High-yield production method of hispidulin
By using Schizophyllum commune for solid-state fermentation and directional transformation of Scutellaria baicalensis, the problems of high production cost and unstable yield of high-plantagin have been solved, realizing efficient and low-cost production of high-plantagin and providing new technologies for new drugs, functional foods and cosmetics.
Patent Information
- Application Number
- CN202610061236.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-16
- Publication Date
- 2026-05-15
AI Technical Summary
Existing methods for producing phytoestrogen have problems such as high cost, unstable output, complicated chemical synthesis steps, and high toxicity, resulting in persistently high market prices.
Solid-state fermentation of Scutellaria baicalensis was carried out using Schizophyllum commune for directional transformation. The fermentation medium was prepared using nutrient solutions such as soybean peptone, urea, and glucose. High-yield production of psyllium husk was achieved through ethanol solvent extraction and silica gel thin-layer separation.
High-yield synthesis of psyllium ether was achieved, with a yield of 48.33 mg/g. The low cost makes it suitable for the development of new drugs, functional foods, and cosmetics.
Smart Images
Figure CN122038499A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of fermentation engineering and pharmaceutical technology, and in particular to a high-yield method for producing high-yield plantago asiatica. Background Technology
[0002] Arachnospermum acetonide (4',5,7-trihydroxy-6-methoxyflavonoid) is a naturally occurring O-methylated flavonoid with a unique structure. Its structural feature is a methoxy group at the 6-position of the flavonoid nucleus, with hydroxyl groups at the 4', 5, and 7-positions. This structure endows it with both hydrophilicity and some lipophilicity, facilitating transmembrane transport and bioavailability. Arachnospermum acetonide possesses multiple pharmacological activities, including antitumor, neuroprotective, antiepileptic, anti-inflammatory, immunomodulatory, anti-osteoporosis, and cardiovascular protective effects, demonstrating significant application potential in pharmaceuticals, functional foods, and cosmetics.
[0003] Currently, there are two main methods for preparing psyllium. The first method is direct extraction from natural plants rich in this component, involving multiple steps: first, the plant material is dried and pulverized; then, it is extracted using a suitable solvent; and finally, a series of separation and purification techniques are used to obtain psyllium with high purity from the complex extract. The second method is chemical synthesis, which involves designing chemical reaction pathways in the laboratory to construct the molecular structure of psyllium from simpler starting materials. The key to the first method is the content of psyllium in natural plants and the extraction process. Natural plants with high psyllium content include those in the Asteraceae family (such as snow lotus), Lamiaceae family (such as plantain and rosemary), and Orchidaceae family (such as dendrobium), but the psyllium content is generally low, usually only 0.1-1 mg / g. Furthermore, the long growth cycle of these plants and their susceptibility to geographical and climatic influences result in high costs and unstable yields with traditional extraction methods. Chemical synthesis can avoid the problem of raw material extraction, but it also suffers from cumbersome steps, high toxicity, numerous byproducts, heavy pollution, and high costs. Due to limitations in the production technology of psyllium oleoresin, its market price remains high. In 2025, the price of 98% pure psyllium oleoresin was as high as 30,000-50,000 yuan / kg. Therefore, developing an efficient, stable, and low-cost production method for psyllium oleoresin is crucial. Summary of the Invention
[0004] Purpose of the invention: To address the problems existing in the prior art, this invention provides a high-yield production method for high-pragmatin content. By utilizing the powerful enzyme system of Schizophyllum commune, the traditional Chinese medicine Scutellaria baicalensis with extremely low (or undetectable) pragmatin content is biotransformed, thereby achieving high-yield production of high-pragmatin. This provides new technologies and methods for the development of new drugs, functional foods, and cosmetics containing high-pragmatin content.
