A method for rapidly separating and preparing high-purity dehydrodiisoeugenol from nutmeg

Abstract

The present invention belongs to the technical field of separation and purification of active ingredients in traditional Chinese medicine, and relates to a method for rapidly separating and preparing high-purity dehydrodiisoeugenol from nutmeg. It uses nutmeg as the raw material for flash extraction with petroleum ether (60-90 °C), uses methanol-water as the mobile phase, and separates with a high-performance liquid chromatography preparative column at a wavelength of 270 nm, and finally obtains a monomer with a purity of ≥98%. The preparation process is rapid, simple, low-cost, has good repeatability and high efficiency. The obtained dehydrodiisoeugenol monomer has a high purity, can be used as a reference substance for content determination, and can also be widely applied in the pharmaceutical and health industries.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification of active ingredients of traditional Chinese medicines, and relates to a method for quickly separating and preparing high-purity dehydrodiisoeugenol from nutmeg. Background Art

[0002] Myristica fragrans Myristica fragrans Houtt. Dried kernels, also known as meat fruit, jade fruit, etc. Originated from the Maluku Islands in Indonesia, it is widely cultivated in tropical areas. In traditional Chinese medicine, nutmeg has the effects of warming the middle and promoting qi, astringing the intestines to stop diarrhea, anorexia and vomiting, and indigestion. The 2020 edition of the "Pharmacopoeia of the People's Republic of China" stipulates that it is mainly used for symptoms such as spleen and stomach deficiency and cold, chronic diarrhea, abdominal distension and pain, anorexia and vomiting. Modern pharmacological studies have found that nutmeg has pharmacological effects such as anti-cardiovascular disease, antibacterial, analgesic, anti-inflammatory, protection of the central nervous system, antidiarrhea, antioxidant and anti-cancer.

[0003] Nutmeg contains a variety of chemical components, including lignans, volatile oils, fatty oils, polyphenols and other components. Current research focuses on lignans and volatile oils. The current quality control indicators are only volatile oils and dehydrogenated diisoeugenol. The 2020 edition of the Pharmacopoeia stipulates that nutmeg contains dehydrogenated diisoeugenol (C 20 H 22 O4) shall not be less than 0.10%. At present, there is no report on the process of rapid extraction and separation of dehydrodiisoeugenol from nutmeg.

[0004] Dehydrodiisoeugenol is a lignan component in the traditional Chinese medicine nutmeg. Studies have shown that the typical lignan functional active ingredients extracted from the dried seeds of nutmeg have very significant anti-inflammatory and anti-allergic effects, among which dehydrodiisoeugenol is a typical high-yield product - benzofuran-type lignans, but there are relatively few studies on the anti-inflammatory effect of dehydrodiisoeugenol.

[0005] Dehydrodiisoeugenol is one of the main components of nutmeg, but there are few studies on its separation and extraction. Therefore, there is an urgent need to study the purification process for the separation and extraction of dehydrodiisoeugenol. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for quickly separating and preparing high-purity dehydrodiisoeugenol from nutmeg. The method of the present invention is simple, rapid, environmentally friendly, and has high product purity.

[0007] A method for rapidly separating and preparing high-purity dehydrodiisoeugenol from nutmeg comprises the following steps:

[0008] Step 1: The nutmeg medicinal materials are pulverized and sieved through a 20-mesh sieve, impregnated with a petroleum ether (60-90 °C) solution, flash-extracted under a certain voltage for multiple times, the supernatant is aspirated, and the petroleum ether is recovered to obtain an extract.

[0009] Step 2: The extract is ultrasonically extracted and dispersed with methanol, white fat is precipitated, and the white fat is filtered off to obtain a methanol extract.

[0010] Step 3: The methanol extract obtained in Step 2 is diluted to a certain concentration (60 mg / ml based on the extract), filtered through a 0.22-mm microporous membrane, and separated and prepared by HPLC (high performance liquid chromatography). Using methanol: water as the mobile phase, under certain mobile phase flow rate, column temperature, and detection wavelength conditions, dehydrodiisoeugenol is prepared.

[0011] Further, in Step 1, the ratio of nutmeg medicinal materials to petroleum ether (g / V) is 1:60-100, preferably 60-fold; the voltage is 95-105 V, preferably 105 V; the extraction is carried out 1-3 times, preferably 1 time; the flash extraction time is 1-2 min, preferably 1 minute.

[0012] Further, in Step 2, the ratio of the extract to methanol (g / V) is 1:3-5, and the dispersion time is 30 minutes.

[0013] Further, in Step 3, isocratic elution with methanol: water is used, and the volume ratio of methanol to water is 78:22.

[0014] Further, in Step 3, the wavelength used for preparative liquid phase is 270 nm, the flow rate is 2 mL / min, and the column temperature is 30 °C.

[0015] Collect the components according to the chromatogram display and perform HPLC detection to obtain dehydrodiisoeugenol with a purity ≥98%.

[0016] Compared with the existing technology, the present invention has the following beneficial effects.

