Preparation method of a mixed extract of Phellinus linteus and Swertia bimaculata

Abstract

The present invention belongs to the technical field of Swertia extract, and particularly relates to a preparation method of a mixed extract of Phellinus linteus and Swertia, and the preparation method at least comprises the following steps: S1, preparing a fermentation broth of Phellinus linteus; S2, fermenting Swertia with the fermentation broth of Phellinus linteus to obtain a ferment of Phellinus linteus and Swertia; S3, mixing the ferment of Phellinus linteus and Swertia with distilled water, performing ultrasonic extraction three times, combining the filtrates and concentrating to obtain a mixed extract of Phellinus linteus and Swertia. The present invention performs microbial fermentation of Swertia with Phellinus linteus, and uses the enzyme substances generated during the fermentation process to destroy the structures such as cell walls and intercellular substances of the raw materials, so that components such as sweroside are fully dissolved, the extraction rate of the bioactive substances of Swertia in the product is improved, the extraction energy consumption is reduced, and the prepared mixed extract can be applied to daily chemical products, and has a wide application range.

Description

Technical Field

[0001] The present invention belongs to the technical field of Swertia extract, and particularly relates to a preparation method of a mixed extract of Phellinus linteus and Swertia. Background Art

[0002] Swertia ( Swertia spp. ) is a medicinal plant of the genus Swertia in the Gentianaceae family, widely distributed in the high-altitude areas of southwestern China. Its whole herb or roots are commonly used in traditional Chinese medicine, with effects such as clearing heat and detoxifying, promoting diuresis and reducing jaundice. From the actual medication experience of the folk and a large number of literature records, the drugs made from Swertia plants and their extracts have unique clinical effects on various inflammations, such as pharyngitis, tonsillitis, conjunctivitis, etc. Its main components are iridoids, such as swertiamarin, gentiopicroside, etc. Modern medical research has confirmed that swertiamarin shows obvious pharmacological activities in aspects such as anti-inflammatory, antioxidant and anti-apoptosis, and shows great therapeutic potential for several common clinical diseases such as diabetes and hyperlipidemia.

[0003] Phellinus linteus ( phellinus linteus ) is a large fungus parasitic on broad-leaved trees and has important medicinal value. Its main bioactive components, such as polysaccharides, flavonoids, triterpenoids, phenols, etc., have the effects of anti-tumor, antibacterial, antiviral, antioxidant and free radical scavenging, and anti-aging.

[0004] Chinese invention application CN1704086A provides a preparation method of swertiamarin raw material medicine, which is prepared by crushing plant medicinal materials and extracting them by cold soaking with ethanol; Chinese patent application CN1966511A provides a method for separating and purifying swertiamarin monomer, using macroporous adsorption resin S-8 as the pre-separation filler and aqueous ethanol as the eluent; then purifying with a silica gel G column chromatography and eluting with ethyl acetate-methanol as the 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 Swertia in the above technologies is not high. Chinese patent application CN112851727A provides an extraction method of swertiamarin, in which the Swertia powder is successively subjected to ultrasonic extraction with distilled water, high-pressure extraction with sodium chloride solution, and extraction with ethanol solution, and the filtrates after the three extractions are filtered and combined, concentrated to obtain an extract, and swertiamarin is obtained through steps such as ultrasonic oscillation adsorption and filtration. However, its process is complex 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 preparation method of a mixed extract of Phellinus linteus and Swertia, which at least includes the following steps:

[0006] S1. Prepare the fermentation broth of Phellinus linteus;

[0007] S2. Use the fermentation broth of Phellinus linteus to ferment Swertia bimaculata to obtain the fermentation product of Phellinus linteus and Swertia bimaculata;

[0008] S3. Take the fermentation product of Phellinus linteus and Swertia bimaculata, mix it with distilled water, extract it ultrasonically 3 times, combine the filtrates and concentrate to obtain the mixed extract of Phellinus linteus and Swertia bimaculata.

