Eleocharis tuberosa peel extract and use thereof in preparing medicine for treating chloasma

Abstract

The present application relates to the technical field of traditional Chinese medicine extraction, and discloses a water chestnut peel extract and application thereof in preparation of a medicine for treating chloasma; during preparation of the extract, low-temperature extraction is carried out by using an acid-alcohol mixed solvent, and enrichment is carried out by using macroporous resin purification, so that the water chestnut peel extract with high purity of active ingredients and complete retention can be prepared, the utilization rate of active ingredients in the water chestnut peel is effectively improved, and resource waste is reduced. The water chestnut peel extract provided by the present application can effectively inhibit tyrosinase activity, and shows excellent treatment potential for chloasma, a pigment deposition disease, thereby providing a solution to the current situation that chloasma is difficult to treat.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction technology, specifically to a water chestnut peel extract and its application in the preparation of drugs for treating melasma. Background Technology

[0002] Melasma is a common acquired hyperpigmentation disorder that commonly affects the face, but can also involve exposed areas such as the neck and forearms. Clinically, it presents as symmetrical yellowish-brown or dark brown patches, often in a butterfly shape or diffusely distributed. Melasma is most prevalent in women of reproductive age, with a male-to-female ratio of approximately 1:9. The global prevalence is about 1%, while the incidence rate among Asian women of reproductive age is as high as 30%. Although the condition does not affect physical health, it significantly impacts facial appearance, causing substantial psychological distress and potentially leading to anxiety, depression, and other mental health problems in severe cases.

[0003] From a pathophysiological perspective, increased tyrosinase (TYR) activity is a key factor in increased melanin synthesis. Tyrosinase is the rate-limiting enzyme in the melanin synthesis pathway, catalyzing the hydroxylation of tyrosine to L-DOPA, which is further oxidized to dopaquinone, ultimately forming melanin. When tyrosinase activity is abnormally elevated, the rate of melanin synthesis accelerates, exceeding the skin's own metabolic clearance capacity, leading to melanin deposition in the epidermis and dermis, forming age spots. Furthermore, tyrosinase-associated protein 2 (TYRP2) and tyrosinase-associated protein 1 (TYRP1) also participate in the regulation of melanin synthesis, while microphthalmia-associated transcription factor (MITF) is an important transcription factor regulating melanocyte proliferation and melanin synthesis.

[0004] Currently, clinically available medications for treating melasma mainly include tyrosinase inhibitors, keratolytic agents, and antioxidants. Hydroquinone is the most widely used tyrosinase inhibitor, but long-term use can cause adverse reactions such as contact dermatitis and skin atrophy, and it also poses a potential carcinogenic risk, leading to its restricted use in several countries. Azelaic acid has limited efficacy, and some patients experience skin irritation after use. Kojic acid is chemically unstable, easily oxidized and discolored, and carries a certain risk of skin sensitization. Retinoic acid drugs have a slow onset of action, are highly irritating, and are contraindicated in pregnant women. α-Arbutin, an anomeric derivative of glycosylated hydroquinone and natural arbutin, is a potent tyrosinase inhibitor and can be used as a powerful skin whitening agent in cosmetics, but its safety still needs further verification. In summary, existing treatments suffer from high irritation, limited efficacy, easy rebound, and questionable safety, necessitating the development of safer and more effective new treatments.

[0005] Water chestnuts are the corms of *Heleocharis dulcis* (Burm.l.) et Henschel, a plant in the Cyperaceae family. Rich in starch, sugars, and proteins, they are an aquatic vegetable used for both food and medicine, widely cultivated in Guangxi, Guangdong, Hunan, Hubei, Jiangsu, Zhejiang, Fujian, and other regions, with abundant resources. Water chestnuts also have the effects of clearing heat and promoting body fluid production, resolving phlegm, and relieving indigestion. They are mainly used to treat thirst due to febrile diseases, sore throat, cough with phlegm and heat, red eyes, diabetes, dysentery, jaundice, urinary tract infections, food stagnation, and warts. During processing, a large amount of water chestnut peel is produced (approximately 20%-25% of the fresh fruit weight). Only a small portion is used as animal feed, with the majority being discarded as waste. Research has found that water chestnut peel contains various active ingredients such as polyphenols, flavonoids, saponins, and sugars. Among them, flavonoids and polyphenols have antibacterial, anti-inflammatory, antioxidant, and antitumor activities, and can be widely used in the biomedical field. Chinese patent application 201710268734.1 discloses an anti-cancer extract from water chestnut peel, its preparation method, and its application. This method uses only an ethanol solution for ultrasonic extraction of water chestnut peel, followed by filtration, concentration, and drying to obtain the extract. This process uses a single solvent, resulting in low extraction efficiency, and the lack of enrichment and purification of the extract leads to a high content of impurities such as pigments and free sugars in the product, resulting in insufficient purity of the active ingredients. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a water chestnut peel extract and its application in the preparation of drugs for treating melasma. The extract is extracted using a mixed acid-alcohol solvent and purified with macroporous resin, exhibiting high extraction efficiency and high purity of active ingredients. It provides a method to effectively improve the utilization rate of water chestnut peel and offers a safe and effective solution for the treatment of melasma.

