Preparation method of acanthopanax root, gastrodia elata and armillaria mellea powder

Abstract

The invention discloses a preparation method of radix acanthopanacis semticosi, gastrodia elata and armillaria mellea powder, relates to the technical field of medicines or health-care products, and aims to solve the problems that the content of secondary metabolites of the gastrodia elata and armillaria mellea powder prepared by the conventional method is lower, and the in-vitro antioxidant activity is also lower. The method comprises the following steps: 1, inoculating gastrodia elata armillaria mellea liquid seeds into a liquid culture medium containing an acanthopanax senticosus leaf extract, and culturing to obtain fermentation liquor; 2, carrying out suction filtration on the fermentation liquor, and taking mycelia; and 3, washing the mycelium, and drying to obtain the acanthopanax and gastrodia elata armillaria mellea powder. The gastrodia elata armillaria mellea powder prepared by the preparation method disclosed by the invention has relatively high contents of uridine, guanosine, adenosine and syringin, and also has relatively high in-vitro antioxidant activity. The method can be used in the fields of food dietary additives, health-care products, medicines and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicines or health products, in particular to a preparation method of Acanthopanax senticosus Armillaria mellea powder. BACKGROUND

[0002] The Armillaria mellea powder is a dry mycelium powder prepared by liquid fermentation culture, separation and extraction of Armillaria mellea (vane. Ex. Fr.) Quel. of the white mushroom family, which is recorded in the Chemical Medicine National Standard Thirteen in 2002, is a nervous system drug, and is commonly used for treating neurasthenia, headache, dizziness, insomnia and the like. The preparation has Armillaria mellea tablets, compound Armillaria mellea tablets, and brain and heart soothing oral liquid. The preparation of Armillaria mellea combined with ginkgo leaves can effectively improve angina pectoris of coronary heart disease. In addition, the Armillaria mellea product preparation is both a medicine and a food, such as Armillaria mellea wine, Armillaria mellea syrup, and Armillaria mellea noodles.

[0003] The Acanthopanax senticosus leaf is the dry leaf of Acanthopanax senticosus (Rupr. et Maxim.) Harms of the Araliaceae family, which is a local medicinal material in Heilongjiang Province and a food raw material. It has the effects of activating blood and resolving stasis, and is used for treating chest pain, chest distress, palpitation and the like caused by blood stasis and obstruction, and is used for treating coronary heart disease and angina pectoris.

[0004] The secondary metabolite content of the Armillaria mellea powder prepared by the existing method is low, and the antioxidant capacity is also low, which limits the use of the Armillaria mellea powder. SUMMARY

[0005] The present application is to solve the problem of low secondary metabolite content and low antioxidant capacity of the Armillaria mellea powder prepared by the existing method, and provides a preparation method of Acanthopanax senticosus Armillaria mellea powder.

[0006] The preparation method of the Acanthopanax senticosus Armillaria mellea powder comprises the following steps:

[0007] I. The activated liquid seed of Armillaria mellea is inoculated into a liquid culture medium containing Acanthopanax senticosus leaf extract, and is subjected to fermentation culture to obtain a fermentation liquid;

[0008] II. The fermentation liquid is suction filtered, and the mycelium is taken;

[0009] III. The mycelium is washed with sterile water and dried to obtain the Acanthopanax senticosus Armillaria mellea powder.

[0010] Further, the Armillaria mellea (vane. Ex. Fr.) Quel. strain in step I is provided by the National Edible Fungus Germplasm Resource Bank.

[0011] Further, the activation culture method in step one is specifically as follows:

[0012] The activation culture method in step one is specifically as follows: the Armillaria mellea strain is transferred to fresh PDA plate culture medium, and is cultured at 24-28°C for 7-10 days until the mycelium grows full of the plate. The Armillaria mellea cake with uniform growth on the PDA plate is taken by a puncher, and is transferred to a liquid seed culture medium. After being oscillation cultured at 24-28°C and 140-150 r / min for 7-10 days, the Armillaria mellea seed liquid is obtained.

[0013] The PDA plate culture medium is prepared as follows: fresh potatoes (200 g) are washed, peeled, cut into pieces, and then poured into boiling water (1 L) to boil (30 min). After filtration, glucose (20 g) and agar (20 g) are added to the filtrate, and water is added to make up to 1 L. The pH is natural, and the medium is divided and sterilized at 121°C for 30 min.

