Millettia specisoa flower extract as well as preparation method and application thereof

Abstract

The invention belongs to the field of development and utilization of beautiful millettia roots, and particularly relates to a beautiful millettia root flower extract and a preparation method and application thereof. Fresh flower buds in the initial flowering period of beautiful millettia roots are adopted as raw materials, fermentation liquor is prepared through fermentation of lactobacillus, lactobacillus casei and lactobacillus acidophilus, after sterilization, cellulase, pectin lyase and cellobiohydrolase are added for enzymolysis, enzymatic hydrolysate is obtained, after enzyme deactivation of the enzymatic hydrolysate, alcohol precipitation is conducted to remove precipitates, and the finished product is obtained. The prepared millettia specisoa flower extract can reduce blood sugar of rats with type 2 diabetes mellitus and regulate intestinal flora of the rats, and a new candidate medicine is provided for treatment of diabetes mellitus.

Description

Technical Field

[0001] This invention belongs to the field of development and utilization of *Smilax glabra*, specifically relating to an extract of *Smilax glabra* flowers, its preparation method, and its application. Background Technology

[0002] *Callerya speciosa* Schot., a plant belonging to the genus *Callerya* in the legume family, is the dried root of *Callerya speciosa*, mainly produced in Guangdong, Guangxi, and Hainan provinces of my country. Traditionally, the perennial root of *Callerya speciosa* is harvested for medicinal use, possessing functions such as tonifying the lungs, strengthening muscles and tendons, and enhancing immunity. *Callerya speciosa* enters its flowering period in June each year, with white flowers adorning the branches, characterized by a long flowering period, abundant flowers, and long flower spikes. The flowers of *Callerya speciosa* are neither medicinal nor edible. Cultivators often allow it to grow, flower, and fruit naturally, completing its life cycle. The medicinal value and large yield of the flowers have not received sufficient attention. Currently, for medicinal plants whose main medicinal part is the root, such as ginseng, notoginseng, and American ginseng, the flowers of their above-ground parts have been developed. These flowers contain rich saponins, amino acids, polysaccharides, and other components, possessing medicinal effects such as enhancing immunity, lowering blood sugar, and anti-tumor properties. They can be used for product development and for the extraction of key active substances such as saponins. Therefore, the high yield of *Smilax glabra* flowers and the chemical substances they contain are noteworthy, and their medicinal value urgently needs to be developed. Summary of the Invention

[0003] This invention provides an extract of *Smilax glabra* flower, its preparation method, and its application.

[0004] The technical solution of this invention is implemented as follows: A method for preparing an extract of *Smilax glabra* flower includes the following steps: Take *Smilax glabra* flowers, add 30-40% water by weight of the material, crush them, add *Lactobacillus acidophilus*, *Lactobacillus casei*, and *Lactobacillus acidophilus*, and ferment for 1-3 days to obtain fermentation liquid; *Lactobacillus acidophilus*, *Lactobacillus casei*, and *Lactobacillus acidophilus* were purchased from Shaanxi Benhe Biotechnology Co., Ltd.

[0005] After boiling the fermentation broth, cool it, then add cellulase, pectin lyase, and hemicellulase, and react at 25-35℃ for 30-60 minutes to obtain the enzymatic hydrolysis product. The enzymatic hydrolysis product is heated and boiled to inactivate microorganisms and enzymes, then cooled and filtered to obtain the filtrate. Add 95% ethanol to the filtrate, let it stand at 4°C for 12-15 hours, filter to remove the precipitate, and dry the supernatant to obtain the final product.

[0006] Furthermore, in step (1), the *Niu Dali* flower buds are fresh buds at the initial flowering stage; the weight ratio of *Lactobacillus*, *Lactobacillus casei*, and *Lactobacillus acidophilus* is 1:1:0.3-0.7; the total weight of the added *Lactobacillus*, *Lactobacillus casei*, and *Lactobacillus acidophilus* is 1%-5% of the weight of the fresh *Niu Dali* flower; the viable count of *Lactobacillus* is ≥10. 9 cfu / g; viable Lactobacillus casei count ≥10 10 cfu / g; Lactobacillus acidophilus ≥10 10 cfu / g.