[0007] The technical solution of this invention is a method for preparing water chestnut peel extract, comprising the following steps: S1. Take water chestnut peel, dry it, crush it, soak it in a mixed solvent, heat and stir it, and then filter it to separate the filtrate and filter residue. S2. Add the filter residue to the mixed solvent again, heat and stir, and mix the filtrate obtained by filtration with the filtrate in S1 for later use. The filtrate of S3 and S2 was concentrated to obtain an extract. The extract was mixed with water and passed through a macroporous resin. It was washed with water and eluted with ethanol in sequence. The ethanol eluent was collected, concentrated to recover the ethanol, and dried to obtain water chestnut peel extract. The mixed solvent is an aqueous solution of glacial acetic acid and ethanol.

[0008] Furthermore, the volume fraction of glacial acetic acid in the mixed solution is 0.5% to 1.5%, and the volume fraction of ethanol is 75% to 90%.

[0009] Furthermore, the amount of mixed solvent added in S1 and S2 is calculated based on water chestnut peel, and the ratio of water chestnut peel to mixed solvent is 1:10~1:20 g / mL.

[0010] Furthermore, the soaking time in S1 is 24~36 h, and the heating and stirring temperature in S1 and S2 is 50~70℃, and the time is 0.5~2 h.

[0011] Furthermore, the macroporous resin in S3 is preferably HPD100 macroporous resin.

[0012] Furthermore, when loading the macroporous resin in S3, the flow rate is 2 BV / h; during elution, it is first eluted with 5 BV of water, and then eluted with 5 BV of 90% ethanol.

[0013] In a further preferred embodiment, the mixed solvent is a mixed aqueous solution of glacial acetic acid and ethanol, wherein the volume fraction of glacial acetic acid is 1.0% and the volume fraction of ethanol is 80%; the ratio of water chestnut peel to the mixed solvent is 1:15 g / mL.

[0014] The present invention also relates to a water chestnut peel extract obtained by the preparation method described above.

[0015] The present invention also relates to the use of water chestnut peel extract in the preparation of drugs for treating melasma.

[0016] This invention also relates to a medicament for treating melasma, wherein the active ingredient of the medicament is water chestnut peel extract. Preferably, the dosage form of the medicament is a cream, ointment, gel, tincture, or spray.

[0017] The present invention has the following beneficial effects: Water chestnut peel is rich in flavonoids such as cyanidin, rutin, apigenin, luteolin, and their derivatives, which possess excellent antioxidant activity. These flavonoids can effectively scavenge free radicals induced by ultraviolet radiation and alleviate skin damage caused by oxidative stress. Simultaneously, water chestnut peel extract can also inhibit skin inflammation induced by ultraviolet radiation and improve the skin microenvironment. Its multi-target synergistic mechanism gives it significant potential medicinal value in the prevention and treatment of melasma.

[0018] This invention employs a synergistic extraction process using an acid-alcohol mixed solvent, combined with macroporous resin purification, to effectively remove impurities such as polysaccharides and pigments, precisely enriching the flavonoids and phenolic acids of the water chestnut peel, thereby improving the purity and quality of the extract. Simultaneously, this invention, starting from the classic α-MSH-MITF-TYR signaling pathway in the pathogenesis of melasma, regulates tyrosinase activity through water chestnut peel extract, inhibiting excessive pigment deposition, thus developing a safe and effective treatment for melasma.

[0019] This water chestnut peel extract is derived from the outer skin of the water chestnut, a plant that is both used in medicine and food. It is abundant, safe, suitable for long-term use, and has no obvious toxic side effects. It provides a new way to make high-value use of water chestnut peel, a waste resource, and has important environmental and economic value. Attached Figure Description

[0020] Figure 1 The effect of different drug concentrations on the viability of B16-F10 cells (n=6, X±SD). Figure 2 Effect of water chestnut peel extract on melanin production in B16-F10 cells (n=6, X±SD). Figure 3 Effect of water chestnut peel extract on tyrosinase activity in B16-F10 cells (n=6, X±SD). Figure 4 Morphological changes in skin tissue of mice in different drug administration groups (HE staining, ×200). Figure 5 Distribution and expression of melanin granules in the skin of mice in different drug administration groups (Masson-Fontana method, ×200). Figure 6 Results of skin tyrosinase content measurement in mice in different drug administration groups (n=6, X±SD). Detailed Implementation

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used are commercially available.

