{"id":21683,"date":"2018-09-21T11:08:47","date_gmt":"2018-09-21T11:08:47","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=21683"},"modified":"2020-04-23T10:57:57","modified_gmt":"2020-04-23T10:57:57","slug":"antioxidant-activity-and-in-silico-analysis-of-centella-asiatica-and-indigofera-aspalathoides-in-psoriasis","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol11no3\/antioxidant-activity-and-in-silico-analysis-of-centella-asiatica-and-indigofera-aspalathoides-in-psoriasis\/","title":{"rendered":"Antioxidant Activity and In Silico Analysis of Centella asiatica and Indigofera aspalathoides in Psoriasis"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Human skin, is the largest organ in the body which covers the whole body and possess the first line defense. However, there are more conditions that may affect the skin and cause many diseases. Among them, psoriasis is one of the most common and frequent skin diseases. Its symptoms were characterized by red, scaly, raised patches, itchiness and bleeding. Psoriasis is an inflammatory disease characterized by the increased rate of epidermal layer associated with the hyperprofileration and malfunctioning mature epidermal keratinocytes. Psoriasis is a common skin condition which can be itchy and painful; between 1.5% and 3% of people in the world have psoriasis.1<\/sup> Day today life, our body have exposed too many external factors, which causes various damages such as irritation or allergies. However, our body produces the defense reaction against the negative effects of these factors is inflammation. In the complex process of inflammation an excess of free radicals was produced. The presents of ROS would trigger the biological responses which activate the transcription factor AP-1 and other nuclear transcription factor Kappa B (TNF-\u03b2).2<\/sup> These factors regulate secretion of few signal molecules known as pro-inflammatory cytokines and interleukins (IL\u2019s). These interleukins cause skin inflammation, which appears redness with surrounded by inflamed and aggregative psoriatic lesions. Another important factor in the pathogenesis of psoarisis, VEGF belongs to the platelet-derived growth represents a family of homodimeric glycoproteins and plays the important role in the angiogenic process3<\/sup><\/p>\n

Around 50% of psoriatic patients in America and Europe use complementary and alternative medicine, including plant-based medicines.4<\/sup> The secondary metabolite in plants plays an important role as antipsoriatic agents. In the present investigation, the antioxidant activity and interaction of the bioactive compound of ethanolic and ethyl acetate of centella asiatica<\/em> and Indigofera aspalathoides <\/em>were analyzed.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Collection of Plant Material<\/strong><\/p>\n

The whole plant of\u00a0Centella asiatica <\/em>Vallarai in Tamil (Eng. Indian Pennywort), and Indigofera aspalathoides<\/em> Sivanar vembu or sivan vembu in Tamil, were provided by collected from chennai and were authenticated by Dr. Jayakumari Prof and Head of the department of Pharmacognosy, Vels college of pharmacy\u00a0 Chennai, India. The voucher specimen is also available in herbarium file of the same Centre.<\/p>\n

Preparation of Crude Extraction<\/strong><\/p>\n

The fresh leaves of\u00a0Centella asiatica\u00a0 <\/em>and Indigofera aspalathoides<\/em> were washed under the running tap water, shade-dried for 5 days and oven-dry at 55 \u00b0C for 24 hrs. The sample was ground to a fine powder using an electrical mixer. The whole part of the plant was taken in 1:3 ratio, suspended ethanol and ethyl acetate respectively. It was kept on a rotatory shaker for continuous agitating for 24 hrs. The extracts were filtered using whatman no.1 filter paper, and the filtrates were dried at ambient temperature in a fume hood in the dark until all solvent evaporated.<\/p>\n

