CN110656130B - Astragalus membranaceus-paecilomyces cicadae fermentation mycoplasm, preparation method and application - Google Patents
Astragalus membranaceus-paecilomyces cicadae fermentation mycoplasm, preparation method and application Download PDFInfo
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Abstract
The invention discloses an astragalus-paecilomyces cicadae fermentation mycoplasm and a preparation method and application thereof. The fermentation mycoplasm is prepared by taking astragalus enzymolysis liquid as a fermentation substrate and paecilomyces cicadae as a fermentation strain through liquid fermentation; the astragalus enzymolysis liquid is obtained by carrying out enzymolysis on astragalus medicinal materials through cellulase and pectinase. The astragalus polysaccharide content in the astragalus-paecilomyces cicadae fermentation mycoplasm is obviously improved, and the astragalus-paecilomyces cicadae fermentation mycoplasm has an obvious effect of treating hyperuricemia and/or hypertriglyceridemia.
Description
Technical Field
The invention relates to a traditional Chinese medicine fermentation mycoplasm, a preparation method and application, in particular to a traditional Chinese medicine astragalus-paecilomyces cicadae fermentation mycoplasm, a preparation method and application.
Background
The traditional Chinese medicine fermentation is characterized in that the original performance of the medicine is changed, new effects are enhanced or generated through the fermentation process under certain environmental conditions (such as temperature, humidity, air, moisture and the like) by means of the action of enzyme and microorganism, and the medicine variety is expanded to adapt to the needs of clinical medication.
Traditional Chinese medicine fermentation is a microbial fermentation technology, and refers to a method for foaming a medicine to generate a yellowish-white mildew coat by virtue of the catalytic decomposition action of microorganisms and enzymes under certain temperature and humidity conditions after the medicine is cleaned or treated. Because the variety and the number of strains participating in fermentation in traditional Chinese medicine fermentation are influenced by geographical environment and seasonal variation, and the fermentation process is judged and controlled by human subjective experience, the safety, the effectiveness, the stability and the controllability of the fermented Chinese medicine product are difficult to ensure.
The modern Chinese medicine fermentation technology is a novel Chinese medicine modern pharmacy technology formed by combining the subjects of modern microecology, bioengineering, fermentation engineering technology and the like on the basis of inheriting the traditional Chinese medicine fermentation processing method. The bidirectional fermentation technology is characterized in that traditional Chinese medicinal materials with active ingredients are used as substrates and are fermented by beneficial medicinal fungi, a series of complex catabolism can occur among the substrates due to the growth process of the fungi to generate new ingredients and new functions, the fermentation effect is developed from the original unidirectional nutrient substrate which is only composed of agricultural and sideline products to provide nutrients such as carbon, nitrogen and the like required by the growth of the fungi to the application of the physiological activities of the fungi to convert the active ingredients in the substrates to generate new ingredients, so that new tastes and functions are generated, and the bidirectional fermentation is realized.
Radix astragali is a common traditional Chinese medicine and has the effects of tonifying qi and invigorating yang, consolidating superficial resistance and arresting sweating, inducing diuresis and relieving swelling, and the like. Modern researches show that astragalus polysaccharide is a main effective component in astragalus for regulating the immunologic function, has various efficacies of resisting fatigue, regulating blood sugar, resisting tumors and the like, and is successfully developed into a product, such as astragalus polysaccharide injection. Regarding modern fermentation research of astragalus, quantan et al reported that after solid fermentation is performed with ganoderma lucidum as a fermentation strain and astragalus dregs as a substrate, the obtained fermentation product has anti-hyperuricemia activity (anti-hyperuricemia activity of astragalus dregs fermentation product, Jiangsu agricultural science [ J ], vol.41, No. 9, 2013, p. 288-. Zhao Chongyan and the like establish a two-way fermentation system of paecilomyces cicadae-astragalus root medicinal material, and find that after fermentation, the content of polysaccharide and total saponin in the mycoplasm of the astragalus root medicinal material is reduced, and the content of flavone is increased (the establishment of the two-way fermentation system of paecilomyces cicadae-astragalus root and the research of components, the world traditional Chinese medicine, 13 th 12 th of 12 months in 2018, and 3195-.
