Application of forsythia suspensa fruit ultrasonic aqueous extract in preparation of skin light injury protection composition

Abstract

The invention discloses application of an ultrasonic aqueous extract of fructus forsythiae fruits in preparation of a composition for protecting skin light injury. The fructus forsythiae fruit ultrasonic water extract disclosed by the invention has excellent oxidation resistance, can effectively remove DPPH and ABTS free radicals, and shows remarkable iron ion reduction capacity; a certain nitric oxide removal rate is achieved; the compound has a light protection effect on cells, and has no cytotoxicity.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extract application, and particularly relates to application of a forsythia fruit ultrasonic water extract in preparing a skin photodamage protection composition. Background Art

[0002] The skin is the largest organ in the human body and also the outermost protective barrier of the body. It plays an important role in resisting external biological invasion and maintaining the stability of the biological environment in the body. Related studies have shown that ultraviolet radiation can cause the skin to produce excessive reactive oxygen species (ROS), which in turn induces oxidative stress, resulting in a series of skin problems, such as inflammation, cell apoptosis, skin aging, and DNA damage. The mechanisms and signaling pathways of skin photodamage are diverse. It is known that UVB can activate signaling pathways including p53, hypoxia-inducible factor, and mitogen-activated protein kinase (MAPK), such as mitogen-activated protein kinase (MAPK) / activator protein-1 (AP-1), nuclear factor κB, and Keap1-nuclear factor E2-related factor 2 (Nrf2) / antioxidant response element (ARE) signaling pathways. At present, many studies have induced oxidative stress or DNA damage by irradiating cells with UVB, and analyzed how to reduce ultraviolet-induced cell apoptosis by detecting cell survival rate and conducting ROS tests, and then evaluated the ability of skin photodamage protection.

[0003] Forsythia suspense is the dried fruit of Forsythia suspense (Thunb.) Vahl, a plant of the Oleaceae family. It was first recorded in Lingshu·Carbuncle. As a major medicinal material, Forsythia suspense is widely used in clinical medicine and is one of the raw materials for Chinese patent medicines such as Lianhua Qingwen Capsules, Shuanghuanglian Oral Liquid, and Qingrejiedu Tablets. Studies have shown that the fruit (dried fruit) of Forsythia suspense has strong antioxidant, anti-inflammatory, antibacterial and anti-tumor activities, and can improve liver function by inhibiting inflammatory cell infiltration. In addition, in vivo experiments on aging mice induced by D-galactose have also confirmed that Forsythia suspense can significantly increase the activity of T-SOD, GSH-Px and CAT, and has an anti-aging effect. At present, studies have shown that plant extracts and related chemicals have a significant and effective effect on the protection of ultraviolet-induced skin photodamage. Although Forsythia suspense has potential therapeutic and preventive effects on some diseases, related research on skin photodamage protection has not been reported. Summary of the invention

[0004] The present invention aims to provide the use of ultrasonic water extract of Forsythia suspensa fruit in preparing a skin photodamage protection composition.

[0005] The technical solution of the present invention is as follows:

[0006] Use of ultrasonic water extract of Forsythia suspensa fruit in preparing a composition for protecting skin from photodamage.

[0007] In a preferred embodiment of the present invention, the method for preparing the ultrasonic water extract of Forsythia suspensa comprises the following steps:

[0008] (1) grinding the fruit of Forsythia suspensa into powder, mixing the powder with pure water, and allowing the powder to stand and soak;

[0009] (2) subjecting the material obtained in step (1) to ultrasonic treatment, followed by centrifugation to obtain a supernatant;

[0010] (3) The supernatant obtained in step (2) is filtered and concentrated under reduced pressure, and then freeze-dried to obtain the product.

[0011] Further preferably, in the step (1), the mass ratio of the powder to pure water is 1:10.

[0012] More preferably, in step (1), the temperature of the static soaking is 4° C. and the time is 24 hours.

[0013] Further preferably, in the step (2), the ultrasonic treatment specifically includes: during the ultrasonic process, the efficacy is set to 85%, an intermittent mode of working for 30 seconds and resting for 90 seconds is adopted, and the total treatment time reaches 1 hour.

[0014] A composition for protecting skin from light damage, the effective component of which comprises ultrasonic water extract of forsythia fruit.

[0015] In a preferred embodiment of the present invention, the method for preparing the ultrasonic water extract of Forsythia suspensa comprises the following steps:

[0016] (1) grinding the fruit of Forsythia suspensa into powder, mixing the powder with pure water, and allowing the powder to stand and soak;

[0017] (2) subjecting the material obtained in step (1) to ultrasonic treatment, followed by centrifugation to obtain a supernatant;

[0018] (3) The supernatant obtained in step (2) is filtered and concentrated under reduced pressure, and then freeze-dried to obtain the product.

[0019] Further preferably, in the step (1), the mass ratio of the powder to pure water is 1:10.

[0020] More preferably, in step (1), the temperature of the static soaking is 4° C. and the time is 24 hours.

[0021] Further preferably, in step (2), the ultrasonic treatment specifically includes: during the ultrasonic process, the efficacy is set to 85%, an intermittent mode of working for 30 seconds and resting for 90 seconds is adopted, and the total treatment time reaches 1 hour.

[0022] The beneficial effects of the present invention are:

[0023] 1. The ultrasonic water extract of the fruit of Forsythia suspensa in the present invention has excellent antioxidant properties, can effectively remove DPPH and ABTS free radicals, and exhibits significant iron ion reducing ability.

[0024] 2. The ultrasonic water extract of the fruit of Forsythia suspensa in the present invention has a certain nitric oxide removal rate.

[0025] 3. The ultrasonic water extract of the fruit of Forsythia suspensa in the present invention has photoprotective effect on cells and is non-cytotoxic. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results show the effects of different concentrations of ultrasonic water extracts of Forsythia suspensa fruit in Example 2 of the present invention on the DPPH free radical scavenging ability.

[0027] Figure 2 The results show the effects of different concentrations of ultrasonic water extract of Forsythia suspensa fruit on the ABTS free radical scavenging ability in Example 3 of the present invention.

[0028] Figure 3 The effect of different concentrations of ultrasonic water extract of Forsythia suspensa fruit on the iron ion reducing power in Example 4 of the present invention is shown.

[0029] Figure 4 The effect of different concentrations of ultrasonic water extract of Forsythia suspensa fruit in Example 5 of the present invention on the scavenging ability of nitric oxide is shown.

[0030] Figure 5 The scanning analysis of the ultrasonic water extract of Forsythia suspensa fruit in Example 6 of the present invention under the UV-visible spectrum is shown.

[0031] Figure 6 The effect of the ultrasonic water extract of Forsythia suspensa in Example 7 of the present invention on the survival rate of HaCaT cells and the analysis of the active oxygen response after UVB radiation (300mJ / cm2, 1h) are shown.

[0032] Figure 7 The results show (A) the effect of ultrasonic water extract of Forsythia suspensa fruit on the viability of HaCaT cells in Example 8 of the present invention and (B) the analysis of cell viability after UVB irradiation.

[0033] Figure 8 The results show the observation of the cell survival rate of HaCaT cells irradiated with UVB by the ultrasonic water extract of Forsythia suspensa in Example 9 of the present invention. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0035] Example 1 Preparation of ultrasonic water extract of Forsythia suspensa

[0036] Weigh 50g of Forsythia fruit (purchased from Taobao, store name: Shenzetang Medicinal Materials Stack), grind it into powder by a grinder (A11, IKA, Germany), and then put it into a beaker for use. Next, the Forsythia fruit powder was mixed with pure water at a material ratio of 1:10 and soaked. The mixture was left to stand at 4°C for 24 hours to ensure sufficient soaking. Subsequently, the mixture was ultrasonically treated using a JY92-IIN ultrasonic instrument (produced by Ningbo Xinzhi Biotechnology Co., Ltd.). During the ultrasonic process, the efficacy was set to 85%, and an intermittent mode of working for 30s and resting for 90s was used, with a total treatment time of 1h. This ultrasonic treatment helps to further extract the active ingredients in Forsythia. After the ultrasonic treatment, the residue in the mixture was precipitated by centrifugation. Then, the supernatant was filtered using filter paper to remove any residual impurities. The filtered supernatant was further concentrated by a N-1210 vacuum concentrator (Tokyo Rika, Japan). Then, the product was freeze-dried using a FDU-1200 freeze dryer (Tokyo Rika, Japan) to obtain the ultrasonic water extract of Forsythia suspensa fruit.

[0037] Example 2 DPPH free radical scavenging rate evaluation

[0038] Weigh 0.0025g DPPH powder, dissolve it in 10mL ethanol, and prepare DPPH mother solution. Subsequently, the mother solution is stored in a refrigerator at 4°C and avoid light to maintain its stability. When in use, take out an appropriate amount of dilution from the mother solution to prepare a DPPH working solution until its absorbance (OD517) at a wavelength of 517nm is between 0.8 and 1.0 to ensure that it is suitable for the experiment. Next, take 20μL of ultrasonic water extract of Forsythia fruit obtained in Example 1 of different concentrations, add 180μL of the prepared DPPH working solution, and use a pipette to gently blow to ensure uniform mixing. After completing the mixing for 5min, use an ELISA instrument (Infinite Plex, Tecan, Switzerland) to measure the absorbance at a wavelength of 517nm, and record the obtained data. When calculating the DPPH free radical scavenging rate, the following formula is used: DPPH free radical scavenging rate (%) = (OD517 absorbance of the control group - OD517 absorbance of the ultrasonic water extract of Forsythia suspensa obtained in Example 1) / OD517 absorbance of the control group × 100.

[0039] like Figure 1As shown in the figure, at a concentration of 0.0625 mg / mL, the scavenging rate of the ultrasonic water extract of Forsythia suspensa fruit prepared in Example 1 on DPPH free radicals was only 22.59%. However, as the concentration increased, its scavenging rate showed a significant upward trend. In particular, under the high concentration condition of 1 mg / mL, the extract showed a DPPH free radical scavenging efficiency of more than 81.04%.

[0040] Example 3 Evaluation of ABTS free radical scavenging rate

[0041] The total antioxidant capacity detection kit (ABTS method) provided by Shanghai Biyuntian Biotechnology Co., Ltd. was used to evaluate the ABTS free radical scavenging ability of the ultrasonic water extract of Forsythia fruit prepared in Example 1 at different concentrations. First, according to the instructions of the kit, the ABTS working solution was accurately prepared. Then, 20 μL of the ultrasonic water extract sample of Forsythia fruit prepared in Example 1 was taken, added to 180 μL of ABTS working solution, and mixed thoroughly. The mixed solution was allowed to stand for 5 minutes to allow the reaction to proceed fully. Next, the absorbance of the solution was measured at a wavelength of 734 nm using an ELISA reader (Infinite Plex, Tecan, Switzerland), and the experimental data was recorded. When calculating the ABTS free radical scavenging rate, the following formula was used: ABTS free radical removal rate (%) = (OD734 absorbance of the control group - OD734 absorbance of the ultrasonic water extract of Forsythia fruit) / OD734 absorbance of the control group × 100.

[0042] like Figure 2 As shown in the figure, at a concentration of 0.05 mg / mL, the scavenging rate of the ultrasonic water extract of Forsythia fruit prepared in Example 1 on ABTS free radicals can reach 56.50%. Similarly, with the increase of concentration, its scavenging rate shows a significant upward trend. At a concentration of 0.1 mg / mL, the extract shows an ABTS free radical scavenging efficiency of more than 90.0%.

[0043] Example 4 FRAP reducing power evaluation

[0044] The total antioxidant capacity detection kit (FRAP method) provided by Shanghai Biyuntian Biotechnology Co., Ltd. was used to evaluate the reducing power of the ultrasonic water extract of Forsythia suspensa fruit prepared in Example 1 at different concentrations. According to the instructions of the kit, the FRAP working solution was prepared. Then, 20 μL of the ultrasonic water extract sample of Forsythia suspensa fruit prepared in Example 1 was taken, added to 180 μL of the FRAP working solution, and mixed thoroughly. The mixed solution was allowed to stand for 5 minutes to allow the reaction to proceed fully. Next, an ELISA reader (Infinite Plex, Tecan, Switzerland) was used to measure the absorbance of the solution at a wavelength of 593 nm, and the experimental data was recorded. Using the FeSO 4 7H 2O was prepared into different concentrations and the standard curve was determined. The antioxidant capacity of the samples was evaluated by the standard curve.

[0045] like Figure 3 As shown in Figure 2, at a concentration of 0.0625 mg / mL, the ultrasonic water extract of Forsythia suspensa obtained in Example 1 has an equivalent activity to 0.33 mM FeSO 4 The antioxidant capacity of the forsythia fruit was 0.5 mg / mL, and its reducing power tended to increase with the increase of concentration. At a concentration of 0.5 mg / mL, the ultrasonic water extract of the forsythia fruit prepared in Example 1 had an antioxidant capacity equivalent to 2.75 mM FeSO 4 antioxidant capacity.

[0046] Example 5 Evaluation of Nitric Oxide Clearance Rate

[0047] Prepare 20mM sodium nitroprusside solution, take 50μL 20mM sodium nitrate solution and 50μL ultrasonic water extract of Forsythia fruit prepared in Example 1 with different dilution gradients, stand for reaction at room temperature for 1h, and use the nitric oxide detection kit provided by Shanghai Biyuntian Biotechnology Co., Ltd. to evaluate the nitric oxide removal rate of ultrasonic water extract of Forsythia fruit prepared in Example 1 with different concentrations. The mixed solution is measured at a wavelength of 540nm using an ELISA reader (Infinite Plex, Tecan, Switzerland) to measure the absorbance of the solution, and record the experimental data. When calculating the nitric oxide clearance rate, the following formula is used: Nitric oxide removal rate (%) = (OD540 absorbance of the control group - OD540 absorbance of ultrasonic water extract of Forsythia fruit prepared in Example 1) / OD540 absorbance of the control group × 100.

[0048] As a core molecule in inflammatory response, excessive production of nitric oxide is closely related to the pathological response of acute or chronic inflammatory diseases. Therefore, effectively reducing the production of nitric oxide has the potential to have anti-inflammatory effects. Figure 4 As shown, at a concentration of 0.0625 mg / mL, the scavenging rate of the ultrasonic water extract of Forsythia suspensa fruit prepared in Example 1 on nitric oxide was only 12.09%. However, as the concentration increased, its scavenging rate showed a significant upward trend. In particular, under the high concentration condition of 1 mg / mL, the extract showed a nitric oxide removal efficiency of more than 51.34%.

[0049] Example 6 Ultraviolet-visible wavelength scanning analysis of the ultrasonic water extract of the fruit of Forsythia suspensa obtained in Example 1

[0050] 0.1 mg / mL of the ultrasonic water extract of Forsythia suspensa obtained in Example 1 was taken and the absorbance was tested using a spectrophotometer (UV-1800, Shimadzu Instrument Co., Ltd., Japan), and the absorbance at each wavelength was recorded.

[0051] The wavelength range of UVA (long-wave ultraviolet rays) is approximately from 320nm to 400nm, the wavelength range of UVB (medium-wave ultraviolet rays) is approximately from 280nm to 320nm, and the wavelength range of UVC (short-wave ultraviolet rays) is approximately from 100nm to 280nm. Before reaching the earth's surface, UVC radiation is almost completely absorbed by the ozone layer in the atmosphere, so it does not reach the ground naturally. In contrast, UVA and UVB, as common types of ultraviolet rays in people's daily environment, have different effects on the skin. Not only do they promote the deposition of melanin in the skin, they may also cause redness and blister formation on the skin, and long-term exposure may even increase the risk of skin cancer. Figure 5 As shown, the spectral characteristics of the ultrasonic water extract of Forsythia suspensa obtained in Example 1 in the wavelength range of 270nm to 370nm, wherein two significant absorption peaks indicate its potential ability to protect against UVA and UVB radiation.

[0052] Example 7 Cytotoxicity Analysis of Ultrasonic Aqueous Extract of Fructus Forsythiae Prepared in Example 1

[0053] Human skin keratinocytes (HaCaT cells) were cultured in a 96-well culture plate using bovine serum DMEM high-glucose culture medium. When the cell density reached about 70-80%, it was confirmed by microscopic observation, and then the culture medium in the culture plate was carefully removed. Next, 195 μL of bovine serum DMEM high-glucose culture medium and 5 μL of different concentrations of the ultrasonic water extract of Forsythia fruit prepared in Example 1 were added to each well, and then the culture plate was placed in a carbon dioxide incubator and cultured for 1 h and 24 h respectively. After the culture was completed, 10 μL of 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) solution (5 mg / mL) was added to each well, and the culture was continued in a carbon dioxide incubator for 4 h. Afterwards, the culture medium in the 96-well plate was carefully removed, and 100 μL of dimethyl sulfoxide (DMSO) was immediately added to each well to allow the cells to fully react with DMSO for 5 min. Finally, the absorbance (OD490) of each well was detected using a microplate reader at a wavelength of 490 nm, and the obtained data was recorded. This result was used to evaluate the effect of the ultrasonic water extract of Forsythia suspensa obtained in Example 1 on the proliferation of HaCaT cells.

[0054] The interaction between UVB radiation and reactive oxygen species (ROS) has significant biological significance. UVB radiation can trigger the generation of reactive oxygen species, and these reactive oxygen molecules can interfere with key signal transduction pathways in cells, causing cell damage, promoting inflammatory responses, and inducing cell apoptosis. Therefore, effectively controlling the level of reactive oxygen species is crucial to preventing cell damage, inhibiting inflammation, and preventing cell apoptosis. Figure 6The effect of the ultrasonic water extract of Forsythia suspensa obtained in Example 1 on cell viability was revealed. After the extract was applied for 1 hour, the cells showed no toxic reaction and the cell survival rate remained at 100%. For HaCaT cells treated with UVB radiation, the level of reactive oxygen species increased significantly. However, as the concentration of the ultrasonic water extract of Forsythia suspensa obtained in Example 1 increased, the content of reactive oxygen species showed a significant downward trend. The results of this example show that the ultrasonic water extract of Forsythia suspensa obtained in Example 1 exhibited significant antioxidant properties in both in vitro biochemical experiments and cell culture models, showing that it has important application prospects in preventing cell inflammation and damage.

[0055] Example 8 Determination of ROS generation in HaCaT cells induced by UVB

[0056] Human skin keratinocytes (HaCaT cells) were cultured in a 96-well culture plate using bovine serum DMEM high-glucose culture medium. When the cell density reached about 70-80%, it was confirmed by microscopic observation, and then the culture medium in the culture plate was carefully removed. Next, 195 μL of bovine serum-free DMEM high-glucose culture medium and 5 μL of different concentrations of the ultrasonic water extract of Forsythia fruit prepared in Example 1 were added to each well, and the culture plate was placed in a carbon dioxide incubator for 1 hour, and then the cells were placed in a cell ultraviolet irradiation system (Bio-Sun, Vilber Bio Imaging, France) and irradiated at 300 mJ / cm 2 Then, the cells were placed in a carbon dioxide incubator for 1 hour, the culture medium was removed, and 100 μL of DMEM high glucose without bovine serum and 1 μL of fluorescent probe (active oxygen detection kit, Shanghai Bio-Tech Biotechnology Co., Ltd.) were added. The cells were placed in a carbon dioxide incubator for 1 hour, washed twice with phosphate buffer, 100 μL of DMEM high glucose without bovine serum was added, the fluorescence value was measured with an enzyme reader, and the data was recorded.

[0057] Figure 7 The effect of the ultrasonic water extract of Forsythia suspensa obtained in Example 1 on cell viability was revealed. After 24 hours of the extract, the cells did not show severe toxicity, and at a concentration of 0.2 mg / mL, the cell survival rate could still be maintained at 89.5%. For HaCaT cells treated with UVB radiation, the cell survival rate was significantly reduced to 38.3% after 24 hours. However, with the increase in the concentration of the ultrasonic water extract of Forsythia suspensa obtained in Example 1, the cell survival rate was significantly improved, and at a concentration of 0.2 mg / mL, the cell survival rate could reach 80.2%. This research result reveals the photoprotective efficacy of the ultrasonic water extract of Forsythia suspensa obtained in Example 1, confirming that it can protect cells from apoptosis induced by UVB radiation.

