Original Article Originalarbeit DOI: 10.1159/000335823 Published online: Formulation and Characterization of a Cream Containing Terminalia chebula Extract Naveed Akhtar a Ali B. Khan a Said Muhammad b Mahmood Ahmed a Haji M. Shoaib Khan a Fatima Rasool c Tariq Saeed c a Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine, Islamia University of Bahawalpur, b Department of Pharmacy, Sarhad University of Sience and Technology, Peshawar, c College of Pharmacy, University of Punjab, Lahore, Pakistan Keywords Terminalia chebula Emulsion Melanin Erythema Skin moisture Skin sebum Summary Background: This study aimed to formulate a water-in-oil emulsion (formulation) of Terminalia chebula versus its vehicle (base) as control, and investigate its effects on skin melanin, skin erythema, skin moisture content, and transepidermal water loss (TEWL). Material and Methods: Base containing no active material, and formulation containing 5% concentrated extract of T. chebula, were developed. Different stability parameters were monitored at 8, 25, and 40 ºC, as well as 40 ºC + 75% relative humidity, for a period of 4 weeks. It was concluded that the creams remained stable at all storage conditions. Both base and formulation were applied to the cheeks of human volunteers for a period of 8 weeks. Different skin parameters were monitored every week to measure any effect produced by these creams. Results: Changes in TEWL produced by base and formulation were insignificant (p > 0.05) with respect to time while significant (p 0.05) with respect to base and formulation. The skin moisture content increased after the application of formulation throughout the study period; this effect was insignificant (p > 0.05) with respect to time while significant (p 0.05) with respect to base and formulation. Both base and formulation showed insignificant (p > 0.05) effects on skin melanin content with respect to time. Skin erythema was reduced by the formulation. Both base and formulation produced statistically insignificant (p > 0.05) effects on skin sebum. Conclusion: Both creams were aesthetic with respect to sensory evaluation. T. chebula topical cream showed a positive rejuvenating effect on human skin. Hopefully, this study will encourage more attention towards the research and utilization of herbal medicines. Schlüsselwörter Terminalia chebula Emulsion Melanin Hautrötung Hautfeuchtigkeit Talg Zusammenfassung Hintergrund: Ziel dieser Studie war es, eine Wasser-in-Öl-Emulsion (Crème) mit Terminalia chebula herzustellen und auf ihre Wirkung auf Hautmelanin, Hautrötung, Hautfeuchtigkeit und transepidermalen Wasserverlust (TEWL) zu prüfen. Die Trägersubstanz diente als Kontrolle. Material und Methoden: Eine wirkstofffreie Trägersubstanz sowie eine Crème mit 5% konzentriertem T. chebula-extrakt wurden hergestellt. Verschiedene Stabilitätsparameter wurden bei 8, 25 und 40 ºC sowie bei 40 ºC + 75% relativer Luftfeuchte über 4 Wochen gemessen. Dabei wurde festgestellt, dass beide Substanzen unter sämtlichen Lagerbedingungen stabil blieben. Sowohl die Trägersubstanz als auch die Crème wurden 8 Wochen lang auf die Wangen von freiwilligen Testpersonen aufgetragen. Die Wirkung der Produkte wurde wöchentlich anhand verschiedener Hautparameter gemessen. Ergebnisse: Die von Trägerstoff bzw. Crème bewirkten Veränderungen des TEWL waren bezüglich der Zeitspanne vernachlässigbar (p > 0,05), aber bezüglich des Vergleichs von Trägerstoff und Crème signifikant (p 0,05). Die Hautfeuchtigkeit konnte durch Auftragen der Crème während der Versuchsperiode gesteigert werden; dieser Effekt war bezüglich der Zeitspanne vernachlässigbar (p > 0,05), aber bezüglich des Vergleichs von Trägerstoff und Crème ebenfalls signifikant (p 0,05). Bezüglich der Zeitspanne zeigten beide Produkte eine vernachlässigbare Auswirkung auf den Anteil an Melanin in der Haut (p > 0,05). Hautrötungen konnten durch Anwendung der Crème gemildert werden. Der Einfluss beider Produkte auf die Talgproduktion war statistisch nicht signifikant (p > 0,05). Schlussfolgerung: Sowohl Trägersubstanz als auch Crème wurden in der sensorischen Bewertung als ästhetisch ansprechend befunden. Die Crème zeigte eine verjüngende Wirkung auf die menschliche Haut. Es bleibt zu hoffen, dass diese Studie Anregung zur verstärkten Erforschung und Anwendung von pflanzenmedizinischen Produkten gibt. Fax +49 761 4 52 07 14 Information@Karger.de www.karger.com 2012 S. Karger GmbH, Freiburg 1661-4119/12/0191- $38.00/0 Accessible online at: www.karger.com/fok Dr. Ali Barkat Khan Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine Islamia University of Bahawalpur 63100 Bahawalpur, Pakistan Tel. +92-629 255 243, Fax -333 973 2578 barki.gold@gmail.com
