Ethanol, methanol, chloroform, petroleum ether and aqueous extracts of leaves of Parrotia persica were evaluated for antibacterial activity. The zone of inhibition varied from 13 to 22 mm. The highest inhibittionwas obtained with methanol and ethanol. Chloroform and petroleum ether extracts did not show any activity. The minimal inhibitory concentration (MIC) value of the methanol extract for the test bacteria ranged between 3.12 and 6.25 mg/ml and that of ethanol extract ranged between 6.25 and 12.5 mg/ml. The results scientifically validate the use of this plant in the traditional medicine of Iran

Azadbakht, M

Mohammad, A

Mohana, DC

Raveesha, KA

English

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African Journal of Biotechnology Vol. 6 (22), pp. 2526-2528, 19 November, 2007 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2007 Academic Journals     Short Communication  Antibacterial potential of extracts of leaves of Parrotia persica  Mohammad Ahanjan1, Mohana, D. C1, Raveesha, K. A.1* and M. Azadbakht2  1Microbiology laboratory, Department of studies in Botany and Microbiology, University of Mysore, Manasagangotri, Mysore 570 006, India. 2Faculty of Pharmacy, University of Mazandaran Medical Sciences, Sary, Iran.  Accepted 6 November, 2007  Ethanol, methanol, chloroform, petroleum ether and aqueous extracts of leaves of Parrotia persica were evaluated for antibacterial activity. The zone of inhibition varied from 13 to 22 mm. The highest inhibit-tion was obtained with methanol and ethanol. Chloroform and petroleum ether extracts did not show any activity. The minimal inhibitory concentration (MIC) value of the methanol extract for the test bacteria ranged between 3.12 and 6.25 mg/ml and that of ethanol extract ranged between 6.25 and 12.5 mg/ml. The results scientifically validate the use of this plant in the traditional medicine of Iran.  Key words: Parrotia persica, antibacterial activity, methanol extract, traditional medicine.   INTRODUCTION  Parrotia persica (DC) CA. Mey (Persian Ironwood) is a deciduous tree of the family Hamamelidaceae, the sole species in the genus Parrotia. It is native to northern Iran, and endemic to the Alborz Mountains. Many herbalists use this plant in the treatment various fevers and respiratory infections. It is also used for food coloring and food flavoring (Rechinger, 1999). There are no scientific reports on the antibacterial properties of the plant. Such lack of scientific knowledge has been major constraint in the use of traditional herbal remedies (Patwardhan et al., 2004). Hence, the aqueous and solvent extracts of leaves of P. persica were quantitatively screened for antibacte-rial activity against five important human pathogenic bacteria, obtained from Microbial Type Culture Collection and Gene bank (MTCC) India.   MATERIALS AND METHODS   Plant material  Apparently healthy leaves of P. persica were collected from the mountainous region of Mazandaran province of I.R. Iran during November 2005. The voucher specimen is deposited in the herba-    *Corresponding author. E-mail: karaveesha@gmail.com. Tel: +91-821-2412418(0). rium of Department of Studies in Botany and Microbiology, Univer-sity of Mysore, Mysore, India.   Extraction of plant materials  The leaves of P. persica were washed thoroughly three times with running water and once with distilled water. The materials were air dried under shade and powdered. The shade dried leaves and powdered samples were hermetically sealed in separate polythene bags until the time of extraction.    Aqueous extraction  Samples (20 g) of thoroughly dried leaves were macerated with 40 ml sterile water in a waring blender for 10 min. The macerate was first filtered through double-layered muslin cloth, and then centri-fuged at 4000 rpm for 10 min. The supernatant was filtered through Whatman No.1 filter paper. The extract was preserved aseptically in a brown bottle at 5ºC until further use.    Solvent extraction  Sample (20 g) dried powder was filled in the thimble and extracted successively with chloroform, ethanol, methanol and petroleum ether in Soxhlet extractor until the extract was clear.    