阅读说明：本技术 一种用于防治急性高原反应的藏药复方及其保健产品 (Tibetan medicine compound for preventing and treating acute altitude stress and health-care product thereof ) 是由 米玛 陈静 雷海民 王鹏龙 于 2021-11-17 设计创作，主要内容包括：本发明公开了一种用于防治急性高原反应的藏药复方及其保健产品的制备方法,它是由以下重量份的藏药组成：芫根0.2～0.4份,沙棘1.2～2.0份,石榴籽1.0～1.5份。本处方是基于石榴普安散处方增减衍生的民间验方,已有三十多年的应用历史,体内外实验证明,本发明具有良好的抗氧化、耐缺氧、抗疲劳作用,且不良反应少、安全性高,对急性高原反应有良好的预防作用,可有效降低急性高原反应的发病率。三味藏药均属药食同源,制备成的速溶茶口味酸甜,绿色健康,携带方便,消费者依从性高,符合三效、三小、五方便的原则,具备较高的使用价值和广阔的开发前景。(The invention discloses a Tibetan medicine compound for preventing and treating acute altitude sickness and a preparation method of a health-care product thereof, wherein the Tibetan medicine compound is composed of the following Tibetan medicines in parts by weight: 0.2-0.4 part of common turnip, 1.2-2.0 parts of sea buckthorn and 1.0-1.5 parts of pomegranate seeds. The prescription is a folk prescription derived based on the increase and decrease of the prescription of the pomegranate Puan powder, has more than thirty years of application history, and in vitro and in vivo experiments prove that the pomegranate extract powder has good effects of resisting oxidation, anoxia and fatigue, has few adverse reactions and high safety, has a good prevention effect on acute altitude reaction, and can effectively reduce the incidence of the acute altitude reaction. The three Tibetan medicines are both homologous in medicine and food, and the prepared instant tea is sour and sweet in taste, green, healthy, convenient to carry, high in compliance of consumers, in line with the principles of three effects, three little effects and five convenience, and has high use value and wide development prospect.)

1. The invention discloses a preparation method of a Tibetan medicine compound for preventing and treating acute altitude stress and a health-care product thereof, which is characterized in that the Tibetan medicine compound is prepared from the following raw materials in parts by weight: 2-4 parts of common turnip, 12-20 parts of sea buckthorn and 10-15 parts of pomegranate seeds.

2. A preparation method of a Tibetan medicine compound for preventing and treating acute altitude sickness and a health-care product thereof is characterized by comprising the following steps:

1) cleaning 2-4 parts of common turnip, 12-20 parts of sea buckthorn and 10-15 parts of pomegranate seeds, crushing, mixing, adding 1000 parts by weight of water for decocting for 1-2 hours, naturally cooling to normal temperature, filtering, and removing filter residues to obtain a crude extract.

2) And (2) after the crude extract is subjected to vacuum freeze drying, adding 50-300 parts by weight of water, uniformly mixing, standing, filtering the obtained solution, sterilizing the filtrate at the temperature of 90-110 ℃ for 5-10 minutes, performing spray drying, and packaging the obtained tea powder into bags to obtain the Tibetan medicine instant tea for preventing and treating acute altitude stress.

3. The use of the health product obtained by the preparation method according to claim 2 in resisting myocardial cell, vascular cell and nerve cell hypoxia injury.

4. The use of the health product of claim 2 for treating hypoxia injury in mice.

5. The use of the health product obtained by the method of claim 2 for the prevention of altitude sickness.

6. The preparation method of the Tibetan medicine compound for preventing and treating acute altitude sickness and the health care product thereof as claimed in claim 2, wherein the preparation method comprises the following steps: firstly, immersing the common turnip, the sea buckthorn and the pomegranate seeds in cold water for 25-35 minutes, draining, drying in a vacuum drying oven until the moisture content is lower than 10%, crushing, and sieving with a 50-mesh sieve.

7. The method for preparing the Tibetan medicine compound for preventing and treating acute altitude sickness and the health care product thereof as claimed in claim 2, wherein the conditions of freeze drying are as follows: working pressure is 40pa, and sublimation temperature is-50 ℃; the spray drying conditions are that the inlet temperature of hot air is 100-110 ℃, the outlet temperature is 55-60 ℃, and the spray drying is carried out at the feeding amount of 1.5-4L per hour.

8. The use of claim 5, wherein the anti-altitude response condition comprises any one of anti-oxidant, anti-hypoxia.

Technical Field

The invention relates to the field of Tibetan medicines and natural medicines, in particular to a Tibetan medicine compound for preventing and treating acute altitude stress and a preparation method of a health-care product thereof.

