Method for extracting and purifying phenols from phellinus igniarius fruiting body in series and application thereof in uric acid regulation

Abstract

The application discloses a method for extracting phenolic active substances from Phellinus baumii fruiting bodies by adopting a series extraction and purification method and application of the phenolic active substances in uric acid regulation, and belongs to the technical field of deep processing of edible fungi. The method is as follows: after the Phellinus baumii fruiting bodies are soaked in an acetone solution, a crude acetone extract is obtained by using a multi-frequency ultrasonic device, then the crude acetone extract is dissolved in ethanol, and a HPD-826 macroporous resin filler and a LX-17 macroporous resin filler are connected in series to carry out adsorption and elution, so that a pure Phellinus baumii polyphenol extract is obtained. The polyphenol content of the phenolic active substances prepared from the Phellinus baumii fruiting bodies is as high as 80% or more, main active substances are protocatechuic acid, protocatechuic aldehyde, coffee acid, p-coumaric acid, dihydroquercetin, Hispidin and the like, and the phenolic active substances can be prepared into medicines for regulating intestinal flora structure and uric acid metabolism.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi deep processing technology. Specifically, it relates to a method for extracting phenolic active substances from Phellinus linteus fruiting bodies using a tandem extraction and purification method and its application in uric acid regulation. Background Technology

[0002] Sanghuang is a precious medicinal fungus, named for its bright yellow fruiting body. It has a slightly bitter taste and is cold in nature, with a long history of medicinal use, earning it the title of "forest gold." Taxonomically, Sanghuang belongs to the phylum Basidiomycota, class Agaricomycetes, order Hymenochaetales, family Hymenochaetaceae, and genus Sanghuangporus. Modern research has found that Sanghuang contains various active substances such as polysaccharides, polyphenols, flavonoids, and triterpenes. In recent years, with the deepening research into the pharmacological effects of Sanghuang and its growing popularity as a natural anti-cancer product, it has become a hot topic in the research and development of pharmaceutical preparations and health products both domestically and internationally.

[0003] Numerous patents have been published regarding the extraction technology of flavonoid active substances from *Sanghuang* (a type of medicinal mushroom), including: CN112138030A, which discloses a method for extracting flavonoids from *Sanghuang* using ethanol extraction and purification with macroporous adsorption resin, and its application in the prevention and treatment of gout; CN115554322A, which discloses a method for extracting flavonoids from *Sanghuang* mycelium using a 55%–85% ethanol solution; CN111096983A, which discloses a method for extracting flavonoids from *Sanghuang* fruiting bodies using ultrasonic ethanol-water-assisted extraction and ethyl acetate extraction; and CN102078339A, which discloses a method for enriching and purifying *Sanghuang* flavonoids using a batch distillation-resin adsorption coupling method. These patents disclose methods for preparing flavonoid compounds from *Sanghuang* from different perspectives. However, due to the significant differences in the chemical structure and physical properties of flavonoids and polyphenols, the extraction and purification methods for polyphenols and flavonoids will inevitably differ considerably. Currently, there are few published reports on the preparation of polyphenolic compounds from Phellinus linteus. The main ones include: CN112656820A, which discloses a method for extracting Phellinus linteus polyphenols from deep fermentation mycelia using a deep eutectic solvent composed of choline chloride and malic acid; and CN114832022B, which discloses a method for stepwise extraction of polyphenolic substances from Phellinus linteus fruiting bodies using organic solvents (a patent applied for by the inventor). The above-mentioned patented technologies suffer from several problems, including the use of large amounts of organic solvents, high reagent costs, and low polyphenol extraction rates.

[0004] Polyphenols are among the most abundant bioactive substances in *Phellinus linteus*, and a key material basis for the development of refined *Phellinus linteus* products. The bioactivity of *Phellinus linteus* polyphenols has attracted considerable attention. Patent CN112791108A discloses a method for extracting active substances from the fermentation broth of *Phellinus linteus* strains using organic solvents; CN101474211A and CN111096983A respectively disclose a method for preparing an ethanol extract from *Phellinus linteus* fruiting bodies and its hypoglycemic activity. CN108379305A and CN112189505A respectively disclose that the aqueous and ethanol extracts of *Phellinus linteus* have uric acid-lowering effects; CN110693921B discloses the application of the ethanol extract of *Phellinus linteus* fruiting bodies in the preparation of drugs for the prevention and treatment of gout and hyperuricemia. Although these extracts all possess some bioactivity, it is impossible for those skilled in the art to determine which, which, or which class of main components are responsible for the hypoglycemic and uric acid-lowering activities, thus hindering drug quality testing. Furthermore, the studies on uric acid-lowering activity (gout) only mentioned alcohol extracts and did not consider the solubility of different active substances in different solvents.

[0005] In summary, there is an urgent need to develop an efficient and inexpensive method for preparing polyphenolic active substances from Phellinus linteus and to clarify their main components and biological activities. Summary of the Invention

[0006] To address the current problems of low extraction rate of polyphenolic active substances from Phellinus linteus fruiting bodies, excessive use of organic solvents, high reagent costs, and mixed functional components, the first objective of this invention is to provide a method for preparing high-purity phenolic active substances from Phellinus linteus fruiting bodies with a polyphenol content of over 80%.

[0007] To achieve the above technical objectives, the inventors, drawing on years of experience in extraction and separation, and through extensive experimental research and relentless exploration, have finally obtained the following technical solution: a method for tandem extraction and purification of polyphenols from Phellinus linteus fruiting bodies, comprising the following steps:

[0008] (1) Take the fruiting bodies of poplar mulberry with a cultivation period of 1 to 3 years, dry them and crush them. Add 5 to 20 times the volume of 30% to 80% acetone solution to soak for 0.5 to 3 hours. Then, use a multi-frequency ultrasonic device to extract 1 to 3 times. The extraction conditions of the multi-frequency ultrasonic device are: the ultrasonic frequency is 40KHz and 80KHz alternately, each time for 8 to 12 minutes, the ultrasonic power is 450 to 600W, and the extraction temperature is 25 to 35℃. After extraction, centrifuge, collect the supernatant, and concentrate it by rotary evaporation to obtain crude acetone extract.

[0009] (2) Dissolve the crude acetone extract obtained in step (1) with 70%-90% ethanol solution and statically adsorb it with HPD-826 macroporous resin packing for 2-12 hours. After the adsorption is completed, separate the macroporous resin packing and adsorb the remaining ethanol solution with LX-17 macroporous resin packing for 2-12 hours. After the adsorption is completed, pack the two macroporous resins into columns respectively.

[0010] (3) First, rinse the two columns with 0.5 to 2 column volumes of pure water respectively. Then, elute the HPD-826 macroporous resin packing column with 30% to 90% ethanol solution by volume, with an elution volume of 2 to 5 column volumes. Collect the eluent. Then, elute the LX-17 macroporous resin packing with the eluent. Collect the eluent and concentrate it by rotary evaporation to obtain pure Sanghuang polyphenol extract.

[0011] More preferably, in the method of extracting and purifying polyphenols from Phellinus linteus fruiting bodies as described above, the Phellinus linteus fruiting bodies described in step (1) are dried and then pulverized through a 20-80 mesh sieve.

[0012] More preferably, in the method for tandem extraction and purification of polyphenols from Phellinus linteus fruiting bodies as described above, the volume percentage of the acetone solution in step (1) is 60% to 80%.

[0013] More preferably, in the method of extracting and purifying polyphenols from Phellinus linteus fruiting bodies as described above, the ultrasonic power in step (1) is 450-500W.

[0014] More preferably, in the method of extracting and purifying polyphenols from Phellinus linteus fruiting bodies as described above, the extraction temperature in step (1) is 28–32 °C.

[0015] More preferably, in the method of extracting and purifying polyphenols from Phellinus linteus fruiting bodies as described above, step (3) involves eluting the HPD-826 macroporous resin packing column with a volume percentage of 70% to 90% ethanol solution.

[0016] The second objective of this invention is to provide the main components of the polyphenols from the fruiting bodies of *Sanghuang* obtained by the above preparation method. The main components of the purified *Sanghuang* polyphenols were identified using HPLC and quantitative analysis with standards, and confirmed by comparison with standards. The main components of the pure *Sanghuang* polyphenol extract are protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid, dihydroquercetin, and hispidin, among others.

