CN113785982A

CN113785982A - Anti-fatigue and anti-radiation capsule for spaceflight and preparation method thereof

Info

Publication number: CN113785982A
Application number: CN202111344045.7A
Authority: CN
Inventors: 简一平
Original assignee: Beijing Xiaoaotiangong Biotechnology Co ltd
Current assignee: Beijing Xiaoaotiangong Biotechnology Co ltd
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2021-12-14
Anticipated expiration: 2041-11-15
Also published as: CN113785982B

Abstract

The invention relates to an anti-fatigue and anti-radiation capsule for spaceflight and a preparation method thereof. The anti-fatigue and anti-radiation capsule for spaceflight is characterized by comprising the following raw materials in parts by mass: 200-300 parts of American ginseng, 800-1000 parts of Fuzhuan tea, 200-300 parts of maca powder, 30-50 parts of xylo-oligosaccharide, 30-50 parts of corn starch and 1-5 parts of sea cucumber peptide. The capsule provided by the invention is prepared from natural extracts, can improve the anti-fatigue and anti-radiation conditions of astronauts at the same time, and has no side effect.

Description

Anti-fatigue and anti-radiation capsule for spaceflight and preparation method thereof

Technical Field

The invention belongs to the technical field of space food, and particularly relates to an anti-fatigue and anti-radiation capsule for space flight and a preparation method thereof.

Background

Weight loss, radiation, noise, narrow closed space and other adverse effects such as space adaptation syndrome (SMS), anorexia, blood head redistribution, hypoimmunity, muscular atrophy, bone density reduction, spleen and stomach function reduction, cognitive function reduction and the like can be brought to the organism under the special aerospace environment. In the process of manned flight tasks, astronauts undertake a lot of scientific research tasks, equipment maintenance and other work, and the astronauts may be in a high-load working strength state for a long time, so that the physical fatigue state of the astronauts occurs, and the working efficiency is influenced. The existing space food relates to seven nutrients including protein, fat, sugar, vitamins, electrolyte, trace elements and water, and has very important functions of ensuring the growth and development of human bodies, maintaining body temperature, supplementing substance consumption, enhancing body resistance and the like, and the food can keep the normal working requirements of the human bodies. However, in the process of space flight, due to the requirements of special environment and working efficiency, a product is required to be convenient to eat, the mental state of astronauts can be changed in a short time, and the flight mission can be efficiently completed. In the prior art, in order to improve the mental state of astronauts and resist fatigue, food compositions containing chemical active ingredients such as caffeine are provided, but the food compositions are not suitable for long-term use.

In addition, because astronauts are in space radiation environments for a long time and the time of each aerospace mission can be long, although the aerospace vehicles and aerospace suits strictly limit space background radiation at present, radiation cannot be completely isolated. Under the radiation background in a short period, cells of an astronaut can be self-repaired, but each time the astronaut goes out of a flight is very long, and even the astronaut can return to the flight for several years, the astronaut body can still be adversely affected after being in the space radiation environment for a long time, and even if the cells cannot be repaired, permanent damage can be caused, for example, damage to the skin, trachea of bone marrow, or cell mutation can be caused.

The prior art also lacks an aerospace food which meets the standards of aerospace foods, is suitable for astronauts to take for a long time, can improve the fatigue resistance degree and radiation resistance simultaneously and has no side effect.

Disclosure of Invention

In order to overcome the defect that the prior art is lack of an effective anti-fatigue food composition for spaceflight, which can simultaneously improve the anti-fatigue and anti-radiation conditions of astronauts and has no side effect. The invention provides an anti-fatigue and anti-radiation capsule for spaceflight, which comprises the following raw materials in parts by mass: 200-300 parts of American ginseng, 800-1000 parts of Fuzhuan tea, 200-300 parts of maca powder, 30-50 parts of xylo-oligosaccharide, 30-50 parts of corn starch and 1-5 parts of sea cucumber peptide.

Preferably, the anti-fatigue and anti-radiation capsule for spaceflight comprises the following raw materials in parts by mass: 280 parts of 220-plus American ginseng, 890-plus 950 parts of Fuzhuan tea, 260 parts of 220-plus maca powder, 40-45 parts of xylo-oligosaccharide, 40-45 parts of corn starch and 1.5-3 parts of sea cucumber peptide

Fuzhuan tea, also called Fuzhuan tea, is one of six major tea types in China, is prepared by taking raw dark green tea as a raw material through two fermentation processing procedures, and belongs to fully fermented tea. Researches show that the Fuzhuan tea contains rich bioactive substances such as catechin, amino acid, caffeine, soluble sugar, soluble protein and the like, is beneficial to relieving physical fatigue, and can excite the central nervous system, enhance the muscle contractility and eliminate the fatigue feeling such as caffeine, theophylline, theobromine and the like. Eurotium cristatum (E.Cristatum) is called as eurotium cristatum in black tea, belongs to Eurotiales, Tricholomataceae and Eurotium cristatum, and consists of ascocarp and hyphae, wherein the diameter of the ascocarp is 150-200 mu m, the ascocarp is generally provided with a yellow spherical or nearly spherical ascocarp, and spherical, nearly spherical or biconvex lens-shaped ascospores with the diameter of 5 mu m can be released after the ascocarp is ruptured. Eurotium cristatum is the dominant bacterium in the processing process of black tea. The Eurotium cristatum contains abundant indole alkaloid metabolites. The indole alkaloid compounds have important biological activities of antibiosis, antianaphylaxis, antiradiation, uvioresistant, antitumor and the like. The black tea can promote metabolism of human body, improve immunity, strengthen physique and delay senility after being drunk for a long time, and has obvious pharmacological health care and pathological prevention effects.

Preferably, the Fuzhuan tea extract used as the raw material in the anti-fatigue and anti-radiation capsule for spaceflight is prepared by a preparation method comprising the following steps of: weighing Fuzhuan tea according to the formula, adding 5-10 times of water, ethyl acetate and ethanol mixed solvent, carrying out warm extraction for the first time, filtering out warm extract, adding 5-10 times of water into tea residues, carrying out warm extraction for the second time, filtering while hot, mixing filtrate with the first extract, concentrating under reduced pressure (-0.06-0.08 Mpa, 60-70 ℃) to obtain a thick extract with the relative density of 1.14-1.20 (55 ℃) for later use.

More preferably, the volume ratio of the water to the ethyl acetate to the ethanol is 10:3-5: 1-1.5.

