In-vitro antioxidant activity of oxalis carniculata linn and investigation of plausible mechanism of photo-protection by molecular modeling study

 

Madhuri Baghel*, Hemant Badwaik, Sangeeta Patil

Rungta College of Pharmaceutical Sciences and Research, Bhilai, Chhattisgarh, India.

*Corresponding Author E-mail: banchhormadhuri@gmail.com

 

 

ABSTRACT:

Oxalis Carniculata Linn belonging to Oxalidaceae family is a small herb with variety of biological activity. The leaves of Oxalis Carniculata was extracted with different solvents and the fractions were screened for antioxidant potential using in-vitro methods such as 2,  2-Diphenyl-2-Picryl  hydrazyl  (DPPH)  and H2O2 radical  scavenging activity, and reducing  power  assay .  Total flavonoid, phenolic content and total antioxidant capacity were determined spectrophotometrically. Among the six different fractions ethanolic fraction exhibited highest total flavonoid content (224.2±0.25 mg/g QAE) and methanolic fraction exhibited highest phenolic content (184.31±0.14 mg/g GAE) and total antioxidant capacity (232.79±0.17mg/g AAE). Further methanolic fraction showed highest DPPH and H2O2 radical scavenging activity with IC50 value 23.43±0.12 and 19.71±0.16 respectively. The major cause of skin photoaging is exposure to ultraviolet light that is coupled with the increased expression of matrix metalloproteinases (MMPs) and decreased collagen synthesis. MMPs (especially MMP-1, MMP-3 and MMP-9) expressions are associated with the decreased elasticity of the dermis due to collagen degradation. In the present study, we have assessed the antioxidant effects of Oxalis Carniculata extract, correlated it with skin photoaging and postulated its mechanism of action by molecular docking study. The molecular docking study of flavones present in O. Carniculata revealed that the phyto-constituents of O. Carniculata species have similar mechanism of binding to that of standard drug with MMP-9 inhibitory action. These results can facilitate the further preclinical models, and clinical study upon skin protective potential against UV-induced skin aging of the O. Carniculata flavones.

 

KEYWORDS: Photoageing, MMPs, phytoconstituents, Molecular Docking, Oxalis Carniculata Linn.

 

 


INTRODUCTION:

Frequent exposure to ultraviolet irradiation (UVR) from solar light causes histologic and clinical changes in human skin1. The photo-damaged skin is characterized by coarse and fine wrinkles, dryness, loss of skin tone and spotty pigmentation2. Collagen and elastin fibrils maintain the strength and flexibility of the skin3. Deterioration of these fibrils with photo-aging decreases the youthful appearance of the skin. Quantitative, qualitative and biochemical alterations were found in the dermal extracellular proteins elastin, interstitial collagen, and glycosaminoglycans in photoaged skin4.

 

The signaling pathways involving ERK, JNK, and p38 are activated by the ROS (Reactive Oxygen Species), followed by activation of AP-1 (activator protein-1) and NF-κB (nuclear factor κB) that triggers the transcription of genes for activation of skin MMPs (matrix metalloproteinases). The MMPs are a family of enzymes responsible for degradation of connective tissue5. These are related to endopeptidases structurally, which provoke the degradation of different macromolecular components of the ECM (extracelluar matrix). The human MMPs are classified into four different subfamilies, these are- collagenases, gelatinases, stromelysins, and membrane MMPs6.

 

Phyto-constituents7 protect the skin against endogenous and exogenous agents and can help to cure many skin conditions; hence theses are gaining popularity in cosmetic formulations8. Oxalis carniculata Linn, which belongs to the family Oxalidaceae, commonly known as yellow sorrel plant is distributed all over Chhattisgarh. The leaves of O. Carniculata are quite edible which has high nutritional value with tangy taste. The plant is rich in vitamin C. It also contains three antioxidant flavones; swerticin, isovitexin and isoorientin9 (Figure 1).

 

Fig. 1. Chemical structure of O. Carniculata phytoconstituents

 

Kaur S. et al10 have performed phytochemical screening and reported the biological potential of methanolic extract of O. Carniculata in different parts of plant. Badrul Alam M. et al11 have investigated the in-vitro and in-vivo antioxidant activity of O. Carniculata in different fractions. Both the authors have not proposed the plausible mechanism of skin antiphotoaging via molecular modeling studies. Hence the objective of present study is to evaluate antioxidant potential of different fractions of O. Carniculata in-vitro. Further we aimed to correlate the antioxidant activity with skin antiphotoaging potential utilizing the molecular modeling methodology. The mechanisms of binding of major phyto-constituent present in the plant with the MMPs were proposed.

