Trichosanthes kirilowii maxim stem and leaf extract with effect of preventing and treating heat stroke and application thereof
By constructing a heat-stress animal model, the effect of preventing and treating heat-stroke diseases of the leuko stem and leaf extract was evaluated, and the leuko stem and leaf extract with significantly improved heat-stroke symptoms was prepared, which solved the problem of lack of effective methods for preventing and treating heat-stroke diseases in the existing technology, and achieved good prevention and treatment effects and functional beverage applications.
Patent Information
- Application Number
- CN202510426733.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-07
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art lacks effective clinical treatment methods to prevent and treat heatstroke, and the importance of preventive measures has been neglected, resulting in a high incidence and mortality rate of heatstroke.
The heat-stressed zebrafish model and the heat-stroke mouse model were constructed by water bath heating. The biological effects of the stem and leaf extract of leuko are evaluated, and the stem and leaf extract of leuko are prepared by reflux extraction method of ethanol and water.
Experimental results show that the extract of 面的游戏 can significantly improve liver index, FIB, PT, and LDH indicators, have good effects on preventing and treating heatstroke, and can be used to make functional tea drinks with antioxidant and anti-aging functions.
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Figure CN120131734A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a trichosanthes stem and leaf extract with the function of preventing and treating heat stroke, belonging to the technical field of traditional Chinese medicine. Background Art
[0002] In recent years, the rising global temperature has led to heat waves, significantly increasing the incidence of heat stroke. Data from the National Weather Service of the United States shows that heat waves cause higher mortality than other extreme weather events. Heat stroke can be divided into exertional heat stroke and classic heat stroke according to the cause of onset and susceptible populations. Statistical data shows that after treatment in the intensive care unit (ICU), the mortality rate of exertional heat stroke is still 26.5%, while the fatality rate of classic heat stroke is as high as 63.2%. Moreover, the prognosis of heat stroke survivors is poor, and they may still experience long-term neurological and cardiovascular complications, facing a high risk of death.
[0003] Currently, there is no specific clinical treatment method except for rapid cooling, and taking reasonable preventive measures is the key to reducing the incidence of heat stroke. Prevention is more effective and relatively easier than treatment, which highly coincides with the core idea of "preventing disease" in traditional Chinese medicine clinical practice. Traditional Chinese medicine has the characteristics of multiple components, multiple targets, and preventing disease. Therefore, it is particularly necessary to make full use of China's traditional Chinese medicine resources and accelerate the transformation of traditional Chinese medicinal materials into heat stroke prevention products.
[0004] The stem and leaf of trichosanthes is the dried stem and leaf of the plant trichosanthes Trichosanthes kirilowii Maxim., sour in taste, cold in nature, and non-toxic. The stem and leaf of trichosanthes contains rich resource-based chemical components, including flavonoids, phenolic acids, triterpenoids, nucleoside amino acids, and polysaccharides, etc. Before the trichosanthes bears fruit, it is often necessary to remove the redundant branches to maintain a reasonable leaf area to increase fruit setting, and this process will produce a part of waste stem and leaf; after the trichosanthes bears fruit, its fruit (trichosanthes fruit), pericarp (trichosanthes peel), and seeds (trichosanthes seeds) can all be used as medicine, but the development and utilization degree of the remaining stem and leaf part is not high. The yield of stem and leaf is huge, with a fresh weight per mu of up to 5000 kg. Except for a small amount of young leaves that can be used as food materials among the people, most of them are discarded, causing great waste of resources. Therefore, it is urgent to accelerate the diversified utilization of the stem and leaf of trichosanthes.
[0005] Both the two ancient books "Supplementary Records of Famous Physicians" and "Justice of Materia Medica" record the medicinal value of the stem and leaf of trichosanthes as "able to treat heat injury in summer". Based on the efficacy of the stem and leaf of trichosanthes in "treating heat injury in summer", the present invention constructs a heat stress zebrafish model by water bath heating and constructs a heat stroke mouse model by a high-temperature and high-humidity climate chamber to evaluate the biological effect of the stem and leaf of trichosanthes in preventing and treating heat stroke. Summary of the Invention
[0006] The purpose of the present invention is to provide a trichosanthes stem and leaf extract with the function of preventing and treating heat stroke and its application on the basis of the traditional efficacy of the stem and leaf of trichosanthes.
[0007] To achieve the above object, the technical solution designed by the present invention is as follows: The present invention provides a Trichosanthes kirilowii Maxim. stem and leaf extract with the function of preventing and treating heat stroke.
[0008] The present invention also provides a preparation method of the above-mentioned Trichosanthes kirilowii Maxim. stem and leaf extract. The Trichosanthes kirilowii Maxim. stem and leaf sample is appropriately pulverized, weighed, ethanol is added, heated under reflux for extraction, filtered while it is hot, the filtrates are combined to obtain an ethanol extract; the filter residue is added with water, heated under reflux for extraction, filtered while it is hot, the filtrates are combined, and combined with the ethanol extract, and concentrated under reduced pressure to obtain the product.
[0009] As a preferred scheme, the Trichosanthes kirilowii Maxim. stem and leaf sample is appropriately pulverized, weighed, 6-12 times the amount of ethanol is added, heated under reflux for extraction 1-3 times, 0.5-2 hours each time, filtered while it is hot, the filtrates are combined to obtain an ethanol extract; the filter residue is added with 6-12 times the amount of water, heated under reflux for extraction 1-3 times, 0.5-2 hours each time, filtered while it is hot, the filtrates are combined, and combined with the ethanol extract, and concentrated under reduced pressure to obtain the product.
[0010] As a preferred scheme, the Trichosanthes kirilowii Maxim. stem and leaf sample is appropriately pulverized, weighed, 10 times the amount of ethanol is added, heated under reflux for extraction 2 times, 1 hour each time, filtered while it is hot, the filtrates are combined to obtain an ethanol extract; the filter residue is taken and added with 10 times the amount of water, heated under reflux for extraction 2 times, 1 hour each time, filtered while it is hot, the filtrates are combined, and combined with the ethanol extract, and concentrated under reduced pressure to obtain the product.
