Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Chenopodium murale effect on atopic dermatitis on mice DOI: https://doi.org/10.31351/vol32iss1pp84-91 84 Evaluation the Effect of Phytosterol Fraction of Chenopodium Murale in Comparison with Tacrolimus on Mice Induced Atopic Dermatitis Zahraa Y. Hassan1*.1, Tuka Y. Hassan** and Ahmed R. Abu Raghif *** * Ministry of Health and Environment, Al-Imamain Al-Kadhimain Medical City, Baghdad, Iraq. ** Ministry of Health and Environment, Public Health Directorate, Baghdad,Iraq. *** Department of Pharmacology and Therapeutics, College of Medicine, AL-Nahrain University, Baghdad, Iraq. Abstract Atopic dermatitis (atopic eczema), is a common familial chronic inflammatory skin disease, determined by xerosis, itching, scaly and erythematous skin lesions, and high serum levels of IgE. Between 10 to 20% of children and 1 to 3% of adults worldwide affected by it and has negative medical and social effect on patients and their families. To evaluate the effectiveness of Phytosterol Fraction of Chenopodium murale on DNCB-induced atopic dermatitis (AD) of mice; Forty mice were included in the study, divided in to four groups (10 mice/group): apparently healthy, induced AD without treatment, induced AD treated with Tacrolimus 0.1% ointment, and induced AD treated with Phytosterol Fraction of Chenopodium murale cream 3% topically. Examination of histopathology was done and skin homogenates levels also measured using Mann Whitney U test to determine mean±SD. Levels of WBC, Eosinophil, skin tissue homogenate of IL-13 and IL-4, serum IgE, and histopathological scores were significantly increased among induced non treated AD group in comparison with control group. Comparisons of non-treated induced AD group with Chenopodium murale or Tacrolimus treated groups; shows a significant reduction in the levels of all studied parameters’ (WBC, Eosinophil, skin tissue homogenate of IL4- and IL-13, serum IgE, observational severity score, and histopathological scores) after the application of Tacrolimus 0.1% ointment or Chenopodium murale cream 3% topically. The comparison between the effect of topical application of tacrolimus and Phytosterol Fraction on the studied variables shows that the level of WBC and thickness of epidermis and inflammatory cells were significantly lower after tacrolimus treatment, while high significant reduction was founded in parakeratosis and score of observational severity among Chenopodium murale treated group in comparison with Tacrolimus treated group. In conclusion, topical application of phytosterol fraction of Chenopodium murale seems to be effective in treatment of atopic dermatitis through their abilities to decrease WBC, eosinophil, s. IgE, skin tissue homogenate of IL4, and IL13; as well as improving histopathology picture and reducing observational severity score. The use of phytosterol fraction of Chenopodium murale that target IgE, IL4, and IL13 could be promising in the treatment of atopic dermatitis. Key words: Phytosterol Fraction, Chenopodium murale, Atopic dermatitis, Tacrolimus, Interleukin-4, Interleukin-13 تقييم فعالية جزيئات الفايتوستيرول لكينوبوديوم ميوريل بالمقارنة مع تاكروليموس على التهاب الجلد التحسسي المستحث في الفئران المختبرية ** رغيف أبو و أحمد **، تقى يونس حسن 1*،زهراء يونس حسن العراق. بغداد، ، االمامين الكاظمين الطبيةوزارة الصحة والبيئة ، مدينة * .الصحة العامة ، بغداد ، العراق وزارة الصحة والبيئة ،دائرة** .كلية الطب ، جامعة النهرين ، بغداد ، العراق*** الخالصة ارتفاع ، و احمرار الجلد وتقشرهبالجفاف ، والحكة ، سمالتهاب الجلد التأتبي )األكزيما التأتبية( ، هو مرض جلدي التهابي مزمن عائلي شائع ، يت واجتماعي صحي به وله تأثير يصابون ٪ من البالغين في جميع أنحاء العالم 3إلى 1٪ من األطفال و 20إلى 10االجسام المضادة أي في الدم. ما بين ى مستو . تم تضمين أربعين المختبرية على التهاب الجلد التأتبي في الفئران ميوريل وبوديوملكين الفايتوستيرول جزيئاتلتقييم فعالية سلبي على المرضى وعائالتهم. فئران / مجموعة(: صحية ، ُمحفَّزة بالتهاب الجلد التأتبي دون عالج ، ُمحفَّزة بالتهاب الجلد التأتبي ُمعالج 10فأًرا في الدراسة ، مقسمة إلى أربع مجموعات ) ٪ . تم إجراء فحص التشريح 3 ميوريل كينوبوديوممن كريم الفايتوستيرول جزيئاتُمحفَّزة بالتهاب الجلد التأتبي معالج ب ٪ مرهم ، و0.1بتاكروليموس واالجسام 4و االنترلوكين 13و في االنترلوكين الخاليا الحمضية و كريات الدم البيضاء المرضي وقياس مستويات تجانس الجلد. وجد زيادة في مستويات في الدم ونتائج األنسجة المرضية بشكل ملحوظ بين المجموعة المستحثة غير المعالجة بالمقارنة مع المجموعة الضابطة. دة أيالمضا ب المعالجة المجموعات مع المعالجة غير التأتبي الجلد بالتهاب الُمحفَّزة المجموعة بين يُظهر ميوريل كينوبوديوممقارنات ؛ تاكروليموس أو أظهرت المقارنة بين تأثير التطبيق الموضعي س.