WO2006108345A1

WO2006108345A1 - Fruit vinegar-egg powder and composition containing vinegar-egg powder

Info

Publication number: WO2006108345A1
Application number: PCT/CN2006/000594
Authority: WO
Inventors: Xinfu Guo; Zhao Wang
Original assignee: Zhejiang Hangzhou Xinfu Pharmaceutical Co., Ltd.
Priority date: 2005-04-01
Filing date: 2006-04-03
Publication date: 2006-10-19
Also published as: CN1669478A; CN100379359C

Abstract

Fruit vinegar-egg powder and process thereof or composition containing vinegar-egg powder and use. The said method comprises immerging eggs into fruit vinegar made of orange or hawthorn in ratio of 3-5:1, then carrying out mincing, filter-pressing and spray drying. The obtained composition containing vinegar-egg powder comprises vinegar egg powder obtained by the said process method, calcium carbonate, pimeddium, drynaria, solomonseal, angelica, notoginseng, casein phosphopeptide.

Description

Fruit vinegar egg powder and composition containing fruit vinegar egg powder

Technical field

The present invention relates to a vinegar egg powder, a process for the preparation thereof, a composition containing vinegar egg powder, and the use of the composition. Background technique

With the significant increase in the population of the old population, osteoporosis has become an important public health problem, seriously threatening the health of the elderly. Osteoporosis is mainly characterized by a decrease in bone mineral density and loosening of the bone, which often leads to compression deformation of the vertebrae and easy fracture of the femoral neck and forearm wrist.

According to epidemiological surveys, about 75 million people in Europe, America and Japan suffer from osteoporosis. According to the findings of the American Osteoporosis Foundation, 54.0% and 30.0% of postmenopausal white women have osteopenia and osteoporosis, respectively, and men older than 50 have 3% to 6% of osteoporosis, 28%. ~47% for bone loss.

At present, the prevalence of osteoporosis in Chinese population over 40 years old is 16.1%. With the increase of age, the incidence of osteoporosis increases. The prevalence rate of people over 60 years old is 22.6%, and the prevalence rate of people over 80 years old is higher than 50%. Moreover, with regional and gender differences, the prevalence of osteoporosis is also significantly different. Cities are higher than rural areas, and women are more likely than men. It is estimated that 84 million osteoporosis patients in China, accounting for 6.6% of the total population.

Many factors are related to osteoporosis, such as genetics, endocrinology, lifestyle, and dietary nutrition. Among the dietary factors, calcium, phosphorus, vitamin D and protein are the most closely related, and the most studied is calcium nutrition. Calcium is one of the major mineral components in bones and a prerequisite for normal bone growth. A large number of clinical trials and epidemiological surveys have shown that bone loss due to inadequate calcium intake is a major risk factor for osteoporosis.

Decreased bone density is an important indicator of osteoporosis. The main method used to increase bone density is calcium supplementation, but there is a problem that it is not easy to absorb or treat the symptoms. Summary of invention

It is an object of the present invention to provide a vinegar egg powder which is produced by the following method:

1) Add pectinase to citrus or hawthorn juice, enzymatically hydrolyze, filter, sterilize, add yeast Fermented with acetic acid bacteria to obtain fruit vinegar;

2) Put the egg into the fruit vinegar prepared in step 1. The ratio of fruit vinegar to egg is 3~5: 1, soak, stir well, and press-filter to obtain fruit vinegar egg liquid;

3) Spraying the fruit vinegar egg liquid to obtain fruit vinegar egg powder.

In another aspect, the present invention also provides a method for preparing the vinegar egg powder, comprising the following steps:

1) adding pectinase to citrus or hawthorn juice, enzymatically disintegrating, filtering, sterilizing, adding yeast and acetic acid bacteria to obtain fruit vinegar;

3) Spraying the fruit vinegar egg liquid to obtain fruit vinegar egg powder.

Another object of the present invention is to provide a composition of vinegar egg powder which is made of the following raw materials: the above-mentioned fruit vinegar powder, calcium carbonate, epimedium, bone crush, huangjing, angelica, and notoginseng , casein phosphopeptide.

Specifically, the composition of the vinegar egg powder of the present invention is prepared from the following raw materials by weight ratio: 0.3-60% of the above-mentioned fruit vinegar powder, 5-50% of calcium carbonate, 5-60% of epimedium, bone Crushed 5-60%, Huangjing 5-55%, Angelica 5-50%, Sanqi 0.5-40%, Casein Phosphopeptide (CPP) 0.1-10%. Preferably, the composition of the vinegar egg powder of the present invention is prepared from the following raw materials by weight ratio: 0.3-40% of the above-mentioned fruit vinegar powder, 5-30% of calcium carbonate, 5-40% of epimedium, bone 5-40% of broken powder, 5-40% of Huangjing, 5-30% of Angelica, 1-30% of Sanqi, 0.1-10% of casein phosphopeptide. More preferably, the composition of the vinegar egg powder of the present invention is prepared from the following raw materials by weight ratio: 1-20% of the above-mentioned fruit vinegar powder, 10-20% of calcium carbonate, and 15-25% of epimedium. 15-25% of broken bones, 15-25% of Huangjing, 10-25% of Angelica, 5-15% of Panax notoginseng, and 0.1-5% of casein phosphopeptides.

Another object of the present invention is to provide the use of the above-described fruit vinegar powder composition for the preparation of a food for increasing bone density and preventing osteoporosis.

Another object of the present invention is to provide the use of the above-described vinegar egg powder composition for the preparation of a food for relieving fatigue and enhancing immunity. Detailed description of the invention

Vinegar powder refers to egg products made by sterilizing eggs after being immersed in vinegar for a certain period of time, being crushed, filtered and dried.

