Preparation method of rhizoma panacis majoris extract and application of rhizoma panacis majoris extract in preparation of medicine for diabetic refractory wounds

Abstract

The invention provides application of rhizoma panacis majoris extract in refractory wounds of diabetes mellitus. The preparation method of the rhizoma panacis majoris extract comprises the following steps: S1, crushing rhizoma panacis majoris rhizomes, performing reflux extraction with 70% ethanol for 3 times, and merging; s2, vacuum concentration: extracting with water-saturated n-butyl alcohol for three times, and then concentrating; s3, adding methanol for dissolving, and then filtering; s4, carrying out chromatographic column purification on macroporous resin D101; s5, performing reduced pressure distillation to obtain the rhizoma panacis majoris extract. The invention discloses the scientific connotation of tonifying deficiency, activating blood, removing slough and promoting tissue regeneration of SRPM for treating diabetes and ulceration, and explores and analyzes the treatment advantage of early regulation and control of the healing process of traditional Chinese medicine. In addition, the invention also explores the additional activity of the characteristic rare'cloud medicine 'rhizoma panacis majoris independent of the hypoglycemic effect, and lays the research and development foundation of'new use of old medicines and special use of common medicines'.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a method for preparing a Chinese medicine extract, in particular to a method for preparing a Panax notoginseng extract and an application of the extract in preparing a medicine for treating refractory diabetic wounds. Background Art

[0002] In the field of plant medicinal resource development, Panax notoginseng[ Panax japonicus CA Mey. var. major (Burk.) CY Wu et KM Feng The rhizome of the Chinese herbal medicine has been documented in authoritative texts such as the Chinese Materia Medica to have the effects of invigorating Qi and nourishing Yin, activating blood circulation and promoting tissue regeneration. It is often used clinically to treat coughs caused by Qi and Yin deficiency, as well as traumatic illnesses. Existing studies have shown that the saponins contained in its rhizomes have anti-inflammatory, antioxidant, and immunomodulatory activities. However, related research has mostly focused on the direct therapeutic effects of the rhizomes and the formulas used in prescriptions and the individual components. The development of active ingredients from non-traditional medicinal parts such as the leaves, especially the systematic research on wound repair in the physiological environment of metabolic diseases, has not yet formed a complete system.

[0003] Traditional treatments for the clinical management of diabetic refractory wounds (DRW) include glycemic control, surgical debridement, dressing therapy, pressure unloading, bioengineered flap transplantation, revascularization, and anti-infective measures. In recent years, new technologies and therapies, such as targeted biological therapy, hyperbaric oxygen therapy, stem cells, exosomes, growth factors, and photodynamic therapy, have emerged. Current treatment strategies have significant limitations: basic therapies require multidisciplinary collaboration and are time-consuming; new biologics pose immunogenicity risks and individual variability in response; tissue engineering techniques face ethical controversies and challenges with cellular heterogeneity; and hyperbaric oxygen therapy is limited by equipment availability and treatment compliance. Although recent studies have confirmed the critical role of cytokines such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in wound repair, their short half-lives, difficulty maintaining local concentrations, and inability to target wounds in hyperglycemic patients remain unresolved. Notably, the synergistic mechanisms of multi-component action in traditional Chinese medicines (TCMs) are potentially compatible with the multifactorial pathological characteristics of DRW. In particular, SRPM, with its multi-target, multi-pathway, safety, low toxicity, and affordability, offers an effective approach for holistic internal and external treatment, potentially addressing the shortcomings of current clinical therapies. This multi-target intervention complements existing single-target drugs, offering new possibilities for developing an integrated treatment model combining "local microenvironmental regulation and systemic metabolic balance."

