International Journal of Drug Development and Research

Key words

Achyranthes aspera, Antibacterial activity, Antifungal activity, Disc diffusion.

Introduction

India is a land of various incredible characteristics in which one is plant diversity. Due to natural origin, cost effectiveness and lesser side effects, our traditional medicinal systems (ayurvedic, unani and siddha systems) totally depend upon medicinal plants [1]. Medicinal herbs based antimicrobial compounds have extensive sources for the development of therapeutic drugs. Various pathogenic microorganisms like bacteria, fungi etc. cause severe diseases in humans and animals in tropical and subtropical countries in world [2]. Today, due to haphazard use of various allopathic antimicrobial drugs, a problem of multiple resistance against several pathogenic strains have became developed [3, 4]. The goal of present study is to offer apt and proficient herbal antimicrobial drug to cure the recent problems of microbial infection and pathogen resistance in patients that can’t afford expensive medicines.

Achyranthes aspera Linn. (Amaranthaceae family) is an annual, stiff erect herb, found as a weed throughout India. The plant has several therapeutic properties like antiviral, anticoagulant, antihypertensive, diuretic, aphrodisiac, antifertility, antispasmodic, antitumor agent [5, 6, 7] and is also used to treat children for ‘colic’ with ‘hydrophobic’ ‘hypoglycemic’ ‘thyroid stimulating brain tonic’ [8, 9, 10, 11]. It also possesses antifungal and antibacterial activity [12] and required minimum amount of extracts to inhibit the growth of pathogenic strains [13]. Further also reported that it contain several phytoconstituents like alkaloids, flavonoids, saponins, steroids and terpenoids that exhibits several remedial properties.

Various phytoconstituents isolated from different medicinal herbs appear to be alternatives to control various resistant pathogens [14]. In view of mentioned properties, the present investigation was carried out to analyze the antibacterial and antifungal potential of different extracts of whole plant parts i.e. leaves, root, stem and inflorescences of Achyranthes aspera. The extract of whole dried plant were tested against 5 pathogenic bacterial strains i.e. Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, Shigella flexineri, Pseudomonas aeruginosa and 3 fungal pathogenic strains i.e. Aspergillus niger, Trichoderma viridae and Fusarium oxysporum by taking standard as antibiotic gentamycin disc in case of bacteria and ketoconazole disc in case of fungi.

Material and Methods

Procurement of Plant Material

Achyranthes aspera was collected in the month of January from the roadsides of the campus, Banasthali University. The plant material was taxonomically identified by Botanist of Krishi Vigyan Kendra, Banasthali, Tonk district, Rajasthan, India.

Preparation of plant extracts by sequential extraction method

All parts of the plant i.e. leaves, root, stem and inflorescence were separated from the whole plant, cleaned, dried and powdered with the help of mixer grinder separately. The powdered parts of plants were then extracted with soxhlet apparatus using various sequential solvents that is petroleum ether, benzene, chloroform, ethyl acetate and ethanol for 16 h. Aqueous extract was also prepared by maceration method. The extracts were than concentrated on a rotary evaporator below 50°C and were stored in air tight containers at 4°C temperature for further experimental studies.

Microbial strains-

The bacterial strains used for the test were Escherichia coli (MTCC NO. 119), Bacillus cereus (MTCC NO. 430), Staphylococcus epidermidis (MTCC NO. 435), Shigella flexineri (MTCC NO. 1457) and Pseudomonas aeruginosa (MTCC NO. 1688). The fungal strains were Aspergillus niger (MTCC NO. 282), Fusarium oxysporum (MTCC NO.2087) and Trichoderma viridae (MTCC NO.167). All the strains were obtained from Microbial Type Culture Collection (IMTECH, Chandigarh, India).

