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International Journal
of Pharmaceutical Sciences and Drug Research ISSN: 0975-248X | DOI Prefix: 10.25004 | CODEN (USA): IJPSPP

Volume No.: 9 (2017) Issue No: 3

International Journal of Pharmaceutical Sciences and Drug Research 2017; 9(3): 100-104 http://dx.doi.org/10.25004/IJPSDR.2017.090301
Research Article

In vitro Anti-Helicobacter pylori Activity and GC-MS Analysis of Enicostemma axillare (Lam). Raynal

Babu A, Sheik Noor Mohamed M, Jaikumar K, Anand D, Saravanan P*

Department of Botany, Ramakrishna Mission Vivekananda College (Autonomous), Mylapore, Chennai - 600 004,

Tamil Nadu, India

*Corresponding author: Dr. P. Saravanan

  Address: P.G. & Research Department of Botany, Ramakrishna Mission Vivekananda College (Autonomous), Mylapore, Chennai - 600 004, Tamil Nadu, India

  Tel.: +91-9444316365

E-mail *: sarviveka@gmail.com

Relevant conflicts of interest/financial disclosures: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Received: 20 March, 2017; Revised: 03 April, 2017; Accepted: 09 April 2017; Published: 29 May 2017 

Copyright © 2017 Babu A et al. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

ABSTRACT

Traditional healers possess a rich knowledge on the use of medicinal plants for the treatment of various diseases. Enicostemma axillare (Lam). Raynal is one plant, used by healer to treat gastric cancer or ulceration. We assessed the phytochemical components of leaf extract for its antibacterial activity, to add value and provide an evidence-base for their traditional use. The antibacterial potential of the leaf extract was tested against Helicobacter pylori using agar well diffusion method. The minimum inhibitory concentration (MIC) of ethanol extract exhibited the (MIC) against H. pylori ranged from 200µg/ml - 250µg/ml followed by chloroform extract. Gas chromatography and mass spectrum (GC-MS) analysis confirms the occurrence of different components in the ethanol and chloroform leaf extract of the studied species.

Keywords: Helicobacter pylori, Enicostemma axillare (Lam). Raynal, GC-MS, Agar well Diffusion.

 

INTRODUCTION

Helicobacter pylori, a Gram-negative microaerophilic and spiral shaped bacterium that has been implicated in gastric, peptic ulcer disease and over long exposure potentially to gastric carcinoma. [1-2] The eradication of H. pylori has thus been utilized as the primary treatment strategy of these diseases for 3 decades. In the past, standard triple therapy consist of a proton pump inhibitor and two broad spectrum antibiotics (usually Amoxicillin, Clarithomycin, Metronidazole) was able to achieve eradication rates > 90%. [3] In view of the incomplete cure achieved with antibiotics-based therapy due to resistant strains. Investigations on naturally occurring antimicrobial compounds found in dietary, medicinal plants and herb extract as alternative source of antibiotics for treating H. pylori infection have excellent potential.

Enicostemma axillare (Lam). Raynal (Gentianaceae), vernacular name (Vellarugu) Indian white head is a perennial herb growing up to 40 cm tall, with 4-angled stems. This species is globally distributed in West Indies, tropical Africa, India and Sri Lanka. Leaves are narrow-oblong, lance shaped. Stalkless white flowers are borne in dense clusters in leaf axils. Plant extracts were reported for the biological activities such as antidiabetic, anti-inflammatory, stimulant, astringent, diuretic and useful in skin disease. [4-5] A number of medicinal preparations in the ayurvedic system of Indian Medicine were recommended for the treatment of gastric ulcers. Though they are claimed to offer significant relief, their usage is in vogue since centuries. However, no effort has been made to identify the phytochemical constituents responsible for the anti helicobacter activity of the plant. A variety of botanical products have been reported to possess antiulcer activity but the documented literature has centered primarily on pharmacological action in experimental animals. Hence an attempt has been made to prove the efficacy of E. axillare in the treatment of gastric ulcer and to assess the phytochemical constituents of the plant.