[0004] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the inventor studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0005] Based on the above technical problems, one of the objectives of the present invention is to provide a method for preparing a Panax notoginseng extract, comprising the following steps: S1: The rhizomes of Panax notoginseng were crushed and extracted with 70% ethanol under reflux for 3 times and then combined; S2: concentrated under reduced pressure, extracted with water-saturated n-butanol three times and then concentrated; S3: add methanol to dissolve and filter; S4: chromatographic column purification using macroporous resin D101; S5: distill under reduced pressure to obtain Panax notoginseng extract.

[0006] According to a preferred embodiment, the "concentration under reduced pressure" in S2 includes the following steps: concentration within a pressure range of 0.06 to 0.1 MPa.

[0007] According to a preferred embodiment, the "extraction with water-saturated n-butanol three times and then concentration" in S2 comprises the following steps: rotary evaporation in a temperature range of 40-60°C.

[0008] According to a preferred embodiment, the "purification" in S4 comprises the following steps: The methanol extract was passed through a D101 macroporous resin column at a flow rate of 2 BV / h, the loading concentration was controlled at 0.3 g / mL, and adsorption was carried out for 2 hours; Elution with distilled water at a flow rate of 3 BV / h was used to remove unadsorbed impurities; Total saponins were eluted by gradient elution with ethanol of 30%, 50%, and 70% by volume, with an elution volume of 5 BV and a flow rate of 3 BV / h.

[0009] One of the purposes of the present invention is to provide the use of the extract in the preparation of a medicine for treating refractory diabetic wounds, wherein the extract is obtained based on the above-mentioned preparation method.

[0010] According to a preferred embodiment, the extract is applied externally to the wound or taken orally.

[0011] According to a preferred embodiment, the oral dosage of the extract is 100-200 mg / kg; the external application dosage of the extract is 50 μL.

[0012] One of the purposes of the present invention is to provide a pharmaceutical composition for treating refractory diabetic wounds, comprising the extract obtained by the above preparation method and excipients acceptable for skin care.

[0013] According to a preferred embodiment, the excipients include carbomer, sodium carboxymethylcellulose, 5% by mass of ethylparaben, triethanolamine and glycerol.

[0014] One of the purposes of the present invention is to provide a method for preparing a gel for treating refractory diabetic wounds, comprising the following steps: S1: The Panax notoginseng extract obtained by the above preparation method is dissolved in sodium carboxymethyl cellulose to obtain a first mixture, wherein the ratio of the Panax notoginseng extract to sodium carboxymethyl cellulose is 1:10 g / mL; S2: Carbomer is fully dissolved in water to obtain a second mixture, wherein the ratio of carbomer to water is 1:20 g / mL; S3: 9.30 parts by weight of the first mixture, 74.83 parts by weight of the second mixture, 0.07 parts by weight of 5% ethylparaben, 1.86 parts by weight of triethanolamine and 12.08 parts by weight of glycerin were mixed.

[0015] One of the purposes of the present invention is to provide a method for preparing a gel for treating refractory diabetic wounds, the method comprising the following steps: Dissolve 2 g of SRPM in 20 mL of sodium carboxymethylcellulose; Carbomer 2.5 g, add 50 mL of water and fully dissolve and swell; The solutions of the above two steps are mixed together; Add 0.15 mL of 5% ethylparaben, 0.4 g of triethanolamine, and 2.6 g of glycerol and mix well.

[0016] The beneficial effects of this technical solution are as follows: By establishing a standardized extraction process and carbomer gel drug delivery system, the transdermal delivery and local sustained release of total saponins of Panax notoginseng (SRPM) in the treatment of refractory diabetic wounds (DRW) were achieved for the first time. The compatibility of the SRPM extract (purity ≥76%) tested for purity and the gel matrix was optimized, which prolonged the maintenance time of drug concentration in the wound and improved the therapeutic effect.

