Method for extracting and separating fingered citron

Abstract

The invention discloses a fingered citron extraction and separation method, which comprises: carrying out alcohol extraction on fingered citron to obtain a medicinal material stock solution, sequentially passing the medicinal material stock solution through a D101 macroporous resin column, an MCI chromatographic column and a 150DAC to prepare a crude component, carrying out separation and gradient elution on the crude component on a 30DAC system, intercepting the required main peak part each time, merging, and carrying out vacuum concentration to obtain the fingered citron extract. After high performance liquid detection, nine compound products are obtained. According to the method, flavonoid and glycoside components, white angelica and angelica brain chemical components are extracted, separated and purified from fingered citron, an unknown new compound (compound 3) is obtained through separation for the first time, the understanding of chemical components of fingered citron is enriched, in addition, the extraction and separation method is low in cost, the large-scale extraction and separation process of fingered citron is facilitated, and the method is suitable for industrial production. And the application range of fingered citron is expanded.

Description

Technical Field

[0001] The invention relates to the technical field of extraction and separation methods, and in particular to a method for extracting and separating bergamot. Background Art

[0002] Buddha's hand (Citri Sarcodactylis Fructus) is the dried fruit of the citrus plant (Citrus medica L. var. sarcodactylis Swingle) of the Rutaceae family. Buddha's hand has a long history of medicinal use in my country and is widely distributed. It is cultivated in Guangxi, Guangdong, Sichuan, Yunnan, Fujian, Zhejiang and other places. Buddha's hand was first recorded as "citron" in Chen Cangqi's "Supplement to Materia Medica", "Illustrated Classic of Materia Medica", "Southern Yunnan Materia Medica", "Compendium of Materia Medica" and other books in the Tang Dynasty. The 2020 edition of the "Chinese Pharmacopoeia" records that it tastes pungent, bitter, sour and warm. It enters the liver, spleen, stomach and lung meridians, and has the effects of soothing the liver and regulating qi, harmonizing the stomach and relieving pain, and drying dampness and resolving phlegm. It is used for liver and stomach qi stagnation, chest and flank distension and pain, fullness in the stomach, poor appetite and vomiting, cough and excessive sputum. Buddha's hand is included in the "Catalogue of Substances that are Traditionally Both Food and Medicinal Materials" issued by the National Health Commission. As a traditional medicinal and edible plant, Buddha's hand also has a very high edible value. At the same time, the Buddha's hand essential oil extracted from it can relieve fatigue and soothe emotions, and is widely used in food, medicine, cosmetics and other fields.

[0003] Modern research has found that bergamot extract contains a variety of bioactive ingredients, mainly including flavonoids, coumarins, polysaccharides and volatile oil compounds. Among them, volatile oils are mainly composed of limonene, γ-terpinene, d-citronene, etc., which give bergamot its unique aroma. Flavonoids include trihydroxydimethoxyflavone, etc., which have antioxidant and anti-inflammatory biological activities. Coumarin compounds: such as dimethoxycoumarin, have certain health benefits for the body. Other bioactive substances: polyphenol compounds, terpenoid compounds, etc., have antioxidant, anti-inflammatory, anti-tumor and other effects.

[0004] Nowadays, metabolic diseases such as cancer and cardiovascular disease and mental illnesses such as anxiety and depression are growing rapidly. Using green and natural plants as raw materials to develop safe and efficient functional foods is an important means to prevent and control the occurrence and development of various diseases. At present, there are few reports on the chemical components isolated from Buddha's hand, and the structural novelty is low. Therefore, the development and separation of compounds in Buddha's hand is in urgent need of solution, and the extraction and separation methods of Buddha's hand are still unclear. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting and separating bergamot, so as to solve the problem that the substances extracted and separated from bergamot are less and the structural novelty is low.

[0006] To achieve the above object, the first aspect of the present invention provides a method for extracting and separating bergamot, comprising the following steps:

[0007] Step 1: Take dried bergamot medicinal materials, crush them, soak them in 10-30 times the amount of methanol at room temperature for 2-5 days, filter them, repeat 3-5 times, combine the extracts, concentrate the extracts under reduced pressure, and recover the solvent in vacuum to obtain the medicinal material stock solution for later use;

[0008] Step 2: dilute the medicinal material stock solution in step 1 with water, pour it into a D101 macroporous resin column after dilution, and perform gradient elution with a mixed solvent of methanol-water to obtain a plurality of first elution fractions;

[0009] After each first eluted fraction is concentrated, freeze-dried, and detected by high performance liquid chromatography, fractions with the same chromatographic peaks and similarity are combined for later use;

