Refining preparation method of polyphenol component in humifuse euphorbia herb

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a preparation method of a refined extract of polyphenol components in traditional Chinese medicines. The traditional Chinese medicine extract is extracted by taking humifuse euphorbia herb as a raw material medicine and taking water as a solvent. The method comprises the following steps: taking humifuse euphorbia herb decoction pieces, adding pure water of which the volume is 6-10 times that of the humifuse euphorbia herb decoction pieces, soaking for 0.5-2 hours, then decocting for 1-3 times at 100 DEG C, heating and extracting for 1-3 hours each time, filtering while hot, combining extracting solutions, and clarifying and filtering through a ceramic membrane and a spiral-wound membrane in sequence. Then, a C18YE column is adopted for purification, a target component is eluted under 30-60% ethyl alcohol, then the eluted target product is subjected to rotary evaporation concentration and freeze-drying, and finally the euphorbia humifusa extract refined aiming at the polyphenol components is obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, and particularly relates to a preparation method of a refined traditional Chinese medicine extract of polyphenol components in Euphorbia humifusa. BACKGROUND

[0002] Traditional Chinese medicine Euphorbia humifusa is the dry whole herb of Euphorbia humifusa Willd. or Euphorbia maculata L. of Euphorbiaceae, and its efficacy is recorded in Chinese Pharmacopoeia. Euphorbia humifusa is considered to have the effects of clearing heat and resolving toxicity, cooling blood and stopping bleeding, and removing dampness and relieving jaundice, and is suitable for treating dysentery, diarrhea, hemoptysis, hematuria, hematochezia, metrorrhagia, furuncles, and carbuncles. In clinical practice, various preparations containing Euphorbia humifusa are used for the treatment of enteritis, such as Euphorbia humifusa capsules. However, these preparations have some shortcomings, such as a large amount of taking, and an adult needs to take 3-6 capsules at a time, 3 times a day. Euphorbia humifusa capsules are obtained by mixing the reduced pressure concentrated decoction and Euphorbia humifusa powder, and then drying and filling into capsules. There are also some shortcomings in using the whole herb extract as medicine, which may contain inactive ingredients, increasing the complexity of the drug and the risk of side effects. This makes it difficult to determine the main active ingredients and mechanism of action of the drug, increasing the uncertainty of the treatment effect. Therefore, the extraction of the extract needs to be refined according to the pharmacological effects and interactions of each component in the whole herb extract, in order to guide the clinical application and drug deployment.

[0003] Polyphenols are the main components in Euphorbia humifusa, mainly including flavonoids, phenolic acids, and tannins, which have antioxidant, immunomodulatory, and anti-inflammatory activities. A large number of studies have shown that polyphenolic compounds have a regulatory effect on intestinal flora, can increase the number of probiotics and reduce the relative abundance of pathogenic bacteria through various pathways such as reducing the secretion of inflammatory-related factors, effectively relieving the symptoms of colitis, and improving the intestinal microecological imbalance caused by the disease (Chinese Traditional and Herbal Drugs 54(10) (2023) 3349). Therefore, the extraction of the extract needs to be refined according to the pharmacological effects of polyphenolic components in Euphorbia humifusa extract, which helps Euphorbia humifusa to better exert its curative effect. However, there is no report on the preparation method of polyphenolic components in Euphorbia humifusa. SUMMARY

[0004] The purpose of the present application is to provide a polyphenol component extract in traditional Chinese medicine and a preparation method thereof. The process includes the following steps: medicinal material extraction, membrane filtration and chromatographic column purification, concentration and drying. The specific steps are as follows:

[0005] (1) Medicinal material extraction: The extraction process has an effect on the components and effects of the final extract. As preferred in the present application, 100°C heating extraction is adopted.

[0006] As a preferred embodiment of the present application, the mass ratio of the material (Euphorbia humifusa) to the liquid (pure water) during extraction is 1:(6-10), such as 1:6, 1:8, or 1:10. In order to further improve the extraction rate, the mass ratio of the material to the liquid is preferably 1:(8-10), and more preferably 1:10. The heating extraction time is not limited, and the extraction time can be 1-3 hours, preferably 1 hour.

