Archives of Medicine

Keywords

Antibacterial activity, Glycyrrhiza glabra, Azadirachta indica, disc diffusion method and typhoid fever

Highlights

• The present study was conducted to analyze in-vitro antibacterial activity of methanolic extract of Glycyrrhiza glabra and Azadirachta indica

• These plant extracts may prove their promising role as antibiotics and may help to overcome the problem of resistivity against many pathogens

• Anti-typhoid activity of these medicinal plants is promising to act as potential agent for treating typhoid fever from natural plant source.

Introduction

Antibiotics provide the main basis for the therapy of bacterial and fungal infection [1]. Bacterial resistance is due to misuse of antibiotics [2]. Bacterial infections are hazardous and responsible for many death in each year [3], and Currently used antimicrobials are failing to bring an end to many bacterial infections due to super resistant strains for this reason research is ongoing for natural anti-microbial activity [4,5]. Herbal medicine was a very ancient art of healing one that use both perceptual and intellectual awareness as well as focus on outcome and anti bacterial activity also reported [6]. The antimicrobial potential of different medicinal plants are being extensively studied all over the world [7]. Chemical agent with general as well as specific activities [8]. Novel phytochemical substance of plant origin has been shown to inhibit the growth of bacteria [7,9]. Recent screening of bioactive compound from plants results in discovery of new medicinal substance [8,10]. The traditionally used medicine, Glycyrrhiza glabra and Azadadirachta indica having antibacterial activity also reported [5,9]. Glycyrrhiza glabra also known as Licorice and sweet wood belonging to family Papilionaceae. It contains Saponin glycoside that is glycyrrhizin, Flavonoids and many other constituents [11]. The Azadirachta indica commonly known as Neem, belonging to family Meliaceae (mehogani family). Neem seeds contain azadirachtin, alkaloids, flavonoids, triperpenoids, phenolic compounds, carotenoids, steroids and ketones. [12]. In 2012, a phytochemical constituents, pharmacological action and medicinal use through the millennia of Glycyrrhiza glabra were conducted and it was accomplished that it is used as a traditional medicine, it contain glycyrrhizin and other chemical constituents extracted from root of licorice. It is used as a prophylactic as well as therapeutic drug for major body ailment sat any age group [13]. In 2013, the phytochemical screening and anti-oxidant potential of Glycyrrhiza root extract was conducted, it was concluded that it have significant anti-bacterial and hydroxyl radical scavenging activities, it is helpful in treating bacterial infection and scavenging hydroxyl radical which are generated during carcinogenesis. In 2012, the antimicrobial activity of leaf extract of neem was conducted against Pseudomonas aeroginosa, Staphylococcus aureus, Salmonella typhii and Baccilus pumillas and concluded that ethanol and methanol extract show maximum inhibition on Baccillus pumillas, Pseudomonas aeruginosa and Staphylococcus aureus in ascending order [14].

The present study was carried out to evaluate plant extract of Glycyrrhiza glabra and Melia azadirachta of different dilution to screen their anti-bacterial activity. These plants are used as traditional medicine to cure common ailment. To give a scientific approach to the use of these plant, this study was carried out to analyze anti-bacterial activity against gram positive (Pseudomonas aerouginosa, Staphylococcus aureus) and gram negative organism (Escherichia coli, Klebsiella and salmonella).

Material and Methods

Material and reagent

Ampicillin, licorice stem, neem seeds, methanol, Nutrient broth and nutrient agar. Chemicals and media of analytical grade were purchased from Merck’s and oxide chemical manufacturer. All the solutions and media were prepared fresh before experiment.

Plant extract

The plant extract were prepared by using maceration method [4]. The crude drug of licorice and neem seeds was grinded into powder and soak required amount of sample in methanol for 2 days and filter the extract with the help of watts man filter paper 1. The plant extract is concerntrated in rotary evaporator and with the help of this extract further dilution of 20 ppm, 30 ppm and 40 ppm were prepared by using methanol as a solvent.

