Preparation method and application of cistanche deserticola extract for promoting wound healing

Abstract

The invention relates to the technical field of medicines, and particularly discloses a preparation method and application of a Xinjiang cistanche deserticola extract for promoting wound healing, and the preparation method comprises the following steps: S1, washing fresh cistanche deserticola, drying, slicing, crushing into particles, and sieving with a 60-mesh sieve to obtain medicinal material particles; s2, drying the medicinal material particles under the condition of 50 DEG C, cooling to room temperature, crushing, drying into medicinal residues, and sieving with a 65-mesh sieve to obtain medicinal material coarse powder; s3, carrying out reflux degreasing on the medicinal material coarse powder for 2 hours by adopting 95% ethanol, and filtering to obtain degreased medicine residues; s4, carrying out reflux extraction on the degreased medicine residues by adopting 80% ethanol for 3 times / 1.5 h, then filtering, carrying out vacuum concentration, then adding absolute ethyl alcohol with the volume being tetraploid of that of the degreased medicine residues, and standing at 4 DEG C for 24 h, so as to obtain a cistanche deserticola concentrated solution; and S5, carrying out suction filtration operation on the cistanche concentrated solution, combining three filtrates, and then carrying out rotary evaporation concentration and freeze drying to obtain cistanche powder, namely the cistanche extract. The cistanche deserticola extract disclosed by the invention is applied to preparation of a medicine for promoting wound healing.

Description

Technical Field

[0001] The patent of this invention relates to the field of medical technology, and specifically, to a preparation method and application of Xinjiang Cistanche extract for promoting wound healing. Background Art

[0002] In the critical link of wound healing, the new blood vessel formation provides oxygen, growth factors and immune support to the healing tissues. Promoting the massive formation of blood vessels during the wound healing stage can enable the tissues to obtain more oxygen and nutrients, thereby improving the efficiency and quality of healing. Conversely, reducing angiogenesis will reduce the healing rate.

[0003] Xinjiang Cistanche deserticola is a parasitic plant, belonging to the tall herbaceous plant. Most of Cistanche deserticola grows underground, and the stems gradually become thinner from bottom to top; the leaves are broadly ovate, and the upper part of the stem is sparse and narrow, generally lanceolate; the inflorescence is spike-shaped, and the edges of the petals are often rolled outward, light yellow-white or light purple, and often turn brown after drying; the fruit is ovoid; the seeds are elliptical or nearly ovate, with a net-like and shiny outside; the flowering period is from May to June; the fruiting period is from June to August. Cistanche deserticola is a fleshy plant with a calm and mild medicinal property, hence the name. Cistanche deserticola has a high medicinal value. According to the "Compendium of Materia Medica" and "Rihuazi Materia Medica", Cistanche deserticola can be used to treat kidney yang deficiency, blood loss, waist and knee cold pain, tinnitus and eye flowers, turbidity, frequent urination, metrorrhagia, infertility and dry bowel constipation. Cistanche deserticola can also be used to make porridge and soak in wine. In addition, Cistanche deserticola is also a precious Chinese medicinal material, known as the "desert ginseng", and has certain economic value.

[0004] Therefore, the pharmacological effects of the active ingredients in Xinjiang Cistanche have attracted extensive attention from researchers. However, there are currently no relevant documents or products that disclose relevant reports on the effects of Xinjiang Cistanche extract on angiogenesis and wound healing. To this end, the inventor of the present application provides a preparation method and application of Xinjiang Cistanche extract that promotes wound healing based on research on Xinjiang Cistanche. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a Xinjiang Cistanche deserticola extract for promoting wound healing.

