Composition with functions of resisting fatigue and preventing heatstroke and application thereof

Abstract

The composition with the functions of resisting fatigue and preventing heatstroke comprises the following components in parts by mass: 8-12 parts of bitter gourd polypeptide, 6-12 parts of ginger polysaccharide, 6-12 parts of seabuckthorn flavone and 4-12 parts of tea polyphenol. The amino acid sequence of the balsam pear polypeptide is shown as SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequences are 11 amino acids and 26 amino acids respectively. According to the invention, a four-factor three-level L9 orthogonal test is used for further screening and combining different doses of the obtained functional factors with the functions of resisting fatigue and preventing heatstroke, so that a functional factor combination with the functions of resisting fatigue and preventing heatstroke and outstanding effects is obtained, and the dual requirements of resisting fatigue and preventing heatstroke can be met. According to the composition, functional drinks, functional glue, chocolates and lozenges with functions of resisting fatigue and preventing heatstroke are preliminarily developed.

Description

Technical Field

[0001] This invention specifically relates to a composition that has both anti-fatigue and heatstroke prevention functions and its application, belonging to the field of food technology. Background Technology

[0002] Heatstroke is a common illness among workers engaged in outdoor high-temperature operations, and it is classified into three levels: mild (heat exhaustion), moderate (heat failure), and severe (heatstroke). Mild heatstroke reduces work efficiency, while moderate and severe heatstroke can lead to varying degrees of multiple organ failure, coagulation disorders, and even death.

[0003] Currently, common methods for preventing heatstroke include comprehensive measures such as heat acclimatization training, hydration and electrolyte replenishment, sun protection and ventilation, planned rest schedules, and traditional Chinese medicine prevention. Among these, traditional Chinese medicines for preventing heatstroke include Huoxiang Zhengqi Water, Rendan, Shidishui, cooling oil, Fengyoujing, Xiguashuang, and Biwensan.

[0004] In the course of heatstroke, disruption of the intestinal barrier function is a key factor, and endotoxemia secondary to intestinal barrier disruption has become a major cause of death from heatstroke. Recent studies have found that dietary supplements can enhance the body's resistance to heat stress (severe heatstroke) and reduce the damage caused by heatstroke. These supplements include proteins, amino acids, vitamins, minerals, probiotics, and herbs (Xu Hui: Research Progress on the Protective Effects and Mechanisms of Dietary Supplements on Heatstroke, Capital Food and Medicine, April 2023).

[0005] Studies by Mu Dong et al. have found that glutamine combined with high-dose vitamin C can significantly reduce the incidence of heatstroke and can be used as a preventive medication for heatstroke (Master's thesis, Third Military Medical University, 2011).

[0006] CN112933151 discloses an oral rehydration salt composition for preventing exertional heatstroke, which combines traditional Chinese and Western medicine. The composition comprises sodium chloride, potassium chloride, calcium citrate and malate, anhydrous glucose, citric acid fruit juice powder, patchouli extract, and tangerine peel extract. Its pleasant taste promotes compliance with hydration and electrolyte replenishment during high-intensity training. Patchouli has a preventative effect against heatstroke, while tangerine peel promotes water absorption and reduces gastrointestinal discomfort.

[0007] CN108272084 discloses a composition for preventing heatstroke and its application, wherein the active ingredients of the composition consist of L-glutamine, glucose, sodium citrate, sodium chloride, and potassium chloride.

[0008] CN107348280 discloses a heatstroke prevention beverage made from Solomon's Seal rhizome, which includes dried Solomon's Seal rhizome, patchouli powder placed inside the Solomon's Seal rhizome, and soda water for soaking the Solomon's Seal rhizome.

[0009] CN1187040 relates to an effervescent beverage with heatstroke prevention function, which is made from citric acid, sodium citrate, NaHCO3, aspartame, flavoring, potassium chloride, calcium chloride, magnesium sulfate, ferric sulfate, zinc sulfate, vitamin B1, vitamin B2, vitamin B6, vitamin A, vitamin E, vitamin C, and niacin in a certain proportion.

[0010] CN104172174 relates to a preparation for preventing heatstroke during outdoor power operations, which is composed of a combination of herbs including Salvia miltiorrhiza, Cornus officinalis, Nelumbo nucifera leaf, Ziziphus jujuba seed, Prunus mume, Houttuynia cordata, Rehmannia glutinosa, Ophiopogon japonicus, Osmanthus fragrans, Paeonia lactiflora, Eupatorium fortunei, Taraxacum mongolicum, Poria cocos, Citrus reticulata peel, Polygala tenuifolia, Acorus tatarinowii, Trichosanthes kirilowii, Morus alba, Campsis grandiflora, Rubus idaeus, Polygonatum odoratum, Schisandra chinensis, Anemarrhena asphodeloides, Morinda officinalis, Plantago asiatica, and Glycyrrhiza uralensis, and has a significant therapeutic effect on heatstroke.

[0011] CN117338878 discloses a traditional Chinese medicine compound and its herbal herbal tea that can prevent summer heatstroke. The raw materials of the traditional Chinese medicine compound include light bamboo leaves, lotus stems, licorice, watermelon rind, jujube seeds and purslane. It can improve the human body's heat tolerance to high temperatures and reduce the damage to organs and tissues caused by high-temperature environments. It can be used to prevent heatstroke caused by high-temperature environments in summer.