Antibacterial activity

The plant extract liquorice and neem were screened for their antimicrobial activity against two gram positive (Pseudomonas aerouginosa, Staphylococcus aureus) and three gram negative (Escherichia coli, Klebsiella and Salmonella typhi) organism by the conventional cylinder plate method [4,15]. The solutions of plant extract for soaking disk were made of different dilution that is 20, 30, and 40 ppm using methanol as a solvent. To ensure that the solution had no effect on bacterial growth, a negative control test was performed with test medium supplemented with methanol and positive control ampicilin at the same dilutions as used in the experiment.

Nutrient agar was prepared, autoclaved at 124°C for 15 minutes cooled and then pour in Petri plates swabbing was done with the help of sterile cotton swab, soaked disks of solution were placed in them and the plates were incubated for 48 hours at 37°C. Finally, the zones of inhibition were carefully measured with the help of scale in millimeter [16].

Result

The result of anti-bacterial activity is shown in Table 1 and Table 2. The diameter of zone of inhibition of extract from 6 mm to 17 mm, whereas standard Ampicilin disc produced zone of inhibition ranging from 7 to 31 mm in diameter. The result shown in Table 1 and 2 and also represent in Graph 1 and 2 shown below:

S.No.	Bacterial strain	Organism name				Zone of inhibition (mm)
			Glycyrrhizaglabra			Positive control Ampicillin			Negative control methanol
			20ppm	30 ppm	40 ppm	20 ppm	30 ppm	40ppm
1	Gram positive	Pseudomonas aeruginosa	13mm	15mm	17mm	23mm	25mm	27mm	NIL
		Staphylococcus aureus	9mm	10mm	11mm	27mm	28mm	31mm	NIL
2	Gram negative	Escherichia coli	7mm	8mm	9mm	11mm	12mm	12mm	NIL
		Klebsella pneumonia	7mm	8mm	10mm	7mm	11mm	12mm	NIL
		Salmonella tymphenia	9mm	11mm	13mm	9mm	11mm	13mm	NIL

Table 1: Anti-bacterial activity of Glycyrrhiza glabra.

S.No.	Bacterial strain	Organism name	Zone of inhibition			Positive control			Negative control
			Azadirachtaindica			Ampicillin			Methanol
			20 ppm	30 ppm	40 ppm	20 ppm	30 ppm	40 ppm
1	Gram positive	Pseudomonas aeroginosa	10mm	12mm	14mm	23mm	25mm	27mm	NIL
		Staphylococcus aureus	6mm	10mm	12mm	27mm	28mm	31mm	NIL
2	Gram negative	Escherichia coli	8mm	11mm	13mm	11mm	12mm	12mm	NIL
		Klebsella pneumonia	8mm	9mm	11mm	7mm	11mm	12mm	NIL
		Salmonella typhi	6mm	8mm	11mm	9mm	11mm	13mm	NIL

Table 2: Anti-bacterial activity of Azadirahta indica.

Graph 1: Graphical representation of micro –organism VS zone of inhibition (mm).

Graph 2 Graphical representation of micro –organism VS zone of inhibition (mm).

Discussion

Plants are renowned to have valuable therapeutic effects. Though bioactive products of Neem and licorice have been used in treatment of various diseases since various decades, role of phytochemical in inhibition of microorganisms growth has provide little evidence [17]. The anti-bacterial activity of plant extract, liquorice and neem were analyzed against a five pathogenic bacterial strains which include both gram positive and gram negative organisms that is Pseudomonas Aerouginosa, Staphylococcus aureus, Escherichia coli, Klebsiella and Salmonellae. They all are pathogenic organisms and cause serious body ailment and infect vital organs.