The invention provides a fruit vinegar egg powder, which is prepared by soaking eggs in citrus or hawthorn juice, The fruit vinegar obtained by fermentation, filtration and the like is prepared by mincing, pressure filtration and spray drying. Specifically, the preparation method of the vinegar egg powder is achieved by the following steps: washing the raw citrus or hawthorn, and processing into a juice by a crusher and a beater (0.5 mm aperture). Add 0.5% food grade pectinase for clarification at a temperature of 35-45 °C and a natural pH of about 4. After reacting for 2 hours, it was pressure-filtered (filter cloth 120 mesh) with a plate and frame filter press, and sterilized by heating at 80 ° C for 15 minutes to obtain a citrus or hawthorn juice. The juice is cooled to 28 ° C, and the activated yeast is added for fermentation for three days. When the fermentation liquid has an alcohol content of 6-7% (v/v) and the total sugar is reduced to less than 10 g/1, the fermentation is stopped to obtain citrus or hawthorn. wine. Add 3% of the cultured acetic acid bacteria to the fermented fruit wine with a stirring speed of 150r/min, aeration ratio of 1:0.8, and fermentation at 32 °C for 4 days, until the total acid (in acetic acid) is raised to 6 The fermentation was stopped at -7%. The cells were sterilized by pressure filtration using a plate and frame filter press, and sterilized by heating at 80 ° C for 15 minutes to obtain a citrus or hawthorn vinegar having a 6-8% acetic acid content. Put the washed, sterilized and sterilized eggs into the above-mentioned fruit vinegar. The ratio of fruit vinegar to egg is 3~5:1, soak for 72 hours, until the pH value is about 5. Stir well and press to obtain fruit vinegar. Egg liquid, spray the fruit vinegar egg liquid to obtain fruit vinegar egg powder, the inlet air temperature is 150-160 ° C, and the outlet air temperature is 80-90 ° C.

According to experimental analysis, the vinegar egg powder of the invention contains protein ≥ 28.0 _§ / 100 _§ , calcium 3. 5 g / 100g, and is rich in B vitamins, amino acids and the like.

The present invention also provides a composition of vinegar egg powder, which is made from the following raw materials: the above-mentioned fruit vinegar powder, calcium carbonate, epimedium, sclerotium, huangjing, angelica, notoginseng, casein phosphate Peptide.

The composition of the vinegar egg powder of the invention is prepared by extracting the formula amount of Epimedium, Rhizoma Drynaria, Polygonatum, Angelica and Sanqi by water extraction and alcohol precipitation, concentrating into a thick paste, and then formulating the amount Made from vinegar egg powder, calcium carbonate, and casein phosphopeptide.

Specifically, the extraction process of the traditional Chinese medicine component of the composition of the vinegar egg powder of the present invention is as follows: Epimedium, Drynaria, Polygonatum, Angelica, Sanqi water soaked, decocted twice, filtered, combined filtrate, concentrated into Clear Cream, add alcohol to the concentration of ethanol to 60%, let stand for 24 hours, filter, the filtrate is recovered ethanol, concentrated into a thick paste.

The present invention also provides the use of a composition of vinegar egg powder for the preparation of a health food product for increasing bone density, preventing osteoporosis, relieving fatigue, and enhancing immunity.

The preparation of the health food composition is usually in the form of a solid preparation, specifically a tablet or capsule or granule or granule.

According to the above ratio, the extraction of the active ingredients is carried out by a conventional production process, and the conventional excipients are added in different dosage forms and thoroughly mixed to form a finished product, which may be a tablet or a capsule or a granule or a granule. The additional materials and the amount of the materials and the specific production processes are all known in the art, and will not be described herein.

The recommended amount of the composition of the present invention is: tablet, capsule preparation is 4.0 g of vinegar egg powder composition / person / day, granules, granules are 8.0 g of vinegar egg powder composition / person / day, orally once a day, Swallow or blister with water according to the dosage form.

The composition of the present invention has no toxic and side effects, has an obvious effect of increasing bone density, and can be used for preparing a health food for increasing bone density.

The protein of the egg is a complete protein, which contains eight kinds of amino acids necessary for the human body. It is decomposed into peptides and amino acids to some extent during the soaking process with vinegar. In addition, there are lecithin, egg butter, vitamins, etc. Calcium absorption has a certain effect. In addition, the vinegar contains sugar, lipids, proteins, amino acids, vitamins, bioflavonoids and minerals, especially the fruit, after the fermentation of vinegar, the B vitamins and other nutrients are significantly increased, which can make calcium in animal food. The substance is dissolved and used by the human body to promote blood metabolism, eliminate fatigue and anti-aging effects. Studies have shown that eggs and egg skins are soaked and dried with 10% acetic acid, which is used as a calcium source to feed rats. Observe the growth and development of calcium acetate on blood calcium, urinary calcium, fecal calcium and femur calcium. Effect, the result is the retention rate of femur calcium in the vinegar egg group, and the absorption rate and retention rate of calcium in the body are not significantly different from those of calcium carbonate and whole milk powder. Animal experimental studies have shown that vinegar egg liquid can significantly promote intestinal peristalsis, promote digestive function, improve cellular immune function, and reduce lipid peroxidation in brain tissue. Fruit vinegar egg powder has a supplemental calcium source in this side, increasing bone density, relieving fatigue, enhancing immunity, and strengthening stomach and digestion.

Calcium carbonate is a calcium supplement which is widely used in adult calcium supplementation. It has a small molecular weight and a high calcium content. It can quickly form soluble calcium ions when neutralizing gastric acid and is easily absorbed by the intestinal tract. Studies have shown that an appropriate increase in calcium intake can increase bone density, reduce bone loss, prevent fractures, and prevent osteoporosis. Animal experiments have shown that calcium carbonate has a good prevention of disuse osteoporosis in rats. It is more economical and reasonable compared with other calcium supplements. Therefore, calcium carbonate is used as a calcium source in this side, and it has calcium supplementation, increases bone density, and improves osteoporosis.

