CN114949024A - Herba Cephalanoploris total flavonoids, preparation method and application thereof in preparing medicine for preventing or treating hyperuricemia - Google Patents
Herba Cephalanoploris total flavonoids, preparation method and application thereof in preparing medicine for preventing or treating hyperuricemia Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, and particularly relates to herba cepbalanoplosis segeti, a preparation method and application thereof in preparation of a medicine for preventing or treating hyperuricemia. The method comprises the steps of degreasing common cephalanoplos herb, carrying out alcohol extraction on the degreased common cephalanoplos herb to prepare common cephalanoplos herb alcohol extract, dispersing the common cephalanoplos herb alcohol extract in water to form dispersion, and carrying out adsorption, elution, purification and drying on the dispersion to prepare common cephalanoplos herb total flavone; proved by hyperuricemia animal experiments, compared with the existing medicine allopurinol for reducing uric acid, the herba cepbalanoplosis segeti total flavone disclosed by the invention can not only reduce the uric acid level of hyperuricemia, but also inhibit renal function indexes such as serum creatinine, urea nitrogen and cystatin C caused by hyperuricemia, and has the effect of improving renal function.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to herba cepbalanoplosis segeti total flavonoids, a preparation method and application thereof in preparation of a medicine for preventing or treating hyperuricemia.
Background
Hyperuricemia (HUA) refers to the condition of purine metabolic disorder of the body, Uric Acid (UA) hypersecretion or renal excretion dysfunction, so that Uric acid is accumulated in the blood, the diagnosis standard is that the Uric acid level of men with fasting blood Uric acid level on different days is more than or equal to 420umol/L, the Uric acid level of women with fasting blood Uric acid level on different days is more than or equal to 360umol/L, and the blood Uric acid can form mono-sodium urate (MSU) crystallization and deposition on the local joints beyond the saturation of the blood Uric acid in blood or tissue fluid, so that gout is caused. Hyperuricemia is a systemic hazard, is an independent risk factor for coronary heart disease, hypertension, diabetes, chronic kidney disease and the like, and the prevalence rate of the hyperuricemia is increased year by year. According to the national health and Nutrition survey (NHANES), the prevalence of hyperuricemia rose from 19.0% in 1988-1994 to 21.5% in 2007-2008 and remained stable in the last decade; in the year 2009-2010, the prevalence of hyperuricemia in adults in China was 8.4%, and increased to 17.4% in 2019. The incidence of hyperuricemia is related to sex, age, heredity and environmental factors, and smoking, drinking, obesity, diabetes, hypertension and dyslipidemia are all important risk factors of hyperuricemia.
In the treatment of hyperuricemia, western medicine therapy mainly takes uric acid reducing medicines (such as allopurinol and benzbromarone), but the uric acid reducing medicines taken for a long time have serious adverse reactions, such as liver and kidney damage, and the uric acid level is easy to rebound after the medicine is stopped, so the long-term curative effect is not ideal. Therefore, there is a need for the development of a drug for treating hyperuricemia which has no or less side effects.
Disclosure of Invention
Aiming at the problem of liver and kidney damage of the existing hyperuricemia treatment medicine, the invention aims to provide herba cepbalanoplosis segeti total flavonoids, a preparation method and application thereof in preparing the medicine for preventing or treating hyperuricemia.
Based on the purpose, the technical scheme adopted by the application is as follows:
in a first aspect, the invention provides a preparation method of herba cepbalanoplosis segeti, which comprises the following steps:
s1: degreasing the common cephalanoplos herb for later use;
s2: extracting the degreased herba cephalanoploris with alcohol to obtain herba cephalanoploris alcohol extract;
s3: dispersing the extract in water to obtain dispersion, adsorbing with macroporous resin, eluting, purifying, and drying to obtain herba Cephalanoploris total flavone.
Preferably, the step of degreasing the herba cephalanoploris in step S1 is as follows:
drying herba Cephalanoploris, pulverizing into herba Cephalanoploris powder, adding diethyl ether, reflux extracting, defatting, and volatilizing diethyl ether.
