In-Vitro Antioxidant, Anti-Inflammatory and Cytotoxic effects of different Solvent Extraction Terminalia chebula, Terminalia billerica, Phyllanthus emblica
Sanakousar K. Patel1, Arun K Shutter2, Ria Patil3, Archana Desangi3,
Vishalakshi Malali3, Jyoti Patil3, Sumangala Patil1, Kusal K. Das1, Prachi P. Parvatikar1*
1Laboratory of Vascular Physiology and Medicine, Department of Physiology, Shri B M Patil Medical College, Hospital and Research Centre, BLDE (DU), Vijayapura, Karnataka, India-586103.
2Cytxon Biosolution Pvt Ltd; 3Karnataka State Akkamahadevi Women’s University, Vijayapura.
*Corresponding Author E-mail: prachisandeepk@gmail.com
ABSTRACT:
Objective: The phytochemical analysis of Terminalia chebula, Terminalia bellirica, Phyllanthus emblica fruit extract as well as to evaluate the potential synergistic activity of screened plant extracts for anti-oxidant, anti-inflammatory and anti-cancer activity on MCF-7 breast cancer cell line. Methodology: In present study phytochemical constituent from selected plant material was extracted in different solvents. Antioxidant activity was evaluated by DPPH and FRAP assay against standard antioxidant. MTT assay was performed to find out cytotoxic and anti-inflammatory effect on MCF- breast cancer cell line. Result: Phytochemical analysis of the performed which showed that alkaloids, phenols, flavonoids and saponins. In context of antioxidant assays almost selected extracts showed moderate activity but ethanol extract of Terminalia chebula, proven to be having significant antioxidant activity over all extracts. Anti-inflammatory activity of MCF-7 breast cancer cell line exhibited that good activity with IC50 value 228.82µg/ml. Whereas Methanol extract of Terminalia bellirica and Aqueous extract of Phyllanthus emblica shown 432.45µg and 259.02µg respectively. Conclusion: The present study concludes that in among three tested extracts, Ethanol extract of Terminalia chebula exhibited prominent anticancer activity against Breast cancer (MCF-7) with significant IC50 value 228.82 µg. However further studies are needed to find out the structural of bioactive compounds and investigate mechanisms of antioxidant and anticancer activities of the bioactive compounds in in-vivo animal study.
KEYWORDS: Terminalia chebula, Phytochemical constitutes, Antioxidant, Breast cancer, MTT assay.
INTRODUCTION:
Since ancient time plants used in traditional medicine, medicinal plants are essential for the well-being and continued survival of man. The traditional knowledge and use of medicinal plants are widespread from various perspectives, like pharmaceuticals.6
Terminalia chebula, a medicinal plant belonging to the family Combretaceae and found in different Indian regions. Some species of family have been traditionally used as folk medicines in the treatment of various diseases7. Terminalia bellirica is such a medicinal plant that has extensive usage as pharmaceutical and nutraceuticals8. Phyllanthus emblica is a tree native to Southeast Asia's tropical climates. The fruit of the tree is known as Indian Gooseberry or Alma. For millennia, the extract from these fruits has been utilized in traditional medicine to cure ailments ranging from constipation to tumor treatment9.
The current study is focused on phytochemical extraction of selected plant material through successive solvent extraction and evaluate the antioxidant, anti-inflammatory and cytotoxic properties against human breast cancer cell line MCF-710.
MATERIALS AND METHODS:
Collection of plant material:
Fresh, dried fruits of Terminalia chebula, Terminalia bellirica and Phyllanthus emblica were collected from the Department of Pharmacology, BLDE AVS Ayurveda Mahavidyalaya, Vijayapura-586108, Karnataka, India. The powder was stored in airtight containers at -20°C for further use for crude solvent extraction.
Preparation of plant extract:
About 25g of powdered material was extracted with 250 ml of Chloroform, Ethyl acetate, Methanol, Ethanol and Distilled water in series extraction method using Erlenmeyer flask and kept at room temperature for 24hours under dark condition. The extract was filtered using what-man filter paper and stored in the airtight bottle at 40C until use.
