Preparation method of hispidin and application of hispidin in resisting colorectal cancer

Abstract

The application belongs to the technical field of biology and specifically relates to a preparation method of Hispidin and application of the Hispidin in resisting colorectal cancer. The application uses the fruiting body of Morus alba Quercus (anamorph - Inonotus hispidus) as raw material, adopts ultrasonic-assisted ethanol extraction and gel column chromatography purification, and obtains high-purity Hispidin. The low-temperature ultrasonic-assisted ethanol extraction avoids the damage of high temperature, strong acid and strong base to the conjugated structure and phenolic hydroxyl group of Hispidin, the extraction condition is mild, the time consumption is short, the energy consumption is low, and the extraction efficiency is significantly higher than that of the traditional method. Combined with gel column chromatography and HPLC real-time quality control, Hispidin monomer with a purity of 97.3% can be stably obtained, which meets the pharmaceutical purity requirement and solves the problems of difficult separation and purification and low purity of natural products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing Hispidin and its application in the treatment of colorectal cancer. Background Technology

[0002] Natural active ingredients refer to chemical components (such as alkaloids, flavonoids, polysaccharides, and volatile oils) from natural sources such as plants, animals, or microorganisms that possess specific physiological activities or application value. These components are mostly encapsulated within dense cell walls and fibrous tissues, and are tightly bound to impurities such as starch, pectin, protein, and cellulose, exhibiting characteristics such as dispersion, low content, poor stability, and difficulty in separation and purification. Therefore, the efficient, green, and complete extraction, separation, and purification of target active substances from natural raw materials is a core technology in the field of deep processing of natural products.

[0003] Existing natural substance extraction processes encompass mainstream technologies such as traditional solvent extraction, physical-assisted extraction, enzymatic extraction, and acid-base modified extraction, which can basically achieve the initial enrichment and preparation of natural active ingredients. However, in actual industrial production and laboratory preparation, these extraction processes generally suffer from numerous technical shortcomings, including low extraction rates, difficulty in balancing extraction efficiency and selectivity, severe activity degradation, and high energy consumption. Furthermore, natural product extraction technologies lack universality and scalability, making it difficult to simultaneously meet the demands of high extraction rates, high activity, high purity, low cost, and environmentally friendly industrial production, significantly limiting the large-scale application of natural active substances and the upgrading of product quality.

[0004] Hispidin is a typical styrylpyranone-based natural polyphenol with a molecular weight of 246.22. Its molecule consists of a 4-hydroxy-α-pyranone core, a trans-ethylene linking chain, and a 3,4-dihydroxyphenyl group, forming a continuous conjugated system. Rich in phenolic hydroxyl groups and lactone structures, it exhibits moderate polarity, is readily soluble in alcohols, and slightly soluble in water. Its structural characteristics limit its physicochemical stability; high temperatures, light, and alkaline environments can all cause molecular structural damage and degradation of the active ingredients. It is stable only in neutral and weakly acidic systems. The ortho-bisphenolic hydroxyl group and the long conjugated system are its core active groups and are the structural units that require careful protection during extraction, separation, and preservation.

[0005] Therefore, existing extraction and purification processes are insufficient to efficiently obtain high-purity active products. Summary of the Invention

[0006] To address the problems raised in the background art, this invention provides a method for preparing Hispidin and its application in the treatment of colorectal cancer.

[0007] The technical solution of the present invention is as follows: This invention provides a method for preparing Hispidin, comprising the following steps: (1) Using dried mulberry fruiting body powder as raw material, ethanol was used as extraction solvent and low temperature extraction was carried out under ultrasonic assistance. The supernatant was obtained by vacuum filtration, centrifugation, vacuum concentration and evaporation to obtain crude Hispidin extract. (2) The crude Hispidin extract was separated by gel column chromatography, eluted with methanol as the mobile phase, and the purity was monitored in real time by high performance liquid chromatography. The high purity components were combined and the solvent was removed under reduced pressure to obtain high purity Hispidin monomer.

[0008] Based on the above-described method for preparing Hispidin, in step (1), the ethanol concentration is 90%-95%, and the ratio of raw material to ethanol solution is 1:10-1:30.

[0009] Based on the Hispidin preparation method described above, the vacuum concentration temperature in step (1) is 40-45℃.

[0010] Based on the Hispidin preparation method described above, the low-temperature extraction temperature in step (1) is 45-55℃.

[0011] Based on the above-described method for preparing Hispidin, in step (2), the gel column is wet-packed after being swelled by elution solvent, ultrasonically or vacuum degassed, and the column bed surface is covered with 0.4-0.6 cm thick quartz sand.

[0012] Based on the above-described method for preparing Hispidin, in step (2), when loading the sample, the crude Hispidin extract is dissolved in methanol, and the amount of methanol used is 1-3 mL for every 1-2 g of crude Hispidin extract.

