GC-MS Analysis of Phytochemical Compounds Present in the Leaves of Citrus medica. L

 

R. Suja Pandian*, A. Thajun Noora

Post Graduate and Research Department of Biochemistry, Rabiammal Ahamed Maiden College for Women, Thiruvarur, Tamilnadu, India

 

ABSTRACT:

Objective: The main objective of the present work is to analyse the phytochemical compounds present in the leaves of Citrus medica L using GCMS. Materials and Methods: The collected fresh young leaves of Citrus medica L were shade dried, grinded to fine powder and kept in dry conditions. The dried powdered leaf material was extracted successively with methanol in an orbital shaker for 24 hrs at room temperature. Whatman No.1 filter paper was used to filter the extract and the extractable substances were removed, at every 3 hrs interval. The combined extracts were then evaporated at 40°C on water bath and the dried extract was used for GCMS analysis Results: The methanolic extract of Citrus medica L confirmed the presence of alkaloids, flavonoids, steroids and glycosides based on the presence or absence of colour changes. The sixteen chemical constituents identified from methanolic leaf extract of Citrus medica. L by GC-MS analysis were found to have various biological activities like anti-microbial, anti-cancer, anti-inflammatory, and hypocholesterolemic activities. Conclusion: Thus the GC-MS analysis of methanolic extract of Citrus medica. L showed a highly complex profile, containing approximately 16 components. This study may be useful to explore the pharmacological and biosynthetic activity of the plants further. The leaves were rich sources of valuable bioactive compounds and can be explored as nutraceuticals.

 

 

 

INTRODUCTION:

Medicinal plants are used in traditional treatments to cure variety of diseases. In the last few decades there has been an exponential growth in the field of herbal medicine. Natural products have been a source of drugs for centuries. Medicinal plants, which form the backbone of traditional medicine, in the last few decades have been the subject for very intense pharmacological studies, this has been brought about by the acknowledgement of the value of medicinal plants as potential sources of new compounds of therapeutic value and as sources of lead compounds in drug development^[1].

 

 

 

In developing countries, it is estimated that about 80% of the population really depends on traditional medicine for their primary healthcare. Hence there is a need to screen medicinal plants for bioactive compounds which is a basis for further pharmacological studies^[2]. In the last few years gas-chromatography mass-spectrometry has become firmly established as a key technological platform for metabolite profiling in plant^[3].  GCMS based metabolome analysis has profound applications in discovering the mode of action of drugs or herbicides and helps unravel the effect of altered gene expression on metabolism and organism performance in biotechnological applications^[4].

 

Citrus medica L. had been used widely as traditional medicine and can be found in various traditional medicine literatures such as Ayurveda^[5]. Citrus plants, belonging to family Rutaceae. They are small trees with smooth, oblong to broadly lanceolate, and narrowly winged short petioled leaves. The fruit has loose skin and leathery pericarp, with a sweet sour juicy pulp which are mainly process as juice drinks. These plants are also considered important medicinal plants. For instance, the flavonoids from Citrus exhibit in vitro and in vivo anti-inflammatory, anticancer, antioxidant, and cardiovascular protective activities^[6].

 

Hence the present investigation was carried out to determine the possible chemical components from Citrus medica L. leaves by Gas Chromatography and Mass Spectrum.

 

MATERIALS AND METHODS:

Plant Material:

The fresh leaves of Citrus medica L were collected from Thiruvarur Tamilnadu, India during the month of January-March 2018. The fresh leaves of Citrus medica L were collected and washed individually under running tap water to remove any traces of soil particles and other dirt and blotted gently with filter paper and shade dried. The dried leaves were powdered using a mechanical grinder and stored in a sterile container at 4°C until future use.

 

Preparation of Extract:

30gms of dried powdered plant material was extracted successively with methanol in an orbital shaker for 24 hrs at room temperature. The extracts were filtered using Whatman No.1 filter paper to remove extractable substances, at every 3 hrs interval. The combined extracts were then evaporated at 40°C on water bath and the dried extract was stored at 4°C in a sterile container.

 

Phytochemical screening:

Phytochemical components were analyzed qualitatively by Harborne method^[7].

