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The biological performance of Crataegus songarica against certain infectious fungal and bacterial diseases.

Introduction

Numerous sources have been practiced to find out new antimicrobial compounds such as micro-organisms from animals and plants and their compounds [1]. During the course of time, biological control has gained incredible significance over synthetic antimicrobials [2]. Since long, medicinal plants continue to be extensively used as major sources of drugs for the treatment of many health disorders all over the world. Pakistan being rich in indigenous herbal resources offers a great scope for ethnobotanical studies [3-6]. Presently we require to convert this need able heritage of plant based therapies into practice, as Pakistani medicinal plants possesses tremendous therapeutic values [7-11] to overcome the increasing demand of indigenous populations in addition to earn foreign exchange from their export.

C. songarica is commonly known as Hawthorn member of genus Crataegus (Rosaceae) encompasses approximately 200 species. In Pakistan, it is common in Boni (Chitral), Swat, Astor, Gilgit and Muree [12]. Additionally, it also found in Afghanistan and Uttarr Pardesh 1500-2700 m [13]. The barriers of the plant possessed antihypertensive and cardio tonic potential. It improved cardiac activity in patients with congestive heart failure [14]. The antispasmodic activity of the plant has already been reported. It relaxed the uterus and intestine smoth vessel however, constricts the bronchi and coronary vessels [15]. The fruits are used as a popular remedy for diarrhea or sligh phlegmasia [16].

It may be used as tincture and has also got antioxidant properties [17]. In the Arab traditional medicine, leaves and unripe fruit has been formulated in the form of decoction for the treatment of cancer, diabetes and sexual weakness [18]. Phytochemically, different groups of compounds have been reported such as vitamin C, flavonoids, glycosides, anthocynaidins, saponins, tannins, antioxidants and phenolics [19,20]. Keeping in view the strong pharmacological and phytochemical backgrounds therefore the current study was aimed to investigate the antibacterial and antifungal activities.

Materials and Methods

Plant material

C. songarica as a whole plant was collected from Upper Boni (Chitral), Khyber Pakhtonkhawa (Pakistan) during the month of October-November 2005. The plant material was authenticated by Prof Dr Abdul Rashid, plant taxonomist Botany Department University of Peshawar.

Extraction

After the preliminary necessary treatments like collection, drying under-shad for three weeks, the plant sample was cut into small pieces and pulverized in to a fine powder. The powdered of fruits of plant material (Berries 10 kg) was macerated in distilled ethanol (80% v/v) with infrequent stirring, at ambient temperature. After 2 weeks, the extract soluble in ethanol was filtered off through filter paper. The procedure was done in triplicate and the filtrate was concentrated under vacuum at low temperature (45[degrees]C) using a Buchi rotary evaporator to offered a dark brown extract. The crude ethanolic extract (40 g) was dissolved in distilled water and sequentially extracted with hexane (11% w/w), chloroform (31.9% w/w), n-butanol (38.8% w/w) and finally the water (18.2% w/w) fraction was obtained.

Microorganisms

The reference bacterial strains in the test were E. coli ATCC 25922, B. subtilis ATCC 6633, S. flexeneri (clinical isolate), S. aureus ATCC 25923, P. aeruginosa ATCC 27853 and S. typhi ATCC 19430. The tested fungal strains includes T. longifusus (clinical isolate), C. albicans ATCC 2091, A. flavus ATCC 32611, M. canis ATCC 11622, F. solani 11712 and C. glaberata ATCC 90030. The pathogens were maintained on agar slant at 4[degrees]C. They were activated at 37[degrees]C for 24 h on nutrient agar (NA) or Sabouraud glucose agar (SGA) respectively for bacteria and fungi, prior to any screening.