[0022] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0023] Example 1 A method for preparing water chestnut peel extract includes the following steps: a. Take dried water chestnut peel, crush it, and soak it in a mixed aqueous solution of 0.5% glacial acetic acid and 75% ethanol at a material-to-liquid ratio of 1:10 g / mL for 24 hours. b. After soaking, heat and stir at 60°C for 1 hour, then filter to obtain filtrate I and filter residue I; c. Add a mixed aqueous solution of 0.5% glacial acetic acid and 75% ethanol at a ratio of 1:10 g / mL to the filter residue I obtained in step b. Heat and stir at 60°C for 1 hour, then filter to obtain filtrate II and filter residue II. d. Combine filtrate I obtained in step b and filtrate II obtained in step c, concentrate under reduced pressure, and recover the solvent to obtain extract I; e. Prepare a 1.5 mg / mL solution of extract I obtained in step d with water, pass it through HPD100 macroporous resin, and load it using the wet method at a flow rate of 2 BV / h. Elute with 5 BV of water first, then with 5 BV of 90% ethanol. Collect the ethanol eluent, concentrate and recover the ethanol, and dry it to obtain water chestnut peel extract A.

[0024] Example 2 A method for preparing water chestnut peel extract includes the following steps: a. Take dried water chestnut peel, crush it, and soak it in a mixed aqueous solution of 1.0% glacial acetic acid and 80% ethanol at a ratio of 1:15 g / mL for 30 h. b. After soaking, heat and stir at 60°C for 1 hour, filter to obtain filtrate I and residue I, for later use; c. Add a mixed aqueous solution of 1.0% glacial acetic acid and 80% ethanol at a ratio of 1:15 g / mL to the filter residue I obtained in step b. Heat and stir at 60°C for 1 hour, filter, and obtain filtrate II and filter residue II for later use. d. Combine filtrate I obtained in step b and filtrate II obtained in step c, concentrate under reduced pressure, and recover the solvent to obtain extract I; e. Prepare a 1.5 mg / mL solution of extract I obtained in step d with water, pass it through HPD100 macroporous resin, wet load the sample at a flow rate of 2 BV / h, elute with 5 BV of water first, then elute with 5 BV of 90% ethanol, collect the 90% ethanol eluent, concentrate and recover the ethanol, and dry to obtain water chestnut peel extract B.

[0025] Example 3 A method for preparing water chestnut peel extract includes the following steps: a. Take dried water chestnut peel, crush it, and soak it in a mixed aqueous solution of 1.5% glacial acetic acid and 90% ethanol at a ratio of 1:20 g / mL for 36 h. b. After soaking, heat and stir at 60°C for 1 hour, filter to obtain filtrate I and residue I, for later use; c. Add a mixed aqueous solution of 1.5% glacial acetic acid and 90% ethanol at a ratio of 1:20 g / mL to the filter residue I obtained in step b. Heat and stir at 60°C for 1 hour, filter, and obtain filtrate II and filter residue II for later use. d. Combine filtrate I obtained in step b and filtrate II obtained in step c, concentrate under reduced pressure, and recover the solvent to obtain extract I; e. Prepare a 1.5 mg / mL solution of extract I obtained in step d with water, pass it through HPD100 macroporous resin, and load it using the wet method at a flow rate of 2 BV / h. First, elute with 5 BV of water, then elute with 5 BV of 90% ethanol. Collect the 90% ethanol eluent, concentrate and recover the ethanol, and dry it to obtain water chestnut peel extract C.

[0026] Example 4 A comparative method for preparing water chestnut peel extract, differing from Example 2 in that it does not involve purification with HPD100 macroporous resin, includes the following steps: a. Take dried water chestnut peel, crush it, and soak it in a mixed aqueous solution of 1.0% glacial acetic acid and 80% ethanol at a ratio of 1:15 g / mL for 30 h. b. After soaking, heat and stir at 60°C for 1 hour, filter to obtain filtrate I and residue I, for later use; c. Add a mixed aqueous solution of 1.0% glacial acetic acid and 80% ethanol at a ratio of 1:15 g / mL to the filter residue I obtained in step b. Heat and stir at 60°C for 1 hour, filter, and obtain filtrate II and filter residue II for later use. d. Combine filtrate I obtained in step b and filtrate II obtained in step c, concentrate under reduced pressure to recover ethanol, and dry to obtain water chestnut peel extract D.