[0013] Based on the above-described method for preparing Hispidin, high-purity Hispidin monomer with a purity of not less than 95% is obtained.

[0014] The present invention also provides the application of Hispidin prepared by the above-described method in the preparation of anti-colorectal cancer products, which is administered by gavage.

[0015] Specifically, Hispidin can inhibit the growth of subcutaneous xenografts of colorectal cancer in a dose-dependent manner by downregulating Ki-67 expression in tumor tissue.

[0016] Furthermore, the dosage is 5-45 mg / kg.

[0017] Beneficial effects This invention uses the fruiting body of *Pheretima aspergillum* (asexual form of *Pheretima salina*) as raw material and employs ultrasound-assisted ethanol extraction and gel column chromatography purification to obtain high-purity Hispidin. The low-temperature ultrasound-assisted ethanol extraction avoids the damage to the conjugated structure and phenolic hydroxyl groups of Hispidin caused by high temperatures, strong acids, and strong alkalis. The extraction conditions are mild, time-efficient, and energy-low, resulting in significantly higher extraction efficiency than traditional methods. Combined with gel column chromatography and real-time quality control by HPLC, Hispidin monomer with a purity of up to 97.3% can be stably obtained, meeting pharmaceutical purity requirements and solving the problems of difficult separation and purification of natural products and low purity.

[0018] This invention applies the prepared Hispidin to the preparation of anti-colorectal cancer products by gavage administration. In vivo animal experiments have confirmed that it has a significant inhibitory effect on colorectal cancer xenografts and has high safety. Attached Figure Description

[0019] Figure 1 This is the standard curve for the Folin-Ciocalteu method.

[0020] Figure 2 This is a photograph of the tumor sample after it was removed.

[0021] Figure 3 This is a weight monitoring curve.

[0022] Figure 4 This is the in vivo tumor growth kinetics curve.

[0023] Figure 5 This is an image showing H&E staining observation.

[0024] Figure 6 This is a graph showing the Ki-67 index analysis by immunohistochemistry. Detailed Implementation

[0025] The following examples are intended to illustrate the present invention, and not to further limit the invention.

[0026] This invention provides a method for preparing Hispidin, comprising the following steps: (1) Using dried mulberry fruiting body powder as raw material, ethanol was used as the extraction solvent and low temperature extraction was carried out under ultrasonic assistance. The supernatant was obtained by vacuum filtration, centrifugation, vacuum concentration and evaporation to obtain crude Hispidin extract.

[0027] The ethanol concentration is 90%-95%, and the ratio of raw material to ethanol liquid is 1:10-1:30.

[0028] Multiple ultrasonic extractions can be performed, with a low-temperature extraction temperature of 45-55℃.

[0029] Furthermore, the extracted supernatants are combined, then solid-liquid separation and concentration are performed, with the concentration under reduced pressure at a temperature of 40-45℃.

[0030] (2) The crude Hispidin extract was separated by gel column chromatography, eluted with methanol as the mobile phase, and the purity was monitored in real time by high performance liquid chromatography. The high purity components were combined and the solvent was removed under reduced pressure to obtain high purity Hispidin monomer.

[0031] First, the gel column is swollen by elution solvent, degassed by ultrasound or vacuum, and then wet-packed. The column bed surface is covered with 0.4-0.6 cm thick quartz sand.

[0032] When loading the sample, the crude Hispidin extract is dissolved in methanol, and the amount of methanol used is 1-3 mL for every 1-2 g of crude Hispidin extract.

[0033] Then, methanol was used as the mobile phase for elution, and the eluent was collected stepwise.

[0034] Finally, high-purity Hispidin monomer was obtained, with a purity of not less than 95%.

[0035] This invention uses the fruiting body of *Phellinus linteus* (asexual form of *Phellinus coarsely hairy fungus*) as raw material, employing ultrasound-assisted ethanol extraction and gel column chromatography purification to obtain high-purity Hispidin. The low-temperature ultrasound-assisted ethanol extraction avoids the damage to the conjugated structure and phenolic hydroxyl groups of Hispidin caused by high temperatures, strong acids, and strong alkalis. The extraction conditions are mild, time-efficient, and energy-low, with significantly higher extraction efficiency than traditional ethanol reflux extraction methods. Combined with gel column chromatography and HPLC real-time quality control, Hispidin monomer with a purity of up to 97.3% can be stably obtained, meeting pharmaceutical purity requirements and solving the problems of difficult separation and purification of natural products and low purity.

