CN115669932A - Composition with function of regulating intestinal flora and preparation method and application thereof - Google Patents
Composition with function of regulating intestinal flora and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a composition with a function of regulating intestinal flora, which comprises the following raw materials: the content of the dendrobium flower is 30-40% (w/w), the content of the cordyceps cicadae is 15-25% (w/w), the content of the seville orange flower is 10-20% (w/w), the content of the sparassis linearis is 10-20% (w/w), the content of the crocodile flower is 10-20% (w/w), and the total amount of all the components is 100%. The composition disclosed by the invention is prepared from entomogenous fungi extract and medicinal and edible raw materials and food management raw materials. Animal experiments show that the composition can realize synergistic interaction, better promote the growth of beneficial bacteria and inhibit the proliferation of harmful bacteria. The composition can be used for preparing health-care food, including hard capsules, soft capsules, tablets, granules, powder, pills, tea bags, oral liquid and other dosage forms. The composition has definite components, no adverse side effects, and obvious effect on regulating intestinal flora.
Description
Technical Field
The invention belongs to the technical field of health-care food, and particularly relates to a composition with a function of regulating intestinal flora, and a preparation method and application thereof.
Background
The intestinal flora is a general term of microbial communities fixedly planted in human intestinal tracts, belongs to human symbiotic microorganisms, is in a dynamic balance state with organisms, forms a system which is mutually regulated, restricted and interdependent, and is known as a 'second genome' of human bodies. A large number of experiments prove that the intestinal flora is an important 'organ' for maintaining the health of a host, influences the host through various ways such as intestine-hepatic axis, intestine-brain axis, intestine-muscular axis and the like, and plays a role in participating in host metabolism, forming systemic immunity, maintaining intestinal homeostasis and influencing the physiological function and behavior of the brain.
Modern researches show that intestinal flora and traditional Chinese medicines have complex action relationship. "Molecular Mechanisms of interaction of the Chinese Herbal Medicines of Understanding the Molecular Mechanisms of the interaction of the intestinal Microbiota with the intestinal Microbiota" (medicinal research review, med Res Rev.2017Sep;37 (5): 1140-1185.) indicates that the intestinal flora can metabolize the Chinese Herbal chemicals, and the resulting metabolites have different bioavailabilities, bioactivities and toxicities. The chemical components of the traditional Chinese medicine can also regulate the composition of intestinal flora, regulate the activity of beneficial bacteria and conditioned pathogenic bacteria, and restore the balance of the intestinal flora in vivo, thereby improving the dysfunction and related pathological conditions.
In addition, the intestinal flora improved by the effective components of the traditional Chinese medicine can promote other components of the traditional Chinese medicine to be fermented by the intestinal flora to generate different activities, so that the synergistic effect or the antagonistic effect of the effective components in the traditional Chinese medicine is formed. Such as intestinal microbial catalyzed deglycosylation, is an important hydrolysis mode, and the secondary glycosides and/or aglycones produced by this process generally have better intestinal absorption and thus better bioavailability. The platycodon grandiflorum or platycodon grandiflorum-liquorice compatible product can inhibit the metabolism of intestinal flora on platycodin D, because the metabolic capability of the intestinal flora is weakened due to multi-component competitive metabolic enzyme and the sterilization or bacteriostasis of certain chemical components. These reports also demonstrate the "multi-component, multi-effect" characteristics of traditional Chinese medicine.
The herba Dendrobii is cultivated product of Dendrobium nobile Lindl, dendrobium chrysotoxum Lindl or Dendrobium fimbriatum hook of Orchidaceae and fresh or dried stem of similar species of plants of the same genus, and its flower (herba Dendrobii flower) is non-medicinal part. Wherein the Dendrobium officinale is dried stem of Dendrobium officinale of Orchidaceae, dendrobium officinale Kimura et Migo, and its flowers (Dendrobium officinale flowers) are managed according to local characteristic food. Researches show that the dendrobium flower has the effects of regulating immunity, reducing blood sugar, regulating intestines and stomach and the like. Along with the popularization of the artificial planting technology of the dendrobium officinale, the planting area is continuously increased, the yield of dendrobium officinale flowers is large, the dendrobium officinale flower resources are fully utilized, and the resource waste is avoided.
The Cordyceps cicadae sporocarp is obtained by inoculating Isaria cicadae Miq (Isaria cicadae Miq) of Isaria cicadae of Isaria of Cordyceps family on a culture medium and performing artificial culture, and has multiple effects of regulating immunity function, improving renal function, reducing blood sugar, etc. The cordyceps sobolifera sporocarp (artificially cultured) is approved as a new food raw material in 2021, and is a medicinal and edible resource with development prospect.
Citrus aurantium L.var.amara Engl, namely Citrus aurantium, citrus aurantium and the like, belongs to a medicinal and edible variety, and is a dried stamen of Citrus subgenus in Rutaceae. The substitute flower is originally recorded in "Kaibao Ben Cao", has the efficacies of regulating qi, soothing liver, stimulating appetite, stopping vomiting and the like, and has the pharmacological actions of resisting oxidation, inflammation, tumors and viruses, promoting gastrointestinal peristalsis and the like. The traditional Chinese medicine composition is mainly used for treating symptoms such as chest and abdomen stuffiness and distending pain, dyspepsia, phlegm and fluid retention and the like in clinic.