Therefore, a method for fermenting astragalus root capable of effectively increasing the yield of astragalus polysaccharide is needed.
Disclosure of Invention
The invention aims to provide astragalus-paecilomyces cicadae fermentation mycoplasm, which has obviously improved astragalus polysaccharide content.
Another object of the present invention is to provide a method for producing the fermentation mycoplasm.
It is a further object of the present invention to provide the use of said fermentation mycoplasm.
The purpose of the invention is realized by the following technical scheme.
The invention provides an astragalus-paecilomyces cicadae fermentation mycoplasm, which is prepared by taking an astragalus enzymolysis liquid as a fermentation substrate and paecilomyces cicadae as a fermentation strain through liquid fermentation; the astragalus enzymolysis liquid is obtained by carrying out enzymolysis on astragalus medicinal materials through cellulase and pectinase.
The invention also provides a preparation method of the astragalus-paecilomyces cicadae fermentation mycoplasm, which comprises the following steps:
(1) the preparation method of the astragalus enzymolysis liquid comprises the following steps: mixing radix astragali with water, adding cellulase and pectinase for enzymolysis, sterilizing, and cooling to obtain radix astragali enzymolysis solution;
(2) a fermentation step: and (3) inoculating the paecilomyces cicadae into the astragalus enzymolysis liquid, and carrying out liquid culture to obtain the paecilomyces cicadae-astragalus fermentation mycoplasm.
In the invention, preferably, in the step (1), the astragalus root medicinal material is used in the form of astragalus root medicinal material powder, and the particle size of the astragalus root medicinal material powder is 60-120 meshes; and the weight ratio of the astragalus medicinal material powder to water is 1: 1.5-10.
In the invention, the astragalus medicinal material is pretreated by an enzymolysis method, and the enzymolysis step has important influence on improving the yield of astragalus polysaccharide and finally exerting the fermentation mycoplasm to treat hyperuricemia and triglyceride.
In the invention, preferably, in the step (1), the dosage of the cellulase is 0.5-5 wt% of the weight of the astragalus; the dosage of the pectinase is 0.5-5 wt% of the weight of the astragalus; the enzymolysis temperature in the enzymolysis process is 40-60 ℃, and the enzymolysis time is 70-110 min.
In the present invention, preferably, in step (2), the paecilomyces cicadae is obtained by subjecting a paecilomyces cicadae strain to activation culture.
In the present invention, preferably, in the step (2), the method for activated culture of paecilomyces cicadae strain comprises: inoculating Paecilomyces cicadae strain on Potato Dextrose Agar (PDA) slant culture medium, placing in a constant temperature and humidity box with 27 deg.C and relative humidity of 80%, activating and culturing for 5 days to obtain activated Paecilomyces cicadae; then, the activated paecilomyces cicadae is picked by an inoculating loop and placed in a potato glucose liquid culture medium at the temperature of 25 ℃ and the temperature of 140 r.min-1Culturing for 7 days under the condition of a shaking table to obtain the paecilomyces cicadae and a seed solution.
In the present invention, preferably, in the step (2), the paecilomyces cicadae seed liquid is further inoculated into the astragalus enzymolysis liquid.
In the invention, in the step (2), the temperature of the liquid culture is preferably 26-30 ℃, and the time of the liquid culture is preferably 3-20 days.
The invention also provides application of the astragalus-paecilomyces cicadae fermentation mycoplasm in preparation of astragalus polysaccharide.
The invention also provides application of the astragalus-paecilomyces cicadae zymophyte in preparing a medicine for treating hyperuricemia and/or hypertriglyceridemia.
Compared with the astragalus medicinal material, the astragalus-paecilomyces cicadae fermentation mycoplasm of the invention has obviously increased total polysaccharide content and can be used for preparing astragalus polysaccharide, thereby improving the yield of the astragalus polysaccharide. In addition, compared with the astragalus root medicinal material, the astragalus root-paecilomyces cicadae fermentation mycoplasma has obviously enhanced effect of treating hyperuricemia and hypertriglyceridemia. The preparation method of the astragalus-paecilomyces cicadae fermentation mycoplasm firstly carries out enzymolysis treatment on the astragalus medicinal material, and has obvious influence on the increase of the yield of astragalus polysaccharide and the drug effect.