More preferably, the warm leaching is carried out for 0.5-1h by decoction and then 1-2h by warm leaching at 50-60 ℃; the second warm-soaking extraction is carried out by soaking for 2-4h at 60-70 ℃.

In order to avoid destruction of active ingredients such as polysaccharides and polyphenols, there has been conventionally used a method of extraction using a eutectic solvent or ultra high pressure, and an extract can be obtained in high yield without destroying the active ingredients. But on the one hand, the production cost is greatly increased and is not paid, in addition, the eutectic solvent is difficult to completely remove, if the residue can influence the product quality, the method is not suitable for space food with more strict requirements, and the ultrahigh pressure extraction needs a special high pressure device and is expensive. Therefore, the above two methods are not suitable for the space food field. The inventor unexpectedly finds that the extract can be obtained in high yield without damaging the active ingredients of the Fuzhuan tea by using the mixed solvent of the water, the ethyl acetate and the ethanol as the extraction solvent, has more obvious beneficial effects on organisms, and can remarkably improve the immunity of human bodies and the anti-fatigue capability. The used solvents of ethyl acetate and ethanol are nontoxic and can be fully removed in the subsequent treatment process.

Maca is a cruciferous plant originally produced in south America, is distributed in the mountain areas with high altitude in south America, is artificially planted in the middle and south of Peru, and in the Yunnan and Xinjiang areas in China, and has a large area of suitable planting land. Has effects in relieving fatigue, improving sexual function, improving sleep, improving blood circulation, enhancing brain nutrition, and relieving anxiety. Maca contains high amounts of iron, protein, amino acid, zinc and other nutrient elements, and can effectively enhance the immune system, improve the disease resistance of the body, resist fatigue and improve anemia symptoms. The sugar content in maca is the only nutrient component of brain and nervous system, and maca is often eaten to benefit brain. The maca can enable the head to be clear and flexible, improve the working efficiency, enable students to make study progress, keep the memory of the middle-aged and the old, and enable the head to be clear and clear in thinking. The maca can effectively improve anxiety, neurasthenia and the like caused by pressure.

The American ginseng has very obvious effects of improving immunity, relieving fatigue, improving the wakened weak constitution and supplementing physical strength, is not only suitable for the old and patients in the recovery period after illness, but also has very obvious treatment and alleviation effects on low-resistance and sub-health people. Can regulate human nervous system, and is suitable for mental workers to relieve brain fatigue. The ginsenoside which is an effective component of the health-care product has the functions of promoting blood oxygen content, strengthening heart rate, promoting blood flow, enabling the brain to be in a state of sufficient blood volume and blood oxygen, promoting the potential of brain cells to play a role to the maximum extent, improving thinking depth and activity and the like.

Extracting and concentrating American ginseng is well known in the art, in one embodiment of the invention, the American ginseng is weighed according to the formula amount, 5-10 times of 40-60wt% edible alcohol is added for heating reflux extraction for 2-5h, an alcohol solution is filtered out, and the alcohol solution is recovered under reduced pressure until no edible alcohol smell exists, so that an alcohol extracting solution is obtained for later use; decocting the residue with 5-10 times of water for 1-3 hr, filtering to obtain decoction, mixing with above ethanol extractive solution, concentrating under reduced pressure (-0.06-0.08 Mpa, 60-70 deg.C) to obtain radix Panacis Quinquefolii soft extract with relative density of 1.14-1.20 (55 deg.C), and keeping.

The intestinal probiotic flora can be regarded as beneficial 'microorganism organs' in the body, and participate in local and systemic physiological processes in various aspects such as regulation and control of metabolism, immunity, endocrine, nerves and the like of a host through mechanisms such as flora self components, metabolites and derivatives, and pathogenic symbiotic bacteria translocation. Xylo-oligosaccharide is one of the varieties with the strongest function for proliferating bifidobacteria in polymeric saccharides, has the efficacy which is about 20 times that of other polymeric saccharides, and has no enzyme for hydrolyzing xylo-oligosaccharide in the gastrointestinal tract of a human body, so that xylo-oligosaccharide can be directly introduced into the large intestine to be preferentially utilized by bifidobacteria, and can promote the proliferation of the bifidobacteria and simultaneously generate a plurality of organic acids. The pH value of the intestinal tract is reduced, the growth of harmful bacteria is inhibited, and the probiotics are greatly proliferated in the intestinal tract to achieve the functions of enhancing the immunity of the organism and the like.

The sea cucumber peptide is mainly micromolecular peptide which is obtained by hydrolyzing and refining fresh and live sea cucumber through protease and has the molecular weight of less than 1000Da and consists of 3-10 amino acids, and has multiple biological activities of oxidation resistance, blood sugar reduction, collagen secretion promotion, tumor resistance, inflammation inhibition, fatigue resistance, bacteria resistance, vascular endothelial cell protection, wound healing promotion and the like.

The capsule obtained by compounding and combining the effective components according to a certain proportion can improve the mental state of astronauts in a short time, can improve the radiation resistance after being taken in space for a long time, and has no side effect. The multiple substances are combined together to obviously achieve the synergistic compounding effect, and the anti-fatigue capability and the anti-radiation capability of the human body are improved. The capsule is a natural product, has no side effect on human body and can be taken for a long time.

Further, the anti-fatigue and anti-radiation capsule for spaceflight provided by the invention is obtained by a preparation method comprising the following steps:

(1) sieving maca powder, xylo-oligosaccharide and corn starch;

(2) extracting and concentrating American ginseng and Fuzhuan tea to obtain an extract; adding maca powder, xylo-oligosaccharide, corn starch and sea cucumber peptide, mixing, drying and crushing to obtain paste fine powder;

(3 granulating and finishing, filling capsules and packaging to obtain the finished product;

further, in the step (1), the maca powder, the xylo-oligosaccharide and the corn starch are sieved by a 60-100-mesh sieve

Further, the step (2) of mixing, drying and crushing is to mix the American ginseng and the two thick extracts of the Fuzhuan tea, add xylo-oligosaccharide and corn starch and stir for 15-30min, add maca powder and sea cucumber peptide and stir for 15-30min, dry in vacuum after being uniform, cool after drying, crush and pass through a sieve of 80-100 meshes for standby.

