In Vitro Antioxidant and Anthelmintic Activity of the Extracts of Whole Plant Crotalaria Biflora (L)
K. I. Anoob Kumar1*, M. Swamivel Manickam2, M. Sreejith3, V. Sebastin4
1Department of Pharmaceutical Chemistry, KVM College of Pharmacy, Cherthala, Alappuzha, Kerala, India
2Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram, Tamil Nadu, India
3Department of Pharmaceutical Chemistry, Nazareth College of Pharmacy, Thiruvalla, Pathanamthitta, Kerala, India
4Department of Pharmaceutical Chemistry, Malik Deenar College of Pharmacy, Seethangoli, Kasaragod, Kerala, India
*Corresponding Author:
K. I. Anoob Kumar, Phone: 7012177726 E-mail: [email protected]
ABSTRACT
Various biological activities of the genus “Crotalaria” was reported in several studies. In this study, the antioxidant and anthelmintic activity of the whole plant Crotalaria biflora was evaluated. The plant material was collected, identified and authenticated and extracted with different solvents such as petroleum ether, chloroform, ethyl acetate and methanol in soxhlet apparatus. Dried extracts thus obtained were used for the evaluation. Antioxidant activity of the extracts was evaluated by DPPH, reducing power assay, superoxide, nitric oxide and hydroxyl free radical scavenging assay methods. Anthelmintic activity was assessed by using the earthworm, Pheretima postuma. From the results of both antioxidant and anthelmintic evaluation, it was found that among the different concentration employed for the evaluation, the highest concentration of test extracts showed their maximum activity. Among these, the methanol extract showed a very significant activity comparing with other tested extracts and standard agents employed for the comparative evaluation followed by ethyl acetate extract showed a significant activity. Findings of this study clearly indicated the potential of methanol extract of the whole plant C. biflora in the tested biological activities. Future studies on this extract may give more significant results.
Keywords: Crotalaria biflora extracts, antioxidant activity,anthelmintic activity Introduction
Nowadays, the global market of medicine reaches the worth of about 1.1 trillion US dollars per annum. About 35% of these drugs originated from natural products directly or indirectly that includes plants (about 25%), microbes (13%) and animals (3%) (Calixto, 2019). A survey about the sources of novel drugs revealed that about thirteen drugs of natural origin were approved during the year 2005-2007 (Katiyar et al., 2012) Products of natural origin are one of the important sources for the pharma companies investing on the development of novel therapeutics.
The systematic evaluation of plants used in the traditional healing system may afford more assuring data about their therapeutic value and may be helpful to face the demand of novel drugs to resist the infections and diseases. With this view, the plant Crotalaria biflora was selected for our research (Kumar et al., 2020).
The first documentation of the genus Crotalaria was done in the year 1753 by Carolus Linnaeus. He documented thirteen species of this genus in his Species Plantarum. Now, the count reaches about 702 species in global distribution particularly in Africa and Madagascar with secondary radiations to the rests of the world (Roux et al., 2013; Samila, 2018; Rokade et al., 2020) The genus Crotalaria is highly diverse and widely distributed that includes perennial aquatic herbs, erect herbs and shrubs, terrestrial creepers as well as trees can be found in different habitats viz., open places, forest and grasslands, plains and hill regions. In India, the Crotalaria is one of the largest legume genera represented by 116 taxa in which fifty two are endemic to the country with the majority of species concentration in the peninsular region (seventy three species restricted to the peninsular region) (Rokade et al., 2020). Many Crotalaria species are under cultivation and consumption by the rural population across the globe for variety of purposes that include medicine, food, green manure, fodder etc (Rouamba et al., 2018). The plant selected for the present study, Crotalaria biflora is commonly found in the south India at an altitude up to 300m mean sea level. The seed of this plant is edible and consumed by rural population because of its protein content (Gritto et al., 2015; Sundararajan et al., 2018).
