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Evaluation of Antibacterial activity of Acacia catechu willd, Azadirachta indica, Aryctostaphylos Uva ursi against Lactobacillus Acidophilus ?An in vitro Comparative Study

José Ignacio López De León1*, Jose Antonio Mata-Marín2, Karen Andrade-Fuentes3, Gloria Huerta-Garcia4, Juan C Domínguez-Hemosillo5 and Jesus Gaytán-Martínez
  1. Reader, Department of Pharmacology, Saveetha Dental College & Hospitals, Chennai
  2. Professor, Department of Oral medicine, SRM Dental College & Hospitals, Kattankulathur
Corresponding Author: Lakshmi.T, Reader, Department of Pharmacology, Saveetha Dental College & Hospitals, Chennai . Email id- lakshmi085@gmail.com
Date of Submission: 22-02-2013 Date of Acceptance: 06-03-2013 Conflict of Interest: NIL Source of Support: NONE
Citation: Lakshmi. T, Vidya Krishnan “Evaluation of Antibacterial activity of Acacia catechu willd, Azadirachta indica, Aryctostaphylos Uva ursi against Lactobacillus Acidophilus –An in vitro Comparative Study” Int. J. Drug Dev. & Res., April- June 2013, 5(2):174-178. doi: doi number
Copyright: © 2013 IJDDR, Lakshmi. T et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Aim: This study was designed to examine the in vitro antibacterial potential of methanolic and acetone leaf extracts of Acacia catechu willd, Azadirachta indica ,Aryctostaphylos Uva ursi against Lactobacillus acidophilus. Materials and Methods: The inhibitory effect of methanolic and acetone extract of three herbal extracts was tested against Lactobacillus acidophilus by using the broth dilution method. Results: The methanolic extract of Acacia catechu willd exhibited antibacterial activity against Lactobacillus acidophilus with minimum bactericidal concentration of 1mg/ml, and appreciable activity with Azadirachta indica methanolic extract at a concentration of 10mg/ml. Preliminary phytochemical analysis of methanolic and acetone extract of the herbal extracts revealed the presence of secondary metabolites such as flavonoids, steroids, and tannins, which may contribute for the antibacterial action . Conclusion: The methanolic extract of Acacia catechu willd was found to be bactericidal in action when compared to the other herbal extracts tested . Clinical Significance: Acacia catechu willd methanolic leaf extract is proved to be an potent antibacterial herb against lactobacillus acidophilus and can be recommended to treat dental caries when compared to Azadirachta indica commonly known as neem which is commercially obtained in the market.

Keywords

Herbal extracts, anti bacterial activity, MBC, Lactobacillus acidophilus

INTRODUCTION

Dental caries is an infectious, communicable disease resulting in destruction of tooth structure by acidforming bacteria found in dental plaque, an intraoral biofilm, in the presence of sugar. The infection results in loss of tooth minerals that begins on the outer surface of the tooth and can progress through the dentin to the pulp, ultimately compromising the vitality of the tooth .various literature has proved that streptococuus mutans and Lactobacillus acidophilus were the main causative agent for dental caries.[1]
Herbal products have been used since ancient times in folk medicine for the management of dental infections.[2] Many plants and plant-derived antimicrobial components are used in folk lore therapeutics for the treatment of dental caries and for the purposes of oral hygiene .[3] Various chewing sticks have been in use since ancient times.
Extracts from the roots and stems of Salvadora persica have been used for the treatment of oral infections in animals.[4] Aqueous and ethanolic extracts of S. persica proved useful in removing the smear layer from dentin surfaces.[5] It had been found that aqueous extracts of S. persica bark, pulp were effective against various bacteria, including Streptococcus mutans ,Lactobacillus acidophilus[.6] The chewing sticks used in India are usually from Azadiracta indica (Neem).
Neem extracts show antimicrobial effects against Streptococcus mutans and S. faecalis. [7] A formulation of mucoadhesive dental gel containing Neem leaves extract reduced both the plaque index and bacterial count .[8] Acacia catechu is also used as an active constituent in toothpastes in India. The components of its bark and gum, mainly tannins, show antimicrobial and astringent affects .[9]
There is growing interest in correlating phytochemical constituents of plants with their pharmacological activity. Since, the earlier reports on the plant leaf for antimicrobial activity are on the preliminary level even without bactericidal values. Hence, the objective of this study was to assess the antibacterial activity of three different phyto chemical extracts (methanolic and acetone ) against the oral bacteria Lactobacillus acidophilus in order to determine the antibacterial component responsible for antibacterial activity.

