Antimicrobial activity of Polygonium hydropiper leaves against plant and human pathogenic fungal and bacterial strains

The present study was conducted to investigate the antimicrobial activities of Polygonium hydropiper L. leaves extract against bacteria and fungi using the disc diffusion method. The extract showed significant antibacterial activities against four strains of gram positive bacteria namely Clavibacter, Staphylococcus aureus, Lactococcous lactis and Bacillus clausii species and four strains of gram negative bacteria namely Escherichia coli, Citrobacter, Proteus and Xanthomonas species. The minimum inhibitory concentration (MIC) values against the bacterial culture were ranged from 16 to 64 ug mL. The antifungal activities were found against six toxic fungal strains namely Penicillium, Fusarium, Trichoderma, Aspergillus niger, Pythium, and Acremonium. It was concluded that Polygonium hydropiper methanolic leaves extract acted as an antibacterial and antifungal agent and can be used in wide range of pharmaceuticals industries. Such as Quinine was the first natural product to be produced on industrial scale for its use against malaria.


Introduction
Polygonium Hydropiper .L. belongs to the family "Polygonaceae" having high medicinal value.Whole Polygonium plant contain large quantity of secondary metabolites and bioactive compound such as 4-Isobutyl-6-methyl-5-oxo-3a,4,5,7a-tetrahydro-1H-inden-13-oic acid (named viscosumic acid) and quercetin 3-O-(6"feruloyl)-beta-D-galactopyranoside, and the known 3',5-dihydroxy-3,4',5',7tetramethoxyflavone have been isolated from Polygonum viscosum and polygonic acid [1].The plant also possesses some natural compound having insecticidal characteristic.[2].Whole plant itself or in combination with some other medicinal herbs are used as remedies against various infectious diseases such as diarrhea, itching skin, dyspepsia, excessive menstrual bleeding and hemorrhoid [3].This species has high pharmacological characteristic and having economical value.Therefore utilization of these herbal plants is increasing day by day among the people.The rate of the pathogenic infectious diseases is increasing day by day worldwide because of exposure to the adverse atmosphere in developing countries and therefore mortality rate is also high in these countries.Thus it is need of the day to make remedies against these pathogenic diseases [4].In order to get rid from this infectious disease many discoveries have been made by using plants against microbial activities on local level as an alternate of synthetic medicines [5].The present study was conducted with the aims to investigate the antimicrobial activities of methanol extract of Polygonium hydropiper leaves.

Plant material collection
The leaves of the Polygonium hydropiper (L) were collected from Tehsil Muree, District Rawalpindi, Punjab, Pakistan.The leaves of the plant were identified at Department of Agronomy Faculty of Agriculture Gomal University Dera Ismail Khan.

Plant material extraction
The leaves were washed with tap water and then were cut with manual cutter and dried at 50 o C [6].After drying they were ground with help of grinding machine and powdered sample was stored in an air tight container.The powder of the leaves sample (250 g) was extracted through cold extraction method by using methanol (1 L) in flat bottom flask.When the mixture was completely dissolved, it was filtrated using Watsmann filter paper and the filtrate was separated from solvent using rotary evaporator to get the crude extract Filter disc (6mm) were placed on the petri dishes pre-inoculated with bacterial pathogen.The plant methanol extract were poured onto the filter disc.Dimethyl sulphoxide (DMSO) was used (10µg/µL) as negative control against the tested bacterial strain while the Vibromycin (30µg/disc) was used as positive control.The experiment was repeated three times.All the Petri plates were held for 20 min at room temperature for the diffusion of the sample extract and then kept into an incubator for 24hours at 37 0 C for bacterial growth.The zone of inhibition was calculated after 24hrs [9].

Antifungal screening
The antifungal screening was performed using disc diffusion method.[10].The antifungal activity was carried out against four pathogenic strains of the fungi at the concentration of 150µg/disc and 300µg/disc.Dimehtyl sulphoxide (DMSO) 10ug/mL was used as negative control while acrobate 20ug/disc was used as positive control against four strains of the fungus.All the petri dishes were held for 20 min at room temperature till to complete diffusion of the extract into the disc and then incubated at 27 o C for 72 hours for fungal growth.The antifungal effect was determined by zone of inhibition and was measured with the help of ruler.

