GC-MS analysis of phyto-constituents and antimicrobial activity of hexane extract of Lanatana camara Linn.

Chanchal Kumari¹, Mrs. P. Meenatchi²

¹Research Scholar, PG and Research Department of Biochemistry, Mohamed Sathak College of Arts and Science, University of Madras, Sholinganallur, Chennai-600119.

²Assistant Professor, PG and Research Department of Biochemistry, Mohamed Sathak College of Arts and Science, University of Madras, Chennai -600119

*Corresponding Author E-mail: chanchal.deepak1992@gmail.com

ABSTRACT:

Nature has been a source of medical agents for thousands of years. Lantana camara linn., family verbenaceae, commonly known as wild sage, is a flowering shrubs native of tropical America and is cultivated throughout the world as an ornamental . The plant based traditional medicine system continues to play an essential role in health care, with about 80% of the world inhabitants. The plant leaves contain a number of medicinally important compounds. The present study was carried out to identify the phytoconstituents and evaluate antimicrobial activity of hexane extract of Lantana camara leaves. The mass spectra of these phytoconstituents were matched with the database available at National Institute of Standard and Technology (NIST) library. The Gas chromatography and Mass spectrometry analysis of terpenes, alkaloids, flavonoids, glycosides, protein etc. lead to identification of 18 compounds. This analysis revealed that the phytoconstituents fraction shows high peak, mainly 2,2,4-trimethyl-3(3,8,12,16-tetramethyl-heptadeca-3,7,11,15-tetraenyl)- cyclohexanol, Cholesta-2,4-diene. The research reveals the potential of hexane extract of Lantana camara leaves as a good source of terpenes, steroids that justify the use of this plant for its various ailments by traditional practitioners. It is used for healing of gastric ulcer, respiratory infections and antipyretic. Lantana camara have therapeutic potential due to the presence of natural agents, Majority of their activity is due to bioactive compounds viz. flavones, isoflavones, flavonoids, anthocyanins, coumarins, lignans, catechins, isocatechins, alkaloids, tannin, saponins and triterpenoids. Bioactive substance from this plant can therefore be employed in the formulation of anti-microbial agents for the treatment of various microbial infection.

KEYWORDS: Lantana camara linn, phytoconstituents, GC-MS, antimicrobial activity.

INTRODUCTION:

Lantan camara linn.introduced as an ornamental, flowering plant but entirely naturalized and found throughout India. However, it is listed as one of the significant medicinal plants of the world (Abu-Shanab 2006). The common names of the lantana camera is Raimuniya, Unnichedi, Arippoo, Pulikampa ,Tantani, samballei, kakke natahu. Lantana camara is a low, erect or subscandent, vigorous shrub which can grow to 2 - 4 meters in height. The leaf is ovate or ovate oblong, 2 - 10 cm long and 2 - 6 cm wide, arranged in opposite pairs. Leaves are bright green, rough, finely hairy, with serrate margins and emit a pungent odour when crushed. The stem in cultivated varieties is often non- thorny and in weedy varieties with recurved prickles. It is woody, square in cross section, hairy when young, cylindrical and up to 15 cm thick as it grows older. Lantana is able to climb to 15 m with the support of other vegetation. Flower heads contain 20 – 40 flowers, usually 2.5 cm across; the colour varies from white, cream or yellow to orange pink, purple and red (Amaral, C.L.F, 2003). Lantana camara have therapeutic potential due to the presence of natural agents, Majority of their activity is due to bioactive compounds viz. flavones, isoflavones, flavonoids, anthocyanins, coumarins, lignans, catechins, isocatechins, alkaloids, tannin, saponins and triterpenoids. Study of the leaves and flowers extract have given an idea about similar carbohydrates and lipid compositions. The carbohydrate levels were higher in the flowers than the leaves, and the lipids higher in the leaves extract (Deepak Ganjewala, 2009). Bioactive substance from this plant can be employed in the formulation of anti-microbial agents for the treatment of various microbial infection such as respiratory infection, liver infection, antipyretic, anti-insectisidal , anti-bacterial, anti-fungal, analgesic, anti- inflammatory.

