INFLUENCE OF DIFFERENT SOLVENTS ON ANTIBACTERIAL POTENTIAL OF THREE SPECIES OF HIMALAYAN OAKSHTML Full Text
INFLUENCE OF DIFFERENT SOLVENTS ON ANTIBACTERIAL POTENTIAL OF THREE SPECIES OF HIMALAYAN OAKS
Suresh Chandra Sati, Amrendra Nath Tripathi * and Parikshit Kumar
Department of Botany, D.S.B. Campus, Kumaun University, Nainital, Uttarakhand, India.
ABSTRACT: The antibacterial property of three Himalayan Oaks species (Quercus floribunda, Q. leucotrichophora and Q. semecarpifolia) in different solvents against eight gram-positive and gram-negative pathogenic bacteria employing disc diffusion method has been investigated. All the test microorganisms were inhibited significantly by Oaks species at 1000 μg/ml. The MIC and MBC values of each extract (where ZOI ≥ 15 mm) were also determined. Q. semecarpifolia exhibited significantly higher antibacterial activity compared to other species. Solvent systems showed species-specific response for extraction of total content (% yield) of dry extract. Maximum yield was found for ethanol extract (3.59-5.79%). Methanol extract of Q. leucotrichophora showed the highest inhibitory activity against all the tested bacteria (16-23 mm) ZOI, followed by acetone extract (17-23 mm), ethanol extract (15-20 mm). Furthermore, methanol and acetone extracts of Q. leucotrichophora showed significant activity against A. tumefaciens and E. chrysanthemi, with MIC values of 15.6-31.25 μg/ml and MBC values of 15.6-125 μg/ml. Q. semecarpifolia showed its highest antibacterial potential against X. phaseoli and E. chrysanthemi whereas extracts of Q. floribunda leaves were found with moderate activity against all the tested bacteria. The chloroform and ethanol extracts have the lowest inhibitory activity (ZOI, 22 mm each). All the tested bacterial strains were found resistant to aqueous extracts of Oaks species. The bacterial inhibitory potential of Q. semecarpifolia was found very close to gentamycin (30 mcg). The present work fully highlighted the utility of three Oaks spp. of Kumaun Himalaya for their antibacterial activities against pathogenic bacteria.
Keywords: Kumaun Himalaya, Oaks spp, Plant extract, Gentamycin, Antibacterial activity
INTRODUCTION: Plants in various forms are being used in different traditional systems of medicine for the treatment of human ailments, particularly those caused by pathogenic microbes since time immemorial 1. These are also valuable resources for the isolation of novel bioactive molecules to combat microbial diseases.
Now a day’s infectious diseases represent a critical problem to health and they are one of the main causes of morbidity and mortality worldwide 2. The discovery of novel active compounds against new targets is a matter of urgency. Almost every part of the plant possesses antimicrobial activity 3 it is due to the presence of phytoalexins formed in plants. The effect of plant extracts on bacteria has been studied by a large number of researchers in different parts of the world 3, 4, 5.
Quercus spp. (Family: Fagaceae) represents an important group of evergreen or semi-deciduous trees from temperate and tropical climatic areas. The genus Quercus is comprised of around 450 species worldwide, which often differ in their flowering and fruiting dynamics 6. Quercus species are used in traditional medicine as astringent, antiseptic and haemostatic and for the treatment of acute diarrhea, hemorrhoid and oral, genital and anal mucosa inflammation 7. The decoction of Q. floribunda plants can be used for burns and added to ointments for the healing of cuts 8. Due to the multipurpose use of Quercus species, it has been the subject of intensive research.
Some recent reports revealed ethnobotanical uses of Q. leucotrichophora in the Himalayan region like, in North Pakistan, fruit and seed powder is taken for the treatment of urinary infection 9, 10 bark is used to cure toothache and piles leaves is used as an astringent and in the treatment of diarrhea while the gum resin paste is used as stomachache, to cure Gonorrhea, asthma, hemorrhages, diarrhea, dysentery 11, 12 and its dry resin with water has been used for stomach pain 13, 14. Some authors reported that, Q. floribunda is also used as an astringent and antidiarrhoeal 15. The seeds are used in the treatment of diarrhoea, menorrhagia, and gastrointestinal hypertrophy. It has been reported that different species of Quercus possess antibacterial activity 16, 17, antioxidant activity and in vivo- hepatoprotective activity 18, 19 and anti-inflammatory effect 20.
