Onosma griffithii was screened for possible pharmacological activities. The crude methanolic extract (MeOH) and its fractions demonstrated parasiticidal activity (IC 50 (ìg/ml ± S.D) = 31.03 ± 0.23) against Leishmania major. Based on the IC 50 values, the potency of the standard drug (Pentamidine) and test fractions were of the order as: Pentamidine > crude extract > n-hexane fraction > ethyl acetate (ETOAc) fraction > chloroform fraction (CHCl3) fraction > n-butanol (BUOH) fraction > aqueous fraction. Similarlymoderate antifungal activity was displayed by the crude methanolic extract against Aspergillus flavus and Fusarium solani. Against the Staphylococcus aureus, the aqueous fraction demonstrated moderate antibacterial activity

Ahmad, B

Ali, N

Azam, S

Bashir, S

Choudhary, MI

Khan, I

English

AJOL - African Journals Online

African Journal of Biotechnology Vol. 8 (19), pp. 5084-5087, 5 October, 2009     Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2009 Academic Journals      Full Length Research Paper  Parasiticidal, antifungal and antibacterial activities of Onosma griffithii Vatke  B. Ahmad1*, N. Ali2, 4, S. Bashir2, M. I. Choudhary3, S. Azam and I. Khan1  1Centre for Biotechnology and Microbiology, University of Peshawar, N.W.F.P. Pakistan. 2Department of Pharmacy, University of Peshawar, N.W.F.P. Pakistan. 3International Centre for Chemical and Biological Sciences, H.E.J Research Institute of Chemistry, University of Karachi, Karachi. Pakistan. 4Department of Pharmacy, University of Malakand, Chakdara Dir, N.W.F.P. Pakistan.  Accepted 13 August, 2009  Onosma griffithii was screened for possible pharmacological activities. The crude methanolic extract (MeOH) and its fractions demonstrated parasiticidal activity (IC 50 (µg/ml ± S.D) = 31.03 ± 0.23) against Leishmania major. Based on the IC 50 values, the potency of the standard drug (Pentamidine) and test fractions were of the order as: Pentamidine > crude extract > n-hexane fraction > ethyl acetate (ETOAc) fraction > chloroform fraction (CHCl3) fraction > n-butanol (BUOH) fraction > aqueous fraction. Similarly moderate antifungal activity was displayed by the crude methanolic extract against Aspergillus flavus and Fusarium solani. Against the Staphylococcus aureus, the aqueous fraction demonstrated moderate antibacterial activity.  Key words: Parasiticidal, antileishmanial, antifungal, antibacterial and IC50 values.    INTRODUCTION   The family Boraginaceae consists of about 100 genera and 2000 species distributed in temperate, especially in Mediterranean and tropical regions (Ali and Nasir, 1983). Onosma is an important genus of the family as extracts of some species of Onosma, except Onosma hispidum, have been used as antioxidant, antibacterial, antiviral and anti-inflammatory agents (Tosun et al., 2008). Traditional-ly, Onosma is used as stimulant in rheumatism, bladder pain, kidney irritation and palpitation of heart (Ahmed, 2004). Hispidone, a new flavanone has been isolated from O. hispidum that have cholinesterase inhibitory acti-vity (Ahmad et al., 2003). O. hispida is also used as colo-rant (Shahina, 2005). Similarly, anticancer activity of Onosma limitaneum (Ahmad et al., 2005), antioxidant and antimicrobial activities of Onosma argenatum have been reported. It has been suggested that the roots of some plant of Boraginaceae contain naphthaquinone derivatives. Shikanins and alkanins do exist in the Onosma species (Ufuk et al., 2004).  Traditionally, O. argentatum is used in the treatment of     *Corresponding author. E –mail: bashirdr2001@yahoo.com. wound healing and burns with well documented anti-oxidant and antimicrobial activity (Sezik et al., 1997; Ozgen et al., 2003). This suggests that this genus is very potent from pharmacological activities point of view. Hence we carried out experiments to screen Onosma griffithii specie for possible pharmacological activities.   MATERIALS AND METHODS  Plant material    Aerial parts of the plant, O. griffithii (Boraginaceae), were collected from the nearby hills of Campus I of University of Malakand, in June-July 2005 and identified by Professor Dr. Jahandar Shah, Plant Taxonomist, University of Malakand, Chakdara Dir N.W.F.P Pakistan. A voucher specimen (OG-05) has been deposited in the herbarium of the University of Malakand.   