 

MATERIALS AND METHODS:

Chemicals

Standards of Quercetin(QA), Ascorbic acid (AA), and Gallic acid (GA) were purchased from CDH fine chemicals Ltd, New Delhi. DPPH (2, 2’ Diphenyl -1-Picrylhydrazyl), Folin’s Ciocalteau reagent, Potassium acetate, Sodium carbonate, Aluminium  chloride, Trichloroacetic  acid, Potassium  ferricyanide, Ammonium molybdate, Ferric chloride, Sodium  phosphate  monobasic were obtained from CDH fine chemicals Ltd, New Delhi. Solvents used for extraction of plants were obtained from Loba chieme Pvt Ltd, Mumbai.

 

Plant Materials

The plant of O. Corniculata was collected from the various places of village Raveli, Funda region, Durg dist. in the month of July and August. The plants were mounted on paper and the sample was indentified and authenticated by Dr. Ranjana Shrivastava, Professor and H.O.D. (Botany Department), Govt. V.V.T. PG College Durg (C.G.). Different parts of plant were first studied for their moisture content using the formula:

 

Where MC= Moisture content; W0  =  weight  (gm)  loss  on  drying,  Wi =  initial  weight  of  sample (gm).

 

Plant extraction

The dried leaves of O. Corniculata were extracted successively with organic solvents ranging from polar to non-polar.  It was extracted with chloroform, ethylacetate, acetone, ethanol, methanol, and water by soxhlation process.  5  gm  of  the  coarsely  powdered  plant  material  was  weighed  and packed into soxhlate extractor containing  1 lit round bottom flask.  Then  the  flask  was  filled  with  different  solvents  (500  ml)  separately.  The plant material was soaked in extractor for 24 hrs at room temperature.  Then the plant material was extracted for 48 hr at the boiling temperature of solvents. The extract collected was cooled to room temperature and the solvents were evaporated to dryness in rotary evaporator. The extractive value in percentage was calculated by using following formula and recorded. The extracts were stored in sealed bottles for further study.

 

Phytochemical analysis

The methanol, ethanol, aqueous, acetone, ethyl acetate and chloroform extract of leaves of O. Carniculata were evaluated for the detection of different active substances12,13.

 

Qualitative analysis

The prepared extracts were  screened  for  the  presence  of  Alkaloids,  Flavanoids, Tannins,  phenolic  components,  terpenoids,  amino  acids  and Glycoside  according  to  standard  procedures  of  analysis  and were identified using characteristic color changes14.

 

Quantitative analysis

Total  flavonoids  content

Total Flavonoid content (TFC) of methanol, ethanol, aqueous, acetone, ethyl acetate and chloroform extract of leaves of O. Carniculata was estimated by aluminium chloride Method15 . The reaction mixture contained 0.5 ml of plant extract, 1.5 ml of methanol, 0.1 ml of (10 %) aluminum Chloride, 0.1 ml of potassium acetate (1 M) and 2.8 ml of distilled water. The mixture was incubated for 30 min at room temperature and absorbance was recorded at 415 nm using UV-Vis Spectrophotometer. The standard curve was prepared using serial dilution of quercetin (200 mg/l).  The concentration of flavonoids of the plant extract was calculated using the standard curve and the amounts were expressed in quercetin equivalent (mg/g QAE).

 

Total Phenolic Content

The  phenols  present  in  plants  are  act  as  highly  effective free  radical  scavengers  and  antioxidants.  The  total phenolic content (TPC) in  various  extracts  of  leaves  was  determined  spectrophotometrically  by  using  Folin-Ciocalteu (FC)  method.  The reaction mixture contained 100 µL of O. Carniculata, 500 µL of the Folin-Ciocalteu reagent, and 1.5 mL of 20% sodium carbonate. The final volume was made up to 10 mL with pure water. After 2h of reaction, the absorbance at 765 nm was measured and used to calculate the phenolic contents using gallic acid as standards. Triplicate reactions were conducted16 and the amounts were expressed in gallic acid equivalent (mg/g GAE). The FC reagent is not specific to phenolic compounds and will react with other reducing species also. 

