CN110893197B - Panax notoginseng extract for treating gout and preparation method thereof - Google Patents
Panax notoginseng extract for treating gout and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a panax notoginseng extract for treating gout and a preparation method thereof, wherein the panax notoginseng extract is formed by mixing extracts of panax notoginseng root stems and leaves, and comprises the following steps: (1) collecting root, stem and leaf of Panax notoginseng separately, oven drying, pulverizing, and sieving to obtain root, stem and leaf powder of Panax notoginseng; (2) respectively extracting the powder of the root, stem and leaf of the panax notoginseng by using a solvent, precipitating with ethanol, extracting and purifying by using column chromatography to obtain an extract of the root, stem and leaf of the panax notoginseng; (3) mixing the root, stem and leaf extracts of Panax notoginseng with the ratio of 1:1:2-1:1:3, and shaking to obtain Panax notoginseng extract, wherein the content of chlorogenic acid is not less than 40%, the content of isochlorogenic acid A, B and C is not less than 3%, and the content of cerebroside is not less than 0.15%. The panax notoginseng extract produced by the invention has good prevention and treatment effects on hyperuricemia and acute gouty arthritis, and has remarkable social and economic benefits.
Description
Technical Field
The invention belongs to the technical field of extraction and application of effective active ingredients of plants, and particularly relates to a panax notoginseng extract for treating gout and a preparation method thereof.
Background
Gout, a painful disease caused by metabolic disorders of uric acid, has afflicted humans for a long time. Once uric acid production and excretion balance are disrupted, blood uric acid levels will rise significantly, and the rise in uric acid levels will lead to urate crystals forming in joints and kidneys, directly leading to gouty arthritis and uric acid kidney stones. At present, gout develops from the past rare diseases to the present common diseases and is closely related to the changes of dietary habits and life styles of people. Gout is mostly seen in middle-aged men, and gout generation tends to be younger. The natural course of gout can be divided into four stages, namely, an asymptomatic hyperuricemia stage, an acute stage, an intermittent stage and a chronic stage. The long-standing gout also affects other tissues and organs of the human body, and causes complications of different degrees, such as hypertension, cardiovascular diseases, diabetes, liver and kidney injury and the like.
In treatment, the western medicines mostly adopt colchicine to relieve pain, allopurinol inhibits the generation of uric acid, and probenecid increases the excretion of uric acid. In the process of taking western medicines, various side effects are generated, including gastrointestinal reactions such as nausea, vomiting and diarrhea, the leucocyte is reduced, the functions of the liver and the kidney are damaged, and the like. The body of the patient is also damaged while the patient is treated. Therefore, natural drugs having low toxicity, low side effects and excellent therapeutic effects are required, and thus, the present inventors have been eagerly eager.
The botanical name of the Gynura procumbens (Gynura procumbens (Lour.) Merr.) is that the Gynura plant is a plant of Compositae, and also is a plant of the genus Panax, namely a Chinese-senna, a senecio and a Shenxiancao, which is pungent, slightly bitter and cool in nature, is a perennial herbal medicine and food dual-purpose plant and is widely distributed in China, Malaysia, Thailand, Indonesia, Korea, Philippine and other countries. Gynura procumbens has wide pharmacological effects, in the first half of 2009, toxicology inspection, hygiene inspection and inspection of various components are completed by relevant units such as the Chinese disease prevention and control center, the national food quality supervision and inspection center, the Chinese food fermentation industry research institute and the like, and the conclusion is that: no toxicity and no teratogenic influence; the effective components of the medicine have the effects of clearing and activating the channels and collaterals, diminishing inflammation and relieving cough, dissipating blood stasis and reducing swelling, promoting blood circulation and promoting tissue regeneration, treating traumatic injury, bronchopneumonia, phthisis and the like; can delay aging, activate immunocyte, improve organism immunity, enhance metabolism and improve dysmnesia; has the obvious effects of reducing blood pressure, blood fat and blood sugar, resisting oxidation and ulcer, preventing chronic nephropathy and inhibiting hepatitis B; has certain curative effect on preventing and treating cardiovascular and cerebrovascular diseases, diabetes and the like; it also has antiviral, antibacterial, and bone marrow cancer and shiga-like toxin cell inhibiting effects. On the other hand, gynura procumbens is a unique plant which can be used as both medicine and food and has extremely good nutritional value, so that the gynura procumbens is widely applied to the fields of food and medicine industry, daily chemical industry and the like and is a plant which can be used as both medicine and food and has extremely high potential and high economic value.
Disclosure of Invention
The invention aims to provide a panax notoginseng extract for treating gout and a preparation method thereof, which extracts active ingredients from panax notoginseng rhizomes and leaves by using methods such as solvent extraction, alcohol precipitation, solvent extraction, column chromatography separation and the like, and investigates the application of the panax notoginseng extract in gout.
In order to achieve the purpose, the invention adopts the following technical scheme:
a Panax notoginseng extract for treating gout is prepared by respectively extracting root, stem and leaf of Panax notoginseng respectively to obtain root extract, stem extract and leaf extract, and mixing the root extract, stem extract and leaf extract at a ratio of 1:1:2-1:1:3 to obtain Panax notoginseng extract.
Further preferably, the content of chlorogenic acid in the extract of panax notoginseng is not less than 40%, the content of isochlorogenic acid A, B and C is not less than 3%, and the content of cerebroside is not less than 0.15%.
