CN110448590B - Oriental millettia root extract with sexual function enhancing effect, and preparation method and application thereof - Google Patents
Oriental millettia root extract with sexual function enhancing effect, and preparation method and application thereof Download PDFInfo
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- CN110448590B CN110448590B CN201910898544.7A CN201910898544A CN110448590B CN 110448590 B CN110448590 B CN 110448590B CN 201910898544 A CN201910898544 A CN 201910898544A CN 110448590 B CN110448590 B CN 110448590B
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- millettia speciosa
- filtrate
- ethanol
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Classifications
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
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- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the field of traditional Chinese medicine extraction technology and application, and aims to disclose an extract with a sexual function enhancing effect. The material is a mixture formed by mixing the following raw materials in parts by weight: 3-5 parts of millettia speciosa champ polysaccharide, 1-3 parts of millettia speciosa champ total flavone and 1-3 parts of millettia speciosa champ alkaloid. The invention also discloses a preparation method of the millettia speciosa champ extract with the function of enhancing sexual function, and the millettia speciosa champ extract prepared by the method can extract the effective components of the millettia speciosa champ to the greatest extent. The invention also provides application of the extract.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine extraction technology and application, in particular to a beautiful millettia root extract with the function of enhancing sexual function; the invention also relates to a preparation method and application of the extract.
Background
Sexual dysfunction is caused by genetic, health condition, hormone level, age, diseases (including chronic diseases, neuropsychiatric diseases, endocrine diseases, and genital lesions), etc., and sexual dysfunction can occur in medicines, and in patients with excessive drinking or drug addiction for a long period of time. Male sexual dysfunction mainly includes sexual desire disorder, penile erection disorder, and sexual dysfunction. The existing commercial sexual function enhancing medicines are various, and the European and American countries mainly adopt synthetic medicines or hormones with strong effects, so that the medicines are easy to generate drug resistance after long-time administration, have large toxic and side effects and seriously damage the liver and kidney functions of patients; yang-strengthening medicines used in some countries in Asia, especially eastern Asia, are mostly natural traditional Chinese medicine preparations with certain curative effects, but the medicines are not suitable for long-term and large-scale administration. Therefore, the provision of a novel medicinal and edible product with sexual function enhancing effect has important significance.
The radix et rhizoma Rhei is dry root of herba Adianti Mimosoidis Millettia speciosa champ of genus Adianti of family Leguminosae, and mainly contains various chemical components such as alkaloid, polysaccharide and flavonoid. The millettia speciosa champ is taken as a famous medicine and food dual-purpose plant in the Ling nan area of China and two wide-area regional medicinal materials, has been recorded in Chinese medicinal materials Standard of Guangxi province and Chinese medicinal materials Standard of Guangdong province, is taken as a soup cooking raw material in folk, has the effects of nourishing and keeping health, is used for symptoms such as limb weakness and postpartum weakness, is also used as medicated diet and medicinal liquor, and has remarkable effects of strengthening yang and strengthening bones and muscles. The Chinese medicine also considers that the cattle has the advantages of sweet and neutral smell, and has the effects of strengthening tendons and activating collaterals, tonifying deficiency and moistening lung, and can be clinically used for treating lumbar muscle strain, cough due to lung heat, impotence, spermatorrhea and other diseases. Modern pharmacological researches have shown that the millettia speciosa champ has the effects of regulating immunity, resisting oxidation, resisting inflammation, resisting fatigue, resisting lung injury and the like. At present, no related research report on the function of the millettia speciosa champ extract in enhancing sexual function is found at home and abroad.
At present, the preparation process of the millettia speciosa champ extract mainly adopts a traditional water extraction or alcohol extraction method, for example, the invention patent with the patent publication number of CN106236825A discloses the millettia speciosa champ and the application of the millettia speciosa champ extract in antidepressant, and the preparation of the extract adopts a simple water reflux extraction method, so that the obtained millettia speciosa champ extract can be used for preparing antidepressant drugs; the invention patent with the publication number of CN103040924A discloses an extract of millettia speciosa and application thereof in preparing osteoporosis drugs, corresponding extracts are obtained by ethanol reflux extraction and organic solvent extraction, and the effect of improving osteoporosis of the extracts is remarkable; in addition, the invention patent with the publication number of CN105432898A discloses a millettia speciosa champ extract compound tea and a preparation method thereof, wherein the preparation process of the millettia speciosa champ extract comprises the processes of continuous countercurrent ultrasonic extraction, enzymolysis, ultrafiltration and the like, and the millettia speciosa champ extract has the problems of incomplete active ingredients, low concentration, low purity or more impurities and the like.
Disclosure of Invention
The invention aims to provide an extract of millettia speciosa champ with a sexual function enhancing effect. The invention also provides a preparation method of the extract, and the millettia speciosa champ extract prepared by the method can extract the effective components of millettia speciosa champ to the greatest extent. The invention also provides application of the extract.
The first technical scheme adopted by the invention is as follows:
a radix millettiae speciosae extract with sexual function enhancing effect is a mixture prepared by mixing the following raw materials in parts by weight: 3-5 parts of millettia speciosa champ polysaccharide, 1-3 parts of millettia speciosa champ total flavone and 1-3 parts of millettia speciosa champ alkaloid.
The second technical scheme adopted by the invention is as follows:
a preparation method of a radix et rhizoma Rhei extract with sexual function enhancing effect comprises the following preparation steps:
(1) Ultrasonic-assisted complex enzyme extraction: pulverizing radix Ardisiae Crispae, sieving to obtain coarse powder, adding water into coarse powder, stirring, adding complex enzyme for enzymolysis, treating the mixed solution in ultrasonic field, inactivating complex enzyme to obtain enzymolysis solution, cooling, and filtering to obtain residue A1 and filtrate A2;
(2) Alcohol precipitation: concentrating the filtrate A2 obtained in the step (1), adding ethanol, standing, centrifuging in a centrifuge, and retaining precipitate to obtain the millettia speciosa champ polysaccharide;
(3) Ethanol sectional extraction: adding acidic ethanol into the filter residue A1 obtained in the step (1), extracting, cooling, filtering to obtain a filter residue B1 and a filtrate B2, adding neutral ethanol into the filter residue B1, extracting, cooling, and filtering to obtain a filter residue C1 and a filtrate C2; adding alkaline ethanol into the filter residue C1, extracting, cooling, and filtering to obtain filter residue D1 and filtrate D2;
(4) Concentrating: mixing the filtrate B2, the filtrate C2 and the filtrate D2 obtained in the step (3), and concentrating to obtain a millettia speciosa champ concentrated solution;
(5) Cation exchange resin adsorption: filtering the concentrated solution obtained in the step (4), fully adsorbing by strong acid cation exchange resin, washing away water-soluble impurities, eluting, collecting eluent E, eluting by using ethanol containing ammonia water, collecting eluent F, concentrating and drying the eluent E to obtain total flavonoids of the radix millettiae speciosae, and concentrating and drying the eluent F to obtain alkaloids of the radix millettiae speciosae;
(6) Mixing: uniformly mixing the millettia speciosa champ polysaccharide obtained in the step (2) and the millettia speciosa champ total flavone and the millettia speciosa champ alkaloid obtained in the step (5) according to a proportion to obtain the millettia speciosa champ extract.
The third technical scheme adopted by the invention is as follows:
an application of the extract of beautiful millettia root with the function of enhancing sexual function is disclosed.
Compared with the prior art, the invention has the following beneficial effects:
the invention relates to a millettia speciosa champ extract with a sexual function enhancing effect, which is a mixture formed by mixing the following raw materials in parts by weight: 3-5 parts of millettia speciosa champ polysaccharide, 1-3 parts of millettia speciosa champ total flavone and 1-3 parts of millettia speciosa champ alkaloid. The millettia speciosa champ extract with the function of enhancing the sexual function is used for treating male sexual dysfunction, has obvious effect and enhances physical strength.
According to the preparation method of the millettia speciosa champ extract with the sexual function enhancing effect, the used composite enzyme components of the cellulase and xylanase can damage cell walls, so that the effective components in the cells can be fully dissolved out, in addition, cellulose and xylan in the cell walls can be degraded into oligosaccharide or glucose, and the polysaccharide yield is improved; proteases degrade proteins, facilitating removal of protein impurities. The method adopts acidic, neutral and alkaline ethanol solution to extract the radix et rhizoma Rhei powder in sections, and can ensure that the alkaline, neutral and acidic effective components in radix et rhizoma Rhei are extracted as far as possible. The ion exchange resin is used for adsorbing and separating flavone and alkaloid, so that the operation is easy, the separation efficiency is high, and the used reagent has no pollution to the environment.
Drawings
FIG. 1 is the effect of the Calotropis gigantea extract on the sexual behavior of mice in the comparative test of the present invention;
FIG. 2 is a graph showing the effect of the Calotropis gigantea extract on the weight bearing swimming time of mice in a comparative experiment according to the present invention.
Detailed Description
The technical scheme of the present invention will be described in further detail below with reference to the specific embodiments, but the present invention is not limited thereto.
A radix millettiae speciosae extract with sexual function enhancing effect is a mixture prepared by mixing the following raw materials in parts by weight: 3-5 parts of millettia speciosa champ polysaccharide, 1-3 parts of millettia speciosa champ total flavone and 1-3 parts of millettia speciosa champ alkaloid.
Wherein the material is a mixture composed of the following raw materials in parts by weight: 4 parts of millettia speciosa champ polysaccharide, 3 parts of millettia speciosa champ total flavone and 2 parts of millettia speciosa champ alkaloid.
A preparation method of a radix et rhizoma Rhei extract with sexual function enhancing effect comprises the following preparation steps:
(1) Ultrasonic-assisted complex enzyme extraction: pulverizing radix Ardisiae Crispae, sieving to obtain coarse powder, adding water into coarse powder, stirring, adding complex enzyme for enzymolysis, treating the mixed solution in ultrasonic field, inactivating complex enzyme to obtain enzymolysis solution, cooling, and filtering to obtain residue A1 and filtrate A2.
Wherein 8-16 times of water is added into the millettia speciosa coarse powder, the added complex enzyme comprises cellulase, xylanase and neutral protease, the addition amounts of the cellulase, the xylanase and the neutral protease are respectively 0.4-0.8%, 0.2-0.6% and 0.6-1.0% of the weight of the millettia speciosa coarse powder, the pH value of the complex enzyme is 5.5-7.0, the enzymolysis temperature is 45-60 ℃, the ultrasonic power is 200-350W, and the treatment time is 1-3 h; the enzymatic hydrolysate was cooled and then filtered through a 0.8 μm filter.
(2) Alcohol precipitation: concentrating the filtrate A2 obtained in the step (1), adding ethanol, standing, centrifuging in a centrifuge, and retaining precipitate to obtain the millettia speciosa champ polysaccharide.
Wherein, the filtrate A2 is concentrated to 1/4 of the original volume, ethanol with the concentration of 95 percent is added into the concentrated solution until the concentration of the final ethanol is 80 percent, and the mixture is slowly added and stirred at the same time, and is kept stand for 6 to 10 hours at the constant temperature of 3 to 6 ℃; the rotational speed of the centrifuge was 3000rpm and the centrifugation time was 20min.
(3) Ethanol sectional extraction: adding acidic ethanol into the filter residue A1 obtained in the step (1), extracting, cooling, filtering to obtain a filter residue B1 and a filtrate B2, adding neutral ethanol into the filter residue B1, extracting, cooling, and filtering to obtain a filter residue C1 and a filtrate C2; adding alkaline ethanol into the filter residue C1, extracting, cooling, and filtering to obtain filter residue D1 and filtrate D2.