The sparrow spalaghus linearis (bark.f.) r.dahlgren is a needle-shaped shrub growing in the Cedarberg mountain area in south africa, and is approved as a new food raw material in 2014 by China. Has effects in invigorating kidney, replenishing vital essence, invigorating qi, invigorating spleen, nourishing yin, promoting fluid production, calming heart, tranquilizing mind, relieving pain in stomach, thirst due to body fluid consumption, listlessness, debilitation, and heaviness of limbs, and has pharmacological effects of resisting oxidation, resisting inflammation, lowering blood sugar, protecting cardiovascular and cerebrovascular system, resisting cancer, and resisting mutagenesis.
Alligator Lindau (N.L. Burman) is a plant of Acanthacce alligator of Acanthaceae, also known as Clinacanthus nutans, etc., and is managed as a common food. The whole herbs are used as herbs for damp-heat jaundice, bi syndrome, irregular menstruation, swelling and pain from injuries and fractures. In Yunnan of China, crocodile flower is used as a national medicine, and is eaten as a wild vegetable in Guangxi and Guangdong areas.
At present, the health care product of intestinal flora on the market is a composite preparation mainly comprising probiotics, probiotics and prebiotics, and a plurality of monosaccharides, and the main action mechanism of the health care product is to promote the growth of beneficial bacteria. The existing probiotics are mostly live bacteria preparations such as bifidobacterium, lactobacillus and the like, and the live bacteria preparations have the problems of difficult guarantee of live bacteria quantity, poor stability, need of refrigeration storage and the like. The Chinese patent medicine composition for regulating intestinal flora is mainly polysaccharide extract of single or multiple medicinal materials or a compound consisting of the polysaccharide extract and probiotics, a composition of the multiple medicinal materials or a compound consisting of the polysaccharide extract and the probiotics, wherein the polysaccharide extract of the single or multiple medicinal materials mainly contains polysaccharide and has a single component; the components of the medicinal compositions are various, but the effect of fully regulating the intestinal flora cannot be achieved.
Disclosure of Invention
The invention aims to provide a composition with a function of regulating intestinal flora, which consists of dendrobium flower, cordyceps sobolifera sporocarp, seville orange flower, gorgonia lineariifolia and alligator flower. The invention provides the following technical scheme:
a composition with the function of regulating intestinal flora comprises the following raw materials: the content of the dendrobium flower is 30-40% (w/w), the content of the cordyceps sobolifera fruiting body is 15-25% (w/w), the content of the seville orange flower is 10-20% (w/w), the content of the gordon malachitum flower is 10-20% (w/w), the content of the crocodile flower is 10-20% (w/w), and the total amount of all the components is 100%.
Preferably, the composition consists of the following raw materials: the content of the dendrobium flower is 35% (w/w), the content of the cordyceps sobolifera fruiting body is 20% (w/w), the content of the seville orange flower is 15% (w/w), the content of the sparassis linearis is 15% (w/w), and the content of the crocodile flower is 15% (w/w).
Preferably, the dendrobium flower is dendrobium officinale flower, and the cicada fungus body is artificially cultivated.
The composition comprises the following effective components in percentage by mass: the content of polysaccharide is 21-25%, the content of flavone is 9-14%, and the content of alkaloid is 0.2-0.7%.
Another object of the present invention is to provide a method for preparing a composition for regulating the function of intestinal flora, comprising the steps of:
step 1, weighing cordyceps sobolifera sporophores according to a formula and crushing; weighing dendrobium flower according to a formula, mixing with crushed cordyceps sobolifera fruiting bodies, and mixing according to a material-liquid ratio of 1:10 to 1: adding water according to the proportion of 40, pre-soaking for 4-8h, performing reflux extraction for 1-2 h in a water bath at the temperature of 100 ℃, extracting for 1-3 times, filtering, and combining filtrate to obtain an extracting solution 1. Preferably, the feed-liquid ratio is 1:20, pre-soaking for 6h, and performing reflux extraction for 1.5h in 100 ℃ water bath for 2 times.
Step 2, weighing the seville orange flower, the gorgon canary flower and the crocodile flower according to the formula, mixing and crushing, wherein the material-liquid ratio is 1:5 to 1: adding 50-70% volume concentration ethanol into 20, refluxing and extracting for 1-2 h in water bath at 80-90 ℃, extracting for 1-3 times, filtering, combining the filtrates to obtain an extract 2, and concentrating under reduced pressure at 50-70 ℃ until no alcohol smell exists to obtain a concentrated solution. Preferably, the ratio of the material to the liquid is 1:10, adding 60 percent ethanol by volume, refluxing in water bath at 90 ℃ for 1.5h, extracting for 2 times to obtain an extracting solution 2, and concentrating under reduced pressure at 60 ℃ until no alcohol smell exists to obtain a concentrated solution.
And 3, combining the extracting solution 1 obtained in the step 1 and the concentrated solution obtained in the step 2, concentrating under reduced pressure at 50-70 ℃, drying and crushing to obtain the composition extract powder.
The invention provides application of the composition in preparing health-care food for regulating intestinal flora.
The health food comprises the composition and pharmaceutic adjuvants, and the dosage form of the health food comprises any one of tablets, capsules, solutions, suspensions, emulsions, oral liquids, syrups, granules, powders and pulvis.
The composition is rich in polysaccharides, flavonoids, alkaloids, lignans and other components, has specific content of active ingredients under specific formula content, namely, after the polysaccharide 21-25% (w/w), the flavonoids 9-14% (w/w) and the alkaloids enter intestinal tracts, the intestinal tract flora can be regulated, and the synergistic effect of the polysaccharides, the flavonoids, the alkaloids and the lignans can promote the growth of probiotics and inhibit the reproduction of pathogenic bacteria, so that the composition has a remarkable regulating effect on the intestinal tract flora, and simultaneously improves the immune function of organisms, thereby achieving the purposes of regulating yin and yang, strengthening the body resistance and eliminating pathogenic factors.
