Functional oral liquid based on scandent hop and capable of promoting secretion of endogenous GLP-1 and preparation method of functional oral liquid

Abstract

The invention provides a humulus japonicus-based functional oral liquid capable of promoting endogenous GLP-1 secretion. The oral liquid is prepared by mixing a humulus japonicus mixed extracting solution, a suspending aid, a preservative and water, wherein the humulus scandens mixed extracting solution is prepared by mixing humulus scandens leaves, humulus scandens roots, humulus scandens stems and radix gentianae and then adding medical alcohol for extraction. The specific preparation method comprises the following steps: S1, taking fresh humulus scandens leaves, humulus scandens roots, humulus scandens stems and radix gentianae, cleaning, drying and crushing, putting mixed powder and medical alcohol into an extractor, starting microwave-assisted extraction, separating and sieving after the extraction is completed, concentrating and centrifuging an extracting solution, and taking supernate, so as to obtain a humulus scandens mixed extracting solution; s2, uniformly mixing the humulus scandens mixed extracting solution, a suspending aid, a preservative and water to obtain the oral liquid. The oral liquid prepared by the invention can remarkably promote secretion of endogenous GLP-1, is a healthy beverage, can play roles in reducing blood sugar, losing weight and the like, and further develops the value of humulus plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of functional oral liquids, and particularly relates to a functional oral liquid based on Humulus scandens that can promote the secretion of endogenous GLP-1 and a preparation method thereof. Background Art

[0002] Plants of the genus Humulus are widely distributed in many regions of China, and common specific varieties include Humulus scandens, Humulus lupulus, Humulus yunnanensis, etc. Most plants of the genus Humulus have rich medicinal value, forage value, and ecological value. In terms of medicinal value: The whole plant can be used as medicine, with a sweet and bitter taste and a cold nature; it has the effects of clearing heat and detoxifying, and promoting diuresis and relieving stranguria, and can treat diseases such as cough due to lung heat, lung abscess, vexation and thirst due to deficiency-heat, stranguria with fever, edema, dysuria, diarrhea due to damp-heat, toxic sores and ulcers, and skin itching. In terms of forage value: It has a high yield and a large amount of leaves, and contains rich crude protein, crude fiber, crude fat, as well as trace elements, vitamins, etc. The nutritional components are rich, and it has a high forage value; it can be used as fresh forage, silage, or dried hay, and can also be used as a feed additive to improve the environmental adaptability of animals, regulate nutritional balance, and enhance digestive and absorption functions. In terms of ecological value: Its carpet-like covering layer can play a significant soil and water conservation benefit in the same year, and it has a high forage yield and good quality. It is an excellent pioneer grass species for slope protection and soil fixation and a good dual-purpose forage grass.

[0003] However, it also has its disadvantages. For example: As a widely distributed field weed, its spread and proliferation pose a serious threat to agricultural production, the ecological environment, and human life. Humulus scandens is widely distributed. After invading farmland, it often becomes a noxious weed, growing mixed with crops, competing for light, space, water, and nutrients, and inhibiting the normal growth of crops, shrubs, and trees. Humulus scandens is covered with barbs, and its vines are flexible, which also brings great trouble to crop harvesting. In addition, Humulus scandens is one of the sensitizing plants for autumn hay fever in China, which will affect people's normal life.

[0004] Therefore, it is urgent to develop more extensive uses for plants of the genus Humulus, reduce its adverse effects, and further improve its commercial value by using this common and widespread plant. Summary of the Invention

[0005] In view of this, the present invention selects Humulus scandens leaves, Humulus scandens roots, Humulus scandens stems, and Gentiana scabra roots for extraction, and further prepares an oral liquid with a fresh fragrance of herbal plants, a bright green color, and a sweet aftertaste. The prepared oral liquid has the function of promoting the secretion of endogenous GLP-1 and has commercial value.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A functional oral liquid based on Humulus scandens that can promote the secretion of endogenous GLP-1, which is prepared by mixing a Humulus scandens mixed extract, a suspending agent, a preservative, and water; Among them, the mixed extract of Humulus scandens is prepared by mixing Humulus scandens leaves, Humulus scandens roots, Humulus scandens stems, and Gentiana scabra roots and then adding medical alcohol for extraction.

