FORMULATION OF A CREAM CONTAINING EXTRACT OF BOMBAX CEIBA AND ITS EX VIVO CHARACTERIZATION UNDER VARIOUS STORAGE CONDITIONS

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1 Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 5 pp. 1465ñ1470, 2017 ISSN Polish Pharmaceutical Society NATURAL DRUGS FORMULATION OF A CREAM CONTAINING EXTRACT OF BOMBAX CEIBA AND ITS EX VIVO CHARACTERIZATION UNDER VARIOUS STORAGE CONDITIONS MASOOD-UR-REHMAN 1 * and NAVEED AKHTAR 1 1 Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine, the Islamia University of Bahawalpur 63100, Bahawalpur, Pakistan Abstract: This study was aimed to formulate a cream (W/O emulsion) containing Bombax ceiba (stem bark) extract and evaluate its stability by exposing it to different storage conditions. The formulation was prepared using ABIL EM90 as an emulsifying agent, liquid paraffin oil and water by heating the aqueous phase at O C and the oil phase at the same temperature and then adding aqueous phase to oil phase with continuous stirring. This newly prepared formulation was stored for a period of 12 weeks at 8 O C, 25 O C, 40 O C and 40 O C/75% RH (relative humidity) and assessed for any change in physical characteristics (color, liquefaction, phase separation) from time to time at certain intervals. The formulation presented better physical stability throughout the study period. Only slight organoleptic changes were observed in the samples stored at higher temperatures. Rheological observations indicated the shear thinning effect (pseudo plastic behavior) of the formulation having the value of flow index less than 1. Microscopy showed that the droplet size of internal phase was increased more in the formulations kept at the higher temperature (40 O C). It can be said that newly developed cream containing B. ceiba extract exhibited good physical characteristics indicating its stability. The cream might be used as a skin care formulation for different cosmetic purposes. Keywords: Bombax ceiba, cream, stability, droplet size, rheology, phase separation A cream is a heterogeneous formulation that is prepared by mixing two liquids that cannot be mixed without the help of a third ingredient known as emulsifying agent. One out of these two liquids serves as the dispersion medium while the other is distributed throughout the dispersion medium in the form of droplets. The range of these droplets in the creams used for pharmaceutical purpose is about 0.1 to 50 µm (1). An oil-in-water (o/w) cream is one in which oil is present as droplets in the dispersion medium that is water while the reverse is true for water-in-oil (w/o) emulsion. When a small amount of oil is required like in shaving or conditioning creams, o/w-emulsions are used while the w/o emulsions are desired where more oily preparation is required like emollient creams and sunscreens. There is a number of emulsifiers that are used to formulate the emulsions but these may cause irritancy and sensitivity to the user. So it is necessary to develop the formulation using emulsifying agents that do not cause such adverse reactions. Nonionic surfactants as emulsifiers are considered safe with regard to such adverse reactions (2). Various processes can account for the instability of the emulsions such as creaming, Ostwald ripening, phase separation, rupture and coalescence (3). Physical and chemical stability of the formulations can be assessed promptly by keeping them at different temperatures for some time. By rheological measurement one can estimate not only the physical stability but also the quality, effectiveness and purpose of system (4). Bombax ceiba belongs to the family Bombacaceae and commonly known as silk cotton tree. It is a remarkable medicinal plant of Indian tropical and subtropical regions including Pakistan. In old-style medicine systems that are still in practice in India like Ayurveda, Siddha and Unani, it is used to cure many diseases like sexual incapacity, vaginal infections and to stop bleeding from wounds (5). Many diseases can be cured by using different parts of B. ceiba. Stem bark acts as acrid, demulcent, * Corresponding author: aarbimasood@gmail.com; tel: ; fax:

2 1466 MASOOD-UR-REHMAN and NAVEED AKHTAR diuretic, anti-inflammatory, slightly astringent and tonic. It can be used for facial illnesses such as marks, acne vulgaris, pigmentation disorder, inflammation, blister and burning sensation. Stem bark contains lupeol, β-sitosterol, shamimicin and apigenin (6). The aim of the present study was to prepare and investigate physical stability of the cream containing B. ceiba extract that could serve as a cosmetic product and contribute to the skin care. The stability of the formulation was evaluated using different parameters by placing at different storage conditions over time. EXPERIMENTAL Plant material identification Bombax ceiba bark was collected locally from Bahawalpur, Pakistan and validated by the Cholistan Institute of Desert Studies (CIDS), The Islamia University of Bahawalpur, Pakistan. A voucher specimen (No. 3524/CIDS/IUB) was issued after depositing the sample in the herbarium of CIDS, The Islamia University of Bahawalpur, Pakistan. ABIL EM 90 or cetyl PEG/PPG-10/1 dimethicone, was obtained from the Franken (Germany) and paraffin oil from Merck (Germany). Extraction and cream preparation Bombax ceiba stem bark was collected locally and extracted with 80% aqueous-methanol. Previously weighed contents of oily phase including paraffin oil (14%) and emulsifier (ABIL-EM 90) (4.5%) were heated up to 75 ± 1 O C. Aqueous phase comprising of water (77.5%) and B. ceiba extract (4%) was also heated to the same temperature (75 ± 1 O C). Subsequently, aqueous phase was added to the oil phase with constant stirring with the help of a stirrer (Euro-Star, IKA D 230, Germany) at a speed of 2000 rpm for 10 min till the complete addition of aqueous phase, 2 to 3 drops of rose oil as fragrant were added during stirring. Mixer speed was adjusted to 1000 rpm after the aqueous phase was added and continued for almost 10 min to homogenize the ingredients. This process of emulsification was further carried on with reduction in speed (500 rpm) for complete homogenization until the emulsion was cooled down to room temperature. Characterization of the cream Examination was done by keeping the samples of the cream at four different conditions of storage i.e., 8 O C, 25 O C, 40 O C and 40 O C + 75% RH (relative humidity) for 12 weeks to assess the stability under these conditions. The cream was evaluated physically (color, liquefaction and phase separation) under these conditions. Centrifugation (Centrifuge Machine, Hettich EBA 20 Germany) was carried out at a speed of 5000 rpm and at 25 O C for 30 min by employing the sample (few grams) in a disposable centrifugal tube. Rheological parameters for the formulations were determined at 25 O C after preparation and at different time intervals for 12 weeks, taking 0.5 g of the sample. Rheology was determined using Table 1. Variation in physical characteristics of the formulation at various time intervals stored at 8 O C, 25 O C, 40 O C and 40 O C/75% RH. Physical Storage Days characteristic temperature 0 Week 4 Week 8 Week 12 Week Liquefaction 8 O C O C O C O C/75% RH Color 8 O C LP LP LP LP 25 O C LP LP LP LP 40 O C LP LP LP LP 40 O C/75% RH LP LP LP LP Phase separation 8 O C O C O C O C/75% RH = No change; + = slight change; LP = light pink.

3 Formulation of a cream containing extract of Bombax ceiba Table 2. Flow index and consistency index values determined at various time intervals after storage at 8 O C, 25 O C, 40 O C and 40 O C/75% RH. Time 8 O C 25 O C 40 O C 40 O C/75% RH FI CI CF FI CI CF FI CI CF FI CI CF 0 Week Week Week Week FI = Flow index; CI = Consistency index; CF = Confidence of fit (%) a CP 41 spindle of a cone-plate rheometer (Brookfield DV-III Ultra). Values for consistency index (related to the system viscosity) and flow index (related to the system pseudo plasticity) were obtained by the power law. Increased shear stresses were applied on the samples and change in viscosity was observed. Power law The power law equation is as shown in Eq. 1. τ = kd n (1) where τ = shear stress, D = yield stress (stress at zero shear rate), k = plastic viscosity, and n = shear rate The calculated parameters for this model are flow index (no units), consistency index (cp) and confidence of fit (%). Microscopic examination Stability of the creams can be established by an essential characteristic that is the droplet size of internal phase. With the help of an optical microscope (Eclipse E200, Nikon, Japan), to which a CCD camera was attached the droplet size was determined. The images were processed using mini see software (V. 1.1). Firstly, a minute quantity of sample stored at different temperatures was taken on the slide then it was diluted with the continuous phase and covered with the cover slip. After that it was observed under the microscope by the 100 lens. The droplet diameter was determined by stage micrometer and graticule. The micrographs obtained are shown (Fig. 2). RESULTS The creams were examined on a physical (color, phase separation and liquefaction) basis and results are shown in Table 1. It was noted that no change in the color of the formulation occurred. There was no phase separation and liquefaction found except to slight phase separation and liquefaction at 40 O C on the 90 th day of study was found. In the present study, centrifugation