The invention has the following beneficial effects:
1. the composition is prepared by combining medicinal and edible raw materials and food management raw materials, and polysaccharides, flavonoids and alkaloids with specific contents in the formula selectively stimulate the growth of symbiotic beneficial bacteria and inhibit the reproduction of pathogenic bacteria and the like by exerting the similar effect of prebiotics. The improved intestinal flora can promote other effective components in the formula to be fermented by the intestinal flora to generate prebiotics similar effect, thereby promoting the synergistic effect among non-carbohydrate micromolecules in the traditional Chinese medicine. The composition takes dendrobium flower and cicada fungus body as monarch and minister drugs, and combines seville orange flower, gordon europaea and crocodile flower as assistant and guide drugs. Wherein the composition obtained by compatibility of the invention contains 21-25% (w/w) of polysaccharide, 9-14% (w/w) of flavone and 0.2-0.7% (w/w) of alkaloid. Animal experiment results show that compared with the prescription (example 6) of the subtractive medicinal material extract with the same concentration and different components, the composition (example 2) obviously promotes the growth of beneficial bacteria bifidobacterium and lactobacillus, inhibits the propagation of harmful bacteria escherichia coli and enterococcus, has lower total amount of effective components and better beneficial bacteria effect, and has good effect of regulating intestinal flora. The content determination and animal test results of different formulations are shown in table 1.
TABLE 1 determination of the contents of different formulations and results of animal experiments
Note: comparison with normal control group: * P <0.01, P <0.05; comparison with model group: # P <0.01; # P <0.05.
2. The fruiting bodies of the dendrobium flowers and the cicada flowers in the formula are rich in polysaccharide components. The inventors have found that conventional aqueous extraction methods result in a large loss of polysaccharide component. The content of polysaccharides in the composition can be increased by hot water extraction or water extraction and alcohol precipitation, insoluble substances are removed, but the content of flavone and alkaloid components is changed, and the function of regulating intestinal flora is reduced. After multiple exploratory attempts by the inventor, the preparation process is improved, the cordyceps sobolifera fruiting body is crushed, then is mixed with the dendrobe flower, is presoaked for 6 hours, and is subjected to water bath extraction to prepare dendrobe flower and cordyceps sobolifera fruiting body extracts, and finally the composition extract powder is obtained.
The results of the content determination and animal tests for the different preparation methods are shown in table 2: the conventional process for increasing the polysaccharide content is adopted: the contents of the components of the composition obtained by microwave-assisted extraction and water extraction and alcohol precipitation are different from the contents of the components of the composition (example 2) obtained by the preparation method, wherein the contents of polysaccharide, flavone and alkaloid in the composition prepared by the microwave extraction method (example 4) are increased; the composition prepared by water extraction and alcohol precipitation (example 5) has a higher polysaccharide content and a lower alkaloid content than the composition of the present invention and example 4. Animal experiment results show that although the total amount of polysaccharide, flavone and alkaloid in the embodiment 4 and the embodiment 5 is higher than that of the composition prepared by the invention under the same formula, the composition has poor effect of regulating intestinal flora for promoting the growth of beneficial bacteria bifidobacterium and lactobacillus and inhibiting the propagation of harmful bacteria escherichia coli and enterococcus. Polysaccharide, flavone and alkaloid are taken as effective components of the composition, and have synergistic beneficial bacteria effect within a specific range of the invention.
TABLE 2 results of assay and animal test for different preparations
Note: comparison with normal control group: * P <0.01, P <0.05; comparison with model groups: # P <0.01; # P <0.05.
3. The components of the composition are all medicinal and edible or food-grade traditional Chinese medicines, are safe and effective, have simple and quick processing technology, are suitable for mass production, have high product quality and have better market application prospect. Moreover, the dendrobium flowers are used as raw materials, so that the dendrobium officinale flower resources are fully utilized, and resource waste is avoided.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
Example 1: composition 1 and preparation of health food thereof
The embodiment provides a composition for regulating intestinal flora function, which comprises the following raw materials: 30g of dendrobe flower, 25g of cordyceps sobolifera sporocarp, 10g of seville orange flower, 15g of sparrow's sinica flower and 20g of crocodile flower. Weighing and crushing cordyceps sobolifera sporophores, mixing dendrobium flowers with the crushed cordyceps sobolifera sporophores according to a material-liquid ratio of 1: adding water in a proportion of 10, pre-soaking for 4h, performing reflux extraction for 2h in a water bath at 100 ℃, extracting for 3 times, filtering, and combining filtrates to obtain an extracting solution 1; weighing the seville orange flower, the gorgon fruit and the crocodile flower, mixing and crushing, wherein the material-liquid ratio is 1:10 adding 50% ethanol, reflux extracting in 90 deg.C water bath for 2 hr for 3 times, filtering to obtain extractive solution 2, and concentrating under reduced pressure at 50 deg.C until no alcohol smell to obtain concentrated solution. Mixing the obtained extractive solution 1 and the concentrated solution, concentrating under reduced pressure at 50 deg.C, and drying to obtain composition 1 extract powder.
