Abstract
Objective
The aim of the present investigation was to assess the antioxidant, antibacterial and antifungal activities of various fractions of Wendlandia exserta (Roxbukan Pansara).
Methods
The methanolic extract of W. exserta (Roxbukan Pansara) was dissolved in water (distilled) and then partitioned with solvents e.g. n-hexane, CHCl3, EtOAc and n-BuOH consecutively. Antioxidant potential of above mentioned fractions and remaining aqueous fraction was checked by four methods. Antimicrobial activity was examined by four bacteria and four fungi by the disc diffusion method by using a standard, as positive control.
Results
The aqueous fraction displayed maximum value of diphenyl picryl hydrazyl (DPPH) radical inhibition (85.94%±0.35) with an IC50 value of 415.31±0.26 μg mL−1. The aqueous fraction also displayed the maximum ferric reducing antioxidant power (FRAP) value [64.5±0.15 μg of trolox equivalents (TE)] as well as maximum inhibition of lipid peroxidation (42.32%±0.59) and maximum value of total phenolic contents (87.10±1.10). However, the n-BuOH fraction was found to display the maximum value of total antioxidant activity (0.529±0.76). The fractions also displayed good antimicrobial activity against various microorganisms.
Conclusions
It was concluded that polar fractions of W. exserta showed good antioxidant and antimicrobial activity, so these might be useful in pharmacological preparations.
Özet
Amaç
Bu araştırmanın amacı, Wendlandia exserta (Roxbukan Pansara) ’nın çeşitli fraksiyonlarının antioksidan, antibakteriyel ve antifungal aktivitelerini değerlendirmektir.
Yöntemler
Wendlandia exserta’nın (Roxbukan Pansara) metanolik ekstraktı suda çözülmüş (damıtılmış) ve daha sonra farklı çözücülerde, ör. n-heksan, CHCl3, EtOAc ve n-BuOH sırayla ayrıştırılmıştır. Yukarıda belirtilen fraksiyonların ve kalan sulu fraksiyonun antioksidan etkisi dört yöntemle kontrol edildi. Antimikrobiyal aktivite, standart olarak pozitif kontrol kullanılarak disk difüzyon yöntemi ile dört bakteri ve dört mantar üzerinde incelendi.
Bulgular
Sulu fraksiyon 415,31±0,26 μg mL−1 IC50 değeri ile DPPH radikal inhibisyonunun maksimum değerini (% 85,94±0,35) gösterdi. Sulu fraksiyon ayrıca maksimum lipoproteinasyon inhibisyonu (% 42,32±0,59) ve toplam fenolik içeriğin maksimum değeri (87,10±1,10) yanı sıra maksimum FRAP değeri (64,5±0,15 μg troloks eşdeğerleri) göstermiştir. Bununla birlikte, n-BuOH fraksiyonunun toplam antioksidan etkinliğinin en yüksek (0,529±0.76) olduğu bulunmuştur. Fraksiyonlar ayrıca çeşitli mikroorganizmalara karşı iyi antimikrobiyal etki göstermiştir.
Sonuçlar
Wendlandia exserta’nın polar fraksiyonlarının iyi antioksidan ve antimikrobiyal aktivite gösterdiği sonucuna varılmış, bu nedenle bu fraksiyonların farmakolojik preparatlarda yararlı olabileceği sonucuna varılmıştır.
Introduction
In the last few decades medicinal importance of traditional plants has been increasing in different parts of the world. Due to potent pharmacological activities, economic viability and low toxicity medicinal plants have been used extensively [1]. Medicinal plants are the class of plants used for therapy and have pharmacological actions for animal and human [2]. The preservative effect in many plants and herbs is due to the presence of antioxidative and antimicrobial compounds in their tissues. It has been studied that there is an opposite correlation between antioxidative status and occurrence of human diseases such as aging, cancer, atherosclerosis, and neurodegenerative disease [3]. For the cure and prevention of cancer, diabetes, stroke, atherosclerosis and Alzheimer’s disease drugs formulation is based on the antioxidants [4]. Some of the plant produced antioxidants like carotenoids, flavonoids, tocotrienols, tocopherols, cinnamic, folic, ascorbic and benzoic acid. Vitamin C (ascorbic acid), α-tocopherol and β-carotene are amongst the commonly used antioxidants [5]. Oxidative stress was produced due to reactive oxygen species (ROS) or by free radicals formation during metabolic activities. In mitochondria molecular oxygen reduction takes place in various steps of cellular respiration, producing the radicals such as HO˙, O2˙−, and H2O2 [6]. As oxidative damage causes chronic diseases, the dietary antioxidants compete against damage and lowered the risk of diseases [7]. Even a low concentration of antioxidants as compared to oxidisable substrate significantly prevents or delays oxidation of substance [8]. In addition to health benefits, antioxidants are used as food in order to delay or prevent oxidation caused by free radicals due to environmental factors e.g. light, temp. and air [9]. Therefore natural antioxidants have been obtained importance due to their ability to neutralize free radicals. Due to toxicological effects of synthetic antioxidants and preservatives, different medicinal plants are being used due to their antibacterial and antioxidant potential [10]. At present most commonly used antioxidants are tert-butylhydroquinone (TBHQ), butylated hydroxyl toluene (BHT), butylatedhydroxyl anisole (BHA) and propyl gallate (PG). However, they are responsible for carcinogenesis and liver damage in lab animals [11]. It has been observed that antioxidants in addition to retard the oxidation process may also exhibit antimicrobial activity [12].
