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Expediency of Flea Tree (Albizia lebbeck L.) in the prevention of known human fungal and bacterial strains

Albizia lebbeck L. is one of the best medicinal plants in the province of Khyber Pakhtunkhwa Pakistan. It possesses different properties but the most important one, it is used in local remedies. The antibacterial and antifungal properties of different fractions were obtained from the selected plant studied. For Antibacterial activity, four strains of bacteria were selected which cause different diseases in humans, these bacteria were Streptococcus mutans, Serratia marcescens, Staphylococcus aureus, and Methicillin resistance staphylococcus aureus. The extract obtained from the Albizia lebbeck L.were fractionated into Chloroform, methanolic, n-hexane, Ethyl acetate, and Aqueous fractions. The highest antibacterial activity was shown by methanolic extracts (3848%) and the lowest activity by an aqueous fraction (19-36%). While in antifungal activity the fractions were tested against four fungal species including Polyspndylium pallidum, Aspergillus flavus, Alternaria alternate, and Fusarium oxysporum. The aqueous extract shows the lowest activity with minimum efficacy in comparison to the standard drug (20-30%), Crude methanolic extract shows a high zone of inhibition followed by n-hexane. The study concluded that A. lebbeck have significant antimicrobial potential and might be helpful in antibiotics.


Introduction
It is globally accepted that the first medicine in human history was the medicinal plants [1]. Different people of a different time with the help of writing or in other ways disclosed the therapeutic properties of medicinal plants [2]. They have many properties in clinics from cough to cancer. From the 18 th century, the pharmacologist was busy to find medicinal plants and knowledge about their constituents to use against different diseases. They consist of two types of metabolites i.e. primary and secondary metabolites [3,4]. The primary metabolites help in growth and development while secondary metabolites run many biochemical pathways and give therapeutic property to plants [5]. The fractions obtained from the selected plant show the therapeutic properties against different pathogens including bacteria and fungi [6]. The medicinal plants were used in different parts of the world but in Asia, it is used about 80%. It is also used in Latin America and Africa with minimal side effects [7]. According to some research such as the World health organization (WHO), in the whole world population, about 80% used traditional medicine which is prepared from medicinal plants [8]. For millennia it is known that medicinal plants have the potential to fight against microorganisms and people of different countries especially China, India, and the northeast were initially known to prepare drugs from the medicinal plants [9]. But with the passage of time and the failure of therapies these drugs were failed against microbes. After that different precuts were used against them which shows a very significant role against them. Fungi and Bacteria have a dual threatening effect because they not only diseased our economic crops but also causing toxicities and allergies. Fungicides that fight against fungal diseases prepared from medicinal plants [10]. In this regard, the present study was held to find anti-fungal and anti-bacterial activity. Albizia lebbeck L. is a dioecious plant with compound leaves fruit is flat and oblong and distributed in Australia, Asia, Africa and South America [11]. The seed is round and colorful and usually grown as a wild plant. It also possesses therapeutic properties. The fractions obtained from the plant showed antifungal and antibacterial activity. These different extracts such as methanolic and Ethanolic fractions possess different properties against certain diseases such as anticancer, hypoglycemic, antiprotozoal, and anti-fertility effect [9,12]. While on the other hand traditionally it is used against pain, inflammatory, analgesic [13]. The drugs which are prepared from the plants have the potential to use against certain diseases of human beings which are caused by microorganism especially bacteria and fungi [14].

Materials and Methods Collection and Processing of the Plant Specimen
A. lebbeck L. collected from different areas of Peshawar, KP, Pakistan. After collection, the plants were washed with water, dried, in newspapers to absorb moisture from the plants. For further drying, they were placed in the oven for 24hrs under 40 0 C. When the plants were completely dried they were ground by grinder for powder formation. The powder was placed in a dried and cool area for further activities.

Preparation of Extract and Fractionation
Active metabolites were extracted using the cold maceration method. In two liters' ethanol one and a half kilogram of powdered which was made from the selected plant was dipped and incubated at 25°C. After that, the filtrate obtained by filtration of mixture thrice using filter paper. For evaporation then it was subjected to a rotary evaporator at 40°C. Then it is again dried and dissolved in 100ml of distilled water. For fractionation different organic solvents such as ethanol, methanol, and n-hexane were used. All the fractions thus obtained were concentrated by rotary evaporator and designated as solventextracted fractions [15].