[0007] The suspending agent includes xanthan gum and locust bean gum; The preservatives include vitamin C and natamycin.

[0008] A preparation method of the above oral liquid includes the following steps: S1. Take fresh Humulus scandens leaves, Humulus scandens roots, Humulus scandens stems, and Gentiana scabra roots, wash, dry, pulverize, place the mixed powder and medical alcohol in an extractor, start microwave-assisted extraction, after extraction is completed, separate and sieve, concentrate the extract, centrifuge, and take the supernatant to obtain the mixed extract of Humulus scandens; S2. Mix the mixed extract of Humulus scandens, the suspending agent, the preservative, and water evenly to prepare the oral liquid.

[0009] Further, the mass ratio of Humulus scandens leaves, Humulus scandens roots, Humulus scandens stems, and Gentiana scabra roots is (5 - 12) : (2 - 3) : (2 - 3) : (2 - 3); The mass ratio of the mixed powder to medical alcohol is 1 : (20 - 30), and the concentration of the medical alcohol is 60% - 95%.

[0010] Further, in step S1, the drying temperature is 80 - 95°C, and the drying time is 2 - 3 h.

[0011] Further, in step S1, the sieve mesh number is 30 - 80 meshes.

[0012] Further, in step S1, the microwave power is 480W - 550W, and the extraction time is 15 - 25 min.

[0013] Further, the concentration conditions in step S1 are: temperature 40 - 75°C, pressure 10 - 100 mbar, rotation speed 60 - 120 rpm; the volume after concentration is 1 / 3 - 1 / 2 of the original volume.

[0014] Further, the centrifugation conditions in step S1 are: rotation speed 700 - 900 r / min, time 7 - 9 min.

[0015] Further, in step S2, the volume of the mixed extract of Humulus scandens accounts for 50 - 75% of the volume of the finished oral liquid; The content of the suspending agent in the finished oral liquid is 0.08 - 0.15%; The content of the preservative in the finished oral liquid is 0.015 - 0.02%.

[0016] Compared with the prior art, the beneficial effects of the present invention are: The present invention uses Humulus scandens leaves, Humulus scandens roots, Humulus scandens stems, and Gentiana scabra roots to prepare an oral liquid with the function of promoting endogenous GLP-1 secretion, reducing the adverse aspects of it as a weed, and fully developing its commercial value. The prepared oral liquid can achieve a GLP-1 concentration of 0.89 pg / mL in in vitro cell experiments and a GLP-1 concentration of 18.26 pg / mL in the serum in in vivo experiments, both far higher than those of the control group. In addition, in the in vivo experiment, the blood glucose also decreased from 16.77 mmol / L to 14.39 mmol / L. Detailed implementation mode

[0017] The following further details the present invention in combination with specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0018] The functional oral liquid based on Humulus scandens provided by the present invention, which can promote endogenous GLP-1 secretion, is prepared according to the following method: S1. Weigh fresh Humulus scandens leaves, Humulus scandens roots, Humulus scandens stems, and Gentiana scabra roots in a mass ratio of (5 - 12):(2 - 3):(2 - 3):(2 - 3) in sequence, wash them, dry them at 80 - 95 °C for 2 - 3 h, and crush them with a wall breaker to obtain a mixed powder; place the mixed powder in an extractor, add medical alcohol with a concentration of 60% - 95% and a volume 20 - 30 times that of the mixed powder, adjust the microwave to 480 W - 550 W for extraction for 15 - 25 min; after extraction, let it stand at room temperature for extraction and sieve through a sieve (30 - 80 mesh), and concentrate the filtrate to 1 / 3 - 1 / 2 of the original volume at a temperature of 40 - 75 °C, a pressure of 10 - 100 mbar, and a rotation speed of 60 - 120 rpm to obtain a concentrated solution; centrifuge the concentrated solution at a rotation speed of 700 - 900 r / min for 7 - 9 min, and take the supernatant to obtain the Humulus scandens mixed extract.