Further, the granulating and grading process of the step (3) is well known in the art, and specifically, the dry paste fine powder prepared in the step (2) is made into soft material by 95% edible alcohol, and is granulated by a 18-mesh sieve; drying, and finishing granules by using a 20-mesh sieve; the capsule filling process is well known in the field and meets the standard regulation of Chinese pharmacopoeia. In one embodiment of the invention, 0 is used^#Packaging the empty capsules by 0.2-1 g/capsule, preferably 0.5-0.7 g/capsule, packaging the food by plastic bottles, externally packaging after inspection, and warehousing the finished products.

Detailed Description

The following detailed description of the preferred embodiments of the invention in its specific application is provided to illustrate and not to limit the scope of the invention as defined by the claims.

The raw materials used in the invention can be obtained from commercial purchase. The Fuzhuan tea is from the Hunan Anhua space raw tea base, the sea cucumber peptide is purchased from Texas blue plum biotechnology limited company, 150 meshes, and the HPLC purity is more than 99.5%.

Preparation example 1

Weighing 890g of Fuzhuan tea according to the formula, adding 8 times of water, ethyl acetate and ethanol according to the volume ratio of 10: 4: 1.2, decocting for 0.5h, soaking at 50 deg.C for 1.5h, filtering to obtain soaking solution, adding 6 times of 70 deg.C water, soaking at 70 deg.C for 1.5h, filtering while hot, mixing the filtrate with the soaking solution, and concentrating under reduced pressure (-0.07 Mpa, 64 deg.C) to relative density of 1.16 (55 deg.C) to obtain FU BRIAN tea thick extract.

Preparation example 2

The other conditions and operations were the same as in preparation example 1 except that the volume ratio of water, ethyl acetate and ethanol was 10: 3: 1.5.

preparation example 3

The other conditions and operations were the same as in preparation example 1 except that the volume ratio of water, ethyl acetate and ethanol was 10: 5: 1.

preparation example 4

The other conditions and operations were the same as in preparation example 1 except that the volume ratio of water to ethyl acetate was 10: 4, no ethanol was added.

Preparation example 5

The other conditions and operations were the same as in preparation example 1 except that the volume ratio of water to ethanol was 10: 1.2, i.e. no ethyl acetate was added.

Example 1

Sieving maca powder, xylo-oligosaccharide and corn starch with a 100-mesh sieve for later use;

weighing 220g of American ginseng according to the formula, adding 8 times of 60% edible alcohol, heating, refluxing and extracting for 2h, filtering out an alcohol solution, and recovering under reduced pressure until no edible alcohol smell exists for later use; decocting the residue with 6 times of water for 1.5 hr, filtering to obtain water decoction, mixing with above ethanol extractive solution, concentrating under reduced pressure (-0.07 Mpa, 64 deg.C) to relative density of 1.16 (55 deg.C) to obtain radix Panacis Quinquefolii soft extract;

mixing the American ginseng thick extract with the Fuzhuan tea thick extract obtained in the preparation example 1, adding 45g of xylo-oligosaccharide and 45g of corn starch, stirring for 15min, adding 220g of maca powder and 1.5g of sea cucumber peptide, stirring for 15min, drying in vacuum after uniform, cooling after drying, crushing, and sieving with a 80-mesh sieve for later use. Making soft mass with 95% edible alcohol, sieving with 18 mesh sieve, and granulating; after drying, the granules are sieved by a 20-mesh sieve. The 0# hollow capsule is used, and meets the standard regulation of Chinese pharmacopoeia: the specification is 0.5 g/grain; packaging with 90 granules/bottle by using a plastic bottle for food packaging; and (5) carrying out external packaging after the inspection is qualified, and warehousing finished products.

Example 2

The other conditions and operations were the same as in example 1 except that the thick extract of Fuzhuan tea was prepared in preparation example 2 instead.

Example 3

The other conditions and operations were the same as in example 1 except that the thick extract of Fuzhuan tea was prepared in preparation example 3 instead.

Example 4

The other conditions and operations were the same as in example 1 except that the thick extract of Fuzhuan tea was prepared in preparation example 4 instead.

Example 5

The other conditions and operations were the same as in example 1 except that the thick extract of Fuzhuan tea was prepared in preparation example 5 instead.

Example 6

The other conditions and the operation are the same as the example 1, and the difference is that the dosage of the raw materials is changed into 260g of maca powder, 40g of xylo-oligosaccharide, 40g of corn starch, 280g of American ginseng, 3g of sea cucumber peptide and the dosage of the Fuzhuan tea thick extract are not changed.

Example 7

The other conditions and the operation are the same as the example 1, and the difference is that the dosage of the raw materials is changed into 300g of maca powder, 40g of xylo-oligosaccharide, 50g of corn starch, 230g of American ginseng, 2g of sea cucumber peptide and the dosage of the Fuzhuan tea thick extract are not changed.

Example 8

The other conditions and the operation are the same as the example 1, and the difference is that the dosage of the raw materials is changed into 200g of maca powder, 50g of xylo-oligosaccharide, 40g of corn starch, 200g of American ginseng, 2.5g of sea cucumber peptide and the dosage of the Fuzhuan tea thick extract is not changed.

Example 9

The other conditions and the operation are the same as the example 1, and the difference is that the dosage of the raw materials is changed into 300g of maca powder, 40g of xylo-oligosaccharide, 40g of corn starch, 200g of American ginseng, 1.8g of sea cucumber peptide and the dosage of the Fuzhuan tea thick extract is not changed.

Example 10

The other conditions and the operation are the same as the example 1, and the difference is that the dosage of the raw materials is changed into 225g of maca powder, 50g of xylo-oligosaccharide, 45g of corn starch, 300g of American ginseng, 2.2g of sea cucumber peptide and the dosage of the Fuzhuan tea thick extract is not changed.

Comparative example 1

The other conditions and operations are the same as those in example 1, except that maca powder is not added to the raw materials, and the dosage of American ginseng is 440g when preparing the American ginseng thick extract.

Comparative example 2

The other conditions and operations are the same as those in example 1, except that the raw materials are not added with the American ginseng thick extract, and the adding amount of the maca powder is changed to 350 g.

Comparative example 3

The other conditions and operations are the same as those in example 1, except that the raw materials are not added with the Fuzhuan tea thick extract, the dosage of the maca powder is changed to 550g, and the dosage of the American ginseng is changed to 665g when the American ginseng thick extract is prepared.

Comparative example 4

The other conditions and operations were the same as in example 1 except that no sea cucumber peptide was added to the starting material.

