H. spicatum (Ham-ex-Smith) is a perennial rhizomatous herb belonging to the family Zingiberaceae. It grows throughout the subtropical Himalaya in the Indian state of Assam, Arunachal Pradesh and Uttarakhand within an altitudinal range of 1000–3000 m.1,2 H. spicatum rhizome is mentioned as Shati in Ayurvedic classics and has been used in various dosage forms to treat cough, wound ulcer, fever, respiratory problems and hiccough. The rhizomes have a strong aromatic odor and bitter taste. In local language, the rhizomes are commonly known as kapur kachari or ban Haldi.3 Rhizomes are also used as perfume in tobacco and insect repellent. The rhizome extract has been reported to contain essential oil, starch, resins, organic acids, glycosides, albumen and saccharides, which has been advocated for blood purification and treatments of bronchitis, indigestion, eye disease and inflammations.4,5 Rhizome is reported to contain sitosterol and its glucosides, furanoid diterpene-hedychenone and 7-hydroxyhedychenone and essential oil contains cineole, terpinene, limonene, phellandrene, p-cymene, linalool and terpeneol. The plant rhizomes possess hypoglycemic, vasodialator, spasmolytic, hypotensive, antioxidant properties.6 Powdered rhizome of H. spicatumhas been used clinically for the treatment of asthma7 and tropical pulmonary eosinophilia 8 and as anti-inflammatory and analgesic.9
An extensive search of the literature reveals no reports on the antimicrobial activity of the plant. Thus, present investigation was planned to find out the therapeutic level of methanolic extract of H. spicatum plant in antimicrobial activity.
MATERIALS AND METHODS
The raw materials of H. spicatum were procured from the supplier (S.S. Herbal, 485/2, Katra Ishwar Bhavan, Khari Baoli, Delhi) and the sample was identified and authenticated. 100 gm of air dried powdered plant material was extracted with methanol in soxlet apparatus for 96 hrs. After that the extract was filtered and again suspended in the above mixture for 24 hrs. Finally extracts was filtered and concentrated over water bath at a temperature of 40ºC. The extract was cooled and kept in desiccator overnight. The extracts was weighed and used for antibacterial and antifungal potentiality.
The test bacteria used were Shigella flexneri type 36 NK 381, Sh. flexneri type 6B 999, Sh. flexneri type BCH 995, Sh. boydii 22461, Sh. boydii 16552, Sh. boydii 8, Sh. soneii BCH 397, Sh. soneii E08869, Sh. soneii NK 840, Sh. soneii BCH 937, Sh. soneii I, Sh. soneii DN3, Sh. soneii F11001, Sh. soneii NK 29, Sh. dysenteriae 1, Sh. dysenteriae 9, Vibrio cholerae 1023, V. cholerae BD 1/81, V. cholerae 1341, V. cholerae 452, V. cholerae 1033, V. cholerae 575, V. cholerae 765, V. cholerae 1311, V. cholerae 756, V. cholerae DN6, V. cholerae A 26, Escherichia. coli AP600, E. coli 383, E. coli RH 07/12, E. coli 18/9, E. coli 597, E. coli 798, E. coli 35B, E. coli 306, E. coli K88, E. coli 872, Enterobacter spp. AP596 , S. typhii Type 2, S. aureus ML 267, Staphylococcus. aureus ATCC 6538, S. aureus MTCC 96, S. aureus 381, Bacillus. subtilis MTCC 441, B. cereus MTCC 1305, B. pumilus 8241, Pseudomonas putida MTCC 2252, P. aeuriginosa AP585 NLF, Klebsiella pneumoniae and Proteus vulgaris AP679 NLF. These microbial strains included various drug resistant hospital isolates collected and characterized in the Department of Pharmaceutical Technology, Jadavpur University, India. All strains were maintained on Nutrient Agar (NA) for bacteria and Sabourauds’s Dextrose Agar (SDA) slants for fungi at 4ºC prior to use for antibacterial and antifungal tests respectively.