وتاكروليم مرهم كريم الفايتوستيرول او ًرا في مستويات جميع المعلمات المدروسة بعد وضعانخفاًضا كبي كانت و الخاليا المضادة لاللتهاب سماكة البشرة كريات الدم البيضاء و على المتغيرات المدروسة أن مستويات الفايتوستيرول جزيئاتو لتاكروليموس عقار ال أقل بشكل و درجة شدة المالحظة نظير التقرن ىبين المجموعات المدروسة ، بينما كانت مستو للتاكروليموس الموضعي التطبيقأقل بشكل ملحوظ بعد الفايتوستيرول لجزيئات الموضعي التطبيق أنك نستتج من ذلبين المجموعات المدروسة. ميوريل لكينوبوديوم الفايتوستيرول جزيئاتملحوظ بعد تطبيق 4االنترلوكين و 13 االنترلوكين و الحمضية الخاليا و كريات الدم البيضاء تقليل على قدرته خالل من التأتبي الجلد التهاب عالج في فعال ميوريل لكينوبوديوم ميوريل لكينوبوديوم الفايتوستيرول جزيئات استخدام يكون لهذا, قد. المالحظة شدة درجة وتقليل سماكة البشرة تحسين وكذلك ؛ الدم في أي المضادة واالجسام . التأتبي الجلد التهاب عالج في واعدًا 13، إنترلوكين 4كينوبوديوم ميوريل ، التهاب الجلد التأتبي ، تاكروليموس ، إنترلوكين ، : جزيئات الفايتوستيرول المفتاحيةالكلمات 1Corresponding author E-mail: zahraahassan793@gmail.com Received: 14/1 /2022 Accepted: 6/4 /2022 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol32iss1pp84-91 Iraqi J Pharm Sci, Vol.32(1) 2023 Chenopodium murale effect on atopic dermatitis on mice 85 Introduction Atopic dermatitis (AD) is an inflammatory skin condition with pruritus; erythema; dryness; and scaly skin. Asthma presented in 30% of patients with AD. It is tending to be chronic with relapsing and remission periods. Patients can be cured during puberty; or persist for long life. (1, 2) Bacterial and viral infections of skin may be a consequencies to AD. Herpetic eczema due to herpes simplex virus is frequently observed among AD patients. Most of patients with AD have Staphylococcus aureus infection. (3) Restoring skin barrier is the gold standard in the management of AD, often through skin moisturizing, decrease itching and inflammation. (4) Tacrolimus, a macrolide lactone of fungal origin (Streptomyces tsukubaensis), is an immunosuppressive drug commonly used in humans. Tacrolimus has been shown to inhibit the granules release of preformed mediators from skin mast cells and basophils and to down regulate the expression of IgE by mast cells, basophils, and Langerhan cells.(5) Its small size make its ability to penetrate skin more powerful; therefore, it can be used in severe cases of AD and improve control of acute attacks and prevention of new ones due its mechanism of action as immune regulation.(6, 7) Tacrolimus has side effects, such as skin burning and itching. (8) Accordingly, effective therapy with fewer side effects is required for treatment of AD. The World Health Organization encourages, promotes and facilitates effective herbal health programs. (9) Extracts from the leaves of various plants have various pharmacological activities, among these antioxidant activities due to their redox properties, which permit them to act as reducing agents, hydrogen donors, and single oxygen quenchers. The crude extract of various plants and its fractions were examined previously against different human pathogens including, Escherichia coli, Klebsiella pneumonia, Bacillus subtilis, and Salmonella typhus, by agar well diffusion method (anti-bacterial activity). Phytosterol fraction seemed to be a potential nutraceutical tool for some diseases such as gastrointestinal inflammatory disease. In addition, combining metabolic systematic and local anti-inflammatory effects (anti-inflammatory activity). Previous research showed that plant also useful as an anthelmintic, stomachic, antispasmodic, diaphoretic, sweaty, for amenorrhea pain, for abortion and for the relief of asthma, cold, and migraine. (10-12) Although the currently used medications in the treatment of AD are