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Oriental Pharmacy and Experimental Medicine

, Volume 15, Issue 2, pp 141–146 | Cite as

Chemical composition and antimicrobial activity of Hyptis suaveolens Poit. seed oil from Uttarakhand State, India

  • R. K. BachhetiEmail author
  • Indra Rai
  • Archana Joshi
  • R. S. Satyan
Research Article

Abstract

The present study was carried out to identify the chemical composition and antimicrobial activity of Hyptis suaveolens Poit. seed oil from Kalagarh region (Uttarakhand State, India). Gas Chromatograph Mass Spectrum (GC-MS) analysis of the seed oil suggested high amounts (86.96 %) of unsaturated fatty acids: linoleic acid (76.13 %), oleic acid (10.83 %) compared to saturated fatty acids i.e. palmitic acid (6.55 %), stearic acid (4.56 %) and heptacosanoic acid (1.94 %) as the main constituents. The oil (1 mg/ml to 0.125 mg/ml) was detrimental to the growth of bacterial and fungal strains tested. Among them Salmonella typhi MTCC 531 (9.92 ± 0.08 mm), Pseudomonas aeruginosa MTCC 424 (10.22 ± 0.11 mm), Lactobacillus plantarum MTCC 2621 (10.92 ± 0.09 mm) and Candida tropicalis MTCC 229 (8.11 ± 0.07 mm) were susceptible at lower concentration (0.125 %). Escherichia coli MTCC 443, Shigella flexnerii MTCC 1457 and Vibrio vulnificus MTCC 1145 were sensitive only at higher concentrations (0.5 and 1 %) tested. The bioactivity of 1 mg/ml seed oil against C. tropicalis MTCC 229 was comparable to Fluconazole control (25 μg). On the contrary Enterococcus fecalis MTCC 439 and Streptococcus fecalis MTCC 3378 were resistant to the seed oil treatment. Minimum Inhibition Concentrations (MIC) were found to be 0.125 mg/ml for S. typhi, and P. aeruginosa, L. plantarum and C. tropicalis, while it was 0.2 mg/ml for L. leishmanii, S. aureus and C. albicans. The observations indicate that the H. suaveolens seed oil possess appreciable antimicrobial properties against Gram negative bacteria tested and can be further studied for the mode of action and further lead as a natural antibiotic.

Keywords

Hyptis suavolens Poit. seed oil GC-MS S. typhi P. aeruginosa C. tropicalis MIC 

Introduction

Hyptis is a genus of Lamiaceae with about 400 species; most of the species are native to the tropical and subtropical regions. One of the species named Hyptis suaveolens Poit. is naturalized in India and is capable of heavy infestations displacing native flora (Raizada 2006). It has now established in Deccan Peninsula, North East India, Vindhyan Highland and Andaman and Nicobar Islands (Wealth of India 1959). H. suaveolens is of common occurrence along the rail tracks, roadsides, foothills of open forests, forest clearings and can heavily infest wastelands particularly arid and rocky substrates. The herb, sometimes attaining a height of 7 ft. Leaves are broadly ovate, very variable, 2.5–11 cm. long, sinuate-denticulate, flowers small, blue, in unilateral axillary or terminal clusters often arranging in panicles, H. suaveolens fruits (nutlets) are about 1.2–1.5 mm long and seeds are protected in spined burr which help in its dispersal and are slightly notched at the end. This species has showed a high variation in composition of volatile constituents and composition of essential oil according to the geographic origin and growth phase (Kodakandla et al. 2012; Tonzibo et al. 2009). Essential oils isolated from aerial parts of this plant have showed antifungal, antibacterial and wound healing activities (Morreira et al. 2010; Iwalokun et al. 2012; Chitra et al. 2009; Sathish et al. 2010). H. suaveolens is an ethnomedicinal plant and having high medicinal value (Basha and Sudarashanam 2010). Much work has been done on ethno medicinal plants in India (Dey et al. 2010; Sumathi et al. 2011; Raja et al. 2011).