[0027] Example 5 A comparative method for preparing water chestnut peel extract, differing from Example 2 in that glacial acetic acid is not added to the extraction solvent and purification with HPD100 macroporous resin is not performed, includes the following steps: a. Take dried water chestnut peel, crush it, and soak it in an aqueous solution of 80% ethanol with a volume fraction of 1:15 g / mL for 30 h. b. After soaking, heat and stir at 60°C for 1 hour, filter to obtain filtrate I and residue I, for later use; c. Add an aqueous solution of 80% ethanol at a ratio of 1:15 g / mL to the filter residue I obtained in step b, heat and stir at 60°C for 1 hour, filter to obtain filtrate II and filter residue II, and set aside. d. Combine filtrate I obtained in step b and filtrate II obtained in step c, concentrate under reduced pressure to recover ethanol, and dry to obtain water chestnut peel extract E.

[0028] Example 6 A comparative method for preparing water chestnut peel extract, differing from Example 2 in that glacial acetic acid is not added to the extraction solvent, includes the following steps: a. Take dried water chestnut peel, crush it, and soak it in an aqueous solution of 80% ethanol with a volume fraction of 1:15 g / mL for 30 h. b. After soaking, heat and stir at 60°C for 1 hour, filter to obtain filtrate I and residue I, for later use; c. Add an aqueous solution of 80% ethanol at a ratio of 1:15 g / mL to the filter residue I obtained in step b, heat and stir at 60°C for 1 hour, filter to obtain filtrate II and filter residue II, and set aside. d. Combine filtrate I obtained in step b and filtrate II obtained in step c, concentrate under reduced pressure, and recover the solvent to obtain extract I; e. Prepare a 1.5 mg / mL solution of extract I obtained in step d with water, pass it through HPD100 macroporous resin, wet load the sample at a flow rate of 2 BV / h, elute with 5 BV of water first, then elute with 5 BV of 90% ethanol, collect the 90% ethanol eluent, concentrate and recover the ethanol, dry it to obtain water chestnut peel extract F.

[0029] Example 7 Study on the effect of water chestnut peel extract on the viability of B16-F10 cells Cell grouping and drug administration: Control group and drug administration groups were set up (α-Arbutin group, water chestnut peel extract groups A, B, C, D, E, and F, with 6 replicates in each group). The control group did not receive any drug, while the drug concentrations of α-Arbutin group, water chestnut peel extract groups A, B, and C, and control water chestnut peel extract groups D, E, and F were 50, 100, 200, 300, 400, and 500 µg / mL, respectively.

[0030] The effect of the drug on the viability of B16-F10 cells was detected using the MTT assay. B16-F10 cells were cultured at 8 × 10⁶ cells / year. 3 Cells were cultured at a density of 1 / well in 96-well plates using RPMI-1640 medium containing 10% FBS. After 6 hours of plating, cells adhered to the plates and were treated with the prescribed drug concentrations for each group. 48 hours after drug treatment, cell viability was determined using the MTT assay, with OD values ​​measured at 490 nm, and cell viability calculated.

[0031] The samples used and the relevant test results are shown in Table 1.

[0032] Table 1. Effects of different drug concentrations on the viability of B16-F10 cells (n=6, X±SD).

[0033]

[0034] Note: Significance compared to the control group is indicated by *, *P<0.05 indicates a significant difference, **P<0.01 indicates an extremely significant difference, and ***P<0.001 indicates an extremely significant difference.

[0035] Table 1 and Figure 1 The effects of different drug concentrations on B16-F10 cell viability were shown (n=6, X±SD). Results showed that, compared to the Control group, the control drug α-Arbutin, and the comparative water chestnut peel extracts D and E at concentrations of 500 µg / mL, water chestnut peel extracts A, B, and C, and comparative water chestnut peel extract F at concentrations of 400 µg / mL all inhibited B16-F10 cell viability. Concentrations of α-Arbutin, water chestnut peel extracts A, B, and C, and comparative water chestnut peel extracts D, E, and F in the range of 50 µg / mL–300 µg / mL had virtually no effect on B16-F10 cell viability, indicating that the optimal drug concentration was 50 µg / mL–300 µg / mL. Therefore, a concentration of 200 µg / mL was selected for subsequent activity testing.

[0036] Example 8 Effects of water chestnut peel extract on melanin production in B16-F10 cells Cell grouping and drug administration: Based on the experimental results of Example 7, the drug concentration was set at 200 µg / mL. Control group, Model group, α-Arbutin group, water chestnut peel extract groups A, B, and C, and control water chestnut peel extract groups D, E, and F were set up, with 6 replicates in each group. Specific treatments are shown in Table 2 below.

