Method for extracting myrobalain from fructus chebulae
Through the combination of ethanol ultrasonic combined with tertiary chromatography, high purity oxinin was successfully extracted, solving the problem of high extraction costs in the existing technology and realizing the possibility of industrial production.
Patent Information
- Application Number
- CN202510494443.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2025-07-22
AI Technical Summary
The lack of a low-cost and easy-to-operate extraction method in the prior art has resulted in low utilization and high cost, making it difficult to achieve industrial production.
The tertiary chromatography combined with ethanol ultrasonic technology combined with macroporous resin, reverse phase chromatography column and ODS column was used, and combined with full preparation liquid chromatography, the Hezining was isolated and purified.
The extraction of high-purity Hozining is achieved, which reduces costs, provides the possibility of industrial preparation, and has broad market prospects.
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Figure CN120349359A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of compound extraction, in particular to a method for extracting terminalia chebula from terminalia chebula. Background Art
[0002] Terminalia, belonging to the Combretaceae family, is widely distributed in tropical areas of both hemispheres. Terminalia is also known as Terminalia chebula, Terminalia fruit, and Terminalia chebula fruit (in Dai language). The 2020 edition of the Pharmacopoeia of the People's Republic of China lists Terminalia chebula Retz. or Terminalia chebula Retz. var. tomentella Kurt., a plant of the Combretaceae family. Terminalia chebula is flat in nature, bitter, sour, and astringent in taste. It belongs to the lung and large intestine meridians. It has the effects of astringing the intestines to stop diarrhea, astringing the lungs to stop coughing, and reducing fire and relieving sore throat. It is mainly used to treat chronic diarrhea and dysentery, rectal prolapse with blood in the stool, asthma and cough due to lung deficiency, chronic cough, sore throat and hoarseness. Modern research shows that the chemical components of Terminalia chebula mainly include phenolic acids, tannins, triterpenes and flavonoids, among which phenolic acids and tannins are its main active ingredients. It has biological activities such as antibacterial, antioxidant, anti-tumor, anti-inflammatory, detoxification, analgesia, cardiotonic, liver protection, blood sugar lowering, inhibition of gastric emptying and small intestinal motility, promotion of angiogenesis, protection of the brain and liver, etc. It can be used to treat a variety of diseases and plays an important therapeutic role in many prescription preparations such as Shiwei Terminalia Tablets, Terminalia Terminalia Five Flavors Capsules and Shiwei Terminalia Decoction Powder.
[0003] Terminalia chebula is a characteristic tannin component of Terminalia chebula, which has been proven to have specific biological activities. Its structural formula is as follows:
[0004]
[0005] At present, the quality control research of terminalia chebula, terminalia chebula and xiqingguo is mainly focused on terminalia chebula. Although terminalia chebula has important medicinal value, its standardized extraction process still has the following technical bottlenecks: At present, the national standard sample of terminalia chebula has not been developed at home and abroad, the terminalia chebula reference substances sold by some companies are too expensive, and there are few reports on the extraction of terminalia chebula monomer compounds from terminalia chebula. Therefore, it is very necessary to provide a low-cost, simple and easy-to-operate terminalia chebula extraction method to improve the utilization rate of terminalia chebula and reduce the cost of terminalia chebula. Summary of the invention
[0006] The object of the present invention is to provide a method for extracting chebulinic acid from Terminalia chebula Retz., so as to solve the problems existing in the above-mentioned prior art. The present invention conducts a directed separation of the marker component chebulinic acid in Terminalia chebula Retz. through ethanol ultrasonic technology, various chromatographic technologies and means, and extracts the monomeric compound of chebulinic acid. This compound has high purity, low extraction cost, and the reagents used are safe and non-toxic, and can be prepared industrially, with broad market prospects.
[0007] To achieve the above object, the present invention provides the following solutions:
[0008] The present invention provides a method for extracting chebulinic acid from Terminalia chebula Retz., comprising the following steps:
[0009] (1) Ultrasonically extract Terminalia chebula Retz. with an ethanol solution, and concentrate to obtain an ethanol extract of Terminalia chebula Retz.;
[0010] (2) Elute the ethanol extract of Terminalia chebula Retz. successively through a macroporous resin, a reversed-phase separation chromatography column, and an ODS column to obtain a crude extract;
[0011] (3) Separate and purify the crude extract by preparative liquid chromatography to obtain chebulinic acid.
[0012] Further, the process of eluting with the macroporous resin is as follows: successively use water and an ethanol solution with a volume fraction of 10% as the eluent, and elute the ethanol extract of Terminalia chebula Retz. with the macroporous resin; the amount of each eluent is 6 column volumes, and collect the eluate of the 1-3 column volumes of the 10% ethanol solution to obtain fraction A.
