Method for preparing four lutein diesters from marigold

Abstract

The invention belongs to the technical field of separation and purification of plant effective components, and relates to a method for preparing four high-purity lutein diesters from marigold. The method comprises the following steps: by taking marigold as a raw material and ethyl acetate as an extraction solvent, carrying out overspeed crushing extraction at room temperature, and then separating by taking ethyl acetate-acetonitrile-methanol as a mobile phase and adopting preparative high performance liquid chromatography to finally obtain four lutein diester monomers of which the purity is greater than or equal to 95%, lutein myristate, lutein myristate-palmitate, lutein dipalmitate, lutein palmitate-stearate, lutein dimyristate, lutein myristate-palmitate The preparation process is simple and rapid, good in stability and high in efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification of plant active ingredients and relates to a method for preparing four high-purity lutein diesters from marigold. Background Art

[0002] Marigold (Tagetes erecta L.) is an annual herbaceous plant of the Asteraceae family. It not only has extremely high ornamental value, but is also commonly used as an important source of lutein extraction. Marigold petals are rich in carotenoids, mainly lutein, which can account for 90% of the total carotenoid content of the petals. Lutein in marigold plants mostly exists in an esterified state to form a variety of lutein esters. It is reported that the lutein esters are mainly lutein diesters, among which lutein dimyristate, lutein myristic acid-palmitate, lutein dipalmitate, and lutein palmitic acid-stearate are the four main lutein diesters in marigold. Research results show that these lutein diesters have high thermal stability and also have antioxidant biological activity and anti-cancer activity.

[0003] In the past few decades, people have been working hard to explore and improve the extraction methods of lutein esters for commercial production. Currently, the commonly used extraction methods include solvent extraction, ultrasound-assisted extraction, microwave-assisted extraction, supercritical CO 2 Extraction and other methods. Previously, lutein esters were extracted and then saponified to generate free lutein, which was used as a functional food and drug additive. It has been reported that lutein can effectively prevent and treat cancer, coronary artery disease and age-related eye diseases. Recently, when we tested eye care products on the market, we found that many products directly added lutein esters. This may be because free lutein is easily decomposed by heat, and lutein esterification has better thermal stability and can be stored for a long time at room temperature. After investigation, it was found that the in vivo bioavailability of free lutein and lutein esters is different, but there is very little research on lutein ester components. High-purity lutein ester compounds are needed as research objects to conduct various biological activities and cellular and in vivo utilization studies. Therefore, it is very necessary to prepare high-purity lutein esters or to isolate lutein esters.

[0004] Ultra-speed crushing extraction is an extraction technology that uses the inner blade to rotate at high speed to drive the outer blade to crush the plant tissue. The medicinal materials are crushed by rapid mechanical cutting, so that the chemical substances in the plant tissue are dissolved in the extraction reagent in a short time. This extraction method has the advantages of short extraction time, safety and energy saving, adjustable scale, high extraction efficiency, and simple operation. There is no literature report on the ultra-speed crushing extraction method for extracting lutein esters from marigold. Ultra-speed crushing method helps to extract a large amount of lutein esters from marigold.

[0005] In view of the above problems, in order to obtain monomer compounds of four lutein diesters with higher content in marigold, an ultra-speed crushing extraction method is adopted, and the present invention provides a method for preparing four lutein diesters from marigold. The method designed by the present invention is simple and rapid, and the obtained product has high purity.

[0006] A method for preparing four lutein diesters from marigold, comprising the following steps:

[0007] Step 1, extracting marigold powder by ultra-speed crushing extraction method, adding a certain amount of ethyl acetate to soak the powder, and ultra-speed crushing extraction at a certain speed;

[0008] Step 2, after a certain period of extraction, centrifuging the extract, recovering the supernatant, repeating the extraction and centrifugation several times, combining the supernatants to form a marigold extract; and performing high performance liquid chromatography analysis on the extract;

[0009] Step 3, the extract obtained in step 2 is concentrated, and separated and prepared by preparative high performance liquid chromatography, using ethyl acetate: acetonitrile: methanol as the mobile phase, at room temperature, at a certain flow rate to prepare lutein dimyristate, lutein myristic acid-palmitate, lutein dipalmitate, and lutein palmitic acid-stearate. After concentration, it is tested that the purity of the four lutein diesters is greater than 95%.