[0012] CN 116637124 discloses a composition and its application for preventing and treating heatstroke. The raw materials include atractylodes, hesperidin, magnolol, poria cocos polysaccharide, succinic acid and curcumin. It also relates to a drop pill preparation for preventing and treating heatstroke.

[0013] In summary, existing technologies disclose that a large number of substances have certain effects in preventing or treating heatstroke, most of which are traditional Chinese medicine formulas. The amino acid category mainly focuses on glutamine, and the mineral category focuses on replenishing salt. However, none of them have the effect of anti-fatigue and preventing heatstroke. Summary of the Invention

[0014] To address the shortcomings of existing technologies, the present invention aims to provide a composition that combines anti-fatigue and heatstroke prevention functions with its application.

[0015] To achieve the above objectives, the present invention adopts the following technical solution:

[0016] A composition with both anti-fatigue and heatstroke prevention functions is made by mixing the following parts by weight: 8-12 parts bitter melon polypeptide, 6-12 parts ginger polysaccharide, 6-12 parts sea buckthorn flavonoids, and 4-12 parts tea polyphenols.

[0017] The amino acid sequence of the bitter melon polypeptide is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0018] The above-mentioned ginger polysaccharide is mainly composed of six different monosaccharides, including mannose, arabinose, galactose, glucose, rhamnose and xylose, with a molar ratio of 26.67:13.33:10.47:4.34:2.67:1.00.

[0019] The preparation method of the above-mentioned bitter melon polypeptide includes the following steps:

[0020] (1) Extraction of bitter melon polypeptides

[0021] S1. Crush the bitter melon and extract the juice.

[0022] S2. Adjust the pH to 8.2-8.5, keep the reaction at 52-56℃ with shaking for 30-45 minutes, centrifuge, and collect the supernatant;

[0023] S3. Adjust the pH to 4.8-5.2, and react at a constant temperature of 38-42℃ for 50-60 min with shaking. Centrifuge, collect the supernatant, ultrafilter, obtain small molecule peptides below 3000 Da, concentrate by vacuum distillation, and spray dry under vacuum.

[0024] S4. Dissolve the bitter melon polypeptide solution in sterile distilled water at 1 / 50 of the volume of the bitter melon juice in step S1.

[0025] (2) Isolation and preparation of bitter melon polypeptides

[0026] The bitter melon polypeptide solution obtained in step (1) was purified by high performance liquid chromatography. Different elution peaks were collected separately, and the last 5 small molecule peptide elution peaks were concentrated and freeze-dried for later use.

[0027] The above-described composition is used in the preparation of solid beverages.

[0028] The above-described composition is used in the preparation of chocolate.

[0029] The above-described composition is used in the preparation of lozenges.

[0030] The above composition is used in the preparation of functional adhesives.

[0031] The advantages of this invention are:

[0032] The composition of this invention comprises bitter melon polypeptides, ginger polysaccharides, tea polyphenols, and sea buckthorn flavonoids. When combined with the daily required complex minerals, water-soluble complex vitamins, and glucose that are depleted through excessive sweating in high temperatures, this composition, when applied to solid beverages, chocolate, functional gums, lozenges, etc., offers the following advantages:

[0033] 1. This invention is formulated based on the mechanism of fatigue and heatstroke in high-temperature and high-humidity environments: Existing technologies for preventing heatstroke mainly focus on physical cooling and rehydration / salt replenishment. However, physical cooling is often impractical in outdoor work, and rehydration / salt replenishment is limited. Heatstroke often begins with gastrointestinal dysfunction, and during heatstroke, increased gastrointestinal permeability allows endotoxins to enter various organs, leading to organ failure. Improving intestinal barrier function and enhancing its tolerance to high temperatures while maintaining physical cooling and rehydration / salt replenishment is a challenge. This invention, formulated based on the mechanism of heatstroke, can effectively delay the progression and harm of heatstroke. Existing anti-fatigue technologies are abundant, but none combine them with heatstroke prevention. Based on the free radical theory of fatigue, this invention recognizes that fatigue is related to the generation of free radicals in the body. Therefore, substances with antioxidant activity possess potential anti-fatigue activity. Adding bitter melon polypeptides, ginger polysaccharides, tea polyphenols, and sea buckthorn flavonoids to the composition can meet the dual needs of anti-fatigue and heatstroke prevention.

[0034] 2. The evaluation method for heatstroke prevention is shifted earlier: Heatstroke is classified into three levels: mild (heat exhaustion), moderate (heat failure), and severe (heatstroke). Existing technologies for heatstroke evaluation do not classify heatstroke levels; most evaluations of preventative effects focus on severe heatstroke (heatstroke), by which time the damage is already significant. As a preventative technology, this invention selects mild heatstroke (heat exhaustion), where the damage has not yet occurred or is minimal, as the evaluation endpoint, thus providing a more objective assessment of the preventative effect.

[0035] 3. Clearly define the effective components of functional factors: Many comparative documents do not refine the raw materials of functional factors, use mixed peptides without sequencing, or simply crush or extract with water, making it difficult to quantify the effective components. The basis for many combinations is not available data and comes from experience. Effective combinations lack scientific data, and the combined effects of anti-fatigue and heatstroke prevention are poor. This invention screens highly effective functional components for anti-fatigue and heatstroke prevention. The effective components of each functional factor are clearly defined. Peptides have amino acid sequences and primary structures, and polysaccharides have clear monosaccharide compositions. Future production can use biosynthetic methods to greatly reduce production costs.