[0037] Table 2 Experimental Groups and Drug Treatments

[0038] Experimental method: 8 × 10⁸ B16-F10 cells in logarithmic growth phase were used as the experimental method. 4 Cells (2.0 mL / mL) were seeded in 6-well plates and cultured for 8 hours. The Control group used culture medium only, the Model group used 200 nM α-MSH to induce melanin synthesis, the α-Arbutin group used 200 nM α-MSH and α-Arbutin, and the experimental groups used 200 nM α-MSH and water chestnut peel extracts A, B, and C, and control water chestnut peel extracts D, E, and F, respectively. After 48 hours of treatment, cells were collected by digestion with 0.25% trypsin. The treated cells were dissolved in 400 μL of 1 mol / L NaOH containing 10% DMSO and reacted at 80℃ for 1 hour. The blank wells were used for zeroing, and the OD value at 405 nm was measured using a multi-mode microplate reader to calculate the relative melanin content.

[0039] The experimental results are shown in Table 3.

[0040] Table 3 Effects of water chestnut peel extract on melanin production in B16-F10 cells (n=6, X±SD)

[0041] Note: Compared with the Control group, significance is indicated by #, where #P<0.05 indicates a significant difference, ##P<0.01 indicates a highly significant difference, and ###P<0.001 indicates an extremely significant difference. Compared with the Model group, significance is indicated by *, where *P<0.05 indicates a significant difference, **P<0.01 indicates a highly significant difference, and ***P<0.001 indicates an extremely significant difference. Compared with the α-Arbutin group, significance is indicated by +, where +P<0.05 indicates a significant difference, ++P<0.01 indicates a highly significant difference, and +++P<0.001 indicates an extremely significant difference.

[0042] Table 3 and Figure 2 This study demonstrates the effects of different water chestnut peel extracts on melanin production in B16-F10 cells. Compared to the Control group, the Model group showed a significant increase in melanin production in B16-F10 cells, indicating successful cell modeling. Compared to the Model group, the positive control drug α-Arbutin and water chestnut peel extracts A, B, and C significantly inhibited α-MSH-induced melanin production. In contrast, water chestnut peel extracts D and E had no inhibitory effect on α-MSH-induced melanin production, while water chestnut peel extract F significantly inhibited α-MSH-induced melanin production. Compared to the positive control drug α-Arbutin group, water chestnut peel extracts A, B, and C significantly reduced melanin levels in B16-F10 cells, while water chestnut peel extracts D, E, and F did not significantly reduce melanin levels in B16-F10 cells. The data on melanin reduction rate show that the water chestnut peel extract prepared by the method of this invention has a significant inhibitory effect on melanin production. In contrast, the comparative water chestnut peel extracts D and E, which were not purified by HPD100 macroporous resin, had no inhibitory effect on α-MSH-induced melanin production. The comparative water chestnut peel extract F, which was extracted with ethanol solution and purified by HPD100 macroporous resin, had a certain inhibitory effect on α-MSH-induced melanin production, but the inhibitory effect was much lower than that of the water chestnut peel extracts A, B, and C prepared by the method of this invention.

[0043] Example 9 The effect of water chestnut peel extract on tyrosinase activity in B16-F10 cells, cell grouping and drug administration are shown in Table 4.

[0044] Table 4 Experimental Groups and Drug Treatments

[0045] Experimental methods: B16F10 cells (8×10) 4Cells (2.0 mL / mL) were seeded in 6-well plates and cultured for 8 hours. The control group used culture medium only, the model group used 200 nM α-MSH to induce melanin synthesis, the α-Arbutin group used 200 nM α-MSH and α-Arbutin, and the experimental groups used 200 nM α-MSH and water chestnut peel extracts A, B, and C, and control water chestnut peel extracts D, E, and F, respectively. After 48 hours of treatment, cells were collected by digestion with 0.25% trypsin. The collected cell pellet was washed twice with PBS and then lysed with RIPA lysis buffer at 4°C for 30 min. The supernatant was obtained by centrifugation at 12000 r / min for 15 min at 4°C. The protein concentration in the lysis buffer was determined using a BCA kit. Then, 40 μL of tyrosinase solution was mixed with 200 μL of 10.0 mL-DOPA and incubated at 37°C for 30 min. The OD value was measured at 475 nm, and the relative activity and inhibition rate of tyrosinase were calculated.

[0046] The experimental results are shown in Table 5: Table 5. Effects of water chestnut peel extract on tyrosinase activity in B16-F10 cells (n=6, X±SD)

[0047] Note: Compared with the Control group, significance is indicated by #, where #P<0.05 indicates a significant difference, ##P<0.01 indicates a highly significant difference, and ###P<0.001 indicates an extremely significant difference. Compared with the model group, significance is indicated by *, where *P<0.05 indicates a significant difference, **P<0.01 indicates a highly significant difference, and ***P<0.001 indicates an extremely significant difference. Compared with the α-Arbutin group, significance is indicated by +, where +P<0.05 indicates a significant difference, ++P<0.01 indicates a highly significant difference, and +++P<0.001 indicates an extremely significant difference.

[0048] Table 5 and Figure 3This study demonstrates the effect of water chestnut peel extract on tyrosinase activity in B16-F10 cells. Compared to the control group, the tyrosinase activity in the model group of B16-F10 cells was significantly increased, indicating successful model establishment. Compared to the model group, the tyrosinase activity in the α-Arbutin group and water chestnut peel extract groups A, B, and C were all significantly decreased, indicating that water chestnut peel extracts A, B, and C could significantly inhibit α-MSH-induced tyrosinase activity. There was no significant difference compared to water chestnut peel extracts D and E, but a significant difference compared to water chestnut peel extract F. Water chestnut peel extracts D and E had no inhibitory effect on tyrosinase activity, while water chestnut peel extract F had a certain inhibitory effect on tyrosinase activity. Compared with the positive control drug α-Arbutin, water chestnut peel extract A significantly reduced tyrosinase activity in B16-F10 cells, while water chestnut peel extracts B and C significantly reduced tyrosinase activity in B16-F10 cells. In contrast, water chestnut peel extracts D, E, and F did not significantly reduce tyrosinase activity in B16-F10 cells.

[0049] In summary, the control water chestnut peel extract without macroporous resin purification could not reduce melanin production by inhibiting tyrosinase activity in B16-F10 cells. The control water chestnut peel extract F, prepared directly from ethanol-water solution and purified by macroporous resin, showed some inhibitory effect on tyrosinase activity in B16-F10 cells and reduced melanin production, but its inhibitory effect was far lower than that of water chestnut peel extracts A, B, and C prepared by the method of this invention. Water chestnut peel extracts A, B, and C prepared by this invention could all reduce melanin production in B16-F10 cells by inhibiting tyrosinase activity. Among them, water chestnut peel extract B showed the best effect, and subsequent experiments will further investigate the effect of water chestnut peel extract B on the skin of mice with melasma.

[0050] Example 10 Treatment of melasma in a mouse model using water chestnut peel extract Experimental animals: Female C57BL / 6J mice, 6 weeks old, weighing 18-20g, purchased from the Animal Experiment Center of Three Gorges University. The C57BL / 6J mice were housed under normal conditions: temperature 25℃±2℃, humidity 50-70%, alternating between 12h light and 12h dark periods. They had free access to food and water, and their wood shavings bedding was changed every two days. Experiments began after they had acclimatized to their environment for 7 days.

[0051] Experimental drug: Prepare cream ingredients, as shown in Table 6.

[0052] Table 6 Cream Ingredients

[0053] Among them, the values ​​of water chestnut peel extract B were low dose (0.2 wt% of the cream), medium dose (0.5 wt% of the cream) and high dose (1.0 wt% of the cream), while α-Arbutin (1.0 wt% of the cream) and blank sample (no active ingredient) were included.

[0054] The preparation steps for the above cream are as follows: For the oil phase preparation, add 1.0g of α-Arbutin, group B of water chestnut peel extract (low dose 0.2g, medium dose 0.5g, high dose 1.0g), then add 15g of liquid paraffin, 25g of white petrolatum, 10g of 3-diisostearic acid polyglycerol ester, and 5g of hydroxylated lanolin, and stir and mix at 75℃.

[0055] For the aqueous phase preparation, add 8g of glycerin, 8g of polyethylene glycol 400, and purified water to make the total amount of cream 100g. Stir and mix at 75℃.

[0056] Emulsify and homogenize by slowly adding the aqueous phase to the oil phase and stirring at 75°C for 5 minutes.

[0057] Stir and cool to room temperature to form a cream.

[0058] Construction of a mouse model of melasma and its application to the skin: Six-week-old C57BL / 6 mice were randomly selected and divided into six groups: Control group, Model group, α-Arbutin group, low-dose water chestnut peel extract B group, medium-dose water chestnut peel extract B group, and high-dose water chestnut peel extract B group, with six mice in each group. Each mouse was anesthetized by intraperitoneal injection of tribromoethanol. A 4cm × 3cm area of ​​skin on the back was shaved using a hair trimmer, and hair removal cream was applied to the back of the mice. Except for the Control group, the other four groups of mice received intramuscular injections of progesterone solution at 20mg / kg in the hind legs, administered once daily in the morning, alternating between legs, for 28 consecutive days. During the injection period, a 320nm wavelength ultraviolet lamp (UVB light source) was placed above the mice's backs at a height of 15cm ± 2cm.

[0059] After modeling, each group of mice had medicated cream and blank matrix applied to their backs for 30 consecutive days. The α-Arbutin group and the low, medium, and high dose groups of water chestnut peel extract B were applied twice daily with 0.1g of 1.0wt% α-Arbutin cream, 0.2wt% water chestnut peel extract B cream, 0.5wt% water chestnut peel extract B cream, and 1.0wt% water chestnut peel extract B cream, respectively. The Control and Model groups were treated with a blank matrix. Mice in each group were euthanized by cervical dislocation after treatment.