[0017] At the same time, through the synergistic administration experiment of oral and topical administration in the DRW rat model, it was confirmed that SRPM can shorten the wound healing cycle (the wound healing rate of the SRPM.L group with external application and oral administration was the highest). Its healing effect is better than that of the positive control recombinant human epidermal growth factor, and it significantly reduces the levels of inflammatory factors such as IL-6 and TNF-α (for example: TNF-α dropped from 1542pg / mL to 1287pg / mL).

[0018] Based on these experimental results, the present invention proposes a multi-target synergistic approach based on total saponins from Panax notoginseng to improve wound microcirculation disorders and glucose metabolism imbalance. This technology overcomes the limitations of existing single-component biologics, which target only one target, and provides a cost-effective and innovative Traditional Chinese Medicine-derived solution for DRW treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The SRPM powder is prepared based on the preparation method proposed in the present invention; Figure 2 The SRPM gel is prepared based on the preparation method proposed in the present invention; Figure 3 is the standard curve of ginsenoside Re; Figure 4 This is a schematic diagram of the wound healing change image acquisition results; Figure 5 This is a line graph showing changes in wound healing rates in rats according to the present invention; Figure 6 This is the transmission electron microscopic ultrapathological observation of the wound surface of rats in the CON group of the present invention; Figure 7 This is the transmission electron microscopic ultrapathological observation of the wound surface of rats in the SRPM.H group of the present invention; Figure 8 Ultramicroscopic pathological observation of wound surface of rats in SRPM.L group under transmission electron microscope; Figure 9 is the relative expression levels of MMP-9 and MMP-2 in the wound surface of each group of mice in the present invention; Figure 10 The expression levels of wound-related inflammation and growth factors in each group of mice of the present invention. DETAILED DESCRIPTION

[0020] In the description of the present invention, terms are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly indicate the number of technical features indicated. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0021] 1. Experimental Design 1. Preparation of SRPM The rhizomes of Panax notoginseng total saponins were crushed, extracted with 70% ethanol reflux for 3 times, combined, concentrated under reduced pressure at 50°C and 0.09 MPa, extracted with water-saturated n-butanol for 3 times, concentrated, dissolved in methanol, filtered, and fixed to volume.

[0022] Purification was performed using a chromatographic column of macroporous resin D101: (1) soaking the D101 macroporous resin in an appropriate amount of ethanol, and then repeatedly washing it with distilled water until there was no alcohol smell, in order to remove impurities in the resin; (2) passing the methanol extract through the D101 macroporous resin column at a flow rate of 2 BV / h, controlling the sample concentration to 0.3 g / mL, and adsorbing for 2 hours; (3) eluting with an appropriate amount of distilled water at a flow rate of 3 BV / h to remove unadsorbed impurities; (4) eluting the total saponins with an ethanol gradient (30%, 50%, 70%) in sequence, with an elution volume of 5 BV and a flow rate of 3 BV / h, and collecting the eluate; and then distilling under reduced pressure to obtain a white powder of total saponins of Panax notoginseng (such as Figure 1 Calculate the yield of SRPM.

[0023] Weigh appropriate amounts of ginsenoside Re and total saponins from Panax notoginseng, add methanol, and prepare a 0.2 mg·mL -1 Ginsenoside Re reference solution and 1 mg·mL -1 Panax ginseng total saponins test solution.

[0024] Take 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of ginsenoside Re reference solution and 0.4 mL of Panax notoginseng total saponins test solution and place them in a test tube, evaporate to dryness in a water bath, add 0.2 mL of 5% vanillin-glacial acetic acid and 0.8 mL of perchloric acid, shake well, heat in a 60°C water bath for 15 min, cool for 3 min, add 5 mL of glacial acetic acid, shake well, and measure the absorbance (A) at 540 nm on a UV-visible spectrophotometer.

[0025] A standard curve was established with mass as the horizontal axis and absorbance as the vertical axis, and the total saponin purity (measured in ginsenoside Re) was obtained by inserting the sample absorbance. Figure 3 shown).