In the literature, many species of the genus Quercus have been studied for their biological activities i.e., antioxidant, antibacterial, and antifungal. However, little is known on the antibacterial and antifungal properties of Himalayan Quercus species. The aim of this study was to investigate the antibacterial potential of various organic extracts of leaves of Q. leucotrichophora, Q. semecarpifolia, and Q. floribunda, occurring at high altitude in Kumaun Himalaya, India, against a wide range of pathogenic bacterial strains, which have not been evaluated earlier.
MATERIALS AND METHODS:
Collection of Plant Material: Green leaves of Q. leucotrichophora (QL), Q. semecarpifolia (QS), and Q. floribunda (QF) were collected in the month of April from Nainital, Kumaun Himalaya, India and authenticated by the plant taxonomist of the department of Botany, Kumaun University Nainital. Voucher specimens were deposited in the herbarium of the department for further references.
Extraction Procedure: Leaves of the plants were thoroughly washed with distilled water and dried at room temperature (20 ± 2°C). The dried material was powdered in an electric grinder. To prepare the stock solution 50 g of this powder was placed in a 500 ml conical flask mixed with 200 ml of solvents (w/v, 50g/ 200 ml). The mouth of flasks is tightly plugged with non-absorbent cotton and tightly wrapped with aluminum foil to prevent evaporation. Solvents used for extraction were methanol, ethanol, chloroform, ethyl acetate, acetone, hexane, and distilled water. All mixtures were shaken on a rotary incubator shaker at 190-220 rpm for 24 h at room temperature 37°C. The mixtures were filtered through Whatman filter paper no. 1 and filtrate collected separately in a clean beaker. The extracts were evaporated, using steam both to dryness at 30°C. The dry extracts were weighed and kept in sterile sample bottles and stored in the refrigerator at 4°C for further use.
Microorganism Used: Five (Gram +ve and –ve) bacteria (Bacillus subtilis MTCC No. 121, Escherichia coli MTCC No. 40, Agrobacterium tumefaciens MTCC No. 609, procured from Institute of Microbial Technology Chandigarh, India, and Ralstonia solanacearum ITCC No. BH0007, Xanthomonas campestris ITCC No. BD0006, Xanthomonas oryzae ITCC No. PI0012 was obtained from Indian Type Culture Collection, IARI, New Delhi, India on the other hand, Xanthomonas phaseoli and Erwinia chrysanthemi obtained from Plant Pathology Department, G. B. Pant University, Pantnagar, India, were used in this investigation. Pathogens obtained from respective stock cultures were inoculated (1% v/v) into nutrient agar broth followed by incubation at 37°C (for 18 h) to activate cultures.
Screening of Antibacterial Activity: Antibacterial tests of selected microorganisms were carried out using the disc diffusion method 21. Nutrient agar plates (90 mm size) were prepared and cool down at room temperature (20 ± 2°C). Test bacterial inoculums containing 106 CFU/ml of test bacteria were spread uniformly. A small sterile swab was dipped into 24 h old test culture of bacteria and was inoculated by streaking the swab over the entire agar surface. The process was repeated by streaking the swab 2 or more times rotating the plates approximately 60° each time to ensure the even distribution of inoculums. Each bacterial culture was inoculated on three plates as replicates. Stock solutions of the tested samples were prepared into 10% v/v aqueous dimethyl sulfoxide solution. The sterile filter paper disc (5 mm) loaded with 20µl of extract were placed on the surface of the bacteria seeded agar plates at equidistance, and it was allowed to diffuse for 5 min then these plates were incubated at 37± 1°C for 24 h of incubation in the bacterial incubator. Gentamycin (30 mcg) was placed in to agar plates used as a positive control, and 10% v/v aqueous dimethylsulfoxide solution was also used as a negative control. After 24 h of incubation, the diameter was observed for the inhibition zone (measured in mm, including disc size). All tests were performed in triplicates, and observed values of ZOI are expressed as mean values with standard error of means (SEM).
Determination of Minimum Inhibitory Concentrations (MIC’s): The most effective plant extracts which exhibiting a strong antibacterial activity with ZOI, 15 mm or more than 15 mm at 1000 μg/ml were tested to determine their MIC using disc diffusion method and evaluate their efficiency in controlling bacterial strains causing plant pathogenic diseases. MIC and MBC were performed at seven concentrations of extracts (500, 250, 125, 62.5, 31.25, 15.625, and 7.8 μg/ml) following two-fold serial dilution technique 22 and loaded their requisite amount over sterilized filter paper discs (6 mm in diameter). Nutrient agar was poured into sterile Petri dishes and seeded with bacterial suspensions of the pathogenic strains. The loaded filter paper discs with different concen-trations of the effective plant extract were placed on the top of the Nutrient agar plates. The plates were incubated at 35ºC for 24 h. The inhibition zones were measured and recorded against the concentrations of the effective plant extracts.