test of freshly prepared sample and samples kept at different storage conditions was done. These evaluations were carried out for a period of 12 weeks at different storage conditions (8 O C, 25 O C, 40 O C, and 40 O C + 75% RH) and at specific time intervals. We also evaluated the influence of storage conditions on the viscosity of the preparation by subjecting the formulations to the rheometer. Figure 1 presents the rheograms of the formulations stored at different temperatures. Measurements were recorded for the flow index and consistency index for the formulations and are shown in Table 2. The droplet size is also an important characteristic of the emulsion so it was measured according to the procedure stated earlier. Figure 2 reveals the microscopic images of the cream containing B. ceiba extract. It was found that the droplet size was not only increased with time but this increase was also more prominent at higher temperature. DISCUSSION Storage at various temperatures is a well-known method to explore the stability of an emulsion. This method is easy to carry out because breaking of the emulsions is accelerated by thermal stress (7). The freshly prepared formulation was light pink in color and sustained the color throughout the study period (12 weeks). This resistance in change of color revealed the stability of the formulation at different storage conditions i.e., 8 O C, 25 O C, 40 O C, and 40 O C + 75% RH throughout the period of investigation. Phytochemical studies on various parts of B. ceiba exposed that it is rich in phenolic compounds (8). These phenolic compounds help retain the original color of the formulations by inhibiting the growth of microbes (9). Flow characteristic of the cream is indicated by its viscosity and stability of emulsion can be assessed by determining the time and temperature effect upon viscosity (10). All samples kept at 8 O C, 25 O C and 40 O C + 75% RH were stable and no liquefaction was

4 1468 MASOOD-UR-REHMAN and NAVEED AKHTAR observed. Very little liquefaction was seen in the sample kept at 40 O C on 90 th day. With the passage of time it is likely that some time and temperature driven processes reduce the viscosity of preparation leading to liquefaction (11). The droplets of internal phase move upward or downward causing creaming or sedimentation due to the density dissimilarity of the two phases (12). Slight phase separation was detected in the sample placed at 40 O C on 90 th day of observation. During creaming/sedimentation the droplets tend to grow leading to decrease in the total interfacial energy. This shift to a larger mean droplet size due to the coalescence of smaller droplets causes separation of the phases in some cases (12) while the emulsion remains stable at low temperatures because viscosity did not change (13). Type of the emulsion can be determined by using different methods. We used electrical conductivity test for this purpose. Emulsion with water as an external phase can conduct the electricity because electricity can pass through the water while oil is a poor conductor of the electric current so when oil is the dispersion medium there will be no electrical conductivity (14). No electrical conductivity was found in the formulation under test so we can say that it was water in oil emulsion. Physical stability of the creams can be evaluated by using the techniques such as centrifugation that can enhance the instability in the formulations (7). It was noted that after centrifugation there was no phase separation found in the samples kept at 8 O C, 25 O C and 40 O C + 75% RH till the end of study. The appropriate speed of the mixer during the process of emulsification can prevent the formulation from breakage under accelerated studies (10). However, the sample exposed to 40 O C exhibited phase separation after 60 days in the centrifugation test. By applying the centrifugal force creaming/sedimentation can be augmented due to change in the droplet size, structure and distribution (12). Figure 1. Rheograms of formulation at various time intervals A) Formulation stored at 8 O C, B) Formulation stored at 25 O C, C) Formulation stored at 40 O C, D) Formulation stored at 40 O C/75% RH