[0014] Further, the liquid culture medium formula containing the Acanthopanax leaf extract is as follows: Acanthopanax leaf extract 2.5 mg / mL-15 mg / mL, glucose 20 g / L, protein peptone 10 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.75 g / L, and vitamin B1 5 mg / L.

[0015] Further, the preparation method of the Acanthopanax leaf extract is as follows:

[0016] The Acanthopanax leaf is dried in the shade, crushed, and sieved to obtain Acanthopanax leaf powder. Water is added to the Acanthopanax leaf powder at a solid-liquid ratio of 1:20, and ultrasonic extraction is performed twice, each for 1 h. After centrifugation, the supernatants are combined, and low-temperature concentration is performed to 50 mg / mL.

[0017] Further, the inoculation amount in step one is 5%-8% (v / v).

[0018] Further, the fermentation culture condition in step one is as follows: 24-28°C, 140-150 r / min, and constant-temperature culture for 7-10 days.

[0019] Further, the washing method in step three is as follows: the mycelium is washed with sterile water, and centrifugation is performed to obtain a precipitate. The washing-centrifugation is repeated for 3 times.

[0020] Preferably, the centrifugation condition is 3000 r / min, 30-35 min.

[0021] Further, the drying method in step three is as follows: drying in a 60-75°C oven.

[0022] The beneficial effects of the present application are as follows:

[0023] The application adds the acanthopanax leaf extract to the fermentation liquor of Gastrodia elata Armillaria, which can not only improve the yield of mycelium and active ingredients, but also improve the mycelium components and enhance the activity of the mycelium.

[0024] The prepared Gastrodia elata Armillaria powder has high contents of uridine, guanosine, adenosine and syringin, and also has high in-vitro antioxidant activity.

[0025] The prepared Armillaria mellea mycelium powder has wide application prospects in food dietary additives, health products, medicines and the like. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Effects of different amounts of acanthopanax leaf extract on the biomass of Armillaria mellea mycelium;

[0027] Figure 2 HPLC analysis results of three kinds of nucleosides in Armillaria mellea mycelium;

[0028] Figure 3 Effects of acanthopanax leaf extract on the yield of three kinds of nucleosides in Armillaria mellea mycelium;

[0029] Figure 4 HPLC analysis results of syringin in Armillaria mellea mycelium;

[0030] Figure 5 Syringin content in Armillaria mellea mycelium;

[0031] Figure 6 DPPH radical scavenging capacity of Armillaria mellea mycelium;

[0032] Figure 7 ·OH radical scavenging capacity of Armillaria mellea mycelium. DETAILED DESCRIPTION

[0033] The technical solution of the application is not limited to the following specific embodiments, and also includes any combination of the specific embodiments.

[0034] Specific embodiment one: the preparation method of the acanthopanax Gastrodia elata Armillaria powder in the embodiment, which comprises the following steps:

[0035] I. inoculating the activated Gastrodia elata Armillaria liquid seed after cultivation into a liquid culture medium containing acanthopanax leaf extract, performing fermentation culture, and obtaining fermentation liquor;

[0036] II. filtering the fermentation liquor and taking mycelium;

[0037] III. washing the mycelium with sterile water, drying, and obtaining the acanthopanax Gastrodia elata Armillaria powder.

[0038] The Armillaria mellea (vane. Ex. Fr.) Quel. strain mentioned in step one was provided by the National Edible Fungus Germplasm Resource Bank.

[0039] The present invention adds Acanthopanax senticosus leaf extract to the fermentation broth of Gastrodia elata and Armillaria mellea, which can not only increase the yield of mycelium and its secondary metabolites, but also improve the composition of mycelium and enhance its activity.

[0040] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the activation and cultivation method described in step one is as follows: Take *Armillaria mellea* spores from *Gastrodia elata* and transfer them to fresh PDA agar plates. Cultivate at 24-28℃ for 7-10 days until the mycelium completely covers the plate. Use a perforator to collect uniformly grown *Armillaria mellea* mycelial cakes from the PDA plates and transfer them to liquid seed culture medium. Cultivate at 24-28℃ and 140-150 r / min with shaking for 7-10 days to obtain *Armillaria mellea* seed culture. Other steps and parameters are the same as in Specific Implementation Method One.

