The Preparation Technology Research and Composition Analysis of Anti-fatigue Yik

Kun TANG, Tong LIN, Chundan ZHANG, Yanxia LU, Shunxing GUO

1.College of Life Sciences, Langfang Normal University, Langfang 065000, China; 2.Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China; 3.Technology Innovation Center for Utilization of Edible and Medicinal Fungi in Hebei Province, Langfang 065000, China; 4.Langfang Key Laboratory of Food Nutrition and Safety, Langfang 065000, China; 5.Beijing Changwei Technology Co., Ltd., Beijing 100085, China

Abstract [Objectives]To optimize the preparation technology of Yikangshu granule, an anti-fatigue health food.[Methods]The process was studied from three aspects(raw material extraction, excipient selection and forming process), and then it was verified in pilot production, and the content of the three components of the product was determined.[Results]The optimum production process of the formula granules was as follows: raw materials were extracted(adding 1∶9 water, extracting twice, 2.5 h each time); vacuum concentration; adding excipients and mixing after vacuum drying, crushing and sieving; granulation of 75% ethanol sifted by a 20-mesh sieve; drying at 60 ℃, sifting through 14-mesh, and packaging.The average content of products obtained was as follows: crude polysaccharides(3 204 mg/100 g), total saponins(2 295 mg/100 g), astragaloside A(21 mg/100 g).[Conclusions]The optimized preparation technology is stable and feasible, and has a quality that can be controlled, which can provide a reference for the development of health food in food industry.

Key words Anti-fatigue, Health food, Formula granules, Technology research, Composition analysis

1 Introduction

Fatigue is a physiological and pathological phenomenon that the body can not sustain at a specific level or organs can not maintain the expected exercise intensity, which is the result of the joint action of many factors inside and outside the body[1].With the increasing pressure of study and work in modern society, people pay more and more attention to the functional food which can relieve physical fatigue, and its market share is also increasing year by year.However, people prefer traditional Chinese medicine health food which can be used for a long time without any side effects on the human body.In traditional Chinese medicine, it is generally believed that human fatigue is caused by "deficiency of healthyqi" or "deficiency ofqiand blood", while syndrome is mostly caused by the imbalance ofqiand blood, yin and yang and viscera and bowels in the body[2].Yikangshu granule is composed of traditional Chinese medicineD.candidum,R.Astragaliand American ginseng, all of which fall into the category of raw materials of health food promulgated by the state.D.candidumis a valuable medicinal material, which has the effect of nourishingyinand clearing heat, benefiting stomach and promoting fluid, and its extract has significant anti-fatigue effect[3-4].R.Astragalihas the effect of invigorating spleen-stomach and replenishingqi, consolidating the exterior to stop sweating, often used as the principal drug in Chinese prescription for tonifyingqi[5], and the contained astragalus polysaccharides can delay physical fatigue[6].American ginseng can tonifyqiand nourishyin, clear heat and promote fluid, which is often used in health food to relieve physical fatigue[7-8].Therefore, the prescription composed of the three raw materials has the health effect of nourishingyinand replenishingqiand regulating the imbalance ofqiand blood,yinand yang in the body.It can alleviate the fatigue caused by "deficiency of vitalqi" or "deficiency ofqiand blood" in the body.

This paper mainly introduces the preparation technology of Yikangshu formula granules from three aspects: raw material extraction technology, excipient selection and forming technology.The technology verification of three batches of pilot products and the quality detection and analysis of components(crude polysaccharides, total saponins and astragaloside A)were carried out.The results showed that the process was stable and the quality was reliable.Therefore, the preparation technology of Yikangshu granules has the characteristics of stable key process parameters and is suitable for industrial large-scale production.This technology will also provide a reference for the development of registered health food in the food industry.

2 Materials

2.1 InstrumentsElectronic balance(1/10 000, Mettler Toledo); spectrophotometer(SmartSpet Plus,Bio-Rad); multifunctional enzymatic analyzer(200pro NanoQuant infinite M200pro,TECAN); high performance liquid chromatograph(2420ELS Detector, Waters); rotary evaporator(Laborota 4000, ITS Experimental Equipment Shanghai Co., Ltd.); numerically controlled ultrasonic cleaner(KQ-700DE, Kunshan Ultrasonic Instrument Co., Ltd.); digital display thermostat water bath(HH-4, Jiangsu Jintan Ronghua Instrument Manufacturing Co., Ltd.); Shanghai Zhicheng thermostat air dry oven(ZXRD-A7140, Shanghai Shengke Instrument Co., Ltd.).The pilot trial equipment for this process is provided by Beijing Yushengtang Health Food Co., Ltd.