Determination of Minimum Bactericidal Concentrations (MBC’s): Streaks were taken from the two lowest concentrations of the plant extract plates exhibiting invisible growth (from inhibition zone of MIC plates) and subcultures onto sterile nutrient agar (NA) plates. The plates were incubated at 35 ± 2 ºC for 24 h. then examined for bacterial growth in corresponding to plant extract concentration. MBC was taken as the concentration of plant extract that did not exhibit any bacterial growth on the freshly inoculated agar plates.
RESULTS AND DISCUSSION:
Plants Extract Yield: The results revealed that different solvent systems showed a variable percentage of yield. The percentage yield of leaves extracts of Q. leucotrichophora (QL), Q. semecarpifolia (QS) and Q. floribunda (QF) in methanol, acetone, ethanol, chloroform, hexane and aqueous solvents are presented in Table 1. The ethanol extracts of QS and QL were showed the highest yielding (5.79% and 5.57%, respectively), while the hexane extracts were observed the least yield (0.81% and 1.53 %, respectively). On the other hand, methanol extract of QF leave was showed its highest yield (4.42%), followed by ethanol extract (3.59%), aqueous extract (3.54%) but hexane extract gave the least yield (0.64%). The polarity of solvent used for extraction and the method of extraction play a vital role in the efficiency (yield) and efficacy (magnitude of bioactivity) of prepared extracts 23. It is interesting to note that the hexane (non-polar) extracts of all selected plants were found lowest yield compare to others solvent extracts (polar solvent).
TABLE 1: PERCENTAGE OF YIELD IN DIFFERENT EXTRACTS OF QUERCUS LEAVES
|Yield of extract (%)|
|Plant extract||Q. leucotrichophora||Q. floribunda||Q. semecarpifolia|
Antibacterial Activity of Plants Extract: Results obtained from disc diffusion method for antibacterial activity of the plant extracts of QL, QS, and QF in different solvents against eight pathogenic microorganisms are shown in Table 2 and 3. Relying upon the results obtained in the present investigation, it is clear that almost all leaves extracts of tested species were potentially effective in suppressing microbial growth of plant pathogenic bacteria with variable potency except aqueous extract. This might be due to the various substances that show activity against bacteria are more soluble in organic solvents than water and might be absent in aqueous extract as suggested by Boer et al. 24 Now- a days a number of micro-organisms acquire several resistance mechanism; making them multi-drug resistance (MDR) and develop novel antimicrobial agents for combating resistant organism 25.
Methanol and acetone extract of all tested Oaks species were found most effective against all bacterial strains Fig. 1, 2, and 3. All the plant extracts were found active against both Gram-positive and Gram-negative bacteria. This is contrary with the earlier reports, which suggest that plant extracts remain more active against Gram-positive bacteria as compared to Gram-negative bacteria 26, 27. While comparing with standard drug (gentamycin), the plant extract demonstrated relatively lesser anti-microbial activity Fig. 1 and Table 2.
These results show the potential utility of natural compounds over synthetic antibiotics considering the reported side effects of synthetic chemicals 28. Also, synthetic drugs have high production costs; therefore, biologically active compounds derived from plants can be an alternative source to combat infectious diseases 29.
In a recent study, Semwal et al., 30 reported that methanol extract of QL leaves has significant inhibitory activity against a panel of bacterial strains, and the result of the present investigation also supported the earlier findings. Out of six fractions tested of QL leaves, the methanol and acetone extracts were found most active against all the tested bacterial strains with a maximum zone of inhibition against E. chrysanthemi (ZOI, 23 mm each) except X. compestris, which was found sensitive to acetone extract of the species Table 2.
On the other hand, the second highest antibacterial activity (ZOI, 22 mm) was recorded at four instances i.e., methanol and acetone extract of QL against E. coli and B. subtilis respectively. Quercetin and its 3‐O -disaccharides were also isolated from the leaves of Quercus sp.31` and recently, some investigators reported the remarkable bacterial inhibitory potential of quercetin against a panel of bacteria 32, 33, 34. Ethanol, methanol and acetone extracts of QS leaves showed significant activity against all studied bacterial strains Fig. 2.
