Method for preparing hydroxytyrosol-containing extract from antrodia camphorata and hydroxytyrosol

Abstract

The invention discloses a method for preparing hydroxytyrosol-containing extract from antrodia camphorata and hydroxytyrosol, and belongs to the technical field of research and development of natural medicines. Antrodia camphorata is used as a strain, and hydroxytyrosol is obtained through microbial fermentation, product extraction, separation and purification. The extract containing hydroxytyrosol and hydroxytyrosol prepared by the invention can be used for developing antrodia camphorata health-care food or medicine with the effects of resisting oxidation, removing wrinkles, resisting aging, resisting cancer, resisting thrombus, regulating blood fat, resisting arteriosclerosis, resisting pathogenic microorganisms and the like. The antrodia camphorata culture medium is prepared by antrodia camphorata microbial fermentation, is wide in raw material source, simple in preparation method and easy to realize industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicine research and development, and particularly relates to a method for preparing an extract containing hydroxytyrosol by utilizing Antrodia cinnamomea and a method for preparing hydroxytyrosol. Background Art

[0002] Hydroxytyrosol (HT) is a natural phenolic compound with strong antioxidant activity. In recent years, some progress has been made in the synthesis of HT in the fields of biotechnology and chemical engineering. (1) Traditional extraction methods mainly extract oleanolic acid from olive oil processing wastewater, olive leaves or fruits, and then obtain HT through acid / base hydrolysis or enzymatic hydrolysis. Ultrasonic-assisted hydrolysis is often used. For example, at an extraction temperature of 50°C, a hydrochloric acid concentration of 1.2 mol / L and a material-liquid ratio of 1:20, the extraction yield can be increased to 3.2 mg / g. Cellulase and pectinase are also used to synergistically hydrolyze olive leaves, with an extraction rate of over 85%. Traditional methods rely on olive resources, have low recovery rates (<5%), are costly, and are easily affected by seasonality and raw material quality. (2) Chemical synthesis: Hydroxytyrosol is synthesized using 4-hydroxyphenylethanol or tyrosine as substrates through multi-step oxidation and substitution reactions. For example, sodium bromide / potassium peroxymonosulfate is used to oxidize 4-hydroxyphenylethanol, combined with cuprous iodide to catalyze debromination, and the final product purity reaches 99%. Photocatalytic synthesis is also used, using nano-TiO2 to catalyze tyrosine conversion, but the conversion rate is only 30%, the product concentration is low, and it has not yet been industrialized. Chemical synthesis is suitable for industrial production and the purity is controllable, but the steps are cumbersome and toxic reagents (such as concentrated hydrochloric acid and organic solvents) are used, which causes serious environmental pollution. (3) Biotechnology synthesis method: The Xinyang Normal University team used microbial metabolic engineering to construct an engineered Escherichia coli containing a multi-enzyme cascade pathway of 5 exogenous enzymes (tyrosine phenol lyase, aldehyde reductase, etc.). Through plasmid optimization and batch feeding strategy, the yield reached 55.3 mM (about 8.5 g / L), which is 89% higher than the traditional method. Xizhenglin Biological Company designed a metabolic pathway with glycerol / glucose as the carbon source, deleted the by-product pathway and optimized the fermentation conditions, and achieved efficient synthesis of hydroxytyrosol without substrate addition, reducing the cost by 40%. Some are also developing novel hydroxylases based on the mechanism of the oxidase (T9αH) in paclitaxel biosynthesis to increase hydroxytyrosol yield. Alternatively, they are using sodium alginate-polysaccharide gel beads to immobilize the hydroxylase, improving enzyme stability and reusability. Biotechnology-based synthesis methods are environmentally friendly, require mild conditions, and are highly sustainable, aligning with the trend of green chemistry.