The main active ingredient of Epimedium is the total flavonoids of Epimedium. Studies have shown that Epimedium aqueous extract inhibits osteoclasts, promotes osteoblast function, and increases the formation of calcified bone, thereby inhibiting osteoporosis.

The sclerotia chinensis mainly contains naringin, sclerotin, and sclerotium. Studies have shown that the bone-crushed extract is Ca++ for the chicken embryo base in tissue culture. The deposition has a significant promoting effect, which improves the activity of alkaline phosphatase (ALP) in the tissue, promotes the synthesis of proteoglycans, and inhibits collagen synthesis. The experimental bone injury model of the lower femur of rats was used to show that the root decoction of the fern can promote the healing of experimental bone injury in rats, and the effect is also enhanced with the increase of dose.

Polygonatum mainly contains saponins, polysaccharides, amino acids and trace elements. Studies have shown that Polygonatum can enhance immune and anti-fatigue effects.

Angelica mainly contains volatile oil. Studies have shown that Angelica can promote hematopoietic function of bone marrow and spleen cells, and significantly increase hemoglobin and red blood cell count. Aqueous solution can promote the recovery of hematopoietic function of bone marrow and spleen cells.

Panax notoginseng mainly contains notoginsenoside, notoginseng, quercetin, volatile oil and so on. Studies have shown that Panax notoginseng can promote the proliferation and differentiation of rat osteoblasts and promote osteoblast OPG.

Casein phosphopeptide (CPP) is a polypeptide extracted from natural casein. It can increase the solubility of calcium and promote the absorption of calcium. Studies have shown that CPP has a positive effect on the absorption and utilization of calcium in humans and animals. As a promoting factor, CPP is a very effective calcium absorption promoting factor and can improve the bioavailability of metal ions such as iron, zinc and magnesium. Currently used as a nutritional supplement in Japan, Europe and China, it is widely used in food, health care, and literary industries.

Studies have shown that the components of the composition of the present invention have a combined calcium source, increase bone density, prevent and treat osteoporosis, relieve fatigue, and enhance immunity. The composition of the present invention is suitable for a group of middle-aged and elderly people who are deficient in calcium and osteoporosis, as well as a frail adult population. Detailed ways

Hereinafter, the present invention will be described in detail by way of examples, and the advantageous effects of the present invention will be further illustrated by animal experiments. The following groups are the weight in grams of the drug substance. Example 1 : Preparation method of vinegar egg powder

Take 2500g of citrus, wash it with water, peel it, use crusher, beater (0.5mm aperture) Processed into juice 1500g. Add 0.1% food grade pectinase (Tianjin Lihua Enzyme Preparation Technology Co., Ltd.) for clarification treatment, temperature 40 ° C, natural pH value of about 4, reaction 2 hours, filter with a plate and frame filter press (filter cloth 120 mesh), and then sterilized by heating at 80 ° C for 15 minutes. The citrus juice was cooled to 28 ° C, added to the activated yeast (Anji Yeast Co., Ltd.), and fermented at 28 ° C for three days, the alcohol content of the fermentation broth was 6-7% (v / v), total sugar When the temperature is reduced to below 1Og/1, the fermentation is stopped to obtain citrus fruit wine. Add 3% cultured acetic acid bacteria (Shanghai Difa Brewing Biological Products Co., Ltd.), stirring speed is 150r/min, ventilation ratio is 1:0.8, fermentation at 32 °C for 4 days, to total acid (calculated as acetic acid) Stop fermentation when rising to 6-7%. After sterilizing the cells with a plate and frame filter press, the mixture was sterilized by heating at 80 ° C for 15 minutes to obtain 1200 g of citrus fruit vinegar having an acetic acid content of 6-8%.

Put the washed, sterilized and disinfected eggs into citrus fruit vinegar. The ratio of fruit vinegar to egg is 3:1, soak for 72 hours, pH value is about 5, stir evenly and then filter with plate and frame filter press. Get the fruit vinegar egg liquid. The vinegar egg liquid is spray-dried to obtain fruit vinegar egg powder, the inlet air temperature is 150-160 ° C, and the outlet air temperature is 80-90 ° C. Example 2: Preparation method of vinegar egg powder

The hawthorn is replaced by hawthorn, and the hawthorn juice is crushed. According to the same procedure, the fruit vinegar egg powder is obtained. Example 3: lg/tablet tablets

Ingredients 1000 tablets

Vinegar powder (vinegar powder prepared in Example 1) 150g

Calcium carbonate 320g

Epimedium 750g

Drynaria 750g

Huang Jing 675g

Angelica 500g

Sanqi 250g

Casein Phosphopeptide (CPP) 30g

Formulation of Epimedium, Drynaria, Rhizoma Polygonati, Angelica, Sanqi, soaked in water for 0.5 hours, decocted twice, the first time adding water 8 times, boiling for 1.5 hours; the second time adding water 6 times, Cook for 1 hour. After filtration, the filtrates were combined and concentrated to a clear paste (relative density of about 1.10 at 60 ° C). Add alcohol to the ethanol concentration of 60%, let stand for 24 hours, filter, the filtrate is recovered ethanol, concentrated to a thick paste (60 ° C phase The density is about 1.30). The thick paste is mixed with a formulated amount of vinegar egg powder, calcium carbonate, casein phosphopeptide, and a tablet-acceptable adjuvant, granulated, 0.3% magnesium stearate, and tableted. Example 4: lg/granule capsule