[0013] Further, in the process of eluting with the macroporous resin, 1 column volume is 7 L; the elution flow rate is 60 ml / min.
[0014] Further, the process of eluting with the reversed-phase separation chromatography column is as follows: successively use water and an ethanol solution with a volume fraction of 10% as the eluent, and elute fraction A with the reversed-phase separation chromatography column; the amount of each eluent is 6 column volumes, and collect the eluate of the 3-6 column volumes of the 10% ethanol solution to obtain fraction B.
[0015] Further, in the process of eluting with the reversed-phase separation chromatography column, 1 column volume is 1.2 L; the elution flow rate is 10 ml / min.
[0016] Further, the process of eluting with the ODS column is as follows: successively use water and methanol solutions with volume fractions of 5% and 10% as the eluent, and elute fraction B with the ODS column; the amount of water used is 6 column volumes, and the amounts of the methanol solutions with volume fractions of 5% and 10% are both 8 column volumes; collect the eluate of the 3-8 column volumes of the 5% methanol solution and the eluate of the 1-2 column volumes of the 10% methanol solution to obtain a crude extract.
[0017] Further, during the elution of the ODS column, 1 column volume is 0.2 L; the elution flow rate is 2 ml / min.
[0018] Optionally, the macroporous resin is HPD-400 macroporous resin.
[0019] Optionally, the reverse-phase separation chromatography column is MCI GEL CHP 20 / P120 column.
[0020] Further, before the preparative liquid chromatography separation and purification, the crude extract is dissolved in an aqueous solution of 30% acetonitrile to form a sample loading solution with a concentration of 500 mg / ml.
[0021] During the preparative liquid chromatography separation and purification, mobile phase A is methanol and mobile phase B is 0.2% formic acid water; the gradient elution program is: 0 - 30 min, 40% - 80% A; the flow rate is 10 mL / min, the elution time is 30 min; the detection wavelength is 275 nm.
[0022] Further, during the preparative liquid chromatography separation and purification, the peak elution time of chebulinic acid is 17.3 min.
[0023] Further, the process of ultrasonic extraction is as follows: the pulverized Terminalia chebula is mixed with an ethanol solution with a volume fraction of 40 - 60% according to a mass ratio of 1:8 - 12, and ultrasonic extraction is carried out 2 - 3 times, 20 - 30 min each time. After suction filtration and ethanol recovery, the ethanol extract of Terminalia chebula is obtained.
[0024] Optionally, the power of the ultrasonic wave is 350 W and the frequency is 53 kHz.
[0025] The present invention discloses the following technical effects:
[0026] Through the combined technology of three - stage chromatography (macroporous resin - reverse - phase chromatography - ODS column) combined with preparative liquid chromatography, the present invention has achieved the following breakthroughs: First, 50% ethanol is used as the extraction solvent, and the ultrasonic extraction method is used to ultrasonically extract Terminalia chebula flesh 3 times and concentrate the extract under reduced pressure to obtain an extract. The HPD - 400 macroporous adsorption resin is used to roughly fractionate the extract, and then the fractions are treated by MCI and ODS column chromatography, and finally purified by preparative high - performance liquid chromatography to obtain monomeric compounds. The monomeric compound of chebulinic acid obtained by the method of the present invention has high purity, low extraction cost, and the reagents used are safe and non - toxic. It can be industrially prepared, has a broad market prospect, and can provide low - cost, sufficient and key reference substance raw materials for the chemical analysis and quality control of Combretaceae plants or medicinal materials. Description of the Drawings
[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.
[0028] Figure 1 It is the process flow chart of the extraction of chebulinic acid in Example 1;
[0029] Figure 2 It is the high-resolution electrospray ionization mass spectrum in negative ion mode of the chebulinic acid extracted in Example 1;
[0030] Figure 3 It is the 1 1H-NMR identification diagram of the chebulinic acid extracted in Example 1;
[0031] Figure 4 It is the 13 13C-NMR identification diagram of the chebulinic acid extracted in Example 1;
[0032] Figure 5 It is the full wavelength scanning spectrum diagram (190 - 400 nm) of the chebulinic acid extracted in Example 1;
[0033] Figure 6 It is the high performance liquid chromatography (HPLC) diagram of the chebulinic acid extracted in Example 1. Specific Embodiments
[0034] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.
[0035] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.
[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.
[0037] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are merely exemplary.
[0038] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.
