PG and Research Department of Biotechnology and Bioinformatics, Bishop Heber College, Tiruchirappalli 620001, India
Received: 10 Jul 2018, Revised and Accepted: 07 Sep 2018
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/)
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 . Traditional plant based medicines for primary health care need is followed in underdeveloped countries of about 80% of world’s population (WHO) . 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 . It is also believed that crude extract from medicinal plants are biologically active than isolated compounds due to their synergistic effects . 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 . 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.
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|
|2||Benzoic acid, silver(1+) salt||10.257||228|
|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|
|11||Phenol, 4-ethenyl-, acetate||10.781||162|
|21||dl-Mevalonic acid lactone||12.326||130|
|22||2-Hexene, 1-methoxy-, (E)-||12.326||114|
|26||2(3H)-Furanone, dihydro-3-hydroxy-4,4-dimethyl-, (.+/-.)-||12.326||130|
|29||Cyclooctyl S-2-(dimethylamino)ethyl propylphosphonofluoridate||12.326||321|
|42||Benzoic acid, 2-(hydroxymethyl)-||14.657||152|
|49||Benzoic acid, 3,5-difluophenyl ester||14.657||234|
|50||. alpha.,. alpha.-Dichloroacetophenone||14.657||188|
|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|
|104||2-Propenoic acid, 3-(2,4-dimethoxyphenyl)-, (E)-||25.966||208|
|107||1H-1,3-Benzimidazole-6-carboxylic acid, 2-mercapto-, methyl est||25.966||208|
|114||Methoxyacetic acid, pentadecyl ester||28.821||300|
|116||1-Dodecanol, 2-methyl-, (S)-||28.821||200|
|117||Isobutyl tetradecyl carbonate||28.821||314|
|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|
|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|
2-Propenoic acid, 3-(2,3-dimethoxyphenyl)-, (E)-
|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|
|138||Octadecanoic acid, 9,10-dichloro-, methyl ester||32.161||366|
|139||14-Methylpentadec-9-enoic acid methyl ester||32.161||268|
|141||Acetic acid, 2-diacetylamino-1-methyl-1-propenyl ester||33.480||213|
|143||1-Nitro-. beta.-d-arabinofuranose, tetraacetate||33.480||363|
|147||1,16-Cyclocorynan-17-oic acid, 19,20-didehydro-, methyl ester,||33.480||322|
|149||9-Oxabicyclo[3.3.1]nonane-2,6-dione, 2-oxime-6-ethylene ketal||33.480||213|
|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|
|172||Heptadecanoic acid, 16-methyl-, methyl ester||38.799||298|
|173||Tridecanoic acid, 12-methyl-, methyl ester||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|
|183||. alpha.-d-6,3-Furanose, methyl-. beta.-d-glucohexodialdo-1,4-fur||40.163||192|
|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|
|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
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.
All the author have contributed equally
CONFLICTS OF INTERESTS
All authors have none to declare
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