Drawings
FIG. 1 is a graph comparing the levels of UA in serum from hyperuricemia rats.
FIG. 2 is a graph showing a comparison of serum BUN levels in hyperuricemia rats.
FIG. 3 is a graph comparing serum CRE levels in hyperuricemia rats.
FIG. 4 is a graph comparing the levels of TG in hyperuricemia rats.
Note: indicated very significant difference (P) compared to normal group<0.001) indicating significant difference (P)<0.01) representing a statistical difference (P)<0.05); in comparison to the set of models,+++shows a very significant difference (P)<0.001)，++Indicates significant difference (P)<0.01)，+Indicates that there is a statistical difference (P)<0.05)。
Detailed Description
The invention provides an astragalus-paecilomyces cicadae fermentation mycoplasm, a preparation method and application thereof, and the embodiment of the invention is specifically explained below.
< preparation method >
A preparation method of astragalus-paecilomyces cicadae fermentation mycoplasm comprises the following steps:
(1) the preparation method of the astragalus enzymolysis liquid comprises the following steps: mixing radix astragali with water, adding cellulase and pectinase for enzymolysis, sterilizing, and cooling to obtain radix astragali enzymolysis solution;
(2) a fermentation step: and (3) inoculating the paecilomyces cicadae into the astragalus enzymolysis liquid, and carrying out liquid culture to obtain the paecilomyces cicadae-astragalus fermentation mycoplasm.
In the step (1), the astragalus root is preferably used in the form of astragalus root powder. According to a preferred embodiment of the invention, the astragalus membranaceus medicinal material powder has a particle size of 60-120 meshes, preferably 80-120 meshes, and more preferably 100 meshes. The granularity of the astragalus medicinal material powder is favorable for enzymolysis of the astragalus medicinal material and dissolution of chemical components, and the dosage of enzyme and the enzymolysis time are saved.
In the step (1), the weight ratio of the astragalus root medicinal material powder to water is 1: 1.5-10, preferably 1: 2-8, and more preferably 1: 3-6. By adopting the proportion, the astragalus enzymolysis liquid with proper concentration can be obtained, so that the paecilomyces cicadae and the astragalus are in full contact, and a better fermentation effect is obtained. Particularly, the weight ratio of the astragalus root medicinal material powder to water is within the range of 1: 3-6, the obtained astragalus root enzymolysis liquid is more suitable for the growth of the paecilomyces cicadae, and the growth rate of the paecilomyces cicadae and the yield of the paecilomyces cicadae-astragalus root fermentation mycoplasm are higher.
In the step (1), the dosage of the cellulase is 0.5-5 wt%, preferably 0.8-4 wt%, and more preferably 1-3 wt% of the weight of the astragalus root medicinal material. The dosage of the pectinase is 0.5-5 wt%, preferably 0.8-4 wt%, and more preferably 1.2-3.5 wt% of the weight of the astragalus root medicinal material. The invention adopts the enzyme dosage, can effectively carry out enzymolysis on the astragalus medicinal material, and simultaneously avoids the waste of enzyme.
In the step (1), the enzymolysis temperature in the enzymolysis process is 40-60 ℃, preferably 45-55 ℃, and more preferably 48-52 ℃; the enzymolysis time is 70-110 min, preferably 80-100 min, and more preferably 85-95 min. According to a preferred embodiment of the invention, the enzymolysis temperature in the enzymolysis process is 48-52 ℃, and the enzymolysis time is 85-95 min. By adopting the enzymolysis conditions, full enzymolysis can be realized, and the time is saved.
And (2) in the step (1), cooling to room temperature.