[0010] Step 3: Separate each of the fractions combined in step 2 through an MCI chromatographic column and perform gradient elution with a mixed solvent of methanol-water to obtain a plurality of second elution fractions;

[0011] After each second elution fraction is concentrated, freeze-dried, and detected by high performance liquid chromatography, fractions with the same chromatographic peaks and similarity are combined for later use;

[0012] Step 4: Prepare a crude fraction by 150 DAC of each fraction combined in step 3, and perform gradient elution with a mixed solvent of methanol-water to obtain several third elution fractions;

[0013] After each third elution fraction is concentrated, freeze-dried, and detected by high performance liquid chromatography, fractions with the same chromatographic peaks and similarity are combined to obtain a mixed sample of the target component;

[0014] Step 5: Concentrate the mixed sample of target components obtained in step 4, separate the crude fraction on a 30DAC system, and perform gradient elution with a mixed solvent of methanol-water. Each time, the required main peak portion is intercepted, combined, and concentrated under reduced pressure. After high performance liquid chromatography detection, 9 compound products are obtained.

[0015] Preferably, the chromatographic conditions for high performance liquid phase detection in steps 2 to 5 are:

[0016] Chromatographic column C18, mobile phase A is 1 / 1000 trifluoroacetic acid aqueous solution and mobile phase B is acetonitrile; 15% B to 100B gradient elution; volume flow rate is 1 mL / min; column temperature is 35°C, injection volume is 8 μL; detection wavelength is 210 nm.

[0017] Preferably, in step 2, the volume ratio of the Chinese medicinal material stock solution to water is 1:1 to 1.3.

[0018] Preferably, the volume ratio of methanol to water during the gradient elution in step 2 is 30:70, 60:40, 80:20, and 100:0, respectively, and each elution gradient is eluted until colorless.

[0019] Preferably, the volume ratio of methanol to water during the gradient elution in step 3 is 5:95 to 100:0, and each elution gradient is eluted until colorless.

[0020] Preferably, the volume ratio of methanol to water during the gradient elution in step 4 is 35:65 to 75:25, and each elution gradient is eluted until colorless.

[0021] Preferably, the volume ratio of methanol to water during the gradient elution in step 5 is 10:90 to 70:30, and each elution gradient is eluted until colorless.

[0022] The second aspect of the present invention provides a bergamot extract separated by the above-mentioned extraction and separation method.

[0023] Preferably, the bergamot extract includes compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, and compound 9, and the chemical structural formulas of the compounds are respectively:

[0024]

[0025]

[0026] The present invention provides a method for extracting and separating flavonoids, coumarins and glycoside compounds from bergamot. The compounds include: 3-(2-glucosyloxy-4-methoxyphenyl) propionic acid (compound 1), coniferin (compound 2), a new compound (compound 3), 4'-O-methylvitanein II (compound 4), cynaroside (compound 5), artemisinin (compound 6), angelica brain (compound 7), white angelica (compound 8) and 5,7-dimethoxycoumarin (compound 9).

[0027] Therefore, the present invention adopts a method for extracting and separating bergamot with the above structure, which has the following beneficial effects:

[0028] (1) The present invention relates to methods for extracting, separating, purifying and identifying coumarins, flavonoids and glycoside compounds from bergamot. An unknown new compound (Compound 3) was separated for the first time. The present invention also separated flavonoids, glycoside components, white angelica and brain chemical components of Angelica dahurica from bergamot for the first time. These discoveries not only enrich the understanding of the chemical composition of bergamot, but also reveal the potential application of bergamot in preventing and treating related diseases. The extraction and separation of these compounds are of great significance for understanding the pharmacological mechanism of bergamot, and also provide a theoretical basis and technical support for the development of new drugs, health products and cosmetics based on bergamot. These compounds may be developed into drugs for treating various diseases, or added as functional ingredients to various products to enhance their health benefits. The present invention has greatly promoted the research progress of the chemical components of bergamot.

[0029] (2) The flavonoids, coumarins and glycoside compounds extracted and separated by the method of the present invention are of high purity. The compounds can be used as reference substances for quality research of bergamot, and are used to detect and identify the presence or absence of these components in bergamot, as well as the level of their content, which is of great significance for ensuring the quality and consistency of bergamot products. In addition, as a traditional herbal medicine, bergamot may have large differences in its chemical composition under different origins, harvest seasons, and processing methods. High-purity standard substances are helpful in establishing the chemical fingerprint of bergamot and achieving standardization and regularization of its quality.