[0010] Furthermore, in step 1, the ratio of marigold powder to ethyl acetate is 1:10-1:50 (g / mL), preferably 1:20 (g / mL); the speed of ultra-speed crushing extraction is 2000-4500 r / min, preferably 3500 r / min.

[0011] Furthermore, in step 2, the ultra-speed crushing extraction is performed 1-3 times, preferably once; and the ultra-speed crushing extraction time is 1-10 minutes, preferably 6 minutes.

[0012] Furthermore, in the step 3, the mobile phase used is ethyl acetate: acetonitrile: methanol, and gradient elution is adopted, wherein methanol is the mobile phase A, and the ratio of ethyl acetate and acetonitrile is fixed, ethyl acetate: acetonitrile = 7:3 (V:V) is the mobile phase B, and within 37 minutes, the ratio of the mobile phase changes with time, 0-10min, 30-10%A; 10-20min, 10-20%A; 20-30min, 20-30%A; 30-37min, 30%A.

[0013] Furthermore, in step 3, the flow rate of the preparative liquid phase is 6-10 mL / min, preferably 8.5 mL; the injection volume is 180-240 μL / time, preferably 210 μL.

[0014] The components were collected according to the chromatogram, and the prepared solution was concentrated at low temperature and dried to obtain a yellow powder. The obtained substance was tested by HPLC, and the purity of lutein dimyristate, lutein myristic acid-palmitate, lutein dipalmitate, and lutein palmitic acid-stearate were all

[0015] ≥95%.

[0016] The present invention has the following beneficial effects:

[0017] The method for extracting and preparing lutein diester from marigold in the present invention sequentially adopts ultra-speed crushing extraction, concentration, and preparative high-performance liquid chromatography preparation. The ultra-speed crushing extraction method adopted in the extraction process is simple to operate, has a short extraction time, and is convenient for process amplification. The purity of the compound obtained by preparative high-performance liquid chromatography separation is relatively high, all greater than 95%, and can be used for subsequent experimental research on biological activity, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The structural formula of lutein dimyristate, lutein myristic acid-palmitate, lutein dipalmitate, lutein palmitic acid-stearate in the present invention is

[0019] Figure 2 HPLC spectrum of marigold extract raw material in Example 1

[0020] Figure 3 LC-MS / MS ion current (A), ion fragments (B), and MALDI-TOF Mass (C) spectra of lutein dimyristate in Example 1

[0021] Figure 4 LC-MS / MS ion current (A), ion fragments (B), and MALDI-TOF Mass (C) spectra of lutein myristic acid-palmitate in Example 1

[0022] Figure 5 LC-MS / MS ion current (A), ion fragments (B), and MALDI-TOF Mass (C) spectra of lutein dipalmitate in Example 1

[0023] Figure 6 LC-MS / MS ion current (A), ion fragments (B), and MALDI-TOF Mass (C) spectra of lutein palmitate-stearate in Example 1 DETAILED DESCRIPTION

[0024] 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.

[0025] Example 1

[0026] A method for preparing four lutein diesters from marigold, comprising the following steps:

[0027] Step 1, crushing marigold and passing it through an 80-mesh sieve, adding ethyl acetate solution with a solid-liquid ratio of 1:20 (g / mL) to impregnate the marigold powder, and overspeed crushing at a speed of 3500 r / min;

[0028] Step 2, ultra-speed crushing extraction once, the extraction time is 6 minutes, centrifugation, solid-liquid separation, recovering the supernatant solution to obtain an extract; concentrating the extract, passing it through a 0.22 mm microporous filter membrane, and obtaining a sample solution to be separated;