Phytochemical screening   The methanol extract, which was antibacterially active, was selec- Ahanjan et al.        2527    Table 1. Antibacterial activity of leaves of Parrotia persica extracts at 100 l.   Zone of Inhibition (mm)   Bacteria Aqueous extract Chloroform extract Ethanol extract Methanol extract Petroleum ether extract Gentamycin 10 (µg) E. coli 13.0±04 0.0 14±0.5 15±0.4 0.0 34±0. K. pneumoniae 16±0.3 0.0 15±0.4 22±0.2 0.0 30±0.4 P. aeruginosa 15±0.4 0.0 18±0.3 17±053 0.0 31±0.5 S. typhi 15±0.6 0.0 22±0.2 21±0.6 0.0 30±0.3 S. aureus 16±0.5 0.0 16±0.5 20±0.5 0.0 33±0.5  Values presented are means of six replicates ± standard error. When subjected to analysis of variance (ANOVA), p < 0.05.    Table 2. Minimal inhibitory concentration (MIC) of methanol extract of P. persica against the test bacteria.   Zone of Inhibition in mm MIC of methanol extract (mg/ml)   Bacteria 50 25 12.5 6.25 3.12 1.56 0.78 Gentamycin  (10 µg) E. coli 9±0.3 5±0.4 3±0.3 2±0.2 0.0 0.0 0.0 34±0.7 K. neumoniae 17±0.5 14±0.4 11±0.2 8±0.4 4±0.4 0.0 0.0 30±0.9 P. aeruginosa 13±0.3 8±0.6 5±0.3 3±0.3 2±0.5 0.0 0.0 31±0.3 S. typhi 15±0.3 11±0.7 6±0.3 4±0.4 2±0.3 0.0 0.0 30±0.3 S. aureus 9±0.2 6±0.2 4±0.7 3±0.2 2±0.4 0.0 0.0 33±0.2  Values presented are means of six replicates ± Standard error. When subjected to analysis of variance (ANOVA), p < 0.05.    ted for phytochemical analysis. The dried extract was first recon-stituted in methanol and subjected to standard phytochemical analysis for the presence of flavonoids, tannins, saponins, carbohy-drates, terpenoids and protein (Harborne, 1988).      Test bacterial strains  Standard type cultures of Escherichia coli (MTCC 443), Klebseilla pneumoniae (MTCC 109), Pseudomonas aeruginosa (MTCC 1688), Salmonella typhi (MTCC 733) and Staphylococcus aureus (MTCC 737) were obtained from MTCC Chandigrah, India. All the test strains were subcultured on Muller Hinton agar (MHA).   Antibacterial activity assay  The extracts were spot checked for antibacterial activity employing the agar well diffusion technique (Okeke et al., 2001). Standardized inoculum (5× 105 cfu/ml) of each test bacterium was spread on to sterile MHA plates so as to achieve a confluent growth. The plates were allowed to dry and a sterile cork borer of diameter 6.0 mm was used to bore wells in the agar plates. The dried extracts were reconstituted in methanol to a concentration of 50 mg/ml. Subse-quently, a 100 µl volume of the extracts were introduced in triplicate wells into the MHA cultures. The plates were allowed to stand for 1 h. for diffusion to take place and then incubated at 37ºC for 24 h. The zone of inhibition was recorded to the nearest mm. Only the extracts exhibiting apparent zone of inhibition were chosen for further evaluation. For comparative evaluation the antibiotic Genta-mycin (10 µg) was used. Determination of minimal inhibitory concentration (MIC)  The MIC was determined only for the methanol extract by agar well diffusion method as it is showed a wider spectrum of antibacterial activity compared to others. A two-fold serial dilution of the extract was prepared by first reconstituting in methanol then diluting in sterile distilled water to achieve a decreasing concentration range of 50 to 0.781 mg/ml. A 100 µl volume of each dilution was introduced in triplicate wells into MHA plates already seeded with the standardized inoculums (5 × 105 cfu/ml) of the test bacterial cells. All test plates were incubated at 37ºC for 24 h. The least concentration of each extract showing a clear zone of inhibition was taken as the MIC.   RESULTS  The results of the antibacterial activity of the extracts are recorded in Table 1. The aqueous extract, ethanol extract and methanol extract of leaves of P. persica showed good activity against all the test bacteria. The chloroform and petroleum ether extracts did not show any activity against the test bacterial strains. The methanol extracts of the leaves recorded activity to all standard cultures with highest mean zone of inhibition. The MIC value of methanol extract was 3.12 mg/ml for K. pneumoniae, P. aeruginosa, S. typhi and S. aureus and 6.25 mg/ml for E. coli (Table 2). The MIC values of ethanol  extract  for  K.  pneumoniae   2528           Afr. J. Biotechnol.    