[0017] The third objective of this invention is to provide the application of the polyphenols from the fruiting bodies of Phellinus linteus obtained by the above preparation method in the preparation of drugs for regulating intestinal flora structure and / or regulating uric acid metabolism. The regulation of uric acid metabolism involves significantly inhibiting xanthine oxidase activity in vitro; reducing xanthine oxidase activity in serum and liver in vivo, decreasing serum uric acid concentration, and increasing uric acid content in urine. The regulation of intestinal flora structure involves selectively reducing the abundance of bacteria such as *Lactobacillus johnsonii* and *Lactobacillus reuteri*, and increasing the abundance of bacteria such as *Muribaculaceae*, *Bacteroides vulgatus*, and *Lachnospiraceae NK4A136*.

[0018] Compared with existing technologies, the polyphenol content of the phenolic active substances in the fruiting bodies of Phellinus linteus prepared by this invention is as high as 80% or more, and the composition of the main active substances is relatively clear, and it has the following beneficial effects:

[0019] (1) In vitro xanthine oxidase inhibition experiments showed that the polyphenols of Phellinus linteus prepared in this invention have significant xanthine oxidase inhibitory activity and a low IC50 value of 38.6 μg / mL.

[0020] (2) Mouse model experiments showed that feeding the phenolic active substances of Phellinus linteus prepared in this invention reduced the concentration of xanthine oxidase in the serum and liver of model mice and the content of uric acid in the serum, while increasing the excretion of uric acid in the urine of mice.

[0021] (3) Mouse model experiments showed that feeding the fruiting body of Phellinus linteus prepared in this invention with phenolic active substances could significantly reduce the abundance of bacteria such as Lactobacillus johnsonii and Lactobacillus reuteri, and increase the abundance of bacteria such as Muribaculaceae, Bacteroides vulgatus, and Lachnospiraceae NK4A136.

[0022] In summary, the preparation method of this invention is simple and efficient, and it produces high-purity polyphenols from Phellinus linteus fruiting bodies. These active substances have beneficial effects on uric acid regulation, such as reducing the uric acid content in mouse urine and increasing beneficial intestinal flora. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the purification process for preparing Phellinus linteus polyphenols using this method;

[0024] Figure 2The diagram shows the composition of the active substances in the *Sanghuang* polyphenols prepared by this method; sample A is the standard and sample B is extract S1. Detailed Implementation

[0025] The present invention will be described in detail through the following embodiments to make its advantages and features more readily understood by those skilled in the art, but without limiting the invention in any way. Any modifications or alterations made using the content of this specification that are easily implemented by those skilled in the art without departing from the technical solutions of the present invention will be included within the scope of patent protection of the present invention.

[0026] Example 1: Preparation of pure Phellinus linteus polyphenol extract

[0027] Fruiting bodies of *Sanghuangporus vaninii*, cultivated for 3 years, were dried, pulverized, and sieved through a 40-mesh sieve. 200g of the powder was soaked in 10 times its volume of 80% acetone solution for 1 hour, followed by extraction three times using a multi-frequency ultrasonic device. The extraction conditions were: alternating ultrasonic frequencies of 40kHz and 80kHz, 5 minutes each time, for a total of 20 minutes; ultrasonic power of 450W; and extraction temperature of 30℃. After extraction, the extract was centrifuged, and the supernatant was collected and concentrated by rotary evaporation to obtain 26.6g of crude acetone extract (extraction rate 13.3%). 3g of the crude acetone extract was dissolved in 100 mL of 80% (v / v) ethanol solution and statically adsorbed using HPD-826 macroporous resin packing material for 12 hours. The macroporous resin packing material was then separated for later use, and the remaining ethanol solution was further adsorbed using LX-17 macroporous resin packing material for 12 hours. After completion, both macroporous resins were packed into columns. (See [link to relevant documentation]). Figure 1 The purification process involved rinsing both columns with two column volumes of pure water, followed by elution with 80% ethanol solution on the HPD-826 macroporous resin column (elution volume: three column volumes). The eluent was collected and then used to elute LX-17 macroporous resin. The eluent was collected again and concentrated by rotary evaporation to obtain 0.94 g of pure Phellinus linteus polyphenol extract (purification rate: 31.2%). This active substance was designated as S1.