Preferably, the step S2 of extracting the defatted herba cephalanoploris with alcohol to obtain the herba cephalanoploris alcohol extract comprises the following steps:
adding 8-12 times of ethanol with mass concentration of 60% -80% into defatted herba Cephalanoploris powder, soaking for 0.5-1h, reflux extracting for 1-2h, collecting ethanol extractive solution, concentrating the ethanol extractive solution to remove ethanol, and making into herba Cephalanoploris ethanol extract.
Preferably, the method of step S3 is as follows:
dispersing the extract in water to obtain dispersion, subjecting the dispersion to macroporous resin, eluting with 1-2 times of column volume of distilled water, and discarding water eluate; eluting with 3-4 times of column volume of 10% -30% ethanol, and discarding ethanol eluate; eluting with 70% -80% ethanol of 5-6 times column volume, collecting the ethanol eluate, recovering ethanol under reduced pressure until no ethanol smell exists, and lyophilizing to obtain herba Cephalanoploris total flavone.
In a second aspect, the present invention provides a total flavonoid from herba Cirsii, which is obtained by the above method.
In a third aspect, the invention provides an application of herba cepbalanoplosis segeti in preparing a medicament for preventing or treating hyperuricemia.
Experiments show that the herba cepbalanoplosis segeti can improve uric acid metabolism and renal function of hyperuricemic mice induced by potassium oxonate and spontaneous hyperuricemic rats knocked out by UOX gene, not only can reduce uric acid level in blood of hyperuricemic mice, but also can inhibit renal function indexes induced by hyperuricemia such as serum creatinine, urea nitrogen and cystatin C, and compared with the existing uric acid-reducing drugs such as allopurinol, the herba cepbalanoplosis segeti disclosed by the invention not only has the effect of reducing serum uric acid level, but also has the effect of improving renal function.
In a fourth aspect, the invention provides an application of herba cepbalanoplosis segeti in preparing a medicament for treating gout, uric acid renal calculus, tophus or uric acid nephropathy related to hyperuricemia.
In a fifth aspect, the invention provides a medicament for preventing or treating hyperuricemia, which comprises the total flavonoids of herba cepbalanoplosis segeti and pharmaceutically acceptable salts or auxiliary materials thereof.
In a sixth aspect, the invention provides an application of herba cephalanoploris total flavone in preparing xanthine oxidase inhibitors.
In a seventh aspect, the present invention provides a xanthine oxidase inhibitor comprising the above total flavonoids of herba Cirsii.
Compared with the prior art, the invention has the following beneficial effects:
compared with the existing uric acid reducing drug allopurinol, the herba cepbalanoplosis segeti total flavonoids can reduce the uric acid level of hyperuricemia, can inhibit the increase of renal function indexes such as serum creatinine, urea nitrogen and cystatin C induced by hyperuricemia, and has the effect of improving the renal function.
Drawings
FIG. 1 shows the effect of total flavonoids from herba Cephalanoploris on uric acid and renal function of hyperuricemic mouse, wherein FIG. 1A shows the result of total flavonoids from herba Cephalanoploris reducing uric acid level in serum of hyperuricemic mouse induced by Potassium Oxonate; FIG. 1B is a graph showing the results of herba Cephalanoploris total flavonoids decreasing the activity of xanthine oxidase in serum of hyperuricemic mice; FIG. 1C and FIG. 1D show the results of the total flavonoids of Cirsium setosum improving renal function in hyperuricemic mice;
FIG. 2 shows the results of the weight and the intake and intake of water of rats in each group, wherein FIG. 2A shows the weight change of rats in each group, and FIGS. 2B and 2C show the intake and intake of water at 3 weeks of intervention of rats in each group, respectively;
FIG. 3 is a graph of the effect of total flavonoids from Cirsium japonicum on uric acid and renal function in UOX-/-rats, wherein FIG. 3A is the result of total flavonoids from Cirsium japonicum reducing uric acid levels in serum from uric acid-rich rats knocked out of the UOX gene; FIGS. 3B, 3C, and 3D show that total flavonoids from Cirsium setosum improved renal function in hyperuricemic rats, indicating serum creatinine, urea nitrogen, and cystatin C levels (Note: P < 0.05, P < 0.01, P < 0.001), respectively.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
Potassium oxonate (Bide pharmaceutical science and technology Co., Ltd., China); allopurinol (shanghai alatin limited, china); sodium carboxymethylcellulose (shanghai alatin limited, china); uric acid detection kit (DIUA-250, Bosch Biotechnology Ltd., USA); xanthine oxidase test kit (A002-1-1, Nanjing institute of bioengineering, China); creatinine kit (C011-2-1, Nanjing, China, institute of bioengineering); urea nitrogen kit (C013-2-1, Nanjing institute of bioengineering, China); cysc Elisa detection kit (Jiangsu Boshen, China); full-automatic biochemical analyzers (Beckman, usa).