[0019] S2. Mix the Humulus scandens mixed extract, suspending agent, preservative, and water evenly to prepare the oral liquid. The prepared oral liquid has the fragrance of herbal plants, presents a bright green color, and has a sweet aftertaste. In the finished oral liquid: the volume of the Humulus scandens mixed extract accounts for 50 - 75% of the volume of the finished oral liquid; the content of the suspending agent in the finished oral liquid is 0.08 - 0.15%; the content of the preservative in the finished oral liquid is 0.015 - 0.02%.

[0020] Among them, the suspending agent includes xanthan gum and locust bean gum; the preservative includes vitamin C and natamycin.

[0021] Example 1 This example provides a functional oral liquid based on Humulus scandens, which can promote endogenous GLP-1 secretion. The specific process is as follows: Place 5 parts of Humulus scandens leaves, 2 parts of Humulus scandens roots, 2 parts of Humulus scandens stems, and 2 parts of Gentiana scabra roots in a drying oven at 80 °C for drying treatment, and the drying time is 3 hours. Subsequently, use a wall breaker for crushing treatment, and place the mixed powder and 90% ethanol in a round-bottom flask at a mass ratio of 1:20. Adopt microwave-assisted extraction method for extraction (microwave power is 480 W, microwave time is 15 minutes). After standing at room temperature, extract and sieve (30 mesh), and then concentrate to 1 / 3 of the original volume at 40 °C, 100 mbar, and 60 rpm to obtain a concentrated solution; centrifuge the concentrated solution at a speed of 700 r / min for 9 min, and take the supernatant to obtain the mixed extract of Humulus scandens.

[0022] Mix the obtained mixed extract of Humulus scandens, xanthan gum, vitamin C and water evenly to prepare the oral liquid. Among them, the volume of the mixed extract of Humulus scandens in the finished oral liquid accounts for 60% of the volume of the finished oral liquid, the mass content of xanthan gum in the finished oral liquid is 0.15%, and the mass content of vitamin C in the finished oral liquid is 0.015%.

[0023] Example 2 This example provides a functional oral liquid based on Humulus scandens that can promote the secretion of endogenous GLP-1, and the specific process is as follows: Place 12 parts of Humulus scandens leaves, 3 parts of Humulus scandens roots, 3 parts of Humulus scandens stems, and 3 parts of Gentiana scabra roots in a drying oven at 95 °C for drying treatment, and the drying time is 2 hours. Subsequently, use a wall breaker for crushing treatment. Place it in a round-bottom flask with 60% ethanol at a ratio of 1:30. Adopt microwave-assisted extraction method for extraction (microwave power is 550 W, microwave time is 15 minutes). After standing at room temperature, extract and sieve (80 mesh), and then concentrate to 1 / 3 of the original volume at 75 °C, 10 mbar, and 120 rpm to obtain a concentrated solution; centrifuge the concentrated solution at a speed of 900 r / min for 7 min, and take the supernatant to obtain the mixed extract of Humulus scandens.

[0024] Mix the obtained mixed extract of Humulus scandens, locust bean gum, natamycin and water evenly to prepare the oral liquid. Among them, the volume of the mixed extract of Humulus scandens in the finished oral liquid accounts for 75% of the volume of the finished oral liquid, the mass content of locust bean gum in the finished oral liquid is 0.08%, and the mass content of natamycin in the finished oral liquid is 0.02%.

