SIGNIFICANT CRUDE EXTRACT USING GC-MS ANALYSIS

MARUTHAMUTHU RAJADURAI1, V. MAITHILI2, R. ARUNAZHAGI3, V. YOGESH4

PG and Research Department of Biotechnology and Bioinformatics, Bishop Heber College, Tiruchirappalli 620001, India
Email: mr.rajadurai@gmail.com

Received: 10 Jul 2018, Revised and Accepted: 07 Sep 2018

ABSTRACT

Objective: The objective of this research is to identify the phytochemical constitutions present in Natural crude extract which obtained from Thumlappati district.

Methods: Kidney stone is one of the most clinical disorder arising nowadays. They are existing due to the depletion of the urine and disproportionate execration of the components such as oxalate, phosphate, uric, cysteine, and struvite. Many alopathy medicine are not effectively curable in the case of kidney stone, consequently people are in need of traditional medicine system. Thus there is a great demand for research on potential inhibitor from natural products for dissolving kidney stone. In present work deals with an unknown crude extract collected from G. Thumlappati, Battalagundu Dindugal district Tamil Nadu. The crude extract of phytochemical are analyzed by using GCMS method.

Results: Thus the sample has some bioactive compound to discharge the stone particles. So we subjected the crude extract sample to GC-MS process which reveals 210 compounds in 21 different peaks.

Conclusion: This studies forms a basis for the biological characterization and importance of bioactive compounds were identified.

Keywords: Kidney stone, Crude extract, GCMS analysis, Bioactive compounds

© 2018 The Authors.. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2018v10i6.30966

INTRODUCTION

History reveals that, every civilization in the world used plants as their derivatives for treatment (or) prevention of diseases. Plants had been used as traditional health care system from the centuries and is a major source of the therapeutic agents for curing the human diseases. In the last few years more than 13000 plants have been studied for the various diseases among these some medicinal properties of plants have been documented by researchers [1-3]. In India the Traditional medicinal system using medicinal plants are Ayurveda, siddha, homeopathy, etc., to treat various diseases [4]. Traditional plant based medicines for primary health care need is followed in underdeveloped countries of about 80% of world’s population (WHO) [5]. A large portion of the world population, especially in developing countries depends on the traditional system of medicine for a variety of disease. Traditional medicine have become more popular in the treatment of many diseases due to popular belief that green medicine is safe, and with less side effects. Traditional medicine is the sum of knowledge skills and practices based on the theories, beliefs, and experience indigenous to different culture that are used to maintain health and also to prevent, diagnose, improve or treat physical and mental illness [6]. It is also believed that crude extract from medicinal plants are biologically active than isolated compounds due to their synergistic effects [7]. Therefore Kidney stones are hard deposits made of minerals and salts on the inner lining of the kidney. The world Health Organization reported 35 million peoples are affected by kidney stone [8]. In India states like Andhra Pradesh, Odisha and Tamil Nadu were worst affected by Kidney stone. The scientific drugs are taken by the affected people are not much benefited only during the time of drugs consumption there are reveling the problem and pain. After few month again they were affected by the stone formation due to their food habits and the environment. So we have a small step to completely cure the stone from the urinary tract and not allowing to form the stone again by our unknown crude sample.

In this study the Gas Chromatography Mass Spectroscopy (GC-MS) method was carried out in the methanol of crude extract for the phytochemical analysis followed by qualitative and quantitative determination of the compounds. This crude possess various medicinal properties, the aim of this study was to identify the phytocompounds in the methanol of crude extract and to identify each specific compound with their concentration by GC-MS analysis. Extraction of several active phytocompounds from these extract leadsto some high activity profile drugs.

MATERIALS AND METHODS

Collection of extract

On every Sunday early morning, the 60 y old folker persons (men and women) providing the mixed plant crude extract to the people affected from kidney stones as a liquid medicine at free of cost, They are so delight to do as a service to the public. By hearing the statement we collected sample G. Thumlappati, Battalagundu Dindugal district Tamil Nadu, India, through interview and questionaries’ from folker peoples, collected the crude sample for further research.