In the step (2), the paecilomyces cicadae is obtained by performing activated culture on a paecilomyces cicadae strain. The paecilomyces cicadae strain is a known strain and can be a strain provided by China forestry microorganism collection center, and the number of the strain is cfcc 81169. The activation culture method may be any activation method conventionally used in the art, and is not limited. According to one embodiment of the present invention, the activation culture method is: inoculating Paecilomyces cicadae strain on Potato Dextrose Agar (PDA) slant culture medium, placing in a constant temperature and humidity box with 27 deg.C and relative humidity of 80%, activating and culturing for 5 days to obtain activated Paecilomyces cicadae; then, the activated paecilomyces cicadae is picked by an inoculating loop and placed in a potato glucose liquid culture medium at the temperature of 25 ℃ and the temperature of 140 r.min-1Culturing for 7 days under the condition of a shaking table to obtain the paecilomyces cicadae and a seed solution.
In the step (2), the inoculation amount of the paecilomyces cicadae is 5-20 wt%, preferably 8-15 wt%, and more preferably 8-12 wt% of the weight of the astragalus mongholicus. By adopting the inoculation amount, the growth rate of the paecilomyces cicadae and the yield of the paecilomyces cicadae-astragalus fermentation mycoplasm are higher, and the using amount of the paecilomyces cicadae is saved.
In the step (2), besides the paecilomyces cicadae is inoculated into the astragalus enzymolysis liquid, the paecilomyces cicadae seed liquid can also be inoculated into the astragalus enzymolysis liquid. The inoculation amount of the seed liquid can be 1-10 vol%, preferably 2-8 vol%, and more preferably 3-6 vol% of the astragalus enzymolysis liquid. Meanwhile, the seed liquid is inoculated, which is beneficial to the growth of the mycoplasm, and the growth rate of the paecilomyces cicadae and the yield of the paecilomyces cicadae-astragalus fermentation mycoplasm are higher.
In the step (2), the temperature of the liquid culture is 26-30 ℃, preferably 27-29 ℃, and more preferably 28 ℃; the liquid culture time is 3-20 days, preferably 5-15 days, and more preferably 6-10 days. According to a preferred embodiment of the present invention, the temperature of the liquid culture is 26 to 30 ℃ and the time of the liquid culture is 6 to 10 days. The liquid culture can be carried out under agitation or shaking table rotation. According to an embodiment of the present invention, the liquid culture is performed on a shaker at a shaker rotation speed of 60-180 r.min-1Preferably 80 to 160 r.min-1More preferably 100 to 140 r.min-1。
< fermentation mycoplasm >
The astragalus-paecilomyces cicadae fermentation mycoplasm for treating hyperuricemia and hypertriglyceridemia is prepared by taking astragalus enzymolysis liquid as a fermentation substrate and paecilomyces cicadae as a fermentation strain through liquid fermentation; the astragalus enzymolysis liquid is obtained by carrying out enzymolysis on astragalus medicinal materials through cellulase and pectinase. Specifically, the fermentation mycoplasm is prepared by the preparation method.
Compared with astragalus root, the astragalus polysaccharide content in the fermentation mycoplasm is obviously increased, and the fermentation mycoplasm can be used for preparing astragalus polysaccharide, so that the yield of the astragalus polysaccharide is improved. In addition, compared with the astragalus root medicinal material, the fermentation mycoplasm of the invention has obviously enhanced effect of treating hyperuricemia and hypertriglyceridemia.
< use >)
Compared with the astragalus medicinal material, the astragalus-paecilomyces cicadae fermentation mycoplasm of the invention has obviously increased total polysaccharide content and can be used for preparing astragalus polysaccharide, thereby improving the yield of the astragalus polysaccharide. Therefore, the invention provides the application of the astragalus-paecilomyces cicadae fermentation mycoplasm in preparing astragalus polysaccharide.
In addition, compared with the astragalus root medicinal material, the astragalus root-paecilomyces cicadae fermentation mycoplasma has obviously enhanced effect of treating hyperuricemia and hypertriglyceridemia. Therefore, the invention also provides the application of the astragalus-paecilomyces cicadae zymophyte in preparing the medicine for treating hyperuricemia and/or hypertriglyceridemia. Preferably, the invention also provides application of the astragalus-paecilomyces cicadae zymophyte in preparing a medicine for treating hyperuricemia complicated with hypertriglyceridemia.