effective in managing the disease; adverse reactions may decrease their usefulness (8). Pharmacological activities of Chenopodium murale: antioxidant activity, anti- bacterial activity, anti-inflammatory and skin disease, Previous research showed that plant also useful as an anthelmintic, stomachic, antispasmodic, diaphoretic, for amenorrhea pain, for abortion and for the relief of asthma, cold, and migraine. (11, 13) This study was carried out to evaluate the effectiveness of Phytosterol Fraction of leafs of Chenopodium murale on treatment of induced atopic dermatitis mice model through their effect on WBC, Eosinophil, serum IgE, tissue homogenate of IL4 and IL13, observational severity score, and histopathological score. The study also aimed to compare the anti-inflammatory effect of Phytosterol Fraction of Chenopodium murale with Tacrolimus on induced atopic dermatitis mice model. Materials and Methods A randomized prospective, controlled animal study was carried out. This study was conducted from December 2020 - June 2021, in the Department of pharmacology-College of Medicine- Al Nahrain University. The protocols for the animal experiment used were carefully reviewed for ethical and scientific care procedures and approved by Al- Nahrain University – College of Medicine review Council (Approval Number 857 in 28/9/2020). Experimental animals and design of study This study included 40 healthy adult male Albino mice weighted 25-30g. They were housed in animal house in a good ventilated isolated place; with a room temperature of 20-24°C. The animals were left for seven days to acclimatize to the animal room conditions and allowed free access to water and Ad libitum feeding. The animals were housed in animal house, at College of Veterinary, and kept light for 12 hours. The practical part of the study was directed at College of Veterinary Medicine, University of Baghdad, Baghdad- Iraq. Ten mice were chosen randomly and considered as a (healthy control) group and compared with other induced groups. Thirty mice treated with 1-Chloro-2, 4- dinitrobenzene (DNCB) induced AD (14) and randomly divided into three groups 10 mice/group (without using anesthetic medication in the AD induction period) (15-17).Induced AD mice non treated (negative control), induced AD mice treated with Tacrolimus 0.1% ointment (positive control), and induced AD mice treated with Phytosterol Friction of Chenopodium Murale cream 3% topically (test compound).(18) Topical treatment was applied once daily at 9:00 AM for 21 days Induction Mouse model of 1-Chloro-2, 4-dinitrobenzene - induced atopic dermatitis Mice described AD skin through shaving hair from dorsal of skin then 150 μL of 1% DNCB in 3:1 (v/v) acetone/olive oil solution was topically applied once to the exposed skin, then after four days, 0.2% DNCB dissolved in an acetone: olive oil mixture (3:1 vol/vol) was applied to the same dorsal skin (150 μL) three times a week for 3 weeks. After Iraqi J Pharm Sci, Vol.32(1) 2023 Chenopodium murale effect on atopic dermatitis on mice 86 the visual confirmation of skin sensitization, mice were treated with test samples. (14) Figure 1. Figure 1. Normal skin lesion without induction (A), Induced atopic dermatitis skin lesion (B) and (C). Plant material Chenopodium murale plant was identified and authenticated by Prof. Dr. Ibrahim S. AlJubori /Department of Pharmacognocy/ College of Pharmacy/ Al-Mustansryiah University .The extraction of herb was executed in Pharmacognocy department, collage of Pharmacy, Al-Mustansryiah University (Iraq). Leaves were washed thoroughly, dried under shade, and ground in a mechanical grinder to coarsely powder. The aerial parts of Chenopodium murale were extracted and authenticated in November, 2020 by Department of Pharmacognocy and medicinal plants / College of Pharmacy/ Al-Mustansiriya University (Iraq). Extraction and fractionation of phytosterol fraction of Chenopodium murale 1. Shade-dried coarsely powdered leaves (250g) plant will extracted with 90% ethanol (500ml) in soxhlet apparatus until complete exhaustion. The alcoholic extract was evaporated under reduced pressure at a temperature not exceeding 40 °C to give a dark green color designated as crude Fraction. 