Herbal composites film production for inflamed acne treatment

Acne is a chronic skin disease caused by the inflammation of sebaceous glands around hair follicles, where could be seen as red blisters, pustules, and scar on the face. These are the effects of bacteria especially Staphylococcus aureus and Propionibacterium acnes infection. Many investigations have been done on acne and scar treatments using Thai herbs as anti-bacterial agents for S. aureus and P. acnes. This research had developed thermoplastic starch films from cassava starch supplemented with herbal extracts from mangosteen pericarps (MP) and centella (CL), which consisted of three main parts; Part (1) Thermoplastic starch preparation, Part (2) MP and CL extraction, formulations and their anti-microbial, anti-oxidant properties, and also total phenolic contents, and Part (3) Preparation of thermoplastic starch films added with selected formulation of MP-CL extracts and the study of extracts releasing profiles and disk diffusion assay of selected formulation.Part (1), Thermoplastic starches were prepared from cassava starch (ST), glycerol (GLY), and sodium alginate (AL). At first stage, the films were prepared only with cassava starch plasticized with glycerol, and measured the mechanical properties of stickiness (SN), adhesiveness (AN), and cohesiveness (CN). The resulted revealed that the ratio of 30:70 (w/w) of ST, polymer part, and GLY, plasticizer part, exhibited high SN, AN, and CN values of 54.31±3.40 g, 0.86±0.06 g.sec, and 0.34±0.03 mm, respectively, at p-value < 0.05. But the appearance of these films revealed that the wrinkle and unsteady films instead.Thus, to improve the quality of the films both appearance and high mechanical properties, sodium alginate (AL) was then added into the polymer part, 30% w/w of the ratio, while GLY, plasticizer part was kept constant at 70% w/w. The ratios between ST and AL in polymer part were varied at 10% (w/w) interval from 100:0 to 0:100 of ST:AL ratio. Ten newly prepared composite films were done on SN, AN, and CN measurement. The results showed that the films with polymer ratio (ST:AL) of 90:10 gave the high SN, AN, and CN values of 68.31±0.62 g, 1.65±0.05 g.sec, and 0.55±0.01 mm, respectively, at p-value < 0.05, with steady and non-wrinkle films. These films were selected to be supplemented with herbal extracts before conducting the third part of this study, extracts releasing profiles of the invented films.Part (2), in this part, MP and CL were extracted by maceration and Soxhlet methods, respectively, and prepared for 7 MP-CL formulations by varying and combining MP and CL extracts at 7 different ratios (w/w), MG0, MG2, MG4, MG5, MG6, MG8, and MG10. These formulations were then measured the anti-oxidant activities, total phenolic contents (TPC), and anti-microbial activities. The anti-oxidant activity was done by using DPPH scavenging assay comparing to the standard ascorbic acid. It was found that MG8 gave the highest anti-oxidant activity with 17.10 µg/mL of IC50 and 69.99±4.67% of radical scavenging among five formulations, MG2 to MG8. However, its activity was lower than that of MP extract alone (MG10), 14.80 µg/mL of IC50 and 75.93±12.16% of radical scavenging, while CL extract alone (MG0) exhibited the lowest anti-oxidant activity of 61.70 µg/mL of IC50 and 31.79±10.54% of radical scavenging. The TPC results also confirmed the effectiveness of MP over the CL, as the TPC for MP and CL was found to be 5,179.64±542.66 and 2,418.81±123.10 mg GAE (Gallic Acid Equivalent)/g extract, respectively. This study also investigated on the anti-microbial activities of all MP-CL formulations by Minimal Inhibitory Concentration (MIC) using broth micro-dilution method. These tests were done to compare the formulations with gentamycin solution against S. aureus DMST 8840 and P. acnes DMST 14916. It was found that MG8 and MG10 had the highest anti-microbial activity with MIC values of 1.95 and 15.63 µg/mL against S. aureus and P. acnes, respectively. The MP-CL formulation MG8 was then selected to add into the anti-acne composite films mentioned in Part (1).Part (3), the composite films supplemented with MG8 were then carried out with 480 minutes herbal extracts releasing profiles. It was found that the selected films gave the cumulative releasing content of 4.74±0.26% (w/w) at 30 minutes and remained approximately constant after that. The disk diffusion assay showed that the inhibition effect against S. aureus DMST 8840 and P. acnes DMST 14916 exhibited the large inhibition zone of 17.56±0.20 and 14.89±0.11 mm, respectively.