Preparation of extract

500 ml of crude extract were heated at the temperature not exceeding the boiling point. The fine paste were obtained. Required quantity of the sample was weighted, transferred to the conical flask, and diluted with methanol in the ratio of 1:2 until the paste was fully immersed, the flask was shaken every hour for the first 6 h and incubated overnight, then filtered through what man No.1 filter paper. The methanol sample may be contains polar and non-polar components of the material and 4 µl of methanol sample was employed in GCMS analysis.

GCMS analysis

The GC–MS analysis was carried out using a Clarus 500 Perkin–Elmer (Auto system XL) Gas Chromatograph equipped and coupled to a mass detector Turbo mass gold–Perkin Elmer Turbo mass 5.1 spectrometer with an Elite–1 (100% Dimethyl poly siloxane), 30m x 0.25 mm ID x 1μm of capillary column. The instrument was set to an initial temperature of 110oC, and maintained at this temperature for 2 min. At the end of this period the oven temperature was rose up to 280oC, at the rate of an increase of 5oC/min, and maintained for 9 min. Injection port temperature was ensured as 250oC and Helium flow rate as one ml/min. The ionization voltage was 70eV. The samples were injected in split mode as 10:1. Mass spectral scan range was set at 45-450 (m/z).

Identification of phytocompounds

Interpretation on Mass-Spectrum GC-MS was conducted using the database of National institute Standard and Technology (NIST) having more 62,000 patterns. The spectrum of the unknown components was compared with the spectrum of known components stored in the NIST library. The name, molecular weight and retention time of the components of the test materials were ascertained.

RESULTS AND DISCUSSION

At present the crude sample was utilized by tribal people residing at different corners of the district and also by rural and urban persons. We observed that the region of G. Thumalappati has lot of traditional utility of medicinal plants and herbs for diseases. But the folker were not willing to reveal the compounds of the crude sample. GCMS is one of the technique to identify the bioactive constituents of long chain branched chain hydrocarbons, acids, alcohols, esters etc. GCMS analysis was done using the organic solvent methanol it shows the presence of different 210 compounds in the crude sample. The spectrum profile of GCMS confirmed the 21 major peaks with the retention time 10.257,10.781,12.326,13.138,14.657,15.222,16.828, 21.892,23.428,24.654,25.966,28.821,31.006,32.161,33.480,36.490,37.941,38.799,40.163,41.134,42.157. The studies on the active principles in the plant crude sample of methanolic extract by GCMS analysis clearly showed the presence of 210 compounds with their retention time (RT), molecular weight (MW) are presented in table 1. The GCMS chromatogram of the 21 peak of the compounds detected was shown in fig. 1. The highest peak area % (15) is 29.742 and the lowest peak area % (1) is 0.010. By comparing the GCMS compound against with traditional plants using Dr. Dukes photochemical and ethanobotanical database, almost maximum number of crude sample compounds are identify insarcostemma acidum, Hymenocardia acida, Cicca acida, Rumex aceosella, Phyllanthus acidus, Citrus auratum, Citrus acida, Uncaria acida, Citrus sinesis, Elephantopus scaber, Tribulus cistoidesplants which has a property of Inhibition formation of uric acid.

