요약AbstractPurposeIt is verified that polysaccharide gel can improve the spleen qi deficiency, especially the skin phenomenon caused by spleen qi deficiency.
MethodsA mouse model of spleen qi deficiency syndrome was established by eating disorder. Four kinds of polysaccharide gels were applied to the skin of the backs of mice, and their weights, spleen-thymus index, skin sections, and skin indexes were used for evaluation.
ResultsThe spleen qi deficiency mice had lower water content, lower gloss, and increased oil secretion, which were significantly different from those of the control group (p<0.01). The polysaccharide gel acts on mice with spleen qi deficiency, compared with the model group, the skin moisture content is increased, the gloss is increased, and the oil secretion is reduced, and it is significant.
Introduction中医学认为“有诸内必形诸于外”。《黄帝内经》曰:“人之气血精神也,所以奉生而周于性命者也。”气血是人体的基本物质,代表人体的正气,是机体内部平衡的基本因素。气血关系到五脏、经络、四肢百骸,气血的平衡,定会表现于面部皮肤(Qiu, 2018)。张仲景也认为,人体肤色红润有光泽,肌肉坚实有弹性及毛发润泽与否等外在表现与营卫气血的功能状态密切相关(Zhu, 2014)。《灵枢•邪气脏腑病形》中写道:“十二经脉,三百六十五络,其血气皆上注于面而走空窍”。由此可见,气血的运行也可影响皮肤的状态。
气血失调的常见为气虚、血瘀。其中,脾气虚是较为常见的气血不足症状。脾为“后天之本”“气血生化之源”,主运化而化生气血,脾胃功能健旺可以滋养人体正气,是增强人体抗
病能力的基础。脾虚证大多由饮食不节、劳倦过度、忧思伤脾或年老体衰、久病耗气所致(Zhang et al. , 2010)。
气血失调对皮肤产生的影响尚未见到有明确报道。本研究通过建立脾气虚的小鼠模型,评价脾气虚对小鼠皮肤指标的影响。同时,在脾气虚小鼠模型皮肤使用多糖凝胶。拟将四个多糖,分别作用于脾气虚动物模型,观察多糖因子对脾气虚小鼠皮肤生理指标及皮肤切片组织的影响,旨在确定多糖是否对脾气虚皮肤有改善情况。
多糖是由10个以上单糖通过糖苷键连接而成的聚糖,在自然界分布广泛,是组成高等植物、动物细胞膜及微生物细胞壁的天然大分子物质田。多糖资源丰富、来源广泛,可以从陆地植物、海藻中提取,也可以通过动物提取和微生物(细菌和真菌)发酵进行生物合成(Qing et al ., 2018)。现代药理学研究表明,多糖在免疫调节、抑制肿瘤、降血糖、降血脂、抗病毒、抗衰老、抗氧化等方面均有显著生物活性(Chen et al ., 2016a)。相比具有如刺激性等安全风险的化学活性成分而言,中药来源的天然成分具有安全、温和、持久、高效等优点,成为现在功效化妆品研究开发的一大趋势(Zhang et al ., 2007)。中药多糖作为一种从传统中草药中提取的一类多糖化合物,大量的研究表明其具有延缓皮肤衰老、美白、晒后修复、祛痘抗炎、促进伤口愈合等生物活性,可以作为功效化妆品活性成分的天然来源(Lü et al ., 2016)。
石斛,甘,微寒。归胃、肾经。具有益胃生津,滋阴清热的功效,俗称“千年润”。现代药理表明石斛具有增强免疫力、抗氧化、抗肿瘤、保肝、抗疲劳等多种功能活性(Chen et al ., 2016b)。其中石斛多糖是石斛的中药活性成分,具有较强的抗肿瘤、抗氧化和增强免疫功能活性(An & He, 2014)。陈默等对精纯的石斛多糖提取物的保湿性能进行了评价,表现出较好的保湿性能,并且有抗干燥损伤的防护作用,可作为一种天然植物类保湿剂应用于化妆品中(Chen et al ., 2015)。