Wendlandia exserta (Roxbukan Pansara) is a small evergreen tree with reddish brown bark, gray tomentose branches and branchlets. It is found in the interior part of Bengal, particularly over the ruins of ancient city of Gour, Henry Creighton, Roxburgh and distributed in Pakistan (Punjab), Kashmir, Nepal. It grows in sub-Himalayan tracts ascending up to 1200 m. The wood is very durable and resistant against white ants. Wendlandia exserta (Roxbukan Pansara) leaves and fruit are used in decoction for treatment of amenorrhea, febrifuge, antispasmodic and for skin diseases (exima, scabies). They are also known to be an excellent blood purifier and flowers are utilized in joint infection [13].
The literature survey conducted on various extracts of W. exserta (Roxbukan Pansara) whole plant showed that the antioxidant and antimicrobial activity of this plant has not been yet reported; hence in the present study these activities have been investigated.
Materials and methods
Plant material
Wendlandia exserta (Roxbukan Pansara) plant was collected in March 2009 from hills of Kashmir. A Taxonomist Mr. Muhammad Ajaib from Lahore, Pakistan recognized the plant. In the herbarium of the GC University Lahore, Pakistan a voucher no. (G.C. Herb. Bot. 864) has been placed on this plant.
Extraction/fractionation
The entire plant was desiccated in shadow and crushed. At room temperature 6.5 kg of crushed plant was extracted with methanol (3 L×4). The residue recovered from the combined methanolic extract was divided into hexane (1 L×4), CHCl3 (1 L×4), EtOH (1 L×4), and BuOH (1 L×4) fractions. Each of these fractions was concentrated on rotary evaporator separately. In vitro antioxidant and antimicrobial activity then performed on these obtained residues. The initial crude methanolic extract as well as remaining aqueous fraction was also tested in order to check their antioxidant and antimicrobial activity.
Chemicals and standards
“1,1-Diphenyl-2-picrylhydrazyl” radical (DPPH˙), “2,4,6-tripyridyl-s-triazine” (TPTZ), Trolox, “Follin Ciocalteu reagent”, “Gallic acid”, and “Butylated hydroxyltoluene” (BHT) were taken from Sigma USA and organic solvents (n-hexane, CHCl3, EtOAc and n-BuOH), “sulfuric acid”, “ammonium molybdate”, “sodium phosphate”, “ferrous chloride” and “ferric chloride” obtained from Merck Germany.
DPPH radical scavenging activity
The DPPH radical scavenging activities of various fractions of plant were examined by comparison with that of known antioxidant, butylated hydroxytoluene (BHT) using the method [14]. Briefly, various amounts of the samples (1000 μg/mL, 500 μg/mL, 250 μg/mL) were mixed with 3 mL of methanolic solution of DPPH (0.1 mM). The mixture was shaken vigorously and allowed to stand at room temperature for one an hour. Then absorbance was measured at 517 nm against methanol as a blank in the spectrophotometer. Lower absorbance of spectrophotometer indicated higher free radical scavenging activity.
The percent of DPPH decoloration of the samples was calculated according to the formula:
Each sample was assayed in triplicate and mean values were calculated.
