A compound morinda officinalis color enhancing liniment for treating vitiligo and a preparation method thereof

Abstract

The application discloses a compound morinda officinalis color-increasing liniment for treating vitiligo and a preparation method thereof, and particularly relates to the technical field of traditional Chinese medicine preparations, wherein the liniment is prepared from morinda officinalis, spilanthes acmella, giant knotweed, caesalpinia sappan, psoralea corylifolia, radix angelicae dahuricae and rhizoma smilacis glabrae as raw medicinal materials. The preparation method comprises the following steps: dynamic reflux extraction, percolation extraction, macroporous adsorption resin purification and preparation molding. Through the classification extraction and purification process, the photosensitive active ingredients are retained, and the photo-toxic impurities are effectively removed, so that the photo-toxicity is completely negative. The pharmacodynamic test shows that the liniment has a significant curative effect on vitiligo. The safety test shows that the liniment has no skin irritation and no allergenicity, and has excellent light safety. The quality research shows that the quality of the liniment is stable and controllable. The liniment has definite curative effect, high safety and stable quality, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, and more specifically, to a compound madder root pigmentation-enhancing liniment for treating vitiligo and its preparation method. Background Technology

[0002] Vitiligo is a common acquired skin depigmentation disorder characterized by localized or generalized white patches on the skin or mucous membranes. The global incidence rate is approximately 0.5%-2%. Although the disease does not directly threaten life, it often causes severe psychological burden and social pressure on patients due to its impact on appearance, seriously affecting their physical and mental health and quality of life. At present, the pathogenesis of vitiligo has not been fully elucidated, but the mainstream view is that it is related to multiple factors such as autoimmunity, genetics, oxidative stress, and neuropsychiatric factors.

[0003] There are various methods for the clinical treatment of vitiligo, including glucocorticoids, immunosuppressants, phototherapy, and photochemotherapy. Among them, photochemotherapy, through the combination of photosensitizing drugs and long-wave ultraviolet irradiation, can activate tyrosinase, promote melanocyte proliferation and melanin production, and is a commonly used method for the clinical treatment of stable vitiligo. Traditional Chinese medicine compound prescriptions have unique advantages in the treatment of vitiligo due to their multi-target and multi-pathway effects, especially photosensitive Chinese medicines such as psoralea corylifolia and angelica dahurica.

[0004] However, existing photosensitive traditional Chinese medicine preparations have significant shortcomings: on the one hand, the traditional decoction or tincture preparation process is crude, the extraction rate of effective ingredients is low, and the quality is difficult to control; on the other hand, photosensitive ingredients (such as psoralen) often bring phototoxicity risks while enhancing photosensitivity, and long-term use may lead to skin erythema, blisters, photoaging, or even skin cancer, which seriously limits their clinical application.

[0005] Therefore, how to develop a topical preparation for vitiligo that retains photosensitivity, has excellent photosensitivity, and has stable and controllable quality has become a technical problem that urgently needs to be solved in this field. In view of this, the present invention provides a compound madder pigmentation-enhancing liniment for the treatment of vitiligo and its preparation method. Summary of the Invention

[0006] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present invention provide a compound madder pigmentation-enhancing liniment for treating vitiligo and its preparation method, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] On the one hand, the present invention provides a compound madder pigmentation-enhancing liniment for treating vitiligo, which is made from the following raw materials in parts by weight: 15-25 parts madder, 20-30 parts purslane, 10-20 parts knotweed, 10-15 parts sappanwood, 5-10 parts psoralea, 5-10 parts angelica dahurica and 8-12 parts clematis.

[0009] Preferably, the raw materials are in the following proportions by weight: 18-22 parts of Rubia cordifolia, 22-28 parts of Portulaca oleracea, 12-18 parts of Polygonum cuspidatum, 12-14 parts of Sappanwood, 6-8 parts of Psoralea corylifolia, 6-8 parts of Angelica dahurica, and 9-11 parts of Clematis chinensis.

[0010] Preferably, the liniment contains 0.010-0.030 mg / ml of hydroxyalizarin and 0.020-0.050 mg / ml of alizarin.

[0011] On the other hand, the present invention provides a preparation method for preparing the above-mentioned compound madder pigmentation-enhancing liniment for treating vitiligo. This method, through graded extraction and purification, effectively removes phototoxic impurities while retaining the photosensitive active ingredients, and includes the following steps:

[0012] S1. Dynamic reflux extraction: Weigh out madder root, purslane, Japanese knotweed, sappanwood and clematis root by weight, pulverize them and add 30%-50% ethanol at 8-12 times the total weight of the medicinal materials. Dynamically reflux extract twice at 60-80℃, 1-2 hours each time. Combine the extracts to obtain extract A.

