Antioxidant and Anti-inflammatory activity of the Terpenoidal fraction of Ethanolic extract of Byttneria herbacea

 

Sanjeeb K. Kar1*,  Prasanna K. Dixit2, Uma S. Mishra3, Chowdhury M. Hossain4

1Department of Pharmacy, Sarala Birla University, Ranchi, Jharkhand - 835103, India.

2Department of Zoology, Berhampur University, Berhampur, Odisha - 760007, India.

3Royal College of Pharmacy and Health Sciences, Berhampur, Odisha - 760002, India.

4Department of Pharmaceutical Technology, Maulana Abul Kalam Azad University of Technology,

Kolkata, West Bengal - 700064, India.

*Corresponding Author E-mail: sanjeeb.bit@gmail.com

 

ABSTRACT:

The goal of this study was to identify the bioactive principle of the plant Byttneria herbacea (Malvaceae) which has a wide range of traditional therapeutic uses.Column chromatography was used to separate the ethanolic extract of aerial parts of the plant with mobile phase Chloroform: Methanol (5:5). The fraction with Rf 0.62 on TLC with mobile phase Toluene:Ethyl acetate (85:15) imparting bluish color under UV 254 nm was giving the test of Terpenoids. The same fraction was assessed for Antioxidant and Anti-inflammatory activity.Antioxidant activity was performed by measuring the decolourizing activity followed by capturing the unpaired electron from the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and compared it with the standard anti-oxidant Gallic acid. To test anti-inflammatory activity in an experimental model of acute inflammation, the carrageenan-induced in rat paw edema method was employed.The antioxidant and the anti-inflammatory activities of the Terpenoid fraction were found to be satisfactory.

 

KEYWORDS: Byttneria herbacea, Terpenoid, Anti-oxidant, Anti-inflammatory, DPPH, Gallic acid, Carrageenan-induced paw edema.

 

 


INTRODUCTION: 

In Indian traditional medicine, herbal treatments are a valuable resource. Both urban and rural inhabitants still employ medicinal plants to treat their most basic health issues. Numerous species that are frequently used in the country to treat conditions involving inflammation are listed in ethnobotanical literature, and they may be a valuable source for discovering medicines with anti-inflammatory characteristics. One such plant is Byttneria herbacea (Malvaceae), different parts of which are used to treat dysentery, leprosy, limb fractures, asthma, and inflammation.

 

To relieve pain, the root paste is applied to wounds and taken orally; the leaf is used to treat dysentery and impaction1. In the present study, one bioactive principle of the plant was identified and characterized. The DPPH method was used to assess antioxidant activity, and the carrageenan-induced paw edoema in rats method was used to evaluate anti-inflammatory activity.

 

MATERIALS AND METHODS:

Crude Drug Extraction and Isolation:

Aerial parts of Byttneria herbacea were shade dried and powdered in a household mixer. In the continuous hot percolator, the coarsely powdered drug was extracted using Petroleum Ether.After that, the residue was dried and extracted with ethanol and the extracts were concentrated in a vacuum rotary evaporator and stored in a refrigerator between 2°C and 4°C. The extract was separated by column chromatography. Silica is used as the stationary phase with Chloroform: Methanol (5:5) as the mobile phase2,3 The fraction with Rf 0.62 on TLC with mobile phase Toluene: Ethyl acetate (85:15) was used for further study.

 

Identification Test of Triterpenoids4–6

Foam Test:

A dilute aqueous solution of the Test sample was agitated for 5 minutes in a test tube. Foam formation indicates the existence of saponins.

 

Sedimentation test:

To the Test sample, few drops of a 10% Lead acetate solution were applied. Precipitate formation indicates the presence of triterpenoids.

 

Color Test:

One drop of 10% copper sulfate solution and one ml of Sulfuric acid were added to the Test sample. The appearance of a blue-green colour on heating indicates the presence of triterpenoids.

 

Antioxidant Assay:

The method used to determine the antioxidant potency of the Test sample was based upon the hydrogen donating capacity or scavenging activity of extracts on the DPPH (Sisco Research Laboratories Pvt. Ltd, Andheri, Mumbai) free radicals. The methanolic solutions of the terpenoidal fraction of the extract of Byttneria herbacea (3 ml) at different concentrations were mixed with 4ml of a DPPH 0.004% methanolic solution Methanolic solution of DPPH (0.0004%) was used as control and methanol as blank.  Absorbance at 517 nm was measured by using UV-Spectrophotometer (UV-2600i, Shimadzu) after 30 min of reaction.7–10.