[0036] 4. This invention uses orthogonal experiments to determine the dosage combination of the composition, and the data source is reliable: compositions with the same product name but different dosages sometimes show highly significant differences in functional results. This invention uses a four-factor, three-level L9 orthogonal experiment to further screen and combine different dosages of the functional factors that have both anti-fatigue and heatstroke prevention functions, obtaining a functional factor combination with outstanding efficacy that has both anti-fatigue and heatstroke prevention functions. This composition consists of polyphenols, flavonoids, polysaccharides, and small molecule peptides.

[0037] 5. Based on this composition, we will initially develop functional drinks, functional gels, chocolates, lozenges, etc. that have both anti-fatigue and heatstroke prevention functions. Detailed Implementation

[0038] The present invention will be described in detail below with reference to specific embodiments.

[0039] Example 1: Screening and amino acid sequence determination of bitter melon polypeptides with anti-fatigue effects.

[0040] Bitter melon has various health benefits, including lowering blood sugar, lowering blood lipids, and anti-tumor effects. Currently known bitter melon polypeptides include polypeptide-P, polypeptide-PA, polypeptide-K, and MC6. The first three polypeptides are large molecules, all longer than 100 amino acids, with molecular weights of 11,000, 38,000, and 18,000 respectively, and have blood sugar-lowering effects. MC6 is a small molecule polypeptide, including MC6.1, MC6.2, and MC6.3, with lengths of 18, 11, and 7 amino acids respectively. The C-terminal 7 amino acid sequences of MC6.1 and MC6.2 are identical to those of MC6.3, and their main function is also to lower blood sugar.

[0041] No bitter melon polypeptides with anti-fatigue effects have been found in current technical literature.

[0042] 1. Materials

[0043] The bitter melon was purchased from a farmers' market and professionally identified as a white-stemmed bitter melon. All reagents were purchased from the Nanjing branch of Sinopharm Group, were of analytical grade, and are within their expiration dates.

[0044] Kunming mice, male, SPF grade, weighing 21.2±2.1g, Nanjing Medical University Experimental Animal Center, License No.: SCXK(Su)2021—0001.

[0045] 2. Methods

[0046] 2.1 Extraction of bitter melon polypeptides

[0047] Bitter melon was crushed using a high-speed blender, and the juice was extracted. The pH was adjusted to 8.2-8.5 with NaOH, and the mixture was reacted at a constant temperature of 52-56℃ with shaking for 30-45 minutes. After centrifugation, the supernatant was collected. The pH was then adjusted to 4.8-5.2 with hydrochloric acid, and the mixture was reacted at a constant temperature of 38-42℃ with shaking for 50-60 minutes. After centrifugation, the supernatant was ultrafiltered through a Merck Millipore 3KD disc ultrafiltration membrane to obtain small molecule peptides below 3000 Da. These peptides were concentrated by vacuum distillation and then spray-dried under vacuum. The peptides were dissolved in 1 / 50th the original volume of sterile distilled water to obtain the bitter melon peptide solution.

[0048] 2.2 Isolation and preparation of bitter melon polypeptides

[0049] Bitter melon polypeptide solution was purified by HPLC using a Waters 2696 high performance liquid chromatograph. Different elution peaks were collected separately, and the last five small molecule peptide elution peaks were concentrated and freeze-dried for later use.

[0050] 2.3 Anti-fatigue effect of bitter melon polypeptides

[0051] The anti-fatigue effects of various bitter melon peptides were screened using the methods of Xia Shulin et al.

[0052] The amphibian toad was selected as the experimental animal. Electrophysiological methods were used to study the contractile properties of the toad's gastrocnemius muscle in vivo. At the same time, the contents of lactic acid and malondialdehyde in isolated gastrocnemius muscle were measured to determine the anti-fatigue effects of various bitter melon polypeptides (Xia Shulin, Wu Qingsong. Extraction of ginger polysaccharide and its anti-fatigue effect. Jiangsu Agricultural Sciences, 2014, 42: 240-242.).

[0053] The Ringer's solution group served as the negative control group, the neostigmine group served as the positive control group, and the experimental groups were bitter melon peptide 1, 2, 3, 4, and 5, with a dose of 0.5 mg / mL.

[0054] 2.4 Amino acid sequence determination

[0055] Based on the contractile properties of the gastrocnemius muscle of toads and the content of lactic acid and malondialdehyde, amino acid sequencing was performed on the elution peak sample of bitter melon polypeptide, which was confirmed to have anti-fatigue effects.

[0056] The mass spectrometer was a 4800 Plus MALDI TOF / TOF, with MS / MS detection parameters of MS / MS 2KV Positive mode, laser intensity of 5800, laser shot count of 2000, and mass error of 0.5u.

[0057] 3. Results

[0058] 3.1 Isolation of Bitter Melon Polypeptides

[0059] HPLC analysis revealed 10 main elution peaks in the purified bitter melon polypeptide solution. Considering production feasibility, only 5 small molecule peptides with a molecular weight of less than 5000 Da were selected for further testing.

[0060] As shown in Tables 1 and 2 below, both bitter melon polypeptides 2 and 4 have anti-fatigue effects.

[0061] Table 1. Effects of various bitter melon polypeptides on the maximum contractile force of gastrocnemius muscle in toads in vivo.

