MATERIALS AND METHODS

Herbs material: Leaves and flowers of H. persicum were collected on mountains of Mazandaran province Kelardasht region (Koohpar village) in northern part of Iran in 2010 and identified at department of plant and agriculture in Islamic Azad University.

Extracts: Leaves and flowers of the herb were collected and dried in shadow separately and mixed together. Then they were grinded powdered mechanically. The amount of 100 g of each powder was extracted by 80% methanol in the room temperature for 24 h. Then filtered by Whatman paper filter number 1 then dried in room temperature for evaporation of methanol solutions and sterilized by flashing of UV in the end of experiment and store in room temperature for further analysis. Related to the mass of dry matter (mg), the concentrations of the extracts for antibacterial investigation (ml) were calculated according to previous studies (Nazemi et al., 2005; Manikandan et al., 2009).

Antibacterial and antifungal activity assay: The disc-diffusion assay (Agar Diffusion Test) was used to determine the bactericidal and fungicidal activity of the plant extracts according to method of Bansod and Rai (2008).

Bacterial and fungal strains: The examined bacterial strains were Gram-negative bacteria consist of: Aeromonas salmonicida, Aeromonas hydrophila, Klebsiella oxytoca, Klebsiella pneumonea, Pseudomonas aeruginosa, Escherichia coli and Gram-positive strains including: Streptococcus iniae, Micrococcus luteus, Staphylococcus aureus (Sittiwet et al., 2008; Sittiwet and Puangpronpitag, 2008; Abdul et al., 2008), Bacillus subtilis (Al-Howiriny, 2002; Abdul et al., 2008; Al-Saghir, 2009) and Bacillus cereus. The two tested fungal strains were Aspergillus niger and Candida albicans (Al-Howiriny, 2002; Abdul et al., 2008). All bacteria and fungal strains were obtained from the stock cultures of the microbiology department of Islamic Azad University.

Evaluation of antibacterial activity: These were maintained at 4°C on nutrient agar plates. Petri plates were prepared by pouring 10 mL of Mueller Hinton agar and allowed to solidify, 0.1 mL of standardized inoculums suspension was added and spread uniformly. The discs were then applied and plates were incubated at 37°C for 24 h. The inhibition zone was measured from the edge of the disc to the inner margin of bacterial colony. DMSO used as a negative control and gentamicin and nystatin used as a positive control. The experiment was done in triplicate. Minimum Inhibitory Concentration (MIC) of the herb extract was tested in Mueller Hinton broth by the two-fold serial dilution method. The culture tubes were incubated in incubators at 37°C for 24 h. The lowest concentration, which did not show any growth of tested organism after microscopic evaluation was determined as minimum inhibitory concentration.

Statistical analysis: Statistical analysis of the results was performed using Excel software (version 12). Numerical results were compiled into Means±SD. The product-moment correlation coefficient was calculated and tested for significance against the no correlation zero hypotheses through the procedure correlation. Data performed to analysis of variance (ANOVA) test to observed variance in a particular variable. The significance level was chosen at p<0.05.

RESULTS AND DISCUSSION

The results revealed that the H. persicum extract showed significantly the antibacterial and antifungal effects (p<0.05). The result indicated there is a direct relation between enhancements of antimicrobial activities with concentration of herbal extract (6.25-50%). The results of diameter zone of inhibition and Minimum Inhibitory Concentration (MIC) are shows in Table 1. According to Table 1 the maximum diameter zones of inhibition for the microorganisms and the amount of MIC were in range of 2.26-51.01 mm and 128-512 mg mL^-1, respectively. Aspergillus niger and Micrococcus luteus showed highest and lowest effect at all ranges, respectively.

The antibacterial activity may be due to several agents such as presence of oil, alkaloids, flavonoids and tannin, reported by Brantner et al. (1996) or the different solvent extract. Nazemi et al. (2005) carried out a study on antibacterial effect of methanol and aquatic extract of H. persicum and published those results in Persian language, the antibacterial activity of extracts in that research showed that the aquatic extract of H. persicum had no effect but the methanolic extract had a significant effect on 5 species of Bacillus polymyxa, Bacillus subtilis, Enterococcus faecalis, Nocardia and Staphylococcus aureus, respectively (Nazemi et al., 2005).

The results of the present survey showed a significant effect (significantly differentiation at p<0.05) of H. persicum extract on different bacterial and fungal strains (Table 1). Bazzaz and Haririzadeh (2003) reported no anti-microbial effect of the leaf of this herb but the present study and unpublished results of inhibitory effect of aquatic extract of Tromsø Palme (H. persicum) in the Arctic area by author, showed some inhibitory effects using in vitro growth inhibition test in all parts of herb.

Some of the other researchers showed that the methanolic extract of leaves of different plants has more active than the aqueous extracts against Bacillus subtilis, Bacillus pumilus, Micrococcus luteus and Staphylococcus aureus (Manikandan et al., 2009), the present study showed significant effects at 50% concentration of all strains especially on Staphylococcus aureus but two strains of Aeromonas hydrophila and Streptococcus iniae showed significant effect at 25 and 12.5% concentrations, respectively.

Table 1:	Extract of H. persicum leaves and flowers bactericidal and fungicidal activity against different strains
Values (Mean±SD) are significantly differentiation at (p<0.05)

Habibi et al. (2010) revealed the maximum inhibitory activity of H. rechingeri species against Gram-positive bacteria especially B. subtilis, at the present study we achieve to same results at 50% concentration with highly significant (p<0.05).

Noudeh et al. (2010) has also recorded the effect of H. persicum (57.16%) inhibition effects on Agrobacterium tumefaciens also inhibited the growth of all tested Gram-positive and Gram-negative strains respectively, present study showed the same effect in different strains and concentrations.

There are numerous studies about of antibacterial activity in some herbs species have high antibacterial activity due to presence of monoterpenoids by the drop diffusion method, showing highly significant inhibition zones for all microorganisms tested, contained Gram-positive bacteria (Staphylococcus aureus) and fungus (Candida albicans) (Oliveira et al., 2007; Kuljanabhagavad et al., 2010), the results of present study showed the same significant results (p<0.05) for Gram-positive bacteria (Staphylococcus aureus) and fungus (Candida albicans), respectively.

Using medicinal herbs as an immunostimulants and acute stress response have been used as feed additives for several years and well documented by several authors (Askarian and Kousha, 2008; Kousha and Askarian, 2008; Faghani et al., 2008; Ringo et al., 2012). Furthermore in vivo, in vitro and ex vivo surveys on effect of Heracleum sp. as an immunostimulant agent, acute and chronic stress response on terrestrial and aquatic animals or human health also need further investigation. Otherwise, antiviral activities of this family merit further investigation.

CONCLUSION

It is concluded that, leaves and flowers extracts of H. persicum have some antimicrobial activity over 4 Gram-negative, 4 Gram-positive bacteria and antifungal effect over 2-fungi’s. Several species or subspecies belong to this family have a huge distribution and well growth around the world specially in Europe and USA as an exotic herbs in different climate, so investigation on similar effects at different regions and climate merit further study.

ACKNOWLEDGMENTS

We would like to thanks our colleagues in the Islamic Azad University for their substantial helps, cooperation of all individuals and everybody who participated in this study. Especially kind people of Koohpar village in Kelardasht region in Mazandaran province of Iran are also gratefully acknowledged.