Table 1: Compound detected in the methanol extract of crude sample

S. No. Compound name Retenton time (min) Molecular weight
1 Benzoic acid 10.257 122
2 Benzoic acid, silver(1+) salt 10.257 228
3 Heptanediamide, N,N'-di-benzoyloxy 10.257 398
4 Benzoic acid 10.257 122
5 Cyclobutane-1,1-dicarboxamide, N,N'-di-benzoyloxy- 10.257 382
6 2,4-Dinitrophennylhydrazone of ribose tetrabenzoate 10.257 746
7 Methanol, oxo-, benzoate 10.257 150
8 4-Piperidinepropanoic acid, 1-benzoyl-3-(2-chloroethyl)-, ethyl 10.257 351
9 Cyclopropanecarboxamide, N-benzoyloxy- 10.257 205
10 1-O-Monoacetyl-2,3-O-dibenzoyl-d-ribofuranose 10.257 400
11 Phenol, 4-ethenyl-, acetate 10.781 162
12 Benzofuran, 2,3-dihydro- 10.781 120
13 4-Ethoxystyrene 10.781 148
14 Benzaldehyde, 4-methyl- 10.781 120
15 Benzene, (ethenyloxy)- 10.781 120
16 Benzaldehyde, 3-methyl- 10.781 120
17 4-tert-Butoxystyrene 10.781 176
18 Benzaldehyde, 2-methyl- 10.781 120
19 6-Methylenebicyclo[3.2.0]hept-3-en-2-one 10.781 120
20 Bicyclo[4.2.0]octa-1,3,5-trien-7-ol 10.781 120
21 dl-Mevalonic acid lactone 12.326 130
22 2-Hexene, 1-methoxy-, (E)- 12.326 114
23 Oxirane, butyl- 12.326 100
24 (2,3-Dimethyloxiranyl)methanol 12.326 102
25 trans-3-Penten-2-ol 12.326 86
26 2(3H)-Furanone, dihydro-3-hydroxy-4,4-dimethyl-, (.+/-.)- 12.326 130
27 2-Nonanone 12.326 142
28 Pentane, 1-(2-propenyloxy)- 12.326 128
29 Cyclooctyl S-2-(dimethylamino)ethyl propylphosphonofluoridate 12.326 321
30 2,6-Octadiene-4,5-diol 12.326 142
31 2-Methoxy-4-vinylphenol 13.138 150
32 4-Hydroxy-2-methylacetophenone 13.138 150
33 Ethanone, 1-(2-hydroxy-5-methylphenyl)- 13.138 150
34 4-Hydroxy-3-methylacetophenone 13.138 150
35 3-Methoxyacetophenone 13.138 150
36 Benzene, 1-ethoxy-4-ethyl- 13.138 150
37 Ethanone, 1-[5-(1-hydroxyethylidene)-1,3-cyclopentadien-1-yl]- 13.138 150
38 Phenol, m-tert-butyl- 13.138 150
39 Phenol, 2-(1,1-dimethylethyl)- 13.138 150
40 1-(4-Hydroxymethylphenyl)ethanone 13.138 150
41 1(3H)-Isobenzofuranone 14.657 134
42 Benzoic acid, 2-(hydroxymethyl)- 14.657 152
43 Benzoyl bromide 14.657 184
44 Ethanone, 2,2-dibromo-1-phenyl- 14.657 276
45 Ethanone, 2,2-dihydroxy-1-phenyl- 14.657 152
46 beta.-Benzilmonoxime 14.657 225
47 Benzhydrazide, N2-(2-methoxy-5-nitrobenzylideno)- 14.657 299
48 N,N'-(4,5-Dimethyl-1,3-phenylene) bisbenzamide 14.657 344
49 Benzoic acid, 3,5-difluophenyl ester 14.657 234
50 . alpha.,. alpha.-Dichloroacetophenone 14.657 188
51 Dodecane, 1-chloro- 15.222 204
52 1-Chloroundecane 15.222 190
53 Decane, 1-chloro- 15.222 176
54 Tetradecane, 1-chloro- 15.222 232
55 Nonane, 1-chloro- 15.222 162
56 Hexadecane, 1,16-dichloro- 15.222 294
57 n-Dodecylpyridinium chloride 15.222 283
58 Hexadecane, 1-chloro- 15.222 260
59 Octane, 1-chloro- 15.222 148
60 1-Octadecanesulphonyl chloride 15.222 352
61 1-Undecanol 16.828 172
62 Cyclodecane, methyl- 16.828 154