[0036] Phellinus linteus is a rare edible and medicinal fungus. Although similar fungi (such as Phellinus linteus) can grow on various broad-leaved trees like poplar and birch, the authentic Phellinus linteus specifically refers to the Phellinus linteus parasitizing mulberry trees. *Fomitopsis coccinea* is the asexual form (mycelial stage) of Phellinus linteus, and its artificial cultivation can be achieved using strains of *Fomitopsis coccinea*. The ethanol extract of Phellinus linteus can inhibit the proliferation and metastasis of colorectal cancer cells, effectively prevent distant invasion of colorectal cancer, and induce mitochondrial apoptosis.

[0037] The present invention also provides the application of Hispidin prepared by the above-described method in the preparation of anti-colorectal cancer products, which is administered by gavage.

[0038] Specifically, Hispidin can inhibit the growth of subcutaneous xenografts of colorectal cancer in a dose-dependent manner by downregulating Ki-67 expression in tumor tissue.

[0039] Furthermore, the dosage is 5-45 mg / kg.

[0040] This invention applies the prepared Hispidin to the preparation of anti-colorectal cancer products by gavage administration. In vivo animal experiments have confirmed that it has a significant inhibitory effect on colorectal cancer xenografts and has high safety.

[0041] Research has revealed that Hispidin is the main polyphenolic component in the ethanol extract of mulberry linteus. Based on the structural characteristics of Hispidin, this invention has developed a targeted extraction technology and confirmed the anti-colon cancer activity of Hispidin, laying a solid research foundation for subsequent application research.

[0042] Example 1 This embodiment provides a method for preparing Hispidin, including the following steps: (1) Extraction of Phellinus linteus (asexual type - Phellinus coarse-haired filamentous fungus, hereinafter referred to as Phellinus linteus) from mulberry tree (11) Take 40g of dried Phellinus linteus fruiting body powder (passed through No. 4 sieve), use 400ml of 95% ethanol (material-liquid ratio 1:20), and ultrasonically extract for 30min at ultrasonic power of 300W and 50℃, stirring continuously during the process. Take the supernatant, add 400ml of ethanol, and ultrasonically extract for 30min. Repeat twice.

[0043] (12) Combine the supernatants from the two extractions, filter them, and finally centrifuge (5000 rpm, 10 min) to collect the supernatant.

[0044] (13) Concentrate under reduced pressure and rotary evaporate at 40°C until no ethanol is expelled.

[0045] (14) The extract was then placed in a 40°C water bath and evaporated to dryness to obtain crude Hispidin extract.

[0046] (2) Purification of crude Hispidin extract (21) Hispidin was purified by gel column chromatography. The gel was fully swollen in the elution solvent, degassed by sonication or vacuum, and then packed into the chromatography column using a wet homogenization method. The mobile phase was turned on and circulated at a constant flow rate until the column bed height was stable and no bubbles were generated. Then, a 0.5 cm thick layer of quartz sand was placed on the resin surface to protect the column bed and ensure stability.

[0047] (22) Weigh 1.6 g of crude Hispidin extract, add 2 mL of methanol to dissolve it completely, and load the sample. Use methanol as the mobile phase for elution, collect the eluent using a fractional collection method, and number the collection tubes sequentially.

[0048] (23) Samples were taken every three eluent tubes, and purity was monitored using high-performance liquid chromatography (HPLC). Chromatographic conditions were as follows: Agilent TC-C18 column, acetonitrile (A) and 0.1% formic acid aqueous solution (B) as mobile phase, flow rate 1.0 mL / min. The gradient elution program was set as follows: 0-10 min, 2% A; 10-15 min, 2%→13% A; 15-55 min, 13%→45% A; 55-60 min, 45% A; 60-68 min, 45%→90% A.

[0049] (24) Based on the HPLC detection results, the eluent fraction containing high-purity Hispidin was combined, and the solvent was removed by rotary evaporation under reduced pressure to finally obtain high-purity Hispidin monomer.

[0050] Application Example 1 The high-purity Hispidin monomer prepared in Example 1 was used in an in vivo anti-colon cancer experiment.

[0051] 1. Animal model construction BALB / c mice (female, 4-5 weeks old) were divided into 5 groups (n=6: control group, model group, low-dose Hispidin group (10 mg / kg), high-dose Hispidin group (40 mg / kg), and 5-fluorouracil (5FU) positive control group (35 mg / kg).

[0052] Subcutaneous injection of 100 μl CT26 cells (1×10⁻⁶) 6 After the tumor reaches a volume of 50-100 mm², (one mouse per tumor) 3 Begin administering the medication.

[0053] 2. Administration and Observation Mice were divided into a control group, a model group, a low-dose Hispidin group (INL), a high-dose Hispidin group (INH) administered by gavage, and a 5FU-positive control group administered by intraperitoneal injection. The administration was once daily for 14 days.

[0054] Tumor volume: The long diameter (L) and short diameter (W) of the tumor are measured every 3 days. Volume = 0.5 × L × W 2 .

[0055] Weight and survival rate: Record weight changes and survival time.