Application example 1

Safety toxicology evaluation test

1. Experimental animals and feed: the experimental animal technology company of Beijing Weitonglihua (license number: SCXK (Jing) 2012-0001] bred SPF-grade Kunming mice and SD rats. The maintenance feed was administered to each experimental group of acute toxicity test and genetic toxicity test, and was prepared from the company "license No.: SCXK (Jing) 2014-. The feed for each dose group and the control group of the maintenance feed in the 30-day feeding test is prepared by Beijing hua fukang biotech GmbH [ license number: SCXK (Jing) 2012-.

2. Test samples: capsules prepared as in example 1, the human recommended dose: 4.5g/60kg BW per day, the contents were taken for testing.

3. Acute toxicity test in mice (maximum tolerated dose method): 18-22g Kunming mouse 20 mice with half of each female and half of each male are selected for test. During the test period, animals had free access to food and water and the test subjects were administered by gavage. The maximum use concentration of the sample is 0.4g/mL, 16.00g of the sample is weighed, and sterile water is added to 40.0 mL. The gavage capacity is 20mL/kgBW, namely, the gavage is performed for 2 times at an interval of 6h at a dosage of 8.0g/kgBW, the gavage dosage is equivalent to 16.00g/kgBW, the continuous observation is carried out for 14d, the state, weight change, toxic signs, death condition and the like of the animals are observed and recorded, and the Maximum Tolerated Dose (MTD) is determined.

4. Mouse genotoxicity test (Ames test): four test strains of Salmonella typhimurium histidine-deficient strain TA97, TA98, TA100 and TA102 which are identified to meet the requirements are selected to carry out a standard plate doping method test without enlarging the liver S9 of the mice. 5 dose groups of 8, 40, 200, 1000, 5000 mug/dish were set. The preparation and sterilization method of the test object comprises the following steps: weighing 1.00g of sample, adding sterile water to prepare 20.0mL of solution, sterilizing at 0.103MPa for 20min, performing sterility test on the test object after sterilization, and culturing at 37 ℃ for 24h for aseptic growth. This is the highest working concentration of 50mg/mL (i.e., 5000. mu.g/dish), and based on this, other concentrations were sequentially diluted with sterile water, 5-fold at equal ratios, while setting positive control, sterile water control, sterile DMSO control, and untreated control. The positive control without S9 was fenaminosulf (50.0. mu.g/dish, applied strain TA97, TA98, TA 102), sodium azide (1.5. mu.g/dish, applied strain TA 100), and the positive control with S9 was 2-aminofluorene (10.0. mu.g/dish, applied strain TA97, TA98, TA 102), 1, 8-dihydroxyanthraquinone (50.0. mu.g/dish, applied strain TA 102). 0.1mL of the enrichment liquid of the test strain, 0.1mL of the test solution and 0.5mL of the mixture of S9 were added to the top agar, mixed well and poured onto bottom culture medium plates, 3 plates per dose. The cells were incubated at 37. + -. 1 ℃ for 48h and the number of colonies transformed per dish was counted. The test object group has more than 2 times of the number of the retrogradation colonies than the spontaneous retrogradation colonies, and is judged to be positive when the test object group has a dose response relationship. The test was repeated twice under the same conditions.

5. Mouse marrow pleochromocyte micronucleus test: 50 mice with the weight of 25-30g are selected and randomly divided into 5 groups, each group comprises 10 mice, and the mice are male and female. Cyclophosphamide (40 mg/kgBW) is used as a positive control, sterile water is used as a solvent control, and the low, middle and high dose groups are respectively 2.00 g/kgBW, 4.00 g/kgBW and 8.00 g/kgBW, which are equivalent to 27, 53 and 107 times of the recommended dose of a human body. 8.00 g/kgBW group: taking a sample of 16.00g, adding sterile water to 40.0 mL; 4.00 g/kgBW group: 8.00g of a sample is taken and sterile water is added to 40.0 mL; 2.00 g/kgBW group: a sample (4.00 g) was taken and sterilized water was added to 40.0 mL. The test substance, the positive control and the solvent control are all administered by oral gavage, and the gavage volume is 20.0 g/kgBW. The test substance is administered by oral gavage twice with an interval of 24h by a 30h administration method. After 6h of the last gavage, the animals were sacrificed by cervical dislocation. The sternum bone marrow is taken and smeared with calf serum dilution, fixed by methanol and stained by Giemsa. Double-blind scoring, under an optical microscope, counting 1000 pleochromophilic erythrocytes (PCE) per animal, and calculating the micronucleus cell rate. Meanwhile, the number of 200 pleochromophilic erythrocytes and mature erythrocytes (NCE) was counted per animal, and the PCE/NCE ratio was calculated. Compared with the control group, the test group can be confirmed to be a positive result when the micronucleus rate of the test result has obvious dose response relation and statistical significance. If the difference is statistically significant but there is no dose response relationship, then repeat tests are performed. The result is reproducible and can be determined as positive.

6. Mouse teratospermia test: 25 sexually mature male mice weighing 25-35g were randomly divided into 5 groups, and cyclophosphamide (40 mg/kgBW) was used as a positive control and sterile water was used as a solvent control. The low, medium and high dose groups are respectively 2.00 g/kgBW, 4.00 g/kgBW and 8.00 g/kgBW, which are equivalent to 27, 53 and 107 times of the recommended dose of human bodies, and the test substance, the positive control and the solvent control are all administered by oral gavage with the gavage capacity of 20.0 g/kgBW. The animals are sacrificed once a day after the last gavage for 5 consecutive days and 30 days after the last gavage, testicles are taken for sectioning, eosin staining is carried out, the sperm morphology of the mice is checked under a high power microscope, 5 animals are counted in each group, 1000 sperms are counted in each animal, the sperm aberration incidence rate is calculated, and statistical analysis is carried out.

