A fermented extract of Rehmannia glutinosa, a composition thereof, its preparation method and application

Abstract

This invention provides a fermented extract of Rehmannia glutinosa, a composition thereof, its preparation method, and its application, belonging to the field of biomedical technology. This invention obtains fermented extracts of black tea and Rehmannia glutinosa by fermenting black tea and Rehmannia glutinosa with a single strain of *Aspergillus cristatus*. Experiments have verified that both extracts, used alone, have good inhibitory effects on dipeptidyl peptidase-4. Further, combining the two extracts and verifying through animal experiments, shows that they have good inhibitory activity against dipeptidyl peptidase-4 in the intestine and feces, while also exhibiting a hypoglycemic effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fermented extract of Rehmannia glutinosa, a composition thereof, its preparation method and application. Background Technology

[0002] Diabetes mellitus (DM) is a typical metabolic disease that not only causes various serious complications but also poses a serious threat to human life. With in-depth research, new hypoglycemic drugs targeting different mechanisms of action in diabetes are constantly emerging, and dipeptidyl peptidase-4 (DPP-4) inhibitors are one such example. DPP-4 inhibitors prolong the duration of action by inhibiting the activity of DPP-4, the enzyme that degrades GLP-1, thereby promoting insulin secretion. Due to their significant blood glucose reduction, fewer gastrointestinal side effects, no weight gain, and ability to improve pancreatic β-cell function, DPP-4 inhibitors have significant advantages in terms of safe medication use, and are therefore generally considered the most promising new drugs for treating type 2 diabetes. However, existing DPP-4 inhibitors are not without their limitations. On the one hand, long-term use of some DPP-4 inhibitors may lead to potential side effects, such as an increased risk of acute pancreatitis; on the other hand, some patients do not respond well to the hypoglycemic effects of existing DPP-4 inhibitors, and their blood glucose control remains unsatisfactory. Furthermore, as research has progressed, it has been discovered that gut microbiota-derived DPP-4 can enter the human body and degrade GLP-1. Commonly used DPP-4 inhibitors (such as sitagliptin) cannot effectively inhibit the activity of this gut microbiota-derived DPP-4, leading to the accumulation of gut microbiota-derived DPP-4 in some patients, which seriously affects the clinical treatment effect of the drug.

[0003] Microbial fermentation technology has received widespread attention in the research and development of traditional Chinese medicine (TCM). Through microbial fermentation, the chemical composition of TCM can be altered, increasing the types and content of active ingredients, and even generating new active substances, potentially significantly enhancing the medicinal value of TCM. Currently, research on obtaining extracts with DPP-4 inhibitory activity from TCM through microbial fermentation is still in its early stages. Current fermentation processes and formulations generally lack systematicity and specificity, resulting in unstable DPP-4 inhibitory activity and low extraction rates in the fermentation products, hindering large-scale industrial production and clinical application. Therefore, developing a microbial fermentation extract that inhibits both animal-derived and bacterial DPP-4, while also possessing hypoglycemic effects, is a key technical challenge that needs to be addressed. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a Rehmannia glutinosa fermented extract, composition, preparation method and application thereof. The Rehmannia glutinosa fermented extract and composition obtained by fermentation with Eurotium cristatum have good inhibitory activity against both animal-derived and bacterial dipeptidyl peptidase-4, and have hypoglycemic effect.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] This invention provides a method for preparing a fermented extract of Rehmannia glutinosa, comprising the following steps: mixing Rehmannia glutinosa with Aspergillus cristatus for fermentation, mixing the fermented Rehmannia glutinosa with water for extraction, and collecting the filtrate.

[0007] Preferably, the mixed fermentation step includes: mixing Rehmannia glutinosa with water, sterilizing, and then inoculating with a suspension of Eurotium cristatum spores; the ratio of Rehmannia glutinosa, water, and Eurotium cristatum spore suspension is 20-35g:6-18mL:1mL, respectively; the concentration of the Eurotium cristatum spore suspension is 1×10⁻⁶. 6 -1×10 8 cfu / mL.

[0008] Preferably, the ratio of fermented Rehmannia glutinosa to water is 1g:45-55mL; the extraction temperature is 60-70℃, the extraction time is 1-2h, and the extraction is performed 1-3 times.

[0009] The present invention provides a fermented extract of Rehmannia glutinosa obtained by the preparation method described above.

