Directional extraction method of ganoderma triterpenes

Abstract

The invention discloses a directional extraction method of ganoderma triterpenes, which is characterized in that 1, 2-propylene glycol-betaine is used as a deep eutectic solvent, and ultrasound is assisted to extract the ganoderma triterpenes. The water content of the eutecticevaporate solvent 1, 2-propylene glycol-betaine is 20%-40%, and the molar ratio of 1, 2-propylene glycol to betaine is 1: (1-3). The extraction method specifically comprises the following steps: crushing and sieving lucid ganoderma, adding a deep-eutectic solvent which is 5-10 times of the amount of the lucid ganoderma, and performing ultrasonic extraction for 15-30 minutes at the temperature of 35-65 DEG C and the power of 500W. The eutecticevaporate solvent 1, 2-propylene glycol-betaine used in the method has the potential of extracting the ganoderma lucidum triterpenes, the reaction condition is mild, tedious purification treatment is not needed, and the content of the total triterpenes in the obtained ganoderma lucidum extract can reach 50% or above.

Description

Technical Field

[0001] The invention belongs to the technical field of ganoderma triterpenoid extraction, and in particular relates to a directional extraction method of ganoderma triterpenoid. Background Art

[0002] Terpenoids are ubiquitous secondary metabolites of medicinal plants, which have attracted extensive attention as a source of drugs for various chronic diseases. The ease of separation and excellent pharmacological properties of triterpenoids are significant reasons for the exponential growth of extensive research on bioactive triterpenoids in the past few decades.

[0003] Ganoderma lucidum is a large fungus that can be used as both medicine and food, and can be used to prevent and treat a variety of diseases. Ganoderma triterpenes are important active ingredients in Ganoderma lucidum, and their content is an important indicator for evaluating the quality of Ganoderma lucidum. Ganoderma triterpenes are tetracyclic triterpenoid compounds with lanostane triterpenes as the basic skeleton. There are many types of Ganoderma triterpenoid compounds, which can be divided into Ganoderma acidic triterpenes and Ganoderma neutral triterpenes according to whether there is a carboxyl group in the structure. In the past 10 years, more than 200 Ganoderma triterpenoid compounds from natural sources have been reported in the literature. The chemical structure of Ganoderma triterpenes determines its diverse biological activities, including anti-tumor, liver protection, anti-inflammatory, antioxidant, anti-radiation, anti-aging, promotion of angiogenesis, regulation of immune function, and regulation of metabolism.

[0004] Ganoderma triterpenes are fat-soluble components and are usually extracted with organic solvents. Common extraction methods include solvent reflux extraction, ultrasonic extraction, supercritical CO2 extraction, microwave-assisted extraction, organic solvent extraction, and enzyme-assisted extraction. Since Ganoderma triterpenes are fat-soluble substances, they can be extracted using traditional organic solvent extraction methods. Based on laboratory environmental conditions, a combination of multiple extraction methods can be used to extract the target material, which can effectively improve the extraction efficiency.

[0005] In recent years, more and more organic solvents have been found to be non-toxic to the environment. Deep eutectic solvent (DES) is a new type of green solvent with low melting point, high thermal stability and good biocompatibility. Traditional organic solvents such as methanol are usually used to extract active ingredients of traditional Chinese medicine, which have defects such as high toxicity, low extraction rate, difficulty in recovery, high volatility and easy pollution of the environment. It is a general trend to find green solvents to replace traditional solvents for the extraction of active ingredients of traditional Chinese medicine. Current research shows that natural deep eutectic solvents can significantly improve the extraction rate of various active ingredients in plants with the help of auxiliary means such as ultrasonic assisted extraction (UAE), and because the components of natural deep eutectic solvents are natural compounds, environmental pollution problems can be avoided. Summary of the invention

[0006] The purpose of the present invention is to provide a method for directional extraction of Ganoderma triterpenes, which uses a non-toxic low eutectic solvent 1,2-propylene glycol-betaine to extract Ganoderma triterpenes. The low eutectic solvent is used as an alternative green solvent, which is beneficial to the further research and development of Ganoderma triterpenes.

[0007] In order to achieve the above object, the present invention adopts the following technical solution: A method for directional extraction of triterpenes of ganoderma lucidum comprises the following steps: crushing and sieving ganoderma lucidum, adding 1,2-propylene glycol, betaine and water, and then ultrasonicating, filtering after the ultrasonication, and collecting the filtrate; and concentrating under reduced pressure and drying under vacuum to obtain a dry extract powder of triterpenes of ganoderma lucidum.

