Efficient preparation and purification method of traditional Chinese medicine active ingredients of rhaponticum uniflorum

Abstract

The invention discloses an efficient preparation and purification method of active pharmaceutical ingredients in rhaponticum uniflorum. The method comprises the steps of pretreatment of raw materials in the flowering stage, vacuum low-temperature countercurrent extraction, membrane coupling purification, resin-crystal combined refining and active ingredient activation. In the pretreatment, ventilating and drying at 40-50 DEG C until the water content is less than or equal to 8%, crushing and sieving with a 40-60-mesh sieve Carrying out dynamic countercurrent extraction in a three-stage series device by using gradient ethanol under the conditions that the temperature is 45 + / -2 DEG C and the pressure is-0.08 to-0.1 MPa; the preparation method comprises the following steps: carrying out microfiltration with a 0.22 [mu] m ceramic membrane and concentration with a 1000Da ultrafiltration membrane, carrying out gradient elution with an HPD-100 resin column, adding a linarin-spinosin (3: 1) seed crystal, crystallizing at 4 DEG C, and finally freeze-drying and activating. According to the present invention, the obtained active component total flavone content is more than or equal to 85%, the iridoid retention rate is more than or equal to 95%, the Nrf2 pathway activation EC50 is less than or equal to 0.12 mg / mL, the APAP liver injury mouse serum ALT is reduced by 55-70% due to the dosage of 80 mg / kg, the active component can be prepared into the enteric capsule for treating the drug-induced liver injury, and the problems of component degradation, low purity and short resin life in the prior art are effectively solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine active ingredient extraction and purification, and particularly relates to a high-efficiency preparation and purification method of active ingredients in radix eupatorium fortunei flowers and application of the active ingredients in preparation of a medicine for treating drug-induced liver injury. BACKGROUND

[0002] Radix eupatorium fortunei flowers are dry inflorescences of a plant in the family Asteraceae, radix eupatorium fortunei, and are commonly used in traditional Chinese medicine for clearing heat and resolving toxins, reducing swelling and draining pus. Modern pharmacological studies have shown that flavonoid glycosides and iridoid compounds rich in radix eupatorium fortunei flowers have significant antioxidant, anti-inflammatory and liver protection activities, and particularly show good application prospects in the treatment of drug-induced liver injury. However, the existing preparation methods of active ingredients in radix eupatorium fortunei flowers have many defects, which restrict their development and utilization.

[0003] In the prior art, the extraction process usually adopts traditional hot reflux extraction or ultrasonic extraction, and has problems such as high extraction temperature (usually 60-80℃), long extraction time (1-2 hours per time), and large solvent consumption, which leads to degradation of heat-sensitive components such as iridoid compounds at high temperatures, and an extraction rate of less than 60%. The purification process usually adopts single macroporous resin adsorption or liquid-liquid extraction, and has low purification precision, and the total flavonoid content is usually only 50-60%, and the impurities cannot be effectively separated and removed, resulting in insufficient purity of the active ingredients.

[0004] At the same time, the existing method has a low retention rate of active ingredients, and particularly, key active ingredients such as geniposide and spinozin are seriously lost during the extraction and purification process, and the retention rate is usually less than 70%. In addition, the resin regeneration method is unreasonable, which leads to rapid decrease of the adsorption capacity of the resin, and the adsorption efficiency is reduced by more than 30% after multiple uses, thereby increasing the production cost. These defects make the active ingredients in radix eupatorium fortunei flowers prepared by the existing method unable to meet the requirements of high purity and high activity for medicinal use, and limit the application thereof in clinical treatment. SUMMARY

[0005] The present application aims to solve the above technical problems, and provides a high-efficiency preparation and purification method of active ingredients in radix eupatorium fortunei flowers.