 

The absorbance of the methanolic solution of the extract was plotted against corresponding concentrations and compared with the standard anti-oxidant Gallic acid11–13

 

The percentage of DPPH decolorization by the sample was determined as:

                               A0 - A

% Decoloration = ----------- x 100

                                   A0

Where A0 represents DPPH absorbance without the sample (control) and A represents DPPH absorbance with the sample at 517nm13.

 

The % decolorizations against the extract concentrations were plotted, to obtain the logarithmic regression line for estimation of the IC50 (Inhibitory concentration 50, µg/ml), the quantity of extract required to reduce DPPH absorbance by 50%. The parabolic equation used to calculate the IC50 was y= ax2 + bx + c, for the value of y=508,14,15

 

 

Antiinflammatory Assay:

The Carrageenan-induced paw edoema method was used to assess the anti-inflammatory activity of the Test sample in Wistar rats weighing 150-200 g16,17. After fasting overnight, the animals were divided into control, standard, and three test groups of six animals each. The test samples were administered orally to the test animals atdoses of 50 mg/kg, 100mg/kg, and 200 mg/kg body weight. Diclofenac was administered orally to the animals in the Standard group at a dose of 10 mg/kg. Control animals were given 0.5% Tween 80 in distilled water at a dose of 10 ml/kg body weight.Thirty minutes after receiving the respective Test samples and Diclofenac, all animals were challenged with 0.1ml of 1% carrageenan in the sub-plantar region of the right hind paw. A digital plethysmometer was used to measure paw volume before and after carrageenan administration at 30 min, 1, 2, 3, and 4 hr intervals.The ability of the test sample to inhibit paw edema was tested in comparison to the control group18–20.

 

Edema volume = final paw volume - initial paw volume

The following formula was used to calculate the percentage inhibition of paw edema:

                                                  Vc - Vt

% Inhibition of paw edema = -------------- x 100

                                                       Vc

Where Vc denotes control group paw edema and Vt denotes treated group paw edema21.

 

The experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC) of Gayatri College of Pharmacy, Sambalpur, Odisha (Vide Ref. No. GCP/L-924/22).

 

RESULTS:

The fraction from the column with Rf 0.62 on TLC with mobile phase Toluene: Ethyl acetate (85:15) imparting bluish color under UV 254nm was given the test of Terpenoids.

 

Antioxidant Assay:

The absorbance of methanolic solutions of DPPH (Control), Gallic acid (Standard), and the the methanolic solutions of the terpenoidal fraction of the extract of Byttneria herbacea (Test) at different concentrations was determined using a UV-Spectrophotometer set to 517 nm and the absorbances are presented in Table 1. The percentage of DPPH decolorization by the Standard and the Testis presented in Table 2. Fig. 1 shows the DPPH radical scavenging activity values for the Test and Standard.

 

The % decolorization of different concentrations of the sample was plotted and the equation of the graph was found to be y = -0.0023x2 + 0.7075x + 26.695.

The IC50 was calculated from the value of y =50 and was found to be 270.09 (µmol/g).

 

The antioxidant activity of the Terpenoidal fraction was found to be satisfactory compared to Gallic acid as the standard.

 

Anti-inflammatory Assay:

Diclofenac was utilised as the reference standard (10 mg/kg body weight), and 0.5% Tween 80 solution served as the control (10ml/kg body weight). Tables 3 and 4 present the findings of the studies. Fig. 2 depicts the anti-inflammatory activity of various concentrations of Test, Standard, and Control. Fig. 3 depicts the inhibition of rat paw edema by Standard and different doses of Test at different times. It is evident that paw volume gradually increased following carrageenan administration, peaking at 2hours and then declining in control groups, but it takes 1h for Standard and Test drugs. At different time intervals, Diclofenac (Standard) at a dose of 10mg/kg body weight showed statistically significant activity (P˂0.001) on comparison with control group. The terpenoidal fraction of the extract (Test) at a dose of 50mg/kg, demonstrated statistically significant activity (P˂0.01) at 1 h, and (P˂0.001) at 2 h, 3h, and 4 h.The 100mg/kg dose demonstrated significant activity (P˂0.05) at 0.5 h, (P˂0.001) at 1h, 2h, 3h, and 4h.When compared to the control, the 200mg/kg dose demonstrated highly significant activity (P˂0.001).

 

Diclofenac, at 10mg/kg dose, significantly reduced the edema formation after 0.5h. At 4h, it showed the greatest percentage reduction (68.06%) in paw edema.

 

At 4 hours, the terpenoidal fraction of the extract (Test) at doses of 50, 100, and 200mg/kg inhibited paw edema by 16.17%, 30.14%, and 53.09%, respectively.

