Abstract
Kattha, which is used as a chewing ingredient, is a resin part of Acacia catechu (L.f.) Willd. (Leguminoseae). The Antimicrobial screening and phytochemical analysis were performed to prove the antibacterial property and presence of active phytochemicals in extracts of Acacia catechu. The agar diffusion method was selected to check antimicrobial activity. A phytochemical analysis was done using a HPTLC instrument. Antimicrobial testing demonstrated excellent results with the petroleum ether extract against Pseudomonas aeruginosa (10 μg/mL), followed by the aqueous extract against Bacillus subtilis (20 μg/mL) and the chloroform extract against Staphylococcus aureus (30 μg/mL). Two major phytochemical constitutents, epicatechin and quercetin, were identified by HPTLC as active ingredients in the extract.
Introduction
Acacia catechu (L.f.) Willd (Leguminoseae) is native to central and east Africa, southern Asia, Bhutan, China, India, Myanmar, Nepal, and Pakistan. It is well known as kattha, and is a crucial ingredient of “pan” which is a beetle leaf preparation chewed in India and Pakistan. A survey shows that branches of this plant are being used as chewing sticks in various parts of the world, due to its antimicrobial effect, and hence it is considered as a valuable ingredient for dental care preparations. It is useful in dental, oral and throat infections, and as an astringent for reducing oozing from chronic ulcers and wounds. Acacia catechu is very valuable for its influential astringent and antioxidant activities. Catechins have significant antioxidant and antimicrobial effects (CitationValsaraj et al., 1997; CitationAhmad et al., 1998; CitationVoravuthikunchai et al., 2004; CitationSingh et al., 2005). The antioxidant capacities are evaluated in terms of ascorbate equivalents by different methods. The extract restores antioxidant enzyme superoxide (SOD) from radiation-inducing damage.
The chief phytoconstituents are catechin and epicatechin. Wood contains 50% tannins, mainly catechutannic acid 20–35%; acacatechin 2-10%, catechins 13-33%, epicatechin 2.5%, isorhamnetin, quercetin, phlobatannins 25-33%, tannic acid 22-50%, catechu-red, gum 20-35% (CitationFelter, 1998).
This paper describes the presence of some phytochemicals in Acacia catechu which are responsible for antimicrobial activity.
Materials and methods
Extraction of plant material
The processed resin part (kattha) of the plant was collected from authorized dealers (LVG) of local markets and finally powdered using a domestic grinder.
For water extract: individual ingredients (20 g) were subjected to boiling in 200 mL doubled distilled water in a 500 mL flask until the total volume reduced to one fourth. The water extract was filtered through a 420 μm stainless steel filter, cooled and transferred to screw-capped glass vials.
For organic solvent extract: 10 g of plant material was powdered and extracted with solvents of different polarities (methanol, chloroform and petroleum ether) by cold maceration for 24 h. The extracts were filtered through Whatman No. 1 filter paper into screw-capped vials. The organic solvents were concentrated to near dryness using a rotary evaporator bath under reduced pressure. The extracts were further diluted with dimethyl sulphoxide (DMSO) for experimentation.
Antimicrobial screening
The standard strains used were Escherichia coli (MTCC 443), Staphylococcus aureus (MTCC 96), Pseudomonas aeruginosa (MTCC 741) and Bacillus subtilis (MTCC 441). A 0.5 mL volume of the standard inoculum (106-107 CFU) of the test bacterial strain was spread on Mueller Hinton Agar (MHA) with a sterile bent glass rod spreader and allowed to dry. Then, 6-mm diameter wells were bored in the MHA. Plant extracts were introduced into each well and allowed to stand for 1 h at room temperature to diffuse before incubation at 37º C for 24 h. The inhibition zone diameter (IZD) was measured by antibiotic zone reader to the nearest mm (CitationOkoli & Iroegbu, 2004). The MIC was determined using the cylinder agar diffusion method as described by CitationFyhrquist et al. (2002).
Phytochemical analysis
HPTLC instrumentation
Quantitative and qualitative analysis was performed with the help of a High Performance Thin Layer Chromatography (HPTLC) instrument. The HPTLC system (Camag, Muttenz, Switzerland) consists of 1) a TLC scanner connected to a PC running WinCATS software under MS Windows NT; 2) Linomat V sample applicator; 3) photo documentation system Camag, Reprostar III.
Spotting of samples
The chromatographic estimation was performed by streaking the extracts in the form of narrow bands of lengths 6 mm on precoated silica gel 60 F254 aluminum TLC plates (10 cm ×10 cm); a constant application rate of 150 μL/s and gas flow 10 s/μL was employed with the help of a Camag 100 μL syringe connected to a nitrogen tank, using a Camag Linomat V (Camag, Muttenz, Switzerland). The space between two bands was kept at 13 mm. For each five extracts (ethyl acetate, methanol, petroleum ether, butanol and chloroform) a volume of 15 μL and concentration of 0.1% were spotted.
Plate development and chromatographic conditions
After spotting, the plate was subjected to linear ascending development up to a distance of about 90 mm using a Camag, twin trough glass chamber, which was presaturated with the solvent system at room temperature. The solvent system consisted of hexane:ethyl acetate:water in the ratio of 1:1:1 v/v.