Antibacterial activity

The crude extract/fractions were screened for antibacterial potential against certain pathogens (E. coli, B. subtilis, K. pneumonae, S. flexenari, S. aurous and S. typhi) by agar using well diffusion method [21]. In the procedure, nutrients broth (10 mL) was inoculated with the test organism and incubated at 37[degrees]C for 24 h. With the help of a sterile pipette, the broth culture of the test organism (0.6 mL) was introduced to a 60 mL of molten agar, which had been cooled to 45[degrees]C the reaction components were mixed and introduced into a sterile petri dish (for the 9 cm Petri dish, 0.2 mL of the culture was added to 20 mL of agar). Three plates were used for test each organism. The agar was permitted to set and become firm. In the medium, the requisite number of wells was dug by means of a sterile metallic cork borer guaranteed appropriate division of the wells in the side-lines and one in the center. Agar stoppers were detached. Stock solutions of the test samples (1 mg/mL) were prepared in the sterile dimethyl sulfoxide (DMSO) and 100 |iL and 200 ||L of each dilution was added in their respective wells. DMSO was used as control while Imipinem as a standard drug in the final concentration of 100 ||L and 200 ||L in each well was employed as standard drug. For diffusion of the samples, the plates were kept at room temperature for 120 min mid incubated face upwards at 37[degrees]C for 24 h. The activity was noted by measuring diameter of the inhibition zone (mm).

Antifungal activity

The antifungal activity of the crude extract/fractions of C. songarica berries were studied using agar tube dilution method [1,22]. The samples in the concentrations of 24 mg/ mL were dissolved in the sterile dimethyl sulfoxide (DMSO). 32.5 g sabouraud, 4% glucose agar and 4.0 g of agar-agar in 500 mL were mixed with distilled water for the preparation of sabouraud dextrose agar (SDA) as medium on a magnetic stirrer. The SDA (4 mL) was spread on screw cap tubes which were autoclaved (120[degrees]C for 15 min) followed by cooling to 15[degrees]C. The uncongealed SDA media was treated with stock solution (66.6 [micro]L) in order to get the final samples concentration of 400 [micro]g per mL of SDA. Later on, the tubes were permitted at room temperature to congeal in the slanted position. The tubes were inoculated with a piece (4 mm diameter) of inoculums obtained from a week old culture of fungi for non-mycelial growth; using an agar surface streak. In the assay, DMSO was used as control while Amphotericin B and Miconazole were standard drugs. After one week incubation at 28 [+ or -] 1[degrees]C and humidity (40-50%), zone of inhibition was calculated.

Determination of MIC (macrodilution method)

In 96 well microplate, samples in the concentrations of 10 mg/mL were dissolved in DMSO followed by serial dilution with sterile water placed in a laminar flow cabinet. Each well was filled with an equal volume of an actively growing culture of the test pathogen. The cultures were grown for 12 h in 100% relative humidity at 37[degrees]C. Each well was added tetrazolium violet and growth was shown by a violet color of the culture. MIC was rated as the lowest concentration of the test solution that inhibited absolute growth [23]. Acetone was used as control that had no effect on the growth even at the highest concentration. Imipinem, standard drugs as shown 1 and 2 Amphotericin B and Miconazole were used as standard drugs.

Results

Effect of antibacterial assay

Antibacterial activity was carried out for the crude extract and subsequent fractions. Zone of inhibition is presented in millimeters and standard drug was Imipenem as shown in Table 1. The crude extract of plant showed potential antibacterial activity against E. coli, B. subtilis and S. flexeneri with MICs 310 [micro]g/mL, 760 [micro]g/mL and 220 [micro]g/mL respectively. The n-hexane fraction was active only against E. coli with MIC 270 [micro]g/mL while the chloroform fraction was active only against B. subtilis with MIC 490 [micro]g/mL. The ethyl acetate fraction exhibited significant activity against E. coli and B. subtilis with MICs 150 [micro]g/ mL and 390 [micro]g/mL, respectively (Table 2). The n-butanol fraction was active against P. aeruginosa with MIC 540 and water fraction was active against S. flexeneri with MIC 470 [micro]g/mL

Effect of antifungal assay

The effect of antifungal activity crude extract and subsequent fractions of the plant is illustrated in Table 3. The crude extract and subsequent fractions of C. songrica showed marked antifungal activity against T. longifusus, A. flavus, M. canis and F. solani with MICs ranges from 670 [micro]g/mL to 110 [micro]g/mL (Table 4). On the other hand, both the Candida species; C. albicans and C. glaberata were neither inhibited by the crude extract nor by the fractions of the plant.

