Preparation method of phellinus linteus and swertia bimaculata mixed extract

Abstract

The invention belongs to the technical field of swertia bimaculata extracts, and particularly relates to a preparation method of a phellinus linteus and swertia bimaculata mixed extract, and the preparation method at least comprises the following steps: S1, preparing a phellinus linteus fermentation broth; s2, performing fermentation treatment on the false Chinese swertia herb by adopting the phellinus linteus fermentation liquor to obtain a phellinus linteus and false Chinese swertia herb fermentation product; s3, mixing the phellinus linteus and false Chinese swertia herb fermented product with distilled water, performing ultrasonic extraction for three times, combining filtrate, and concentrating to obtain the phellinus linteus and false Chinese swertia herb mixed extract. According to the method disclosed by the invention, phellinus linteus microbial fermentation is performed on the swertia, and enzyme substances generated in the fermentation process are utilized to destroy structures of cell walls, intercellular substances and the like of the raw materials, so that components such as swertiamarin and the like are fully dissolved out, the extraction rate of bioactive substances of the swertia in the product is increased, and the extraction energy consumption is reduced; the prepared mixed extract can be applied to daily chemical products and is wide in application range.

Description

Technical Field

[0001] The invention belongs to the technical field of Swertia japonica extracts, and particularly relates to a method for preparing a mixed extract of Familiar porphyra and Swertia japonica. Background Art

[0002] Swertia japonica Swertia spp. ) is a medicinal plant of the genus Swertia in the Gentianaceae family. It is widely distributed in the high-altitude areas of southwest China. Its whole plant or root is often used in traditional Chinese medicine, with the effects of clearing away heat and detoxifying, promoting dampness and relieving jaundice. According to the actual experience of folk medication and a large number of literature records, the drugs made from Swertia plants and their extracts have unique clinical efficacy for various inflammations, such as pharyngitis, tonsillitis, conjunctivitis, etc. Its main components are cyclopentadiene ether terpenes, such as swertiamarin and gentiopicroside. Modern medical research has confirmed that swertiamarin has obvious pharmacological activities in anti-inflammatory, antioxidant and anti-apoptotic aspects, and has shown great therapeutic potential for several common clinical diseases such as diabetes and hyperlipidemia.

[0003] Phyllostachys schizophylla linen cloth ) is a large fungus that parasitizes on broad-leaved trees and has important medicinal value. Its main biologically active ingredients, such as polysaccharides, flavonoids, triterpenes, phenols, etc., have anti-tumor, anti-bacterial, anti-viral, antioxidant, free radical elimination and anti-aging effects.

[0004] Chinese invention application CN1704086A provides a method for preparing a raw material of swertiamarin, which is prepared by crushing the plant medicinal material and extracting it with ethanol cold soaking; Chinese patent application CN1966511A provides a method for separating and purifying swertiamarin monomers, using macroporous adsorption resin S-8 as a pre-separation filler and aqueous ethanol as an eluent; then purifying by silica gel G column chromatography, analyzing with ethyl acetate-methanol as an eluent, and the chromatographic product is subjected to crystallization and decolorization treatment, and finally freeze-dried to obtain 98% pure swertiamarin. However, the extraction rate of the effective components of swertiamarin in the above-mentioned technology is not high. Chinese patent application CN112851727A provides a method for extracting swertiamarin, which is subjected to ultrasonic extraction of distilled water, high-pressure extraction of sodium chloride solution, and leaching of ethanol solution on swertiamarin powder, and the filtrates after three extractions and filtrations are combined, and the extract is concentrated to obtain an extract, and swertiamarin is obtained through ultrasonic oscillation adsorption, filtration and other steps, but its process is complicated and the extraction energy consumption is high. Summary of the invention

[0005] In view of the problems in the prior art, the present invention provides a method for preparing a mixed extract of Schizodactylum schizophyllum and Swertia japonica, which comprises at least the following steps: S1, preparing a fermentation liquid of Phyllostachys schizophylla; S2, fermenting Swertia japonica with the fermentation liquid of F. schizodactylifera to obtain a fermentation product of F. schizodactylifera and Swertia japonica; S3. Mix the fermented product of Schizodactylum schizophyllum and Swertia truncatum with distilled water, perform ultrasonic extraction three times, combine the filtrate and concentrate to obtain a mixed extract of Schizodactylum schizophyllum and Swertia truncatum.