Wherein, adding 4-16 times volume of ethanol with pH value of 1-6 and concentration of 50-90% into the filter residue A1, extracting at 70-80 ℃ for 0.5-2 h, cooling and filtering with a filter membrane with 0.8 μm; adding 4-16 times of ethanol with the volume, the pH value of 6-8 and the concentration of 50-90% into the filter residue B1, extracting at 70-80 ℃ for 0.5-2 h, cooling and filtering with a filter membrane with the thickness of 0.8 mu m; adding 4-16 times of ethanol with the volume, the pH value of 8-12 and the concentration of 50-90% into the filter residue C1, extracting at 70-80 ℃ for 0.5-2 h, cooling and filtering with a filter membrane with the thickness of 0.8 mu m; the pH value of the ethanol is regulated by adopting disodium hydrogen phosphate-citric acid buffer solution.
(4) Concentrating: mixing the filtrate B2, the filtrate C2 and the filtrate D2 obtained in the step (3), and concentrating to obtain the millettia speciosa champ concentrated solution. Wherein, the temperature of the mixed solution of the filtrate B2, the filtrate C2 and the filtrate D2 is 40-50 ℃ when the mixed solution is concentrated, and the steam pressure is less than 0.3MPa.
(5) Cation exchange resin adsorption: filtering the concentrated solution obtained in the step (4), fully adsorbing by strong acid cation exchange resin, washing away water-soluble impurities, eluting, collecting eluent E, eluting by using ethanol containing ammonia water, collecting eluent F, concentrating and drying the eluent E to obtain total flavonoids of the millettia speciosa champ, and concentrating and drying the eluent F to obtain alkaloids of the millettia speciosa champ.
Wherein, distilled water is used for washing water-soluble impurities, the flow rate is 4-6 Bv/h, ethanol with the weight of 5-10 times and the concentration of 50-90% is used for eluting, then eluent E is collected, the flow rate is 1-3 Bv/h, ammonia water with the concentration of 1-5% and ethanol with the concentration of 50-90% are used for eluting, eluent F is collected, the flow rate is 1-3 Bv/h, and the used strong acid cation exchange resin is 001X 7 type or D001 type strong acid cation exchange resin. The ion exchange resin adsorption method is used for replacing the traditional organic solvent extraction method to separate flavone and alkaloid, so that the problems of complex operation, low separation efficiency and large organic solvent consumption can be solved.
(6) Mixing: uniformly mixing the millettia speciosa champ polysaccharide obtained in the step (2) and the millettia speciosa champ total flavone and the millettia speciosa champ alkaloid obtained in the step (5) according to a proportion to obtain the millettia speciosa champ extract. The mixing proportion of the millettia speciosa champ polysaccharide, the millettia speciosa champ total flavone and the millettia speciosa champ alkaloid is 3-5: 1 to 3:1 to 3.
According to the preparation method of the millettia speciosa champ extract with the sexual function enhancing effect, the used composite enzyme components of the cellulase and xylanase can damage cell walls, so that the effective components in the cells can be fully dissolved out, in addition, cellulose and xylan in the cell walls can be degraded into oligosaccharide or glucose, and the polysaccharide yield is improved; proteases degrade proteins, facilitating removal of protein impurities. The method adopts acidic, neutral and alkaline ethanol solution to extract the radix et rhizoma Rhei powder in sections, and can ensure that the alkaline, neutral and acidic effective components in radix et rhizoma Rhei are extracted as far as possible. The ion exchange resin is used for adsorbing and separating flavone and alkaloid, so that the operation is easy, the separation efficiency is high, and the used reagent has no pollution to the environment.
An application of the extract of beautiful millettia root with the function of enhancing sexual function is that the extract of the beautiful millettia root is applied to the medicine for enhancing sexual function. The radix millettiae speciosae extract is tablets, capsules or granules. The invention discloses the functions of strengthening yang, nourishing kidney, tonifying deficiency, strengthening tendons and activating collaterals and the like of the beautiful millettia root, and the related research or application of the beautiful millettia root in the functions is not found at present. The invention aims to solve the technical problem of providing the millettia speciosa champ extract for enhancing the sexual function and relieving physical fatigue, which has the effects of remarkably improving the sexual capacity and relieving the physical fatigue, has the advantages of no toxic or side effect, convenient administration, easiness in popularization and application and the like, can be used for treating male sexual dysfunction, has obvious effect and enhances the physical strength, and provides a potential medicament for enhancing the sexual function.
Example 1
(1) Ultrasonic-assisted complex enzyme extraction: crushing and sieving radix millettiae speciosae to obtain radix millettiae speciosae coarse powder, adding 12 times of water into the radix millettiae speciosae coarse powder, stirring uniformly, adding compound enzyme for enzymolysis, placing the mixed liquor in an ultrasonic field for treatment, wherein the added compound enzyme comprises cellulase, xylanase and neutral protease, the addition amount of the cellulase, the xylanase and the neutral protease is 0.8%, 0.4% and 1.0% of the mass of the radix millettiae speciosae coarse powder in sequence, the pH value of the compound enzyme is 6.0, the enzymolysis temperature is 50 ℃, the ultrasonic power is 350W, the treatment time is 2 hours, inactivating the compound enzyme after the treatment is finished to obtain enzymolysis liquor, and filtering the enzymolysis liquor by a 0.8 mu m filter membrane after cooling to obtain filter residues A1 and filtrate A2.
(2) Alcohol precipitation: concentrating the filtrate A2 obtained in the step (1) to 1/4 of the original volume, adding 95% ethanol to the concentrated solution until the concentration of the final ethanol is 80%, slowly adding, stirring while keeping the temperature at 4 ℃ for 10 hours, centrifuging at 3000rpm for 20 minutes in a centrifuge, and retaining precipitate to obtain the millettia speciosa champ polysaccharide.
