STUDY ON THE POTENTIAL EFFECT OF COMBINATION OF AGERATUM CONYZOIDES L. EXTRACT AND EGGSHELL MEMBRANE HYDROLYSATE IN PSORIASIS ANIMAL MODEL

• Main Author: Nar Vikasari, Suci
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/86777
• Institution: Institut Teknologi Bandung

Psoriasis is an autoimmune disease characterized by hyperproliferation and thickening of skin keratinocytes. Clinical manifestations that can arise during psoriasis include skin redness (inflammation), dry and scaly skin, and the risk of anemia and bleeding. The goal of psoriasis therapy is to reduce symptoms and suppress recurrence. Psoriasis treatment through the BPJS scheme is only given to patients with lesions >10%. Therefore, alternative psoriasis therapies are needed. Babadotan (Ageratum conyzoides) is an herb that is beneficial for the skin. The need for chicken eggs in Indonesia reaches 395,187 tons per month, causing eggshells to become abundant waste. Eggshells are traditionally used for skin hygiene. This study aims to develop a combination of babadotan leaf extract and eggshell membrane hydrolysate in reducing clinical manifestations of psoriasis. The study was conducted by examining the quality of the test material, testing the efficacy in vivo, testing the mechanism of action in vitro and in silico, testing the safety of the test material in vitro and in silico, and optimizing the manufacture of topical preparations. The eggshell test material was collected from food industry waste and the collection of babadotan leaf simplicia from Pancoran Village, Kab. Bondowoso, East Java. Determination of the quality of babadotan leaf and eggshell membrane simplicia includes determination of water content, total ash content, water-soluble ash content, acid-insoluble ash content, water-soluble essence content and ethanol- soluble essence content. The preparation of eggshell membrane extract was carried out using 40% ethanol solvent pH 12, then heated at 70oC for 2 hours, then acetic acid was added to neutralize the pH and activated carbon was added for 20 minutes to remove the color. The yield of eggshell membrane extract produced was 7.99%. The preparation of 50% ethanol extract of babadotan was carried out by reflux and the yield obtained was 15.17%. The 50% ethanol extract of babadotan leaves made using the reflux method contains, among others, 2-hydroxycinnamic acid, nobiletin, coumarin, proline, choline, tangeritin, stramamide, pheninalanine, steariolglycerol and isoleucine. Babadotan leaf extract contains total flavonoids of 2.7±0.1% equivalent to quercetin, and total phenols of 9.6±0.5% equivalent to gallic acid. Analysis of eggshell membrane hydrolysate shows that the highest amino acid content is glutamic acid, aspartic acid, leucine, serine and proline. Based on data obtained by Scanning Electron Microscopy and FTIR, eggshell membrane hydrolysate is indicated to contain hyaluronic acid and collagen. Efficacy test of babadotan extract and eggshell membrane hydrolysate was conducted on acute anti-inflammatory effects using carrageenan induction, chronic anti-inflammatory test using cotton pellet induction, and anti-psoriasis test with imiquimod induction. The results of acute anti-inflammatory test of ethanol extract of babadotan leaves showed that extract doses of 22.5, 45 and 90 mg/kg bw significantly inhibits the inflammation and comparable to sodium diclofenac (p>0.05). The results of acute anti-inflammatory test of eggshell membrane hydrolysate showed that doses of 9, 22.5, 45 and 90 mg/kg bw significantly inhibits the inflammation and comparable to sodium diclofenac (p>0.05). Based on these results, a dose of 45 mg/kg bw of babadotan extract and a dose of 22.5 mg/kg bw of eggshell membrane hydrolysate were selected for combination testing. Chronic anti-inflammatory testing was conducted using the cotton pellet induction method. The test animals were divided into control group, comparison of sodium diclofenac, egg shell membrane hydrolysate group with a dose of 22.5 mg/kg bw, babadotan leaf ethanol extract group with a dose of 45 mg/kg bw, combination of egg shell membrane hydrolysate and babadotan leaf ethanol extract: combination 1 (100%:100%), combination 2 (75%:25%), combination 3 (50%:50%) and combination 4 (25%:75%). The main parameter measured was the inhibition of granuloma formation. The results showed that all test groups had chronic anti- inflammatory effects when compared to the control group, with inhibition of granuloma formation in combination group 1 of 61.41%, combination 2 of 26.53%, combination 3 of 34.19% and combination 4 of 56.53%. In addition, hematological profile observations were also carried out. Based on observations of the hematological profile, the use of the combination can increase the number of leukocytes and their derivatives, decrease the number of erythrocytes and their derivatives and decrease the number of platelets when compared to single use. Testing on an animal model of psoriasis was carried out using topical induction of 62.5 mg of imiquimod in male Balb/c mice. The test group in the antipsoriasis test was like the chronic anti-inflammatory test, except for the methotrexate comparison group. The parameters measured were the psoriasis area severity index (PASI), hematological profile, the amount of IL-17A and IL-22 cytokines in the back and ear skin, the spleen organ index, spleen and skin histopathology. The results showed that eggshell membrane hydrolysate, babadotan extract, and the combination were significantl able to prevent crusts, back redness, ear redness