In vitro antioxidant activity of crude protein of Coleus aromaticus

Introduction

Free radicals by virtue of their high reactivity induce oxidative stress. By supplying antioxidants externally, one can reduce the toxic effect of free radicals in the body. Though, many synthetic antioxidants such as Butylated Hydroxy Anisole (BHA), Butylated Hydroxy Toluene (BHT) and Tert-Butylhydroquinone (TBHQ) etc., play a key role in this aspect, but due to the potential health hazards, they are under strict regulation. This leads to a great research interest in natural sources. Therefore, in recent years, the interest is growing towards naturally available antioxidants.^1-3

Coleus aromaticus plant leaves are used as traditional medicine for treatment of many ailments, ranging from simple cold and cough to convulsions and epilepsy.⁴  In-vitro studies showed that the oils of Coleus aromaticus have significant antioxidant activity due to bioactive components like total phenols, flavonoids, chlorophyll and are subjected to natural drug formulations.^5,6 However role of proteins of Coleus aromaticus as an antioxidant has not been studied till date.

The present study includes investigating effective antioxidant activity of crude proteins of Coleus aromaticus using in vitro models such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), Nitric Oxide and Superoxide radical scavenging activity. This would reveal the antioxidant activity of crude proteins of Coleus aromaticus in order to ascertain the scientific proof for its traditional use.

Material & Methods

All the chemicals and reagents used were of analytical grade were purchased from Chetana chemicals, Mysuru and the Merck Co, and S.d. fine chem., Mumbai, India.

Extraction of protein from Coleus aromaticus leaves

Coleus aromaticus leaves collected from authentic source, cleaned with 0.1% KMnO4 solution, followed with double distilled water, crushed, shade dried and powdered (British Pharmacopoeia 100 mesh) and stored in glass bottle. The 4gm of Coleus aromaticus leaves powder mixed with 100 ml of double distilled water and vortexed for 4 hours at 20oC using magnetic stirrer. The vortexed mixture is centrifuged at 8000 rpm for 20 minutes at -4oC, the supernatant was separated. The supernatant was subjected to 65% ammonium sulphate precipitation and vortexed over night. The mixture was centrifuged at 8000 rpm for 20 minutes at -4oC. The precipitated proteins was collected and subjected to dialysis using 2.5kDa molecular cutoff bio-membrane against double distilled water for 76 hours with an interval of 6 hours. The dialyzed proteins was separated and stored at -10OC for further analysis.

Phytochemical analysis

The crude protein of Coleus aromaticus extract was subjected to phytochemical analysis using following standard protocols and present work is continuous work of Santhosh et.al, march 2018.⁷ The proteins estimation was carried according to Bradford’s method⁸ using BSA as standard and absorbance was read at 535nm. Total phenols were determined according to the method of Folin Ciocalteu reaction⁹ using Gallic acid as a standard and absorbance was read at 750 nm. Flavonoids estimation was done using Quercetin¹⁰ as a standard; absorbance was measured at 415 nm. Total Sugars estimation was done according to Dubois method,¹¹ the absorbance was read at 520 nm. The concentrations were calculated accordingly using standard graph.

Antioxidant activity DPPH

Radical scavenging activity¹²

DPPH radical scavenging activity was assessed according to the method of Shimada et al. The crude protein of Coleus aromaticus extract at concentrations ranging from 20 to 100 µg mixed in 1 ml of freshly prepared 0.5 mM DPPH ethanolic solution and 2 ml of 0.1 M sodium acetate buffer pH 5.5. The resulting solutions then incubated at 37°C for 30 min. Ascorbic acid and BHA (20 to 100 µg) used as standards under the same assay conditions. Control was without any standards or crude protein. The % DPPH radical scavenging activity of crude protein was calculated from the decrease in absorbance at 517 nm and compared with control. The % DPPH radical scavenging activity was calculated using the following formula.