Discussion

The current study revealed significant antibacterial and fungal activity of fruits (berries) of Crataegus songarica against various pathogenic test microorganisms.

B. subtilis was the only sensitive bacterium among the tested Gram positive bacteria. Clinical studies declared B. subtilis as a nonpathogenic or less pathogenic bacterium and only few cases of its infections are reported. Researchers therefore paid little importance to the study of its resistance. However, in an immunocompromised patient recurrent septicemia has been reported due to probiotic strains of B. subtilis [24]. The extract/fractions of the plant showed significant susceptibility against B. subtilis and thus it could be a significant natural healing agent infections caused by it.

The extract/fractions of the plant were more susceptible to Gram negative tested pathogens (E. coli, S. flexeneri and P. aeruginosa). Escherichia coli (E. coli) is a very harmful human pathogen which involved in the pathogenicity of several infection namely urinary tract infections, gastroenteritis, neonatal meningitis, hemolyticuremic syndrome, peritonitis, mastitis, septicemia and Gram-negative pneumonia [25]. Shigella flexneri is a gram-negative bacterium which causes the most communicable of bacterial dysenteries, shigellosis. Shigellosis causes 1.1 million deaths and over 164 million cases each year, with the majority of cases occurring in the children of developing nations. The pathogenesis of S. flexneri is based on the bacteria's ability to invade and replicate within the colonic epithelium, which results in severe inflammation and epithelial destruction [26]. P. aeruginosa typically the causative agent of pulmonary tract, urinary tract, burns, wounds, and of the outer ear (otitis externa), and is the most frequent colonizer of medical devices (e.g., catheters) [27]. The clinical utility of synthetic antibiotic has been reduced by resistant developed against these strains [28-30]. Therefore, the significant sensitive of extract/ fractions of the fruits of the plant could offer a potential natural healing agent against infections caused by these Gram negative bacteria.

In antifungal bioassay, the crude extract and subsequent fractions of the plant offered potential activity against tested fungi including T. longifusus, Candida albicans, Aspergillus flavus, Microspoum canis, Fusarium solani and Candida glaberata as presented in Table 3. The crude extract and subsequent fractions of C. songrica showed marked antifungal activity against T. longifusus, A. flavus, M. canis and F. solani with MICs ranges from 670 [micro]g/mL to 110 [micro]g/mL. On the other hand, both the Candida species; C. albicans and C. glaberata were neither inhibited by the crude extract nor by the fractions of the plant.

Antimicrobial resistance is indeed a global challenge for clinicians. In order to address the current emergence of resistance to various bacterial and fungal strains, appropriate and rapid measures has been suggested in terms of focusing new and effective antimicrobial agents [31-34]. For this purpose, multiple agents of different sources are testing worldwide including medicinal plants being time tested and therefore could be the ideal therapeutic option.

Conclusion

It can be concluded on the basis of our findings in the present antimicrobial study that this plant species has great potential. In the light of outstanding outcomes, the plant can be subjected to further detail studies for designing clinically effective antimicrobial of plant origin especially isolation of pure secondary metabolites.

References

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Shafiq Ahmad Tariq (1) *, Muhammad Nisar (2), Haroon Khan (3) * and Muhammad Raza Shah (4)

(1) Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan

(2) Institute of Chemical Sciences, University of Peshawar, 25120, Peshawar, Pakistan

(3) Department of Pharmacy, Abdul Wali Khan University Mardan, Pakistan

(4) HEJ Research Institute, International Center for Chemical and Biological Sciences, Karachi University, Karachi, Pakistan

* Corresponding author: Dr. Haroon Khan, Principal, Department of Pharmacy, Abdul Wali Khan University Mardan, Pakistan, E-mail: hkdr2006@gmail.com

Received October 05, 2013; Accepted October 21, 2013; Published October 28, 2013

doi: 10.4172/0974-8369.1000194
Table 1: Antibacterial activity of crude extract and the fractions
of Crateagus songrica. Activity is represented in zones of inhibition
of bacterial growth (in mm).