[0013] S2. Percolation extraction: Take Psoralea corylifolia and Angelica dahurica, crush them, use 70%-85% ethanol as solvent, soak for 20-28 hours, then perform percolation extraction, collect 8-12 times the amount of percolate to obtain extract B.

[0014] S3. Purification and enrichment: Combine extract A and extract B, concentrate under reduced pressure until there is no alcohol odor, dilute with water and pass through a macroporous adsorption resin column. First wash with water to remove impurities, then elute with 50%-70% ethanol and collect the ethanol eluent.

[0015] S4. Formulation: Concentrate the ethanol eluent under reduced pressure to a thick paste, add excipients, adjust the pH to 5.0-7.0, add solvent to adjust to the specified volume, and fill into containers to obtain the final product.

[0016] Preferably, 40% ethanol, which is 10 times the total weight of the medicinal materials, is added and the extraction is performed twice under dynamic reflux at 70°C, each time for 1.5 hours.

[0017] Preferably, the percolation extraction conditions in step S2 are as follows: using 75% ethanol as a solvent, soaking for 24 hours, then performing percolation extraction, and collecting 10 times the amount of percolate.

[0018] Preferably, the macroporous adsorption resin in step S3 is AB-8 type, D101 type or HPD-100 type macroporous adsorption resin.

[0019] Preferably, in step S3, the sample loading concentration is equivalent to 0.5-1.5g of the original drug per milliliter, and the elution flow rate is 1-3 times column volume / hour.

[0020] Preferably, the excipients in step S4 include a transdermal absorption promoter and a preservative. The transdermal absorption promoter is azone, menthol, or a combination of both, and the preservative is ethylparaben or sodium benzoate.

[0021] Preferably, in step S4, the pH value is adjusted to 5.5-6.5.

[0022] The technical effects and advantages of this invention are as follows:

[0023] 1. The liniment of this invention can effectively promote melanin production and significantly increase tyrosinase activity, thus having a good therapeutic effect on vitiligo;

[0024] 2. The topical agent of this invention is non-irritating and non-allergenic to the skin, and has excellent photosensitivity. Through graded extraction and purification process, phototoxic impurities are effectively removed while retaining the photosensitive active ingredients, achieving complete phototoxicity negativity and solving the phototoxicity risk of traditional photosensitive vitiligo preparations.

[0025] 3. The liniment of this invention uses hydroxyalizarin and alizarin as quality control indicators, with a clear content range, good formulation stability, and a scientific and reasonable process, making it suitable for industrial production. Attached Figure Description

[0026] Figure 1 This is a flowchart of the preparation method of the present invention. Detailed Implementation

[0027] The present invention will be further illustrated below with specific embodiments, but these should not be construed as limiting the scope of protection of the present invention. The raw materials mentioned in the embodiments are all commercially available Chinese medicinal herbs, and the reagents are all of analytical grade or chromatographic grade.

[0028] Example 1: Preparation of Compound Madder Color-Enhancing Liniment

[0029] In this embodiment, the raw materials are weighed according to the following parts by weight: 20 parts of madder root, 25 parts of purslane, 15 parts of knotweed, 13 parts of sappanwood, 7 parts of psoralea, 7 parts of angelica dahurica, and 10 parts of clematis.

[0030] This embodiment also provides a preparation method for preparing the above-mentioned compound madder pigmentation-enhancing liniment for treating vitiligo, comprising the following steps:

[0031] S1. Dynamic reflux extraction: Rubia cordifolia, Portulaca oleracea, Polygonum cuspidatum, Sappanwood and Clematis chinensis are pulverized and passed through a 10-mesh sieve. 40% ethanol with 10 times the total weight of the medicinal materials is added. Dynamic reflux extraction is performed twice at 70℃, 1.5h each time. The extracts are combined to obtain extract A.

[0032] S2. Percolation extraction: Psoralea corylifolia and Angelica dahurica are pulverized and passed through a 40-mesh sieve. Using 75% ethanol as a solvent, they are soaked for 24 hours and then percolated. Ten times the amount of percolate is collected to obtain extract B.

