A method for extracting tripterygium wilfordii quinone A from the root of jasmine

Abstract

The present invention relates to the technical field of extracting active ingredients from medicinal plants. Specifically, a method for separating and extracting tripterygium wilfordii quinone A from the root of the Chinese herb "Torreya grandis" is disclosed. The method uses the root of the Chinese herb "Torreya grandis" as the raw material and obtains tripterygium wilfordii quinone A monomer through pulverization, reflux extraction, extraction, reverse-phase medium-pressure C18 column chromatography, and reverse-phase high-pressure C18 column chromatography. The method is simple, rapid, and has high socioeconomic value. The obtained tripterygium wilfordii quinone A has a high purity (99.72%), laying the foundation for subsequent research on its pharmacological activity and the development of new immunotherapy drugs.

Description

Technical Field

[0001] The invention relates to the technical field of extraction of active ingredients of medicinal plants, and in particular to a method for separating and extracting tripterygium wilfordii quinone A from the root of Ficus microcarpa. Background Art

[0002] Torch Flower Root is the peeled, dried root core of Tripterygium hypoglaucum (Levl.) Hutch, a plant of the Euonymus family. It can be harvested year-round, removed of impurities, the root bark removed, crushed, and dried. It has a bitter, warm flavor and is toxic. It enters the spleen and kidney meridians. It dispels wind and dampness, relaxes tendons and activates collaterals, and clears heat and detoxifies. It is primarily used for rheumatic arthritis (rheumatoid arthritis, rheumatic arthritis), chronic nephritis, lupus erythematosus, traumatic injuries, and itchy skin.

[0003] Tripterygium wilfordii quinone A is one of the active ingredients in the root of the jasmine plant. Relevant studies have shown that Tripterygium wilfordii quinone A has good anti-inflammatory and immune-regulating physiological activities, and can significantly inhibit the release of interleukin-1 (IL-1). Interleukin-1 (IL-1) is a cytokine that is widely present in animals. It mainly exists in the forms of IL-1α and IL-1β. It can regulate immune responses, inflammatory responses, and other physiological activities, and is an important active substance. It is a multifunctional cytokine that can promote cell differentiation and proliferation, affect immune responses, regulate the body's metabolism, fight viruses and tumors, and participate in other important physiological activities of the body. Excessive IL-1 levels can lead to abnormal immune function in the body, easily induce diseases such as arthritis, hepatitis, nephritis, and even have the possibility of tumor development. Torch Flower Root Tablets, made from the root of Torch Flower (Pyrrhiza uralensis) through water extraction and alcohol precipitation, have demonstrated promising therapeutic effects on immune disorders such as rheumatoid arthritis, lupus erythematosus, and kidney disease. One of the active ingredients, Tripterygium wilfordii quinone A, exhibits significant inhibitory activity against interleukin-1 (IL-1) release. Currently, the extraction and purification of Tripterygium wilfordii quinone A has not been reported. To obtain high-purity Tripterygium wilfordii quinone A for research on its pharmacological activity, it is necessary to develop a simple, rapid, and efficient extraction method. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for extracting tripterygium wilfordii quinone A from the root of the medicinal material of the torch flower. The method has high extraction efficiency and the obtained tripterygium wilfordii quinone A has high purity (99.72%), providing a basis for the preparation and production of a reference substance of tripterygium wilfordii quinone A.

[0005] To achieve the above object, the present invention comprises the following steps:

[0006] Step S10: Grinding 10 kg of the root of the Chinese torch flower medicinal material, passing it through a 50-60 mesh sieve to obtain a coarse powder of the root of the Chinese torch flower medicinal material, adding 75%-90% ethanol solution with a weight of 10-30 times the powder, heating and refluxing at 65-95° C. for 2-5 hours, extracting 2-3 times, and filtering to obtain a root of the Chinese torch flower extract;

[0007] Step S20: The extract of the jasmine root in step S10 is concentrated under reduced pressure until there is no alcohol taste, and the ethanol solution is recovered to obtain a jasmine root concentrated solution;

[0008] Step S30: extracting the concentrated liquid of the root of the Ficus microcarpa with equal volumes of petroleum ether, dichloromethane, and n-butanol three times, combining the petroleum ether, dichloromethane, and n-butanol extracts, and recovering the corresponding solvents under reduced pressure to obtain the petroleum ether, dichloromethane, and n-butanol extracts of the root of the Ficus microcarpa;

[0009] Step S40: enriching the dichloromethane extract of the root of the jasmine flower in step S30 by reverse phase medium pressure C18 column chromatography, eluting with a gradient of 30%, 40%, 50%, and 60% acetonitrile, with each gradient elution lasting 4 column volumes. The eluted fractions were detected by HPLC, combined, and concentrated. Triptolide quinone A was enriched in the LF04 fraction, wherein the LF04 fraction was the fraction eluted with 60% acetonitrile water.

