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PHARMACOLOGICAL STUDY
Year : 2014  |  Volume : 35  |  Issue : 2  |  Page : 207-210  

Microbial evaluation of Limnophila rugosa Roth. (Merr) leaf


1 Department of Dravyaguna, Institute for Postgraduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
2 Department of Pharmacognosy Laboratory, Institute for Postgraduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
3 Department of Pharmaceutical Chemistry Laboratory, Institute for Postgraduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India

Date of Web Publication5-Dec-2014

Correspondence Address:
Rabinarayan Acharya
Department of Dravyaguna, I.P.G.T. and R.A., Gujarat Ayurved University, Jamnagar - 361 008, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-8520.146259

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   Abstract 

Background: Limphonia rugosa Roth. (Merr.), family-Scrophulariaceae is considered as a botanical source of classical Ayurvedic drug Bhringaraja by the traditional practitioners of Odisha and is being used for the management of various disorders. Aim: To study the antimicrobial activity of leaf of L. rugosa. Materials and Methods: Methanol extract of L. rugosa leaf (LRLM) has been studied, at various (5, 25, 50, 100, 250 μg/ml) dilutions, against medically important human pathogenic bacteria (two Gram-positive Staphylococcus aureus, Streptococcus pyogenes and two Gram-negative-Escherichia coli, Pseudomonas aeruginosa) and two fungal strains (Aspergillus niger, A. clavatus, Candida albicans) by using the agar disc diffusion method. A zone of inhibition of extract was compared with that of different standards such as ampicillin, ciprofloxacin, norfloxacin and chloramphenicol for antibacterial activity and nystatin and griseofulvin for antifungal activity. Results: The antibacterial and antifungal activities of the LRLM increased linear with the increase in concentration of extracts. When compared with standard drugs, the results revealed that, for bacterial activity S. pyogenes and S. aureus were more sensitive and in fungal activity C. albicans was more inhibited. The range of growth inhibition zone for all the sensitive bacteria was 11-20 mm and 13-19 mm for fungal strains. Conclusion: Methanolic extract of L. rugosa leaf is having antibacterial and antifungal activities.

Keywords: Antifungal activity, Bhringaraja, Gandhamardan hills, in vitro antibacterial activity, Limnophila rugosa leaf, microbial load


How to cite this article:
Acharya R, Padiya RH, Patel ED, Harisha C R, Shukla VJ. Microbial evaluation of Limnophila rugosa Roth. (Merr) leaf. AYU 2014;35:207-10

How to cite this URL:
Acharya R, Padiya RH, Patel ED, Harisha C R, Shukla VJ. Microbial evaluation of Limnophila rugosa Roth. (Merr) leaf. AYU [serial online] 2014 [cited 2020 Oct 21];35:207-10. Available from: https://www.ayujournal.org/text.asp?2014/35/2/207/146259


   Introduction Top


Limnophila rugosa Roth. (Merr.) of Scrophulariaceae family is an erect herbaceous, aromatic annual, 30-60 cm. high, found in aquatic situations and moist lands almost throughout India, ascending to 1800 m in the Himalayas. Locally known as Bhringaraja[1] [Figure 1], one of the famous drugs of Ayurveda, it is reported to be used in hair oil preparation. [2] The plant is claimed for its carminative and tonic action; and used for the management of diarrhea, dysentery, dyspepsia, in pestilent fever, elephantiasis and as a flavoring agent of food and perfuming. [3],[4],[5] Different types of functional groups such as alkaloid, tannin, triterpenoid (steroid), flavonoid, phenols and essential oil has been reported from L. rugosa.[4],[6],[7],[8],[9] The plant though highlighted for its use in many diseases, caused due to microbes, but has not been evaluated for its antimicrobial activities. Hence, the present study was designed to evaluate the antimicrobial properties of Limnophila rugosa leaf.
Figure 1: Photograph of Bhringaraja (Limnophila rugosa)

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Collection and authentication of plant material

L. rugosa Roth. (Merr.) (Scrophulariaceae), locally called as Bhringaraja, was collected from its natural habitat of Gandhamardan hill ranges, Bargarh, Odisha, India, in fully matured condition, with the help of local traditional healers. The correct identity and authenticity of the plant L. rugosa was done by studying its morphological characters and comparing them with the characters mentioned in various floras. [2],[10],[11],[12] Plants were washed properly under running water to make it free from foreign matter such as sand, soil etc., Whole plant was dried under shade, powdered to 60# and few were preserved in solution of AAF (70% Ethyl alcohol: Glacial acetic acid: Formalin) in the ratio of (90:5:5). [13] Herbarium was also prepared and submitted to Pharmacognosy laboratory museum of Institute for Postgraduate Teaching and Research in Ayurveda, Jamnagar vide Herbarium no. 6003.