Introduction An emulsion is a thermodynamically unstable system consisting of at least 2 immiscible liquid phases one of which is dispersed as globules (the dispersed phase) in the other liquid phase (continues phase), stabilized by the presence of a third, emulsifying, agent [1]. Emulsions are widely used by the pharmaceutical, food, and cosmetic industry. Their advantages include: complete protection of the incorporated drug, increased therapeutic properties and spreadability of the constituents, more controlled drug absorption and penetration, prolonged action and greater emollient effect compared to other preparations, and use of water as an inexpensive diluent and good solvent for many drugs and flavors [2]. Terminalia chebula (myrobalan) contains phenolic depigmenting agents which show a melanin-inhibitory effect. Since some depigmenting agents such as kojic acid have been found to have carcinogenic effects, safe agents should be developed for use in the cosmetic industry [3]. Furthermore, T. chebula extract exhibits a significant cytoprotective effect against UVBinduced oxidative damage [4]. Materials and Methods Identification of the Plant The identification of T. chebula (chebulic myrobalan) was performed by Dr. Muhammad Arshad at the Cholistan Institute of Desert Studies, The Islamia University of Bahawalpur, Pakistan. The specimen was deposited in the Pharmacognosy section of the Faculty of Pharmacy and Alternative Medicine. The voucher number is Pharm 169/Apr. 2009. Materials T. chebula fruits were purchased from the local market of Bahawalpur, Pakistan. Paraffin oil (dynamic viscosity 110 mpas, kinematic viscosity 34 cst) was obtained from Merck (Darmstadt, Germany). Abil EM90 (cetyl dimethicone copolyol, HLB 5) was purchased from Franken Chemical (Germany). Methanol was provided by Merck, and lemon oil was obtained from the Chemoflor Manufacturing Corporation (Karachi, Pakistan). Apparatuses The following technical equipment was used: EBA 20 centrifuge (Hettich, Tuttlingen, Germany), Sanyo MIR-153 cooled incubator (Sanyo, Osaka, Japan), ProfiLine Cond 197i conductivity meter (WTW, Weilheim, Germany), Corneometer MPA 5 (Courage-Khazaka, Cologne, Germany), Mexameter MPA 5 (Courage-Khazaka), Sebumeter MPA 5 (Courage-Khazaka), TEWA meter MPA 5 (Courage-Khazaka), digital humidity meter (TES Electronic Corp., Taipei, Taiwan), BJ-210 electronic balance (Precisa Gravimetrics AG, Dietikon, Switzerland), Ika EUROSTAR homogenizer D 230 (Sigma-Aldrich Chemie Gmbh, Munich, Germany), MIR-162 heated incubator (Sanyo), ProfiLine ph-197i ph meter (WTW), refrigerator (Dawlance, Karachi, Pakistan), rotary evaporator (Eyela-Tokyo Rikakikai Co., Ltd., Tokyo, Japan), UV-1601 ultraviolet spectrophotometer (Shimadzo, Kyoto, Japan). Antioxidant Activity of Terminalia chebula The free radical scavenging activity of T. chebula was determined in accordance with the Marsden S. Blois method using DPPH (1, 1-diphenyl-2-picrylhydrazyl) which is a stable free radical [5]. Equal volumes of diluted extract were mixed with equal volumes of DPPH (0.5 ml) in analytical grade methanol, and the obtained mixtures were kept at room temperature for 30 min. Then, absorption at 517 nm of the mixtures was measured and compared with the control solution (maximum absorption). Vitamin C was used as standard. The activity of free radicals was calculated in % inhibition according to the following equation: % Inhibition = (A control A test) 100 (1). A control The free radical scavenging activity of T. chebula was 78%. Formulation Development In this study, water-in-oil (w/o) emulsions were prepared by adding the aqueous phase to the oily phase under continuous agitation [6]. The oily phase consisted of paraffin oil (14%). The surfactant Abil EM90 (5%) was heated up to 75 ± 5 C. At the same time, the aqueous phase consisting of water (q.s.) was heated to the same temperature before adding the T. chebula extract (5%). After that, the aqueous phase was added to the oil phase drop by drop. Stirring with the mechanical mixer was continued at 2,000 rpm for about 15 min until all of the aqueous phase was added; a few drops of lemon oil were added during this stirring time to give the formulation a pleasant fragrance. The speed of