[0011] The present invention also provides an application of the above-mentioned Trichosanthes kirilowii Maxim. stem and leaf extract or the Trichosanthes kirilowii Maxim. stem and leaf extract prepared by the above preparation method in drugs and functional foods for preventing and treating heat stroke.
[0012] A functional tea drink of Trichosanthes kirilowii Maxim. stem and leaf, which includes 10% of the stem and leaf extract after Trichosanthes kirilowii Maxim. bears fruit, 2% of the compound sweetener, 0.05% of citric acid, and the rest is water. The compound sweetener is white granulated sugar and stevioside with a mass ratio of 40:1.
[0013] The above-mentioned functional tea drink of Trichosanthes kirilowii Maxim. stem and leaf has good antioxidant function.
[0014] Beneficial effects: The present invention has the following advantages compared with the prior art: The Trichosanthes kirilowii Maxim. stem and leaf extract provided by the present invention. The results of animal model experiments show that the Trichosanthes kirilowii Maxim. stem and leaf extract provided by the present invention can play a role in preventing and treating heat stroke by significantly improving the liver index, FIB, PT, and LDH indexes, and has a good improvement effect on heat stroke. The tea drink made with the Trichosanthes kirilowii Maxim. stem and leaf extract as the main raw material has good antioxidant and anti-aging functions. Description of the Drawings
[0015] Figure 1 LD of the stem and leaf of Trichosanthes kirilowii Maxim. before bearing fruit50 Results
[0016] Figure 2 It is a bar chart showing the effects of the stems and leaves of Trichosanthes kirilowii Maxim. before fruit setting and the stems and leaves after fruit setting on the activities of ALT, AST, and LDH in heat-stressed zebrafish.
[0017] Figure 3 It is a bar chart showing the effects of the stems and leaves of Trichosanthes kirilowii Maxim. after fruit setting on the coagulation indexes (APTT, FIB, PT) and blood routine indexes (HGB, RBC, HCT) in heatstroke model mice.
[0018] Figure 4 It is a bar chart showing the effects of the stems and leaves of Trichosanthes kirilowii Maxim. after fruit setting on the activities of AST and AKP in the liver tissue, the activity of LDH in the heart tissue, and the activity of SOD in the brain tissue of heatstroke model mice.
[0019] Figure 5 It is a graph showing the effects of the addition amounts of Trichosanthes kirilowii Maxim. stems and leaves, citric acid, and sweeteners on the sensory scores.
[0020] Figure 6 It is a line graph showing the scavenging rates of DPPH radicals, ABTS radicals, and hydroxyl radicals and the total reducing ability of a functional beverage. Specific implementation methods
[0021] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent forms of modification by those skilled in the art fall within the scope defined by the appended claims of this application.
[0022] The following describes in detail the specific implementation manners of the technical solutions of the present invention.
[0023] Example 1: Preparation of extracts from the stems and leaves of Trichosanthes kirilowii Maxim. Respectively take the stems and leaves of Trichosanthes kirilowii Maxim. before fruit setting and the stems and leaves after fruit setting, dry them at 40 °C, and cut them into small sections of 2 - 3 cm. Respectively take 10 g of the stems and leaves of Trichosanthes kirilowii Maxim. before fruit setting and after fruit setting, add 10 times the amount of 95% ethanol for reflux extraction twice, each time for 1 h, then add 10 times the amount of water for reflux extraction twice, each time for 1 h, combine the four filtrates, and concentrate them under reduced pressure to obtain an extract of the stems and leaves of Trichosanthes kirilowii Maxim. before fruit setting and an extract of the stems and leaves after fruit setting with a crude drug mass concentration of 1 g / mL, and reserve them for use.
[0024] Example 2: Screening the biological activities of extracts from the stems and leaves of Trichosanthes kirilowii Maxim. before and after fruit setting using a heat-stressed zebrafish model 1. Experimental animals and reagents Stem and leaf extracts of Trichosanthes kirilowii Maxim. before fruiting; Stem and leaf extracts of Trichosanthes kirilowii Maxim. after fruiting; Alanine aminotransferase (ALT / GPT) kit; Aspartate aminotransferase (AST / GOT) kit; Lactate dehydrogenase (LDH) kit; BCA protein concentration assay kit.
[0025] AB wild-type zebrafish were purchased from Nanjing Yishulihua Biotechnology Co., Ltd. The breeding temperature of zebrafish was 26 °C, and the environment was controlled by alternating 14 h of light / 10 h of darkness. Artemia nauplii were fed once in the morning and once in the evening every day. Healthy zebrafish were selected the previous night and placed in a breeding tank at a male-female ratio of 2:2 for paired spawning. The partition was removed before the start of light the next morning, and zebrafish embryos were collected 2 h later. After being cultured into larvae, they were used for subsequent experiments.
[0026] 2. Experimental methods 2.1 Preparation of the test drugs The extracts of the stems and leaves of Trichosanthes kirilowii Maxim. before and after fruiting in Example 1 were used.
[0027] 2.2 Investigation of the administration doses of the stems and leaves of Trichosanthes kirilowii Maxim. before and after fruiting Zebrafish larvae at 4 dpf after fertilization with good status were randomly selected and placed in a 12-well plate, 10 tails per well. The liquid medicines of the stems and leaves of Trichosanthes kirilowii Maxim. before and after fruiting were diluted to 100, 200, 400, 800, 1200, 1600, 2000, 2400, 2800, 3200 μg / mL with embryo culture medium respectively. 4 mL of the liquid medicine was added to each well, and the same volume of embryo culture medium was used as the blank control group. Six replicates were set for each concentration. After administration for 24 h, the mortality of zebrafish in each group was observed and recorded.
[0028] 2.3 Grouping and administration of zebrafish Six doses lower than LC 0 were selected as the administration concentrations. Healthy zebrafish larvae at 4 dpf were collected in a 12-well plate and divided into a blank group, a model group, a group of the stems and leaves of Trichosanthes kirilowii Maxim. before fruiting (GQ) (18.75, 37.5, 75, 150, 300, 600 μg / mL), and a group of the stems and leaves of Trichosanthes kirilowii Maxim. after fruiting (GH) (18.75, 37.5, 75, 150, 300, 600 μg / mL), 10 per well, with a total volume of 4 mL. The blank control group and the model group were given 4 mL of embryo culture water. Three replicates were set for each concentration. After culturing in a constant temperature incubator for 24 h, the drug-containing culture medium was removed, and the zebrafish were washed 3 times with embryo culture water.
[0029] 2.4 Establishment of a heat stress zebrafish larvae model After the administration was completed, the juvenile zebrafish in the pre-fruiting stem and leaf group of Trichosanthes kirilowii, the post-fruiting stem and leaf group of Trichosanthes kirilowii, and the model group were transferred to a beaker containing 200 mL of pre-warmed embryo culture water (30 juvenile zebrafish were placed in each beaker), and were subjected to acute heat stress at 42 °C for 30 min, with the temperature controlled by a constant temperature water bath. After the model was established, the beaker was quickly taken out, and a large amount of ice was added to euthanize them. The blank control group was placed in a constant temperature incubator at 28 °C and euthanized by ice bath at the same time point.
[0030] 2.5 Determination of biochemical indexes of juvenile zebrafish After acute heat stress on juvenile zebrafish, 30 juvenile zebrafish in each drug treatment group with different concentrations were sampled at the end point of model establishment. After being washed 3 times with ultrapure water to remove moisture, they were collected in 1.5 mL centrifuge tubes. 30 juvenile zebrafish were taken as a sample, and physiological saline was added for homogenization according to the ratio of weight (g): volume (mL) = 1:9. After centrifugation at 2500 r / min for 10 min, the supernatant was collected. Subsequently, the contents of ALT and AST in the zebrafish tissue homogenate were determined by the Reitman method according to the steps of the kit instructions, and the enzyme activity of LDH in the zebrafish tissue homogenate was determined by the microplate method. The experiment was repeated 3 times.
[0031] 2.6 Statistical analysis All experimental results are expressed as ±SD. Statistical processing and plotting were performed using Graphpad Prism 10.1.2 software. The Dunnett method in one-way ANOVA was used for comparison among multiple groups, P and <0.05 was considered statistically significant.
[0032] 3. Experimental results 3.1 Investigation results of the drug administration doses of the pre-fruiting stems and leaves and post-fruiting stems and leaves of Trichosanthes kirilowii Juvenile zebrafish at 4 dpf were given 10 different concentrations of the liquid medicine of the pre-fruiting stems and leaves of Trichosanthes kirilowii and the liquid medicine of the post-fruiting stems and leaves of Trichosanthes kirilowii (100, 200, 400, 800, 1200, 1600, 2000, 2400, 2800, 3200 μg / mL). When the concentration of the liquid medicine of the pre-fruiting stems and leaves of Trichosanthes kirilowii increased, the mortality rate of juvenile zebrafish also showed an upward trend. At 3200 μg / mL, the mortality rate was as high as 96.7%. Through analysis with Graphpad software, the LD of the pre-fruiting stems and leaves of Trichosanthes kirilowii for 4 dpf juvenile zebrafish 50 was 1701 μg / mL (see Figure 1). In the range of 100 - 3200 μg / mL, when zebrafish at 4 dpf were given the liquid medicine of the stems and leaves after the trichosanthes fruit set, no death was observed. When the concentration of the liquid medicine was increased to 6 mg / mL, the mortality rate could reach 16.7%. However, for the liquid medicine with a concentration higher than this, after being placed in an incubator at 28°C for 24 hours, there was an unpleasant smell and a small amount of precipitation. Limited by the concentration and properties of the liquid medicine, it was impossible to continue exploring the LD of the stems and leaves after the trichosanthes fruit set. 50 .
[0033] To screen the best period for the stems and leaves before and after the trichosanthes fruit set to intervene in heat - stressed zebrafish, the intersection of their safe dose ranges was selected for the next experiment. Therefore, six concentrations of 18.75, 37.5, 75, 150, 300, and 600 μg / mL were selected as the dosing doses. 3.2 Test results of biochemical indexes Compared with the model group, the stems and leaves before the trichosanthes fruit set had a significant improvement effect on the LDH level when the dosing concentrations were 75, 150, 300, and 600 μg / mL ( P < 0.01, P < 0.001), and had a significant improvement effect on the ALT level when the dosing concentrations were 75, 150, 300, and 600 μg / mL ( P < 0.05, P < 0.01, P < 0.001). When the dosing concentrations were 75 and 150 μg / mL, there was a tendency for the AST level to decline. The stems and leaves after the trichosanthes fruit set had a significant improvement effect on the LDH level when the dosing concentrations were 75, 150, 300, and 600 μg / mL ( P < 0.01), and significantly reduced the ALT level of zebrafish when the concentrations were 75, 300, and 600 μg / mL ( P < 0.05). For the AST index, all six dosing concentration groups had a tendency to decline towards the blank group (see Figure 2 ).
[0034] 3.3 Screening results of the stems and leaves before and after the trichosanthes fruit set Compared with the stems and leaves before the trichosanthes fruit set, the safe range of the stems and leaves after the trichosanthes fruit set was wider. When the dosing concentration was 6 mg / mL, the mortality rate of zebrafish was 16.7%, while when the dosing concentration of the stems and leaves before the trichosanthes fruit set was 3.2 mg / mL, the mortality rate of zebrafish was as high as 96.7%. The LD 50 value of the stems and leaves after the trichosanthes fruit set was much larger than that of the stems and leaves before the trichosanthes fruit set, and in the actual cultivation process, the stems and leaves after the trichosanthes fruit set were more easily obtained. Therefore, through the comprehensive analysis of the drug efficacy, safety range, and yield of the stems and leaves before and after the trichosanthes fruit set, the stems and leaves after the trichosanthes fruit set were selected for subsequent research.