Extraction   The shade dried plant material was crushed into small pieces and finally pulverized into fine powder. The plant material (7 kg) was soaked in methanol with occasional shaking, at room temperature. After 15 days, the methanol soluble materials were filtered off. The filtrate was concentrated under vacuum at  low  temperature  (below  Ahmad et al.         5085    Table 1. Antileishmanial activities of crude methanolic extract and fractions of Onosma griffithii against the susceptible strains of Leishmania major promastigotes (Pakistani isolates). IC50 values in µg/ml ± S.D obtained from three separate experiments are shown.   Control Extract / Fraction / standard* Negative Control (% Survivors) Positive control * (% Inhibition)  IC50 values (µg/ml ± S.D) Mean relative index (IC50 Sample / IC50 Standard) Pentamidine* 100% 100% 2.5 ±  0.09 1 Crude methanolic extract 100% 100% 31.03 ± 0.23 12.41 n-Hexane 100% 100% 31.36 ± 0.34 12.54 Chloroform 100% 100% 42.60 ±0.465 16.24 Ethyl acetate 100% 100% 38.39 ± 0.37 15.32 n-Butanol 100% 100% 41.87 ± 0.39 16.748 Aqueous 100% 100% 60.35 ± 0.87 24.14    40°C) using rotary evaporator. A blackish crude extract (150 g) was obtained.   Fractionation   The crude methanolic extract (150 g) was suspended in distilled water (400 ml) and sequentially partitioned with n-hexane (3 x 350 ml), chloroform (3 x 350 mL), ethyl acetate (3 x 350 ml) and n-buta-nol (3 x 350 ml) to yield n- hexane (30 g), chloroform (20 g), ethyl acetate (2g), n-butanol (2 g) and aqueous (65 g) fractions, respec-tively. About 25-gram crude methanolic was reserved for other pharmacological screenings.   Statistical analysis  All the experiments were performed in triplicate. Standard devia-tions and IC50 values of compounds possessing antileishmanial activity were calculated by Software Ezfit 5.03 Perella Scientific. P values of 0.05 or less was considered significant using student’s “t” test.     Parasiticidal activity  The antileishmanial activity was performed according to the proce-dure of Rehman et al. (1991) described as follows:   Parasite culture   Leishmania major parasite’s Promastigotes were grown in bulk, early in modified NNN biphasic medium, using normal physiological saline. Then the Promastigotes were cultured with RPMI 1640 me-dium supplemented with 10% heat inactivated fetal bovine serum (FBS). The parasites, at log phase, were centrifuged at 2000 rpm for 10 min and washed three times with saline at same speed and time. Finally the parasites were diluted with fresh culture medium to give a final density of 106 cells/ml. In a 96 -well micro titer plate, 180 µl of medium was added in dif-ferent wells. 20 µl of the experimental compound was added in medium and serially diluted so that the minimum concentration of compound was one 1 µg/ml. 100 µl of parasite culture (final density of 106 cells/ml) was added in all wells. 2 rows were left one for negative and other for positive control. Negative controls received the medium while the positive control received Pentamidine as standard antileishmanial compound. The plate was incubated at 21 - 22°C for 72 h. The culture was examined microscopically for cell viability by counting the number of motile cells on an improved neubauer counting chamber and IC50 values of compounds pos-sessing antileishmanial activity were calculated by Software Ezfit 5.03 Perella Scientific. IC50 values of different fractions against the test pathogen are mentioned in Table 1.   Antifungal activity   Similarly antifungal activity was evaluated by agar tube dilution method as per our reported procedure (Bashir et al., 2007). Des-cribing the procedure, the test samples in the concentration of 24 mg/ml were dissolved in the sterile (autoclaved) dimethyl sulfoxide (DMSO, Merck), which served as stock solution. Sabouraud Dextrose Agar (SDA, Sigma-Aldrich, Germany) was prepared by mixing 32.5 g sabouraud, 4% glucose agar and 4.0 g of agar-agar in 500 ml distilled water and the mixture was mixed thoroughly with magnetic stirrer. Then 4 ml amount was dispensed into screw cap tubes, which were autoclaved at 120ºC for 15 min and then cooled to 15ºC. The non-solidified SDA media was mixed with stock solu-tion (66.6 µl) giving the final concentration of 400 µg of the extract per ml of SDA. Tubes were then allowed to solidify in the slanted position at room temperature. Each tube was inoculated with a piece (4 mm diameter) of inoculums removed from a seven days old culture of fungi for non-mycelial growth; an agar surface streak of Aspergillus flavus and Fusarium solani was employed. Other media supplemented with dimethyl sulfoxide (DMSO) and standard antifungal drugs served as negative and positive control respec-tively (Figure 1). Inhibition of fungal growth was observed after 7 days of incubation at 28 ± 1ºC. Humidity (40 - 50%) was controlled by placing an open pan of water in the incubator.    