[0006] In one embodiment, the preparation of the fermentation broth of Phyllostachys schizophylla comprises the following steps: S11, preparing a solid plate culture medium, inoculating the strain of Schizothrix schizophylla into the solid plate culture medium for cultivation, and scraping the mycelium after the mycelium covers the entire plate; S12, preparing a liquid culture medium, inoculating the mycelium into the liquid culture medium for liquid fermentation culture, and obtaining a fermentation liquid of the schizopyrite fungus.

[0007] In one embodiment, the strain of Phyllostachys schizophylla is derived from TS327899 of Ningbo Testol Biotechnology Co., Ltd.

[0008] In one embodiment, the raw materials of the solid plate culture medium include 200-300 parts of potato pieces, 20-30 parts of glucose, 5-10 parts of peptone, 1-2 parts of potassium dihydrogen phosphate, 0.5-1 part of magnesium sulfate, 18-20 parts of agar powder, and 1000 parts of water, by weight. The solid plate culture medium is sterilized at 121° C. for 20 minutes before use.

[0009] In one embodiment, the culture conditions in step S11 are: culture temperature 28° C., culture 7-10 days.

[0010] In one embodiment, the culture conditions in step S11 are: culture temperature 28° C., and culture for 7 days.

[0011] In one embodiment, the inoculation amount of the mycelium is 5-10wt% of the liquid culture medium, and may be 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, or 10wt%.

[0012] In one embodiment, the raw materials of the liquid culture medium include 4-8 parts of soluble starch, 20-30 parts of glucose, 5-10 parts of peptone, 1-2 parts of potassium dihydrogen phosphate, 0.5-1 parts of magnesium sulfate, and 1000 parts of water, by weight. The liquid culture medium is sterilized at 121° C. for 20 minutes before use.

[0013] This application does not further limit peptone and soluble starch, which can be purchased from Sinopharm Chemical Reagent Co., Ltd.

[0014] In one embodiment, the conditions of the liquid fermentation culture are: culture temperature 28° C., aeration or shaking culture for 5-7 days.

[0015] In one embodiment, the conditions of the liquid fermentation culture are: culture temperature 28° C., shaking culture for 7 days.

[0016] In one embodiment, the fermentation treatment of Swertia japonica with the fermentation liquid of Schizothrix truncatula specifically comprises the following steps: S21, breaking the Swertia japonica into inch-long segments and passing steam into the segments to obtain pretreated Swertia japonica; S22, spraying the fermentation liquid of Schizodactylum sphaerocephalum uniformly on the pretreated Swertia japonica for fermentation and culturing, steam-inactivating after fermentation and allowing static sedimentation for 15 days, taking the supernatant and filtering to obtain the fermentation product of Schizodactylum sphaerocephalum and Swertia japonica.

[0017] In one embodiment, the step S21 is specifically as follows: the Swertia japonica is crushed into 1-2 cm pieces and placed in a material tank or a high temperature resistant sealed plastic tent, steam is introduced to raise the temperature to 80-90°C, maintained for at least 20 minutes, and then cooled to below 30°C to obtain pretreated Swertia japonica.

[0018] In one embodiment, the water content of the pretreated Swertia japonica is 50-60%.

[0019] In one embodiment, the inoculation amount of the fermentation broth of Schizodactylum sphaerocephalum is 10wt% of the pretreated Swertia japonica.

[0020] In one embodiment, the fermentation conditions in step S22 are fermentation temperature 28-32° C. and fermentation time 5-7 days.

[0021] In one embodiment, the fermentation culture conditions in step S22 are a fermentation temperature of 28° C. and a fermentation time of 7 days.

