In-vitro Evaluation of Antioxidant and Anti-diabetic activity of Canavalia gladiata (Jacq.) DC.
Mrinalini Pal1,2, Nem Kumar Nagpoore1,2, Sushil Agrahari2, Bhramanand Singh2,
Vashist Narayan Pandey1*
1Experimental Botany and Nutraceutical Laboratory,
Deen Dayal Upadhyaya Gorakhpur University, Gorakhpur - 273009, UP, India.
2Pharmacogynosy, Phytochemistry and Product Development, CSIR NBRI, Lucknow - 226001, UP, India.
*Corresponding Author E-mail: vnpgu@yahoo.co.in
ABSTRACT:
The present study was to investigate antioxidant and anti diabetic activities of Canavalia gladiata. The plant extract from different parts of C. gladiata was investigated for its TPC and TFC content. the seed showed highest TPC content 77.015±0.21μg GAE/mg extract and leaf showed highest TFC content 150.350±0.22μg Rutin E/mg dry extract. Antioxidant activity was investigated by DPPH and ABTS methods based on IC50 value, the seed showed highest DPPH and ABTS value 275.125±0.58μg/ml and 7.848±0.17μg/ml respectively. The anti diabetic activity was investigated by α- amylase and α- glucosidase the result showed highest activity by stem i.e. 838.057±1.00μg/ml and 659.638±0.89μg/ml respectively. Result revealed the positive effect of plant extract for anti diabetic activity which could be potentially used for development of herbal medicine or food supplement i.e. nutraceutical in near future.
KEYWORDS: Canavalia gladiata, Antioxidant, Diabetes mellitus, Anti diabetic, α- amylase, α- glucosidase.
INTRODUCTION:
Diabetes mellitus is a life style disease which increases at a faster pace in the world including India1. Diabetes mellitus is a health disorder which become chronic, if handled carelessly. In this condition the pancreas cells either lessen or completely stop the production of insulin. The condition termed as hyperglycemia which further obstruct most of the metabolic pathways2,3,4. According to recent report of ICMR (2023), New Delhi 101.3 million diabetes, 136.0 million prediabetes and 236.4 million dysglycaemic people were present in India5. Antioxidants are those substances present in the plant foods have ability to nullify and neutralize the free radicals which occur in our body and injurious to our health6,7,8. There are many antioxidants in the studied plants which gives protection to our body8,9.
Canavalia gladiata (Jacq.) DC. belongs to family Fabaceae and subfamily Papilionoideae, commonly known as sword bean in English, badi sem in Hindi and mahasimbi in Sanskrit. It is a underutilized cultivated climber bean species not known in wild10,11. Phytochemical analysis of sword bean showed antioxidant, antidiabetic, antimicrobial and anti-inflammatory activities10,12,13.
In the current scenario, our attention is to be given to the supplement of plant based nutraceuticals to control and manage the life style disease which occur in our society especially diabetes and dysglycaemia14. Which was evidently controlled and managed during the Covid-19 throughout the world. The present study aims at the evaluation of antioxidant and anti-diabetic effect of extract from different parts of the sword bean.
MATERIAL AND METHODS:
The plant samples viz., leaf, stem, immature pod, mature pod and seed were collected from the local area of Gorakhpur, Uttar Pradesh, India. The Collected samples were thoroughly cleaned, shade dried and grounded in waring blender. A precisely weighed 5.0g of homogenized powder was extracted with 100ml 70% hydro-alcoholic solvent overnight. The solution obtained was filtered and further concentrated using rotary evaporator. The 20mg concentrated extract was dissolved in 1.0ml of methanol and sonicated for 2 minutes each for further analysis of antioxidant and anti-diabetic activities.
Phytochemical Screening:
Total Phenolic Content (TPC):
The TPC of the plant sample extract was determined using Folin-Ciocalteu method15. The 200μL of Canavalia gladiata (CG) extract was taken from stock solution (1.0mg/ml) and was diluted in 3.0ml of distilled water, carefully mixed with 0.5ml Folin-Ciocalteu reagent for 5 minutes. Thereafter, 2.0ml of 20% sodium carbonate was added. The test solution was incubated in dark for 30 minute. Thereafter, absorbance was measured at 715nm. The total phenolic content was calculated using plotted calibration curve, the results were expressed as mg gallic acid equivalent per gram of dry extract.