[0009] In one embodiment, the preparation of the fermentation broth of Phellinus linteus includes the following steps:

[0010] S11. Prepare a solid plate medium, inoculate the Phellinus linteus strain in the solid plate medium for cultivation. After the mycelium covers the plate, scrape the mycelium;

[0011] S12. Prepare a liquid medium, inoculate the mycelium in the liquid medium for liquid fermentation cultivation to obtain the fermentation broth of Phellinus linteus.

[0012] In one embodiment, the Phellinus linteus strain is derived from TS327899 of Ningbo Taisituo Biotechnology Co., Ltd.

[0013] In one embodiment, by weight, the raw materials of the solid plate 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. The solid plate medium is sterilized at 121 °C for 20 min before use.

[0014] In one embodiment, the cultivation conditions in step S11 are: cultivation temperature 28 °C, cultivation for 7 - 10 days.

[0015] In one embodiment, the cultivation conditions in step S11 are: cultivation temperature 28 °C, cultivation for 7 days.

[0016] In one embodiment, the inoculation amount of the mycelium is 5 - 10 wt% of the liquid medium. Examples include 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%.

[0017] In one embodiment, by weight, the raw materials of the liquid 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. The liquid medium is sterilized at 121 °C for 20 min before use.

[0018] This application does not further limit peptone and soluble starch, which can be purchased from Sinopharm Chemical Reagent Co., Ltd.

[0019] In one embodiment, the conditions for liquid fermentation culture are as follows: the culture temperature is 28 °C, and aeration or shaking culture is carried out for 5 - 7 days.

[0020] In one embodiment, the conditions for liquid fermentation culture are as follows: the culture temperature is 28 °C, and shaking culture is carried out for 7 days.

[0021] In one embodiment, the fermentation treatment of Swertia mussotii Franch. with the fermentation broth of Phellinus linteus specifically includes the following steps:

[0022] S21. Crush Swertia mussotii Franch. into segments of about an inch and introduce steam to obtain pretreated Swertia mussotii Franch.

[0023] S22. Uniformly spray and inoculate the fermentation broth of Phellinus linteus into the pretreated Swertia mussotii Franch. for fermentation culture. After fermentation is completed, inactivate with steam and let it stand and precipitate for 15 days, then take the supernatant and filter to obtain the fermentation product of Phellinus linteus and Swertia mussotii Franch.

[0024] In one embodiment, the specific operation of step S21 is as follows: crush Swertia mussotii Franch. into segments of 1 - 2 cm, put them into a material tank or a heat-resistant sealed plastic tent, introduce steam to raise the temperature to 80 - 90 °C, keep it for at least 20 minutes, and then cool it down to below 30 °C to obtain pretreated Swertia mussotii Franch.

[0025] In one embodiment, the water content of the pretreated Swertia mussotii Franch. is 50 - 60%.

[0026] In one embodiment, the inoculation amount of the fermentation broth of Phellinus linteus is 10 wt% of the pretreated Swertia mussotii Franch.

[0027] In one embodiment, the conditions for fermentation culture in step S22 are as follows: the fermentation temperature is 28 - 32 °C, and the fermentation time is 5 - 7 days.

[0028] In one embodiment, the conditions for fermentation culture in step S22 are as follows: the fermentation temperature is 28 °C, and the fermentation time is 7 days.

[0029] In one embodiment, the step of steam inactivation in S22 is to introduce steam to raise the temperature to 80 - 90 °C and keep it for 20 minutes.

[0030] In one embodiment, the specific operation of step S3 is as follows:

[0031] Mix the fermentation product of Phellinus linteus and Swertia mussotii Franch. with distilled water at a weight ratio of 1:3 of material to water, perform ultrasonic extraction at 40 - 50 °C for 10 - 15 minutes, and filter to obtain the first filter residue;

[0032] Mix the first filter residue with distilled water at a weight ratio of 1:3 of material to water, perform ultrasonic extraction at 40 - 50 °C for 5 - 10 minutes, and filter to obtain the second filter residue;

[0033] Mix the second filter residue with distilled water at a material-to-water weight ratio of 1:3, perform ultrasonic extraction at 40 - 50 °C for 5 min, and then filter; combine the filtrates from three extractions and concentrate to a solid mass fraction of 5% to obtain the mixed extract of Phellinus linteus and Swertia bimaculata.

