Phloretin-matrine co-crystal, and preparation method and application thereof

Abstract

The application discloses a phloretin-matrine co-crystal and a preparation method and application thereof, relates to the technical fields of natural active ingredient crystal modification, daily chemical raw materials and skin external drugs, and has the characteristic diffraction peaks of X-ray powder diffraction patterns at diffraction angles 2theta of 7.2°, 10.4°, 14.8°, 18.2°, 22.6° and 26.0°, and the relative peak intensities of the peaks are 100%, 89%, 81%, 76%, 65% and 58% respectively, wherein the error tolerance of the diffraction angle is ±0.2°; the molar ratio of phloretin to matrine in the co-crystal is 1:1; and the co-crystal is a yellow crystalline solid; the liquid-assisted grinding method and the anti-solvent crystallization method are used to construct the co-crystal with a fixed molar ratio of phloretin to matrine, the solubility and stability are improved, the co-crystal is uniformly dispersed, the co-crystal has the effects of anti-inflammation, soothing and antioxidation, the two mild processes can be flexibly prepared, and the co-crystal is suitable for the fields of daily chemicals and medicines.

Description

Technical Field

[0001] This invention relates to the fields of natural active ingredient crystal modification, daily chemical raw materials and topical skin drugs, specifically to a phloretin-matrine cocrystal and its preparation method and application. Background Technology

[0002] Phloretin and matrine are both natural substances with excellent biological activity and high application value in skincare and topical dermatological medicine. Phloretin has outstanding antioxidant capacity, which can resist damage to the skin caused by external environmental factors, while also regulating skin condition and soothing discomfort. Matrine has good anti-inflammatory effects and can improve various skin problems. With the increasing demand for products containing natural active ingredients, combining these two types of active substances has become a mainstream research direction in the industry. The two substances are often directly compounded and added to cosmetics or topical preparations, relying on their synergistic effect to enhance the overall efficacy of the product. Crystal modification is a common technique for improving the application performance of natural active ingredients. Co-crystallization technology, as an important method in crystal engineering, can change the original crystal structure of substances and optimize physicochemical properties. It is also increasingly being applied to the modification research of natural active components, providing new technical ideas for improving the application performance of these two types of substances.

[0003] The current method of simply combining phloretin and matrine physically has several significant drawbacks. The crystal structures of the two raw materials remain unchanged, limiting their inherent solubility. Solubility varies considerably in media with different pH levels, easily leading to incomplete dissolution and preventing the active ingredients from functioning properly. The resulting system exhibits poor stability, easily resulting in uneven component distribution and crystal precipitation during product storage and processing, impacting overall product quality and user experience. Furthermore, conventional mixing methods struggle to achieve a stable bond between the two active components, failing to fully realize their synergistic effects. Traditional preparation methods are often simplistic, requiring specific equipment and environmental adaptability, making it difficult to balance small-scale preparation with industrial-scale production. Additionally, the unmodified raw materials exhibit poor dispersion in various formulation matrices, further limiting their widespread use in multiple product categories. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies by providing a phloretin-matrine cocrystal, its preparation method, and its applications. This cocrystal combines two active components in a fixed molar ratio to form a novel crystal structure, effectively improving the solubility of the raw materials. It exhibits good solubility in various acidic and alkaline environments and maintains stable crystal properties. This invention employs liquid-assisted grinding and anti-solvent crystallization methods for preparation. Both processes are mild and standardized, and through refined process control, products with uniform quality can be obtained, making it suitable for experimental research and development as well as industrial production. This cocrystal combines the efficacy advantages of both raw materials, possessing antioxidant, anti-inflammatory, and soothing effects. It can be applied to various skincare products and topical skin preparations, and is compatible with multiple product dosage forms.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: Firstly, a phlorizin-matrine eutectic, wherein the X-ray powder diffraction pattern of the eutectic exhibits characteristic diffraction peaks at diffraction angles 2θ of 7.2°, 10.4°, 14.8°, 18.2°, 22.6°, and 26.0°, with relative peak intensities of 100%, 89%, 81%, 76%, 65%, and 58%, respectively, and the diffraction angles have an error tolerance of ±0.2°; the molar ratio of phlorizin to matrine in the eutectic is 1:1; the eutectic is a yellow crystalline solid.

[0006] Furthermore, the equilibrium solubility of the eutectic in pure water at 37°C is 68.5±4.2 μg / mL; the equilibrium solubility in hydrochloric acid solution at 37°C and pH 1.2 is 102.6±6.8 μg / mL; and the equilibrium solubility in phosphate buffer solution at 37°C and pH 6.8 is 78.3±5.1 μg / mL.

[0007] Secondly, a method for preparing phloretin-matrine eutectic using a liquid-assisted grinding method, the specific steps of which are as follows: a1. Weigh out phloretin and matrine in a 1:1 molar ratio and place them in a mortar. a2, add anhydrous ethanol, wherein the amount of anhydrous ethanol is 0.1 to 0.4 ml per gram of total solid mass; a3, grind at room temperature for 20-30 minutes; a4, the obtained solid is dried at 40-50℃ for 8-12 hours.

[0008] Furthermore, in step a1, phloretin and matrine are placed in a vacuum drying oven and dried at 40℃ and -0.08MPa for 2 hours before weighing; the purity of phloretin is ≥98% and the moisture content is ≤0.5%; the purity of matrine is ≥98% and the moisture content is ≤0.5%; the weighing is performed using an electronic analytical balance with an accuracy of 0.1mg, and the molar ratio deviation does not exceed ±1%.

[0009] Furthermore, in step a1, phloretin and matrine are placed in a vacuum drying oven and dried at 40℃ and -0.08MPa for 2 hours before weighing; the purity of phloretin is ≥98% and the moisture content is ≤0.5%; the purity of matrine is ≥98% and the moisture content is ≤0.5%; the weighing is performed using an electronic analytical balance with an accuracy of 0.1mg, and the molar ratio deviation does not exceed ±1%.

[0010] A method for preparing phlorizin-matrine eutectic using an anti-solvent crystallization method, the specific steps of which are as follows: b1, mix phloretin and matrine in a 1:1 molar ratio, add anhydrous ethanol, wherein the amount of anhydrous ethanol is 8 to 15 times the total solid mass by volume mass ratio; b2, cool to room temperature, add deionized water dropwise, 10 μL each time, shake well after each addition and let stand for 30 seconds until the volume ratio of ethanol to water is 1:1.5 to 1:3. b3, allow the solvent to evaporate and crystals to grow at room temperature for 24–48 hours; b4. Centrifuge at 10,000 rpm for 10 minutes to collect the solid, and dry at 40-50°C for 8-12 hours.

[0011] Furthermore, in step b1, the oscillation speed of the constant temperature water bath shaker is 150 rpm, and the oscillation time is 20 to 40 minutes; in step b2, the ambient temperature for adding deionized water is 20 to 25°C, and the relative humidity is ≤60%; after each drop of deionized water is added, the water is shaken for 10 seconds; when the addition is stopped, the volume ratio of ethanol to water is 1:1.5 to 1:3.

[0012] Furthermore, in step b3, the solution obtained in step b2 is transferred to a glass crystallizing dish, with a solution thickness not exceeding 5 mm; the mouth of the crystallizing dish is covered with plastic wrap and 10-15 small holes with a diameter of 0.5 mm are punched in the plastic wrap; the crystal growth process is carried out in a horizontal, shockproof, and light-proof environment; in step b4, the drying is carried out in a vacuum drying oven, and after drying, the vacuum state is maintained and cooled to room temperature, and the resulting solid is ground and passed through an 80-mesh standard sieve; before centrifugation, the suspension is stirred evenly, and the centrifuge tube balancing error does not exceed 0.1 g.

[0013] Thirdly, an application of a phloretin-matrine cocrystal, wherein the cocrystal can be used to prepare anti-inflammatory, soothing, and antioxidant cosmetics or topical medications; the cosmetics include toners, serums, lotions, creams, masks, eye creams, and acne treatment products; the topical medications include ointments, creams, gels, and lotions for treating eczema, dermatitis, acne, and pruritus; the cocrystal is added in cosmetics at a rate of 0.1%-5%; the cocrystal is added in topical medications at a rate of 0.5%-10%.

[0014] Compared with existing technologies, this phlorizin-matrine eutectic, its preparation method, and its application have the following advantages: I. This invention optimizes the binding morphology of the two active components by constructing a co-crystal structure of phloretin and matrine with a fixed molar ratio, effectively improving the solubility shortcomings inherent in single raw materials. The co-crystal system significantly enhances the solubility of the active ingredients in various acidic and alkaline environments. Whether in neutral water or acidic and weakly acidic media, it maintains a stable and excellent solubility, laying the foundation for the active ingredients to fully exert their effects. Simultaneously, the regular crystal morphology makes the physicochemical properties of the substances more stable, reducing the likelihood of component precipitation or changes in properties during storage and use. Compared to using the two raw materials alone, the co-crystal form allows the active substances to be more uniformly dispersed in various matrices, adapting to the production and processing requirements of cosmetics and topical preparations, and ensuring the continuity and uniformity of efficacy release during product use.

[0015] II. This invention prepares the target eutectic through two different process routes, adaptable to different production scenarios and equipment conditions, thus improving the flexibility and practicality of the preparation method. Both preparation processes are simple to operate, with mild reaction conditions, requiring no complex high-temperature and high-pressure environment throughout, and the overall process is easy to control, facilitating large-scale application. The preparation process involves standardized control of raw material purity and proportions, combined with meticulous operational details, ensuring the stable production of uniform crystalline products. This eutectic combines the efficacy characteristics of the two raw materials, possessing anti-inflammatory, soothing, and antioxidant effects. Its application scope covers a wide range of skincare products and topical skin preparations. A reasonable addition range ensures efficacy while avoiding discomfort caused by excessive ingredient intake, broadening the application space of these two natural active ingredients in the daily chemical and pharmaceutical fields.

[0016] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0018] Figure 1 Flowchart of the liquid-assisted milling process for preparing eutectic crystals; Figure 2 Flowchart of the antisolvent crystallization process for preparing eutectic crystals; Figure 3 X-ray powder diffraction patterns of matrine, phloretin, and phloretin-matrine eutectics prepared by different methods and proportions. Detailed Implementation

[0019] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0020] Example 1: The preparation of phlorizin-matrine eutectic by liquid-assisted grinding method is as follows: Figure 1 As shown.

[0021] Phloretin and matrine with a purity of ≥98% and a moisture content of ≤0.5% were selected and placed in a vacuum drying oven. The temperature was set at 40℃ and the vacuum degree at -0.08MPa. The mixture was vacuum dried at a constant temperature for 2 hours to remove residual moisture from the raw materials.

[0022] Using an electronic analytical balance with an accuracy of 0.1 mg, the two raw materials were weighed according to the molar ratio of phloretin to matrine = 1:1, and the molar ratio deviation was controlled to be ≤ ±1%. The weighed raw materials were then transferred together into a clean mortar.

[0023] Add anhydrous ethanol dropwise to the mortar, at a rate of 0.1–0.4 mL per gram of total solid mass. After each addition of anhydrous ethanol, manually grind for 1.5 minutes until all powder is uniformly wetted. The entire process should be carried out at room temperature of 20–25°C and relative humidity ≤60%, with continuous grinding for 25 minutes. Every 5 minutes, use a scraper to scrape off the powder adhering to the inner wall and bottom of the mortar and mix thoroughly to ensure uniform grinding.

[0024] The ground yellow wet solid was transferred to a tray and placed in a forced-air drying oven. The temperature was set at 45℃ and dried at a constant temperature for 10 hours to obtain a yellow crystalline solid, which is the phloretin-matrine eutectic.

[0025] The product yield in this embodiment was 84.6%; the obtained eutectic molar ratio was phloretin:matrine = 1:1, and the appearance was uniform yellow crystals.

[0026] Example 2: The preparation of phlorizin-matrine eutectics by antisolvent crystallization (1:1, 1:2, 2:1 molar ratio) is as follows: Figure 2 As shown.

[0027] Take the pretreated phloretin and matrine from Example 1 and mix them in molar ratios of 1:1, 1:2, and 2:1, respectively. Add anhydrous ethanol to the mixture, with the amount of ethanol being 8-15 times the total solid mass. Place the system in a constant temperature water bath shaker at 40°C and 150 rpm for 30 minutes until the solid is completely dissolved, yielding a clear solution.

[0028] Cool the solution naturally to room temperature (20~25℃, relative humidity ≤60%). Add deionized water dropwise using a pipette, with a single drop volume of 10μL. Shake the container for 10s after each drop and let it stand for 30s. Continue adding until the volume ratio of ethanol to deionized water is 1:1.5~1:3. At this point, the solution will be slightly turbid. Stop adding the solution.

[0029] Transfer the above suspension to a flat-bottomed glass crystallizer, controlling the solution layer thickness to ≤5mm; cover the mouth of the crystallizer with plastic wrap, and use a fine needle to poke 12 small holes with a diameter of 0.5mm in the plastic wrap for ventilation. Place the crystallizer in a horizontal, shockproof, and light-proof environment, and allow it to stand at room temperature for 36 hours to evaporate the solvent and grow crystals.

[0030] The crystallized suspension was thoroughly stirred and dispensed into centrifuge tubes, ensuring the tube balancing error was ≤0.1g. The tubes were centrifuged at 10000 rpm for 10 minutes, and the bottom solid precipitate was collected. The solid was then transferred to a vacuum drying oven and dried under vacuum at 45℃ for 10 hours. After drying, the solid was allowed to cool naturally to room temperature while still under vacuum. It was then removed, ground, and passed through an 80-mesh standard sieve to obtain the final eutectic crystal.

[0031] In this embodiment, phlorizin-matrine eutectic was prepared using an anti-solvent crystallization method. The yield was approximately 92.5% when the ratio of phlorizin to matrine was 1:1; approximately 59.8% when the ratio was 1:2; and approximately 63.9% when the ratio was 2:1. The phlorizin:matrine ratio of 1:1 was the optimal ratio for the anti-solvent method, and the product appeared as a yellow crystalline solid.

[0032] Example 3: X-ray powder diffraction (XRD) structural characterization of the phloretin-matrine eutectic.

[0033] The eutectic samples prepared in Examples 1 and 2, as well as pure root bark extract and pure matrine, were subjected to XRD tests to verify the eutectic crystal structure.

[0034] 1. Testing conditions X-ray source: Cu-Kα radiation (λ=1.5406Å); tube voltage 40kV, tube current 30mA; scanning range 5°~30° (2θ), scanning step 0.02°, scanning speed 5° / min; detection temperature 25℃.

[0035] 2. Test Results Characteristic diffraction peaks (2θ) of pure root dermalin: 6.8°, 13.6°, 20.4°, 27.2°; Characteristic diffraction peaks (2θ) of pure matrine: 8.4°, 12.2°, 18.6°, 23.4°; Eutectic samples of the present invention (Example 1, Example 2): as follows Figure 3 As shown, unique characteristic diffraction peaks appear at diffraction angles of 2θ = 7.2°, 10.4°, 14.8°, 18.2°, 22.6°, and 26.0°, with an allowable error of ±0.2° for the diffraction angles. The relative peak intensities are 100%, 89%, 81%, 76%, 65%, and 58%, respectively.

[0036] 3. Conclusion The diffraction pattern of the product differs significantly from that of the single raw material, proving the successful formation of a novel phlorizin-matrine eutectic.

[0037] Example 4: Determination of the equilibrium solubility of phloretin-matrine eutectic.

[0038] The 1:1 eutectic, pure root bark extract, and 1:1 physical mixture prepared in Example 2 were selected as samples, and their equilibrium solubility in different media at 37°C was determined.

[0039] 1. Experimental medium Pure water, pH 1.2 hydrochloric acid solution, pH 6.8 phosphate buffer.

[0040] 2. Experimental Procedure Take an excess of the sample to be tested and add it to 5 mL of each of the three media mentioned above. Place the mixture in a 37°C constant temperature water bath and stir magnetically for 72 h to reach dissolution equilibrium. The supernatant was filtered through a 0.22 μm microporous membrane, and the concentration of the active ingredient in the solution was determined by HPLC. HPLC detection conditions: C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol-0.1% phosphoric acid aqueous solution = 65:35; flow rate 1.0 mL / min; detection wavelength 288 nm; column temperature 30 ℃.