Ingredients 1000 tablets

Vinegar powder (vinegar powder prepared in Example 1) 150g

Calcium carbonate 320g

Epimedium 750g

Drynaria 750g

Huang Jing 675g

Angelica 500g

Sanqi 250g

Casein Phosphopeptide (CPP) 30g The amount of Epimedium, Rhizoma Dry, Huang Jing, Angelica, and Sanqi, soaked in water for 0.5 hours, decocted twice, the first time adding water 8 times, and boiling for 1.5 hours; Add water 6 times for the second time and cook for 1 hour. After filtration, the filtrates were combined and concentrated to a clear paste (relative density of about 1.10 at 60 ° C). Add alcohol to the concentration of ethanol to 60%, let stand for 24 hours, filter, and recover the ethanol from the filtrate and concentrate to a thick paste (the relative density is about 1.30 at 60 °C). The thick paste is mixed with a formulated amount of fruit vinegar powder, calcium carbonate, casein phosphopeptide, and a capsule acceptable auxiliary agent, granulated, and encapsulated. Example 5: 4 g/pack of granules Ingredients 1000 packs

Vinegar egg powder (vinegar powder prepared in Example 1) 300g

Calcium carbonate 640g

Epimedium 1500g

Drynaria 1500g

Huang Jing 1350g

Angelica 1000g

Sanqi 500g

Casein Phosphopeptide (CPP) 60g Formulation of Epimedium, Drynaria, Rhizoma Polygonati, Angelica, Sanqi, soaked in water for 0.5 hours, decocted twice, the first time adding water 8 times, boiling for 1.5 hours; the second time adding water 6 times, Cook for 1 hour. After filtration, the filtrates were combined and concentrated to a clear paste (relative density of about 1.10 at 60 ° C). Add alcohol to the ethanol concentration of 60%, let stand for 24 hours, filter, and recover the ethanol from the filtrate and concentrate to a thick paste (relative density about 1.30 at 60 ° C). The thick paste is prepared by mixing and granulating the formulated amount of fruit vinegar powder, calcium carbonate, casein phosphopeptide, and granule acceptable auxiliary.

6: 4g/pack of granules

Ingredients 1000 packs

Vinegar powder (vinegar powder prepared in Example 2) 300g

Calcium carbonate 640g

Epimedium · 1500g

Drynaria 1500g

Huang Jing 1350g

Angelica lOOOg

Sanqi 500g

Casein Phosphopeptide (CPP) 60g The formula amount of Epimedium, Rhizoma Drynariae, Polygonatum, Angelica, Sanqi, soaked in water for 0.5 hours, decocted twice, the first time adding water 8 times, boiling for 1.5 hours; Add water 6 times for the second time and cook for 1 hour. After filtration, the filtrates were combined and concentrated to a clear paste (relative density of about 1.10 at 60 ° C). Add alcohol to the concentration of ethanol to 60%, let stand for 24 hours, filter, and recover the ethanol from the filtrate and concentrate to a thick paste (the relative density is about 1.30 at 60 °C). The thick paste is prepared by mixing and granulating the formulated amount of fruit vinegar powder, calcium carbonate, casein phosphopeptide, and an auxiliary agent acceptable for the granule. Example 7: Safety Toxicology Test

1 Materials and methods

1.1 Test substance: A tablet produced by the formulation of Example 3 of the present invention is hereinafter referred to as a test substance. 1.2 Test animals: (The following 2 and 3 experiments) ICR mice, SD rats were provided by the Zhejiang Experimental Animal Center, medical laboratory animal certificate No. 22001001, clean grade. (4 feeding experiment) SD rats were provided by Shanghai Experimental Animal Center of Chinese Academy of Sciences. The certificate number is No. 003 of Chinese Medicine, and the clean grade is 60-80g. 2 Acute toxicity test

SD rats weighed 180-220 g, 20 males and 20 females; ICR mice weigh 18-22 g, 20 males and 20 females. SD rats and ICR mice were randomly divided into four dose groups of 1.00 g/kg, 2.15 g/kg, 4.64 g/kg, and 10.00 g/kg body weight according to Horn's method, with 5 males and 5 females in each group.经 By oral gavage, the test substance is administered at a dose of lml/100g (10.00g/kg dose group is 5.00g/kg and 2ml/100g is given to the test substance).

The general condition, symptoms of poisoning and death of rats and mice were observed. The observation period was 2 weeks, and the median lethal dose LD _{5Q of} rats and mice was determined, and the acute toxicity classification was determined. Acute toxicity of test substances to rats and mice

Dosage female male LD ₅₀

(g/kg) Number of animals Number of deaths Number of animals Number of deaths Reaction (g/kg body weight)

1.00 5 0 5 0 Normal

SD

2.15 5 0 5 0 Normal

〉10

Rat 4.64 5 0 5 0 normal

10.0 5 0 5 0 Normal

1.00 5 0 5 0 Normal

ICR 2.15 5 0 5 0 Normal

> 10

Mouse 4.64 5 0 5 0 Normal

10.0 5 0 5 0 Normal

According to the acute toxicity grading standards, the oral toxicity of rats and mice is actually non-toxic.

3 Genetic toxicity test

3.1 Ames test: Select histidine auxotrophic Salmonella typhimurium TA _97A , TA,

TA touch, TA ₁₀₂ , set 5000, 1000, 200, 40, 8 g / dish five dose groups, each dose of parallel dish, repeat the test once. At the same time, a blank control group and a positive control group (dikesone, daunorubicin, diaminoguanidine, 1, 8-dihydroxyindole) were set.

The number of returned colonies of each strain on the culture medium was observed, and the test results are shown in Table 2 and Table 3.