[0022] In one embodiment, the steam inactivation step in S22 is to introduce steam to raise the temperature to 80-90° C. and maintain it for 20 minutes.

[0023] In one embodiment, the step S3 is specifically as follows: The fermented products of Phyllostachys schizodactylifera and Swertia japonica were mixed with distilled water in a weight ratio of 1:3, ultrasonically extracted at 40-50° C. for 10-15 min, and filtered to obtain the first filter residue; The first filter residue is mixed with distilled water in a material-water weight ratio of 1:3, ultrasonically extracted at 40-50° C. for 5-10 min, and filtered to obtain a second filter residue; The second filter residue was mixed with distilled water at a material-water weight ratio of 1:3, ultrasonically extracted at 40-50° C. for 5 min, and filtered; the three filtrates were combined and concentrated to a solid mass fraction of 5% to obtain a mixed extract of Schizodactylum and Swertia japonica.

[0024] Beneficial Effects 1. Before extracting the effective ingredients from Swertia japonica, the present invention first performs a microbial fermentation treatment on it, which is beneficial to the transformation or modification of the effective ingredients; under the combined action of various enzymes produced by microbial fermentation, the cell walls of plant tissues are decomposed and broken, which is more conducive to the dissolution and release of cell contents, improves the extraction rate of effective ingredients, and reduces extraction energy consumption, thereby realizing a green and environmentally friendly production model that reduces production costs.

[0025] 2. The present invention controls the process parameters of the fermentation treatment of Swertia japonica so that the effective ingredients are fully converted, which is beneficial to the dissolution of the effective ingredients.

[0026] 3. The present invention conducts microbial fermentation of Swertia japonica with Schizodactylum sphaerocephalum, and Schizodactylum sphaerocephalum uses Swertia japonica as a culture matrix to complete its own mycelium solid fermentation, thereby realizing the production of a mixed extract of Schizodactylum sphaerocephalum and Swertia japonica.

[0027] 4. The present invention adopts water extraction method, which is simple to operate, has a short preparation cycle, a stable and safe extraction process, is pollution-free, has low cost, and can realize industrial production.

[0028] 5. The mixed extract of Schizodactylum sphaerocephalum and Swertia japonica prepared by the preparation method of the present invention can be used in most daily chemical products and has a wide range of applications. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental methods in the examples without specifying specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without specifying the manufacturer are all conventional products that can be purchased commercially.

[0030] Example 1 This embodiment provides a method for preparing a mixed extract of Schizodactylum schizophyllum and Swertia japonica, comprising the following steps: S1, preparing a fermentation liquid of Phyllostachys schizophylla; S2, fermenting Swertia japonica with the fermentation liquid of F. schizodactylifera to obtain a fermentation product of F. schizodactylifera and Swertia japonica; S3. Mix the fermented product of F. schizodactylifera and Swertia japonica with distilled water, perform ultrasonic extraction for 3 times at a power of 300 W, combine the filtrate and concentrate it to obtain a mixed extract of F. schizodactylifera and Swertia japonica.

[0031] The preparation of the fermentation liquid of Phyllostachys schizophylla comprises the following steps: S11, preparing a solid plate culture medium, inoculating the strain of Schizothrix schizophylla into the solid plate culture medium for cultivation, and scraping the mycelium after the mycelium covers the entire plate; S12, preparing a liquid culture medium, inoculating the mycelium into the liquid culture medium for liquid fermentation culture, and obtaining a fermentation liquid of the schizopyrite fungus.

[0032] The strain of Phyllostachys schizophylla is derived from TS327899 of Ningbo Testo Biotechnology Co., Ltd.

[0033] The raw materials of the solid plate culture medium include 200 parts of potato pieces, 20 parts of glucose, 5 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.5 parts of magnesium sulfate, 18 parts of agar powder and 1000 parts of water in terms of weight. The solid plate culture medium is sterilized at 121° C. for 20 minutes before use.