[0026] 2. Preparation of SRPM Gel Component A: Dissolve 2 g of SRPM in 20 mL of sodium carboxymethylcellulose.

[0027] Component B: Add 2.5 g of carbomer to 50 mL of water and allow to swell freely overnight.

[0028] Component A + 16.1 g component B + 0.15 mL 5% ethyl paraben + 0.4 g triethanolamine + 2.6 g glycerol, stir with a magnetic stirrer to obtain a light yellow gel (such as Figure 2 shown).

[0029] 3. Animal efficacy verification 3.1 Animal model establishment 3.1.1 Diabetes model establishment SPF♂SD rats, weighing (200±20 g), were fed a high-fat diet for 4 weeks and then fasted (without water) for 16 h except for the normal group.

[0030] Streptozocin (STZ) was prepared with 0.1 mol / L citric acid buffer at pH 4.4 to a concentration of 0.25% and injected intraperitoneally at 40 mg / kg. The normal group was injected intraperitoneally with an equal volume of 0.1 mol / L citric acid buffer.

[0031] During the experiment, all DM model rats (diabetic rats) were fed a high-fat diet, while the normal control rats were fed a normal diet. Model establishment success was determined by screening rats with fasting blood glucose ≥11.0 mmol / L and insulin sensitivity index (insulin sensitivity index = 22.5 / [fasting blood glucose × insulin], HOMA) ≤ the mean of normal animals 72 hours after STZ injection to define the DM model.

[0032] 3.1.2 Establishment of a refractory wound model The animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg / kg). The backs were shaved, and the hair removal site was marked with a sterile 2-cm-diameter circular plastic sheet. The full-thickness skin was excised, avoiding the underlying fascia. Bleeding areas were gently pressed with a cotton swab, covered with dry gauze, and secured with medical tape. One day before surgery and two days after surgery, 40,000 units of penicillin were injected intramuscularly per animal. After surgery, the animals were housed individually with free access to food and water.

[0033] 3.1.3 Animal grouping and drug administration Eighty model rats were randomly divided into four groups: model group (MOD), SRPM high-dose group (SRPM.H), SRPM low-dose group (SRPM.L), and sitagliptin oral administration + human epidermal growth factor gel topical application group (SIT). Normal SD rats were set as blank group (CON), with 20 rats in each group.

[0034] The treatment groups received oral administration of 100 mg / kg and 200 mg / kg SRPM suspensions, respectively, and topical application of 50 μL / rat of SRPM gel. The positive group received oral administration of 10 mg / kg SIT solution and topical application of 50 μL / rat of human epidermal growth factor gel. The blank and model groups received oral administration of equal volumes of distilled water and topical application of 50 μL / rat of normal saline gel. Disposable sterile dressings were applied externally, and oral administration and dressing changes were performed once daily. Skin healing and infection were observed on days 0, 3, 7, and 14 after injury, and wound healing rates were calculated using digital camera images.

[0035] 3.1.4 Specimen collection Rats were killed 14 days after injury, and arterial blood was collected. Two skin tissue samples from the wound edge were fixed with 2.5% glutaraldehyde and stored at -80°C for testing.

[0036] 4. Study on the healing mechanism of SRPM in DRW rats 4.1 Effect of SRPM on wound ultrastructure Skin samples fixed with 2.5% glutaraldehyde were post-fixed with 1% osmium tetroxide for 1–1.5 hours. After fixation, the tissue was dehydrated and embedded in epoxy resin. The embedded tissue blocks were cut into ultrathin sections and stained with uranyl acetate. The stained sections were imaged under an electron microscope.

[0037] 4.2 Effects of SRPM on Matrix Metalloproteinases (MMPs) Skin samples stored at -80°C were obtained and homogenized using a tissue grinder. The supernatant was collected and the expression levels of MMP-9 and MMP-2 in the skin were determined according to the ELISA procedure.