[0006] The above-mentioned purpose of the present invention is achieved like this:

[0007] The present invention provides a method for preparing a Xinjiang Cistanche extract for promoting wound healing, comprising the following steps:

[0008] S1. Wash the fresh Cistanche deserticola, dry it, slice it, and crush it into granules, and pass it through a 60-mesh sieve to obtain medicinal material granules;

[0009] S2, drying the medicinal material particles in step S1 at 50° C., cooling to room temperature, crushing and drying into medicinal residues, and passing through a 65-mesh sieve to obtain a coarse medicinal material powder;

[0010] S3, defatting the crude powder of medicinal materials in step S2 by refluxing with 95% ethanol for 2 hours, and filtering to obtain defatted medicinal residues;

[0011] S4, refluxing and extracting the defatted residue in step S3 with 80% ethanol, 3 times / 1.5h, filtering, concentrating under reduced pressure, and then adding four times the volume of anhydrous ethanol and standing at 4°C for 24h to obtain a Cistanche deserticola concentrate;

[0012] S5. Filter the Cistanche deserticola concentrate, combine the three filtrates, and then concentrate by rotary evaporation and freeze-dry to obtain Cistanche deserticola powder, that is, Cistanche deserticola extract.

[0013] Furthermore, in step S3, the material-liquid ratio of 95% ethanol reflux degreasing is 1:3.

[0014] Furthermore, the freeze-drying temperature in step S5 is -80°C.

[0015] The present invention also provides an application of a method for preparing a Xinjiang Cistanche deserticola extract for promoting wound healing, and an application of the Cistanche deserticola extract extracted by the method in preparing a medicine for promoting wound healing.

[0016] The difficulty and significance of the technical problem solved by the present invention are:

[0017] Wound healing is a pathological process after trauma, and wound healing can be achieved through autoregulatory functions under normal circumstances. However, clinically, cases of wounds that do not heal for a long time often occur due to infection, poor blood circulation, and other reasons. Current medical research believes that Chinese medicine can promote wound healing through the interactive regulation of multiple cells and cell growth factors on the wound surface, strengthen the oxidative metabolism function of immune active cells on the wound surface, improve wound microcirculation, and regulate the pH value of the wound surface. The inventor of this application has studied the pharmacological efficacy of the active ingredients in Xinjiang Cistanche, and confirmed through experiments that the extract of Xinjiang Cistanche has a promoting effect on angiogenesis, which can significantly promote the capillary formation of skin wounds and improve the angiogenesis ability of wound healing. The present invention proposes a new research direction and new use of the extract of Xinjiang Cistanche, which has important research significance in angiogenesis and wound healing.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The preparation method of the Xinjiang Cistanche extract provided by the present invention is simple and convenient to operate, and can realize the extraction of active ingredients of Cistanche;

[0020] 2. The present invention provides an application of Xinjiang Cistanche extract in the preparation of a drug for promoting wound healing, develops a new use of Xinjiang Cistanche extract, and is of great significance for promoting angiogenesis and wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 1 is a flow chart of the preparation method of Xinjiang Cistanche deserticola extract in an embodiment of the present invention;

[0022] Figure 2 It is the chemical identification mass spectrum of the main components of Cistanche deserticola in the embodiment of the present invention;

[0023] Figure 3 It is the chemical component identification result of the Cistanche deserticola extract based on the UNIFI platform in the embodiment of the present invention;

[0024] Figure 4 It is the local structure of the docking of core proteins such as IL 10 and HIF-1α with some molecules of different Cistanche deserticola ligands in the embodiment of the present invention;

[0025] Figure 5 The effect of the Xinjiang Cistanche deserticola extract on the viability and migration activity of human umbilical vein endothelial cells in the embodiment of the present invention;

[0026] Figure 6 The effect of the Xinjiang Cistanche deserticola extract on HUVECs cell angiogenesis in the embodiment of the present invention;

[0027] Figure 7 The Xinjiang Cistanche extract in the embodiment of the present invention promotes wound healing in vivo;

[0028] Figure 8 HE staining of the wound surface 12 days after surgery in the embodiment of the present invention;

[0029] Fig. 9 This is the CD31 immunohistochemical staining of the wound surface 12 days after surgery in the embodiment of the present invention;

[0030] Fig.10 In the embodiment of the present invention, Western blot quantitatively detected the protein expression changes of vascular endothelial growth factor (VEGF) and platelet-endothelial cell adhesion molecule (CD31);

[0031] Fig.11 It is the mRNA expression level related to angiogenesis verified by RT-PCR in the embodiment of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0034] As shown in the drawings, a preferred embodiment of the present invention is provided.

