Essential oil constituents of Illicium griffithii and its antimicrobial activity

)-1,3-benzodioxole was characterized as the major constituent, followed by linalool amongst the volatile constituents. The essential oil was found to be effective against Aspergillus niger , Penicillium spp. and Saccharomyces cerevisiae and possessed considerable activity against Staphylococcus aureus and was inactive against Klebsiella pnemoniae , Pseudomonas aureginosae , Proteus vulgari s and Escherichia coli .


Plant material
The dried fruit of I. griffithii was procured from Arunachal Pradesh, India, and was identified by National Botanical Research Institute, Lucknow. A voucher specimen (no. I/145ICMR88) was deposited in the museum of this institute.

Instrument
Shimadzu GC 2010 was the instrument used for gas chromatography-mass spectroscopy (GC-MS) analysis. The constituents were identified by comparison of the mass fragments with the spectrum library NIST/EPA/NIH.

Test organisms
Organisms such as Klebsiella pnemoniae (ATCC 700603), Pseudomonas aureginosae (ATCC 27853), Escherichia coli (ATCC 25922), Proteus vulgaris (ATCC 9484) and Staphylococcus aureus (ATCC 25923) were used for the study. The organisms were procured from Christian Medical College, Vellore, and were maintained by serial sub-culturing every month on nutrient agar slants and incubating at 37ºC for 18-24 hours. The cultures were stored under refrigerated condition. The antifungal activity of the oil was tested against Aspergillus niger, Penicillium spp. (both were isolated from soil) and Saccharomyces cerevisiae (isolated from dough). [7][8][9] Isolation and identification of the fungus was done by the microbiologist of this institute.

Extraction of essential oil
Exactly 200 g of the dried fruit of I. griffithii was coarsely powdered and transferred in to a 2-l round bottom flask. Sufficient amount of water was added and fixed with Clevenger's apparatus. This was boiled for 4 h and the steam-distilled essential oil (yield 0.85%) was collected, then dried over anhydrous sodium sulfate, transferred into an airtight sample tube and stored at 8ºC.

Gas chromatography-mass spectroscopy analysis of the essential oil
One microliter of the essential oil of the fruit was injected into GC. The injector temperature was maintained at 250ºC. The detector used was flame ionization detector which was maintained at 280ºC. The pressure of the carrier gas, nitrogen, was kept at 10 psi. The oven temperature was set at 60-280ºC with a gradual increment of 10ºC/min. The injected oil was eluted in the DB-5 MS column of 30 m length and 0.25 mm inner diameter and the eluted constituents were detected by flame ionization detector and the GC chromatogram was recorded [ Figure 1].

Antibacterial activity
Antibacterial activity was determined by the well diffusion method. [10] Petri plates containing 25 ml of nutrient agar medium were seeded with a 24-h culture of the bacterial strains. The inoculum's size was adjusted so as to deliver a final inoculum of approximately 108 colony-forming units (CFU/ml). Wells (6 mm diameter) were made on solidified inoculated nutrient agar plates by using sterile plunger. Ten and 20 μl of the oil were transferred aseptically to the subsequent wells and labeled. Standard disc of ampicillin 30 μg (positive control) was placed on the inoculated plate to evaluate the potency of the oil. The plates were left undisturbed for 15 min at room temperature to aid in seeping of the oil and then the plates were incubated at 37ºC for 24 h. The zone of inhibition was measured in millimeters.

Antifungal activity
The antifungal activity was tested by agar diffusion method. [11][12][13] Petri plates containing 25 ml of Sabouraud dextrose agar (SDA) medium were seeded with a 7-day old culture of fungus organisms. Wells (6 mm diameter) were made on solidified inoculated SDA plates by using sterile plunger. Ten and 20 μl of the oil were transferred aseptically to the subsequent wells and labeled. Standard disc of 30 µg of amphotericin B was loaded as a reference antifungal drug. The plates were incubated at 25 ± 2ºC for 5 days. The zone of inhibition was measured in millimeters.

RESULTS
The volatile oil constituents along with their retention time and percentage obtained from the GC-MS analyzer are given in Table 1. The spectrum obtained is shown in
Ten and 20 μl of the oil showed antibacterial activity only against St. aureus and the inhibition zone was 14 mm and the positive control showed the inhibition zone ranging from 13 to 14 mm against K. pneumoniae, Ps. aureginosae, E. coli, Pr. vulgaris and St. aureus. There was no considerable change in inhibition found by increasing the concentration of the oil and thus the bacterial strains like K. pneumoniae, Ps. aureginosae, E. coli, Pr. vulgaris were susceptible to the oil.
Ten and 20 μl oil showed appreciable antifungal activity against A. niger, Penicillium spp. and Sa. cerevisiae. The inhibition zone of the oil for both the concentrations varied from 9 to 13 mm while for the positive control it was in the range of 10-12 mm. The results of the antifungal activity are shown in Table 3.

CONCLUSION
The essential oil isolated from I. griffithii was found to contain 41 volatile constituents, of which 4-methyl-6-(2propenyl)-1,3-benzodioxole and linalool were the major compounds. The oil has significant activity against spoilage   fungus A. niger, Penicillium spp. and Sa. cerevisiae and was active against the bacterial strain St. aureus. Therefore, it can be concluded that the oil can effectively be used as an antifungal agent and a food preservative, after detailed research.