[0006] To solve the above technical problems, the technical scheme provided by the present application is as follows: a high-efficiency preparation and purification method of active ingredients in radix eupatorium fortunei flowers, comprising the following steps: (a) Pre-treatment of raw materials in the flowering period: collecting radix eupatorium fortunei inflorescences in the flowering period (August), drying at 40-50℃ until the water content is ≤8%, and crushing to pass through a 40-60 mesh sieve; (b) Vacuum low-temperature countercurrent extraction: the raw material was mixed with 60-70% ethanol at a ratio of 1:10-12 solid to liquid, and was extracted in a three-stage series device at 45±2°C under a vacuum of-0.08~-0.1MPa for 3 times, 40 minutes each time; (c) Membrane coupling purification: the extraction liquid was combined and micro-filtered through a 0.22μm ceramic membrane, and was then concentrated to 1 / 5 of the original volume through a 1000Da ultrafiltration membrane (operating pressure 0.4MPa, material temperature ≤40°C); (d) Resin-crystal combined refining: the concentrated liquid was loaded onto an HPD-100 resin column, and was eluted with 5BV water, 3BV 20% ethanol, and 5BV 60% ethanol (flow rate 1.8BV / h) in sequence, and the eluate of the 60% ethanol stage was collected, concentrated under reduced pressure, and then 0.2 volumes of seed crystals were added, and the product was crystallized at 4°C for 18 hours; (e) Active ingredient activation: the crystallized product was freeze-dried (-45°C pre-freezing for 4h→-20°C sublimation for 10h→25°C desorption for 6h), and an active ingredient with total flavonoids ≥85% and iridoid retention rate ≥95% was obtained.

[0007] Further, the seed crystals in step (d) are mixed crystals of geniposide and spinooside at a ratio of 3:1, and the content of geniposide in the product after crystallization is ≥15%, and the content of spinooside is ≥10%.

[0008] Further, the ethanol gradient for the three-stage countercurrent extraction in step (b) is as follows: First stage: 70% ethanol, solid to liquid ratio 1:12; Second stage: 60% ethanol, solid to liquid ratio 1:10; Third stage: 95% ethanol, solid to liquid ratio 1:8.

[0009] Further, the biological activity of the active ingredient meets the following requirements: Nrf2 pathway activation EC 50 ≤0.12mg / mL (HepG2 cell model); Reduces serum ALT of APAP liver injury mice by 55-70% at a dose of 80mg / kg.

[0010] An active ingredient of Echinacea purpurea L. medicine, the component content is: flavonoid glycosides 38-42% (including geniposide 15-18%); Iridoid 18-22% (including spinooside 9-12%); Organic acids 10-12% (including chlorogenic acid 6-8%); The HPLC fingerprint contains characteristic peaks at retention times of 12.3min (chlorogenic acid), 21.7min (rutin), and 34.5min (geniposide).

[0011] The application of a medicinal active ingredient in radix eupatorium fortunei in the preparation of a medicine for treating drug-induced liver injury, wherein the liver injury is induced by paracetamol (APAP) or carbon tetrachloride (CCl4). The medicine dosage form is an enteric capsule, and the oral dose is 50-150 mg / kg / day.

[0012] Further, the regeneration method of the HPD-100 resin column is as follows: sequentially washing with 0.1M NaOH, pure water, 0.05M citric acid and pure water, and the flow rate is 1BV / h; after regeneration, the adsorption capacity retention rate of the resin to loganin is greater than or equal to 98%.

[0013] Compared with the prior art, the application has the following advantages: The application combines low-temperature extraction, membrane separation, resin purification and freeze-drying technologies, solves the defects of easy degradation of active ingredients, low purity, insufficient retention rate and short service life of resin in the prior art, and the total flavonoids obtained are greater than or equal to 85%, the iridoid retention rate is greater than or equal to 95%, the resin adsorption capacity retention rate is high, and the liver protection activity is significant. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is a process flow chart of the high-efficiency preparation and purification method of the medicinal active ingredient in radix eupatorium fortunei. DETAILED DESCRIPTION

[0015] 4, working principle of the application: The technical scheme of the application is based on the principle of multi-step synergistic optimization, and realizes the high-efficiency preparation and purification of the medicinal active ingredient in radix eupatorium fortunei by the organic combination of the following steps: raw material pretreatment, vacuum low-temperature countercurrent extraction, membrane coupling purification, resin-crystallization combined refining and active ingredient activation.

