Abstract
The present study was designed to investigate antimicrobial and anti-inflammatory activities of four plant species from Indian origin. The antimicrobial activity of petroleum ether, chloroform, ethyl-acetate and methanol extracts of barks of Acacia nilotica, Berberis chitria, Terminalia paniculata and Madhuca longifolia were tested against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis using agar well diffusion and micro dilution assays. In the well diffusion assay, 27 out of the 64 extracts showed good activity. Significant activity was observed in the micro dilution assay with all extracts. Highest activity against Escherichia coli and Staphylococcus aureus was observed in the ethyl acetate and methanol extracts of Berberis chitria with minimum inhibitory concentration values of 5.5 and 6.5 mg/ml, respectively. Methanolic extracts of Berberis chitria and Terminalia paniculata showed significant inhibitory effect against Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentration values 3.25 and 3.5 mg/ml. The effects of the plant extract were compared with those of Chloromphenicol and Streptomycin. For the investigation of anti-inflammatory activity of the methanol extracts of the selected plants, carrageenan induced rat hind paw edema model was employed. All the selected plant extracts exhibited significant, dose-dependent anti-inflammatory activity and produced a significant inhibition of carrageenan-induced paw edema in rats at the end of 3 h, which was comparable to the standard drug, indomethacin.
Introduction
Plant derived drugs have been used for various ailments due to less side effects and potential efficacy. Plants have potent ability to synthesize aromatic substances, most of which include alkaloids, Quinones, Flavones, Tannins, phenols or their oxygen-substituted derivatives (1). In many cases, these substances serve as plant defense mechanisms against predation by microorganisms, insects and herbivores. Phenolics and Polyphenols show toxicity against microorganisms due to its hydroxyl groups and Quinones provide stable free radicals which target adhesins on microbial cell. Flavones, flavanoids and flavanols are synthesized by plants in response to microbial infection (2). Four plants have been screened or selected for preliminary antimicrobial and anti-inflammatory activities; based on the literature in Ayurveda and ethnobotanical studies. The investigation of certain indigenous plants for their Antimicrobial and anti inflammatory activities can play an important role in drug development program in the pharmaceutical industries.
Acacia nilotica tree colloquially called is the Babbula of Sanskrit writers, who mention the use of young leaves and pods as an astringent. Acacia nilotica contains gallic acid, m-digallic acid, (+)-catechin, chlorogenic acid, gallolyated flavan-3, 4-diol, robidandiol (7, 3, 4, 5-tetrahydroxyflavan-3-4-diol), androstene steroid, D-pinitol carbohydrate, catechin-5-galloyl ester. Acacia nilotica has anticancer and antimutagenic, anti-inflammatory, antiplasmodial, antidiarrhoeal, antihypertensive, antiplatelet aggregatory, molluscicidal, antifungal, antimicrobial activity, and inhibitory activity against Hepatitis C(3-5).
Berberis chitria is an erect, spiny shrub up to 3.3 m height with obovate or elliptic-acute, spinere-serrate leaves, deep yellow flowers in loose corymbose panicles and dark red brown oblong-ellipsoid berries. The root and the stem bark are the richest source of alkaloids (5% and 4.2% respectively calculated as berberine and can be used for commercial manufacture of berberine. Plant contains Berberine, jatrorrhizine, o-methylcorydine-N-oxide palmitine, oxyacanthine (6). Hyperaccumulator and Antimicrobial (7) activities of Berberis chitria have been exploited.
Terminalia paniculata is most frequently found in valleys and on lower slopes preferring fairly moist situations. From the heartwood, Ellagic acid, 3, 3’-O-dimethylellagic acid-4- glucoside, O-pentamethyl flavellagic acid, 3, 4, 3’-O-trimethyl flavellagic acid (8) and ? sitosterol have been isolated. The bark contains 14% tannins which have a pyrogallol nucleus along with gallic acid. Saponins in Terminalia paniculata have spermicidal activity (9).
