CN116138455A - Panax notoginseng polysaccharide extract and its application in the preparation of anti-fatigue products - Google Patents

The invention belongs to the technical field of medicines or foods, and relates to a pseudo-ginseng polysaccharide extract and application thereof in preparing an anti-fatigue product. The pseudo-ginseng polysaccharide extract is prepared by taking pseudo-ginseng dregs from which the total saponins of pseudo-ginseng are extracted as raw materials and adopting a water extraction and alcohol precipitation method. Animal experiments show that the pseudo-ginseng polysaccharide extract prepared by the preparation method has obvious anti-fatigue effect for the first time. The preparation method of the extract is simple and easy to obtain, is suitable for industrial production, has definite curative effect, provides another thought for preparing products with anti-fatigue function, and has very wide application prospect. The invention extracts the notoginseng polysaccharide extract from the notoginseng residues waste after extracting the notoginseng total saponins, thereby reducing the environmental pollution and avoiding the waste of notoginseng resources. The Notoginseng radix polysaccharide extract can be used as raw material of food, functional food, health food or medicine.

三七多糖提取物及其在制备抗疲劳产品中的应用Panax notoginseng polysaccharide extract and its application in preparing anti-fatigue products

技术领域Technical Field

本发明属于食品及医药技术领域，涉及一种三七多糖提取物及其在制备抗疲劳产品中的应用。The invention belongs to the technical field of food and medicine, and relates to a notoginseng polysaccharide extract and application thereof in the preparation of an anti-fatigue product.

背景技术Background Art

随着现代社会生活节奏的加快，社会竞争的加剧，生存压力加大，“亚健康”状态成为困扰人们的大敌。WHO提出亚健康状态最典型的表现为疲劳。疲劳是一种生理性现象，对人来说是一种保护性的机制，这是身体向我们发出应该休息的信号。运动性疲劳包括生理性疲劳(机体能量消耗，表现为肌肉酸痛疲倦、无力等，运动能力下降)和心理性疲劳(精神疲惫，主要表现为学习与工作效率低，抑郁和焦虑)。运动性疲劳产生的中枢机制：主要有脑细胞内糖的大量消耗，氨产生增加和清除能力下降；脑内抑制性神经递质γ-氨基丁酸(GABA)、5-羟色胺(5-HT)浓度升高，多巴胺(DA)、乙酰胆碱(Ach)浓度下降等，从而引起中枢疲劳。With the acceleration of the pace of life in modern society, the intensification of social competition, and the increase in survival pressure, the "sub-health" state has become a major enemy that troubles people. WHO proposed that the most typical manifestation of sub-health is fatigue. Fatigue is a physiological phenomenon and a protective mechanism for people. It is a signal from the body that we should rest. Sports fatigue includes physiological fatigue (energy consumption of the body, manifested as muscle soreness, fatigue, weakness, etc., and decreased athletic ability) and psychological fatigue (mental fatigue, mainly manifested as low learning and work efficiency, depression and anxiety). The central mechanism of sports fatigue: mainly includes a large amount of sugar consumption in brain cells, increased ammonia production and decreased clearance ability; increased concentrations of inhibitory neurotransmitters γ-aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT) in the brain, and decreased concentrations of dopamine (DA) and acetylcholine (Ach), which cause central fatigue.

目前，市场上抗疲劳的功能产品很多，如红牛、启力、力保健等，应用这类产品可以迅速缓解人体的疲劳感，使人感觉头脑清醒，精神兴奋。这类产品以饮料制品居多，一般多含有咖啡因，咖啡因具有兴奋中枢的作用，其含量为每100ml饮料中约含咖啡因15-100mg。在应用这类含咖啡因的产品后，短时间内精神得到一定的兴奋，然而当饮用一定量后，兴奋度并不随饮用量的增加而增加，反而在大量饮用这类饮料后，会使人体感到更加疲惫，甚至形成深度疲劳。西医采用补充氨基酸、肌醇等营养能源物质，起到暂时舒缓疲劳的作用，作用效果不持久。还有一些缓解体力疲劳的药物或补品含有激素，毒副作用较大，而且不方便服用，或没有经过药理验证，迄今为止，人们一直在寻找一种制作简单、功效确切、服用方便、无副作用的抗疲劳的药物。At present, there are many anti-fatigue functional products on the market, such as Red Bull, Qili, Libaojian, etc. The application of such products can quickly relieve the fatigue of the human body, make people feel clear-headed and excited. Most of these products are beverage products, which generally contain caffeine. Caffeine has the function of exciting the central nervous system, and its content is about 15-100mg of caffeine per 100ml of beverage. After using such caffeinated products, the spirit will be excited to a certain extent in a short time. However, after drinking a certain amount, the excitement does not increase with the increase in the amount of drinking. On the contrary, after drinking a large amount of such beverages, the human body will feel more tired and even form deep fatigue. Western medicine uses supplementary amino acids, inositol and other nutritional energy substances to temporarily relieve fatigue, but the effect is not lasting. There are also some drugs or supplements that relieve physical fatigue that contain hormones, have large toxic and side effects, and are inconvenient to take, or have not been pharmacologically verified. So far, people have been looking for an anti-fatigue drug that is simple to make, has a definite effect, is easy to take, and has no side effects.