The polarity of a solvent plays an important role on the composition of an extract and hence on its potential antibacterial activity. Fig. 2 showed that chloroform extract of QS has the least inhibitory activity against maximum tested bacterial strains. But it was interesting to note that, the promising inhibitory activity of QS was recorded in chloroform and acetone extract against X. phaseoli and E. chrysanthemi (ZOI, 22 mm each) respectively Table 2 and Fig. 2. The present study clearly demonstrated that chloroform extract of QS showed the comparable inhibition potential with used positive control (gentamycin) against X. phaseoli Table 2 and Fig. 2. Hexane extracts of QS and QF were also showed promising antibacterial activity against all the studied bacterial strains.
On the other hand Jamil et al., 35 studied the antibacterial activity of QF (Syn. Q. dilatata) and reported that polar fractions of the aerial part showed remarkable antibacterial activity with the highest against Bordetella bronchiseptica. Although, in the present study, polar extract (methanol and acetone) showed moderate inhibitory activity and ethanol has the least activity Fig. 3. This inhibitory effect might be influence by climatic conditions i.e., CO2 and temperature 36. Recently, Top et al., 37, studied the climatic influence on content and chemical composition of Quercus rubra tissues and reported that climatic factors directly influenced the biologically active polyphenolic and tannin content of Q. rubra tissues. As evident from Table 2, it was notable that all tested bacterial strains have resistant potential against aqueous extracts of all studied plants and chloroform extract of QL. Chloroform extract of QF was also found least active against E. coli and X. compestris with ZOI, 19 and 15 mm respectively, while it has no activity against other tested strains Table 2 and Fig. 3. Also, hexane extracts of QL did inhibit no growth of A. tumefaciens, E. coli and X. phaseoli. Similar results were also reported by Berahou et al., 38 that only ethyl acetate, butanol and aqueous phases of methanol extract of Quercus ilex bark showed antibacterial activity against all the bacterial strains, whereas the hexane and dichloromethane phases were found almost inactive. This suggests that the quality of active compounds responsible for antimicrobial activity varies extracts to extracts Table 2.
The polar organic solvents exhibited greater antimicrobial activity Fig. 1, 2, and 3 and strengthening the fact that antimicrobial agents are polar in nature; therefore, can be extracted through polar organic solvents 7. The lipophilic solutes extracted by hexane and chloroform extract (non-polar solvent) are, therefore, not much effective against Gram-negative bacteria. Generally, Gram-positive was found comparatively more susceptible than Gram-negative bacteria, which might be due to the presence of outer peptidoglycan layer, which is not an effective permeability barrier 39. However, in this study, more sensitivity of Gram-negative bacterial strains might be due to other possible mechanisms of extracted constituents which are directly associated with membrane disruption 40.
FIG. 1-3: ANTIMICROBIAL ACTIVITY OF DIFFERENT EXTRACTS OF Q. FLORIBUNDA LEAVES AGAINST BACTERIAL STRAINS, ATU- A. tumefaciens; BSU- B. subtilis; ECH- E. chrysanthemi; ECO- E. coli; XPH- X. phaseoli; RSO- R. solanacearum; XOR- X. oryzae; XCO- X. compestris
TABLE 2: ANTIBACTERIAL ACTIVITY OF DIFFERENT EXTRACTS OF Q. LEUCOTRICHOPHORA Q. SEMECARPIFOLIA AND Q. FLORIBUNDA LEAVES
|Plant extracts||Diameter of Inhibition Zone (mm)*|
|Gentamycin (Positive Control)||26±0.9||21±1.9||28±1.3||26±1.8||22±1.0||23±1.1||25±0.9||22±0.3|
ATU- A. tumefaciens; BSU- B. subtilis; ECH- E. chrysanthemi; ECO- E. coli; XPH- X. phaseoli; RSO- R. solanacearum; XOR- X. oryzae; XCO- X. compestris; na- not active
Minimum Inhibitory Concentrations (MIC’s) and Minimum Bactericidal Concentrations (MBC’s): The MIC and MBC results are summarized in Table 3. Organic extracts from the leaf part of QL appeared to be more potent than that of QS and QF. In many instances, MIC and MBC values of QL and QS extracts were lesser than QF extracts, it showed that higher MIC and MBC values represents the least bioactivity. Among all the extracts tested lowest MBC values demonstrated by methanol and acetone extracts of QL against E. chrysanthemi (15.6 and 31.25 µg/ml, respectively). Methanol extract of QL also had significant MBC value against E. coli (31.25 µg/ml). Lower limit of MIC values (15.6 µg/ml) was also demonstrated by chloroform, ethanol and acetone extracts of QS against X. phaseoli, E. coli and E. chrysanthemi, respectively Table 3.