In our previous studythe whole plant of Crotalaria biflora was collected from the Mekkarai, the village located near the foothills of Western Ghats, Tirunelveli District, Tamil Nadu, India. The collected material was subjected to extraction, preliminary phytochemical
evaluation and spectral characterization (Kumar et al., 2020). The present study was aimed to evaluate the in vitro antioxidant and anthelmintic activity of the extracts of whole plant Crotalaria biflora.
Materials and Methods Plant material collection and extraction
The whole plant, Crotalaria biflora was collected from the Mekkarai, a village in the close proximity of Western Ghats foothills in the Tirunelveli District of Tamil Nadu, India. The collected plant material was identified and authenticated, shade dried and powdered by mechanical grinding. The coarse powder thus obtained was extracted by soxhelationusing the solvents such as petroleum ether, chloroform, ethyl acetate, and methanol. The dried extracts thus obtained were used for the experiments.
In vitro antioxidant activity
In vitro antioxidant evaluation of all the prepared extracts of Crotalaria biflora was evaluated by different approaches such as DPPH, reducing power assay, superoxide, nitric oxide and hydroxyl free radical scavenging assay methods.
DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay
Antioxidant evaluation by this approach was done with the slightly modified procedure of Baba et al., 2015 and Ruskin et al., 2017.
Test extracts (1ml) in different concentration (100, 200, 400 and 800µg/ml) was mixed with methanolic solution of DPPH (1ml; 0.1mM). This reaction mixture was incubated at room temperature in dark condition for 30min. After that, the absorbance of reaction mixtures was measured spectrophotometrically (517nm). Methanol was used as blank; DPPH in methanol was employed as control and the ascorbic acid as standard control. The percentage inhibition of DPPH radical by the Crotalaria biflora extracts was determined by
Percentage inhibition = Control Abs. −Sample Abs.
Control Abs. X 100
Percentage inhibition Vs Concentration was plotted as a graph. The IC50 values of the tests were assessed from the regression equation of the graph.
Reducing power assay
This approach of antioxidant evaluation of test extracts was carried out by using the modified procedure of Moonmun et al., 2017 and Batool et al., 2019. Test extracts (500µl) in different concentration (100, 200, 400 and 800µg/ml) was mixed with sodium phosphate buffer (1.5ml; 0.2M; pH 6.6) and potassium ferricyanide (1.5ml; 1%) This mixture was kept for incubation (50°C; 20min). Then, trichloroacetic acid (5ml; 10%) was added to the incubated mixture and subjected to centrifugation (3000rpm; 6min; 4°C). Upper layer of the centrifuged was collected. Collected upper layer solution (1.5ml) was mixed with equal volume of distilled water and fresh ferric chloride solution (300μl; 0.1%) and kept for 10min. Then, the absorbance of reaction mixture was measured spectrophotometrically (700nm). Ascorbic acid was employed as standard control for comparative evaluation.
Superoxide radical scavenging assay
The superoxide radical scavenging activity of the test extracts was assessed by using the modified procedure of Fazilatun et al., 2004 and Awah et al., 2010. The reaction mixture (3ml) contains test extracts in different concentration (100, 200, 400 and 800µg/ml), Nitro blue tetrazolium (0.1ml; 1.5mM) solution, EDTA (0.2ml; 0.1M), riboflavin (0.05ml;
0.12mM) and phosphate buffer (2.55ml; 0.067M). Control tubes were prepared wherein DMSO was added instead of sample. The reaction mixture was kept (30 min) in front of fluorescent light (34 W) and then the absorbance was measured spectrophotometrically (560nm). Ascorbic acid was used as the standard control. All the tests were performed in triplicate and the results were averaged. The percentage inhibition superoxide radical was calculated by
% inhibition = Control Abs. −Sample Abs.
Control Abs. X 100 The IC50 indicated the concentration of the tests that inhibited 50% of radical.