Total antioxidant activity by phosphomolybdenum method
The total antioxidant activities of various fractions of plant were evaluated by phosphomolybdenum complex formation method [15]. Briefly, 500 μg/mL of each sample was mixed with 4 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) in sample vials. The blank solution contained 4 mL of reagent solution. The vials were capped and incubated in water bath at 95°C for 90 min. After the samples had been cooled to room temperature, the absorbance of mixture was measured at 695 nm against blank. The antioxidant activity was expressed relative to that of butylated hydroxytoluene (BHT). All determinations were assayed in triplicate and mean values were calculated.
Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was done according to method [16] with some modifications. The stock solutions included 300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM hydrochloric acid, and 20 mM ferric chloride hexahydrate solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution and 2.5 mL ferric chloride hexahydrate solution and then warmed at 37°C before using. The solutions of plant samples and that of trolox were prepared in methanol (250 μg/mL). Ten microliter of each of sample solution were taken in separate test tubes and 2990 μL of FRAP solution was added in each to make total volume up to 3 mL. The plant samples were allowed to react with FRAP solution in the dark for 30 min. Absorbance of the colored product [ferrous tripyridyltriazine complex] was checked at 593 nm. The FRAP values were expressed as micromoles of trolox equivalents (TE) per mg of the sample using the standard curve constructed for different concentrations of trolox. Results were expressed in TE μM/mL.
Total phenolic contents
Total phenolics of various fractions of plant were determined reported method [17]. The 0.1 mL (0.5 mg/mL) of sample was combined with 2.8 mL of 10% sodium carbonate and 0.1 mL of 2N Folin-Ciocalteu’s phenol reagent. After 40 min absorbance at 725 nm was measured by UV-visible spectrophotometer. Total phenolic contents were expressed as micrograms of gallic acid equivalents (GAE) per gram of sample using the standard calibration curve constructed for different concentrations of gallic acid. The curve was linear between 50 μg/mL and 400 μg/mL of gallic acid. Results were expressed in GAE μg/g.
Ferric thiocyanate (FTC) assay
The antioxidant activities of various fractions of plant on inhibition of linoleic acid peroxidation were assayed by thiocyanate method [18]. The 0.1 mL of each of sample solution (0.5 mg/mL) was mixed with 2.5 mL of linoleic acid emulsion (0.02 M, pH 7.0) and 2.0 mL of phosphate buffer (0.02 M, pH 7.0). The linoleic emulsion was prepared by mixing 0.28 g of linoleic acid, 0.28 g of Tween-20 as emulsifier and 50.0 mL of phosphate buffer. The reaction mixture was incubated for 5 days at 40°C. The mixture without extract was used as control. The mixture (0.1 mL) was taken and mixed with 5.0 mL of 75% ethanol, 0.1 mL of 30% ammonium thiocyanate and 0.1 mL of 20 mM ferrous chloride in 3.5% HCl and allowed to stand at room temperature. Precisely 3 min after addition of ferrous chloride to the reaction mixture, absorbance was recorded at 500 nm. The antioxidant activity was expressed as percentage inhibition of peroxidation (IP%) [IP%={1−(abs. of sample)/(abs. of control)}×100]. The antioxidant activity of BHT was assayed for comparison as reference standard.
Statistical analysis
All the measurements were done in triplicate and statistical analysis was performed by Microsoft excel 2003. Results are presented as average±SEM.
Antibacterial and antifungal assay
Microbial strains
The samples both irradiated and un-irrdiated were tested separately against two strains of Gram-positive bacteria: Bacillus subtilis JS 2004 and Staphylococcus aureus, API Staph TAC 6736152, and two strains of Gram-negative bacteria: Pasteurella multocida (local isolate) and Escherichia coli ATCC 25922 and four fungal strains Aspergillus niger, Aspergillus flavus, Ganoderma lucidum and Alternaria alternata. Pure bacterial and fungal strains were collected from the Dept. of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan. Verification of the identity and purity of the strains was done by the Institute of Microbiology of the same university. In the Nutrient agar (NA, Oxoid), the bacterial strains were cultivated at 37°C overnight.
Disc diffusion method
Disc diffusion method was used to check antibacterial and antifungal activities of plant extracts. Suspension of the tested microorganisms (100 μL), contained 107 CFU/mL (colony-forming units) of bacteria cells on the nutrient agar medium. The 9 mm diameter filter discs were impregnated separately with extracts’ solution and placed on agar plates that were already inoculated with tested microorganisms. Streptomycin sulfate (Oxoid, UK) (30 μg/mL/dish) was taken as positive reference for bacterial strains to compare the sensitivity of isolate/strain in the analyzed microbial species. The discs with no samples were taken as negative control. Plates were kept at 4°C for 2 h and then incubated for 18 h at 37°C for bacterial strains. Evaluation of the antibacterial and antifungal activity for the organisms was done by measurement of diameter of the growth inhibition zones in millimeters and by comparing to that of positive and negative controls [19].
