A total triterpene of gomphrena celosiae root, extraction method, device and application

Abstract

The patent application belongs to the technical field of skin disease treatment, and particularly relates to an extraction method and device of total triterpenoids of Malanthera incana root and application, which comprises the following steps: S1, after the Malanthera incana root medicinal material is crushed, ethyl acetate is added for heating reflux extraction, and the extracted extract is filtered and concentrated to Malanthera incana root extract; S2, the Malanthera incana root extract after mixing is treated by adopting a silica gel wet method column packing and dry method sample loading technology, eluted by petroleum ether-ethyl acetate twice, and the petroleum ether-ethyl acetate eluent is collected; S3, the petroleum ether-ethyl acetate eluent is treated by vacuum concentration and vacuum drying, and the total triterpenoids of Malanthera incana root are obtained; the composition can produce more comprehensive treatment effect on the complex multifactor pathogenesis of vitiligo through the synergistic effect of multiple components and multiple targets.

Description

Technical Field

[0001] This invention relates to the field of skin disease treatment technology, specifically to a method, apparatus, and application for extracting total triterpenes from the roots of Torchflower. Background Technology

[0002] Vitiligo is a common primary depigmentation disorder that primarily affects the skin and mucous membranes, manifesting as localized or widespread complete loss of pigmentation. Also known as "leukoderma," its clinical features include decreased skin color and whitening, and patients typically experience no subjective symptoms. From a pathogenesis perspective, vitiligo is closely related to multiple factors, including genetics, neuropsychiatric factors, autoimmune reactions, and melanocyte self-destruction. Epidemiological surveys show that the global incidence of vitiligo is approximately 0.5%–2%, with a total incidence of approximately 0.56% in China, including both adults and children. Its incidence has been gradually increasing in recent years. Due to its long course, high recurrence rate, and frequent occurrence in visible areas, it affects appearance and can be disfiguring, often causing psychological stress for patients in their daily lives and social interactions, negatively impacting their mental health, normal life, and quality of life.

[0003] The root of *Tripterygium wilfordii* is the dried, peeled root core of *Tripterygium kunmingense*, a plant belonging to the Celastraceae family. The process involves removing impurities, removing the root bark, crushing, and drying. It is bitter and warm in nature; it is toxic. It enters the spleen and kidney meridians. It has the effects of dispelling wind and dampness, relaxing muscles and tendons, clearing heat and detoxifying. Currently, it is mainly used for rheumatic arthritis (rheumatoid arthritis, rheumatoid arthritis), chronic nephritis, lupus erythematosus, traumatic injuries, and pruritus. Many components in *Tripterygium wilfordii* root have been proven to have anti-inflammatory, immunosuppressive, and anti-tumor pharmacological effects, such as diterpenoids, alkaloids, and triterpenoids. In particular, triterpenoids show good activity in regulating immune cell function and inhibiting the release of inflammatory factors, providing a theoretical basis for its application in the treatment of immune-related diseases such as vitiligo. Therefore, further extraction and analysis of the medicinally active components of *Tripterygium wilfordii* root, and the separation and extraction of new pharmacologically active chemical components, is an urgent problem to be solved in current technology, and has significant research value for the development of new drugs based on *Tripterygium wilfordii* root extracts.

[0004] The existing patent (CN202110457683.3) on the application of norzelamin in vitiligo medications and its ointment has confirmed the effective therapeutic effect of the Tripterygium wilfordii monomer norzelamin in treating vitiligo. The active ingredient norzelamin in the medication inhibits the production and secretion of keratinocyte chemotactic factors CXCL9 / 10 / 16 under inflammatory stimulation and oxidative stress. This drug targets the migration, activation, proliferation, and differentiation of CD8+ T cells in the skin to treat vitiligo. It can be formulated into oral, injectable, or topical formulations for oral administration. The prepared ointment is low in toxicity and highly effective, laying the foundation for future research and development of targeted therapies for vitiligo.

[0005] Existing patented single-component drugs often suffer from inherent drawbacks such as limited target areas, a clear ceiling to efficacy, susceptibility to drug resistance, or significant individual variability in efficacy. Therefore, discovering a composition from the roots of *Torchbearer chinensis* that consists of multiple active ingredients and can enhance efficacy through synergistic effects across multiple targets is a key issue in improving its therapeutic value. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a composition composed of multiple active ingredients that can enhance the therapeutic effect through multi-target synergistic action, namely total triterpenes from the root of Torchflower, which can play a role in treating vitiligo.