[0035] Example:

[0036] The solution of the embodiment of the present invention provides a preparation method and application of Xinjiang Cistanche extract for promoting wound healing. The process of the method is as follows: Figure 1 As shown, the following steps are included:

[0037] S1. Wash the fresh Cistanche deserticola, dry it, slice it, and crush it into granules, and pass it through a 60-mesh sieve to obtain medicinal material granules;

[0038] S2, drying the medicinal material particles in step S1 at 50° C., cooling to room temperature, crushing and drying into medicinal residues, and passing through a 65-mesh sieve to obtain a coarse medicinal material powder;

[0039] S3, defatting the crude powder of medicinal materials in step S2 by reflux with 95% ethanol (solid-liquid ratio is 1:3) for 2 hours, and filtering to obtain defatted medicinal residues;

[0040] S4, refluxing and extracting the defatted residue in step S3 with 80% ethanol, 3 times / 1.5h, filtering, concentrating under reduced pressure, and then adding four times the volume of anhydrous ethanol and standing at 4°C for 24h to obtain a Cistanche deserticola concentrate;

[0041] S5. Filter the Cistanche deserticola concentrate, combine the three filtrates, and then concentrate by rotary evaporation and freeze-dry (-80° C.) to obtain Cistanche deserticola powder, that is, Cistanche deserticola extract.

[0042] The embodiment of the present invention also provides the use of the above-mentioned Cistanche deserticola extract in preparing a medicine for promoting wound healing.

[0043] The following is a specific experiment of the implementation scheme of the present invention:

[0044] 1. Sample preparation

[0045] Preparation of reference solution: Accurately weigh appropriate amount of tubuloside A, cistancheside A, and echinacoside standard, add 70% methanol to dissolve, and prepare 1 mg / mL standard solution. Preparation of test solution: Accurately weigh 0.5 g of Cistanche extract powder, 50 mL of 70% methanol, plug, weigh, reflux in a water bath for 1 hour, centrifuge for 15 minutes (3000 r·min-1), make up the weight with methanol, take the supernatant, filter with 0.22 μm microporous membrane, and the filtrate is the sample solution.

[0046] 2. UPLC-QTOF-MS detection conditions

[0047] (1) Chromatographic conditions: ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm), column temperature 40°C, sample manager temperature 10°C, injection volume 1 μL, flow rate 0.3 mL / min, mobile phase: 0.1% formic acid water (A)-acetonitrile (B).

[0048] (2) Mass spectrometry conditions: Electrospray ionization (ESI), MSEcontinuum mode, positive and negative ion detection modes. Full scan range: m / z 100-1600. Capillary voltage: 3 kV (ESI+) or 2.5 kV (ESI-), cone gas flow rate: 50 L·h-1, cone voltage: 40 V, desolvation nitrogen flow rate: 900 L·h-1, desolvation gas temperature: 450 °C, source temperature: 120 °C, high energy channel collision energy: 20-45 V. The instrument was calibrated with sodium formate solution, and leucine enkephalin [M+H]+=556.2771, [MH]-=554.2615 was used as the Lock Spray calibration standard solution (50 pg·mL-1), and the real-time calibration flow rate was 5 μL·min-1). The data acquisition control and analysis software was MassLynx (V4.2), which included the MarkerLynx data analysis module.

[0049] 3. Data collection and data processing

[0050] First, UPLC-QTOF-MS and UNIFI natural product analysis platform were used to screen, identify and analyze the chemical components of Cistanche deserticola: ① Self-built Cistanche compound database: Referenced literature on the chemical components of Cistanche deserticola, based on literature data and online databases such as CNKI, PubMed, TCMSP, etc., the chemical structures of the compounds were saved as .mol2 and .sdf format files, and the molecular formulas, chemical names and other information of the compounds mentioned in the literature were entered into Excel tables, a total of 71 items. As a supplement to the built-in database. ② The data were imported into the UNIFI natural product analysis platform for processing, screening, matching and identification. The minimum area of ​​2D peak detection was set to 200, the peak intensity of the high-energy channel of 3D peak detection was greater than 100 and the peak intensity of the low-energy channel was greater than 500, the error of identifying the compound was allowed to be within 10ppm, the summed ion selection of +H, +Na, +K for the positive spectrum, and the summed ion selection of -H, +COOH for the negative spectrum; ③ The self-built Cistanche deserticola database was used as a screening database based on the UNIFI information platform. During the processing, the error between the theoretical molecular weight and the actual molecular weight was set to 10ppm, and all samples were subjected to chemical qualitative analysis. Low collision energy and high collision energy were switched in the UNIFI platform. The molecular ion peak of the compound could be observed at low energy, and the corresponding fragment ion information could be obtained under high energy channels. The information of the two could be automatically matched and screened. Through database search and comparison with standard products, the appropriate compound information was displayed, including name, compound structure, molecular mass deviation, actual molecular mass and measured molecular mass, etc.