[0029] Step 3, the sample solution to be separated obtained in step 2 is separated and prepared by preparative high performance liquid chromatography, with ethyl acetate: acetonitrile: methanol as the mobile phase gradient elution, wherein methanol is the mobile phase A, ethyl acetate: acetonitrile = 7:3 (V: V) is the mobile phase B; 0-5min, 0-20% A; 5-10min, 20-25% A; 10-20min, 25-30% A; 20-40min, 30% A. The flow rate is 8.5mL / min, the detection wavelength is 445nm, and the injection volume is 210μL / time. The components are collected according to the chromatogram display, and the preparation liquid is concentrated to obtain a yellow powder. Four lutein diesters are prepared, namely lutein dimyristate, lutein myristic acid-palmitate, lutein dipalmitate, and lutein palmitic acid-stearate.

[0030] HPLC detection of the four substances showed that the purity of lutein dimyristate was 96.12% and the preparation rate was 54.23 (mg / g), the purity of lutein myristic acid palmitate was 98.37% and the preparation rate was 57.23 (mg / g), the purity of lutein dipalmitate was 97.68% and the preparation rate was 48.23 (mg / g), and the purity of lutein palmitic acid stearate was 97.03% and the preparation rate was 22.04 (mg / g).

[0031] After mass spectrometry analysis, the four lutein diester molecules prepared were determined to be lutein dimyristate (C 68 H 108 O 4 ), Lutein myristic acid palmitate (C 70 H 114 O 4 ), lutein dipalmitate (C 72 H 116 O 4 ), Lutein palmitic acid-stearate (C 74 H 122 O 4), and the LC-MS / MS fragment ion data are shown in Table 1.

[0032] Table 1. Ion fragmentation data of four lutein esters

[0033]

[0034] Example 2

[0035] A method for preparing four lutein diesters from marigold, comprising the following steps:

[0036] Step 1, crushing marigold medicinal materials and passing through an 80-mesh sieve, adding ethyl acetate solution with a solid-liquid ratio of 1:20 (g / mL) to soak the medicinal materials, and overspeed crushing at a speed of 3500 r / min;

[0037] Step 2: Ultra-speed crushing and extraction for 2 times, each extraction time is 6 minutes, centrifugation, solid-liquid separation, recovery of solution, and obtaining extract; concentrating the extract, dissolving the solid with ethyl acetate, and filtering through a 0.22 mm microporous membrane.

[0038] obtaining a sample solution to be separated;

[0039] Step 3, the sample solution to be separated obtained in step 2 is separated and prepared by a preparative liquid chromatography instrument, and ethyl acetate: acetonitrile: methanol is used as the mobile phase gradient elution, wherein methanol is the mobile phase A, and ethyl acetate: acetonitrile = 7:3 (V: V) is the mobile phase B; 0-10min, 30-10% A; 10-20min, 10-20% A; 20-30min, 20-30% A; 30-37min, 30% A. The flow rate is 8.5mL / min, the column temperature is 30°C, the detection wavelength is 445nm, and the injection volume is 210μL / time, and lutein dimyristate, lutein myristic acid-palmitate, lutein dipalmitate, and lutein palmitic acid-stearate are prepared. And through the detection of analytical high performance liquid chromatography instrument, the purity of lutein dimyristate is 96.35%, and the preparation rate is 53.81 (mg / g), the purity of lutein myristic acid palmitate is 98.01%, and the preparation rate is 57.76 (mg / g), the purity of lutein dipalmitate is 97.78%, and the preparation rate is 47.28 (mg / g), and the purity of lutein palmitic acid stearate is 97.67%, and the preparation rate is 22.71 (mg / g).