7. Rat 30-day feeding trial: weaned rats 80 in half female and half male. The group was randomly divided into 4 groups, and the feed control group and the low, medium and high three dose groups were maintained. The maintenance feed control group was given maintenance feed (protein content 21.0%). The low, medium and high dose groups are respectively 1.88 g/kgBW, 3.75 g/kgBW and 7.50 g/kgBW, which are equivalent to the recommended daily intake of 25, 50 and 100 times of human body. Feeds incorporating 1.88%, 3.75%, 7.50% of the sample (protein content 17.2%) and different proportions of casein (protein content 83%) were administered separately. The protein content of each dose group was 21.0%. High dose: sample 1.42kg + maintenance feed meal 17.49kg + casein 0.09 kg; the medium dose group: sample 0.71kg + maintenance feed meal 18.25kg + casein 0.04 kg; low dose: sample 0.36kg + maintenance feed meal 18.62kg + casein 0.02 kg. Animals were housed in single cages and observed and recorded daily for general performance, toxic performance and mortality. Weighing the body weight, the food scattering amount and the remaining amount twice a week, and calculating the food intake and the food utilization rate every week. After fasting for 16h on day 30, the fasting body weight is called, and blood indicators such as hemoglobin, red blood cell count, white blood cell count and classification are measured. Serum alanine transaminase, glutamic-oxalacetic transaminase, urea nitrogen, creatinine, glucose, albumin, total protein, total cholesterol, triglyceride, etc. Gross dissection was performed, and the absolute weights of the organs of liver, kidney, spleen, and testis were weighed and the organ/body ratio calculated. The results were statistically analyzed. When the animals in each dose group are generally inspected to find no obvious lesion and no significant change of biochemical indexes, the liver, kidney, stomach, intestine, spleen, testis and ovary of the high dose group and the maintenance feed control group are taken to carry out histopathological examination. If lesions are found, the lower dose groups are examined accordingly. Liver tissue, kidney tissue, spleen tissue, stomach tissue, duodenum tissue, ovary tissue or testis tissue are cut from each animal, fixed by formaldehyde, dehydrated and transparent by alcohol and xylene, embedded by paraffin, and made into 5 μm thick slices, stained by hematoxylin and eosin, and observed by optical microscope.

8. Data processing: data processing was performed using SPSS11.0 software. Adopting single-factor analysis of variance to the measurement data, but firstly carrying out the homogeneity test of variance according to the analysis of variance procedure, if the variance isOverall comparisons were made using one-way anova, and differences were found using Dunnett to make pairwise comparisons between the mean of multiple dose groups and a control group. Carrying out proper variable conversion on the data with uneven variance, and counting by using the converted data after the requirement on the uniformity of the variance is met; if the variance uniformity is not achieved after the variables are converted, statistics is carried out by using a rank sum test. For counting datax ²And (6) checking.

Second, experimental results

1. Acute toxicity (maximum tolerated dose) test in mice

TABLE 1 mouse acute toxicity test results

As can be seen from the results in Table 1, the mice of both sexes were gavaged twice with a gavage dose of 16.00g/kgBW, and the animals were observed to be normal during 14 days without abnormal conditions such as toxic signs and death. At 15d, the test animals were sacrificed for gross anatomical examination and no major organs were visibly abnormal. The maximum oral tolerated dose (MTD) of the test subjects was greater than 15g/kgBW in both male and female mice. The contents of the capsules prepared according to the invention in example 1 were of non-toxic grade according to the acute toxicity classification criteria.

2. Genotoxicity test

2.1 Ames test

Table 2 units of first Ames test results: one/one dish

Note: the results above are the mean ± standard deviation of three plates.

Table 3 units of second Ames test results: one/one dish

Note: the results above are the mean ± standard deviation of three plates.

As can be seen from the results in tables 2 and 3, the number of the mutator colonies of each dose group of the test substance does not exceed the number of the mutator colonies of the solvent control group and the untreated control group by more than 2 times, and no dose-response relationship exists.

And (4) conclusion: the Ames test results of the capsule contents prepared according to the invention in example 1 were negative with and without the liver microsomal enzyme activation system.

2.2 mouse bone marrow pleochromocyte micronucleus test

TABLE 4 bone marrow pleochromatic erythrocyte micronucleus test results in female mice

Note that^#: compared with the solvent control group, the positive control group has very significant difference (P is less than 0.01).

TABLE 5 Male mouse bone marrow pleochromocyte micronucleus test results

As can be seen from the results in tables 4 and 5, each dose of the test substance has no obvious inhibition effect on the proliferation of bone marrow cells of mice of two sexes, and the PCE/NCE ratio of the low, medium and high dose groups indicates that the test substance has no cytotoxicity. The bone marrow eosinophilic polycythemic micronucleus rate of each dose group of mice with two sexes has no significant difference compared with a solvent control group (P is more than 0.05). The micronucleus rate of the myelophilia polystaining erythrocytes of the mice of the positive control group is obviously higher than that of the solvent control group (P is less than 0.01), which indicates that the tested animals are sensitive and the test is reliable.

And (4) conclusion: within the tested dose range, the encapsulated mice prepared according to example 1 of the present invention were negative in myelopleochromocyte micronucleus test.

2.3 mouse teratospermia test

TABLE 6 mouse teratospermia test results

As can be seen from Table 6, the incidence of mouse teratospermia in each dose group of the test object has no significant difference (P is more than 0.05) compared with the solvent control group, and the incidence of mouse teratospermia in the positive control group is significantly higher than that in the solvent control group (P is less than 0.01), which indicates that the test object is sensitive and the test is reliable.

And (4) conclusion: within the tested dose range, the encapsulated mice prepared according to example 1 of the present invention were negative for teratospermia.

2.4 rat 30 day feeding test

2.4.1 general examination, organ weighing, blood biochemistry examination, and hematology examination results

TABLE 7 examination results of body weight, body weight gain, food intake, and food utilization of female rats

TABLE 8 examination results of body weight, body weight gain, food intake and food utilization in male rats

As can be seen from tables 7 and 8, after the rats are given different doses of the test substances for 30 days, the body weight, the weight gain, the food intake and the food utilization rate of the rats have no significant difference (P > 0.05) when compared with the control group of the maintenance feed in each dose group of the experimental animals, namely, the capsules prepared according to the embodiment 1 of the invention have no significant influence on the weight gain, the food intake, the food utilization rate and the like of the rats.

TABLE 9 fasting body weight in rats

TABLE 10 examination result of organ coefficients of rat-1

TABLE 11 examination result of organ coefficients of rat-2

As can be seen from tables 9, 10 and 11, after the test substances with different doses are administered to the rats for 30 days, the organ coefficients of the rats have no significant difference (P > 0.05) in comparison between each dose group of the experimental animals and the control group of the maintenance feed, i.e., the capsules prepared according to the embodiment 1 of the invention have no significant influence on the organs of the rats.