[0010] Step S50: The fraction LF04 obtained in step S40 is subjected to preparative purification by reverse phase high pressure C18 column chromatography, with a gradient elution of 50% to 60% acetonitrile water, and the fraction containing tripterygium wilfordii quinone A is collected, concentrated, and freeze-dried to obtain tripterygium wilfordii quinone A.

[0011] The structural formula of the obtained tripterygium wilfordii quinone A is as follows:

[0012]

[0013] Triptoquinone A, molecular formula: C 20 H 24 O4; HR-ESI-MSm / z329.1738[M+H] +;1H NMR(Chloroform-d,500MHz)δH 6.37(1H,s), 2.99(1H,hept,J=6.9Hz), 2.81~2.72(2H,m), 2.62~2.41(2H,m), 2.44~2.33(1H,m), 2.25~2. 18(2H,m), 2.10(3H,s), 1.53~1.39(2H,m), 1.17(3H,s), 1.11(3H,d,J=6.9Hz), 1.10(3H,d,J=6.9Hz); 13C NMR(125MHz,Chloroform-d)δC 187.97, 187.74, 174.31, 153.35, 148.83, 147.84, 142.63, 131.94, 124.84, 47.46, 36.70, 31.97, 26.54, 25.31, 24.69, 21.52, 21.48, 19.30, 18.85, 18.5.

[0014] Furthermore, in step S10, the amount of 90% ethanol added is 10 times, and the heating reflux extraction conditions are as follows: extraction temperature is 80-85° C.; reflux extraction time is 2-3 hours; extraction times are 2 times.

[0015] Furthermore, the reverse phase medium pressure C18 silica gel used in step S40 is 100-200 mesh.

[0016] Furthermore, a small amount of the obtained tripterygium wilfordii quinone A sample was taken, dissolved in methanol and prepared into a solution of about 0.5 mg / mL, and detected by HPLC high performance liquid chromatography.

[0017] Furthermore, the HPLC high performance liquid chromatography method was used, and the chromatographic conditions were as follows: chromatographic column: Agilent Extend-C 18 ,4.6x250mm,5μm; mobile phase: water (A)-acetonitrile (B) gradient elution (0-25min, 70%A-20%A; 25-26.1min, 20%A-0%A; 26.1-30min, 0%A); flow rate: 1mL / min; column temperature: 30℃; detection wavelength: 260nm; injection volume: 10μL.

[0018] The beneficial effects of the present invention are: this preparation method is the first to perform reflux extraction on tripterygium wilfordii quinone A, and use HPLC technology to track the flow components, medium and high pressure chromatography separation technology, this method has high extraction efficiency, saves reagents, and the obtained tripterygium wilfordii quinone A is high in purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the HPLC chromatogram of tripterygium wilfordii quinone A;

[0020] Figure 2 is the MS of tripterygium wilfordii quinone A 1 Spectrum;

[0021] Figure 3 Tripterygium wilfordii quinone B 1 H-NMR spectrum (test frequency is 125 MHz, test solvent is CDCl3);

[0022] Figure 4 Tripterygium wilfordii quinone B 13 C-NMR spectrum (test frequency is 125 MHz, test solvent is CDCl3);

[0023] Figure 5 This is a diagram of the separation and purification process of tripterygium wilfordii quinone A; DETAILED DESCRIPTION

[0024] A method for extracting tripterygium wilfordii quinone A from the root of the medicinal material of the torch flower, the present invention includes the following embodiments:

[0025] Example 1: 10 kg of the medicinal material of the root of the torch flower was crushed and passed through a 50-mesh sieve to obtain the medicinal material powder of the torch flower root. 100 L of a 90% ethanol solution was added, and the mixture was heated under reflux at 75° C. for 2 h, extracted three times, and filtered to obtain a torch flower root extract. The torch flower root extract was concentrated under reduced pressure until there was no alcohol taste, and extracted three times with equal volumes of petroleum ether, dichloromethane and n-butanol, respectively. The petroleum ether, dichloromethane and n-butanol extracts were combined, and the corresponding solvents were recovered under reduced pressure to obtain petroleum ether, dichloromethane and n-butanol extracts of the torch flower root, thereby obtaining a dichloromethane extract of the torch flower root.

[0026] Example 2: 10 kg of the medicinal material of the root of the torch flower was crushed and passed through a 60-mesh sieve to obtain the medicinal material powder of the root of the torch flower, 120 L of a 75% ethanol solution was added, and the mixture was heated under reflux at 85° C. for 2 hours, extracted three times, and filtered to obtain a root extract of the torch flower; the root extract of the torch flower was concentrated under reduced pressure until there was no alcohol taste, and extracted three times with equal volumes of petroleum ether, dichloromethane and n-butanol, respectively. The petroleum ether, dichloromethane and n-butanol extracts were combined, and the corresponding solvents were recovered under reduced pressure to obtain petroleum ether, dichloromethane and n-butanol extracts of the root of the torch flower, thereby obtaining a dichloromethane extract of the root of the torch flower.