3.2 Mouse bone marrow cell micronucleus test: ICR mice weigh 25-30g. The mice were randomly divided into three groups of 2.5, 5.0, 10.0 g/kg body weight, one negative control group (distilled water) and one positive control group (cyclophosphamide 60 mg/kg body weight), 10 in each group, 5 males and 5 females. The sample was administered twice at intervals of 24 hours, and the amount of gastric perfusion was 0.1 ml/10 g body weight (10.00 g/kg dose group was formulated into 5.00 g/kg and 0.2 ml/10 g was administered to the test substance). The mice were sacrificed 6 hours after the second gavage, bone marrow was taken from the sternum bone marrow, fixed in methanol, and stained with Giemsa. A total of 1000 polychromatic red blood cells (PCE) were counted per animal during the microscopic examination. Table 2 Test results of Ames test (first time)

Dose reverting colony number (X Shi SD)

(μ^ TA _97a ΤΛ98 ΤΑιοο TAi02

皿) -S ₉ +S ₉ -S ₉ +S ₉ -S ₉ +S ₉ -S ₉ +S ₉ subject to 8 115±16.3 118±21.8 35±3.1 36±2.9 162±16.3 170±11.6 273 6.2 261 ±15.7

40 125±8.7 119±12.5 40±6.1 36±7.8 179±27.8 162±15.1 261 ±14.0 266±19.1 Test 200 121 ±17.7 122°22.1 32±9.1 31±8.0 163 ±16.8 166±15.6 259±8.7 263± 13.0

1000 107±25.2 117±15.5 38±5.0 37±9.8 166±16.8 162±9.5 269±22.9 266+18.6 Object 5000 132±17.7 119±20.5 39±5.5 32±5.5 155±6.2 170±20.7 257±20.6 268± 14.8 Blank control 126±21.5 119±20.5 32±5.6 35±5.0 170±15.7 175±18.1 255±14.0 248±12.3 Dixon 50 2925 ±152.7 976±81.2 547±85.5 1312±170.3

Sodium azide 1.25 1131±125.0

Dinomycin 10 1261 ±186.8

Diaminoguanidine 40 920+105.8 893 ±209.3 872 + 71.1

1.8-Dihydroxyguanidine ±128.' Calculate the micronucleus rate (MN%.:), and the results are shown in Table 4. Table 3 Test results of Ames test (second time)

Dosage Reversion colony number (X±SD)

TA _97i TA ₉₈ TA ₁₀₀ TA ₁₀₂ dishes) -S ₉ +s ₉ -S ₉ +s ₉ -s ₉ +S ₉ -S ₉ +s ₉

8 126±23.3 119±12.1 34±10.7 43±3.2 177±9.1 163±13.3 262±18.3 272±23.3

40 122±22.3 124±13.1 34±5.7 33±7.1 171±16,2 164±11.6 268±16.1 267 士 24.1.

200 115±26.8 125±25.7 37±6.1 33±9.0 172±15.8 164±12.5 263±16.9 260 士 16.7

1000 114±9.8 123±27.1 38±2.6 35±3.8 189±10.1 162±17.2 262±19.1 259±15.9

5000 106±9.5 119±13.0 38±3.1 42±3.8 171±19.5 170 soil 9.8 260±16.3 277 soil 9.8 blank control 114+21.0 121 + 13.7 33±4.9 35±4.4 167±23.7 164±15.9 253±25.1 264 ± 23 Dixon 50 2912±154.5 1080± 173.3 557±40.6 1197+184.1 Azide sodium 1.25 1165±197.5

Dinomycin 10 1261 ±186.8

Diaminoguanidine 40 984士 119.5 829±252.5 872±71.1

1.8-dihydroxyindole 1051±128.1 Table 4 Effect of test substance on micronucleus rate in mice

Dosage polychromatic red micronucleus rate gender group number of mice micronucleated cells

(g/kg) Cell number (%o) Negative control 0.0 5 5000 7 1.4

2.5 5 5000 5 1.0

Female test substance 5.0 5 5000 9 1.8

10.0 5 5000 8 1.6

Cyclophosphamide 60 5 5000 105 21.0* Negative control 0.0 5 5000 9 1.8

2.5 5 5000 7 1.4

Male test substance 5.0 5 5000 7 1.4

10.0 5 5000 7 1.4

Cyclophosphamide 60 5 5000 111 22.2*

*P<0.01 (compared to the negative control group)

3.3 Mouse sperm abnormality test: ICR male mice, weighing 26-34g. Three dose groups of 2.5, 5.0, 10.0 g/kg body weight, one negative control group (distilled water) and one positive control group (mizomycin C 2.0 mg/kg body weight) were set, with 7 rats in each group. Each group of samples was administered orally by O.lml/lOg body weight (5.00 mg/kg 5.00 g/kg to 0.2 ml/10 g to the test substance) for 5 consecutive days, and the control group was treated similarly. After the first 35 days after the first gavage, the mice were sacrificed by dislocation. Five mice were randomly selected to take the epididymis on both sides, and placed in a plate containing appropriate amount of normal saline. The epididymis was cut with an ophthalmic scissors, and four layers of fluoroscopic paper were suction filtered. Direct smear, naturally dry, fixed in methanol, stained with 1% eosin. The sperm morphology was observed under high power microscope. Each mouse counted 1000 intact spermatozoa, the type and number of abnormal sperm were recorded, and the incidence of sperm abnormality (%) was calculated. The results are shown in Table 5.