Nitric oxide radical scavenging assay
This method of antioxidant evaluation of test extracts was done by using the modified procedure of Awah et al., 2010 and Habu et al., 2015. 1ml of Griess reagent (1%
sulphanilamide in 5% phosphoric acid and 0.1% naphthyl ethylene diamine dihydro chloride) was added to the reaction mixture (5ml) which was prepared by mixing different concentration of test extracts (100, 200, 400 and 800µg/ml) with sodium nitroprusside (5mM) in phosphate buffer (pH 7.3). This reaction mixture was kept in 25°C (in front of 25W
tungsten lamp) for 3h. The nitric oxide radical thus formed was interacted with oxygen to produce nitrite ion which was measured spectrophotometrically (546nm). Normal and standard control (Ascorbic acid) were prepared. The percentage inhibition of nitric oxide radical formation was determined by
% inhibition = Control Abs. −Sample Abs.
Control Abs. X 100 Hydroxyl radical scavenging assay
Evaluation of antioxidant activity by hydroxyl radical scavenging assay was done with the modified procedure of Habu et al., 2015 and Batool et al., 2019. Test extracts (100µl) in different concentration (100, 200, 400 and 800µg/ml) was mixed with deoxyribose (0.1ml;
2.8mM) prepared in potassium phosphate buffer (20mM; pH 7.4), EDTA (0.1mM), ferric chloride (200µl; 100mM), H2O2 (100µl; 200mM) and ascorbic acid (100µl; 300mM). This mixture was kept for 1h at 37°C. Then, trichloroacetic acid (1ml; 2.8%w/v) and thiobarbituric acid (1ml; 1%w/v) prepared in NaOH (50mM) were added to it and the whole reaction mixture was kept (15min) in water bath for boiling. After cooling, the absorbance of reaction mixture was measured spectrophotometrically (532nm). Normal and standard control (Quercetin) were prepared. Inhibition of deoxyribose degradation was calculated by
% inhibition = Control Abs. −Sample Abs.
Control Abs. X 100
In vitro anthelmintic activity
Evaluation of anthelmintic activity was carried out in accordance with the procedure of Ajaiyeoba et al., 2001 and Das et al., 2017 with slight modification on adult Indian earthworms, Pheretima postuma. Different concentration (25, 50, 100 and 200mg/ml) of petroleum ether, chloroform, ethyl acetate and methanol extracts of the whole plant C. biflora was subjected to the evaluation. Normal saline was used as the control and Piperazine citrate (10mg/ml) was used as the standard control. The test and standard drug solution were freshly prepared in carboxy methyl cellulose (1%). Two worms per each group were employed for the evaluation. Observations were made for paralysis and death of individual worms. Time for paralysis was noted when no movement of any sort could be observed except when the worms were shaken vigorously. Time for death of worms were recorded after ascertaining that the worms neither moved when shaken vigorously nor when dipped in warm water at 50°C followed with fading of their body colour. Three sets of experiments were done
statistical significance. Results are presented as mean ± SEM (Standard error of Mean). The results were analyzed by ANOVA and Dunnet’s test.
Results and Discussion
The results of in vitro antioxidant evaluation of all the prepared extracts of the whole plant C.
biflora by DPPH method is shown in Table 1.
Table 1. Antioxidant activity of test extracts by DPPH assay
% Inhibition
Con. Test extracts
(µg/ml) Pet. ether Chloroform Ethyl acetate Methanol Std.
100 12.74±0.12 21.10±0.60 24.73±1.26 45.23±1.58 42.10±0.12 200 17.56±0.31 32.70±0.75 37.38±1.47 58.85±1.75 53.72±1.73 400 23.30±0.55 42.25±0.87 45.29±1.77 70.80±1.90 64.91±1.52 800 36.53±0.69 58.48±1.10 65.83±1.50 85.21±2.03 76.52±2.10
IC50 850.10 720.59 435.80 115.64 285.70
Con. – Concentration; Std. – Standard (ascorbic acid)
From the results, a concentration dependent rise of antioxidant activity was found in all the tested extracts. In the highest test concentration, (800µg/ml), all the extracts showed their maximum activity, however, the methanol followed by ethyl acetate extract of C. biflora showed a significantly higher activity comparing with other tested extracts. The methanol extract showed a percentage inhibition of 85.21±2.03 in 800µg/ml concentration, Next to that, the ethyl acetate extract showed a percentage inhibition of 65.83±1.50. The standard agent, ascorbic acid used for the comparative evaluation showed a percentage inhibition of 76.52±2.10 in the same concentration. Thus the results clearly indicated the antioxidant potential of the methanol extract of the C. biflora.