[0034] The culture conditions in step S11 are: culture temperature 28° C., culture 7 days.

[0035] The inoculation amount of the mycelium is 5wt% of the liquid culture medium.

[0036] According to weight, the raw materials of the liquid culture medium include 4 parts of soluble starch, 20 parts of glucose, 5 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.5 parts of magnesium sulfate, and 1000 parts of water. The liquid culture medium is sterilized at 121° C. for 20 minutes before use.

[0037] The conditions of the liquid fermentation culture are: culture temperature 28° C., shaking culture for 7 days.

[0038] The method of fermenting Swertia japonica with the fermentation liquid of Schizothrix schizophylla specifically comprises the following steps: S21. Crush the Swertia japonica into 1 cm pieces and put them into a material tank. Pass steam to raise the temperature to 80° C., maintain the temperature for 20 min, then cool it down to 28° C. to obtain pretreated Swertia japonica.

[0039] S22, spraying the fermentation liquid of Schizodactylum occidentalis uniformly on the pretreated Swertia japonica for fermentation and culturing, introducing steam after the fermentation is completed to raise the temperature to 80°C, maintaining it for 20 minutes and allowing it to settle for 15 days, taking the supernatant and filtering it to obtain the fermentation product of Schizodactylum occidentalis and Swertia japonica.

[0040] The water content of the pretreated Swertia japonica is 50%.

[0041] The inoculation amount of the fermentation liquid of Schizodactylum sphaerocephalum is 10wt% of the pretreated Swertia japonica.

[0042] The fermentation conditions in step S22 are fermentation temperature 28° C. and fermentation time 7 days.

[0043] The S3 step is specifically as follows: The fermented products of Phyllostachys schizodactylifera and Swertia japonica were mixed with distilled water in a weight ratio of 1:3, and ultrasonically extracted at 300W and 40°C for 10 minutes, and filtered to obtain the first filter residue; The first filter residue was mixed with distilled water at a material-water weight ratio of 1:3, ultrasonically extracted at 300W and 40°C for 5 minutes, and filtered to obtain a second filter residue; The second filter residue was mixed with distilled water at a material-water weight ratio of 1:3, ultrasonically extracted at 300W and 40°C for 5 minutes, and filtered; the three filtrates were combined and concentrated to a solid mass fraction of 5% to obtain a mixed extract of Schizodactylum and Swertia japonica.

[0044] Example 2 The specific implementation of this embodiment is the same as that of Example 1, except that, in terms of weight, the raw materials of the solid plate culture medium include 300 parts of potato chunks, 30 parts of glucose, 10 parts of peptone, 2 parts of potassium dihydrogen phosphate, 1 part of magnesium sulfate, 20 parts of agar powder, and 1000 parts of water.

[0045] The inoculation amount of the mycelium is 10 wt % of the liquid culture medium.

[0046] In terms of weight, the raw materials of the liquid culture medium include 8 parts of soluble starch, 30 parts of glucose, 10 parts of peptone, 2 parts of potassium dihydrogen phosphate, 1 part of magnesium sulfate, and 1000 parts of water.

[0047] The method of fermenting Swertia japonica with the fermentation liquid of Schizothrix schizophylla specifically comprises the following steps: S21. Crush the Swertia japonica into 2 cm pieces and put them into a material tank. Pass steam to raise the temperature to 90° C., maintain the temperature for 20 min, then cool it down to 28° C. to obtain pretreated Swertia japonica.

[0048] S22, spraying the fermentation liquid of Schizodactylum occidentalis uniformly on the pretreated Swertia japonica for fermentation and culturing, introducing steam after the fermentation is completed to raise the temperature to 90°C, maintaining it for 20 minutes and allowing it to settle for 15 days, taking the supernatant and filtering it to obtain the fermentation product of Schizodactylum occidentalis and Swertia japonica.