 

Test for alkaloids:

To 2 ml of the extract, few drops of Dragendorff’s reagent was added, formation of orange brown precipitate indicates the presence of alkaloids.

 

Test for flavonoids:

Few drops of extract were mixed with 1 ml of 5% lead acetate solution. Appearance of white precipitate confirms the presence of flavonoids.

 

Test for Tannins:

To 2 ml of the extract, 2-3 drops of ferric chloride solution was added, formation of dark blue or greenish black colour indicates the presence of tannins.

 

Test for Glycosides:

Dissolve the extracts in pyridine and added few drops of sodium nitroprusside and 2% alcoholic sodium hydroxide. Appearance of red colour indicates the presence of glycosides.

 

Test for Triterpenoids:

To 5 ml of the extract, 2ml of chloroform and 3ml of conc. H2SO4 was added; a monolayer of reddish brown coloration indicates the presence of triterpenoids.

Test for steroids:

To 1 ml of the extract, 1 ml of chloroform, 2 ml of acetic anhydride and 2 drops of conc. H2SO4 was added. Appearance of dark red colour indicates the presence of steroids.

 

Test for Saponins:

The extract was diluted with 10 ml of distilled water and it was shaken for 15 minutes. Formation of foam indicates the presence of saponins.

 

GC Condition and Identification of Compounds:

The sample was investigated through Gas Chromatography Mass Spectrometry/Mass Spectrometry Electron Ionization (GC-MS/EI) mode. The GC-MS/MS is a Scion 436- GC Bruker model coupled with a Triple quadruple mass spectrophotometer with fused silica capillary column BR-5MS (5% Diphenyl/95% Dimethyl polysiloxane) and Length: 30m; Internal diameter: 0.25 mm; Thickness: 0.25 µm. Helium gas (99.999%) was used as the carrier gas at a constant flow rate of 1 ml/min and an injection volume of 2 µl was employed (split ratio of 10:1). The injector temperature was 250°C; ion-source temperature 280°C. The oven temperature was programmed from 110°C (isothermal for 2 min), with an increase of 10°C/min, to 200°C, then 5°C/min to 280°C, ending with a 9 min isothermal at 280°C and total GC running time was 41 min. This last increase was to clean the column from any residues. The mass spectrometer was operated in the positive electron ionization (EI) mode with ionization energy of 70eV. The solvent delay was 0-3.0 min. A scan interval of 0.5 seconds and fragments from m/z 50 to 500 Da was programmed. The inlet temperature was set at 280 °C, source temperature 250°C.

 

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 MS Work station 8.

 

Table 1: Phytochemical screening of Citrus medica.L leaves

(+) and (-) signs indicates the presence and absence of the compound

 

The NIST Version 2.0 library database of National Institute Standard and Technology (NIST) having more than 62,000 patterns was used for identifying the chemical components. The GC-MS/MS was performed by Food Safety and Quality Testing Laboratory, Institute of crop processing technology, Thanjavur.

 

 

 

RESULTS:

The phytochemical active compounds of Citrus medica L leaves were qualitatively analyzed and the results are presented in Table-1. The methanolic extract of Citrus medica L confirmed the presence of alkaloids, flavonoids, steroids and glycosides based on the presence or absence of colour changes. For the pharmacological as well as pathological discovery of novel drugs, the essential information regarding the chemical constituents are generally provided by the qualitative phytochemical screening of plant extract ^[8]. The phytochemical screening of methanolic leaf extract of Citrus medica L is depicted in Table 1. The result indicates the presence of alkaloids, flavonoids, phenols, tannins, steroids, triterpenoids, saponins and glycosides in the methanolic leaf extract of Citrus medica L. These bioactive compounds synthesized by secondary metabolism are chemically and taxonomically diverse compounds with obscure function. They are widely used in the human therapy, veterinary, agriculture, scientific research and countless other areas^[9].