5 Formulation of a cream containing extract of Bombax ceiba Figure 2. Micrographs of formulation A) Freshly prepared B) Formulation after 12 weeks storage at 8 O C, C) Formulation after 12 weeks storage at 25 O C, D) Formulation after 12 weeks storage at 40 O C/75% RH E) Formulation after 12 weeks storage at 40 O C Right consistency (rheology) for a good sensation of the skin and better spreading is desired standard for the emulsions used as personal care products. Rheology can be used to display the information about the stability of the emulsion (15). In the current study it was observed that the formulations depicted shear thinning effect because viscosity of the formulations decreased with the increase in shear stress as can be incurred from the values of the flow index that are less than 1. It is the property that is very much required for good application and good feel of the formulation. It is necessary to expose the formulation to different temperatures and obtain the rheological data to get the valuable information about the product stability and consistency (15). The slight decrease in the consistency index was found in the formulations. Consistency index represents the viscosity of the formulations and it is known that consistency index normally decreases upon storage (4). Physicochemical characteristics including the droplet size and rheological characteristics can be used to assess the emulsion stability. The droplet size of the disperse systems like emulsions can be achieved by using numerous methods (16). In the current investigation micrographs indicated that spherical globules of 2-4 µm were present in the freshly prepared formulations. These values are within the given range as mentioned in the literature for the pharmaceutical emulsions (1). After keeping the formulations on different storage conditions it was seen that the droplet size increased differently at various temperatures. The droplet size was increased up to 10.4 µm and 12.2 µm, respectively, at 8 O C and 25 O C after 12 weeks of storage. While the droplet size of the formulations kept at 40 O C and 40 O C/75% RH were found to be 17 µm and 25 µm after 12 weeks. It can be seen that at higher temperature the droplet size of the formulation was increased more as compared to the formulations kept at lower temperature. There is some evidence that the stability of the emulsion at lower temperatures may be because of the stable film of emulsifier around the droplets that allows less contact of globule during storage. Slight decrease in the viscosity with time at higher temperatures, 40 O C and 40 O C/75% RH allows more probability of the droplet size increase by coalescence (17). CONCLUSION From the current study, we can conclude that the cream containing Bombax ceiba stem bark extract in concentration of 4% exhibited promising stability and physicochemical characteristics at different storage conditions. This formulation showed no change in color or any liquefaction however a slight phase separation was seen at 40 O C. The stabil-

6 1470 MASOOD-UR-REHMAN and NAVEED AKHTAR ity of the cream can be increased by storing at low temperatures while higher temperature can affect it negatively. The formulation can offer a good delivery system for skin rejuvenation agents. Further in vivo studies have to be done to discover the benefits of the cream as a phytocosmetic agent. Acknowledgment The authors thank to the Chairman and Dean of the Faculty of Pharmacy & Alternative Medicine, The Islamia University of Bahawalpur, Pakistan for providing the lab facilities to conduct the study and the moral support given to them. Conflict of interest There is no conflict of interest associated with this work. REFERENCE 1. James S., James C.B., Encyclopedia of Pharmaceutical Technology. p. 1066, Second Edition, Marcel Dekker, Inc. (USA) Chain I.P., Wan-GU C., Seong J.L.: Aust. Rheol J. 15, 125 (2003). 3. Roland I., Piel G., Delattre L., Evrard B.: Int. J. Pharm. 263, 85 (2003). 4. Guaratini T., Gianeti M.D., Campos P.M.B.G.M.: Int. J. Pharm. 327, 12 (2006). 5. Anandarajagopal K., Sunilson J.A.J., Ajaykumar T.V., Ananth R., Kamal S.: Eur. J. Med. Plants 3, 99 (2013). 6. Verma V., Jalalpure S.S., Sahu A., Bhardwaj L.K., Prakesh Y.: Int. Pharm. Sci. 1, 62 (2011). 7. Masmoudia H., Dr eaua Y.L., Piccerelleb P., Kister J.: Int. J. Pharm. 289, 117 (2005). 8. Dar A., Faizi S., Naqvi S., Roome T., Rehman S.Z.U. et al.: Biol. Pharm. Bull. 28, 596 (2005). 9. Ali A., Akhtar N.: Trop. J. Pharm. Res. 13, 677 (2014). 10. Waqas M.K., Akhtar N., Khan H.M.S., Mustafa R., Murtaza G.: Lat. Am. J. Pharm. 33, 731 (2014). 11. Herbert A.L., Martin M.R., Gilbert S.B.: Pharmaceutical dosage, Disperse Systems. p. 199 and 285, Vol. 1, Marcel Dekker, New York Badolato G.G., Aguilar F., Schuchmann H.P., Sobisch T., Lerche D.: Prog. Colloid Polym. Sci. 134, 66 (2008). 13. Nour A.H., Yunus R.M.: J. Appl. Sci. 6, 2895 (2006). 14. Khan B.A., Akhtar N., Khan H.M.S., Waseem K., Mahmood T. et al.: Afr. J. Pharm. Pharmacol. 5, 2715 (2011). 15. Tadros T.: Adv. Colloid Interface Sci. 108, 227 (2004). 16. Baby A.R., Santoro D.M., Velasco M.V.R., Serra C.H.D.R.: Int. J. Pharm. 361, 99 (2008). 17. Tcholakova S., Denkov N., Ivanov I.B., Marinov R.: Bulg. J. Phys. 31, 96 (2004). Received: