[0031] Example 2-Experiment of the effect of the total flavonoids of Aina serrata on the photodamage of mouse skin caused by UVB radiation
[0032] 1. Experimental method
[0033] (1) Experimental animals and groups
[0034] Take SPF-grade healthy female Balb/C mice (4-6 weeks), 120, weighing 18-22g, provided by Hubei Experimental Animal Research Center (production license: SCXK (E) 2015-0018).
[0035] The animals were reared in cages according to the standard conditions of clean animals and bred adaptively for 7 days. After that, they were randomly divided into 6 groups according to their body weight, each with 20 animals, which were the blank control group (Normal Control, NC) and the UVB-irradiation model group (UVB-irradiation, UVB), 85% ethanol solvent control group (Vehicle Control, VC), compound menthol ointment positive control group (PositiveControl, PC), 100mg/mL Aina Geranium total flavonoids treatment group prepared in Example 1 (Total Flavonoids 1 , TFE1), the 200 mg/mL Aina Geranium total flavonoids treatment group (Total Flavonoids 2, TFE2) prepared in Example 1. The NC group did not receive any treatment, and the UVB group was only irradiated with UVB and labeled with picric acid.
[0036] (2) Preparation and administration of animal models
[0037] 2 days before the modeling experiment, cut off the long hair on both sides of the back spine of the mouse, with an area of about 3.0cm×3.0cm, and depilate with 8% sodium sulfide. Before each irradiation, after fixing the mouse with a modified fixator, first observe the movement of the mouse's head and whether there is blood congestion on the back skin, and then fix the mouse fixator to expose the shaved area and the rest of the non-irradiated area Cover with anti-radiation cloth, adjust the height and fix the UV lamp.
[0038] The mouse skin photoinjury model was used. Except for the NC group, the other groups of mice were treated as follows: turn on the UVB ultraviolet light, preheat for 15 minutes, the distance between the light source and the back of the mouse was about 20 cm, the irradiation time was 40 minutes, and the radiation intensity was 0.25mW/cm 2 , The irradiation amount is 600mJ/cm 2 , 30min after irradiation, evenly apply the drug to the irradiated area. VC, TFE1 and TFE2 groups are given 0.25mL/head, PC group, 0.1g/head, external coating area 3.0cm×3.0cm, once a day, continuous administration 11d.
[0039] At 5 time points 1, 2, 4, 7, and 11 days after UVB irradiation, 4 mice in each group were sacrificed each time, and the back skin and blood of the mice were taken. After rinsing, weighing, homogenizing and centrifuging the skin tissue, the supernatant is reserved for future use. After the whole blood is allowed to stand at room temperature for 20-30 minutes, it is centrifuged at 4500 rpm for 5 minutes, and the supernatant is taken to obtain the serum, which is stored in a refrigerator at -80°C for use.
[0040] (3) Observation of the thickness of mouse skin epidermis
[0041] Cut mouse skin tissue specimens to make paraffin sections. After HE staining, the thickness of the skin epidermis was observed under a microscope, and the thickness of the skin tissue epidermis was measured by Axio Vision Rel.4.8 software.
[0042] (4) Observation of skin reaction in mice
[0043] After 1 hour of daily administration and before re-administration, observe the changes of the wound with naked eyes, including erythema, edema, crusting, cracking, wrinkling, desquamation, etc., keep recording, and score erythema and edema, and all mice Image acquisition was performed on the skin of the UVB-irradiated area in the depilatory area until the eschar on the wound fell off, and the time when the scab fell off completely was recorded as the time for the mouse to scab off the wound.
[0044] Table 1 Scoring criteria for skin reactions
[0045]
[0046] (5) Measurement of mouse skin thickness and tissue water content
[0047] Take the skin of the same part of the back of each mouse, about 100mg, filter paper to absorb the blood, and weigh the wet tissue weight with an electronic balance. Use a spiral micrometer (minimum accuracy: 0.01mm) to measure the thickness of the skinfolds at 10 points on the center and back of the shaved area, then put it in an oven at 80°C for 24 hours, take out the tissue and weigh the dry weight of the tissue, and calculate the tissue water content by wet and dry method . The relevant formula is as follows:
[0048]
[0049] (6) Statistical analysis methods
[0050] Using SPSS 19.0 statistical software, all data use mean ± standard deviation Indicates that the ANOVA method is used to compare the statistics between groups, and P <0.05 is considered statistically significant.