 

Gas Chromatography and Mass spectroscopy analysis of compounds was carried out in methanolic leaf extract of Citrus medica L, shown in Table-1. In the GC-MS analysis, 16 bioactive phytochemical compounds were identified in the methanolic extract of Citrus medica L. The identification of phytochemical compounds is based on the peak area, retention time molecular weight and molecular formula. The active compounds identified in the methanolic leaf extract of Citrus medica L by GC-MS analysis was shown in Figure-1. The composition and identification of the main components present in the leaves of Citrus medica L are shown in Table 2.

 

Table 2: Phytochemical components of Citrus medica.L Leaf extract

S. No.	RT	Name of the compound	Molecular Formula	Molecular Weight	Peak Area%
1.	5.08	Tetradecane	C14H30	198	8.44
2.	8.20	Nonadecane	C19H40	268	6.29
3.	9.05	Isopinocarveol	C10H16O	152	10.73
4.	10.88	1-Dodecanol, 3,7,11-trimethyl-	C15H32O	228	3.69
5.	11.20	2-Bromotetradecanoic acid	C14H27BrO2	306	9.52
6.	12.89	1-Aminononadecane, N-trifluoroacetyl-	C21H40F3NO	379	7.84
7.	13.21	1-Hexadecanol, 2-methyl-	C17H36O	256	1.34
8.	14.63	2-Myristynoyl pantetheine	C25H44N2O5S	484	8.05
9.	16.50	Ethyl iso-allocholate	C26H44O5	436	5.01
10.	18.53	5β,7βH,10α-Eudesm-11-en-1α-ol	C15H26O	222	6.35
11.	20.40	9-Octadecenoic acid, (2-phenyl-1,3-dioxolan-4-yl)methyl ester, cis-	C28H44O4	444	4.43
12.	22.43	trans-13-Octadecenoic acid	C18H34O2	282	4.89
13.	22.58	Strychane, 1-acetyl-20α-hydroxy-16-methylene-	C21H26N2O2	338	2.30
14.	23.02	Propanamide, 2-(2,4-dichlorophenoxy)-N-(2,6-diethylphenyl)-	C19H21Cl2NO2	365	13.40
15.	24.24	Dasycarpidan-1-methanol, acetate (ester)	C20H26N2O2	326	5.58
16.	24.66	2-Methyl-E,E-3,13-octadecadien-1-ol	C19H36O	280	2.14

 

 

Fig 1: GCMS Spectra of methanolic extract of Citrus medica.L leaves

 

It  reports the phytoconstituents present in leaves of Citrus medica.L. Sixteen  constituents have been identified in leaf essential oil which are dominated by Propanamide, 2-(2,4-dichlorophenoxy)-N-(2,6-diethylphenyl)-(13.40%),Isopinocarveol(10.73%),2-Bromotetradecanoic acid (9.52%), Tetradecane (8.44%), 2-Myristynoyl pantetheine (8.05%), 1-Aminononadecane, N-trifluoroacetyl-(7.84%), 5β,7βH,10α-Eudesm-11-en-1α-ol (6.35%), Nonadecane(6.29%), Dasycarpidan-1-methanol, acetate (ester) (5.58%), Ethyl iso-allocholate (5.01%), rans-13-Octadecenoic acid (4.89%), 9-Octadecenoic acid, (2-phenyl-1,3- dioxolan-4-yl)methyl ester, cis- (4.43%), 1-Dodecanol, 3,7,11-trimethyl- (3.69%), Strychane, 1-acetyl-20α-hydroxy-16-methylene- (2.30%), 2-Methyl-E,E-3,13-octadecadien-1-ol (2.14%) and 1-Hexadecanol, 2-methyl- (1.34%).

 

DISCUSSION:

The presence of phytoconstituents like alkaloids, saponins and glycosides in the methanolic extract of Citrus medica L were reported to have various biological functions which include anticancer, anti-inflammatory and antimicrobial activities. Higher plants as sources of bioactive compounds continue to play a dominant role in the maintenance of human health. Reports available on green plants represent a reservoir of effective chemo-therapeutants, these are non-phytotoxic, more systemic and easily biodegradable^[10]. Plants are a rich source of secondary metabolites with interesting biological activities. In general, these secondary metabolites are an important source with a variety of structural arrangements and properties^[11]. 