三七[Panax notoginseng(Burk.)F.H.Chen]，又名田七、血参、田三七、滇三七、参三七，系五加科人参属多年生草本植物，主要药用部位为根部，产地主要为云南文山，广西、江西、四川等地也有种植。三七为我国的传统名贵中药材，享有“金不换”、“南国神草”的美称，可见其活血化瘀、消肿止痛药效之显著，主要用于治疗外伤出血、咯血、吐血、便血、胸腹刺痛、跌打肿痛等。三七皂苷为三七的主要活性成分，具有抗炎、抗肿瘤、提高免疫力、保护心血管、保肝、保肾等药理作用。三七多糖作为三七研究另一个热门的活性成分，药理研究表明三七多糖具有增强机体免疫力的作用，近几年来，中药多糖的提取工艺、药理活性研究是一个热门，有研究表明中药多糖为一类天然的高分子化合物，具有增强机体免疫力、抗肿瘤、抗辐射、降血糖、抗心肌缺血、抗炎、抗氧化等多种药理活性。同时，植物多糖又具有来源广泛、毒副作用小的特点，对多糖的开发利用越来越受研究者的重视。Panax notoginseng (Burk.) F.H.Chen, also known as Tianqi, blood ginseng, Tiansanqi, Yunnan Sanqi, Shensanqi, is a perennial herbaceous plant of the genus Panax in the Araliaceae family. The main medicinal part is the root. It is mainly produced in Wenshan, Yunnan, and is also planted in Guangxi, Jiangxi, Sichuan and other places. Panax notoginseng is a traditional precious Chinese medicinal material in my country. It enjoys the reputation of "golden incomparable" and "shencao of the south", which shows that it has significant effects of promoting blood circulation, removing blood stasis, reducing swelling and relieving pain. It is mainly used to treat traumatic bleeding, hemoptysis, hematemesis, blood in the stool, chest and abdominal pain, bruises and swelling, etc. Panax notoginseng saponins are the main active ingredients of Panax notoginseng, which have pharmacological effects such as anti-inflammatory, anti-tumor, improving immunity, protecting cardiovascular, liver and kidney. Panax notoginseng polysaccharide is another popular active ingredient in Panax notoginseng research. Pharmacological studies have shown that Panax notoginseng polysaccharide has the effect of enhancing the body's immunity. In recent years, the extraction process and pharmacological activity research of Chinese medicine polysaccharides has been a hot topic. Studies have shown that Chinese medicine polysaccharides are a class of natural high-molecular compounds with multiple pharmacological activities such as enhancing the body's immunity, anti-tumor, anti-radiation, hypoglycemic, anti-myocardial ischemia, anti-inflammatory, and antioxidant. At the same time, plant polysaccharides have the characteristics of a wide range of sources and few toxic and side effects, and the development and utilization of polysaccharides has received more and more attention from researchers.

研究表明，三七皂苷提取后的药渣中的三七多糖在三七药渣中的含量约为2％，每年弃去三七多糖近30吨，因此，将提取后的药渣用于制备三七多糖提取物，不仅节约了原料，而且经皂苷提取后的药渣由于组成成分不同，发挥的药效也不完全相同。Studies have shown that the content of Panax notoginseng polysaccharide in the residues after Panax notoginseng saponin extraction is about 2%, and nearly 30 tons of Panax notoginseng polysaccharide are discarded every year. Therefore, using the extracted residues to prepare Panax notoginseng polysaccharide extract not only saves raw materials, but also the residues after saponin extraction have different compositions, so the efficacy is not exactly the same.

发明内容Summary of the invention

本发明的目的是提供一种疗效确切、制备方法简单的三七多糖提取物。The purpose of the present invention is to provide a Panax notoginseng polysaccharide extract which has definite curative effect and simple preparation method.

本发明的另一目的是提供一种三七多糖提取物在制备抗疲劳产品中的应用。Another object of the present invention is to provide a use of a Panax notoginseng polysaccharide extract in the preparation of an anti-fatigue product.

为达到上述目的，本发明通过以下技术方案来实现：To achieve the above object, the present invention is implemented by the following technical solutions:

本发明提供一种三七多糖提取物，所述的三七多糖提取物是以提取过三七总皂苷的三七药渣作为原料，采取水提醇沉的方法制备的三七多糖提取物：The present invention provides a Panax notoginseng polysaccharide extract, wherein the Panax notoginseng polysaccharide extract is prepared by using Panax notoginseng drug residues from which Panax notoginseng total saponins have been extracted as raw materials and adopting a water extraction and alcohol precipitation method:

所述三七多糖提取物的制备方法依次包括如下步骤：The preparation method of the Panax notoginseng polysaccharide extract comprises the following steps in sequence:

(1)对三七药渣进行水提，得到提取液C；(1) extracting the notoginseng residue with water to obtain an extract C;

所述“水提”包括如下步骤：The “water extraction” comprises the following steps:

①取三七药渣，加入水，加热回流，收集液体，即为提取液A；① Take the residue of Panax notoginseng, add water, heat and reflux, and collect the liquid, which is the extract A;

②向步骤①的固体残余物中加入水，加热回流，收集液体，即为提取液B；② Add water to the solid residue of step ①, heat to reflux, and collect the liquid to obtain the extract B;