[0033] S3. Purification and enrichment: Combine extract A and extract B, concentrate under reduced pressure until there is no alcohol odor, dilute with pure water to 1.0 g of raw drug per milliliter, pass through AB-8 macroporous adsorption resin column, loading at a flow rate of 2 column volumes / hour, first wash with 3 column volumes of water to remove impurities, then elute with 4 column volumes of 60% ethanol, and collect the ethanol eluent.

[0034] S4. Formulation: Concentrate the ethanol eluent under reduced pressure to a thick paste, add 0.5% menthol and 1% azone as transdermal absorption enhancers, add 0.1% ethylparaben as a preservative, adjust the pH to 6.0 with triethanolamine, add pure water to adjust to the specified volume, and fill into vials to obtain the compound madder color-enhancing liniment.

[0035] Example 2: Compound Madder Color-Enhancing Liniments with Different Proportions

[0036] In this embodiment, the raw materials are weighed according to the following parts by weight: 15 parts of madder root, 20 parts of purslane, 10 parts of knotweed, 10 parts of sappanwood, 5 parts of psoralea, 5 parts of angelica dahurica, and 8 parts of clematis.

[0037] Alternatively, use 25 parts madder root, 30 parts purslane, 20 parts Japanese knotweed, 15 parts sappanwood, 10 parts psoralea corylifolia, 10 parts angelica dahurica, and 12 parts clematis chinensis.

[0038] The remaining preparation process is the same as in Example 1.

[0039] Example 3: Preparation with different process parameters

[0040] Adjust the ethanol concentration in step S1 to 30% or 50%, the extraction temperature to 60℃ or 80℃, and the extraction time to 1h or 2h.

[0041] In step S2, the ethanol concentration is adjusted to 70% or 85%, and the soaking time is adjusted to 20h or 28h.

[0042] In step S3, the macroporous adsorption resin is replaced with D101 or HPD-100, and the elution ethanol concentration is adjusted to 50% or 70%.

[0043] In step S4, the pH value is adjusted to 5.0 or 7.0.

[0044] The rest is the same as in Example 1, and a suitable liniment can be prepared in all cases.

[0045] Comparative Example 1: Preparation using conventional water decoction process

[0046] Using the same raw material ratio as in Example 1 (20 parts Rubia cordifolia, 25 parts Portulaca oleracea, 15 parts Polygonum cuspidatum, 13 parts Sappanwood, 7 parts Psoralea corylifolia, 7 parts Angelica dahurica, and 10 parts Clematis chinensis), add 10 times the total weight of the medicinal materials in water, soak for 1 hour, and then reflux extract twice, 1.5 hours each time. Filter, combine the filtrates, concentrate to the specified volume, and add 0.1% ethylparaben to obtain the conventional decoction sample.

[0047] Comparative Example 2: Prepared without macroporous resin purification process

[0048] Following the same raw material formulation and extraction method as in Example 1 (steps S1 + S2), extracts A and B were combined, concentrated under reduced pressure until no alcohol odor was detected, diluted with water, and then directly concentrated to a thick paste. Excipients were added to adjust the pH to 6.0 to obtain the final product. This example omits the S3 macroporous resin purification step.

[0049] Experimental Example 1: Content Determination

[0050] The liniment prepared in Example 1 was used to determine the contents of hydroxyalizarin and alizarin by high performance liquid chromatography. The chromatographic conditions were: Agilent ZORBAX-SB C18 column (250 mm × 4.6 mm, 5 μm), mobile phase: methanol-acetonitrile-0.2% phosphoric acid (25:50:25), flow rate: 1.0 ml / min, detection wavelength: 250 nm, column temperature: 30 °C. The results showed that the content of hydroxyalizarin was 0.0106 mg / ml and the content of alizarin was 0.0229 mg / ml.

[0051] Experimental Example 2: Pharmacodynamic Experiment (Guinea Pig Vitiligo Model):

[0052] Thirty black guinea pigs were randomly divided into a normal group, a model group, a positive control group (vitiligo-removing tincture), and low, medium, and high dose groups of compound madder root pigment-enhancing liquid (75, 150, and 300 μg / ml, respectively, based on the content of polygalactoside), with 5 rats in each group. Except for the normal group, the other groups applied 5% hydroquinone cream to the hair-removed area on their backs twice daily for 50 consecutive days to establish a vitiligo model. After successful model establishment, each treatment group was given the corresponding drug twice daily, and UVA irradiation (starting dose was the minimum erythema dose, once daily) was combined after the second administration for 30 consecutive days. After the treatment, the repigmentation of the skin on the back was observed and scored. The results are shown in the table below:

[0053] Animal number Valid animal count Overall effectiveness (%) normal group 5 5 100 Model group 5 0 0 Positive control group 5 3 50 low-dose group 5 3 50 medium dose group 5 5 100 High-dose group 5 5 100

[0054] Pathological sections showed that the number of melanin-containing hair follicles, melanocytes, and epidermal cells containing melanin granules in the treatment group were significantly increased in a dose-dependent manner. Immunohistochemistry showed that the activity of tyrosinase in the treatment group was significantly enhanced, indicating that the topical agent of the present invention has a good therapeutic effect on vitiligo.

[0055] Test Example 3: Skin Irritation Test

[0056] Eight New Zealand rabbits were randomly divided into an intact skin group and a damaged skin group, with four rabbits in each group, half male and half female. Hair was removed from both sides of the spine on the back of the rabbits. 0.5 ml of the ointment in Example 1 was applied to the left side, and an equal amount of physiological saline was applied to the right side as a control. The treatment was carried out once a day for 14 consecutive days. Skin irritation was observed 4 hours after each administration and 1, 24, 48 and 72 hours after the last administration. The results were evaluated according to the scoring criteria.

[0057] Results: No obvious erythema or edema was observed in either the intact skin group or the damaged skin group, and the irritation response scores were all less than 0.5, indicating that the liniment of the present invention is non-irritating to both intact and damaged skin of rabbits.

[0058] Test Example 4: Skin Allergy Test

[0059] Eighteen Hartley guinea pigs were randomly divided into a negative control group (physiological saline), a positive control group (1-chloro-2,4-dinitrobenzene), and a compound madder color-enhancing liquid group (the topical application in Example 1), with six guinea pigs in each group. An active skin allergy test was conducted, with sensitization (application on the left back) on days 1, 8, and 15, and challenge (application on the right back) on day 29. Skin allergy reactions were observed at 1, 24, 48, and 72 hours after challenge.

[0060] Results: The skin reaction score of the positive control group was 0.83 1 hour after stimulation, with a sensitization rate of 67% (highly sensitized); no allergic reaction was observed in the topical application group of this invention, with a sensitization rate of 0, indicating no sensitization.

[0061] Experimental Example 5: Animal Phototoxicity Test

[0062] Thirty guinea pigs were randomly divided into a negative control group (physiological saline), a positive control group (8-methoxypsoralen), and a compound madder color-enhancing liquid group (the topical solution in Example 1), with 10 guinea pigs in each group. Hair was removed from both sides of the back. The test substance was applied to the left side and then the area was shielded from light. The test substance was applied to the right side and then the area was irradiated with UVA (dose 10000mJ / cm²). Skin reactions were observed at 1, 24, 48, and 72 hours after irradiation.

[0063] Results: The positive control group showed obvious erythema and edema on the right side with a score ≥2. No abnormal reactions were observed on both sides in the liniment group of this invention, indicating that there is no phototoxicity.

[0064] Experimental Example 6: In vitro 3T3 cell phototoxicity assay (including control)

[0065] Neutral red uptake assays were performed in BALB / c 3T3 cells according to OECD Guideline 432. Cells were seeded in 96-well plates, and different concentrations of the test sample were added, dividing the cells into a light-illuminated group (+Irr, UVA 5 J / cm²) and a dark-protected group (-Irr). After culture, neutral red uptake was measured, and IC50, photostimulation factor (PIF), and mean light effect (MPE) were calculated. The test samples included: the topical solution of the present invention prepared in Example 1, Comparative Example 1 (conventional water decoction process), and Comparative Example 2 (without macroporous resin purification). The results are shown in the table below:

[0066] IC50 (-Irr) (μg / ml) IC50 (+Irr) (μg / ml) PIF value MPE value Phototoxicity conclusions Example 1 >1000 >1000 *1 -0.088 Negative Comparative Example 1 >1000 312 3.2 0.18 Mild phototoxicity Comparative Example 2 >1000 556 1.8 0.12 suspicious

[0067] The results showed that the liniment of the present invention did not exhibit cytotoxicity at the highest concentration of 1000 μg / ml, with PIF=*1 and MPE=-0.088<0.1, indicating negative phototoxicity. In contrast, samples prepared by conventional water decoction process showed mild phototoxicity, and samples not purified by macroporous resin were in the phototoxicity questionable range. This indicates that the present invention, through a combination of fractional extraction and macroporous resin purification, effectively removed phototoxic impurities that could not be removed by conventional processes while retaining the photosensitive active ingredients, achieving completely negative phototoxicity and obtaining unexpected technical effects.