2. Crude fraction was acidified with hydrochloric acid (5%) to pH2 and partitioned (three times) with equal volume of ethyl acetate to get two layers. Aqueous acidic (F1, F2) was left and get ethyl acetate layer. 3. The ethyl acetate layer of the original alcoholic extract (crude fraction) was evaporated to dryness under reduced pressure and basified with 300ml of sodium hydroxide 5% to pH 10 and extracted with chloroform in the separator funnel to get two layers, the aqueous basic layer (F3) was left and chloroform layer was collected. 4. Chloroform layer was also separated and evaporated to dryness under reduced pressure then two types of solvents: methanol 80% and petroleum ether were added to chloroform layer to obtain phytosterol in petroleum ether layer (fraction F4, used in the study) (15) . Preparation of phytosterol fraction 3% cream Three gm of phytosterol fraction extracted from Chenopodium murale was weighted and dissolved in 3 ml of alcohol and shaking it for 4 minutes until it dissolved completely and became clear, after that we complete the weight to 100 gram with aquasoft cream (Ajanta Company) and shake the combination for 5 minutes by spatula (16) . Preliminary qualitative phytochemical analysis Chemical tests were carried out using ethanol extracts using standard procedures to identify the phytosterol fraction of Chenopodium murale (15) (I) Liebermann-Burchard test: extract (3ml) was treated with chloroform, acetic anhydride and drops of sulphuric acid were added. The formation of dark pink or red color indicates the presence of steroids. (II) H2SO4 test: The development of a greenish color was considered as indication for the presence of steroids, when 2 ml of the organic extract was treated with sulphuric and acetic acids). Qualitative and quantitative estimation of phytosterol fraction of Chenopodium murale using High performance liquid chromatography (HPLC) (19) High performance liquid chromatography was used for identification of quantitative and qualitative estimation of phytosterol fraction in the plant. The identifications will made by detection of retention time obtained at identical chromatographic conditions of steroid fraction and the standards. Experimental condition of HPLC • Mobile phase: ethyl acetate: water (7:30 ratio) • Column: hyper clone ODCC C18 V-25cm ODS C18 • Column temperature: 25ºC • Flow rate: 0.5ml/min • Injection concentration 0.5mg/1ml. • Injection volume: 20μl • Detection wavelength: 280 nm Treatment protocols, parameters, and animal sacrificing The topical applications of Tacrolimus 0.1% ointment(17) and Phytosterol Fraction of Chenopodium murale cream 3% (18) were applied on atopic dermatitis area of animal for 21 days once daily at 9 AM starting from the fifth day of induction. Parameters are used to compare the results were WBC, eosinophil, serum IgE, IL4 IL13, and histopathology of AD skin lesion and compared with those of controls, and then we determine the observational severity score. After 21 days of treatment, we took whole number of mice from each study groups and anesthetized through a piece of cotton socked with ether put with the mouse inside a closed jar for few minutes to ensure be anesthetized by inhalation. Before sacrificed; blood sample collected (1ml) in EDTA tube for CBC and serum IgE, then sacrificed Iraqi J Pharm Sci, Vol.32(1) 2023 Chenopodium murale effect on atopic dermatitis on mice 87 by cervical dislocation; (blood sample collected by Cardiac puncture (one ml) by using (three ml syringe) in EDTA tube for CBC and serum IgE. After that Cervical dislocation for the mice was done, then atopic dermatitis skin area was cut by sharp blade (no.15). This skin wound was dissection into two equal pieces one for the histological analysis and the second for the preparation of skin homogenate. The remaining mice from each group were subjected to the same procedure at the 21th day of the treatment. Dorsal skin samples were collected from each animal in study groups and fixed in 10% formaldehyde paraffin embedded and cut into 6 μm sections. Deparaffinized sections were stained