Total Flavonoid content (TFC):
The TFC in CG extract was determined using aluminum chloride method16. The 500μL of stock extract (1.0 mg/ml) was taken and made the volume upto 1.0ml of methanol, then mixed with 4.0ml of distilled water. Incubated for 5 minutes and added 0.3ml of 5% NaNO2 solution. After 5 minutes incubation, 0.3ml of 10% AlCl3 solution was added. Mixture was allowed to stand for 6 minutes and then 2.0ml of 1M NaOH solution was added. The final volume of test mixture was diluted to 10.0ml with distilled water. After 15minutes, the absorbance was measured at 510nm. The total flavonoid content was calculated using plotted calibration curve, the results were expressed as mg of rutin equivalent per gram of dry extract.
Antioxidant Activity:
DPPH Radical Scavenging Assay:
The antioxidant of sample is determined by assessment of polyphenols and flavanoids, which scavenge the formed DPPH radicals, resulting in reduced colour intensity17. In a 96 well microtiter plate (MTP), 0.280 ml of DPPH solution and 0.020ml of plant extract was combined, the test mixture was vortexed for 20 seconds and allowed to stand in dark room for 30 minutes. Absorbance was measured at 515nm. Control solution methanol was used, and ascorbic acid was used as reference antioxidant compound.
ABTS Radical Scavenging Assay:
The ABTS radical scavenging assay was used for determination of antioxidant18. ABTS solution was prepared by dissolving 2 tablets of ABTS in 5.0ml distilled water and 3.0mg of potassium persulfate in 5.0 ml of distilled water, resulting in total volume of 10ml. The solution is allowed to stand for overnight. The 3.0 ml of the ABTS solution was mixed with 90.0ml of methanol to make a working solution. The test solution of extract in different concentration and added to appropriate volume of ABTS solution and absorbance was measured at 734nm using 96 well plates in ELISA reader.
In-vitro Antidiabetic Activity:
Inhibitory Assay of α- Amylase:
The α- amylase inhibitory activity was evaluated by the protocol of Okolo et al.19 with slight modifications. Canavalia hydro-alcoholic extracts were incubated with pancreatic α- amylase in a sodium acetate buffer (0.1 mol/ml, pH 6.9). The different concentrate of CG extracts were incubated with α- amylase (0.5ml, 5.0 μl/ml) for 20 minutes at 37°C. Then 0.5ml starch solution (0.5%w/v) was added, to the mixture and incubated for 15 minutes at 37°C. To this mixture 0.5ml DNS solution was added. Then the solution was boiled in water bath for 20 minutes. After boiling 3.0 ml of distilled water was added, and was allowed to stand and cool at room temperature. The absorbance was measured at 540nm using spectrophotometer. The untreated control sample without extract was used for comparision. Pure Acarbose was compared as the experimental reference control.
Inhibitory activity by α- Glucosidase :
The inhibitory activity of α- glucosidase was evaluated according to Gowri et al.20 with slight modification. CG extract were expressed with α- glucosidase dissolved in 67mmol-1 potassium phosphate buffer (pH 6.8) containing 0.2% bovine serum albumin and 0.02% sodium azide. Different concentration of test solutions were added to 20μL of α- glucosidase (0.15 U ml-1) and incubated for 20 minutes at 37°C. The reaction was stopped by adding sodium carbonate (100μL, 0.2 mol/ml). The absorbance was measured at 405 nm using UV- SYNERGY HTX multi-mode reader. Acarbose were used as positive control.
RESULTS AND DISCUSSION:
The total phenol content (TPC) of Canavalia gladiata in 70% hydro-alcoholic extract from different plant parts viz., stem, young pod, mature pod, leaf and seeds were estimated. The study showed that the plant parts are rich in phenolic contents. The seed contain maximum phenol (77.015±0.21μg GAE/mg extract) followed by mature pod, leaf, young pod and stem respectively. The flavonoid of different plant parts were estimated. The leaf contain maximum flavonoid (150.350±0.22μg Rutin E/mg dry extract) followed by mature pod, seed, young pod and stem (Table 1).