2.2 ReagentsR.Astragali(No.15030902, Beijing Minghui Hengtong Pharmaceutical Co., Ltd.);D.candidum(it was purchased from Huoshan Shantangchun Biotechnology Co., Ltd., identified by Guo Shunxing, a researcher from Institute of Medicinal Plants, Chinese Academy of Medical Sciences, the company did not provide a batch number, but had a testing report); American ginseng(it was purchased from Jilin Shennong Chinese Herbal Medicine Co., Ltd., the company did not provide a batch number, but had a testing report); D-anhydrous glucose standard substance(No.110833-201205, content: 99.5%, National Institute for Food and Drug Control); standard glucose stock solution: 10.0 mg/mL; Astragaloside A reference substance(No.150709, content: 99%, Shanghai Ronghe Pharmaceutical Technology Co., Ltd.); Ginsenoside Re standard substance(No.110754-201626, National Institute for Food and Drug Control); methanol(chromatographically pure), acetonitrile(chromatographically pure).

3 Methods and results

3.1 Design of technology process(Fig.1)

Fig.1 Technology process

3.2 Study on extraction processThe effective parts of the three kinds of raw materials are mainly water-soluble chemical components, so in the part of extraction process research, the extraction rate and crude polysaccharides were mainly taken as the investigation indexes.Three key technology parameters affecting the extraction effect(extraction time, extraction frequency and water addition)were optimized by orthogonal test.

3.2.1Orthogonal test method.The effects of extraction time(A), extraction frequency(B)and water addition(C)on the extraction effect were investigated.The water extraction method was used to extract 9 times, and the amount of raw materials extracted each time was 240 g.The orthogonal experiment was carried out according to L9(34)table(Table 1).The extract powder was obtained by decompression and concentration of filtrate after water extraction(60 ℃).Taking the amount of extract and the content of crude polysaccharide as the evaluation index, the comprehensive scoring method was used.The highest value of amount of extract accounts for 40% of the total score(100 points), and the highest value of crude polysaccharide content accounts for 60% of the total score(100 points).The sum of the two scores is calculated as the comprehensive score value.According to the comprehensive evaluation value, the visual analysis and variance analysis were carried out, and the best extraction process parameters of the product were selected.

Table 1 Level of factor by water extraction

3.2.2Determination of investigation index.Determination of the content of crude polysaccharides: the content of crude polysaccharides was measured according to the method of "determination of the content of crude polysaccharides by phenol-sulfuric acid spectrophotometry" inDetectionMethodofFunctionalComponentsofHealthFood[9].Amount of extract: the dry extract powder of each sample was weighed.

3.2.3Results of orthogonal test.The results of orthogonal test by water extraction are shown in Table 2.For factor A,K2>K3>K1; for factor B,K2>K3>K1; for factor C,K2>K1>K3.According to the visual analysis, the range showed that the effect of different factors was in the order of A>B>C, and the best level was A2B2C2.The results of analysis of variance showed that the extraction time and extraction frequency had significant influence(P<0.05).However, the effect of water addition was not significant(P>0.05).Therefore, the factors of production cost and water saving can be taken into account, and the best process was A2B2C1(Table 3).

Table 2 The results of L9(34)orthogonal test by water extraction

Table 3 Analysis of variance

Process verification: 1 000 g of raw materials for the preparation of granules were weighed and extracted according to the optimum process above.A total of 354.0 g of extract powder was obtained, the extraction rate was 26.5%, and the crude polysaccharide content of extract powder was 95.34 mg/g.Therefore, the key technological parameters for the extraction of this product are as follows: adding 1∶9 water to raw materials, decocting and extracting twice, 2.5 h each time.