[0003] Antrodia cinnamomea ( Antrodiacamphorata ) is also known as Antrodia camphorata and Antrodia camphorata. It is an edible fungus unique to Taiwan, China, and only grows on the Antrodia camphorata tree unique to Taiwan, China. Summary of the Invention

[0004] The present invention aims to provide a method for preparing an extract containing hydroxytyrosol and hydroxytyrosol using Antrodia cinnamomea. The hydroxytyrosol extract and hydroxytyrosol prepared by the present invention adopt microbial fermentation technology, the culture medium raw materials are widely available and inexpensive, the preparation method is simple, and industrial production is easily achieved.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A method for preparing an extract containing hydroxytyrosol and hydroxytyrosol by using Antrodia cinnamomea, wherein Antrodia cinnamomea is used as a strain, an extract containing hydroxytyrosol is obtained by microbial fermentation and product extraction, and hydroxytyrosol is further separated and purified to obtain hydroxytyrosol; the molecular formula of the hydroxytyrosol is C8H 10 O3, chemical structure is as follows: .

[0006] The method comprises the following steps: (1) Microbial fermentation: Activate the Antrodia cinnamomea strain and then perform solid-state or liquid fermentation; (2) Product extraction: the fermentation product obtained in step (1) is leached or extracted with a solvent, and the solvent is removed to obtain an extract containing hydroxytyrosol; (3) Isolation and purification: The extract containing hydroxytyrosol obtained in step (2) is dissolved in methanol or ethanol, and then purified by chromatography to obtain hydroxytyrosol.

[0007] Preferably, the solvent in step (2) is one or more of ethanol, methanol, ethyl acetate, n-butanol, and chloroform.

[0008] Preferably, the chromatographic technique in step (3) comprises a combination of reverse phase silica gel column chromatography, gel column chromatography and normal phase silica gel column chromatography.

[0009] Preferably, there is no fixed order in which reverse phase silica gel column chromatography, gel column chromatography and normal phase silica gel column chromatography are used.

[0010] Preferably, the chromatographic chromatography technique in step (3) is reverse phase silica gel column chromatography, gel column chromatography, reverse phase silica gel column chromatography, and normal phase silica gel column chromatography in sequence.

[0011] The beneficial effects of the present invention are as follows: the hydroxytyrosol prepared by the present invention has the effects of anti-oxidation, anti-wrinkle, anti-aging, anti-cancer, anti-thrombotic, lipid-regulating, anti-arteriosclerotic and anti-pathogenic microorganisms, has a large market prospect, and is prepared by liquid fermentation of Antrodia cinnamomea, the culture medium raw materials are widely available, the preparation method is simple, and industrial production is easy to achieve. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound hydroxytyrosol.

[0013] Figure 2 is the carbon nuclear magnetic resonance spectrum of the compound hydroxytyrosol.

[0014] Figure 3 This is a high-resolution mass spectrum of the compound hydroxytyrosol.

[0015] Figure 4 is the chemical structure of the compound hydroxytyrosol.

[0016] Figure 5 This is an experiment on the antioxidant activity of the compound hydroxytyrosol (Note: a) is vitamin C; b) is the compound hydroxytyrosol). DETAILED DESCRIPTION

[0017] A method for preparing an extract containing hydroxytyrosol and hydroxytyrosol using Antrodia cinnamomea: Antrodia cinnamomea is used as a strain, and an extract containing hydroxytyrosol is obtained through microbial fermentation and product extraction, and hydroxytyrosol is obtained after separation and purification. The chemical structure characteristics of the hydroxytyrosol are as follows: The method for preparing an extract containing hydroxytyrosol and hydroxytyrosol by using Antrodia cinnamomea comprises the following steps: Step 1: Add Antrodia cinnamomea ( Antrodiacamphorata ) After the strain is activated, solid-state fermentation or liquid fermentation is carried out under appropriate culture conditions using a culture medium suitable for fungal growth; Step 2: The solid fermentation product obtained by culturing in step 1 is extracted with an organic solvent, and solids are removed by filtration, centrifugation or membrane filtration. The solvent is concentrated to obtain a crude extract; Step 3: The crude extract obtained in step 2 is dissolved in a solvent such as methanol or ethanol, and then subjected to purification methods such as reverse phase silica gel column chromatography, gel column chromatography, and normal phase silica gel column chromatography. The mixture is analyzed by high performance liquid chromatography or thin layer chromatography to obtain the target component and pure hydroxytyrosol. Step 4: The hydroxytyrosol obtained in step 3 is subjected to nuclear magnetic resonance spectroscopy and mass spectrometry detection.