MATERIALS AND METHODS:

Collection and Authentication certificate number of Lantana camara:

The plant was collected from Thandalam, Kancheepuram, Tamilnadu, India in the month of December 2014. The collected plant was identified and authenticated through Visual inspection, Experience, Education and training, organoleptic characteristics, Morphology, Taxonomy and Microscopical method as lantana camara linn.(verbenaceae). It has been authenticated Dr.D.Aravind Assistant Professor, Department of Medicinal Botany , National Institute of Siddha, An Autonomous body under the Ministry of AYUSH, Govt. of India, Tambaram, Sanatorium, Chennai-600047, Tamil Nadu, India.

Preparation of Extract:

The leaves were cut into small piece and shade dried at room temperature. The dried leaves were subjected to size reduction to a coarse powder with the help of grinder. The dried powders of wild plant material (10g) of lantana camera was soaked individually at room temperature in hexane, methanol, chloroform, diethyl-ether for 72hr . After 3 days, the suspension was filtered through a fine muslin cloth and was evaporated to dryness at low temperature (< 40 ºC) under reduced pressure in a rotary evaporator. The crude extract thus obtained was stored in an air-tight container and used for further analysis. The extracts obtained from air dried plant material of lantana camera were carried out for further analytical and pharmacological activities(Parekh J. et. al 2007).

Qualitative phytochemical analysis

The preliminary phytochemical screening of methanol, hexane, diethyl-ether and chloroform were carried out for the detection of chemical constituents of Lanatana camara using following standard methods (Mamta Saxena., et al 2012) .

Test for carbohydrates

To 2ml of the extract, added 1ml of α-naphthol solution, and concentrated sulphuric acid though the side of test tube. Purple or reddish violet colour at the junction of two liquids revealed the presence of carbohydrates.

Test for fatty acids

5ml of the extract was mixed 5ml of ether, these extract was allowed to evaporate on a filter paper, after dried the appearance of transparence of filter paper indicates the presence of fatty acid.

Test for proteins

To 1ml of the extract added 1ml 40% sodium hydroxide solution and 2 drops of 1% copper sulphate solution formation of violet colour indicated the presence of proteins.

Test for amino acids

Added two drops of freshly prepared 0.2% ninhydrin reagent to the extract solution and heated development of blue colour revealed the presence the proteins, peptides or amino acids.

Test for saponins

1g of dried extract was allowed to boil with 10ml distilled of water in a water bath for 10mins. The mixture was filtered while hot and allowed to cool.2.5ml of filtrate was diluted to 10ml with distilled water and shaken vigorously for 2 minutes, frothing indicated the presence of saponins in the filtrate.

Test for tannins

1g of each dried extract was boiled with 20ml distilled water for five minutes in water bath and was filtered while hot.1ml of cool filtrate was added to 5ml with distilled water follow few drops (2-3) of 10% ferric chloride and observed for any formation of precipitates and colour change a bluish-black or brownish-green precipitate indicated the presence of tannins.

Test for carotenoids

1g of each dried sample was extracted with 10ml of chloroform in a test tube with vigorous shaking. The resulting mixture was filtered and 85% sulphuric acid was added. A blue colour indication showed the presence of carotenoids.

Test for flavonoids

1g of the dried extract of was boiled with 10ml of distilled water for 5mins and filtered while hot few drops of 20%sodium hydroxide solution were added to 1ml of the cooled filrate. A change to yellow colour which on addition of acid changed to colorless solution depicted the presence of flavonoids.

Test for alkaloids

1g of dried extract was dissolved in 10ml of 1% hydrochloric acid. Then they wereg kept in a water bath for few minutes and filtered. The pH of the filtrate was adjusted to 6-7 with ammonia. To the 0.5ml filtrate six drops of the wagner’sreagent (iodo potassium iodide), was added, reddish brown precipitate indicates the presence of alkaloids.