[0007] Furthermore, in step (2), the fermentation liquid is boiled for 30 minutes and cooled to 30-35°C; the weight ratio of cellulase, pectin lyase and hemicellulase is 2-3:1-2:0.5-1.5; the total amount of enzymes added is 3-5% of the weight of fresh smilax glabra flowers.

[0008] Furthermore, in step (3), the mixture is heated and simmered for 30 minutes, then cooled to 20-30°C.

[0009] Furthermore, in step (4), the supernatant is first concentrated under reduced pressure to remove ethanol and obtain a concentrated solution, which is then freeze-dried at -80°C.

[0010] A preparation containing *Smilax glabra* flower extract, comprising the *Smilax glabra* flower extract obtained by the above method.

[0011] The application of the extract of *Smilax glabra* obtained by the above preparation method or the above preparation in the preparation of preparations for lowering blood sugar and / or regulating intestinal flora.

[0012] In the above applications, the extract or preparation can lower blood glucose levels in type 2 diabetic rats and also regulate the gut microbiota of type 2 diabetic rats.

[0013] Furthermore, the extract of *Smilax glabra* obtained by the preparation method or the preparations described above can increase the proportion of Firmicutes, Lactobacillus and / or *Acetobacter acetaminophen* in the intestine.

[0014] The beneficial effects of this invention are: This invention uses fresh flower buds from the early flowering stage of *Smilax glabra* as raw material. The broth is fermented with *Lactobacillus casei*, *Lactobacillus acidophilus*, and *Lactobacillus acidophilus*. After sterilization, cellulase, pectin lyase, and cellobiase are added for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The hydrolysate is then inactivated by enzyme inactivation and precipitated with alcohol to remove the precipitate. The resulting *Smilax glabra* flower extract can lower blood glucose levels in type 2 diabetic rats and regulate the intestinal flora, providing a new candidate drug for the treatment of diabetes. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1 A method for preparing an extract of *Smilax glabra* flower includes the following steps: (1) Take *Smilax glabra* flowers, add 30% water by weight of the material, crush them, add *Lactobacillus citrinum*, *Lactobacillus casei* and *Lactobacillus acidophilus*, ferment for 1 day to obtain fermentation liquid; the weight ratio of *Lactobacillus citrinum*, *Lactobacillus casei* and *Lactobacillus acidophilus* is 1:1:0.3; the total weight of the added *Lactobacillus citrinum*, *Lactobacillus casei* and *Lactobacillus acidophilus* is 1% of the weight of fresh *Smilax glabra* flowers; the viable count of *Lactobacillus citrinum* is 10. 9 cfu / g; Lactobacillus casei viable count 10 10 cfu / g; Lactobacillus acidophilus 10 10 cfu / g. In this embodiment, the *Smilax glabra* flower buds are fresh flower buds in their initial flowering stage.

[0017] Lactobacillus, Lactobacillus casei, and Lactobacillus acidophilus were purchased from Shaanxi Benhe Biotechnology Co., Ltd.

[0018] (2) After boiling the fermentation liquid for 30 minutes to sterilize it, cool it to 30°C, and then add cellulase, pectin lyase and hemicellulase. React at 25°C for 60 minutes to obtain the enzymatic hydrolysis product. The weight ratio of cellulase, pectin lyase and hemicellulase is 2:1:0.5. The total amount of enzymes added is 3% of the weight of fresh smilax glabra flowers.

[0019] (3) Heat the enzymatic hydrolysis product to boiling for 30 minutes to inactivate the enzyme, cool to 20°C, and filter to remove insoluble matter to obtain filtrate; (4) Add 95% ethanol to the filtrate, let it stand at 4°C for 12 hours, filter to remove the precipitate, concentrate the supernatant under reduced pressure, evaporate the ethanol to obtain the concentrate, and freeze dry the concentrate at -80°C to obtain the final product.