[0010] The present invention provides a composition comprising a Rehmannia glutinosa fermented extract obtained by the preparation method and a Fuzhuan tea protease hydrolysate; wherein the weight ratio of the Rehmannia glutinosa fermented extract to the Fuzhuan tea protease hydrolysate is 1:0.5-3.5.

[0011] Preferably, the preparation of the Fu tea protein hydrolysate includes the following steps: mixing *Aspergillus cristatus* with black tea for fermentation to obtain Fu tea; mixing Fu tea with sodium hydroxide solution, bathing in water, adjusting the pH to 3.0-4.0, centrifuging to collect the precipitate, mixing the precipitate with water, adjusting the pH to 6.5-7.5, and drying to obtain crude fermented protein from black tea; mixing the crude fermented protein from black tea with water, keeping warm in a water bath, adjusting the pH to 8-10, adding protease for enzymatic hydrolysis, centrifuging to remove the precipitate, and collecting the protein hydrolysate.

[0012] Preferably, the concentration of the *Eurotium cristatum* spore suspension is 1 × 10⁻⁶. 6 -1×10 8 cfu / mL; the inoculation amount of the *Aspergillus cristatus* spore suspension is 0.8-2 mL / g of the black tea mass; the mass-volume ratio of the Fu tea to the sodium hydroxide solution is 1g:20-30mL; the protease includes one or more of trypsin, pepsin, and alkaline protease.

[0013] The present invention provides a method for preparing the composition, comprising the following steps: mixing Rehmannia glutinosa fermented extract and Fuzhuan tea protein hydrolysate according to the weight ratio.

[0014] The present invention provides the use of the fermented extract of Rehmannia glutinosa obtained by the preparation method, or the fermented extract of Rehmannia glutinosa, or the composition thereof, in the preparation of a product that inhibits dipeptidyl peptidase-4.

[0015] The present invention provides the application of the fermented Rehmannia glutinosa extract obtained by the preparation method, or the fermented Rehmannia glutinosa extract or the composition thereof, in the preparation of hypoglycemic products.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention is the first to use a single strain of *Aspergillus cristatus* to ferment and extract *Rehmannia glutinosa*. This fermentation extraction can improve some functional components. The obtained fermented extract of *Rehmannia glutinosa* has high inhibitory activity against dipeptidyl peptidase-4, which can be of animal or bacterial origin.

[0018] Furthermore, this invention utilizes the fermentation of black tea by *Aspergillus cristatus* or the fermentation of *Rehmannia glutinosa* to extract the fermented extracts. Combining these two fermented extracts results in better inhibitory activity against dipeptidyl peptidase-4, exhibiting a certain hypoglycemic effect. Simultaneously, it also demonstrates good inhibitory activity against dipeptidyl peptidase-4 in the intestines and feces. Detailed Implementation

[0019] This invention provides a method for preparing a fermented extract of Rehmannia glutinosa, comprising the following steps: fermenting Rehmannia glutinosa with *Aspergillus cristatus*, extracting the fermented Rehmannia glutinosa with water, and collecting the filtrate. The *Aspergillus cristatus* strain used in this invention was purchased from the Beijing Center for Biological Preservation, with strain accession number AS3.462. Unless otherwise specified, all Rehmannia glutinosa used are commercially available products well-known to those skilled in the art. This invention utilizes *Aspergillus cristatus* fermentation of Rehmannia glutinosa to enhance some of its functional components.

[0020] In this invention, *Aspergillus cristatus* is cultured on PDA medium at 23-30°C for 2-6 days, and the spore suspension is collected and adjusted to a concentration of 1×10⁻⁶ using a hemocytometer. 6 -1×10 8 cfu / mL. The preferred temperature for culturing *Aspergillus cristatus* on PDA medium is 25-29℃, and the preferred time is 3-5 days; further preferably, the temperature is 28℃, and the preferred time is 4 days. The preferred concentration of the *Aspergillus cristatus* spore suspension is 5 × 10⁻⁶. 6 -7×10 7 cfu / mL, further preferably 1×10⁻⁶ 7 cfu / mL.

[0021] In this invention, the mixed fermentation step includes: mixing Rehmannia glutinosa with water, sterilizing, and then inoculating with a suspension of Aspergillus cristatus spores; the ratio of Rehmannia glutinosa, water, and Aspergillus cristatus spore suspension is 20-35g:6-18mL:1mL, preferably 22-33g:9-16mL:1mL, and more preferably 28g:12mL:1mL; the concentration of the Aspergillus cristatus spore suspension is 1×10⁻⁶. 6 -1×10 8 cfu / mL, selected as 5×10 6 -7×10 7 cfu / mL, further preferably 1×10⁻⁶ 7 cfu / mL.