[0030] (3) Since bergamot can be used as both medicine and food and has a large demand for its use, the extraction and separation method of the present invention is low-cost to extract and separate flavonoids, coumarins and glycoside compounds from bergamot, which is conducive to the scale-up of the bergamot extraction and separation process and expands the scope of use of bergamot.

[0031] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the H NMR spectrum of compound 1;

[0033] Figure 2 is the C NMR spectrum of compound 2;

[0034] Figure 3 is the H NMR spectrum of compound 2;

[0035] Figure 4 is the H NMR spectrum of compound 3;

[0036] Figure 5 is the H NMR spectrum of compound 4;

[0037] Figure 6 is the C NMR spectrum of compound 5;

[0038] Figure 7 is the H NMR spectrum of compound 5;

[0039] Figure 8 is the H NMR spectrum of compound 6;

[0040] Fig. 9 is the H NMR spectrum of compound 7;

[0041] Fig.10 is the H NMR spectrum of compound 8;

[0042] Fig.11 is the H NMR spectrum of compound 9. DETAILED DESCRIPTION

[0043] The present invention will be further described below. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and a specific operation process, but the present invention is not limited to this embodiment.

[0044] The instruments used in the present invention are: SHZ-D (III) circulating water vacuum pump (Henan Yuhua Instrument Co., Ltd.); Bruker Avance II-400 MHz nuclear magnetic resonance instrument (Bruker, Germany); MCI (inner diameter 70 mm×460 mm) column chromatography column (Beijing Yuanbaoshan Chromatography Technology Co., Ltd.); D101 macroporous resin column (Xi'an Lanxiao Technology New Materials Co., Ltd.); SPD-20A high performance liquid chromatograph (Shimadzu Corporation); ZF-20D dark box ultraviolet analyzer (Gongyi Yuhua Instrument Co., Ltd.).

[0045] Example 1

[0046] A method for extracting and separating bergamot comprises the following steps:

[0047] Step 1: Take 5 kg of dried bergamot, crush it, and soak it in 10 times the amount of pure methanol for 3 times, each time for 36 hours, combine the 3 extracts, and concentrate the extract under reduced pressure to obtain a 1 mg / mL extract;

[0048] Dissolve the remaining precipitate with 1 volume of clean water and mix it with the concentrated bergamot extract for later use.

[0049] Step 2: Dilute the extract from step 1 with 5 times the volume of water, load it into a D101 macroporous resin column to filter out larger impurities and sand, load the filtered sample into the D101 macroporous resin column again, collect fractions, concentrate each fraction, freeze-dry, and perform HPLC detection, combine fractions with the same chromatographic peaks and similarity, and obtain five fractions: F1, F2, F3, F4, and F5.

[0050] Step 3: Pour the five combined fractions from step 2 into five D101 macroporous resin columns respectively, dissolve them with appropriate methanol (sample to methanol ratio 1:1), and perform preliminary elution and separation.

[0051] According to the chromatographic test results, a methanol: water system (30:70, 60:40, 80:20, 100:0) was used for gradient elution. Each elution gradient was eluted until colorless to obtain several fractions.

[0052] Each fraction was subjected to high performance liquid chromatography detection, and the chromatographic column conditions were: SPD-20A-C18, mobile phase (A) acid water (1 / 1000 trifluoroacetic acid water) solution and (B) acetonitrile; 15% B to 100B gradient elution; volume flow rate was 1 mL / min; column temperature was 35°C, injection volume was 8 μL; detection wavelength was 210 nm; and the spectral results were combined and concentrated to 10% of the original volume.

[0053] Step 4: The five fractions combined in step 3 were separated by a preparative MCI column (inner diameter 70 mm, column height 460 mm).

[0054] The concentrated sample was diluted with methanol in a ratio of 1:1 and loaded into an MCI column for gradient elution with methanol-water (5:95, 25:75, 50:50, 75:25, 100:0). Each gradient elution was performed until the sample became colorless to obtain several elution fractions.

[0055] Each fraction was detected by high performance liquid chromatography, and the chromatographic column conditions were: SPD-20A-C18, mobile phase (A) acid water (1 / 1000 trifluoroacetic acid water) solution and (B) acetonitrile; 15% B-100B gradient elution; volume flow rate was 1 mL / min; column temperature was 35°C, injection volume was 8 μL; detection wavelength was 210 nm; similar fractions were concentrated and combined according to the chromatographic peaks to obtain 14 pressure components, namely F1-1, F1-2, F1-3, F1-4, F2-1, F2-2, F2-3, F3-1, F3-2, F3-3, F4-1, F4-2, F5-1, and F5-2.