[0041] The PDA plate culture medium formula of this embodiment is as follows: Wash and peel fresh potatoes (200 g), cut them into pieces, pour them into boiling water (1 L), boil (30 min), filter, add glucose (20 g) and agar (20 g) to the filtrate, add water to make up to 1 L, set the pH to natural, dispense, and autoclave at 121 ℃ for 30 min.

[0042] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One in that the liquid culture medium containing Acanthopanax senticosus leaf extract described in step one is formulated as follows: Acanthopanax senticosus leaf extract 2.5 mg / mL~15 mg / mL, glucose 20 g / L, peptone 10 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.75 g / L, and vitamin B1 5 mg / L. Other steps and parameters are the same as in Specific Implementation Method One or Two.

[0043] Specific Implementation Method Four: This implementation method differs from Specific Implementation Method One in that the preparation method of the Acanthopanax senticosus leaf extract is as follows:

[0044] After the leaves of Acanthopanax senticosus are dried in the shade, they are pulverized and sieved to obtain Acanthopanax senticosus leaf powder. Water is added to the Acanthopanax senticosus leaf powder at a material-to-liquid ratio of 1:20, and the mixture is extracted twice by ultrasonication, 1 hour each time. After centrifugation, the supernatants are combined and concentrated at low temperature to 50 mg / mL. Other steps and parameters are the same as in one of the specific implementation methods one to three.

[0045] Acanthopanax senticosus (Rupr. et Maxim.) Harms, a plant in the Araliaceae family, is a dried leaf with the effects of tonifying the liver and kidneys, and strengthening bones and essence. It is suitable for transient ischemic attacks, cerebral arteriosclerosis, and cerebral embolism, and can also be used for coronary heart disease, angina pectoris, neurasthenia, and menopausal syndrome. Acanthopanax senticosus leaves are rich in saponins, flavonoids, coumarins, lignans, organic acids, phenolic acids, amino acids, and other components. Based on research showing that adding certain herbal extracts to the fermentation broth of edible and medicinal fungi can significantly increase the content of secondary metabolites and may even promote the generation of entirely new secondary metabolites, adding different doses of Acanthopanax senticosus leaf extract to the fermentation broth of Armillaria mellea not only increases the yield of mycelium and its secondary metabolites but also improves the composition of the mycelium and enhances its activity.

[0046] Specific Implementation Method Five: This implementation method differs from Specific Implementation Method One in that the inoculation amount in step one is 5%~8% (v / v). Other steps and parameters are the same as in any of Specific Implementation Methods One to Four.

[0047] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method One in that the fermentation culture conditions described in step one are: 24~28℃, 140~150 r / min, and constant temperature culture for 7~10 days. Other steps and parameters are the same as in any of Specific Implementation Methods One to Five.

[0048] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method One in that the washing method in step three is as follows: wash the mycelium with sterile water, centrifuge to collect the precipitate, and repeat the washing-centrifugation process three times. Other steps and parameters are the same as in any of Specific Implementation Methods One through Six.

[0049] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Method One in that the centrifugation conditions are 3000 r / min for 30-35 min. Other steps and parameters are the same as in any of Specific Implementation Methods One through Seven.

[0050] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Method One in that the drying method in step three is: drying in an oven at 60~75℃. Other steps and parameters are the same as in Specific Implementation Methods One through Eight.

[0051] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0052] Example 1:

[0053] The preparation method of Acanthopanax senticosus, Gastrodia elata, and Armillaria mellea powder in this embodiment includes the following steps:

[0054] In this embodiment, the Armillaria mellea (vane. ex. Fr.) Quel. strain was provided by the National Edible Fungi Germplasm Bank. Adenosine, uridine, guanosine, and syringin reference standards were purchased from the National Institutes for Food and Drug Control; all reagents used for HPLC analysis were of chromatographic grade, and all other reagents were of analytical grade.

[0055] I. Preparation of Acanthopanax senticosus leaf extract

[0056] Acanthopanax senticosus leaves were harvested from Mishan City, Heilongjiang Province. After being air-dried, they were pulverized and passed through a 20-mesh sieve to obtain Acanthopanax senticosus leaf powder. The Acanthopanax senticosus leaf powder was then mixed with water at a material-to-liquid ratio of 1:20 and extracted twice by ultrasonication (1 h / time). After centrifugation, the supernatants were combined and concentrated at low temperature to obtain Acanthopanax senticosus leaf extract at a concentration of 50 mg / mL.