③将提取液A和提取液B合并，得到提取液C；③ Combine the extract A and the extract B to obtain the extract C;

所述方法中，In the method,

步骤①中的三七药渣或步骤②中的固体残余物与加入的水的质量体积比为：1:6-16；The mass volume ratio of the notoginseng residue in step ① or the solid residue in step ② to the added water is: 1:6-16;

优选地，Preferably,

步骤①中的三七药渣与加入的水的质量体积比为：1:8-10；The mass volume ratio of the notoginseng residue in step ① to the added water is: 1:8-10;

步骤②中固体残余物与加入的水的质量体积比为：1：6-8；In step ②, the mass volume ratio of the solid residue to the added water is: 1:6-8;

(2)将步骤(1)得到的提取液C进行减压浓缩：提取液C经70-80℃减压浓缩至原体积的1/(8-10)，冷却、过滤，取滤液，缓缓加入3-5倍量无水乙醇，边加边搅拌，放置过夜，抽滤得滤渣；(2) Concentrating the extract C obtained in step (1) under reduced pressure: Concentrate the extract C under reduced pressure at 70-80° C. to 1/(8-10) of the original volume, cool and filter, take the filtrate, slowly add 3-5 times the amount of anhydrous ethanol, stir while adding, let stand overnight, and filter to obtain the residue;

(3)滤渣干燥即得三七多糖提取物；(3) drying the filtered residue to obtain the notoginseng polysaccharide extract;

所述的三七多糖提取物的制备方法具体为：The preparation method of the Panax notoginseng polysaccharide extract is specifically as follows:

三七药渣加入水，药渣与水的质量体积比为1：6-1：16，热回流提取4-6h，过滤得三七多糖一次浸提液；过滤后的药渣与水按照1:6-1：16(W/V)混合，热回流4-6h，过滤得三七多糖二次浸提液。合并两次滤液，70-80℃减压浓缩至原体积的1/8-1/10，放置至室温，过滤，取滤液，缓缓加入3-5倍量无水乙醇，边加边搅拌，放置过夜，抽滤得滤渣，减压干燥，即得三七多糖提取物。Add water to the residue of Panax notoginseng, the mass volume ratio of the residue to water is 1:6-1:16, heat reflux extraction for 4-6 hours, filter to obtain the first extract of Panax notoginseng polysaccharide; the filtered residue is mixed with water at 1:6-1:16 (W/V), heat reflux for 4-6 hours, and filter to obtain the second extract of Panax notoginseng polysaccharide. Combine the two filtrates, reduce pressure and concentrate to 1/8-1/10 of the original volume at 70-80℃, let it stand to room temperature, filter, take the filtrate, slowly add 3-5 times the amount of anhydrous ethanol, stir while adding, let it stand overnight, filter to obtain the residue, and reduce pressure and dry to obtain Panax notoginseng polysaccharide extract.

所述的三七药渣为提取过三七总皂苷的药渣；The Panax notoginseng medicinal residues are the medicinal residues from which Panax notoginseng total saponins have been extracted;

所述的三七药渣通过如下方法获得：取三七粉碎成粗粉，用70-80％的乙醇提取，滤过后的药渣经干燥得三七药渣。The Panax notoginseng medicinal residue is obtained by the following method: Panax notoginseng is crushed into coarse powder, extracted with 70-80% ethanol, and the filtered medicinal residue is dried to obtain Panax notoginseng medicinal residue.

所述的三七药渣中三七多糖占三七药渣的含量不低于1.5％。The content of notoginseng polysaccharide in the notoginseng medicinal residue is not less than 1.5%.

所述三七多糖提取物中三七多糖的含量为40-70％：其中采用硫酸蒽酮法测定的中性多糖含量为20-50％，采用间羟基联苯法测定的酸性多糖含量为10-30％。The content of Panax notoginseng polysaccharide in the Panax notoginseng polysaccharide extract is 40-70%, wherein the content of neutral polysaccharide measured by the sulfuric acid anthrone method is 20-50%, and the content of acidic polysaccharide measured by the m-hydroxybiphenyl method is 10-30%.

本发明提供了所述三七多糖提取物在制备抗疲劳产品中的应用。The present invention provides application of the notoginseng polysaccharide extract in preparing an anti-fatigue product.

所述的产品为食品、保健食品、药品或食品添加剂。The product is food, health food, medicine or food additive.

进一步地，所述的疲劳为运动性疲劳。Furthermore, the fatigue is sports fatigue.

所述的食品可以为三七多糖提取物与食品添加剂制成的饮料、饼干等。The food can be beverages, biscuits, etc. made from Panax notoginseng polysaccharide extract and food additives.

所述的药品可以为三七多糖提取物与药学上可接受的载体或赋形剂制备的片剂、胶囊、颗粒、滴丸、口服液等。The medicine can be tablets, capsules, granules, pills, oral liquids, etc. prepared from Panax notoginseng polysaccharide extract and pharmaceutically acceptable carriers or excipients.

所述三七多糖提取物的剂量可以为100-400mg/kg。The dosage of the Panax notoginseng polysaccharide extract can be 100-400 mg/kg.