[0056] Step 5: F1-3, F2-2, F3-2, and F4-1 obtained in step 4 were diluted with 1-fold water and injected multiple times, separated and purified by preparation 150DAC (Ruihe Technology 600ml preparative high performance liquid chromatograph 150DAC), eluted with 35%, 45%, 55%, 65%, and 75% methanol-water gradient, and the required main peak was intercepted each time, combined and concentrated under reduced pressure to 20 times the original volume. According to the test results of each fraction by high performance liquid phase, the condition chromatographic column is: SPD-20A-C18, mobile phase (A) acid water (1 / 1000 trifluoroacetic acid water) solution and (B) acetonitrile; 15% B~100B gradient elution; volume flow rate is 1mL / min; column temperature is 35°C, injection volume is 8μL; detection wavelength is 210nm; the crude compound is obtained>60%. There are 10 sections, namely F1-3-1, F1-3-2, F1-3-4, F2-2-1, F2-2-2, F2-2-3, F3-2-1, F3-2-2, F4-1-1, F4-1-2.

[0057] Step 6: Dissolve F1-3-1 obtained in step 5 in 15 ml of methanol and inject it several times, separate it by 30DAC (Beijing Qingbohua preparative high performance liquid chromatograph 30DAC), intercept the required main peak part each time, combine and concentrate under reduced pressure to obtain compound 1 (306 mg) and compound 2 (172 mg). Chromatographic conditions are: Daisogel C18-10μm-120A column, UV detector, detection wavelength 210nm, column temperature 20°C, mobile phase 40% MeOH / H2O, flow rate 15ml / min, injection volume 1ml.

[0058] The chemical structures of compound 1 and compound 2 are:

[0059]

[0060] from Figure 1-3 It can be seen that compound 1 and compound 2 have been successfully separated.

[0061] Step 7: Dissolve F1-3-2 obtained in step 5 in 20 ml methanol and inject it several times, separate and purify it by 30DAC preparation, intercept the required main peak part each time, combine and dry under reduced pressure to obtain crude compound 3 (purity of about 80%). Then, dissolve the crude compound 3 and inject it several times, perform 30DAC preparation three times, and further separate and purify it to obtain compound 3 (70 mg). The chromatographic conditions are: Daisogel C18-10μm-120A chromatographic column, UV detector, detection wavelength 210nm, column temperature 20℃, mobile phase 40% MeOH / H2O, flow rate 15ml / min, injection volume 1ml.

[0062] The chemical structural formula of compound 3 is:

[0063]

[0064] from Figure 4 It can be seen that compound 3 has been successfully isolated. Compound 3 has many similarities with the compound structure of 2-beta-D-pyranose glucose-4-methoxy cis-cinnamic acid. It may play a certain role in gynecological anti-cancer and circadian rhythm control of hernia.

[0065] Step 8: Dissolve F2-2-2 obtained in step 5 in 20 ml of methanol and inject it several times, separate it by 30DAC preparation, intercept the required main peak part each time, combine and dry under reduced pressure to obtain compound 4 (1 g) and compound 5 (125 mg). The chromatographic conditions are: Daisogel C18-10 μm-120A column, UV detector, detection wavelength 210 nm, column temperature 20 ° C, mobile phase 40% MeOH / H2O, flow rate 15 ml / min, injection volume 1 ml.

[0066] The chemical structures of compound 4 and compound 5 are:

[0067]

[0068] from Figure 5-7 It can be seen that compounds 4 and 5 have been successfully separated.

[0069] Step 9: Dissolve the F3-2-1 obtained in step 5 in 20 ml of methanol and inject it several times, separate it by 30DAC preparation, intercept the required main peak part each time, combine and dry under reduced pressure to obtain compound 6 (209 mg), compound 7 (604 mg) and compound 8 (89 mg). The chromatographic conditions are: Daisogel C18-10μm-120A chromatographic column, UV detector, detection wavelength 210nm, column temperature 20°C, mobile phase 40% MeOH / H2O, flow rate 15ml / min, injection volume 1ml.

[0070]

[0071] from Figure 8-10 It can be seen that compounds 6, 7 and 8 have been successfully separated.

[0072] Step 10: Dissolve F4-1-1 obtained in step 5 in 20 ml of methanol and inject it several times, separate it by 30DAC preparation, cut the required main peak part each time, combine and dry under reduced pressure to obtain compound 9 (3.27 g). Chromatographic conditions are: Daisogel C18-10μm-120A chromatographic column, UV detector, detection wavelength 210nm, column temperature 20°C, mobile phase 40% MeOH / H2O, flow rate 15ml / min, injection volume 1ml.