Culture media and inoculum preparation

Nutrient agar broth (Sisco Research laboratory pvt. Ltd., Mumbai) were used as the media for bacterial culturing and Potato dextrose brothagar (SRL, Mumbai) for fungal strains. To 1 ml of mother culture, respective bacterial strains were inoculated in nutrient broth in aseptic condition and then incubated at 37°C for 24 h. For fungal strains culturing, a loop full of all the fungal strains were inoculated in the Potato dextrose broth separately and then incubated at 28-30°C for 7-15 days.

Test Sample preparation

Three different concentrations that is 10 mgml, 12.5 mgml and 25 mgml of all six extracts (petroleum ether, benzene, chloroform, ethyl acetate, ethanol and aqueous) of all plant parts were prepared for screening of antibacterial and antifungal activity.

Screening of antibacterial and antifungal activity-

The antimicrobial activity of Achyranthes aspera was screened by disc diffusion method.

Disc diffusion method

Firstly, prepared the sterile extract disc by using Whatman No-1 filter paper, and soaked in already prepared different concentrations of extracts. Spreaded the bacterial and fungal strains on their respective agar media. Test extract loaded discs were placed on respective bacterial and fungal lawn and then incubated at suitable temperature. After incubation, different zone of inhibitions were measured and recorded. Similarly, standard antibiotic discs of Gentamycin and ketoconazole in case of bacteria and fungi respectively were used instead of test extract for comparative study of test extracts.

Results

Achyranthes aspera is an important medicinal plant. Various sequential extracts were used to determine the antibacterial and antifungal activity of plant.

Percentage Yield of the Extracts

Table 1 represented the different average extraction values (in percentage) of dried parts i.e. leaves, root, stem and inflorescence of A. aspera in different solvents from nonpolar to polar i.e. petroleum ether, benzene, chloroform, ethyl acetate, ethanol and aqueous. The values were high in aqueous for 3 parts i.e. 17.09% in root, 14.354% in stem and 8.04% in inflorescence but in case of leaves, the high value of extraction was obtained in petroleum ether i.e. 14.42%. The minimum extraction values were obtained in ethyl acetate extract of root (0.08%), in chloroform extract of leaves (1.6%), ethyl acetate extract of stem and inflorescence i.e. 0.46% and 0.24% respectively.

Antibacterial activity of leaves extracts of A. aspera

Table 2 depicted that petroleum ether extract of leaves part of A. aspera exhibited broad spectrum antibacterial potential compared to other extracts like chloroform, benzene, ethyl acetate and aqueous. All extracts of A. aspera showed high antibacterial activity against E. coli, then for P. aeruginosa, then S. epidermidis, then S. flexineri and finally for B. cereus. The different concentrations of petroleum ether extract showed predominant inhibition of gram negative bacteria E. coli (20, 26 & 31 mm for 10, 12.5 and 25 mg/ml concentrations of plant extracts respectively), P. aeruginosa (10, 9.5 & 10mm) and S. flexineri (7.5, 8.5 & 7.5mm) in comparison to gram positive bacteria strain i.e. B. cereus (8, 8 & 8mm) and S. epidermidis (7.5, 11.5 & 9.5mm). These results showed that the 25 mg/ml concentration of extract showed high antibacterial activity for both gram positive and gram negative bacteria in comparison to other concentrations.

The benzene extract showed high antibacterial activity for S. epidermidis (14.5, 9.5 and 15.5 mm for 10, 12.5 and 25 mg/ml plant extract respectively) and minimum for E. coli (gram negative). The chloroform extract had maximum efficacy to inhibit the growth of E. coli (gram negative) which showed different zone of inhibitions i.e. 20, 13.5 and 15.5 mm, then for P. aeruginosa (gram negative) i.e. 11.5, 10.5 and 11.5 mm as compared to gram positive bacterial strains, B. cereus, i.e. 6.5, 14 and 12.5 mm and S. epidermidis that showed 7, 8.5 and 9.5 mm zone of inhibitions for 10, 12.5 and 25 mg/ml concentration of plant extract respectively. The descending order was P. aeruginosa > B. cereus > E. coli > S. epidermidis > Shigella flexineri. Ethanolic and ethyl acetate extracts also showed moderate antibacterial activity. The aqueous extract of A. aspera exhibited highest zone of inhibition for B. cereus (11, 12 and 9) and minimum for E. coli (7, 8 and nil for 10, 12.5 and 25 mg/ml concentration of plant extract. When we compared the various zone of inhibitions of different extract against pathogenic strains with the standard Gentamycin antibiotic, it was shown that the complete leaves part of A. aspera exhibits potential antibacterial activity.

