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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access  Full Text Article                                                                                                                                               Research Article 

To perform Gas chromatography and Mass spectroscopy (GC-MS) analysis of Achyranthes aspera L. leaf extract

Dr. Ved Prakash* 

Assistant Professor, Department of Botany, Bhagat Singh Govt. P.G. College, Jaora (Ratlam) Madhya Pradesh 457226 (India)

INTRODUCTION

Due to the development of new and sophisticated techniques, phyto-chemical studies have attracted the attention of scientists throughout the world. These techniques played a significant role in the search for additional resources/materials of raw material for pharmaceutical industries¹. Knowledge of the chemical constituents or bio-active components of plants is desirable because such information will be of value for the synthesis of complex chemical substances. 

One of the most popular methods of studying phytochemical composition of plant extract is Gas Chromatography and Mass Spectroscopy (GC-MS) which is a very compatible and the most commonly used technique for the identification and quantification purpose. The unknown organic compound in a complex mixture can be determined by interpretation and also by matching the spectra with the reference spectrum². GC-MS method used for the analysis of prepared plant extracts can be used as an interesting tool for testing the amount of some active principles in different plants used in cosmetics, drugs, pharmaceutical or food industry. Because of its very good separation ability which can produce a chemical fingerprint of high quality, GC-MS technique has become a prevalent technique for the analysis of phyto-chemicals. Moreover, with the coupled mass spectroscopy and the corresponding mass spectra database, the qualitative and the quantitative composition information/knowledge of the herb under investigation could be provided by GC-MS which will be extremely useful for elucidating the relationship between chemical constituents in herbal medicine and its pharmacology in further research³. Therefore, GC-MS technique can be a valuable tool in natural product research assisting in the separation and identification of biologically active volatile organic compounds⁴. This method also finds application in certain other areas such as medical, environmental, chemical engineering and law-enforcement fields.

Approximately 122 biologically active compounds have been identified only from 94 species of different plants. A conservative estimate of the number of flowering plants occurring on the planet is around 2,50,000. Of these, only about 6% have been screened for biological activity and 15% have been evaluated phyto-chemically by different methods. Consistent findings should be carried out to discover a probable abundance of medicinal constituents in these plants/plant parts⁵.

Achyranthes aspera L., an erect or procumbent, annual or perennial herb of about 1-2 m is found on road sides, field boundaries and waste places as a weed throughout India, tropical Asia and other parts of the world up to an altitude of 2100 m. It grows throughout the tropical and warmer regions of the world and is also widespread in Baluchistan, Ceylon, Tropical Asia, Africa, Australia, America and Northern Bangladesh^6,7.                 

Traditionally, A. aspera is used in indigenous system of medicine as emenagogue, anti-arthritic, anti-fertility, anti-phlegmatic, anti-periodic, purgative, laxative, ecbolic, abentifacient, anti-helminthic, aphrodisiac, antiviral, anti-plasmodic, anti-hypertensive, anticoagulant, antitumor agent etc.^8,9. It is also effective in controling or treating cough, asthma, oedema, renal dropsy, fistula, scrofula, skin rash, nasal, infection, chronic malaria, impotence, fever, piles and snake bites^10,11. Because of above mentioned useful properties, GC-MS analysis was carried out of this plant to find out the exact chemical constituents responsible for producing medicinal effects.

MATERIALS AND METHODS

Collection and processing of plant material 

Fresh leaves of Achyranthes aspera L. were plucked, collected and were brought to the laboratory for further examinations. A. aspera leaves were washed thoroughly under tap water and then treated with 2% Mercuric chloride. Thereafter, the parts were cut into smaller pieces for quick drying. The plant material thus obtained after drying was crushed into fine powder with the help of pestle mortar and stored in air tight containers at room temperature.