[0062]

[0063] Note: Due to individual differences, all test results are compared with those before administration in the same group. A difference greater than 10% indicates a significant difference "*", and a difference greater than 20% indicates an extremely significant difference "**".

[0064] Table 2. Effects of various bitter melon polypeptides on lactic acid and malondialdehyde content in isolated gastrocnemius muscle of toads.

[0065]

[0066] Note: "*" indicates a significant difference compared to the Ringer's solution group, and "**" indicates an extremely significant difference compared to the Ringer's solution group.

[0067] Mass spectrometry analysis:

[0068] Bitter melon polypeptide 2 is a 26-amino acid polypeptide, as shown in SEQ ID NO.2, with the following amino acid sequence:

[0069] Leu-Glu-Ala-Pro-Val-Val-Ala-Leu-Lys-Lys-Pro-Pro-Trp-His-Glu-Trp-Arg-Trp-Phe-Leu-Val-Gln-Gln-Ile-Arg-Arg (LEAPVVALKKPPWHEWRWFLVQQIRR), molecular weight is 3624Da.

[0070] Bitter melon polypeptide 4 is an 11-amino acid polypeptide, as shown in SEQ ID NO.1, with the following amino acid sequence:

[0071] Trp-Arg-Trp-Phe-Leu-Val-Gln-Gln-Ile-Arg-Arg (WRWFLVQQIRR) has a molecular weight of 1768 Da.

[0072] Searching for this amino acid sequence in the Uniprot full sequence database and performing Blast alignment did not detect the same amino acid sequence. The 11 amino acids of bitter melon polypeptide 4 completely overlap with the last 11 amino acids of bitter melon polypeptide 2.

[0073] Example 2: Screening of functional factor combinations with both anti-fatigue and heatstroke prevention effects

[0074] Based on the mechanism of heatstroke, and through literature review and previous research data, four food-grade functional factors that have anti-fatigue and heatstroke prevention effects, either individually or in combination, were selected for testing: bitter melon polypeptide, ginger polysaccharide, sea buckthorn flavonoids, and tea polyphenols.

[0075] 1. Materials

[0076] 1.1 Functional Factors

[0077] The bitter melon polypeptide (11-peptide) is the 11-peptide of bitter melon polypeptide 4 screened in Example 1, purified by HPLC with a purity of 99.66%.

[0078] Ginger polysaccharides were extracted using the compound enzyme method reported by Ma Lihua et al. (Ma Lihua, Qin Weidong, et al. Food Science, 2008, 29(8): 369-371). The polysaccharide content was determined by the sulfuric acid-anthrone method, and the purity was 96.7%. Capillary electrophoresis analysis showed that ginger polysaccharides were mainly composed of six different monosaccharides, including mannose, arabinose, galactose, glucose, rhamnose, and xylose, with a molar ratio of 26.67:13.33:10.47:4.34:2.67:1.00. Tea polyphenols were purchased from Shaanxi Xiazhou Biotechnology Co., Ltd., with a polyphenol content of 99.8%.

[0079] Sea buckthorn flavonoids, with a total flavonoid content of 40.3%, were purchased from Beijing Baoderui Health Industry Co., Ltd.

[0080] Aspartic acid, various vitamins, and mineral salts were all purchased from Nanjing Pharmaceutical Company.

[0081] 1.2 Test Reagents

[0082] The blood lactate and urea test kits are products of Nanjing Jiancheng Bioengineering Institute.

[0083] The mouse endotoxin (ET) Limulus amebocyte lysate (LAL) reagent kit was purchased from Xiamen Limulus amebocyte lysate Reagent Biotechnology Co., Ltd.

[0084] 1.3 Animals

[0085] Kunming mice, male, SPF grade, weighing 21.2±2.1g, Nanjing Medical University Experimental Animal Center, License No.: SCXK(Su)2021—0001.

[0086] 2. Methods

[0087] 2.1 Establishment of a mouse model of combined stress for fatigue prevention and heatstroke prevention under high temperature and humidity

[0088] The artificial climate chamber was set with a temperature of 35±1℃ and a relative humidity of 75±1%. A mouse treadmill was loaded inside to create a fatigue stress model under high temperature and high humidity conditions.

[0089] This invention uses mild heatstroke (heat exhaustion) as the evaluation endpoint. After working in a high-temperature workplace for a certain period of time, the human body will experience symptoms such as dizziness, headache, thirst, excessive sweating, general fatigue, palpitations, poor concentration, and uncoordinated movements, with normal or slightly elevated body temperature. Therefore, a core body temperature of <38℃ is used as the standard. The core body temperatures for moderate and severe heatstroke are 38-40℃ and ≥40℃, respectively.

[0090] Mouse treadmills were set up in the artificial climate chamber (Shanghai Tazheng Technology Co., Ltd.): each track was 58cm long, 8.5cm wide, and 12cm high; the stimulation voltage was 50V.

[0091] During the last three days of gavage, the experimental mice underwent a 3-day treadmill adaptation experiment, once a day for 30 minutes each time, with the experimental environment temperature consistent with the feeding temperature.

[0092] The day after gavage, mice in each group were placed in an artificial climate chamber for a treadmill test. The treadmill speed was 20 m / min and the inclination angle was 15°. The treadmill test was terminated when either the mouse was exhausted from running or its core body temperature reached 38°C.