[0035] Example 3: Evaluation of the Effect of Stem and Leaf Intervention of Trichosanthes kirilowii Maxim. on Heat Stroke Mice after Fruit Setting 1. Drugs, Reagents and Experimental Animals Stem and leaf extract of Trichosanthes kirilowii Maxim. after fruit setting; Huoxiang Zhengqi Oral Liquid; Sodium citrate anticoagulant; Glutamic oxaloacetic transaminase (AST / GOT) kit; Alkaline phosphatase (AKP) kit; Lactate dehydrogenase (LDH) kit; Superoxide dismutase (SOD) kit; BCA protein concentration assay kit; Fibrinogen assay kit; Activated partial thromboplastin time assay kit; Prothrombin time assay kit. SPF-grade male C57BL / 6J mice weighing 18 - 22 g.
[0036] 2. Experimental Methods 2.1 Preparation of Test Drugs Weigh 40 g of the stem and leaf of Trichosanthes kirilowii Maxim. after fruit setting, add 10 times the amount of 95% ethanol and reflux extract twice, each time for 1 h, then add 10 times the amount of water and reflux extract twice, each time for 1 h. Combine the four filtrates and concentrate under reduced pressure to a crude drug concentration of 1 g / mL for standby.
[0037] 2.2 Animal Grouping and Modeling After 7 days of adaptive feeding of C57BL / 6J mice, they were randomly divided into a blank group, a model group, a Huoxiang Zhengqi Oral Liquid group, and high- and low-dose groups of the stem and leaf of Trichosanthes kirilowii Maxim. after fruit setting (converted to crude drug amounts of 2 g / kg and 6 g / kg), with 8 mice in each group. Weighing showed that there was no statistically significant difference in body weight among the groups. After grouping, continuous intragastric preventive administration was given for 12 days, once a day, and the administration volume was 0.2 ml / 10 g body weight. The mice were fasted for 12 h before modeling but allowed to drink water. Except for the blank group, the mice in the other groups were placed in a high-temperature and high-humidity environment to induce heat stroke. Specifically, the mice were placed in an artificial climate chamber at a temperature of 41°C ± 1°C and a relative humidity of 60% ± 5% for heat stress. During this period, the mice were fasted and water-deprived. After the mice entered the artificial climate chamber, the rectal temperature was measured every 30 min. After 120 min of heat shock, the measurement was changed to every 20 min. When the rectal temperature reached 40°C and the mice had no spontaneous activity time (mild painless stimulation could not drive the animals to crawl or change position) > 5 s, the modeling was successful.
[0038] 2.3 Pharmacodynamic Evaluation Method 2.3.1 Determination of Organ Index The body weight of the mice was weighed before modeling. After anesthesia and sacrifice with sodium pentobarbital, the liver, heart, spleen, lung, kidney, and thymus were dissected, and the excess fat was removed and then weighed.
[0039] 2.3.2 Determination of Blood Routine and Coagulation Indexes After the mouse model was established, blood was collected from the fundus venous plexus and placed in an anticoagulant tube containing sodium citrate for anticoagulation. 0.1 mL of whole blood was taken to measure the blood routine with an automatic modular blood and body fluid analyzer, and the remaining whole blood was centrifuged at 3000 rpm for 10 min. Then the upper plasma was taken to measure the activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen (FIB) with a semi-automatic coagulation analyzer.
[0040] 2.3.3 Determination of biochemical indexes and oxidative stress indexes The content of AST in liver tissue was detected by the Reitman-Frankel method according to the steps of the kit instructions. The content of AKP in liver tissue was detected by the microplate enzyme-labeling method according to the steps of the kit instructions. The content of LDH in heart tissue was measured by the microplate method according to the steps of the kit instructions. The content of SOD in brain tissue was measured by the WST-1 method according to the steps of the kit instructions.
[0041] 2.3.4 Statistical analysis All experimental results are expressed as ±SD. Statistical processing and plotting were performed using Graphpad Prism 10.1.2 software. Dunnett's method in one-way analysis of variance was used for comparison among multiple groups. P P < 0.05 was considered statistically significant.
[0042] 3. Experimental results 3.1 Effects on the organ indexes of HS mice Compared with the blank group, the liver index of mice in the model group was significantly increased ( P P < 0.01), while the organ indexes of the heart and thymus were significantly decreased ( P P < 0.05, P P < 0.01). There was no significant difference in the changes of spleen index, lung coefficient, and kidney index. Compared with the model group, the Huoxiang Zhengqi Koufuye group, the low-dose and high-dose groups of Trichosanthes kirilowii Maxim. stem and leaf could significantly reduce the liver index of mice ( P P < 0.01, P P < 0.001). Although there was no significant difference in the heart index and thymus index of mice in each drug administration group compared with the model group, there was a tendency to return to the normal group. The results of the organ indexes of mice in each group are shown in Table 1.