[0005] Technical solution: On one hand, the present invention provides a high-yield production method for high-grade plantago asiatica, comprising the following steps: 1) Nutrient solution preparation: Mix soybean peptone, urea, glucose and water in a certain proportion and set aside. 2) Preparation of solid fermentation conversion medium: Mix Scutellaria baicalensis with the nutrient solution in step 1) in a certain proportion, sterilize and set aside; 3) Solid-state fermentation directional transformation: Take the solid-state fermentation transformation medium from step 2), aseptically inoculate it with Schizophyllum commune seed liquid at a volume-to-mass ratio of 8-16%, stir evenly, and ferment solid-state at 25-32℃ for 12-20 days to obtain solid-state fermentation transformation product; 4) Extraction of psyllium: Total flavonoids were extracted from the solid fermentation product in step 3) using ethanol solvent extraction, followed by extraction with ethyl acetate. The supernatant was concentrated and separated by silica gel thin layer chromatography. The psyllium bands were recovered with ethanol, and the solvent was evaporated to obtain psyllium monomer.
[0006] Further, in step 1), the specific proportions of the nutrient solution are as follows: soybean peptone concentration of 4-6%, urea concentration of 0.5-3%, glucose concentration of 0.5-2%, and the remainder is water.
[0007] Preferably, in step 1), the sources of soybean peptone, urea, and glucose are not limited; the mass concentration of soybean peptone is 4.73%, the mass concentration of urea is 1.66%, the mass concentration of glucose is 0.77%, and the water is deionized water.
[0008] Further, in step 2), the mass ratio of Scutellaria baicalensis to the nutrient solution in step 1) is 1:0.5 to 1:2.
[0009] Preferably, in step 2), the mass ratio of Scutellaria baicalensis to the nutrient solution in step 1) is 1:1.8. Furthermore, the Scutellaria baicalensis is dried.
[0010] Further, in step 3), the specific preparation method of the Schizophyllum commune seed solution is as follows: Add 100 mL of potato glucose solution to a 250 mL Erlenmeyer flask, sterilize it according to routine procedures, inoculate 2-3 pieces of Schizophyllum commune preservation slant, and incubate at 28℃ for 3-5 days on a shaker at 150 rpm. Then, transfer the solution at a volume ratio of 10% to a 500 mL Erlenmeyer flask containing 200 mL of potato glucose solution and continue incubation for 4 days.
[0011] Preferably, in step 3), the solid fermentation conversion medium from step 2) is taken, and the *Schizophyllum commune* seed liquid is aseptically inoculated at a volume-to-mass ratio of 10%. After stirring evenly, it is placed in an environment with a temperature of 32°C and a humidity of 90% for solid fermentation for 16 days to obtain the solid fermentation conversion product.
[0012] Furthermore, in step 4), the developing agent used for the silica gel thin-layer separation is toluene:ethyl acetate:acetic acid = 20:5:1, and the colorimetric agent used is 1% aluminum trichloride.
[0013] On the other hand, the present invention provides the application of psyllium husk prepared by any of the above methods in pharmaceuticals, functional foods and cosmetics. Beneficial effects:
[0014] 1. This invention selects Scutellaria baicalensis, a traditional Chinese medicine, as the raw material for the production of plantago asiatica, which has the advantages of easy availability and relatively low price, thus improving the applicability and economy of this invention.
[0015] 2. Nutrient solution was added to the solid fermentation conversion medium, which contains soybean peptone, urea and glucose. The source of the nutrient solution is readily available and the cost is low, so it is also highly economical.
[0016] 3. High-yield synthesis of plantago-acid was achieved through solid-state fermentation of Scutellaria baicalensis by Schizophyllum commune, with a yield of 48.33 mg / g, which is 8.8 times the yield of plantago-acid obtained by non-directional transformation of Scutellaria baicalensis by Schizophyllum commune alone.
[0017] In summary, this invention achieves high-yield production of psyllium husk through solid-state fermentation and directional conversion of Scutellaria baicalensis using Schizophyllum commune. This invention features high directional conversion efficiency, simple operation, and inexpensive raw materials, providing new technologies and methods for the development of new drugs, functional foods, and cosmetics containing high levels of psyllium husk, and has significant application prospects and substantial economic benefits. Attached image description:
[0018] Figure 1 The HPLC chromatogram of the extract of Scutellaria baicalensis solid-state fermentation directional conversion product obtained by the method of the present invention is shown below. Figure 2 This invention illustrates the effect of different fermentation times of Scutellaria baicalensis on the yield of high-propargyl oleoresin in the method of the present invention. Figure 3 The image shows the HPLC chromatogram of the extract from Scutellaria baicalensis. Figure 4 This is a silica gel plate thin-layer chromatogram of psyllium obtained by the method of the present invention; Figure 5 This is the HPLC chromatogram of the purified plantago asiatica obtained by the method of the present invention; Figure 6 The HPLC chromatogram of the non-directional conversion product of Scutellaria baicalensis solid-state fermentation is shown. Figure 7 This is the HPLC chromatogram of the extract of Ganoderma lucidum transformed into Scutellaria baicalensis. Detailed Implementation
[0019] The present invention will now be described in detail with reference to the embodiments.