[0028] Comparative Example 1: Single-frequency ultrasonic extraction process

[0029] The fruiting bodies of *Phellinus linteus* from poplar trees cultivated for 3 years were dried, pulverized, and passed through a 40-mesh sieve. 100g of the *Phellinus linteus* fruiting body powder was added to 10 times the volume of 80% acetone solution and soaked for 1 hour. Then, it was extracted 3 times using a single-frequency ultrasonic device. The extraction conditions of the ultrasonic device were: ultrasonic frequency 80KHz, ultrasonic power 450W, extraction temperature 30℃, and extraction time 20min. After extraction, the extract was centrifuged, the supernatant was collected, and concentrated by rotary evaporation to obtain 9.1g of crude acetone extract (extraction rate 9.1%). This active substance was designated as D1.

[0030] Comparative Example 2: Extraction of Phellinus linteus polyphenols from Phellinus linteus using ethanol as a solvent

[0031] The fruiting bodies of *Sanghuangporus vaninii*, cultivated for 3 years, were dried, pulverized, and passed through a 40-mesh sieve. 100g of the fruiting body powder was soaked in 10 times its volume of 80% ethanol solution for 1 hour. The mixture was then extracted three times using a multi-frequency ultrasonic device. The extraction conditions were: alternating ultrasonic frequencies of 40kHz and 80kHz, each used for 5 minutes, for a total of 20 minutes; ultrasonic power of 450W; and extraction temperature of 30℃. After extraction, the mixture was centrifuged, and the supernatant was collected. The supernatant was then concentrated by rotary evaporation to obtain 11.8g of crude ethanol extract (extraction rate 11.8%), which was designated as D2.

[0032] Comparative Example 3: Column purification of crude ethanol extract

[0033] Take 3g of the crude ethanol extract prepared in Comparative Example 2, dissolve it in 100 mL of 80% ethanol solution, and perform static adsorption using HPD-826 macroporous resin packing material. After adsorption for 12 h, separate the macroporous resin packing material for later use. The remaining ethanol solution is then adsorbed using LX-17 macroporous resin packing material for another 12 h. After completion, pack the two macroporous resins into columns separately. Wash both columns with 2 column volumes of pure water, and then elute the HPD-826 macroporous resin packing column with 80% ethanol solution for 3 column volumes. Collect the eluent, and then use the eluent to elute the LX-17 macroporous resin packing material. Collect the eluent again, and concentrate it by rotary evaporation to obtain 0.81g of pure Phellinus linteus polyphenol extract (purification rate 27.0%). This active substance is designated as D3.

[0034] Example 2: Identification of the active substance composition of Phellinus linteus polyphenol extract

[0035] Total polyphenol content determination: A standard curve was prepared using gallic acid as a standard (Y=9.1782X+0.0191, R2=0.9935). The total polyphenol value was expressed as gallic acid equivalent per gram of dry mass. 1 mL of each 0.3 g / L *Sanghuang* extract polyphenol solution was transferred to a 10 mL centrifuge tube, 1.5 mL of Folin-phenol reagent was added, and the mixture was allowed to stand in the dark for 5 min. Then, 1.5 mL of 6% sodium carbonate solution was added, and the mixture was heated in a 75 ℃ water bath for 10 min. The reaction was immediately stopped using an ice bath. The volume was then adjusted to the mark with distilled water, and the absorbance was measured using a UV-Vis spectrophotometer at a wavelength of 750 nm.

[0036] The polyphenol components of different extracts were determined by high performance liquid chromatography (HPLC). The analytical conditions are shown in Table 1. The standards were protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid, dihydroquercetin, and Hispidin.