MATERIALS AND METHODS

Plant material
The Herbal extracts were obtained from Green Chem, Herbal Extracts & Formulations, Bangalore.
Preliminary phytochemical analysis
The refined methanolic and acetone leaf extract s of three herbs was subjected to qualitative chemical screening for identification of various classes of active chemical constituents such as carbohydrates, tannins, saponins, cardiac glycosides, steroids/triterpenoids, flavonoids, anthraquinones, and alkaloids. The phytochemical analysis was done according to standard methods.[ 10, 11]
Microbiological procedures
Test microorganism
Pure strain of Lactobacillus acidophilus(ATCC 53103) were obtained from Department of Microbiology , Saveetha Dental College & Hospitals, Chennai.
Medium used
Trytic soy broth is used as a medium
Preparation of different concentratins of herbal extracts
The herbal extracts each 200mg were weighed aseptically into a sterile tube and dissolved in 2ml of sterile Tryptic soy Broth (TSB).From the stock solution various concentrations were prepared,viz.,62μg,125 μg,250 μg,500 μg/100μl ,1mg,5mg,10mg/100μl respectively in to wells of micro plates.The tested organism was grown in (TSB)Tryptic soy broth medium [MHA-Hi media ,Mumbai] for 24hrs at 37°C and concentration was adjusted to 0.5 Mac farland Standard.[12-14]
The different concentrations of extracts were taken in 100μl quantities in a U bottom micro culture plates. Control well received plain broth without plant extract. the plates were kept in sealed covers and incubated at 37°C overnight and growth/no growth was detected. All the tests were done in triplicate to minimize the test error.
Minimum Inhibitory Concentration (MIC)
Minimum inhibitory concentration of herbal extracts against tested microorganism was determined by macro broth dilution method . [15]A series of two- fold dilution of each extract ( 62 μg/100μl to 10mg/100μl) was made in to which 100μl of the standardized bacterial suspension containing 106 organisms was made in Tryptic soy broth as specified by National Committee for Clinical Laboratory Standards (NCCLS, 1990).[16]The control well received plain broth without herbal extract .The plates were incubated at 37°C for 24 hours and observed for visible growth. As the extracts were colored, MIC could not be read directly by visual methods.Hence subcultures from all the wells were made and growth/nogrowth is detected.then the MBC were obtained.
Minimum Bactericidal Concentration (MBC)
The MBCs were determined by selecting wells that showed no growth . The least concentration, at which no growth was observed, were noted as the MBC.

RESULT AND DISCUSSION

Azadirachta indica was reported to be used widely in oral care formulations. Acacia catechu appear to be synergistic to the antibacterial activity of Azadirachta indica in maintenance of oral hygiene. Acacia catechu and Uva ursi were selected because of their astringent and antioxidant properties in addition to antibacterial activity.
Extraction was done for individual herbs and was evaluated for antibacterial activity against Lactobacillus acidophilus. The results showed that methanolic extract of Acacia catechu willd used in the study have significant antibacterial activity comparatively. Similar study was conducted by Geetha and coworkers to evaluate the antibacterial activity of Acacia catechu willd heartwood extract against oral microbes including Lactobacillus acidophilus. The results indicates that the extract is found to be bactericidal in action.[17]
Various studies were conducted to assess the antibacterial efficacy of neem and its active compounds.
Ravi Kant Upadhyay in his study concludes that neem oil as an effective agent against many gram positive organisms including L.acidophilus.[18]
Salvadora persica and Miswak chewing sticks is found be as effective anti plaque and anti cariogenic agent. due to this property it is recommended as a oral hygiene tool in developing countries for management of dental caries/plaque.[7] Neem sticks or bark inhibits oral microbes like streptococcus mutans, streptococcus salivarius, streptococcus mitis and streptococcus sanguis which are involved in development of dental caries.[19]
Venka A et al conducted a study related to the antibacterial effect of Neem mouthwash against salivary levels of streptococcus mutans and Lactobacillus Acidophilus which has been tested over a period of two months, along with it's effect in reversing incipient carious lesions were assessed. he found that streptococcus mutans was inhibited by Neem mouthwashes, with or without alcohol as well as chlorhexidine, lactobacillus growth was inhibited by chlorhexidine alone. [20]
The antibacterial effects of various concentrations were tabulated (Table 1)the solvent extracts of three phytochemicals was studied for the antibacterial activity in the range of concentration from 62μg/ml to 10 mg/ml of extract. In the invitro study it was found that the Acacia catechu willd methanolic and Azadirachta indica acetone extract at a concentration of 1mg/ml and 10mg/ml exhibited antibacterial activity against the bacterial strain tested.
The methanolic leaf extract of Acacia catechu exhibited a high degree of activity against the organism tested when compared to acetone extract. Azadirachta indica methanolic leaf extract does not exhibit antibacterial activity whereas acetone leaf extract found to possess moderate antibacterial potency against Lactobacillus acidophilus. methanolic and acetone leaf extract of Uva ursi does not showed significant antibacterial activity against the dental pathogen tested
No Growth indicates high effectiveness of the extract whereas presence of Growth indicates less effectiveness of the extract.
Preliminary phytochemical analysis of Acacia catechu willd, Azadirachta indica, Uva ursi leaf extract was performed as per the standard procedure of Trease and Evans. this revealed the presence of tannins, saponins, alkaloids, Terpenoids, Flavonoids which are responsible for the antibacterial property.

CONCLUSION

The results showed that the methanolic extract of Acacia catechu willd leaf was able to inhibit the oral bacteria that cause dental caries used in this study with different degree of concentrations. The information obtained may provide validation for its reported medicinal uses. In conclusion, the Acacia catechu willd methanolic extract is found to be very effective against the tested bacterial strain when compared to Azadirachta indica commonly known as Neem- A widely used herbal product in several tooth powder, paste and mouthwashes available in the market.

Tables at a glance

Table icon
Table 1

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