the mixer was reduced to 1,000 rpm for homogenization for a period of 5 min, and then to 500 rpm for 5 min for complete homogenization and until the emulsion had cooled to room temperature. The base was prepared by the same method and with the same ingredients except T. chebula extract. Properties of the Formulation Stability tests were performed at 8 ± 0.1 ºC (refrigerator), 25 ± 0.1 ºC, 40 ± 0.1 ºC, and 40 ± 0.1 ºC (incubator) with 75% relative humidity (RH). Physical characteristics (color, creaming, liquefaction), electrical conductivity, and ph of the formulation were measured at various intervals for 4 weeks. Product Evaluation on the Skin A total of 11 healthy, male volunteers with a mean age of 30 years were selected for the study. Patch tests were performed on the forearms of each volunteer on the first day of sampling to determine any reactions to the creams. After 48 h, each volunteer was provided with 2 creams. One was the base and the other the active formulation. The volunteers were instructed to apply the cream provided for a period of 8 weeks, and to come for measurement at the end of week 1, 2, 3, 4, 6 and 8. Study Design A single-blinded study was designed for the comparison of the 2 creams, i.e. the active formulation containing T. chebula extracts, and the base. The creams were named A (active formulation) and B (base formulation) and given to the volunteers with instructions of application. Results were measured in a controlled room at 20 ± 1 C and 40 ± 2% RH [7]. Ethical Standards This study was approved by the Board of Advanced Study and Research (BASR), The Islamia University of Bahawalpur, and the institutional ethical committee in compliance with NIH Principles of Laboratory Animal Care, 1985. The reference no. is Pharm 640/09. Burchard Test (Patch Test) Patch tests were performed on the forearms of each volunteer. The patch (bandage disc) for the right forearm was saturated with 1.0 g of base while the patch for left forearm was saturated with 1.0 g of formulation. Each was applied to a 5 4 cm marked region separately on each forearm. The regions were covered with a surgical dressing after application. The patches were removed after 48 h, and the forearms were washed with physiological saline. After 48 h, scores were recorded for the presence of Formulation of a Cream Containing Terminalia chebula Extract 3
erythema (skin reddening) using a 0 3 scale (0 = absent, 1 = mild, 2 = moderate, 3 = severe). Each volunteer was asked to note the level of irritation/itching arising from the patches and then assign a score from the scale. Average scores are given in table 1 [7]. Panel Test Every individual was provided with a form prepared previously to test the sensory values of the creams. This form consisted of 7 parameters to be evaluated, and every parameter was assigned 11 values from 5 to +5 indicating very bad to very good. This form was to be completed independently by each volunteer. The average points were calculated from the points assigned to each question for the 2 creams. It was concluded that there was no variation between base and formulation regarding the sensory evaluation. Both creams scored similarly from a sensory point of view. Mathematical Analysis The percent changes for the individual values of the different parameters taken every week for each volunteer were calculated with the following formula: Percent Change = ((A B) / B) 100 (2), where A = the individual value of any parameter of the 1st, 2nd, 3rd, or 4th week, and B = the zero hour value of that parameter. Statistical Analysis The measured values obtained for different parameters (skin moisture, sebum, melanin, erythema, transepidermal water loss (TEWL)) were analyzed using SPSS 12.0 software (SPSS Inc., Chicago, IL, USA). The paired samples t-test was used for variation between the 2 preparations, and 2-way analysis of variance (ANOVA) for variation between different time intervals. A 5% level of significance was applied. Results Organoleptic Tests (Color, Liquefaction, Phase Separation) In this study, base and formulation were divided into 4 separate samples which were kept at 8 ºC in the refrigerator, at 25 and 40 ºC, and at 40 ºC + 75% RH in stability chambers. The samples were observed organoleptically with respect to Table 1. Scores given by volunteers to base and formulation on the basis of itching/irritation Volunteers, n score 0 score 1 score 2 score 3 Base 7 3 1 0 Formulation 8 1 2 0 Table 2. Average ph