Test for steroid and terpenoids

1g of dried extract with 1ml of chloroform, 2-3ml of acetic anhydride and 1-2 drop of concentrated sulphuric acid. Dark green colouration of the solution indication the presence of steroids and dark pink or the solution indicates the presence of terpenoids.

Test for Polyphenols

Extracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black colour indicates the presence of polyphenols.

Test for glycosides

Equal volume of organic extract and water were added with 0.5 ml of lead acetate solution shaken and filtered. Filtrate was extract with equal volume of chloroform and the chloroform extract was evaporated for dryness. The residue was dissolved in 2ml of pyridine and 2ml of sodium prusside followed by the addition of sodium hydroxide solution to make alkaline formation of pink colour indicate the presence glycosides.

Antimicrobial activity of lantana camera

Microorganism :

The test organism used in this study were antimicrobial activity against human pathogenic gram positive and gram negative bacteria are Bacillus subtilis (MTCC-441), Staphylococcus aureus (MTCC-98), Escherichia coli (1687), Salmonella typhi (MTCC-733) and Vibrio cholerae (MTCC-3906). The test organism used in this study were one fungal strain namely Candida krusei (ATCC-24408) is budding yeast involved in chocolate production. C. krusei is an emerging fungal nosocomial pathogen primarily found in the immuno compromised and those with hematological malignancies of different solvent (Haxane) extracts of leaf of Lantana camera .

Preparation of Inoculum

Fresh cultures were prepared by inoculating the organisms in nutrient broth (NB) for bacteria and Sabouraud Dextrose Broth (SDB) for fungi in incubating at room temperature for 24 hours. Each organism was suspended in sterile broth and diluted with water. The culture thus obtained was the standardized bacterial and fungal suspension(Navon-Venezia S.et.al 2005).

Evaluation of antimicrobial activity by agar well diffusion method.

The in vitro antimicrobial activities of test compounds were determined by the well-diffusion method described by Arjun et al., 2012. Muller Hinton Agar(MHA) (Beef infusion,300 g/L; casein acid hydrolysate, 17.5 g/l; starch, 1.5 g/l ph 7.3). For bacterial studies and sabouraud Dextrose Agar (SDA) for fungi (meat peptone 5g, casein peptone 5g, dextrose 40g, agar15g and distilled water 1000 ml) medium was preparation of plates. The medium was poured onto sterile petridishes of 90 mm diameter. The agar was allowed to set at ambient temperature. Fresh bacterial cultures of B. subtilis, S. aureus, E.coli, s. typhi, were spread on the surface of MHA; fresh fungal culture was spread on surface of the SDA plate with swabs. After incubation, using a sterile cork borer, well were cut from the agar in plate. The concentrated leaf of different solvent extracts of L. camera was weighed and dissolved in dimethyl sulfoxide (DMSO) to prepare extract solution of 10 mg/ml of DMSO. To each well, concentration ranging from 25,50,75 micro litre of this extract solution was dispensed using a sterile micropipette. The inoculated plates were incubated within 15 min of inoculation at 37degree centigrade for 24 hr. Turbidity was adjusted with sterile broth so as to corresponds to 0.5 McFarland standards. The inoculated plates were examined for every zone of inhibition. Inhibition zones were as the diameter of growth free zones including the diameter of the well in mm at the end of incubation period (Chavan SR, et. al 1982).

I(diameter of the inhibition zones )

% of inhibition =---------------------------------------- x 100

90 (diameter of petri plates in mm)

GC-MS identification of bioactive constituents

GC-MS analysis of the extract was performed using a Perkin Elmer GC Claurus 500 system and Gas Chromatograph interfaced to a Mass Spectrometer equipped with an Elite 5MS fused silica capillary column (30 × 0.25 mm ID. ×1 Mm df, composed of 5% Diphenyl/ 95% Dimethyl poly siloxane). For GC-MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas (99.999%) was used as the carrier gas at a constant flow rate of 1 ml/min and an injection volume of 3 Ml was employed (split ratio of 10:1). Injector temperature 250ºC; Ion-source temperature 280ºC. The oven temperature was programmed from 110°C (isothermal for 2 min) with an increase of 10ºC/min to 200°C, then 5ºC/min to 280 ºC ending with a 9 min isothermal at 280°C. Mass spectra were taken at 70 eV, a scan interval of 0.5seconds and fragments from 45 to 450 Da. 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 Turbo Mass V. 5.2.0. ( P. Maria Jancy Rani 2011).