[0022] In this invention, the ratio of fermented Rehmannia glutinosa to water is 1g:45-55mL, preferably 1g:47-52mL, and more preferably 1g:50mL; the extraction temperature is 60-70℃, the time is 1-2h, and the number of extractions is 1-3 times, preferably 62-68℃, the time is 1.2-1.8h, and the number of extractions is 2 times, and more preferably 65℃, the time is 1.5h, and the number of extractions is 2 times.

[0023] The present invention also provides a fermented extract of Rehmannia glutinosa obtained by the preparation method described above.

[0024] The present invention also provides a composition comprising a Rehmannia glutinosa fermented extract obtained by the preparation method and a Fuzhuan tea protease hydrolysate; wherein the weight ratio of the Rehmannia glutinosa fermented extract to the Fuzhuan tea protease hydrolysate is 1:0.5-3.5, preferably 1:0.8-3.0, and more preferably 1:1, 1:1.5, 1:2 or 1:2.5.

[0025] In this invention, the preparation method of the Fu brick tea protease hydrolysate includes the following steps: Fu brick tea is obtained by mixing *Aspergillus cristatus* with black tea and fermenting; the Fu brick tea is mixed with sodium hydroxide solution, placed in a water bath, and the pH is adjusted to 3.0-4.0; the precipitate is collected by centrifugation; the precipitate is mixed with water, the pH is adjusted to 6.5-7.5, and the mixture is dried to obtain crude fermented protein from black tea; the crude fermented protein from black tea is mixed with water, kept warm in a water bath, the pH is adjusted to 8-10, protease is added for enzymatic hydrolysis, the precipitate is removed by centrifugation, and the protease hydrolysate is collected to obtain the Fu brick tea protease hydrolysate. This invention uses *Aspergillus cristatus* fermentation to extract black tea, which can improve some functional components, such as catechins and theabrownins. The *Aspergillus cristatus* strain used in this invention was purchased from the Beijing Center for Biological Preservation, with strain preservation number AS3.462. Unless otherwise specified, the black tea used is a commercially available product well known to those skilled in the art.

[0026] In this invention, the controlled moisture content of the black tea is 25-35%, preferably 28-33%, and more preferably 30%. The black tea of ​​this invention needs to be sterilized before being inoculated with *Aspergillus cristatus*. The sterilization process of this invention involves steaming at 100°C for 8-12 minutes in an autoclave, preferably for 10 minutes at 100°C.

[0027] In this invention, the concentration of the *Aspergillus cristatus* spore suspension in fermented black tea is 1 × 10⁻⁶. 6 -1×10 8 cfu / mL, selected as 5×10 6 -7×10 7 cfu / mL, further preferably 1×10⁻⁶ 7 cfu / mL. The inoculation amount of the *Aspergillus cristatus* spore suspension described in this invention is 0.8-2% (v / w, mL / g) of the weight of the black tea, preferably 1-1.8%, and more preferably 1.25%. After inoculation with *Aspergillus cristatus*, the tea is piled at 65-75℃ for 1-3 hours, then allowed to develop its characteristic bloom at 25-30℃, and finally dried to obtain Fu brick tea.

[0028] In this invention, the mass-to-volume ratio of Fu brick tea to sodium hydroxide solution is 1g:20-30mL, preferably 1g:22-28mL, and more preferably 1g:25mL. The concentration of the sodium hydroxide solution in this invention is 0.1-0.3mol / L, preferably 0.2mol / L. The temperature of the water bath for mixing Fu brick tea and sodium hydroxide solution in this invention is 45-55℃ for 1-3 hours, preferably 48-52℃ for 1.3-2.5 hours, and more preferably 50℃ for 1.5 hours. After adjusting the pH, the precipitate collected by centrifugation is a protein precipitate, which needs to be repeatedly washed with deionized water with a pH of 3-4, rinsing 2-3 times. After rinsing, the precipitated protein is dissolved in deionized water, and the pH is adjusted to 6.5-7.5 to obtain crude protein from fermented black tea.