[0020] The preparation method of the *Schizophyllum commune* seed culture used in this invention is as follows: 100 mL of potato glucose solution is added to a 250 mL Erlenmeyer flask. After routine sterilization, 2-3 pieces of *Schizophyllum commune* culture slant are inoculated and cultured at 28°C for 3-5 days on a shaker at 150 r / min. Then, the culture is transferred at a volume ratio of 10% to a 500 mL Erlenmeyer flask containing 200 mL of potato glucose solution and cultured for another 4 days before use. Example 1
[0021] The high-yield production method of phytoestrogen is as follows: Scutellaria baicalensis roots were dried to constant weight in a 60 ℃ oven and set aside. A nutrient solution was prepared with the following formula: soybean peptone (4.73% by mass), urea (1.66% by mass), and glucose (0.77% by mass), dissolved in deionized water. 15 g of dried Scutellaria baicalensis roots were weighed and placed in a 150 mL Erlenmeyer flask, and 27 g of nutrient solution was added to completely immerse the roots and allow them to fully absorb the nutrient solution. The flask was then autoclaved at 121 ℃ for 20 min. After sterilization and cooling, 4.2 mL of Schizophyllum commune seed culture was aseptically inoculated and stirred thoroughly. The flask was then placed in a mold incubator at 32 ℃ and 90% humidity and allowed to ferment statically for 16 days to obtain the Scutellaria baicalensis solid-state fermentation directed transformation product.
[0022] The above-mentioned Scutellaria baicalensis solid-state fermentation directional conversion product was extracted and analyzed by HPLC. The content of psyllium husk in the solid-state fermentation conversion product reached 48.33 mg / g. Example 2
[0023] The high-yield production method of phytoestrogen is as follows: Scutellaria baicalensis roots were dried to constant weight in a 60 ℃ oven and set aside. A nutrient solution was prepared with the following formula: soybean peptone (4% by mass), urea (0.5% by mass), and glucose (0.5% by mass), dissolved in deionized water. 15 g of dried Scutellaria baicalensis roots were weighed and placed in a 150 mL Erlenmeyer flask, and 8 g of nutrient solution was added to completely immerse the roots and allow them to fully absorb the nutrient solution. The flask was then autoclaved at 121 ℃ for 20 min. After sterilization and cooling, 3.5 mL of Schizophyllum commune seed culture was aseptically inoculated and aseptically stirred until homogeneous. The flask was then placed in a mold incubator at 25 ℃ and 90% humidity and allowed to ferment statically for 20 days to obtain the Scutellaria baicalensis solid-state fermentation directed transformation product.
[0024] The above-mentioned Scutellaria baicalensis solid-state fermentation directional conversion product was extracted and analyzed by HPLC. The content of high psyllium husk in the solid-state fermentation conversion product reached 35.82 mg / g. Example 3
[0025] The high-yield production method of phytoestrogen is as follows: Scutellaria baicalensis roots were dried to constant weight in a 60 ℃ oven and set aside. A nutrient solution was prepared with the following formula: soybean peptone (6% by mass), urea (3% by mass), and glucose (2% by mass), dissolved in deionized water. 15 g of dried Scutellaria baicalensis roots were weighed and placed in a 150 mL Erlenmeyer flask, and 30 g of nutrient solution was added to completely immerse the roots and allow them to fully absorb the nutrient solution. The flask was then autoclaved at 121 ℃ for 20 min. After sterilization and cooling, 4 mL of Schizophyllum commune seed culture was aseptically inoculated and stirred thoroughly. The flask was then placed in a mold incubator at 32 ℃ and 90% humidity and allowed to ferment statically for 14 days to obtain the Scutellaria baicalensis solid-state fermentation directed transformation product.