[0025] Example 3 This example provides a functional oral liquid based on Humulus scandens that can promote the secretion of endogenous GLP-1, and the specific process is as follows: 8 parts of Humulus leaves, 3 parts of Humulus roots, 3 parts of Humulus stems, and 2 parts of Gentiana roots were placed in a drying oven at 90°C for 2.5 hours. Subsequently, they were crushed using a wall breaker. They were placed in a round-bottom flask with 75% ethanol at a ratio of 1:25. Microwave-assisted extraction was used for extraction (microwave power was 500W and microwave time was 15 minutes). After standing at room temperature, the extract was sieved (50 mesh) and then rotary evaporated at 60°C, 50mbar, and 100rpm. After concentration, the volume was 1 / 2 of the original volume, and a Humulus composite extract was obtained.

[0026] The obtained Humulus japonicus mixed extract, locust bean gum, vitamin C and water are uniformly mixed to prepare an oral liquid, wherein the volume of the Humulus japonicus mixed extract in the finished oral liquid accounts for 65% of the volume of the finished oral liquid, the mass content of the locust bean gum in the finished oral liquid is 0.1%, and the mass content of the vitamin C in the finished oral liquid is 0.02%.

[0027] Comparative Example 1 This embodiment provides a functional oral liquid based on Humulus japonicus that can promote the secretion of endogenous GLP-1. The specific process is basically the same as that of Example 3, except that: "extraction is carried out using a microwave-assisted extraction method, the microwave power is 550 W, the temperature is 120°C, and the microwave time is 15 minutes" is replaced by "reacting in a constant temperature water bath at 45°C for 55 minutes", and the rest remains unchanged.

[0028] Comparative Example 2 This embodiment provides a functional oral liquid based on Humulus japonicus that can promote the secretion of endogenous GLP-1. The specific process is basically the same as that of Example 3, except that: "extraction is carried out using a microwave-assisted extraction method, the microwave power is 550 W, the temperature is 120° C., and the microwave time is 15 minutes" is replaced by "reacting in a constant temperature water bath at 60° C. for 55 minutes", and the rest remains unchanged.

[0029] Comparative Example 3 This comparative example provides a functional oral liquid based on Humulus japonicus that can promote the secretion of endogenous GLP-1 and a preparation method. The raw materials and the preparation method are basically the same as those in Example 3, except that: no gentian root is added, the weight proportion of Humulus japonicus leaves is 10 parts, the weight proportion of Humulus japonicus roots is 3 parts, the weight proportion of Humulus japonicus stems is 3 parts, and the rest remain unchanged.

[0030] Comparative Example 4 This comparative example provides a functional oral liquid based on Humulus japonicus that can promote the secretion of endogenous GLP-1 and a preparation method. The raw materials and the preparation method are basically the same as those in Example 3, except that: Humulus japonicus roots and Humulus japonicus stems are not added, the weight proportions of Humulus japonicus leaves are 8 parts, the weight proportions of Gentiana scabra roots are 2 parts, and the rest remain unchanged.

[0031] Comparative Example 5 This comparative example provides a functional oral liquid based on Humulus scandens that can promote the secretion of endogenous GLP-1 and its preparation method. The raw materials and preparation method are the same as those in Example 3, except that: the gentian root is replaced with an equal mass of Dendrobium officinale, and the rest remains unchanged.

[0032] Furthermore, in order to understand the effects of the oral liquids prepared in the above examples and comparative examples, the following tests were also carried out: 1. Intestinal fermentation of different oral liquids using in vitro intestinal simulation fermentation technology In vitro intestinal simulation: Dissolve arabinogalactan (1 g), apple pectin (2 g), xylan (1 g), potato starch (3 g), glucose (0.4 g), yeast extract (3 g), peptone (1 g), mucin (4 g) and L-cysteine (0.5 g) in 1 L of physiological saline to prepare the culture medium. Adjust the pH value of the culture medium to 6 using sodium hydroxide and perform autoclaving at 120 °C for 20 minutes. Subsequently, divide the culture medium into three series-connected fermenters to simulate the ascending colon (80 mL, pH = 5.6), transverse colon (100 mL, pH = 6.2) and descending colon (120 mL, pH = 6.8), and the flow rate of the device is 6.25 mL / h.