[0049] The S3 step is specifically as follows: The fermented products of Phyllostachys schizodactylifera and Swertia japonica were mixed with distilled water in a weight ratio of 1:3, and ultrasonically extracted at 300W and 50°C for 15 minutes, and filtered to obtain the first filter residue; The first filter residue was mixed with distilled water at a material-water weight ratio of 1:3, and ultrasonically extracted at 300W and 50°C for 10 minutes, and filtered to obtain a second filter residue; The second filter residue was mixed with distilled water at a material-water weight ratio of 1:3, ultrasonically extracted at 300W and 50°C for 5 minutes, and filtered; the three filtrates were combined and concentrated to a solid mass fraction of 5% to obtain a mixed extract of Schizodactylum and Swertia japonica.

[0050] Comparative Example 1 The present comparative example provides a method for preparing a Swertia japonica extract, which does not use a fermentation liquid of the phylloporin of Schizotrichia cochinchinensis to ferment Swertia japonica. The specific preparation method comprises the following steps: cutting the Swertia japonica into sections, mixing it with distilled water at a material-water weight ratio of 1:3, ultrasonically extracting it at 300W and 40°C for 10 minutes, and filtering to obtain a first filter residue; further mixing the first filter residue with distilled water at a material-water weight ratio of 1:3, ultrasonically extracting it at 300W and 40°C for 5 minutes, and filtering to obtain a second filter residue; further mixing the second filter residue with distilled water at a material-water weight ratio of 1:3, ultrasonically extracting it at 300W and 40°C for 5 minutes, and filtering. The three filtrates are combined and concentrated to a solid mass fraction of 5%, thereby obtaining a Swertia japonica extract.

[0051] Comparative Example 2 The specific implementation of this comparative example is the same as that of Example 1, except that the fermentation culture conditions in step S22 are a fermentation temperature of 20° C. and a fermentation time of 7 days.

[0052] Comparative Example 3 The specific implementation of this comparative example is the same as that of Example 1, except that the fermentation culture conditions in step S22 are a fermentation temperature of 28° C. and a fermentation time of 3 days.

[0053] Comparative Example 4 The specific implementation of this comparative example is the same as that of Example 1, except that the step S3 is specifically as follows: The fermented products of Phyllostachys schizodactylifera and Swertia japonica were mixed with distilled water in a weight ratio of 1:3, and ultrasonically extracted at 300W and 25°C for 10 minutes, and filtered to obtain the first filter residue; The first filter residue was mixed with distilled water at a material-water weight ratio of 1:3, and ultrasonically extracted at 300W and 25°C for 5 minutes, and filtered to obtain a second filter residue; The second filter residue was mixed with distilled water at a material-water weight ratio of 1:3, ultrasonically extracted at 300W and 25°C for 5 minutes, and filtered; the three filtrates were combined and concentrated to a solid mass fraction of 5% to obtain a mixed extract of Schizodactylum and Swertia japonica.

[0054] Comparative Example 5 The specific implementation of this comparative example is the same as that of Example 1, except that in step S3, ultrasonic extraction is performed twice, and the time of each ultrasonic extraction is 10 minutes.

[0055] Performance Testing The contents of polysaccharides, total flavonoids, swertiamarin and gentiopicroside in the samples prepared in the examples and comparative examples were tested. The test results are shown in Table 1 for details.

[0056] Table 1

[0057] It can be seen from the above table that compared with the comparative example, the preparation method provided in the present application can fully dissolve the effective ingredients such as scutellaria glycoside and gentiopicroside, and the effective ingredients can be transformed and modified, thereby increasing the content of bioactive substances of scutellaria glycoside in the product.

Claims

1. A method for preparing a mixed extract of Schizodactylum schizophyllum and Swertia japonica, characterized in that: At least the following steps are included: S1, preparing the fermentation liquid of Phyllostachys schizophylla; S2, fermenting Swertia japonica with the fermentation liquid of F. schizodactylifera to obtain a fermentation product of F. schizodactylifera and Swertia japonica; S3. Mix the fermented product of Schizodactylum schizophyllum and Swertia truncatum with distilled water, perform ultrasonic extraction three times, combine the filtrate and concentrate to obtain a mixed extract of Schizodactylum schizophyllum and Swertia truncatum.

