Anti-fatigue effect of small molecular weight sea cucumber peptide on mice

Anti-fatigue effect of small molecular weight sea cucumber peptide on mice

Abstract: Different concentrations of low molecular weight sea cucumber peptide were administered to mice, and the anti-fatigue effect of sea cucumber peptide on mice was studied. The results showed that low molecular weight sea cucumber peptide had no significant effect on the body weight of mice, but it could significantly prolong the weight-bearing swimming time and rotating rod time of mice, significantly reduce the blood urea nitrogen content of mice after exercise, and increase the liver glycogen content.

Key words: sea cucumber; sea cucumber peptide; anti-fatigue; liver glycogen sea cucumber (Holothurian, sea cucumber), belonging to invertebrates

The animal echinoderma gate (Echinode-rmata) is a generic term for the Holothuroidea animal. The organic component of the dried sea cucumber has up to 90% protein, about 4% lipid, 6% polysaccharide, and a small amount of nucleic acid. The inorganic components contain calcium, magnesium salts and trace elements such as iron, manganese, zinc, copper, molybdenum and selenium [1], which are typical high-protein, low-fat, very low-cholesterol, mineral-rich and vitamin-rich foods. The protein of sea cucumber contains all the amino acids required by the human body, and the proportion of essential amino acids is very high [2], which is a veritable high-quality protein. As an important food resource and drug resource, sea cucumber has long been used in health care, health care and treatment. Yao Ke, the author of the book "Food Materia Medica" published in the Ming Dynasty, pointed out that sea cucumber has the function of supplementing vitality, benefiting the internal organs and sputum deficiency [3]. In the Qing Dynasty, "Chinese herbal medicine" and "Compendium of Materia Medica" and other Chinese medicine classics listed sea cucumber as a supplemental drug [4].

The sea cucumber peptide refers to a protein hydrolysate which is mainly composed of small molecular peptides and has multiple functional components obtained by hydrolysis and purification of sea cucumber by protease. It has many physiological functions such as lowering blood pressure, anti-fatigue, improving immunity and delaying aging. Compared with sea cucumber, sea cucumber peptide has good physical and chemical properties such as good solubility, stability and low viscosity. Therefore, sea cucumber peptide prepared by enzymatic hydrolysis has easy digestion and absorption, high bioavailability, no antigenicity, and safe consumption. The advantages are higher bio-valence and more important physiological functions than ordinary sea cucumber products. Fresh sea cucumber is used as raw material, and high-purity low molecular weight sea cucumber peptide is obtained by enzymatic hydrolysis and ultrafiltration. In this paper, the anti-fatigue effects of sea cucumbers on weight-bearing swimming and rod-turning time, blood urea nitrogen and liver glycogen content of mice after exercise were analyzed, and the theory of further development of functional foods with significant anti-fatigue function was established. basis.

1 Materials and methods

1.1 Materials

1.1.1 Experimental Animals Kunming breeds male and female mice, weighing 18g ~ 22g.

1.1.2 Experimental material Sea cucumber peptide, peptide with molecular weight less than 1500D accounted for more than 90%.

1.1.3 Experimental instruments and reagents TH-86- 340- LA Tianhan ultra-low temperature refrigerator; semi-automatic biochemical analyzer; YLS- 4C rotating rod fatigue meter; swimming box; analytical balance; blood urea nitrogen determination kit, liver glycogen determination kit Other test reagents are of the AR grade.

1.2 Experimental methods

1.2.1 Animal grouping and intragastric dose design The mice were randomly divided into 4 groups according to body weight, 10 rats in each group, half male and half female, as the control group, low dose group, medium dose group and high dose group. After 7 days of adaptive feeding, the stomach was administered by gavage. The daily doses of the low, medium and high dose groups were 2.703mg/0.5mL/20g body weight, 5.406mg/0.5mL/20g body weight, 6.758mg/0.5mL/20g body weight; Double distilled water, weighed once a week. The diet, spirit, hair, and urine of the mice were observed daily. After the test article was continuously administered for 28 days, each index was measured.