[0041] 3. Test Results Table 1. Equilibrium solubility of phlorizin, matrine and eutectic (37°C, μg / mL)

[0042] 4. Conclusion The cocrystal has a solubility in pure water that is 4.5 times that of the phloretin raw material, and its solubility is increased by about 4.5 times and 4.2 times in pH 1.2 and pH 6.8 media, respectively. Compared with the physical mixture, the solubility is increased by 2.3 to 2.4 times, and the cocrystal significantly improves the water solubility of phloretin.

[0043] Example 5: Evaluation of the anti-inflammatory activity of cocrystals and examples of their application in cosmetics and topical medications.

[0044] Part 1: In vitro anti-inflammatory activity test (RAW264.7 macrophage model): 1. Experimental grouping and modeling An inflammation model was established by inducing RAW264.7 mouse macrophages with LPS (1 μg / mL). The mice were divided into a blank group, an LPS model group, a phloretin group, a matrine group, a physical mixture group, and a cocrystallized low / medium / high dose group (calculated as phloretin).

[0045] 2. Detection indicator 1: CCK-8 cell viability Cell seeding density 5×10 3 / well, after drug administration and culture for 24h, CCK-8 reagent was added, incubated for 2h, absorbance at 450nm was measured, and cell viability was calculated.

[0046] Table 2. Survival rate of RAW264.7 cells in each group (%)

[0047] 3. Detection index 2: NO release inhibition rate (Griess method) The NO content in the cell supernatant was measured, and the inhibition rate was calculated. The results are as follows: Table 3. NO release inhibition rate (%) in RAW264.7 cells of each group

[0048] 4. Activity Conclusion The co-crystal exhibits synergistic anti-inflammatory effects, with 10 μM being the optimal dosage. The medium-dose group showed significantly better cell protection and NO inhibition compared to single raw materials and physical mixtures. However, at high doses, activity decreased due to the slight toxicity of matrine. The high-dose group may experience decreased activity due to the toxic side effects of matrine. Part Two: Examples of Product Application Formulations (1) Anti-inflammatory and soothing essence (cosmetics, eutectic addition 2.0%) Formula (mass fraction): Phloretin-matrine eutectic 2.0%, Glycerin 5.0%, Sodium Hyaluronate 0.2%, Panthenol 1.0%, Propylene Glycol 3.0%, Preservative 0.1%, Deionized Water to 100%. Preparation: Stir at room temperature until completely dissolved, homogenize and filter, fill into containers. The finished product has antioxidant, anti-inflammatory, and skin-soothing effects.

[0049] (2) Acne gel (topical medication, eutectic addition 5.0%) Formula (mass fraction): Phloretin-matrine cocrystal 5.0%, Carbomer 1.0%, Triethanolamine 0.8%, Ethanol 8.0%, Propylene Glycol 5.0%, Purified Water to 100%. Preparation: Prepared using conventional gel processing techniques. For topical treatment of acne, skin inflammation, and pruritus.

[0050] Summary of application scope: Cosmetics: Toners, serums, lotions, creams, masks, eye creams, acne treatment products, with co-crystal addition of 0.1%~5%; Topical medications: ointments, creams, gels, and lotions for eczema, dermatitis, acne, and pruritus, with eutectic addition of 0.5% to 10%.

Claims

1. A phloretin-matrine eutectic, characterized in that, The X-ray powder diffraction pattern of the eutectic exhibits characteristic diffraction peaks at diffraction angles 2θ of 7.2°, 10.4°, 14.8°, 18.2°, 22.6°, and 26.0°, with relative peak intensities of 100%, 89%, 81%, 76%, 65%, and 58%, respectively. The diffraction angles have an error tolerance of ±0.2°. The molar ratio of phlorizin to matrine in the eutectic is 1:

1. The eutectic is a yellow crystalline solid.

2. The phlorizin-matrine eutectic according to claim 1, characterized in that, The equilibrium solubility of the eutectic in pure water at 37°C is 68.5±4.2 μg / mL; the equilibrium solubility in hydrochloric acid solution at 37°C and pH 1.2 is 102.6±6.8 μg / mL; and the equilibrium solubility in phosphate buffer solution at 37°C and pH 6.8 is 78.3±5.1 μg / mL.

