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Studies on antimicrobial and antifungal activities of Ziziphus mauritiana against human clinical bacterial and fungal pathogens.

Introduction

Ziziphus mauritiana Lam (Family Rhamnaceae) is a common medicinal plant in the tropical and subtropical parts of the world. Commonly it is referred as Indian plump, Chinese date (Morton, 1987) or Bair in Pakistan and India. The extract of Z. mauritiana is well known for its alkaloids, flavonoids, glycosides, saponins and volatile oil (Dahiru et al., 2006), vitamin C, mucilage, protein (Adzu et al., 2003), caffeic acid, ferulic acid andp-coumaric acid (Muchuweti et al., 2005). The Z. mauritiana whole plant has an array of uses for management of ample human ailments (Dubey et al., 2010), including healing of wounds, ulcers (Adzu et al., 2001), hepatic disorders (Dahiru et al., 2005; Michel, 2002), pregnancy associated abdominal discomforts (Kaaria, 1998), gouty arthritis (Morton, 1987) and skin infections (Adzu et al., 2001), sperm immobilization (Dubey et al., 2010), antioxidant (Dahiru and Obidoa, 2008) and antitumor activity (Mishra et al., 2011). However, insufficient information is available about its antifungal and antimicrobial activities (Abalaka et al., 2010; Mahesh and Satish, 2008). This research intended to determine the antimicrobial and antifungal potential of Z. mauritiana.

Materials and Methods

Plant material. Fresh plant leaves were collected locally in district D.I.Khan, Khyber Pukhtunkhwa (KPK) Pakistan. Plant identification was established in Department of Pharmacognosy, Faculty of Pharmacy, Gomal University, where the plant specimens were deposited.

Preparation of crude extracts. The plant material (leaves) were collected at early morning hours and subjected to sun shade drying (15-20 days). The leaves were subsequently powdered by Wiley Mill (300 mm mesh). About 200 g of dry powdered leaves were extracted with 300 mL of 95% ethanol using rotary shaker (190-200 rpm) overnight, followed by filtration and concentrating it to one-fifth of the total volume. Afterwards the dried crude extract was prepared by subjecting plant material (10g) to slow heat (oven) for 6-8 h (Vlietinck and Vanden, 1991).

Preparation of fractions. About 100 g of dried plant material was extracted with n-hexane, chloroform, butanol and methanol using soxhlet apparatus. The solvent was evaporated under reduced pressure and the semisolid mass obtained was stored in silica gel beads. (Ieven et al., 1979).

Microorganisms. Six clinical bacterial species; Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter, Staphylococcus aureus (methicilline resistant), Micrococcus luteus and one fungal strain; Aspergillus niger were used for antimicrobial and antifungal assay. These clinical strains were obtained from Pakistan Institute of Medical Sciences (PIMS) Islamabad, Pakistan. The strains were further identified and characterized in microbiology research lab (MRL) Microbiology Department, Quaid-i-Azam University-Islamabad, Pakistan. The strains were maintained on agar slants at 4 C[degrees] in Gomal Center of Biochemistry and Biotechnology (GCBB) for susceptibility tests. Microorganisms were incubated overnight at 37[degrees]C in Mueller-Hinton broth (Oxoid) at pH 7.4.

Antimicrobial screening. Agar well diffusion assay. The antibacterial activity was determined using agar well diffusion method (Boakye et al., 1977). All bacterial cultures were first grown in nutrient broth at 37[degrees]C for 24 h and turbidity of inoculum was matched with McFarland's turbidity standard (0.5 x 108 cfu/mL). The inocula of the respective bacteria were applied on to the surface of sterilized Muller Hinton agar (Oxoid) plates with a sterilized cotton swab to make certain uniform thickness of bacterial growth after incubation and sterilized cork borer (6 mm diameter) was employed to figure well on agar plates. About 100 [micro]L of each plant extract fraction was applied in respective well and plates were subsequently allowed to stay for 1-2 h at room temperature for proper diffusion. The plates were later incubated at 37[degrees]C for 18-24 h and observed for zone of inhibition (mm). The streptomycin (10 [micro]g) (Oxoid) was used as reference antibiotic.