Background

Acute altitude sickness is a common disease in high altitude areas, and if the disease cannot be controlled, the disease can be developed into high altitude pulmonary edema, high altitude cerebral edema and high altitude pulmonary hypertension, which seriously harms tourists and common people in the Tibetan area to be healthy and influences economic development and safe work of the Tibetan area. At present, the anti-altitude stress medicament is mainly divided into Chinese herbal medicines, Tibetan medicines, western medicines, Chinese and western medicine combined medicines and the like, and mainly aims at reducing the oxygen consumption speed of an organism under an anoxic condition, increasing the oxygen production speed of each tissue organ, reducing the anoxic injury of the organism, improving the content of superoxide dismutase in the organism and the like. Due to the pressure of traffic and resources in the Tibetan region, development of a simple, portable, safe and effective health-care product for preventing and treating acute altitude stress is urgently needed.

The Tibetan medicine is one of the important components of the traditional Chinese medicine, is great essence bred by combining the wisdom of traditional Chinese pharmacology, Indian pharmacology and bulk pharmacology, and proved by hundreds of thousands of years of clinical practice, the Tibetan medicine with the characteristics of the Tibetan medicine is diligently worked and brave, and the wisdom and the labor of the Tibetan medicine compete with the severe natural environment. Altitude sickness is one of the common diseases in the Tibetan region, and the Tibetan medicine treatment has a long history. The common turnip, named Niuma in Tibetan language, is recorded in the Tibetan medicine famous book of four medical classics, and has the effects of nourishing, increasing oxygen, stimulating appetite, promoting digestion, resisting anoxia, resisting fatigue, reducing blood fat, relieving water and soil incoordination and the like, and has extremely high regulation and supplement effects. Pomegranate seed, known as "saizhu" in Tibetan language, is sour and sweet in property, moistens middle energizer and strengthens stomach, and can treat stomachache, dyspepsia, vomiting and the like caused by stomach cold. Seabuckthorn fruit is firstly recorded in the Yue Wang Yao Dian (diagnosis of drug by Yue Wang), can treat hyperlipemia and promote blood circulation, and has good curative effect on tracheitis and emphysema. The three Tibetan medicines are mutually assisted and commonly used in compatibility, are applied to the patent medicines such as common turnip seabuckthorn tablets, seventeen gypsum rubrum pills, pomegranate praepam pills and the like, and have the synergistic effect on symptoms such as headache, vomit, chest distress and the like caused by altitude stress. The traditional Chinese medicine is prepared from a Tibetan medicine prepared preparation pomegranate prian powder, also called as 'saizhuyiedai', can benefit stomach fire, remove phlegm and dampness and warm kidney, and is used for treating diseases such as 'pegen jiabu', 'wood cloth', stomach fire, weakness, dyspepsia, waist cold pain, difficult urination, female blood disease and the like. The folk prescription relies on a large amount of pomegranate seeds and sea buckthorn two medicines in pomegranate prance powder, is used for warming stomach and tonifying yang and strengthening lung qi to deal with symptoms such as nausea, vomiting, inappetence, oliguria, chest distress, short breath, hypodynamia and the like caused by acute altitude reaction, is supplemented with yuenkanin for nourishing, oxygenation, appetizing and digestion promoting, can obviously relieve acute symptoms caused by water and soil inappetence, has been clinically practiced for many years, has a long application history but related products are in a vacancy stage at present, and on the basis of specific medicine resources in a Tibetan region, the Tibetan medicine product with high quality and good curative effect is developed, so that the Tibetan medicine has a very wide market prospect.

Disclosure of Invention

Based on the problems, the Tibetan medicine compound for preventing and treating acute altitude stress and the health-care product thereof have the advantages of dual purposes of medicine and food, high safety, good taste, enhanced effect after compatibility, simple process and easy portability, and fill the blank of the Tibetan medicine in preventing and treating acute altitude stress and solve the problem of non-portability of medicines.

The preparation process of the product comprises the following steps:

1) cleaning 2-4 parts of common turnip, 12-20 parts of sea buckthorn and 10-15 parts of pomegranate seeds, crushing, mixing, adding 1000 parts by weight of water for decocting for 1-2 hours, naturally cooling to normal temperature, filtering, and removing filter residues to obtain a crude extract.