(3) Ethanol sectional extraction: adding 10 times of ethanol with the volume of 4 and the pH value and the concentration of 70% into the filter residue A1 obtained in the step (1) as an acidic solvent for extraction, wherein the extraction temperature is 75 ℃, the extraction time is 1.5h, and filtering the mixture with a 0.8 mu m filter membrane after cooling to obtain filter residue B1 and filtrate B2. Adding 10 times of ethanol with pH of 7 and concentration of 70% into the residue B1 as neutral solvent, extracting at 75deg.C for 1.5 hr, cooling, and filtering with 0.8 μm filter membrane to obtain residue C1 and filtrate C2. Adding 10 times volume of ethanol with pH value of 10 and concentration of 70% into the filter residue C1 as alkaline solvent, extracting at 75deg.C for 1.5 hr, cooling, and filtering with 0.8 μm filter membrane to obtain filter residue D1 and filtrate D2. The pH value of the acidic, neutral and alkaline ethanol solution is regulated by adopting disodium hydrogen phosphate-citric acid buffer solution.
(4) Concentrating: uniformly mixing the filtrate B2, the filtrate C2 and the filtrate D2 obtained in the step (3), and concentrating to 1/5 of the original volume at the temperature of 45 ℃ and the steam pressure of less than 0.3MPa to obtain the radix millettiae speciosae concentrated solution.
(5) Cation exchange resin adsorption: filtering the concentrated solution obtained in the step (4), fully adsorbing by using strong acid cation exchange resin, washing water-soluble impurities with distilled water at a flow rate of 5Bv/h, eluting with ethanol with a concentration of 80% by weight of 8 times at a flow rate of 2Bv/h, collecting the eluent E, eluting with ammonia water with a concentration of 3% and ethanol with a concentration of 80% at a flow rate of 2Bv/h, collecting the eluent F, concentrating and drying the eluent E under a reduced pressure state to obtain total flavonoids of the radix millettiae speciosae, and concentrating and drying the eluent F under a reduced pressure state to obtain alkaloids of the radix millettiae speciosae. The strongly acidic cation exchange resin used was a 001×7 strongly acidic cation exchange resin.
(6) Mixing: uniformly mixing the millettia speciosa champ polysaccharide obtained in the step (2) and the millettia speciosa champ total flavone and the millettia speciosa champ alkaloid obtained in the step (5) according to a proportion to obtain the millettia speciosa champ extract. The mixing ratio of the millettia speciosa champ polysaccharide, the millettia speciosa champ total flavone and the millettia speciosa champ alkaloid is 4:3:2.
example 2
(1) Ultrasonic-assisted complex enzyme extraction: crushing and sieving radix millettiae speciosae to obtain radix millettiae speciosae coarse powder, adding 8 times of water into the radix millettiae speciosae coarse powder, stirring uniformly, adding compound enzyme for enzymolysis, placing the mixed liquor in an ultrasonic field for treatment, wherein the added compound enzyme comprises cellulase, xylanase and neutral protease, the addition amount of the cellulase, the xylanase and the neutral protease is 0.4%, 0.6% and 0.6% of the mass of the radix millettiae speciosae coarse powder in sequence, the pH value of the compound enzyme is 6.5, the enzymolysis temperature is 45 ℃, the ultrasonic power is 200W, the treatment time is 1h, inactivating the compound enzyme after the treatment is finished to obtain enzymolysis liquid, and filtering the enzymolysis liquid by a 0.8 mu m filter membrane after cooling to obtain filter residues A1 and filtrate A2.
(2) Alcohol precipitation: concentrating the filtrate A2 obtained in the step (1) to 1/4 of the original volume, adding 95% ethanol to the concentrated solution until the concentration of the final ethanol is 80%, slowly adding, stirring while keeping the temperature at 3 ℃ for 6 hours, centrifuging at 3000rpm for 20 minutes in a centrifuge, and retaining precipitate to obtain the millettia speciosa champ polysaccharide.
(3) Ethanol sectional extraction: adding 4 times of ethanol with the volume of 1, the pH value of 1 and the concentration of 50% into the filter residue A1 obtained in the step (1) as an acidic solvent for extraction, wherein the extraction temperature is 70 ℃, the extraction time is 0.5h, and filtering the mixture with a 0.8 mu m filter membrane after cooling to obtain filter residue B1 and filtrate B2. Adding 4 times of ethanol with pH value of 6 and concentration of 50% into the filter residue B1 as neutral solvent, extracting at 70deg.C for 0.5 hr, cooling, and filtering with 0.8 μm filter membrane to obtain filter residue C1 and filtrate C2. Adding ethanol with volume of 4 times, pH value of 8 and concentration of 50% into the filter residue C1 as alkaline solvent, extracting at 70deg.C for 0.5 hr, cooling, and filtering with 0.8 μm filter membrane to obtain filter residue D1 and filtrate D2. The pH value of the acidic, neutral and alkaline ethanol solution is regulated by adopting disodium hydrogen phosphate-citric acid buffer solution.
(4) Concentrating: concentrating the filtrate B2, the filtrate C2 and the filtrate D2 obtained in the step (3) to 1/4 of the original volume at the temperature of 45 ℃ and the steam pressure of less than 0.3MPa to obtain the millettia speciosa champ concentrated solution.
(5) Cation exchange resin adsorption: filtering the concentrated solution obtained in the step (4), fully adsorbing by using strong acid cation exchange resin, washing water-soluble impurities with distilled water at a flow rate of 4Bv/h, eluting with ethanol with a concentration of 50% by weight of 5 times at a flow rate of 1Bv/h, collecting a liquid-removed solution E, eluting with ammonia water with a concentration of 1% and ethanol with a concentration of 50% at a flow rate of 1Bv/h, collecting an eluent F, concentrating and drying the eluent E under a reduced pressure state to obtain total flavonoids of the radix millettiae, and concentrating and drying the eluent F under a reduced pressure state to obtain alkaloids of the radix millettiae. The strongly acidic cation exchange resin used was the D001 type strongly acidic cation exchange resin.