[0008] Furthermore, the molar ratio of 1,2-propylene glycol to betaine is 1:1 to 1:3.

[0009] Furthermore, the water content of the 1,2-propylene glycol / betaine solution is 20% to 40%.

[0010] Furthermore, the solid-liquid ratio of Ganoderma lucidum to 1,2-propylene glycol / betaine solution is 1g:5mL~1g:10mL.

[0011] Furthermore, the temperature of the ultrasound is 35-65°C.

[0012] Furthermore, the power of the ultrasound is 500W.

[0013] Furthermore, the ultrasonic time is 15 to 30 min.

[0014] The beneficial effects of the present invention are: (1) The present invention mixes betaine as a hydrogen bond acceptor with 1,2-propylene glycol as a hydrogen bond donor to form DES. The hydrogen bond donor in the low eutectic solvent forms a hydrogen bond with the hydrogen bond acceptor, thereby reducing the interaction force between molecules, making it easier for the mixture to be transformed into a liquid state, and performing non-polar material ultrasonic-assisted extraction of Ganoderma lucidum triterpenes. The extraction is carried out at a temperature range of 35-65°C, and the extract is analyzed using high performance liquid chromatography. 1,2-propylene glycol-betaine DES has potential as a solvent for extracting Ganoderma lucidum, and the conditions are mild.

[0015] (2) Without complicated purification treatment, the total triterpene content of the obtained Ganoderma lucidum extract can reach more than 50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : HPLC spectrum of Ganoderma lucidum extract; Figure 2 :Total ion chromatogram of LC-MS analysis of Ganoderma lucidum extract; Figure 3 : Ultraviolet spectrum of ganoderic acid A; Figure 4 : Ultraviolet spectrum of ganoderic acid B; Figure 5 : Ultraviolet spectrum of ganoderic acid H; Figure 6 : Ultraviolet spectrum of ganoderic acid D; Figure 7 : Ultraviolet spectrum of Ganoderic acid F. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described in detail below through specific embodiments.

[0018] The instruments and equipment used in the following examples are as follows: 1.1 Instrument A set of Shimadzu LC-20AT high performance liquid chromatograph and SPD-20AV detector, Waters QuattroMicro / Alliance 2695 triple quadrupole liquid chromatograph-tandem mass spectrometer, equipped with electrospray ionization source (ESI), online vacuum degasser, Waters 2996 Photodiode Array Detector, autosampler, constant temperature column box and LC-MS masslynx4.0 workstation. Electronic analytical balance (Shanghai Precision Scientific Instrument Co., Ltd., model FA2104N), rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, model RE-2000), Chinese herbal medicine crusher (Tianjin Test Instrument Co., Ltd.), SHB-Ⅲ circulating water multi-purpose vacuum pump (Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd.), ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0019] 1.2 Materials Medicinal materials: Ganoderma lucidum was purchased from Nanping, Fujian.

[0020] Acetonitrile (chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.); methanol (chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.); acetic acid (analytical grade, Shanghai Lianshi Chemical Reagent Co., Ltd.); n-butanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.), pure water is ultrapure water with a resistivity of 18.2Ω, 1,2-propylene glycol (Sigma-Aldrich); betaine (Shanghai Yuanye Biotechnology Co., Ltd.).

[0021] Example 1 A specific step of directional extraction of triterpenes of Ganoderma lucidum: (1) Ganoderma lucidum was crushed and passed through a 20-mesh sieve to obtain Ganoderma lucidum powder, and a low eutectic solvent with a solid-liquid ratio of 1 g: 6 mL (wherein: the molar ratio of 1,2-propylene glycol to betaine was 1:1, and the water content was 20%) was added for ultrasonic extraction. The ultrasonic temperature was 35°C, the power was 500 W, and the time was 30 min; (2) After the extraction is completed, filter and collect the filtrate; concentrate under reduced pressure to near dryness, and dry under vacuum to obtain the Ganoderma lucidum extract powder.

[0022] Example 2 A specific step of extracting triterpenes of Ganoderma lucidum: (1) Ganoderma lucidum was crushed and passed through a 20-mesh sieve to obtain Ganoderma lucidum powder, and a low eutectic solvent with a solid-liquid ratio of 1 g:8 mL (wherein: the molar ratio of 1,2-propylene glycol to betaine was 1:2, and the water content was 30%) was added for ultrasonic extraction. The ultrasonic temperature was 50°C, the power was 500 W, and the time was 20 min; (2) After the extraction is completed, filter and collect the filtrate; concentrate under reduced pressure to near dryness, and dry under vacuum to obtain the Ganoderma lucidum extract powder.