Both plant exhibits anti-bacterial activities against both Gram positive and Gram negative microorganism. The liquorice and neem both are used as a traditional medicine all over the world. The concept of alternative medicine increases day by day, especially herbal medicine because of no or least harmful effects. In our study, the control (methanol) showed no zone of inhibition. The diameters of zone of inhibition of both extract are of different concentrations that are 20 ppm, 30 ppm and 40 ppm and the results were compared with the activity of the well known antibiotic, Ampicillin. It exhibited strong anti bacterial activity against selected organism, shown in Table -1 and Graph -1 represent the comparative analysis of methanolic liquorice extract with the reference drug of Ampicillin. The Azadirachta indica also used in cosmetic products as well as a home remedies. It has antiseptic effect and used to treat many skin diseases, also exhibit remarkable antibacterial activity. They contain bioactive components which are responsible for anti bacterial activity, shown in Table 2 and Graph 2. From the result it is evidence that methanolic extracts of these plants has remarkable zone of inhibition against salmonella typhi. The zone of inhibition is nearest to the controlled drug that is ampicillin. Salmonella typhi is causative agent of typhoid fever. Currently, typhoid fever is a major health problem in developing countries with limited success of treatment with antimicrobial agent [18,19]. The previous study evaluated the anti Salmonella typhi activity of ethanol, hot and cold crude water extracts of Vitex doniana (root), Cassia tora (Leaf), Alstonia boonei (bark), Stachytarpheta jamaicensis (leaf), and Carica papaya (leaf) used as traditionally medicine in Ebonyi state, Nigeria [9]. The neem seeds extract and glycyrrhiza extract shows comparable same result as compare to ampicillin. The plant extract as naturally synthesize mostly preferable because of having no destructive effects. The frequent use of antimicrobial agent is restricted because the frequent use may cause resistivity, G I upset and loss of appetite. The antimicrobial activity of Glycyrrhiza glabra and Azadirachta indica has shown in Table 1 and 2. The tested methanol extracts of Glycyrrhiza glabra and Azadirachta indica have profound antimicrobial activity against both gram positive and gram negative bacteria. The valuable zone of inhibition of both extract is especially against salmonella typhi. The result clearly shows that the herbal extract of both plant shows the same zone of inhibition as compare to controlled drug i.e. ampicilin. The quantitative and qualitative differences in inhibition variation are influenced by the method of extraction and concentration. In this study the anti-bacterial activity was tested by using the disc diffusion method, which is widely acceptable for preliminary screening of plant extracts. It is essentially an initial test indicating the sensitivity and resistivity of microorganisms to the test sample [20-22]. It became also clear in the current study that concentrations affect the degree of antimicrobial activity, as the concentration increases the antibacterial activity also increases. Various plant shows inhibitory effect on the growth of bacteria and also effective for systemic diseases, and our finding also prove that both plant extracts have great potential as antimicrobial activity against enteric bacterial pathogens and they can be used in the treatment of communicable diseases with no destructive effects. The data obtained in this study justify the use of herbal preparations of neem and liquorice in typhoid. It is concluded from the result that both plant extracts have anti-bacterial activity against Salmonella typhi and they can be used in typhoid fever as alternate medicine in order to prevent antimicrobial resistivity. Currently, we reported experimental proof of the same. Further research should be proceeded to go into more detail studies to find out the bioactive constituents [23,24].

Conclusion

The present study showed the traditional roles of plants in curing typhoid. Methanolic extract of Glycyrrhiza glabra and Azadirachta indica have broad spectrum antibacterial activity against grampositive and gram-negative bacteria. The study support the use of these herbal extracts as to prevent or control the typhoid, and anti-typhoid activity of these medicinal plants are promising to act as potential agent for treating typhoid fever from natural plant source.