[0018] Among them, the solid-state fermentation described in step 1 can be carried out using rice as the culture medium; the liquid fermentation can use a culture medium suitable for fungal growth, including a culture medium formula composed of: 5-25% potato, 1-5% glucose, and the balance water; the suitable culture conditions are that the culture medium has a natural pH, is sterilized at 0.1 MPa and 100-140°C for 15-40 minutes, is shaken at a temperature of 25-33°C, has a rotation speed of 150-250 rpm, and is cultured for 5-30 days.

[0019] Wherein, the organic solvent described in step 2 is one or more of ethanol or methanol, ethyl acetate, n-butanol, and chloroform with different concentrations.

[0020] In the reversed-phase silica gel column chromatography described in step 3, the chromatographic medium is reversed-phase silica gel (RP-C18), and the chromatography parameters are as follows: using methanol water, ethanol water or acetone water of different concentrations as the eluent, the flow rate is 5-50 mL / min, collecting 5-200 mL in each tube, sampling each test tube for thin layer chromatography: the developing solvent is chloroform: methanol = 10:1, the color developer is 10% ethanolic sulfuric acid, and similar components are combined.

[0021] Wherein, the gel column chromatography described in step 3, the chromatography medium is gel Sephadex LH-20, the chromatography parameters are: methanol, ethanol or acetone as the eluent, the flow rate is 5-30s / drop, 3-20mL is collected in each tube, and each test tube is sampled for thin layer chromatography: the developing solvent is chloroform: methanol = 10:1, the color developer is 10% ethanolic sulfuric acid, and similar components are combined.

[0022] Wherein, the normal phase silica gel column chromatography described in step 3, the chromatography medium is silica gel, and the chromatography parameters are: using chloroform, dichloromethane, ethyl acetate, petroleum ether, acetone, and methanol in different volume ratios as eluents, wherein the volume ratio of dichloromethane or petroleum ether to ethyl acetate or acetone is 500:1~50, and the volume ratio of chloroform to methanol is 300:1~30.

[0023] Among them, the purification methods such as reverse phase silica gel column chromatography, gel column chromatography and normal phase silica gel column chromatography described in step 3 can be adjusted in the order of using these methods; Wherein, the nuclear magnetic resonance spectrum detection described in step 4 is mainly to detect the hydrogen spectrum ( 1 H-NMR) and carbon spectroscopy ( 13 C-NMR), mass spectrometry can be high-resolution mass spectrometry or low-resolution mass spectrometry.

[0024] According to the antioxidant activity test results of the compounds, it was found that hydroxytyrosol has strong antioxidant activity (Example 3).

[0025] The present invention is further illustrated by the following examples, but the protection scope of the present invention is not limited to the following examples.

[0026] Example 1 The preparation method of hydroxytyrosol provided by the present invention comprises the following steps: 80 g of rice and 120 mL of water were added to a 500 mL Erlenmeyer flask and sterilized at 121°C for 20 minutes. After cooling, the mixture was inoculated with Antrodia cinnamomea for solid-state fermentation and incubated at 28°C for 41 days. After fermentation, the mixture was extracted with 1 L of extraction solvent (ethyl acetate:methanol:acetic acid = 85:10:5, volume ratio), sonicated for 30 minutes, and allowed to stand at room temperature for 12 hours. After filtration and solvent removal, 2.6 g of crude extract was obtained. The crude extract was initially separated on an RP-18 reverse-phase silica gel column (packing weight: 200 g) using a methanol-water mobile phase and eluted using a 1 L gradient from pure water to 100% methanol (0%, 30%, 50%, 70%, 100%). Fraction A (68.2 mg) containing the target compound was obtained from 30% methanol-water. Fraction A (68.2 mg) was then separated by Sephadex LH-20 gel chromatography using methanol as the eluent at a rate of 15-20 seconds per drop, with one tube collected every 60 minutes. Fractions were analyzed by TLC, and similar fractions were combined based on color development and mobility to obtain Fraction A1 (28.4 mg) containing the target compound. Fraction A1 was further purified by RP-C18 reverse-phase silica gel chromatography (packing weight: 40 g). The sample was dissolved in 1 mL of methanol and loaded at a flow rate of 10 mL / min and a column pressure below 1.6 MPa using a methanol-water system (pure water, 40% methanol, 50% methanol, and methanol) as the mobile phase. Similar fractions were combined by TLC analysis, and subfraction A11 (13.4 mg) containing the target compound was obtained by eluting with 30% methanol in water. Subfraction A11 was purified by normal phase silica gel column chromatography (packing amount: 0.536 g) and eluted with dichloromethane-methanol in a volume ratio of 100:1 to obtain pure hydroxytyrosol (4.9 mg).