[0026] The above-mentioned Scutellaria baicalensis solid-state fermentation directional conversion product was extracted and analyzed by HPLC. The content of high psyllium husk in the solid-state fermentation conversion product reached 41.04 mg / g. Example 4
[0027] The HPLC analysis of the products from the directional conversion of Scutellaria baicalensis through solid-state fermentation is as follows: 1. Total flavonoid extraction The solid-state fermentation product of Scutellaria baicalensis was dried and crushed. 0.5 g of the sample powder was accurately weighed and placed in a 50 mL centrifuge tube. 20 mL of 56% ethanol was added, and the tube was placed in a 50 ℃ water bath with shaking for 1 hour. The tube was then further centrifuged at 8000 r / m for 10 minutes. The supernatant was collected to obtain the extract of solid-state fermentation product of Scutellaria baicalensis, which was stored in a refrigerator at 4 ℃ for later use.
[0028] Using Scutellaria baicalensis root instead of Scutellaria baicalensis solid-state fermentation directional conversion product, the Scutellaria baicalensis raw material extract was obtained by following the above steps and stored in a refrigerator at 4 ℃ for later use.
[0029] 2. HPLC analysis The extracts of the above-mentioned Scutellaria baicalensis solid-state fermentation directional conversion product and the extract of Scutellaria baicalensis raw material were filtered through a 0.22 μm filter membrane and then analyzed by HPLC. The conditions were as follows: Shimadzu LC-16 liquid chromatography system, Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm), detection wavelength 396 nm, flow rate 1.0 mL / min, injection volume 20 μL, column temperature 27 ℃, mobile phase consisting of methanol-0.1% phosphoric acid aqueous solution, and gradient elution for 35 min. The elution program was: 0-15 min, 35% methanol; 25 min, 80% methanol; 32 min, 35% methanol, hold for 3 min.
[0030] The HPLC chromatogram of the extract of Scutellaria baicalensis solid-state fermentation directional conversion product obtained in Example 1 is shown in Figure 1. Figure 1It can be seen that Scutellaria baicalensis, through solid-state fermentation and directed transformation by Schizophyllum commune, produced high-purine and an unknown compound, with the high-purine yield reaching 48.33 mg / g; the effect of different transformation times on high-purine is shown in [the figure]. Figure 2 It can be seen that with the extension of the directed transformation time of Scutellaria baicalensis by Schizonepeta tenuifolia, the yield of physalisin increased from zero to present, showing a trend of first increasing and then decreasing, reaching its maximum at 16 days of directed transformation; the HPLC chromatogram of the Scutellaria baicalensis raw material extract is shown in [Figure number missing]. Figure 3 It can be seen that the raw material of Scutellaria baicalensis is rich in baicalin, wogonin and a small amount of baicalein and wogonin, and no high-plantagorin was detected. Example 5
[0031] The isolation, purification, and analysis of physalisin are detailed below: Take 20 mL of the solid-state fermentation directed conversion product obtained in Example 1 and extract it according to Example 4. Remove ethanol by rotary evaporation, extract with ethyl acetate, and retain the supernatant after separation. Concentrate the supernatant appropriately and perform silica gel thin-layer chromatography separation. The developing solvent used is toluene:ethyl acetate:acetic acid = 20:5:1, and the colorimetric reagent is 1% aluminum trichloride. Scrape off the bands containing psyllium oleoresin from the silica gel plate with a blade, dissolve the psyllium oleoresin in the silica gel powder with ethanol, and obtain the purified psyllium oleoresin product by centrifugation and low-temperature drying. The purity of the psyllium oleoresin prepared after separation and purification reaches over 95%. See the silica gel plate thin-layer chromatogram below. Figure 4 The chromatogram of the purified product of *Platycodon grandiflorus* is shown below. Figure 5 . Comparative Example 1
[0032] The non-directional transformation of Scutellaria baicalensis by solid-state fermentation using Schizophyllum commune is as follows: By replacing the nutrient solution in Example 1 with deionized water, and keeping the other solid-state fermentation conversion conditions the same, a non-directional conversion product of Scutellaria baicalensis solid-state fermentation was obtained.
