In Vitro Hydrogen Peroxide Scavenging Activity and Alpha Amylase Inhibitory Activity of Croton tiglium extract

 

Dr. V. P. Karthik¹, Dr. Punnagai², Mr. Parepalli Suresh³, Dr. Darling Chellathai David⁴

¹Assistant Professor in Pharmacology, Sri Ramachandra Medical College & Research Institute, Chennai, TN

²Professor in Pharmacology, Sri Ramachandra Medical College & Research Institute, Chennai, TN

³Demonstrator in Pharmacology, Sri Ramachandra Medical College & Research Institute, Chennai, TN

⁴Professor and HOD of Pharmacology, Sri Ramachandra Medical College & Research Institute, Chennai, TN

 

ABSTRACT:

Diabetes mellitus is a metabolic disorder which has emerged as a global public health threat in the present century. The present oral hypoglycemic agents produce undesirable side effects and oxidative stress. Thus there is a need for a safe, cost effective and complementary therapy for Diabetes mellitus. Objective: To evaluate the hydrogen peroxide scavenging activity and Alpha amylase inhibitory activity of Croton tiglium extract. Materials & Methods: Different concentrations of the plant extract (1.5, 3, 7, 15.30, 60, 125, 250, 500 and 1000 µg/ml) were prepared in ethanol and subjected to α amylase inhibitory and nitric oxide radical scavenging activity. The absorbance was read at 540nm and 546nm respectively using spectrophotometer. The ability of ethanolic extract of Croton tiglium to scavenge hydrogen peroxide was determined according to the method of Ruch et al (1989). Alpha-amylase inhibitory activity was evaluated according to the method of Bernfield (1955). Results: % Inhibition of hydrogen peroxide by Croton tiglium extract at 20, 40, 60, 80, 100µg/ml was 12.24±0.80, 26.35±0.13, 42.52±0.97, 62.97±0.32, 81.27±0.28 respectively. % inhibition of Alpha amylase by extract of Croton tiglium at 20, 40, 60, 80, 100µg/ml are 23.52±0.28, 32.54±0.12, 42.82±0.46, 55.09±0.09 and 74.15±0.86 respectively. Conclusion: Based on the results we conclude that Croton tiglium as strong in vitro antioxidant and alpha amylase inhibitory activity. Thus, Croton tiglium is a potential antidiabetic agent.

 

 

 

INTRODUCTION:

Living cells produce free radicals as a by-product of biochemical processes. Free radicals are the cause for number of disorders in humans like diabetes, tumor, cardiovascular accidents, arthritis etc^[1]. Diabetes as emerged as a major health problem worldwide. Oral hypoglycemic agents produce adverse effects like hypoglycemia, hypersensitivity, lactic acidosis, weight gain, GI side effects etc. The transition to herbal drugs seems necessary considering their efficacy, safety and cost^[2].

 

 

Antioxidant property of plants products are mainly attributed to the phytochemical constituents present in the plant. The treatment of diabetes these days is focused on decreasing glucose level by inhibiting α-amylase^[3]. Therefore ideal antidiabetic properties in herbs are high flavonoids, tannins contents and presence of amylase inhibitory activity

 

AIM:

To evaluate the hydrogen peroxide scavenging activity and alpha amylase inhibitory activity of Croton tiglium extract.

 

MATERIALS AND METHODS:

MATERIAL

The plant material (Croton tiglium) was procured from Green Chem. Herbal Extract & Formulations, Bangalore. The Hydrogen peroxide was purchased from Sisco Research Laboratories Pvt. Ltd, Pallikaranai, Chennai.

 

Fig 1. Pterocarpus marsupium plant

 

PREPARATION OF PLANT EXTRACT:

Leaves were washed with running tap water thoroughly, rinsed with distilled water, sun dried, powdered and made as extract with chloroform, ethanol, petroleum ether, ethyl acetate and water using soxhlet equipment.

 

The obtained extract was filtered using whateman no:1 filter paper, condensed using rotary flash vaporator and stored in an airtight container^[4].

 

 

Fig 2. Dried leaves of Pterocarpus marsupium

 

 

Fig 3. Croton tiglium extract

 

EXTRACTION OF WHEAT α-AMYLASE:

500mg of wheat flour was added to 1 liter of 0.2% calcium acetate solute at room temperature and was continuously stirred for a while. The suspension was then centrifuged. The obtained extract was stored at 3oC. β-amylase activity was inactivated by heating the extract at 70oC for 10 minutes at a pH of 6.6. The extract was cooled to 4oC until use^[5].

 

 

Fig 4. Wheat α-amylase extract

 

HYDROGEN PEROXIDE SCAVENGING ACTIVITY

The ability of Croton tiglium to scavenge hydrogen peroxide was determined according to the method of Ruch et al (1989). Hydrogen peroxide solution (40mM) was prepared in phosphate buffer (pH 7.4). Croton tiglium extract (100 µg/mL) in distilled water was added to hydrogen peroxide solution (0.6 mL, 40mM). Absorbance of hydrogen peroxide at 230 nm was determined against a blank solution containing the phosphate buffer without hydrogen peroxide ^[6].