Table 3. MIC of ethanol extract of P. persica against the test bacteria.   Zone of Inhibition in mm MIC of ethanol extract (mg/ml)   Bacteria 50 25 12.5 6.25 3.12 1.56 0.78 Gentamycin (10 µg) E. coli 8.0±0.3 4.5±0.4 3±0.3 0.0 0.0 0.0 0.0 33±0.7 K. neumoniae 11±0.5 8±0.4 4±0.2 2.0±03 0.0 0.0 0.0 29±0.9 P. aeruginosa 12±0.3 7±0.6 4±0.3 2.0±02 0.0 0.0 0.0 30±0.3 S. typhi 13±0.3 8±0.7 4±0.3 2.0±03 0.0 0.0 0.0 30±0.3 S. aureus 7±0.2 4±0.2 2±02 0.0 0.0 0.0 0.0 30±0.2  Values presented are means of six replicates ± Standard error.  When subjected to analysis of variance (ANOVA), p < 0.05.    Table 4. Phytochemical analysis of methanol extract of P. persica.  Constituent Methanol extract Alkaloids - Carbohydrates + Flavonoids + Gum + Phenolics + Proteins + Saponins + Tannins + Terpenoids + Waxes -  + Present, - absent.    was 6.25 and 12.5 mg/ml for E. coli and S. aureus (Table 3). The methanol extract of leaves of P. persica was anti-bacterially active to K. pneuminiae with highest mean zone of inhibition (22 mm). The phytochemical analysis showed that the methanol extract of P. persica contained tannin, flavonoid, protein, saponin and carbohydrate (Table 4).    DISCUSSION  Review of literature reveals lack of information on the antibacterial potential of P. persica. Aqueous and differ-rent solvent extracts of leaves of P. persica evaluated for antibacterial potential, revealed highly significant activity of methanol extract against all the test bacteria followed by ethanol extract. None of the earlier reports have demonstrated the antibacterial potential of this plant (Okoli et al., 2002; Khan et al., 2002; Ahmet et al., 2005). In the present investigation the antibacterial activity of this plant has been demonstrated for the first time.  It is also evident from the results that methanol extract has significantly high degree of antibacterial activity, sug-gesting that methanol is the appropriate solvent for extraction of the antibacterial principle (Nkere and Iroegbu, 2005). Thus methanol is recommended for the large-scale extraction of the active principle (Taous et al., 2005).  Saponins and tannins are reported to possess antibac-terial activity (Newman et al., 2000). Preliminary phyto-chemical analysis of the methanol extract of leaves of P. persica revealed the presence of saponin, tannin and flavonoid, which could be the active principle. Further investigations are in progress to isolate and characterize the active principle. This study scientifically validates the use of this plant for treatment of fever and other respira-tory infection in traditional medicine of Iran.   REFERENCES  Ahmet A, Medine G, Meryam S, Hatice O, Fikrettin S, Isa K (2005). Antimicrobial Effects of Ocimum basillicum (Labiatae) Extract.  Turk J. biol. 29: 155-160 Harborne JB (1998).  Phytochemical methods: A guide to modern techniques of plant analysis. 3rd edition. Chapman and Hall. London.  Khan MR, Kihara M, Omoloso AD (2002). Antimicrobial activity of Calophyllum soulattri. Fitoprapia, 73: 741-743. Newman DJ, Cragg GM, Snader KM (2000). The influence of natural products upon drug discovery. Nat. Prod. Res. 17: 215-234. Nkere CK, Iroegbu CU (2005). Antibacterial screening of the root, seed and stembark extracts of Picralima nitida. Afr. J. Biotechnol. 4: 522-526. Okeke MI, Iroegbu CU, Eze EN, Okoli AS and Esimone CO (2001). Evaluation of extracts of the root of Landolphia owerrience for antibacterial activity. J. Ethnopharmacol. 78: 119-127 Okoli AS, Okeke MI, Iroegbu CU, PU (2002). Antibacterial activity of Harungana madagascariensis leaf extracts. Phytother. Res. 16: 174-179. Patwardhan B, Vaidya ADB, Chorghade M (2004). Ayurveda and natural products drug discovery. Curr. Sci. 86: 789-799. Rechinger KH (1999). Flora Iranica. Akademische Druk-u.Verlagsanstalt. Graz, Austria. Taous k, Mansoor A, Hamayun K, Mir AK (2005). Biological activities of aerial parts of Paeonia emodi wall.  Afr. J. 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Antibacterial potential of extracts of leaves of Parrotia persica

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Antibacterial potential of extracts of leaves of Parrotia persica

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