[0037] Table 1. HPLC Detection Conditions for Polyphenol Components

[0038]

[0039] Example 3: In vitro xanthine oxidase inhibitory activity experiment of polyphenols from Phellinus linteus fruiting body

[0040] Assay for xanthine oxidase inhibitory activity: 0.6 mL of phosphate buffer (0.1 mol / L, pH=8.5), 0.2 mL of xanthine solution (2 mmol / L), and 0.1 mL of different sample solutions (3.0 mg / mL, prepared in DMSO) were vortexed in a 1.5 mL centrifuge tube. Then, 0.2 mL of xanthine oxidase solution (0.1 U / mL) was added, and the mixture was reacted in a water bath at 25 ℃ for 30 min. After the reaction was completed, 0.2 mL of HCl solution (1.0 mol / L) was added to terminate the reaction. Allopurinol was used as a positive control, and the absorbance was measured at a wavelength of 290 nm. The formula for calculating the xanthine oxidase inhibition rate is as follows:

[0041] Inhibition rate (%) = {[(AB) - (CD)] / (AB)} × 100

[0042] Where A represents the absorbance value with and without enzyme; B represents the absorbance value without enzyme and without sample tube; C represents the absorbance value with and with enzyme; and D represents the absorbance value with and without enzyme.

[0043] Example 4: Experimental study on the regulation of uric acid metabolism in vivo by polyphenols from Phellinus linteus fruiting body

[0044] (1) Mouse feeding and modeling: Sixty Kunming mice weighing 20-22 g were prepared and randomly divided into 6 groups of 10 mice each. 1 was the blank control group, 2 was the model group, 3 was the S1 sample (80 mg / kg) in Example 1, 4 was the D1 sample (80 mg / kg) in Comparative Example 1, 5 was the D2 sample (80 mg / kg) in Comparative Example 2, and 6 was the D3 sample (80 mg / kg) in Comparative Example 3. Except for the blank control group, the other groups were administered 200 mg / kg potassium oxonate and 200 mg / kg hypoxanthine by gavage daily to establish a mouse model of hyperuricemia for 7 consecutive days. The blank control group was administered an equal volume of 0.5% CMC-Na by gavage. Starting from day 8, in addition to administering 200 mg / kg potassium oxonate and 200 mg / kg hypoxanthine by gavage, the corresponding drugs were administered by gavage. The blank control group and the model group were administered an equal volume of 0.5% CMC-Na by gavage for 28 consecutive days. The gavage volume was 0.1 mL / 10g. On the last day of feeding, intestinal feces of mice were collected for sequencing.

[0045] (2) The xanthine oxidase activity in mouse serum and liver tissue and the uric acid content in mouse urine were determined using a kit from Nanjing Jiancheng Company.

[0046] (3) Analysis of microbial community structure and diversity: Intestinal microbial sequencing analysis of mouse rectal feces was performed by Shanghai Meiji Company (n=6).

[0047] Results analysis:

[0048] (1) Polyphenol content and in vitro xanthine oxidase inhibitory activity of different samples.

[0049] Table 2. Polyphenol content and in vitro xanthine oxidase inhibitory activity of different samples

[0050]

[0051] The content of *Sanghuang* polyphenols and the in vitro xanthine oxidase inhibition rate obtained in Examples 1, 1, 2, and 3 are shown in Table 2. In Example 1, the total phenol content of S1 was significantly higher than that in Comparative Examples 1, 2, and 3. In Example 1, the in vitro xanthine oxidase inhibitory activity of S1 was significantly higher than that in Comparative Examples 1, 2, and 3, indicating that the *Sanghuang* polyphenols prepared by this invention have a high total phenol content and good xanthine oxidase inhibitory activity.

[0052] (2) Determination of the composition of the polyphenols prepared by the present invention.

[0053] Table 3. Composition determination results of S1, the polyphenol extract of Phellinus linteus prepared in this invention.

[0054]

[0055] The component identification results of the Sanghuang polyphenol S1 prepared by this invention are as follows: Figure 2 As shown in Table 3. Figure 2 The standard substances 1-7 are gallic acid, protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid, dihydroquercetin, and Hispidin. The main components of the *Sanghuang* polyphenol S1 prepared in this invention are protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid, dihydroquercetin, and Hispidin. Among these, protocatechuic acid, protocatechuic aldehyde, and dihydroquercetin are present in higher amounts and may be key compounds.

[0056] (3) Effects of different samples on xanthine oxidase in mouse serum and liver and uric acid content in urine

[0057] Table 4. Effects of different samples on xanthine oxidase and uric acid levels in mice.