Minimum inhibitory concentration
The minimum inhibitory concentration of the plant extract was determined by the method described [11].In order to calculate the MIC the extract of the sample was diluted with nutrient broth of the concentration 50, 25, 12.5, 6.75, 3.12 and 1.56 mg/mL prepared from stock solution (500mg/mL) in test tubes and the control tubes were also prepared.The tubes were incubated at 37 o C for 24 hours.The minimum concentration of the leaves extract where no bacterial growth was seen was considered as MIC.

Antibacterial screening
The antibacterial screening of methanol extract were presented in Table 1.The maximum MIC (24.2 mm) of plant methanol extract was recorded against Calvibacter while lowest MIC (21.1 mm) was observed against Proteus at 300µg/disc.The concentration 150ug/disc showed maximum activity (16.3 mm) against Bacillus clausii while minimum zone of inhibition (12.3 mm) was found against Citrobacter.When compared with standard (Vibromycin) at concentration of 30µg/disc.It was noticed that the standard (Vibromycin) showed maximum zone of inhibition (4µg/mL) against gram positive bacteria Bacillus clausii and Lactococcus followed by Styphylococcus aureus and Clavibacter (2µg/mL).Similarly it was also found that Standard (Vibromycin) showed maximum inhibitory zone against gram negative bacteria Citrobacter and Proteus (8µg/mL) followed by Xanthomonas sp and Escherichia Coli at 4µg/mL.In the present research it was also observed that methanol extract showed good result against positive and negative bacterial strains and confirmed the investigation of the previous findings.The figure one shows the zone of inhibition of antibacterial activities.(Figure 1) [12].The Dimethylsulphooxide was used negative control having no zone of inhibition.All the data are as mean of three test± SE of the standard grou

Antifungal screening
The antifungal screening of the methanolic extract of Polygonium hydropiper L leaves were investigated and results are presented in Table 2. Two different concentrations 150µg/disc and 300µg/disc were tested against six fungal strains i.e.Penicillium, Fusarium oxysporum, Trichoderma, Aspergillus niger, Pythium, and Acremonium.The highest activity (18.8 mm) was observed against Fusarium oxysporum followed by Pythium (18.3 mm) and Penicillium while the minimum zone of inhibition (15.2±0.4) was showed by Acremonium at 300ug/disc.The antifungal activity was decreased by decreasing the amount of concentration for all the six fungal species.It was observed that the extract showed maximum activity (13.3±0.0)against Aspergillus niger while it showed minimum activity (11.0±0.3)against Pythium at 150µg/disc.The standard Vibromycin (20µg/disc) significantly inhibited the growth of all the six different fungi.Previously literature showed that activities of the various extract have been carried out against various strains of pathogenic fungus [13].Overall result showed that leaves extract of Polygonium hydropiper has significant effect against tested fungal strains.The figure one shows the zone of inhibition of antifungal activities.(Fig.2).

Table 2. Results of antifungal activity of Polygonium hydropiper leaves Diameter of zone of inhibition (nm)
The Dimethylsulphooxide was used Positive control having no zone of inhibition.All the data are represented as mean of three test± SE of the standard group

Minimum Inhibitory Concentration Calculation
Data in Table 3 represent the Minimum Inhibitory Concentration (MIC) value of the sample plant extract against selected strains of bacteria which were 16, 32, 16, 16, 32, 32 and 64µg/ml against bacterial culture.The minimum inhibitory concentration against four gram positive tested bacterial strains were ranged from 16µg/ml to 32µg/ml and then (MIC) value against the four strains of gram negative bacteria were ranged from 16µg/ml to 64µg/ml.The (MIC) value of the extract showed that methanol extract of leaves of Polygonium hydropiper L is more effective against positive bacterial cultural as compared to the gram negative bacteria at minimum concentration.The figure two shows MIC value of MeOH extract against bacterial isolates.(Figure 2).

Figure 2 .
Figure 2. Minimum inhibitory concentration of MeOH extract against bacterial isolates