天山雪莲,是维吾尔族常用药,具有补肾活血、强筋骨通经络的功效。雪莲中的多糖对紫外线具有很强的吸收作用(Guo et al ., 2015)。
银耳,是一种大型高等真菌,属于有隔担子菌亚纲银耳科,重要的药食同源物种。研究发现,银耳多糖具有促进机体免疫功能、抗衰、抗氧化的作用,可作为保湿美白剂应用于化妆品中(Huang et al ., 2013)。
Methods1. 实验材料2)实验试剂本文所使用实验试剂如下:薇婷脱毛膏(depilatory cream, Veet, China)、猪脂、生理盐水、石斛多糖(Dendrobium polysaccharides, Foshan Allan Conney Biotechnology Co., Ltd., China)、雪莲多糖(Saussurea ivolucrata polysaccharides, Foshan Allan Conney Biotechnology Co., Ltd.)、银耳多糖(Tremella polysaccharides, Foshan Allan Conney Biotechnology Co.,Ltd.)、去离子水(deionized water, Aladdin, China)、丁二醇(butanediol, Aladdin, China)、EDTA二钠(EDTA-Na2, Aladdin, China)、甘油(glycerinum, Aladdin, China)、丙烯酸(酯)类/C10-30烷醇丙烯酸酯交联聚合物(acrylates/c10-30 alkanol acrylate crosslinked polymers, Aladdin, China)、氢氧化钠(NaOH, Aladdin, China)
3)仪器本文所使用的实验仪器如下:皮肤水分含量测试仪及测试探头(Corneometer® CM825; Courage+Khazaka electronic GmbH, Germany)、皮肤水分流失测试仪及测试探头(Tewamater® TM300, Courage+Khazaka electronic GmbH)、皮肤油脂分泌测试及油脂测试盒(SubumeterSM810)、皮肤红黑色素测试仪及测试探头(Mexameter MX18; Courage+Khazaka electronic G m b H ) 、皮肤颜色测试仪及测试探头( C o l o r i m e t e r , Courage+Khazaka electronic GmbH)、皮肤光泽度测试探头(GL200; Courage+Khazaka electronic GmbH)。
2. 实验方法1)多糖凝胶的制备(1)在称重过的250 mL烧杯中称取去离子水,用称量纸称取U20,将其均匀撒在水中。静置约5 min(注意:不要搅拌,自然分散润湿),分散浸润至白色粉末完全润湿。
(2)在浸润后体系中,依次称取丁二醇、甘油,搅拌加热升温至80-85℃,搅拌5-10 min,转速35-40转/min,搅拌均匀后降温。体系中有絮状物属正常现象。
(3)用50 mL 烧杯依次称取B相EDTA-2Na、离子水,搅拌5-10
min,转速35-40转/min搅拌溶解至透明(可适当加热,加快溶解,温度55-60℃)待用。
(4)分别取雪莲糖脂0.4 g,银耳多糖3 g,石斛多糖3 g,雪莲糖脂、银耳多糖、石斛多糖各0.8 g混合。用适量去离子水将多糖溶解,作为样品一、样品二、样品三和样品四。
(5)A相降温至45℃,搅拌混合均匀后加入NaOH(10%)中和(用量为U20的2.5倍),(注意:边搅拌边加,不能过快),加入待用溶液B相和多糖样品,搅拌形成透明啫喱。加入MTI搅拌混合均匀。
(6)降至室温后称量,装入已经灭菌的样品瓶。样品一:0.4%雪莲糖脂凝胶,样品二:3%银耳多糖凝胶,样品三:3%石斛多糖凝胶,样品四:0.8等比混合多糖凝胶。制备完成。详见Table 1。
2)分组、建模及给药(2)脾气虚建模模型组:隔日灌胃猪油20 mL/kg 1次,每日喂饲甘蓝(Li, 2011)。
对照组:隔日灌胃生理盐水20 mL/kg 1次,每日喂饲料。
雪莲组:隔日灌胃猪油20 mL/kg 1次,每日喂甘蓝。每日涂抹样品1,涂抹量2 mg/cm2。