Antioxidant Activity Determination<\/strong><\/p>\n

Super Oxide Anion Radical Scavenging Activity <\/strong><\/p>\n

The Superoxide anion scavenging activity was determined by the method of.5<\/sup> About 1 ml NBT solution containing 156 \u03bcM NBT dissolved in 1.0 ml 100 mM phosphate buffer, pH 7.4, 1 ml NADH solution containing 468 \u03bcM NADH dissolved in 1 ml 100 mM phosphate buffer, pH 7.4, and 0.1 ml various concentration of sample and reference compounds (25, 50, 100, 200, and 500 \u03bcg) were mixed well and the reaction was started by adding 100 \u03bcl phenazinemethosulfate solution containing 60 \u03bcM phenazinemethosulfate in 100 mM phosphate buffer, pH 7.4. the reaction mixture was incubated at 25\u00b0C for 5 min and absorbance at 560 nm was measured against control samples. BHT were used as reference compounds. Percent inhibition was calculated by comparing the results of control and test samples.<\/p>\n

Nitric Oxide Radical Scavenging Activity<\/strong><\/p>\n

The nitric oxide scavenging ability of the plant extracts were measured by the method of Griess reaction.6<\/sup> The reaction mixture (3ml) containing 10 mM sodium nitroprusside in phosphate buffered saline and \u00a0<\/em>the reference compound at different concentrations were incubated at 25\u00b0C for 150 min. A 0.5-ml aliquot of the incubated sample was removed at 30-min intervals and 0.5 ml Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihyrochloride in 2% H3PO4) was added. The absorbance of the chromophore formed was measured at 546nm. All tests were performed in triplicate. Percent inhibition of the nitric oxide generated was measured by comparing the absorbance values of control and test preparations. Curcumin was used as a positive control.<\/p>\n

NO Scavenged (%) = (A cont – A test)\/A cont \u00d7 100<\/p>\n

Hydroxyl Radical Scavenging Activity<\/strong><\/p>\n

The ability of the plant extracts to scavenge the H2<\/sub>O2<\/sub> was determined by.7<\/sup> Various concentrations of the plant extracts were dissolved in One milliliter of iron-EDTA solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 mL of EDTA (0.018%), and 1 mL of DMSO(0.85% v\/v in 0.1 M phosphate buffer, pH 7.4) were added to these tubes, and the reaction was initiated by adding 0.5 mL of 0.22% ascorbic acid., the absorbance of H2<\/sub>O2<\/sub> was measured at 412 nm. BHT was used as the reference compound. Percent inhibition was calculated by comparing the results of control and test samples.<\/p>\n

Percent inhibition = (1-(Control OD\/test OD))*100<\/p>\n

DPPH method<\/strong><\/p>\n

DPPH radical scavenging activity of extract was determined according to the method reported by.8<\/sup> An aliquot of 0.5 ml of sample solution in methanol was mixed with 2.5 ml of 0.5 mM Methanolic solution of DPPH. The mixture was shaken vigorously and incubated for 30 min in the dark at room temperature. The absorbance was measured at 517 nm using UV spectrophotometer. Ascorbic acid was used as a positive control. DPPH free radical scavenging ability (%) was calculated by using the formula. % of inhibition = absorbance of control \u2013 absorbance of sample \/ absorbance of control \u00d7100.<\/p>\n

Cytotoxicity Studies<\/strong><\/p>\n

The fibroblast cells (L929) were plated separately using 96 well plates with the concentration of 1\u00d7105<\/sup>cells\/well in DMEM media with 1X Antibiotic Antimycotic Solution and 10% fetal bovine serum (Himedia, India) in CO2<\/sub> incubator at 37\u02daC with 5% CO2. The cells were washed with 200 \u03bcL of 1X PBS, then the cells were treated with various test concentration (25, 50, 100, 250 and 500 \u03bcg\/ml) of compound in serum free media and incubated for 24 hrs. The medium was aspirated from cells at the end of the treatment period. 0.5mg\/mL MTT prepared in 1X PBS was added and incubated at 37\u02daC for 4 hrs using CO2<\/sub> incubator. After incubation period, the medium containing MTT was discarded from the cells and washed using 200 \u03bcL of PBS. The formed crystals was dissolved with 100 \u03bcL of DMSO and thoroughly mixed. The development of color intensity was evaluated at 570nm. The formazan dye turns to purple blue color. The absorbance was measured at 570 nm using microplate reader.<\/p>\n