[0029] In this invention, the mass-to-volume ratio of fermented crude protein from black tea to water is 1-2 g: 450-550 mL, preferably 1.1-1.8 g: 480-520 mL, and more preferably 1.25 g: 500 mL. The fermented crude protein from black tea is mixed with water and kept in a water bath at 50-60°C for 10-30 min, preferably at 55°C for 20 min. The solution after water bath incubation is adjusted to a pH of 8.5-9.5 using NaOH solution, preferably to pH 9. A protease is added to the pH-adjusted solution, with an enzyme-to-substrate weight ratio of 1:45-55, preferably 1:48-53, and more preferably 1:50. The enzymatic hydrolysis time of the present invention is 4-6 hours and the pH is 8-10, preferably 4.5-5.5 hours and the pH is 8.5-9.5, and even more preferably 5 hours and the pH is 9; the protease of the present invention includes one or more of trypsin, pepsin, and alkaline protease, preferably trypsin, pepsin, or alkaline protease.

[0030] The present invention provides a method for preparing the composition, comprising the following steps: mixing Rehmannia glutinosa fermented extract and Fuzhuan tea protein hydrolysate according to the weight ratio.

[0031] This invention also provides the application of the Rehmannia glutinosa fermented extract obtained by the preparation method, or the Rehmannia glutinosa fermented extract, or the composition thereof, in the preparation of a product inhibiting dipeptidyl peptidase-4. The Rehmannia glutinosa fermented extract of this invention has a significant inhibitory effect on dipeptidyl peptidase-4. In mouse experiments, the composition significantly reduced the activity of DPP-4 in the intestine and feces of mice, increased plasma GLP-1 activity, and controlled postprandial 4-hour blood glucose and body weight. The fecal DPP-4 content of this invention reflects the level of intestinal DPP-4 to some extent, since it is mainly excreted from the intestine. The reduced DPP-4 activity in feces and the intestine also demonstrates the inhibitory effect of the composition on DPP-4 and the specific mechanism of its hypoglycemic effect. The DPP-4 of this invention can degrade GLP-1, and GLP-1 can promote insulin secretion, thereby lowering blood glucose. Increased GLP-1 activity can promote blood glucose reduction, demonstrating the effectiveness of DPP-4 inhibition in lowering blood glucose and the specific mechanism of the composition's hypoglycemic effect, and also indirectly demonstrating the inhibitory effect of the composition on DPP-4.

[0032] This invention also provides the application of the Rehmannia glutinosa fermented extract obtained by the preparation method, or the Rehmannia glutinosa fermented extract, or the composition thereof, in the preparation of hypoglycemic products. In mouse experiments, the composition of this invention effectively controlled blood glucose and body weight in mice 4 hours after a meal.

[0033] In this invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0034] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0035] The materials used in the following embodiments are as follows:

[0036] The strain of *Eurotium cristatum* has the accession number AS3.462 and was purchased from the Beijing Biotechnology Collection Center.

[0037] The Rehmannia glutinosa was purchased from Xi'an Kaimei Pharmaceutical Technology Co., Ltd.

[0038] The black tea was purchased from Jingchangsheng Fuzhuan Tea Co., Ltd.

[0039] Example 1: Fermentation and Extraction of Rehmannia glutinosa

[0040] (1) Incubate *Eurotium cristatum* on PDA medium at 28°C for 4 days, collect the spore suspension, and adjust the concentration to 1×10⁻⁶ using the hemocytometer method. 7 cfu / mL.

[0041] (2) First, slice 28g of Rehmannia glutinosa, mix it with 12mL of ultrapure water, sterilize it at 121℃ for 20min, cool it, and then inoculate it with 1mL of the above 1×10 under sterile conditions. 7 A CFU / mL suspension of *Eurotium cristatum* spores was incubated at 28°C for 14 days. 2g of fermented *Rehmannia glutinosa* tablets were placed in a beaker, 100mL of distilled water was added, and the mixture was extracted in a 65°C water bath for 1.5 hours. The filtrate was collected, and the extraction was repeated. The filtrates from both extractions were combined to obtain the fermented *Rehmannia glutinosa* extract. This extract was then freeze-dried to obtain the fermented freeze-dried *Rehmannia glutinosa* powder for later use.

[0042] Example 2

[0043] (1) Incubate *Eurotium cristatum* on PDA medium at 23°C for 2 days, collect the spore suspension, and adjust the concentration to 1×10⁻⁶ using the hemocytometer method. 6 cfu / mL.

