*Corresponding author: Vazirian_m@tums.ac.ir © 2020. Open access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/) Research Journal of Pharmacognosy (RJP) 7(2), 2020: 61-69 Received: 29 July 2019 Accepted: 4 Mar 2020 Published online: 10 Mar 2020 DOI: 10.22127/RJP.2020.104701 Original article Potential of Trachyspermum ammi (ajwain) Gel for Treatment of Facial Acne vulgaris: a Pilot Study with Skin Biophysical Profile Assessment and Red Fluorescence Photography Ziba Talebi1 , Gholamreza Kord Afshari2, Saman Ahmad Nasrollahi3, Alireza Firooz3, Maedeh Ghovvati1, Aniseh Samadi3, Mehrdad Karimi2, Sima Kolahdooz2, Mahdi Vazirian4,5* 1International Campus, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. 2Department of Iranian Traditional Medicine, School of Iranian Traditional Medicine, Tehran University of Medical Sciences, Tehran, Iran. 3Center for Research & Training in Skin Diseases & Leprosy, Tehran University of Medical Sciences, Tehran, Iran. 4Department of Pharmacognosy, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. 5Medicinal Plants Research Center, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. Abstract Background and objectives: Acne vulgaris is one of the most common dermatologic conditions. The available anti-acne treatments are not satisfactory and safe. In this regard, searching for new treatments, especially natural materials with reasonable side effects and satisfactory effectiveness, could be promising. The aim of the present study was to explore the safety and efficacy of a topical formulation containing Trachyspermum ammi (ajwain) fruits essential oil in patients with facial acne. Methods: The essential oil of the fruits was extracted by hydrodistillation method and formulated as a 1% gel. In this open-labeled, uncontrolled clinical trial, 20 patients with mild to moderate acne received topical ajwain gel twice daily for 8 weeks. The outcomes of acne lesion count, red fluorescence parameters and biophysical skin profiles were evaluated at baseline, 4th and 8th weeks. Any adverse reaction was recorded during the study. Results: All patients completed the study. Two months after treatment, the mean reduction in the total (8.2±3.36; P=0.000) and non-inflammatory (7.3±4.53; p=0.000) lesions was statistically significant. Furthermore, a significant reduction in the size and quantity of red fluorescence spots was also observed. Biophysical skin profile measurements revealed a significant reduction in erythema (p=0.033) and sebum (p=0.026) and a significant increase in pH (p=0.005). No serious adverse events were reported. Conclusion: The results of this pilot study provided a basis for the effectiveness of topical ajwain gel for the treatment of mild to moderate facial acne. Conducting further double blind clinical trials are necessary to confirm the efficacy and safety of the product. Keywords: acne vulgaris; ajwain; clinical trial; essential oil; Trachyspermum ammi Citation: Talebi Z, Kord Afshari Gh, Ahmad Nasrollahi S, Firooz A, Ghovvati M, Samadi A, Karimi M, Kolahdooz S, Vazirian M. Potential of Trachyspermum ammi (ajwain) gel for treatment of facial acne vulgaris: a pilot study with skin biophysical profile assessment and red fluorescence photography. Res J Pharmacogn. 2020; 7(2): 61-69. Introduction Acne vulgaris is one of the most common causes of referring to dermatologists [1]. This disease is characterized by non-inflammatory black and white comedones and inflammatory papules, pustules, nodules and cysts on the face, neck, and the trunk [2]. The chronic nature of the disease and its appearance on face and body may lead to anxiety, depression, decreased self-esteem and in https://orcid.org/0000-0003-4526-3384 https://orcid.org/0000-0002-8386-2225 Talebi Z. et al. 62 Res J Pharmacogn 7(2): 61-69 severe cases, suicidal thoughts in patients [3]. Acne is considered a pilosebaceous unit disorder which the following factors are involved in its pathogenesis: pilosebaceous canal obstruction due to hypercratization, increased sebum production, Propionibacterium acne, inflammation and oxidative stress [4]. Topical therapy is the mainstay of mild to moderate acne treatment. Retinoids and antimicrobial drugs such as benzoyl peroxide and antibiotics are the most common topical medications administered for this disease [2]. These medications target one or several pathogenetic factors via reducing follicular hyperkeratosis and anti-inflammatory, antioxidant, antimicrobial, keratolytic and sebostatic activities [5]. Increased bacterial resistance to antibiotics and skin irritation are the most common side effects of the available anti-acne treatments [6]; therefore, there