 

MATERIALS & METHODS

Collection of plant material

The healthy mature plants of Enicostemma axillare were collected during the month of April-May in 2016, from Thuthipattu and Karuvatchi village of Villupuram District, Tamil Nadu. Collected plant was authenticated as E. axillare (Lam). Raynal at the Botanical Survey of India, Southern Regional Centre, Tamil Nadu. Herbarium of the plant specimen was deposited in the Department of Botany, Ramakrishna Mission Vivekananda College (Autonomous), Chennai, Tamil Nadu, India.

Extraction of Bioactive compound from plant material

Fresh leaves from healthy plants were shade dried, and pulverized to fine powder using mechanical grinder. A portion of the dried powdered leaves (50 g) was placed in a soxhlet apparatus and the extraction was performed with 750 ml of different solvents like ethanol and chloroform for 8 h separately at a temperature not exceeding the boiling point of the respective solvent. Extract obtained was filtered through a 45µm filter. The resulting solution was concentrated in a vacuum to dryness to give ethanol and chloroform extract. The extract was stored in a refrigerator at 4°C until further use.

GC-MS analysis

The chemical composition of the ethanol and chloroform extract was established by GC-MS analysis. The analysis was performed on a Clarus 680 GC-MS system in Mass Spectrometer clarus 600 (EI), Software Turbo Mass ver 5.4.2. The injector temperature was set at 260°C during the chromatographic run.  The 1μl of extract sample injected into a split ratio of 1/10, the instrument the oven temperature was as follows: 60°C (2 min); followed by 300°C at the rate of 10°C min−1; and 300°C, where it was held for 6 min.  The mass detector conditions were: transfer line temperature 240°C; ion source temperature 240°C; and ionization mode electron impact at 70 eV, a scan time 0.2 sec and scan interval of 0.1 sec. The fragments from 40 to 600 Da. The spectrums of the components were compared with the database of spectrum of known components stored in the GC-MS NIST (2008) library. The name, molecular weight and structure of the components of the test materials were ascertained.

Bacteria and Cultural Conditions

Clinically isolated strains of Helicobacter pylori culture were identified and used for this experiment. H. pylori were cultivated on Columbia blood agar (Oxoid, UK) at 37°C for 72 h. Plates were incubated in anaerobic conditions.

Agar Well Diffusion Method

The agar diffusion assay was performed on Columbia blood agar as described earlier. [6] The test organism (H. pylori) was swabbed over the medium using sterile cotton buds. Aliquots of 85μl of each test-sample solution of different concentrations (100µg, 150µg, 200µg and 250µg) were applied into 10 mm diameter agar wells. After incubation in anaerobic conditions at 37°C for 72 h, the diameter of the clear zone (no growth) around the well in the bacterial lawn was measured and used to express the antimicrobial activity. The inhibition zone diameter was measured in millimeters (mm). The tests were performed in triplicate and the final results were presented as the arithmetic average.

Statistical analysis

The results were expressed as mean ± SD of the triplicates. One way ANOVA was applicable and used to analyze level of statistical significance. P<0.05 were considered statistically significant. [7]

 

Fig. 1: GC-MS profile of chloroform leaf extract of Enicostemma axillare (Lam). Raynal

Fig. 2: GC-MS profile of ethanol leaf extract of Enicostemma axillare (Lam). Raynal

RESULT & DISCUSSION

GC-MS analysis

The components present in the chloroform and ethanol leaf extracts of E. axillare was identified by GC-MS analysis. The mass spectra of all the phytochemicals identified in both chloroform and ethanol leaf extract of E. axillare were presented in Figure (1 and 2). The GC-MS analysis revealed the presence of 12 and 10 compounds in ethanol and chloroform leaf extract respectively (Table 1 and 2). Premiere phytochemical analyses of the methanolic extract of E. axillare leaves revealed the presence of alkaloid, flavonoids, tannins, glycosides. [8] The various phytochemicals which contribute to the medicinal activities of the plant were shown in Table 2 are based on Dr. Duke’s phytochemical and ethnobotanical Databases. [9] Out of the bioactive components identified, the ethanol leaf extract showed the presence of Erythrocentaurin (RT 15.839) has antimicrobial properties as reported earlier. It was found that Erythrocentaurin was identified in GC-MS analysis of methanolic extract of E. axillare, which supported the work. [10] Erythrocentaurin is a metabolite of swertiamarin which is a secoiridoid glycoside.