The following further illustrates embodiments of the invention by way of specific examples.
In the following examples, the Paecilomyces cicadae strain is provided by China forestry microbiological Collection center, with the number cfcc 81169. The paecilomyces cicadae strain is inoculated on a Potato Dextrose Agar (PDA) slant culture medium and is placed in a constant temperature and humidity box with the temperature of 27 ℃ and the relative humidity of 80 percent for activation culture for 5 days to obtain the activated paecilomyces cicadae. The activated paecilomyces cicadae is picked by an inoculating loop and placed in a potato glucose liquid culture medium at the temperature of 25 ℃ and the temperature of 140 r.min-1Culturing for 7 days under the condition of a shaking table to obtain the paecilomyces cicadae and a seed solution.
Reagent: potassium oxonate (Potasisonate) was purchased from Sigma, USA; benzbromarone tablets (Narcarin) were purchased from Excella GmbH, germany. The calibration solution, the quality control solution and the detection kit are all purchased from Beijing Lidemann Biochemical technology GmbH. Triglyceride assay kit, batch number: 112293K. Uric acid assay kit, batch number: 00275. urea nitrogen assay kit, batch No.: 01049; creatinine assay kit, lot No.: 04210.
animals: sprague Dawley (SD) rats, male, with a body mass of 220-250 g, purchased from Beijing Wintolite laboratory animal technology, Inc., with a license number of SCXK (Jing) 2016-.
The instrument comprises the following steps: the CX-4Pro type full-automatic biochemical analyzer is a product of Beckman company in America; an electronic analytical balance (one hundred thousand) model R200D is a product of Sartorius, germany; the Millipore Synergy UV type ultra-pure water machine is a product of Millipore company in America; the BXM-30R high-pressure steam sterilization pot is a product of laboratory instruments and equipment Limited of the Xian apparatus; the UV-2000 ultraviolet visible spectrophotometer is a product of Beijing Rayleigh analysis instruments company; the FW-100 high-speed pulverizer is a product of Tensted instruments, Inc. of Tianjin; the LHS-80 HC-II type constant temperature and humidity incubator is a product of Shanghai-constant technology limited company.
Example 1
Pulverizing radix astragali, and sieving with 100 mesh sieve to obtain radix astragali powder. Uniformly mixing the astragalus powder with distilled water which is 5 times (by weight) of the astragalus powder, sequentially adding cellulase and pectinase which are 1.5 wt% of the weight of the astragalus powder, performing enzymolysis at 50 ℃ for 90min, placing in a steam sterilization pot at 121 ℃ for sterilization for 30min, and cooling to room temperature to obtain an astragalus enzymolysis liquid; inoculating Paecilomyces cicadae into the radix astragali enzymolysis solution, wherein the inoculation amount is 10 wt% of the weight of the radix astragali medicinal material, and the temperature is 28 ℃, and the speed is 120 r.min-1Culturing for 7 days under shaking table condition to obtain Paecilomyces cicadae-radix astragali fermentation mycoplasm A (hereinafter referred to as "mycoplasm A").
Example 2
Pulverizing radix astragali, and sieving with 100 mesh sieve to obtain radix astragali powder. Uniformly mixing the astragalus powder with distilled water which is 5 times (by weight) of the astragalus powder, sequentially adding cellulase and pectinase which are 1.5 wt% of the weight of the astragalus powder, performing enzymolysis at 50 ℃ for 90min, placing in a steam sterilization pot at 121 ℃ for sterilization for 30min, and cooling to room temperature to obtain an astragalus enzymolysis liquid; inoculating Paecilomyces cicadae and seed liquid into radix astragali enzymolysis liquid, wherein the inoculation amount of Paecilomyces cicadae is 10 wt% of the weight of radix astragali, the inoculation amount of seed liquid is 4 vol%, and the inoculation amount is 120 r/min at 28 deg.C-1Culturing for 7 days under shaking table condition to obtain Paecilomyces cicadae-radix astragali fermentation mycoplasm B (hereinafter referred to as "mycoplasm B").