[0027] Example 3: 10 kg of the medicinal material of the root of the torch flower was crushed and passed through a 60-mesh sieve to obtain the medicinal material powder of the torch flower root. 200 L of an 85% ethanol solution was added, and the mixture was heated under reflux at 90°C for 2 h, extracted three times, and filtered to obtain a torch flower root extract. The torch flower root extract was concentrated under reduced pressure until there was no alcohol taste, and extracted three times with equal volumes of petroleum ether, dichloromethane and n-butanol, respectively. The petroleum ether, dichloromethane and n-butanol extracts were combined, and the corresponding solvents were recovered under reduced pressure to obtain petroleum ether, dichloromethane and n-butanol extracts of the torch flower root, thereby obtaining a dichloromethane extract of the torch flower root.

[0028] The obtained dichloromethane extract of the root of the jasmine flower was enriched by reverse phase medium pressure C18 column chromatography, and gradient elution was performed with 30%, 40%, 50%, and 60% acetonitrile-water system, with each gradient elution lasting 4 column volumes. The eluted fractions were detected by HPLC, combined and concentrated, and tripterygium wilfordii quinone A was enriched in the LF04 fraction (approximately 60% acetonitrile-water);

[0029] The LF04 fraction was subjected to preparative purification by reverse-phase high-pressure C18 column chromatography with a gradient elution of 50% to 60% acetonitrile in water. The fractions containing tripterygium wilfordii quinone A were collected, concentrated, and lyophilized to obtain tripterygium wilfordii quinone A. Finally, the obtained tripterygium wilfordii quinone A was analyzed by HPLC (the analysis conditions are shown in Tables 1 and 2).

[0030] Table 1. Chromatographic analysis conditions

[0031]

[0032] Table 2. Mass spectrometry analysis conditions

[0033]

[0034]

[0035] The beneficial effects of the present invention are: this preparation method is the first to extract, separate and purify tripterygium wilfordii quinone A, and use HPLC technology to track the flow components, medium and high pressure chromatography separation technology, this method has high extraction efficiency, and the obtained tripterygium wilfordii quinone A has a high purity (99.72%), laying the foundation for subsequent research on its pharmacological activity and the development of new immune drugs.

[0036] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalent features for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for extracting tripterygium wilfordii quinone A from the root of the medicinal material of the torch flower, comprising the following steps: Step S10: Grinding 10 kg of the root of the Chinese torch flower medicinal material, passing it through a 50-60 mesh sieve to obtain a coarse powder of the root of the Chinese torch flower medicinal material, adding 75%-90% ethanol solution with a weight of 10-30 times the powder, heating and refluxing at 65-95° C. for 2-5 hours, extracting 2-3 times, and filtering to obtain a root of the Chinese torch flower extract; Step S20: The extract of the jasmine root in step S10 is concentrated under reduced pressure until there is no alcohol taste, and the ethanol solution is recovered to obtain a jasmine root concentrated solution; Step S30: extracting the concentrated liquid of the root of the Ficus microcarpa with equal volumes of petroleum ether, dichloromethane, and n-butanol three times, combining the petroleum ether, dichloromethane, and n-butanol extracts, and recovering the corresponding solvents under reduced pressure to obtain the petroleum ether, dichloromethane, and n-butanol extracts of the root of the Ficus microcarpa; Step S40: enrich the dichloromethane extract of the root of the torch flower in step S30 on a reverse phase medium pressure C18 column chromatography, eluting with a gradient of 30%, 40%, 50%, and 60% acetonitrile, with each gradient elution lasting 4 column volumes. The eluted fractions were detected by HPLC, combined, and concentrated. Triptolide quinone A was enriched in the LF04 fraction, wherein, The LF04 fraction is the fraction eluted with 60% acetonitrile water; Step S50: The fraction LF04 obtained in step S40 is subjected to preparative purification by reverse phase high pressure C18 column chromatography, with a gradient elution of 50% to 60% acetonitrile water, and the fraction containing tripterygium wilfordii quinone A is collected, concentrated, and freeze-dried to obtain tripterygium wilfordii quinone A.

2. The method for extracting tripterygium wilfordii quinone A from the root of Psoralea corylifolia as claimed in claim 1, characterized in that: In step S10, the amount of 90% ethanol added is 10 times, and the heating reflux extraction conditions are as follows: the extraction temperature is 80-85° C.; the reflux extraction time is 2-3 hours; and the extraction times are 2 times.

3. A method for extracting tripterygium wilfordii quinone A from the root of Pyrola truncatum as claimed in claim 1 or 2, characterized in that: The reverse phase medium pressure C18 silica gel used in step S40 is 100-200 mesh.

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