[0044] (2) First, mix 20g of Rehmannia glutinosa slices with 6mL of ultrapure water, sterilize at 121℃ for 20min, cool, and then inoculate 1mL of the above 1×10 under sterile conditions. 6 A CFU / mL suspension of *Eurotium cristatum* spores was incubated at 23°C for 10 days. 2g of fermented *Rehmannia glutinosa* tablets were placed in a beaker, 90mL of distilled water was added, and the mixture was extracted in a 60°C water bath for 1 hour. The filtrate was collected, and the extraction was repeated. The filtrates from both extractions were combined to obtain the fermented *Rehmannia glutinosa* extract. This extract was then freeze-dried to obtain the fermented freeze-dried *Rehmannia glutinosa* powder for later use.

[0045] Example 3

[0046] (1) Incubate *Eurotium cristatum* on PDA medium at 30°C for 6 days, collect the spore suspension, and adjust the concentration to 1×10⁻⁶ using the hemocytometer method. 8 cfu / mL.

[0047] (2) First, mix 35g of Rehmannia glutinosa slices with 18mL of ultrapure water, sterilize at 121℃ for 20min, cool, and then inoculate 1mL of the above 1×10 under sterile conditions. 8 A CFU / mL suspension of *Eurotium cristatum* spores was incubated at 30°C for 18 days. 2g of fermented *Rehmannia glutinosa* tablets were placed in a beaker, 110mL of distilled water was added, and the mixture was extracted in a 70°C water bath for 2 hours. The filtrate was collected, and the extraction was repeated. The filtrates from both extractions were combined to obtain the fermented *Rehmannia glutinosa* extract. This extract was then freeze-dried to obtain the fermented freeze-dried *Rehmannia glutinosa* powder for later use.

[0048] Comparative Example 1

[0049] Take 2g of unfermented Rehmannia glutinosa tablets in a beaker, add 100mL of distilled water, and extract in a water bath at 65℃ for 1.5h. Collect the filtrate, repeat the extraction, and combine the two filtrates to obtain the non-fermented Rehmannia glutinosa extract. Then freeze-dry to obtain the non-fermented Rehmannia glutinosa freeze-dried powder for later use.

[0050] Experimental Example 1

[0051] The Rehmannia glutinosa fermented lyophilized powder prepared in Example 1 was dissolved in ultrapure water to prepare a Rehmannia glutinosa fermentation broth with a final test concentration of 0.1-1 mg / mL. The Rehmannia glutinosa non-fermented lyophilized powder prepared in Comparative Example 1 was dissolved in ultrapure water to prepare a Rehmannia glutinosa non-fermentation broth with a concentration of 0.5 mg / mL. The inhibition rate of DPP-4 by the Rehmannia glutinosa fermented lyophilized powder and the Rehmannia glutinosa non-fermented lyophilized powder was tested using a dipeptidyl peptidase-4 inhibitor screening kit (Biovision, K780-100).

[0052] DPP-4 inhibition rate (%) = (control well activity - sample well activity) / control well activity × 100%.

[0053] Table 1. Inhibition results of DPP-4 by Rehmannia glutinosa fermented broth or non-fermented broth.

[0054]

[0055] Table 1 shows that when the concentration of Rehmannia glutinosa fermentation broth is higher than 0.5 mg / mL, although the inhibition rate of DPP-4 still increases, the increase is not significant. Therefore, a concentration of 0.5 mg / mL was selected as the experimental concentration. The inhibition rate of DPP-4 in the non-fermentation broth of Rehmannia glutinosa was 24.52%, which was lower than the 49.07% after fermentation by *Aspergillus cristatus*.

[0056] Example 4

[0057] 1. Fermentation and extraction of black tea

[0058] Fu brick tea is made using *Eurotium cristatum* as raw material and black tea through processes such as steaming, piling, fermentation, and drying. The production steps are as follows:

[0059] (1) Incubate *Eurotium cristatum* on PDA medium at 28°C for 4 days, collect the spore suspension, and adjust the concentration to 1×10⁻⁶ using the hemocytometer method. 7 cfu / mL.

[0060] (2) Take 1 kg of black tea, control the moisture content to 30%, place it in an autoclave and steam at 100℃ for 10 min, then inoculate with 1.25% (v / w, 1.25 ml of spore suspension added to 100 g of black tea) 1×10 7 CFU / mL spore suspension was piled at 70℃ for 1.5 hours, then allowed to bloom at 28℃, and finally dried to obtain Fu brick tea.