银耳组:隔日灌胃猪油20 mL/kg 1次,每日喂甘蓝。每日涂抹样品2,涂抹量2 mg/cm2。
石斛组:隔日灌胃猪油20 mL/kg 1次,每日喂甘蓝。每日涂抹样品3,涂抹量2 mg/cm2。
混合组:隔日灌胃猪油20 mL/kg 1次,每日喂甘蓝。每日涂抹样品4,涂抹量2 mg/cm2。
Results and Discussion1.生物表征观察通过观察脾气虚组动物生物表征(Zhou et al ., 2001),判断造模成功。脾气虚证型的生物表征如Table 2.所示。
脾气虚模型组小鼠开始出现明显精神倦怠。嗜卧,懒动,弓背,扎堆,咪眼。有泄泻的现象,肛门周围污秽。体重下降,体温降低。具体生物表征观察对比结果见Table 3。对照组小鼠毛色光泽饱满,模型小鼠毛色枯槁稀疏。经统计软件分析,与对照组相比,模型组体重具有极显著性差异(p <0.01)。造模时间七天。
2. 脾和胸腺指数脾气虚模型组小鼠胸腺指数和脾指数均高于对照组,且呈显著性差异,表明造模成功。脾指数、胸腺指数结果见Table 4。与对照组相比,p <0.05,*表示差异显著;p <0.01,**表示差异极显著。
3. 皮肤测试指标按照脾气虚造模方式喂养第七天,进行皮肤生物指标测试。皮肤指标的测定于每只小鼠背部同一部位,水含、光泽、颜色、红黑色素测定5次,取平均值;水散、油脂测一次。统计发现,模型组水分含量极显著降低,光泽度显著降低,油脂含量极显著升高。多糖给药组皮肤指标相比于模型组,皮肤水分含量明显升高,光泽度明显升高,油脂含量极显著降低。测试结果见Table 5。与模型组相比,p <0.05,*表示差异显著;p <0.01,**表示差异极显著。
Conclusion1. 模型评价脾虚模型建立成功的标准主要是通过表征观察、体重、皮肤指标测定、脾和胸腺指标和对照组进行对比来判断。从表征观察上看,模型组的毛发明显变稀疏,且神情倦怠、便溏泄泻。体重上,模型组与对照组间的差异极显著(p <0.01)。皮肤指标的测定上,模型组与对照组在水分含量、光泽度、油脂上具有极显著差异(p <0.01)。脾脏和胸腺,模型组明显小于对照组;计算其脾指数和胸腺指数,在统计学上,具有极显著差异(p <0.01)。
2. 多糖凝胶改善脾气虚状态相关性涂抹多糖凝胶组小鼠从毛发上看,仍比对照组稀疏,同时会出现便溏的现象。体重上,给药组小鼠的体重明显低于对照组具有极显著差异(p <0.01),与模型组无明显差异。在皮肤指标上,水含、光泽度明显高于模型组,但低于对照组,两两间差异极显著;油脂低于模型组,高于对照组,且具有极显著差异。脾指数和胸腺指数给药组都介于模型组与对照组之间,且具有极显著性差异(p <0.01)。由皮肤组织切片的结构观察情况,表明在皮肤角质细胞、毛囊发育及分泌油脂情况,多糖组相比于模型组都有一定的改善。
3. 四种多糖凝胶改善脾气虚状态的比较涂抹多糖凝胶主要对小鼠的皮肤指标有显著的改善作用。混合多糖与雪莲糖脂对皮肤光泽度改善效果最为明显,其次是银耳,石斛多糖次之。模型组油脂远高于正常组。雪莲多糖明显改善因脾虚而导致的油脂分泌过多的现象。银耳多糖对油脂分泌的改善情况也十分显著,混合多糖次之,石斛多糖效果最弱,但也具有明显的改善作用。脾气虚模型皮肤水含的改善情况,雪莲组最优,其次是混合多糖组,最后是银耳、石斛多糖。
多糖凝胶对脾气虚模型的水含、光泽、油脂含量、皮肤组织结构形态有明显的改善效果。但对脾虚小鼠的体重改善不明显。故,外用多糖凝胶虽对皮肤部分测试指标有所改善,但对脾气虚的体质以及肉眼可见的表征改变不明显。
四个多糖凝胶对脾气虚皮肤指标改善情况,雪莲组>混合组>银耳组>石斛组。对皮肤指标改善的主要方面为皮肤的水含、油脂和光泽度。
NOTESAuthor's contribution
BC designed, performed experiments, analyzed data, and wrote the manuscript. JRL designed all experimental investigations, and developed the process. LNL performed histology experiments and collected data. LNL, JRL and LL performed mouse experiments. HM and JL oversaw the study. All figures are created by the author and the co-authors.