[0017] The present invention relates to a method for extracting and separating dehydrodiisoeugenol, a lignan compound derived from nutmeg. Flash extraction, concentration, methanol purification to remove fatty oil, and preparative liquid phase preparation are sequentially adopted. The flash extraction operation adopted in the extraction and separation process is simple, time-saving, environmentally friendly, and the purity of the compound separated by this method is relatively high, all greater than 98%.

[0018] Based on the principle of tissue fragmentation, the flash extractor can quickly break the materials into appropriate particle sizes. Meanwhile, with the functions of high-speed stirring, vibration, negative pressure percolation, etc., extraction can be achieved, which can maximize the retention of plant active ingredients without thermal damage, with the advantages of small solvent consumption, short extraction time, high efficiency, etc. There is no literature report on the extraction of nutmeg with petroleum ether. Dehydrodiisoeugenol is highly lipophilic and easily soluble in petroleum ether. However, petroleum ether can also extract a large amount of fat in nutmeg, and fat is insoluble in methanol. Therefore, methanol is used for further purification.

[0019] In addition, studies have shown that this compound has anti-inflammatory and antioxidant effects. Therefore, dehydrodiisoeugenol can participate in the development of new drugs and be used to prepare anti-inflammatory drugs. Brief Description of the Drawings

[0020] Figure 1 This is the structural formula of dehydrodiisoeugenol of the present invention.

[0021] Figure 2 For the 1H-NMR spectrogram of dehydrodiisoeugenol in Example 1 1 1H-NMR spectrogram.

[0022] Figure 3 This is the HPLC chromatogram of the extraction raw materials of dehydrodiisoeugenol in Example 1.

[0023] Figure 4 This is the high-resolution mass spectrogram of dehydrodiisoeugenol in Example 1.

[0024] Figure 5 This is the HPLC chromatogram of the dehydrodiisoeugenol product obtained in Example 1. Detailed Embodiments

[0025] The following examples will help to understand the present invention, but these examples are only for illustrating the present invention, and the present invention is not limited to these contents. The operation methods in the examples are all conventional operation methods in the technical field.

[0026] A method for quickly separating and preparing high-purity dehydrodiisoeugenol from nutmeg specifically includes the following steps:

[0027] Step 1: After the nutmeg medicinal materials are crushed and passed through a 20-mesh sieve, they are impregnated with a petroleum ether (60-90 °C) solution, and flash extraction is carried out under a certain voltage for multiple times. The supernatant is sucked, and the petroleum ether is recovered to obtain an extract.

[0028] Step 2: The extract is ultrasonically extracted and dispersed with methanol to precipitate white fat, and the white fat is filtered off to obtain a methanol extract.

[0029] Step 3: Dilute the methanol extract obtained in Step 2 to a certain concentration (60 mg / ml based on the extract), filter through a 0.22 mm microporous membrane, and separate and prepare it by HPLC (high performance liquid chromatography). Using methanol: water as the mobile phase, under certain mobile phase flow rate, column temperature, and detection wavelength conditions, dehydrodiisoeugenol is prepared.

[0030] Furthermore, in Step 1, the ratio of nutmeg medicinal material to petroleum ether (g / V) is 1:60 - 100, preferably 60 times the amount; the voltage is 95 - 105 V, preferably 105 V; extract 1 - 3 times, preferably 1 time; the flash extraction time is 1 - 2 min, preferably 1 minute.

[0031] Furthermore, in Step 2, the ratio of the extract to methanol (g / V) is 1:3 - 5, and the dispersion time is 30 minutes.

[0032] Furthermore, in Step 3, isocratic elution with methanol: water is used, where the volume ratio of methanol to water is 78:22.

[0033] Furthermore, in Step 3, the wavelength used for preparative liquid chromatography is 270 nm, the flow rate is 2 mL / min, and the column temperature is 30°C.

[0034] Example 1.

[0035] A method for rapidly separating and preparing high-purity dehydrodiisoeugenol from nutmeg, specifically including the following steps:

[0036] Step 1: After pulverizing the nutmeg medicinal material and passing it through a 20-mesh sieve, impregnate the medicinal material with 60 times the amount of petroleum ether (60 - 90°C) solution. Under a voltage of 105 V, perform flash extraction for 1 min, extract 1 time, suck the supernatant, and recover petroleum ether to obtain an extract.

[0037] Step 2: Add 3 times the amount of methanol to the extract, perform ultrasonic extraction and dispersion for 30 minutes, precipitate white fat, and filter to obtain a methanol extract.

[0038] Step 3: Dilute the methanol extract obtained in Step 2 to a certain concentration (60 mg / ml based on the extract), filter through a 0.22 mm microporous membrane, and separate and prepare it by HPLC (high performance liquid chromatography). Using methanol: water (78:22) as the mobile phase, with a mobile phase flow rate of 2 mL / min, a column temperature of 30°C, and a detection wavelength of 270 nm, prepare dehydrodiisoeugenol.

[0039] Collect the components according to the chromatogram display and perform HPLC detection to obtain dehydrodiisoeugenol with a purity of 98.6%.