Madhuca longifolia is member of Sapotaceae family. It is a large, evergreen tree with spreading crown and dark grey or brownish scaly bark. Leaves are thin broadly ovate-lanceolate, acute, glabrous and are clustered near the ends of branches. Flowers are pale yellow and fleshy. Fruits are ovoid and yellow when ripe. Flowers are the rich source of sugars, vitamin A, ascorbic acid, thiamine, riboflavine, Ca, P, Fe, Mg, Cu, anthocyanins, betains, salts of malic and succinic acid (10). Bark contains 17% tannins (11). Bark is used for rheumatism, ulcers, itches, bleeding and spongy gums. The bark is good remedy for inflammations, sprains and pruritus.
The aim of this study was to investigate these plant species to determine their antimicrobial and anti-inflammatory activity and to determine whether the ethnobotanical approach in finding anti-infectives is a useful approach.
Materials and Methods
Plant Materials
Stem Bark of the plant Acacia nilotica (AN, family: Mimosaceae), Terminalia paniculata (TP, family: Combretaceae), Madhuca longifolia (ML, family: Sapotaceae) and root of Berberis chitria (BC, family: Berberidaceae) were collected in and around Haridwar, during September 2007.
Drugs and Chemicals
Carrageenan was purchased from SD Fine Chemicals, Mumbai; Gum Acacia was obtained from Indocap, Faridabad; Indomethacin was obtained as a gift sample from Jagsonpal Pharmaceuticals, Faridabad; Chloramphenicol, Neomycin and Streptomycin were procured from Sigma Aldrich, USA. All other solvents and reagents were of AR grade and were obtained from SD Fine Chemicals, Mumbai.
Microorganisms
The lyophilized vials for the strains of the test organisms were procured from Institute of Microbial Technology (IMTECH), Chandigarh. The microorganisms used were Staphylococcus aureus (MTCC 96), Pseudomonas aeruginosa (MTCC 2453), Escherichia coli (MTCC 1687), and Bacillus subtilis (MTCC 441).
Animals
Wistar rats (150-250 g) and albino mice (20-27 g) of either sex, maintained in the institutional animal house, were used for the anti-inflammatory study.
Preparation of Extracts
The plants parts were shade dried, powdered and extracted individually with methanol by hot continuous percolation, using Soxhlet apparatus. The extracts were filtered and concentrated and kept in a vacuum desicator for complete removal of the solvent. The extracts were stored at 4°C in air tight glass vials until further use.
For antimicrobial activity, dried, ground plant materials were extracted sequentially with petroleum ether (PE), chloroform (CHL), Ethyl Acetate (EA) and methanol (MeOH) using a Soxhlet apparatus. The crude extracts were filtered through Whatman filter paper and concentrated at 40°C under vacuum using a rotary evaporator. The concentrated extracts were dried at room temperature and stored in air tight glass vials until further use.
Phytochemical Screening
The methanol extracts were screened for the presence of various phytoconstituents such as alkaloids, tannins, flavonoids phenolics and glycosides by employing standard phytochemical tests (12).
Antimicrobial Activity
Initially, the antimicrobial potential of different extracts was determined by using zone of inhibition assay using well diffusion method. The extracts that exhibited potential activity in this test were further subjected to the determination of minimum inhibitory concentration (MIC) against selected Gram +ve and Gram –ve bacteria, using micro dilution assay (13-16).
The activated cultures of each test organisms were prepared by inoculating one loop full of the pure culture from the preserved slants into 25 ml of sterile nutrient broth. The inoculated flasks were incubated under shaking conditions at 37°C and allowed to grow them up to their exponential phase. The bacterial culture (200 µl A660 0.8 to 1.0) mixed properly with 25 ml melted agar medium (45°C) and poured on sterile empty petridish and allowed to solidify. After solidification a well (0.75 cm) was prepared in the plates with a cup borer and 100 µl of the test compound was inoculated directly into the well. For the reference solution of standard antibiotics was also tested against the test organisms. The plates were incubated overnight at 37°C. The diameter of zone of inhibition surrounding the well was measured. Chloromphenicol and Streptomycin were used as standards for comparison.