Scanning of plate
TLC plates were dried in a flow of air at room temperature. Densitometric scanning was carried out using a Camag TLC Scanner III in the absorbance mode from wavelength 200-450 nm with a slit dimension of 6.00 × 0.30 mm, micro and scanning speed of 20 mm/s, data resolution 100 μm/step. The source of radiation utilized was deuterium and tungsten lamps. All remaining measurement parameters were left at default settings. The chromatograms were integrated and regression analysis and statistical data were generated using WinCATS evaluation software (Version 1.4.2.8121).
Photo documentation of plate
After scanning, the image of the plate was taken at three different wavelengths of light (254 nm by UV lamp, 366 nm by mercuric lamp, 400- 800 nm by white lamp) with the help of photo documentation system Camag, Reprostar III.
Results and discussion
Antimicrobialm activity
The extract yields were very minute in petroleum ether and chloroform solvents, but increased in methanol and water (). Phytochemical studies of this plant found catechin and epicatechin in high amounts which are responsible for antibacterial activity. The results ( and ) indicate that the highest inhibition is found with chloroform extracts against P. aeruginosa (MIC: 10 μg/mL), followed by water extracts against B. subtilis (MIC: 20 μg/mL) and chloroform extracts against S. aureus (MIC: 30 μg/mL). The water extracts generally displayed very good inhibition (MIC: 330, 40, 220 and 20 μg/mL) against E. coli, S. aureus, P. aeruginosa and B. subtilis. Other extracts also demonstrated good antibacterial against all the screened pathogens. Antibacterial activity of the A. catechu has been studied by many researchers (CitationValsaraj et al., 1997; CitationAhmad et al., 1998; CitationVoravuthikunchai et al., 2004; CitationSingh et al., 2005). Some contradictory and equivocal reports of plant extracts against screened bacteria are found in the literature. Experimental results are in agreement with the results of CitationVoravuthikunchai et al. (2004) in which enteropathogenic strains of the E. coli were inhibited by the ethanol extract; results are equivocal in comparison with those reported by CitationSingh et al. (2005) in which B. subtilis and E. coli were inhibited and P. aeruginosa was not inhibited. Equivalent results were found in the case where an 80% ethanol extract showed a growth suppressive for all pathogens (CitationValsaraj et al., 1997). An aqueous extract has been found effective against B. subtilis and S. aureus, whereas an alcoholic extract has been found highly active against all four bacteria (CitationAhmad et al., 1998) which is in good agreement with experimental results. Staphylococcus aureus and Pseudomonas aeruginosa are common pathogens causing serious infections (CitationGnanamani et al., 2003), and Escherichia coli is an opportunistic pathogen. S. aureus expresses surface proteins that promote attachment to host proteins forming part of the extracellular matrix on epithelial and endothelial cell surfaces, as well as being a component of blood clots (CitationGnan & Demello, 1999; CitationBaie & Sheikh, 2000). Of the two million nosocomial infections each year, 10% are caused by P. aeruginosa. Infections caused by Pseudomonas aeruginosa are among the most difficult to treat with conventional antibiotics (CitationGnan & Demello, 1999; CitationBaie & Sheikh, 2000; CitationKumar et al., 2006). Bacillus species are common microbes found in most natural environments, including soil, water, plant and animal tissues. While most Bacillus species are regarded as having little pathogenic potential, both Bacillus cereus and Bacillus subtilis are known to act as primary invaders or secondary infectious agents in a number of diseases and have been implicated in some cases of food poisoning (CitationKumar et al., 2006). Many of the plants in the food and medicine of the indigenous people may help to combat these microbes.
Table 1. Antimicrobial activity of Acacia catechu extracts (1 mg/mL).
Table 2. Minimum inhibitory concentration (MIC; in μg/mL) of Acacia catechu extracts against bacteria.
Phytochemical analysis
For TLC plate development, the plant material was extracted in five solvents to get isolated band. The TLC scanning of the Acacia catechu is shown in . A literature survey showed that the chief phytoconstituents found in the plant are catechutannic acid; acacatechin, catechins, epicatechin, isorhamnetin, quercetin, phlobatannins, tannic acid, quercetin, catechu-red and gum. But in the present phytochemical analysis two known compounds (catechin/epicatechin and quercetin) are identified based on their Rf values. The Rf value of catechin is ∼0.15 and that of quercetin is 0.6-0.7, and there was no overlap with any other component in the analyzed sample at 200–450 nm.
Figure 1. Chromatograms obtained with different extracts of Acacia catechu. TLC synopsis: (A) UV lamp image at 254 nm; (B) Mercuric lamp image at 254 nm; and (C) White lamp image at 400- 800 nm. Five spots of five different extracts were made. Spot abbreviations are described below with their application volume:
a = ethyl acetate extract 15 μL, b = methanol extract 15 μL, c = petroleum ether extract 15 μL, d = butanol extract 15 μL, e = chloroform extract 15 μL. Catechin/epicatechin is visible at Rf value of ∼ 0.15 and quercetin at 0.6-0.7.
Acknowledgements
We are thankful to Professor Anil K. Gupta for honorary supervision and support to carry out this research. The authors are also thankful to Sadbhav Foundation Mumbai for financial support.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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