[0051] In the above experiment of the embodiment of the present invention, the inventor of the present application first extracted the active ingredients from Cistanche deserticola. The specific extraction steps are shown in Figure 1 As shown, the UHPLC-QTOF-MS detection method of high performance liquid chromatography and quadrupole time of flight tandem multi-stage mass spectrometry was then used to qualitatively analyze the Cistanche deserticola extract. The inventors of the present application found a total of 38 chemical components in the Cistanche deserticola extract, including 7 iridoids, 4 lignins, 22 phenylethanoid glycosides, 1 ferulic acid, 1 fatty acid, 1 menthone, and 2 terpenoids ( Figure 2 and Figure 3 ).

[0052] Subsequently, PyMoL was used to determine the interaction profile along the protein-ligand binding pattern. Figure 4As shown, it shows the local structure of the docking of core proteins such as IL 10 and HIF-1α with some molecules of different Cistanche ligands. For example, PTGS2 (PDB id 5f19), the compound bound to it is Poliumoside. The binding affinity is contributed by the hydrogen bonds of ASN-68, GLN-42, GLU-465, and LYS-468. The compound with STAT 3 (PDB id 6tlc) is Cistanoside A, and the corresponding amino acids forming hydrogen bonds are THR-443, GLU-444, LEU-362, LEU-358, TYR-446, GLY-449, GLU-357, and GLN-361 residues. The compound that binds to mTOR (PDB id 6zpn) is 2'-acetyl verbascoside, which can form hydrogen bonds with residues such as LEU-1332, GLU-1290, PHE-1289, ASP-1233, THR-1190, PHE-1191, PHE-1473, and THR-1471. Through conformational analysis of molecular docking structures, it was found that the receptors can form relatively good docking pockets, all ligand molecules are located in the corresponding docking pockets, and hydrogen bonds can be formed between the receptors and the ligands. The inventors of this application confirmed the high accuracy of the target prediction of Cistanche deserticola from the perspective of molecular docking.

[0053] 4. Effects of Xinjiang Cistanche deserticola extract on viability and migration activity of human umbilical vein endothelial cells

[0054] HUVECs were treated with Xinjiang Cistanche extract (0.5, 1, 10, 100, 200, 400 μg / mL) or ECM medium. Figure 5 As shown, Figure 5 (A) The results of the cell proliferation activity test of HUVEC treated with different concentrations of Cistanche deserticola extract. Figure 5 B and C) The results of the migration activity test of HUVEC treated with different doses of Xinjiang Cistanche extract. The scratch width is expressed as a percentage. Representative pictures of HUVEC cell migration are shown under a 5x microscope. The data represent the mean ± standard deviation (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001. NS: not significant.).

[0055] 5. Effect of Xinjiang Cistanche deserticola extract on HUVECs cell angiogenesis (e.g. Figure 6 (shown)

[0056] Compared with the control group, when the drug concentration was 1-100 μg / mL, the tube-forming ability of HUVECs was significantly enhanced, the number of meshes and nodes increased significantly, and the ability to form tubes increased in a dose-dependent manner. The 100 μg / mL drug group had the largest number of tubes formed by HUVECs. Data represent mean ± standard deviation (n = 3) (p < 0.001).

[0057] 6. Xinjiang Cistanche extract promotes wound healing in vivo (such as Figure 7 (shown)

[0058] (A) Gross photograph of the full-thickness wound model.

[0059] (B) Representative images of the wound healing process of rats treated with Xinjiang Cistanche extract at different time points.

[0060] (C) Wound healing rate at different time points. Data represent mean ± SD (n = 6) (*p < 0.05).