values of base and formulation kept at different storage conditions for a period of 28 days changes in color, liquefaction, and phase separation for a period of 28 days at definite time intervals. No change in color occurred in any of the base or formulation samples. While no liquefaction was observed in any of the samples kept at 8 ºC and 25 ºC during the observation period, slight liquefaction occurred in the base and formulation samples kept at 40 ºC and 40 ºC + 75% RH on days 21 and 28. A slight degree of phase separation occurred on day 28 in the base samples stored at 40 ºC and 40 ºC + 75% RH; the samples of formulation remained stable at all temperatures. Centrifugation Tests Centrifugation tests for both freshly prepared base and formulation samples and the samples kept under different storage conditions were performed, and phase separation in these samples was observed for 28 days at different time intervals. No phase separation was found in any sample throughout the study period. Electrical Conductivity Test The electrical conductivity values of the fresh base and formulation as well as the samples kept under different storage conditions for 28 days were determined. No electrical conductivity was found in any sample throughout the study period. ph Tests The ph of freshly prepared base and formulation was 5.34 and 6.0, respectively. Average changes in ph values of both base and formulation from the time of preparation up to week 4 of the study period were determined and are shown in table 2. The ph values were measured immediately after preparation, then after 12, 24, 36, 48 and 72 h, and then after 1, 2, 3 and 4 weeks. Skin Erythema and Melanin Skin erythema and skin melanin content were measured before application of the creams (0 hour readings) and then after week 1, 2, 3, 4, 6 and 8 of the study period, using a Mexameter MPA 5 (Courage-Khazaka). The percent changes that occurred in the values of 10 of the volunteers are presented in figure 1. Skin Moisture Skin moisture content was measured before application of the creams (0 h readings) and then after week 1, 2, 3, 4, 6 and 8 of ph value (mean ± standard error of mean) at different storage conditions 8 o C 25 o C 40 o C 40 o C + 75% RH Base 5.19 ± 0.075 5.4 ± 0.067 4.92 ± 0.098 4.98 ± 0.088 Formulation 4.95 ± 0.099 4.75 ± 0.111 5.09 ± 0.112 4.58 ± 0.109 RH = Relative humidity. 4 Akhtar/Khan/Muhammad/Ahmed/Khan/ Rasool/Saeed
Fig. 1. Percent changes in skin melanin after application of base and formulation. Fig. 2. Percent changes in skin moisture content after application of base and formulation. the study period, using a Corneometer MPA 5 (Courage- Khazaka). The percent changes that occurred in the values of the 11 volunteers are presented in figure 2. Skin Sebum Skin sebum secretion was measured before application of the creams (0 hour readings) and then after week 1, 2, 3, 4, 6 and 8 of the study period, using a Sebumeter MPA 5 (Courage- Khazaka). The percent changes that occurred in the values of the 11 volunteers are presented in figure 3. Transepidermal Water Loss The percent changes in TEWL values before and after application of base and formulation are presented in figure 4. Discussion During the observation period of 28 days, no change in color occurred in any of the base or formulation samples, showing that both base and formulation are stable under those conditions for up to 28 days. While no liquefaction was observed in any of the samples kept at 8 ºC and 25 ºC, slight liquefaction oc- Fig. 3. Percent changes in skin sebum after application of base and formulation. Fig. 4. Percent changes in transepidermal water loss (TEWL) after application of base and formulation. curred in the base and formulation samples kept at 40 ºC and 40 ºC + 75% RH on days 21 and 28. Also, slight phase separation occurred on day 28 in the base samples stored at 40 ºC and 40 ºC + 75% RH; however, the samples of formulation remained stable at all temperatures. Furthermore, the centrifugation test revealed no phase separation in any of the samples, indicating stability of the samples at all storage conditions for 28 days. The ph of human skin ranges from 4.5 to 6.0, and 5.5 is considered to be the average ph of the skin. Therefore, formulations intended for application to the skin should have a ph close to this range. In this study, the ph of the freshly prepared base and formulation was 5.34 and 6.0, respectively. The ph values of the samples of base kept at different storage conditions was found to be increasing gradually in the first 3 days and then declining continuously until day 28 with some variations. In contrast, the ph of the samples of formulation kept under different storage conditions showed gradual reduction from the beginning with slight variations. The twoway ANOVA technique at a 5% level of significance revealed the change in ph at different levels of time and temperature to be significant in the different samples of both base and formulation. The decrease in the ph of the formulation might be due to the production of an acidic metabolite. Formulation of a Cream Containing Terminalia chebula Extract 5