[0043] Table 1 Results of organ indexes of mice in each group Group Liver index / % Heart index / % Spleen index / % Lung coefficient / % Kidney index / % Thymus index / % KB 4.07±0.20 0.57±0.04 0.25±0.04 0.59±0.04 1.24±0.10 0.19±0.02 Mod <![CDATA[4.50±0.34 △△ > <![CDATA[0.50±0.04 △ > 0.21±0.02 0.63±0.05 1.21±0.10 <![CDATA[0.13±0.01 △△ > Y <![CDATA[4.05±0.22 ** > 0.54±0.07 0.22±0.02 0.61±0.07 1.21±0.06 0.15±0.02 G-L <![CDATA[3.97±0.20 *** > 0.52±0.05 0.21±0.04 0.58±0.04 1.26±0.08 0.14±0.01 G-H <![CDATA[3.92±0.17 *** > 0.52±0.04 0.20±0.03 0.61±0.10 1.22±0.08 0.15±0.03 3.2 Effects on blood routine and coagulation indexes of HS mice It can be seen from Figure 3 that compared with the blank group, the hemoglobin concentration (HGB), red blood cell count (RBC), and hematocrit (HCT) of mice in the model group were significantly increased ( P< 0.001, P < 0.01), while the prothrombin time (PT) and activated partial thromboplastin time (APTT) were significantly shortened ( P < 0.001), and the fibrinogen (FIB) content was significantly increased ( P < 0.001) (the time was negatively correlated with the FIB content), indicating that the heatstroke mice were in a hypercoagulable state, and due to dehydration leading to blood concentration, the blood routine indexes were abnormal. Compared with the model group, Huoxiang Zhengqi Oral Liquid group significantly reduced the hemoglobin concentration, red blood cell count, hematocrit and fibrinogen content of heatstroke mice ( P < 0.01), and significantly prolonged the prothrombin time ( P < 0.01); both the high-dose and low-dose groups of Trichosanthes kirilowii Maxim. stems and leaves could reduce the fibrinogen content of heatstroke mice ( P < 0.01), and both could prolong the prothrombin time ( P < 0.01), and the high-dose group of Trichosanthes kirilowii Maxim. stems and leaves could also significantly prolong the activated partial thromboplastin time ( P < 0.05).
[0044] 3.3 Effects on Biochemical Indexes and Oxidative Stress Indexes of HS Mice It can be seen from Figure 4 that compared with the blank group, the AST level and LDH level in the model group were significantly increased ( P < 0.001, P < 0.05), and the SOD level was significantly decreased ( P < 0.001). Compared with the model group, Huoxiang Zhengqi Oral Liquid group significantly reduced the AST level of heatstroke mice ( P < 0.001), and significantly increased the SOD level ( P < 0.05); both the high-dose and low-dose groups of Trichosanthes kirilowii Maxim. stems and leaves had improvement effects on the LDH level of heatstroke mice ( P < 0.001), and the low-dose group could also significantly increase the SOD level ( P < 0.05).
[0045] Example 4: Preparation Research of Functional Tea Drink from Trichosanthes kirilowii Maxim. Stems and Leaves 1. Experimental Materials and Drugs Stems and leaves of Trichosanthes kirilowii Maxim. after fruit setting; gallic acid reference substance; rutin reference substance; Folin-Ciocalteu reagent; neutral red; sodium carbonate; sodium nitrite; sodium hydroxide; aluminum nitrate; glucuronic acid; yeast extract; agar powder; oxgall salt; sodium tetraborate; carbazole; anhydrous magnesium sulfate; rose bengal; BCA protein concentration assay kit; tryptone.
[0046] 2. Experimental Methods 2.1 Preparation Process of a Functional Drink Containing Trichosanthes kirilowii Maxim. Stems and Leaves: Trichosanthes kirilowii Maxim. stem and leaf pretreatment → extraction → concentration → centrifugation → blending → sterilization → canning → finished product.
[0047] Trichosanthes kirilowii Maxim. stem and leaf extract: Weigh 40 g of Trichosanthes kirilowii Maxim. stems and leaves after fruit setting, add 10 times the amount of 95% ethanol for reflux extraction twice, 1 h each time, then add 10 times the amount of water for reflux extraction twice, 1 h each time. Combine the filtrates from the four extractions and concentrate under reduced pressure to obtain the Trichosanthes kirilowii Maxim. stem and leaf extract for standby.
[0048] 2.2 Single-factor experiments Using the Trichosanthes kirilowii Maxim. stem and leaf extract as the main raw material, adding sweeteners (granulated sugar, stevioside) and citric acid, determine the optimal formula through single-factor experiments and orthogonal experiments.
[0049] 2.2.1 Screening of the addition amount of Trichosanthes kirilowii Maxim. stem and leaf Fix the addition amount of the compound sweetener at 2% and the addition amount of citric acid at 0.1%, and investigate the influence of adding 10%, 15%, 20%, 25%, 30% of Trichosanthes kirilowii Maxim. stem and leaf on the sensory score.
[0050] 2.2.2 Screening of the addition amount of citric acid Fix the addition amount of Trichosanthes kirilowii Maxim. stem and leaf at 15% and the addition amount of sweetener at 2%, and investigate the influence of adding 0.05%, 0.1%, 0.15%, 0.2%, 0.25% of citric acid on the sensory score.
[0051] 2.2.3 Screening of the addition amount of sweetener (granulated sugar: stevioside = 40:1): Fix the addition amount of Trichosanthes kirilowii Maxim. stem and leaf at 15% and the addition amount of citric acid at 0.1%, and investigate the influence of adding 1%, 1.5%, 2%, 2.5%, 3% of the compound sweetener on the sensory score.
[0052] 3. Beverage evaluation criteria The quality of the Trichosanthes kirilowii Maxim. stem and leaf functional beverage mainly consists of four aspects: color, odor, taste and texture. Therefore, these four factors are selected as the sensory evaluation criteria, and weight values are assigned to each factor according to their importance in the sensory evaluation. With a total score of 100 points, finally, 20 points are assigned to each of color, odor and texture, and 40 points are assigned to taste. In this experiment, 10 test subjects are selected to form an evaluation group to ensure that the test subjects have good judgment on each index of the sensory evaluation. After each sample test, the test subjects need to rest for a while and gargle to prevent the taste residue of the previous sample. During the evaluation process, the test subjects do not communicate with each other. The sensory evaluation criteria for the Trichosanthes kirilowii Maxim. stem and leaf functional beverage are shown in Table 2.