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 and the molecular weight and structure of the components of the test materials were ascertained.

RESULTS:

Preliminary phytochemical screening:

The prepared Haxane extract was subjected to qualitative chemical test. Various extract of dried leaves of Lantana camera indicated the presence of most of the phytochemical is shown in Table-1. These phytochemicals ultimately contribute to the anticancer property of plant.

Table-1.

Phytochemical constituents	e-Haxane extract	Methanol extract	Diethyl Ether extract	CHCl₃ extract
Carbohydrates	+++	+++	+++	+++
Fatty acid	+++	+++	+++	+++
Protein	+++	+++	+++	+++
Saponins	+++	+++	+++	+++
Tannins	+++	+++	+++	+++
Carotenoids	+++	+++	+++	+++
Polyphenols	+++	+++	+++	+++
Flavonoids	+++	+++	+++	+++
Glycoside	+++	+++	+++	+++
Steroids	+++	+++	+++	+++
Terpenoids	+++	+++	+++	+++
Alkaloids	+++	+++	+++	+++

Gas Chromatography and Mass Spectroscopy identification of bioactive constituents:

The e-Haxane extract of L. camera contains rich phytochemical constituents which in turn resulted in the identification of eighteen different compounds by GC-MS analysis. The prevailing bioactive components with their Retention time (RT), Molecular formula, Molecular weight and nature of the compounds were presented in figure 1 and table 2. These bioactive constituents found to be 18 was listed below.

Fig-1. GC-MS Chromatogram of hexane extracts of Lantana camara L.

Table-2. Listed Below Name of Compounds, Retention Time, Molecular Formulae, Molecular Weight And Nature of The Compounds As Follows.

S.NO.	NAME OF THE COMPOUNDS	RT	Molecular Formulae	Molecular Weight	Nature of the compounds
1.	1-Naphthalenol 1,2,3,4,4a,7,8,8a- octahydro1,6-dimethyl4-(1-methylethyl), (1R-(1a,4a,4aa,8aa)	12.27	C₁₅H₂₄O	246.68	Terpenes
2	2-Methyl -4- (2,6,6- trimethyl cyclohex-1-enyl)but-2-en-1-ol	12.53	C₁₄H₂₄O	208.34	Flavonoids
3.	1-Oxaspiro[2.5]octane, 5,5-dimethyl-4-(3-methyl-1,3-butadienyl)	13.37	C₁₅H₂₄O	206.32	Saponins
4.	Trans-z alpha bisabolene epoxide	14.05	C₁₅H₃₀O₂	220.35	Steroids
5.	Tridecanoic acid-12 methyl, methyl ester	14.93	C₁₆H₃₂O₂	242.39	Steroids
6.	Pentadecanoic acid, methyl ester	16.03	C₁₇H₃₂O₂	256.42	Isoflavones
7.	E-15- Heptadecenoic acid	16.93	C₁₇H₃₄O₂	268.43	Caretonoids
8.	Pentadecanoic acid, 13-methyl-, methyl ester	17.18	C₁₇H₃₂O₃	270.45	Alkaloids
9.	Methyl 10-oxohexadecanoate	17.93	C₁₉H₃₆O₂	284.43	Ketones
10.	2-Hexadecenoic acid 2,3-dimethyl-, methyl ester	18.92	C₁₉H₃₈O₂	296.48	Terpenes
11.	Heptadecanoic acid, 16-methyl-, methyl ester	19.17	C₂₃H₄₆	298.50	Alkaloids
12.	5-Methyl-Z-5-docosene	19.87	C₂₀H₃₆O₃	322.61	Terpenes
13.	8-(2-Octylcylopropyl)-8-oxooctanoicacid, methyl ester	20.73	C₁₇H₂₈O₂	324.49	Flavonoids
14.	4,7,10-Hexadecatrieonic acid, methyl ester	22.17	C₁₆H₃₀O₃	264.40	Flavonoids
15.	15-Hydroxypentadecanoic acid	23.03	C₁₅H₃₀O₃	258.39	Alkaloids
16.	2,2,4a,6a,8a,9,12b,14a-Octamethyl-1,2,3,4,4a,5,6,6a,7,8,8a,9,12,12a,12b,13,14,14a,14b-eicosahydropicene	28.28	C₃₀H₅₀	410.71	Steroids
17.	Cholesta-2,4-diene	29.67	C₂₇H₄₄	368.64	Carotenoids
18.	2,2,4-trimethyl-3(3,8,12,16-tetramethyl-heptadeca-3,7,11,15-tetraenyl)- cyclohexanol	31.08	C30H52	428.733	Saponins

These Phytochemicals Ultimately Contribute to the Antimicrobial Property. Shown in Figure 2.

Fig.-2. Antimicrobial activity of Lantana camera leaf Hexane Extract

A. Bacillus subtilis B. Staphylococcus aureus C. Salmonella typhi D. Escherichia coli E. Vibrio cholera F. Candida krusei

SUMMARY AND DISCUSSION:

The hexane extract of Lantana camara L effectively identifies the presence of various phytochemicals when compared with other solvents used for extract preparation. The factors affecting the choice of solvent are; quantity of phytochemicals to be extracted, rate of extraction, diversity of different compounds extracted, ease of subsequent handling of the extracts, toxicity of the solvent in the bioassay process, potential health hazard of the extractant (Tiwari et al., 2011). The present study results also substantiate Lantana camara L as source of pharmaceutically important phytochemicals via saponins, tannins, carotenoids, polyphenols, flavonoids, glycosides, steroids, terpenoids and alkaloids. Alkaloids play some metabolic role and control development in living system (Lalitha and Jayanthi, 2012). The results indicated that the most sensitive organisms were Staphylococcus aureus, Salmonella typhi, Vibrio cholera, Candida krusei and Escherichia coli. DMSO showed no activity against any of the bacterial and fungal strains, while chloramphenicol showed the activity of all the tested strains.

CONCLUSION :

During my study of the phytochemical study reveals it contains many phytoconstituents like alkaloids, glycosides, flavanoids, tannins etc. The anti-microbial activity of hexane extract of Lantana Camara were investigated. During the study the zone of inhibition of bacteria and fungi were compared with positive standard drug chloramphenical and negative standard DMSO respectively. The antimicrobial activity of crude hexane extracts of Lantana camara, a traditional medicinal plant was determined against five test bacteria and one test fungal strains (Bishnu Joshi.,et.al;2011). The solvent extracted extracts inhibited the growth of Staphylococcus aureus Vibrio cholerae to the maximum. The fungi toxic spectrum of the test plant’s leaf shows maximum percentage growth of inhibition at 75 µL concentration against Candida krusei.

Thus, the broad spectrum anti-microbial activities of the plant extract, possibly due to identified alkaloids, terpenoides, flavonoides, saponins etc., further confirm its use as a health remedy in folklore medicine. Bioactive substance from this plant can therefore be employed in the formulation of anti-microbial agents for the treatment of various microbial infection (Dipak Koche et.al 2000). Isolation, identification and purification of these phyto-constituents and determination of their respective anti-microbial potencies are help to formulating novel chemotherapeutic agents should be the future direction for investigation.

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Received on 22.04.2017 Modified on 05.05.2017

Res. J. Pharmacognosy and Phytochem. 2017; 9(2): 115-120.

DOI: 10.5958/0975-4385.2017.00021.8