[0057] 2. Add 2.5 mg / mL of Acanthopanax senticosus leaf extract to the basic culture medium, autoclave at 121℃ for 30 min to obtain a liquid culture medium containing Acanthopanax senticosus leaf extract.

[0058] The basic culture medium formula is as follows: glucose 20 g / L, peptone 10 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.75 g / L, and vitamin B1 5 mg / L.

[0059] 3. The activated and cultured Gastrodia elata liquid seed was inoculated into a liquid culture medium containing Acanthopanax senticosus leaf extract (60 mL in a 100 mL shake flask), with an inoculation amount of 8% (v / v), and fermented at 140 r / min on a shaker for 8 days at a constant temperature of 28℃ to obtain the fermentation broth.

[0060] 4. Filter the fermentation broth and collect the mycelium.

[0061] 5. Wash the mycelium three times with sterile water, centrifuge at 3000 r / min for 30 min to collect the precipitate, and dry the mycelium at low temperature to obtain Acanthopanax senticosus and Armillaria mellea powder.

[0062] Example 2:

[0063] The difference between this embodiment and Embodiment 1 is that: in step two, 5.0 mg / mL of Acanthopanax senticosus leaf extract is added to the basal culture medium, while the other steps and parameters are the same as in Embodiment 1.

[0064] Example 3:

[0065] The difference between this embodiment and Example 1 is that: in step two, 7.5 mg / mL of Acanthopanax senticosus leaf extract is added to the basal culture medium, while the other steps and parameters are the same as in Example 1.

[0066] Example 4:

[0067] The difference between this embodiment and Embodiment 1 is that: in step two, 10.0 mg / mL of Acanthopanax senticosus leaf extract is added to the basal culture medium, while the other steps and parameters are the same as in Embodiment 1.

[0068] Example 5:

[0069] The difference between this embodiment and Embodiment 1 is that: in step two, 12.5 mg / mL of Acanthopanax senticosus leaf extract is added to the basal culture medium, while the other steps and parameters are the same as in Embodiment 1.

[0070] Example 6:

[0071] The difference between this embodiment and Example 1 is that: in step two, 15.0 mg / mL of Acanthopanax senticosus leaf extract is added to the basal culture medium, while the other steps and parameters are the same as in Example 1.

[0072] Comparative Example 1:

[0073] 1. The activated and cultured Armillaria mellea liquid seed was inoculated into the basic culture medium (60 mL in a 100 mL shake flask) at an inoculation volume of 8% (v / v) and fermented. The shaker speed was 140 r / min and the culture was carried out at a constant temperature of 28℃ for 8 days to obtain the fermentation broth.

[0074] The basic culture medium formula is as follows: glucose 20 g / L, peptone 10 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.75 g / L, and vitamin B1 5 mg / L.

[0075] 2. Filter the fermentation broth and collect the mycelium.

[0076] 3. Wash the mycelium three times with sterile water, centrifuge at 3000 r / min for 30 min to collect the precipitate, and dry the mycelium at low temperature to obtain Gastrodia elata honey fungus powder.

[0077] The following experiments were conducted on the *Gastrodia elata* and *Armillaria mellea* powder prepared in the above examples:

[0078] (I) Effects of different concentrations of Acanthopanax senticosus leaf extract on Armillaria mellea mycelial biomass

[0079] The dried mycelium was weighed, and the effect of different doses of Acanthopanax senticosus leaf extract on the biomass of Armillaria mellea mycelium was analyzed. The results are shown in […]. Figure 1 .

[0080] Depend on Figure 1It was found that different doses of Acanthopanax senticosus leaf extract had different effects on the mycelial biomass of Armillaria mellea, all of which increased the mycelial dry weight. With the increase of Acanthopanax senticosus leaf extract in the basal medium, the mycelial biomass of Armillaria mellea showed a trend of first increasing and then decreasing. When the amount of Acanthopanax senticosus leaf extract in the basal medium was 10 mg / mL, the mycelial dry weight of Armillaria mellea reached its maximum value of 2.639 ± 0.164 g / L. Afterward, with the increase of Acanthopanax senticosus leaf extract, the mycelial dry weight of Armillaria mellea showed a decreasing trend.