[0033] Intestinal simulation of diabetic patients: Before the start of the experiment, inoculate 20 g / mL of the suspension prepared from the feces of diabetic patients into each fermenter, and control the temperature at 37 °C. Each fermenter is continuously stirred with a magnetic stirrer at 200 rpm to simulate intestinal peristalsis. Nitrogen is filled to maintain the anaerobic environment in the fermenter. Each fermenter is independently connected to a bottle containing alkaline liquid (0.1 g / L sodium hydroxide), and the alkaline liquid is evenly delivered to the fermenter through a constant flow pump to maintain the preset pH value of each fermenter.

[0034] The specific operation process is as follows: First, inoculate fresh fecal solution (20 g / mL) into 3 colon reactors respectively. After inoculation, run the system for 14 days to maintain the stability of microorganisms. Subsequently, apply 5 mL of the oral liquid to the 3 reactors continuously for 7 days. Collect the substances in the reactors on the 7th day, and centrifuge to collect the supernatant for subsequent cell experiments.

[0035] Cell culture: Routinely passage GLUTag cells (a cell line that can secrete GLP-1) using high-glucose medium. Seed 10,000 cells in a 6-well plate, and apply the culture medium containing different fermentation broths after 24 hours of culture. After treatment for 24 hours, perform freeze-thaw treatment on the cells.

[0036] ELISA analysis: Use the ELISA method to analyze the GLP-1 secretion ability of GLUTag cells in response to different fermentation broths.

[0037] The GLP-1 secretagogue effects of different oral liquids after intestinal fermentation were compared through in vitro cell experiments, and the results are shown in Table 1 (the reason for such an experimental setup is that the secretion of GLP-1 is mainly carried out by enteroendocrine cells. Enteroendocrine cells are exposed to the intestinal environment, and only the drug components after microbial metabolism can affect the GLP-1 secretion function of enteroendocrine cells). The control group did not add oral liquid.

[0038] Table 1

[0039] As can be seen from Table 1, after being treated with the intestinal fermentation broth derived from Example 3, GLUTag cells had the highest GLP-1 secretion ability.

[0040] 2. In vivo experiments to verify the effects of different oral liquids The groups were set as follows: a. Control group (drinking water without any treatment); b. Oral liquid prepared in Example 3 c. Oral liquid prepared in Comparative Example 3 d. Oral liquid prepared in Comparative Example 4 e. Oral liquid prepared in Comparative Example 5 The experimental design was as follows: The diabetic mice were evenly distributed into each group according to their physical conditions and fasting blood glucose levels. The experimental period was 35 days, and the fasting blood glucose levels were monitored weekly (the results are shown in Table 2). At the end of the experiment, serum was collected by eye blood sampling to analyze the GLP-1 concentration (the results are shown in Table 3). The colon tissues were isolated, and the fluorescence intensity of the cells with GLP-1 secretion ability in the colon tissues was analyzed using immunofluorescence method (the results are shown in Table 2).

[0041] Table 2 Results of fasting blood glucose (mmol / L) levels of experimental mice in each group

[0042] As can be seen from Table 2: Compared with the control group, the oral liquids ingested by diabetic mice could reduce the fasting blood glucose levels on the 21st, 28th, and 35th days. In addition, on the 28th day and after, the mice that ingested the oral liquid of Example 3 had significantly lower fasting blood glucose levels than those that ingested the oral liquids of Comparative Examples 3-5. It can be seen that with the extension of time, the oral liquid of Example 3 had the best effect in improving the fasting blood glucose levels of diabetic mice.

[0043] Table 3 Serum GLP-1 levels of experimental mice in each group

[0044] As can be seen from Table 3: In terms of serum GLP-1 levels, compared with the control group, each oral liquid can increase the circulating GLP-1 level; among them, the concentration of Example 3 is the highest, and this oral liquid has the best effect in increasing the serum GLP-1 level of diabetic mice.