[0039] Unless otherwise specified, the test methods involved in the following examples of the present invention are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are all reagents and materials that can be obtained commercially.
[0040] Example 1
[0041] This example provides a method for extracting chebulanin from Terminalia chebula Retz., and the extraction process flow is as Figure 1 shown. The specific process is as follows:
[0042] Raw materials: The Terminalia chebula Retz. decoction pieces were purchased from Sichuan Hongkangyuan Pharmaceutical Co., Ltd. (batch number: 230501, production date: May 20, 2023, origin: Yunnan), and were identified by Researcher Guo Baolin of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences as the pitted flesh of the dried ripe fruit of Terminalia chebula Retz., a plant of the genus Terminalia in the Combretaceae family.
[0043] (1) Take 1.0 kg of Terminalia chebula Retz. fruit samples with residues, pits, etc. removed, crush them, pass the powder through a No. 3 sieve, add 10 times the mass of 50% ethanol solution, and perform ultrasonic extraction 3 times under the conditions of a power of 350 W and a frequency of 53 kHz, with each extraction for 30 min. Filter the extraction liquid by suction, combine the filtrates, and concentrate under reduced pressure to recover ethanol to 0.95 L (without the smell of alcohol). Then use HPD-400 macroporous adsorption resin (the packing dosage is 7 L) for separation, and elute successively with water, 10%, 15%, 20%, 30%, 50%, and 95% ethanol solutions in a gradient manner. The elution flow rate is 60 ml / min, and each eluent is eluted for 6 column volumes, where 1 column volume is 7 L. After detection by thin-layer chromatography (TLC) (developer: 1% ferric chloride ethanol, developing agent: ethyl formate - formic acid - water (35:4:2)), the target fraction is further detected by HPLC. Combine the fractions of each column volume with the same or similar components, and freeze-dry to obtain the macroporous resin elution fractions H1 - 8. Take the H2 fraction for subsequent purification.
[0044] Among them, H1, H4 - H8 are obtained by combining 6 column volumes eluted with water, 15%, 20%, 30%, 50%, and 95% ethanol solutions respectively; H2 is obtained by combining the first 3 column volumes eluted with 10% ethanol solution; H3 is obtained from the last 3 column volumes eluted with 10% ethanol solution.
[0045] (2) The H2 fraction is 93.8 g in total. For convenience of subsequent processing, take half of it (47 g) for purification. Dissolve H2 in 0.5 L of water, and separate it through an MCI GEL CHP 20 / P120 column (the packing dosage is 1.2 L). Elute successively with water, 10%, 15%, 20%, 30%, 50%, and 95% ethanol solutions in a gradient manner. The elution flow rate is 10 ml / min, and each eluent is eluted for 6 column volumes, where 1 column volume is 1.2 L. After thin-layer identification by TLC (developer: 1% ferric chloride ethanol, developing agent: ethyl formate - formic acid - water (35:4:2)), the target fractions are further detected by HPLC. Combine the fractions of each column volume with the same or similar components, and freeze-dry to obtain the MCI elution fractions H2-1 - H2-9. Take the H2-3 fraction for subsequent purification.
[0046] Among them, H2-1, H2-7 - H2-9 are obtained by combining 6 column volumes eluted with water, 30%, 50%, and 95% ethanol solutions respectively; H2-2 is obtained by combining the first 2 column volumes eluted with 10% ethanol solution; H2-3 is obtained by combining the 3rd - 6th column volumes eluted with 10% ethanol solution; H2-4 is obtained by combining the first 3 column volumes eluted with 15% ethanol solution; H2-5 is obtained by combining the 4th - 6th column volumes eluted with 15% ethanol and the 1st column volume eluted with 20% ethanol; H2-6 is obtained by combining the 2nd - 6th column volumes eluted with 20% ethanol.
[0047] (3) The H2-3 portion was 8.56 g in total. After dissolving H2-3 in 30 ml of water, it was separated by ODS column chromatography and eluted successively with water, 5%, 10%, 20%, 30%, 50%, and 95% methanol solutions in a gradient manner. The elution flow rate was 2 ml / min. Water was used for elution for 6 column volumes, 5% and 10% methanol solutions were used for elution for 8 column volumes each, and 20%, 30%, 50%, and 95% methanol solutions were used for elution for 3 column volumes each. One column volume was 0.2 L. After detection by thin-layer chromatography (TLC) and then by HPLC, the target fraction with the same or similar components was combined and freeze-dried to obtain H2-3-3 and H2-3-4. The purity of the H2-3-3 portion was relatively high, and subsequent purification was carried out.
