1*Department of Chemistry, Vimala College, Thrissur, Kerala, 680009, India, 1Department of Statistics, Mercy College, Palakkad, Kerala, India.
Email: drbeenajose@gmail.com
Received: 23 Apr 2014 Revised and Accepted: 28 May 2014
ABSTRACT
Objective: Wrightia tinctoria has been extensively used in Folk medicine. It has been reported to have good analgesic, anti-inflammatory, anthelmintic, antiulcer, antidysentric, antidiabetic, anticancer, antipyretic activities and also effective in the treatment of psoriasis. The present investigation was undertaken to statistically evaluate the antimicrobial activity of the leaf and bark extracts of Wrightia tinctoria.
Methods: The antibacterial and antifungal activities of leaf coconut oil extracts (1, 4, 7 and 15 days exposure to sunlight), bark and leaf methanol, ethyl acetate, chloroform and petroleum ether extracts of Wrightia tinctoria (Apocynaceae) against nine pathogenic bacteria such as Bacillus cereus, Enterobacter faecalis, Salmonella paratyphi, Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens and two fungi viz. Aspergillus niger and Penicillium chrysogenum were statistically analyzed so as to evaluate the medicinal potential of these plant extracts.
Results: Wrightia tinctoria leaf and bark methanol extracts were found to be potent extracts and their activity is quite comparable with the standard antibiotics such as tobramycin and gentamycin sulphate screened under similar conditions.
Conclusion: Methanol extracts of the leaves and bark of Wrightia tinctoria can be used as a potential external antiseptic and can be incorporated into drug formulations.
Keywords: Wrightia tinctoria, Antibacterial activity, Statistical analysis, Standard antibiotics, Drug formulation.
INTRODUCTION
The universal role of plants in the treatment of diseases is established by their employment in all important systems of medicine. There are many herbs on earth which lies unexplored in the field of medicine or Science. Wrightia tinctoria (Syn. Pala indigo plant) of Apocyanaceae family is widely used in skin diseases, liver disorders and broad spectrum biological activities [1]. Wrightia tinctoria flower has been reported to have a good anti-inflammatory activity. Many compounds of plant origin have been identified that inhibit different stages in the replication cycle of virus [2, 3]. Wrightia tinctoria is an important medicinal plant used in the Indian system of medicine for the treatment of variety of diseases [4] and it was reported to possess analgesic [5] and cytotoxic activities. It has anti-dandruff properties and hence is used in hair oil preparations. The leaves are a fodder for the cattle, goat and sheep. In south India the plant is used for green manuring rice fields. The major active constituents of the plant are saponins, β-sitosterol, triterpenoids, ursolic acid, lupeol, oleanolic acid, α and β-amyrins [6]. The bark of W. tinctoria is considered for antidiarrhoeal, aphrodisiac, anthelmintic, febrifuge, stomachic, toothache, tonic and dog bite [7, 8]. It is employed in seminal weakness and flatulence, also used in piles and skin diseases [9].
Ethnomedically, the bark of this plant is used as a galactagogue to treat abdominal pain, skin diseases and wounds [10] as an anti-pyretic [11] anti-dysenteric, anti-diarrheal- and anti-hemorrhagic [12] agents, and as an antidote for snake poison[13]. Seeds of this plant are also used as an aphrodisiac [14].
MATERIALS AND METHODS
Plant material
The leaves and bark of Wrightia tinctoria were collected from Thrissur district of Kerala, South India and authenticated by Dr. Kochuthressia M.V., HOD, Department of Botany, Vimala College, Thrissur. Voucher specimen is deposited in the specially maintained herbarium, Department of Botany, Vimala College, Thrissur.
Preparation of Plant Extracts
Fifty grams of the powered plant material were extracted successively with 150ml of petroleum ether, chloroform, ethyl acetate and methanol as solvents for 24hours by Soxhlet equipment. Leaf coconut oil extract was prepared by exposing the fresh leaves of Wrightia tinctoria in coconut oil to sunlight for 1, 4, 7 and 15 days.
Test microorganisms
The microorganisms used for antibacterial and antifungal activity evaluation were obtained from Microbial Type Culture Collection and gene bank (IMTECH, Chandigarh, India). They were bacteria such as Bacillus cereus (MTCC-1305), Enterobacter faecalis (MTCC-5112), Salmonella paratyphi, (MTCC-735), Staphylococcus aureus (MTCC-96), Escherichia coli (MTCC-729), Proteus vulgaris (MTCC-426), Klebsiella pneumoniae (MTCC-109), Pseudomonas aeruginosa (MTCC-647), Serratia marcescens (MTCC-86) and fungi such as Aspergillus niger (MTCC-2425) and Penicillium chrysogenum (MTCC-5108).