63 Cyclopropane, nonyl- 16.828 168
64 E-11,13-Tetradecadien-1-ol 16.828 210
65 Cyclodecane 16.828 140
66 1-Decanol 16.828 158
67 3-Tetradecene, (Z)- 16.828 196
68 Cyclooctane, 1,2-dimethyl- 16.828 140
69 3-Dodecene, (E)- 16.828 168
70 Cyclooctane, methyl- 16.828 126
71 3-tert-Butyl-4-hydroxyanisole 21.892 180
72 Ethanone, 1-(3,4-dimethoxyphenyl)- 21.892 180
73 3',5'-Dimethoxyacetophenone 21.892 180
74 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol 21.892 180
75 Phenol, 3-(1,1-dimethylethyl)-4-methoxy- 21.892 180
76 (+)-s-2-Phenethanamine, 1-methyl-N-vanillyl- 21.892 271
77 Ethanone, 1-(2,5-dimethoxyphenyl)- 21.892 180
78 (+-)-2-Phenethanamine, 1-methyl-N-vanillyl- 21.892 271
79 2,5-Dimethoxy-4-ethylamphetamine 21.892 223
80 1,2,4-Cyclopentanetrione, 3-(2-pentenyl)- 21.892 180
81 Tetradecane, 1-chloro- 23.428 232
82 Dodecane, 1-chloro- 23.428 204
83 Hexadecane, 1-chloro- 23.428 260
84 1-Chloroundecane 23.428 190
85 Decane, 1-chloro- 23.428 176
86 Hexadecane, 1,16-dichloro 23.428 294
87 1-Octadecanesulphonyl chloride 23.428 352
88 Nonadecane, 1-chloro- 23.428 302
89 Octadecane, 1-chloro- 23.428 288
90 Nonane, 1-chloro- 23.428 162
91 1-Hexadecanol 24.654 242
92 n-Tridecan-1-ol 24.654 200
93 Cyclotetradecane 24.654 196
94 Hexadecen-1-ol, trans-9- 24.654 240
95 3-Hexadecene, (Z)- 24.654 224
96 5-Octadecene, (E)- 24.654 252
97 7-Hexadecene, (Z)- 24.654 224
98 n-Heptadecanol-1 24.654 256
99 1-Undecanol 24.654 172
100 Cetene 24.654 224
101 2-Propenoic acid, 3-(4-hydroxy-3-methoxyphenyl)-, methyl ester 25.966 208
102 2-Propenoic acid, 3-[4-(acetyloxy)-3-methoxyphenyl]-, methyl es 25.966 208
103 1,2-Dimethoxy-4-(3-methoxy-1-propenyl)benzene 25.966 208
104 2-Propenoic acid, 3-(2,4-dimethoxyphenyl)-, (E)- 25.966 208
105 3,5-Dimethoxycinnamic acid 25.966 208
106 2,3-Dimethoxycinnamic acid 25.966 208
107 1H-1,3-Benzimidazole-6-carboxylic acid, 2-mercapto-, methyl est 25.966 208
108 2,5-Dimethoxycinnamic acid 25.966 208
109 4-Methyl-3,5-dinitrobenzamide 25.966 225
110 3,5-Dimethoxy-4-hydroxycinnamaldehyde 25.966 208
111 1-Octanol, 2-butyl- 28.821 186
112 2-Ethyl-1-dodecanol 28.821 214
113 2-Dodecanol 28.821 186
114 Methoxyacetic acid, pentadecyl ester 28.821 300
115 2-Methyl-1-undecanol 28.821 186
116 1-Dodecanol, 2-methyl-, (S)- 28.821 200
117 Isobutyl tetradecyl carbonate 28.821 314
118 1-Hexadecanol, 2-methyl- 28.821 256
119 2-Hexyl-1-octanol 28.821 214
120 2-Hexadecanol 28.821 242
121 2-Propenoic acid, 3-(4-hydroxy-3-methoxyphenyl)-, methyl ester 31.006 208
122 2-Propenoic acid, 3-[4-(acetyloxy)-3-methoxyphenyl]-, methyl es 31.006 250
123 1,2-Dimethoxy-4-(3-methoxy-1-propenyl)benzene 31.006 208
124 1H-1,3-Benzimidazole-6-carboxylic acid, 2-mercapto-, methyl est 31.006 208
125 2-Propenoic acid, 3-(2,4-dimethoxyphenyl)-, (E)- 31.006 208
126 2,5-Dimethoxycinnamic acid 31.006 208
127