[0051] 2. Experimental results and analysis
[0052] (1) The effect of the total flavonoids of Aina Geranium on the thickness of skin epidermis in mice with UVB-induced light damage
[0053] Table 2 The effects of different treatments on the thickness of the epidermal layer of mice damaged by UVB radiation ( n=4)
[0054]
[0055]
[0056] Note: Compared with NC group, # P <0.05, ## P <0.01; Compared with UVB group, *P <0.05, **P <0.01; Compared with VC group, Δ P <0.05, ΔΔ P <0.01, same as Table 3-9.
[0057] As shown in Table 2 above, the thickness of the skin layer in the UVB and VC groups increased significantly by 476% and 446% compared with the NC group. On the 1st day, the PC group, TFE1 and TFE2 groups significantly inhibited the increase in photodamaged skin thickness caused by UVB radiation, which decreased by 20.00%, 17.02% and 15.45% compared with the UVB group. On 7d and 11d, TFE1 and TFE2 were better than PC in inhibiting epidermal thickening. The formation of sunburned cells is another major indicator of UV radiation damage. Sunburned cells were easily observed in the UVB group, while the PC, TFE1 and TFE2 groups were few in number, while the NC group had sunburned cells. The above results indicate that the use of compound brain ointment and total flavonoids of Aina Geranium can reduce the photodamage of the skin induced by UVB radiation.
[0058] (2) The effect of the total flavonoids of Aina odoratum on the skin erythema and edema scores of UVB-induced light-damaged mice
[0059] Such as figure 1 with figure 2 As shown, the effects of different treatments on the skin erythema and edema scores of mice with light damage caused by UVB radiation. Through the observation of this experiment, it was found that the skin erythema and edema scores of UVB-induced light-damaged mice showed an increase in the whole process. The trend of decline after high, reached the peak on the 4th day, and then the score gradually decreased, which indicates that the degree of skin damage is gradually recovering. Each treatment group can improve the skin erythema and edema of the UVB group mice to varying degrees, especially on the 11th day Obviously, compared with the UVB group, PC, TFE1 and TFE2 significantly reduced the skin erythema and edema scores of mice (P <0.05, P <0.01).
[0060] (3) The effect of the total flavonoids of Aina serrata on the skin scab shedding time of UVB-induced light-damaged mice
[0061] Such as image 3 It shows the effect of different treatments on the skin scab shedding time of the photodamaged mice caused by UVB radiation. After the observation of this experiment, it was found that on the first day after UVB radiation treatment, the mice in the NC group had tighter skin, soft texture, and skin on the back There are almost no wrinkles, no photo-damaged wounds and skin peeling phenomenon. The skin texture of the mice in the UVB group became hard, thickened, uneven, and hyperkeratosis after UVB radiation. From the 2nd day, the mice in the different treatment groups were exposed to light. The wounds and skin peeling were obvious. Compared with other groups, PC, TFE1 and TFE2 can effectively reduce the time of wound scab shedding (P <0.01)( image 3 ), indicating that the total flavonoids of Aina serrata can effectively promote the healing of light-damaged skin wounds, reduce UV-induced skin shedding, and maintain normal skin conditions.
[0062] (4) The effect of the total flavonoids of Aina Geranium on the water content of the skin tissue of mice with UVB-induced light damage
[0063] Table 3 Different treatments on the water content of skin tissue of mice with light damage caused by UVB radiation ( n=4)
[0064]
[0065] As shown in Table 3, compared with other treatment groups, UVB radiation caused different degrees of reduction in the water content of mouse skin tissue. On the second day, the skin tissue water content of the PC, TFE1 and TFE2 groups was significantly higher than that of the UVB group (P <0.05). At other time points, the skin tissue water content of the PC group was significantly higher than that of the UVB group (P <0.01), the water content of skin tissue in TFE1 and TFE2 groups was significantly higher than that in UVB and VC groups (P <0.01). The above results show that the total flavonoids of Aina Gerani can increase the water content of skin tissue.
[0066] (5) The effect of the total flavonoids of Aina Geranium on the skin tissue thickness of UVB-induced light-damaged mice Table 4 Comparison of different treatments on the skin tissue thickness of UVB-induced light-damaged mice ( n=4)
[0067]
[0068]
[0069] As shown in Table 4, compared with NC, UVB radiation extremely significantly increased the thickness of mouse skin tissue (P <0.01). On the 11th day, the skin tissue thickness of the mice in the TFE1 and TFE2 groups was not significantly different from that of the UVB and VC groups. At other time points, it was extremely significantly lower than the UVB group (P <0.01). The results showed that the total flavonoids of Aina Gerani could inhibit the increase of skin tissue thickness in mice after UVB radiation.