[0068] Test Example 7: Stability Test

[0069] The liniment prepared in Example 1 was subjected to accelerated testing for 6 months at 40℃±2℃ and 75%±5% relative humidity. Samples were taken at 0, 1, 2, 3 and 6 months to examine its properties, pH value, hydroxyalizarin content, and lysimachia content. The results showed that there were no significant changes in any of the indicators, and the RSD of hydroxyalizarin and lysimachia content was less than 2%, indicating that the preparation had good stability.

[0070] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound madder root pigmentation-enhancing liniment for treating vitiligo, characterized in that: It is made from the following raw materials in parts by weight: 15-25 parts madder root, 20-30 parts purslane, 10-20 parts Japanese knotweed, 10-15 parts sappanwood, 5-10 parts psoralea corylifolia, 5-10 parts angelica dahurica, and 8-12 parts clematis chinensis.

2. The compound madder pigmentation-enhancing liniment for treating vitiligo according to claim 1, characterized in that: The raw materials are in the following weight proportions: Rubia cordifolia 18-22 parts, Portulaca oleracea 22-28 parts, Polygonum cuspidatum 12-18 parts, Sappanwood 12-14 parts, Psoralea corylifolia 6-8 parts, Angelica dahurica 6-8 parts, and Clematis chinensis 9-11 parts.

3. The compound madder pigmentation-enhancing liniment for treating vitiligo according to claim 1, characterized in that: The liniment contains 0.010-0.030 mg / ml of hydroxyalizarin and 0.020-0.050 mg / ml of alizarin.

4. A preparation method for preparing the compound madder pigmentation-enhancing liniment for treating vitiligo as described in any one of claims 1-3, characterized in that: This method, through graded extraction and purification, effectively removes phototoxic impurities while retaining the photosensitizing active ingredients, and includes the following steps: S1. Dynamic reflux extraction: Weigh out madder root, purslane, Japanese knotweed, sappanwood and clematis root by weight, pulverize them and add 30%-50% ethanol at 8-12 times the total weight of the medicinal materials. Dynamically reflux extract twice at 60-80℃, 1-2 hours each time. Combine the extracts to obtain extract A. S2. Percolation extraction: Take Psoralea corylifolia and Angelica dahurica, crush them, use 70%-85% ethanol as solvent, soak for 20-28 hours, then perform percolation extraction, collect 8-12 times the amount of percolate to obtain extract B. S3. Purification and enrichment: Combine extract A and extract B, concentrate under reduced pressure until there is no alcohol odor, dilute with water and pass through a macroporous adsorption resin column. First wash with water to remove impurities, then elute with 50%-70% ethanol and collect the ethanol eluent. S4. Formulation: Concentrate the ethanol eluent under reduced pressure to a thick paste, add excipients, adjust the pH to 5.0-7.0, add solvent to adjust to the specified volume, and fill into containers to obtain the final product.

5. The preparation method according to claim 4, characterized in that: The conditions for dynamic reflux extraction in step S1 are as follows: add 40% ethanol at 10 times the total weight of the medicinal materials, and perform dynamic reflux extraction twice at 70℃, each time for 1.5 hours.

6. The preparation method according to claim 4, characterized in that: The conditions for percolation extraction in step S2 are as follows: use 75% ethanol as solvent, soak for 24 hours, then perform percolation extraction and collect 10 times the amount of percolate.

7. The preparation method according to claim 4, characterized in that: In step S3, the macroporous adsorption resin is AB-8 type, D101 type or HPD-100 type macroporous adsorption resin.

8. The preparation method according to claim 4, characterized in that: In step S3, the loading concentration is equivalent to 0.5-1.5g of the original drug per milliliter, and the elution flow rate is 1-3 column volumes per hour.

9. The preparation method according to claim 4, characterized in that: The excipients in step S4 include transdermal absorption enhancers and preservatives. The transdermal absorption enhancers are azone, menthol, or a combination of both, and the preservatives are ethylparaben or sodium benzoate.

10. The preparation method according to claim 4, characterized in that: In step S4, adjust the pH value to 5.5-6.5.

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