1.2.2 Measurement of mouse body weight The mice were weighed before administration, 1 week after administration, 2 weeks after administration, 3 weeks after administration, and 4 weeks after administration, and the average value of each group was calculated. recording.

1.2.3 Weight-bearing swimming experiment [5]

After the last administration of the test substance for 30 minutes, the mice were given a weight of 5% by weight of the lead wire, and the mice were placed in a swimming pool having a water depth of 30 cm and a water temperature of (25 ± 1) °C. Record the time from the start of swimming until the head is completely submerged in water for 8s and cannot rise to the surface as swimming time.

1.2.4 Rotating rod test The training was carried out for two days under the condition of a speed of 36 r/min and a time of 30 min. On the third day, 30 minutes after the administration of the test substance, the mouse was placed on a rod of a rotary argon apparatus, and a rotating rod experiment was performed at (rotation speed: 36 r/min, time: 45 min). The rotary bar will automatically record the time the mouse has dropped from the start to the time of the stick, ie as the time of the wand.

1.2.5 Determination of blood urea nitrogen content [5]

Principle: Under the conditions of heating and strong acid, urea nitrogen and diacetyl condensed to synthesize red dipyridazine, Fearon reaction, and the urea nitrogen content can be calculated according to the color depth.

METHODS: After the last administration of the test substance for 30 min, the mice were placed in a swimming pool with a water depth of 30 cm and a water temperature of (25 ± 1) °C for 90 min. After 60 min of rest, blood was taken from the fundus and 0.5 μL and 150 μL of anticoagulant. (Trisodium citrate dihydrate solution) was added together to a 1 mL microtube, and each reagent was added to each tube according to the blood urea nitrogen test kit instructions. After the addition, mix well, place the water in the boiling water for 15 minutes, and immediately cool with tap water. The absorbance was measured by a semi-automatic biochemical analyzer at a wavelength of 520 nm, and the content of urea nitrogen in the bleeding was calculated.

1.2.6 Determination of hepatic glycogen content [6]

Principle: Glycogen can be dehydrated under the action of concentrated sulfuric acid to form furfural derivatives, which in turn react with anthrone (C4H10O) to form a blue compound, which is colorimetrically quantified with the standard glucose solution treated in the same way. The glycogen is very stable in concentrated sulfuric acid solution, so the tissue is placed in concentrated sulfuric acid to heat to destroy other components and retain glycogen before color development.

Method: After the last administration of the test substance for 30 minutes, the mice were placed in a swimming pool with a water depth of 30 cm and a water temperature of (25 ± 1) °C for 90 minutes. The mice were sacrificed immediately by the cervical vertebrae method [18], and the liver was rinsed with physiological saline. Weigh 100mg and 300μL of lye [by sample mass (mg): lye volume (μ L) = 1: 3] into the test tube, puncture each tube with plastic wrap, and tie a small tube on the film The hole (preservative film prevents moisture from evaporating, punctures the hole for the gas to expand and contract), and is placed in a boiling water bath for 20 minutes and then cooled. The liver glycogen hydrolysate is further prepared into a glycogen detection solution. Each reagent was added to each tube according to the hepatic glycogen assay kit. After mixing, the mixture was boiled for 5 minutes in boiling water. After cooling, the absorbance was measured by a semi-automatic biochemical analyzer at a wavelength of 620 nm, and the liver glycogen content was calculated.

1.3 Data statistics and analysis [7]

Data were statistically analyzed using SPSS software, and the significance test was performed using an independent sample t test. The data was expressed as ± SD.

2 Results and discussion

2.1 Effect of sea cucumber peptide on mouse status

According to the observation of 4 weeks: the mice had no abnormalities in mental state, diet, hair state, urine and so on during the experiment. In the initial stage of the experiment, occasional mouse death was caused by inadvertent gavage.