[0061] (3) After crushing the Fu tea using a high-speed blender, pass it through an 80-mesh sieve to obtain Fu tea powder. Weigh 5g of Fu tea powder, add 125mL of 0.2mol / L NaOH solution, and heat in a 50℃ water bath for 1.5h, stirring once every 15min. Add hydrochloric acid to the solution to adjust the pH to 3.5, then centrifuge at 10000r / min for 25min, remove the supernatant, and collect the precipitate. Wash the precipitate repeatedly with deionized water with a pH of 3.5, twice, then dissolve the precipitated protein in deionized water, adjust the pH to 7, and freeze-dry to obtain crude protein from fermented black tea, which is then ready for use.

[0062] (4) Accurately weigh 1.25g of crude protein from fermented black tea, dissolve it in 500mL of ultrapure water to prepare a crude protein solution of 2.5mg / mL, and keep it in a water bath at 55℃ for 20min. Then adjust the pH value to 9 with 2mol / L NaOH solution, add trypsin (specification: 2500U / mg) at a ratio of 1:50 (w / w) of enzyme to substrate, and enzymatically hydrolyze for 5h, keeping the pH stable at 9 during the enzymatic hydrolysis. After the enzymatic hydrolysis is completed, keep it in a water bath at 95℃ for 20min to inactivate the trypsin, and then centrifuge at 4℃ and 10000r / min for 10min to remove the precipitate, thereby obtaining the protease hydrolysate. Freeze-dry to obtain Fuzhuan tea protease hydrolysate powder for later use.

[0063] Example 5

[0064] The difference from Example 4 is that the protease is pepsin.

[0065] Example 6

[0066] The difference from Example 4 is that the protease is an alkaline protease.

[0067] Comparative Example 2

[0068] Purchase finished Fu brick tea (purchased from Jingchangsheng Fu Brick Tea Co., Ltd.), and enzymatically hydrolyze it using trypsin at a concentration of 0.5 mg / mL (specification: 2500 U / mg) in the same manner as in Example 4 to obtain a protein hydrolysate. Freeze-dry the hydrolysate to obtain finished Fu brick tea protein hydrolysate powder for later use.

[0069] Experimental Example 2

[0070] The Fu brick tea protein hydrolysate powders prepared in Examples 4-6 were dissolved in ultrapure water to prepare Fu brick tea protein hydrolysate solutions with a final test concentration of 0.1-2 mg / mL. The finished Fu brick tea protein hydrolysate powder prepared in Comparative Example 2 was dissolved in ultrapure water to prepare a finished Fu brick tea protein hydrolysate solution with a final test concentration of 0.5 mg / mL. The inhibition rate of different protease hydrolysate products against DPP-4 was tested using a DPP-4 inhibitor screening kit (Biovision, K780-100).

[0071] Table 2. Inhibition results of DPP-4 by Fuzhuan tea protein hydrolysate or finished Fuzhuan tea protein hydrolysate.

[0072]

[0073] Table 2 shows that when the concentration of trypsin-hydrolyzed Fu brick tea protein fermentation broth is higher than 0.5 mg / mL, although the inhibition rate of DPP-4 still increases, the increase is not significant. Furthermore, the enzymatic hydrolysis effect of trypsin is higher than that of pepsin or alkaline protease at the same concentration. Therefore, a concentration of 0.5 mg / mL was selected as the experimental concentration. The inhibition rate of DPP-4 by the finished Fu brick tea protein hydrolysate was 47.34%, lower than the 52.68% inhibition rate of *Aspergillus cristatus* single-strain fermentation.

[0074] Example 7

[0075] The Rehmannia glutinosa fermented freeze-dried powder prepared in Example 1 and the Fuzhuan tea protease hydrolysate powder prepared in Example 4 were mixed in a ratio of 1:2 (w:w) to obtain a composition.

[0076] Examples 8-11

[0077] The difference from Example 7 is that in Examples 8-11, the Rehmannia glutinosa fermented freeze-dried powder prepared in Example 1 and the Fuzhuan tea protease hydrolysate powder prepared in Example 4 were mixed in weight ratios of 4:1, 2:1, 1:1 and 1:4, respectively, to obtain the composition.

[0078] Experimental Example 3

[0079] The compositions prepared in Examples 7-11 were dissolved in water to prepare concentrations of 0.5 mg / mL, and the inhibition rate of the compositions against dipeptidyl peptidase-4 was determined using a DPP-4 inhibitor screening kit (Biovision, K780-100).