 

Results showed that the leaves oils were a complex mixture of numerous compounds; many of which were present in trace amounts. It is worth mentioning here that the chemical composition of the leaf of C. medica.L contain Propanamide, 2-(2, 4-dichlorophenoxy)-N-(2, 6-diethylphenyl) - and Isopinocarveol as the most important and main components in leaf and we can conclude that Propanamide, 2-(2, 4-dichlorophenoxy)-N-(2, 6-diethylphenyl) - and Isopinocarveol are the reported component in C. medica. L leaf. It is worth mentioning that the oil of C. medica. L has been reported to be used in folk medicine in the treatment of dysentery, rheumatism, diabetes and Alzheimer’s disease. Various parts of Citrus medica. L plants have been used traditionally to treat illness. Citrus medica. L belong to Rutaceae family which is known, used and exploited for its essential oil content. The study reveals that stereochemistry play an important role in the effect of biological active essential oil which sometimes short-sighted.

 

The sixteen chemical constituents identified from methanolic leaf extract of Citrus medica. L by GC-MS analysis were found to have various biological activities like anti-microbial, anti-cancer, anti-inflammatory, and hypocholesterolemic activities. Thus presence of various bioactive compounds justifies the use of Citrus medica. L leaf extracts for various ailments by traditional practitioners and further studies on isolation and identification of individual constituents is needed.

 

 

 

 

CONCLUSION:

Thus the GC-MS analysis of methanolic extract of Citrus medica.L showed a highly complex profile, containing approximately 16 components. This study may be useful to explore the pharmacological and biosynthetic activity of the plants further.

 

REFERENCES:

1.      Khalaf NA, Shakya AK, Al-othman A, Akbar Z, Farah H. Antioxidant activity of some common plants, Turk. J. Biol, 31, 2007:1-5.

2.      Kalaisezhiyen P, Sasikumar V. GC- MS evaluation of chemical constituents from methanolic leaf extract of Kedrostis foetidissima (jacq.) Cogn. Asian J Pharm Clin Res. 5, 2012: 77-81.

3.      Jayapriya G, Shoba FG. GC-MS analysis of bio-active compounds in methanolic leaf extracts of Justicia adhatoda (Linn.). Journal of Pharmacognosy and Phytochemistry.4,  2015:113-117.

4.      Sen A, Batra A. Chemical composition of methanol extract of the leaves of Melia azedarach L. Asian J Pharm Clin Res. 5, 2012: 42-45.

5.      Tundis R, Calabria U, Bonesi M, Calabria U and Calabria U. Chemical composition and bioactivity of Citrus medica L. cv . Diamante essential oil obtained by hydrodistillation. Nat. Prod. Res. 25; 2011: 789-99.

6.      Milton R, Dulay R., Ellenita GM. Castro De. Antibacterial and Antioxidant Activities of Three Citrus Leaves Extracts. Der Pharmacia Lettre. 8; 2016:167-170.

7.      Harborne, J. B. 1973. Phytochemical methods, London, Chapman and Hall, Ltd., P. 49-188.

8.      Ganatra SH, Ramteke AM, Durge SP and Patil SU. Phytochemicals investigation and TLC profiling of Cyamopsis tetragonoloba L. seeds (Fabaceae) - Pea Family. Int J Pharm Sci Res 4(4); 2013:1551-1555.

9.      Uma C and Sekar KG. Phytochemical analysis of a folklore medicinal plant Citrullus colocynthis L (bitter apple). Journal of Pharmacognosy and Phytochemistry. 2 (6); 2014: 195-202.

10.   Sermakkani m and Thangapandian V.  GC-MS analysis of Cassia italica leaf methanol extract Asian J Pharm Clin Res. 5(2); 2012: 90-94.

11.   de-Fatima A, Modolo LV, Conegero LS, Pilli RA, Ferreira CV, Kohn LK, de-Carvalho JE. Lactones and their derivatives: biological activities, mechanisms of action and potential leads for drug design. Curr. Med. Chem 13; 2006: 3371-3384.

 

 

 

 

 

 

Received on 05.10.2018           Modified on 26.11.2018

Accepted on 21.12.2018         © RJPT All right reserved