Determination of Minimum Inhibitory Concentration (MIC)
Overnight cultures (incubated at 37°C in a water bath with an orbital shaker) of four bacterial strains, two Gram positive (Bacillus subtilis MTCC 441 and Staphylococcus aureus MTCC 96) and two Gram-negative (Escherichia coli MTCC 1687 and Pseudomonas aeruginosa MTCC 2453), were diluted with sterile Mueller-Hinton (MH) broth (1 ml bacterial suspension/ 50 ml MH). The crude plant extracts were re-dissolved in methanol to make 40 mg/ ml. One hundred microliters of each extract were serially diluted with sterile distilled water in a 96-well micro-plate for each of the four bacteria. A similar serial dilution of neomycin (0.1mg/ ml) was used as a positive control against each bacterium. One hundred microliters of each bacterial culture were added separately to each well. The methanol solvent and bacteria-free controls were included as negative controls. The plates were covered with parafilm and incubated overnight at 37°C. To indicate bacterial growth, 50 µl of 0.2 mg/ ml p-iodonitrotetrazolium chloride (INT) was added to each well and the plates were incubated at 37°C for 30 min. Bacterial growth in the wells was indicated by a change in color, whereas clear wells indicated inhibition by the tested extracts. MIC values were recorded as the lowest concentrations of extracts showing clear wells. The assay was repeated twice with three replicates each.
Anti-inflammatory Activity
Wistar rats and albino mice were the animals used for this study. The animals were maintained in the institutional animal housing facility, under standard husbandry conditions and had free access to food and water. The animals were acclimatized to the laboratory environment for a period of one week prior to the experiment. All the experiments were performed after obtaining prior approval from Institutional Animal Ethics Committee (Approval No. MIET / IAEC / PhD / 001 / 2008-09).
The animals, after overnight fasting, were divided into six groups. Group I (n=6) served as control and received the vehicle (1% Carboxymethylcellulose, 10 ml/kg oral). Group II (n=6) served as Standard, received Indomethacin at dose of 1 mg/kg body weight. Group III (18 animals in subgroups of 6), Group IV (18 animals in subgroups of 6), Group V (18 animals in subgroups of 6), and Group VI (18 animals in subgroups of 6) served as test, received methanolic extract of AN, BA, TP and ML at doses of 100, 200 and 400 mg/kg body weight, oral respectively.
Carrageenan induced paw oedema was used to determine the anti-inflammatory activity of the extracts (17). The animals pretreated with extract or Indomethacin one hour before were injected with 0.1 ml of 1% carrageenan (in 1% CMC) solution into the sub-plantar region of right hind paw. Paw volume was measured by dislocation of the water column in a Plethysmometer (Ugo Basile, Italy) immediately after carrageenan application at 0, 1, 2, 3, 4 and 6 h after the stimulus. Reduction in the paw volume compared to the vehicle-treated control animals was considered as anti-inflammatory response.
The percentage inhibition of edema was calculated as follows:
Percent Inhibition = 100 * {[(Vt – V0)Control – (Vt – V0)Treated] / (Vt – V0)Control}
Where, V0 = paw volume of the rat before administration of carrageenan, Vt = paw volume of the rat after the administration of carrageenan at different time intervals. The percentage inhibition of paw edema is proportional to anti-inflammatory activity.
Results and Discussion
Methanol extract of Acacia nilotica (AN), Terminalia paniculata (TP), Madhuca longifolia (ML) and Berberis chitria (BC) were obtained in the yield of 10.6%, 7.58%, 3.85% and 9.2% w/w respectively. The percent yields of other extracts are given in Illustration 1. After extraction, the extracts were subjected to qualitative phytochemical tests. The results are shown in Illustration 2.
The results of antimicrobial activity of different extracts of the selected plants are presented in Illustrations 3 and 4. All the extracts showed good antibacterial activity in the well diffusion method. A maximum inhibitory effect against Escherichia coli and Pseudomonas aeruginosa was exhibited by methanol extracts of all the four plants. Ethyl acetate and methanol extracts of Berberis chitria showed maximum activity against Staphylococcus aureus. Ethyl acetate extract of Acacia nilotica showed maximum inhibitory zone against Bacillus subtilis.