Antimicrobial activity assay
The agar diffusion method is used for the antimicrobial evaluations. Wells of 8mm (0.8cm) diameter were dug on the inoculated nutrient agar medium (antibacterial assay) and on potato dextrose agar medium (antifungal assay) with sterile cork borer and 50µl of the petroleum ether, chloroform, ethyl acetate and methanol extracts of the bark and leaf and coconut oil extract of the leaf of Wrightia tinctoria were added in each well. Wells introduced with 50µl of pure petroleum ether, chloroform, ethyl acetate, methanol and coconut oil served as negative controls. The plates were incubated at 370C over night and examined for the zone of inhibition. The diameter of the inhibition zone was measured in mm. The standard antibiotic drugs such as tobramycin, gentamicin sulphate, ofloxacin and ciprofloxacin were used for antibacterial evaluation and standard drug chloramphenicol was used for antifungal study. An extract was classified as active when the diameter of the inhibition was equal to or larger than 8mm [15]. All the assays were performed in triplicate and expressed as average values.
RESULTS AND DISCUSSION
The antimicrobial activities of methanol, ethyl acetate, chloroform and petroleum ether extracts of the bark and leaves of Wrightia tinctoria and leaf coconut oil extracts (1, 4, 7 and 15 days exposure to sunlight) against nine pathogenic bacteria such as Bacillus cereus, Enterobacter faecalis, Salmonella paratyphi, Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens and two fungi viz. Aspergillus niger and Penicillium chrysogenum were statistically evaluated.
Statistical analysis
Let us assume that the mean activity of W. tinctoria leaf methanol extract (Wtla) and W. tinctoria bark methanol extract (Wtba) are same. Also the alternate hypothesis is that the mean activity of Wtla is greater than Wtba. By applying student’s t-test, it can be seen that the calculated t-value is less than tabled value at twenty degrees of freedom with significance level 0.05. Hence the assumption that two extracts having same activity is accepted and the mean activity of Wtba against eleven pathogenic microorganisms is found to be comparable with Wtla.
Similarly by applying t-test, the mean activities of W. tinctoria bark and leaf extracts in various solvents are compared. The mean activity of Wtba>Wtbd (activity of Wtba greater than Wtbd), Wtba>Wtbc, Wtba>Wtlc, Wtba>Wtld, Wtba>Wt15, Wtba>Wt 7, Wtba>Wt 4, Wtba>Wt 1 while that of Wtba-Wtla and Wtba-Wtbb are almost same.
The interval estimation of the mean activity of W. tinctoria bark and leaf extracts in various solvents are given in table 1.
Table 1: Interval estimation of the mean antimicrobial activity of W.tinctoria bark and leaf extracts
| W. tinctoria bark and leaf extracts | Mean activity | Standard Deviation | Interval estimation at 5% level of significance |
| Wtba Wtbb Wtbc Wtbd Wtla Wtlb Wtlc Wtld Wt15 Wt7 Wt4 Wt1 |
20 17.45 14.82 12.91 19 16.82 14.55 12.55 14.36 13.09 11.82 10.18 |
2.79 2.21 2.14 2.12 5.23 4.69 3.86 2.94 1.91 1.70 1.47 1.54 |
20 + 1.65 17.45 + 1.30 14.82 + 1.26 12.91 + 1.25 19 + 3.1 16.82 + 2.77 14.55 + 2.28 12.55+ 1.74 14.36 + 1.13 13.09 + 1.00 11.82 + 0.87 10.18 + 0.91 |
Wtb: W.tinctoria bark; Wtl: W.tinctoria leaf
Wt1, Wt4, Wt7, Wt15: W.tinctoria leaf coconut oil extract with 1, 4, 7 &15 days of exposure to sunlight
a: methanol extract; b: ethyl acetate extract; c: chloroform extract d: petroleum ether extract The correlation studies suggest that Wtla-Wtlb (0.9824), Wtla-Wtlc (0.9263), Wtlb-Wtlc (0.9356), Wtlc-Wtld (0.9661) and Wt1-Wt15 (0.9619) are highly correlated and hence the mean activity of these extracts against eleven pathogenic microorganisms increases simultaneously.
Ethyl acetate (b) and chloroform (c) extracts of W. tinctoria bark and leaf also showed appreciable positive correlation (table 2).
From table 3, it can be seen that the mean activity of Wtla-Wtlb, Wtlb-Wtlc, Wtlc-Wtld and Wtla-Wtba are almost same as the t-value is less than tabled value. The correlation studies showed that standard antibiotics and bark extracts of W.tinctoria Oflo-Wtba, Oflo-Wtbb, Oflo-Wtbc, Oflo-Wtbd, Cip-Wtba, Cip-Wtbb, Cip-Wtbc and Cip-Wtbd showed low positive correlation. Gen- Wtba, Gen- Wtbb, Gen- Wtbc and Gen- Wtbd showed negative correlation and the mean activity of these extracts varies in an inverse manner.
The standard antibiotics and leaf extracts of W.tinctoria Cip-Wtla, Cip-Wtlb, Cip-Wtlc, Cip-Wtld, Gen- Wtla, Gen- Wtlb, Gen- Wtlc, Gen- Wtld, Oflo-Wtla, Oflo-Wtlb and Tob-Wtla exhibited negative correlation and they have inverse relationship.