Determination of minimum inhibitory concentration.

The minimum inhibitory concentration (MIC) of the crude extract, defined as the lowest concentration of the sample, that produced visible growth (90%) of a microorganism, was determined by agar dilution method (Mukherjee, 2002; EUCAST, 2000). The sterilized Muller Hinton agar (oxoid) was allowed to cool to 50[degrees]C and about 19 mL of this was added to sterilized test tubes containing 1 mL of different concentration of crude extract. This mixture was thoroughly mixed and poured into sterilized petri plates. The concentrations of the extracts used in this test ranged from 30 mg to 0.007 mg/mL. The microbial suspension with density adjusted to 0.5 McFarland turbidity standard were inoculated (0.05 [micro]L) onto the series of agar plates using micropipette. The plates were thereafter incubated at 37[degrees]C for 24 h.

Determination of minimum bactericidal concentration. Minimum inhibitory concentration (MBC) of the selected plant parts was calculated by the viable cell count method (Toda et al., 1989), and the results were expressed as number of viable cells as a percentage of the control.

Screening for antifungal activity. The requisite amount of fungal strain (A. niger) was suspended in 2 mL of Sabauraud dextrose broth. This suspension was consistently stretched on petri plates containing Sabauraud dextrose agar media using sterilized cotton swabs. Wells were prepared using sterilized cork borer (6 mm). The crude plant extracts (100 [micro]L) were applied into the wells using micropipette and incubated at 25[degrees]C for 3 days. The plates were then examined for zones of inhibition around each well. Itraconazole (30 mg/mL) was used as a positive control.

Results and Discussion

The antimicrobial resistance pattern of all human clinical bacterial and fungal pathogens was determined (Table 1) that are admissible of multidrug resistance (MDR). The results obtained in the present study manifested that the butanol fraction showed a considerable antimicrobial activity against M. luteus and possessed very little or no activity against other bacterial strains. Conversely the methanol and chloroform fractions proclaimed proportionately ailing antimicrobial activities against M. luteus but little or no activity against other bacterial strains. None of crude fractions were reported as active against E. coli and P. aeruginosa (Table 2). With the analogous susceptibility pattern, consequent MIC and MBC of all fractions were disseminated as significantly high (15-30 mg/mL) (Table 3-4).

The data pertaining to susceptibility of all four crude extract fractions of Z. mauritiana leaves against A. niger affirmed potential antifungal activities, with the exception of the methanol fraction (0.00 mm). The n-hexane fraction demonstrated aberrant zones of inhibition (11 [+ or -] 0.05) at concentration of 30 mg/mL followed by butanol (8.0 [+ or -] 0.02) and chloroform (7 [+ or -] 0.02) fraction (Table 5).

The bacterial and fungal infections are threatening human health around the globe regardless of the emergence of new therapeutic regimens to address such infections (Youngabi et al., 2000). People all over the world are spinning back towards the use of medicinal plants to manage the situation anticipated by development of resistance in microorganism. Like many medicinal plants numerous therapeutic claims are associated with Ziziphus species that is an integral part in Ayuveda (Dafni et al., 2005).

During present investigation the susceptibility test outcomes established the butanol fraction as most active fraction in comparison with other fractions (hexane, chloroform and methanol) against all clinical strains whose resistance pattern has not been established previously. Nearly all fractions exhibited placid activity against M. luteus while very little or no activity was observed against E. coli, K. pneumoniae, P. aeruginosa, Enterobacter and S. aureus (methicillin resistant). These provisions are not surprising in analogy with earlier reports (Abalaka et al., 2010; Mahesh and Satish, 2008). Nevertheless, the non responsiveness of majority of clinical strains to crude leaf fractions is an apparent manifestation of sprite of resistance among clinical isolates (Bouchillon et al., 2004) as apparent from resistance pattern of clinical strains (Table 1). The consequent bactericidal and bacteriostatic concentrations (MIC and MBC) reported were radically higher against and considered as indicators of presence of less potent active constituents in crude leaf fractions (Adamu et al., 2000). These observations limit the use of Z. mauritiana leaf extract confronting multiresistant bacterial pathogens (Bouchillon et al., 2004).