2) And (2) after the crude extract is subjected to vacuum freeze drying, adding 50-300 parts by weight of water, uniformly mixing, standing, filtering the obtained solution, sterilizing the filtrate at the temperature of 90-110 ℃ for 5-10 minutes, performing spray drying, and packaging the obtained tea powder into bags to obtain the Tibetan medicine instant tea for preventing and treating acute altitude stress.

Analyzing the prepared Tibetan medicine instant tea, and determining that the content of isorhamnetin in the solid beverage is 1.31mg/g, the complete dissolution time is 17-20s, and the solubility determination conditions are as follows: 5g of the Tibetan medicine instant tea is poured into 200mL of water with the temperature of 45-50 ℃ and continuously stirred.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

It is to be noted that the experimental procedures described in the following examples are conventional ones unless otherwise specified, and the reagents and materials described therein are commercially available without otherwise specified.

Example 1: preparation of compound instant tea of Tibetan medicine of the invention

1. 20g of yuenkan, 150g of sea buckthorn and 100g of pomegranate seeds.

2. Preparation method

1) Screening fresh yuenkanin, sea buckthorn and pomegranate seeds with high quality, cleaning, draining, cutting yuenkanin into slices of about 5mm, and placing in a vacuum drier (vacuum degree: 100 pa; temperature: -28 ℃ to-30 ℃; the temperature of the heating plate; 65-70 ℃; time: 16h) drying, collecting dried radix Genkwa pieces, fructus Hippophae and semen Granati, weighing, pulverizing, sieving with 50 mesh sieve, mixing to obtain crude Tibetan medicinal composition (water content less than 10%), adding 10L water, decocting for 1 hr, naturally cooling to room temperature, filtering, and removing residue to obtain crude extractive solution 9.64L.

2) And (3) after the crude extract is subjected to vacuum freeze drying (working pressure is 40pa, sublimation temperature is-50 ℃, and resolution temperature is 60 ℃), adding 3L of water, stirring and mixing uniformly, standing, filtering the obtained solution, sterilizing the filtrate at the temperature of 90-110 ℃ for 5-10 minutes, performing spray drying (hot air inlet temperature is 100-110 ℃, outlet temperature is 55-60 ℃, and feeding amount is 1.5-4L per hour), performing dry granulation, sieving and grading to obtain tea powder, and packaging into bags to obtain the Tibetan medicine instant tea for preventing and treating acute altitude stress.

3) Analyzing the prepared Tibetan medicine instant tea, and determining that the content of isorhamnetin in the solid beverage is 1.24mg/g, the complete dissolution time is 17-20s, and the solubility determination conditions are as follows: 5g of the Tibetan medicine instant tea is poured into 200mL of water with the temperature of 45-50 ℃ and continuously stirred.

4) Analyzing the prepared Tibetan medicine instant tea, and performing content determination by adopting a high performance liquid chromatography standard curve method to determine that the content of isorhamnetin in the solid beverage is 1.47mg/g and the complete dissolution time is 15-20 s.

The pharmacodynamics of the present invention is exemplified in particular by the following experiments.

Example 2: in vitro antioxidant assay

DPPH radical scavenging experiment

1.1 Experimental methods

1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) is precisely weighed, 0.2mM DPPH solution is prepared by absolute ethyl alcohol, then 100 microliter DPPH solution is absorbed and added into a 96-pore plate, 100 microliter of sample solution to be detected with different concentrations is added into each pore, three multiple pores are arranged under each concentration, after shaking and shaking uniformly, the sample solution is placed at room temperature (20-25 ℃) for incubation in a dark place for 30min, absolute ethyl alcohol is used for replacing the sample solution to be detected for determination of a control group, the three multiple pores are also arranged, the absorbance value A517 of the system at the wavelength of 517nm is determined on an enzyme-labeling instrument, and the experiment is repeated for three times in parallel.

The calculation formula is as follows:

DPPH free radical clearance (%) - (control A-sample A)/control A. times.100%

Control group: DPPH + absolute ethanol;

blank (sample): adding absolute ethyl alcohol into the sample solution to be detected;

blank (positive drug): positive drug + ethanol

1.2 results of the experiment

TABLE 1 DPPH radical scavenging rate of instant tea of the present invention

OH free radical scavenging experiment

2.1 Experimental methods

Precisely measuring 30 mu L of sample solution to be detected with different concentrations in a 96-well plate, and then sequentially adding 30 mu L of 5.0mM phenanthroline solution and 50 mu L of 5.0mM FeSO in each well₄The solution, 50. mu.L of 15.0mM EDTA-2Na solution, and 30. mu.L of 0.2m MPBS buffer (pH7.4) were mixed, and 30. mu.L of 1.0% H was added₂O₂And (3) incubating the solution in a constant-temperature incubator at 37 ℃ for 1h, measuring the absorbance value A536 of the solution system at the wavelength of 536nm by using a microplate reader, and setting three multiple holes at each concentration. The damaged group uses deionized water to replace the sample solution to be measured, and the non-damaged group uses deionized water to replace the sample solution to be measured and 1.0% H₂O₂The solution (six duplicate wells for the injured and non-injured groups, respectively) was repeated in parallel three times.