[0007] The technical solution adopted in this invention is as follows: A total triterpenoid from the root of Torchflower, extracted and separated from the medicinal material of Torchflower root, includes desmethylazinaldehyde, triptolide, and styracil.

[0008] As a preferred embodiment of the present invention, by weight, 23-25 ​​parts of demethylazine, 17-20 parts of triptolide, and 28-30 parts of styrax oleoresin. This invention also provides a method for extracting and separating total triterpenoids from the roots of Torchflower, used to prepare the above-mentioned total triterpenoids from Torchflower roots, comprising the following steps: S1. After crushing the root of Torchflower, add ethyl acetate and heat under reflux to extract. Filter and concentrate the extract to Torchflower root extract. S2. After dissolving the Torchflower root extract in ethyl acetate, mix the sample and treat the mixed Torchflower root extract using silica gel wet packing and dry loading techniques. Elute sequentially with a petroleum ether-ethyl acetate mixture at a volume ratio of 20:1 and a petroleum ether-ethyl acetate mixture at a volume ratio of 1:1. Collect the eluent obtained by eluting with the petroleum ether-ethyl acetate mixture at a volume ratio of 1:1. S3. The eluent was concentrated under reduced pressure and dried under vacuum to obtain total triterpenes from the roots of Torchflower.

[0009] In a preferred embodiment of the present invention, in step S1, the extraction material-to-liquid ratio is 1:5 to 1:20; the number of heating and reflux cycles is 2 to 3; the extraction time is 0.5 to 1.0 h; and the extraction temperature is 75℃ to 80℃.

[0010] In a preferred embodiment of the present invention, in step S2, the silica gel is 100-200 mesh, the amount of dry loading is 1 times, and the amount of wet packing is 5-20 times.

[0011] In a preferred embodiment of the present invention, in step S2, the column volume eluted with a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 20:1 is 1 to 4 column volumes; the column volume eluted with a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 1:1 is 4 to 10 column volumes.

[0012] This invention also provides an extraction and separation device for total triterpenoids from the roots of *Torchbearer chinensis*, applied to the above-mentioned extraction and separation method for total triterpenoids from the roots of *Torchbearer chinensis*. The device includes an extraction and concentration assembly, comprising a condenser, a switching mechanism, a herbal extraction container, and a self-rotating concentration container. The herbal extraction container and the concentration container are heated by a water bath. During reflux extraction, the condenser is connected to the herbal extraction container. During concentration, the switching mechanism allows the extract in the herbal extraction container to flow into the concentration container, and the condenser is connected to the concentration container.

[0013] This invention also provides an application of total triterpenoids from the roots of *Torchbearer chinensis* for the preparation of drugs for vitiligo.

[0014] The beneficial effects of this invention are as follows: 1. This invention extracts and isolates a composition with norzelamin, triptolide, and styracil as the main triterpenoid components from the roots of Torchflower. All components of this composition are derived from Torchflower roots, resulting in high utilization of the medicinal material and suitability for large-scale production. Compared to the single-component (norzelamin) drugs mentioned in the background art, this composition may produce a more comprehensive therapeutic effect on the complex multifactorial pathogenesis of vitiligo (such as autoimmunity, oxidative stress, melanocyte apoptosis, etc.) through the synergistic effect of multiple components and multiple targets.

[0015] 2. The main components of this composition can bind well to multiple potential therapeutic target proteins for vitiligo, such as BCL2, ESR1, CASP3, SIRT1, MTOR, and PARP1. They can also exert their effects by regulating related pathways such as "MicroRNAs in cancer" and specific microRNAs (such as hsa-miR-7-5p). This provides preliminary theoretical basis and mechanistic insights for the clinical application of this composition and lays a solid foundation for its development into a novel drug for the treatment of vitiligo. Attached Figure Description

[0016] Figure 1 This is a flowchart of the steps in the extraction and separation method of total triterpenoid composition according to an embodiment of the present invention; Figure 2This is a diagram illustrating the separation and purification process in the extraction and separation method of total triterpenoid compositions according to embodiments of the present invention; Figure 3 This is a schematic diagram illustrating the docking of the main components of total triterpenoids from Torchflower root with target proteins in the preparation of vitiligo drugs according to an embodiment of the present invention. Figure 4 This is a bubble diagram showing the KEGG pathway enrichment of total triterpenes from the roots of *Torchbearer chinensis* in the preparation of vitiligo drugs, according to an embodiment of the present invention. Figure 5 This is a schematic diagram of the structure of the extraction and separation device for total triterpenoid composition in Torchflower root during reflux extraction according to an embodiment of the present invention; Figure 6 This is a schematic diagram of the structure of the extraction and separation device for total triterpenoid composition in Torchflower root during filtration and concentration according to an embodiment of the present invention.