Table 1: Analysis of Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) in Different Plant Parts Extracts
|
Activity |
Stem |
Young pod |
Mature pod |
Leaf |
Seed |
|
TPC (μg GAE/mg dry extract) |
53.333±0.89 |
55.355±0.12 |
76.393±0.24 |
67.377±0.67 |
77.015±0.21 |
|
TFC (μg Rutin E/mg dry extract) |
97.225±0.32 |
105.850±0.36 |
133.100±0.69 |
150.350±0.22 |
114.225±0.31 |
Table 2: DPPH Radical Scavenging Assay and ABTS Radical Scavenging Assay in Different Plant Parts Extracts
|
Activity |
Stem |
Young pod |
Mature pod |
Leaf |
Seed |
|
DPPH (μg/ml) |
706.875±0.43 |
506.055±0.45 |
275.985±0.67 |
357.995±0.29 |
275.125±0.58 |
|
ABTS (μg/ml) |
51.471±0.99 |
30.748±0.89 |
11.331±0.89 |
11.587±0.49 |
7.848±0.17 |
Table 3: Inhibitory Assay of α- Amylase and α- Glucosidase in Different Plant Parts Extracts
|
Activity |
Stem |
Young pod |
Mature pod |
Leaf |
Seed |
|
Inhibitory Assay of α- Amylase IC50 (μg/ml) |
838.057±1.00
|
1506.680±1.02
|
1282.27±1.10
|
1835.591±1.09
|
1265.838±0.98
|
|
Inhibitory activity by α- Glucosidase IC50 (μg/ml) |
659.638±0.89
|
1153.991±0.65
|
1060.402±0.19
|
829.59±0.44
|
911.447±0.73
|
The antioxidants decrease the risk of heart disease, diabetes, Alzheimer’s disease, atherosclerosis, alzheimer’s disease even cancer21,22,23. The antioxidant activity of C. gladiata was determined by DPPH (2,2-diphenyl-1-picrylhydrazyl) assay and ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) assay (Table 2). The polyphenols and flavanoid present in C. gladiata showed high antioxidant activity24. In DPPH assay, the polyphenols and flavanoids scavange the formed DPPH radicals which result in lower colour intensity. The low IC50 values indicate high antioxidant activity of the seed (275.125±0.58μg/ml) followed by mature pod (275.985±0.67μg/ml). ABTS assay showed high antioxidant activity in the seed (7.848±0.17μg/ml) followed by mature pod (11.331±0.89). However, leaf, young pod, stem also contain measurable amount of polyphenols and flavanoids.
The anti diabetic effect of C. gladiata by inhibitory Assay of α- Amylase and inhibitory Assay of α- Glucosidase were measured (Table 3). The 70% hydro- alcoholic extract of stem, young pod, mature pod, leaf and seed were used to estimate the α- Amylase IC50 value for stem (838.057±1.00μg/ml) which was found to be effective, the IC50 value of standard drug acarbose (52.08±0.26μg/ml ) and in α- Glucosidase IC50 value of stem (659.638±0.89μg/ml), the IC50 value of standard drug acarbose (95.45±1.28μg/ml) was found to be effective.
This study indicates that Canavalia gladaiata is a potentially rich in biomolecules especially polyphenols and flavonoids, in addition to primary health metabolites.
CONCLUSION:
The radical scavenging activities of different plant parts of Canavalia gladiata exhibit the potential antioxidant and anti-diabetic effect. The contents of phenol and flavonoids are present in the plant parts in a substantial amount which is responsible to control and manage ageing, antioxidants and general health6,13. The plant can be easily incorporated in the daily diet of individuals to provide protection against different disease and has a significant potential for the development of food supplements i.e. nutraceuticals and herbal medicine for therapy of diabetes and healthcare.
ACKNOWLEDGMENT:
Authors are very thankful to the Department of Botany, DDU Gorakhpur University, Gorakhpur and CSIR-NBRI, Lucknow for providing the necessary facilities for this research work.
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Received on 18.01.2024 Modified on 16.05.2024
Accepted on 24.07.2024 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(9):4515-4518.