[0034] Beneficial effects

[0035] 1. Before extracting the active ingredients from Swertia bimaculata, the present invention first performs microbial fermentation treatment on it, which is beneficial to the transformation or modification of the active ingredients; under the combined action of various enzymes produced by microbial fermentation, the plant tissue cell wall is decomposed and broken, which is more conducive to the dissolution and release of the cell contents, improves the extraction rate of the active ingredients, reduces the extraction energy consumption, and thus realizes a green and environmentally friendly production mode with reduced production costs.

[0036] 2. By controlling the process parameters of the fermentation treatment of Swertia bimaculata, the present invention enables the full transformation of the active ingredients, which is beneficial to the dissolution of the active ingredients.

[0037] 3. The present invention performs microbial fermentation of Swertia bimaculata with Phellinus linteus, and Phellinus linteus uses Swertia bimaculata as the culture medium to complete the solid fermentation of its own mycelium, realizing the production of the mixed extract of Phellinus linteus and Swertia bimaculata.

[0038] 4. The present invention adopts the water extraction method, which has the advantages of simple operation, short preparation cycle, stable and safe extraction process, no pollution, low cost, and can be industrially produced.

[0039] 5. The mixed extract of Phellinus linteus and Swertia bimaculata prepared by the preparation method of the present invention can be applied to most daily chemical products, and has a wide range of applications. Specific embodiments

[0040] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The experimental methods without specific conditions in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments without indicating the manufacturer are all conventional products that can be obtained through commercial purchase.

[0041] Example 1

[0042] This example provides a preparation method for the mixed extract of Phellinus linteus and Swertia bimaculata, including the following steps:

[0043] S1. Prepare the fermentation broth of Phellinus linteus;

[0044] S2. Use the fermentation broth of Phellinus linteus to ferment Swertia bimaculata to obtain the fermented product of Phellinus linteus and Swertia bimaculata;

[0045] S3. Mix Phellinus linteus and Swertia mileensis ferment with distilled water, extract by ultrasonic wave three times at the power of 300W, combine the filtrate and concentrate it to obtain the mixed extract of Phellinus linteus and Swertia mileensis.

[0046] The preparation of the fermentation broth of Phellinus linteus comprises the following steps:

[0047] S11. Prepare a solid plate medium, inoculate the Phellinus linteus strain in the solid plate medium for cultivation, and scrape the mycelium after the mycelium covers the plate.

[0048] S12. Prepare a liquid medium, inoculate the mycelium in the liquid medium for liquid fermentation culture to obtain the fermentation broth of Phellinus linteus.

[0049] The Phellinus linteus strain is derived from TS327899 of Ningbo Taisituo Biotechnology Co., Ltd.

[0050] By weight, the raw materials of the solid plate medium include 200 parts of potato pieces, 20 parts of glucose, 5 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.5 part of magnesium sulfate, 18 parts of agar powder and 1000 parts of water. The solid plate medium is sterilized at 121°C for 20 min before use.

[0051] The culture conditions in step S11 are: culture temperature 28°C, culture for 7 d.

[0052] The inoculation amount of the mycelium is 5 wt% of the liquid medium.

[0053] By weight, the raw materials of the liquid medium include 4 parts of soluble starch, 20 parts of glucose, 5 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.5 part of magnesium sulfate and 1000 parts of water. The liquid medium is sterilized at 121°C for 20 min before use.

[0054] The conditions for the liquid fermentation culture are: culture temperature 28°C, shake culture for 7 d.

[0055] The fermentation treatment of Swertia mileensis with the fermentation broth of Phellinus linteus specifically comprises the following steps:

[0056] S21. Crush Swertia mileensis into 1 cm segments and put them into a feed tank, introduce steam to raise the temperature to 80°C, keep it for 20 min and then cool down to 28°C to obtain the pretreated Swertia mileensis.

[0057] S22. The fermentation liquid of F. schizodactyla is evenly sprayed and inoculated on the pretreated Swertia japonica for fermentation and culture. After the fermentation is completed, steam is introduced to raise the temperature to 80° C., and the temperature is maintained for 20 minutes and allowed to settle for 15 days. The supernatant is collected and filtered to obtain the fermentation product of F. schizodactyla and Swertia japonica.