[0073] The chemical structural formula of compound 9 is:

[0074]

[0075] from Fig.11 It can be seen that compound 9 has been successfully isolated.

[0076] Table 1 Compound related information

[0077]

[0078] Therefore, the present invention adopts the above-mentioned extraction and separation method of bergamot, not only extracts, separates and purifies flavonoids, glycoside components, white angelica and angelica brain chemical components from bergamot, but also separates an unknown new compound (compound 3) for the first time, enriching the understanding of the chemical composition of bergamot, wherein the flavonoids, coumarins and glycoside compounds are high in purity and can be used as reference substances for bergamot quality research, for detecting and identifying the presence and content of these components in bergamot. In addition, the extraction and separation method of the present invention has low cost, is conducive to the scale-up of the bergamot extraction and separation process, and expands the scope of use of bergamot.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for extracting and separating bergamot, characterized in that: The following steps are involved: Step 1: Take dried bergamot medicinal materials, crush them, soak them in 10-30 times the amount of methanol at room temperature for 2-5 days, filter them, repeat 3-5 times, combine the extracts, concentrate the extracts under reduced pressure, and recover the solvent in vacuum to obtain the medicinal material stock solution for later use; Step 2: dilute the medicinal material stock solution in step 1 with water, pour it into a D101 macroporous resin column after dilution, and perform gradient elution with a mixed solvent of methanol-water to obtain a plurality of first elution fractions; After each first eluted fraction is concentrated, freeze-dried, and detected by high performance liquid chromatography, fractions with the same chromatographic peaks and similarity are combined for later use; Step 3: Separate each of the fractions combined in step 2 through an MCI chromatographic column and perform gradient elution with a mixed solvent of methanol-water to obtain a plurality of second elution fractions; After each second elution fraction is concentrated, freeze-dried, and detected by high performance liquid chromatography, fractions with the same chromatographic peaks and similarity are combined for later use; Step 4: Prepare a crude fraction by 150 DAC of each fraction combined in step 3, and perform gradient elution with a mixed solvent of methanol-water to obtain several third elution fractions; After each third elution fraction is concentrated, freeze-dried, and detected by high performance liquid chromatography, fractions with the same chromatographic peaks and similarity are combined to obtain a mixed sample of the target component; Step 5: Concentrate the mixed sample of target components obtained in step 4, separate the crude fraction on a 30DAC system, and perform gradient elution with a mixed solvent of methanol-water. Each time, the required main peak portion is intercepted, combined, and concentrated under reduced pressure. After high performance liquid chromatography detection, 9 compound products are obtained.

2. The method for extracting and separating bergamot according to claim 1, wherein: The chromatographic conditions for high performance liquid phase detection in steps 2 to 5 are: Chromatographic column C18, mobile phase A is 1 / 1000 trifluoroacetic acid aqueous solution and mobile phase B is acetonitrile; 15% B to 100B gradient elution; volume flow rate is 1 mL / min; column temperature is 35°C, injection volume is 8 μL; detection wavelength is 210 nm.

3. The method for extracting and separating bergamot according to claim 1, characterized in that: In step 2, the volume ratio of the Chinese medicinal material stock solution to water is 1:1 to 1.

3.

4. The method for extracting and separating bergamot according to claim 1, characterized in that: In step 2, the volume ratios of methanol and water during the gradient elution process are 30:70, 60:40, 80:20, and 100:0, respectively, and each elution gradient is eluted until colorless.

5. The method for extracting and separating bergamot according to claim 1, characterized in that: In step 3, the volume ratio of methanol to water during the gradient elution process is 5:95 to 100:0, and each elution gradient is eluted until colorless.

6. The method for extracting and separating bergamot according to claim 1, characterized in that: In step 4, the volume ratio of methanol to water during the gradient elution process is 35:65 to 75:25, and each elution gradient is eluted until colorless.

7. The method for extracting and separating bergamot according to claim 1, characterized in that: In step 5, the volume ratio of methanol to water during the gradient elution process is 10:90 to 70:30, and each elution gradient is eluted until colorless.

8. A bergamot extract, characterized in that: Separated by the extraction and separation method according to any one of claims 1 to 7.

9. The bergamot extract according to claim 8, characterized in that: The bergamot extract includes compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8 and compound 9, and the chemical structural formulas of the compounds are:

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