is a need to develop new, safer and more effective therapies [7]. Natural medicines, by their multi-component nature and long-term use by humans, may be good candidates for this purpose [8]; however, there is not enough evidence for this claim and further studies are needed [9]. Persian medicine (PM) is one of the oldest traditional medicine systems in the world with a vast majority of experiences in treatment of diseases with medicinal herbs [10]. PM Scholars believed that skin has an important role in the secretion of waste products and any disturbance in this process may cause skin rashes and facial spots similar to acne. In traditional literatures, many kinds of topical and oral preparations are mentioned for skin cleansing in different dermatologic conditions [11]. Trachyspermum ammi L. (synonym: Carum copticum), commonly named ajwain and known as “Zenyan” or “Nankhah” in PM, belongs to the Apiaceae family [12]. In PM resources, topical use of ajwain fruit has been recommended for treatment of a variety of skin conditions including acne [13]. Furthermore, the anti- inflammatory [14,15], antioxidant [16-21] and antibacterial [22,23] properties of these fruits have been confirmed in pharmacologic studies. Despite traditional use and related evidences for probable anti-acne properties of ajwain fruits, no study has evaluated their safety and efficacy for treatment of acne. This pilot study aimed to evaluate the safety and efficacy of a topical formulation of ajwain essential oil for treatment of facial acne in a phase 2A clinical trial. To provide more objective results, we used fluorescence digital photography and skin biophysical parameters assessment. Material and Methods Ethical considerations The study protocol was approved by the Research Ethics Committee of Tehran University of Medical Sciences (approval number IR.TUMS.REC.1394.2063 on 26/02/2016) and the trial was registered in the Iranian Registry of Clinical Trials (www.irct.ir, registration number: IRCT2016031126938N3). All patients provided written informed consent before entering the study. Plant material Ajwain fruits were purchased from a medicinal plant market (Tehran, Iran). They were ground in a mechanical grinder for 60 s, just before extraction of the essential oil. Essential oil extraction The essential oil was obtained from 500 g of ground powder of ajwain fruits by hydro- distillation method using Clevenger apparatus for 2.5 h. The obtained essential oil was yellow in color, had a pungent odor (because of thymol) and a density of 0.89 mg/mL. It was collected in a glass container and kept at refrigerator (4±1°C) and protected from light until used. Gas chromatography analysis The essential oil was diluted 1:100 in n-hexane (HPLC grade, Merck, Germany) before injection to Gas chromatography column. The identification of essential oil components was carried out by a GC/MS system (Agilent 6890 for Gas Chromatograph, and 5973 for Mass spectrophotometer, USA) equipped with a BPX5 column (30 m × 0.25 mm internal diameter, 0.25 µm film thickness). Electron ionization (70 eV) was used for system detection. The carrier gas was helium with a flow rate of 1 mL/min. The injection room temperature was set at 250 ° C. The initial column temperature was set at 50 ° C for 1 min, which was then raised to a final temperature of 280 ° C by a rate of 5 ° C/min. The operation was done in 56 min. The identification of components was made by calculation of Kovats indices, calculated by injection of a mixture of Trachyspermum ammi gel for treatment of facial acne vulgaris 63 homologous n-alkanes (C8-C25) on GC column and further confirmation of proposed structure (by software library, Wiley nl7) with standard spectra [24]. Quantitative analysis of each compound was based on the relative area under the curve of each peak in the spectrum. Preparation of topical gel Based on maximum dermal use of 1.4% (w/w) for ajwain essential oil [24], a 1% (w/w) gel, was prepared by dissolving 5 g ajwain oil in hydro- alcoholic solution (containing 50 g propylene glycol, 320.8 g 96% ethanol and 119 g distilled water) and then pre-hydrated gelling agent (5 g carbomer 941) was added to this mixture under stirring (400 rpm). Finally, triethanolamine was added slowly until the final gel was formed. Physicochemical evaluation of formulation Physical features of the ajwain gel were assessed based on the ICH stability guideline. The prepared gel was stored at 25±2 °C and 60±5% relative humidity for 6 months. During this period, the color, odor, consistency, and homogeneity of the ajwain gel was analyzed visually. The viscosity of gel was examined by a DV1TM digital