[0053] Table 2 Sensory evaluation criteria for the Trichosanthes kirilowii Maxim. stem and leaf functional beverage Scoring index Scoring criteria Score Bright brownish yellow, harmonious, uniform and stable 15-20 Color and luster (20 points) Light brownish yellow, not soft, not transparent 10-14 Light yellow, light color 0-9 The smell is harmonious, with the unique aroma of Trichosanthes kirilowii stems and leaves 15-20 Smell (20 points) The smell is basically harmonious, without obvious peculiar smell 10-14 The smell is disharmonious, with peculiar smell and unacceptable 0-9 Sour and sweet are moderate, and the taste is good 30-40 Taste (40 points) Sour and sweet are relatively moderate, and the taste is good 15-29 Sour and sweet are out of balance, either too sour or too sweet 1-14 The quality is uniform and stable, without floccules and precipitation 15-20 Morphology of tissue (20 points) There are a small amount of floccules and precipitation, and the tissue is relatively uneven 10-14 There are floccules and a large amount of precipitation 0-9 4. Detection of Physicochemical Indexes of Trichosanthes kirilowii Maxim. Stem and Leaf Functional Beverage (1)Total phenols: The total phenol content in the Trichosanthes kirilowii Maxim. stem and leaf functional beverage was determined by the Folin-Ciocalteu method.
[0054] (2)Total flavonoids: The total flavonoid content in the Trichosanthes kirilowii Maxim. stem and leaf functional beverage was determined by the NaNO 2 -Al(NO 3 ) 3 -NaOH method.
[0055] (3)Neutral polysaccharides: The neutral polysaccharide content in the Trichosanthes kirilowii Maxim. stem and leaf functional beverage was determined by the phenol-sulfuric acid method.
[0056] (4)Acidic polysaccharides: The acidic polysaccharides in the Trichosanthes kirilowii Maxim. stem and leaf functional beverage were determined by the carbazole-sulfuric acid method.
[0057] (5)Protein: The protein concentration of the Trichosanthes kirilowii Maxim. stem and leaf functional beverage was determined according to the method described in the BCA kit instruction manual.
[0058] 5. Detection of Microbiological Indexes of Trichosanthes kirilowii Maxim. Stem and Leaf Functional Beverage (1)Determination of total colony count: The total colony count was determined according to the inspection procedure in GB 4789.2-2022.
[0059] (2)Determination of coliform group: The number of coliforms was determined by the plate count method in GB 4789.3-2016.
[0060] (3)Determination of mold and yeast counts: The mold and yeast counts were determined by the plate count method in GB 4789.15-2016.
[0061] 3. Experimental Results 3.1 Results of Single-Factor Experiments The experimental results showed that (as Figure 5 ), when the addition amount of Trichosanthes kirilowii Maxim. stem and leaf was in the range of 10% - 30%, the sensory score of the Trichosanthes kirilowii Maxim. stem and leaf functional beverage showed a trend of first increasing and then decreasing. When the addition amount of Trichosanthes kirilowii Maxim. stem and leaf reached 15%, the sensory score reached the peak, and the color, odor, and taste of the beverage were all in the best state.
[0062] The experimental results showed that (as Figure 5 ), when the addition amount of the compound sweetener (sucrose: stevioside = 40:1) was in the range of 1% - 3%, the sensory score of the Trichosanthes kirilowii Maxim. stem and leaf functional beverage showed a trend of first increasing and then decreasing. When the addition amount reached 2.5%, the sour and sweet taste of the beverage was more coordinated, and the sensory score reached the peak.
[0063] The experimental results showed that (as Figure 5), when the addition amount of citric acid is in the range of 0.05% - 0.25%, the sensory score of the Trichosanthes kirilowii Maxim. stem and leaf functional beverage shows a trend of first increasing and then decreasing. When the addition amount reaches 0.1%, the sour and sweet taste of the beverage is more coordinated and the sensory score reaches the peak.
[0064] 3.2 Orthogonal experiment Taking the addition amount of Trichosanthes kirilowii Maxim. stem and leaf, the addition amount of citric acid, and the addition amount of compound sweetener as orthogonal factors, an L9(3 3 ) orthogonal factor table was designed to determine the optimal addition amounts of the original solution and the flavoring agent. The orthogonal factor level table is shown in Table 3.
[0065] Table 3 Orthogonal factor level table Level A: Addition amount of Trichosanthes kirilowii stems and leaves B: Addition amount of compound sweetener C: Addition amount of citric acid 1 10% 2% 0.05% 2 15% 2.5% 0.1% 3 20% 3% 0.15% As can be seen from Table 4, the influence degree of each factor on the sensory score of the Trichosanthes kirilowii Maxim. stem and leaf functional beverage is A > C > B, that is, the addition amount of Trichosanthes kirilowii Maxim. stem and leaf > the addition amount of citric acid > the addition amount of compound sweetener. Among the three factors, the addition amount of Trichosanthes kirilowii Maxim. stem and leaf (factor A) has the greatest influence on the sensory score ( P <0.05). In addition, according to the K value, the optimal combination of the Trichosanthes kirilowii Maxim. stem and leaf functional beverage can be determined as A1B1C1, that is, the addition amount of Trichosanthes kirilowii Maxim. stem and leaf extract is 10%, the addition amount of compound sweetener is 2%, and the addition amount of citric acid is 0.05%.
[0066] Table 4 Results of orthogonal design Group Addition amount of A Trichosanthes kirilowii stems and leaves Addition amount of B compound sweetener Addition amount of C citric acid Sensory score 1 1 1 1 89.5 2 1 2 2 88.2 3 1 3 3 85.8 4 2 1 2 85.2 5 2 2 3 82.8 6 2 3 1 87.0 7 3 1 3 78.5 8 3 2 1 79.3 9 3 3 2 79.1 k1 87.83 84.40 85.27 k2 85.00 83.43 84.17 k3 78.97 83.97 82.57 R 8.86 0.97 2.70 3.3 Detection results of physical and chemical indexes of Trichosanthes kirilowii Maxim. stem and leaf functional beverage The contents of total phenols, total flavonoids, neutral polysaccharides, acidic polysaccharides, and proteins in the Trichosanthes kirilowii Maxim. stem and leaf functional beverage are shown in Table 5. It can be seen that the Trichosanthes kirilowii Maxim. stem and leaf functional beverage contains relatively rich chemical components.