[0048] Among them, H2-3-3 was obtained by combining the 3rd to 8th column volumes eluted with 5% methanol and the 1st to 2nd column volumes eluted with 10% methanol, and H2-3-4 was the combined fraction of the 1st to 2nd column volumes eluted with 5% methanol and the 3rd to 6th column volumes eluted with 10% methanol.
[0049] (4) The H2-3-3 portion was 1.96 g in total. One gram of it was taken and dissolved in 2 ml of 30% aqueous acetonitrile solution to prepare a sample solution with a concentration of 500 mg / ml, and then separated and purified by preparative liquid chromatography. The chromatographic column was Daisogel C18 10u 100A from Beijing Innovation Tongheng Technology Co., Ltd. The mobile phase was methanol (A)-0.2% formic acid water (B), and the gradient elution program was as follows: 0 - 30 min, 40% - 80% A; the elution time was 30 min, the flow rate was 10 mL / min, the detection wavelength was 275 nm, and the operation was carried out at room temperature. Finally, the compound chebulinic acid (267.5 mg, tR = 17.3 min) was obtained.
[0050] Example 2
[0051] In this example, the chebulinic acid compound prepared in Example 1 was identified, and the identification process was as follows:
[0052] 1. Morphological identification
[0053] The chebulinic acid prepared in Example 1 was a white amorphous powder.
[0054] 2. Structural identification
[0055] The compound prepared in Example 1 was detected by HR-ESI-MS in the negative ion mode for the first-stage mass spectrum, and the results were as Figure 2 shown. The results showed that: m / z [M-H]-651.0834, and the molecular formula was C 27 H 24 O 19 , and the degree of unsaturation was 16.
[0056] 1The results of the \(^1H\)-NMR (600 MHz, \(CD_3OD\)) spectrum are as follows Figure 3 shown: δ 7.13 (2H, s, Galloyl, C-2, C-6), δ 7.46 (Chebubyl, 1H, s, H-3'), δ 6.36 (1H, d, J = 2.83 Hz, glc-1-H), δ 5.21 (1H, d, J = 2.99 Hz, glc-2-H), δ 4.78 (1H, br.s, glc-3-H), δ 4.81 (1H, br.s, glc-4-H), δ 4.31 (1H, t, J = 6.55 Hz, glc-5-H), δ 4.07 (1H, dd, J = 11.43, 6.56 Hz, glc-6a), δ 4.00 (1H, dd, J = 11.44, 6.43 Hz, glc-6b), δ 4.93 (Chebubyl, 1H, d, J = 7.14 Hz, H-2), δ 5.09 (Chebubyl, 1H, dd, J = 7.13 Hz, H-3), δ 3.81 (Chebubyl, 1H, td, J = 11.88, 3.47 Hz, H-4), δ 2.18 (Chebubyl, m, H-5).
[0057] 13 The results of the \(^{13}C\)-NMR (150 MHz, \(CD_3OD\)) spectrum are as follows Figure 4As shown, the results showed: δ93.1 (Clucose-1), δ74.4 (Clucose-2), δ61.8 (Clucose-3), δ72.1 (Clucose-4), δ79.6 (Clucose-5), δ63.6 (Clucose-6), δ120.5 (Galloyl, C-1), δ110.5 (Galloyl, C-2, C-6), δ146.6 (Galloyl, C-3, C-5), δ140.2 (Galloyl, C-4), δ166.5 (Galloyl, C-7); δ170.8 (Chebubyl, C-1), δ67.1 (Chebubyl, C-2), δ41.6 (Chebubyl, C-3), δ40.2 (Chebubyl, C-4), δ31.0 (Chebubyl, C-5), δ174.7 (Chebubyl, C-6), δ175.2 (Chebubyl, C-7), δ166.7 (Chebubyl, C-8), δ116.1 (Chebubyl, C-1'), δ119.5 (Chebubyl, C-2'), δ117.4 (Chebubyl, C-3'), δ147.3 (Chebubyl, C-4'), δ140.5 (Chebubyl, C-5'), δ141.4 (Chebubyl, C-6').
[0058] The above results were compared with the spectral data reported in the literature (Lee J, Nho Y H, Yun S K, et al. Use of ethanol extracts of Terminalia chebula to prevent periodontal disease induced by dental plaque bacteria[J]. BMC Complement Alter Med, 2017, 17(1):110; Rahimi V B, Askari V R, Shirazinia R, et al. Protective effects of hydro-ethanolic extraet of Terminalia chebula on primary microglia cells and their polarization (M1 / M2 balance)[J]Mult Scler Relat Dis, 2018, 25:5-3), and they were basically consistent (see Table 1). Therefore, it was determined that the compound obtained by extraction in Example 1 was Chebulanin, with a molecular weight of 652 and a molecular formula of C27 H 24 O 19 。The full wavelength scanning of the chebulinine was carried out, and the results are as Figure 5 shown.