with ordinary hematoxylin and eosin (H&E) to determine inflammatory degree and histological changes associated with atopic dermatitis (20). Histopathological follow-up procedures were used for the skin samples taken from each group on the 21 days of treatment. Histopathology of skin of each specimen were evaluated and scored by semi quantitative scoring systems. Histopathology included epidermal thickness, hyperkeratosis, parakeratosis, erosion, inflammatory cell infiltration, and extracellular edema, each scored from 0 to 3 (0 no abnormality, 1+ slight, 2+ mild, and 3+ moderate)(14), the sections examined by pathologist and carried out in histopathology department /Ibn Sina University of Medical and Pharmaceutical Sciences to observe the changes in tissues. Skin tissue homogenate preparation The second piece of skin obtained were washed with normal saline, and rinsed with chilled phosphate buffer saline (1X PBS), with filter paper and weighed. Each 100 mg of skin wound tissue was homogenized with 1 ml of (1X PBS) with the aid of tissue homogenizer (21) for 1 minute at 4 °C, and must be stored overnight at 20°C. Two freeze-thaw cycles must be performed to break the cell membrane; the homogenate were centrifuged for 10 minute at 2000 RPM at 2-8 °C. The supernatant was obtained and stored at –20°C to the assay of IL-4 and IL-13 levels in the tissue. Serum IgE: The enzyme-linked immunosorbent assay) (ELISA) Kit for the estimation of IgE was obtained from CUSABIO\China Kit. Specific different antibodies can be measured quantitatively by the enzyme- linked immunosorbent assay (ELISA). After incubating the tested serum in an antigen-coated polystyrene plat or tube, enzyme specifically labeled anti-immunoglobulin is then added and the remaining in the plate after washing will gives a measure to the quantity of specifically related antibody in the serum. The procedure depends on the insolubilization of specific antigens by passive adsorption to a solid phase (plate), example polystyrene phase (22). Skin tissue homogenate of IL4 and IL13: ELISA Kit for the estimation of IL4 and IL13 was obtained from CUSABIO\China Kit was established on the base of sandwich enzyme-linked immunosorbent assay technology. Anti- IL4 and Anti- IL13 antibodies were pre-coated onto 48-well plates. And as detection antibodies, the biotin conjugated Anti- IL4 and anti- IL13 antibodies were used. We added; the standards, test samples and biotin conjugated detection antibodies to the wells subsequently, and washed with wash buffer. HRP- Streptavidin was added and unbound conjugates were washed away with wash buffer. The concentration of IL4 and IL13 in each sample was expressed in pg/ml for comparison of results with those of controls concentration (23). Assessment of observational severity score The severity of AD on the dorsal area was evaluated for each group on the 21th days of treatment. The evaluation of erythema, dryness, erosion and edema scored as 0 (none), 1 (mild), 2 (moderate), and 3 (severe). Clinical skin score was defined as the summation of each individual scores, range from 0 to 12 (24) . Statistical analysis Microsoft Excel 2016 and SPSS 24 were used for data entry and analysis. Numerical variables were expressed as mean ± SD while categorical variables were expressed by frequencies and percentages, then represented by Figures and tables. All statistical comparisons were made using one -way ANOVA test. P <0.05 was considered statistically significant. Results High performance liquid chromatography (HPLC) for examination of phytosterol fraction of Chenopodium murale Qualitative and quantitative estimations of active constituents of Chenopodium murale fraction was done, in which identifications was made by comprising the retention times obtained at identical chromatographic conditions of analyzed samples; the results show the presence of Beta sitosterol as a major constituent. Table 1. Table 1. Retention time of standard and sample of Beta sitosterol of Chenopodium murale subject Retention Time of Stander/min Retention Time of Sample/min Area of Sample Beta sitosterol 2.547 2.517 822409 Iraqi J Pharm Sci, Vol.32(1) 2023 