| With the development of life science and biotechnology,many natural bio-active factors,for instance,epidermal growth factor(EGF)and superoxide dismutase(SOD1),have been applied to the cosmetic and skin care industry.The bio-activity of functional factors in skin care products is the key to its effectiveness.However,the formula contains a variety of excipients that might affect the biological activities of functional factors.How to select appropriate excipients and proportion for skin care formula is the focus of this thesis.In this study,25 of excipients,extensively used in skin care products,were selected and set up in three concentration gradients of high,medium and low according to the recommended proportion in governmental guideline.The pyrogallol autoxidation method is used to screen the excipients that have an influence on the biological activity of rh-SOD1.Balb/3T3 cell line was used as a cell culture model by MTT assay to screen the excipients that have an influence on the biological activity of rh-EGF.Finally,based on the healing function of rh-EGF in epidermal wounding,the New Zealand rabbits were used to establish an incised wounding skin model and to further clarify the influence of excipients on rh-EGF bioactivity in wound healing.The main conclusions are as follows:(1)The experimental results of pyrogallol autoxidation method showed that 0.20% EDTAˇ2Na had no effect on rh-SOD1 activity within 7 days,0.50% EDTAˇ2Na decreased rh-SOD1 activity to66.53% of the control at d7,and 1.00% EDTAˇ2Na decreased its activity to 60.09% of the control at d3,and 34.52% at d7.Other tested excipients had no effect on the activity of rh-SOD1.As a common chelating agent,EDTA can chelate Cu2+ and Zn2+ from rh-SOD1,leading to the loss of its bio-activity.The results suggested that the reduced proportion of EDTAˇ2Na to an appropriate level can maintain the activity of rh-SOD1 in the products.(2)Cell model experiments showed that 0.15% and 0.40% SOD1 ium hyaluronatethe with a large molecular weight decreased rh-EGF activity to 50% and 45%,respectively,of the control.0.20% and0.40% xanthan gum decreased rh-EGF activity to 38% and 21%,respectively.Other tested excipients had no effect on the activity of rh-EGF.Data suggested that the concentration of sodium hyaluronate with a large molecular weight could be adjusted in the formula to reduce its effect on rh-EGF activity,the combination of sodium hyaluronate with different molecular weights could play a better role,and Xanthan gum could be substituted by other excipient with the same function.(3)The results of animal experiments showed that rh-EGF could promote the healing of rabbit skin wound and the average healing time was 1~3d in advance compared to the saline group.Along with the dose increase,the healing time was further reduced.The most obvious healing effect of wound was observed at 100 ?g/m L of rh-EGF..At the same time,it was also observed that rh-EGF treatment could recover hair growth.It implied that rh-EGF could promote the increment and division of hair follicle stem cells and accelerate the formation of new hair follicles at scar.The healing time of 0.40% xanthan gum group and 10?g/m L rh-EGF plus0.40% xanthan gum group was 1d later than that of normal saline group.Xanthan gum could inhibit the healing effect of rh-EGF on skin wound,consistent with the results from cell culture experiment.The wound healing time of both 0.40% sodium hyaluronate group and 10?g/m L rh-EGF plus 0.40% hyaluronate group was2 d ahead of the saline group,which was as same as the healing time of 10?g/m L rh-EGF group.This was inconsistent with the result from cell culture experiment.This is probably related to the specific physiological function of hyaluronic acid,one of the main components of the extra-cellular matrix that has the function of promoting wound healing. |
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