Comparative example 1
Pulverizing radix astragali, and sieving with 100 mesh sieve to obtain radix astragali powder. Mixing radix astragali powder with 15 times (by weight) of distilled water, and sequentially adding 1.5 wt% of fiberPerforming enzymolysis with pectinase 1.5 wt% of the weight of the powder of radix astragali at 50 deg.C for 90min, sterilizing in a steam sterilization pot at 121 deg.C for 30min, and cooling to room temperature to obtain radix astragali enzymolysis solution; inoculating Paecilomyces cicadae into the radix astragali enzymolysis solution, wherein the inoculation amount is 10 wt% of the weight of the radix astragali medicinal material, and the temperature is 28 ℃, and the speed is 120 r.min-1Culturing for 7 days under shaking table condition to obtain Paecilomyces cicadae-radix astragali fermentation mycoplasm C (hereinafter referred to as "mycoplasm C").
Comparative example 2
Pulverizing radix astragali, and sieving with 100 mesh sieve to obtain radix astragali powder. Uniformly mixing the astragalus powder with distilled water which is 5 times (by weight) of the astragalus powder, sequentially adding cellulase and pectinase which are 1.5 wt% of the weight of the astragalus powder, performing enzymolysis at 50 ℃ for 90min, placing in a steam sterilization pot at 121 ℃ for sterilization for 30min, and cooling to room temperature to obtain an astragalus enzymolysis liquid; inoculating Paecilomyces cicadae into the radix astragali enzymolysis solution, wherein the inoculation amount is 4 wt% of the weight of the radix astragali medicinal material, and the temperature is 28 ℃, and the speed is 120 r.min-1Culturing for 7 days under shaking table condition to obtain Paecilomyces cicadae-radix astragali fermentation mycoplasm D (hereinafter referred to as "mycoplasm D").
Comparative example 3
Pulverizing radix astragali, and sieving with 100 mesh sieve to obtain radix astragali powder. Uniformly mixing the astragalus powder with distilled water which is 5 times (by weight) of the astragalus powder, sequentially adding cellulase and pectinase which are 1.5 wt% of the weight of the astragalus powder, performing enzymolysis at 50 ℃ for 90min, placing in a steam sterilization pot at 121 ℃ for sterilization for 30min, and cooling to room temperature to obtain an astragalus enzymolysis liquid; inoculating Paecilomyces cicadae into the radix astragali enzymolysis solution, wherein the inoculation amount is 20 wt% of the weight of the radix astragali medicinal material, and the temperature is 28 ℃, and the speed is 120 r.min-1Culturing for 7 days under shaking table condition to obtain Paecilomyces cicadae-radix astragali fermentation mycoplasm E (hereinafter referred to as "mycoplasm E").
Comparative example 4
Pulverizing radix astragali, and sieving with 100 mesh sieve to obtain radix astragali powder. Mixing radix astragali powder with 5 times (by weight) of distilled water, and steaming at 121 deg.CSterilizing in a sterilizing pot for 30min, and cooling to room temperature to obtain radix astragali substrate; inoculating Paecilomyces cicadae into radix astragali substrate, wherein the inoculation amount is 10 wt% of the weight of radix astragali material, and the temperature is 28 ℃, and the speed is 120 r.min-1Culturing for 7 days under shaking table condition to obtain Paecilomyces cicadae-radix astragali fermentation mycoplasm F (hereinafter referred to as "mycoplasm F").
Comparative example 5
Pulverizing radix astragali, and sieving with 100 mesh sieve to obtain radix astragali powder.
Experimental example 1 determination of growth rate of mycoplasm
Sampling on the 7 th day of fermentation, measuring the wet weight of mycelia, filtering the fermentation liquor with 8 layers of gauze to obtain mycelia, washing the mycelia with distilled water for 3 times, drying in a 60 ℃ oven to constant weight, and weighing. The results are shown in Table 1.