Example 1
The embodiment provides a preparation method of herba cepbalanoplosis segeti total flavonoids, which comprises the following steps:
drying herba Cephalanoploris at 60 deg.C, pulverizing, and sieving with 40 mesh sieve to obtain herba Cephalanoploris powder; adding 8-12 times of diethyl ether into herba Cephalanoploris powder, reflux extracting for 1-2 hr, defatting, and discarding diethyl ether; after ether in the degreased herba Cirsii powder is completely volatilized, adding 8-12 times of ethanol with mass concentration of 60% -80% into the degreased herba Cirsii powder, soaking for 0.5-1h, reflux extracting for 1-2h, filtering, and collecting ethanol extract; recovering ethanol from the ethanol extractive solution under reduced pressure until no ethanol smell exists to obtain herba Cephalanoploris ethanol extract; adding distilled water into herba Cephalanoploris alcohol extract to obtain dispersion solution containing crude drug 0.3-0.5g/mL, purifying with AB-8 macroporous adsorbent resin, eluting with 1-2 times of column volume of distilled water in order of polarity from large to small, and discarding water washing solution; eluting with 3-4 times column volume of 10% -30% ethanol, and discarding the ethanol eluate; eluting with 5-6 times column volume of 70% -80% ethanol, collecting the ethanol eluate, recovering ethanol under reduced pressure until no ethanol smell exists, lyophilizing, and pulverizing to obtain herba Cephalanoploris total flavone.
Example 2
In this example, the effect of total flavonoids of herba cepbalanoplosis on uric acid metabolism of hyperuricemic mice induced by potassium oxonate is analyzed, and the specific test method and test results are as follows:
1. design of experiments
40 Kunming mice (20-25g) were randomly divided into a control group (CTR group, n: 10), a model group (MOD group, n: 10), a common cephalanoplos herb total flavone group (CIR group, n: 10) and an allopurinol group (ALP, n: 10), and were acclimatized for 1 week under rearing conditions: the breeding temperature is 21-25 deg.C, relative humidity is 60 + -10%, and the light and shade alternate illumination is performed for 12h, so that the people can freely eat and take water.
After the adaptive feeding is finished, (1) the pretreatment of the medicines is carried out for 3 days: CIR group and ALP group respectively perfuse herba Cephalanoploris total flavone suspension (200mg/kg) and allopurinol suspension (20mg/kg), continuously administer for 3 days, perfuse equal volume 0.5% sodium carboxymethylcellulose solution (0.5% CMC-Na) in CTR group and MOD group; (2) potassium oxonate induced mouse hyperuricemia model: after 1 hour of intragastric administration of herba Cephalanoploris total flavonoids and allopurinol, mice in MOD group, CIR group and ALP group were injected with Potassium Oxonate suspension (PO 300mg/kg) intraperitoneally, and mice in CTR group were injected with equal volume of 0.5% CMC-Na intraperitoneally for 7 days with continuous intervention. Fasting is carried out 8 hours before the last administration without water prohibition, and eyeball blood is removed after PO is injected into abdominal cavity for 1 hour for the last time. Standing the blood at room temperature for 60min, centrifuging at 4000rpm for 10min at 4 deg.C, collecting supernatant, and storing at-80 deg.C. The activity of uric acid and xanthine oxidase in the serum of each group of mice, and the levels of creatinine and urea in the serum are measured.