[0023] Example 3 A specific step of extracting triterpenes of Ganoderma lucidum: (1) Ganoderma lucidum was crushed and passed through a 20-mesh sieve to obtain Ganoderma lucidum powder, and a low eutectic solvent with a solid-liquid ratio of 1 g:10 mL (wherein: the molar ratio of 1,2-propylene glycol to betaine was 1:3, and the water content was 40%) was added for ultrasonic extraction. The ultrasonic temperature was 65°C, the power was 500 W, and the time was 15 min; (2) After the extraction is completed, filter and collect the filtrate; concentrate under reduced pressure to near dryness, and dry under vacuum to obtain Ganoderma lucidum triterpenoid dry extract powder.

[0024] Comparative Example 1 A specific step of extracting triterpenes of Ganoderma lucidum: (1) Ganoderma lucidum was crushed and passed through a 20-mesh sieve to obtain Ganoderma lucidum powder; a low eutectic solvent (wherein: the molar ratio of 1,2-propylene glycol to betaine was 2:1, and the water content was 15%) was added for ultrasonic extraction, and the ultrasonic extraction temperature was 40°C, the power was 500W, and the time was 15 min; (2) After the extraction is completed, filter and collect the filtrate; concentrate under reduced pressure to near dryness, and dry under vacuum to obtain the Ganoderma lucidum extract powder.

[0025] Comparative Example 2 A specific step of extracting triterpenes of Ganoderma lucidum: (1) Ganoderma lucidum was crushed and passed through a 20-mesh sieve to obtain Ganoderma lucidum powder; a low eutectic solvent (wherein the molar ratio of 1,2-propylene glycol to betaine was 2:1 and the water content was 15%) was added to perform ultrasonic extraction at a temperature of 40°C, a power of 500 W, and a time of 30 min; (2) After the extraction is completed, filter and collect the filtrate; concentrate under reduced pressure to near dryness, and dry under vacuum to obtain the Ganoderma lucidum extract powder.

[0026] Results Analysis 2.1 Detection of triterpenoid content in Ganoderma lucidum extract: S1. Reference solution and standard curve Accurately weigh 5 mg of the standard of oleanolic acid and dilute to volume with methanol to obtain a reference stock solution with a mass concentration of 0.20 mg / mL. Accurately measure 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the reference stock solution, heat in a water bath to evaporate the solvent, cool it, add 0.2 mL of the newly prepared 5% vanillin-glacial acetic acid and 0.8 mL of perchloric acid, and shake it quickly; heat in a 70°C water bath for 15 minutes, cool in an ice bath for 5 minutes, and finally dilute to 5 mL with glacial acetic acid. Use glacial acetic acid as a blank, measure the absorbance at a wavelength of 546 nm in a spectrophotometer, and draw a standard curve with oleanolic acid as the horizontal coordinate and absorbance as the vertical coordinate.

[0027] S2. Determination of total triterpenoid content in Ganoderma lucidum extract a) Accurately weigh the dry extract powder of triterpenoids of Ganoderma lucidum, use 30 mL of methanol, perform ultrasonic extraction 3 times, 15 min each time, and recover the methanol under reduced pressure until dry. Dissolve the residue in 30 mL of water, and then extract it four times with n-butanol (30, 30, 15, 15 mL), separate the n-butanol layer, and recover the n-butanol under reduced pressure until dry. Dissolve the residue in chromatographic methanol under ultrasonic pressure, make up to volume in a 5 mL volumetric flask, filter through a 0.45 μm filter membrane, and set aside.

[0028] b) The determination of the content of triterpenoids in Ganoderma lucidum was carried out by vanillin-perchloric acid colorimetric reaction. Take a certain volume of the test solution, heat it in a water bath to evaporate the solvent, cool it down, add 0.2 mL of newly prepared 5% vanillin-glacial acetic acid and 0.8 mL of perchloric acid, and shake it quickly; heat it in a 70℃ water bath for 15 min, cool it in an ice bath for 5 min, and finally dilute it to 5 mL with glacial acetic acid, shake it well, and measure the absorbance at a wavelength of 546 nm. The content of triterpenoids in the extract was calculated by regression equation.

[0029]

[0030] The total triterpenoid content of the dry extract powder of the Ganoderma lucidum extract obtained in the best embodiment of the extraction method is 50.77%, while the total triterpenoid content of the dry extract powder of the Ganoderma lucidum extract obtained in the comparative embodiment is less than 50%.