2.2 Effect of sea cucumber peptide on body weight of mice

From the 28d weight gain of the control group and the experimental group, it can be seen that the weight of the mice in the control group and the experimental group generally showed an increasing trend, and the growth was slightly different. After t test, there was no significant difference between low dose, medium dose, high dose and control group (P>0.05). The above results indicate that sea cucumber peptide has no significant effect on the body weight of mice.

2.3 Effect of sea cucumber peptide on swimming and rod time in mice

The improvement of exercise endurance is the most direct and objective indicator of strengthening anti-fatigue ability [8]. The length of swimming time and rotating rod time can reflect the degree of animal sports fatigue and the device is simple and can objectively reflect the physical fitness of the body. There are many factors involved in exercise experiments. In the swimming experiment, the weight should be moderate. When the wound is too tight, the blood circulation of the rat tail is not smooth, which affects the experimental results. It is easy to fall off when too loose; some mice are easy to float on the water surface, so that they can keep moving. The temperature and swimming space are also very important. In the rotating rod experiment, it is necessary to train at a certain speed for 2 days and 30 minutes per day, so that they can get used to this sport. Compared with the control group, the rats in the experimental group had significantly longer weight-bearing swimming and swinging time. After t test, the difference was significant between the different dose groups and the control group (P<0.05 or P<0.01). The swimming time in the middle dose group and the rotating rod time in the low dose group were more than doubled compared with the control group. The above results indicate that sea cucumber peptide can significantly prolong the test

The weight-bearing swimming time and the turning time of the mouse. The medium and low dose effects are more obvious.

2.4 Effects of sea cucumber peptide on blood urea nitrogen and liver glycogen content in mice after exercise

During the exercise, as the long-term exercise progresses, the body can not obtain sufficient energy through saccharification of sugar and fat, and the catabolism of protein and amino acids will be strengthened. The serum urea nitrogen content of the body increases with the increase of exercise load, and the body adapts to the load. The worse the ability, the more obvious the increase in serum urea nitrogen. After vigorous exercise, serum urea nitrogen is significantly increased. The serum urea nitrogen content can reflect the decomposition of muscle protein, anabolic status and damage and recovery of muscle cells after intense training. Therefore, serum urea nitrogen can evaluate the body's movement. The ability to withstand lower physical loads. Glycogen is an important source of exercise energy. The storage of glycogen can directly affect the body's ability to exercise. Increasing the amount of glycogen reserves can improve endurance and exercise capacity, and help to resist fatigue. The exhaustion of physical strength during prolonged intense exercise is always accompanied by the depletion of muscle glycogen. At the same time as the muscle glycogen is depleted, in order to maintain blood sugar levels, the liver glycogen reserve will be reduced, and the test substance can increase the liver glycogen reserve or/and Reduce the consumption of liver glycogen during exercise to provide more energy, so the content of hepatic glycogen can indicate the speed or degree of fatigue [9]. It can be seen from Table 3 that the blood urea nitrogen content of the experimental group was significantly lower than that of the control group, and the liver glycogen content was significantly higher than that of the control group (P<0.05 or P<0.01). Especially in the low-dose group, the effect was better. Compared with the control group, the difference between the two indexes was extremely significant (P<0.01). The above experimental results show that for the mice after exercise, the sea cucumber peptide can significantly reduce the blood urea nitrogen content, increase the liver glycogen content, enhance the adaptability of the body to the load, accelerate the elimination of fatigue, and show a strong anti-fatigue effect. Improve the endurance of the body.

3 Conclusion

3.1 Sea cucumber peptide had no significant effect on the body weight of mice, which can significantly prolong the weight-bearing swimming time and rotating rod time of mice; significantly reduced the blood urea nitrogen content and increased the liver glycogen content of mice after exercise.

3.2 Comprehensive analysis can be known: Sea cucumber peptide has the effect of increasing exercise tolerance, promoting glycogen storage, and accelerating the body's urea nitrogen metabolism, and has anti-fatigue effect.