痤疮是一种因毛囊周围皮脂腺炎症引发的慢性皮肤病，临床表现为面部出现红色水疱、脓疱及瘢痕，其致病机制与细菌感染密切相关，尤以金黄色葡萄球菌（Staphylococcus aureus）和痤疮丙酸杆菌（Propionibacterium acnes）感染为主。目前已有诸多研究探索以泰国草药作为抗金黄色葡萄球菌、痤疮丙酸杆菌的抗菌剂，用于痤疮及瘢痕的治疗。本研究以木薯淀粉为基材，添加山竹果皮（mangosteen pericarps, MP）与积雪草（centella, CL）提取物制备热塑性淀粉膜，研究内容主要分为三部分：（1）热塑性淀粉制备：以木薯淀粉（ST）、甘油（GLY）及海藻酸钠（AL）为原料制备热塑性淀粉。首先制备仅以甘油塑化木薯淀粉的薄膜，并测定其黏性（SN）、粘附性（AN）与内聚性（CN）等力学性能。结果显示，当聚合物组分木薯淀粉与增塑剂组分甘油的质量比为30:70时，薄膜的黏性、粘附性与内聚性分别为54.31±3.40 g、0.86±0.06 g·sec与0.34±0.03 mm，且差异具有统计学意义（p<0.05），但该薄膜外观存在褶皱且平整度不佳。为改善薄膜外观与力学性能，向聚合物组分中添加占比30%（w/w）的海藻酸钠，同时保持增塑剂组分甘油占比为70%（w/w）不变；聚合物组分中木薯淀粉与海藻酸钠的质量比以10%（w/w）为梯度，设置100:0至0:100共10组配比，制备复合薄膜并测定其SN、AN与CN。结果表明，当聚合物组分质量比ST:AL为90:10时，薄膜的SN、AN与CN分别为68.31±0.62 g、1.65±0.05 g·sec与0.55±0.01 mm，差异具有统计学意义（p<0.05），且薄膜平整无褶皱。该组薄膜将用于后续添加草药提取物的实验，即本研究第三部分的提取物释放特性研究。（2）山竹果皮与积雪草提取物的制备、配方及其抗菌、抗氧化性能与总酚含量：分别采用浸渍法与索氏提取法提取山竹果皮与积雪草提取物，并以7种不同质量比组合MP与CL提取物，制备MG0、MG2、MG4、MG5、MG6、MG8、MG10共7种MP-CL复合配方。随后对各配方开展抗氧化活性、总酚含量（TPC）与抗菌活性测定。抗氧化活性采用DPPH自由基清除法测定，并以抗坏血酸作为标准对照。结果显示，在MG2至MG8的5种配方中，MG8的抗氧化活性最高，其半数抑制浓度（IC50）为17.10 μg/mL，自由基清除率为69.99±4.67%；但其活性低于单独使用山竹果皮提取物的MG10组（IC50=14.80 μg/mL，自由基清除率75.93±12.16%），而单独使用积雪草提取物的MG0组抗氧化活性最低，IC50为61.70 μg/mL，自由基清除率仅为31.79±10.54%。总酚含量测定结果也证实山竹果皮提取物的总酚含量高于积雪草提取物，二者分别为5179.64±542.66 mg GAE（没食子酸当量）/g提取物与2418.81±123.10 mg GAE/g提取物。本研究同时采用肉汤微量稀释法测定最低抑菌浓度（MIC），以评价各MP-CL配方对金黄色葡萄球菌DMST 8840与痤疮丙酸杆菌DMST 14916的抗菌活性，并以庆大霉素溶液作为对照。结果显示，MG8与MG10组抗菌活性最强，其对金黄色葡萄球菌与痤疮丙酸杆菌的MIC值分别为1.95 μg/mL与15.63 μg/mL。最终选择MG8配方添加至（1）中制备的复合薄膜中，用于后续实验。（3）添加MG8配方的复合薄膜的制备及其提取物释放特性与纸片扩散法抗菌实验：对添加MG8配方的复合薄膜开展时长480分钟的提取物释放特性研究。结果显示，该薄膜在30分钟时的累积释放量为4.74±0.26%（w/w），此后释放量基本保持稳定。纸片扩散法抗菌实验结果表明，该薄膜对金黄色葡萄球菌DMST 8840与痤疮丙酸杆菌DMST 14916的抑菌圈直径分别为17.56±0.20 mm与14.89±0.11 mm。