A. Chloroform leaf extract of Enicostemma axillare (Lam). Raynal

B. Ethanol leaf extract of Enicostemma axillare (Lam). Raynal

Fig. 3: In vitro Antiulcer activity of ethanol and chloroform leaf extract of Enicostemma axillare (Lam). Raynal

Table 1: GC-MS profile of chloroform leaf extract of Enicostemma axillare (Lam). Raynal

S. No

Name of the compound

Retention Time

Peak %

Molecular Weight

Molecular formula

1

TRICHLOROMETHANE

2.823

32.389

118

CHCl3

2

3,7,11,15-TETRAMETHYL-2-HEXADECEN-1-OL

16.544

9.829

296

C20H40O

3

HEXADECANOIC ACID, ETHYL ESTER,

18.095

8.507

284

C18H36O2

4

1-HEXYL-2-NITROCYCLOHEXANE

19.600

2.492

213

C12H23O2N

5

9,12,15-OCTADECATRIENOIC ACID, METHYL ESTER, (Z,Z,Z)-,

19.700

5.094

292

C19H32O2

6

9-TRICOSENE, (Z)-

19.760

3.744

322

C23H46

7

1-HEXYL-2-NITROCYCLOHEXANE

19.825

8.598

213

C12H23O2N

8

2,6,10,14,18,22-TETRACOSAHEXAENE, 2,6,10,15,19,23-HEXAMETHYL-,(ALL-E)-,

24.637

6.615

410

C30H50

9

TRITETRACONTANE

25.207

2.921

604

C43H88

10

2H-CYCLOPROPA[A]NAPHTHALEN-2-ONE, 1,1A,4,5,6,7,7A,7B-OCTAHYDRO-1,1,7,7A-TETRAMETHYL-, (1A.ALPHA.,7.

26.368

3.845

218

C15H22O

11

HEPTACOSANE, 1-CHLORO

26.603

4.530

414

C27H55Cl

12

2,4,4-TRIMETHYL-3-HYDROXYMETHYL-5A-(3-METHYL-BUT-2-ENYL)-CYCLOHEXENE

26.723

11.436

222

C15H26O

Table 2: GC-MS profile of ethanol leaf extract of Enicostemma axillare (Lam). Raynal

S. No

Name of the compound

Retention Time

Peak%

Molecular Weight

Molecular formula

1

1,3,6-HEPTATRIENE, 2,5,5-TRIMETHYL

15.613

3.198

136

C10H16

2

ERYTHROCENTAURIN

15.839

2.988

176

C10H8O3

3

BICYCLO[4.2.0]OCTA-1,3,5-TRIENE-7-CARBOXYLIC ACID

15.909

2.992

148

C9H8O2

4

BENZAMIDE, N-(4-CYANOMETHYLPHENYL)-2-METHYL

15.974

7.030

250

C16H14ON2

5

3,7,11,15-TETRAMETHYL-2-HEXADECEN-1-OL

16.384

12.732

296

C20H40O

6

EICOSANOIC ACID

17.979

29.297

312

C20H40O2

7

9,12-OCTADECADIENOYL CHLORIDE, (Z,Z)-

19.560

17.619

298 l

C18H31OC

8

1-HEXYL-2-NITROCYCLOHEXANE

19.720

6.743

213

C12H23O2N

9

2,6,10,14,18,22-TETRACOSAHEXAENE, 2,6,10,15,19,23-HEXAMETHYL-, (ALL-E)-

24.607

6.657

410

C30H50

10

2R-ACETOXYMETHYL-1,3,3-TRIMETHYL-4T-(3-METHYL-2-BUTEN-1-YL)-1T-CYCLOHEXANOL

26.698

5.050

282

C17H30O3

Table 3: Biological activity of compounds in leaf extract of Enicostemma axillare (Lam). Raynal