Chenopodium murale effect on atopic dermatitis on mice 88 The Comparison between ALL study groups (healthy control, negative control, test compound and Positive control) regarding WBC, Eosinophil, serum IgE, and skin tissue homogenate of IL-4 and IL-13, histopathology changes and scores In comparison between the effect of topical Tacrolimus and phytosterol fruction among all study groups, the level of WBC, eosinophil. IgE, IL4 were significantly lower after tacrolimus treatment among studied groups, (P<0.05) Table 5. The effect of positive control (topical Tacrolimus) on the epidermal thickness, Extracellular Edema, and inflammatory cells were significantly reduced, p<0.05. While high significant reduction in IL3, parakeratosis, erosion and score of observational severity was observed among test compound (Chenopodium murale treated group). P<0.001 and p=0.028 respectively. Hyperkeratosis shows high significant reduction among both positive control and test compound (Tacrolimus and Chenopodium murale treated groups), P<0.001. Table 2, Figure 2, 3 and 4. Table 2 . Comparison between ALL study groups (Healthy mice, Negative control, test compound, and positive control treated groups) regarding WBC, Eosinophil, serum IgE, and skin tissue homogenate of IL-4 and IL-13, histopathological changes and score Variables Groups (Mean±SD) P* Healthy control Negative control Test compound Positive control WBC (x109 /L) 3.3 ± 2 10±2.1 7.06± 2.01 6.03± 2.02 0.01* Eosinophil (x109 /L) 0.0 2.5±0.02 0.025±0.09 0.020± 2.02 0.001* IgE(ng/ml) 15.59±8.65 26.62±5.15 16.50±6.61 16.0±6.08 0.003* IL13 (pg/ml) 22.3±68.76 57.8±10.53 31.63±12.31 31.82±21.3 <0.001* IL4 (pg/ml) 6.68±3.01 22.11±6.21 9.68±2.88 9.05±4.03 <0.001* Epidermal Thickness 0.0 3.50±0.52 2.20±0.78 1.20±1.22 <0.001* Hyperkeratosis 0.0 3.00±0.81 1.60±0.51 1.60±0.51 <0.001* Parakeratosis 0.0 3.40±0.69 1.00±0.003 1.20±0.78 <0.001* Erosion 0.0 1.50±0.52 0.20±0.35 0.20±0.42 <0.001* Inflammatory Cell 0.0 2.60±0.51 1.80±0.42 1.70±0.42 <0.001* Extracellular Edema 0.0 2.50±0.52 1.23±0.42 1.20±0.51 <0.001* Observational Severity Score 0.0 10.00±0.81 3.50±0.97 4.50±1.08 <0.001* *One way ANOVA test where p significant at < 0.05 and high significant at <0.001 Iraqi J Pharm Sci, Vol.32(1) 2023 Chenopodium murale effect on atopic dermatitis on mice 89 Figure 2. Histopathology changes in negative control group (B) in comparison with healthy controls (A), test compound group (C), and positive control group (D) (10x): ordinary Hematoxylin and eosin stain. Figure 3 Comparison between negative control, healthy controls, test compound, and positive control groups regarding WBC, Eosinophil, serum IgE, and skin tissue homogenate of IL-4 and IL-13 in mice; Results are expressed as mean ± SD by one way ANOVA test, P is significant at < 0.05. Figure 4. Comparison between negative control, healthy controls, test compound, and positive control groups regarding histological changes and observational score in mice; Results are expressed as mean ± SD by one way ANOVA test, P is significant at < 0.05 Discussion Atopic dermatitis (AD) is the most common chronic inflammatory and chronically relapsing skin disease. The disease leads to a significantly reduced quality of life. The pathogenesis of AD is not well understood but appears to be associated with the activation of innate immune responses, including inflammation (25, 26). Apparently, AD induced untreated group shows significant inflammation signs and significant increase in thickness and in the level of observational severity score among AD induced untreated group. Similarly; a study reported that significant increase in all types of WBC was found among AD induced untreated group (27). Histopathology changes and observational scores after application of 3% cream topically of phytosterol fraction show a significant decrease when compared with untreated induced AD group. This indicates that the anti-atopic effect of β- Sitosterol (HPLC for examination of phytosterol fraction of Chenopodium murale was done, and the results show the presence of Beta sitosterol as a major constituent) is similar to the effect of tacrolimus. β-Sitosterol is attributable