Mouse sperm abnormality test results

Malformation rate, number of abnormal spermatozoa (a) subtotal (a)

Rat test (%) group

Number of numbers (units) No fixed No folding Fat Xiang Double head /

Hook stacking head

Negative control ( 0) 5 5000 50 46 0 2 4 0 102 2.04 by 2500 5 5000 58 40 0 0 2 1 101 2.02 Test 5000 5 5000 67 32 0 3 5 0 107 2.14 Object 10000 5 5000 44 58 0 4 2 1 109 2.18 Mitomycin C2.0 5 5000 152 124 0 9 14 6 305 6.10*

H 0.806

P 0.848 compared with the negative control group *P <0.01 4 Rat 30-day feeding test

SD rats were randomly divided into 4 groups, 20 in each group, half male and half female. Test set to control group, test substance 1.67, 3.33, 6.67g/kg body recombination, equivalent to 25 times, 50 times and 100 times the recommended amount of human (the recommended amount is 4.0g / day). Each dose of the experiment was uniformly mixed into the basic feed according to the weight of 10%, and was fed by feeding means, free to eat and drink. The observation was continued for 30 days, and the body weight and food intake were recorded and the food utilization rate was calculated. At the end of the experimental period, blood was taken from the tail vein for hematological examination, and blood was taken for decapitation to perform blood biochemical examination. The organs were generally observed for each organ, and the liver, kidney, spleen, testis (ovary) were weighed and the body was counted. And take liver, kidney, spleen, stomach, intestine, testis (ovary) for histopathological examination. The experimental results are shown in Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12.

ο

Table 7 Effect of total food utilization rate of rats in test subjects (X soil SD) dose rat weight gain food intake food utilization sex

(g/kg) (only) (g) (g) (g) Control group 10 140.5±18.9 617.2±71·7 22.7 ±1.4

1.67 10 136·9±15·1 613·1±50·9 22.4±2.1 Female

3.33 10 129.7+16.2 612.7±66.3 21.2±2.1

6.67 10 135.7±13.0 614.5±41.1 22.1 ±2.2

F 0.790 0.012 1.089

P 0.507 0.366 Control group 10 192.9±25.6 656.0±54.3 29.3 ±2.0

1.67 10 192.1 ±14.2 657.4±41.4 29.3 ±2.4 male

3.33 10 194.5±16.6 657.0 ± 67.5 29.8 ± 2.9

6.67 10 176.7±16.3 645.3±35.1 27.4±2.0

F 2.261 0.128 2.083

P 0.098 0.943 0.120 The results are shown in Table 2. The original data accorded with the requirement of variance (P>0.05). There was no significant difference in body weight gain, food intake, food utilization and control group in each dose group (analysis of variance, P>0.05). Effect of test substance on rat HB, RBC, WBC (X Shi SD)

Effect of test substance on rat WBC (X soil SD)

Dose rat number lymphocyte monocyte granulocyte sex

(g/kg) (only) (%) (%) (%) Control group 10 77·7±8·1 2.1±0.3 20.2±8·1

1.67 10 76.0±5.4 2.4±0.3 21.6±5.4 female

3.33 10 76.5±8.1 2·2±0·6 21.4±7.9

6.67 10 75.9 ± 6.4 2.3 ± 0.5 21.9 ± 6.2

F 0.141 0.848 0.111

P 0.935 0.477 0.953 Control group 10 76.7±7.7 2.4±0.4 21.0±7.6

1.67 10 76.5±5.5 2.1 ±0.3 21.4±5.3 male

3.33 10 76.3±8·4 2.3 ±0.3 21.4±8.4

6.67 10 75.2±8.0 2.3±0.5 22.5±7.8

F 0.077 0.992 0.080

P 0.972 0.407 0.971 Table 10 Effect of test substance on blood biochemistry of rats (X soil SD)

Sexually dosed mouse, alanine, transamin, transaminase, urea, creatinine, cholesterol, triglyceride (g/kg) (only) enzyme (u/L) (iVL) (mmol/L) (umol/L) (mmol/L) (mmol/L) Control group 10 37.9±11·3 194.6±37.2 8.4±1·7 46.8±6.4 1.89±0.2 0·75±0.2 Female 1.67 10 41.6±13.0 196.3±35.3 8.6±1.2 46·7±3.5 2.02 + 0.3 0.95 ±0.3

3.33 10 39.9±10.6 204.1 ±32.5 9.4±1.9 47.6±4.6 1.95±0.3 0.82±0.2

6.67 10 36.1 ±9.9 175.2±23.5 9.1±1.7 46.6±5.7 1·95±0.3 0.98±0.2

F 0.450 1.422 0.737 0.353 2.027

P 0.719 0.252 0.537 0.971 0.787 0.128 Control group 10 40.4 + 8.9 194.7 soil 37.1 7.8±2.0 47.0±5.4 1.80 + 0.4 0.67 + 0.2 Male 1.67 10 39.3±4.1 198.2±31.4 6.7 ±0.6 43.1 ±3.5 1.76±0.4 0.69 + 0.2

3.33 10 41.1±7.2 202.2±29.6 7.8±1.6 43.1±3.6 1.82±0.3 0.68±0.2

6.67 10 35.7±6.2 184.5±28.1 7.3±1.8 45.4±2.8 1.97±0.3 0.66±0.2

F 1.241 0.570 1.126 2.365 0.757 0.654

P 0.309 0.638 0.352 0.087 0.526

ο

ο Effect of test substance on blood biochemistry of rats (X Shi SD)

Sex dose rat number blood glucose total protein albumin globulin white / ball

(g/kg) (only) (mmol/L) (g/L) (g/L) (g/L)