TABLE 1
Experimental example 2 measurement of content of active ingredient
Respectively extracting total polysaccharides from mycoplasm A of example 1, mycoplasm F of comparative example 4 and astragalus root powder of comparative example 5, and comparing the method with the method for measuring content of astragalus polysaccharide in astragalus root by using sulfuric acid-phenol colorimetric method (the method is the same as the following documents: Yangli et al, the comparison of the method for measuring content of astragalus polysaccharide in astragalus root, China journal of medical industry [ J],2005, 36(9): 562-2＝0.9972。
Extracting total flavone from mycoplasm A of example 1, mycoplasm F of comparative example 4 and astragalus powder of comparative example 5 by using ethyl acetate, and adopting NaNO2-A1(NO3)3Colorimetric method (the same as the following documents: House Tree mark, etc.), ultraviolet spectrophotometry method for determining total flavone content in Mongolian radix astragali, Chinese medicine and clinic [ J],2007,7(12): 899-901) to obtain a regression equation of y-13.839 x +0.0297, R2＝0.9997。
Three samples are taken in parallel for the above determination, the content is determined, and the average value is taken.
The results are shown in Table 2.
TABLE 2
Note: indicates that the powder group of the astragalus has significant difference (P) compared with the powder group of the astragalus<0.05); indicates that the powder group of the astragalus has extremely significant difference (P) compared with the powder group of the astragalus<0.001)；+++Shows a very significant difference (P) compared with the fermentation mycoplasma A group<0.001)
Compared with the astragalus medicinal material powder group, the total polysaccharide content in the mycoplasm A is obviously increased and is 1.37 times of that of the astragalus medicinal material powder group; the content of flavone is obviously reduced and is only 1/3 of the astragalus group. Compared with the astragalus root medicinal material powder group, the content of total polysaccharide in the mycoplasm F is increased, the content of total flavone is reduced, and the mycoplasm F is very obviously different from the mycoplasm A group (P is less than 0.001).
Experimental example 3 drug efficacy test
1. Molding and administration
SD rats are taken and randomly divided into a normal group, a HUA model group, a positive drug control group, a mycoplasm A group and a mycoplasm F group according to the body mass, each group comprises 8 animals, and the animals are subjected to adaptive feeding for 1 week and then are used for experiments.
Each group was given normal feed. In addition, the normal group was gavaged with distilled water; the other groups are administered with 300 mg/kg by intragastric administration-1Molding potassium oxonate, and after 1 hour, feeding 20 mg/kg of the positive drug control group by intragastric administration-1The gavage of the benzbromarone and mycoplasm A group is 300 mg/kg-1The mycoplasm A and the mycoplasm F are administered to the group of 300 mg/kg-1Mycoplasm F is continuously administrated for 14 days twice a day, and the intragastric administration amount is 15 mL/kg-1。
2. Collection of serum samples
After 4 hours after the last day of administration treatment, the rats were subjected to orbital venous plexus blood sampling, and after standing overnight at 4 ℃ in a blood sample, serum was collected by centrifugation and used for measurement of biochemical indexes.
3. Determination of biochemical indices
After conventional calibration and quality control, a full-automatic biochemical analyzer is adopted to detect the content of Uric Acid (UA), urea nitrogen (BUN), Creatinine (CRE) and Triglyceride (TG) in serum.
4. Data analysis
All experimental results are expressed as "mean ± standard deviation". Excel and SPSS17.0 software is adopted for calculation and statistics, after normal distribution and homogeneity of variance test, single-factor variance analysis is carried out on all data, t test is adopted for the difference between two groups of means, and the difference is considered to have statistical significance when P is less than 0.05.
5. Results
5.1 Effect on the serum UA level in hyperuricemic rats
As shown in fig. 1, the serum UA level of rats in the hyperuricemia model group induced by potassium oxonate was extremely significantly increased (P <0.001) compared to the normal group, indicating successful modeling of hyperuricemia model in rats; compared with the model group, the positive drugs benzbromarone group, the mycoplasm A group and the mycoplasm F group all remarkably reduce the serum UA level of the model rat, and the action effect of the mycoplasm A group is strongest.