(6) Mixing: uniformly mixing the millettia speciosa champ polysaccharide obtained in the step (2) and the millettia speciosa champ total flavone and the millettia speciosa champ alkaloid obtained in the step (5) according to a proportion to obtain the millettia speciosa champ extract. The mixing ratio of the millettia speciosa champ polysaccharide, the millettia speciosa champ total flavone and the millettia speciosa champ alkaloid is 5:1:1.
example 3
(1) Ultrasonic-assisted complex enzyme extraction: crushing and sieving radix millettiae speciosae to obtain radix millettiae speciosae coarse powder, adding 16 times of water into the radix millettiae speciosae coarse powder, stirring uniformly, adding compound enzyme for enzymolysis, placing the mixed liquor in an ultrasonic field for treatment, wherein the added compound enzyme comprises cellulase, xylanase and neutral protease, the addition amount of the cellulase, the xylanase and the neutral protease is 0.6%, 0.2% and 0.8% of the mass of the radix millettiae speciosae coarse powder in sequence, the pH value of the compound enzyme is 7.0, the enzymolysis temperature is 60 ℃, the ultrasonic power is 300W, the treatment time is 3 hours, inactivating the compound enzyme after the treatment is finished to obtain enzymolysis liquor, and filtering the enzymolysis liquor by a 0.8 mu m filter membrane after cooling to obtain filter residues A1 and filtrate A2.
(2) Alcohol precipitation: concentrating the filtrate A2 obtained in the step (1) to 1/4 of the original volume, adding 95% ethanol to the concentrated solution until the concentration of the final ethanol is 80%, slowly adding, stirring while keeping the temperature at 6 ℃ for 8 hours, and centrifuging at 3000rpm for 20 minutes in a centrifuge to obtain the millettia speciosa champ polysaccharide.
(3) Ethanol sectional extraction: adding 16 times of ethanol with the volume, the pH value of 6 and the concentration of 90% into the filter residue A1 obtained in the step (1) to be extracted as an acidic solvent, wherein the extraction temperature is 80 ℃, the extraction time is 2 hours, and filtering the mixture by using a 0.8 mu m filter membrane after cooling to obtain filter residue B1 and filtrate B2. Adding 16 times of ethanol with pH value of 8 and concentration of 90% into the filter residue B1 as neutral solvent, extracting at 80deg.C for 2 hr, cooling, and filtering with 0.8 μm filter membrane to obtain filter residue C1 and filtrate C2. Adding ethanol with volume of 16 times, pH value of 12 and concentration of 90% into the filter residue C1 as alkaline solvent, extracting at 80deg.C for 2 hr, cooling, and filtering with 0.8 μm filter membrane to obtain filter residue D1 and filtrate D2. The pH value of the acidic, neutral and alkaline ethanol solution is regulated by adopting disodium hydrogen phosphate-citric acid buffer solution.
(4) Concentrating: uniformly mixing the filtrate B2, the filtrate C2 and the filtrate D2 obtained in the step (3), and concentrating to 1/6 of the original volume at the temperature of 45 ℃ and the steam pressure of less than 0.3MPa to obtain the radix millettiae speciosae concentrated solution.
(5) Cation exchange resin adsorption: filtering the concentrated solution obtained in the step (4), fully adsorbing by using strong acid cation exchange resin, washing water-soluble impurities with distilled water at a flow rate of 6Bv/h, eluting with ethanol with a concentration of 95% by weight of 10 times at a flow rate of 3Bv/h, collecting the eluent E, eluting with ammonia water with a concentration of 5% of ammonia water and ethanol with a concentration of 95% at a flow rate of 3Bv/h, collecting the eluent F, concentrating and drying the eluent E under a reduced pressure state to obtain total flavonoids of the radix millettiae, and concentrating and drying the eluent F under a reduced pressure state to obtain alkaloids of the radix millettiae. The strongly acidic cation exchange resin used was a 001×7 strongly acidic cation exchange resin.
(6) Mixing: uniformly mixing the millettia speciosa champ polysaccharide obtained in the step (2) and the millettia speciosa champ total flavone and the millettia speciosa champ alkaloid obtained in the step (5) according to a proportion to obtain the millettia speciosa champ extract. The mixing ratio of the millettia speciosa champ polysaccharide, the millettia speciosa champ total flavone and the millettia speciosa champ alkaloid is 3:2:3.
comparative test
The following experiments were performed using the millettia speciosa champ extracts of examples 1, 2, 3:
1 study method
1.1 grouping and administration
100 ICR pure line male mice with the weight of (25+/-2) g are selected, and after 2 days of adaptive breeding, the ICR pure line male mice are randomly divided into five groups of blank control group, positive control group and beautiful millettia root extract 1, 2 and 3 groups, and 20 groups each. The positive control group mice are filled with the capsule of the yang-leading rope every day, the Sichuan province is produced by the pharmaceutical company of the Jilin, inc. (national standard Z20080094), the main production medicinal materials are epimedium herb and schisandra chinensis, the capsule of the yang-leading rope is removed from the capsule shell, the medicinal powder is dissolved by a proper amount of physiological saline, the administration dosage is 4g/kg, the cow-horn-leaf extract obtained in the embodiment 1 of the invention is filled with the cow-leaf extract 1 group, the cow-leaf extract obtained in the embodiment 2 of the invention is filled with the cow-leaf extract 2 group, the cow-leaf extract 3 group is filled with the cow-leaf extract obtained in the embodiment 3 of the invention, the administration dosage is 4g/kg, and the blank control group is filled with the physiological saline of equal volume at the same time. The administration is carried out once a day, the gastric lavage amount is 0.2ml/10g, and the administration is carried out continuously for 30 days.
1.2 mating test
After 1h of last dose, 10 mice were randomly selected for sexual function related test in each group, the test was performed in a low light, quiet environment with an observation time of 20: 00-23: female mice were injected subcutaneously with 5 μg estradiol benzoate 48h prior to the test and 5 μg progesterone to induce estrus 4h prior to the test. When the mating experiment is carried out, 1 male mouse is put into a mating box (30 cm multiplied by 15 cm), and 1 female mouse entering estrus is put into the mating box immediately after 5min of adaptation. The time of occurrence of the first ride from the cage (i.e., the ride-on latency), the time of occurrence of the first mating from the cage (i.e., the mating latency), and the number of rides and mating occurred within 20 minutes were recorded.