[0061] As shown in the figure, HE staining of the wound surface 12 days after surgery. After H&E staining, the number of blood vessels in the wound of the drug-treated group was significantly higher than that of the control group, indicating that Xinjiang Cistanche extract can significantly promote the formation of capillaries in skin wounds and improve the angiogenesis ability of wound healing in rats. Original magnification: (A), ×40; (B), ×100.

[0062] like Figure 8 As shown, HE staining of the wound surface 12 days after surgery. After H&E staining, the number of blood vessels in the wound of the drug-treated group was significantly higher than that of the control group, indicating that Xinjiang Cistanche extract can significantly promote the formation of capillaries in skin wounds and improve the angiogenesis ability of wound healing in rats. Original magnification: (A), ×40; (B), ×100.

[0063] like Fig. 9 As shown in the figure, CD31 immunohistochemical staining of the wound surface 12 days after surgery. After immunohistochemical staining, the number of blood vessels in the wound surface of the drug-treated group was significantly higher than that of the control group, indicating that Xinjiang Cistanche extract can significantly promote the formation of capillaries in skin wounds and improve the angiogenesis ability of wound healing in rats. The epidermal regeneration of the skin in the high-concentration drug group (MH group) was in good condition, the inflammatory cells had been greatly reduced, and the recovery effect was the best. From the perspective of the graphic characteristics of the tissue section, it was closest to normal skin tissue and had the best ability to promote wound repair. Original magnification: (A), ×40; (B), ×100.

[0064] like Fig.10As shown in the figure, in order to further clarify the effect of Xinjiang Cistanche extract on mouse skin wounds, we used Western blot to quantitatively detect the changes in protein expression of vascular endothelial growth factor (VEGF) and platelet-endothelial cell adhesion molecule (CD31) (as shown in Figure: B, C). On the 12th day, the expression of VGEF and CD31 in the wound tissue of the MH group was significantly higher than that of the NG group (p < 0.01), and this result was consistent with the immunohistochemistry results. The results showed that drug treatment promoted the expression of VGEF and CD31 in skin wound tissue during wound healing, promoted wound angiogenesis, and accelerated wound healing.

[0065] like Fig.11 As shown in the figure, RT-PCR was used to verify the expression level of mRNA related to angiogenesis. Compared with the NG group, after adding Xinjiang Cistanche extract, the MH group significantly upregulated the expression levels of VEGF, FGF, and CD31 (P<0.01), indicating that the drug can significantly promote the expression of angiogenesis factors. In summary, Xinjiang Cistanche extract can accelerate the speed of wound healing.

[0066] 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 and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a Xinjiang Cistanche extract for promoting wound healing, characterized in that: The following steps are involved: S1. Wash the fresh Cistanche deserticola, dry it, slice it, and crush it into granules, and pass it through a 60-mesh sieve to obtain medicinal material granules; S2, drying the medicinal material particles in step S1 at 50° C., cooling to room temperature, crushing and drying into medicinal residues, and passing through a 65-mesh sieve to obtain a coarse medicinal material powder; S3, defatting the crude powder of medicinal materials in step S2 by refluxing with 95% ethanol for 2 hours, and filtering to obtain defatted medicinal residues; S4, refluxing and extracting the defatted residue in step S3 with 80% ethanol, 3 times / 1.5h, filtering, concentrating under reduced pressure, and then adding four times the volume of anhydrous ethanol and standing at 4°C for 24h to obtain a Cistanche deserticola concentrate; S5. Filter the Cistanche deserticola concentrate, combine the three filtrates, and then concentrate by rotary evaporation and freeze-dry to obtain Cistanche deserticola powder, that is, Cistanche deserticola extract.

2. The method for preparing a Xinjiang Cistanche extract for promoting wound healing according to claim 1, characterized in that: In step S3, the material-liquid ratio of 95% ethanol reflux degreasing is 1:

3.

3. The method for preparing the Xinjiang Cistanche extract for promoting wound healing according to claim 1, characterized in that: The freeze-drying temperature in step S5 is -80°C.

4. The use of the preparation method of the Xinjiang Cistanche extract for promoting wound healing according to claim 1, characterized in that: The Cistanche deserticola extract extracted by the method is used in preparing medicine for promoting wound healing.

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