进一步地，本发明提供了三七多糖提取物的功能性饮料及其制备方法：Furthermore, the present invention provides a functional beverage of Panax notoginseng polysaccharide extract and a preparation method thereof:

所述的功能性饮料按重量份计，每100份三七多糖提取物运动饮料中包括以下重量份的原料：三七多糖提取物2-5份、赤藓糖醇9-10份、甜菊糖苷0.038-0.040份，余量为纯净水。The functional beverage comprises the following raw materials in parts by weight per 100 parts of Panax notoginseng polysaccharide extract sports beverage: 2-5 parts of Panax notoginseng polysaccharide extract, 9-10 parts of erythritol, 0.038-0.040 parts of stevioside, and the balance is purified water.

其制备方法包括如下步骤：The preparation method thereof comprises the following steps:

步骤一：按重量份，在50-60℃纯净水中溶解三七多糖提取物、赤藓糖醇、甜菊糖苷，不断进行搅拌，使液体混合均匀，放冷，过滤；Step 1: Dissolve the notoginseng polysaccharide extract, erythritol and stevioside in 50-60°C pure water according to weight, stir continuously to mix the liquid evenly, cool and filter;

步骤二：将步骤一中混合均匀的液体进行灌装，先将液体温度加热到60-65℃，排除管内空气，再进行密封；Step 2: Fill the liquid mixed evenly in step 1, heat the liquid temperature to 60-65℃, remove the air in the tube, and then seal it;

步骤三：将灌封后液体置于110-130℃高温下灭菌15-25min，冷却后得到三七多糖提取物运动饮料。Step 3: sterilize the encapsulated liquid at a high temperature of 110-130°C for 15-25 minutes, and obtain a Panax notoginseng polysaccharide extract sports drink after cooling.

本发明的有益效果在于：本发明公开了一种三七多糖提取物的制备方法，该方法制得的三七多糖提取物经动物试验，具有显著的抗疲劳的功效，功效确切，无毒副作用；制作方法简单，适合工业化生产。The beneficial effects of the present invention are as follows: the present invention discloses a method for preparing a Panax notoginseng polysaccharide extract; the Panax notoginseng polysaccharide extract prepared by the method has significant anti-fatigue effect through animal experiments, has definite effect, and has no toxic side effects; the preparation method is simple and is suitable for industrial production.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1三七多糖提取物对小鼠爬杆时间影响的实验结果图。Figure 1. Experimental results of the effect of Panax notoginseng polysaccharide extract on the climbing time of mice.

***：与阴性组相比，P＜0.001；***: P < 0.001 compared with the negative group;

图2三七多糖提取物对小鼠游泳时间影响的实验结果图。Figure 2 shows the experimental results of the effect of Panax notoginseng polysaccharide extract on the swimming time of mice.

**：与阴性组相比，P＜0.01；***：与阴性组相比，P＜0.001；**: compared with the negative group, P < 0.01; ***: compared with the negative group, P < 0.001;

图3三七多糖提取物对小鼠脏器组织病理学检测的实验结果图。Figure 3 shows the experimental results of the histopathological test of mouse organs by using Panax notoginseng polysaccharide extract.

图4三七多糖提取物对小鼠肝肾功能(BUN、ALT、AST)影响的实验结果图。Figure 4 is a graph showing the experimental results of the effect of Panax notoginseng polysaccharide extract on liver and kidney function (BUN, ALT, AST) in mice.

*：与阴性组相比，P＜0.05；**：与阴性组相比，P＜0.01；***：与阴性组相比，P＜0.001；*: compared with the negative group, P < 0.05; **: compared with the negative group, P < 0.01; ***: compared with the negative group, P < 0.001;

图5三七多糖提取物对小鼠血清指标LDH、CK含量影响的实验结果图。Figure 5 shows the experimental results of the effect of Panax notoginseng polysaccharide extract on the levels of LDH and CK in mouse serum.

**：与阴性组相比，P＜0.01；***：与阴性组相比，P＜0.001；**: compared with the negative group, P < 0.01; ***: compared with the negative group, P < 0.001;

图6三七多糖提取物对小鼠糖原含量影响的实验结果图。Figure 6 shows the experimental results of the effect of Panax notoginseng polysaccharide extract on the glycogen content in mice.

**：与阴性组相比，P＜0.01；***：与阴性组相比，P＜0.001；**: compared with the negative group, P < 0.01; ***: compared with the negative group, P < 0.001;

图7三七多糖提取物对小鼠全血乳酸含量影响的实验结果图。Figure 7 Experimental results of the effect of Panax notoginseng polysaccharide extract on the whole blood lactic acid content of mice.

***：与阴性组相比，P＜0.001；***: P < 0.001 compared with the negative group;

图8三七多糖提取物对小鼠肝脏中SOD、CAT、GSH-Px、MDA影响的实验结果图。Figure 8 shows the experimental results of the effect of Panax notoginseng polysaccharide extract on SOD, CAT, GSH-Px and MDA in mouse liver.

**：与阴性组相比，P＜0.01；***：与阴性组相比，P＜0.001。**: compared with the negative group, P < 0.01; ***: compared with the negative group, P < 0.001.