[0007] As a preferred embodiment of the present application, the material is soaked for 0.5-2 hours before heating extraction, and the soaking time is preferably 0.5 hours.

[0008] As a preferred embodiment of the present application, the extraction is performed at least twice. Multiple extractions refer to the extraction of the filtered residue with water after each extraction, and the final filtrate obtained after each extraction is combined. The number of extractions can be 2, 3, or 4, and the number of extractions is preferably 2, to obtain a crude extract of Euphorbia humifusa.

[0009] (2) Clarification filtration: In order to filter and remove impurities from the Euphorbia humifusa extract, as a preferred embodiment of the present application, the crude extract of Euphorbia humifusa obtained in step (1) is sequentially filtered through one or more of ceramic membranes, microporous membranes, and ultrafiltration membranes, preferably ceramic membranes and spiral membranes, and preferably 500 nm ceramic membranes and 50 kD spiral membranes.

[0010] (3) Column purification: When the traditional Chinese medicine extract contains more extraction components, the medicinal effect is relatively better. In order to refine the extraction rate, as a preferred embodiment of the present application, the permeate obtained in step (2) is purified through C18YE filler.

[0011] As a preferred embodiment of the present application, 30-60% ethanol, preferably 50% ethanol, is used for elution of the target component.

[0012] (4) Concentration and drying: After the target product is eluted, it is concentrated by rotary evaporation and vacuum dried to obtain a refined extract of the prepared Euphorbia humifusa polyphenolic component.

[0013] The method of the present application can be applied to the refined extraction of the Euphorbia humifusa polyphenolic component.

[0014] Advantages of the present application

[0015] A method for refining and extracting the polyphenolic component of Euphorbia humifusa is provided. This method can effectively increase the content of polyphenolic compounds used for treating enteritis, thereby reducing the risk of complexity and side effects during drug treatment. Through this technology, Euphorbia humifusa can better exert its therapeutic effect, and a reliable technical solution is provided for the preparation of Euphorbia humifusa drugs. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1The chromatogram of the extract of Euphorbia humifusa is analyzed. Characteristic compounds: peak 1: breynin and / or its isomer; peak 2: breynin and / or its isomer; peak 3: quercetin 3-O-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside; peak 4: ellagic acid; peak 5: quercetin 3-O-α-L-arabinoside.

[0017] Figure 2 The chromatogram of the extract of Euphorbia humifusa is analyzed. Characteristic compounds: peak 1: breynin and / or its isomer; peak 2: breynin and / or its isomer; peak 3: quercetin 3-O-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside; peak 4: ellagic acid; peak 5: quercetin 3-O-α-L-arabinoside.

[0018] Figure 3 The chromatogram of the extract of Euphorbia humifusa is analyzed. Characteristic compounds: peak 1: breynin and / or its isomer; peak 2: breynin and / or its isomer; peak 3: quercetin 3-O-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside; peak 4: ellagic acid; peak 5: quercetin 3-O-α-L-arabinoside.

[0019] Figure 4 The chromatogram of the extract of Euphorbia humifusa is analyzed. Characteristic compounds: peak 1: breynin and / or its isomer; peak 2: breynin and / or its isomer; peak 3: quercetin 3-O-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside; peak 4: ellagic acid; peak 5: quercetin 3-O-α-L-arabinoside.

[0020] Table 1 is an experimental parameter taking 200g of Euphorbia humifusa extract as an example.

[0021] Table 2 is the main parameter before and after the filtration of the 500nm ceramic membrane.

[0022] Table 3 is the main parameter before and after the filtration of the 50kD rolling membrane.

[0023] Table 4 is the transfer rate of each characteristic compound after the filtration of the 50kD rolling membrane. Characteristic compounds: peak 1: breynin and / or its isomer; peak 2: breynin and / or its isomer; peak 3: quercetin 3-O-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside; peak 4: ellagic acid; peak 5: quercetin 3-O-α-L-arabinoside.

[0024] Table 5 is the main parameter of each eluent in the purification. DETAILED DESCRIPTION

[0025] The present application will be further described in conjunction with examples. The examples are only intended to illustrate the present application, but not to limit the present application.