[0058] The water content of the pretreated Swertia japonica is 50%.

[0059] The inoculation amount of the fermentation liquid of Schizothrix truncatula is 10 wt % of the pretreated Swertia japonica.

[0060] The fermentation conditions in step S22 are a fermentation temperature of 28° C. and a fermentation time of 7 days.

[0061] The S3 step is specifically as follows:

[0062] The fermented products of F. schizodactylum and Swertia japonica were mixed with distilled water at 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;

[0063] 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;

[0064] 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 Schizothrix schizophylla and Swertia japonica.

[0065] Example 2

[0066] The specific implementation of this embodiment is the same as that of Example 1, except that, in parts by 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.

[0067] The inoculation amount of the mycelium is 10 wt % of the liquid culture medium.

[0068] In parts by 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.

[0069] The method of fermenting Swertia japonica with the fermentation liquid of Phellinus schizophylla specifically comprises the following steps:

[0070] 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 it for 20 minutes, and then cool it to 28° C. to obtain pretreated Swertia japonica.

[0071] S22. Uniformly spray and inoculate the fermentation broth of Phellinus linteus on the pretreated Swertia mussotii, and conduct fermentation culture. After fermentation, introduce steam to raise the temperature to 90 °C, maintain for 20 min and let it stand for sedimentation for 15 d, then take the supernatant and filter to obtain the fermentation product of Phellinus linteus and Swertia mussotii.

[0072] The specific steps of the S3 step are as follows:

[0073] Mix the fermentation product of Phellinus linteus and Swertia mussotii with distilled water according to the weight ratio of 1:3 of material to water, and perform ultrasonic extraction at 300 W and 50 °C for 15 min, then filter to obtain the first filter residue;

[0074] Mix the first filter residue with distilled water according to the weight ratio of 1:3 of material to water, and perform ultrasonic extraction at 300 W and 50 °C for 10 min, then filter to obtain the second filter residue;

[0075] Mix the second filter residue with distilled water according to the weight ratio of 1:3 of material to water, and perform ultrasonic extraction at 300 W and 50 °C for 5 min, then filter; Combine the three filtrates and concentrate to a solid mass fraction of 5%, thus obtaining the mixed extract of Phellinus linteus and Swertia mussotii.

[0076] Comparative Example 1

[0077] This comparative example provides a preparation method of Swertia mussotii extract without using the fermentation broth of Phellinus linteus to ferment Swertia mussotii. The specific preparation method includes the following steps: Cut Swertia mussotii into sections, mix it with distilled water according to the weight ratio of 1:3 of material to water, perform ultrasonic extraction at 300 W and 40 °C for 10 min, then filter to obtain the first filter residue; Continue to mix the first filter residue with distilled water according to the weight ratio of 1:3 of material to water, perform ultrasonic extraction at 300 W and 40 °C for 5 min, then filter to obtain the second filter residue; Continue to mix the second filter residue with distilled water according to the weight ratio of 1:3 of material to water, perform ultrasonic extraction at 300 W and 40 °C for 5 min, then filter. Combine the three filtrates and concentrate to a solid mass fraction of 5%, thus obtaining the Swertia mussotii extract.

[0078] Comparative Example 2

[0079] The specific implementation manner of this comparative example is the same as that of Example 1, the difference is that in the S22 step, the fermentation culture conditions are a fermentation temperature of 20 °C and a fermentation time of 7 d.

[0080] Comparative Example 3

[0081] The specific implementation manner of this comparative example is the same as that of Example 1, the difference is that in the S22 step, the fermentation culture conditions are a fermentation temperature of 28 °C and a fermentation time of 3 d.