The conductivity test performed at definite time intervals for all samples of base and formulation showed no electrical conductivity in any of the samples, also indicating that the creams were stable at different storage conditions. Melanin is a biopolymer produced by melanocytes, which determines, mostly, the color of the skin. Excessive production of melanin and its accumulation in the skin can cause pigmenting disorders including melasma, solar lentigo, and post-inflammatory hyperpigmentation. Melanogenesis is the process of melanin production and its subsequent distribution by melanocytes within the skin and hair follicles. Melanocytes have melanosomes which contain several enzymes that mediate the production of melanin [3]. The ANOVA test showed the melanin effects of base and formulation to be insignificant with respect to time. By applying the paired sample t-test, insignificant differences were observed between the melanin effects of base and formulation throughout the study period (except for week 5). Like kojic acid, which is a depigmenting ingredient used in cosmetic products, the methanol extract of T. chebula has melanin-inhibitory properties [3]. In this study 5% of T. chebula extract were used in the formulation which decreased the skin melanin contents throughout the study period, but statistically this decrease in skin melanin was insignificant. Erythema is an acute cutaneous inflammatory reaction and the best-known response to ultraviolet radiation. It is characterized by signs of inflammation including redness, heat, tenderness, and edema. The degree of erythema depends on host and ecological factors [8]. An irregular increase in erythema was observed after application of the base throughout the study period. In contrast, application of formulation decreased the level of erythema throughout the study period. The ANOVA test showed that the effects on skin erythema produced by both base and formulation were insignificant with respect to time. However, the paired sample t-test revealed significant variations. It was concluded that the base had variable results, while formulation had decreased the level of erythema in the skin by the end of the study period. Hence, it is safe to use without causing any significant skin irritation. Anti-inflammatory activity may be attributed to the high tannin content in T. chebula. One of the chief constituents of tannin is gallic acid which was found to possess antiinflammatory properties [9]. Moisturizing the skin involves repairing the skin barrier, retaining/increasing the water content, reducing the TEWL, restoring the lipid barriers ability to attract, hold and redistribute water, and maintaining skin integrity and appearance [10]. A regular increase in skin moisture was found after application of the formulation throughout the study period. ANOVA showed both base and formulation to have produced insignificant effects on the moisture content with respect to time. However, the paired sample t-test showed significant differences in the moisture values after application of base and formulation except for week 1, 5 and 6. The improvement in skin moisture after application of formulation may be due to the vitamin C content of T. chebula, since vitamin C is known to enhance the rate of collagen biosynthesis in the dermis, which subsequently leads to improved hydration [11]. The sebaceous glands, located in each hair follicle, produce an oily substance (sebum) to lubricate and protect the skin [12]. In this study, the effects of the base and formulation on the sebum contents of human cheeks were investigated. Sebum was measured every week in all individuals. It was found that both the base and formulation samples increased the sebum contents throughout the study period. ANOVA showed these effects to be