[0020] Example 2 A method for preparing an extract of *Smilax glabra* flower includes the following steps: (1) Take *Smilax glabra* flowers, add 35% water by weight of the material, crush them, add *Lactobacillus citrinum*, *Lactobacillus casei* and *Lactobacillus acidophilus*, ferment for 2 days to obtain fermentation liquid; the weight ratio of *Lactobacillus citrinum*, *Lactobacillus casei* and *Lactobacillus acidophilus* is 1:1:0.5; the total weight of *Lactobacillus citrinum*, *Lactobacillus casei* and *Lactobacillus acidophilus* added is 3% of the weight of fresh *Smilax glabra* flowers; the viable count of *Lactobacillus citrinum* is 10. 10 cfu / g; Lactobacillus casei viable count 1011 cfu / g; Lactobacillus acidophilus 10 11 cfu / g. In this embodiment, the *Smilax glabra* flower buds are fresh flower buds in their initial flowering stage.

[0021] Lactobacillus, Lactobacillus casei, and Lactobacillus acidophilus were purchased from Shaanxi Benhe Biotechnology Co., Ltd.

[0022] (2) After boiling the fermentation liquid for 30 minutes to sterilize it, cool it to 35°C; then add cellulase, pectin lyase and hemicellulase, and react at 30°C for 50 minutes to obtain the enzymatic hydrolysis product; the weight ratio of cellulase, pectin lyase and hemicellulase is 2.5:1.5:1; the total amount of enzymes added is 3-5% of the weight of fresh smilax glabra flowers.

[0023] (3) Heat the enzymatic hydrolysis product to boiling for 30 minutes to inactivate the enzyme, cool to 25°C, and filter to remove insoluble matter to obtain filtrate; (4) Add 95% ethanol to the filtrate, let it stand at 4°C for 13 hours, filter to remove the precipitate, concentrate the supernatant under reduced pressure, evaporate the ethanol to obtain the concentrate, and freeze dry the concentrate at -80°C to obtain the final product.

[0024] Example 3 A method for preparing an extract of *Smilax glabra* flower includes the following steps: (1) Take 40% of the mass of water from the flowers of *Smilax glabra*, crush them, add lactobacillus, *Lactobacillus casei* and *Lactobacillus acidophilus*, and ferment for 3 days to obtain fermentation liquid; the weight ratio of lactobacillus, *Lactobacillus casei* and *Lactobacillus acidophilus* is 1:1:0.7; the total weight of the added lactobacillus, *Lactobacillus casei* and *Lactobacillus acidophilus* is 5% of the weight of fresh *Smilax glabra*; the viable count of lactobacillus is 10. 9 cfu / g; Lactobacillus casei viable count 10 11 cfu / g; Lactobacillus acidophilus 10 11 cfu / g. In this embodiment, the *Smilax glabra* flower buds are fresh flower buds in their initial flowering stage.

[0025] Lactobacillus, Lactobacillus casei, and Lactobacillus acidophilus were purchased from Shaanxi Benhe Biotechnology Co., Ltd.

[0026] (2) After boiling the fermentation liquid for 30 minutes to sterilize it, cool it to 35°C; then add cellulase, pectin lyase and hemicellulase, and react at 35°C for 60 minutes to obtain the enzymatic hydrolysis product; the weight ratio of cellulase, pectin lyase and hemicellulase is 3:2:1.5; the total amount of enzymes added is 3-5% of the weight of fresh smilax glabra flowers.

[0027] (3) Heat the enzymatic hydrolysis product to boiling for 30 minutes to inactivate the enzyme, cool to 30°C, and filter to remove insoluble matter to obtain filtrate; (4) Add 95% ethanol to the filtrate, let it stand at 4°C for 15 hours, filter to remove the precipitate, concentrate the supernatant under reduced pressure, evaporate the ethanol to obtain the concentrate, and freeze dry the concentrate at -80°C to obtain the final product.

[0028] The extract of *Smilax glabra* obtained by the above method has significant hypoglycemic activity and can be used directly as an adjunct to hypoglycemic preparations. Alternatively, certain excipients, such as flavoring agents and excipients, can be added as needed to prepare corresponding preparations.