Phytochemical analysis:
The crude solvent extracts of Terminalia chebula, Terminalia bellirica and Phyllanthus emblica was qualitatively tested for the presence of different phytochemical constituents.11
Determination of antioxidant activity by using in-vitro methods:
Ferric ion reducing antioxidant power assay (FRAP):
Ferric ions reducing power assay was measured according to standard method with slight modification12. Different solvent extracts of Terminalia chebula, Terminalia bellirica and Phyllanthus emblica taken in different concentrations ranging from 100µl to 500µl. The absorbance was measured at 700nm using a UV-VIS Spectrophotometer. Ascorbic acid was used as the reference standard. All samples were assayed in triplicates.
Hydrogen peroxide scavenging assay (H2O2):
The antioxidant activity of selected plant material was carried with ascorbic acid as a standard was assessed based on their ability to scavenge the hydrogen peroxide 13. 0.6ml of 4mM H2O2 solution (pH-7.4) was added to 0.5ml of known concentration of standard ascorbic acid and tubes containing different concentrations ranging from 100µl to 500µl of plant extracts in phosphate buffer (pH-7.4). The absorbance of the solution was measured at 230nm after 10min against the blank solution containing phosphate buffer without hydrogen peroxide. Control was prepared by replacing the sample or standard with phosphate buffer.
DPPH free radical-scavenging ability assay
According to the standard method, radical scavenging activities of solvent extracts of plant extract were determined using the DPPH radical as a reagent, according to the standard method [14]. 100µL of a DPPH radical solution in ethanol (60 µM) were mixed with 100µL of sample solution. The mixture was incubated for 30 min in the dark at room temperature, and then absorbance was measured at 517nm using a UV-VIS Spectrophotometer. Ascorbic acid was used as a reference standard. The DPPH scavenging activity of each sample was calculated using the following equation:
% inhibition= Ac-At/Ac x 100
Where Ac- the absorbance of the control; At- the absorbance of the test. The IC50 value was calculated for all the samples used.
Evaluation of In-vitro anti-inflammatory activity:
Anti-inflammatory activity of Different solvent extracts of selected plant was evaluated by protein denaturation method15. The reaction mixture consisting of 2ml of known concentration of Selected plant extracts (100 µg/ml) with standard Diclofenac sodium (100µg/ml) and 2.8ml of phosphate buffered saline (pH 6.4) was mixed with 2ml of egg albumin (from fresh hens egg) and incubated at (27±1)°C for 15 min. Denaturation was induced by keeping the reaction mixture at 70°C in a water bath for 10 min. After cooling, the absorbance was measured at 660nm by using double distilled water as blank. The percentage inhibition of protein denaturation was calculated.
Determination of cell viability by MTT Assay:
The effect of plant extract on the viability of breast cancer (MCF-7) cells was determined using the standard colorimetric MTT assay using the 3-(4,5-dimethylthiazol- 2-yl)-2,5-dimethyl tetrazolium bromide dye (Sigma, St. Louis, MO, USA)[16]. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (IC50) values is generated from the dose-response curves for each cell line17.
Inhibition Percentage = OD of Test sample ÷ OD of control × 100
Statistical analysis:
All the tests were carried out in triplets (n = 3) and statistically analyzed and is presented as mean±S.E. using ANOVA statistical programme.
RESULTS AND DISCUSSION:
Phytochemical analysis:
The qualitative analysis of the various extract of selected plant material was found to possess more phytoconstituents which was revealed by qualitative analysis indicating alkaloids, flavonoids, glycosides, phenols, saponins, tannins, terpenoids, steroids and carbohydrates.
Antioxidant assay:
FRAP Assay
In the present study, different concentrations of plant extract were subjected to FRAP assay. Ethanol extract of T.chebula ethanol extract of known concentration (500µl) showed good antioxidant activity with absorbance values (1.232). In the case of T.bellirica, methanol extract exhibited higher activity among other sections (1.262), an aqueous extract of P.emblica showed higher activity. (Table.1)
Hydrogen peroxide scavenging assay:
H2O2 assay is one of the common methods used to investigate the antioxidant capacity of the extracts. The results revealed that aqueous extract of P emblica showed highest scavenging activity (80.8667) than standard and among all other plant extracts (Table 2).