[0016] In the raw material pretreatment stage (step a) in the flowering period, the inflorescences of radix eupatorium fortunei in Qizhou in the flowering period (August) are selected as raw materials, and the content of active ingredients is the highest at this time. 40-50 DEG C ventilation drying method is adopted to avoid the damage of high temperature to the active ingredients, and the moisture content is ≤8% to ensure the stability of the raw materials. The crushed material is passed through a 40-60 mesh sieve to increase the specific surface area of the raw material and provide a good foundation for subsequent extraction.

[0017] The vacuum low-temperature countercurrent extraction step (step b) is crucial for the efficient extraction of active ingredients. The pretreated raw material is mixed with 60-70% ethanol at a solid-liquid ratio of 1:10-12 and extracted in a three-stage series apparatus. A low-temperature condition of 45±2℃, combined with a vacuum environment of -0.08 to -0.1 MPa, is used to lower the solvent boiling point and reduce degradation of active ingredients due to high temperatures. The three-stage countercurrent extraction employs a gradient of ethanol concentrations: 70% ethanol (solid-liquid ratio 1:12) in the first stage, 60% ethanol (solid-liquid ratio 1:10) in the second stage, and 95% ethanol (solid-liquid ratio 1:8) in the third stage. This gradient in solvent concentration and solid-liquid ratio achieves stepwise and efficient dissolution of active ingredients with different polarities. Dynamic countercurrent extraction is performed three times, each time for 40 minutes, significantly improving extraction efficiency.

[0018] In the membrane coupling purification step (step c), the combined extracts are first microfiltered through a 0.22 μm ceramic membrane to remove large molecular impurities such as suspended particles and colloids, reducing the burden on subsequent purification. The microfiltered filtrate is then concentrated through a 1000 Da ultrafiltration membrane at an operating pressure of 0.4 MPa and a feed temperature ≤40℃ to 1 / 5 of its original volume, achieving the separation of small molecule active ingredients from some water-soluble impurities, and simultaneously completing the preliminary concentration of the extract, laying the foundation for subsequent purification.

[0019] A resin-crystallization purification step (step d) further improves the purity of the active ingredient. The concentrated solution is loaded onto an HPD-100 resin column and eluted sequentially with 5 BV of water, 3 BV of 20% ethanol, and 5 BV of 60% ethanol (flow rate 1.8 BV / h). This gradient elution with different ethanol concentrations separates impurities from the target active ingredient. The 60% ethanol eluent is collected, which is rich in flavonoid glycosides and iridoids. After concentration under reduced pressure, 0.2 times the volume of seed crystals (a 3:1 mixture of stigmosiderin and spinosin) is added, and crystallization is performed at 4°C for 18 hours. Directional crystallization further enriches the target active ingredient and improves product purity.

[0020] The active ingredient activation step (step e) adopts a freeze-drying process, first pre-freezing at -45℃ for 4 hours, then sublimating at -20℃ for 10 hours, and finally desorption at 25℃ for 6 hours. This avoids the damage to the structure of the active ingredient caused by high temperature during conventional drying, ensures the biological activity of the active ingredient, and finally obtains an active ingredient with a total flavonoid retention rate of ≥85% and an iridoid retention rate of ≥95%.

[0021] In addition, the HPD-100 resin column is regenerated by sequentially rinsing with 0.1M NaOH → pure water → 0.05M citric acid → pure water (flow rate 1 BV / h), which can effectively restore the resin's adsorption performance, ensuring that the regenerated resin retains ≥98% of the adsorption capacity of strychnine, extending the resin's service life and reducing production costs.