The results of determination of MIC are given in Illustration 4. In this test, the methanol extracts showed the highest activity followed by the ethyl-acetate extracts in all the plants. EA and MeOH extracts of Berberis chitria gave the best antibacterial activity against Escherichia coli with MIC values of 5.5 mg/ml and 6.5mg/ml, respectively. MeOH extract of Acacia nilotica was inhibitorier on the Pseudomonas aeruginosa than the methanol extract of Berberis chitria with MIC values of 7.25 and 7.5 mg/ml. All the four plant extracts had higher activity on Gram-positive bacteria than Gram-negatives. In some cases there were lower activities against Gram-negative bacteria, which could be due to the thick murein layer in their structure preventing the entry of inhibitors (18). MeOH extract of Berberis chitria and EA extract of Terminalia paniculata gave the best antibacterial activity against Staphylococcus aureus with MIC values of 3.25 mg/ml and 3.5 mg/ml, respectively. Almost all the plants and all the extracts showed inhibitory activity against Bacillus subtilis. Methanol extract of Terminalia paniculata showed significant MIC value of 3.25 mg/ml against Bacillus subtilis.
Phytochemical analysis of all the four plants revealed the presence of alkaloids, glycosides, tannins, phenolics, coumarin and xanthoproteins (Illustration 2). Highest in vitro antioxidant activity and positive phytochemical test for phenolics, coumarin and lignin and reported ethanopharmacological properties such as antidiarrhoeal properties supports the antimicrobial activity of AN, TP. It is well known that phytoconstituents have antimicrobial properties (19). All phenolics are high oxidation state compounds and the sites and number of hydroxyl groups on the phenol group is thought to be related to their relative toxicity to microorganisms, with evidence showing that the increased hydroxylation results in increased toxicity (20). The mechanisms thought to be responsible for phenolic toxicity to microorganisms include enzyme inhibition by the oxidized compounds, possibly through reaction with sulfhydryl groups or through more nonspecific interactions with the proteins (19). Tannins which are group of polymeric phenolic substances interact with proteins through so-called nonspecific forces such as hydrogen bonding and hydrophobic effects, as well as by covalent bond formation (20). Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, enzymes, cell envelope transport proteins, etc. BC, whose total phenol content was 2.73 ?g/ml, which is lower than AN (9.88 ?g/ML), showed the highest activity against the microorganisms among the selected plants. Apparently, the broad antimicrobial activity of BC is correlated with the presence of the alkaloids found in the phytochemical screening and its traditional use in infectious disease.
The anti-inflammatory effect of the extract and the reference drug in carrageenan induced paw edema model in rats is shown in Illustration 5. After carrageenan administration, paw edema in rats reached to a peak value at 4 h and various doses of methanolic extract of all four plants produced a significant inhibition in the edema volume (P < 0.001) at the end of 3 h. Maximum percent inhibition of edema exhibited with 400 mg/kg of methanolic extract of Acacia nilotica at was 74% and the effect was comparable to that of the standard drug 85%.
It is well-known that in chronic and sub-acute inflammation, reactive oxygen species (ROS) play an important role in modulating the extent of inflammatory response and consequent tissue and cell injury (21) and antioxidants are considered as possible protection agents reducing oxidative damage of human body from ROS and retard the progress of many diseases (21). In our earlier in vitro antioxidant studies, AN showed 91.1% DPPH radical inhibition at the 10 ?g/mL conc, 98.41% ABTS radical inhibition at the 25 ?g/mL conc, 31.91% NO radical inhibition at the 40 ?g/mL conc, 11.64% H2O2 radical inhibition at the 25 ?g/mL conc, and 43.54% OH radical inhibition at the 20 ?g/mL concentration (22). The results of previous in vitro antioxidant studies and quantitative determination of phenols, strongly support the high anti-inflammatory activity of AN due to free radical scavenging activity in carrageenan-induced paw edema model (22). Phytochemical analysis of AN, which revealed the presence of phenolic compounds and tannins, support its highest anti-inflammatory activity among the group.
Conclusion
The methanol extracts of all the selected plants exhibited good antibacterial and anti-inflammatory activities. Among the selected plants, Acacia nilotica exhibited excellent antibacterial as well as anti-inflammatory activities, which might be attributed to the presence of high concentration of phenolic compounds in this plant. This study supports the traditional use of this plant in infections and inflammations. Further studies for isolation and identification of the phytochemicals responsible for the activities are needed to establish it as a herbal medicine for infectious diseases and therapy of inflammation.
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Source(s) of Funding
No funding was obtained for this project from any agency
Competing Interests
Nil
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