2-Propenoic acid, 3-(2,3-dimethoxyphenyl)-, (E)-

8 166872

 31.006 208
128 trans-2,5-Dimethoxycinnamic acid 31.006 208
129 3,5-Dimethoxycinnamic acid 31.006 208
130 1,3-Benzenedicarboxylic acid, 4-methyl-, dimethyl ester 31.006 208
131 6,9,12,15-Docosatetraenoic acid, methyl ester 32.161 346
132 Cyclopropanepentanoic acid, 2-undecyl-, methyl ester, trans- 32.161 310
133 Oxiraneundecanoic acid, 3-pentyl-, methyl ester, cis- 32.161 312
134 Cyclopropanedodecanoic acid, 2-octyl-, methyl ester 32.161 366
135 Oxiraneundecanoic acid, 3-pentyl-, methyl ester, trans- 32.161 312
136 Methyl 11-hexadecenoate 32.161 268
137 Butyl 6,9,12-hexadecatrienoate 32.161 306
138 Octadecanoic acid, 9,10-dichloro-, methyl ester 32.161 366
139 14-Methylpentadec-9-enoic acid methyl ester 32.161 268
140 Methyl 9-eicosenoate 32.161 324
141 Acetic acid, 2-diacetylamino-1-methyl-1-propenyl ester 33.480 213
142 6,6-Dimethyl-1,4-dioxa-spiro[4.5]dec-7-ene 33.480 168
143 1-Nitro-. beta.-d-arabinofuranose, tetraacetate 33.480 363
144 1-Nitro-2-acetamido-1,2-dideoxy-d-glucitol 33.480 252
145 N,N-Diethyl-N'-(1-naphthyl)ethylenediamine 33.480 242
146 DL-Leucine, N-DL-leucyl- 33.480 244
147 1,16-Cyclocorynan-17-oic acid, 19,20-didehydro-, methyl ester, 33.480 322
148 1-Nitro-2-acetamido-1,2-dideoxy-d-mannitol 33.480 352
149 9-Oxabicyclo[3.3.1]nonane-2,6-dione, 2-oxime-6-ethylene ketal 33.480 213
150 Malonodihydrazide, 2-(3-butoxy-2-hydroxypropyl)- 33.480 262
151 trans-13-Octadecenoic acid, methyl ester 36.490 296
152 11-Octadecenoic acid, methyl ester 36.490 296
153 6-Octadecenoic acid, methyl ester, (Z)- 36.490 296
154 10-Octadecenoic acid, methyl ester 36.490 296
155 6-Octadecenoic acid, methyl ester 36.490 296
156 cis-13-Octadecenoic acid, methyl ester 36.490 296
157 13-Octadecenoic acid, methyl ester 36.490 296
158 16-Octadecenoic acid, methyl ester 36.490 296
159 9-Octadecenoic acid (Z)-, methyl ester 36.490 296
160 9-Octadecenoic acid (Z)-, methyl ester 36.490 296
161 trans-13-Octadecenoic acid, methyl ester 37.941 296
162 11-Octadecenoic acid, methyl ester 37.941 296
163 10-Octadecenoic acid, methyl ester 37.941 296
164 cis-13-Octadecenoic acid, methyl ester 37.941 296
165 13-Octadecenoic acid, methyl ester 37.941 296
166 16-Octadecenoic acid, methyl ester 37.941 296
167 6-Octadecenoic acid, methyl ester 37.941 296
168 14-Octadecenoic acid, methyl ester 37.941 296
169 6-Octadecenoic acid, methyl ester, (Z)- 37.941 296
170 9-Octadecenoic acid (Z)-, methyl ester 37.941 296
171 Methyl stearate 38.799 298
172 Heptadecanoic acid, 16-methyl-, methyl ester 38.799 298
173 Tridecanoic acid, 12-methyl-, methyl ester 38.799 242
174 Methyl tetradecanoate 38.799 242
175 Hexadecanoic acid, 15-methyl-, methyl ester 38.799 284