具体实施方式DETAILED DESCRIPTION

为了进一步了解本发明的技术特征，下面结合具体实施例对本发明进行详细地阐述。实施例子对本发明具有示例性的作用，而不具有任何限制性的作用，本领域的技术人员在本发明的基础上做出的任何非实质性的修改，都应属于本发明的保护范围。In order to further understand the technical features of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The embodiments are exemplary of the present invention and do not have any restrictive effect. Any non-substantial modifications made by those skilled in the art on the basis of the present invention should fall within the scope of protection of the present invention.

实施例1：三七多糖提取物的提取Example 1: Extraction of Panax notoginseng polysaccharide extract

将三七药渣1kg与超纯水1：10重量体积比混合，热回流6h，过滤得提取液；继续加入8倍量超纯水，热回流6h，过滤得提取液，混合两次提取液，80℃减压浓缩至原体积的1/10，放冷至室温，过滤，取滤液，向滤液中缓缓加入3倍量无水乙醇，边加边搅拌，放置过夜，过滤得滤渣，减压干燥，粉碎，过100目筛，即得三七多糖提取物68.33g，提取率为6.83％。Mix 1 kg of Panax notoginseng residue with ultrapure water in a weight-to-volume ratio of 1:10, reflux for 6 hours, and filter to obtain an extract; continue to add 8 times the amount of ultrapure water, reflux for 6 hours, filter to obtain an extract, mix the two extracts, concentrate at 80°C under reduced pressure to 1/10 of the original volume, cool to room temperature, filter, take the filtrate, slowly add 3 times the amount of anhydrous ethanol to the filtrate, stirring while adding, let it stand overnight, filter to obtain the residue, dry under reduced pressure, crush, and pass through a 100-mesh sieve to obtain 68.33 g of Panax notoginseng polysaccharide extract with an extraction rate of 6.83%.

其中，三七药渣中三七多糖的含量为76.8％；采用硫酸蒽酮法测定的中性多糖含量为20-30％，采用间羟基联苯法测定的酸性多糖含量为10-20％；The content of Panax notoginseng polysaccharide in the Panax notoginseng residue is 76.8%; the content of neutral polysaccharide determined by the anthrone sulfate method is 20-30%, and the content of acidic polysaccharide determined by the m-hydroxybiphenyl method is 10-20%;

三七多糖提取物中三七多糖的含量为：70％；The content of Panax notoginseng polysaccharide in Panax notoginseng polysaccharide extract is: 70%;

三七药渣为提取过三七总皂苷的药渣。The notoginseng residues are the residues from which notoginseng total saponins have been extracted.

本实验使用的三七药渣由云南三七科技有限公司、昆明制药集团有限公司提供：The Panax notoginseng residue used in this experiment was provided by Yunnan Panax notoginseng Technology Co., Ltd. and Kunming Pharmaceutical Group Co., Ltd.:

三七药渣的获得方法：取三七粉碎成粗粉，用70％的乙醇提取，滤过后的药渣经干燥即得。Method for obtaining Panax notoginseng residue: Grind Panax notoginseng into coarse powder, extract with 70% ethanol, and dry the filtered residue to obtain the residue.

实施例2Example 2

试验例Test example

一、动物试验1. Animal Testing

实验分组及处理：80只SPF级KM小鼠，雄性，标准体重(18-22g)，随机分成五组(n＝16)，具体分组为阴性组(生理盐水，NS)、阳性组(Vc)、三七多糖提取物低(100mg/kg)、中(200mg/kg)、高(400mg/kg)剂量组。Experimental groups and treatments: 80 SPF-grade KM mice, male, standard body weight (18-22 g), were randomly divided into five groups (n=16), specifically divided into a negative group (normal saline, NS), a positive group (Vc), and a low (100 mg/kg), medium (200 mg/kg), and high (400 mg/kg) dose group of Panax notoginseng polysaccharide extract.

实验方法：按表1对小鼠连续灌胃给药。给药21d后，每组随机取8只小鼠进行爬杆实验，爬杆实验结束后，各组小鼠继续灌胃给药，给药28d，进行力竭游泳实验，记录力竭游泳实验时间。力竭游泳实验结束后，处死小鼠，快速解剖后腿腓肠肌和肝脏进行组织病理学观察。各组剩余的8只小鼠，第28d最后一次给药完成30min后，不负重游泳90min，休息30min后，小鼠眼眶后静脉丛采血，收集全血，取50μl全血，测定全血乳酸，其余血液，离心得血清，用于肝肾功能的测定和乳酸脱氢酶和肌酸激酶的测定。肝脏和腓肠肌用于后续生化组织测定，并快速分离心、肝、脾、肺、肾、胸腺等脏器，生理盐水漂洗后，吸干水分，称重并记录，用于后续脏器指数的测定。Experimental methods: Mice were continuously gavaged and administered according to Table 1. After 21 days of administration, 8 mice were randomly selected from each group for the pole climbing test. After the pole climbing test, mice in each group continued to be gavaged and administered for 28 days. The exhaustive swimming test was performed and the exhaustive swimming test time was recorded. After the exhaustive swimming test, the mice were killed and the gastrocnemius muscle and liver of the hind legs were quickly dissected for histopathological observation. The remaining 8 mice in each group swam for 90 minutes without weight after the last administration on the 28th day. After resting for 30 minutes, blood was collected from the retro-orbital venous plexus of the mice, whole blood was collected, 50 μl of whole blood was taken, and whole blood lactate was measured. The remaining blood was centrifuged to obtain serum for the determination of liver and kidney function and the determination of lactate dehydrogenase and creatine kinase. The liver and gastrocnemius were used for subsequent biochemical tissue determination, and the heart, liver, spleen, lung, kidney, thymus and other organs were quickly separated. After rinsing with physiological saline, the water was absorbed, weighed and recorded for the subsequent determination of organ indexes.