[0067] Table 5 Physical and chemical indexes of Trichosanthes kirilowii Maxim. stem and leaf functional beverage Total phenol content (mg / 100 mL) Total flavonoid content (mg / 100 mL) Neutral polysaccharide content (mg / 100 mL) Acidic polysaccharide content (mg / 100 mL) Protein content (mg / 100 mL) 19.59±0.05 3.60±0.48 171.13±3.99 98.43±2.06 527.52±9.19 3.4 Detection results of microbial indexes of Trichosanthes kirilowii Maxim. stem and leaf functional beverage The Trichosanthes kirilowii Maxim. stem and leaf functional beverage was sterilized by the method of sterilizing at 121℃ for 20 min. The results in Table 6 show that the total number of colonies is less than 100 CFU / mL in the national standard, and the test results are qualified. Coliform bacteria, molds, and yeasts were not detected in this test. In the above four microbial detections, the sample test was qualified.
[0068] Table 6 Microbial indexes of Trichosanthes kirilowii Maxim. stem and leaf functional beverage Microbial index Detection method Detection result Detection conclusion Total number of colonies (CFU / mL) GB 4789.2-2022 <100 CFU / mL Qualified Coliform group (MPN / 100 mL) GB 4789.3-2016 Not detected Qualified Mold count (CFU / mL) GB 4789.15-2016 Not detected Qualified Yeast count (CFU / mL) GB 4789.15-2016 Not detected Qualified Example 5: Evaluation of antioxidant activity of Trichosanthes kirilowii Maxim. stem and leaf functional beverage 1. Drugs and reagents Functional beverage of Trichosanthes kirilowii Maxim. stems and leaves prepared in Example 4 (the addition amount of the extract of Trichosanthes kirilowii Maxim. stems and leaves after fruit setting is 10%, the addition amount of the compound sweetener is 2%, and the addition amount of citric acid is 0.05%); 2, 2-diphenyl-1-picrylhydrazyl; 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); potassium ferricyanide; ferric chloride hexahydrate; ferrous sulfate heptahydrate; potassium persulfate; ascorbic acid; salicylic acid; disodium hydrogen phosphate dodecahydrate; sodium dihydrogen phosphate dihydrate; trichloroacetic acid; 6% hydrogen peroxide.
[0069] 2. Experimental method 2.1 Determination of antioxidant activity of the functional beverage of Trichosanthes kirilowii Maxim. stems and leaves 2.1.1 Determination of DPPH free radical scavenging rate Take 100 μL of sample solutions with different concentrations in a 96-well plate, add 100 μL of 0.1 mg / mL DPPH ethanol solution, mix well, react at room temperature in the dark for 30 min, measure the absorbance at 517 nm, and use absolute ethanol as the blank control, V C as the positive control.
[0070]
[0071] In the formula, A 1 is the absorbance value of the sample solution with different concentration gradients after reacting with the DPPH solution, A 2 is the absorbance value of the sample solution with different concentration gradients after reacting with absolute ethanol, A 0 is the absorbance value of absolute ethanol reacting with DPPH.
[0072] 2.1.2 Determination of ABTS free radical scavenging rate Take 100 μL of sample solutions with different concentrations in a 96-well plate, add 100 μL of ABTS working solution, shake and mix well, react at room temperature in the dark for 30 min, measure the absorbance at 734 nm, and use pure water as the blank control, V C as the positive control.
[0073]
[0074] In the formula, A 1 is the absorbance value of the sample solution with different concentration gradients after reacting with the ABTS working solution, A 2 is the absorbance value of the sample solution with different concentration gradients after reacting with pure water, A 0 is the absorbance value of pure water reacting with DPPH.
[0075] In the formula, A 1 is the absorbance value of the sample solution with different concentration gradients after reacting with the ABTS working solution, A 2A is the absorbance value after the reaction of sample solutions with different concentration gradients and pure water. 0 A is the absorbance value after the reaction of pure water and DPPH.
[0076] 2.1.3 Determination of Hydroxyl Radical Scavenging Rate Take 50 μL of sample solutions with different concentrations into a 96-well plate, add 50 μL of 6 mmol / L ferrous sulfate solution, 50 μL of 6 mmol / L hydrogen peroxide solution, incubate at 37 °C for 10 min, then add 50 μL of 6 mmol / L salicylic acid ethanol solution, incubate at 37 °C for 30 min, measure the absorbance at 510 nm, and use pure water as the blank control, V C is the positive control.
[0077]
[0078] In the formula, A 1 is the absorbance value after the reaction of sample solutions with different concentration gradients and ferrous sulfate solution, hydrogen peroxide solution, and salicylic acid ethanol solution, A 2 is the absorbance value after the reaction of pure water replacing hydrogen peroxide solution and sample solutions with different concentration gradients, A 0 is the absorbance value after the reaction of pure water replacing sample solutions with different concentration gradients and ferrous sulfate solution, hydrogen peroxide solution, and salicylic acid ethanol solution.
[0079] 2.1.4 Determination of Total Reducing Power Take 0.2 mL of sample solutions with different concentrations into a 1.5 mL centrifuge tube, add 0.2 mL of 0.2 mol / L phosphate buffer with pH 6.6 and 0.2 mL of 1% potassium ferricyanide solution, vortex and mix well, then incubate in a 50 °C water bath for 20 min. After the reaction, quickly cool with running water, add 0.2 mL of 10% trichloroacetic acid solution, mix well, and centrifuge at 3000 r / min for 10 min. Take 80 μL of the supernatant into a 96-well plate, add 80 μL of pure water and 20 μL of 0.1% ferric chloride solution, shake and mix well, let stand for 15 min, then measure the absorbance at 700 nm, and use pure water as the blank control, V C is the positive control.
[0080]
[0081] In the formula, A 1 is the absorbance value after the reaction of sample solutions with different concentration gradients and potassium ferricyanide solution, trichloroacetic acid solution, and ferric chloride solution, A 2 is the absorbance value after the reaction of pure water replacing the sample and potassium ferricyanide solution, trichloroacetic acid solution, and ferric chloride solution.