The calculation formula is as follows:

OH free radical clearance (%) - (sample a-a lesion group)/(non-a lesion group) × 100%

Blank (sample): adding 190 mu L of water into the sample solution to be detected;

blank (positive drug): positive drug + 190. mu.L water

2.2 results of the experiment

The concentration gradient was set to 20, 16, 8, 4, 2, 1mg/mL, and experiments were performed

TABLE 2 OH free radical scavenging table for instant tea of the present invention

3. Superoxide anion (O)₂ ^-) Free radical scavenging experiments

3.1 Experimental methods

50mM Tris-HCl solution is prepared by mixing 50mL of 0.1M Tris (hydroxymethyl) aminomethane (Tis) solution with 19.9mL of 0.1mM HCl solution, and diluting to 100mL with deionized water. 80 mu L of sample solutions to be measured with different concentrations (0.05, 0.1, 0.5, 1.0, 5.0 and 10.0mg/mL) are precisely measured and added into a 96-well plate, 80 mu L of Tris-HCl solution (50mM PH8.3) containing 1.0mM EDTA and 40 mu L of 0.01mM HCl solution containing 1.5mM pyrogallol are sequentially added into each well, three multiple wells are arranged at each concentration, an absorbance value A320 of the solution system at the wavelength of 320nm is measured by a microplate reader every 1min at room temperature, A320 at 0, 1, 2, 3, 4 and 5min is continuously measured, a control group uses Tris-HCl solution (50mM PH8.3) containing 1.0mM EDTA to replace the sample solution to be measured, and six multiple wells are arranged. The polymerization rate of pyrogallol induced by superoxide anion is expressed by the change of absorbance value (delta A/min) at 320nm, and the experiment is repeated three times in parallel

The calculation formula is as follows:

O₂-free radical clearance (%) ═ Δ a_control/min-△A_sample/min)/△A_contro/minX 100% (where. DELTA.A)_control/minThe induction rate Delta A of the sample solution to be measured_samole/minThe induction rate of the control solution. )

3.2 results of the experiment

TABLE 3 superoxide anion radical scavenging table for instant tea of the present invention

Example 3: evaluation of anti-hypoxia Activity of HUVEC, H9C2, SY5Y cells

1. Materials and methods

1.1 reagents and instruments

DMEM medium, Fetal Bovine Serum (FBS) purchased from Gibco; penicillin, streptomycin and PBS were purchased from Beijing Solaibao technologies, Inc.; trypsin was purchased from carnot biopharmaceutical technologies, inc; MTT kit (bodham, deld bioengineering, ltd); the instrument included a BIO-RAD microplate reader (Shandong Boke scientific instruments, Inc., model IMARK).

1.2 cell lines

HUVEC (human umbilical vein endothelial cells), H9C2 (cardiac myocytes) and SY5Y (human neuroblastoma cells) were purchased from ATCC (American society for culture and transportation).

1.3 Experimental methods

1.3.1 cell grouping and establishment of hypoxia model

HUVEC, H9C2 and SY5Y cells were divided into 4 groups, which were a control group, a model group, an instant tea low concentration group and an instant tea high concentration group, respectively. Culturing the cells of the control group in a DMEM culture solution containing 10% fetal calf serum, and changing the culture solution for 1 time within 3-5 days; placing the model group cells in a three-gas culture box, keeping the temperature in the box at 37 ℃, and continuously introducing 5% CO₂+95％N₂Mixed gas of (2) to (2)HUVEC, H9C2, SY5Y cells were hypoxic for 12H, the culture medium was rapidly changed, HUVEC, H9C2, SY5Y cells were transferred to a cell culture box (37 ℃, 5% CO)₂) Continuously culturing for 12h, and recovering oxygen supply to the cells; after the instant tea low-concentration group and the instant tea high-concentration group are respectively treated with 1.25mg/mL and 20mg/mL of instant tea for 12 hours, the cells are treated in the same manner as the model group.