[0033] Extraction and HPLC analysis of the non-directional transformation products from the solid-state fermentation of Scutellaria baicalensis revealed that the yield of physalisin reached its maximum at 24 days of fermentation, reaching 5.47 mg / g. The chromatogram of the extract after 24 days of fermentation is shown below. Figure 6 . Comparative Example 2
[0034] The Ganoderma lucidum fungus was used for solid-state fermentation and directional transformation of Scutellaria baicalensis, as detailed below: Ganoderma lucidum seed liquid was prepared by culturing Ganoderma lucidum to replace Schizophyllum commune in the seed liquid. The prepared Ganoderma lucidum seed liquid was then used to replace the Schizophyllum commune seed liquid in Example 1, while the other solid-state fermentation and transformation conditions remained the same, to obtain the Ganoderma lucidum-transformed Scutellaria baicalensis product.
[0035] Extraction and HPLC analysis of the above-mentioned Ganoderma lucidum-transformed Scutellaria baicalensis product revealed the following HPLC chromatogram of the Ganoderma lucidum-transformed Scutellaria baicalensis extract (… Figure 7 ) and HPLC chromatogram of Scutellaria baicalensis raw material ( Figure 3 The high similarity between the two indicates that Ganoderma lucidum cannot biotransform the flavonoids of Scutellaria baicalensis.
[0036] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.
Claims
1. A high-yield production method for high-yield plantago asiatica, characterized in that, Includes the following steps: 1) Nutrient solution preparation: Mix soybean peptone, urea, glucose and water in a certain proportion and set aside. 2) Preparation of solid fermentation conversion medium: Mix Scutellaria baicalensis with the nutrient solution in step 1) in a certain proportion, sterilize and set aside; 3) Solid-state fermentation directional transformation: Take the solid-state fermentation transformation medium from step 2), aseptically inoculate it with Schizophyllum commune seed liquid at a volume-to-mass ratio of 8-16%, stir evenly, and ferment solid-state at 25-32℃ for 12-20 days to obtain solid-state fermentation transformation product; 4) Extraction of psyllium: Total flavonoids were extracted from the solid fermentation product in step 3) using ethanol solvent extraction, followed by extraction with ethyl acetate. The supernatant was concentrated and separated by silica gel thin layer chromatography. The psyllium bands were recovered with ethanol, and the solvent was evaporated to obtain psyllium monomer.
2. The high-yield production method of high-yield plantago asiatica according to claim 1, characterized in that: In step 1), the specific proportions of the nutrient solution are as follows: soybean peptone concentration of 4-6%, urea concentration of 0.5-3%, glucose concentration of 0.5-2%, and the remainder is water.
3. The high-yield production method of high-yield plantago asiatica according to claim 1, characterized in that: In step 2), the mass ratio of Scutellaria baicalensis to the nutrient solution in step 1) is 1:0.5 to 1:
2.
4. The high-yield production method of high-yield plantago asiatica according to claim 3, characterized in that: The Scutellaria baicalensis was dried.
5. The high-yield production method of high-yield plantago asiatica according to claim 1, characterized in that: In step 3), the specific preparation method of the Schizophyllum commune seed solution is as follows: Add 100 mL of potato glucose solution to a 250 mL Erlenmeyer flask, sterilize it according to routine procedures, inoculate 2-3 pieces of Schizophyllum commune preservation slant, and incubate at 28℃ for 3-5 days on a shaker at 150 rpm. Then, transfer the solution at a volume ratio of 10% to a 500 mL Erlenmeyer flask containing 200 mL of potato glucose solution and continue incubation for 4 days.
6. The high-yield production method of high-yield plantago asiatica according to claim 1, characterized in that: In step 3), take the solid fermentation conversion medium from step 2), aseptically inoculate it with Schizophyllum commune seed liquid at a volume-to-mass ratio of 10%, stir evenly, and place it in an environment with a temperature of 32℃ and a humidity of 90% for solid fermentation for 16 days to obtain solid fermentation conversion product.
7. The high-yield production method of high-yield plantago asiatica according to claim 1, characterized in that: In step 4), the developing agent used for the silica gel thin-layer separation is toluene:ethyl acetate:acetic acid = 20:5:1, and the colorimetric agent used is 1% aluminum trichloride.
8. The use of psyllium argentide prepared by any one of claims 1-7 in pharmaceuticals, functional foods and cosmetics.