[0059] The chebulinine extracted in Example 1 was identified by HPLC. The specific process is as follows: Take chebulinine and make a solution with 70% methanol solution at a concentration of 100 μg / mL for injection analysis. The HPLC column is: Welch Utimate XB C 18 column (4.6×250 mm, 5 μm); the mobile phase is: methanol (A) - 0.2% phosphoric acid aqueous solution (B); the gradient elution degree is: 0 - 30 min, 24% - 27% A; 30 - 50 min, 27% - 42% A; 50 - 65 min, 42% - 90% A; the column temperature is 35°C, and the flow rate is 1.0 mL·min -1 , the detection wavelength is 275 nm, and the injection volume is 10 μL. The HPLC chromatogram is as Figure 6 shown. Calculated by the peak area normalization method, the purity of chebulinine can reach over 95%.
[0060] Table 1 Comparison of the compounds in Example 1 with the 13 C-NMR, 1 H-NMR data
[0061]
[0062]
[0063] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.
Claims
1. A method for extracting chebulinic acid from Terminalia chebula Retz., characterized in that, It includes the following steps: (1) Ultrasonically extract Terminalia chebula Retz. with an ethanol solution, and concentrate to obtain an ethanol extract of Terminalia chebula Retz.; (2) Elute the ethanol extract of Terminalia chebula Retz. successively through a macroporous resin, a reversed-phase separation chromatography column, and an ODS column to obtain a crude extract; (3) Separate and purify the crude extract by preparative liquid chromatography to obtain chebulinic acid.
2. The extraction method according to claim 1, wherein The process of eluting with the macroporous resin is as follows: Use water and an ethanol solution with a volume fraction of 10% as eluents successively to elute the ethanol extract of Terminalia chebula Retz. with the macroporous resin; the amount of each eluent is 6 column volumes, and collect the eluate of the 1st - 3rd column volumes of the 10% ethanol solution to obtain fraction A.
3. The extraction method according to claim 2, wherein The process of eluting with the reversed-phase separation chromatography column is as follows: Use water and an ethanol solution with a volume fraction of 10% as eluents successively to elute fraction A with the reversed-phase separation chromatography column; the amount of each eluent is 6 column volumes, and collect the eluate of the 3rd - 6th column volumes of the 10% ethanol solution to obtain fraction B.
4. The extraction method according to claim 3, wherein The process of eluting with the ODS column is as follows: Use water and methanol solutions with volume fractions of 5% and 10% as eluents successively to elute fraction B with the ODS column; the amount of water is 6 column volumes, and the amounts of the methanol solutions with volume fractions of 5% and 10% are both 8 column volumes; collect the eluate of the 3rd - 8th column volumes of the 5% methanol solution and the eluate of the 1st - 2nd column volumes of the 10% methanol solution to obtain a crude extract.
5. The extraction method according to claim 1 or 2, characterized in that, The macroporous resin is HPD - 400 macroporous resin.
6. The extraction method according to claim 1 or 3, characterized in that, The reversed-phase separation chromatography column is MCI GEL CHP 20 / P120 column.
7. The extraction method according to claim 1, wherein Before the preparative liquid chromatography separation and purification, dissolve the crude extract with an aqueous solution of 30% acetonitrile to prepare a sample loading solution with a concentration of 500 mg / ml; During the preparative liquid chromatography separation and purification, mobile phase A is methanol, and mobile phase B is 0.2% formic acid water; the gradient elution program is: 0 - 30 min, 40% - 80% A; the flow rate is 10 mL / min, the elution time is 30 min; the detection wavelength is 275 nm.
8. The extraction method according to claim 7, characterized in that During the preparative liquid chromatography separation and purification, the peak emergence time of chebulinic acid is 17.3 min.
9. The extraction method according to claim 1, wherein The process of ultrasonic extraction is as follows: Mix the pulverized Terminalia chebula Retz. with an ethanol solution with a volume fraction of 40 - 60% according to a mass ratio of 1:8 - 12, perform ultrasonic extraction 2 - 3 times, each time for 20 - 30 min, and after suction filtration and ethanol recovery, obtain the ethanol extract of Terminalia chebula Retz.
10. The extraction method according to claim 9, wherein, The power of the ultrasonic wave is 350 W, and the frequency is 53 kHz.