[0080] Table 3. Inhibition results of each composition on DPP-4

[0081]

[0082] Table 3 shows that by studying the inhibition rate of DPP-4 by different ratios of Rehmannia glutinosa fermented freeze-dried powder and Fuzhuan tea protein hydrolysate powder, it was found that the inhibition rate of DPP-4 was the highest when the weight ratio of the two was 1:2.

[0083] Test Example 4

[0084] 1. Study on the inhibitory effect of Rehmannia glutinosa fermented freeze-dried powder and Fuzhuan tea protease hydrolysate powder on bacterial DPP-4.

[0085] The DPP-4 encoding gene was synthesized by a third-party gene synthesis company and cloned into a vector. A C-terminal label was added, and the plasmid was transformed into *E. coli* to induce recombinant protein expression. The bacterial protein was then collected and purified. The collected bacterial DPP-4 protein replaced the human DPP-4 protein in the DPP-4 inhibitor screening kit (Biovision, K780-100), and the inhibition rate of the combination of Fu brick tea protein hydrolysate and Rehmannia glutinosa fermentation broth against bacterial DPP-4 was determined using the same method.

[0086] Table 4. Inhibition results of each group of compositions on bacterial DPP-4.

[0087]

[0088] Table 4 shows that by studying the inhibition rate of different ratios of Rehmannia glutinosa fermented freeze-dried powder and Fuzhuan tea protein hydrolysate on DPP-4, it was found that the inhibition rate of DPP-4 was the highest when the mass ratio of the two was 1:2.

[0089] Furthermore, it was found that when the mass ratio of local yellow ginseng fermented freeze-dried powder to Fu brick tea protein hydrolysate was 4-2:1, the inhibition rate of dipeptidyl peptidase-4 was lower than that of Fu brick tea protein hydrolysate alone. As the proportion of Fu brick tea protein hydrolysate gradually increased, the inhibitory activity of dipeptidyl peptidase-4 also gradually increased. The inhibition rate was highest when the ratio reached 1:2. After that, the inhibition rate decreased with the increase of the ratio, but all of them exceeded the inhibition rate of Fu brick tea protein hydrolysate alone, indicating that the inhibition of dipeptidyl peptidase-4 by the combination is a synergistic effect.

[0090] 2. Study on the inhibitory effect of finished Fu brick tea protein hydrolysate powder and unfermented Rehmannia glutinosa freeze-dried powder on DPP-4 microbial inoculum.

[0091] The protease hydrolysate obtained from the purchased finished Fu brick tea and the obtained unfermented Rehmannia glutinosa freeze-dried powder were prepared into a liquid with a final concentration of 0.5 mg / mL at a ratio of 2:1 (w / w). The inhibition rates of the liquid against dipeptidyl peptidase-4 and bacterial dipeptidyl peptidase-4 were determined using the same method. The results showed that the inhibition rates were 55.74% and 57.96%, respectively, which were lower than those of the Rehmannia glutinosa fermented freeze-dried powder and the Fu brick tea protease hydrolysate combination after Aspergillus cristatus fermentation.

[0092] Experimental Example 5

[0093] Thirty-two C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and divided into control group, low-dose (50 mg / kg), high-dose (100 mg / kg), and model group, with eight mice in each group.

[0094] The normal control group was fed a normal diet (15% fat content). The remaining groups were fed a high-fat diet (60%) to establish an obesity model in mice aged 6-8 weeks, and fed continuously for 10 weeks until their weight reached 42-46g. Blood glucose and body weight were measured, and the mice were averaged. Mice in the low-dose and high-dose groups were fed the composition prepared in Example 7 (50mg / kg for low dose, 100mg / kg for high dose) for 6 consecutive weeks; the model group was fed the same amount of distilled water for 6 consecutive weeks. After the last administration, the intestinal and fecal DPP-4 activity, plasma GLP-1 activity, 4-hour postprandial blood glucose level, and body weight of the mice were measured.

[0095] Fecal DPP-4 activity assay: 60 mg of mouse feces was accurately weighed, placed in a 1.5 mL EP tube, and 1 mL of ultrapure water was added. After thorough homogenization, the mixture was centrifuged at 13000 rpm, and the supernatant was collected. The DPP-4 activity was detected using a DPP-4 activity assay kit (BioVision, K779-100).