and reduce PASI when compared to the control group. Administration of babadotan leaves, eggshell membrane hydrolysate and their combination were able to reduce crusts, back and ear skin redness, PASI scores, the amount of IL-17A and IL-22 cytokines, and improve the hematological profile and spleen histology compared to the control group in imiquimod-induced test animals. The order of the best chronic anti- inflammatory and antipsoriasis effects of the combination of babadotan leaf extract and eggshell membrane hydrolysate was combination 1, combination 4, combination 3, and combination 2. The chronic anti-inflammatory and antipsoriasis effects of the combination of babadotan leaf extract and eggshell membrane hydrolysate were additive. In vitro and in silico mechanism of action tests were conducted through the approach of inflammation inhibition, enzyme activity, signal transduction pathways and cytokines. In vitro anti-inflammatory mechanism of action tests was conducted by protein denaturation inhibition, proteinase activity and membrane stabilization tests. The protein denaturation inhibition activity of babadotan leaf extract and eggshell membrane hydrolysate was moderate, with IC50 values of 76.83 and 93.34 ?g/mL. The proteinase inhibition activity of babdotan leaf extract and eggshell membrane hydrolysate was weak, with IC50 values of 136.14 and 249.45 ?g/mL. The ability of babadotan leaf extract and eggshell membrane hydrolysate to stabilize membranes was categorized as strong, with IC50 values of 36.35 and 39.10 ?g/mL in heat induction, and IC50 values of 16.64 and 27.42 ?g/mL in hypotonic induction. In vitro enzyme inhibition test was conducted on the inhibition of collagenase and neutrophil elastase enzyme activity. Collagenase enzyme inhibition activity is indicated by the ability of the test material to inhibit collagenase enzyme to decompose FALGPA substrate. Babadotan leaf extract actively inhibits collagenase enzyme activity (IC50 60.30±3.02 ?g/mL), while the activity of eggshell membrane hydrolysate as a collagenase inhibitor is low (IC50 106.08±6.02 ?g/mL). Neutrophil elastase enzyme inhibition activity is indicated by the ability to inhibit neutrophil elastase enzyme to decompose SucAla3-pNA substrate. The potential of babadotan leaf extract and eggshell membrane hydrolysate in inhibiting neutrophil elastase is relatively weak with IC50 114.56±1.07 and 215.23±13.96 ?g/mL. Prediction using computation shows the ability of compounds contained in babadotan leaf extract and eggshell membrane hydrolysate to inhibit chymase enzyme activity in the skin, affect signal transduction pathways in protein kinase C and p38-MAPK, and are able to bind to the IL-17A binding site. Antioxidant activity is carried out through scavenging the 2,2-Diphenyl-1-1-picryl hydral (DPPH) radical. Babadotan leaf extract and eggshell membrane hydrolysate have moderate antioxidant activity with IC50 values of 153.63 and 148.38 ?g/mL. It is suspected that the antioxidant activity of eggshell membrane hydrolysate is a synergistic effect of hyaluronic acid and collagen which have weak antioxidant activity with IC50 values of more than 200 ?g/mL. Acute toxicity test of babadotan leaf extract and eggshell membrane hydrolysate in vivo showed LD50 value more than 5000 mg/kg bb. This shows that babdotan leaf extract and eggshell membrane hydrolysate are safe for consumption. Prediction of drug properties and pharmacokinetic profiles of all amino acids in eggshell membrane hydrolysate and several compounds in babadotan leaf extract (monohydroxy-penta-methoxy-flavone, nobiletin, eupalestin, luteolin, kaempferol- 7-O-rhamnopyranoside, and apigenin) have properties similar to drugs. This study also carried out optimization of the manufacture of cream preparations containing 1% combination of babadotan leaf extract and eggshell membrane hydrolysate. Additional ingredients for the cream formula are decyl glucoside, propylene glycol and cetyl alcohol, and optimization of the formulation using the Response Surface Methodology (RSM) Box Behnken method. The RSM Box- Behnken Design (BBD) model was successfully used to evaluate and optimize the viscosity response variables consisting of decyl glucoside (15, 20, 25%), propylene glycol (10, 15, 20%) and cetyl alcohol (7, 8.5, 10%). The results obtained from the analysis of variance (ANOVA) showed that propylene glycol (p = 0.016) and cetyl alcohol (p = 0.004) were the variables that had the most influence on the viscosity response. Optimization of the viscosity response of 5000 cP obtained a formulation of decyl glucoside 15.82%, propylene glycol 11.21% and cetyl alcohol 8.84%. At optimum conditions, a cream with a viscosity of 4675 cP was obtained, where the viscosity was close to the predicted value (5007 cP), with an error value of 4.83%. The information that can be provided from the results of this study is that the oral use of a combination of babadotan leaf extract and eggshell membrane hydrolysate has the potential as an antipsoriasis with various working mechanisms including inhibiting inflammation, enzyme activity (collagenase, neutrophil elastase, chymase), influencing the signal transduction pathway of protein kinase C and p38- MAPK, and being an antioxidant. The combination of babadotan leaf extract and eggshell membrane hydrolysate can be developed into a preparation to reduce psoriasis symptoms during a relapse and has been proven safe to use. In addition, this study has tried to make a cream preparation that can later be used for topical use in psoriasis patients.