The hexane extract of QF had a significant MIC value against R. solanacearum (15.6 µg/ml). It is evident from Table 3 that X. oryzae had more resistant potential for different tested organic extracts (polar and non-polar) of QS and QF as compare to QL. Only acetone extract of QS had significant MIC and MBC to inhibit the X. compestris growth (62.5 and 125 µg/ml respectively) while other tested plant extracts were more sensitive for X. compestris different concentrations Table 3.
It was noteworthy that different organic extracts of QF leaves had moderate MIC values range (125-500 µg/ml) but hexane extract showed significant MIC (62.5-250 µg/ml) and MBC (62.5-500 µg/ml) values against tested bacterial strains except E. coli and X. oryzae Table 3. Jamil et al., 35 reported that methanol extract of QF had effective MIC (range 200-500 µg/ml) against tested pathogenic bacterial strains and they reported that bacterium E. coli most sensitive with lowest MIC value (200 µg/ml) for methanol extract. In the present investigation acetone extract also had close proximity of MIC (250 µg/ml) against E. coli Table 3. This proximity of inhibitory potential in acetone and methanol extracts might be possible due to the propinquity of polarity in observed solvent system.
It was also notable that, in some instances studied plant extracts showed significant MIC against tested bacteria but do not have MBC against the same bacteria i.e., ethanol extract of QL and QS against X. phaseoli, acetone extract of QS against R. solanacearum, hexane extract of QF against B. subtilis, methanol extract of QS and chloroform extract of QF against X. compestris Table 3. MIC and MBC observations clearly showed that the studied plant extracts had less bactericidal potential as compare to bacteriostatic potential against gram-negative bacteria. The antibacterial activity of selected Quercus species had never been checked before against plant pathogenic bacteria. Thus, this is the first report on antibacterial activity of leaves extracts of Kumaun Himalayan Quercus species i.e., QL, QS and QF against a panel of plant pathogenic bacteria. The results of the present investigation are largely in agreement with the earlier studies on antimicrobial activity of other Quercus species 7, 16, 17, 35, 38, 41 and provide a lead towards searching for new source of antibacterial substances. The results of this study indicate valuable findings for promoting the use of such species as a potential supplement in drug development. On the basis of available literature, it is observed that there is no previous record on the sensitivity of these plants' pathogenic bacterial strains E. chrysanthemi, A. tumefaciens, and X. phaseoli which are responsible for various plant disease.
TABLE 3: MIC AND MBC VALUES OF DIFFERENT EXTRACTS OF Q. LEUCOTRICHOPHORA, Q. SEMECARPIFOLIA AND Q. FLORIBUNDA LEAVES
|Plant extracts||MIC and MBC values (μg/ml)|
ATU- A. tumefaciens; BSU- B. subtilis; ECH- E. chrysanthemi; ECO- E. coli; XPH- X. phaseoli; RSO- R. solanacearum; XOR- X. oryzae; XCO- X. compestris; na- not active; nt- not test
CONCLUSION: The antimicrobial activity of different organic extracts of Q. leucotrichophora (QL), Q. semecarpifolia (QS) and Q. floribunda (QF) leaves were studied and indicated that the tested Quercus species have good antibacterial potentiality against all tested bacterial strains, especially against gram-negative bacteria. Furthermore, methanol and acetone extracts of Q. leucotrichophora showed more significant activity against A. tumefaciens and E. chrysanthemi, with MIC values of 15.6-31.25 μg/ml and MBC values of 15.6-125 μg/ml. The efficacy of each species differs depending on the chemical profile of the plants. The results suggest that these plant extracts should be further analyzed, as it might provide a new compound that is effective against multi-resistant infections.
ACKNOWLEDGEMENT: The authors wish to thank the Department of Plant Pathology, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand (India), and Microbial Type Culture Collection (MTCC), Chandigarh (India) for providing bacterial strains. One of us (ANT) is also thankful to University Grant Commission (UGC), New Delhi, for financial support under UGC-JRF Fellowship.
DECLARATION OF CONFLICT OF INTEREST: No potential conflict of interest was reported by the authors.
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How to cite this article:
Sati SC, Tripathi AN and Kumar P: Influence of different solvents on antibacterial potential of three species of Himalayan oaks. Int J Pharm Sci & Res 2022; 13(1): 197-05. doi: 10.13040/IJPSR.0975-8232.13(1).197-05.
All © 2022 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Suresh Chandra Sati, Amrendra Nath Tripathi * and Parikshit Kumar
Department of Botany, D.S.B. Campus, Kumaun University, Nainital, Uttarakhand, India.
14 February 2021
01 June 2021
09 June 2021
01 January 2022