[0022] 5. Implementation method: (1) Raw material pretreatment Inflorescences of *Eupatorium fortunei* from Qizhou during its peak flowering period in August were collected, impurities were removed, and the inflorescences were placed in a ventilated drying oven and dried at 45°C until the moisture content of the raw material dropped to 7%. The dried raw material was then pulverized in a grinder and passed through a 50-mesh sieve to obtain dried *Eupatorium fortunei* flower powder, which was then sealed and stored for later use.

[0023] (2) Vacuum low-temperature countercurrent extraction The dried powder of *Echinops latifolius* flowers and ethanol were added to a three-stage countercurrent extraction apparatus (1) in a specific ratio. 70% ethanol was added to the first extraction tank (solid-liquid ratio 1:12); 60% ethanol was added to the second extraction tank (solid-liquid ratio 1:10); and 95% ethanol was added to the third extraction tank (solid-liquid ratio 1:8). The extraction temperature was set to 45℃ and the vacuum degree to -0.09MPa. The stirring device was started, and dynamic countercurrent extraction was performed three times, each extraction lasting 40 minutes. After extraction, the extracts from the three extraction tanks were combined to obtain a crude extract of *Echinops latifolius* flowers.

[0024] (3) Membrane coupling purification The combined crude extract was pumped into a 0.22 μm ceramic membrane filter (2) for microfiltration to remove suspended impurities, and the microfiltrate was collected. The microfiltrate was then sent to a 1000 Da ultrafiltration membrane separation system (3), with the operating pressure set at 0.4 MPa and the feed temperature controlled at 35 °C, for ultrafiltration concentration until the volume of the concentrate was 1 / 5 of the original microfiltrate volume, and the concentrate was collected.

[0025] (4) Refining by resin-crystallization The concentrate was loaded onto a pretreated HPD-100 resin column (4) at a flow rate of 1.8 BV / h. After loading, the column was eluted sequentially with 5 BV purified water, 3 BV 20% ethanol solution, and 5 BV 60% ethanol solution at the same flow rate, and the 60% ethanol eluent was collected. The 60% ethanol eluent was concentrated under reduced pressure at 45℃ and -0.08 MPa to a paste with a relative density of 1.15. 0.2 times the volume of strychnosine-spinosine (3:1) mixed seed crystals (5) were added to the paste, stirred evenly, and then placed in a refrigerator at 4℃ for 18 hours to crystallize. The precipitated crystals were collected by filtration.

[0026] (5) Activation of active ingredients The collected crystals were placed in a freeze dryer (6), pre-frozen at -45°C for 4 hours, then sublimated at -20°C for 10 hours, and finally decomposed at 25°C for 6 hours to obtain the active ingredient dry powder of Luluhua.

[0027] (6) Resin regeneration When the adsorption capacity of the HPD-100 resin column decreased, regeneration was performed. The resin column was flushed sequentially with 0.1M NaOH solution, purified water, 0.05M citric acid solution, and purified water at a flow rate of 1 BV / h until the pH of the effluent was neutral, thus completing resin regeneration. After regeneration, the resin retained 98.5% of the adsorption capacity of strychnine.

[0028] Analysis revealed that the active ingredients of *Echinops latifolius* prepared in this embodiment contained 40% flavonoid glycosides (including 16% scutellarin), 20% iridoids (including 10% spinosin), 11% organic acids (including 7% chlorogenic acid), a total flavonoid content of 86%, and an iridoid retention rate of 96%. HPLC fingerprinting showed characteristic peaks at retention times of 12.3 min (chlorogenic acid), 21.7 min (rutin), and 34.5 min (scutellarin). Biological activity assays indicated that the Nrf2 pathway activation EC50 was 0.11 mg / mL (HepG2 cell model); at a dose of 80 mg / kg, it reduced serum ALT by 62% in mice with APAP-induced liver injury. This active ingredient can be formulated into enteric-coated capsules for the treatment of APAP or CCl4-induced drug-induced liver injury, with an oral dose of 100 mg / kg / day.