S. No

Name of the Compound

Compound Nature

Activity *

1

TRICHLOROMETHANE

Chloroform

anti-virus, anti-cancer, anti-mutagenic, anti-allergic and anti-ulcer [11]

2

3,7,11,15-TETRAMETHYL-2-HEXADECEN-1-OL

Terpene alcohol

Anti-inflammatory Fragrance compound, Antimicrobial, Anti-inflammatory [12]

3

HEXADECANOIC ACID, ETHYL ESTER,

Ester Compound

Antioxidant, Flavor, Hypocholesterolemic, Nematicide, Pesticide, Lubricant, Antiandrogenic, Hemolytic, 5-Alpha reductase inhibitor [13]

4

1-HEXYL-2-NITROCYCLOHEXANE

Ketone

Antioxidant, antimicrobial, anti-inflammatory [14]

5

9,12,15-OCTADECATRIENOIC ACID, METHYL ESTER, (Z,Z,Z)-,

Fatty acid ester compound

Anti-inflammatory, Hypocholesterolemic, Cancer preventive, Hepatoprotective, Nematicide, Insectifuge, Antihistaminic, Antiarthritic, Anticoronary, Antieczemic, Antiacne, 5-Alpha reductase inhibitor, Antiandrogenic [12]

6

9-TRICOSENE, (Z)-

Aliphatic hydrocarbon

Antibacterial, antioxidant [15]

8

2,6,10,14,18,22-TETRACOSAHEXAENE, 2,6,10,15,19,23-HEXAMETHYL-,(ALL-E)-,

Triterpene compound

Antibacterial, Antioxidant, Antitumor, Cancer preventive, Immunostimulant, Chemopreventive, Lipoxygenase-inhibitor, Pesticide [14]

9

TRITETRACONTANE

Alkane hydrocarbon

No activity reported [16]

10

2H-CYCLOPROPA[A]NAPHTHALEN-2-ONE, 1,1A,4,5,6,7,7A,7B-OCTAHYDRO-1,1,7,7A-TETRAMETHYL-, (1A.ALPHA.,7.

-

-

11

HEPTACOSANE, 1-CHLORO

Alkenes

Antioxidant [17]

12

2,4,4-TRIMETHYL-3-HYDROXYMETHYL-5A-(3-METHYL-BUT-2-ENYL)-CYCLOHEXENE

Essential oil component

Antibacterial, Antioxidant [18]

13

1,3,6-HEPTATRIENE, 2,5,5-TRIMETHYL

Hydrocarbons

Antioxidant [18]

14

ERYTHROCENTAURIN

-

Cytotoxicity against human [19]

15

BICYCLO[4.2.0]OCTA-1,3,5-TRIENE-7-CARBOXYLIC ACID

-

-

16

BENZAMIDE, N-(4-CYANOMETHYLPHENYL)-2-METHYL

-

-

17

EICOSANOIC ACID

-

Anticancer activity (Pubcem)

18

9,12-OCTADECADIENOYL CHLORIDE, (Z,Z)-

Linolenic acid

Anti-inflammatory, Insectifuge, Hypocholesterolemic, Cancer preventive, Nematicide, Hepatoprotective, Antihistaminic, Antieczemic, Antiacne, 5-Alpha reductase inhibitor [13]

19

2R-ACETOXYMETHYL-1,3,3-TRIMETHYL-4T-(3-METHYL-2-BUTEN-1-YL)-1T-CYCLOHEXANOL

Fragrance industry

antibacterial, anti inflammatory activities [20]

* Source: Dr. Duke’s phytochemical and Ethanobotanical Database.