[0096] Intestinal DPP-4 activity assay: 20 mg of mouse intestinal tissue was accurately weighed, placed in a 1.5 mL EP tube, and 1 mL of protein lysis buffer was added. After thorough homogenization, the mixture was centrifuged at 13000 rpm, and the supernatant was collected. DPP-4 activity was detected using a DPP-4 activity assay kit (BioVision, K779-100).

[0097] Plasma GLP-1 level determination: Blood was collected after the last administration, centrifuged at 3000 rpm at 4℃, and 50 μL of serum was collected. The GLP-1 level was detected using an active GLP-1 detection kit (IBL, RE53121).

[0098] Blood glucose level measurement 4 hours after meal: Blood was collected from the tail tip 4 hours after the last dose and the blood glucose value was measured using a Roche blood glucose meter (Roche Diagnostics Products (Shanghai) Co., Ltd., ACCU-Chek performa).

[0099] Table 5 Effects of the composition on DPP-4 activity in mice

[0100]

[0101] Table 6. Effects of the composition on plasma GLP-1 activity in mice fed a high-fat diet.

[0102]

[0103] Table 7 Effects of the composition on 4-hour postprandial blood glucose in mice fed a high-fat diet

[0104]

[0105] Table 8. Effects of the composition on body weight in mice fed a high-fat diet.

[0106]

[0107] The results in Table 5-8 show that, compared with the control group, the low-dose group (50 mg / kg) mice had significantly reduced intestinal and fecal DPP-4 activity, increased plasma GLP-1 activity, and controlled postprandial blood glucose and body weight. The high-dose group also had similar effects, and the effects were more significant.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a fermented extract of Rehmannia glutinosa, characterized in that, The process includes the following steps: mixing and fermenting *Eurotium cristatum* with *Rehmannia glutinosa*, extracting the fermented *Rehmannia glutinosa* with water, and collecting the filtrate. The mixed fermentation steps include: mixing Rehmannia glutinosa with water, sterilizing, and then inoculating with a suspension of Eurotium cristatum spores; the ratio of Rehmannia glutinosa, water, and Eurotium cristatum spore suspension is 20-35g: 6-18mL: 1mL, respectively; the concentration of the Eurotium cristatum spore suspension is 1×10⁻⁶. 6 -1×10 8 cfu / mL; The ratio of fermented Rehmannia glutinosa to water is 1g:45-55mL; the extraction temperature is 60-70℃, the extraction time is 1-2h, and the extraction is performed 1-3 times.

2. The Rehmannia glutinosa fermented extract obtained by the preparation method described in claim 1.

3. A composition, characterized in that, The preparation includes the Rehmannia glutinosa fermented extract and Fuzhuan tea protein hydrolysate obtained by the preparation method of claim 1; the weight ratio of the Rehmannia glutinosa fermented extract to the Fuzhuan tea protein hydrolysate is 1:0.5-3.5; The preparation of the Fu tea protein hydrolysate includes the following steps: Fu tea is obtained by mixing and fermenting *Aspergillus cristatus* with black tea; Fu tea is mixed with sodium hydroxide solution, placed in a water bath, and the pH is adjusted to 3.0-4.0; the precipitate is collected by centrifugation; the precipitate is mixed with water, the pH is adjusted to 6.5-7.5, and the mixture is dried to obtain crude fermented protein from black tea; the crude fermented protein from black tea is mixed with water, kept warm in a water bath, the pH is adjusted to 8-10, enzymatic hydrolysis is performed with protease, the precipitate is removed by centrifugation, and the protein hydrolysate is collected. The concentration of the *Eurotium cristatum* spore suspension was 1 × 10⁻⁶. 6 -1×10 8 cfu / mL; the inoculation amount of the *Aspergillus cristatus* spore suspension is 0.8-2% v / w, mL / g of the weight of the black tea; the mass-volume ratio of the Fu tea to the sodium hydroxide solution is 1g:20-30mL; the protease includes one or more of trypsin, pepsin, and alkaline protease.

4. The method for preparing the composition according to claim 3, characterized in that, The process includes the following steps: mixing Rehmannia glutinosa fermented extract and Fuzhuan tea protein hydrolysate in a specific weight ratio.

5. The application of the Rehmannia glutinosa fermented extract obtained by the preparation method of claim 1, or the Rehmannia glutinosa fermented extract of claim 2, or the composition of claim 3 in the preparation of hypoglycemic products.

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