Control group 10 5.56 ± 0.6 65.1 ± 2.5 32.2 ± 3.2 32.9 ± 2.1 0.98 ± 0.1

1.67 10 5.72 ± 0.3 65.5 ± 2.0 32.2 ± 2.2 33.3 ± 2.8 0.99 ± 0.2 female o

3.33 10 5.39±0.5 66.2±2.3 32.4±2.4 33.8 soil 2.5 0.98±0.1

6.67 10 5.84±0.8 65.5±1.9 32.4±2.0 33.1±2.4 1.00±0.1

F 1.154 0.392 0.016 0.234 0.046

P 0.341 0.760 0.997 0.872 0.987

Control group 10 5.66±0.5 63.3±4.2 31·6±3·5 31.7±2.4 0.99±0.2

1.67 10 5.47±0.8 63.8±3.0 31.3±2.3 32.4±2.5 0.98±0.1 Male

3.33 10 5.27±0.7 64.4 + 2.6 31.9±2.4 32.5±2.8 0.99±0.1

6.67 10 5·47±1.0 62.8±3.1 30.9±2.4 31.9±3.1 0.99±0.2

F 0.422 0.400 0.228 0.204 0.012

P 0.739 0.754 0.876 0.893 0.998 Effect of test substance on rat's visceral ratio (X soil SD)

5 Conclusion

According to the results of the test, the composition of the present invention has an oral LD _5Q of more than 10.00 g/kg body weight for both male and female mice, and is practically non-toxic. Ames test, mouse bone marrow micronucleus test, mouse sperm abnormality test results were negative. The results of the 30-day feeding test in rats showed no symptoms of poisoning in the experimental animals, and no abnormal pathological changes were observed in the gross anatomy. There were no damaging effects on blood routine and biochemical indicators. Example 8: Increased bone density test

In order to verify the efficacy of the composition of the present invention, the Chinese Center for Disease Control and Prevention was commissioned to complete an animal efficacy test.

1 Materials and methods

1.1 Sample: The tablets produced according to the formula 3 of the present invention are hereinafter referred to as the test materials, and are provided by Hangzhou Xinfu Pharmaceutical Co., Ltd., and the recommended intake is 4 tablets per person per time, once a day.

1.2 Test animals: 50 clean-ranked weaned milk with an initial body weight of 60-75 g WISTAR male rats, provided by the Institute of Laboratory Animals, Chinese Academy of Medical Sciences, license number: SCXK (Beijing) 2000-0006.

1.3 Test methods: Rats were randomly divided into 5 groups, namely low calcium feed control group, low, medium and high dose test groups, which were equivalent to 5 times, 15 times and 30 times the recommended amount per kilogram of body weight for adults. (According to the average rat eating 100g per kilogram of body weight, a semi-synthetic feed was prepared at a dose of 0.335g/kg BW, 1.005g/kg BW, 2.010g/kg BW, and the content of the test substance in the feed was 3.35g/kg, 10.05g, respectively. /kg, 20.10g/kg), and then set a high-dose calcium carbonate control group according to the calcium content of the high-dose test group feed. 10 in each group, single cage feeding, free eating, drinking deionized water, weighing weekly, record Food intake. The test period is 12 weeks.

1.4 Observation indicators:

After 12 weeks of feeding, the head was killed and the right femur was removed.

To constant weight, weigh the bones. Using the SD-1000 bone density meter to measure I

density.

2 test results

2.1 Effect of test substance on rat body weight (Table 13) Table 13 Effect of test substance on rat body weight (M±SD)

Weight (g)

Group only

The fourth week of the fourth week of the eighth week of the twelfth week of the low calcium control group 10 85.4 ± 5.3 195.4 ± 11.5 274.8 ± 20.2 326.8 ± 24.9 high dose calcium carbonate group 10 85 · 2 ± 5.3 199.5 ± 11.6 278.8 ± 20 · 4 323.0 ±26.0 Low-dose experimental group 10 85.6±5.4 203.2±11.4 285.4±15·2 333·7±20·2 Medium dose experimental group 10 85.0±5.3 206.6±13.1 287.0 soil 28.8 339.8±33.6 High-dose experimental group 9 85.2±4.8 210.7 ± 13.2 300.4 ± 20.5 344.6 ± 21.4

2.2 Effects of test substances on femur length, weight and bone calcium content (Table 14) Table 14 Fecal length, weight and bone calcium content of rats in the test article (M±SD) Group femur length Femur weight Femur total Calcium content of femur calcium content only

(cm) (g) (mg) (mg/g) Low Calcium Control Group 10 3.60 ± 0.09 0.553 ± 0.044 118.8 ± 9.9 214.7 ± 5.2 High dose calcium carbonate group 10 3.61 ± 0.06 0.601 ± 0.041 144.4 ± 11.8 * 240.2 ± 3.6 * Low dose experimental group 10 3.63 ± 0.11 0.553 ± 0.038 127.3 ± 12.4 230.0 ± 9.0 * medium dose experimental group 10 3.63 ± 0.99 0.591 ± 0.051 136.0 ± 12.9 * 230.1 ± 6.0 * high dose experimental group 9 3.68 ± 0.06 0.614 ± 0.039 * 145.3 ±11.5* 236.6±4.6* compared with low calcium control group *P (0.05

2.3 Effect of test substance on bone mineral density in rats (Table 15)

High dose calcium carbonate group 10 0.261 ±0.008* 0.258 ±0.009* Low dose experimental group 10 0.253 ±0.010 0.247 ±0.008 medium dose experimental group 10 0.261 ±0.010* 0.249 ±0.008 high dose experimental group 9 0.267±0.013* 0.255 ±0.004* 3 test conclusion

According to the results of this test, the femur weight of rats was significantly higher than that of the low calcium control group (P<0.05) by administering 2.010 g/kg BW test substance; 0.335 g kgBW, 1.005 g/kg BW, 2.010 g/kg BW test substance were administered. The femur calcium content in rats was significantly higher than that in the low-calcium control group (P<0.05). The administration of 1.005g/kg BW and 2.010g/kg BW test specimens significantly increased the femoral telecentric end bone density of rats. The control group (P < 0.05); the bone mineral density at the midpoint of the femur was significantly higher in the rat femur at the midpoint of the 2.010 g/kg BW test (P < 0.05 =. Example 9: Relieving fatigue, enhancing immunity) Functional test