[0045] Fluorescence intensity of GLP-1 secreting cells in the colon of experimental mice in each group in Table 4

[0046] As can be seen from Table 4: Compared with the control group, only the group of Example 3 can significantly increase the fluorescence intensity of cells with GLP-1 secreting function in the colon. It shows that this oral liquid has the best effect in improving the fluorescence intensity of cells with GLP-1 secreting function in the colon.

[0047] 3. Acute toxicity experiment The toxicity test of the oral liquid was carried out on female mice through the Up-and-Down procedure (https: / / www.oecd.org / content / dam / oecd / en / publications / reports / 2022 / 06 / test-no-425-acute-oral-toxicity-up-and-down-procedure_g1gh2953 / 9789264071049-en.pdf).

[0048] Experimental results: During the 14-day period, we administered the highest dose (2000 mg / 100 g body weight) to the mice according to the procedure, and observed parameters such as the physical condition, appetite, and state of the mice after ingesting the drug, and no signs of toxicity were found in the mice.

[0049] Through the above verification experiments, it can be shown that the oral liquid prepared in this application can play a role in promoting the secretion of endogenous GLP-1, and it is a healthy drink that can play roles such as reducing blood sugar and losing weight.

[0050] In the present invention, the specific raw materials not described are all existing substances and can be directly purchased from the market.

[0051] The above are only the preferred implementation schemes of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A functional oral liquid based on Humulus japonicus that can promote the secretion of endogenous GLP-1, characterized in that: The oral liquid is prepared by mixing Humulus japonicus mixed extract, suspending agent, preservative and water; The mixed extract of Humulus japonicus is prepared by mixing Humulus japonicus leaves, Humulus japonicus roots, Humulus japonicus stems and Gentiana scabra roots and then adding medical alcohol for extraction.

2. The oral liquid according to claim 1, characterized in that The suspending agent includes xanthan gum and locust bean gum; The preservatives include vitamin C and natamycin.

3. A method for preparing the oral liquid according to any one of claims 1 or 2, characterized in that: The following steps are involved: S1. Take fresh Humulus lappa leaves, Humulus lappa roots, Humulus lappa stems, and Gentiana lappa roots, wash, dry, and crush, place the mixed powder and medical alcohol in an extractor, start microwave-assisted extraction, separate and sieve after the extraction is completed, concentrate the extract, centrifuge, and take the supernatant to obtain a Humulus lappa mixed extract; S2. Evenly mix the Humulus japonicus mixed extract, suspending agent, preservative and water to prepare the oral solution.

4. The method according to claim 3, characterized in that The mass ratio of Humulus leaf, Humulus root, Humulus stem and Gentiana root is (5-12): (2-3): (2-3): (2-3); The mass ratio of the mixed powder to the medical alcohol is 1:(20-30), and the concentration of the medical alcohol is 60%-95%.

5. The method according to claim 3, characterized in that: In step S1, the drying temperature is 80-95° C. and the drying time is 2-3 hours.

6. The preparation method according to claim 3, characterized in that: The sieving mesh number in step S1 is 30-80 meshes.

7. The preparation method according to claim 3, characterized in that: The microwave power in step S1 is 480W-550W, and the extraction time is 15-25min.

8. The preparation method according to claim 3, characterized in that: The concentration conditions in step S1 are as follows: temperature 40-75° C., pressure 10-100 mbar, and rotation speed 60-120 rpm; the volume after concentration is 1 / 3-1 / 2 of the original volume.

9. The preparation method according to claim 3, characterized in that: The centrifugal conditions in step S1 are: rotation speed 700-900 r / min, time 7-9 min.

10. The preparation method according to claim 3, characterized in that: The volume of the mixed extract of Humulus japonicus in step S2 accounts for 50-75% of the volume of the finished oral solution; The content of the suspending agent in the finished oral solution is 0.08-0.15%; The content of the preservative in the finished oral liquid is 0.015-0.02%.

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