[0040] Example 3

[0041] A method for preparing four lutein diesters from marigold, comprising the following steps:

[0042] Step 1, crushing marigold medicinal materials and passing through an 80-mesh sieve, adding ethyl acetate solution with a solid-liquid ratio of 1:20 (g / mL) to soak the medicinal materials, and overspeed crushing at a speed of 3500 r / min;

[0043] Step 2: Ultra-speed crushing extraction 3 times, each extraction time is 6 minutes, centrifugation, solid-liquid separation, recovery of solution, and extraction liquid; Concentrate the extraction liquid, dissolve the solid with ethyl acetate, filter through a 0.22 mm microporous membrane,

[0044] obtaining a sample solution to be separated;

[0045] Step 3, the sample solution to be separated obtained in step 2 is separated and prepared by a preparative liquid chromatography instrument, and ethyl acetate: acetonitrile: methanol is used as the mobile phase gradient elution, wherein methanol is the mobile phase A, and ethyl acetate: acetonitrile = 7:3 (V: V) is the mobile phase B; 0-10min, 30-10% A; 10-20min, 10-20% A; 20-30min, 20-30% A; 30-37min, 30% A. The flow rate is 8.5mL / min, the column temperature is 30°C, the detection wavelength is 445nm, and the injection volume is 210μL / time, and lutein dimyristate, lutein myristic acid-palmitate, lutein dipalmitate, and lutein palmitic acid-stearate are prepared. HPLC detection was also performed, and the purity of the obtained lutein dimyristate was 96.71%, and the preparation rate was 54.27 (mg / g), the purity of lutein myristic acid palmitate was 98.76%, and the preparation rate was 57.19 (mg / g), the purity of lutein dipalmitate was 97.23%, and the preparation rate was 48.39 (mg / g), and the purity of lutein palmitic acid stearate was 97.45%, and the preparation rate was 22.68 (mg / g).

[0046] In Example 4-7, other steps are the same as those in Example 1-3.

[0047]

[0048] Examples 8-10 are detection processes using different mobile phases, and the steps are the same as those of Examples 1-3.

[0049]

[0050]

Claims

1. A method for preparing four lutein diesters from marigold, characterized in that: The following steps are included: Step 1, extracting marigold powder by ultra-speed crushing extraction method, adding a certain amount of ethyl acetate to soak the powder, and ultra-speed crushing extraction at a certain speed; Step 2, after a certain period of extraction, centrifuging the extract, recovering the supernatant, repeating the extraction and centrifugation several times, combining the supernatants to form a marigold extract; and performing high performance liquid chromatography analysis on the extract; Step 3, the extract obtained in step 2 is concentrated, and separated and prepared by preparative high performance liquid chromatography, using ethyl acetate: acetonitrile: methanol as the mobile phase, at room temperature, at a certain flow rate to prepare lutein dimyristate, lutein myristic acid-palmitate, lutein dipalmitate, and lutein palmitic acid-stearate. After concentration, it is tested that the purity of the four lutein diesters is greater than 95%.

2. The method for preparing four lutein diesters from marigold according to claim 1, characterized in that: In the step 1, the ratio of marigold powder to ethyl acetate is 1:10-1:50 (g / mL), preferably 1:20 (g / mL); the rotation speed is 2000-4500 r / min, preferably 3500 r / min.

3. The method for preparing four lutein diesters from marigold according to claim 1, characterized in that: In step 2, the extraction times are 1-3 times, preferably 1 time; and the ultra-speed crushing extraction time is 1-10 minutes, preferably 6 minutes.

4. The method for preparing four xanthophyll diesters from marigold according to claim 1, characterized in that: In step 3, the gradient elution used is ethyl acetate: acetonitrile: methanol, wherein methanol is the mobile phase A, and ethyl acetate: acetonitrile = 7:3 (V:V) is the mobile phase B; 0-10 min, 30-10% A; 10-20min, 10-20%A; 20-30min, 20-30%A; 30-37min, 30%A.

5. The method for separating and preparing four high-purity lutein esters from marigold according to claim 1, characterized in that: In step 3, the flow rate used for preparative high performance liquid chromatography is 6-10 mL / min, preferably 8.5 mL; the injection volume is 180-240 μL / time, preferably 210 μL.

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