Antibacterial activity of root extracts of A. aspera

According to table 3, in all root extracts, only petroleum ether was effective for both gram positive and gram negative bacteria. The maximum zone of inhibition for B. cereus (gram positive) was 10.5 mm at 12.5 mg/ml concentration and minimum for also B. cereus at 10 mg/ml. Chloroform and benzene extracts showed less antibacterial activity than petroleum ether for B. cereus and S. epidermidis. Chloroform extract had no activity against P. aeruginosa but both (benzene and chloroform) had high antibacterial activity for Shigella flexineri (7.5, 9.5 and 9.5 mm for chloroform and 7.5, 10 and 7.5 mm for benzene extract at 10, 12.5 and 25 mg/ml extract concentration). The ethyl acetate and ethanol extracts showed also less activity than chloroform and benzene extracts. Aqueous extract had no bacterial activity for B. cereus, S. epidermidis and P. aeruginosa, so had less potent activity.

Antibacterial activity of stem extracts of A. aspera

Table 4 represented that the benzene extract of stem part of A. aspera exhibited maximum potential to inhibit growth of microbial strains and showed maximum zone of inhibition (9, 8.5 and 9 mm) for S. flexineri, then E. coli (both are gram negative) (9, 7 and 7 mm) but expressed less activity for gram positive bacteria i.e. 7, 7 and 7 mm and 7, 6.5 and 6.5 mm for S. epidermidis at 10, 12.5 and 25 mg/ml concentration of plant extract.

The aqueous and ethanolic extract of stem part of A. aspera showed high antibacterial activity for E. coli and S. flexineri but no activity for B. cereus and S. epidermidis and P. aeruginosa (only at concentration of 12.5 and 25 mg/ml). The petroleum ether and ethyl acetate extract showed no activity for S. epidermidis (gram positive), less activity for B. cereus (only petroleum ether) and high activity for B. cereus (only ethyl acetate).

Antibacterial activity of inflorescence extracts of A. aspera

Table 5 indicates that chloroform extract showed highest antibacterial activity against P. aeruginosa (13, 14 and 10 mm) whereas benzene extract showed minimum antibacterial activity or zone of inhibition (6, 6 and 9 mm at the concentration of 10. 12.5 and 25 mg/ml concentration of plant extract.

The ethyl acetate showed minimum antibacterial activity for E. coli and highest for S. epidermidis. So, we can say that it is effective for mainly gram positive bacteria.

Antifungal activity of leaves extracts of A. aspera

According to table 6, the ethanolic extract of A. aspera exhibited broad spectrum antifungal activity compared to other solvents like petroleum ether, benzene, ethyl acetate, aqueous and chloroform. The maximum zone of inhibition of ethanolic extract against F. oxysporum was 10.5 mm at 12.5 mg/ml concentration, for T. viridae was 8 mm at 25 mg/ml and 5 mm at 25 mg/ml for A. niger.

The minimum zone of inhibition showed by ethanolic extract was 10 mm for F. oxysporum, nil at 10 mg/ml for T. viridae (no effect) and 3.5 mm for A. niger at 10 mg/ml concentration. All extracts showed high antifungal potential against F. oxysporum. Petroleum ether extract of A. aspera showed 7.5 mm minimum zone of inhibition at 10 mg/ml concentration, benzene showed 6.5 mm at 25 mg/ml concentration, chloroform showed 7.5 mm at 10 mg/ml, ethyl acetate showed 5.5 mm at 25 mg/ml and aqueous extract showed 8 mm at 10 mg/ml concentration. Aqueous and benzene extract showed no activity against T. viridae and A. niger but petroleum ether, ethyl acetate and ethanolic extracts showed less antifungal activity but chloroform extract exhibited higher antifungal activity against T. viridae. For A. niger, ethanolic extract was highly effective than compared to other solvents.