[0056] Histopathology: After drug administration, the tumor was dissected, and HE staining was performed to observe tumor necrosis in order to preliminarily assess the in vivo safety of the drug, and Ki-67 (proliferation) was detected by immunohistochemistry.

[0057] 3. Experimental Results Depend on Figure 1It can be seen that Hispidin with a purity of up to 97.3% (based on GAE) was obtained by optimizing the extraction process.

[0058] Depend on Figure 2 The images of the tumor samples after dissection (each small square represents 3 cm) show that, compared with the MOL group (model group) which has the most vigorous growth and the largest volume, Hispidin treatment significantly reduced the tumor size in a dose-dependent manner, and the tumor-suppressing effect of the high-dose Hispidin group (INH group) has shown the potential to approach that of the 5-fluorouracil positive control group (5FU group).

[0059] Depend on Figure 3 The weight monitoring curves showed that, compared with the 5-fluorouracil positive control group (5FU group) which showed significant weight loss and drug toxicity, the weight of mice in the Hispidin intervention group (INL and INH groups) remained relatively stable throughout the entire administration period, demonstrating that it has good biocompatibility while achieving tumor suppression effect.

[0060] Depend on Figure 4 The in vivo tumor growth kinetic curves showed that Hispidin's inhibitory effect on colorectal cancer xenografts was significantly dose-dependent; among them, the INH group effectively inhibited the exponential expansion of tumor volume.

[0061] Depend on Figure 5 It was found that the tumor tissue in the MOL group (model group) had an intact structure, extremely densely packed parenchymal cells, large and deeply stained nuclei, a significantly increased nucleus-cytoplasmic ratio, and almost no obvious necrotic areas, showing vigorous malignant proliferative characteristics. In both the low-dose Hispidin group (INL group) and the high-dose Hispidin group (INH group), the tumor tissue showed significant regressive changes with increasing dosage. Specifically, the DH group showed significant cell vacuolation, loose tissue structure, and focal cell necrosis, suggesting that medium- to high-dose Hispidin had begun to disrupt the integrity of the tumor tissue. The 5-fluorouracil positive control group (5FU group) also showed a clear tumor-suppressive effect, with widened interstitial spaces, decreased cell density, and scattered necrotic areas.

[0062] Depend on Figure 6 Dense, dark brown positive granules (Ki-67 protein is mainly located in the cell nucleus) were visible in the MOL group. The proportion of positive cells was extremely high, and the staining intensity was strong. With increasing polyphenol dosage, the brown area in the DH group decreased significantly, while the proportion of blue (negative cell nuclei) increased significantly. This indicates that Hispidin effectively arrests tumor cells in the quiescent phase by downregulating Ki-67 expression, thus inhibiting tumor expansion at its source.

Claims

1. A method for preparing Hispidin, characterized in that, Includes the following steps: (1) Using dried mulberry fruiting body powder as raw material, ethanol was used as extraction solvent and low temperature extraction was carried out under ultrasonic assistance. The supernatant was obtained by vacuum filtration, centrifugation, vacuum concentration and evaporation to obtain crude Hispidin extract. (2) The crude Hispidin extract was separated by gel column chromatography, eluted with methanol as the mobile phase, and the purity was monitored in real time by high performance liquid chromatography. The high purity components were combined and the solvent was removed under reduced pressure to obtain high purity Hispidin monomer.

2. The method for preparing Hispidin according to claim 1, characterized in that, In step (1), the ethanol concentration is 90%-95%, and the ratio of raw material to ethanol liquid is 1:10-1:

30.

3. The method for preparing Hispidin according to claim 1, characterized in that, In step (1), the vacuum concentration temperature is 40-45℃.

4. The method for preparing Hispidin according to claim 1, characterized in that, In step (1), the low-temperature extraction temperature is 45-55℃.

5. The method for preparing Hispidin according to claim 1, characterized in that, In step (2), the gel column is swollen by elution solvent, degassed by ultrasound or vacuum, and then wet-packed. The column bed surface is covered with 0.4-0.6 cm thick quartz sand.

6. The method for preparing Hispidin according to claim 1, characterized in that, In step (2), when loading the sample, the crude Hispidin extract is dissolved in methanol, and the amount of methanol used is 1-3 mL for every 1-2 g of crude Hispidin extract.

7. The method for preparing Hispidin according to claim 1, characterized in that, High-purity Hispidin monomer, with a purity of not less than 95%.

8. The application of Hispidin prepared by the method of Hispidin as described in claim 1 in the preparation of anti-colorectal cancer products, wherein the administration is carried out by gavage.

9. The application according to claim 8, characterized in that, Hispidin can inhibit the growth of subcutaneous xenografts of colorectal cancer in a dose-dependent manner by downregulating Ki-67 expression in tumor tissue.

10. The application according to claim 8, characterized in that, The dosage is 5-45 mg / kg.

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