[0058]

[0059] The levels of xanthine oxidase in the serum and liver of mice in each experimental group are shown in Table 4. Compared with Comparative Examples 1, 2, and 3, the levels of xanthine oxidase in the serum and liver of mice in Example 1 were relatively low, indicating that the polyphenolic substance prepared in this invention has good in vivo inhibitory activity against xanthine oxidase. The levels of uric acid in the serum and urine of mice in each experimental group are shown in Table 4. It can be seen that the *Sanghuang* polyphenol prepared in this invention can inhibit serum uric acid production and increase uric acid excretion in urine, thus having a regulatory effect on uric acid metabolism in mice.

[0060] (4) Effects of different samples on the composition of mouse gut microbiota

[0061] Table 5. Effects of different samples on the composition of the gut microbiota in mice.

[0062]

[0063] Table 5 shows that the dominant gut microbiota in mice were Bacteroides and Firmicutes. Compared with the control group and the model group, the Firmicutes content was significantly reduced and the Bacteroides content was significantly increased in Examples 1, Comparative Examples 1, 2, and 3. At the genus level, the content of Muribacterium and Bacteroides was significantly increased and the content of Lactobacillus was significantly reduced in Examples 1, 1, 2, and 3. At the key species level, the abundance of Lactobacillus johnsonii and Lactobacillus reuteri was significantly reduced and the abundance of Bacteroides vulgatus and Lachnospiraceae NK4A136 was significantly increased in Examples 1, 1, 2, and 3. Compared with Comparative Examples 1, 2 and 3, Example 1 showed a consistent trend in regulating gut microbiota with the other two examples, but Example 1 had a significantly better regulatory effect on gut microbiota than Comparative Examples 1, 2 and 3.

[0064] The above research results show that the polyphenolic substance prepared by this invention has a high total phenol content and good in vitro xanthine oxidase inhibitory activity; in vivo, it can effectively reduce the content of xanthine oxidase in mouse serum and liver, reduce serum uric acid content, and increase uric acid excretion; in addition, the polyphenolic substance prepared by this invention can also effectively regulate the intestinal flora structure. The research results indicate that the polyphenolic substance prepared by this invention can serve as a potential regulator of uric acid metabolism.

Claims

1. A method for tandem extraction and purification of polyphenols from Phellinus linteus fruiting bodies, characterized in that, The polyphenols from the fruiting body of Phellinus linteus are used to treat hyperuricemia, and the method includes the following steps: (1) Take the fruiting bodies of poplar mulberry with a cultivation period of 1 to 3 years, dry them, crush them and pass them through a 20 to 80 mesh sieve. Add 5 to 20 times the volume of 60% to 80% acetone solution to soak for 0.5 to 3 hours. Then, use a multi-frequency ultrasonic device to extract 1 to 3 times. The extraction conditions of the multi-frequency ultrasonic device are: the ultrasonic frequency is 40KHz and 80KHz alternately, each time for 8 to 12 minutes, the ultrasonic power is 450 to 500W, and the extraction temperature is 28 to 32℃. After extraction, centrifuge, collect the supernatant, and concentrate it by rotary evaporation to obtain crude acetone extract. (2) Dissolve the crude acetone extract obtained in step (1) with 70% to 90% ethanol solution by volume, and then use HPD-826 macroporous resin packing for static adsorption for 2 to 12 hours. After adsorption, separate the macroporous resin packing and use LX-17 macroporous resin packing to adsorb the remaining ethanol solution for another 2 to 12 hours. After adsorption, pack the two macroporous resins into columns respectively. (3) First, rinse the two columns with 0.5 to 2 column volumes of pure water respectively. Then, elute the HPD-826 macroporous resin packing column with 70%-90% ethanol solution by volume, with an elution volume of 2 to 5 column volumes. Collect the eluent. Then, elute the LX-17 macroporous resin packing with the eluent. Collect the eluent and concentrate it by rotary evaporation to obtain pure Sanghuang polyphenol extract.

2. A polyphenol derived from the fruiting body of Phellinus linteus obtained by the method according to claim 1, comprising protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid, dihydroquercetin, and Hispidin.

3. The use of the polyphenols from the fruiting body of Phellinus linteus according to claim 2 in the preparation of a medicament for treating hyperuricemia.

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