[0082] Comparative Example 4

[0083] The specific implementation manner of this comparative example is the same as that of Example 1, the difference is that the specific steps of the S3 step are as follows:

[0084] Mix Phellinus linteus, Swertia mileensis ferment and distilled water according to the weight ratio of 1:3 of material to water, perform ultrasonic extraction at 300 W and 25 °C for 10 min, and filter to obtain the first filter residue;

[0085] Mix the first filter residue with distilled water according to the weight ratio of 1:3 of material to water, perform ultrasonic extraction at 300 W and 25 °C for 5 min, and filter to obtain the second filter residue;

[0086] Mix the second filter residue with distilled water according to the weight ratio of 1:3 of material to water, perform ultrasonic extraction at 300 W and 25 °C for 5 min, and filter; Combine the three filtrates and concentrate to a solid mass fraction of 5% to obtain the mixed extract of Phellinus linteus and Swertia mileensis.

[0087] Comparative Example 5

[0088] The specific implementation manner of this comparative example is the same as that of Example 1, the difference is that in step S3, ultrasonic extraction is performed 2 times, and the ultrasonic extraction time each time is 10 min.

[0089] Performance test

[0090] Detect the component contents of polysaccharides, total flavonoids, swertiamarin and gentiopicroside in the samples prepared in each example and comparative example, and the test results are shown in Table 1 for details.

[0091] Table 1

[0092]

[0093] It can be seen from the above table that the preparation method provided by this application can make the active ingredients such as swertiamarin and gentiopicroside dissolve sufficiently compared with the comparative example, the active ingredients are transformed and modified, and the content of the bioactive substances of Swertia in the product is increased.

Claims

1. A preparation method of a mixed extract of Phellinus linteus and Swertia mussotii, characterized in that, Comprising at least the following steps: S1. Prepare the fermentation broth of Phellinus linteus; S2. Crush Swertia bimaculata into sections of about one inch in length and introduce steam to obtain pretreated Swertia bimaculata; evenly spray and inoculate the fermentation broth of Phellinus linteus into the pretreated Swertia bimaculata, ferment and culture at 28 - 32 °C for 5 - 7 d, inactivate with steam and allow to stand and precipitate for 15 d after fermentation is completed, and take the supernatant and filter to obtain the fermentation product of Phellinus linteus and Swertia bimaculata; S3. Mix the fermentation product of Phellinus linteus and Swertia bimaculata with distilled water according to a weight ratio of material to water of 1:3, perform ultrasonic extraction at 40 - 50 °C for 10 - 15 min, and filter to obtain the first filter residue; Mix the first filter residue with distilled water according to a weight ratio of material to water of 1:3, perform ultrasonic extraction at 40 - 50 °C for 5 - 10 min, and filter to obtain the second filter residue; Mix the second filter residue with distilled water according to a weight ratio of material to water of 1:3, perform ultrasonic extraction at 40 - 50 °C for 5 min, and filter; combine the three filtrates and concentrate to a solid mass fraction of 5%, thus obtaining the mixed extract of Phellinus linteus and Swertia bimaculata; The preparation of the fermentation broth of Phellinus linteus comprises the following steps: S11. Prepare a solid plate medium, inoculate the Phellinus linteus strain in the solid plate medium for culture, and scrape the mycelium after the mycelium covers the plate; S12. Prepare a liquid medium, inoculate the mycelium in the liquid medium for liquid fermentation culture to obtain the fermentation broth of Phellinus linteus.

2. The preparation method of the mixed extract of Phellinus linteus and Swertia mussotii according to claim 1, wherein, Calculated by weight parts, the raw materials of the solid plate 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.

3. The preparation method of the mixed extract of Phellinus linteus and Swertia mussotii according to claim 1, wherein, The culture conditions in step S11 are: culture temperature 28 °C, culture for 7 - 10 d.

4. The preparation method of the mixed extract of Phellinus linteus and Swertia mussotii according to claim 1, characterized in that, Calculated by weight parts, the raw materials of the liquid 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.

5. The preparation method of the mixed extract of Phellinus linteus and Swertia mussotii according to claim 1, characterized in that, The conditions for liquid fermentation culture are: culture temperature 28 °C, aeration or shaking culture for 5 - 7 d.

6. The preparation method of the mixed extract of Phellinus linteus and Swertia mussotii according to claim 1, characterized in that, The inoculation amount of the fermentation broth of Phellinus linteus is 10 wt% of the pretreated Swertia bimaculata.

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