to the regulation of inflammatory mediator as in study concluded that the anti-inflammatory activities of β- Sitosterol could be attributed to the inhibition of inflammatory cytokine in AD-like skin lesion and reported the effect of β-Sitosterol as a therapeutic use in inflammatory skin diseases such as AD. (28) A study on animals have indicated that β-Sitosterol reduces the secretion of pro-inflammatory cytokines, as well as edema and increases anti- inflammatory cytokines (29). Similar to the effect of phytosterol fraction of Chenopodium murale topical treatment, application of Tacrolimus 0.1% topically was significantly associated with reduction; as compared with non-treated induced AD group, in the levels of WBC, eosinophil, skin tissue homogenate of IL4- A B C D Iraqi J Pharm Sci, Vol.32(1) 2023 Chenopodium murale effect on atopic dermatitis on mice 90 and IL-13, serum IgE, observational severity score, and histopathological changes. A significant improvement in erythema, pruritus, sleep pattern, and quality of life was reported by 4 week tacrolimus treatment study (30). In addition to its ability in to preventing, delay, and reduce the occurrence of disease exacerbation in adult and children. (31, 32) The levels of parakeratosis and observational Severity Score after phytosterol fraction of Chenopodium murale treatment were lower than after Tacrolimus treatment. While WBC, epidermal thickness and inflammatory cells shows more significant decrease among Tacrolimus treated group. (18, 28) These results indicate that phytosterol fraction of Chenopodium murale that targeting IgE, IL4, and IL13 resemble the effectiveness of previously approved drug Tacrolimus ointment 1%; therefore it may be promising a good treatment for atopic dermatitis. Conclusion Topical application of phytosterol fraction of leafs of Chenopodium murale seems to be effective in treatment of atopic dermatitis through their abilities to decrease WBC, eosinophil, s. IgE, skin tissue homogenate of IL4, and IL13; as well as improving histopathology picture and reducing observational severity score. The use of phytosterol fraction of Chenopodium murale that target IgE, IL4, and IL13 could be promising in the treatment of atopic dermatitis. References 1. Fuxench ZCC. Atopic dermatitis: disease background and risk factors. In Management of Atopic Dermatitis. Springer, Cham, 2017;1027:11-19. 2. Spergel JM. Epidemiology of atopic dermatitis and atopic march in children. Immunol Allergy Clin North Am. 2010;30(3):269-280. 3. Nutten S. Atopic Dermatitis: Global Epidemiology and Risk Factors. Ann Nutr Meta, 2015; 66(1):8-16. 4. Tanei, R. Atopic Dermatitis in Older Adults: A Review of Treatment Options. Drugs Aging 2020:37(3):149-160. 5. Astellas Pharma US. Patient information sheet: Protopic 0.1% (tacrolimus), NDC 2006; 0469- 5202-30. 6. Lee JH, Son SW, Cho SH. A comprehensive review of the treatment of atopic eczema. Allergy Asthma Immunol Res, 2016; 8(3):181– 190. 7. Nghiem P, Pearson G, Langley RG: Tacrolimus and pimecrolimus: from clever prokaryotes to inhibiting calcineurin and treating atopic dermatitis. J Am Acad Dermatol, 2002; 46 (2):228-241. 8. Kang S, Lucky AW, Pariser D, Lawrence I, Hanifin JM. Long-term safety and efficacy of tacrolimus ointment for the treatment of atopic dermatitis in children. J Am Acad Dermatol , 2001;44(1):58–64 9. Singh KP, Dwevedi AK, Dhakre G, Evaluation of antibacterial activities of Chenopodium album. IJABPT, 2011;2(3):398-401. 10. Ahmed A A, Abu-Raghif A R. Effect of topical phytosterol fraction of chenopodium murale on induced hypertrophic scar in rabbits. Journal of Global Pharma Technology, 2020;12(02):115- 124 11. Aldini R, Micucci M, Cevenini M, Fato R, Bergamini C, Nanni C, et al. Anti - inflammatory effect of phytosterols in experimental murine colitis model: prevention, induction, remission study. PloS one, 2014;9(9), e108112. 12. Ahmad B, Jan Q, Bashir S, Nisar M, Shaheen F, Ahmad M. Pharmacological and biological investigations of Chenopodium murale Linn. Asian Journal of Plant Sciences, 2003;2(15-16), pp.1107-1111. 