[0017] The reference numerals in the attached drawings include: first cylinder 1, cover 11, first annular plate 12, second cylinder 2, filter plate 21, second annular plate 22, third cylinder 3, bottom plate 31, limiting ring 32, compression spring 33, and lifting rod 4. Detailed Implementation

[0018] Typical embodiments embodying the features and advantages of the present invention will be specifically described in the following description. It should be understood that the present invention can have various variations in different embodiments without departing from the scope of the present invention, and the descriptions and illustrations herein are for illustrative purposes only and not intended to limit the present invention.

[0019] In the description of this application, the terms "first", "second", etc. are used only for the convenience of describing this application and simplifying the description, and are not intended to indicate or imply that the structure referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0020] Example 1 See Figure 1 and Figure 2 As shown, this embodiment discloses a total triterpenoid from the root of Torchflower, which is extracted and separated from Torchflower root medicinal material, including desmethylazinaldehyde, triptolide, and styracil.

[0021] This embodiment also discloses a method for extracting and separating total triterpenoids from the roots of Torchflower, used to prepare the above-mentioned total triterpenoids from Torchflower roots, comprising the following steps: S1. After crushing the root of Torchflower, add ethyl acetate and heat under reflux to extract. Filter and concentrate the extract to Torchflower root extract. S2. After dissolving the Torchflower root extract in ethyl acetate, mix the sample and treat the mixed Torchflower root extract using silica gel wet packing and dry loading techniques. Elute sequentially with a petroleum ether-ethyl acetate mixture at a volume ratio of 20:1 and a petroleum ether-ethyl acetate mixture at a volume ratio of 1:1. Collect the eluent obtained by eluting with the petroleum ether-ethyl acetate mixture at a volume ratio of 1:1. S3. The eluent was concentrated under reduced pressure and dried under vacuum to obtain total triterpenes (extract) from the roots of Torchflower.

[0022] In this embodiment, 10 kg of *Torchbeareria fraseri* root was pulverized, and 15 times the amount of ethyl acetate was added. The mixture was heated to reflux at 80°C twice, for 1 hour each time. The extracts were combined, filtered, and the filtrate was concentrated to obtain a paste. The paste was dissolved in ethyl acetate and mixed with 1 times the amount of silica gel (100-200 mesh). The sample was packed into a column using a wet method with 10 times the amount of silica gel (100-200 mesh). After dry loading, the sample was eluted for 3 column volumes with a petroleum ether-ethyl acetate mixture at a volume ratio of 20:1, followed by 7 column volumes with a petroleum ether-ethyl acetate mixture at a volume ratio of 1:1. The eluent was collected, concentrated under reduced pressure, and dried under vacuum to obtain total triterpenes from *Torchbeareria fraseri* root.

[0023] like Figure 3 , Figure 4 As shown, Figure 3 This is a schematic diagram of the docking of the main components of total triterpenoids from *Tortuosa Root* in the preparation of vitiligo drugs according to an embodiment of the present invention with target proteins (A-binding mode of norzelamin and CASP3; B-binding mode of triptolide and mTOR; C-binding mode of styraxin and CASP3). Figure 4 This is a bubble diagram showing the KEGG pathway enrichment of total triterpenes from the roots of *Torchbearer chinensis* in the preparation of vitiligo drugs, according to an embodiment of the present invention. Based on bioinformatics analysis, the mechanism of action of total triterpenoid extract from Torchflower root in improving vitiligo was predicted. The experimental steps are as follows: (1) Screening of effective targets for the components: Three triterpenoid components, namely norzelamin, triptolide, and styracil, were searched in the Pubchem database (https: / / pubchem.ncbi.nlm.nih.gov / ), and their corresponding 2D structures were downloaded. The targets of the components were predicted by the Swiss Target Prediction database (http: / / www.swisstargetprediction.ch / ) and the Pharmmapper database (https: / / www.lilab-ecust.cn / pharmmapper / ). The targets were converted into standard Gene Symbols by the UniProt database (https: / / www.uniprot.org / ). Targets that did not match Gene Symbols were removed. At the same time, the corresponding targets of the components were searched in the HERB database (herb.ac.cn / ). The target information from each database was integrated as the effective targets of the components.

