Enzymatic Preparation of 3-Hydroxyphloridzin and Its Therapeutic Activity in Vitiligo
OBJECTIVE To design and optimize the enzymatic preparation process of 3-hydroxyphloridzin and investigate the in vivo anti-vitiligo activity of 3-hydroxyphloridzin,thus to provide scientific basis for achieving the mass production of 3-hydroxyphloridzin and vitiligo prevention and treatment.METHODS 3-hydroxyphloridzin was prepared in a system containing L-ascorbic acid and crude polyphenol oxidase(PPO)solution,with phloridzin from the aqueous extract of young leaves of Malus hupehensis being the sub-strate.The molar yield of 3-hydroxyphloridzin was taken as the index,and factors including phloridzin concentration,pH,tempera-ture,reaction time,L-ascorbic acid concentration,enzyme concentration,and stirring speed were selected to optimize the preparation process conditions of 3-hydroxyphloridzin by a complete randomized design,Placket-Burman(PB)test,path of steepest ascent meth-od,and central composite design(CCD)-effect surface method.To verify the feasibility of the preparation process,the reaction system was scaled up to 250 L.Besides,Vitiligo model was constructed by applying hydroquinone cream to the back skin of C57BU6 mice for 75 consecutive days,and the mice were randomly divided into blank group,model group,positive group(8-methoxypsoralen(8-mop),4.25 mg·kg-1),and 3-hydroxyphloridzin low,medium,and high dosage group(10,20,and 40 mg·kg-1)on the 20th day of modeling,and were administered with 55 days of gavage.The mice in each group were depilated every 5 days,while the modeling area was videotaped and the hair decoloration was scored.At the end of the administration,blood was taken by removing the mice eye-balls,and the serum tyrosinase(TYR),cholinesterase(CHE)activity,and malondialdehyde(MDA)content of mice were detected using the kit.In addition,skin was taken from the dorsal lesions of mice and sections were stained with hematoxylin-eosin(HE),then under a light microscope,50 hair follicles were observed to count those containing melanin RESULTS The optimal preparation process of 3-hydroxyphloridzin was as follows:phloridzin concentration of 1.70 mmol·L-1,reaction time of 6.0 h,and PPO concen-tration of 25.0 U·mL-1.In the amplified experiment,the reaction solution was decontaminated by a macroporous resin,concentrat-ed,and dried to obtain 334.80 g of crude 3-hydroxyphloridzin with a purity of 70.32％,and the extraction rate of 8.37％,and the pu-rity of 98.10％of the 3-hydroxyphloridzin was obtained by further isolation and purification,which yielded a pure product of 3-hydroxy-phloridzin with a purity of 98.10％and the yield of 68.50％.In vivo activity experiments showed that compared with the model group,the hair decoloration of mice in the 3-hydroxyphloridzin low,medium and high dose groups was significantly improved,with a signifi-cant decrease in hair decoloration scores,serum MDA content and CHE activity(P＜0.05)and a significant increase in the number of melanin-containing hair follicles and serum TYR activity(P＜0.05).CONCLUSION 3-hydroxyphloridzin can be prepared by enzy-matic extraction of the aqueous extract of young leaves of Malus hupehensis,and the method has the feasibility of quantitative produc-tion.3-hydroxyphloridzin has therapeutic activity for vitiligo,and the mechanism of action is related to enhancement of tyrosinase activ-ity to increase melanin synthesis and improvement of oxidative stress damage to slow down the development of vitiligo.
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