Name of         Zones of inhibition of bacterial growth (in mm) by
Bacteria        various samples

                Std. drug   SI-1   SI-2   SI-3   SI-4   SI-5   SI-6

E. coli            24        14     14     --     17     --     --
B. subtilis        23        9      --     12     13     17     --
S. flexeneri       28        15     --     16     --     --     12
S. aureus          27        --     --     --     --     --     --
P. aeruginosa      20        --     --     --     --     11     9
S. typhi           26        --     --     --     --     --     --

Std. drug: Imipenem, SI-1 = Crude extract; SI-2 = n-hexane fraction;
SI-3 = Chloroform (basic) fraction; IS-4 = Ethyl acetate fraction;
SI-5 = n-Butanol fraction and SI-6 = Aqueous fraction.

Table 2: Antibacterial activity of crude extract and the fractions
of Crateagus songrica represented in Minimum Inhibitory Concentration
(MIC).

Name of         Minimum Inhibitory Concentration (MIC, [micro]g/mL)
Bacteria

                Std. drug   SI-1   SI-2   SI-3   SI-4   SI-5   SI-6

E. coli           0.19      310    270           150     --     --
B. subtilis       0.22      760     --    490    390    159     --
S. flexeneri      0.13      220     --    161     --     --    470
S. aureus         0.17       --     --     --     --     --     --
P. aeruginosa     0.31       --     --     --     --    540     --
S. typhi          0.17       --     --     --     --     --     --

Std. drug: Imipenem, SI-1 = Crude extract; SI-2 = n-hexane fraction;
SI-3 = Chloroform (basic) fraction; IS-4 = Ethyl acetate fraction;
SI-5 = n-Butanol fraction and SI-6 = Aqueous fraction.

Table 3: Antifungal activity of the crude extract and fractions of
Crateagus songrica represented in % inhibition of fungal growth.

Name of Fungi   % Inhibition of Fungal Growth By Various Samples

                Std. drug   SI-1   SI-2   SI-3   SI-4   SI-5   SI-6

T. longifusus     100 (1)    50     --     --     50     --     30
C. albicans       100 (1)
A. flavus         100 (2)    50     60     40     60     20     50
M. canis          100 (2)    50     80     40     10     70     50
F. solani         100 (1)    40     70     50     30     40     60
C. glaberata      100 (1)    --     --     --     --     --     --

(1) Standard Drug = Miconazole, (2) Standard Drug = Amphotericin B,
SI-1 = Crude extract; SI-2 = n-hexane fraction; SI-3 = Chloroform
(basic) fraction; IS-4 = Ethyl acetate fraction; SI-5 = n-Butanol
fraction and SI-6 = Aqueous fraction.

Table 4: Antifungal activity of crude extract and the fractions of
Crateagus songrica represented in Minimum Inhibitory Concentration
(MIC).

Name of Fungi   Minimum Inhibitory Concentration (MIC, [micro]g/mL)

                 Std.     SI-1   SI-2   SI-3   SI-4   SI-5   SI-6
                 drug

T. longifusus   1.4 (1)   230                  220           670
C. albicans     1.8 (1)
A. flavus       2.3 (2)   220    190    270    180    430    230
M. canis        1.6 (2)   230    110    260    490    150    250
F. solani       1.1 (1)   280    160    210    310    270    180
C. glaberata    0.5 (1)

(1) Standard Drug = Miconazole, (2) Standard Drug = Amphotericin B,
SI-1 = Crude extract; SI-2 = n-hexane fraction; SI-3 = Chloroform
(basic) fraction; IS-4 = Ethyl acetate fraction; SI-5 = n-Butanol
fraction and SI-6 = Aqueous fraction.
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Title Annotation:Research Article
Author:Tariq, Shafiq Ahmad; Nisar, Muhammad; Khan, Haroon; Shah, Muhammad Raza
Publication:Biology and Medicine
Article Type:Statistical data
Geographic Code:9PAKI
Date:Jan 1, 2014
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