Measurement of MIC
The MIC (minimum inhibitory concentration) was stated as the lowest concentration of sample that has capability to inhibit complete growth of bacterial strain being tested [20]. It was calculated graphically as an extrapolation of linear relationship to zero value.
Results and discussion
Antioxidant activities
DPPH radical scavenging activity
Antioxidant activities of all fractions of W. exserta was checked by DPPH. Scavenging ability of DPPH free radical is used in order to assess antioxidant activities in shorter period. At 517 nm a rapid decrease in the optimal density was observed as the DPPH solution was added to extracts. The antioxidant capacity of extracts was measured by the degrees of decolorization. In food due to autoxidation of unsaturated lipids free radicals are produced. It was thought that radical scavenging activity or hydrogen donating ability of antioxidant effects on DPPH radical. Antioxidants cease the oxidation of free radical chain and donate hydrogen from phenolic hydroxyl group [21]. At different concentrations reduction in absorbance of the DPPH radical was observed and results in color change from violet to yellow. It was observed in the assay that the activity was increased with increasing concentration of the fraction as mentioned in Table 1. In comparison to other fractions aqueous fraction exhibited highest inhibitory power of DPPH radical (85.94±0.35 at 1000 μg/mL), n-hexane fraction exhibited very low percent inhibition of DPPH radical (48.35±0.40 μg/mL). The IC50 value for each fraction was also calculated. IC50 is that concentration of a substance which results 50% loss of the DPPH action [22]. The result of IC50 values of each fraction revealed that lesser the IC50 value, greater the scavenging power Table 2. In comparison to other fractions aqueous fraction exhibited lowest IC50 value 415.31±0.26 μg/mL. IC50 values of BuOH and EtOH soluble fraction were compared with a reference standard; butylated hydroxyltoluene (BHT) and it were found to be 461.23±0.69 μg/mL and 685±0.38 μg/mL, respectively. IC50 was not observed in n-hexane and chloroform soluble fraction.
S. no | Sample | Concentration in assay (μg/mL) | Percentage scavenging of DPPH radical±SEMa |
---|---|---|---|
2 | n-Hexane soluble fraction | 1000 | 48.35±0.40 |
500 | 35.84±0.62 | ||
250 | 21.47±0.94 | ||
3 | Chloroform soluble fraction | 1000 | 52.43±0.233 |
500 | 36.05±0.368 | ||
250 | 23.89±0.18 | ||
4 | Ethyl acetate soluble fraction | 1000 | 60.07±0.27 |
500 | 50.12±0.60 | ||
250 | 27.61±0.12 | ||
5 | n-Butanol soluble fraction | 1000 | 71.39±0.73 |
500 | 57.83±0.78 | ||
250 | 36.67±0.88 | ||
6 | Aqueous fraction | 1000 | 85.94±0.35 |
500 | 62.30±0.12 | ||
250 | 34.70±1.01 | ||
7 | BHTb | 60 | 91.25±0.13 |
30 | 75.56±0.07 | ||
15 | 42.67±0.04 | ||
8 | 23.57±0.31 |
aStandard mean error of three assays. bStandard antioxidant.
Sample | IC50 of DPPH assay (μg/mL)±SEMa | Total antioxidant activity±SEMa | FRAP value TE (μM/mL)±SEMa | Total phenolics (GAE mg/g of extract)±SEMa | Inhibition of lipid peroxidation (%)±SEMa |
---|---|---|---|---|---|
n-Hexane soluble fraction | 0.00 | 0.116±0.03 | 18±0.31 | 8.5±1.12 | 5.13±0.19 |
Chloroform soluble fraction | 0.00 | 0.236±0.25 | 34.5±0.65 | 22.58±0.04 | 9.24± 0.43 |
Ethyl acetate soluble fraction | 685±0.38 | 0.451±0.01 | 26±0.58 | 40.25±1.5 | 18.63±0.03 |
n-Butanol soluble fraction | 461.23±0.69 | 0.529±0.76 | 37±0.37 | 60.35±1.45 | 31.44±0.67 |
Aqueous fraction | 415.31±0.26 | 0.338±0.01 | 64.5±0.15 | 87.10±1.10 | 42.32±0.59 |
BHTb | 12.1±0.92 | 1.219±0.07 | – | 62.73±0.96 |
aStandard mean error of three assays. bStandard antioxidant.