表1实验分组(n＝16)Table 1 Experimental groups (n=16)

给药周期结束后对各项指标进行测定After the administration period, various indicators were measured

(1)小鼠疲劳爬杆实验：给药21d，休息30min后，每组随机取8只小鼠进行爬杆实验，将小鼠置于爬杆装置(上方固定的长玻璃棒)上进行爬杆实验，记录小鼠从开始到掉落的时间，即为小鼠爬杆时间。(1) Mouse fatigue pole climbing test: After 21 days of drug administration and 30 minutes of rest, 8 mice were randomly selected from each group for the pole climbing test. The mice were placed on a pole climbing device (a long glass rod fixed on top) for the pole climbing test. The time from the start to the fall of the mouse was recorded, which was the mouse climbing time.

(2)小鼠力竭游泳实验：通过游泳运动建立力竭运动模型，评估三七多糖提取物对小鼠运动能力的影响。第28d最后一次给药完成30min后，在小鼠尾部缠绕自身体重5％的铅丝(铅丝松紧度应以不脱落不紧绕为宜)，在水深不低于30cm，水温25±1℃的游泳箱中进行负重游泳实验，观察并用秒表记录小鼠力竭游泳时间(小鼠沉入水中10s不浮出水面的时间)。(2) Exhaustive swimming test in mice: An exhaustive exercise model was established by swimming exercise to evaluate the effect of Panax notoginseng polysaccharide extract on the exercise capacity of mice. 30 minutes after the last administration on the 28th day, a lead wire weighing 5% of the mouse's body weight was wrapped around the mouse's tail (the lead wire should be neither loose nor tightly wound), and a weighted swimming test was performed in a swimming box with a water depth of not less than 30 cm and a water temperature of 25±1°C. The exhaustive swimming time of the mouse was observed and recorded with a stopwatch (the time the mouse sank into the water for 10 seconds without surfacing).

(3)安全性评价：分离力竭游泳小鼠的腓肠肌、肾脏，制作4μm厚的切片，采用苏木精和伊红(HE)染色试剂盒对小鼠腓肠肌、肾脏进行染色，并评估小鼠腓肠肌、肾脏损伤情况。应用光学显微镜在×400倍放大倍数下观察各组小鼠的腓肠肌、肾脏组织病理改变；并称量不负重游泳小鼠心、肝、脾、肺、肾、胸腺重量，按下列公式计算脏器指数。(3) Safety evaluation: The gastrocnemius and kidneys of exhausted swimming mice were separated and slices of 4 μm thick were made. The gastrocnemius and kidneys of mice were stained with a hematoxylin and eosin (HE) staining kit, and the damage of the gastrocnemius and kidneys of mice was evaluated. The pathological changes of the gastrocnemius and kidney tissues of mice in each group were observed under an optical microscope at a magnification of ×400; the weights of the heart, liver, spleen, lung, kidney, and thymus of the unweighted swimming mice were weighed, and the organ index was calculated according to the following formula.

脏器指数＝脏器(g)/体重(g)×100％Organ index = organ (g) / body weight (g) × 100%

(4)小鼠肝肾功能的测定：取血清，按照试剂盒说明书要求测定小鼠尿素氮(BUN)、谷丙转氨酶(ALT)、谷草转氨酶(AST)水平。(4) Determination of liver and kidney function in mice: Serum was collected and the levels of mouse urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined according to the instructions of the kit.

(5)乳酸脱氢酶和肌酸激酶的测定：取血清，按照试剂盒说明书要求测定乳酸脱氢酶(LDH)、肌酸激酶(CK)等生化指标。(5) Determination of lactate dehydrogenase and creatine kinase: Take serum and determine biochemical indicators such as lactate dehydrogenase (LDH) and creatine kinase (CK) according to the instructions of the kit.

(6)肝糖原和肌糖原的测定：分别准确称取肝脏和后腿腓肠肌，制备糖原检测液，按试剂盒说明书测定肝糖原和肌糖原含量。(6) Determination of liver glycogen and muscle glycogen: Accurately weigh the liver and hind leg gastrocnemius muscle, prepare glycogen detection solution, and determine the liver glycogen and muscle glycogen content according to the kit instructions.

(7)全血乳酸的测定：取小鼠全血0.05mL与0.3mL蛋白沉淀剂制备乳酸上清液，按全血乳酸检测试剂盒说明书测定乳酸含量。(7) Determination of whole blood lactate: Take 0.05 mL of mouse whole blood and add 0.3 mL of protein precipitant to prepare lactate supernatant, and determine the lactate content according to the instructions of the whole blood lactate detection kit.