[0093] Criteria for determining exhaustion in mice running on the platform: When a mouse falls off the electric grid repeatedly and becomes short of breath, it is considered unable to complete the running platform test, and the time of exhaustion is recorded.

[0094] The core body temperature of mice was measured 5 minutes after starting on the treadmill, and recorded once per minute. The measurement was stopped when the core body temperature reached 38°C.

[0095] After the treadmill test, serum and gastrointestinal samples were collected from mice. The four groups with the longest core body temperature of 38°C were tested for exercise fatigue-related biochemical indicators, endotoxins, and bacterial abundance.

[0096] 2.2 Anti-fatigue heatstroke test in mice under high temperature and humidity

[0097] All experimental mice were fed at an ambient temperature of 22±1℃ and a relative humidity of 65±1% and underwent treadmill training. Mice that passed the training were then moved to the next stage of the experiment.

[0098] Mice were randomly divided into groups of 10 each, each mouse was numbered, and provided with experimental food and water. At a fixed time each day (9:00 AM), mice were administered different doses of the test substance via gavage for 10 consecutive days, once daily, at a dose of 0.4 ml each time; the control group received an equal volume of physiological saline.

[0099] One hour after the last administration, a combined stress model test of fatigue heatstroke (heat exhaustion) under high temperature and high humidity was conducted.

[0100] 2.3 Anti-fatigue function test

[0101] In accordance with the requirements of the "Technical Specifications for Inspection and Evaluation of Health Foods (2020)," mouse blood lactate, urea, and liver glycogen were tested. The detection methods are detailed on pages 17-29 of the technical specifications.

[0102] 2.4 Endotoxin Detection

[0103] During the course of heatstroke, intestinal permeability increases, allowing bacteria and endotoxins to enter the bloodstream through intestinal capillaries or lymphatic vessels, leading to endotoxemia.

[0104] The changes in serum endotoxin levels in mice were compared between those of the present invention and the control group by detecting endotoxins in mouse serum. Endotoxin content was determined according to the instructions of the endpoint colorimetric matrix Limulus amebocyte lysate (LAL) reagent kit.

[0105] 2.5 Methods for determining bacterial abundance

[0106] After the treadmill test, fecal samples were collected from the rectum of mice in the orthogonal experimental control group and the A2B1C2D3, A2B2C3D1, A2B3C1D2, and A3B2C1D3 combination mice. The fecal samples from each group were mixed thoroughly, and 0.2g of the mixture was placed in a sterile centrifuge tube, frozen in liquid nitrogen, and stored at -80°C. These samples were then sent for Illumina MiSeq sequencing.

[0107] The I-Sanger cloud platform conducted a survey and analysis of the 16S rRNA gene of the entire bacterial community to detect the abundance of the bacterial community in mice.

[0108] 2.6 Screening for anti-fatigue and heatstroke prevention functions using single-factor and pairwise combinations

[0109] A total of 12 groups were set up: a control group (physiological saline), 5 single-factor groups (bitter melon polypeptide 2, dose 80 mg / kg / day; bitter melon polypeptide 4, dose 80 mg / kg / day, denoted as A; ginger polysaccharide, dose 100 mg / kg / day, denoted as B; sea buckthorn flavonoids, dose 100 mg / kg / day, denoted as C; tea polyphenols, dose 100 mg / kg / day, denoted as D), and 6 pairwise combination groups (groups A and B consisted of bitter melon polypeptide 4 and ginger polysaccharide, with doses of...). The dosages were 80 and 100 mg / kg / day for group AC (bitter melon polypeptide 4 and sea buckthorn flavonoids, respectively); for group AD (bitter melon polypeptide 4 and tea polyphenols, respectively); for group BC (ginger polysaccharide and sea buckthorn flavonoids, respectively); for group BD (ginger polysaccharide and tea polyphenols, respectively); and for group CD (sea buckthorn flavonoids and tea polyphenols, respectively).

[0110] The mouse running test was conducted using the combined stress model of high temperature and high humidity for fatigue prevention and heatstroke prevention established in 2.1 and the experimental method in 2.2. The test was terminated when either the mouse was exhausted or its core body temperature reached 38°C. The results are shown in Table 3.

[0111] The results showed that the time it took for mice in all experimental groups to reach a core body temperature of 38°C was faster than the time it took for them to exhaustion while treading the table. Therefore, the time to reach a core body temperature of 38°C was used as the criterion. Table 3 shows that the time to reach a core body temperature of 38°C in the five single-factor groups (bitter melon polypeptide 2, bitter melon polypeptide 4, ginger polysaccharide, sea buckthorn flavonoids, and tea polyphenols) was 10.1%, 14.9%, 3.5%, 12.2%, and 4.8% longer than that in the control group (saline group), respectively. The time to reach a core body temperature of 38°C in the pairwise combinations AB, AC, AD, BC, BD, and CD was 17.5%, 19.3%, 12.2%, 13.2%, 8.3%, and 16.7% longer than that in the control group (saline group), respectively.

[0112] Table 3. Time to exhaustion or core body temperature of 38°C in mice using univariate and pairwise factors.

[0113]

[0114] 2.7 Orthogonal Experiment

[0115] Based on the results in 2.6, and considering the stomachic and digestive effects of bitter melon, ginger, and sea buckthorn, the above four functional factors were selected as a combination, considering three levels, and the L9(34) four-factor three-level table was used.