[0029] Effects of different preparation methods on hypoglycemic activity Healthy, clean-grade male SD rats, weighing 200–220 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After a week of acclimatization feeding, they were fed a normal diet and a high-fat, high-sugar diet, respectively. After 8 weeks, they were fasted for 12 hours but allowed free access to water, and were administered a dose of 35 mg / kg. -1 The control group received an intraperitoneal injection of STZ (pH 4.4), while the control group received an equal volume of citrate buffer. Three days after STZ injection, fasting blood glucose (FBG) was measured via tail vein sampling. A fasting blood glucose level ≥7.8 mmol / L was required. -1 To establish a standard diabetic model, successfully modeled rats were randomly divided into groups of 5 for the experiment. Metformin hydrochloride tablets, streptozotocin, citric acid, and trisodium citrate were purchased from Shenzhen Nord Biotechnology Co., Ltd. The high-sugar, high-fat diet was purchased from Beijing Botai Hongda Biotechnology Co., Ltd., with the following composition: 10% lard, 15% sucrose, 7% egg yolk, 66.5% ordinary feed, 0.5% bile salts, and 1% cholesterol.

[0030] Sample 1: The extract of *Smilax glabra* obtained in Example 2 of this invention.

[0031] Sample 2: Prepared according to the method in Example 2 of this invention, except that no bacterial agent is added during fermentation.

[0032] Sample 3: Prepared according to the method in Example 2 of this invention, except that no enzyme is added for enzymatic hydrolysis.

[0033] Sample 4: The preparation method is the same as in Example 2 of this invention, except that boiling is not performed after fermentation.

[0034] Sample 5: Prepared according to the method in Example 2 of this invention, except that boiling is not performed after enzymatic hydrolysis.

[0035] Samples 1-5 were administered at a daily dose of 0.5 g·kg. -1 The samples were administered according to body weight. The positive control group received 0.2 g / kg daily. -1 Metformin was administered based on body weight. The model group and the normal control group received 0.5 g / kg daily. -1Rats were given physiological saline to monitor body weight. Samples 1-5, the positive control group, and the model group consisted of successfully modeled rats, while the normal group consisted of rats fed a standard diet. After 4 weeks of continuous feeding, various indicators were measured. All rats were fasted but allowed free water for 12 hours, and blood was collected from the tail vein to measure fasting blood glucose (FBG). On the morning of the day before the experiment, rats were placed in sterile cages, secured, and their tails were lifted. The lower abdomen was gently pressed with fingers, and feces were collected using a toothpick or sterile forceps into a 2 ml sterile EP tube. The tube was carefully closed. The tube containing feces was immediately stored at -80℃ and transported on dry ice. The intestinal microbiota composition was determined using 16S RNA technology.

[0036] Statistical analysis was performed using SPSS 21.0 software. Data are expressed as mean ± standard deviation. The results are shown in Tables 1 and 2.

[0037] Table 1. Effects of *Smilax glabra* flower extract on fasting blood glucose (FBG) in type 2 diabetic rats.

[0038] Grouping <![CDATA[Blood glucose before administration (mmol·L -1 )]]> <![CDATA[Blood glucose in the fourth week (mmol·L -1 )]]> normal group 4.50±0.18 4.63±0.23 Model group 17.28±1.64 22.27±2.84 Positive control group 17.13±2.15 15.23±2.45 Sample 1 16.84±2.37 13.81±2.48 Sample 2 17.82±1.86 17.40±3.12 Sample 3 17.68±2.81 16.72±3.25 Sample 4 18.07±3.05 19.57±4.76 Sample 5 18.23±2.73 16.58±2.63 The results showed that the FBG level in the model group rats was significantly increased compared with the normal group. Compared with the model group, the positive control group and samples 1-5 could reduce FBG in rats to varying degrees. Among them, sample 1 (Example 2) showed the most significant reduction in blood glucose, which was not only significantly lower than that in the model group but also lower than that in the positive control group. Although samples 2-5 also had hypoglycemic activity, their hypoglycemic activity was significantly reduced compared with sample 1.

[0039] Table 2. Effects of *Smilax glabra* flower extract on key gut microbiota in type 2 diabetic rats.