Reported pharmacological activities of the H. suaveolens plant include antiulcer (Das et al. 2009), antioxidant (Gavani and Paarakh 2008; Ghaffari et al. 2011), insecticidal and herbicidal (Ajibaye et al. 2009; Kapoor 2011; Adda et al. 2011), gastroprotection and antidiarrhoeal (Carlos et al. 2012; Shaikat et al. 2012), anthelmintic, hepatoprotactive and Antilithiatic activities (Nayak et al. 2010; Babalola et al. 2011; Shashi et al. 2010). The leaf extract of H. suaveolens was reported for its anti-hyperglycemic activity (Shanti et al. 2011). Methanoilc extract of leaf used in treatment of renal, urinary calculi and urolithiasis (Kumkum and Ranjana 2012). Phytohormonal potential and effect of leaf extract on seed germination and seedling growth shows importance of H. suaveolens in agricultural aspects (Okukpe et al. 2012; Boonrod et al. 2012). Several studies were performed on the chemical composition and activities of the essential oil. Nevertheless much research work has been done on seed oil of H. suaveolens. Thus main objective of this study is to examine the chemical composition and antimicrobial activities of the seed oil of H. suaveolens from Kalagarh region, Uttarakhand state, India (29.5° North latitude, 78.75° East longitude and 248 meters elevation above the sea level) against selected clinically significant bacteria and fungi.

Materials and methods

Reagents

All reagents and acids used were of analytical grade and procured from Merck® (Darmatadt, Germany) and Sigma Aldrich®. All solutions were stored in well cleaned high density polypropylene bottles. All glassware were cleaned by soaking in nitric acid and then rinsed with distilled water and dried at 105 °C.

Collection and extraction of H. suaveolens seed oil

The fruits of H. suaveolens collected from forest of Kalagarh region, Uttarakhand, India. Seeds were identified and authenticated in National Institute of Science Communication and Information Resources, New Delhi, India. Seeds were dried and separated from fruit by breaking into two parts. The seed samples were stored in airtight amber colored glass bottles and kept in a refrigerator prior to analysis. One hundred grams of the seeds was grounded into powder form and then oil was extracted with Petroleum ether (boiling point 40–60 °C) by Soxhlet method for 6 to 8 h. The extracted solvent was distilled off at 60 °C by rotary evaporator to concentrate the oil. Oil content was calculated from weight of oil and weight of seed taken for extraction. Extracted oil collected and preserved in amber colored glass bottle for further analysis.

Gas Chromatography/mass spectrometry analysis (GC/MS)

GC/MS analysis was performed as per standard procedure described for FAME (Hisil 1988). Derivatized fatty acids methyl esters was analyzed by using a Shimadzu GC-2010® equipped with a Shimadzu GCMS-QP2010 Plus® mass selective detector having Rtx-5 MS capillary column (30 m × 0.25 mm, film thickness 0.25 μm (stationary phase: 5 % diphenyl/ 95 % dimethyl polysiloxane) (AOAC 1990). The initial column oven temperature was 140 °C, 5 min kept then programmed at 4 °C/min to final oven temperature 240 °C and hold for 10 min at this temperature, injector temperature was 270 °C. Helium was used as carrier gas with column flow rate 1.21 ml/min. and the split ratio 1:20. For GC/MS detection, an electron ionization system with ionization energy of 70 eV was used, Ion source temperature was 230 °C and Interface temperature was 280 °C. The components were identified by comparing their relative retention times and mass spectra with those of standards (main components), Wiley 8® library data of the main system.

Microbial strains

Gram positive bacterial species Staphylococcus aureus (MTCC 737), Enterococcus fecalis (MTCC 439), Staphylococcus fecalis (MTCC 3378), Lactobacillus plantarum (MTCC 2621), Lactobacillus leishmanii (MTCC 911), Gram negative bacterial species Escherichia coli (MTCC 443), Salmonella typhi (MTCC 531), Shigella flexneri (MTCC 1457), Vibrio vulnificus (MTCC 1145), Pseudomonas aeruginosa (MTCC 424) and two fungal species Candida albicans (MTCC 227), and Candida tropicalis (MTCC 227). Microorganisms were provided by the Institute of Microbial Technology (IMTECH), Chandigarh, India. The Candida isolates were identified using standard taxonomic procedures, including microscope appearance on Corn meal agar with Tween-80.

Antimicrobial screening

Disc diffusion method (Bauer et al. 1959) and dilution broth method were used to determine the antibacterial activity. Twenty hours bacterial culture incubated at 37 °C was used for the assay. The fungi were reseeded on potato-dextrose agar and cultured for 72 h at 25 °C. The oil was diluted in 10 % dimethylsulfoxide (DMSO) to different concentrations (mg/ml). Antimicrobial activities was carried out with a suspension containing 108 colony forming units/ml of bacteria spread on nutrient agar and 106 colony forming units /ml of fungi spread on potato dextrose agar. Different concentrations (0.05–1 mg/ml) of diluted extract were soaked in filter paper discs the well (6 mm in diameter) and allowed to soak in, specific inoculated medium plates. Amikacin (0.125 mg/ml) and Fluconazole (25 μg/ml) were used as ‘positive controls’ against the tested bacteria and fungi. The inoculated plates were incubated at 37 °C for 24 h for bacterial strains and at 25 °C for 72 h for fungus. Antimicrobial activity was evaluated by measuring the inhibition zone (in millimeters) against test microorganisms.