The tablets produced according to the formula 3 of the present invention are hereinafter referred to as the test materials, and are provided by Hangzhou Xinfu Pharmaceutical Co., Ltd., and the recommended intake is 4 tablets per person per time, once a day, set low, medium, The three high-dose groups were equivalent to 5, 10, and 30 times the actual intake of humans, and the negative control group (distilled water) was used to perform functional tests to relieve fatigue and enhance immunity. The results are as follows:

1 fatigue function test

1. 1 Mouse weight-bearing swimming test The test object can significantly prolong the weight-bearing swimming time of mice. The difference between the middle and high dose groups and the water control group is statistically significant (P<0.05). '

Table 16 Effect of test substance on swimming time in mice

Group only swimming time (S)

Water control group 10 453.40± 112.58

Low dose experimental group 10 532.08 ± 157.45

Medium dose experimental group 10 829.68±280.10

High dose experimental group 10 872.06 ±231.24

1. 2 Serum urea nitrogen content after exercise in mice The serum urea nitrogen content of the mice in the test and high dose groups was significantly lower than that in the water control group (P<0, 05). Table 17 Effect of test substance on serum urea nitrogen content after exercise in mice

Group only serum urea nitrogen (mmol/L)

Water control group 10 10.262±0.968

Low dose experimental group 10 9.429 ± 1.085

Medium dose experimental group 10 8.533 ± 1.182

High dose experimental group 10 8.296 ± 1.268

1. 3 After the exercise, the blood lactate value of the high-dose group of mice was decreased after 10 minutes of exercise, and the difference was statistically significant (P<0.05). Table 18 Differences in blood lactic acid between mice before and after exercise

Group only The difference in blood lactate before and after exercise (mmol/L)

Water control group 10 2.532 ±0.622

Low dose experimental group 10 2.306 ± 1.028

Medium dose experimental group 10 2.298 ± 1.042

High dose experimental group 10 0.984 ±0.469

Conclusion: The results of the above three experiments prove that the composition has the function of relieving fatigue

2 enhanced immunity function test

2.1 ConA-induced spleen lymphocyte transformation in mice The high-dose group can increase the proliferation of spleen lymphocytes induced by ConA in mice. The difference is statistically significant (P<0.05). . '

Table 19 Effect of test substances on ConA-induced spleen lymphocyte transformation in mice

Lymphocyte transformation

Group only

(expressed by MTT OD difference)

Water control group 10 0.16±0.03

Low dose experimental group 10 0.18 ±0.02

Medium dose experimental group 10 0.21 ±0.01

High dose experimental group 10 0.22 + 0.02

2.2 Mice carbon deposition profile experiment The phagocytosis of the high and middle dose groups of the test group was statistically significant compared with the water control group (P<0.05).

Table 20 Effect of test substance on carbon profile clearance ability of mice

Group only number of phagocytic index (a)

Water control group 3.99 + 0.48

Low dose experimental group 4.09 ±0.63

Medium dose experimental group 4.64±0.58

High dose experimental group 4.67±0.61

2.3 Determination of NK cell activity The NK cell activity of the high dose group was significantly different from that of the water control group (P<0.05).

Table 21 Effect of test substance on NK cell activity

Group only NK cell activity (%)

Water control group 10 17.83 ± 5.92

Low dose experimental group 10 17.43 ±3.63

Medium dose experimental group 10 18.64±5.58

High dose experimental group 10 22.03 ±5.41

Conclusion: The above three test results prove that the test substance has the function of enhancing immunity ₍ In summary, the composition of the invention has no toxic and side effects, has a significant function of increasing bone density, and has the functions of relieving fatigue and enhancing immunity, and can be made to increase bone density, prevent and treat osteoporosis, relieve fatigue, and enhance immunity. Healthy food.

Claims

Rights request

1. A fruit vinegar egg powder which is produced by the following method -

3) Spraying the fruit vinegar egg liquid to obtain fruit vinegar egg powder.

2. A method for preparing a fruit vinegar egg powder, comprising the following steps:

3) Spraying the fruit vinegar egg liquid to obtain fruit vinegar egg powder.

3. A composition of fruit vinegar egg powder, which is prepared from the following raw materials: fruit vinegar powder, calcium carbonate, epimedium, bone crush, huangjing, angelica, and notogins according to claim 1. , casein phosphopeptide. '

The composition of vinegar egg powder according to claim 3, which is prepared from the following raw materials by weight ratio: 0.3-60% of vinegar egg powder according to claim 1, calcium carbonate 5-50 %, Epimedium 5-60%, sclerotium supplement 5-60%, Huang Jing 5-55%, Angelica 5-50%, Panax notoginseng 0.5-40%, Casein Phosphopeptide 0.1-10%.

The composition of vinegar egg powder according to claim 3, which is prepared from the following raw materials by weight ratio: 0.3-40% of vinegar egg powder according to claim 1, calcium carbonate 5-30 %, Epimedium 5-40%, Drynaria supplement 5-40%, Polygonatum 5-40%, Angelica 5-30%, Panax notogins 1-30%, Casein Phosphopeptide 0.1-10%.

The composition of vinegar egg powder according to claim 3, which is prepared from the following raw materials by weight ratio: 1-20% of vinegar egg powder according to claim 1, calcium carbonate 10-20 %, Epimedium 15-25%, smashed 15-25%, Polygonatum 15-25%, Angelica 10-25%, Panax notar 5-15%, Casein Phosphopeptide 0.1-5%.

The use of the vinegar egg powder composition according to any one of claims 3 to 6 for the preparation of a food for increasing bone density and preventing osteoporosis.

Use of the vinegar egg powder composition according to any one of claims 3 to 6 for the preparation of a food for relieving fatigue and enhancing immunity.