[0038] 6.4.3 Regulation of wound-related inflammatory factors by SRPM Serum samples stored at -80°C were taken and the levels of pro-inflammatory factors TNF-α and IL-1β, anti-inflammatory factors IL-10 and TGF-β1, and epidermal growth factor PDGF-BB in the serum were determined according to the ELISA method.

[0039] 2. Experimental Results 1. The yield of SRPM is 8.2% and the purity reaches 76.27% 500 g of Panax notoginseng rhizome was extracted according to the above method to obtain 41 g of white SRPM powder with a calculated yield of 8.2%.

[0040] The purity of the total saponins of Panax notoginseng was determined by the UV spectrophotometric method described above. The sample absorbance was measured to be 0.9346, and substituting it into the standard curve, the purity of SRPM was calculated to be 76.27%.

[0041] According to the above gel preparation method, light yellow SRPM carbomer gel was obtained.

[0042] 2. SRPM can significantly promote wound healing in DRW rats According to the above-mentioned DRW modeling, grouping and administration methods, the animal efficacy of SRPM on DRW was verified, and the wound healing rate of rats in each group was used as the observation indicator.

[0043] like Figure 4 、 4 As shown in Table 1 , the healing rate of MOD was significantly slower than that of CON ( P<0.001), the healing of SIT was significantly slow on the 3rd and 7th days ( P <0.05, P <0.01), SRPM.H was significantly slow on the 7th day ( P <0.05); compared with MOD, SRPM.L healed significantly faster ( P <0.001), SIT healing was significantly faster on the 10th and 14th days ( P <0.001), SRPM.H healed significantly faster on the 3rd, 10th, and 14th days ( P <0.05, P <0.01).

[0044] Table 1 shows the statistical data of changes in wound healing rate in rats.

[0045] Table 1

[0046] Note: Compared with CON, * P <0.05, ** P <0.01, *** P <0.001; compared with MOD, # P <0.05, ## P <0.01, ### P <0.001.

[0047] 3. SRPM can significantly improve ultramicroscopic pathological damage on the wound surface like Figure 6 As shown in the model group (MOD), the tissue showed abundant and neatly arranged mitochondria (red arrows), slight swelling (green arrows), and mostly intact membrane structures. The matrix was dissolved and cristae were broken. Lamellar bodies were abundant and structurally intact.

[0048] like Figure 7 As shown, in the high-dose group of Panax notoginseng (SRPM.H), abundant mitochondria (red arrows) were visible in the tissue, arranged neatly, with intact membrane structure, a small amount of matrix dissolution, and some cristae broken (green arrows).

[0049] like Figure 8 As shown, in the low-dose group of Panax notoginseng (SRPM.L), abundant mitochondria (red arrows) were visible in the tissue, with a moderate number, slightly loose arrangement, intact membrane structure, a small amount of matrix dissolution, and mild swelling (green arrows) in some areas.

[0050] 4. SRPM can reduce the accumulation of matrix metalloproteinases (MMPs) in the skin like Figure 9 As shown, compared with MOD, SRPM.H and SRPM.L can be significantly reduced ( P <0.01) accumulation of MMP-2.

[0051] like Figure 9 As shown, compared with MOD, SRPM.H can reduce MMP-9 ( P <0.05) accumulation, SRPM.L could significantly reduce the MMP-9 ( P <0.01) accumulation.

[0052] 5. SRPM can significantly regulate the levels of related inflammatory and growth factors like Figure 10 As shown in Table 2, compared with MOD, SRPM.H can downregulate the pro-inflammatory factor TNF-α ( P <0.05), IL-1β( P <0.001) and up-regulate the anti-inflammatory factor IL-10 ( P <0.01), TGF-β1( P <0.01) level; SRPM.L can also downregulate the pro-inflammatory factor TNF-α ( P <0.01), IL-1β( P <0.01) and up-regulate the anti-inflammatory factor TGF-β1 ( P <0.01), epidermal growth factor PDGF-BB ( P <0.01). Compared with STI, SRPM.H can upregulate the anti-inflammatory factor IL-10 ( P <0.01), SRPM.L can upregulate epidermal growth factor PDGF-BB ( P <0.001).