Dilution broth assay was used for calculating the MIC of the essential oil sample. The oil sample was diluted with 5 % DMSO at 10−2 to 10−5 successive levels, negative and positive controls were prepared in same medium. One milliliter of each dilution and 0.5 ml of tested culture strains are added to 8 ml of sterile nutrient broth and incubated for 24 h at 37 °C for bacteria and in Sabouraud Dextrose (SD) broth for 5 days at 25 °C for fungal cultures. Results calculated on the basis of intensity of turbidity due to microbial growth and MIC was calculated.

Results

Among the components detected, H. suaveolens seed oil (Yield: 17.44 %) contained linoleic acid (76.13 %), oleic acid (10.83 %), palmitic acid (6.55 %), stearic acid (4.56 %) and heptacosanoic acid (1.94 %) as the major constituents (Table 1, Figs. 1 and 2). The seed oil exhibited moderate to appreciable antimicrobial activities against all the bacteria tested, except E. fecalis and S. fecalis. Among the susceptible species S. typhi MTCC 531 (9.92 ± 0.08 mm), P. aeruginosa MTCC 424 (10.22 ± 0.11 mm), L. plantarum MTCC 2621 (10.92 ± 0.09 mm) and C. tropicalis MTCC 229 (8.11 ± 0.07 mm) were susceptible at lower concentration (0.125 %). E. coli MTCC 443, S. flexnerii MTCC 1457 and V. vulnificus MTCC 1145 were sensitive only at higher concentrations (0.5 and 1 %). The bioactivity of 1 % seed oil against C. tropicalis MTCC 229 was comparable to Fluconazole control (25 μg/ml). (Table 2). The MIC value for S. typhi, P. aeruginosa and C. tropicalis was 0.125 mg/ml. whereas it was 0.25 mg/ml for C. albicans MTCC 227, S. aureus MTCC 737 and L. leishmania (MTCC 911) (Table 3).
Table 1

Fatty acid profile of H. suaveolens seed oil

Fatty acids

Area %

Palmitic acid

6.55

Stearic acid

4.56

Oleic acid

10.83

Linoleic acid

76.13

Heptacosanoic acid

1.94

Fig. 1

GCMS chromatogram of H. suaveolens seed oil

Fig. 2

Mass spectrum (m/z) of H. suaveolens seed oil with peak report

Table 2

Antimicrobial activity of H. suveolens seed oil against tested organisms

Organism

Inhibition zone (mm) Concentration of sample (mg/ml)

Amikacin (mg/ml)

Negative control

1.0

0.5

0.25

0.125

0.1

0.05

0.125

E. coli (MTCC 443)

11.48 ± 0.07

10.78 ± 0.11

*

*

*

*

17.5 ± 0.08

*

S. typhi (MTCC 531)

14.81 ± 0.13

13.47 ± 0.09

12.31 ± 0.11

9.92 ± 0.08

*

*

16.81 ± 0.11

*

S. flexneri (MTCC 1457)

12.01 ± 0.11

9.22 ± 0.12

*

*

*

*

17.81 ± 0.12

*

V. vulnificus (MTCC 1145)

9.22 ± 0.12

8.91 ± 0.09

*

*

*

*

23.72 ± 0.11

*

P. aeruginosa (MTCC 424)

14.22 ± 0.11

13.56 ± 0.14

12.21 ± 0.08

10.22 ± 0.11

*

*

16.58 ± 0.09

*

L. plantarum (MTCC 2621)

15.89 ± 0.08

14.32 ± 0.11

12.23 ± 0.11

10.92 ± 0.09

*

*

17.23 ± 0.11

*

L. leishmania (MTCC-911)

13.04 ± 0.12

12.0 ± 0.08

10.02 ± 0.08

*

*

*

16.55 ± 0.09

*

S. aureus (MTCC 737)

11.12 ± 0.09

9.81 ± 0.08

8.03 ± 0.11

*

*

*

14.98 ± 0.08

*

E. fecalis (MTCC 439)

*

*

*

*

*

*

15.88 ± 0.09

*

S. fecalis (MTCC 3378)

*

*

*

*

*

*

15.32 ± 0.11

*

C. tropicalis (MTCC 229)

13.1 ± 0.11

12.09 ± 0.08

11.02 ± 0.12

8.11 ± 0.07

*

*

13.65 ± 0.12

*

C. albicans (MTCC 227)

16.81 ± 0.09

14.04 ± 0.11

13.52 ± 0.11

*

*

*

14.23 ± 0.12

*

* No activity detected. MTCC Microbial Type Culture Collection. Fluconazole (25 μg/ml) was used as antifungal antibiotic control