[0060] The day after the last treatment, mice in each group were euthanized by cervical dislocation, and the skin tissue on their backs was quickly excised and divided into two parts with an area of ​​2cm×1cm. One part was used for paraffin embedding, and the other part was used for the detection of tyrosine and melanin content.

[0061] Histological HE staining observation: Skin from the back of mice was taken, laid flat in an embedding clip, and placed in 4% paraformaldehyde for 24 hours. It was then dehydrated with a gradient of alcohols, cleared with xylene, and embedded in paraffin using an embedding machine to form a paraffin block. The paraffin block was sectioned into 4μm sections, attached to a glass slide, baked at 60℃ for 4 hours, and stained in an automatic staining machine. After completion, it was mounted with neutral resin and the histopathological changes were observed under a microscope.

[0062] HE staining procedure: 1) Immerse the tissue in xylene I to dewax for 10 minutes.

[0063] 2) Remove from xylene I and place in xylene II for dewaxing for 10 minutes.

[0064] 3) Place in anhydrous ethanol, 95% ethanol and 80% ethanol solutions for 5 minutes each.

[0065] 4) Rinse the tissue three times with PBS solution, each rinse lasting 5 minutes.

[0066] 5) Add hematoxylin staining solution to the slide to cover the tissue, incubate at room temperature for 1 minute, and rinse the tissue with PBS solution for 1 minute.

[0067] 6) Place the tissue slide in hydrochloric acid-alcohol solution for 5 seconds to differentiate, then rinse with PBS solution for 1 minute.

[0068] 7) Add eosin staining solution to the slide to cover the tissue, incubate at room temperature for 30 seconds, and rinse the tissue with PBS solution for 10 seconds.

[0069] 8) Place in 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II solutions for 1 minute each.

[0070] 9) Clear the film twice with xylene solution, each time soaking for 2 minutes, and then seal it with neutral resin.

[0071] Figure 4HE staining results. Histological observation results: The skin of mice in the Control group felt very smooth, thin, and highly elastic, with intact hair follicles and sebaceous glands. In the Model group, the skin at the shaved area showed significant melanin deposition, with obvious spots and patches, significant epidermal thickening, disordered arrangement of fibrous tissue in the dermis, and enlarged outlines of hair follicles and sebaceous glands. In the α-Arbutin group, the epidermal thickness was close to normal, the fibrous tissue arrangement in the dermis was relatively regular, and the hair follicles and sebaceous glands were relatively intact. In the 0.2wt% water chestnut peel extract B group, the epidermal thickness was slightly increased, the fibrous tissue arrangement in the dermis was improved, and the hair follicles and sebaceous glands were relatively normal. In the 0.5wt% and 1.0wt% water chestnut peel extract B groups, the epidermal thickness was close to normal, the fibrous tissue arrangement in the dermis was relatively neat, and the hair follicles and sebaceous glands were relatively intact.

[0072] Observation of melanin staining: Mouse back skin was taken, laid flat in an embedding clip, and placed in 4% paraformaldehyde for 24 hours. It was then dehydrated with a gradient of alcohols, cleared with xylene, and embedded in paraffin using an embedding machine to prepare a wax block. The wax block was sectioned into 4μm sections, attached to a glass slide, baked at 60℃ for 4 hours, and then stained.

[0073] Masson-Fontana staining procedure: 1) Immerse the tissue in xylene I to dewax for 10 minutes.

[0074] 2) Remove from xylene I and place in xylene II for dewaxing for 10 minutes.

[0075] 3) Place in anhydrous ethanol, 95% ethanol and 80% ethanol solutions for 5 minutes each.

[0076] 4) Rinse the tissue with distilled water for 5 minutes.

[0077] 5) Soak in Fontana silver ammonia solution for 12 hours at room temperature in the dark, and wash 5 times with distilled water for 2 minutes each time.

[0078] 6) Treat the slices with sodium hypochlorite solution for 5 minutes, then wash with tap water for 5 minutes.

[0079] 7) Counterstain with neutral red staining solution for 5 min, then wash with distilled water for 5 min.

[0080] 8) Place in 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II solutions for 1 minute each.

[0081] 9) Clear the film twice with xylene solution, each time soaking for 2 minutes, and then seal it with neutral resin.