A peer literature review reveals little knowledge covering antifungal activities of Ziziphus species (Mahesh and Satish, 2008). Conversely unlike inadequate antibacterial activities, nearly all fractions of Z. mauritiana L. illuminated comparatively enhanced antifungal activities with exception of methanol fraction. These findings are comparatively better than earlier reports (Abalaka et al., 2010; Mahesh and Satish, 2008; Adamu et al., 2006).

Conclusion

The findings of present research ascertain the limited medicinal potential of Z. mauritiana against human bacterial and fungal pathogens.

References

Abalaka, M.E., Daniyan, S.Y., Mann, A. 2010. Evaluation of the antimicrobial activities of two Ziziphus species (Ziziphus mauritiana L. and Ziziphus spinachristi L.) on some microbial pathogens. African Journal Pharmacy and Pharmacology, 4: 135-139.

Adamu, H.M., Abayeh, O.J., Ibok, N.N.E., Kafu, S.E. 2006. Antifungal activities of extracts of some cassia detarium and Zizipus species against dermatophytes. Natural Products Radiance, 5: 357-360.

Adamu, H.M., Bilar, A., Odis, M., Matchave, C. 2000. Antimicrobial activity and phytochemical screening of some selected medicinal plants in Buchi (Nigeria). Journal of Economics and Taxonomic Botany, 24: 123-127.

Adzu, B., Amos, S., Amizan, M.B., Gamaniel, K. 2003. Evaluation of the antidiarrhoeal effects of Ziziphus spina-christi stem bark in rats. Acta Tropica, 87: 245-250.[right arrow]

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Boakye, Y.K., Fiagbe, N., Ayim, J. 1977. Antimicrobial properties of some west African medicinal plants. Journal of Natural Product, 40: 543-545.

Bouchillon, S.K., Jonson, B.M., Hoban, D.J., Johnson, J.L., Dowzicky, M.J., Wu, D. H., Visalli, M.A., Bradford, P.A. 2004. Determining incidence of extended spectrum [beta]-lactamase producing Enterobacteriaceae, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus in 38 centers from 17 countries: the PEARLS Study 2001-2002. International Journal of Antmicrobial Agents, 24: 119-124.

Dafni, A., Levy, S., Lev, E. 2005. The ethnobotany of Christ's Thorn Jujube (Ziziphusspina-christi) in Israel. Journal of Ethnobiology and Ethnomedicine, 1: 8.

Dahiru, D., Obidoa, 0.2008. Evaluation of the antioxidant effects of Ziziphus mauritiana Lam. Leaf extracts against chronic ethanol induced hepatotoxicity in rat liver. African Journal Traditional Complementary and Alternative Medicine, 5: 39-45.

Dahiru, D., Sini, J., Jhon, M., John, A.L. 2006. Anti-diarrhoeal activity of Ziziphus mauritiana root extract in rodents. African Journal of Biotechnology, 5: 941-945.

Dahiru, D., William, E.T., Nadro, M.S. 2005. Protective effect of Ziziphus mauritiana leaf extract on carbon tetrachloride-induced liver injury. African Journal of Biotechnology, 4: 1177-1179.

Dubey, R., Kushagra, D., Sridhar, C., Jayaveera, K.N. 2010. Sperm immobilization activity of aqueous, methanolic and saponins extract of bark of Ziziphus mauritiana. Der Pharmacia Sinica, 1: 151-156.

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Ieven, M., Berghe, V., Mertens, D.A., Vlietinck, F., Lammens, A.E. 1979. Screening of higher plants for biological activities. Planta Medica, 36: 311-321.

Kaaria, 1.1998. Seed production, dispersal and germination in Cryptostegia grandifolia and Ziziphus mauritiana, two invasive shrubs in tropical woodlands of Northern Australia. Australian Journal of Ecology, 21: 324-331.