[0116] The three levels for each substance are determined as follows:

[0117] Bitter melon polypeptides A1: 40 mg / kg, A2: 80 mg / kg, A3: 120 mg / kg;

[0118] Ginger polysaccharides B1: 60 mg / kg, B2: 90 mg / kg, B3: 120 mg / kg;

[0119] Sea buckthorn flavonoids C1: 60 mg / kg, C2: 90 mg / kg, C3: 120 mg / kg;

[0120] Tea polyphenols D1: 40mg / kg, D2: 80mg / kg, D3: 120mg / kg.

[0121] Table 4. Factors and levels of bitter melon polypeptides, ginger polysaccharides, sea buckthorn flavonoids, and tea polyphenols.

[0122]

[0123] Table 5. Orthogonal experimental design for bitter melon polypeptides, ginger polysaccharides, sea buckthorn flavonoids, and tea polyphenols.

[0124] Serial Number A B C D 1 A1 B1 C1 D1 2 A1 B2 C2 D2 3 A1 B3 C3 D3 4 A2 B1 C2 D3 5 A2 B2 C3 D1 6 A2 B3 C1 D2 7 A3 B1 C3 D2 8 A3 B2 C1 D3 9 A3 B3 C2 D1

[0125] 3. Results and Analysis of Orthogonal Experiments

[0126] 3.1 Mouse treadmill test

[0127] The test was terminated when either the mice reached exhaustion from running on the platform or their core body temperature reached 38°C. The results showed that the time for the mice in all experimental groups to reach a core body temperature of 38°C was faster than the time for them to reach exhaustion from running on the platform. Therefore, the time for the mice to reach a core body temperature of 38°C was used as the criterion.

[0128] The results are shown in Table 6 below:

[0129] Table 6. Time to exhaustion or core body temperature reaching 38°C in mice under different combinations of orthogonal experiments.

[0130]

[0131]

[0132] As shown in the table above, the combined mice of A2B3C1D2, A2B2C3D1, A3B2C1D3, and A2B1C2D3 took the longest to reach a core body temperature of 38℃, at 36.9±4.6, 35.3±2.9, 34.6±3.7, and 33.5±3.8 min respectively. These times were 59.1%, 52.4%, 48.7%, and 44.4% longer than the control group (23.2±4.1 min), respectively, and all were highly significant (P<0.01).

[0133] 3.2 Contents of lactic acid, blood urea nitrogen, liver glycogen, and endotoxin in mice

[0134] Lactic acid is an intermediate product of glucose metabolism. Long-term high-intensity work in emergency environments will inevitably lead to excessive accumulation of lactic acid, which in turn can induce severe muscle soreness and fatigue.

[0135] Urea nitrogen is the final product of protein and amino acid metabolism. When engaging in prolonged or strenuous exercise, the catabolism of proteins and amino acids is significantly enhanced, leading to an increase in serum urea nitrogen levels.

[0136] During exercise, liver glycogen is continuously broken down into glucose and enters the bloodstream to replenish the constantly consumed blood sugar, thereby achieving the effect of anti-fatigue. After exercise, the body's glycogen content will generally decrease, of which most of the endogenous glucose (about 75%) comes from the breakdown of liver glycogen. The increase in liver glycogen content improves fatigue tolerance.

[0137] During the course of heatstroke, intestinal permeability increases, allowing bacteria and endotoxins to enter the bloodstream through intestinal capillaries or lymphatic vessels, leading to elevated endotoxin levels in the blood and even endotoxemia. Endotoxins are an important factor in heatstroke-induced elevated body temperature and multiple organ failure.

[0138] Therefore, blood lactate, blood urea nitrogen, and liver glycogen are the main biochemical indicators for measuring the degree of fatigue in the body; endotoxin content is an important indicator of the degree of heatstroke.

[0139] The results are shown in Table 7 below:

[0140] Table 7 Results of serum lactate (BLA), blood urea nitrogen (BUN), liver glycogen, and endotoxin assays. n=12)

[0141] Group Lactic acid (mg / 100ml) Blood urea nitrogen (mmol / L) Liver glycogen (g / 100g) Endotoxin (EU / mL) control group 36.52±4.68 43.12±3.81 0.88±0.08 1.98±0.16 <![CDATA[A2B1C2D3]]> 25.33±1.22 33.27±3.14 1.37±0.21 1.22±0.11 <![CDATA[A2B2C3D1]]> 28.31±2.13 34.16±387 1.63±0.27 0.76±0.15 <![CDATA[A2B3C1D2]]> 26.69±5.32 32.61±4.02 1.77±0.31 0.69±0.10 <![CDATA[A3B2C1D3]]> 32.62±3.35 38.77±3.52 1.28±0.28 1.16±0.17

[0142] As shown in Table 7, the lactic acid content of combinations A2B1C2D3, A2B2C3D1, A2B3C1D2, and A3B2C1D3 was reduced by 11.19 (P<0.01), 8.21 (P<0.01), 9.83 (P<0.01), and 3.90 (P<0.05) mg / 100ml, respectively, compared with the control group.

[0143] The urea nitrogen levels decreased by 9.85 (P<0.01), 8.96 (P<0.01), 10.51 (P<0.01), and 4.35 (P<0.05) mmol / L compared to the control group, respectively.