GCMS -IIT\u00a0\u00a0 <\/strong><\/p>\n

The JEOL GCMATE II GC-MS with data system is a high resolution, double focusing instrument. Maximum resolution: 6000 Maximum calibrated mass: 1500 Daltons. Source options: Electron impact (EI); Chemical ionization (CI)9<\/sup><\/p>\n

Molecular Docking \u2013Patchdock<\/strong><\/p>\n

Docking was performed for the compounds present in the FSE extract. Both ligand and receptor were converted to the pdb format and processed for the docking\u00a0 protocol using\u00a0 patchdock.10<\/sup><\/p>\n

Statistical Analysis<\/strong><\/p>\n

In the present study, all the experiments were conducted in triplicate, and data analysis was done by mean\u00b1SEM.<\/p>\n

Results and Discussion<\/strong><\/p>\n

Two plants extract namely, Centella asiatica <\/em>(FVEA, FVE) and Indigofera aspalathoides <\/em>(FSEA, FSE) of ethanol and ethyl acetate used respectively. The yield calculated and shown in table 1. These extracted were stored and used for further investigation.<\/p>\n

Table 1: Yield of the plant extracts<\/strong><\/p>\n\n\n\n\n\n\n\n
Extract<\/td>\nYield (g)<\/td>\n<\/tr>\n
Centella asiatica-<\/em>Ethyl acetate (FVEA) <\/em><\/td>\n1.13<\/td>\n<\/tr>\n
Centella asiatica-<\/em>Ethanol(FVE)<\/td>\n5.77<\/td>\n<\/tr>\n
Indigofera aspalathoides- <\/em>Ethyl acetate (FSEA)<\/td>\n0.96<\/td>\n<\/tr>\n
Indigofera aspalathoides-<\/em>Ethanol (FSE)\u00a0<\/em><\/td>\n1.65<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\nFigure 1: Hydroxyl radical scavenging activity of FSEA, FVEA, FVE and FSE, values were reported as mean \u00b1 SD(n=3)<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 2: Superoxide anion radical scavenging activity of FSEA, FVEA, FVE and FSE, values were reported as mean \u00b1 SD(n=3)<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\nFigure 3: Nitric oxide scavenging activity of FSEA, FVEA, FVE and FSE, values were reported as mean \u00b1 SD(n=3)<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 4: DDPH radical scavenging activity of FSEA, FVEA, FVE and FSE, values were reported as mean \u00b1 SD(n=3)<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\nFigure 5: Control assay for antioxidant activity, values were reported as mean \u00b1 SD(n=3)<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Table 2: IC50 <\/sub>values for plant extracts<\/strong><\/p>\n\n\n\n\n\n\n\n
Free Radical Assay<\/strong><\/td>\nExtract<\/strong><\/td>\nIC 50<\/sub> \u00b5g<\/strong><\/td>\n<\/tr>\n
Hydroxyl radical scavenging activity<\/td>\nFSEA<\/p>\n