5.2 Effect on serum BUN levels in hyperuricemic rats
As shown in fig. 2, serum BUN levels were significantly increased in rats in the hyperuricemia model group induced by oxonate potassium (P <0.01) compared to the normal group; compared with the model group, the mycoplasm A group and the mycoplasm F group both reduce the serum UA level of the model rat; wherein, the mycoplasma A group remarkably reduces the serum UA level of a model rat (P <0.001), is obviously superior to the mycoplasma F group (P <0.05), and the positive drug benzbromarone has no obvious influence on the serum BUN level.
5.3 Effect on serum CRE levels in hyperuricemic rats
As shown in FIG. 3, there was no significant difference in serum creatinine levels in rats of different groups. However, compared with the model group, the positive drugs benzbromarone group, mycoplasm A group and mycoplasm F group have the tendency of reducing the serum CRE level, and the mycoplasm A group has the strongest effect.
5.4 Effect on the level of TG in hyperuricemia rats
Hyperuricemia is often complicated by hypertriglyceridemia. As shown in fig. 4, the serum TG levels of rats in the oteracil potassium salt-induced hyperuricemia model group were very significantly increased (P <0.001) compared to the normal group; compared with the model group, the positive drug benzbromarone group, the mycoplasm A group and the mycoplasm F group can reduce the serum TG level (P <0.01 or P <0.05) of the model rat, and the TG reducing effect of the mycoplasm A group is strongest and is obviously better than that of the mycoplasm F group.
The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.
Claims (7)
1. A preparation method of astragalus-paecilomyces cicadae fermentation mycoplasm comprises the following steps:
(1) the preparation method of the astragalus enzymolysis liquid comprises the following steps: mixing radix astragali with water, adding cellulase and pectinase for enzymolysis, sterilizing, and cooling to obtain radix astragali enzymolysis solution; the weight ratio of the astragalus medicinal material powder to water is 1: 2-8; the dosage of the cellulase is 0.5-5 wt% of the weight of the astragalus; the dosage of the pectinase is 0.5-5 wt% of the weight of the astragalus; the enzymolysis temperature in the enzymolysis process is 40-60 ℃, and the enzymolysis time is 70-110 min;
(2) a fermentation step: inoculating paecilomyces cicadae into the astragalus enzymolysis liquid, and carrying out liquid culture to obtain paecilomyces cicadae-astragalus fermentation mycoplasm; the inoculation amount of the paecilomyces cicadae is 8-12 wt% of the weight of the astragalus mongholicus; the temperature of the liquid culture is 26-30 ℃, and the time of the liquid culture is 3-20 days.
2. The preparation method according to claim 1, wherein in the step (1), the astragalus root is used in the form of astragalus root powder, and the particle size of the astragalus root powder is 60-120 meshes; and the weight ratio of the astragalus medicinal material powder to water is 1: 3-6.
3. The method according to claim 1, wherein the Paecilomyces cicadae is obtained by subjecting a Paecilomyces cicadae strain to activation culture in the step (2).
4. The process according to claim 3, wherein the culture of Paecilomyces cicadae in step (2) is carried out by the activation culture method comprising: inoculating Paecilomyces cicadae strain on Potato Dextrose Agar (PDA) slant culture medium, placing in a constant temperature and humidity box with 27 deg.C and relative humidity of 80%, activating and culturing for 5 days to obtain activated Paecilomyces cicadae; then, the activated paecilomyces cicadae is picked by an inoculating loop and placed in a potato glucose liquid culture medium at the temperature of 25 ℃ and the temperature of 140r ∙ min-1Culturing for 7 days under the condition of a shaking table to obtain the paecilomyces cicadae and a seed solution.
5. The method according to claim 4, wherein in the step (2), the Paecilomyces cicadae seed solution is further inoculated into the astragalus enzymolysis solution; the inoculation amount of the seed liquid is 2-8 vol% of the astragalus enzymolysis liquid.
6. Use of the fermentation mycoplasm of astragalus membranaceus-paecilomyces cicadae prepared by the preparation method according to any one of claims 1 to 5 in preparation of astragalus polysaccharides.
7. Use of the fermentation mycoplasma from astragalus membranaceus to paecilomyces cicadae prepared by the preparation method according to any one of claims 1 to 5 in preparation of medicines for treating hyperuricemia and hypertriglyceridemia.
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