The next method employed for the evaluation of antioxidant activity, the reducing power assay method also showed a concentration dependent increase of antioxidant activity in all the tested extracts. In this method also, the methanol extract proved its antioxidant potential by showing a percentage inhibition of 96.50±1.78 followed by the ethyl acetate extract showed a percentage inhibition of 86.80±1.50. Both these two extracts of C. biflora revealed a significant antioxidant activity comparing with the standard agent, ascorbic acid, employed for the comparative evaluation (Table 2).
Table 2. Antioxidant activity of test extracts by reducing power assay
% Inhibition Con.
(µg/ml)
Test extracts
Pet. ether Chloroform Ethyl acetate Methanol Std.
100 17.20±0.24 27.80±0.80 39.75±1.30 59.38±1.10 22.64±0.58 200 28.32±0.77 32.85±0.92 48.86±2.05 69.01±1.71 31.50±0.75 400 37.70±1.12 54.96±1.36 65.60±1.65 76.80±1.59 42.14±0.98 800 46.07±1.25 66.20±1.52 86.80±1.50 96.50±1.78 63.53±1.23
IC50 788.86 380.23 296.25 80.27 420.30
A similar kind of results was found in superoxide radical scavenging assay, another one method employed for assessing the antioxidant ability of the extract of the whole plant C.
biflora. In this method also, comparing with other tested extracts and the standard agent (ascorbic acid), the methanol extract revealed a maximum activity by showing a percentage inhibition of 73.50±0.72 in 800µg/ml, the highest concentration of evaluation (Table 3).
Table 3. Antioxidant activity of test extracts by Superoxide radical scavenging assay
Con.
(µg/ml)
% Inhibition Test extracts
Pet. ether Chloroform Ethyl acetate Methanol Std.
100 09.21±0.78 10.63±1.03 12.44±0.15 28.21±0.60 69.57±0.35 200 11.58±0.47 16.32±0.20 18.38±0.20 34.20±0.10 90.60±0.55 400 13.20±0.89 21.65±0.34 27.58±0.86 57.24±0.15 92.18±0.23 800 19.26±0.69 31.20±1.10 43.65±0.09 73.50±0.72 98.08±0.28
IC50 1270 1100 1010 390 80
The other two methods, nitric oxide and hydroxyl radical scavenging assay utilized for the evaluation of antioxidant activity also revealed a same sort of results (Table 4 & 5). Results of these evaluations clearly indicated the antioxidant potential of methanol extract comparing with other tested extracts and standard agent (ascorbic acid in nitric oxide radical scavenging assay and quercetin in hydroxyl radical scavenging assay) used for the comparative evaluation.
Table 4. Antioxidant activity of test extracts by nitric oxide radical scavenging assay
Con.
(µg/ml)
% Inhibition Test extracts
Pet. ether Chloroform Ethyl acetate Methanol Std.
100 12.35±1.10 16.58±0.60 18.69±0.13 40.58±08 25.38±0.90 200 14.25±0.91 19.42±0.50 23.12±0.15 50.21±0.13 30.52±0.40 400 21.49±0.83 26.37±0.97 30.22±0.36 59.50±0.10 60.65±0.32 800 25.53±0.72 32.80±0.63 37.63±0.26 65.60±0.54 55.63±0.30
IC50 1310 1270 1160 260 420
Table 5. Antioxidant activity of test extracts by hydroxyl radical scavenging assay
Con.
(µg/ml)
% Inhibition Test extracts
Pet. ether Chloroform Ethyl acetate Methanol Std.