2. Test results
The uric acid and renal function conditions of the mice in each group are shown in figure 1, wherein figure 1A shows the result that the total flavonoids of herba cepbalanoplosis reduce the uric acid level in the serum of hyperuricemic mice induced by potassium oxonate, and figure 1B shows the result that the total flavonoids of herba cepbalanoplosis reduce the activity of xanthine oxidase in the serum of hyperuricemic mice; compared with the MOD group, the mice in the MOD group have relatively higher uric acid level and xanthine oxidase activity, which shows that the method successfully constructs a hyperuricemic mouse model, and compared with the MOD group, the method can reduce the serum uric acid level of the mice and inhibit the serum xanthine oxidase activity after CIR intervention, the two have statistical difference (P is less than 0.05), and the effect of CIR in reducing uric acid is similar to that of allopurinol.
Fig. 1C and fig. 1D show the results of total cirsium flavonoids improving the renal function serum creatinine and urea nitrogen in mice with high uric acid respectively, compared to MOD group, after CIR intervention, the mice serum creatinine and urea nitrogen levels can be reduced, and renal function can be improved, and compared to allopurinol group, the total cirsium flavonoids treatment has better effect of inhibiting the serum creatinine and urea nitrogen increase of mice induced by high uric acid, i.e. the total cirsium flavonoids have better effect of improving the renal function of mice induced by high uric acid compared to allopurinol group.
Example 3
In this example, the effect of total flavonoids of herba cepbalanoplosis on uric acid metabolism and renal function of a UOX knockout spontaneous hyperuricemic rat is analyzed, and the specific test method and the test result are as follows:
1. design of experiments
The experimental rat used in the experiment is an SD rat using a CRISPR-Cas9 system to knock out a UOX gene, purchased from Nanjing biomedical research institute, and a homozygote UOX gene knock-out high uric acid SD rat is obtained by repeatedly crossing F1 generation uricase gene knock-out heterozygotes (Uox-/+) rats. 30 high uric acid SD rats (UOX) with 6-7 weeks of age obtained by gene identification -/- ) The rat was randomly divided into a hyperuricemia model group (MOD, n ═ 10), a CIR total flavone pretreatment group (CIR, n ═ 10), an allopurinol pretreatment group (ALP, n ═ 10), and a littermate wild type SD rat was used as a blank control group (CTR, n ═ 10). CIR group and ALP group are separately irrigated with stomach herba Cephalanoploris total flavone suspension (130mg/kg), allopurinol suspension (20mg/kg), CTR group and MOD group are irrigated with equal volume of 0.5% sodium carboxymethylcellulose solution (0.5% CMC-Na), continuous intervention is carried out for 4 weeks, fasting is carried out for 8 hours before the last administration without water prohibition, 1 hour after the last administration, 1% sodium pentobarbital solution is injected into abdominal cavity for anesthesia, blood is taken from abdominal main artery, standing is carried out for 60min at room temperature, centrifugation is carried out for 10min at 4000rpm under 4 ℃ low-temperature environment, supernatant is collected, and the supernatant is stored at 80 ℃. The full-automatic biochemical analyzer is used for measuring the serum uric acid, serum creatinine and serum urea nitrogen levels, and the Cysc Elisa detection kit is used for detecting the level of serum cystatin C.
2. Results of the experiment
(1) Effect of total flavonoids of Cirsium setosum on body weight and dietary intake of UOX-/-rats
The weight change, food intake and water intake of the rats in each group are shown in fig. 2, and the results in fig. 2A show that the weights of the rats in the four groups have no obvious difference, and the comparative differences among the groups have no statistical significance. The food intake and water intake of rats in each group are respectively shown in fig. 2B and 2C, the food intake of rats in each group has no obvious difference between every two groups, the water intake of rats in the MOD group is obviously higher than that of rats in the CTR group, and the water intake of rats in the CIR group and the ALP group is slightly lower than that of rats in the MOD group, but the difference has no statistical significance. The above results indicate that CIR has less effect on body weight and on food intake in UOX-/-rats.