Sieving the extract powder of the composition 1 by a 60-mesh sieve, adding a proper amount of microcrystalline cellulose, granulating by using ethanol, drying at 70 ℃, finishing granules, adding a proper amount of magnesium stearate, uniformly mixing, and encapsulating or tabletting to obtain the capsule or tablet of the health food.
The composition proportion of the health food provided by the embodiment can also be prepared into various dosage forms in pharmaceutics, such as powder or granules and the like.
Example 2: composition 2 and preparation of health food thereof
The embodiment provides a composition for regulating intestinal flora function, which comprises the following raw materials: 35g of dendrobe flower, 20g of cordyceps sobolifera sporocarp, 15g of seville orange flower, 15g of gordon malachitum and 15g of alligator flower. Weighing and crushing cordyceps sobolifera sporophores, mixing dendrobium flowers with the crushed cordyceps sobolifera sporophores according to a material-liquid ratio of 1: adding water at a ratio of 20, pre-soaking for 6h, extracting under reflux in 100 deg.C water bath for 1.5h for 2 times, filtering, and mixing filtrates to obtain extractive solution 1; weighing the seville orange flower, the sparrow flower and the crocodile flower, mixing and crushing, and mixing according to a material-liquid ratio of 1:10 adding 60% ethanol, reflux-extracting in 80 deg.C water bath for 1.5 hr for 2 times, filtering, mixing filtrates to obtain extractive solution 2, and vacuum-concentrating at 60 deg.C until no alcohol smell exists to obtain concentrated solution. Mixing the obtained extractive solution 1 and the concentrated solution, concentrating at 70 deg.C under reduced pressure, and drying to obtain composition 2 extract powder.
And (3) sieving the extract powder of the composition 2 by a 60-mesh sieve, adding a proper amount of microcrystalline cellulose, granulating by using ethanol, drying at 60 ℃, finishing granules, adding a proper amount of magnesium stearate, uniformly mixing, and encapsulating or tabletting to obtain the capsule or tablet of the health food.
The composition proportion of the health food provided by the embodiment can also be prepared into various dosage forms in pharmaceutics, such as powder or granules and the like.
Example 3: composition 3 and preparation of health food thereof
The embodiment provides a composition for regulating intestinal flora function, which comprises the following raw materials: 40g of dendrobe flower, 15g of cordyceps sobolifera fruiting body, 20g of seville orange flower, 10g of sparrow's broom cypress flower and 15g of crocodile flower. Weighing and crushing cordyceps sobolifera sporophores, mixing dendrobium flowers with the crushed cordyceps sobolifera sporophores according to a material-liquid ratio of 1: adding water in a proportion of 40, pre-soaking for 8h, performing reflux extraction for 1h in a water bath at 100 ℃, extracting for 1 time, and filtering to obtain an extracting solution 1; weighing the seville orange flower, the sparrow flower and the crocodile flower, mixing and crushing, and mixing according to a material-liquid ratio of 1: adding 70% ethanol into 20, reflux extracting for 1 hr in 85 deg.C water bath, extracting for 1 time, filtering to obtain extractive solution 2, and concentrating under reduced pressure at 65 deg.C until no alcohol smell to obtain concentrated solution. Mixing the obtained extractive solution 1 and the concentrated solution, concentrating under reduced pressure at 65 deg.C, and drying to obtain composition 3 extract powder.
And (3) sieving the extract powder of the composition 3 by a 60-mesh sieve, adding a proper amount of microcrystalline cellulose, granulating by using ethanol, drying at 65 ℃, finishing granules, adding a proper amount of magnesium stearate, uniformly mixing, and encapsulating or tabletting to obtain the capsule or tablet of the health food.
The composition proportion of the health food provided by the embodiment can also be prepared into various dosage forms in pharmaceutics, such as powder or granules and the like.
Example 4: preparation of composition 4 by microwave extraction
The embodiment provides a composition for regulating intestinal flora function, which comprises the following raw materials: 35g of dendrobe flower, 20g of cordyceps sobolifera sporocarp, 15g of seville orange flower, 15g of sparrow's flabellate flower and 15g of crocodile flower. Mixing dendrobe flowers and crushed cordyceps sobolifera sporophores according to a material-liquid ratio of 1: adding water at a ratio of 30, pretreating with microwave power at middle fire for 8min, leaching at 80 deg.C for 1.5 hr for 2 times, filtering, and mixing filtrates to obtain extractive solution 1; weighing the seville orange flower, the sparrow flower and the crocodile flower, mixing and crushing, and mixing according to a material-liquid ratio of 1:10 adding 60% ethanol, reflux-extracting in 80 deg.C water bath for 1.5 hr for 2 times, filtering, mixing filtrates to obtain extractive solution 2, and vacuum-concentrating at 60 deg.C until no alcohol smell exists to obtain concentrated solution. Mixing the obtained extractive solution 1 and concentrated solution, concentrating at 70 deg.C under reduced pressure, and drying to obtain composition 4 extract powder.