3.3 Study on screening of excipientsThe effects of sucrose, dextrin, starch, lactose and D-mannitol on the sense and taste about granules were compared in the experiment.Results showed that the properties of lactose and D-mannitol were the most stable and the granulation effect was the best.Therefore, according to the extraction rate of the extract and the edible method of the product, the ratio of extract powder to excipients was determined to be 1∶2 after selection and comparison, and the use ratio of lactose and D-mannitol was optimized as follows:

800 g of raw material was weighed.After extraction, the weight of the extract powder was 210 g, and the extraction rate was 26.2%.The extract powder was divided into 3 parts, with each part weighing 68 g, and the total weight of excipients added to each sample was 136 g.The experiment was carried out according to three schemes A, B and C.Taking the color, taste, granule size and solubility of granules as the main indexes, the results showed that scheme B had the best effect(Table 4), that is, the ratio of lactose to D-mannitol was 1∶1.

Table 4 Screening of the amount of excipients

3.4 Study on forming process1∶9 water was added to the three kinds of raw materials in the prescription before decoction and extraction twice, 2.5 h each time, and the extract powder was obtained after optimization, extraction and concentration.Then lactose and D-mannitol were mixed evenly, and the granules were obtained by granulation, drying and breaking.This part mainly studies the key technology parameters of granulation and breaking.

3.4.1Granulation.85% ethanol, 75% ethanol and 65% ethanol were selected as wetting agents, respectively, and the mixed powder was added to the wetting agent to make soft material, and the preparation effect of soft material and the granule yield obtained by different wetting agents were compared.The optimized soft materials were sifted through 12-mesh, 14-mesh and 20-mesh sieves, respectively, and the granulation effect was observed after drying at 60 ℃.Results showed that 75% ethanol was the most suitable wetting agent for preparing the granules(Table 5).The grain size of the soft material sifted by 20-mesh sieve was the best, and the granule yield of 88% was the highest.

Table 5 Selection of wetting agents

3.4.2Breaking.After granulation and drying, the granules were sifted with 10-mesh, 12-mesh, 14-mesh and 20-mesh sieves respectively for breaking, and the results showed that it was the best when sifted with a 14-mesh sieve.The following is the test of granule moisture absorption:

7 parts of the prepared granules(each 3 g)were put in weighing bottle, respectively.After drying at 60 ℃ to constant weight, it was placed in a closed dryer with different relative humidity.After being dried to constant weight, it was taken out, weighed precisely, and the moisture absorption rate was calculated.The results are shown in Table 6.Taking the relative humidity(RH, %)as the x-axis and the moisture absorption(%)of the granules as the y-axis, the moisture absorption equilibrium curve was drawn.The x-axis corresponding to the intersection of the tangents at both ends of the curve is the critical relative humidity(CRH)of the granules, and the results showed that the CRH of the granules was 72%(Fig.2).

Table 6 Test results of moisture absorption of granules

Fig.2 Equilibrium curve of moisture absorption of granules

3.5 Process verification and detection of componentsYikangshu granules were produced in three batches(materials input in each batch was 23 times of the raw material needed to prepare 1 000 g of granules), and the pilot production was carried out in Beijing Yushengtang Health Food Co., Ltd.Results showed that the finished product rates of the three batches of pilot products were 94.20%, 94.48% and 94.20%, respectively, with an average of 94.29%.Then three batches of samples were tested for the quality of crude polysaccharides, total saponins and astragaloside A according to the technical requirements of Yikangshu granules.Results showed that the average content of crude polysaccharides(calculated by D-anhydrous glucose)was 3 204 mg/100 g, the content of total saponins(calculated by ginsenoside Re)was 2 295 mg/100 g, and the content of astragaloside A was 20.8 mg/100 g(Table 7).

Table 7 Testing of components in three batches of samples mg/100 g

The trial verification showed that the process was stable and feasible, and the quality can be controlled.The linear relationship and content determination methods of the three components are described as follows:

(i)Crude polysaccharides: according to the method described inTechnicalRequirements-CrudePolysaccharidesof this product, different concentrations of glucose standard solutions(5.3-42.4 μg/mL)were prepared, and then 1.000 g of sample powder was precisely weighed for crude polysaccharide sample treatment.The absorbance value of the sample solution was measured at the wavelength of 485 nm, and substituted into the standard curve equationY=0.006 5X+0.088 7(absorbance value:Y; standard solution concentration:X),r=0.999 7.The concentration in the tested sample solution was measured, and then the content of crude polysaccharides in the sample was calculated[9].