All parameters were applied on rhizome physicochemical analysis i.e., percentage of ash values and extractive values, were performed according to the official methods prescribed in Indian Pharmacopoeia, 1996 and the WHO guidelines on quality control methods for medicinal plant materials (WHO/QCMMPM guidelines).10
Preliminary Phytochemical Screening
Chemical tests were performed in the preliminary phytochemical screening to identify various secondary metabolites such as tannins and phenols, carbohydrates, glycosides, saponins, alkaloids, flavonoids and sterols using standard methods.11,12
Determination of MIC by Serial Dilution technique
The rhizome extract (stock solution) was reconstituted with a minimum amount of dimethyl sulfoxide (DMSO). This solvent did not possess any antimicrobial activity of its own. Calculated volumes of this stock solution were dispensed in a series of McCartney bottles previously containing calculated volume of sterile cooled molten nutrient agar media (40-45ºC) to prepare final volume of 30 ml each with dilutions of 5, 10, 25, 50, 100, 200, 400, 800 and1000 μg/ml. The stock solution were dispensed into molten SDA to prepare varying dilutions of 100, 200, 400, 800, 1500 and 2000 μg/ml while determining the MIC against the fungi. Then these molten media containing varying concentration of extract were poured aseptically in pre-sterilized Petri dishes (70 mm) to give sterile nutrient agar plates with varying dilution of extract. These plates were then kept in refrigerator at 4ºC for 24hrs to ensure uniform diffusion of the extract. Then these plates were dried at 37ºC for bacteria and 25ºC for fungi for 2 hours before spot inoculations. One loopful (loop diameter: 3mm) of an overnight grown bacterial strain suspension (105 CFU/ml) was added in each quadrant as marked by checker board technique. The spotted plates were incubated at 37ºC and 25ºC for 24 hours for bacteria and fungi respectively, in an incubator and MIC values were obtained.13,14,15
Determination of Mode of Action of the Extract
To determine whether the extract was bacteriostatic or fungistatic and bactericidal or fungicidal in nature, plugs from the zone of inhibition were taken out and reincubated into fresh media which were then examined for their growth after 96 hours incubation at 37ºC and 25ºC in an incubator, respectively.15,16
RESULTS AND DISCUSSION
Ash value of a drug gives an idea of the earthy matter or the inorganic composition and other impurities present along with the drug. Extractive values are primarily useful for the determination of exhausted or adulterated drugs. The alcohol soluble extractive was high in rhizomes of H. spicatum. The results of physicochemical constants of the drug powder are presented in (Table 1).
Preliminary Phytochemical Screening
Preliminary phytochemical screening of the extract revealed the presence of tannins, saponins, flavonoids, steroids, terpenoids, cardiac glycosides and reducing sugar in it (Table 2).
|Acid insoluble value||1.03% w/v|
|Water soluble Ash||2.61% w/v|
|Water insoluble Ash||2.10 w/v|
|Extractive value(water soluble)||13.9 w/v|
|Extractive Value (alcohol soluble)||5.24 w/v|
|pH of 1% suspension||5.41 w/v|
The result in Table 3 depicted the MIC values of the methanolic extract of the rhizomes of H. spicatum against various tested bacterial pathogens.
It was evident from the results shown in the Table 3 that the extract was highly active against Shigella boydii, Sh. soneii, Shigella flexneri, B. cereus, Vibrio cholera, E. coli, S. aureus, Ps. aeruginosa and K. pneumoniae. The result of determination of zone of inhibition of the crude extract of the rhizomes of the plant and their comparison with those of standard antibacterial agent Ciprofloxacin against the tested bacterial strains is recorded in Table 4.
The sensitivity pattern of the bacterial organisms to the extract was found to decrease in the following order: E. coli 358, B. subtilis MTCC441, V. cholera 1341, Pseudomonas putida MTCC 2252, V. cholerae 1311, V. cholerae 756, V. cholerae 575, V. cholera 1023, E. coli 872, E. coli 798, E. coli 18/9, Ps. aeruginosa AP585 NLF, as evident from table 5.
The pharmacognostical and phytochemical evaluation of H. spicatum (Zingiberaceae)) rhizome provided useful information for identification and authentication of the plant. The antibacterial study of the methanolic extract of the rhizomes of H. spicatum showed the maximum activity against Shigella boydii, Sh. soneii, Shigella flexneri, B. cereus, Vibrio cholera, E. coli, S. aureus, Ps. aeruginosa and K. pneumoniae. The results of phytochemical analysis and antimicrobial activity studies of the plants extracts confirmed its therapeutic usage, as depicted in the literature. The active plant extract may be further subjected to biological and pharmacological investigations for isolation of antibacterial and therapeutic compounds.
Hedychium spicatum is a good source for flavonoid compounds.
Methanolic extract had shown strong positivity in preliminary phytochemical screening.
Antimicrobial activity performed by determining its MIC (by checker board method) and zone of inhibition (by cup plate method)
Antibacterial study of the methanolic extract of the rhizomes of H. spicatum showed the maximum activity against Shigella boydii, Sh. soneii, Shigella flex neri, B. cereus, Vibrio cholera, E. coli, S. aureus, Ps. aeruginosa and K. pneu moniae.