[0040] Grouping Firmicutes (%) Lactobacillus (%) Myxophilic Ekkermansia (%) normal group 2.59±0.63 1.55±0.41 1.03±0.24 Model group 1.22±0.75 0.19±0.17 0.08±0.01 Positive control group 1.69±0.14 0.64±0.04 0.23±0.02 Sample 1 2.14±0.56 1.03±0.15 0.88±0.02 Sample 2 1.57±0.51 0.57±0.03 0.51±0.03 Sample 3 1.61±0.38 0.60±0.02 0.47±0.11 Sample 4 1.58±0.46 0.51±0.01 0.50±0.06 Sample 5 1.47±0.30 0.54±0.02 0.34±0.05 16S-RNA sequencing revealed the greatest changes in Firmicutes, Lactobacillus, and Acetobacter xanthipes. Compared with the normal group, the proportions of all three bacterial groups in the model group were significantly reduced; compared with the model group, the proportions of all three bacterial groups in the positive control group and samples 1-5 were increased to varying degrees, with sample 1 showing the most significant increase, not only significantly higher than the positive control group but also significantly higher than samples 2-5.

[0041] The above data demonstrate that the extract of *Smilax glabra* obtained by this invention can not only reduce FBG in type 2 diabetic rats, but also regulate the intestinal flora of rats.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an extract of Mirabilis jalapa, characterized by, The method comprises the following steps: (1) taking the cow's strength flower and adding 30-40% of the mass of the material water, crushing, then adding lactobacillus, lactobacillus casei and lactobacillus acidophilus, and fermenting for 1-3 days to obtain a fermentation liquor; the lactobacillus, lactobacillus casei and lactobacillus acidophilus are purchased from Shaanxi Benhe Biological Engineering Co., Ltd. (2) after boiling the fermentation liquor, cooling, then adding cellulase, pectin lyase and hemicellulase, and reacting at 25-35℃ for 30-60min to obtain an enzymatic product; (3) heating and decocting the enzymatic product to inactivate the microorganisms and enzymes, cooling, and filtering to obtain a filtrate; (4) adding 95% volume concentration ethanol to the filtrate, standing at 4℃ for 12-15h, filtering to remove the precipitate, and drying the supernatant to obtain the product.

2. The production method according to claim 1, wherein In the step (1), the fresh flower buds of Millettia speciosa Benth. are in early flowering stage; the weight ratio of Lactobacillus, Lactobacillus casei and Lactobacillus acidophilus is 1:1:0.3-0.7; the total weight of Lactobacillus, Lactobacillus casei and Lactobacillus acidophilus added is 1%-5% of the weight of the fresh Millettia speciosa Benth.; the viable count of Lactobacillus is ≥10 9 cfu / g; the viable count of Lactobacillus casei is ≥10 10 cfu / g; and the viable count of Lactobacillus acidophilus is ≥10 10 cfu / g.

3. The production method according to claim 1, wherein In the step (2), the fermentation liquor is boiled for 30min and cooled to 30-35℃; the weight ratio of cellulase, pectin lyase and hemicellulase is 2-3:1-2:0.5-1.5; and the total amount of the added enzymes is 3-5% of the mass of the fresh cow's strength flower.

4. The production method according to claim 1, wherein In the step (3), heating and decocting for 30min, and cooling to 20-30℃.

5. The production method according to claim 1, wherein In the step (4), the supernatant is first concentrated under reduced pressure, and the ethanol is distilled off to obtain a concentrated solution, which is freeze-dried at-80℃.

6. A preparation containing a cow's strength flower extract, which contains the cow's strength flower extract prepared by the above method.

7. Use of the cow's strength flower extract prepared by the preparation method of any one of claims 1-5 or the preparation of claim 6 in the preparation of a preparation for reducing blood sugar and / or regulating intestinal flora.

8. Use according to claim 7, wherein the compound is ###0002### The extract or the preparation can reduce the blood sugar of type 2 diabetic rats.

9. The use according to claim 7, wherein the compound is ###00003### 8 or ###00004### 9. The extract or the preparation can regulate the intestinal flora of type 2 diabetic rats.

10. The use according to claim 7, wherein the compound is ###00006### or a pharmaceutically acceptable salt thereof. The extract or the preparation can increase the proportion of the intestinal Firmicutes, lactobacillus and / or mucinophilic-Akkermansia bacteria. The extract or the preparation can increase the proportion of the intestinal Firmicutes, lactobacillus and / or mucinophilic-Akkermansia bacteria.