[0053] Table 2 shows the statistical results of wound-related inflammation and growth factor levels.

[0054] Table 2

[0055] Note: Compared with CON, # P <0.05, ## P <0.01, ### P <0.001; compared with MOD, * P <0.05, ** P<0.01, *** P <0.001, compared with SIT, ▲▲ P <0.01, ▲▲▲ P <0.001.

[0056] It should be noted that the above-described specific embodiments are illustrative only. Those skilled in the art may devise various solutions based on the disclosure of the present invention, and such solutions fall within the scope of the present invention and are intended to be protected by the present invention. Those skilled in the art should understand that the present description and its accompanying drawings are intended to be illustrative only and are not intended to limit the scope of the claims. The scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A method for preparing a Panax notoginseng extract, characterized in that: The following steps are involved: S1: The rhizomes of Panax notoginseng were crushed and extracted with 70% ethanol under reflux for 3 times and then combined; S2: concentrated under reduced pressure, extracted with water-saturated n-butanol three times and then concentrated; S3: add methanol to dissolve and filter; S4: chromatographic column purification using macroporous resin D101; S5: distill under reduced pressure to obtain Panax notoginseng extract.

2. The preparation method according to claim 1, wherein The "decompression concentration" in S2 includes the following steps: concentration within the pressure range of 0.06~0.1 MPa.

3. The preparation method according to claim 1 or 2, characterized in that The step of "extracting three times with water-saturated n-butanol and then concentrating" in S2 includes the following steps: rotary evaporation at a temperature range of 40-60°C.

4. The preparation method according to any one of claims 1 to 3, characterized in that The "Purification" step in S4 includes the following steps: The methanol extract was passed through a D101 macroporous resin column at a flow rate of 2 BV / h, the loading concentration was controlled at 0.3 g / mL, and adsorption was carried out for 2 hours; Elution with distilled water at a flow rate of 3 BV / h was used to remove unadsorbed impurities; Total saponins were eluted by gradient elution with ethanol of 30%, 50%, and 70% by volume, with an elution volume of 5 BV and a flow rate of 3 BV / h.

5. Use of the extract in the preparation of a medicament for treating refractory diabetic wounds, characterized in that: The extract is an extract according to the preparation method according to any one of claims 1 to 4.

6. The use according to claim 5, characterized in that The extract can be applied externally on wound surfaces or taken orally.

7. The use according to claim 5, characterized in that The oral dosage of the extract is 100-200 mg / kg; the external application dosage of the extract is 50 μL.

8. A pharmaceutical composition for treating refractory diabetic wounds, characterized in that: The invention comprises the extract involved in the preparation method according to any one of claims 1 to 7 and excipients acceptable for skin care.

9. The pharmaceutical composition according to claim 8, characterized in that The auxiliary materials include carbomer, sodium carboxymethyl cellulose, 5% by mass of ethylparaben, triethanolamine and glycerol.

10. A method for preparing a gel for treating refractory diabetic wounds, characterized in that: The following steps are involved: S1: dissolving the Panax notoginseng extract involved in the preparation method according to any one of claims 1 to 4 in sodium carboxymethyl cellulose to obtain a first mixture, wherein the ratio of the Panax notoginseng extract to the sodium carboxymethyl cellulose is 1:10 g / mL; S2: Carbomer is fully dissolved in water to obtain a second mixture, wherein the ratio of carbomer to water is 1:20 g / mL; S3: 9.30 parts by weight of the first mixture, 74.83 parts by weight of the second mixture, 0.07 parts by weight of 5% ethylparaben, 1.86 parts by weight of triethanolamine and 12.08 parts by weight of glycerin were mixed.

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