3. A method (a) for preparing a phlorizin-matrine eutectic, comprising a liquid-assisted grinding method, wherein the method is used to prepare the phlorizin-matrine eutectic according to any one of claims 1 and 2, characterized in that, The specific steps of this method are as follows: a1. Weigh out phloretin and matrine in a 1:1 molar ratio and place them in a mortar. a2, add anhydrous ethanol, wherein the amount of anhydrous ethanol is 0.1 to 0.4 ml per gram of total solid mass; a3, grind at room temperature for 20-30 minutes; a4, the obtained solid is dried at 40-50℃ for 8-12 hours.

4. The method for preparing a phloretin-matrine eutectic according to claim 3, characterized in that, In step a1, phloretin and matrine were placed in a vacuum drying oven before weighing and dried at 40℃ and -0.08MPa for 2 hours; the purity of phloretin was ≥98% and the moisture content was ≤0.5%; the purity of matrine was ≥98% and the moisture content was ≤0.5%.

5. The method for preparing a phloretin-matrine eutectic according to claim 3, characterized in that, In step a2, anhydrous ethanol is added dropwise to the mortar in 3 to 4 portions, and after each drop, the mortar is ground for 1 to 2 minutes until all the powder is uniformly wetted. In step a3, the grinding process is carried out at room temperature of 20 to 25°C and relative humidity of ≤60%. During the grinding process, the powder on the inner wall and bottom of the mortar is scraped off and mixed evenly every 5 minutes.

6. A method (b) for preparing a phlorizin-matrine cocrystal, comprising an anti-solvent crystallization method, wherein the method is used to prepare the phlorizin-matrine cocrystal according to any one of claims 1 and 2, characterized in that, The specific steps of this method are as follows: b1, mix phloretin and matrine in a 1:1 molar ratio, add anhydrous ethanol, wherein the amount of anhydrous ethanol is 8 to 15 times the total solid mass by volume mass ratio; b2, cool to room temperature, add deionized water dropwise, 10 μL each time, shake well after each addition and let stand for 30 seconds until the volume ratio of ethanol to water is 1:1.5 to 1:

3. b3, allow the solvent to evaporate and crystals to grow at room temperature for 24–48 hours; b4. Collect the solid by centrifugation at 10,000 rpm for 10 minutes, and dry it at 40-50°C for 8-12 hours.

7. The method for preparing a phloretin-matrine eutectic according to claim 6, characterized in that, In step b1, the oscillation speed of the constant temperature water bath shaker is 150 rpm, and the oscillation time is 20 to 40 minutes; in step b2, the ambient temperature for adding deionized water is 20 to 25°C, and the relative humidity is ≤60%; shake for 10 seconds after each drop of deionized water is added; when the addition is stopped, the volume ratio of ethanol to water is 1:1.5 to 1:

3.

8. The method for preparing a phloretin-matrine eutectic according to claim 6, characterized in that, In step b3, the solution obtained in step b2 is transferred to a glass crystallizing dish, with a solution thickness not exceeding 5 mm; the mouth of the crystallizing dish is covered with plastic wrap and 10-15 small holes with a diameter of 0.5 mm are punched in the plastic wrap; the crystal growth process is carried out in a horizontal, shockproof, and light-proof environment; in step b4, the drying is carried out in a vacuum drying oven, and after drying, the vacuum state is maintained and cooled to room temperature, and the resulting solid is ground and passed through an 80-mesh standard sieve.

9. The application of the phloretin-matrine eutectic according to claims 1 and 2, characterized in that, The eutectic can be used to prepare anti-inflammatory, soothing, and antioxidant cosmetics or topical medications; the cosmetics include toners, serums, lotions, creams, masks, eye creams, and acne treatment products; the topical medications include ointments, creams, gels, and lotions for treating eczema, dermatitis, acne, and itchy skin; the amount of the eutectic added to the cosmetics is 0.1%-5%; the amount of the eutectic added to the topical medications is 0.5%-10%.

Images