% Inhibition of DPPH radical scavenging activity = 

                                  (Abs of control - Abs of samples)                                                                                                                                              X 100                                                                                                                                     Abs of control

Nitric oxide radical scavenging activity^13,14

Nitric oxide scavenging activity was determined according to the method reported by Marocci et al. Crude protein of Coleus aromaticus extract, BHA and Ascorbic acid as standards in the range of 2 –10 µg were taken in respective tubes containing phosphate buffer saline, so that the volume in each tube was made up to 1ml.  For controls, volume was made up to 3ml with phosphate buffer saline. Then 2ml of 10mM sodium nitroprusside added to all the tubes except the controls. Nitric oxide radicals were generated from the samples spontaneously during the incubation period of 150 min. 0.5ml of the solution taken from each tube to their respective tubes. To this 1ml of 0.33% sulphanilamide added and allowed to stand for 5 min for completing diazotization, followed by the addition of 1ml of NED (0.1%) to each tube. Then incubate for 30 min at room temperature. The nitrite ions released were measured at 516nm and % Nitric Oxide radical scavenging activity was calculated using the following formula.

% Inhibition of Nitric Oxide radical activity=            

                                 (Abs of control - Abs of samples)                                                                                                                                              X 100                                                                                                                                     Abs of control

Superoxide scavenging activity¹⁵

The Superoxide radical scavenging activity of crude protein of Coleus aromaticus was measured according to the method of Lee et al. (2002) with minor modifications. The reaction mixture (containing 100μl of 30mM EDTA (pH 7.4), 10μl of 30mM hypoxanthine in 50mM NaOH, and 200μl of 1.42mM nitro blue tetrazolium) with or without dialyzed Coleus aromaticus protein extract and standards (Ascorbic acid & BHA) at various concentrations ranging from 2-10μg. After the solution was pre-incubated at ambient temperature for 3min. 100μl of xanthine oxidase solution (0.5U/ml) was added to the mixture and incubated for 1 hour at 37⁰C, and the volume was made up to 3ml with 20mM phosphate buffer (pH 7.4). The solution was allowed to stand for 20min. Absorbance was measured at 560 nm against a control (without any inhibitor) to determine the quantity of formazan generated. Decreased absorbance indicates increased superoxide anion scavenging activity. The % inhibition was determined as below.

% Super Oxide radical activity =

                                  (Abs of control - Abs of samples)                                                                                                                                              X 100                                                                                                                                     Abs of control

Statistical analysis:

All data are expressed as mean ± standard deviation of five replicate (n=5). The significance of the experimental observation was checked by student’s t-test and P<0.05 was considered as statistically significant when compared to relevant controls.

Results

The proteins were isolated from Coleus aromaticus as explained in methods. The isolated proteins were analyzed for their antioxidant activity by DPPH radicals, Nitric Oxide radicals and Superoxide radical scavenging activity, where BHA and ascorbic acid were used as standards at different concentrations. The results are expressed as percentage (%) inhibition exhibited by the test extract and the standard antioxidants.

The percentage inhibition of radicals activity increased with increase in concentration of crude protein Coleus aromaticus extract is comparable with standard antioxidants as shown in table 1-3. The results show that, the Coleus aromaticus proteins have good antioxidant activity when compared to standards.

Discussion

The results show the percentage inhibition of DPPH radicals increased with increase in concentrations of crude protein of Coleus aromaticus extract (Table 1). DPPH assay was based on the ability of DPPH, a stable free radical, to decolorize in the presence of antioxidants, which denotes the extract has significant antioxidant potential.

The results show nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH reacts with O2 to form nitrite ion (Table 2).  Aqueous extract inhibited nitrite formation in concentration dependent manner. Antioxidant nature of the extract complete with oxygen to react with nitric oxide. It was observed that, percentage (%) inhibition was increased with the increase in concentration of the extract of crude protein of Coleus aromaticus with various concentrations.

The percentage inhibition of superoxide radical scavenging activity of crude protein of Coleus aromaticus was increased with increasing concentrations (Table 3). In this method, superoxide anion plays an important role in plant tissues and it involved in the formation of other cell-damaging free radicals.¹⁶ Potent antioxidant capability of the extract is detected by the scavenging potential of the superoxide anion, with their electron donation. This observation has also been reported by other researchers.^17,18 

Conclusion

Plant proteins have been investigated in the search for novel antioxidants in the past few years but generally there is still a demand to find more information concerning the antioxidant potential of plant species as they are safe and also bioactive. Crude protein of Coleus aromaticus extract showed a significant antioxidant activity and might be an alternate to synthetic antioxidants available in the market. Hence further studies are needed to purify the protein of Coleus aromaticus and to evaluate the in-vivo antioxidant activity on animal models.