[0024] (2) Acquisition of vitiligo disease targets: Vitiligo-related targets were obtained by searching the GeneCard (https: / / www.genecards.org / ), OMIM (https: / / www.omim.org / ), and Drugbank (https: / / go.drugbank.com / ) databases using "vitiligo" as the keyword. Among them, genes with scores greater than twice the median were selected from the GeneCard database.

[0025] (3) Construction of the protein-protein interaction network (PPI network) and screening of key targets: Venny analysis was performed on the effective targets of the components and the targets of vitiligo disease. The intersection targets of the two were selected as potential targets for the treatment of vitiligo by the three triterpenoid components. The intersection targets were imported into the "MultipleProteins" section of the STRING database (https: / / string-db.org / cgi / input.pl). The species was selected as "Homo sapiens". Data with a confidence level higher than 0.7 were selected and a list of protein-protein interaction relationships was exported. The PPI network diagram was drawn using Cytoscape 3.10.1 software. The network topology parameters were analyzed. The median of "Degree", "Betweenness Centrality" and "Closeness Centrality" were used as the chi-square value. Genes that simultaneously met more than 3 chi-square values ​​were selected as key targets for the treatment of vitiligo by the three triterpenoid components.

[0026] (4) Processing of component structure: The 2D structural formulas of the three triterpenoid components were imported into Chem 3D 19.0 software, and the MM 2 force field was selected for energy minimization to obtain the most stable spatial conformation.

[0027] (5) Collection and processing of target proteins: Human crystal structures of key targets were screened through the PDB database (http: / / www.rcsb.org). The protein crystal complex structure containing the protoligand with the highest score was selected. The protoligand was extracted using Pymol software and pretreated by removing excess protein conformation and dehydration, and then used as the acceptor for molecular docking.

[0028] (6) Definition of active pocket: Use AutoDock Tools vina to re-dock the proligand extracted from the protein, and use PyMOL software to calculate the root mean square deviation (RMSD) of the molecular conformation before and after docking. If it is less than 2.0, it indicates that the site is suitable for molecular docking and the active pocket is successfully defined.

[0029] (7) Obtaining the location parameters of the active pocket: Use AutoDock Tools to determine the center and size of the active pocket. Take the center of the protein crystal structure protoligand as the center of the active pocket, and the size should include all key residues of the active site where the protoligand is located.

[0030] (8) Docking of triterpenoid components with key target proteins: AutoDock Tools vina was used to dock triterpenoid components with key target proteins. After preprocessing the receptor protein by hydrogenation and charge calculation, the active pocket position parameters were set, the active component was docked to the active pocket, and the binding energy was calculated. If the binding energy is less than 0 kcal / mol, it indicates that the ligand molecule can spontaneously bind to the receptor molecule, and the component can be considered to have successfully docked with the target protein.

[0031] (9) KEGG enrichment analysis of key targets: KEGG analysis of key targets of triterpenoid components in the treatment of vitiligo was performed using the DAVID database (https: / / davidbioinformatics.nih.gov / ). With P<0.05 as the screening condition, the signaling pathways with significant differences were obtained.

[0032] (10) Screening of candidate key microRNAs: The TargetScan database (http: / / www.targetscan.org / vert_72 / ) and the miRDB database (http: / / www.mirdb.org / miRDB / ) were used to predict microRNAs for key targets. The most representative transcripts were selected to obtain microRNAs that bind at conserved sites, and microRNAs with a seed match of 8 mer were selected and retained. MicroRNAs with a context score percentile in the TargetScan database and a miRDB predicted value of not less than 95 were selected as candidate key microRNAs for the treatment of vitiligo with triterpenoid components.

[0033] Experimental results: As shown in Table 1, the binding energies of the main triterpenoid components of *Tortuosa rotundifolia* root—norzelamin, triptolide, and stylosin—to the six predicted key targets BCL2, ESR1, CASP3, SIRT1, MTOR, and PARP1 were all less than 0 kcal / mol, indicating successful binding. Among the related pathways enriched by KEGG, the pathway involving the most genes and with the most significant significance was MicroRNAs in cancer, suggesting that the treatment of vitiligo by triterpenoid components is likely closely related to the regulation of microRNAs. The 17 microRNAs listed in Table 2, including hsa-miR-7-5p, hsa-miR-133a-5p, and hsa-miR-4795-3p, all had contextual score percentiles and miRDB predicted values ​​of no less than 95 in the TargetScan database, and were selected as candidate key microRNAs for the treatment of vitiligo by triterpenoid components. This indicates that the total triterpenoids of *Tortuosa rotundifolia* root can bind well to the key targets of vitiligo and play a role in the treatment process through the regulation of microRNAs.