 

Phosphomolybdenum assay

The total antioxidant capacities of the extracts were determined by the phosphomolybdenum assay17, which is based  on  the  reduction  of  Mo  (VI)  to  Mo  (V)  by  the  extract  and  subsequent  formation of a green phosphate-Mo (V) complex in acidic condition. 0.1 ml of each extract was combined with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate).  The reaction mixture was incubated at 95şC for 90 min. After cooling  to  room  temperature,  the  absorbance  of  the solution  was  measured  at  695  nm  using  a  UV-visible spectrophotometer,  0.1  ml  methanol  was  used  as  the blank. The total antioxidant capacity was expressed as the number of gram equivalents of ascorbic acid per ml of the extract.

 

In-vitro Antioxidant activity

DPPH radical scavenging assay

The  free  radical  scavenging  capacity  of  the  extracts  was  determined  using  DPPH  (2,  2-diphenyl  1-picryl  hydrazyl)  radical  as  described  by  Barros  et  al.18 with  minor  alterations. Each sample stock solution (1.0 mg/mL) was diluted to final concentrations of 15-55 µg/mL. with 95 % methanol. Various concentrations of plant extracts (0.3 mL) were mixed with freshly prepared methanolic solution containing DPPH radicals (0.004% (w/v), 2.7 mL).  The mixture was shaken vigorously and allowed to stand for 60 min in the dark (until stable absorption values were obtained).  The extent of reduction of the DPPH radical was determined by measuring the absorption at 517 nm.  Ascorbic  acid  was  used  as  a  reference  standard  and  DPPH  solution  was used  as  the  control. 

The  percentage  radical  scavenging  activity  (%  RSA)  was  calculated  as  a percentage of DPPH discoloration using the equation:

 

Where:  A0  represents  the  absorbance  of  the  control  and  A1 represents  absorbance  containing  different  extracts  of O. Corniculata.

 

Percent  (%)  scavenging  activity  was  plotted  against  concentration  and  IC50  (the  extract concentration  providing  50  %  of  radicals  scavenging  activity)  value  was  calculated  from  the graph by linear regression analysis.

 

Hydrogen peroxide (H2O2) scavenging activity

The ability of the methanolic extracts of O. corniculata to scavenge hydrogen peroxide was evaluated by hydrogen peroxide.  A solution of hydrogen peroxide (40mM) was prepared by using phosphate buffer (pH 7.4). Extracts of different parts of plant with various concentrations (15-55µg/mL) were added to a hydrogen peroxide solution (0.6 mL, 40mM). Absorbance was measured at 230 nm after 10 minutes against a blank solution containing the phosphate buffer without extract using spectrophotometer.  Ascorbic acid was used as a positive control.

 

The percentage of hydrogen peroxide scavenging of extracts of different plant parts were calculated using following equation: 

 

Where:  A0  represents  the  absorbance  of  the  control  and  A1 represents  absorbance  containing  different  extracts  of O. Corniculata.

 

Reducing power assay

The reducing power of plant extracts was determined according to the method of Oyaizu19. The concentration of the extracts ranged from (15 to 55 µg/ml).  0.5  ml  extract  made  to  1  ml  with  distilled  water  and  mixed  with  2.5  ml  of Phosphate buffer (0.2 M, pH 6.6), 2.5 ml 1% Potassium ferricyanide [K3Fe(CN)6]. The mixture was incubated at 50 °C for 20  minutes  and  centrifuged  at  5000  ×  g  after  addition  of  2.5  ml  of  10  %  trichloroacetic  acid.  A 2.5 ml aliquot of upper layer (supernatant) was collected and mixed with 0.5 ml of 0.1 % ferric chloride. The absorbance was measured at 700 nm against a blank using UV-Vis spectrophotometer. Ascorbic acid at various concentrations was used as a standard. Increase in absorbance was directly correlated to increase in reducing power.