[0081] (II) Effects of Acanthopanax senticosus leaf extract on the yield of three nucleoside components in Armillaria mellea mycelium

[0082] 1. Preparation of the test solution

[0083] Accurately weigh 0.5 g of mycelium powder, add 50% methanol (solid-to-liquid ratio 1:10), sonicate for 1 h, and filter through a 0.22 μm filter membrane to obtain the mycelium powder test solution.

[0084] 2. Preparation of reference solution

[0085] Accurately weigh appropriate amounts of adenosine, uridine, and guanosine reference standards, dissolve them in 50% methanol, and dilute to volume to prepare a mixed reference solution containing 0.2 mg, 0.2 mg, and 0.5 mg per 1 mL, respectively.

[0086] 3. HPLC analysis of three nucleoside components in Armillaria mellea mycelium: Mobile phase: methanol-0.2% phosphoric acid water (15:85); Detection wavelength: 260 nm; Injection volume: 20 μL; Flow rate: 1.0 mL·min -1 Column temperature: 25 ℃.

[0087] HPLC analysis results of three nucleoside components in Armillaria mellea mycelium are as follows: Figure 2 As shown, Figure 2 1: Urate, 2: Guanosine, 3: Adenosine; A: Mixed control of urate, guanosine, and adenosine; B: Basal culture medium; C: Acanthopanax senticosus leaf extract; D: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf at a concentration of 2.5 mg / mL; E: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf at a concentration of 5 mg / mL; F: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf at a concentration of 7.5 mg / mL; G: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf at a concentration of 10 mg / mL; H: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf at a concentration of 12.5 mg / mL; I: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf at a concentration of 15 mg / mL; J: Armillaria mellea powder sample cultured in basal culture medium.

[0088] Depend on Figure 2It can be seen that the three nucleoside components were not detected in the basal medium (B) and the three nucleoside components were also not detected in the Acanthopanax senticosus leaf extract (C). However, the three nucleoside compounds were detected in the Armillaria mellea mycelium cultured in the medium with Acanthopanax senticosus leaf extract, and their peak heights were higher than those of the Armillaria mellea mycelium powder cultured in the basal medium (J) without Acanthopanax senticosus extract.

[0089] The effect of Acanthopanax senticosus extract on the yield of three nucleoside compounds in Armillaria mellea mycelium, as follows: Figure 3 As shown. By Figure 3 It can be seen that as the amount of Acanthopanax senticosus leaf extract added to the Armillaria mellea culture medium increases, the yield of the three nucleoside compounds in the Armillaria mellea mycelium shows a trend of first increasing and then decreasing. When the dosage of Acanthopanax senticosus extract added reaches 10 mg / mL, the yield of the three nucleoside compounds in the Armillaria mellea mycelium reaches the highest value, of which the content of guanosine is 0.046±0.009 mg / g, the content of adenosine is 0.069±0.005 mg / g, and the content of uridine is 0.100±0.006 mg / g.

[0090] (III) Determination of syringin content in Armillaria mellea mycelium

[0091] 1. Preparation of the test solution

[0092] Accurately weigh 0.5 g of mycelium powder, add methanol (solid-to-material ratio 1:10), sonicate for 1 h, and filter through a 0.22 μm filter membrane to obtain the mycelium test solution.

[0093] 2. Preparation of reference solution

[0094] Accurately weigh an appropriate amount of syringin reference standard, dissolve it in methanol, and dilute to a final volume to prepare a 0.5 mg / mL reference solution.

[0095] 3. HPLC analysis of syringin content in Armillaria mellea mycelium: Mobile phase: methanol-0.2% phosphoric acid water (35:65); Detection wavelength: 360 nm; Injection volume: 20 μL; Flow rate: 1.0 mL·min -1 Column temperature: 25 ℃.

[0096] Using HPLC, with syringin, an active ingredient in Acanthopanax senticosus leaves, as a control, we determined whether Armillaria mellea mycelium powder contained syringin at different dosages of Acanthopanax senticosus leaf extract.

[0097] HPLC analysis results of syringin in Armillaria mellea mycelium powder are as follows: Figure 4 As shown. Figure 4A: Syringin reference standard; B: Basal culture medium; C: Acanthopanax senticosus leaf extract; D: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf extract at a concentration of 2.5 mg / mL; E: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf extract at a concentration of 5 mg / mL; F: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf extract at a concentration of 7.5 mg / mL; G: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf extract at a concentration of 10 mg / mL; H: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf extract at a concentration of 12.5 mg / mL; I: Armillaria mellea powder sample cultured with Acanthopanax senticosus leaf extract at a concentration of 15 mg / mL; J: Armillaria mellea mycelium powder cultured in basal culture medium.