1.3.2 cell proliferation Rate detection

Culturing HUVEC, H9C2 and SY5Y cells in a 96-well plate, adding 20 mu L of MTT solution into each well, mixing uniformly, culturing for 4H, carefully sucking off supernatant in each well, adding 150 mu L of dimethyl sulfoxide into each well, mixing uniformly, and detecting the absorbance value at 570nm by using an enzyme labeling instrument.

2. Results

TABLE 4 Table of activity of HUVEC, H9C2 and SY5Y cells under anoxia promoting condition of instant tea

P < 0.05(1) compared to control; p < 0.05(2) compared to control.

Example 4: mouse hypoxia tolerance test

1. Materials and methods

1.1 reagents and instruments

The instant tea of the invention is prepared by the method of the first embodiment. Soda lime, electronic balance, electronic stopwatch, and the like.

1.2 Experimental animals

40 ICR mice with the weight of 18-22g are provided by Beijing Wittingli laboratory animal technology, the temperature of the animal room is controlled at 20-25 ℃, and the relative humidity is 45-55%. Day and night is 12h/12h, and water and diet can be freely taken.

1.3 Experimental methods

1.3.1 dose setting

Four groups of three experimental groups, one control group, and 9 mice per group were set up for the experiment. The dosages of the instant tea in the three experimental groups are 0.15, 0.65 and 1.30g/kg respectively, the control group is given distilled water, and the three experimental groups are administrated by intragastric administration 1 time per day for 30 days continuously, and the intragastric volume is 0.1mL/10g of body weight.

1.3.2 Normal pressure anoxia test

30min after the last dose, each group of mice was placed in 500mL ground flasks (1 per flask) with 10g of soda lime, and mice survival time was recorded as a death characteristic of respiratory arrest.

2. Results

TABLE 5 Effect of instant tea of the present invention on the survival time of mice to withstand hypoxia at normal pressure (i + -s)

Example 5: acute toxicity test in mice

1. Materials and methods

1.1 reagents and instruments

The instant tea concentrate of the present invention was prepared by the method of example one. Electronic balance, etc.

1.2 Experimental animals

20 ICR mice with the weight of 18-22g and half of the weight are provided by Beijing Witonglihua experimental animal technology, and the temperature of an animal room is controlled to be 20-25 ℃ and the relative humidity is 45-55 percent. Day and night is 12h/12h, and water and diet can be freely taken.

1.3 Experimental methods

After fasting for 12 hours, the Tibetan medicine instant tea suspension is subjected to single intragastric administration at the ratio of 6 g/kg.

2. Results

Within the observation period of 2 weeks, the mice were in good mental status and had no toxic reaction or death. The final weight of female mice (31.2 + -3.6) g, and male mice (37.1 + -3.8) g. The Tibetan medicinal instant tea belongs to a nontoxic grade according to the evaluation of acute toxicity grading standards.

Example 6: population taste evaluation method

48 healthy volunteers were selected as subjects, the acidity and sweetness of the instant tea were evaluated, warm water was rinsed before evaluation, rinsing was performed in the mouth during tasting, the instant tea was spitted out after staying for 10 seconds, the mouth feel was classified into mouth-in, taste within 10 seconds and residual aftertaste in the mouth, scoring was performed according to the mouth feel, rinsing was performed after tasting one solution until no taste remained, and the next solution was tasted.

TABLE 6 taste evaluation grades

TABLE 7 evaluation results of the instant tea of the present invention in taste

According to the results, more than 91.6% of the subjects consider the sour taste to be slightly sour, more than 95.8% of the subjects consider the sweet taste to be slightly sweet, and the taste is better and proper in sweet and sour in the whole.

Pharmacodynamic experiments prove that the Tibetan medicine instant tea has extremely strong scavenging effect on DPPH, OH free radicals and superoxide anions and has obvious in-vitro antioxidant effect; can effectively resist the damage of myocardial cells, vascular endothelial cells and neuroblastoma cells induced by hypoxia on a cell model; in vivo animal experiments prove that the traditional Chinese medicine composition can obviously improve the survival rate of anoxic mice and has no toxicity to normal mice. Pharmaceutical experiments prove that the Tibetan medicine instant tea has the advantages of high dissolving speed, quick response, good flavor and strong adaptability to consumers. In conclusion, the Tibetan medicine compound and the health-care product thereof, namely the Tibetan medicine instant tea, have the characteristics of good curative effect, quick response and high safety on acute altitude sickness, and in addition, the raw materials are easy to obtain, the transportation is convenient, the market is wide, and the Tibetan medicine compound and the health-care product thereof are suitable for large-scale development and production.