Antibacterial activity  The crude extract along with fractions was screened against various human pathogens including Escherichia coli, Bacillus subtilis, Shigella flexenari, Staphylococcus aureus and Salmonella typhi as per our reported procedure (Bashir et al., 2007). In this method, 10 ml aliquots of nutrients broth (Sigma-Aldrich, Germany) was inoculated with the test organism and incubated at 37ºC for 24 h. Using a sterile pipette, 0.6 ml of the broth culture of the test organism was added to 60 ml of molten agar, which had been cooled to 45ºC, mixed well and poured into a sterile petri dish (for the 9 cm petri dish, 0.2 ml of the culture was added to 20 ml of agar). Duplicate plates of each organism were  prepared. The  agar  5086         Afr. J. Biotechnol.      Figure 1. Antifungal activities of crude extract and fractions of Onosma griffithii against Aspergillus flavus and Fusarium solani using Amphotericin B (20 µg/ml) and Miconazole (98.4 µg/ml) as standards respectively.    was allowed to set and harden and the required numbers of wells were dug in the medium with the help of a sterile metallic cork borer ensuring proper distribution of the wells in the periphery and one in the center. Agar plugs were removed. Stock solutions of the test samples in the concentration of 1 mg/ml were prepared in the sterile dimethyl sulfoxide (DMSO) and 100 and 200 µl of each dilution was added in their respective wells. The control well re-ceived only 100 and 200 µl of DMSO. Imipenem was used as standard drug. The plates were left at room temperature for 2 h to allow diffusion of the samples then incubated face upwards at 37ºC for 24 h. The diameter of the zones of inhibition was measured (mm).   RESULTS AND DISCUSSION  Parasiticidal activity  Leishmaniasis particularly cutaneous leishmaniasis is a parasitic disease with diverse clinical manifestations that depends on both infecting species of Leishmania and the immune response of the host (WHO, 1984). The results of the crude methanolic extract and its fractions are sum-marized in Table 1. The lower the IC50 value of a drug against a particular pathogen, the more will be its potency and vice versa. Based on the IC50 values (Table 1), the crude methanolic extract and its fractions showed statistically significant (P < 0.05) antileishmanial activities against the L. major, Pakistani isolates. In comparison with the Pentamidine as a standard drug used, the crude  methanolic  extract  and      its various fractions demonstrated the order of potency as: Pentamidine > crude extract > n-hexane fraction > ethyl acetate > chloroform fraction > n-butanol > aqueous fraction. Cutaneous leishmaniasis being a challenge and threat to Pakistani society as its incidence increases day by day. There were 350 patients in 1995 - 1997, 1210 patients in 1996-2001 and 1640 patients reported in 2008 in Pakistan (Bhutto et al., 2008). Therapy results of al-ready existing topical preparation of paromomycin and gentamicin combination are not so much satisfactory be-cause of 50% relapse and resistance (Grogl et al., 1999; El-On et al., 1991). Therefore, search for new antile-ishmanial drugs is necessary.  Very few medicinal plants have been reported for their antileishmanial activities (Kvist et al., 2006), hence the plant specie could be a potential target for activity guided isolation of bioactive compounds as the crude methanolic extract and its fractions are very potent because of their low IC50 values.   Antifungal activity  The crude methanolic extract of O. griffithii displayed moderate antifungal activity against Aspergillus flavus (55%) and Fusarium solani (40%). Whereas, the chloro-form fraction showed good antifungal activity against A. flavus (59%) and Fusarium solani (60%). While rest of the fractions showed no antifungal activity against the stated fungal strains (Figure 1). This confirms the folkloric uses of the species of the onosma genus in the treatment of wounds (Sezik, et. al, 1997).   