7708

References

1. Harbottle H, Thakur S, Zhao S, White DG (2006) Genetics of antimicrobial resistance.  AnimBiotechnol 17: 111-124.
2. Uniyal SK, Singh KN, Jamwal P, Lal B (2006) Traditional use of medicinal plants among the tribal communities of ChhotaBhangal, Western Himalaya.  J EthnobiolEthnomed 2: 14.
3. Betoni JE, Mantovani RP, Barbosa LN, Di Stasi LC, Fernandes Junior A (2006) Synergism between plant extract and antimicrobial drugs used on Staphylococcus aureus diseases.  MemInstOswaldo Cruz 101: 387-390.
4. Lewis K, Ausubel FM (2006) Prospects for plant-derived antibacterials.  Nat Biotechnol 24: 1504-1507.
5. Bonjar S (2004) Evaluation of antibacterial properties of some medicinal plants used in Iran.  J Ethnopharmacol 94: 301-305.
6. Nassiri-Asl M, Saroukhani S, Zamansoltani F (2007) Anticonvulsant effects of aqueous extract of Glycyrrhizaglabra root in PTZ-induced seizure in mice. Int. J. Pharmacolog 3: 432–434.
7. Lewis K, Ausubel FM (2006) Prospects for plant-derived antibacterials.  Nat Biotechnol 24: 1504-1507.
8. Varsha S, Agarwal RC, PandeySonam ND (2013) Phytochemical Screening and determination of Anti-bacterial and anti-oxidant potential of glycyrrhizaglabraroot extract.
9. Ates DA, Erdourul ZT (2003)  Antimicrobial activities of various medicinal and commercial plant extracts. Turk. J. Biol 27: 157- 162.
10. Usmanghani K (1997) Indyusenic medicine, Traditional medicine of Herbal , Animal and mineral origion in Pakistan.
11. Brindha MS, kariyarasi S, Annadurai NS, Gangwar SK (2012) Antimicrobial activity in leaf extractofNeem( Azadirachtaindica ). International journal of science and Natural  3: 110.
12. Roshan A (2012) A phytochemical constituents pharmacological activities and medicinal plant use through millennia the Glycyrrhizaglabra.International research journal of pharmacy.
13. Brindha MS, KariyarasiS, Annadurai NS, Gangwar SK (2012) Antimicrobial activity in leaf extractofNeem (Azadirachtaindica). International Journal of Science and Natural 3: 110.
14. Bonjar S (2004) Evaluation of antibacterial properties of some medicinal plants used in Iran.  J Ethnopharmacol 94: 301-305.
15. Arayne MS, Sultana N, Gul S, Khan A (2014) Novel derivatives of 5-amino-1-cyclopropyl-7-[(3R, 5S) 3, 5-dimethylpiperazine-1-yl]-6, 8-difluoro-4-oxo-quinoline-3-carboxylic acid: their synthesis, antimicrobial, antifungal, and urease inhibitory studies. Medicinal Chemistry Research 23: 1248-1256.
16. SaiRam M, Ilavazhagan G, Sharma SK, Dhanraj SA, Suresh B, et al. (2000) Anti-microbial activity of a new vaginal contraceptive NIM-76 from neem oil (Azadirachtaindica).  J Ethnopharmacol 71: 377-382.
17. Otegbayo JA, Daramola OO, Onyegbutulem HC, Balogun WF, Oguntoye OO (2002) Retrospective analysis of typhoid fever in a tropical tertiary health facility.  Trop Gastroenterol 23: 9-12.
18. Lin FY, Vo AH, Phan VB, Nguyen TT, Bryla D, et al. (2000) The epidemiology of typhoid fever in the Dong Thap Province, Mekong Delta region of Vietnam.  Am J Trop Med Hyg 62: 644-648.
19. Bensky D (2004) Chinese Herbal Medicine:MateriaMedica (3rdedn), Eastland Press, Seattle.
20. Ahmad I, Beg AZ (2001) Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens.  J Ethnopharmacol 74: 113-123.
21. Lee SB, Cha KH, Kim SN, Altantsetseg S, Shatar S, et al. (2007) The antimicrobial activity of essential oil from Dracocephalumfoetidum against pathogenic microorganisms.  J Microbiol 45: 53-57.
22. Di Mambro VM, Fonseca MJ (2005) Assays of physical stability and antioxidant activity of a topical formulation added with different plant extracts.  J Pharm Biomed Anal 37: 287-295.
23. Bhattarai S, Chaudhary RP, Taylor RSL (2008) Screening of selected ethnomedicinal plants of Manang district, Central Nepal for antibacterial activity. Ethnobotany20: 9-15.