Antifungal activity of root A. aspera extract:

Table 7 represented that all extracts showed high antifungal activity against F. oxysporum, maximum zone of inhibition was exhibited by benzene extract at 25 mg/ml i.e. 11 mm and lowest was nil means no activity by petroleum ether extract at 12.5 mg/ml concentration. Petroleum ether and benzene had no antifungal activity against T. viridae but chloroform and aqueous extracts had little activity but on the other hand, it was shown that ethyl acetate and ethanolic extract had higher potential and showed maximum zone of inhibition of 4 mm at 10 mg/ml and 5 mm at 12.5 mg/ml concentration respectively. All extracts had low antifungal activity against A. niger.

Antifungal activity of stem A. aspera extract

Table 8 depicted that petroleum ether, benzene, chloroform, ethyl acetate and aqueous extracts showed high antifungal activity against F. oxysporum but ethanolic extract showed less antifungal activity than other solvents. The maximum zone of inhibition was shown by petroleum ether and chloroform i.e. 9.5 mm at 10 mg/ml and 25 mg/ml concentrations respectively against F. oxysporum and minimum zone of inhibition was nil (no activity) at 12.5 mg/ml by ethanolic extract. Very little activity had been seen against T. viridae and A. niger but aqueous extract showed potential activity against A. niger.

Antifungal activity of inflorescences A. aspera extract

Table 9 indicates that ethanolic extract of inflorescences part of A. aspera showed maximum zone of inhibition that was 18 mm at 12.5 mg/ml concentration and minimum zone of inhibition was 7 mm at 10 mg/ml concentration of ethanolic and chloroform extracts against F. oxysporum. Aqueous extract was also highly effective for F. oxysporum and showed maximum zone of inhibition at 10.5 mm at 12.5 mg/ml concentration.

The decreasing order of various extracts on the basis of zone of inhibition are ethanolic extract > aqueous extract > benzene extract > chloroform extract > petroleum ether extract > ethyl acetate. Petroleum ether, benzene, chloroform and ethyl acetate extract showed no antifungal activity against A. niger but aqueous extract and ethanolic extract were effective for preventing the growth of A. niger.

DISCUSSION:

Available literature indicates that the antimicrobial activity of plant is due to the presence of different bioactive compounds in various types of extracts such as flavanoids, triterpenoids, and some essential oils like thymol and natural phenolic compounds that are classified as ‘active antimicrobial compounds’ [15].

Successful anticipation of various herbal chemical compounds from plant is largely reliant on the type of solvent that were used in the extraction procedure. The customary practitioners in our medicinal system suggested water primarily as a solvent, but our studies showed that organic solvents are much better and powerful due to better solubility of bio-active compounds in organic solvents thus verified the high antimicrobial properties of therapeutic plant in organic solvents in comparison to aqueous that were also supported by many investigators [16, 17, 18, 19]. Flavonoids are least soluble in water which is the primary phenolic compound in plants and responsible for several therapeutic activity of plant [20]. It was reported that Achyranthes aspera possesses antimicrobial potential against several pathogenic microorganism [21, 22]. Our studies also proved that A. aspera have potent antibacterial and antifungal activity and thus have possible therapeutic values in pharmaceutical field.

Conclusion:

The present study concluded that A. aspera possesses high antimicrobial activity and thus may be used for the preparation of various pharmacological formulations. The most active extracts can also be subjected for the isolation of the active compounds that could be used for pharmacological evaluation or for the therapy of infectious diseases which are caused by pathogen.

Acknowledgements

The authors are grateful to the authorities of Banasthali University for providing support and the Department of Biotechnology (DBT), New Delhi, India, for giving financial assistance to complete the study.

Tables at a glance

Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9

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