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Michel, A. 2002. Tree, Shrub and Liana of West African Zone. 440 pp., Margraf Publishers GMBH, Paris, France.

Mishra, T., Madhu, K., Aruna, B. 2011. Anticancer potential of aqueous ethanol seed extract of Ziziphus mauritiana against cancer cell lines and ehrlich ascites carcinoma. Evidance based complementry and Alternative Medicine, 6: 1-11.

Morton, J. 1987. Indian Jujube. In: Fruits of Warm Climates. J. F. Morton (ed.), Miami, Florida. 272-275. Last updated: 23/4/2004. Accessed on 5/5/2004 at http://www. Indian Jujube. htm.

Muchuweti, M., Gretchen, Z., Ashwell, R.N., Abisha, K. 2005. Sugars, organic acid and phenolic compounds of Ziziphus mauritiana Fruit. European Food Research and Technology, 221: 570-574.

Mukherjee, P.K., 2002. Quality Control of Herbal Drugs, an Approach to Evaluation of Botanicals. 256 pp., Business Horizons, New Delhi, India.

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Youngabi, K.A., Dhukku, U.H., Agho, M.O., Chindo, I.H. 2000. Studies on the antifungal properties of Urtica dioica urticaceae (stinging nettle). Journal of Phytomedicine and Therapeutics, 5: 39-43.

Adnan Amin (a) *, Swahid Shah (a), Saadia Andaleeb (b), Muhammad Mohibullah Khan (c) and Muhammad Ayaz Khan (a)

(a) Gomal Center of Biochemistry and Biotechnology (GCBB), Gomal University, D.I. Khan, KPK, Pakistan

(b) NCVI, NUST, Islamabad, Pakistan

(c) Department of Plant Breeding and Genetics, Faculty of Agriculture, Gomal University, D.I. Khan, KPK, Pakistan

(received April 20, 2011; revised November 3, 2011; accepted March 2, 2012)

* Author for correspondence; E-mail: adnan_amin@gu.edu.pk, dani_amin79@yahoo.com
Table 1. Resistance pattern of human clinical bacterial
and fungal pathogens

Microorganism              Resistance pattern

Bacterial pathogen
  Staphylococcus aureus    MET,AML,ATM,AMC,CRO,CIP,CE,
  Micrococcus luteus       MET,AML,FEP,STP,ATM,AMC,CRO
  Escherichia coli         AML,FEP,STP,AMC,CRO,CE,ATM,MET
  Pseudomonas aeroginosa   AML,STP,FEP,ATM,AMC,CRO
  Enterobacter             STP,ATM,AMC,CRO,MET
  Klebsiellapneumoniae     AML,STP,FEP,ATM,AMC,CRO,CE
Fungal pathogen
  Aspergillus niger        KET, MY

AMC = amoxicillin/clavulanic acid; AML = amoxicillin;
ATM = azteronam; CE = cephradine; CIP = ciprofloxacin;
CRO = ceftrioxone; FEP = cefepime; KET = ketoconazole;
MET = methicillin; MY = myconazole; STP = streptomycin

Table 2. Zone of inhibition of Z. mauritiana crude extract
fractions against microorganisms

Fraction       Ec                   Ps

               Zone of inhibition (mm)

Methanol       na                   na
Butanol        na                   na
n-Hexane       na                   na
Chloroform     na                   na
Streptomycin   9.0 [+ or -] (0.1)   9 [+ or -] (0.0.5)

Fraction       Kp                     Ent

               Zone of inhibition (mm)

Methanol       na                     na
Butanol        2.03 [+ or -] (0.05)   2.0 [+ or -] (0.05)
n-Hexane       na                     2.0 [+ or -] (0.05)
Chloroform     2.06 [+ or -] (0.1)    2.0 [+ or -] (0.05)
Streptomycin   11 [+ or -] (0.1)      10 [+ or -] (0.1)

Fraction       Sta                    Ml

               Zone of inhibition (mm)