FVEA<\/p>\n

FVE<\/p>\n

FSE<\/p>\n

BHT<\/td>\n

>500<\/p>\n

264 \u00b114.21<\/p>\n

212 \u00b121.4<\/p>\n

244.4 \u00b135.99<\/p>\n

40.97\u00b13.575<\/td>\n<\/tr>\n

Nitric oxide radical\u00a0 scavenging\u00a0\u00a0 activity<\/td>\nFSEA<\/p>\n

FVEA<\/p>\n

FVE<\/p>\n

FSE<\/p>\n

Curcumin<\/td>\n

><\/strong>500<\/p>\n

485.21\u00b134.2<\/p>\n

185.6 \u00b131.24<\/p>\n

175.9 \u00b129.65<\/p>\n

36.52 \u00b12.36<\/td>\n<\/tr>\n

Superoxide anion radical scavenging activity<\/td>\nFSEA<\/p>\n

FVEA<\/p>\n

FVE<\/p>\n

FSE<\/p>\n

BHT<\/td>\n

> 500<\/p>\n

> 500<\/p>\n

492.21\u00b123.25<\/p>\n

474.32\u00b129.82<\/p>\n

33.79 \u00b14.06<\/td>\n<\/tr>\n

DPPH scavenging assay<\/td>\nFSEA<\/p>\n

FVEA<\/p>\n

FVE<\/p>\n

FSE<\/p>\n

Ascorbic acid<\/td>\n

327.9\u00b142.77<\/p>\n

487.39\u00b132.3<\/p>\n

183.9 \u00b1 43.91<\/p>\n

96.29 \u00b1 12.07<\/p>\n

23.11 \u00b1 2.055<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Among the four extracts, increased percentage of inhibition for free radical scavenging activity was found in FSE extract as compared with other extracts (Figure 1, 2, 3, 4, 5).The decreased IC50<\/sub>value for FSE was found in scavenging the nitric oxide , super oxide and DPPH however, FVE showed decreased IC50<\/sub>value only in scavenging the hydroxyl radical (Table 2).Overall scavenging the free radical assay, FSE was observed to have potent antioxidant activity.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 6: GC-MS of FSE Extract<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

In silico<\/em> analysis performed using patch dock server to support the in vitro<\/em> analysis. Eighteen \u00a0compounds found in the FSE extract, one of the compound Dodecanoic acid,10 methyl-,methyl ester, a fatty acid produced high affinity -26.47 with VEGF \u00a0and Pregnan-18-oic acid,20-hydroxy-,[5alpha] compound produced high affinity -30.56 with IL-17. The hydrogen bond interaction of VEGF and IL-17 was observed with the respective compounds (Table 3).<\/p>\n

Table 3: Ligand \u2013Protein interaction by Patch Dock Server<\/strong><\/p>\n\n\n\n\n\n\n\n
S.No<\/strong><\/td>\nCompounds of FSE extract<\/strong><\/td>\nHydrogen bond interaction <\/strong><\/td>\nDocking score <\/strong><\/p>\n

VEGF <\/strong><\/td>\n

Hydrogen bond interaction <\/strong><\/td>\nDocking score <\/strong><\/p>\n

IL-17<\/strong><\/td>\n<\/tr>\n

1<\/td>\nDodecanoic acid,10 methyl-,methyl ester<\/td>\nArg 159<\/td>\n-26.47<\/td>\n–<\/td>\n-71.24<\/td>\n<\/tr>\n
2<\/td>\nPregnan-18-oic acid,20-hydroxy-,[5alpha]<\/td>\n–<\/td>\n-69.45<\/td>\nGly 583,<\/p>\n

Glu644,<\/p>\n

Arg 614<\/td>\n

-30.56<\/td>\n<\/tr>\n
3<\/td>\nMethotrexate<\/p>\n

(PubchemCID:126941)<\/td>\n

Arg 159, His 182<\/td>\n-91.91<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
4<\/td>\nCrucumin<\/p>\n

(Pubchem CID:969516)<\/td>\n

–<\/td>\n-56.11<\/td>\nGlu 624, Glu 644,Trp 633<\/td>\n-56.11<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\nFigure 7: Interaction between dodecanoic acid,10 methyl-,methyl ester with VEGF(Ligplot) and Pymol<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 8: Interaction between Methotrexate (control)with VEGF(Ligplot)<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\nFigure 9: Interaction between Pregnan-18-oic acid ,20-hydroxy, 5alpha with IL-17<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 10: Interaction between Crucumin (control) with IL-17 (Ligplot)<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 11: Effect of ethanolic extract of FSE against L929 fibroblast cell line \u2013MTT Assay<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 12: Percentage viability of L929 fibroblast cell line treated with ethanolic FSE extract<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Discussion<\/strong><\/p>\n