 

Molecular Docking studies

Molecular docking20-24 is carried out in order to provide a binding site with a population of probable ligand orientations and conformations25,26. It has been employed to validate the findings of screening studies, and understand the mechanism of skin anti photoaging property. A receptor-ligand interactions of phytoconstituents and standard Styraxjaponoside with MMP-9 enzymes (PDB Code: 2ow0) was studied using Argus lab 4.0. The ligand structures were generated using the Chemdraw software.  Three-dimensional optimizations of the ligand structures were done and saved as PDB file format. Geometry optimizations of the ligands were performed using ArgusLab 4.0.1 (Mark A. Thompson, Planaria Software LLC, Seattle, WA, USA.) software27. MMP-9 enzymes was docked against three phytoconstituents viz isoorentin, isovitexin and swerticin, the interaction was carried out to find the favorable binding geometries of the ligand with the protein. The protein-ligand interaction was visualized by using Discovery studio 2016 software28.

 

RESULTS AND DISCUSSION:

Plant extraction and phytochemical screening

The  moisture content (table 1), extractive  value (table 3)  and  color  of  extracts  of  Oxalis Carniculata  was  investigated.  From  the  present  study  it  was  found  that,  the  extractive  value  of Oxalis Carniculata in methanolic extract was maximum followed by ethanol and aqueous solvents. The  ethanolic  extract  showed  slightly  lower  extractive  value  than  methanolic  extract. The chloroform extract showed least extractive value. The  color  of  extracts  observed were  yellowish  green  in  methanol,  green  in  ethanol,  dark  brown  in  aqueous,  light  brown  in acetone and ethyl acetate, and  colorless  in  chloroform.

 

Table 1: moisture content of different plant parts of O. Carniculata

Sample

Leaves

Stems

Initial weight (in Grams)

13.12

11.89

Final weight (in grams)

2.0

2.64

Loss in weight

11.2

9.25

Moisture content

85.36

77.79

 

The extracts of leaves of O. Carniculata were  evaluated  to  various  chemical  tests  for  the  detection  of  different active substances. The results were presented in table 2.

 

Quantitative analysis

Total flavonoids content

The  qualitative estimation of  total  flavonoids  in  different  extracts  of  O. Carniculata  was carried out  by aluminium  chloride  method and the  results (table 3)  were  expressed  in  terms  of  mg/g of quercetin  equivalents.  The TFC was highest for ethanolic fraction followed by methanol and ethyl acetate. Chloroform extract had lowest TFC value while TFC value for acetone and aqueous extracts were found to be 61.1±0.20 mg/g  and 50.99±0.18 mg/g of QE respectively.

 

Total Phenolic Content (TPC)

The total phenolics in diferent extraxts of O Carniculata was quantitatively estimated using Folin-Ciocalteau method and the results were expressed in terms of mg/g of gallic acid equivalents (table 3). The TPC was found to be highest in methanolic extract and lowest in chloroform extract. The TPC was found to increase in the order of chloroform>aqueous>acetone>Ethyl acetate > etanol>Methanol.

 

 

Table 2: Qualitative phytochemical analysis of O. Carniculata in different solvents

S. No

Secondary Metabolite

Phytochemical test

H2O

EtOH

MeOH

Acetone

EtAC

CHCl3

1.         

Carbohydrates

Molisch’s Test

-

-

-

-

-

-

2.         

Proteins

Millon’s Reagent Test

+

+

+

+

+

+

3.         

Amino acid

Ninhydrin Test

-

-

-

-

-

-

4.         

Steriod

Liebermann Burchard

Reaction

+

+

+

+

+

+

5.         

Glycosides

a)Cardiac

Legal’s Test

-

-

-

-

-

-

b)Anthraquinone

Borntrager’s Test

-

-

-

-

-

-

6.         

Saponin

Foam Test

+

+

+

+

+

+

7.         

Flavonoid

Sodium hydroxide test

+

+

+

+

+

+

Shinoda’s Test

+

+

+

+

+

+

8.         

Phenolics

Ferric chloride (FeCl3) Test

+

+

+

+

+

+

Lead (Pb) Acetate Test

+

+

+

+

+

+

9.         

Alkaloids

Mayer’s Test

+

+

+

+

+

+

Wagner’s Test

+

+

+

+

+

+

Hager’s Test

+

+

+

+

+

+

10.       

Tannins

Dilute Nitric acid Test

+

+

+

+

+

+

 


Table 3: Extractive value, total flavonoid aontent, total phenolic content, and total antioxidant capacity of Oxalis carniculata  in different solvents.