Methanol       na                     7.03 [+ or -] (0.05)
Butanol        2.0 [+ or -] (0.05)    15.0 [+ or -] (0.02)
n-Hexane       2.03 [+ or -] (0.05)   2.0 [+ or -] (0.11)
Chloroform     na                     7.0 [+ or -] (0.05)
Streptomycin   12 [+ or -] (0.1)      12 [+ or -] (0.1)

Ec = E. coli; Ps = P. aeruginosa; Kp = K. pneumoniae; Ent =
Enterobacter; Sta = S. aureus; Ml = M. luteus (methicilline
resistant); na = not active

Table 3. Minimum inhibitory concentrations (MIC) of
Z. mauritiana fractions

Fraction       Ec                  Ps

               Microorganism/MIC (mg/mL)

Methanol       na                  na
Butannol       na                  na
n-Hexane       na                  na
Chloroform     na                  na
Streptomycin   0.93 [+ or -] (0)   0.93 [+ or -] (0)

Fraction       Kp                  Ent

               Microorganism/MIC (mg/mL)

Methanol       na                  na
Butannol       30 [+ or -] (0)     30 [+ or -] (0)
n-Hexane       na                  >30 [+ or -] (0)
Chloroform     >30 [+ or -] (0)    >30 [+ or -] (0)
Streptomycin   0.46 [+ or -] (0)   0.46 [+ or -] (0)

Fraction       Sta                 Ml

               Microorganism/MIC (mg/mL)

Methanol       na                  30 [+ or -] (0)
Butannol       30 [+ or -] (0)     15 [+ or -] (0)
n-Hexane       30 [+ or -] (0)     30 [+ or -] (0)
Chloroform     na                  30 [+ or -] (0)
Streptomycin   0.93 [+ or -] (0)   0.46 [+ or -] (0)

Ec = E. coli; Ps = P. aeruginosa; Kp = K. pneumoniae;
Ent = Enterobacter; Sta = S. aureus; Ml = M. Luteus
(methicilline resistant); na = not active

Table 4. Minimum bactericidal concentrations (MBC)
of Z. mauritiana fractions

Fraction       Ec                  Ps

               Microorganism/MBC (mg/mL)

Methanol       na                  na
Butannol       na                  na
n-Hexane       na                  na
Chloroform     na                  na
Streptomycin   3.72 [+ or -] (0)   3.72 [+ or -] (0)

Fraction       Kp                  Ent

               Microorganism/MBC (mg/mL)

Methanol       na                  na
Butannol       >30 [+ or -] (0)    >30 [+ or -] (0)
n-Hexane       na                  >30 [+ or -] (0)
Chloroform     >30 [+ or -] (0)    >30 [+ or -] (0)
Streptomycin   1.84 [+ or -] (0)   1.84 [+ or -] (0)

Fraction       Sta                 Ml

               Microorganism/MBC (mg/mL)

Methanol       na                  >30 [+ or -] (0)
Butannol       >30 [+ or -] (0)    15 [+ or -] (0)
n-Hexane       >30 [+ or -] (0)    >30 [+ or -] (0)
Chloroform     na                  >30 [+ or -] (0)
Streptomycin   3.72 [+ or -] (0)   1.84 [+ or -] (0)

Ec = E. coli; Ps = P. aeroginosa; Kp = K. pneumoniae;
Ent = Enterobacter; Sta = S. aureus; Ml = M. luteus
(methicilline resistant)

Table 5. Antifungal activities of Z.
mauritiana fractions against A. niger

Fraction       Zones of inhibition (mm)

Methanol       0.00
Butannol       8.0 [+ or -] (0.02)
n-Hexane       11 [+ or -] (0.05)
Chloroform     7 [+ or -] (0.02)
Itraconazole   12 [+ or -] (0.05)
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Author:Amin, Adnan; Shah, Swahid; Andaleeb, Saadia; Khan, Muhammad Mohibullah; Khan, Muhammad Ayaz
Publication:Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences
Article Type:Report
Geographic Code:6NIGR
Date:Mar 1, 2012
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