[0098] Depend on Figure 4 It was found that syringin was not detected in the basal culture medium (blank control group), and similarly, it was not detected in the *Armillaria mellea* mycelium powder cultured in the basal culture medium. However, syringin, the active ingredient from *Eleutherococcus senticosus* leaves, was detected in *Armillaria mellea* mycelium cultured in a medium supplemented with *Eleutherococcus senticosus* leaf extract. These results indicate that *Armillaria mellea* mycelium can accumulate syringin.

[0099] The effect of Acanthopanax senticosus leaf extract on the syringin content in Armillaria mellea mycelium is as follows: Figure 5 As shown. By Figure 5 It can be seen that as the amount of Acanthopanax senticosus leaf extract added to the Armillaria mellea culture medium increases, the syringin content in the Armillaria mellea mycelium powder shows a trend of first increasing and then decreasing. When the dosage of Acanthopanax senticosus leaf extract reaches 10 mg / mL, the syringin content in the Armillaria mellea mycelium powder reaches the highest value of 0.014±0.001 mg / g.

[0100] (iv) In vitro antioxidant activity assay

[0101] 1. Determination of DPPH free radical scavenging ability

[0102] Dissolve an appropriate amount of DPPH in methanol and bring the volume to 100 mL to prepare a solution with a mass concentration of 0.15 mmol·L⁻¹. -1 Prepare a DPPH solution; dissolve an appropriate amount of vitamin C in methanol and dilute to 10 mL to prepare a 200 μg·mL⁻¹ solution. -1 The reference solution;

[0103] Armillaria mellea mycelium powder was dissolved in an appropriate amount of methanol to prepare a solution with a concentration of 200 μg·mL. -1 The test solution.

[0104] The above reference standard and test solution were diluted sequentially to a concentration gradient of 100 μg·mL. -1 50 μg·mL -1 25 μg·mL -112.5 μg·mL -1 6.25 μg·mL -1 Accurately pipette 2 mL of each solution into a test tube, using methanol as a blank control group. Add 0.15 mmol·L⁻¹ to each test tube. -1 4 mL of DPPH solution was added and placed in the dark for 30 min. The absorbance of each sample was measured at 517 nm (n=3). The formula for calculating the DPPH free radical scavenging rate is as follows:

[0105]

[0106] In the formula A i Indicates the absorbance value when the test sample and DPPH solution are added simultaneously; A j A0 indicates the absorbance when the test sample and blank control are added simultaneously; A0 indicates the absorbance when the blank control solution (methanol) and DPPH solution are added simultaneously.

[0107] The scavenging ability of Armillaria mellea mycelium against DPPH free radicals was observed under different amounts of Acanthopanax senticosus leaf extract. Figure 6 .from Figure 6 It can be seen that the DPPH scavenging ability of the mycelium powder is positively correlated with the concentration when the concentration is between 6.25 μg / mL and 100 μg / mL. With the increase of the amount of Acanthopanax senticosus leaf extract, the DPPH free radical scavenging rate of Armillaria mellea mycelium powder increases. The DPPH scavenging rate of Armillaria mellea mycelium powder is highest when the amount of Acanthopanax senticosus leaf extract is 15 mg / mL. These results indicate that Acanthopanax senticosus leaf extract can enhance the DPPH free radical scavenging ability of Armillaria mellea mycelium powder.

[0108] 2. Determination of ·OH free radical scavenging ability

[0109] Accurately pipette 2 mL of each of the above-mentioned test solutions of different concentrations into a test tube, and add 2 mL of 6 mmol·L⁻¹ solution to each tube. -1 FeSO4, 2 mL 6 mmol·L -1 Add H2O2, shake to mix well, let stand for 10 min, then add 2 mL of 6 mmol·L⁻¹ H₂O₂. -1 The salicylic acid-ethanol solution was allowed to stand at 37℃ for 30 min, and the absorbance of each sample was measured at 510 nm (n=3). The formula for calculating the ·OH free radical scavenging rate is as follows:

[0110]

[0111] In the formula, A1 represents the absorbance of the added test sample; A2 represents the absorbance of distilled water instead of H2O2 to eliminate the color effect of the reagent itself in the solution; and A0 represents the absorbance of methanol instead of the test sample.