[0026] Example 1: Extraction of Euphorbia humifusa

[0027] Euphorbia humifusa decoction pieces 200 g were loaded into a 5 L single-necked flask, and 2 L of pure water was added to soak for 30 minutes. Then, an intelligent magnetic stirrer was used to heat and raise the temperature to 100 °C for the first extraction. The 100 °C decoction was performed for 1 hour, and then a 200-mesh filter cloth was used to collect the first extraction liquid while hot. Then, 2 L of pure water was added to the flask containing the solid residue, and the temperature was raised to 100 °C for the second extraction. The 100 °C decoction was performed for 1 hour, and then a 200-mesh filter cloth was used to collect the second extraction liquid while hot. The two extraction liquids were combined to obtain the crude extract of Euphorbia humifusa.

[0028] Taking the extraction experiment of 200 g of Euphorbia humifusa as an example, the process parameters are shown in Table 1, and the liquid chromatogram analysis spectrum and representative components of the Euphorbia humifusa extraction liquid are shown in Figure 1 .

[0029] Example 2: Membrane filtration of the crude extract of Euphorbia humifusa

[0030] The crude extract of Euphorbia humifusa obtained in Example 1 was sequentially subjected to 500 nm ceramic membrane and 50 kD roll-type membrane through a ceramic membrane small-scale test device CeraMem0100-015, according to the molecular weight cutoff (3 L of water was added for washing and filtration during the process to improve the recovery rate). The comparative chromatograms before and after the 500 nm ceramic membrane and the 50 kD roll-type membrane are shown in Figure 2 and Figure 3 , respectively. The main parameters and the peak transfer rate of the characteristic compounds before and after the filtration are shown in Tables 2-4. The transfer rate of polyphenolic compounds basically reached more than 94%.

[0031] Example 3: Chromatographic column purification

[0032] The 50 kD roll-type membrane permeate liquid sample of Euphorbia humifusa obtained in Example 2 was loaded onto a C18YE column (specifications: 30 x 240 mm, filler particle size: 30 μm, Huapu) for purification. 3 column volumes of equilibration treatment were performed in pure water, and after 20 minutes, 6% column volumes of sample loading were used for loading. 3 column volumes of elution were performed in pure water for 20 minutes. Then, 3 column volumes of 50% ethanol by volume were used for the first elution, and then 3 column volumes of ethanol were used for the second elution. The chromatograms of each eluent before and after the C18YE purification experiment are shown in Figure 4 , and the main parameters in each eluent are shown in Table 5.

[0033] Table 1

[0034]

[0035] Table 3

[0036]

[0037] Table 4

[0038]

[0039]

[0040] Table 5

[0041]

[0042] As can be seen from the figures and examples, the method has a good refining effect on polyphenolic components in Euphorbia humifusa.

Claims

1. A method for purifying and preparing polyphenolic components from Euphorbia humifusa, characterized in that: Using *Euphorbia humifusa* as the raw material, water was used as the solvent for extraction. The extract was purified by membrane filtration and chromatographic column to obtain polyphenolic components.

2. The method according to claim 1, characterized in that: Extraction process: The material-to-liquid mass ratio is 1:(6-10), and the water extraction is carried out under heating conditions; optionally or not, soaking for 0.5-2 hours before heating extraction, preferably 0.5-1 hour; The heating and extraction temperature is 90-100℃, preferably 98-100℃, and the heating and extraction time is 1-3 hours, preferably 1-2 hours; The extraction is performed 1 to 3 times, and the solid and liquid are separated and the extracts are combined.

3. The method according to claim 1, characterized in that: The extract is passed through one or more of ceramic membranes, microporous membranes, and ultrafiltration membranes, preferably one or two of ceramic membranes and spiral wound membranes, to obtain a permeate (or membrane permeate).

4. The method according to claim 1, characterized in that: The permeate from the membrane filtration was purified using C18YE packing material and eluted with 30-60% ethanol (v / v), preferably 50-55% ethanol. The target product was concentrated by rotary evaporation and then freeze-dried to obtain a refined extract of the polyphenolic components of Euphorbia humifusa.

5. The method according to claim 1, characterized in that: The above-mentioned extracts can be made into one of the following: capsules, powders, granules or tablets, and can be used to prepare drugs for treating enteritis.

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