Author's contribution
Bill Che (Director), Department of Research and Development Daily Chemicals Cosmetology, Infinitus Co. Ltd., 17th floor, Infinity center, 12 Zhujiang west road, Zhujiang new town, Tianhe district, Guangzhou 510000, China; Linan Li (Engineer), Department of Research and Development Daily Chemicals Cosmetology, Infinitus Co. Ltd., 17th floor, Infinity center, 12 Zhujiang west road, Zhujiang new town, Tianhe district, Guangzhou 510000, China; Jiarui Li (Graduate student), Department of Cosmetology, Beijing Technology and Business University, 11 Fucheng Road, Haidian District, Beijing 100048, China; Li Li (Professor), Department of Cosmetics, Beijing Technology and Business University, 11 Fucheng Road, Haidian District, Beijing 100048, China; Hong Meng (Professor), Department of Cosmetics, Beijing Technology and Business University, 11 Fucheng Road, Haidian District, Beijing, 100048, China; Jim Liu (Manager), Department of Research and Development Daily Chemicals Cosmetology, Infinitus Co. Ltd., 17th floor, Infinity center, 12 Zhujiang west road, Zhujiang new town, Tianhe district, Guangzhou 510000, China.
Figure 1.Model group skin tissue sectionKeratinocyte hyperplasia, hair follicle hypoplasia, more hair follicle oil, thicker dermis, eosinophil invasion, and less vascular endothelial cells.
![]() Figure 2.Normal group skin tissue sectionNo keratinocyte proliferation, normal hair follicle development, less hair follicle fat, no thickening of the dermis, no infiltration of eosinophils, and normal distribution of vascular endothelial cells.
![]() Figure 3.Skin tissue section of saussurea involucrate groupKeratinocyte hyperplasia basically recovered, hair follicles developed more normally, hair follicles had less oil, and the dermis layer was slightly thickened. No infiltration of eosinophils and normal vascular endothelial cell density were observed.
![]() Figure 4.Skin tissue section of Tremella groupThe skin keratinocytes are proliferated, the hair follicles are normal, the hair follicles have less oil, and the dermis is thicker. A small amount of eosinophils and multinucleated cells infiltrate, and the density of vascular endothelial cells is close to normal.
![]() Figure 5.Skin tissue section from Dendrobium groupKeratinocyte proliferation is heavier, the hair follicles are less developed, the hair follicles have more oil, the dermis is thicker, the borders are unclear, infiltration of eosinophils and multinucleated cells, and high density of vascular endothelial cells are seen.
![]() Figure 6.Mixed group skin tissue sectionsKeratinocyte hyperplasia is lighter, hair follicles are slightly more vigorously developed, hair follicles have less oil, and the dermis layer is close to normal. A small amount of multinucleated cells infiltrate and the density of vascular endothelial cells is slightly lower.
![]() Table 1.Gel preparation formula Table 2.Observation of spleen Qi deficiency type biological characterization Table 3.Observation results of spleen Qi deficiency type biological characterization Table 4.Comparison of spleen and thymus indexes
Table 5.Comparison of skin indicators
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