[0112] The scavenging ability of Armillaria mellea mycelium powder against ·OH free radicals was observed under different amounts of Acanthopanax senticosus leaf extract. Figure 7 .from Figure 7 It can be seen that the scavenging ability of Armillaria mellea mycelium powder against ·OH free radicals is positively correlated with the concentration when the concentration is between 6.25 μg / mL and 100 μg / mL. With the increase of the amount of Acanthopanax senticosus leaf extract, the scavenging rate of Armillaria mellea mycelium powder against ·OH free radicals increases. The scavenging rate of Armillaria mellea mycelium powder against ·OH free radicals is highest when the dosage of Acanthopanax senticosus leaf extract is 15 mg / mL. These results indicate that Acanthopanax senticosus leaf extract can enhance the ability of Armillaria mellea mycelium powder to scavenge ·OH free radicals.

Claims

1. A method for preparing Acanthopanax senticosus, Gastrodia elata, and Armillaria mellea powder, characterized in that, The method includes the following steps:

1. The activated and cultured Armillaria mellea liquid seed was inoculated into a liquid culture medium containing Acanthopanax senticosus leaf extract and fermented to obtain fermentation broth; 2. Filter the fermentation broth and collect the mycelium; 3. Wash the mycelium with sterile water and dry it to obtain the mycelial powder of Acanthopanax senticosus and Armillaria mellea.

2. The method for preparing Acanthopanax senticosus, Gastrodia elata, and Armillaria mellea powder according to claim 1, characterized in that, The activation culture method described in step one is as follows: Take the Armillaria mellea inoculum from Gastrodia elata and transfer it to a fresh PDA plate culture medium. Culture it at 24-28℃ for 7-10 days until the mycelium fills the entire plate. Use a punch to take the uniformly growing Armillaria mellea mycelial cake from the PDA plate and transfer it to a liquid seed culture medium. Culture it at 24-28℃ and 140-150 r / min for 7-10 days with shaking to obtain the Armillaria mellea seed liquid.

3. The method for preparing Acanthopanax senticosus, Gastrodia elata, and Armillaria mellea powder according to claim 1, characterized in that, The liquid culture medium containing Acanthopanax senticosus leaf extract is formulated as follows: Acanthopanax senticosus leaf extract 2.5 mg / mL~15 mg / mL, glucose 20 g / L, peptone 10 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.75 g / L, and vitamin B1 5 mg / L.

4. A method for preparing Acanthopanax senticosus, Gastrodia elata, and Armillaria mellea powder according to claim 1 or 3, characterized in that, The preparation method of the Acanthopanax senticosus leaf extract is as follows: After the leaves of Acanthopanax senticosus are dried in the shade, they are crushed and sieved to obtain Acanthopanax senticosus leaf powder. Water is added to the Acanthopanax senticosus leaf powder at a material-to-liquid ratio of 1:20, and ultrasonic extraction is performed twice, each time for 1 hour. After centrifugation, the supernatants are combined and concentrated at low temperature to 50 mg / mL.

5. The method for preparing Acanthopanax senticosus, Gastrodia elata, and Armillaria mellea powder according to claim 1, characterized in that, The inoculation amount in step one is 5%~8% (v / v).

6. The method for preparing Acanthopanax senticosus, Gastrodia elata, and Armillaria mellea powder according to claim 1, characterized in that, The fermentation conditions described in step one are: 24~28℃, 140~150 r / min, and constant temperature culture for 7~10 days.

7. The method for preparing Acanthopanax senticosus, Gastrodia elata, and Armillaria mellea powder according to claim 1, characterized in that, The washing method in step three is as follows: wash the mycelium with sterile water, centrifuge to collect the precipitate, and repeat the washing-centrifugation process three times.

8. The method for preparing Acanthopanax senticosus, Gastrodia elata, and Armillaria mellea powder according to claim 7, characterized in that, The centrifugation conditions were 3000 r / min for 30-35 min.

9. The method for preparing Acanthopanax senticosus, Gastrodia elata, and Armillaria mellea powder according to claim 1, characterized in that, The drying method in step three is to dry the food in an oven at 60~75℃.

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