(2) Effect of total flavones of Cirsium setosum on uric acid and renal function in UOX-/-rats
Dry prognosis the change of uric acid and related indexes of renal function of rats in each group is shown in figure 3, wherein the blood uric acid level is shown in figure 3A, and the blood uric acid level of MOD group is significantly higher than that of CTR group (P < 0.05); compared with MOD group, serum uric acid of CIR group and ALP group are both reduced significantly (P < 0.05), but uric acid of rats in ALP group is reduced more significantly (P < 0.05); the detection results of rat renal function indexes serum creatinine, urea nitrogen and cystatin C are respectively shown in fig. 3B, fig. 3C and fig. 3D, and the serum creatinine, urea nitrogen and cystatin C in the MOD group are all obviously higher than those in the CTR group; after the dry prognosis of the herba cepbalanoplosis, serum creatinine, urea nitrogen and cystatin C in the CIR group are obviously reduced compared with those in the MOD group, and the differences have statistical significance (P is less than 0.05), while the indexes in the ALP group do not show obvious reduction, which shows that the herba cepbalanoplosis not only can reduce UOX -/- Uric acid levels in rats and has therapeutic effects on protecting renal function.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A preparation method of herba cepbalanoplosis segeti total flavonoids is characterized by comprising the following steps:
s1: degreasing the common cephalanoplos herb for later use;
s2: extracting the degreased herba cephalanoploris with alcohol to obtain herba cephalanoploris alcohol extract;
s3: dispersing the extract in water to obtain dispersion, adsorbing with macroporous resin, eluting, purifying, and drying to obtain herba Cephalanoploris total flavone.
2. The method of claim 1, wherein the step S1 is to defat the Cirsium setosum as follows:
drying herba Cephalanoploris, pulverizing into herba Cephalanoploris powder, adding diethyl ether, reflux extracting, defatting, and volatilizing diethyl ether.
3. The method of claim 1, wherein the step S2 is to extract the defatted herba Cirsii with ethanol to obtain an ethanol extract of herba Cirsii, and the method comprises the following steps:
adding 8-12 times of ethanol with mass concentration of 60% -80% into defatted herba Cephalanoploris powder, soaking for 0.5-1h, reflux extracting for 1-2h, collecting ethanol extractive solution, concentrating the ethanol extractive solution, and removing ethanol to obtain herba Cephalanoploris ethanol extract.
4. The method of claim 1, wherein the step S3 is performed by the following steps:
dispersing the extract in water to obtain dispersion, subjecting the dispersion to macroporous resin, eluting with 1-2 times of column volume of distilled water, and discarding water eluate; eluting with 3-4 times column volume of 10% -30% ethanol, and discarding the ethanol eluate; eluting with 5-6 times column volume of 70% -80% ethanol, collecting the ethanol eluate, recovering ethanol under reduced pressure until no ethanol smell exists, lyophilizing, and pulverizing to obtain herba Cephalanoploris total flavone.
5. Herba cepbalanoplosis segeti total flavonoids prepared by the method of any one of claims 1 to 4.
6. Application of herba Cephalanoploris total flavonoids in preparing medicine for preventing or treating hyperuricemia is provided.
7. Application of herba Cephalanoploris total flavonoids in preparing medicine for treating gout, uric acid renal calculus, tophus or uric acid nephropathy related to hyperuricemia is provided.
8. A medicament for preventing or treating hyperuricemia, comprising the total flavonoids of Cirsium setosum according to claim 5 and pharmaceutically acceptable salts or excipients thereof.
9. Application of herba Cephalanoploris total flavonoids in preparing xanthine oxidase inhibitor is provided.
10. A xanthine oxidase inhibitor comprising the total flavonoids of herba cephalanoploris of claim 5.
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| CN116832127B (en) * | 2023-08-22 | 2024-01-23 | 时珍本草生物科技(中山)有限公司 | Composite plant extract with uric acid reducing effect and preparation method thereof |
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