(ii)Total saponins: according to the method described in "Technical Requirements-Total Saponins" of this product, different concentrations of ginsenoside Re standard solutions(40-400 μg/mL)were prepared, and then 1.000 g of sample powder was precisely weighed for sample treatment of total saponins.The absorbance value of the sample solution was determined at the wavelength of 550 nm, and substituted into the standard curve equationY=0.001 9X+0.001 4(absorbance value:Y; standard solution concentration:X),r=0.999 7.The total saponins concentration in the tested sample solution was measured, and then the total saponins content in the sample was calculated[10].

(iii)Astragaloside A: according to the method described in "Technical Requirements-Astragaloside A" of this product, the standard solution of astragaloside A(0.50 mg/mL)was prepared by high performance liquid chromatography-evaporative light scattering detector(HPLC-ELSD).The logarithmic equation of astragaloside A standard solution was obtained by external standard two-point method: lgY=1.413 5lgX+5.326 6(peak area:Y; injection quality:X).5.0 g of sample was precisely weighed for astragaloside A sample treatment, 20 μL of sample solution was pipetted, injected into liquid chromatograph, determined, and calculated by logarithmic equation of external standard two-point method[11].

Through the study of the above technology, the optimum production process of the formula granules was as follows: raw materials were extracted(adding 1∶9 water, extracting twice, 2.5 h each time); vacuum concentration; adding excipients and mixing after vacuum drying, crushing and sieving; granulation of 75% ethanol sifted by a 20-mesh sieve; drying at 60 ℃, sifting through 14-mesh, and packaging.

3 Discussion

In the research and development of Yikangshu granule as a health food product, it needs to go through the test of pilot production and the future large-scale production process, so the technology design can not be too complex, and it is necessary to be scientific, reasonable and stable.In the extraction of raw materials, we should not only fully consider the characteristics of each raw material, but also ensure that the effective parts relieving fatigue can be extracted as completely as possible, for example, the crude polysaccharides and total saponins in the effective parts ofD.candidum,R.Astragaliand American ginseng are soluble in water, so the raw materials were still extracted using the traditional water extraction method.The method was simple, convenient, safe and easy for large-scale production.The pilot product was tested by the Health Analysis and Testing Center of Nanjing Medical University, and results showed that it had the function of relieving physical fatigue(testing acceptance number: GZ01020160119), which proved that the preparation technology of Yikangshu granules was scientific, reasonable and stable.

The detection of the components of Yikangshu granules is also the key to measuring whether the process is stable and controllable.and the product testing method involves the enterprise’s internal control standard, so it was described briefly in this paper, but the selection of the components was based on the following:

(i)Crude polysaccharides: the chemical components ofD.candidummainly include polysaccharides and stilbenes, and their pharmacological effects mainly include regulating immunity, relieving fatigue, antioxidation and so on[12].American ginseng polysaccharides can increase the non-specific immunity and cellular immunity of mice[13-14], so it also has auxiliary role in relieving physical fatigue.R.Astragalipolysaccharides can also prolong the exhaustive swimming time of mice, reduce the accumulation of blood lactic acid, increase the reserve of liver glycogen, delay the occurrence of fatigue and improve exercise ability[15].Therefore, the detection of crude polysaccharide content is the main index to judge the quality and function of the product.

(ii)Total saponins:Panaxquinquefoliumsaponins(PQS)is one of the main effective components of American ginseng, and it is also the most physiologically active substance.At present, 49 kinds of PQS saponins have been isolated and identified, and the ginsenoside of damarane type is the main active component of American ginseng.Ginsenosides in American ginseng can reduce the accumulation of serum urea nitrogen and increase the level of liver glycogen in mice after exercise-induced fatigue, so as to effectively give play to the effect of anti-fatigue[16].

(iii)Astragaloside A: it is stipulated in Chinese Pharmacopoeia that astragaloside A is a major component to evaluate the quality ofR.Astragali.This component has a wide range of biological effects, such as immune regulation, antioxidation and neuroprotection[17-18].

Therefore, crude polysaccharides, total saponins and astragaloside A were selected as the major components to control the product quality of for Yikangshu granules.