viscometer (Brookfield, Spain) using spindle 5 at 100 rpm with the runtime of 15 seconds. About 2 g of the gel was dissolved in 20 mL distilled water and the pH was determined by calibrated pH meter (Metrohm 827, Swiss). Study design This open-label uncontrolled clinical trial was conducted between December 2017 and April 2018 in Center for Research and Training in Skin Diseases and Leprosy (CRTSDL) of Tehran University of Medical Sciences, Tehran, Iran. Inclusion and exclusion criteria The inclusion criteria was: age of 20-58 years; mild to moderate acne affection without cystic acne; having at least 15 inflamed and 15 non- inflamed acne lesions on the face. Acne severity was determined according to the number of papules and pustules per half face: 0-5 = mild, 6- 20 = moderate, 21-50 = severe and more than 50 = very severe [26]. The following conditions were considered as exclusion criteria: (1) the presence of nodulocystic acne lesion, (2) pregnancy, lactation, (3) excessive sun or UV light exposure, (4) allergy or sensitivity to product ingredients, (5) use of topical anti-acne treatments or procedures four weeks prior to the study, and (6) use of systemic anti-acne medications six months prior to the study. The dermatologist referred eligible patients to researcher. Finally, twenty volunteers aged 20-58 years with mild to moderate facial acne from both sexes were chosen through convenience sampling by a dermatologist Intervention After obtaining informed consent from the patients, they were instructed to use the topical gel on their face, twice daily (morning and night) for 8 weeks. Outcome measurement To determine the therapeutic efficacy, clinical assessment, digital photography, fluorescence photography, and biophysical skin profile measurement were evaluated at baseline and at the week 4, and week 8 follow-up visits. For clinical evaluations, the count of acne lesions (inflammatory, non-inflammatory, and total) was determined by a dermatologist. Additionally, Patients satisfaction from the treatment efficacy was assessed using a Visual Analog Scale (VAS) of 0 (totally unsatisfied) to 10 (totally satisfied). Local adverse effects such as erythema, itching, edema, scaling, dryness and burning/stinging were recorded during the study. Digital photographs of the face were captured using an identical camera and photographic conditions at baseline and each follow-up session. A consent form was obtained from each individual to use their photograph for research purposes. Fluorescence digital photography was performed using the Visiopor® PP 34 camera (Courage & Khazaka, Germany), with a narrow-band UVA light of 375 nm. Photographs were taken from the right cheek. The quantity and size (percentage of the area covered) of orange-red spots in a measured area of 6×8 mm were analyzed. Six skin biophysical parameters (stratum corneum hydration, sebum content, trans-epidermal water loss (TEWL), erythema index, melanin and pH value) were measured using the Cutometer® dual MPA 580 (Courage-Khazaka, Germany) with the following probes: Corneometer CM 825, Sebumeter SM 815, Tewameter TM 300, Mexameter MX 18 and pH-Meter pH 908. These parameters were assessed by the same investigator on the right cheeks of the face in a laboratory with Talebi Z. et al. 64 Res J Pharmacogn 7(2): 61-69 20-25 °C (room temperature) and a relative humidity of 35-50%. Statistical analysis Statistical analysis was performed using SPSS 16 software (SPSS Inc., USA). The descriptive data were expressed as the mean ± SD. The paired T- test with a significance level of 0.05 was used for comparison between baseline and post-treatment values. Results and Discussion The main components of the oil were identified as thymol (50.17%), gamma-terpinene (16.95%) and para-cymene (27.11%). The results of this section have been presented in table 1. The dermatologist conducted the primary screening on thirty patients with facial acne vulgaris and referred a group of twenty eligible patients (2 males and 18 females) with mild to moderate disease to the researcher. Ten patients were excluded due to acne severity and concurrent use of anti-acne treatments. All patients recruited completed the study. All patients had moderate acne. The average age of the patients was 21.4 ± 3.4 years (range, 20-40 years). Nine patients declared a family history of acne. Twelve patients had persistent acne (began in adolescence) and rest of them had late-onset acne (first appears after 25 years old). The baseline demographic and clinical characteristics of patients have been presented in table 2. 