[0144] Liver glycogen levels increased by 0.49 (P<0.01), 0.75 (P<0.01), 0.89 (P<0.01), and 0.40 (P<0.01) g / 100g, respectively, compared to the control group.

[0145] Endotoxin levels decreased by 0.76 (P<0.01), 1.22 (P<0.01), and 1.29% compared to the control group, respectively.

[0146] (P<0.01), 0.82 (P<0.01)EU / mL.

[0147] In summary, the combinations A2B2C3D1 and A2B3C1D2 are relatively better (P<0.01).

[0148] 3.3 Abundance of fecal microbiota in mice

[0149] Based on the results of measuring the levels of lactic acid, blood urea nitrogen, liver glycogen, and endotoxin in mice, mouse fecal samples from combinations A2B2C3D1 and A2B3C1D2, which showed better performance, were selected for bacterial abundance detection.

[0150] Sequencing of fecal samples from experimental mice yielded 437,655 valid sequences, which were clustered into 4,341 operational taxonomic units (OTUs) with 97% sequence identity. The sequencing coverage of all fecal samples exceeded 99%. This indicates that the sequences obtained in this experiment represent over 99% of bacterial phylogenetics in the microbial world, demonstrating good sequencing coverage and the observation of a rich variety of species.

[0151] At the phylum level, the nine dominant bacterial phyla in mouse feces are Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, Actinobacteria, Saccharibacteria, Tenericutes, Cyanobacteria, and Deferribacteres, accounting for more than 99% of the bacterial community classification.

[0152] The results are shown in Table 8 below:

[0153] Table 8. Determination of Gut Microbiota Abundance in Mice

[0154]

[0155] Note: When comparing the data in the table with the control group, 'a' indicates a significant difference (P<0.05); 'b' indicates an extremely significant difference (P<0.01).

[0156] In terms of abundance, Firmicutes, Bacteroidetes, and Proteobacteria are the dominant bacterial phyla. Literature indicates that reducing the ratio of Firmicutes and Bacteroidetes can effectively prevent the occurrence of low-grade inflammation.

[0157] Firmicutes is the largest group of bacteria, mostly Gram-positive, appearing as spherical or rod-shaped organisms. Many members of Firmicutes are beneficial bacteria, such as lactobacilli. However, there are also pathogenic bacteria within Firmicutes, such as certain anaerobic clostridiums, Staphylococcus aureus, and pathogenic streptococci.

[0158] Bacteroidetes are a type of bacterium that participates in many important metabolic activities in the human colon and is a very successful competitor in the intestinal ecosystem.

[0159] Proteobacteria includes many pathogenic bacteria, including Escherichia coli, Salmonella, Vibrio cholerae, and Helicobacter pylori.

[0160] Table 8 shows:

[0161] The ratios of Firmicutes / Bacteroidetes in the control group mice and the A2B2C3D1 and A2B3C1D2 combinations were 3.47, 1.13 and 1.19, respectively. The number of Bacteroidetes in the mice with the two combinations was significantly higher than that in the control group, being 2.23 and 2.61 times that of the control group, respectively.

[0162] The number of Proteobacteria in the combination of A2B2C3D1 and A2B3C1D2 was significantly reduced, with ratios of 0.450 and 0.52 compared to the control group, respectively.

[0163] Tenericutes (soft-walled bacteria) are Gram-negative bacteria that are generally harmless to humans. Table 7 shows that the A2B2C3D1 and A2B3C1D2 combinations of soft-walled bacteria were also significantly more abundant than the control group.

[0164] Although Saccharibacteria is not a dominant species, it can form a biofilm on intestinal epithelial cells, protecting them from harmful substances. Table 7 shows that the Saccharibacteria in the A2B2C3D1 and A2B3C1D2 combinations were 5.8 and 4.5 times that of the control group, respectively.

[0165] In summary, both the A2B2C3D1 and A2B3C1D2 combinations effectively promoted the growth of beneficial intestinal bacteria and inhibited the growth of harmful bacteria, which is consistent with the fact that the endotoxin content in the two groups was significantly lower than that in the control group.

[0166] Based on the results of the above treadmill test, lactic acid, blood urea nitrogen, liver glycogen, endotoxin levels, and fecal microbiota abundance, the two combinations A2B2C3D1 and A2B3C1D2 are preferred.

[0167] Specifically, bitter melon polypeptide 80mg / kg, ginger polysaccharide 90mg / kg, sea buckthorn flavonoids 120mg / kg and tea polyphenols 40mg / kg;

[0168] In addition, it contains 80 mg / kg of bitter melon polypeptide, 120 mg / kg of ginger polysaccharide, 60 mg / kg of sea buckthorn flavonoids, and 80 mg / kg of tea polyphenols.

[0169] The dosage for humans is calculated based on 1 / 10 of the weight of mice, which is the industry standard. Assuming an average human weight of 60 kg, the A2B2C3D1 combination is 1.98g, which contains 480mg of bitter melon polypeptide, 540mg of ginger polysaccharide, 720mg of sea buckthorn flavonoids, and 240mg of tea polyphenols. The A2B3C1D2 combination is 2.04g, which contains 480mg of bitter melon polypeptide, 720mg of ginger polysaccharide, 360mg of sea buckthorn flavonoids, and 480mg of tea polyphenols.