TABLE 12 Biochemical examination of rats-1

TABLE 13 Biochemical examination of rats-2

TABLE 14 Biochemical examination of rats-3

TABLE 15 results of hematological examination of rats-1

TABLE 16 results of hematological examination of rats-2

It can be seen from tables 12 to 16 that, after the rats are given different doses of the test substances for 30 days, the biochemical and blood indicators of the rats have no significant difference (P > 0.05) when compared with the dose groups of the experimental animals and the control group of the maintenance feed, i.e. the capsules prepared according to the embodiment 1 of the invention have no significant influence on the blood indicators, liver and kidney functions, lipid, sugar and protein metabolism of the rats.

3. Results of histopathological examination

3.1 general examination description

80 experimental animals (a feed control group, a low dose group, a medium dose group and a high dose group are maintained, and each group comprises 10 male and female animals), the liver, the spleen, the stomach, the duodenum, the bilateral kidney, the testis or the ovary and the like are generally examined, and no obvious pathological changes are seen in the appearance, the volume, the color and the structure of the experimental animals.

3.2 histopathological examination description

40 experimental animals (maintenance feed control group and high dose group, 10 each for male and female).

Liver: the liver cells of female rats in the feed control group are maintained to be 1 spotted necrosis, and the liver cells of male rats are maintained to be 1 spotted necrosis. The hepatic cells of the male and female rats in the high-dose group are shown in 1 case of punctate necrosis and 1 case of focal necrosis, and the hepatic cells of the male rats are shown in 1 case of punctate necrosis.

Kidney: the kidney of the female rat and the male rat in the feed control group and the high-dose group is maintained to be normal.

Gastrointestinal tract: the stomach and intestine of female rats and male rats in the control group and the high-dose group of the maintenance feed are normal.

Spleen: the spleens of female rats and male rats in the feed control group and the high-dose group are maintained to be normal.

Testis/ovary: the testis/ovary of the feed control group and the testis/ovary of the high dose group are maintained to be normal.

Three, small knot

1. Oral toxicity test

The test result of the maximum tolerance of the mice shows that the capsules prepared according to the embodiment 1 of the invention have the maximum oral tolerance dose of more than 15g/kgBW for the mice of two sexes, and belong to non-toxic grade according to acute toxicity grade.

2. Genotoxicity test

The Ames test, the mouse myelophagocytophilic erythrocytic micronucleus test and the mouse teratospermia test show that the capsule prepared according to the embodiment 1 of the invention has no mutagenic activity in the range of the tested dosage.

3. Rat 30-day feeding test

The results of rat weight, weight gain, food intake, food utilization rate, visceral organ examination, visceral organ weight, visceral/body ratio, hematology and blood biochemistry examination show that the test items have no significant difference (P is more than 0.05) when each dosage group is compared with the maintenance feed control group. The pathological histological examination result shows that no obvious damage pathological changes are found in liver, kidney, stomach, intestine, spleen, ovary and testis of the female and male rats in the control group and the high-dose group of the maintenance feed. No significant toxic effect was observed in the 30-day feeding test for the capsules prepared in example 1.

Application example 2Physical fatigue relieving function evaluation test

Experimental methods and conditions

1. Experimental animals and feed: selecting Beijing Huafukang biotech GmbH [ license number: 16g-19g Kunming clean male mice bred by SCXK (Jing) 2014-0004] are divided into four batches for experiment, each batch is randomly divided into 4 groups, and each group of the experiment is 12. The test batch is used for carrying out a load swimming test, the test batch is used for measuring serum urea, the test batch is used for measuring liver glycogen, and the test batch is used for measuring blood lactic acid. The experimental animals were raised in the SPF-level animal room of the health food function testing center of the university of joint culture of north jing [ license number: SYXK (Kd) 2012 and 0031 ]. The maintenance feed was prepared by the company "ao synergetic feed limited of australia of beijing, co: SCXK (Jing) 2014-.

2. Dosage: the test was divided into low, medium and high dose groups, i.e. 0.38g/kgBW, 0.75g/kgBW and 2.25g/kgBW per day, using the capsules prepared in example 1, wherein the medium dose group of 0.75g/kgBW is the recommended dose for adults (60 kg body weight): 4.5g per day, corresponding to 0.075g/kg BW. High dose test article: weighing 13.50g of sample, and adding sterile water to 60.0 mL; medium dose test article: weighing 4.5g of sample, and adding sterile water to 60.0 mL; low dose test article: sample 2.28g was weighed and sterile water was added to 60.0 mL. Orally administered once a day, and continuously gavage for 31 days to measure various indexes. The gavage volume of the mice was 10 mL/kgBW. A blank control (0 g/kgBW) was also included. The daily gavage volume was the same for each subject group. Maintenance feed was administered to each dose group.

3. Experimental methods

3.1 weight bearing swimming test

30min after the test object is given to the mouse for the last time, the mouse with 5% weight lead wire loaded at the tail root is placed in a swimming box for swimming. The water depth is 30cm, the water temperature is 25 ℃ +/-1.0 ℃, and the time from the beginning of swimming to death of the mouse is recorded as the weight swimming time of the mouse.

The swimming time is measured data, if the swimming time of the test object group is obviously longer than that of the blank control group, and the difference is significant (P is less than 0.05), the test object can be judged to have the effect of prolonging the weight swimming time of the mouse.

3.2 serum Urea assay

30min after the test object is given to the mouse for the last time, the mouse swims in water with the temperature of 30 ℃ for 90min, and 0.5mL of blood is collected by pulling out eyeballs immediately after the mouse has a rest for 60 min. Placing in a refrigerator at 4 deg.C for about 3h, centrifuging at 3000r/min for 15min after blood coagulation, collecting serum, and determining serum urea content.

The obtained data is measurement data, and if the urea content in the serum of the test object group is obviously lower than that of the blank control group and the difference is significant (P is less than 0.05), the test object can be judged to have the effect of reducing the urea content of the fatigue mice.