Total antioxidant activities
Phosphomolybdenum process was used to find out total antioxidant activity of each separated fraction. Antioxidants of the fractions reduce the molybdenum (VI) to molybdenum (V) and at acidic pH a green colored complex of phosphate Mo (V) is formed. In this assay based on structure of antioxidant electron is transferred [15] and BHT standard is used in order to compare total antioxidant activities of these fractions. n-Butanol fraction displayed highest values of total antioxidant activity (0.529±0.76). Ethyl acetate soluble fraction had 0.451±0.01 of the total antioxidant activity, while that of aqueous fraction and chloroform soluble fraction had 0.338±0.01 and 0.236±0.25 of the total antioxidant activity. The n-hexane soluble fraction possessed lowest amount of antioxidant contents (0.116±0.03).
FRAP test
Oxidative contents of fractions is measured by a simple direct method FRAP test. This process was originally planned to check capacity of plasma antioxidant, but now used for plant extracts [22]. The reducing capacity of antioxidants which effects on reactive oxygen species is measured by FRAP assay. Reductants are electron donating antioxidants and in redox reactions inactivation of oxidants by reductants is measured. In this assay antioxidants reduces Fe3+ to Fe2+ result in formation of deep blue Fe2+–TPTZ complex at 593 nm [23]. Increasing absorbance of a fraction indicates rise in reductive capacity. The aqueous fraction exhibited maximum FRAP value (64.5±0.15 TE μM/mL) in comparison to other fractions while poor source of FRAP value (18±0.31 TE μM/mL) was n-hexane fraction.
Total phenolic contents
Important part of plant constituents is phenolic compounds. They also have scavenging power due to hydroxyl groups. It also acts as a powerful chain breaking antioxidants. Antioxidant activity of phenolic compounds play significant role in lipid oxidation. Phenolic compounds participate in anti-oxidative action. In humans these compounds stopped down carcinogenesis and mutagenesis [24]. Phenolic compounds are important part of plants constituents have antioxidant activities, these phenolic compounds are quercetin, narigin, rutin, catechins, gallic acid, chlorogenic acid and caffeic acid [25]. In the fractions, phenolic concentrations are showed as micrograms of GAEs per gram of fraction. Results of our fractions showed, aqueous fraction have highest amount of total phenolics value (87.10±1.10 GAE μg/g), then n-butanol soluble fraction (60.35±1.45 GAE μg/g), ethylacetate soluble fraction (40.25±1.5 GAE μg/g), chloroform soluble fraction (22.58±0.04 GAE μg/g) while lowest phenolic contents (8.5±1.12 GAE μg/g) was present in n-hexane soluble fraction.
% Inhibition of lipid peroxidation
Reactive and cytotoxic products are produced both in vivo and in vitro due to peroxidation of lipids. These products give modified or damaged DNA due to malfunctioning of the normal cell. Lipid hydroperoxides and free radicals are generates when oxygen reacts with double bond of unsaturated lipid. Hydrogen donating antioxidants breakdown the process of production of new radicals when react with lipid peroxyl radicals. In FTC method peroxide will react with FeCl2 and form Fe3+ which in turn react with NH4SCN and produce reddish ferric thiocyanate pigment. This process is used to calculate the quantity of peroxide in lipid peroxidation [26]. It was observed that aqueous fraction possessed the highest value of lipid peroxidation inhibition (42.32±0.59%). n-Hexane fraction displayed the lowest percentage of lipid peroxidation inhibition (5.13±0.19%). CHCl3, EtOAc and n-BuOH also displayed % inhibition of lipid peroxidation having value of 9.24±0.43%, 18.63±0.03% and 31.44±0.67%, respectively. The result was compared with a standard BHT having inhibition of lipid peroxidation value 62.91±0.60%.
Antimicrobial activities
Antibacterial activity
In the therapeutic treatments use of plant extracts and phytochemicals are well known due to their antimicrobial properties. Many plants have been synthesized antimicrobial product during the secondary metabolism of the plant. These products are their active substances, like phenolic compounds in essential oils and in tannin [27].