8 weeks after treatment, a mean reduction of 8.2 ± 3.36 (p = 0.000) in total lesion count, 7.3 ± 4.53 (p = 0.000) in non-inflammatory lesion count and 0.9 ± 2.77 (p = 0.16) in inflammatory lesion count was observed. The reduction in the total and non- inflammatory lesions was statistically significant (p≤0.05). The Mean (SD) count of different lesion types has been summarized in table 3. In fluorescence digital photography, the mean decrease in the size (0.46 ± 0.7; P = 0.009) and quantity (8.1 ± 6.6; P = 0.000) of red spots versus baseline were significant. The effect of ajwain gel on red fluorescence parameters has been depicted in figure 3. Two months after using ajwain gel, the mean value of sebum decrease (23.05 ± 42.6; p = 0.026) and pH increase (0.48 ± 0.68; p = 0.005) were significant. Additionally, the results showed a decrease of 6.56 ± 18.99 in skin hydration and an increase of 2.66 ± 11.43 in trans-epidermal water loss (TEWL). Finally, Melanin index had an average increase of 2.17 ± 27.25 and erythema index had an average decrease of 37.71 ± 73.46 versus baseline. The value of skin biophysical parameters at baseline and follow-up visits has been shown in table 4. At the final visit, the mean VAS score of patient satisfaction was 5.98 ± 1.14 (ranged 4-7). Ajwain gel was well tolerated and no adverse event was reported. The clinical effectiveness of the ajwain gel has been shown in figure 1. Acne vulgaris is a very common and chronic pilosebaceous unit disorder with a complex pathogenesis [27]. None of the available treatments for this disease are completely satisfactory and safe. Studies on natural substances with anti-acne properties could potentially help in developing efficient alternative treatments with fewer side effects [28]. Table 1. Chemical composition of Trachyspermum ammi essential oil, obtained by hydrodistillation method Percentage RT3 KI2 RI1 Compound No. 1.4 10.17 980 981 β-Pinene 1 0.27 10.76 991 992 β-Myrcene 2 27.11 12.00 1026 1031 p-Cymene 3 16.95 13.17 1062 1063 γ-Terpinene 5 0.2 24.71 1177 1190 Terpinen-4-ol 6 0.31 19.78 1270 1275 Cinnamaldehyde 9 50.17 20.457 1290 1306 Thymol 10 0.86 20.52 1298 1312 Carvacrol 11 7.9 Monoterpenes hydrocarbons 0.5 Oxygenated monoterpenes 90.7 Aromatic monoterpenes 99.1 Total identified 1Relative index; 2Kovats index (based on literature); 3Retention time Table 2. Baseline demographic and clinical characteristics of patients involved in trial Demographic/clinical parameter Characteristic Age (year) Gender Severity of Acne Mean Min/Max Male Female Mild Moderate Severe 21.4 20/40 2 18 0 20 0 Trachyspermum ammi gel for treatment of facial acne vulgaris 65 Table 3. Mean counts of total, inflammatory and non-inflammatory lesions at baseline, 4th and 8th week after treatment and percenage reduction versus baseline Type of lesions Baseline (n=20) Week 4 (n=20) Week 8 (n=20) Mean (SD) Mean (SD) % reduction Mean (SD) % reduction Non-inflammatory 12.35 (6.4) 8.10 (5) 34 5.05 (4.09) 61 Inflammatory 3.10 (2.12) 3.00 (1.91) 3 2.20 (2.04) 29 Total 15.45 (5.62) 11.10 (5.24) 28 7.25 (4.62) 55 Figure 1. Digital photographs, at baseline (a & c) and 2 months (b & d) after application of 1% (w/w) ajwain gel depicts a decrease in the facial acne lesions Figure 2. Mean value of red fluorescence parameters at baseline, 4th week and 8th week after treatment with Trachyspermum. ammi gel Table 4. Mean value of skin biophysical parameters at baseline, 4th and 8th week after treatment Parameters Baseline (n=20) Week 4 (n=20) Week 8 (n=20) p value* Maen (SD) Maen (SD) Maen (SD) Hydration 59.44 (13.56) 58.95 (11.33) 52.87 (16.86) 0.138 TEWL 20.02 (5.52) 19.41 (5.48) 22.68 (10.41) 0.311 Sebum content 74.3 (31.5) 52.15 (52.23) 51.25 (26.51) 0.026 Skin pH 6.44 (0.56) 6.56 (0.53) 6.93 (0.36) 0.005 Erythema index 411.33 (95.44) 378.75 (90.74) 373.61 (101.11) 0.033 Melanin index 179.45 (26.03) 178.84 (30.43) 181.61 (34.84) 0.726 *p value of paired t-test between baseline and 8th week Time (weeks) S iz e o f sp o ts ( m m ) N u m b e r o f sp o ts Time (weeks) Talebi Z. et al. 66 Res J Pharmacogn 7(2): 61-69 In this study we investigated the clinical efficacy and safety of a topical gel made from Trachyspermum ammi fruits in patients with acne vulgaris. Our pilot study showed that ajwain gel was clinically efficient for treatment of mild to moderate facial acne. After two months, a statistically significant decrease was observed in the mean number of total and non-inflammatory acne lesions. Furthermore, size and quantity of red fluorescence spots, skin sebum and erythema index declined significantly after treatment. Porphyrins are metabolic products of P. acnes and are strongly fluorescent [29]. Significant decrease in fluorescence parameters in our study