Antibacterial activity  Crude methanolic extract and all fractions of O. griffithii were screened for antibacterial activity. The results are summarized in Figure 2. It is evident that against S. flex-enari the fractionated extracts showed antibacterial activity of the order as CHCl3 Fraction (33.3%) > EtOAc Fraction = Aqueous Fraction (30.55%) > n-hexane Fraction (27.77%). Against B. substillus n-hexane Frac-tion and CHCl3 Fraction showed 29.72 and 33.42 % antibacterial activity respectively. Against the S. aureus, the aqueous fraction demonstrated moderate anti-bacterial activity (42.3%) and the EtOAc fraction showed 38.46% antibacterial activity. The rest of the fractions showed no activity against the tested pathogens.    Conclusion  O. griffithii has potent antileishmanial and moderate anti-fungal and antibacterial activities that strongly encourage the activity guided isolation of biologically active com-pounds.      0102030405060708090100 % InhibitionCrude Extractn-hexane ChloroformEthyl acetateButanolAqueousImipenemB. subitilis S. flexenari S. aureus  Figure 2. Antibacterial activity of fractions of Onosma griffithii extracts    ACKNOWLEDGMENT  The author wants to thank the Sponsor Higher Education Commission, Islamabad for Research Grant No: 1-23/HEC/RandD-   2007/CASE/34-Local.   REFERENCES  Ahmad I, Anis I, Malik A, Nawaz SA, Choudhary MI (2003). Cholinesterase Inhibitory Constituents from Onosma hispida Chem. Pharm. Bull. 51(4): 412-414. Ahmad VU, Kousar F, Khan A, Zubair M, Iqbal S, Tareen RB (2005). A New Ketone and a known anticancer triterpenoid from the leaves of Onosma  limitaneum, Helv. Chim. Acta. 88(2): 309-311. Ali SI, Nasir YJ (1983). Onosma griffithii Vatke (Boraginaceae) (Eds.) In: Flora of Pakistan vol. 191, Shamim Printing Press Karachi p.103. Bashir A, Shah SMH, Bashir S, Nisar M, Chaudry MI (2007). Antibacterial and antifungal activities of Andrachne cordifolia muell. J. Enzyme Inhib. Med. Chem. 22(6): 726-729.                     Ahmad et al.         5087    Bhutto AM, Soomro FR, Katakura K (2008). Leishmaniasis in Sindh, Pakistan: Outbreak and review of literature. J. Pak. Assoc. Dermatol. 18: 212-219. El-On J, Sulitzeanu A, Shnur LF (1991). Leishmania major: resistance of promastigotes to paromomycin, and susceptibility of amastigotes to paromomycin- methylbenzethonium chloride ointment. Ann. Trop. Med. Parasitol. 85(3): 323-328. Grogl M, Schuster BG, Ellis WY, Berman JD (1999). Successful topical treatment of murine cutaneous leishmaniasis with a combination of paromomycin (Aminosidine) and gentamicin. J. Parasitol. 85(2): 354-359. Kvist LP, Christensen SB, Rasmussen HB, Mejia K, Gonzalez A (2006). Identification  and evaluation of Peruvian plants used to treat malaria and leishmaniasis. J. Ethnopharmacol. 106(3): 390-402. Dr. Ahmed T (2004). Medicinal Herbs and products, page no xxi and xxii; a printed version of herbal medicines from online drug information system: www.druginfosys.com.  Rehman AU, Choudhary MI, Thomsen WJ (1991). In: (eds.) Manual of Bioassay Techniques for Natural Product Research, Harward Academic Press, Amsterdam, p. 17. Sezik E, Silada EY, Tabata M, Honda G, Takaishi Y, Fujita T, Tanaka T, Takeda Y  (1997). Traditional Medicine in Turkey VIII. Folk Medicine in East Anatolia; Erzurum, Erzincan, Agri, Kars, Igdir Provinces Ekrem. Econ. Bot. 51: 195-212. Shahina N (2005). Structure and functionality of the pigments isolated from Onosma hispidum (ratanjot) Terminalia catappa (jangli badam) and other tropical plant. Ph. D thesis, University of    Karachi, Karachi.  Ozgen U, Houghton PJ, Ogundipe Y, Coskun M (2003). Antioxidant and antimicrobial activities of Onosma argenatum and Rubia peregrina. Fitoterapia, 74(7-8): 682-685  Tosun A, Akkol EK, Bahadır O, Yeilada E (2008). Evaluation of anti-inflammatory and antinociceptive activities of some Onosma L. species growing in Turkey. J. Ethnopharmacol. 120(3): 378-831.  Ufuk O, Maksut CK, Cavit K, Hasan SK (2004). Naphthoquinones from the Roots of Onosma argentatum Hub. Mor. (Boraginaceae). Turk. J. Chem. 28: 451-454 World Health Organization (1984). The leishmaniasis. Report of a WHO expert committee. Tech. Rep. Series WHO No 701, Geneva.          

Parasiticidal, antifungal and antibacterial activities of Onosma griffithii Vatke

https://www.ajol.info/index.php/ajb/article/download/65228/52930

Parasiticidal, antifungal and antibacterial activities of Onosma griffithii Vatke

Abstract

Similar works

Full text

Available Versions

AJOL - African Journals Online