The folklore of medicinal plants has been widely used in the allergies, skin diseases, inflammations and scavenging free radicals. The known secondary metabolite of the extracts are the response for the antioxidant activity and also essential to reduces the pathogenesis of several skin diseases. In the present study, selected whole plants of Centella asiatica <\/em>(FVEA, FVE) and Indigofera aspalathoides <\/em>(FSEA, and FSE) of ethanol and ethyl acetate extract were used for free radical scavenging activities. FSE showed better activity in scavenging the hydroxyl radical (\u2022OH), nitric oxide (NO\u2022) and super oxide (O2<\/sub>\u2212.<\/sup>), when compare to other three extracts. The presences of hydroxyl ion are known to initiate the lipid peroxidation and cause DNA damage.11<\/sup> Nitric oxide (NO\u2022), plays a major role in autoimmunity and inflammation process. It has been reported, in the case of undue production of nitric oxide causes many inflammatory disorders which includes psoriasis.12<\/sup> The free radical superoxide has been implicated one of the harmful ROS. It affects the cellular components in the biological system by indirectly commence the lipid oxidation. It has been reported the toxicity of nitric oxide (NO\u2022) increases greatly when it reacts with superoxide radical (O2<\/sub>\u2212.<\/sup>), forming the highly reactive peroxynitrite anion, toxic for living cells.13<\/sup> DPPH assay widely used to test the ability of the plant compounds as free radical scavengers or hydrogen donor. Among the extracts, FSE extract showed the better reduction in DPPH and thus proves the presence of antioxidant properties.<\/p>\n

Psoriasis is commonly associated with a prominent permeability barrier abnormality and excess dermal vascularity in VEGF production.14<\/sup> Angiogenesis is initiated by the activation of vascular endothelial cells through several factors. Thus, VEGF protein selected for in silico<\/em> analysis and it has been found that dodecanoic acid, 10 methyl-, methyl ester have higher affinity. The supported report of inhibiting the VEGF -2 with ligand axitinib showed lowest binding free energy -54.68Kcal\/mol.15<\/sup>\u00a0However, there still has no proper inhibitor for controlling the VEGF expression. In general, methotrexate has been prescribed for the psoriatic patients, which is an anticancer drug and causes major dysfunction to various organs. Thus, natural bioactive compounds would be new anti-VEGF agents without side effect to control the angiogenesis. In the pathogenesis of psoriasis, dendritic cells (DC), T- helper (Th)17 and keratinocytes are involved in various stimuli factors produce and allow to secrete TNF-alpha and interleukin IL-23. The induced expression of IL-23 differentiates the native T cells to Th17. Thus the activated Th17 cells produce over expressed IL-17, in-turn it activates keratinocytes and also promotes epidermal hyperplasia.<\/strong>16<\/sup> In the present study, Pregnan-18-oic acid,20-hydroxy, 5alpha has produced high affinity towards IL-17 as compared to other interaction patterns. According to author,17<\/sup> IL-17 expressed 30 times more for the people with psoriatic lesions than the normal people. Thus, IL-17 could unlock psoriatic lesions and clear the skin. Therefore, these potential VEGF and IL-17 inhibitors are required for the future treatment of psoriatic patients to improve their quality of life. Further, ethanolic extract of FSE was assessed for cytotoxicity studies in L929 fibroblasts cell lines. The cell viability showed 85% at the concentration of 500\u00b5g\/ml and found to be non-cytotoxicity.<\/p>\n

From the analysis, ethanolic FSE extract showed better antioxidant scavenging activity. The bioactive compounds in the ethanolic FSE extract showed control in the growth of fibroblast by using cell viability assay and also it proves the inhibitory action against vascular endothelial growth factor (VEGF) and IL-17 in silico <\/em>model. However, the common side effects are known during the treatment. Therefore, the natural character and high effectiveness of compounds are promising features for developing novel topical formulations that might replace hitherto known remedies with limited application.<\/p>\n

References<\/strong><\/p>\n

    \n
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  17. Bagel. https:\/\/www.psoriasis.org\/advance\/features\/interleukin-17-could-unlock-psoriasis-treatments. 2012.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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