[0034] In summary, with the help of the above-mentioned technical solutions of the present invention, the total triterpenes extracted from the roots of *Torchella oleracea* provided by the present invention have been found in bioinformatics studies to have the potential to act on target proteins BCL2, ESR1, CASP3, SIRT1, MTOR, and PARP1, and to potentially regulate the levels of microRNAs in cancer pathways such as hsa-miR-7-5p, hsa-miR-133a-5p, and hsa-miR-4795-3p, and have potential therapeutic effects on vitiligo.

[0035] Example 2 Total triterpenes from *Torchella oleracea* root, LF3 and LF4 fractions from *Torchella oleracea* root (LF3 and LF4 fractions were isolated and purified according to the method published in patent publication number: CN121021611A), triptolide, norzelamin (triptolide and norzelamin were isolated and purified according to the method published in patent publication number: CN121021611A, purity ≥99%), tacrolimus ointment (clinical use, 0.1%), hematoxylin-eosin (HE) staining solution, DOPA staining solution, anti-tyrosinase (TYR) antibody, and immunohistochemical reagents were obtained. The experimental subjects were male SPF-grade C57BL / 6 mice, 6-8 weeks old, weighing 18-22g, without skin pigmentation abnormalities, and acclimatized for one week before being used in the experiment.

[0036] I. The experimental steps are as follows: (1) Preparation of ointment: Take appropriate amounts of total triterpenes from Torchflower root, LF3 component, LF4 component, triptolide, and desmethyl zelamal, and prepare them according to the ointment preparation method as follows: Torchflower root total triterpenes ointment (high dose: 150mg / g, medium dose: 100mg / g, low dose: 50mg / g); triptolide ointment (4mg / g); desmethyl zelamal ointment (4mg / g); LF3 ointment (15mg / g), LF4 ointment (15mg / g); the content of each component of triptolide ointment, desmethyl zelamal ointment, LF3 ointment, and LF4 ointment is equivalent to the high dose of Torchflower root total triterpenes ointment.

[0037] (2) Establishment of vitiligo mouse model: The back of C57BL / 6 mice was shaved, the shaved area was about 2cm×2cm. After the skin recovered, 2.5% hydroquinone was applied to the back for 60 days until obvious white depigmented spots appeared on the back. Histological analysis showed that compared with the control group, the number of basal melanocytes and melanin-containing epidermal cells in the hydroquinone-treated mice was reduced. (3) Grouping and administration: The mice that successfully developed the model were randomly divided into 9 groups of 5 mice each. Five normal mice were set up as a blank group (hair was removed only on the back, without hydroquinone). All groups were given topical medication on the depigmented spots on the back once a day for 4 consecutive weeks. The groups were as follows: blank group, physiological saline was applied to the hair removal area; model group, physiological saline was applied to the depigmented spots; tacrolimus positive control group, 0.1% tacrolimus ointment (0.1g / 10g body weight) was applied; total triterpenoids from Torchflower Root group, total triterpenoid ointment (high dose: 150mg / g, medium dose: 100mg / g, low dose: 50mg / g) was applied; single group, LF3 (15mg / g) / LF4 (15mg / g) / tripterpinen (4mg / g) / norzelamaldehyde (4mg / g) ointment was applied.

[0038] (4) Detection indicators: General observation: Observe the mice’s mental state, diet and activity daily. Record the mice’s weight and the area and degree of pigment recovery of the depigmented spots on their backs weekly. The degree of pigment recovery is scored as follows: 0 points (complete depigmentation, white), 1 point (minor pigmentation, light pink), 2 points (moderate pigmentation, light brown), 3 points (most pigmentation recovered, brown), 4 points (complete recovery, consistent with normal skin). Skin tissue pathological examination (HE staining): Four weeks after administration, mice were sacrificed, and skin tissue from the back was taken to prepare pathological sections. After HE staining, the thickness of the epidermis, the number of melanocytes, and the infiltration of inflammatory cells in the dermis were observed under an inverted microscope. Melanocyte detection (DOPA staining): After skin tissue sections are stained with DOPA, the number of DOPA-positive melanocytes in the basal layer of the epidermis (cells / field) is counted to reflect the activity and number of melanocytes; Tyrosinase (TYR) protein expression detection (immunohistochemistry): Following the immunohistochemistry kit procedure, the expression of TYR protein in skin tissue was detected. The positive expression area and optical density (IOD) were calculated using an image analysis system to reflect the expression level of TYR protein.