[0031] 2.2 HPLC analysis of Ganoderma lucidum extract The sample solution obtained in step S1 of 2.1 was used for HPLC analysis; HPLC conditions: Dalian Yilite C 18 Chromatographic column, flow rate of 1 ml / min, mobile phase of acetonitrile-0.1% acetic acid aqueous solution binary gradient elution, detection wavelength of 260nm, column temperature of 25℃, injection volume of 20μL. Elution program is shown in Table 1, and liquid chromatography (HPLC) diagram is shown in Figure 1 The distribution of chromatographic peak areas is shown in Table 2.

[0032] Table 1. Gradient elution program of HPLC fingerprint of Ganoderma lucidum extract

[0033] Table 2 Distribution of chromatographic peak areas of various components of Ganoderma lucidum extract Table 2-1

[0034] Table 2-2

[0035] Table 2-3

[0036] Table 2-4

[0037] Table 2-5

[0038] The triterpene content in the extract can be calculated using the area normalization method according to the following formula.

[0039]

[0040] 2.3 LC-MS Identification of Triterpenoid Characteristic Peaks in Ganoderma Lucidum Extract Total ion chromatogram of LC-MS analysis of Ganoderma lucidum extract is shown in Figure 2 , and the chromatographic peak components were identified by mass spectrometry.

[0041] Peak 5 in ESI-MS negative ion mode gave m / z 513 [MH] - 、m / z495[MH-H2O] + , combined with the UV spectrum, it is inferred to be ganoderic acid A, see Figure 3 .

[0042] Peak 6 gave m / z 539 [M+Na] in ESI-MS positive and negative ion modes respectively. + ,m / z497[MH-H2O] - , combined with the UV spectrum, it is inferred to be ganoderic acid B, see Figure 4 .

[0043] Peak 8 gave m / z 571[MH] in ESI-MS negative ion mode. - , m / z553[MH-H2O] - , combined with the UV spectrum, it was preliminarily identified as ganoderic acid H, see Figure 5 .

[0044] Peak 11 gave m / z 553 [M+K] in ESI-MS positive and negative ion modes respectively. + ,m / z537[M+Na] + ,m / z497[M+H-H2O] + , m / z513[MH] - ,m / z495[MH-H2O] - , combined with the UV spectrum, it was preliminarily identified as ganoderic acid D, see Figure 6 .

[0045] Peak 12 gave m / z 593 [M+Na] in ESI-MS positive and negative ion modes, respectively. + ,m / z571[M+H] + ,m / z569[MH] - , m / z551[MH-H2O] - Combined with the UV spectrum, it was preliminarily identified as 12-acetoxy-ganoderic acid F. Figure 7 .

[0046] The Ganoderma triterpenoid extract was analyzed by UHPLC / MS. The components with retention time in the range of 40-55min were Ganoderma triterpenoids. In the positive and negative ion modes of the ESI source, a total of 5 compounds with peak areas greater than 2% were identified, including Ganoderic acid A, Ganoderic acid B, Ganoderic acid D, Ganoderic acid F, and Ganoderic acid H. According to the normalized method, Ganoderma triterpenoids accounted for 52.39% of the total extract.

[0047] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core ideas of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for directional extraction of triterpenes from Ganoderma lucidum, characterized in that: The method comprises the following steps: crushing and sieving ganoderma lucidum, adding 1,2-propylene glycol, betaine and water, and then ultrasonicating, filtering after the ultrasonication, and collecting the filtrate; and concentrating under reduced pressure and drying under vacuum to obtain ganoderma lucidum triterpene dry extract powder.

2. The method for directional extraction of triterpenes from Ganoderma lucidum according to claim 1, characterized in that: The molar ratio of 1,2-propylene glycol to betaine is 1:1-1:

3.

3. The method for directional extraction of triterpenes from Ganoderma lucidum according to claim 1, characterized in that: The water content of the 1,2-propylene glycol / betaine solution is 20%~40%.

4. The method for directional extraction of triterpenes from Ganoderma lucidum according to claim 1, characterized in that: The solid-to-liquid ratio of Ganoderma lucidum to 1,2-propylene glycol / betaine solution is 1g:5mL~1g:10mL.

5. The method for directional extraction of triterpenes from Ganoderma lucidum according to claim 1, characterized in that: The temperature of the ultrasound is 35-65°C.

6. The method for directional extraction of triterpenes from Ganoderma lucidum according to claim 1, characterized in that: The power of the ultrasound is 500W.

7. The method for directional extraction of triterpenes from Ganoderma lucidum according to claim 1, characterized in that: The ultrasonic time is 15 to 30 min.

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