Table 2: Student’s t-test: Mean activities of W. tinctoria bark and leaf extracts
| W.tinctoria bark and leaf extracts | Calculated value of ‘t’ | Correlation coefficient |
| Wtba-Wtbb Wtba-Wtbc Wtba-Wtbd Wtbb-Wtbc Wtbb-Wtbd Wtbc-Wtbd Wtla-Wtlb Wtla-Wtlc Wtla-Wtld Wtlb-Wtlc Wtlb-Wtld Wtlc-Wtld Wt1-Wt15 Wtla-Wtba |
2.261 4.66 6.40 2.71 4.70 2.00 0.982 2.167 3.40 1.184 2.441 1.303 5.390 0.533 |
0.8759 0.7039 0.5950 0.8675 0.8141 0.8434 0.9824 0.9263 0.8303 0.9356 0.8338 0.9661 0.9619 0.1984 |
Wtb: W.tinctoria bark; Wtl: W.tinctoria leaf; Wt1, Wt15: W. tinctoria leaf coconut oil extract with 1 &15 days of exposure to sunlight, a: methanol extract; b: ethyl acetate extract, c: chloroform extract d: petroleum ether extract
Table 3: Mean activities of W. tinctoria bark and leaf extracts
| W.tinctoria leaf extracts and standard antibiotics | Correlation coefficient | W.tinctoria bark extracts and standard antibiotics | Correlation coefficient |
| Tob-Wtla Tob-Wtlb Tob-Wtlc Tob-Wtld Gen- Wtla Gen- Wtlb Gen- Wtlc Gen- Wtld Oflo-Wtla Oflo-Wtlb Oflo-Wtlc Oflo-Wtld Cip-Wtla Cip-Wtlb Cip-Wtlc Cip-Wtld |
-0.0392 0.030 0.240 0.320 -0.290 -0.30 -0.210 -0.180 -0.12 -0.12 0.10 0.25 -0.52 -0.52 -0.38 -0.25 |
Tob-Wtba Tob-Wtbb Tob-Wtbc Tob-Wtbd Gen- Wtba Gen- Wtbb Gen- Wtbc Gen- Wtbd Oflo-Wtba Oflo-Wtbb Oflo-Wtbc Oflo-Wtbd Cip-Wtba Cip-Wtbb Cip-Wtbc Cip-Wtbd |
0.09 0.24 0.35 0.35 -0.02 -0.18 -0.34 -0.25 0.37 0.20 0.14 0.30 0.58 0.44 0.17 0.23 |
Wtb: W.tinctoria bark; Wtl: W.tinctoria leaf a: methanol extract; b: ethyl acetate extract c: chloroform extract d: petroleum ether extract Tob: tobramycin; Gen: gentamicin sulphate; Oflo: ofloxacin; Cip: ciprofloxacin
CONCLUSION
Wrightia tinctoria bark methanol extract (Wtba) was found to be a potent extract and its activity is quite comparable with the standard antibiotics such as tobramycin screened under similar conditions. W.tinctoria leaf methanol extract (Wtla) was also found to be effective against Staphylococcus aureus and Bacillus cereus and its activity is comparable with the standard antibiotics tobramycin, gentamicin suphate, ofloxacin and ciprofloxacin (10µg each). Methanol extracts of the Wrightia tinctoria bark and leaf can be used as a potential external antiseptic in pharmaceutical preparations. The antimicrobial potency of the W.tinctoria leaf methanol extract can be attributed to the presence of flavonoids, phenolic compounds and saponins and the activity of bark methanol extract is due to the presence of phenolic compounds, saponins and tannins [16, 17].
It is interesting to note that even crude extract of this plant showed prominent activity against various pathogenic bacteria where modern therapy has failed. The variation of the susceptibility of the tested microorganisms could be attributed to their intrinsic properties that are related to the permeability of their cell surface to the extracts [18]. The results of this study support the use of this plant for human diseases and reinforce the ethnobotanical importance of plant as a potential source of bioactive substances.
ACKNOWLEDGEMENT
One of the authors (Beena Jose), sincerely acknowledge University Grants Commission (UGC), New Delhi, for providing financial assistance to carry out the study.
ABBREVATIONS
Wtba: W.tinctoria bark methanol extract; Wtla: W.tinctoria leaf methanol extract
Wtlc: W.tinctoria leaf chloroform extract; Wtlb: W.tinctoria leaf ethyl acetate extract
Wtbc: W.tinctoria bark chloroform extract
Wtbb: W.tinctoria bark ethyl acetate extract
Wtbd: W.tinctoria bark petroleum ether extract
Wtld: W.tinctoria leaf petroleum ether extract
Wt1: W.tinctoria leaf coconut oil extract (1d)
Wt4: W.tinctoria leaf coconut oil extract (4d)
Wt7: W.tinctoria leaf coconut oil extract (7d)
Wt15: W.tinctoria leaf coconut oil extract (15d)
CONFLICT OF INTERESTS
Declared None
REFERENCES