1.3 detection of organ index and Biochemical index related to sexual function
The eyes are removed, the eyes are immediately killed after blood collection, organs such as testis, epididymis, prostate, seminal vesicle gland and prepuce gland are separated, the organs are weighed and recorded by an analytical balance, and corresponding indexes (the weight of the organs is mg/the body weight of a mouse is 10 g) are calculated; taking epididymis, shearing and repeatedly washing with PBS buffer solution to enable sperms to be fully dissociated, incubating for 30min in a 37 ℃ water bath, sucking 20 μl of sperm suspension, adding into 380 μl of PBS, shaking uniformly, and counting the number of sperms in the epididymis suspension by a white blood cell counting method under a microscope; stripping the cavernous body at two sides of the penis of the mouse, adding tissue lysate for full cleavage, and measuring contents of cyclic guanosine monophosphate (cGMP) and phosphodiesterase 5 (PDE 5) in the cavernous body; the serum testosterone and Nitric Oxide (NO) levels are measured, and the related operation steps are all carried out according to the instruction of the kit.
1.4 anti-fatigue test
After the last administration for 2 hours, 10 mice are randomly selected for anti-fatigue test, and lead blocks which are 5% of the body mass are placed at the tail of each mouse, the mice are placed in a swimming groove for swimming, the water depth is 40cm, the water temperature is 25+/-1 ℃, and the time from the beginning of swimming to the time when the heads of the mice enter water for 8 seconds and cannot swim out of the water surface is recorded. The mice with load swimming to exhaustion are routinely raised for 2 days without drug administration, after the water temperature (25+/-1) DEG C and no load swimming for 30min, the mice are immediately sacrificed after eyeball blood collection, thigh muscle tissues are taken, and the thigh muscle tissues are washed with normal saline, homogenized and centrifuged to determine the activity of succinate dehydrogenase in the muscle tissues; the urea nitrogen content and lactic dehydrogenase activity in serum are measured, and the related operation steps are carried out according to the instruction of the kit.
2 results of the study
2.1 Calotropic extract to improve sexual behavior
The riding frequency and mating frequency of the blank control group, the positive control group and the millettia speciosa champ extracts 1, 2 and 3 groups are statistically analyzed to obtain the figure 1. As can be seen from fig. 1, compared with the blank control group, the positive control group, the extract groups have significantly increased riding frequency and mating frequency, and the differences have statistical significance (P < 0.05); the riding latency of the positive control group and the extract groups is obviously shorter than that of the blank control group (P < 0.05); the extract showed no significant change in the number of rides, mating number, ride latency, mating latency (P > 0.05) for each group compared to the positive control group. The results show that the radix millettiae speciosae extract can obviously improve the sexual behavior of mice, increase mating times, shorten mating latency, and has the effect equivalent to that of the Yingyang cable capsule.
2.2 Effect of radix Millettiae Dillenii extract on organ index of sexual organ
The testis index, epididymal index, prostate-seminal vesicle index, prepuce index of the sexual organs of mice in the blank control group, positive control group, and groups 1, 2, and 3 of the Millipore extracts were analyzed and counted as follows:
TABLE 1 influence of Millettia speciosa champ extract on the organ index of the mouse (mg/10 g, n=10)
| Group of | Testis | Epididymis type epididymis | Prostate + seminal vesicle gland | Prepuce gland |
| Blank control group | 58.3±2.9 | 11.6±0.8 | 84.2±5.3 | 27.4±1.5 |
| Positive control group | 59.6±3.1 | 10.7±0.9 | 83.7±4.9 | 27.6±1.2 |
| Extract 1 group | 57.1±3.2 | 11.8±0.7 | 85.1±4.8 | 27.1±1.7 |
| Extracts 2 groups | 58.8±2.7 | 11.3±0.9 | 84.6±5.1 | 26.8±1.4 |
| Extracts 3 groups | 57.4±3.5 | 10.9±0.8 | 84.9±4.6 | 27.5±1.6 |
As is clear from Table 1, the testis index, epididymal index, prostate-seminal vesicle index, and prepuce gland index of each of the positive control group and extract group did not significantly change (P > 0.05) as compared with the blank control group. The results show that the millettia speciosa champ extract has no obvious influence on the organ index of the sexual organs.
2.3 action of the Calf extract to improve penile erection and related mechanisms
Testosterone secreted by testicular interstitial cells can promote synthesis of nitric oxide synthase (Nitric Oxide Synthase, NOS) through expression of genes related to regulatory sexual function, thereby catalyzing conversion of L-arginine to NO, and NO can reduce hydrolysis of cGMP in vivo through inhibition of PDE5 production, promotion of guanylate cyclase (Guanosine Cyclase, GC) synthesis, relaxation of arterioles of penis and cavernous smooth muscle, rapid blood pumping into cavernous sinuses to cause erection, and indirect reflection of erection ability through measurement of key factor content thereof. The serum testosterone, NO levels and epididymal sperm numbers of mice in the blank control group, positive control group, and groups 1, 2, and 3 of the millettia speciosa extract were statistically analyzed as follows:
table 2 effect of the beautiful millettia root extract on serum testosterone, NO level and epididymal sperm count of mice (n=10)
Note that: p <0.05 compared to the blank.
As can be seen from table 2, the serum testosterone, NO levels and epididymal sperm numbers of the positive control group, the extract groups were significantly increased, and the differences were statistically significant (P < 0.05) compared to the blank control group; the extract showed NO significant changes in serum testosterone, NO content and epididymal sperm count (P > 0.05) for each group compared to the positive control group. The results show that the radix millettiae speciosae extract can obviously enhance the sexual function of mice, increase the number of sperms and has the effect equivalent to that of the capsule of the catgut.
The cavernous cGMP and PDE5 content of mice in the blank, positive control, and millettia speciosa extracts 1, 2, 3 groups were statistically analyzed as follows:
table 3 effect of the millettia speciosa champ extract on the mouse cavernous cGMP and PDE5 content (n=10)
| Group of | cGMP(nmol/L) | PGE5(pg/mL) |
| Blank control group | 25.3±2.1 | 341.6±25.7 |
| Positive control group | 32.1±2.7* | 258.1±19.4* |
| Extract 1 group | 33.0±3.2* | 237.2±17.3* |
| Extracts 2 groups | 31.5±2.9* | 265.4±20.1* |
| Extracts 3 groups | 32.6±2.4* | 252.8±21.6* |
Note that: p <0.05 compared to the blank.