Sample extract

Extractive value

(%)

Total flavonoid content in plant extract (mg/g) QAE

Total phenol content in plant extract (mg/g) GAE

Total antioxidant capacity in plant extract (mg/g) AAE

Aqueous

16.78

50.99±0.18

69.28±0.11

194.56±0.19

Methanol

19.42

205.18±0.2

184.31±0.14

232.79±0.17

Ethanol

17.14

224.2±0.25

173.85±0.16

181.39±0.26

Acetone

11.46

61.1±0.20

66.66±0.23

 

172.25±0.25

Ethyl acetate

10.36

132.73±0.14

116.01±0.14

90.9±0.26

Chloroform

6.1

12.58±0.28

20.58±0.27

8.71±0.2

 


Phosphomolybdenum assay

The  total antioxidant  capacity  of  different  extracts  was  determined  spectrophotometrically  at maximum absorption of 695 nm, using  phosphomolybdenum method. The antioxidant capacity was found to be highest in methanolic extract followed by aqueous, ethanol and then acetone. The antioxidant capacity is found to be lowest in chloroform. The results are shown in table 3.

 

DPPH radical scavenging assay

Higher antioxidant activity is correlated with the lower IC50 value. Lowest radical scavenging activity was observed in methanolic extract (23.43±0.12) while the chloroform extract showed highest (more than 500) radical scavenging activity. The IC50 value in ethanol, methanol and aqueous extracts were lower than the positive control gallic acid. The IC50 value of ethyl acetate extract was found to be similar to that of standard gallic acid. The results are shown in table 4.

 

Hydrogen peroxide (H2O2) scavenging activity

The H2O2 scavanging activity was eastimated in different extracts of O. Carniculata.  The IC50 value were found in the order of methanol>ethanol>aqueous>ethylacetate>acetone> chloroform. The IC50 value in ethanol, methanol and aqueous extracts were lower than the positive control gallic acid. The IC50 value of ethyl acetate extract was found to be similar to that of standard gallic acid. The results are shown in table 4.

 

Reducing power assay

The reducing ability (reduction of iron  (III)  to  iron  (II)) of different fractions of O. Carniculata  was estimated and compared with the strong reducing agent ascorbic acid (table 5). At 55 µg/ml concentration, the absorbance of methanol, ethanol, aqueous, acetone, ethyl acetate and chloroform extracts were found to be of 0.97, 0.872, 0.578, 0.528, 0.51and 0.19 respectively.

 

Table 4:  DPPH and H2O2 free radical scavenging activity

Sample

DPPH IC50 in µg/ml)

H2O2 IC50 in µg/ml

Aqueous

35.06±0.16

26.80±0.44

Methanol

23.43±0.12

19.71±0.16

Ethanol

31.13±22

25.92±41

Acetone

70.29±25

41.10±33

Ethyl acetate

37.95±28

35.31±21

Chloroform

569.80±72

614.23±0.86

Ascorbic Acid

37.65±61

31.44±0.32

 

 

Molecular Docking Study

In this research, the phytoconstituents produced improved effects, which led us to consider its mechanism of action. We have, therefore, chosen the docking studies as an instrument to support our results. MMP-9 enzymes inhibition has been the most powerful method for managing photoageging to date. In this analysis, we contrasted the findings with Styraxjaponoside for the docking of our phytoconstituents. The binding data obtained from Argus Lab revealed, phytoconstituents have a good affinity towards MMP-9 enzymes with binding energy -10.175 and -10.289 kcal/mol. Analysis of residual interaction between the inhibitor and target enzyme at the active binding site are shown in Figure 2.  Results revealed that the Styraxjaponoside and phytoconstituents bind with MMP-9 enzymes by van der Waals interaction, conventional hydrogen bond, carbon-hydrogen bond, Pi-sigma interaction, alkyl, and Pi-alkyl interaction (Figure 3 to 5)


 

Table 5:  Reducing power of Oxalis carniculata (L) in different solvents at 700 nm

Conc (µg/ml)

Absorbance (nm)

0

Methanol

Ethanol

Aqueous

Acetone

Ethyl acetate

Choloroform

15

0.188±0.15

0.155±28

0.095±12

0.102±32

0.082±39

0.02±53

25

0.376±0.23

0.332±32

0.209±16

0.215±26

0.161±41

0.06±46

35

0.602±16

0.519±14

0.324±24

0.33±19

0.281±40

0.102±61

45

0.801±11

0.701±22

0.471±20

0.461±24

0.351±35

0.14±55

55

0.97±12

0.872±11

0.578±26

0.528±33

0.51±27

0.19±67

 