   Materials and Methods Top


Preparation of plant extract

Powdered sample was extracted with methanol for 24 h (shaking for 18 h frequently and then kept aside as a stand by for 6 h). Then extracts were filtered and methanol was added to prepare solutions with different concentrations 5, 25, 50, 100 and 250 μg/ml. The extract was coded as Limnophila rugosa leaf methanol (LRLM).

Test microorganisms and growth media

The microorganisms and growth media were selected following standard guidelines. [14],[15],[16],[17],[18]

Selection of microorganisms

Staphylococcus aureus (MTCC 96), Streptococcus pyogenes (MTCC 442),  Escherichia More Details coli (MTCC 443), Pseudomonas aeruginosa (MTCC 424) and fungal strains Aspergillus niger (MTCC 282), Aspergillus clavatus (MTCC 1323), Candida albicans (MTCC 227) were chosen based on their clinical and pharmacological importance. The bacterial strains, obtained from Institute of Microbial Technology, Chandigarh, were used for evaluating antimicrobial activity. The bacterial and fungal stock cultures were incubated for 24 h at 37°C on nutrient agar and potato dextrose agar (PDA) medium (Microcare laboratory, Surat, Gujarat, India) respectively following refrigeration storage at 4°C. The bacterial strains were grown in Mueller-Hinton agar plates at 37°C (The bacteria were grown in the nutrient broth at 37°C and maintained on nutrient agar slants at 4°C), whereas the yeasts and molds were grown in sabouraud dextrose agar and PDA media, respectively, at 28°C. The stock cultures were maintained at 4°C.

Antimicrobial activity

Determination of zone of inhibition method


In vitro antimicrobial activity testing was carried out by using Agar cup method. Each purified extracts were dissolved in dimethyl sulfoxide, sterilized by filtration using sintered glass filter and stored at 4°C. For the determination of zone of influence, pure Gram-positive, Gram-negative and fungal strain antibiotics were taken as a standard for comparison of the results. All the extracts were screened for their antibacterial and antifungal activities against the E. coli, P. aeruginosa, S. aureus, S. pyogenes and the fungi C. albicans, A. niger, and A. clavatus. The sets of five dilutions (5, 25, 50, 100 and 250 μg/ml) of LRL extract and standard drugs were prepared in double distilled water using nutrient agar tubes. Muller Hinton sterile agar plates were seeded with indicator bacterial strains (108 cfu) and allowed to stay at 37°C for 3 h. Control experiments were carried out under similar condition by using ampicillin, chloramphenicol, ciprofloxacin and norfloxacin for antibacterial activity; nystatin and griseofulvin for antifungal activity as standard drugs. The zones of growth inhibition around the disks were measured after 18-24 h of in incubation at 37°C for bacteria and 48-96 h for fungi at 28°C, respectively. The sensitivity of the microorganism species to the plant extracts were determined by measuring the sizes of inhibitory zones (including the diameter of disk) on the agar surface around the disks and values <8 mm were considered as not active against microorganisms.


   Results and Discussion Top


Microbial load

The observations on the microbial load of LRLM showed that the tasted samples, when collected from their natural sources, are either free or within prescribed limit of the microbes [Table 1].
Table 1: Microbial load report of LRLM

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Antimicrobial activity

The result of antimicrobial activity of LRLM extract studied in different concentrations (5, 25, 50, 100 and 250 μg/ml) are presented in [Table 2] and [Table 3] and antibacterial and antifungal potential of standard drugs presented in [Table 4].
Table 2: Antibacterial activity of LRLM, ampicillin and chloramphenicol against gram +ve and gram −ve organisms

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Table 3: Antibacterial activity of LRLM, ciprofloxacin and norfloxacin against gram +ve and gram −ve organisms

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Table 4: Antifungal activity of LRLM, griseofulvin and nystatin against fungal strains organisms

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The antibacterial and antifungal activities of the LRLM increased linearly with the increase in concentration of extracts (μg/ml). When compared with standard drugs, the results revealed that in the extracts for bacterial activity, S. pyogenes and S. aureus were more sensitive when compared to E. coli and P. aeruginosa, and for fungal activity C. albicans was more inhibited as compare to A. niger and A. clavatus. The growth inhibition zone measured ranged from 11-20 mm for all the sensitive bacteria and ranged from 13 to 19 mm for fungal strains [Table 2],[Table 3] and [Table 4].