[0082] 3. Experimental Results 3.1 Results of DPPH Radical Scavenging Rate As shown in Figure 6 A, in the range of 0.03125 - 4 mg / mL, the DPPH radical scavenging rate of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves showed a dose-dependence. Specifically, as the concentration of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves increased, its ability to scavenge DPPH radicals also enhanced, but it was always less than that of Vc at the corresponding concentration. When the concentration of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves was higher than 2 mg / mL, the increase in the scavenging rate slowed down. At a concentration of 4 mg / mL, its scavenging rate reached 97.06%, slightly higher than the scavenging rate of Vc at 96.59%. The half scavenging rate IC 50 of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves for DPPH radicals was 0.82 ± 0.10 mg / mL.
[0083] 3.2 Determination of ABTS Radical Scavenging Rate As shown in Figure 6 B, in the range of 0.03125 - 1 mg / mL, the ABTS radical scavenging rate of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves showed a dose-dependence. Specifically, as the concentration of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves increased, its ability to scavenge ABTS radicals also enhanced, but it was always less than that of Vc within this range. When the concentration of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves was higher than 2 mg / mL, its ability to scavenge ABTS radicals was comparable to that of Vc, reaching 99.24%. The half scavenging rate IC 50 of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves for ABTS radicals was 0.25 ± 0.04 mg / mL.
[0084] 3.3 Determination of Hydroxyl Radical Scavenging Rate As shown in Figure 6 C, in the range of 0.03125 - 4 mg / mL, the hydroxyl radical scavenging rate of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves showed a dose-dependence. Specifically, as the concentration of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves increased, its ability to scavenge hydroxyl radicals also enhanced. In the range of 0.03125 - 0.125 mg / mL, the ability of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves to scavenge hydroxyl radicals was slightly stronger than that of Vc at the same concentration. However, as the concentration increased, its scavenging ability increased more slowly and was much less than that of Vc. The half scavenging rate IC 50 of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves for hydroxyl radicals was 5.73 ± 0.55 mg / mL.
[0085] 3.4 Determination of Total Reducing Power As shown in Figure 6As can be seen from Table D, in the range of 0.03125 - 4 mg / mL, the total reducing power of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves showed a dose-dependence. Specifically, as the concentration of the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves increased, its reducing power and antioxidant activity enhanced, but it was always less than the reducing power of Vc at the same concentration. At 0.5 mg / mL, the reducing power of Vc was the strongest. From the results, in the concentration range of 0.03125 - 4 mg / mL, there was a good linear relationship between the functional beverage from Trichosanthes kirilowii Maxim. stems and leaves and the reducing power (R 2 = 0.9989).
[0086] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.
Claims
1. An extract of Trichosanthes kirilowii stems and leaves having the effect of preventing and treating heat stroke, characterized in that: It is prepared by the following method: The stem and leaf samples of Trichosanthes kirilowii are appropriately crushed, ethanol is added after weighing, the mixture is heated under reflux for extraction, filtered while hot, and the filtrates are combined to obtain an alcohol extract; water is added to the filter residue, the mixture is heated under reflux for extraction, filtered while hot, and the filtrates are combined with the alcohol extract and concentrated under reduced pressure to obtain the product.
2. The extract of Trichosanthes kirilowii stems and leaves having the effect of preventing and treating heat stroke according to claim 1, characterized in that: It is prepared by the following method: The stem and leaf samples of Trichosanthes kirilowii are appropriately crushed, and after weighing, 6 to 12 times the amount of ethanol are added, and the mixture is heated under reflux for extraction for 1 to 3 times, each time for 0.5 to 2 hours. The mixture is filtered while hot, and the filtrates are combined to obtain an alcohol extract. 6 to 12 times the amount of water is added to the residue, and the mixture is heated under reflux for extraction for 1 to 3 times, each time for 0.5 to 2 hours. The mixture is filtered while hot, and the filtrates are combined with the alcohol extract and concentrated under reduced pressure to obtain the product.
3. The extract of Trichosanthes kirilowii stems and leaves having the effect of preventing and treating heat stroke according to claim 2, characterized in that: It is prepared by the following method: The stem and leaf samples of Trichosanthes kirilowii were properly crushed, and 10 times the amount of ethanol was added after weighing, and the mixture was heated under reflux and extracted twice for 1 hour each time. The mixture was filtered while hot, and the filtrates were combined to obtain an alcohol extract. 10 times the amount of water was added to the residue, and the mixture was heated under reflux and extracted twice for 1 hour each time. The mixture was filtered while hot, and the filtrates were combined with the alcohol extract and concentrated under reduced pressure to obtain the product.
4. The extract of Trichosanthes kirilowii stems and leaves having the effect of preventing and treating heat stroke according to any one of claims 1 to 3, characterized in that: The stems and leaves of the Trichosanthes kirilowii are the stems and leaves after the Trichosanthes kirilowii bears fruit.
5. Use of the Trichosanthes kirilowii stem and leaf extract having the effect of preventing and treating heat stroke as claimed in claim 4 in medicines or functional foods for preventing and treating heat stroke.
6. The use according to claim 5, characterized in that: The heat stroke includes pre-heatstroke, mild heatstroke and severe heatstroke.
7. A pharmaceutical preparation or functional food having the effect of preventing and treating heat stroke, characterized in that: The invention comprises the extract of stem and leaf of Trichosanthes kirilowii with the function of preventing and treating heat stroke as claimed in any one of claims 1 to 3.
8. A functional tea drink made from the stems and leaves of Trichosanthes kirilowii, characterized in that: The invention comprises 10% of the extract of stems and leaves of Trichosanthes kirilowii after fruiting, 2% of the compound sweetener, 0.05% of citric acid and the rest of water.
9. The functional tea drink of Trichosanthes kirilowii stems and leaves according to claim 8, characterized in that: The compound sweetener is white sugar and stevioside in a mass ratio of 40:
1.
10. Use of the functional tea of Trichosanthes kirilowii stems and leaves according to claim 8 in anti-oxidation and anti-aging.