 

Fig.2. 3D representation of a) MMP-9 enzymes, b) docked structure of Styraxjaponoside with MMP-9 enzymes, c) docked structure of isoorientin with MMP-9 enzymes, d) docked structure of swerticin with MMP-9 enzymes

 

Fig.3. Receptor-ligand interactions of Styraxjaponoside as (a) 2D pose, (b) 3D pose

 

 

Fig.4. Receptor-ligand interactions of isoorientin as (a) 2D pose, (b) 3D pose

 


Discussion

Phenolics and flavones usually found in plants have proven variety of biological effects including antioxidant activity. The redox property of these phyto-constituents is responsible for antioxidant activity that absorbs and neutralizes the free radicals generated due to UV radiation. In the present study the total flavonoid content, total phenolic content and total antioxidant activity was found to be highest in ethanol, methanol and methanol extract respectively.

 

Free radicals contain one or more unpaired electrons and they are highly unstable. They are highly reactive and to attain stability, they extract electrons from other molecules causing potential damage to the transient chemical species.The radical scavenging activity was determined by DPPH and H2O2 radical scavaging Assay. In this study it was observed that both DPPH and H2O2 radical scavenging activity of methanolic fraction was significantly lower than the standard ascorbic acid. It was revealed that the polar organic fraction of O. Carniculata showed the electron donating property and served as free radical scavenger and primary antioxidant that can reacts with the free radicals and protects the skin from free radical damage.

 

The potential antioxidant activity of a compound can be indicated by it’s reducing ability. The reductones present in compound or extract breaks the free radical chain reaction by donating the proton, thus it produces antioxidant action29. In the present study, the methanolic and ethanolic extract produces significant antioxidant activity and significantly contributes towards the observed antioxidant activity.

MMPs   play crucial role in skin photoaging mediated by UVR.  The connective tissue of the skin damages due to   over expression of MMPs on exposure to UV B radiation.  Initially  types  I,  III,  VII,  VIII,  and  X  collagens are cleaved by MMP-1 that  are further degraded  by  MMP-2  and  MMP-930 .  The  gelatinase  activity   of  MMP-2  and MMP-9  play  a  important  role  in  the  formation of  wrinkles  by  UV  irradiation ,  while  MMP-3  activates   pro MMP-1.  hence,  topical  MMP   inhibitors  may be  effective  at  preventing  UVB-induced photoageing  and formation  of  wrinkles. Results of molecular docking revealed constituents of species O. Carniculata have similar antioxidant activity to that of standard drug with MMP-9 inhibitory action. Out of 14 amino acid residue which interacts with Styraxjaponoside, 12 were found common with phytoconstituent swerticin (Figure 3, 4, 5). While In case of isorientin 8 amino acid residue found similar to that Styraxjaponoside binding pocket. Noteworthy that out 3 hydrogen bond formed by standard with MMP-9 enzyme, two were found similar in isorientin (with LEU-188 and ALA-189). These interaction patterns give a strong impression that constituents of species O. Carniculata possess good inhibitory potential against MMP-9 enzyme.



 


Fig.5. Receptor-ligand interactions of swerticin as (a) 2D pose, (b) 3D pose

 


CONCLUSION:

The results presented in the present study indicate that the methanolic and ethanolic extracts exhibit significant in vitro antioxidant activity. Further the molecular docking study of flavonoid present in O. Carniculata reveleaed constituents of species O. Carniculata have similar mode of binding to that of standard drug with MMP-9 inhibitory action. These results can facilitate the further preclinical models, and clinical study upon skin protective potential against UV-induced skin aging of the O. Carniculata flavones therefore,  O. Carniculata could  be  a  promising  agent  for  the  prevention   of  photoageing.

 

ACKNOWLEDGEMENT:

The authors like to thank Chhattisgarh Swami Vivekanand Technical University, Bhilai and TEQIP-III (REF NO:CSVTU/CRP/TEQIP-III/105) for providing financial support. 

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 06.03.2021                Modified on 20.07.2021

Accepted on 16.10.2021               © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(4):1843-1851.

DOI: 10.52711/0974-360X.2022.00310