GC-MS analysis

GC-MS analysis was carried out using thermo GC model TRACE 1300 and thermo MS model TSQ 8000 (triple quadrupole) equipment employing the following conditions/features: Thermo TG 5MS Column having dimensions of “30 m × 0.25 mm × 0.25 μm”, operated in electron impact mode at 70 eV. Helium (99.99%) was used as carrier gas at a constant flow rate of 1 mL per minute with an injection volume of 1 μL. An injector temperature of 250^oC and an ion-source temperature of 280^oC were regularly employed. The Oven program consisted of maintaining the temperature at 60^oC for 2 minutes followed by an increase to 250^oC at the rate of 15^oC/minute and maintaining this temperature of 250^oC for a further 15 minutes duration. Samples or extracts which dissolved in methanol were run fully at a range of 50-1000 m/z. The mass spectra were detected in 26 minutes and the percentage of each chemical constituent was calculated by comparing the average peak area to the total area. 

Identification of component/s 

Interpretation of mass spectrum of GC-MS was done by 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 finally.

RESULTS AND DISCUSSION

GC-MS analysis of leaves of Achyranthes aspera revealed the presence of 12 major peaks in methanol extract. Phytol (21.99%), 9,12-Octadecadienoicacid (Z,Z) (13.74%) and 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z) (13.35%) were among the major compounds of methanol leaf extract. Phytol is a key acyclic diterpene alcohol that is a precursor for vitamins E and K and generally used along with simple sugar or corn syrup as a hardener in candies. Varadharaj and Kuppan¹² studied Achyranthes aspera whole plant extract for its phytochemical constituents by using GC-MS analysis method and revealed the occurence of phenol, 4-(3-hydroxy-1-propenyl)-2-methoxy (2.95%), dl-(2-fluorophenyl)-glycine (2.18%), hexadecanoic acid, ethyl ester (3.28%), phytol (22.13%) and 9,12-octadecadienoic acid (Z,Z) (12.74%) as the major chemical constituents.

Phytochemical examination of the seeds of A. aspera revealed the presence of α-L-rhamnopyranosyl-(14)-(β-Dglucopyranosyluronicacid)-(13)-oleanolic acid, α-L-rhamnopyranosyl-(14)-(β-Dglucopyranosyluronicacid)-(13)-oleanolic acid-28-O-β-D- glucopyranoside and α-L-rhamnopyranosyl-(14)-(β-Dglucopyranosyluronic acid)-(13)-oleanolic acid-28-O-β-Dglucopyranosyl-(14)-β-Dglucopyranoside¹³. Hariharan & Rangaswami¹⁴ and Ali¹⁵ reported the isolation & identification of Saponins A and B in the seeds of A. aspera. Hence, Saponin A was identified as D-glucuronic acid and Saponin B was identified as β-D-galactopyranosyl ester of D-glucuronic acid. Along with these chemical constituents, certain other constituents were also isolated such as oleanolic acid, amino acids and hentriacontane. Chemical constituents like 10-tricosanone, 10-octacosanone and 4-tritriacontanone were also found in this plant by Ram et al.¹⁶. Danial¹⁷ also reported the presence of many compounds such as polysaccharides, ecdysterone (harmone), achyranthine, betaine (alkaliods), vanillic acid, syringic acid, p-coumaric acid (phenolic acids), saponin A, saponin B (saponins), protein and carbohydrates in A. aspera.

Table 1: GC-MS spectral analysis of methanol extract of Achyranthes aspera

Figure 1: GC-MS chromatogram of methanol extract of A. aspera

CONCLUSIONS

The present study was carried out to find out the bio-active constituents present in methanol extract of Achyranthes aspera L. leaves by using Gas chromatography and Mass spectroscopy. GC-MS chromatograms of plant extracts revealed the presence of numerous phytochemicals by showing different peaks. GC-MS analysis of A. aspera revealed the presence of 12 major peaks in methanol leaf extract. The major chemical compounds were reported to be Phytol (21.99%), 9,12-Octadecadienoicacid (Z,Z) (13.74%) and 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z) (13.35%). The presence of these phyto-compounds in this plant may be directly or indirectly responsible for its valuable medicinal activities.

ACKNOWLEDGEMENT

Author greatly acknowledges the financial support provided by Indian Council of Medical Research (ICMR), New Delhi to conduct this work smoothly and efficiently.

CONFLICT OF INTERESTS

Author hereby declares no conflict of interest.

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