3.3 liver glycogen assay: anthrone process

The test object of the mouse is immediately killed after 30min of the last time, the liver is taken and rinsed by normal saline, then the liver is sucked dry by filter paper, 100mg of the liver is accurately weighed, 4.0mL of TCA is added, the homogenate is homogenized for 1min, the homogenate is poured into a centrifuge tube, the centrifuge tube is centrifuged for 15min at 3000r/min, 0.5mL of supernatant is taken, 2mL of 95% ethanol is added, the mixture is fully mixed until no interface is left between the two liquids, and the mixture is plugged by a clean plug and is vertically placed at room temperature overnight. The next day, centrifuge at 3000r/min for 15 min. Carefully pour off the supernatant and place the tube upside down for 10 min. Glycogen was dissolved in 2.00mL of distilled water and assayed. 0.25mL of the solution to be tested and 0.25mL of distilled water are put into a test tube and mixed evenly. Reagent blank tube: 0.5mL of distilled water was aspirated into a clean tube. Standard tubes: 0.125mL of glucose standard (50 mg/dL) and 0.375mL of distilled water were pipetted into a test tube and mixed well.

2.5mL of anthrone reagent was added to each tube vigorously, boiled for 15min, then transferred to an ice-water bath, cooled, and then the absorbance was measured after zeroing with a reagent blank tube at a wavelength of 620 nm. Liver glycogen content was calculated as follows:

DU: sample tube absorbance, DS: standard tube absorbance, G: liver tissue weight (g), V: extract volume (mL), 16: dilution factor, 0.0625: is the glucose content in 0.125mL glucose standard, 0.9: the glucose is converted to a coefficient of glycogen.

The obtained data is measurement data, if the liver glycogen content of the tested object group is obviously higher than that of the blank control group, and the difference is significant (P is less than 0.05), the tested object can be judged to have the effect of promoting the liver glycogen storage of the mice.

3.4 blood lactic acid assay

Preparing a high blood lactic acid model and preparing a blood specimen: 30min after the test object is administered last time, before swimming, 20 μ L of blood is taken from the angular vein plexus of the mouse eye, added into 0.48mL of 1% NaF solution, added with 1.5mL of protein precipitant, and shaken and mixed evenly; then swimming in water at 30 ℃ for 10min without load, stopping, immediately taking 20 mu L of blood, adding into 0.48mL of 1% NaF solution, adding 1.5mL of protein precipitant, oscillating and mixing uniformly; after resting for 20min, 20 μ L of the blood was immediately collected and added to 0.48mL of 1% NaF solution, 1.5mL of protein precipitant was added, and the mixture was shaken and mixed. Centrifuging at 3000r/min for 10min, collecting supernatant, and performing the operations in Table 17.

TABLE 17 blood lactic acid measurement sample pretreatment

After the above steps are completed, the mixture is shaken up and placed in a water bath at 30 ℃ for 30min (shaken every 10 min). Taking out, heating in boiling water bath for 90s, taking out, cooling to room temperature, performing color comparison at wavelength of 560nm, and zeroing in blank tube.

The blood lactate content and the area under the blood lactate curve were calculated according to the following calculation:

the obtained data is measurement data, and if the area under the blood lactic acid curve of the test object group is obviously lower than that of the blank control group and the difference is significant (P is less than 0.05), the test object can be judged to have the effect of reducing the area under the blood lactic acid curve after the swimming of the mouse.

4. Statistics of test data

Data processing was performed with SPSS software. Performing anova, wherein anova is required to be performed according to an anova program, performing anova, calculating an F value, wherein the F value is less than F_0.05And the conclusion is that: the difference between the average numbers of all groups is not significant; f value > F_0.05P is less than or equal to 0.05, and statistics is carried out by a pairwise comparison method of mean values between a plurality of experimental groups and a control group; carrying out proper variable conversion on the data which are not just too big or uneven in variance, and counting by using the converted data after meeting the requirements of normal or uniform variance; if the variable still does not reach the goal of being normal or uniform in variance after conversion, the statistics is carried out by using the rank sum test.

5. Determination of results

If the result of the load swimming test is positive and any two indexes of three biochemical indexes of blood lactic acid, serum urea and liver glycogen are positive, the tested object can be judged to have the function of relieving physical fatigue.

Second, experimental results

1. Effect on mouse body weight

As can be seen from tables 18 and 19, the initial body weights of the mice were not significantly different (P > 0.05) when compared between the four experimental animal dose groups and the 0g/kgBW group. I.e. the initial body weight of the mice was more balanced between the groups.

After 31 days of oral administration of the mice with different doses of the samples, the body weights of the mice were compared between the four experimental animal dose groups and the 0g/kgBW group, and the difference was not significant (P > 0.05). I.e. each dose sample had no adverse effect on mouse body weight.

2. Influence on mouse weight bearing swimming time

As can be seen from Table 20, the mice in the 0.75g/kgBW group and the 2.25g/kgBW group had prolonged swimming time with significant difference (P < 0.01) compared to the 0g/kgBW group 31 days after the oral administration of the samples at different doses to the mice. Namely, 5 groups of samples can prolong the weight swimming time of the mice, and compared with comparative examples 1-3, the test object of the experimental group of example 1 has obvious synergistic effect on the exercise endurance under each dosage group condition, and is obviously superior to the test object of the comparative examples 1-3 (P is less than 0.05), and the comparative example 4 has no obvious difference from the example 1.

3. Effect on serum Urea levels after swimming of mice

As can be seen from Table 21, after the mice are orally administered with different dosages of samples for 31 days, the serum urea content of the mice after swimming is reduced under each dosage group condition, and compared with the 0g/kgBW group, the tested substances have significant difference (P < 0.05), namely, each tested substance can reduce the serum urea content of the fatigue mice under different dosage group conditions, wherein the effect of the comparative example 4 and the example 1 is the best.

4. Effect on liver glycogen content in mice

As can be seen from Table 22, 31 days after the oral administration of different doses of the samples to the mice, there was no significant difference (P > 0.05) in the liver glycogen content of the mice in each dose group compared with the 0g/kgBW group, i.e., there was no effect on the liver glycogen content of the mice in each dose group of the test substances.

5. Influence on the blood lactic acid value of mice after swimming

As can be seen from Table 23, after 31 days of orally administering different doses of samples to mice, the area under the blood lactic acid curve of mice after swimming of 5 groups of test objects was reduced, compared to the 0g/kgBW group, and there was a significant difference (P < 0.05) between the conditions of the middle dose group and the high dose group, wherein example 1 was able to significantly reduce the area under the blood lactic acid curve of mice after swimming under different dose groups compared to comparative examples 1-3.