[0029] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A highly efficient method for preparing and purifying the active pharmaceutical ingredients from *Echinops latifolius* flowers, characterized in that: Includes the following steps: (a) Pre-treatment of raw materials during flowering period: collect the inflorescences of Qizhou Loulu during the peak flowering period (August), dry them in a ventilated environment at 40-50℃ until the moisture content is ≤8%, and then crush them through a 40-60 mesh sieve; (b) Vacuum low-temperature countercurrent extraction: The raw material is mixed with 60-70% ethanol at a material-to-liquid ratio of 1:10-12 and dynamically countercurrently extracted three times in a three-stage series device at 45±2℃ and a vacuum degree of -0.08~-0.1MPa, each time for 40 minutes; (c) Membrane coupling purification: The combined extracts were microfiltered through a 0.22 μm ceramic membrane and then concentrated to 1 / 5 of the original volume through a 1000 Da ultrafiltration membrane (operating pressure 0.4 MPa, material temperature ≤40℃); (d) Resin-crystallization purification: The concentrated solution was loaded onto an HPD-100 resin column and eluted sequentially with 5 BV water, 3 BV 20% ethanol, and 5 BV 60% ethanol (flow rate 1.8 BV / h). The 60% ethanol eluent was collected, concentrated under reduced pressure, and then 0.2 times the volume of seed crystals were added. The solution was crystallized at 4°C for 18 hours. (e) Activation of active ingredients: The crystalline product was freeze-dried (pre-frozen at -45℃ for 4h → sublimated at -20℃ for 10h → decomposed at 25℃ for 6h) to obtain active ingredients with a total flavonoid retention rate of ≥85% and a iridoid retention rate of ≥95%.

2. The method for efficient preparation and purification of active pharmaceutical ingredients from *Echinops latifolius* according to claim 1, characterized in that: The seed crystals in step (d) are a mixture of styrax and spinosin in a 3:1 ratio, and the crystallized product contains ≥15% styrax and ≥10% spinosin.

3. The method for efficient preparation and purification of active pharmaceutical ingredients from *Echinops latifolius* according to claim 1, characterized in that: The ethanol gradient for the three-stage countercurrent extraction in step (b) is as follows: Grade 1: 70% ethanol, solid-liquid ratio 1:12; Secondary: 60% ethanol, solid-liquid ratio 1:10; Grade III: 95% ethanol, solid-liquid ratio 1:

8.

4. The method for efficient preparation and purification of active pharmaceutical ingredients from *Echinops latifolius* according to claim 1, characterized in that: The biological activity of the active ingredient satisfies: Nrf2 pathway activates EC 50 ≤0.12 mg / mL (HepG2 cell model); At a dose of 80 mg / kg, serum ALT levels in APAP-injured mice decreased by 55-70%.

5. The active pharmaceutical ingredient in *Echinops latifolius* according to claims 1-4, characterized in that: Component content: Flavonoid glycosides 38-42% (of which buddleja glycosides 15-18%); Iridoids 18-22% (of which spinosin 9-12%); Organic acids 10-12% (of which chlorogenic acid 6-8%); The HPLC fingerprint spectrum contains characteristic peaks with retention times of 12.3 min (chlorogenic acid), 21.7 min (rutin), and 34.5 min (monotropin).

6. The application of the active pharmaceutical ingredient from *Echinops latifolius* according to claim 5 in the preparation of a drug for treating drug-induced liver injury, characterized in that: The liver injury was induced by acetaminophen (APAP) or carbon tetrachloride (CCl4); The drug is in the form of enteric-coated capsules, with an oral dose of 50-150 mg / kg / day.

7. The method for efficient preparation and purification of active pharmaceutical ingredients from *Echinops latifolius* according to claim 1, characterized in that: The regeneration method of the HPD-100 resin column involves sequential rinsing with 0.1M NaOH → pure water → 0.05M citric acid → pure water at a flow rate of 1 BV / h; after regeneration, the resin retains ≥98% of the adsorption capacity for strychnine.

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