 

DISCUSSION:

The current research aims to investigate and assess the bioactivity of Byttneria herbacea which is used in folklore in Odisha and India and has significant medicinal importance. The plant extracts were biologically screened to determine antioxidant,and anti-inflammatory activities, as well as related biochemical parameters, with their ethnomedicinal and folklore uses in the background. However, there are only a few reports in the available literature that support their scientific validity. As a result, studies on folklore claims were conducted to provide scientific evidence and support for the use of these herbal drugs. Antioxidant activity was assessed by measuring decolorizing activity, then capturing the unpaired electron from the DPPH and comparing it to the standard anti-oxidant Gallic acid. In an acute inflammation experimental model, anti-inflammatory activity was tested using the  Carrageenan-induced edoema in rat paws.

 

CONCLUSION:

The antioxidant and anti-inflammatory actions of the Terpenoid fraction were found to be satisfactory. As a result, it can be used as a natural source of antioxidant and anti-inflammatory with great potential as a therapeutic agent in preventing or slowing the progression of aging and age-related oxidative stress-related degenerative diseases.

 

Table 1: Absorbance of Methanolic solutions of DPPH (Control), Gallic acid (Standard), and the Sample at different concentrations at 517nm

Sample

Concentration (µg/ml)

Absorbance

Control (DPPH)

4

0.118

Standard

(Gallic acid)

with 4 ml methanolic DPPH solution (0.004%)

5

0.094

10

0.071

20

0.044

30

0.024

40

0.012

50

0.010

100

0.009

TEST

(Terpenoidal fraction of the Ethanol extract)

with 4 ml methanolic DPPH solution (0.004%)

10

0.086

20

0.069

40

0.055

50

0.043

100

0.032

150

0.028

200

0.023

 

Table 2: % of DPPH decolorization at 517nm

Sample

Concentration (µg/ml)

% of DPPH decolorization

Standard
 (Gallic acid)

5

20.34

10

39.83

20

62.71

30

79.66

40

89.83

50

91.53

100

92.37

Terpenoidal fraction of the

Ethanol extract

10

27.12

20

41.53

40

53.39

50

63.56

100

72.88

150

76.27

200

80.51


 

Table 3: Anti-inflammatory action of the terpenoidal fraction of ethanol extract of the aerial parts of Byttneria herbacea by Carrageenan-induced paw edoema in rats.

Groups

Dose (mg/kg)

Initial paw volume

Paw volume at different time intervals in ml.

0.5 hr

1h

2h

3h

4h

Control

(Tween 80)

10

0.77 ±0.05

1.37 ±0.04

1.70 ±0.04

1.71±0.04

1.68 ±0.03

1.67±0.04

Standard

(Diclofenac)

10

0.65±0.03

0.73±0.04***

0.82±0.03***

0.65±0.04***

0.58±0.03***

0.53±0.02***

Test

(Terpenoidal fraction of the Ethanol extract)

50

0.75±0.03

1.28±0.03

1.52±0.04**

1.47±0.05***

1.43±0.03***

1.40±0.04***

100

0.73±0.04

1.20±0.04*

1.45±0.04***

1.37±0.04***

1.25±0.04***

1.17±0.03***

200

0.77±0.06

0.92±0.03***

1.08±0.03***

0.93±0.04***

0.83±0.04***

0.78±0.03***

All values are presented as Mean±SEM, n=6. Between the control and other groups, there is a significant difference at ***P < 0.001, **P < 0.01, and *P < 0.05 significant levels.

 

Table 4: Percentage inhibitions of rat paw edema by the different doses of the terpenoidal fraction of the ethanol extract of Byttneria herbaceaat different times

Groups

Dose (mg/kg p.o.)

% Inhibition of paw edema in rat at different time

0.5 h

1 h

2 h

3 h

4 h

Standard

Diclofen (Diclofenac)

 

10

 

46.34

 

51.96

 

62.43

 

65.35

 

68.06

Test

(Terpenoidal fraction of the Ethanol extract)

50

6.1

10.59

14.04

14.85

16.17

100

12.2

14.71

21

25.74

30.14

200

32.93

36.27

46.05

50.5

53.09

 


 

Fig. 1: Activity (Decolorization %)

 

Fig. 2: Anti-inflammatory action of the terpenoidal fraction of the extract of Byttneria herbaceaby carrageenan-induced paw edema in rat.

 

Figure 3: Inhibition of rat paw edemaby Standard and different doses of the terpenoidal fraction of the ethanol extract of Byttneria herbaceaat different times

 

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Received on 19.03.2023            Modified on 10.08.2023

Accepted on 09.11.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(4):1481-1485.

DOI: 10.52711/0974-360X.2024.00234