As can be seen from table 3, compared with the blank control group, cGMP content of the positive control group and the extract group was significantly increased, and the difference was statistically significant (P < 0.05); the PGE5 content of the positive control group and the extract group is significantly lower than that of the blank control group (P < 0.05); compared with the positive control group, the cGMP and PGE5 levels of each extract group were not significantly changed (P > 0.05). The results show that the radix millettiae speciosae extract can enhance the penile erection function of mice to a certain extent, and the effect is equivalent to that of the yang-guiding capsule.
2.4 anti-fatigue action and mechanism of the Calotropis gigantea extract
Urea nitrogen is a metabolic product generated by protein decomposition due to insufficient metabolism energy of sugar and fat in the organism, and serum urea nitrogen level is increased when the organism is tired. Long-term endurance exercise may cause damage to muscle cell membranes and increase in permeability, so that lactate dehydrogenase permeates into blood, and the activity of lactate dehydrogenase in blood is greatly increased, and the stronger the activity of lactate dehydrogenase, the more serious the damage to muscle tissues. Succinate dehydrogenase is a key enzyme in the aerobic metabolism of tricarboxylic acid cycle, the improvement of the aerobic metabolism ability of muscles is a key for enhancing the fatigue resistance of organisms, and the aerobic metabolism ability of muscles can be reflected by detecting the activity of the aerobic metabolism related enzyme. The time of the mice in the blank control group, the positive control group, the millettia speciosa extract 1, 2 and 3 groups were subjected to load swimming, the serum urea nitrogen content after the load swimming, the lactate dehydrogenase activity and the muscle succinate dehydrogenase activity index were subjected to statistical analysis, and fig. 2 and table 4 were obtained, respectively.
Table 4 effect of the extract of millettia speciosa on serum urea nitrogen content, lactate dehydrogenase activity and muscle succinate dehydrogenase activity after mouse load swimming (n=10)
Note that: p <0.05 compared to the blank.
As can be seen from fig. 2 and table 4, compared with the blank control group, the positive control group and the extract groups have significantly prolonged swimming time under load, reduced serum urea nitrogen content, reduced serum lactate dehydrogenase activity, and enhanced muscle succinate dehydrogenase activity, and the difference comparison has statistical significance (P < 0.05); compared with the positive control group, the extract has no obvious change of the weight-bearing swimming time, serum urea nitrogen content, serum lactate dehydrogenase activity and muscle succinate dehydrogenase activity (P > 0.05). The results show that the radix millettiae speciosae extract can prolong the load swimming time of mice, strengthen the anti-fatigue capability of the mice, and has the effect equivalent to that of the yang-guiding capsule.
Conclusion of the study
The millettia speciosa champ extract obtained by the invention can increase the serum testosterone and NO content, the cavernous cGMP level, the cavernous PDE5 level and the sperm quantity of a male mouse, shorten the riding latency period and mating latency period of the mouse, increase the riding frequency and mating frequency within 20min, prolong the load swimming time of the mouse, reduce the serum urea nitrogen content, weaken the serum lactate dehydrogenase activity and strengthen the muscle succinate dehydrogenase activity. The results show that the radix et rhizoma Rhei extract can improve sexual behavior, enhance penile erection and fatigue resistance, and the above effects are equivalent to those of the kidney-invigorating yang-strengthening medicine capsule.
The foregoing description is only of the preferred embodiments of the invention, and it is therefore intended that all modifications, equivalents, and improvements made within the spirit and scope of the principles of the invention.
Claims (9)
1. The millettia speciosa champ extract with the sexual function enhancing effect is characterized by being a mixture formed by mixing the following raw materials in parts by weight: 3-5 parts of millettia speciosa champ polysaccharide, 1-3 parts of millettia speciosa champ total flavone and 1-3 parts of millettia speciosa champ alkaloid;
the preparation method of the beautiful millettia root extract comprises the following preparation steps:
(1) Ultrasonic-assisted complex enzyme extraction: pulverizing radix Ardisiae Crispae, sieving to obtain coarse powder, adding water into coarse powder, stirring, adding complex enzyme for enzymolysis, treating the mixed solution in ultrasonic field, inactivating complex enzyme to obtain enzymolysis solution, cooling, and filtering to obtain residue A1 and filtrate A2;
(2) Alcohol precipitation: concentrating the filtrate A2 obtained in the step (1), adding ethanol, standing, centrifuging in a centrifuge, and retaining precipitate to obtain the millettia speciosa champ polysaccharide;
(3) Ethanol sectional extraction: adding acidic ethanol into the filter residue A1 obtained in the step (1), extracting, cooling, filtering to obtain a filter residue B1 and a filtrate B2, adding neutral ethanol into the filter residue B1, extracting, cooling, and filtering to obtain a filter residue C1 and a filtrate C2; adding alkaline ethanol into the filter residue C1, extracting, cooling, and filtering to obtain filter residue D1 and filtrate D2;
(4) Concentrating: mixing the filtrate B2, the filtrate C2 and the filtrate D2 obtained in the step (3), and concentrating to obtain a millettia speciosa champ concentrated solution;
(5) Cation exchange resin adsorption: filtering the concentrated solution obtained in the step (4), fully adsorbing by strong acid cation exchange resin, washing away water-soluble impurities, eluting, collecting eluent E, eluting by using ethanol containing ammonia water, collecting eluent F, concentrating and drying the eluent E to obtain total flavonoids of the radix millettiae speciosae, and concentrating and drying the eluent F to obtain alkaloids of the radix millettiae speciosae;
(6) Mixing: uniformly mixing the millettia speciosa champ polysaccharide obtained in the step (2) and the millettia speciosa champ total flavone and the millettia speciosa champ alkaloid obtained in the step (5) according to a proportion to obtain the millettia speciosa champ extract.