Example 5: water extraction and alcohol precipitation method for preparing composition 5
The embodiment provides a composition for regulating intestinal flora function, which comprises the following raw materials: 35g of dendrobe flower, 20g of cordyceps sobolifera sporocarp, 15g of seville orange flower, 15g of gordon malachitum and 15g of alligator flower. Mixing dendrobe flowers and unbroken cordyceps sobolifera sporophores, and mixing the dendrobe flowers and the unbroken cordyceps sobolifera sporophores according to a material-liquid ratio of 1: adding water in a proportion of 30, stirring and extracting for 2h at 40 ℃, extracting for 2 times, mixing extracting solutions, concentrating under reduced pressure at 60 ℃ to 100mL, centrifuging for 10min at 10000r/min, taking supernate, precipitating with ethanol with the final volume fraction of 80% for 24h, centrifuging, taking alcohol precipitate, deproteinizing by a Sevag method, dialyzing (the molecular weight cutoff is 3500D), and freeze-drying to obtain an extract 1; weighing the seville orange flower, the gorgon fruit and the crocodile flower, mixing and crushing, wherein the material-liquid ratio is 1:10 adding 60% ethanol, reflux extracting in 80 deg.C water bath for 1.5 hr for 2 times, filtering, mixing filtrates to obtain extractive solution 2, and concentrating under reduced pressure at 60 deg.C until no alcohol smell exists to obtain concentrated solution. Mixing the obtained extractive solution 1 and the concentrated solution, concentrating at 70 deg.C under reduced pressure, and drying to obtain composition 5 extract powder.
Example 6: preparation of over-the-counter composition 6
The embodiment provides a composition for regulating intestinal flora function, which comprises the following raw materials: 50g of dendrobe flower, 30g of cordyceps sobolifera fruiting body, 10g of seville orange flower, 5g of sparrow's broom cypress flower and 5g of crocodile flower. Weighing and crushing cordyceps sobolifera sporophores, mixing dendrobium flowers with the crushed cordyceps sobolifera sporophores according to a material-liquid ratio of 1: adding water at a ratio of 20, pre-soaking for 6h, extracting under reflux in 100 deg.C water bath for 1.5h for 2 times, filtering, and mixing filtrates to obtain extractive solution 1; weighing the seville orange flower, the sparrow flower and the crocodile flower, mixing and crushing, and mixing according to a material-liquid ratio of 1:10 adding 60% ethanol, reflux-extracting in 80 deg.C water bath for 1.5 hr for 2 times, filtering, mixing filtrates to obtain extractive solution 2, and vacuum-concentrating at 60 deg.C until no alcohol smell exists to obtain concentrated solution. Mixing the obtained extractive solution 1 and the concentrated solution, concentrating at 70 deg.C under reduced pressure, and drying to obtain composition 6 extract powder.
Example 7: component determination
1. Determination of polysaccharide content
Preparation of polysaccharide control solution: taking a proper amount of anhydrous glucose reference substance, precisely weighing, and adding water to obtain a solution containing 90 μ g per 1 mL.
Preparation of a standard curve: precisely measuring reference substance solutions 0.2mL, 0.4mL, 0.6mL, 0.8mL and l.0mL, respectively placing in 10mL test tubes with plugs, respectively adding water to supplement to 1.0mL, precisely adding 5% phenol solution 1mL (prepared for clinical use), shaking, precisely adding sulfuric acid 5mL, shaking, heating in boiling water bath for 20min, taking out, placing in ice bath for cooling for 5min, measuring absorbance at wavelength of 488nm with corresponding reagent as blank, and drawing standard curve with absorbance as ordinate and concentration as abscissa.
Preparation of a test solution: weighing about 50mg of the extract powder of the embodiments 1-6 respectively, precisely weighing, placing in an eggplant-shaped bottle, precisely adding 25mL of water, weighing, heating and refluxing for 1h, cooling, weighing again, complementing the weight loss with water, shaking uniformly, filtering, precisely weighing 2mL of filtrate, placing in a 10mL centrifuge tube, precisely adding 8mL of absolute ethyl alcohol, shaking uniformly, refrigerating for 1h, taking out, centrifuging (the rotating speed is 4000 rpm) for 20min, discarding the supernatant, washing the precipitate with 80% ethanol for 2 times, 8mL each time, centrifuging, discarding the supernatant, dissolving the precipitate with hot water, transferring to a 25mL measuring bottle, cooling, adding water to the scale, and shaking uniformly to obtain the product.
The determination method comprises the following steps: precisely measuring 1mL of the test solution, placing the test solution in a 10mL test tube with a plug, measuring the absorbance according to the method under the preparation item of the standard curve, reading the amount of the anhydrous glucose in the test solution from the standard curve, and calculating to obtain the glucose sensor.
In addition, the extract liquid 1 of the dendrobium flower and cordyceps sobolifera sporophore in the step 1 and the concentrated liquid of the seville orange flower, the broom cypress flower and the crocodile flower in the step 2 are respectively concentrated at 70 ℃ under reduced pressure and dried to obtain extract powder of the dendrobium flower and cordyceps sobolifera sporophore and extract powder of the seville orange flower, the broom cypress flower and the crocodile flower. The solutions were prepared and their polysaccharide content was determined according to the method under the item for the preparation of the test solutions.
2. Measuring flavone content
Preparation of control solutions: accurately weighing 25mg of rutin control substance dried at 120 deg.C to constant weight, placing in 50mL measuring flask, adding appropriate amount of ethanol, ultrasonic treating to dissolve, cooling, adding ethanol to scale, and shaking. Precisely measuring 20mL, placing in a 50mL measuring flask, adding water to the scale, and shaking to obtain the final product (each 1mL contains anhydrous rutin 0.2 mg).
Preparation of a standard curve: precisely measuring reference substance solutions 1mL, 2mL, 3mL, 4mL, 5mL and 6mL, respectively placing in 25mL measuring bottles, adding water to 6mL, adding 5% sodium nitrite solution 1mL, shaking up, standing for 6min, adding 10% aluminum nitrate solution 1mL, shaking up, standing for 6min, adding sodium hydroxide test solution 10mL, adding water to scale, shaking up, standing for 15min, immediately irradiating by ultraviolet-visible spectrophotometry with corresponding reagents as blank, measuring absorbance at 500nm wavelength, and drawing a standard curve with absorbance as ordinate and concentration as abscissa.