[0082] Figure 5Masson-Fontana staining results. The results showed that no melanin granules were deposited in the epidermis, basal cell layer, and spinous layer of the skin in the Control group; however, a large number of melanin granules were deposited in the epidermis, basal cell layer, and spinous layer of the skin in the Model group, with a significant increase in the number of melanin granules; the α-Arbutin group, the 0.2wt% water chestnut peel extract B group, the 0.5wt% water chestnut peel extract B group, and the 1.0wt% water chestnut peel extract B group all showed significant improvement in melanin granule deposition in the epidermis, basal cell layer, and spinous layer, with a significant decrease in the number of melanin granules. The 1.0wt% water chestnut peel extract B group showed an effect on improving epidermal melanin granule deposition that was comparable to the α-Arbutin group.

[0083] Measurement of tyrosinase content in mouse skin: Skin samples were taken from the back, accurately weighed, homogenized, and the tyrosinase content in the homogenate was determined using ELISA. The determination steps are as follows: The blank well is used for zeroing only by adding colorimetric reagent and stop solution without adding sample; Standard wells: Add 50 μL of diluted standard and 50 μL of biotin antigen working solution to each well. Sample wells: Add 50 μL of sample and 50 μL of biotin antigen working solution to each well. Incubate at 37 ℃ for 30 min.

[0084] First wash: Discard the liquid, spin dry, fill with washing buffer, let stand for 30 s, discard, repeat 5 times, and pat dry. Add 50 μL of avidin-HRP to the standard wells and sample wells, and incubate at 37 ℃ for 30 min.

[0085] Second wash: Discard the liquid, spin dry, fill with detergent and let stand for 30 seconds, then discard. Repeat 5 times, then pat dry.

[0086] Color development: Add 50 μL of colorimetric reagent and incubate at 37 ℃ in the dark for 10 min. Add 50 μL of stop solution. Zero the blank well and measure the absorbance at a wavelength of 490 nm.

[0087] Table 7. Results of skin tyrosinase content measurement in mice under different drug administration groups (n=6, X±SD)

[0088] Note: Compared with the Control group, significance is indicated by #, where #P<0.05 indicates a significant difference, ##P<0.01 indicates a highly significant difference, and ###P<0.001 indicates an extremely significant difference. Compared with the Model group, significance is indicated by *, where *P<0.05 indicates a significant difference, **P<0.01 indicates a highly significant difference, and ***P<0.001 indicates an extremely significant difference. Compared with the α-Arbutin group, significance is indicated by +, where +P<0.05 indicates a significant difference, ++P<0.01 indicates a highly significant difference, and +++P<0.01 indicates an extremely significant difference.

[0089] Table 7 and Figure 6 The results of skin tyrosinase content determination in mice under different drug administration groups are shown (n=6, X±SD). Compared with the control group, the tyrosinase content in the model group was significantly increased, indicating successful animal model establishment. Compared with the model group, the tyrosinase content in the α-Arbutin group and the low, medium, and high dose groups of water chestnut peel extract B were all significantly decreased. Compared with the α-Arbutin group, the tyrosinase content in the medium and high dose groups of water chestnut peel extract B was significantly decreased. It is evident that the low, medium, and high dose groups of water chestnut peel extract B can effectively inhibit the activity of tyrosinase, the key rate-limiting enzyme in skin melanin synthesis.

[0090] The above embodiments describe preferred embodiments of the present invention, but the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other way. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. The application of a water chestnut peel extract in the preparation of a drug for treating melasma, characterized in that, The preparation of this extract includes the following steps: S1. Water chestnut peel is dried and pulverized, soaked in a mixed solvent for 24-36 hours, heated and stirred, and then filtered to separate, yielding filtrate I and filter residue I; S2. Add the mixed solvent to filter residue I again, heat and stir, and filter to obtain filtrate II and filter residue II; S3. Mix filtrate I and filtrate II and concentrate to obtain an extract. Add water to the extract and pass it through a macroporous resin. Elute with water and ethanol in sequence. Collect the ethanol eluent, concentrate and recover the ethanol, and dry to obtain water chestnut peel extract. The above-mentioned mixed solvent is a mixed aqueous solution of glacial acetic acid and ethanol, wherein the volume fraction of glacial acetic acid is 0.5%~1.5% and the volume fraction of ethanol is 75%~90%; the amount of mixed solvent added in S1 and S2 is based on water chestnut peel, and the material-to-liquid ratio of water chestnut peel to mixed solvent is 1:10 - 1:20 g / mL; the heating and stirring temperature in S1 and S2 is 50~70 ℃, and the time is 0.5~2 h; the macroporous resin in S3 is HPD100 macroporous resin. When loading the sample, the flow rate is 2 BV / h. During elution, it is first eluted with 5 BV of water, and then eluted with 5 BV of 90% ethanol.

2. The application according to claim 1, characterized in that: The mixed solvent contains 1.0% glacial acetic acid and 80% ethanol by volume; the ratio of water chestnut peel to the mixed solvent is 1:15 g / mL.

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