Test Microorganism
A total of four bacterial and four fungal species were tested. The bacteria species Streptococcus mutans, Serratia marcescens, Staphylococcus aureus, and Methicillin resistance staphylococcus aureus (MRSA) which cause diseases in human beings and the fungal species are Polyspndylium pallidum, Fusarium oxysporum, Alternaria alternate and Aspergillus flavus [15].

Antibacterial bioassay
Antibacterial activity of crude extracts was carried out in Petri dishes by disc diffusion method [12]. 6mg/ml of dilute culture was transferred to the petri dish and placed at 37°C for 30 min. After transferring 8mm diameter disc was used for soaking of differently concentrated solutions. For positive control, Gentamicin, 20 µg, and Gatifloxacin 20 µg was used while for negative control soaked disc with distilled water is used. The disc was placed in plates and incubated for 24 hr at 37°C. The inhibition zone was measured after the completion of incubation [15]. Antifungal bioassay During antifungal activity four fungal strains Polyspndylium pallidum, Fusarium oxysporum, Alternaria. alternate and Aspergillus flavus were studied. Sabouraud agar medium was used in Petri dishes for such activity. 6mg/ml fractions were poured into each plate and the same size disc which was used for antibacterial activity was used. For incubation, such plates were placed for one day at human body temperature (37°C). When the incubation period was completed inhibition zone was measured [16].

Statistical analysis
The statistical analysis was done through ANOVA and SPSS data analysis.

Results
The focused antimicrobial activities of different fractions of A. lebbeck L. revealed momentous effects against different bacterial and fungal strains of humans. The biochemical must be followed to isolate substantial compounds for the preparation of antibiotics [17]. For this purpose, antimicrobial activities were carried out.

Antibacterial activity
In our results the antibacterial properties ( Fig.  1 & 2)  The statistical analysis of data shows that one factor ANOVA of antibacterial contains the value of P= 1.94x 10 -09 which is very much significant. Antifungal Activity of Albizia lebbeck L.
The statistical analysis of data shows that one factor ANOVA of antifungal contains the value of P= 2.71x 10 -10 which is very much significant. The trend of results showed that efficacy against selected was more dependent on the kinds of fraction rather than the type of pathogen. In our results, the antifungal properties (Fig. 3)

Antibacterial activity
The selected bacterial pathogens were Serratia marcescens, Staphylococcus aureus, Methicillin resistance staphylococcus aureus (MRSA), and Streptococcus mutans. A 10 mg/ml of the selected medicinal plant fractions were tried out on selected pathogens. The results indicated that all fractions of the selected plant were significantly effective against the selected human pathogens. The statistical analysis of data shows that one factor ANOVA of antibacterial contains the value of P= 1.94x 10 -09 (Table 1) which is very much significant. The trend of results showed that efficacy against selected was more dependent on the kinds of fraction rather than the type of pathogen.

Antifungal activity
The fractionated extracts were tested on common human bacterial and fungal pathogens, which were collected from hospitals. The selected bacterial pathogens were Polyspndylium pallidum, Fusarium oxysporum, Alternaria. alternate and Aspergillus flavus . A 10 mg/ml of the selected medicinal plant fractions were tried out on selected pathogens. The results indicated that all fractions of the selected plant were significantly effective against the selected human pathogens which are following findings of [18]. The statistical analysis of data shows that one factor ANOVA of antibacterial contains the value of P= 1.94x 10 -09 which is very much significant. The trend of results showed that efficacy against selected was more dependent on the kinds of fraction rather than the type of pathogen [19,20]. The statistical analysis of data shows that one factor ANOVA of antifungal contains the value of P= 2.71x 10 -10 ( Table 2) which is very much significant [21-23]. The trend of results showed that efficacy against selected was more dependent on the kinds of fraction rather than the type of pathogen [24,25]. In conclusion, it is very much clear now the plant has a strong antimicrobial potential [26] and has very significant results against bacteria and fungi.