The antibacterial activity of isolated plant fractions was examined against two gram-positive bacteria and two gram-negative bacteria by the disc diffusion method. Inhibition zones were measured in mm and results have been given in Table 3. Results of the disc-diffusion assays showed that ethyl acetate fraction showed good activity against Bacillus subtilis and P. multocida (22 and 24 mm, respectively). The n-butanol and remaining aqueous fraction exhibited good activity against Bacillus subtilis (22 and 25 mm, respectively) and E. coli (24 and 20 mm, respectively). n-Hexane and chloroform fraction did not show good antibacterial activity. MIC was also calculated and results have been given in Table 3. Ethyl acetate fraction shown lowest MIC against Bacillus subtilis and P. multocida i.e. 99 and 92 mg/mL, respectively. The n-butanol fraction also showed good value of MIC against Bacillus subtilis and E. coli i.e. Ninety seven and 92 mg/mL, respectively. While aqueous fraction showed lowest MIC against Bacillus subtilis i.e. 90 mg/mL.
Sample | Zones of inhibition (mm) | MIC (μg/mL) | ||||||
---|---|---|---|---|---|---|---|---|
S. aureus | B. subtilis | P. multocida | E. coli | S. aureus | B. subtilis | P. multocida | E. coli | |
n-Hexane fr. | 6 | 5 | – | – | 300 | 361 | – | – |
CHCl3 fr. | 18 | 16 | 15 | 12 | 202 | 238 | 219 | 230 |
EtOAc fr. | 16 | 22a | 24a | 12 | 242 | 99 | 92 | 234 |
n-BuOH fr. | 15 | 22a | 14 | 24a | 268 | 97 | 285 | 92 |
Aqueous fr. | 12 | 25a | 14 | 20a | 342 | 90 | 248 | 102 |
Streptomycin sulfate | 33 | 35 | 32 | 33 | 42 | 20 | 50 | 38 |
ap<0.05 comparative to blank i.e. negative control (p<0.05 was considered as significant).
Antifungal activity
In the present study antifungal activity of all the isolated fractions of W. exsertawas checked against A. flavus, A. niger, A. alternata and G. lucidum by measurements of zones of inhibition in mm and the results have been illustrated in Table 4. It was observed that Chloroform, n-BuOH and aqueous fraction showed good activity against A. alternata i.e. 20, 22 and 24 mm, respectively. n-BuOH and aqueous fraction also showed good activity against A. flavus i.e. 20 and 24 mm, respectively. All the other fractions showed very less or no activity.
Sample | Zones of inhibition (mm) | MIC (mg/mL) | ||||||
---|---|---|---|---|---|---|---|---|
A. flavus | A. niger | A. alternata | G. lucidum | A. flavus | A. niger | A. alternate | G. lucidum | |
n-Hexane fr. | – | – | – | – | – | – | – | – |
CHCl3 fr. | 18 | 10 | 20a | 18 | 175 | 351 | 132 | 165 |
EtOAc fr. | 22a | 14 | 16 | 18 | 122 | 296 | 194 | 164 |
n-BuOH fr. | 24a | 12 | 22a | 14 | 102 | 324 | 110 | 322 |
Aqueous fr. | – | 10 | 24a | 16 | – | 298 | 98 | 197 |
Fluconazole | 30 | 30 | 34 | 32 | 50 | 52 | 31 | 42 |
ap<0.05 comparative to blank i.e. negative control (p<0.05 was considered as significant).
MIC was also calculated and results have been given in Table 4. Ethylactate and n-BuOH fraction showed good MIC values against A. flavus i.e. 122 and 102 mg/mL, respectively. While Chloroform, n-BuOH and aqueous fraction showed good MIC values against A. alternata i.e. 132, 110 and 198 mg/mL, respectively.
Conclusions
This study indicated that polar fractions of W. exserta (Roxbukan Pansara) exhibited good antioxidant activity. The order of antioxidant activity was aqueous Fraction>Ethylacetate fraction>n-BuOH fraction>Chloroform fraction. The polar fractions of plant also showed the good antimicrobial activity against selected pathogenic microbes. Hence it was concluded that polar fractions of plant is a natural source of an antioxidants that is effective in preventing free radical formation but also effective against pathogenic microbes, so further studies may be conducted on the isolation and identification of specific phytochemical which can limit the growth of microbes and can be a source of some important drugs.
Acknowledgement
Special thanks are paid to Dr. Muhammad Shahid, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan, to provide antimicrobial activity results.
Conflict of interest: The authors have no conflict of interest for this manuscript.
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