13. Batcha O, Gnatoulma K, Gérard T, Laura L, Efui G, Manuel R, et al. Anti-inflammatory, antibacterial and antioxidant activities of Chenopodium ambrosioides L. (Chenopodiaceae) extracts 2021;162: 16764 - 16794 . 14. Kim H, Kim JR, Kang H, Choi J, Yang H, Lee P, et al. 7,8,49-Trihydroxyisoflavone attenuates dncb-induced atopic dermatitis-like symptoms in NC/Nga mice. PLoS ONE 2014;9(8):e104938. 15. Harborne J.B. Phytochemical Methods. A guide to modern techniques of plant analysis.1st ed. London: Chapman and Hall, New York, 1979;278. 16. Mohammed NJ, Wisam A. Ameen W A. 2015. The effect of topical finasteride in treatment of idiopathic hirsutism. AJBM 2015; 3(9):552 – 566 17. Han JS, Won KH, Chang SE, Kim JE. Tacrolimus 0.1% ointment in the treatment of allergic contact dermatitis: a new approach. Int J Dermatol, 2014;53: e470-e471. 18. TrivellatoGrassi L, Malheiros A, Meyre-Silva C, Buss Z, Monguilhott E D, Fröde T S, et al. From popular use to pharmacological validation: A study of the anti-inflammatory, anti-nociceptive and healing effects of Chenopodium ambrosioides extract, Journal of Ethnopharmacology 2013;145(1):127-138. 19. Sheng, Y., & Chen, X. B. (2009). Isolation and identification of an isomer of β-sitosterol by HPLC and GC-MS. Health, 1(03), 203. 20. Ghasemzadeh A, Ghasemzadeh N. Flavonoids and phenolic acids: role and biochemical activity in plants and human. J. Med. Plants Res., 2011; 5 (31P): 6697-6703. 21. 21.Fernandez F, Shridas P, Jiang S, Aebi M, Waechter C. Expression and characterization of a human cDNA that complements the Iraqi J Pharm Sci, Vol.32(1) 2023 Chenopodium murale effect on atopic dermatitis on mice 91 temperature-sensitive defect in dolichol kinase activity in the yeast sec59-1 mutant: the enzymatic phosphorylation of dolichol and diacylglycerol are catalyzed by separate CTP- mediated kinase activitiesin Saccharomycescerevisiae, Glycobiology.2002 ;12(9):555562. 22. Singh MP, Nagori BP, Shaw NR, Tiwari M, Jhanwar B. Formulation development & evaluation of topical gel formulations using different gelling agents and its comparison with marketed gel formulation. International Journal of Pharmaceutical Erudition, 2013; 3(3),110 23. Attia M.A, El-Gibaly I, Shaltout SE, Fetih GN. Transbuccal permeation, antiinflammatory activity and clinical efficacy of piroxicam formulated in different gels. Int. J. Pharm. 2004;276(1-2):11-28. 24. Khadim EJ, Abdulrasool AA, Awad ZJ. Phytochemical investigation of alkaloids in the Iraqi Echinops heterophyllus (Compositae). Iraqi J Pharm Sci 2014; 23 (1): 26-34. 25. Kapur S, Watson W, Carr S. Atopic dermatitis. Allergy Asthma Clin Immunol 2018;14(2):52. 26. Shirinbak S, Taher YA, Maazi H, Gras R, van Esch BC, Henricks PA, et al. Suppression of Th2-driven airway inflammation by allergen immunotherapy is independent of B cell and Ig responses in mice. J Immunol 2010;185(7):3857–65. 27. Vimalkumar CS, Hosagaudar VB, Suja SR, Vilash V, Krishnakumar NM, Latha PG. Comparative preliminary phytochemical analysis of ethanolic extracts of leaves of Olea dioica Roxb., Infected with the rust fungus Zaghouania oleae (E.J. Butler) Cummins and non-infected plants. J Pharmacogn Phytochem 2014; 3(4):69-72. 28. Su-Jin Kim. The Ameliorative Effect of β- sitosterol on DNCB-induced. Atopic Dermatitis in Mice Biomedical Science Letters.2017;23(4): 303~309. 29. Valerio M, Awad AB. B-sitosterol down- regulates some pro-inflammatory signal transduction pathways by increasing the activity of tyrosine phosphatase SHP-1 in J774A. 1 murine macrophages. Int. Immunopharmacol. 2011;11:1012–1017. 30. Kaiko GE, Phipps S, Angkasekwinai P, Dong C, Foster PS. NK cell deficiency predisposes to viral-induced Th2-type allergic inflammation via epithelialderived IL-25. J Immunol. 2010; 185(8):4681–90. 31. Ohtsuki M, Morimoto H, Nakagawa H. Tacrolimus ointment for the treatment of adult and pediatric atopic dermatitis: Review on safety and benefits. The Journal of dermatology, 2018;45(8), 936-942 32. Yousif AD, Abu-Raghif AR. The Effect of Topical Dapsone in Comparison with Tacrolimus on Dncb Induced Atopic Dermatitis in Mice, Int. J. Res. Pharm. Sci, 2020;11(4):2050-2062 This work is licensed under a Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/