[0170] Application Example 1

[0171] A solid beverage that has both anti-fatigue and heatstroke prevention effects

[0172] The solid beverage in this application example weighs approximately 10-15g per package. Each package contains the following ingredients: a combination of functional factors with heatstroke prevention and fatigue relief effects (1.98g or 2.04g), glucose (1-3g), sucrose (3-6g), whey protein powder (1-3g), water-soluble vitamins (64mg, including 1mg each of vitamins B1, 2, 6, and 12, 10mg niacin, and 50mg vitamin C), minerals (0.9g, including 0.3g calcium pantothenate, 0.3g salt, 0.2g potassium chloride, and 0.1g magnesium sulfate), sweet orange flavoring (0.01-0.05g), silicon dioxide (0.3g), and citric acid (0.5g).

[0173] Among them, the functional factor combination is one of the A2B2C3D1 or A2B3C1D2 combinations screened in Example 2.

[0174] Produced using standard solid beverage manufacturing processes.

[0175] Application Example 2

[0176] A type of chocolate that also has anti-fatigue and heatstroke prevention effects

[0177] The chocolate in this application example weighs 7-15g per piece, and each piece's formula includes the following ingredients: 0.5-2.5g white sugar, 1.0-2.5g lactose, 1.5-4.5g cocoa powder, 2.5-4.5g cocoa butter, 1.9-2.8g skim milk powder, 0.2-0.5g anhydrous butter, and 0.99g or 1.02g of functional factors that have anti-fatigue and heatstroke prevention effects.

[0178] The functional factor combination is one of the A2B2C3D1 or A2B3C1D2 combinations screened in Example 2, and is produced according to the conventional chocolate process.

[0179] Application Example 3

[0180] A functional gel with both anti-fatigue and heatstroke prevention effects

[0181] The functional adhesive used in this application example weighs 20-35g per package. Each package contains the following ingredients: 1.5-2.5g colloidal powder, 10-25g water, 0.5-1.0g whey protein powder, 2.0-4.5g glucose, 0.5-2g complex minerals, 0.1-0.5g multiple vitamins, 0.99g or 1.02g of a functional factor composition with anti-fatigue and heatstroke prevention effects, and appropriate amounts of sweetener and flavoring. Alternatively, it may contain 1-3g glucose, 3-6g sucrose, 1-3g whey protein powder, 64mg water-soluble vitamins (1mg each of vitamins B1, 2, 6, and 12, 10mg niacin, and 50mg vitamin C), 0.9g minerals (0.3g calcium pantothenate, 0.3g salt, 0.2g potassium chloride, and 0.1g magnesium sulfate), 0.01-0.05g sweet orange flavoring, and 0.5g citric acid.

[0182] The functional factor combination described in the formula is one of the combinations A2B2C3D1 or A2B3C1D2 selected in Example 2, and the colloidal powder is agar powder and konjac gum mixed in a 5:2 ratio.

[0183] Produced using standard functional adhesive processes.

[0184] Application Example 4

[0185] A lozenge with both anti-fatigue and heatstroke prevention effects

[0186] The functional lozenges used in this application example weigh 3-5g per package. Each package contains the following ingredients: 1.5-3.5g glucose, 0.3-0.6g complex minerals, 0.1-0.2g multivitamins, and 0.99g or 1.02g of a functional combination that also has anti-fatigue and heatstroke prevention effects.

[0187] The functional factor combination described in the formula is one of the A2B2C3D1 or A2B3C1D2 combinations screened in Example 2, and is produced according to the conventional process for lozenges.

[0188] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A composition that combines anti-fatigue and heatstroke prevention functions, characterized in that, It is a mixture of the following parts by weight, including 8-12 parts bitter melon polypeptide, 6-12 parts ginger polysaccharide, 6-12 parts sea buckthorn flavonoids, and 4-12 parts tea polyphenols; The amino acid sequence of the bitter melon polypeptide is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The composition according to claim 1, characterized in that, The ginger polysaccharide comprises mannose, arabinose, galactose, glucose, rhamnose, and xylose, with a molar ratio of 26.67:13.33:10.47:4.34:2.67:1.00 for each monosaccharide.

3. The composition according to claim 1, characterized in that, The preparation method of the bitter melon polypeptide includes the following steps: (1) Extraction of bitter melon polypeptides S1. Crush the bitter melon and extract the juice. S2. Adjust the pH to 8.2-8.5, keep the reaction at 52-56℃ with shaking for 30-45 minutes, centrifuge, and collect the supernatant; S3. Adjust the pH to 4.8-5.2, and react at a constant temperature of 38-42℃ for 50-60 min with shaking. Centrifuge, collect the supernatant, ultrafilter, obtain small molecule peptides below 3000 Da, concentrate by vacuum distillation, and spray dry under vacuum. S4. Dissolve the bitter melon polypeptide solution in sterile distilled water at 1 / 50 of the volume of the bitter melon juice in step S1. (2) Isolation and preparation of bitter melon polypeptides The bitter melon polypeptide solution obtained in step (1) was purified by high performance liquid chromatography. Different elution peaks were collected separately, and the last 5 small molecule peptide elution peaks were concentrated and freeze-dried for later use.

4. The use of the composition according to any one of claims 1-3 in the preparation of solid beverages.

5. Use of the composition according to any one of claims 1-3 in the preparation of chocolate.

6. Use of the composition according to any one of claims 1-3 in the preparation of lozenges.

7. The use of the composition according to any one of claims 1-3 in the preparation of functional adhesives.

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