2. The method for preparing the mixed extract of Schizothrix truncatula and Swertia japonica according to claim 1, characterized in that: The preparation of the fermentation liquid of Phyllostachys schizophylla comprises the following steps: S11, preparing a solid plate culture medium, inoculating the strain of Schizothrix schizophylla into the solid plate culture medium for cultivation, and scraping the mycelium after the mycelium covers the entire plate; S12, preparing a liquid culture medium, inoculating the mycelium into the liquid culture medium for liquid fermentation culture, and obtaining a fermentation liquid of the schizopyrite fungus.

3. The method for preparing the mixed extract of Schizothrix truncatula and Swertia japonica according to claim 2, characterized in that: According to weight percentage, the raw materials of the solid plate culture medium include 200-300 parts of potato pieces, 20-30 parts of glucose, 5-10 parts of peptone, 1-2 parts of potassium dihydrogen phosphate, 0.5-1 part of magnesium sulfate, 18-20 parts of agar powder and 1000 parts of water.

4. The method for preparing the mixed extract of Schizothrix truncatula and Swertia japonica according to claim 2, characterized in that: The culture conditions in step S11 are: culture temperature 28° C., culture 7-10 days.

5. The method for preparing the mixed extract of Schizothrix truncatula and Swertia japonica according to claim 2, characterized in that: In terms of weight, the raw materials of the liquid culture medium include 4-8 parts of soluble starch, 20-30 parts of glucose, 5-10 parts of peptone, 1-2 parts of potassium dihydrogen phosphate, 0.5-1 part of magnesium sulfate, and 1000 parts of water.

6. The method for preparing the mixed extract of Schizothrix truncatula and Swertia japonica according to claim 2, characterized in that: The conditions of the liquid fermentation culture are: culture temperature 28° C., aeration or shaking culture for 5-7 days.

7. The method for preparing the mixed extract of Schizodactylum sphaerocephalum and Swertia japonica according to claim 1, characterized in that: The method of fermenting Swertia japonica with the fermentation liquid of Phyllostachys schizophylla specifically comprises the following steps: S21, breaking the Swertia japonica into inch-long segments and passing steam into the segments to obtain pretreated Swertia japonica; S22, spraying the fermentation liquid of Schizodactylum sphaerocephalum uniformly on the pretreated Swertia japonica for fermentation and culturing, steam-inactivating after fermentation and allowing static sedimentation for 15 days, taking the supernatant and filtering to obtain the fermentation product of Schizodactylum sphaerocephalum and Swertia japonica.

8. The method for preparing the mixed extract of Schizothrix truncatula and Swertia japonica according to claim 7, characterized in that: The fermentation conditions in step S22 are fermentation temperature 28-32° C. and fermentation time 5-7 days.

9. The method for preparing the mixed extract of Schizothrix truncatula and Swertia japonica according to claim 7, characterized in that: The inoculation amount of the fermentation liquid of Schizodactylum sphaerocephalum is 10wt% of the pretreated Swertia japonica.

10. The method for preparing the mixed extract of Schizodactylum sphaerocephalum and Swertia japonica according to claim 1, characterized in that: The S3 step is specifically as follows: The fermented products of Phyllostachys schizodactylifera and Swertia japonica were mixed with distilled water in a weight ratio of 1:3, ultrasonically extracted at 40-50° C. for 10-15 min, and filtered to obtain the first filter residue; The first filter residue is mixed with distilled water in a material-water weight ratio of 1:3, ultrasonically extracted at 40-50° C. for 5-10 min, and filtered to obtain a second filter residue; The second filter residue was mixed with distilled water at a material-water weight ratio of 1:3, ultrasonically extracted at 40-50° C. for 5 min, and filtered; the three filtrates were combined and concentrated to a solid mass fraction of 5% to obtain a mixed extract of Schizodactylum and Swertia japonica.

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