[0040] The dehydrodiisoeugenol prepared in this example has the molecular formula C 20 H 22O4, named 4-(2,3-dihydro-7-methoxy-3-methyl-5-propenyl-2-benzofuranyl)-2-methoxyphenol. The structural formula is as Figure 1 shown.

[0041] The NMR data of this compound are as follows:

[0042] 1H NMR (600 MHz, CDCl3) 1.38 (d, J = 6.7 Hz, 3H) and 3.45 (s, 1H) prove the presence of the fragment CH3-CH- in the structure, 3.89 (d, J = 9.4 Hz, 6H) are the proton signals of two methoxy groups, 5.10 (d, J = 9.4 Hz, 1H) is the proton signal on the carbon attached to oxygen, 1.87 (d, J = 6.5Hz, 3H) and 6.36 (d, J = 15.7Hz, 1H) indicate the presence of a propenyl group in the structure, 5.65 (s, 1H) is a labile proton signal, and 6.11 (dd, J =15.6, 6.7 Hz, 1H), 6.90 (d, J = 1.9Hz, 2H), 6.98 (s, 1H) indicate the presence of an ABX coupling system on the benzene ring.

[0043] Example 2.

[0044] A method for rapidly separating and preparing high-purity dehydrodiisoeugenol from nutmeg, specifically including the following steps:

[0045] Step 1: After the nutmeg medicinal material is crushed and passed through a 20-mesh sieve, it is impregnated with 80 times the amount of petroleum ether (60-90 °C) solution. Under a voltage of 105V, flash extraction is carried out for 1 min, and the extraction is carried out 2 times. The supernatant is sucked out, combined, and the petroleum ether is recovered to obtain an extract;

[0046] Step 2: Add 4 times the amount of methanol to the extract, ultrasonically extract and disperse for 30 minutes, white fat is precipitated, and filtered to obtain a methanol extract;

[0047] Step 3: Dilute the methanol extract obtained in Step 2 to a certain concentration (calculated based on the extract, 60 mg / ml), pass through a 0.22 mm microporous filter membrane, and separate and prepare by HPLC (high performance liquid chromatography). Using methanol: water (78:22) as the mobile phase, the flow rate of the mobile phase is 2 mL / min, the column temperature is 30 °C, and dehydrodiisoeugenol is prepared under the condition that the detection wavelength is 270 nm. The components are collected according to the chromatogram display and subjected to HPLC detection, and dehydrodiisoeugenol with a purity of 98.2% can be obtained.

[0048] Example 3.

[0049] A method for rapidly separating and preparing high-purity dehydrodiisoeugenol from nutmeg, specifically comprising the following steps:

[0050] Step 1: The nutmeg medicinal materials are crushed and passed through a 20-mesh sieve, and the medicinal materials are impregnated with 60 times the amount of petroleum ether (60-90 °C) solution. Under a voltage of 105 V, flash extraction is carried out for 1 min, and extraction is carried out 3 times. The supernatant is sucked, combined, and petroleum ether is recovered to obtain an extract;

[0051] Step 2: 5 times the amount of methanol is added to the extract, and ultrasonic extraction and dispersion are carried out for 30 minutes to precipitate white fat, and then filtration is carried out to obtain a methanol extract;

[0052] Step 3: The methanol extract obtained in Step 2 is diluted to a certain concentration (calculated according to the extract, 60 mg / ml), passed through a 0.22 mm microporous filter membrane, and separated and prepared by HPLC (high performance liquid chromatography). Using methanol: water (78:22) as the mobile phase, the flow rate of the mobile phase is 2 mL / min, the column temperature is 30 °C, and dehydrodiisoeugenol is prepared under the condition that the detection wavelength is 270 nm. The components are collected according to the chromatogram display and subjected to HPLC detection, and dehydrodiisoeugenol with a purity of 98.8% can be obtained.

Claims

1. A method for rapidly separating and preparing high-purity dehydrodiisoeugenol from nutmeg, characterized in that: The specific steps include: Step 1, crushing the nutmeg medicinal material and passing it through a 20-mesh sieve, impregnating the medicinal material with a petroleum ether solution, flash extracting at a certain voltage, extracting multiple times, absorbing the supernatant, recovering the petroleum ether, and obtaining an extract; the g / V ratio of the nutmeg medicinal material to the petroleum ether is 1:60-100, the voltage is 95-105V, the extraction multiple times is 1-3 times, and the flash extraction time is 1-2min; Step 2, using methanol to ultrasonically extract and disperse the extract, precipitate white fat, filter and remove the white fat, and obtain a methanol extract; the ratio of the extract to methanol is 1:3-5 g / V, and the dispersion time is 30 minutes; Step 3, diluting the methanol extract obtained in step 2 to a concentration of 60 mg / ml, passing through a 0.22 mm microporous filter membrane, and separating and preparing by HPLC, using methanol: water as the mobile phase, and preparing dehydrodiisoeugenol under certain mobile phase flow rate, column temperature, and detection wavelength conditions; the methanol: water used isocratic elution, wherein the volume ratio of methanol to water is 78:22; the wavelength used for the preparation of the liquid phase is 270 nm, the flow rate is 2 mL / min, and the column temperature is 30°C.

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