[0039] II. The experimental results are as follows (1) General condition and weight of mice: During the administration period, the mice in each group were in good mental condition, with normal diet and activity, no death or obvious adverse reactions, and their weight showed a slow growth trend. There was no significant difference in weight among the groups (P>0.05), indicating that the total triterpenes of the root of Torchflower and each single component ointment applied externally had no obvious systemic toxicity to mice.

[0040] (2) Area of ​​depigmented patches and pigment recovery score (after 4 weeks of drug administration), the experimental results are shown in the table below: (3) Skin tissue pathology and melanocyte detection, the experimental results are shown in the table below: (4) TYR protein expression in skin tissue (IOD value), the experimental results are shown in the table below: III. Analysis of Animal Experiment Results (1) Effect on skin pigmentation recovery: Compared with the model group, the area of ​​depigmented spots on the back of mice in all treatment groups was significantly reduced and the pigmentation recovery score was significantly increased (P<0.01). The total triterpenes of Torchflower root showed a dose-dependent effect, and the high-dose group was significantly better than tacrolimus (P<0.01). Among the individual components, triptolide, norzelamin, and LF4 component showed significantly better pigmentation recovery effects than LF3 component (P<0.01), indicating that the total triterpenes of Torchflower root and each effective component can effectively promote skin pigmentation recovery in vitiligo model mice.

[0041] (2) Effects on skin pathology: The model group mice had thickened epidermis and a large number of inflammatory cells infiltrated the dermis, indicating the presence of skin inflammation. All treatment groups could significantly reduce epidermal thickness and reduce inflammatory cell infiltration. Among them, high-dose total triterpenes, triptolide, norzelamin, and LF4 components could basically eliminate inflammatory infiltration. Moreover, the high-dose total triterpenes were significantly better than those in other groups (P<0.01), indicating that they have significant anti-inflammatory effects and can improve skin pathological damage in vitiligo model mice.

[0042] (3) Protective effect on melanocytes: The number of DOPA-positive melanocytes in the basal layer of the skin of mice in the model group was significantly reduced, while the number of melanocytes in each treatment group was significantly increased. Among them, the number of melanocytes in the high-dose total triterpenoids group was close to that in the blank group, indicating that the total triterpenoids of Torchflower root can effectively protect melanocytes, reduce their apoptosis, and promote melanocyte survival and proliferation.

[0043] (4) Effect on TYR protein expression: Compared with the model group, the expression level of TYR protein in the skin tissue of all drug-treated groups was significantly increased (P<0.01). The TYR expression in the high-dose total triterpenoid group was close to that in the blank group and significantly higher than that in the tacrolimus group (P<0.01), indicating that the total triterpenoids of Torchflower root can promote melanin synthesis by upregulating the expression of TYR protein in the skin tissue, thereby achieving pigmentation recovery.

[0044] (5) Toxicity and safety: No obvious adverse reactions were observed in mice during the administration period, and their weight increased normally, indicating that the total triterpenes of the root of Torchflower and each single component ointment have good local and systemic safety.

[0045] IV. Conclusion (1) The total triterpenes of Torchflower root can effectively promote melanin synthesis and restore skin pigmentation at the animal level. It is non-toxic to melanocytes and has good systemic safety. Its mechanism of action is mainly related to increasing tyrosinase (TYR) activity and protein expression, protecting melanocytes, and inhibiting skin inflammatory response.

[0046] (2) The anti-vitiligo effect of total triterpenes from the roots of Torchflower is dose-dependent, with the highest dose (150 mg / g in animal experiments) showing the best effect and significantly superior to the commonly used clinical drug tacrolimus, thus having potential clinical application value.

[0047] (3) The core effective components of the anti-vitiligo activity of Torchflower root are triptolide, desmethyl zelamin, and styracin, followed by LF4 component. LF3 component has a relatively weak effect, indicating that the anti-vitiligo effect of total triterpenes in Torchflower root is the result of the synergistic effect of each component.

[0048] Example 3 Based on Example 1, this example also discloses an extraction and separation device for total triterpenoid compositions from the roots of Torchflower, including an extraction and concentration component. The extraction and concentration component includes a condenser, a switching mechanism, a herbal extraction container, and a self-rotating concentration container. The herbal extraction container and the concentration container are heated by a water bath. During heating and reflux extraction, the condenser is connected to the herbal extraction container. During concentration, the switching mechanism allows the extract in the herbal extraction container to flow into the concentration container, and the condenser is connected to the concentration container.