Table 3

Minimal Inhibitory Concentration (MIC) of H. suveolens seed oil against tested organisms

Organism

MIC (mg/ml)

Bacterial strains

E. coli (MTCC 443)

0.5

S. typhi (MTCC 531)

0.125

S. flexneri (MTCC 1457)

0.5

V. vulnificus (MTCC 1145)

0.5

P. aeruginosa (MTCC 424)

0.125

L. plantarum (MTCC 2621)

0.125

L. leishmania (MTCC 911)

0.25

S. aureus (MTCC 737)

0.25

E. fecalis (MTCC 439)

S. fecalis (MTCC 3378)

Fungal strains

C. tropicalis (MTCC 229)

0.125

C. albicans (MTCC 227)

0.25

(−) No activity

Discussion

Components of H. suaveolens seed oil were comparable to other commercially used oils (Maidment et al. 2009). Unsaturated fatty acids like linoleic acid and oleic acid were high in percent abundance compared to saturated fatty acid like palmitic acid and stearic acid. The high % of linoleic acid (76.13 %) content is comparable with sunflower & soya bean seed oils which are already in use for edible purposes (Bachheti et al. 2012). Long-chain unsaturated fatty acids, such as linoleic acid, show antibacterial activity and are the key ingredients of antimicrobial food additives and some antibacterial herbs. Zheng et al. (2005) reported that linoleic acid inhibited bacterial enoyl-acyl carrier protein reductase (Fabl), an essential component of bacterial fatty acid synthesis, which has served as a promising target for antibacterial drugs. Additional unsaturated fatty acids including palmitoleic acid, oleic acid, linolenic acid, and arachidonic acid also exhibited the inhibition of Fabl. In another study, Agoramoorthy et al. (2007) proved that Fatty acid Methyl Esters (FAME) from Excoecaria agallocha L. that was detrimental to Bacillus subtilis and S. aureus (MIC: 0.125 mg/ml) contained less of linoleic and oleic acids (3.13 and 1.71 %) ) and more of palmitic acid (56.02 %) when compared to our study. It has been proved that unsaturated fatty acids (especially PUFAs) help to reduce cholesterol formation or deposition and hence to decrease the risks of atherosclerosis and other heart disease (Penumetcha et al. 2012). The susceptibility of Gram positive bacteria to H. suaveolans oil compared to Gram negative species might be due to the presence of hydrophilic outer membrane in the latter, mainly with regard to the presence of lipoproteins and lipopolysaccharides in the cell wall which form a barrier to hydrophobic compounds. When compared to other agents the oil was found to be more detrimental to C. tropicalis and C. albicans (8.11 ± 0.07 to 16.8 ± 0.09 mm) at different concentration levels. Apart from plant-derived fatty acids rich in linoleic acid, novel synthetic linoleic acid-dipeptide-spermidine conjugates exhibited potent antibacterial activity against drug resistant Gram positive and Gram negative bacteria (Joshi et al. 2012). From the results of the present study, it was clearly observed the efficacy of oil which could support and provide scope for the application of H. suaveolens seed oil as a source of antimicrobial ingredients and natural preservatives for the food industries, cosmetics industries and pharmaceutical industries in the prevention and treatment of bacterial infections caused by pathogenic microorganisms, moulds and fungus. Further assays to identify the mode of action and toxicity would pave way for the feasibility of the oil as a sustainable product.

Notes

Acknowledgments

The authors are thankful to Graphic Era University, Dehradun, Uttarakhand, India for providing the necessary facilities for the research work.

Conflict of Interest

We declare that we have no conflict of interest.

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Copyright information

© Institute of Korean Medicine, Kyung Hee University and Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • R. K. Bachheti
    • 1
    • 2
    Email author
  • Indra Rai
    • 1
  • Archana Joshi
    • 1
    • 3
  • R. S. Satyan
    • 3
  1. 1.Department of ChemistryGraphic Era UniversityDehradunIndia
  2. 2.Department of Chemistry, College of Natural and Computational Sciences (CNCS)Haramaya UniversityEthiopiaNorth East Africa
  3. 3.Department of Biology, College of Natural and Computational Sciences (CNCS)Haramaya UniversityEthiopiaNorth East Africa

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