Table.1. FRAP Assay of selected of plant extracts. (C-chloroform, E-ethyl acetate, M-methanol, Et-ethanol, A-aqueous)
|
Name of the plants |
Terminalia chebula |
Terminalia bellirica |
Phyllanthus emblica |
||||||||
|
Sl. No |
Concentration |
Std Ascorbic acid |
E
|
M
|
A
|
M |
E |
A |
M |
E |
A |
|
1 |
100µl |
0.4650 ± 0.00300 |
0.5527± 0.00503 |
0.4367± 0.00416 |
0.2220± 0.01000 |
0.3490± 0.00265 |
0.2687± 0.00503 |
0.2917± 0.00351 |
0.2653± 0.00321 |
0.3327 ± 0.00702 |
0.3890± 0.00300 |
|
2 |
200 µl |
0.7530 ± 0.00458 |
0.7177± 0.00351 |
0.6133± 0.00802 |
0.3303± 0.01550 |
0.4310± 0.00361 |
0.3947± 0.00306 |
0.4433± 0.00416 |
0.5543± 0.00321 |
0.4513 ± 0.00833 |
0.5783± 0.00252 |
|
3 |
300 µl |
0.9943 ± 0.00416 |
0.8433± 0.00611 |
0.8167± 0.00611 |
0.4907± 0.00611 |
0.6737± 0.01457 |
0.5120± 0.00200 |
0.5797± 0.00153 |
0.7463± 0.00379 |
0.5287 ± 0.00902 |
0.6870± 0.00458 |
|
4 |
400 µl |
1.1353 ± 0.00416 |
0.9467± 0.00416 |
0.8743± 0.00950 |
0.5793± 0.00416 |
0.8617± 0.00751 |
0.6433± 0.00416 |
0.7710± 0.00964 |
0.8767± 0.00451 |
0.6680 ± 0.00872 |
1.0417± 0.00252 |
|
5 |
500 µl |
1.4647 ± 0.00306 |
1.2320± 0.00755 |
1.0247± 0.00503 |
0.6427± 0.00503 |
1.2620± 0.00917 |
0.7160± 0.00529 |
0.8867± 0.00379 |
1.0113± 0.00404 |
0.7820 ± 0.00400 |
1.1533± 0.00416 |
Table.2. DPPH activity of selected plant extract of plant extracts. (C-chloroform, E-ethyl acetate, M-methanol, Et-ethanol, A-aqueous)
|
Name of the plants |
Terminalia chebula |
Terminalia bellirica |
Phyllanthus emblica |
||||||||
|
Sl. No |
Concentration |
Std Ascorbic acid |
M |
E |
A |
M |
E |
A |
M |
E |
A |
|
1 |
10µg |
67.7867±0.17898 |
51.6300±0.35000 |
57.0633±0.52596 |
42.7350±0.81862 |
73.8867±0.46501 |
58.2300±0.29206 |
68.7200±0.41146 |
57.8800± 0.35679 |
46.0761±0.58662 |
68.2167± 0.35572 |
|
2 |
20 µg |
73.6533±0.23502 |
57.4500±0.35000 |
58.9300±0.41146 |
55.6332±0.48524 |
77.3067±0.35572 |
65.6500±0.35679 |
72.8533±0.32347 |
67.2033±0.35572 |
59.4794±0.59808 |
70.1167± 0.37434 |
|
3 |
30 µg |
78.2000±0.30806 |
69.8900±0.29206 |
69.4233±0.44287 |
62.9370±0.23310 |
80.7100±0.19975 |
68.2700±0.15100 |
74.8967±0.48840 |
72.5700±0.29206 |
60.5112±0.48524 |
75.9067± 0.27135 |
|
4 |
40 µg |
80.8000±0.35679 |
73.1100±0.35679 |
75.3667±0.24420 |
66.9386±0.52558 |
84.0200±0.73980 |
71.6367±0.24420 |
77.4233±0.17898 |
78.3567±0.40624 |
72.4941±0.46620 |
80.3333± 0.24420 |
|
5 |
50 µg |
83.9100±0.35000 |
77.2700±0.35000 |
78.9000±0.42297 |
74.8251±0.46620 |
86.0100±0.30806 |
73.6267±0.56889 |
78.7833±0.23502 |
81.0733±0.17786 |
78.0108±0.48524 |
83.0200± 0.24021 |
Figure 1: Percentage of inhibition of different solvent extracts of selected plant extracts by hydrogen peroxide assay at 100mg/ml
DPPH free radical-scavenging ability assay:
The antioxidant capacity of the extracts was compared with ascorbic acid as a standard antioxidant17. In the case of T.chebula, all solvent extract shown good scavenging activity but inhibition percentage (83.9100). The DPPH assay of T Billerica shows that methanol extract exhibited more significance than others (86.0100). In case of P.emblica among all three sections showed promising antioxidant activity with the percentage of inhibition (Table 3).