[0049] The herbal extraction container includes a first cylinder 1, a cap 11 fixedly connected to the top of the first cylinder 1, a second cylinder 2 slidably installed on the lower part of the inner wall of the first cylinder 1, and a filter plate 21 fixedly connected to the bottom of the second cylinder 2.

[0050] A first annular plate 12 is fixedly connected to the lower part of the outer wall of the first cylinder 1. The concentration container includes a third cylinder 3. A bottom plate 31 is fixedly connected to the lower part of the third cylinder 3. The radius of the third cylinder 3 is larger than that of the second cylinder 2. The filter plate 21 is located above the bottom plate 31.

[0051] In this embodiment, fluororubber sealing rings can be installed at each connection point to ensure its sealing performance. The herbal extraction container and the concentration container are heated by a water bath. During concentration, a toothed ring is installed on the outer side of the third cylinder 3, which can be driven by a motor to rotate the third cylinder 3 through gear and toothed ring transmission. In other embodiments, a rotating platform can be provided, and the entire device can be placed on the rotating platform for rotation.

[0052] The switching mechanism includes an electric push rod, and a lifting rod 4 is fixedly connected to the moving end of the electric push rod. The lifting rod 4 passes through the top plate and the bottom plate 31 and is fixedly connected to the filter plate 21. A limit ring 32 is threadedly connected to the bottom plate 31 below the lifting rod 4. A compression spring 33 is fixedly connected between the limit ring 32 and the bottom plate 31.

[0053] In the initial state, the second cylinder 2 and the third cylinder 3 of this device are separated from the first cylinder 1, which facilitates the placement of medicinal materials into the filter plate 21 and the second cylinder 2. After the medicinal materials are placed, the lifting rod 4 drives the second cylinder 2 and the third cylinder 3 to rise until the bottom plate 31 is pressed against the bottom wall of the second cylinder 2 and the third cylinder 3 is pressed against the bottom wall of the first annular plate 12 (due to the pressing force generated by the compression spring 33). A certain pressing force can better ensure the sealing performance.

[0054] Hot water is added to the outer container, above the first cylinder 1, and a 78°C water bath is started. Ethyl acetate is introduced into the container, and the condenser is activated for reflux extraction.

[0055] After reflux extraction is complete, the lifting rod 4 continues to rise, as do the second cylinder 2 and filter plate 21. However, the bottom plate 31 and the third cylinder 3 are blocked by the first annular plate 12 and cannot rise further. The compression spring 33 then compresses the cylinder, separating the medicinal material from the extract. The extract enters the third cylinder 3. At this point, a vacuum pump reduces the pressure inside the container, causing the third cylinder 3 to rotate and be heated in a 50°C water bath for concentration. Due to the larger surface area of ​​the third cylinder 3, a liquid film can be better formed on its outer wall during rotation (the centrifugal force generated by rotation causes the liquid to form a film on the inner wall of the container, while the low pressure lowers the boiling point of ethyl acetate, allowing it to evaporate quickly and preventing prolonged high temperatures from deactivating the active ingredients). This increases the contact area, resulting in higher concentration efficiency and eliminating the need to transfer the medicinal material and extract, thus improving production efficiency.

[0056] The lower part of the outer wall of the second cylinder 2 is provided with a second annular plate 22. The second annular plate 22 is in contact with the inner wall of the third cylinder 3. During the concentration process, it can be used as a scraper. By raising and lowering the lifting rod 4, the extract on the inner wall of the third cylinder 3 is scraped downward to avoid the extract formed on the inner wall of the third cylinder 3 affecting the formation of the liquid film.

[0057] The above-mentioned device integrates the two processes of heating reflux extraction and vacuum concentration into one set of equipment, avoiding intermediate transfer and reducing volatilization loss and pollution risk caused by material transfer; during concentration, the centrifugal force generated by rotation causes the extract to form a uniform liquid film on the inner wall of the cylinder, improving evaporation efficiency; the lifting rod 4 realizes the switching of different containers and the separation of medicinal materials and extract, with a high degree of automation and suitable for large-scale production.

[0058] Example 4 The total triterpenoid extract from the roots of Torchflower obtained in Example 1 was then made into tablets according to the following method, each tablet consisting of the following components: 100 mg of total triterpenoid extract from the roots of Torchflower, appropriate amounts of starch, talc, and magnesium stearate were prepared into tablets using conventional tablet manufacturing methods.