[0027] Example 2 The pure compound was subjected to nuclear magnetic resonance spectroscopy ( 1 H-NMR and 13 C-NMR) determination, the structure of the compound can be determined according to the nuclear magnetic spectrum data (Table 1). 1 H NMR (600 MHz, Methanol- d 4): δ 6.65 (d, J =2.0 Hz, H-2), 6.67 (d, J = 8.0 Hz, H-5), 6.53(dd, J = 8.0, 2.1 Hz, H-6), 2.66 (t, J =7.2 Hz, H2-7), 3.67 (t, J = 7.3 Hz, H2-8) ( Figure 1 ).13 C NMR (151 MHz, Methanol- d 4): δ 131.8 (C-1), 117.1 (C-2), 146.2 (C-3), 144.6 (C-4), 116.3 (C-5), 121.2 (C-6), 39.7 (C-7), 64.6 (C-8)( Figure 2 The compound was further subjected to high-resolution mass spectrometry detection, and the molecular formula of the compound was determined by HRQ-TOFMS to be C8H 10 O3, mass spectrometry signals of compounds can be detected m / z [M + H] + 155.0705 (theoretical value is C8H 11 O3, 155.0708) and m / z [M + Na] + 177.0541 (theoretical value is C8H 10 NaO3, 177.0528) Figure 3 ). Combined 1 H-NMR and 13 The chemical shift values of C-NMR and the literature identified the compound as hydroxytyrosol ( Figure 4 ).

[0028] Table 1 NMR spectra of hydroxytyrosol (H 600 MHz, C 151 MHz, deuterated methanol) Example 3 The antioxidant activity of the compound was determined using thin-layer chromatography-bioautography. The compound and vitamin C were prepared at a concentration of 1 mg / mL, and 10 μL was spotted onto a thin-layer chromatography plate. The plate was then sprayed with a 0.04% DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) ethanol solution and heated at 40°C for 30 minutes. Similar to the positive control vitamin C, the compound showed distinct white spots at the corresponding positions on the blue thin-layer plate ( Figure 5 ). The results showed that the compound had significant antioxidant activity.

[0029] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing an extract containing hydroxytyrosol and hydroxytyrosol using Antrodia cinnamomea, characterized in that: Antrodia cinnamomea is used as the strain, and an extract containing hydroxytyrosol is obtained through microbial fermentation and product extraction, and hydroxytyrosol is further separated and purified. The chemical structure of the hydroxytyrosol is as follows: 。 2. The method according to claim 1, wherein: The following steps are involved: (1) Microbial fermentation: Activate the Antrodia cinnamomea strain and then perform solid-state or liquid fermentation; (2) Product extraction: the fermentation product obtained in step (1) is leached or extracted with a solvent, and the solvent is removed to obtain an extract containing hydroxytyrosol; (3) Isolation and purification: The extract containing hydroxytyrosol obtained in step (2) is dissolved in methanol or ethanol, and then purified by chromatography to obtain hydroxytyrosol.

3. The method according to claim 2, wherein: The solvent in step (2) is one or more of ethanol, methanol, ethyl acetate, n-butanol, and chloroform.

4. The method according to claim 2, wherein: The chromatographic technique in step (3) includes a combination of reverse phase silica gel column chromatography, gel column chromatography and normal phase silica gel column chromatography.

5. The method according to claim 4, characterized in that: There is no fixed order for using reverse phase silica gel column chromatography, gel column chromatography and normal phase silica gel column chromatography.

6. The method according to claim 4, characterized in that: The chromatographic chromatography technique in step (3) is to sequentially perform reverse phase silica gel column chromatography, gel column chromatography, reverse phase silica gel column chromatography, and normal phase silica gel column chromatography.

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