100 08.12±0.74 11.55±0.76 14.96±0.31 36.85±0.08 21.19±0.20 200 10.80±1.16 14.63±0.57 19.22±0.50 46.12±0.31 26.25±0.40 400 13.26±0.80 20.15±0.48 26.44±0.63 55.05±0.08 48.56±0.23 800 19.88±0.55 27.45±0.50 33.36±0.262 61.16±0.45 51.36±0.32
IC50 1160 920 860 220 340
Results of the evaluation of anthelmintic activity are shown in Table 6. In this evaluation, all the tested extracts showed different level of activity based on its concentration. However, all the tested extracts revealed a maximum of their activity in the highest concentration (200mg/ml). But the methanol extract showed a significant activity (58.78 ± 1.0 minutes taken for paralysis and 63.43 ± 0.7 minutes for death) comparing with other tested extracts and the standard drug (Piperazine citrate) for the comparative evaluation.
Table 6. Anthelmintic activity of test extracts Treatment group Concentration
(mg/ml)
Paralysis time (min)
Death Time (min)
Normal Control - - -
Standard control 10 21.80 ± 0.6 27.51± 0.3 Petroleum ether extract 25 98.32 ± 0.8 105.05 ± 0.5
50 93.60 ± 1.1 98.30 ± 0.4
100 89.40 ± 0.3 96.42 ± 1.3
200 88. 45 ± 0.6 95.55 ± 1.0
Chloroform Extract 25 92.25 ± 1.7 97.15 ± 1.2
50 86.31 ± 0.3 91.30 ± 0.7
100 82.07 ± 0.5 87.84 ± 0.8
200 75.28 ± 0.9 81.48 ± 0.1
Ethyl acetate Extract 25 81.32 ± 1.1 86.10 ± 1.0
50 80.50 ± 1.2 85.26 ± 0.7
100 77. 34 ± 1.6 83.03 ± 1.1
200 75.24 ± 1.3 80.21 ± 0.8
Methanol Extract 25 73.28 ± 1.8 76.52 ± 0.2
50 69.81 ± 0.8 73. 12 ± 0.2
100 64.40 ± 1.1 69.40 ± 1.5
200 58.78 ± 1.0 63.43 ± 0.7
Regarding with antioxidant activity of tested extracts of the whole plant C. biflora, a concentration dependent rise of activity was found in the results. A similar type of observation was reported in the studies of Habu et al., 2015 and Sebastin et al., 2021. In the present study it was found that the methanol extract showed a very significant antioxidant activity comparing with other tested extracts. It may be due to the presence of phenolic compounds in this extract which was confirmed in our previous study on preliminary phytochemical evaluation (Kumar et al., 2020). Relation of phenolic compounds and the antioxidant activity was reported by several studies such as Awah et al., 2010, Baba et al., 2015 Ruskin et al., 2017 and Sebastin et al., 2021. A very significant activity of methanol extract was found in the evaluation of anthelmintic activity. It was reported that the presence of secondary metabolites such as alkaloids, flavonoids and terpenoids in the plants significantly contribute to its anthelmintic activity (Das et al., 2017). Our previous study on preliminary phytochemical evaluation (Kumar et al., 2020) found the presence of above said secondary metabolites in the methanol extract. Collectively, our studies on the whole plant,
Conclusion
In the present study, the antioxidant and anthelmintic activity of the Crotolaria biflora was evaluated. For that, the whole plant was collected. Properly identified and authenticated material was dried in shade, powdered in mechanical grinder, extracted in soxhlet apparatus using the solvents such as petroleum ether, chloroform, ethyl acetate, and methanol. Dried extracts thus obtained was screened for the in vitro antioxidant and anthelmintic activity.
Among all the tested extracts, the methanol extract of the whole plant C. biflora revealed a very significant activity in both antioxidant and anthelmintic evaluation. Next to that the ethyl acetate extract showed a significant activity. Outcome of the present study is encouraging for further studies in these extracts in the future.
Acknowledgement
Authors are thankful to Mr. Kumaran J., M. Pharm., (Pharmaceutical Biotechnology), for his help in the preparation of this manuscript.
Conflicts of interests: None Funding: None
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