Three, small knot

After the test object is orally given to a mouse for 31 days in different doses, compared with a 0g/kgBW group, the test object can prolong the weight-bearing swimming time of the mouse (P is less than 0.05), can reduce the serum urea content of a fatigue mouse (P is less than 0.05), can reduce the area under a blood lactic acid curve of the mouse after swimming (P is less than 0.05), and has a certain quantity-effect relationship, and the test object has no adverse effect on the weight gain of the mouse. The composition of American ginseng-Fuzhuan tea-maca has obvious synergistic effect on anti-fatigue effect, and is obviously superior to the compositions of American ginseng-Fuzhuan tea, Fuzhuan tea-maca, American ginseng-maca and the like. According to the judgment standards of health food inspection and evaluation technical specifications (2003 edition) on the health food for relieving physical fatigue, the capsule prepared in example 1 has the function of relieving physical fatigue.

Application example 3Evaluation test of radiation resistance function

The capsules obtained in the examples and the comparative examples are taken for carrying out a small radiation damage resistance test, SPF male Kunming mice are selected and randomly divided into 6 groups, 12 mice in each group are respectively a blank control group, a model control group and an experimental group (examples 1-10, comparative examples 1-4 and 14 groups in total), and the mice in the model control group and the experimental group are subjected to whole-body radiation with 2Gy dose of gamma rays every day except that the blank control group is not irradiated. 1 hour before irradiation, the blank control group and the model control group are filled with physiological saline with the stomach of 0.75g/kgBW, the experimental groups are filled with the capsules of examples 1-10 and comparative examples 1-4 respectively, and the dosage is 0.75 g/kgBW. Irradiating continuously for 7 days, carrying out cervical dislocation after 7 days to kill the mouse, collecting tail peripheral blood, and testing the number of leucocytes; the femur was dissected, total bone marrow cells were flushed out using Hank's solution, the cell suspension passed through a 4-gauge needle syringe, and after the cells were well dispersed in the suspension, the OD values at 260nm were measured using an ultraviolet spectrophotometer to show DNA content, as shown in table 24 below:

watch 24

As can be seen from the data in Table 24, the number of leukocytes in the mice was significantly reduced in the model control group compared to the blank group, indicating that the mice were injured by irradiation. After the capsules prepared by the invention are infused, the number of leucocytes and the DNA content of bone marrow cells are recovered. The results of examples 4 and 5 are slightly inferior, and the possible reason is that the extraction rate and the damage degree of the chemical structure of the active ingredients are different when different solvent systems are used for extracting the active ingredients of the Fuzhuan tea. According to the invention, the Fuzhuan tea is extracted by using a mixed solvent of water, ethyl acetate and ethanol according to a volume ratio, and the capsule prepared from the Fuzhuan tea extract has the optimal radiation resistant effect. The effect of the comparative example 3 and the comparative example 4 is poor, when the capsule components lack the Fuzhuan tea active ingredients or the sea cucumber peptide, the radiation resistant effect of the capsule is greatly reduced, and a certain synergistic compounding effect exists between the Fuzhuan tea active ingredients and the sea cucumber peptide.

Claims

1. An anti-fatigue and anti-radiation capsule for spaceflight is characterized by comprising the following raw materials in parts by mass: 200-300 parts of American ginseng, 800-1000 parts of Fuzhuan tea, 200-300 parts of maca powder, 30-50 parts of xylo-oligosaccharide, 30-50 parts of corn starch and 1-5 parts of sea cucumber peptide.

2. The anti-fatigue and anti-radiation capsule for spaceflight according to claim 1, which is characterized by comprising the following raw materials in parts by mass: 280 parts of American ginseng, 890 parts of 950 parts of Fuzhuan tea, 260 parts of maca powder, 40-45 parts of xylo-oligosaccharide, 40-45 parts of corn starch and 1.5-3 parts of sea cucumber peptide.

3. The anti-fatigue and anti-radiation capsule for spaceflight according to claim 1, wherein the Fuzhuan tea is used as a component in the form of an extract, and the Fuzhuan tea extract is obtained by a preparation method comprising the following steps: weighing Fuzhuan tea according to the formula, adding a mixed solvent of 5-10 times of water, ethyl acetate and ethanol, carrying out warm extraction for the first time, filtering out warm extract, adding 5-10 times of water into tea residues, carrying out warm extraction for the second time, filtering while hot, mixing filtrate with the first extract, and concentrating under reduced pressure to obtain a thick extract with the relative density of 1.14-1.20 at 55 ℃.

4. The anti-fatigue and anti-radiation capsule for spaceflight according to claim 3, wherein the volume ratio of the water, the ethyl acetate and the ethanol in the mixed solvent is 10:3-5: 1-1.5.

5. The capsule of claim 3, wherein the first warm-dipping extraction is carried out by decocting for 0.5-1h, and then warm-dipping at 50-60 ℃ for 1-2 h; the second warm-soaking extraction is carried out by soaking for 2-4h at 60-70 ℃.

6. The capsule of claim 1, wherein the American ginseng is in the form of an extract, the American ginseng extract is prepared by weighing American ginseng according to the formula, adding 5-10 times by mass of 40-60wt% edible alcohol, heating and refluxing for 2-5h, filtering out an alcohol solution, and recovering under reduced pressure until no edible alcohol smell exists, thereby obtaining an alcohol extract for later use; decocting the residue with 5-10 times of water for 1-3 hr, filtering to obtain water decoction, mixing with above ethanol extractive solution, and concentrating under reduced pressure to obtain radix Panacis Quinquefolii soft extract with relative density of 1.14-1.20 at 55 deg.C.

7. A process for the preparation of an anti-fatigue, anti-radiation capsule for aerospace use according to any of claims 1 to 6, comprising the steps of:

(1) sieving maca powder, xylo-oligosaccharide and corn starch;

(3) granulating and grading, filling hollow capsules, and packaging to obtain the finished product.

8. The preparation method according to claim 7, wherein the maca powder, the xylo-oligosaccharide and the corn starch in the step (1) are sieved by a 60-100-mesh sieve.

9. The preparation method of claim 7, wherein the mixing, drying and crushing in the step (2) are carried out by mixing the two thick extracts of American ginseng and Fuzhuan tea, adding xylo-oligosaccharide and corn starch, stirring for 15-30min, adding maca powder and sea cucumber peptide, stirring for 15-30min, vacuum drying after uniform, cooling after drying, crushing and sieving with a 80-100 mesh sieve for later use.

10. The method according to claim 7, wherein the empty capsules in the step (3) are packed at 0.5-0.7 g/capsule.