[0059] Example 5 The total triterpenoid extract from the roots of Torchflower obtained in Example 1 was then made into granules according to the following method. Each bag of granules consists of the following components: 100 mg of total triterpenoid extract from the roots of Torchflower, appropriate amounts of sucrose and dextrin were prepared into granules using conventional granule preparation methods.

[0060] Example 6 The total triterpenoid extract from the roots of Torchflower obtained in Example 1 was then made into capsules according to the following method, with each capsule consisting of the following components: 100 mg of total triterpenoid extract from the roots of Torchflower, appropriate amounts of starch, talc, and magnesium stearate were prepared into capsules using conventional capsule manufacturing methods.

[0061] Example 7 The total triterpenoid extract from the roots of *Torchbearer* obtained in Example 1 was further prepared into an ointment according to the following method, wherein each 100 g of the ointment consists of the following components: 10 g of total triterpenoid extract from Torchflower root, along with appropriate amounts of octadecanol, petrolatum, liquid paraffin, glycerin, methylparaben, menthol, and citric acid / sodium citrate, are prepared into an ointment using conventional latex preparation methods.

[0062] Example 8 The total triterpenoid extract from the roots of Torchflower obtained in Example 1 was then prepared into an ointment according to the following method, wherein each 100 g of the ointment consists of the following components: 10 g of total triterpenoid extract from the roots of Torchflower, appropriate amounts of polyethylene glycol 400, polyethylene glycol 3350, benzyl alcohol, menthol or lanolin, paraffin, propylene glycol, and vitamin E are prepared into an ointment according to conventional methods for making ointments.

[0063] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A total triterpenoid from the roots of Torchflower, characterized in that, Extraction and separation of medicinal materials from the roots of Torchflower include demethylazin, triptolide, and styracil.

2. The total triterpenes from the roots of *Torchbearer* according to claim 2, characterized in that, Included by weight parts: 23-25 ​​parts of demethylazine, 17-20 parts of triptolide, and 28-30 parts of styraxol.

3. A method for extracting and separating total triterpenoids from the roots of Torchflower, used to prepare the total triterpenoids from Torchflower roots according to any one of claims 1-2, characterized in that, Includes the following steps: S1. After crushing the root of Torchflower, add ethyl acetate and heat under reflux to extract. Filter and concentrate the extract to Torchflower root extract. S2. After dissolving the Torchflower root extract in ethyl acetate, mix the sample and treat the mixed Torchflower root extract using silica gel wet packing and dry loading techniques. Elute sequentially with a petroleum ether-ethyl acetate mixture at a volume ratio of 20:1 and a petroleum ether-ethyl acetate mixture at a volume ratio of 1:

1. Collect the eluent obtained by eluting with the petroleum ether-ethyl acetate mixture at a volume ratio of 1:

1. S3. The eluent was concentrated under reduced pressure and dried under vacuum to obtain total triterpenes from the roots of Torchflower.

4. The method for extracting and separating total triterpenoids from the roots of *Torchbearer* according to claim 2, characterized in that, In step S1: The extraction material-to-liquid ratio is 1:5 to 1:20; the number of heating and reflux cycles is 2 to 3; the extraction time is 0.5 to 1.0 h; and the extraction temperature is 75℃ to 80℃.

5. The method for extracting and separating total triterpenoids from the roots of *Torchbearer* according to claim 4, characterized in that, In step S2: The silica gel should be 100-200 mesh. For dry loading, the sample volume should be 1 times the volume of the silica gel, and for wet loading, the sample volume should be 5-20 times the volume of the silica gel.

6. The method for extracting and separating total triterpenoids from the roots of *Torchbearer* according to claim 4, characterized in that, In step S2: The column volume for elution with a petroleum ether-ethyl acetate mixed solvent at a volume ratio of 20:1 is 1 to 4 column volumes; the column volume for elution with a petroleum ether-ethyl acetate mixed solvent at a volume ratio of 1:1 is 4 to 10 column volumes.

7. An apparatus for extracting and separating total triterpenoids from the roots of Torchflower, applied to the method for extracting and separating total triterpenoids from the roots of Torchflower as described in claim 4, characterized in that: The device includes an extraction and concentration assembly, which comprises a condenser, a switching mechanism, a herbal extraction container, and a self-rotating concentration container. The herbal extraction container and the concentration container are heated by a water bath. During heating and reflux extraction, the condenser is connected to the herbal extraction container. During concentration, the switching mechanism allows the extract in the herbal extraction container to flow into the concentration container, and the condenser is connected to the concentration container.

8. An application of total triterpenes from the roots of Torchflower, characterized in that, Used in the preparation of drugs for vitiligo.

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