insignificant with respect to time, but the paired sample t-test revealed that both base and formulation produced significant variations regarding the skin sebum content except for week 1. The increase in sebum may be attributed to the oily nature of w/o emulsions containing a thick viscous oily liquid such as paraffin oil [13]. TEWL is the outward transmission of water through the skin. An increase in TEWL indicates an impaired water barrier. TEWL measurements allow parametric evaluation of the effect of barrier creams against irritants, and characterization of skin functionality. TEWL measurements can be affected by the anatomical site, sweating, skin surface temperature, interand intra-individual variations, air convection, ambient air temperature, ambient air humidity, and instrument-related variables [14]. According to ANOVA, the changes in TEWL produced by base and formulation were insignificant with respect to time. However, the paired sample t-test showed significant variations in TEWL for both base and formulation. Paraffin oil may reduce TEWL by as much as 30% [15]. The significant reduction in TEWL seen after application of the formulation may again be due to the vitamin C content of T. chebula [11]. Conclusion From our preliminary study, we concluded that a stable topical cream (w/o emulsion) containing T. chebula extract can produce a decrease in the melanin content of the skin and hence exert a skin whitening effect. The formulation was observed to decrease skin erythema, which indicates an antiinflammatory effect. Furthermore, the cream has skin moisturizing properties, and the formulation was observed to decrease TEWL significantly which translates into an anti-wrinkle effects. The T. chebula extract formulation is not suitable for people with oily skin as it causes an increase in skin sebum which may potentiate acne. A targeted study needs to be conducted in patients with freckles/melasma, psoriasis, and dry wrinkled skin so that the actual potential of T. chebula can be further explored. 6 Akhtar/Khan/Muhammad/Ahmed/Khan/ Rasool/Saeed
Acknowledgement Disclosure Statement The authors thank the Vice Chancellor and Chairman of the Department of Pharmacy, The Islamia University of Bahawalpur, for their moral support to accomplish this task, and The Islamia University of Bahawalpur, Pakistan, for financial support. References 1 Sinko PJ: Martin s Physical Pharmacy and Pharmaceutical Sciences, 5th ed. London, Lippincott Williams & Wilkins, 2006, pp 509 510. 2 Gennaro AR: Remington: The Science and Practice of Pharmacy, 19th ed. London, Lippincott Williams & Wilkins, 1995, pp 1510 1515. 3 Jin YZ, Li GH, Ahn SY, et al.: Extraction and purification of depigmenting agent from Chinese plants. Chem Res Chinese Universities 2006;22:162 167. 4 Naik GH, Priyadarsini KI, Naik DB, et al.: Studies on the aqueous extract of Terminalia chebula as a potent antioxidant and a probable radioprotector. Phytomedicine 2004;11:530 536. 5 Blois MS: Antioxidant determinations by the use of a stable free radical. Nature 1958;18:199 201. The authors declare no conflicts of interest. 6 Khan BA, Akhtar N, Mahmood T, et al.: Formulation and pharmaceutical evaluation of a W/O emulsion of Hippophae rhamnoides fruit extract. J Pharm Res 2010;13:1342. 7 Akhtar N, Khan BA, Mahmood T, et al.: Formulation and evaluation of antisebum secretion effects of sea buckthorn w/o emulsion. J Pharm Bioll Sci 2010;2:13. 8 Hönigsmann H: Phototherapy, photomedicine and pigmentary disorders. J Cutan Med Surg 2002;18: 75 81. 9 Chattopadhyay RR, Bhattacharyya SK: Plant review. Terminalia chebula: an update. Pharmacogn Rev 2007;1:151 156. 10 Kraft, JN, Lynde, et al.: Moisturizers: what they are and a practical approach to product selection. Skin Ther Letter 2006;122:13 20. 11 Sharma, SR, Poddar R: Effect of vitamin C on collagen biosynthesis and degree of birefringence in polarization sensitive optical coherence tomography (PS-OCT). Afr J Biotechnol 2008;7:2049 2054. 12 Knaggs HE, Wood EJ, Rizer EL, et al.: Post-adolescent acne. Int J Cosmet Sci 2004;26:129 138. 13 Raymond CR (ed): Handbook of Pharmaceutical Excipients, 4th ed. London, Pharmaceutical Press, 2003, pp 395 396. 14 Levin J, Maibach H: The correlation between transepidermal water loss and percutaneous absorption: an overview. J Control Release 2005;103:291 299. 15 Draelos ZD: Therapeutic Moisturizers. Dermatol Clin 2000;18:597 607. Formulation of a Cream Containing Terminalia chebula Extract 7