Preparation of a test solution: respectively weighing 1g of the extract powder of the embodiments 1-6, precisely weighing, placing in a conical flask with a plug, weighing, precisely adding 50mL of diluted ethanol, carrying out ultrasonic treatment (power 200W and frequency 40 kHz) for 30min, cooling, weighing again, complementing the weight loss with the diluted ethanol, shaking up, and filtering. Accurately measuring 5mL of the subsequent filtrate, placing the subsequent filtrate in a 25mL measuring flask, adding water to dilute the subsequent filtrate to a scale, and shaking up the subsequent filtrate.
The determination method comprises the following steps: precisely measuring 2mL of the test solution, placing the test solution in a 25mL measuring flask, measuring the absorbance according to the method under the preparation item of the standard curve, reading the weight of rutin in the test solution from the standard curve, and calculating to obtain the rutin compound.
In addition, the extract liquid 1 of the fruiting bodies of the dendrobium flower and the cicada flower in the step 1 and the concentrated solution of the seville orange flower, the gordon europaea and the alligator flower in the step 2 are respectively concentrated under reduced pressure at 70 ℃ and dried to obtain extract powder of the dendrobium flower and the cordyceps sobolifera fruiting bodies and extract powder of the seville orange flower, the gordon europaea and the alligator flower. The solution was prepared and the flavone content was measured according to the method under the item for the preparation of the test solution.
3. Determination of the content of alkaloids
Preparation of alkaloid control solutions: an appropriate amount of atropine sulfate control dried at 120 deg.C to constant weight is weighed precisely, and water is added to make into solution containing hyoscyamine 75 μ g per 1mL (hyoscyamine weight = atropine sulfate weight × 0.8551).
Preparation of a test solution: respectively weighing 0.5g of the extract powder of the embodiments 1-6, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of citric acid-disodium hydrogen phosphate buffer solution (pH 4.0), shaking for 5min, standing overnight, filtering with dry filter paper, and taking the subsequent filtrate.
The determination method comprises the following steps: precisely measuring 2mL of each of the test solution and the reference solution, respectively placing the test solution and the reference solution in a separating funnel, precisely adding 10mL of citric acid-disodium hydrogen phosphate buffer solution (pH 4.0) into each of the test solution and the reference solution, precisely adding 2mL of 0.04% bromocresol green solution prepared by the buffer solutions, shaking up, extracting for 5min by shaking up 10mL of trichloromethane, after the solutions are completely layered, respectively taking trichloromethane solution, filtering the trichloromethane solution into a 25mL measuring flask by using filter paper wetted by trichloromethane, extracting for 3 times by using trichloromethane, extracting for 5mL each time, sequentially filtering into the measuring flask, washing the filter paper by using trichloromethane, filtering into the measuring flask, adding the trichloromethane to the scale, shaking up, respectively measuring the absorbance at the wavelength of 415nm by using an ultraviolet-visible spectrophotometry (generally 0401), and calculating to obtain the test solution.
In addition, the extract liquid 1 of the fruiting bodies of the dendrobium flower and the cicada flower in the step 1 and the concentrated solution of the seville orange flower, the gordon europaea and the alligator flower in the step 2 are respectively concentrated under reduced pressure at 70 ℃ and dried to obtain extract powder of the dendrobium flower and the cordyceps sobolifera fruiting bodies and extract powder of the seville orange flower, the gordon europaea and the alligator flower. The solutions were prepared and the alkaloid content was determined according to the method under the test solution preparation. The test results of the index content of the effective components are shown in the following table 3.
TABLE 3 determination of polysaccharide, flavone and alkaloid content in each group
| Group of | Polysaccharide content (%) | Content of flavone (%) | Alkaloid content (%) |
| Example 1 | 22.04±0.27 | 10.60±0.30 | 0.25±0.04 |
| Example 2 | 24.60±0.36 | 12.92±0.38 | 0.54±0.04 |
| Example 3 | 23.45±0.27 | 9.93±0.31 | 0.62±0.04 |
| Example 4 | 26.56±0.30 | 15.08±0.68 | 0.55±0.03 |
| Example 5 | 27.76±0.55 | 14.51±0.17 | 0.36±0.03 |
| Example 6 | 26.03±0.45 | 10.82±0.28 | 0.31±0.02 |
| Dendrobium flower and cicada fungus body | 36.81±0.53 | 6.71±0.32 | 0.10±0.01 |
| Seville orange flower, gorgon flower and crocodile flower | 4.42±0.34 | 16.13±0.15 | 0.76±0.04 |
Experimental results show that under the same formula, the composition prepared by the method for preparing the fruiting bodies of dendrobium flowers and cicada flowers by crushing and pre-soaking is different from the composition prepared by microwave-assisted extraction (example 4) and water extraction and alcohol precipitation (example 5). Wherein the contents of polysaccharides, flavones and alkaloids of the composition prepared by the microwave extraction method (example 4) are all increased compared with the invention; the composition prepared by the water extraction and alcohol precipitation method (example 5) has higher polysaccharide content and lower alkaloid content compared with the composition prepared by the invention and example 4. Under the condition of adopting a subtractive formula, namely a formula of dendrobium flower, cordyceps sobolifera sporocarp, seville orange flower, gorgonia leucocephala and alligator flower, the contents of polysaccharide, flavone and alkaloid are different. In the embodiment 2, the sum of the contents of the dendrobium flower, the cordyceps sobolifera sporophore and the seville orange flower, the alligator flower and the gordon malachite flower is equal to the content of each component in the extract powder of the composition in the embodiment 2. Wherein, the yields of the extract powders obtained in the two steps are different, so the component contents of the extract finished product of the composition in the embodiment 2 can be obtained without simply adding the component contents in the two steps.