176 Pentadecanoic acid, 15-bromo-, methyl ester 38.799 334
177 Pentadecanoic acid, methyl ester 38.799 256
178 Cyclopentaneundecanoic acid, methyl ester 38.799 268
179 Tetradecanoic acid, 12-methyl-, methyl ester 38.799 256
180 Octadecanoic acid, 17-methyl-, methyl ester 38.799 312
181 Myo-Inositol, 4-C-methyl- 40.163 194
182 Myo-Inositol, 2-C-methyl- 40.163 194
183 . alpha.-d-6,3-Furanose, methyl-. beta.-d-glucohexodialdo-1,4-fur 40.163 192
184 3-O-Methyl-d-glucose 40.163 194
185 D-Epi-Inositol, 4-C-methyl- 40.163 194
186 3-Methylmannoside 40.163 194
187 2-O-Methyl-D-mannopyranosa 40.163 194
188 Scyllo-Inositol, 1-C-methyl- 40.163 194
189 Methyl 4-O-methyl-d-arabinopyranoside 40.163 178
190 Hydroperoxide, 1,4-dioxan-2-yl 40.163 120
191 Heptacosane, 1-chloro- 41.134 414
192 Tritetracontane 41.134 604
193 2-methyloctacosane. 41.134 408
194 Tetracosane, 11-decyl- 41.134 478
195 Tetratetracontane 41.134 618
196 Sulfurous acid, butyl heptadecyl ester 41.134 376
197 Sulfurous acid, butyl tridecyl ester 41.134 320
198 Sulfurous acid, butyl tetradecyl ester 41.134 334
199 Sulfurous acid, pentadecylpentyl ester 41.134 362
200 Sulfurous acid, butyl pentadecyl ester 41.134 348
201 Sulfurous acid, butyl heptadecyl ester 42.157 376
202 Sulfurous acid, butyl octadecyl ester 42.157 390
203 Sulfurous acid, butyl hexadecyl ester 42.157 362
204 Tritetracontane 42.157 604
205 Heptacosane, 1-chloro- 42.157 414
206 Sulfurous acid, butyl pentadecyl ester 42.157 348
207 Sulfurous acid, octadecylpentyl ester 42.157 404
208 Sulfurous acid, butyl tetradecyl ester 42.157 334
209 Sulfurous acid, hexadecylpentyl ester 42.157 376
210 Sulfurous acid, butyl tridecyl ester 42.157 320

Fig. 1: GCMS analysis of crude extract

CONCLUSION

This shows that the crude sample may be the mixture of these plant extract. Gas Chromatography and mass spectroscopy analysis put on view the available of various compound with variable molecular weight. This experiment showed that the stronger extraction capacity of methanol could have produced number of bioactive constituents which are plays vital role for many biological activities. This various bioactive compounds might be utilized for the expansion for the drug development which used to treat the kidney stone formation without no side effects, purely in traditional way. At this end it can be concluded that the in vivo studies on the crude extract open up to new ways for natural drug that can be employed for clinical trials which may generate successful results in future.

AUTHORS CONTRIBUTIONS

All the author have contributed equally

CONFLICTS OF INTERESTS

All authors have none to declare

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