The inhibitory effect of LRLM showed at (25, 50, 100, 250 μg/ml) were (12, 15, 16, 17) against E. coli MTCC 443, (13, 14, 15, 17) against P. aeruginosa MTCC 424, (13, 16, 17, 19) against S. aureus MTCC 96, (12, 13, 16, 18) against S. pyrogenes MTCC 442, (13, 14, 17, 19) against A. niger MTCC 282 and (12, 14, 17, 18) against C. albicans MTCC 227 [Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6] and [Figure 7].
Figure 2: Effect of standard drugs and test drug against Escherichia coli

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Figure 3: Effect of standard drugs and test drug against Pseudomonas aeruginosa

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Figure 4: Effect of standard drugs and test drug against Staphylococcus aureus

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Figure 5: Effect of standard drugs and test drug against Streptococcus pyrogenes

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Figure 6: Effect of standard drugs and test drug against Aspergillus niger

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Figure 7: Effect of standard drugs and test drug against Candida albicans

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The results showed that the extracts of all samples were found to be more effective against all the microbes tested, which may be due to the reported phyto constituents present in the plant.


   Conclusion Top


The present study justified the claimed ethnic uses of L. rugosa in the preparation of hair oil, to treat various infectious disease caused by the microbes. However, further studies are needed, on different extract and concentrations, to better evaluate the potential effectiveness of the crude extracts as antimicrobial agents. The present results will form the basis for selection of plant species for further investigation in the potential discovery of new natural anti-microbial drugs.

 
   References Top

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Saxena HO, Brahman M. The Flora of Orissa. Vol. III. Bhubaneshwar, Orissa, India: Regional Research Laboratory, Orissa Forest Development Corporation Ltd.; 1995. pp. 1237.  Back to cited text no. 1
    
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Brahman M, Saxena HO. Ethnobotany of Gandhamardan Hills - Some noteworthy folk-medicinal uses, regional research laboratory Bhubaneshwar-13. Ethnobotany 1990;2:71.  Back to cited text no. 2
    
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Anonymous. The Wealth of India (Raw Materials). Vol. 4. J-Q. Reprinted ed. New Delhi: CSIR; 2003. pp. 40.  Back to cited text no. 3
    
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Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants. 1 st ed. New Delhi: CSIR; 1956. pp. 55.  Back to cited text no. 4
    
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Mukherjee KS, Gorai D, Sohel SM, Chatterjee D, Mistri B, Mukherjee B, et al. A new flavonoid from Limnophila rugosa. Fitoterapia 2003;74:188-90.  Back to cited text no. 7
    
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Mukherjee KS, Brahmachari G, Manna TK, Laha S. A new triterpene from Limnophila rugosa (Roth.) Merrill. Abstract: Cheminform 1996;27 (50).  Back to cited text no. 8
    
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Acharya RN, Padiya RH, Patel ED, Harisha CR, Shukla VJ. Preliminary phyto-chemical study on the leaf of an ethno-medicinal plant Limnophila rugosa Roth.(Merr.). Ayurpharm Int J Ayur Alli Sci. 2012;1 (7):138-43.  Back to cited text no. 9
    
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Haines HH. The Botany of Bihar and Orissa. Part. III-IV. Dehradun: Bishen Singh Mahendra Palsingh; 1988. pp. 625.  Back to cited text no. 10
    
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Desai NC, Shihora PN, Moradia DL. Synthesis and characterization of new quinazolines as potential antimicrobial agents. Indian J Chem 2007;46b: 550-3.  Back to cited text no. 15
    
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Anonymous. Indian Pharmacopoeia. 1 st ed., Vol. 1. Part. 1. New Delhi: Ministry of Health and Family Welfare, Controller of Publications, Government of India; 1996. pp. 37-49.  Back to cited text no. 16
    
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Shadomy S, Albert B. Manual of Clinical Microbiology. Washington: ASM Press; 1991. pp. 1173.  Back to cited text no. 17
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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