Example 8: intestinal flora regulation test
The test results of the effects of the health foods prepared according to the above examples 1 to 6 on the function of regulating intestinal flora are as follows:
test animals: BALB/c mice, 6-8 weeks old, 20. + -.2 g body weight, and 110 mice each male and female.
The test method comprises the following steps: after the animals are adaptively raised for 1 week, 10 mice are randomly selected as normal control groups, the rest 100 mice are modeled, and lincomycin hydrochloride (300 mg/mL) is irrigated 2 times a day, 0.2mL/10g each time, and continuously for 3 days. After 3 days, the mice in the model group are listened, the weight is reduced, the anus is reddish, and diarrhea with different degrees appears, which indicates that the modeling is successful.
The model-built mice are divided into a model group, an example 1 group, an example 2 group, an example 3 group, an example 4 group, an example 5 group, an example 6 group, a dendrobe flower + cordyceps sobolifera sporophore group, a seville orange flower + broom cypress flower + crocodile flower group and a commercial brand control group, wherein 10 mice in each group are respectively irrigated with physiological saline, and the solution prepared from the extract powder obtained in examples 1-6, the dendrobe flower + cordyceps sobolifera sporophore extract powder and the seville orange flower + gorge orange flower extract powder. Weighing the extract powder obtained in the embodiments 1-6, dendrobium flower, cordyceps sobolifera sporophore extract powder, seville orange flower, sparrow's sinica and crocodile flower extract powder, dissolving with distilled water, preparing into solution with concentration of 2.5mg/mL respectively, and carrying out continuous intragastric administration for 1 time for 14 days, wherein the administration dose is 50mg/kg, and each time is 0.2mL/10 g. The normal control group and the model group were each administered with the same volume of physiological saline. The activity of the mice was observed daily.
After a mouse is dissected, aseptically collecting 0.1g of cecum content, placing the cecum content into a sterile 1.5mL centrifuge tube, adding 0.9mL of sterile normal saline, uniformly mixing the cecum content and the sterile normal saline on a vortex oscillator, performing l 0-time gradient serial dilution, selecting a proper concentration, coating 4 selective culture media, culturing and counting, and after bacterial colonies grow out, identifying the bacterial colonies by bacterial colony morphology, gram staining and biochemical reaction so as to determine the number of target bacterial colonies.
TABLE 4 bacterial culture Medium species and culture conditions
| Name of flora | Culture medium | Culture conditions |
| Bifidobacterium | TPY | Anaerobic, 37 +/-1 ℃ and 48h |
| Lactobacillus strain | LBS | Anaerobic reaction at 37 +/-1 ℃ for 48h |
| Enterobacter | EMB | Aerobic, 37 +/-1 ℃ and 24h |
| Enterococcus | EC | Aerobic at 37 +/-1 ℃ for 48h |
1. Observation of apparent characteristics of mice
The normal group mice had a normal diet and were active. After the lincomycin hydrochloride is infused for 3 days, the food intake of the mice is obviously reduced, the mice are cachectic, slow in movement, dilute in stool and diarrhea, and the phenomena of cold tail, lassitude and prickling are caused. After drenching treatment, the state of the mice is changed, and after 7 days of treatment, diarrhea symptoms are obviously improved, and most of the mice are recovered to be normal; after 14 days of treatment, the mice had good mental status, smooth and glossy hair.
2. Body weight changes in mice of each group
The results of the body weight of the mice in each group are shown in Table 5, and the body weight of the mice in example 2 and example 3 after treatment is not obviously different from that of the mice in a normal control group, namely the composition has no adverse effect on the body weight gain of the mice.
TABLE 5 Effect of groups on mouse body weight
Note: comparison with normal control group: * P <0.01, P <0.05; comparison with model groups: # P <0.01; # P <0.05.
3. Change in intestinal flora in groups of mice
The results of measuring the contents of lactobacillus, bifidobacterium, enterobacter and enterococcus in the cecal contents of the mice are shown in Table 6.
Table 6 variation of the number of intestinal flora (lgCFU/g) in the mice for each group.
| Group of | Bifidobacterium | Lactobacillus strain | Escherichia coli | Enterococcus |
| Normal control group | 9.09±0.43 | 8.90±0.80 | 6.74±0.76 | 6.86±0.54 |
| Model set | 8.52±0.44 ** | 7.87±0.90 * | 7.63±1.07 * | 7.70±0.95 * |
| Example 1 | 8.96±0.77 | 8.61±1.25 | 7.07±0.34 | 7.38±0.88 |
| Example 2 | 9.82±0.66 **## | 9.92±0.60 **## | 6.38±0.79 ## | 6.71±0.56 # |
| Example 3 | 9.65±0.70 *## | 9.75±0.82 *## | 6.65±0.55 # | 6.88±0.86 |
| Example 4 | 9.41±0.60 ## | 9.43±0.91 ## | 6.76±0.98 | 6.98±1.01 |
| Example 5 | 9.21±0.71 # | 8.89±0.95 # | 6.85±0.70 | 7.06±0.52 |
| Example 6 | 9.07±0.84 | 8.48±0.77 | 7.18±1.00 | 7.27±0.94 |
| Dendrobium flower and cicada fungus body | 8.94±0.92 | 8.78±0.93# | 7.01±0.54 | 7.10±1.24 |
| Seville orange flower, gorgon flower and crocodile flower | 8.87±0.93 | 8.59±0.75 | 7.33±0.79 | 7.23±1.09 |
| Positive control group | 9.66±0.63 *## | 9.72±0.51 *## | 7.07±0.62 | 6.86±0.88 |
Note: comparison with normal control group: * P <0.01, P <0.05; comparison with model group: # P <0.01; # P <0.05.