Table 4: Antibacterial activity of Enicostemma axillare (Lam). Raynal against Helicobacter pylori using agar well diffusion method.

S. No

Concentration µg/ml

Ethanol extract (Zone of inhibition in mm)

Chloroform extract (Zone of inhibition in mm)

1.

250

21.76 ± 0.68

20.33 ± 0.57

2.

200

18.00 ± 1.00

17.33 ± 0.57

3.

150

15.66 ± 1.15

-

4.

100

13.00 ± 1.00

-

5.

Clarithromycin (Control)

30.66 ± 0.57

28.33 ± 1.15

The data plotted represent Mean ± SD of triplicate experiments (n-3)

Anti-ulcer activity of Enicostemma axillare (Lam).Raynal against Helicobacter pylori

The ethanol and chloroform extract of E. axillare inhibited the growth of clinical isolates of H. pylori in agar well diffusion method. [21] The results of the anti pylori activity of the investigated extracts are shown in Table 4. Different solvents are used for extraction of antimicrobial compounds form plants, and the success is largely dependent on the type of solvent used. [22] In E. axillare, the effect of ethanol extract reported in this study is in line with many others that have documented the good extracting ability of methanol from plants.

It was observed in E. axillare that chloroform extract, ethyl acetate and hydro alcoholic extracts showing prominent antimicrobial activity against Bacillus subilis, Staphylococcus aureus, E. coli, Shigella sonni, Pseudomonas aeruginosa, Proteus vulgaris, Aspergillus niger, Candida albicans as compared to aqueous and methanol extracts. However our results also indicate that the chloroform extract showed prominent activity in line with ethanol extract at 250µg/ml concentration. Our result, in comparison of chloroform and ethanol observed ethanol to be the best in terms of the diversity of compounds extracted. [23] This antimicrobial activity may be due to the presence of compound ‘Erythrocentavrin’ in ethanol extract, which is already reported to possess antimicrobial properties. According to duke data base many compounds isolated also possess antimicrobial properties.

Earlier it was reported that anti H. pylori activity in vitro has been estimated with fruit juices using agar well diffusion method seemed to possess marked antibacterial activity, with a maximum growth inhibition diameter of 19 mm whereas E. axillare exhibited considerable growth inhibition with diameter ranging above 20 mm. [6] In General, among the investigated extracts, the ethanol fraction exhibited a higher antiulcer activity effect followed by the chloroform extract. The maximum inhibition zone of 21.76 ± 0.68 mm was shown by ethanol extract at a concentration of 250µg/ml. When the concentrations of the extracts were decreased from 250 - 100µg/ml, a slight decrease in inhibition zone was observed in both the solvent case. Despite all the maximum zone of inhibition zone attained at a concentration of 250 mg/ml by plant extract was in near similar to the standard antibiotic Clarithromycin.

Thus the present investigation reveals that the non-polar and polar extracts of E. axillare possess significant bactericidal activity which was analyzed by GC-MS showed the presence of phytoconstituents belonging to the Triterpene, Ketone, Aliphatic hydrocarbon, esters, alcohols, etc. The presence of such a variety of phytochemicals may be attributed to the medicinal characteristics of the plant. Further Isolation and characterization of the bioactive compounds may provide novel or lead compounds, which could become a template for the synthesis of alternate anti H. pylori drugs.

ACKNOWLEDGEMENT

The authors are thankful to the Secretary and the Principal, Ramakrishna Mission Vivekananda College (Autonomous), Mylapore, Chennai, India for providing all facilities. Authors also thank Botanical survey of India (BSI), Coimbatore, Tamil Nadu for identification and authentication of Plants. We thank SAIF VIT, Vellore for contribution of GC-MS analysis and Armats Biotek Training and Research Institute, Gundy, Chennai – 600 032 for anti H. pylori studies.

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