(8)肝脏中SOD、CAT、GSH-Px、MDA生化指标的测定：取小鼠肝脏100mg，加入0.9mL生理盐水，研磨，4500r/min离心10min，取上清，即为10％肝匀浆检测液。用肝匀浆测定超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)等生化指标。(8) Determination of biochemical indices of SOD, CAT, GSH-Px, and MDA in the liver: 100 mg of mouse liver was taken, 0.9 mL of physiological saline was added, ground, centrifuged at 4500 r/min for 10 min, and the supernatant was taken as 10% liver homogenate test solution. Biochemical indices such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were determined using liver homogenate.

试验结果：Test results:

(1)三七多糖提取物可显著延长小鼠爬杆时间：如表2和图1所示，与阴性组相比，给药组爬杆时间显著延长，具有统计学意义(P＜0.001)。说明三七多糖提取物具有抗疲劳作用。(1) Panax notoginseng polysaccharide extract can significantly prolong the time mice can climb the pole: As shown in Table 2 and Figure 1, compared with the negative group, the time mice could climb the pole was significantly prolonged in the drug-treated group, which was statistically significant (P < 0.001). This indicates that Panax notoginseng polysaccharide extract has an anti-fatigue effect.

表2三七多糖提取物对小鼠爬杆时间影响Table 2 Effect of Panax notoginseng polysaccharide extract on the climbing time of mice

(2)三七多糖提取物可显著延长小鼠力竭游泳时间：如表3和图2所示，与阴性组相比，给药组游泳时间显著延长，具有统计学意义(P＜0.01)。说明三七多糖提取物具有抗疲劳作用。(2) Panax notoginseng polysaccharide extract can significantly prolong the exhaustive swimming time of mice: As shown in Table 3 and Figure 2, compared with the negative group, the swimming time of the drug-treated group was significantly prolonged, with statistical significance (P < 0.01), indicating that Panax notoginseng polysaccharide extract has an anti-fatigue effect.

表3三七多糖提取物对游泳时间影响Table 3 Effect of Panax notoginseng polysaccharide extract on swimming time

(3)三七多糖提取物HE染色各组无明显差异：如图3所示，各组肾脏和腓肠肌的组织切片未见明显病理改变；脏器指数如表4所示，各组之间心、肝、脾、肺、肾、胸腺等脏器指数无统计学差异，说明三七多糖提取物对脏器无毒副作用。(3) There was no significant difference in the HE staining of Panax notoginseng polysaccharide extract among the groups: As shown in Figure 3, no obvious pathological changes were found in the tissue sections of the kidney and gastrocnemius muscle of each group; the organ indexes are shown in Table 4, and there was no statistical difference in the organ indexes of heart, liver, spleen, lung, kidney, thymus, etc. among the groups, indicating that Panax notoginseng polysaccharide extract has no toxic side effects on the organs.

表4三七多糖提取物对小鼠脏器指数/％的影响(mean±SD，n＝10)Table 4 Effects of Panax notoginseng polysaccharide extract on mouse organ index/% (mean±SD, n=10)

(4)三七多糖提取物可显著降低血清中尿素氮(BUN)、谷丙转氨酶(ALT)、谷草转氨酶(AST)的积累：如图4所示，与阴性组相比，给药组显著降低了血清中BUN、ALT、AST含量，表明三七多糖提取物可降低肝、肾中有害代谢物的积累。BUN含量中，与阴性组比，三七多糖提取物100mg/kg和VC组具有统计学意义(P＜0.01，P＜0.05)，三七多糖提取物200mg/kg、400mg/kg组差异极显著(P＜0.001)。ALT含量中，与阴性组比，各给药组都降低了ALT含量，其中200mg/kg、400mg/kg、VC组具有统计学差异(P＜0.01，P＜0.001，P＜0.001)。AST含量中，与阴性组相比，100mg/kg组具有统计学差异(P＜0.05)，200mg/kg组与VC组有显著性差异(P＜0.01)，400mg/kg组差异极显著(P＜0.001)。(4) Panax notoginseng polysaccharide extract can significantly reduce the accumulation of serum urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST): As shown in Figure 4, compared with the negative group, the drug-treated group significantly reduced the levels of BUN, ALT, and AST in serum, indicating that Panax notoginseng polysaccharide extract can reduce the accumulation of harmful metabolites in the liver and kidney. In terms of BUN content, compared with the negative group, the 100 mg/kg Panax notoginseng polysaccharide extract and VC groups were statistically significant (P < 0.01, P < 0.05), and the differences between the 200 mg/kg and 400 mg/kg Panax notoginseng polysaccharide extract groups were extremely significant (P < 0.001). In terms of ALT content, compared with the negative group, each drug-treated group reduced the ALT content, among which the 200 mg/kg, 400 mg/kg, and VC groups were statistically significant (P < 0.01, P < 0.001, P < 0.001). In terms of AST content, compared with the negative group, the 100 mg/kg group had a statistically significant difference (P < 0.05), the 200 mg/kg group had a significant difference with the VC group (P < 0.01), and the 400 mg/kg group had an extremely significant difference (P < 0.001).