[0058] Example 9 Determination of cell viability after UVB irradiation of HaCaT cells

[0059] Human skin keratinocytes (HaCaT cells) were cultured in a 96-well culture plate using bovine serum DMEM high-glucose culture medium. When the cell density reached about 70-80%, it was confirmed by microscopic observation, and then the culture medium in the culture plate was carefully removed. Next, 195 μL of bovine serum-free DMEM high-glucose culture medium and 5 μL of different concentrations of the ultrasonic water extract of Forsythia fruit prepared in Example 1 were added to each well, and the culture plate was placed in a carbon dioxide incubator for 1 hour, and then the cells were placed in a cell ultraviolet irradiation system (Bio-Sun, Vilber Bio Imaging, France) and irradiated at 225 mJ / cm 2 , and then continue to culture in a carbon dioxide incubator for 24 hours. After the culture is completed, take pictures using an optical microscope to observe the cell growth status. Then add 10μL of MTT solution (5mg / mL) to each well and continue to culture in a carbon dioxide incubator for 4 hours. After that, carefully remove the culture medium in the 96-well plate, and immediately add 100μL of dimethyl sulfoxide to each well to allow the cells to fully react with DMSO for 5 minutes. Finally, use a spectrophotometer at a wavelength of 490nm to detect the absorbance (OD490) of each well, and record the obtained data.

[0060] Figure 8 The results of observation of HaCaT cells treated with UVB radiation under an optical microscope after 24 hours were revealed. Compared with the control group that was not treated with UVB radiation and did not add the ultrasonic water extract of Forsythia fruit prepared in Example 1, the cells of the latter almost covered the entire bottom of the culture dish. In contrast, the number of cells that were only treated with UVB radiation without adding the ultrasonic water extract of Forsythia fruit prepared in Example 1 was significantly reduced. Under UVB radiation treatment, as the concentration of the ultrasonic water extract of Forsythia fruit prepared in Example 1 increased, cell growth showed an increasing trend, which is consistent with the cell survival rate results detected by the MTT experiment.

[0061] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. Use of ultrasonic water extract of Forsythia suspensa fruit in preparing a composition for protecting skin from photodamage.

2. The use according to claim 1, characterized in that: The preparation method of the ultrasonic water extract of Fructus Forsythiae comprises the following steps: (1) grinding the fruit of Forsythia suspensa into powder, mixing the powder with pure water, and allowing the powder to stand and soak; (2) subjecting the material obtained in step (1) to ultrasonic treatment, followed by centrifugation to obtain a supernatant; (3) The supernatant obtained in step (2) is filtered and concentrated under reduced pressure, and then freeze-dried to obtain the product.

3. The use according to claim 2, characterized in that: In the step (1), the mass ratio of the powder to pure water is 1:

10.

4. The use according to claim 3, characterized in that: In the step (1), the temperature of the static soaking is 4° C. and the time is 24 hours.

5. The use according to claim 2, characterized in that: In the step (2), the ultrasonic treatment specifically includes: during the ultrasonic process, the efficacy is set to 85%, an intermittent mode of working for 30 seconds and resting for 90 seconds is adopted, and the total treatment time reaches 1 hour.

6. A composition for protecting skin from photodamage, characterized in that: Its active ingredients include ultrasonic water extract of Forsythia suspensa fruit.

7. A skin photodamage protection composition according to claim 6, characterized in that: The preparation method of the ultrasonic water extract of Fructus Forsythiae comprises the following steps: (1) grinding the fruit of Forsythia suspensa into powder, mixing the powder with pure water, and allowing the powder to stand and soak; (2) subjecting the material obtained in step (1) to ultrasonic treatment, followed by centrifugation to obtain a supernatant; (3) The supernatant obtained in step (2) is filtered and concentrated under reduced pressure, and then freeze-dried to obtain the product.

8. A skin photodamage protection composition according to claim 7, characterized in that: In the step (1), the mass ratio of the powder to pure water is 1:

10.

9. A skin photodamage protection composition according to claim 8, characterized in that: In the step (1), the temperature of the static soaking is 4° C. and the time is 24 hours.

10. The skin photodamage protection composition according to claim 7, characterized in that: In the step (2), the ultrasonic treatment specifically includes: during the ultrasonic process, the efficacy is set to 85%, an intermittent mode of working for 30 seconds and resting for 90 seconds is adopted, and the total treatment time reaches 1 hour.

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