In-vitro anti-inflammatory assay:
Anti-inflammatory study of known concentrations (100µg) of plant extracts was subjected for anti-inflammatory activity through protein denaturation assay. The results revealed that methanol extract of T.bellirica exhibited significant anti-inflammatory activity with the percentage of inhibition 86.9133% than standard drug and all the quotes. In comparison, the standard drug Diclofenac sodium showed 94.2467% of inhibition of protein.
Figure 2: Ani-inflammatory activity of different solvent extracts of selected plant extracts at 100mg/ml
Cell Viability and Morphological observation
The cell viability assay was performed on a triple-negative MCF-7 cell line taken as the control group. In the present study, different concentrations (100µg - 500 µg) of standard drug and plant extract were taken to study morphological changes and cell growth inhibition in a cell line.
An increase in the cell viability was observed at minimum concentration only. As the concentration increases, the viability of cells was decreased. Among three tested extracts, the Ethanol extract of Terminalia chebula exhibited prominent activity with a significant IC50 value of 228.82µg. Whereas Methanol extract of Terminalia bellirica and Aqueous extract of Phyllanthus emblica shown 432.45µg and 259.02µg, respectively.
Table.3. Comparison of Effect of different solvent extracts of experimental plants on MCF-7 (Breast Cancer) Cell viability
|
Treatment |
Cell Line |
Concentration in µg |
Cell Viability in Percentage (%) |
IC50 in µg |
|
Ethanol extract of Terminalia chebula |
MCF-7 |
100 |
68.7142±0.015 |
228.82 |
|
200 |
56.0645±0.0165 |
|||
|
300 |
32.4310±0.0215 |
|||
|
400 |
13.4825±0.0185 |
|||
|
500 |
6.9234±0.0015 |
|||
|
Methanol extract of Terminalia bellirica |
MCF-7 |
100 |
94.3779±0.005 |
432.45 |
|
200 |
85.4763±0.0545 |
|||
|
300 |
70.0676±0.0650 |
|||
|
400 |
53.9781±0.0380 |
|||
|
500 |
39.6147±0.0305 |
|||
|
Aqueous extract of Phyllanthus emblica |
MCF-7 |
100 |
87.4544±0.0035 |
259.02 |
|
200 |
67.0900±0.0105 |
|||
|
300 |
34.0551±0.0420 |
|||
|
400 |
17.7782±0.0125 |
|||
|
500 |
5.4138±0.0090 |
|||
|
Standard Drug Cisplatin |
MCF-7 |
20 |
6.9755±0.0050 |
≥15 |
CONCLUSION:
In the present study, the phytochemical analysis helped detect the secondary metabolites in the selected extracts of the plant. Phytoconstituents were evaluated by different quantitative biochemical tests. Phytochemical analysis of plant extract contains a broad spectrum of bioactive compounds that included alkaloids, glycosides, saponins, tannins, phenols and flavonoids. In the context of antioxidant assays, almost all selected extracts showed moderate activity, but ethanol extract of T. chebula, methanol extract of T. bellirica and aqueous extract of P.emblica proven to be having significant antioxidant activity overall sections. The in-vitro anti-inflammatory activity of the extracts was comparable to the reference drug. The results revealed that methanol extract of T.bellirica exhibited effectiveness with the percentage of inhibition 86.9133%.In anticancer activity, comprehensive studies showed that ethanol extract of T. chebula has solid anticancer activity against Breast cancer cells MCF-7. However, further studies are needed to determine the structure of bioactive compounds and investigate the antioxidant and anticancer activities of the bioactive compounds in the in-vivo animal study.
CONFLICT OF INTEREST:
Authors have no conflict of interest to declare.
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Received on 15.03.2021 Modified on 29.07.2021
Accepted on 06.10.2021 © RJPT All right reserved
Research J. Pharm. and Tech. 2022; 15(7):2940-2944.
DOI: 10.52711/0974-360X.2022.00490