(5)三七多糖提取物显著降低血清中乳酸脱氢酶(LDH)、肌酸激酶(CK)的含量：如图5所示，与阴性组相比，给药组显著降低血液中LDH、CK的含量，表明三七多糖提取物给药可显著缓解疲劳。LDH含量中，与阴性组相比，给药组具有极显著差异(P＜0.001)。CK含量中，与阴性组相比，100mg/kg、200mg/kg、VC组具有显著性差异(P＜0.01)，400mg/kg组差异极显著(P＜0.001)。(5) Panax notoginseng polysaccharide extract significantly reduces the levels of lactate dehydrogenase (LDH) and creatine kinase (CK) in serum: As shown in Figure 5, compared with the negative group, the administration group significantly reduced the levels of LDH and CK in the blood, indicating that the administration of Panax notoginseng polysaccharide extract can significantly relieve fatigue. In terms of LDH content, compared with the negative group, the administration group had a very significant difference (P < 0.001). In terms of CK content, compared with the negative group, the 100 mg/kg, 200 mg/kg, and VC groups had significant differences (P < 0.01), and the 400 mg/kg group had a very significant difference (P < 0.001).

(6)三七多糖提取物升高肝脏和腓肠肌中的糖原含量：如图6所示，阴性组肝糖原含量3.2403mg/g，100mg/kg组肝糖原含量升高至5.6068，与阴性组相比，具有显著性差异(P＜0.01)，200mg/kg、400mg/kg、VC组分别升高至6.5955、12.0540、11.1136，与阴性组相比，差异极显著(P＜0.001)；肌糖原含量给药组与阴性组相比，具有统计学差异(P＜0.001)。(6) Panax notoginseng polysaccharide extract increases the glycogen content in the liver and gastrocnemius muscle: As shown in Figure 6, the liver glycogen content of the negative group was 3.2403 mg/g, and the liver glycogen content of the 100 mg/kg group increased to 5.6068, which was significantly different from the negative group (P < 0.01). The 200 mg/kg, 400 mg/kg, and VC groups increased to 6.5955, 12.0540, and 11.1136, respectively, which were extremely significantly different from the negative group (P < 0.001). The muscle glycogen content of the drug-treated group was statistically different from that of the negative group (P < 0.001).

(7)三七多糖提取物可显著降低血液中乳酸含量：如图7所示，与阴性组相比，给药组显著降低了血液中乳酸含量，有显著统计学差异(P＜0.001)。(7) Panax notoginseng polysaccharide extract can significantly reduce the lactic acid content in the blood: As shown in Figure 7, compared with the negative group, the drug-treated group significantly reduced the lactic acid content in the blood, with a significant statistical difference (P < 0.001).

(8)三七多糖提取物升高肝脏中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)等抗氧化酶的酶活力，降低MDA水平：如图8所示，与阴性组相比，给药组显著升高抗氧化酶SOD、CAT、GSH-Px的活力，具有统计学意义(P＜0.001，P＜0.01，P＜0.001)；降低了MDA水平(P＜0.01)，具有较好的抗氧化效果且呈剂量相关关系。(8) Panax notoginseng polysaccharide extract increases the enzyme activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in the liver, and reduces the MDA level: As shown in Figure 8, compared with the negative group, the drug-treated group significantly increased the activities of antioxidant enzymes SOD, CAT, and GSH-Px, which was statistically significant (P < 0.001, P < 0.01, P < 0.001); reduced the MDA level (P < 0.01), with a good antioxidant effect and a dose-related relationship.

上述实验结果证实，三七多糖提取物可以延长小鼠爬杆时间和游泳时间，降低血尿素氮、乳酸、乳酸脱氢酶、丙氨酸氨基转移酶、天冬氨酸氨基转移酶、肌酸激酶水平，升高糖原水平，增强抗氧化酶活力，减少氧化代谢物积累，具有良好的抗疲劳功效，尤其是抗运动性疲劳。The above experimental results confirm that Panax notoginseng polysaccharide extract can prolong the climbing and swimming time of mice, reduce the levels of blood urea nitrogen, lactic acid, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, and creatine kinase, increase glycogen levels, enhance the activity of antioxidant enzymes, reduce the accumulation of oxidative metabolites, and has good anti-fatigue effects, especially anti-sports fatigue.

实施例2Example 2

三七多糖提取物功能饮料的制备Preparation of functional beverage from Panax notoginseng polysaccharide extract

将三七多糖提取物2.4份、赤藓糖醇9份、甜菊糖苷0.04份，纯净水88.56份，混匀，加热至50-60℃，不断进行搅拌，使液体混合均匀，放冷，过滤。将混合均匀的液体进行灌装，先将液体温度加热到60-65℃，排除管内空气，再进行密封。将灌封后液体至于110-130℃高温下灭菌15-25min，冷却后得到三七多糖提取物运动饮料。Mix 2.4 parts of Panax notoginseng polysaccharide extract, 9 parts of erythritol, 0.04 parts of stevioside, and 88.56 parts of purified water, heat to 50-60°C, stir continuously to mix the liquid evenly, cool, and filter. Fill the evenly mixed liquid, first heat the liquid temperature to 60-65°C, remove the air in the tube, and then seal it. Sterilize the sealed liquid at a high temperature of 110-130°C for 15-25 minutes, and obtain a Panax notoginseng polysaccharide extract sports drink after cooling.