may be explained by anti-bacterial effect of ajwain essential oil. Propionibacterium acnes is an anaerobic, gram- positive commensal bacterium that normally lives on the skin. Excessive sebum and keratinocytes derbies could plug hair follicles and make a favorable anaerobic environment for the growth of this bacterium in the pilosebaceous unit [30]. Release of porphyrins, chemotactic factors and enzymes by P. acnes potentiate the oxidation of sebum lipids and neutrophils accumulation at acne prone sites. All of these events result in cutaneous oxidative stress, inflammatory cascades, tissue injury and acne lesion formation [31]. Significant therapeutic response after using ajwain gel in our study may be explained by antimicrobial, anti-inflammatory and antioxidant properties of T. ammi fruits. Among the normal skin flora related to acne vulgaris, antimicrobial activity of T. ammi against Staphylococcus aureus [32-36] and Staphylococcus epidermidis [35] have been documented in several studies. Alpha-pinene, a minor ajwain essential oil constituent, has been found to have antimicrobial activity against S. aureus, S. epidermidis and P. acnes [37]. Known mechanisms for anti-inflammatory effects of T. ammi fruits are free radicals scavenging activity [38] and suppressing the production of nitric oxide and pro-inflammatory cytokines i.e. IL-18 and TNF-α [39,40]. These therapeutic effects are mainly attributed to major phenolic compounds of T. ammi named thymol and carvacrol [41]. The topical preparation of our study was well tolerated and no adverse events were reported. Acidic pH of the skin is important for keratinization, skin barrier regeneration and cutaneous antimicrobial defense [42]. Although at the end of the study, skin pH was still in acidic range, a significant rise in pH (6.44 ± 0.56 to 6.93 ± 0.36) compared with baseline was observed. Other unwanted effects of ajwain gel on skin barrier properties were mild non-significant increase in TEWL and melanin index and decrease in hydration two months after treatment. To our knowledge, this is the first clinical trial evaluating the effect of ajwain fruit in acne vulgaris. Tea tree oil is one of the proposed herbal treatments for acne vulgaris. In a study by Malhi et al. mean percent decrease in total lesion count after applying tea tree oil gel and face wash at week 4 and 8 were respectively 25% and 37% in comparison with 28% and 55% in our study [43], Compared with the results of a similar study on the use of cinnamon gel, ajwain gel was more efficient in reduction of the total (55% vs.47%) and non- inflammatory (61% vs.48%) lesion counts after 8 weeks. Applying cinnamon gel caused a significant decrease in fluorescence spots size whereas ajwain gel could significantly decrease both the size and quantity of the spots. Changes of skin biophysical profile after applying ajwain and cinnamon gel was similar as follow: both preparations increased TEWL, melanin index and decreased hydration, erythema and sebum [44]. In conclusion, our results suggest that twice daily use of topical ajwain gel for two months could be safe and effective in the treatment of mild to moderate facial acne vulgaris. This is the first study about ajwain application for treatment of acne vulgaris and double-blind randomized clinical trials with larger sample size are needed to confirm these findings. In addition, further studies are warranted to elucidate the action mechanism of T. ammi fruits on acne pathogenic factors and its long-term effects on skin barrier properties. Acknowledgments This research project was supported by Tehran University of Medical Sciences (TUMS) (grant no. 31055). Author contributions Mahdi Vazirian designed the study and revised the manuscript; Sima Kolahdooz prepared the manuscript and was involved in data analysis; Trachyspermum ammi gel for treatment of facial acne vulgaris 67 Ziba Talebi and Maedeh Ghovvati conducted the experiments related to pharmacognosy and pharmaceutical and clinical parts; Gholamreza kord Afshari and Mehrdad Karimi were involved in patient recruitment; Saman Ahmad Nasrollahi formulated the gel; Alireza Firooz supervised the clinical part; Aniseh Samadi was involved in data analysis. Declaration of interest The authors declare that there is no conflict of interest. The authors alone are responsible for the accuracy and integrity of the paper content. References [1] Chaudhary SS, Tariq M, Zaman R, Imtiyaz S. The in vitro anti-acne activity of two unani drugs. Anc Sci Life. 2013; 33(1): 35-38. [2] Kraft J, Freiman A. Management of acne. Can Med Assoc J. 2011; 183(7): 430-435. 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