The results of the mouse intestinal bacteria culture are shown in table 6, and after 14d of the test object is given, the colony numbers of bifidobacterium and lactobacillus in examples 1 to 3 are obviously increased compared with those of a normal control group and a model group; the colony number of the escherichia coli and the enterococcus is not different from that of a normal control group, and is obviously reduced compared with a model group, and particularly, the effect of the composition obtained in the example 2 is better than that of a positive control group. The same formula as that in example 2 is adopted, and the microwave-assisted extraction example 4 of the dendrobe flower and cicada fungus fruiting bodies and the water extraction and alcohol precipitation example 5 of the dendrobe flower and cicada fungus fruiting bodies can promote the growth of beneficial bacteria and inhibit the propagation of harmful bacteria, but the effect is lower than that in example 2. The antibacterial effect obtained by adopting a formula (example 6) outside the protection range of the invention, namely dendrobium flower, cordyceps sobolifera sporophore, seville orange flower, gorgon canadensis flower and alligator flower, is obviously lower than that of the composition.
The composition defined by the invention can generate a synergistic effect, obviously promote the growth of beneficial bacteria bifidobacteria and lactobacilli, and obviously inhibit the propagation of harmful bacteria escherichia coli and enterococcus, so that the composition has a good function of regulating intestinal flora.
Claims (10)
1. The composition with the function of regulating intestinal flora is characterized by comprising the following raw materials: the content of the dendrobium flower is 30-40% (w/w), the content of the cordyceps sobolifera fruiting body is 15-25% (w/w), the content of the seville orange flower is 10-20% (w/w), the content of the gordon malachitum flower is 10-20% (w/w), the content of the crocodile flower is 10-20% (w/w), and the total amount of all the components is 100%.
2. The composition according to claim 1, characterized in that it consists of the following raw materials: the content of the dendrobium flower is 35% (w/w), the content of the cordyceps sobolifera fruiting body is 20% (w/w), the content of the seville orange flower is 15% (w/w), the content of the malachite flabellatus is 15% (w/w), and the content of the crocodile flower is 15% (w/w).
3. The composition of claim 1, wherein the dendrobium flowers are dendrobium officinale flowers.
4. The composition as claimed in claim 1, wherein the fruiting body of cicada fungus is artificially cultivated.
5. The composition according to claim 1, wherein the composition comprises the following medicinal ingredients: the polysaccharide content is 21-25% (w/w), the flavone content is 9-14% (w/w), and the alkaloid content is 0.2-0.7% (w/w).
6. A process for the preparation of a composition according to any one of claims 1 to 5, characterized in that it comprises the following specific steps:
step 1, crushing cordyceps sobolifera sporocarp, weighing dendrobium flower according to a formula, mixing with the cordyceps sobolifera sporocarp, and mixing according to a material-liquid ratio of 1: 10-1: adding water in a proportion of 40, pre-soaking for 4-8h, performing reflux extraction for 1-2 h in a water bath at 100 ℃, extracting for 1-3 times, filtering, and combining filtrates to obtain an extract 1;
step 2, replacing the flowers instead of the sparrow, the gorgon canary and the crocodile flowers according to the prescription amount, mixing and crushing according to the material-liquid ratio of 1:5 to 1:20 adding 50-70% volume concentration ethanol, refluxing and extracting for 1-2 h in water bath at 80-90 ℃, extracting for 1-3 times, filtering, combining filtrates to obtain an extracting solution 2, and concentrating under reduced pressure at 50-70 ℃ until no alcohol smell exists to obtain a concentrated solution;
and 3, combining the extracting solution 1 obtained in the step 1 and the concentrated solution obtained in the step 2, concentrating under reduced pressure at 50-70 ℃, drying and crushing to obtain the composition extract powder.
7. The preparation method according to claim 6, wherein the feed-liquid ratio in the step 1 is 1:20, pre-soaking for 6h, and extracting for 1.5h in 100 ℃ water bath under reflux for 2 times.
8. The preparation method according to claim 6, wherein the feed-liquid ratio in the step 2 is 1:10, adding 60 percent ethanol by volume, refluxing in water bath at 80 ℃ for 1.5h, extracting for 2 times to obtain an extracting solution 2, and concentrating under reduced pressure at 60 ℃ until no alcohol smell exists to obtain a concentrated solution.
9. Use of the composition according to any one of claims 1 to 5 for the preparation of an oral health food for regulating intestinal flora.
10. A health food comprising the composition of any one of claims 1 to 5, wherein the health food is composed of the composition and pharmaceutical excipients, and the health food is in the form of any one of tablets, capsules, solutions, suspensions, emulsions, oral liquids, syrups, granules, powders and powders.
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| CN105942511A (en) * | 2016-05-26 | 2016-09-21 | 惠州同富康生物科技有限公司 | Green-plum-contained health care food composition capable of regulating intestinal flora, and preparation method |
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