2. The millettia speciosa champ extract with sexual function enhancing effect according to claim 1, which is characterized by being a mixture composed of the following raw materials in parts by weight: 4 parts of millettia speciosa champ polysaccharide, 3 parts of millettia speciosa champ total flavone and 2 parts of millettia speciosa champ alkaloid.
3. A method for preparing an extract of millettia speciosa having sexual function enhancing effect according to claim 1 or 2, comprising the steps of:
(1) Ultrasonic-assisted complex enzyme extraction: pulverizing radix Ardisiae Crispae, sieving to obtain coarse powder, adding water into coarse powder, stirring, adding complex enzyme for enzymolysis, treating the mixed solution in ultrasonic field, inactivating complex enzyme to obtain enzymolysis solution, cooling, and filtering to obtain residue A1 and filtrate A2;
(2) Alcohol precipitation: concentrating the filtrate A2 obtained in the step (1), adding ethanol, standing, centrifuging in a centrifuge, and retaining precipitate to obtain the millettia speciosa champ polysaccharide;
(3) Ethanol sectional extraction: adding acidic ethanol into the filter residue A1 obtained in the step (1), extracting, cooling, filtering to obtain a filter residue B1 and a filtrate B2, adding neutral ethanol into the filter residue B1, extracting, cooling, and filtering to obtain a filter residue C1 and a filtrate C2; adding alkaline ethanol into the filter residue C1, extracting, cooling, and filtering to obtain filter residue D1 and filtrate D2;
(4) Concentrating: mixing the filtrate B2, the filtrate C2 and the filtrate D2 obtained in the step (3), and concentrating to obtain a millettia speciosa champ concentrated solution;
(5) Cation exchange resin adsorption: filtering the concentrated solution obtained in the step (4), fully adsorbing by strong acid cation exchange resin, washing away water-soluble impurities, eluting, collecting eluent E, eluting by using ethanol containing ammonia water, collecting eluent F, concentrating and drying the eluent E to obtain total flavonoids of the radix millettiae speciosae, and concentrating and drying the eluent F to obtain alkaloids of the radix millettiae speciosae;
(6) Mixing: uniformly mixing the millettia speciosa champ polysaccharide obtained in the step (2) and the millettia speciosa champ total flavone and the millettia speciosa champ alkaloid obtained in the step (5) according to a proportion to obtain the millettia speciosa champ extract.
4. The method for preparing the millettia speciosa extract with the sexual function enhancing effect according to claim 3, wherein in the step (1), 8-16 times of water is added to millettia speciosa coarse powder, the added complex enzyme comprises cellulase, xylanase and neutral protease, the addition amounts of the cellulase, the xylanase and the neutral protease are respectively 0.4-0.8%, 0.2-0.6% and 0.6-1.0% of the weight of the millettia speciosa coarse powder, the pH value of the complex enzyme is 5.5-7.0, the enzymolysis temperature is 45-60 ℃, the ultrasonic power is 200-350W, and the treatment time is 1-3 h; the enzymatic hydrolysate was cooled and then filtered through a 0.8 μm filter.
5. The method for preparing an extract of Oriental millettia root having sexual function enhancing effect according to claim 3, wherein in the step (2), the filtrate A2 is concentrated to 1/4 of the original volume, 95% ethanol is added to the concentrated solution until the concentration of the final ethanol is 80%, and the mixture is kept at a constant temperature of 3-6 ℃ for 6-10 hours; the rotational speed of the centrifuge was 3000rpm and the centrifugation time was 20min.
6. The method for preparing an extract of Oriental millettia root having sexual function enhancing effect according to claim 3, wherein in the step (3), 4-16 times of ethanol with a pH value of 1-6 and a concentration of 50-90% is added to the residue A1, the extraction temperature is 70-80 ℃, the extraction time is 0.5-2 hours, and the mixture is filtered by a 0.8 μm filter membrane after cooling; adding 4-16 times of ethanol with the volume, the pH value of 6-8 and the concentration of 50-90% into the filter residue B1, extracting at 70-80 ℃ for 0.5-2 hours, cooling, and filtering with a filter membrane with the thickness of 0.8 mu m; adding 4-16 times of ethanol with the volume, the pH value of 8-12 and the concentration of 50-90% into the filter residue C1, extracting at 70-80 ℃ for 0.5-2 hours, cooling, and filtering with a filter membrane with the thickness of 0.8 mu m; the pH value of the ethanol is regulated by adopting disodium hydrogen phosphate-citric acid buffer solution.
7. The method for producing an extract of Oriental millettia root having sexual function enhancing effect according to claim 3, wherein in the step (4), the temperature at which the mixture of filtrate B2, filtrate C2 and filtrate D2 is concentrated is 40 to 50 ℃ and the vapor pressure is less than 0.3MPa.
8. The method for producing an extract of Oriental millettia root having sexual function enhancing action as claimed in claim 3, wherein in the step (5), distilled water is used to wash out water-soluble impurities at a flow rate of 4 to 6Bv/h, 5 to 10 times by weight of 50 to 90% ethanol is used to elute, then eluent E is collected at a flow rate of 1 to 3Bv/h, ammonia water containing 1 to 5% concentration is used to elute at a flow rate of 50 to 90% ethanol, eluent F is collected at a flow rate of 1 to 3Bv/h, and the strongly acidic cation exchange resin used is a strongly acidic cation exchange resin of type 001X 7 or type D001.
9. The method for preparing a beautiful millettia root extract with sexual function enhancing effect according to claim 3, wherein in the step (6), the mixing ratio of the beautiful millettia root polysaccharide, the beautiful millettia root total flavone and the beautiful millettia root alkaloid is 3-5: 1-3: 1-3.
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Application publication date: 20191115 Assignee: Guangxi green recycling new material technology Co.,Ltd. Assignor: Yulin Normal University Contract record no.: X2023980045321 Denomination of invention: A Niu Da Li extract with enhancing sexual function and its preparation method and use Granted publication date: 20230825 License type: Common License Record date: 20231101 |