 

DETERMINATION OF α-AMYLASE INHIBITORY ACTIVITY

The mixture containing 20µl of enzyme, 200µl of 0.02M sodium phosphate buffer and plant extract of concentration ranging between 20-100μg/ml was incubated in room temperature for 10minutes followed by adding 200µl of starch in all dilution. The reaction is terminated by adding 400μl of DNS. Absorbance is measured at 540nm. Acarbose was used as reference^[7].

 

Percentage inhibition = Abs control - Abs sample x 100

                                                            Abs control

 

Acarbose was used as the reference α-amylase inhibitor

 

 

Fig 5.α-amylase inhibitory activity

 

RESULTS:

Table 1. Hydrogen peroxide scavenging activity

Concentration (μg/ml)	% Inhibition Quercetin	% Inhibition Croton tiglium
20	49.86 ± 1.14	12.24±0.80
40	61.29 ± 0.14	26.35±0.13
60	74.09 ± 0.26	42.52±0.97
80	84.04 ± 2.48	62.97±0.32
100	94.14 ± 1.07	81.27±0.28

Table 2. Amylase inhibitory activity

Concentration (μg/ml)	% Inhibition Acarbose	% Inhibition Croton tiglium
20	46.66 ± 0.10	23.52±0.28
40	59.54 ± 0.52	32.54±0.12
60	68.25 ± 0.12	42.82±0.46
80	76.14 ± 1.01	55.09±0.09
100	82.69 ± 1.02	74.15±0.86

 

 

Fig 6. % Inhibition Hydrogen Peroxide Croton tiglium Vs Quercetin

 

 

Fig 7. % Inhibition α-amylase Croton tiglium Vs Acarbose

 

DISCUSSION:

Antioxidant Activity: Antioxidants present in herbal plants are accountable for prevention of damage caused by free radicals. Flavonoids and tannins present in plants are potent free radical scavengers. Nitric oxide is widely used chemical compound for assessing free radical scavenging activity. The above results shows that Croton tiglium has significant antioxidant property under in vitro condition. Moreover the graph indicates a dose dependent inhibition of nitric oxide.

 

α-Amylase Inhibition Activity: α-amylase is a key enzyme in carbohydrate metabolism. Inhibition of α-amylase is one of the strategy of treating diabetes. Inhibiting α-amylase will lower post prandial blood sugar [8]. The result suggest that ethanoic extract of Croton tiglium exhibit good αamylase activity under in vitro condition. Dose dependent % inhibitory activity against α-amylase was noted.

Our study indicates that Croton tiglium could be useful in the treatment of post prandial hyperglycaemia. The Antioxidant and anti-diabetic activity may be attributed to the presence of flavonoids, tannins & anti α-amylase activity.

 

CONCLUSION:

Based on the above results we conclude that ethanolic leaf extract of Croton tiglium exhibit potent α-amylase inhibitory activity and could be exploited in the management of post prandial hyperglycemia in treatment of Type 2 diabetes mellitus. However pharmacokinetic and safety profile of Croton tiglium requires pre-clinical testing prior to its application on humans.

 

ACKNOWLEDGEMENT(S):

The authors would like to acknowledge Sri Ramachandra Institute of Higher Education and Research for providing infrastructure to conduct the research work

 

CONFLICT OF INTEREST:

Nil.

 

FUNDING SOURCE:

Self-funded.

 

REFERENCES:

1.     Muhammad, Bashir. Evaluation of Antioxidant & Cytotoxic Capacity of Croton bonplandianum. Baill. American J of Plant Sciences, 2013, 4, 1709-1712.

2.     G. Keerthana, M.K. Kalaivani. In-vitro alpha amylase inhibitory & anti-oxidant activities of ethanolic leaf extract of croton bonplandianum. Asian J Pharm Clin Res, Vol 6, Suppl 4, 2013, 32-36.

3.     Govindappa. A Review on Role of Plant(s) Extracts and it’s Phytochemicals for the Management of Diabetes. J Diabetes Metab, 6, 2015, 7.

4.     ChunmeiLi and, Myeong-Hyeon Wang In vitro biological evaluation of 100 selected methanol extracts from the traditional medicinal plants of Asia. Nutrition Research and Practice, 8(2), 2015, 151-157.

5.     Kondo NK, Ida EI. Extraction, purification and some partial characterization of alpha-amylase inhibitors from wheat Iapar 28-Igapo. Arch Latinoam Nutr, 45(4), 1995, 310-6.

6.     Sindhu. S. Nair, Vaibhavi K and Anshu M. In vitro studies on alpha amylase and alpha glucosidase inhibitory activities of selected plant extracts. European Journal of Experimental Biology, 3(1), 2013, 128-132.

7.     Sakthidevi G and Mohan VR. Total Phenolic, Flavonoid Contents and In vitro Antioxidant Activity of Dioscoreaalata l. Tuber. J. Pharm. Sci. & Res, 2013, 5(5), 115-119.

8.     Paloma M, Paula Monteiro de Souza, Luiz Alberto Simeoni, Pérola de Oliveira Magalhães, Dâmaris Silveira. α-Amylase Inhibitors: A Review of Raw Material and Isolated Compounds from Plant Source. J Pharm Pharmaceut Sci, 5(1), 2012, 141-183.

 

 

 

 

Received on 12.02.2019          Modified on 14.03.2019

Accepted on 02.04.2019        © RJPT All right reserved