A preparation method of a panax notoginseng extract for treating gout is characterized by comprising the following steps:
step 1): collecting root, stem and leaf of Panax notoginseng respectively, pulverizing, extracting with ethanol water solution at room temperature under ultrasonic condition, filtering, and concentrating under reduced pressure until no alcohol smell exists;
step 2): adding ethanol aqueous solution into the solutions obtained in the step 1), precipitating with ethanol, centrifuging, collecting supernatant, and concentrating under reduced pressure until no ethanol smell exists to obtain root, stem and leaf extractive solutions of Panax notoginseng;
step 3): extracting the panax notoginseng root extract with an organic solvent for three times, combining the extracts, and concentrating under reduced pressure to obtain a panax notoginseng root extract; extracting stem extract of Panax notoginseng with organic solvent for three times, mixing extractive solutions, and concentrating under reduced pressure to obtain Panax notoginseng stem extract;
step 4): subjecting the obtained extract to macroporous resin column chromatography, collecting the same fractions, mixing, concentrating under reduced pressure, and drying to obtain leaf extract of Panax schinseng;
step 5): mixing root, stem and leaf extracts of Panax notoginseng in proportion, and shaking to obtain Panax notoginseng extract.
Further, in the step 5), root, stem and leaf extracts of the panax notoginseng are mixed and shaken uniformly according to the mass ratio of 1:1:2-1:1:3 to prepare the panax notoginseng extract.
Furthermore, the content of chlorogenic acid in the extract of panax notoginseng is not less than 40%, the content of isochlorogenic acid A, B and C is not less than 3%, and the content of cerebroside is not less than 0.15%.
Further, in the step 1), the extraction solvent is 50-90% ethanol water solution with pH of 2-7, the liquid-material ratio is 1:10-20, the extraction temperature is normal temperature and ultrasonic extraction is carried out for 2 hours, and the filtrate is concentrated under reduced pressure until no alcohol smell exists.
Further, step 2) adding 80% -100% ethanol aqueous solution with volume 3-6 times of that of the solution obtained in step 1) respectively, and putting the solution into a refrigerator at 4 ℃ overnight for alcohol precipitation.
Further, in the step 2), 100% ethanol aqueous solution with 4 times volume of the solution obtained in the step 1) is respectively added into the solution, the solution is placed in a refrigerator at 4 ℃ overnight for alcohol precipitation, supernatant fluid is collected by centrifugation and is decompressed and concentrated until no alcohol smell exists, and root, stem and leaf extracting solutions of the panax notoginseng are respectively obtained.
Further, the organic solvent used in the step 3) is any one of petroleum ether, n-hexane, dichloromethane, ethyl acetate, n-butanol, dichloromethane/methanol and petroleum ether/ethyl acetate.
Further, preferably, the organic solvent used in step 3) is selected from dichloromethane/methanol (3:1) for the roots of Panax notoginseng and ethyl acetate for the stems of Panax notoginseng.
Further, the macroporous resin D101 used in the step 4) is washed by pure water, and then eluted and collected by 30% ethanol water solution, concentrated under reduced pressure and dried to obtain the extract of the leaves of the panax notoginseng.
Further, in the step 5), the root, stem and leaf extracts of the panax notoginseng are mixed and uniformly shaken according to the mode of 1:1:2-1:1:3 to prepare the panax notoginseng extract, wherein the content of chlorogenic acid is not lower than 40%, the content of isochlorogenic acid A, B and C is not lower than 3%, and the content of cerebroside is not lower than 0.15%.
The invention also provides application of the prepared panax notoginseng extract in preparing a medicine for treating gout.
The invention also provides application of the prepared panax notoginseng extract in preparing a medicine for treating hyperuricemia.
Due to the adoption of the technical scheme, the invention has the following advantages:
the invention relates to a panax notoginseng extract for treating gout and a preparation method thereof, which are used for obtaining maximum active ingredients by respectively extracting three organs according to different active ingredients contained in roots, stems and leaves of panax notoginseng, aiming at solving the problem that the extracted active ingredients cannot fully contain three active ingredients of chlorogenic acid, isochlorogenic acid A, B, C and cerebroside after the roots, stems and leaves of panax notoginseng are mixed and extracted. The prepared panax notoginseng extract contains chlorogenic acid, isochlorogenic acid A, B, C, cerebroside and other effective components, and explains the material basis of resisting gout. The prepared panax notoginseng extract has particularly good effect on patients with hyperuricemia and acute gouty arthritis, gout of patients who use the extract basically does not relapse, and the extract is obviously better than the root, stem and leaf extract and the panax notoginseng mixed extract of the panax notoginseng.
Drawings
FIG. 1 is a flow chart of the present invention.
FIG. 2 is a liquid chromatogram of the leaf extract of Panax schinseng C.A.Meyer of the present invention.
FIG. 3 is a liquid chromatogram of an extract of Panax notoginseng root of the present invention.
Fig. 4 is a liquid chromatography and high resolution mass spectrum of the stem extract of panax notoginseng of the present invention.
FIG. 5 is a graph showing the results of an anti-mouse hyperuricemia experiment, FIG. 5A is the effect of MSQ on the mouse blood uric acid content, FIG. 5B is the effect of MSQ on the XO activity in the mouse serum, FIG. 5C is the effect of MSQ on the mouse liver XO activity, FIG. 5D is the effect of MSQ on the hyperuricemia mouse serum SOD activity, FIG. 5E is the effect of MSQ on the hyperuricemia mouse serum GSH-Px activity, and FIG. 5F is the effect of MSQ on the hyperuricemia mouse serum ROS activity.
FIG. 6 is a graph showing the results of an experiment on the activity against acute gouty arthritis in rats, FIG. 6A is the effect of MSQ on the ankle joint swelling rate of rats with acute gouty arthritis, FIG. 6B is the effect of MSQ on the IL-1. beta. content in the serum of rats with acute gouty arthritis, FIG. 6C is the effect of MSQ on the IL-6 content in the serum of rats with acute gouty arthritis, FIG. 6D is the effect of MSQ on the IL-10 content in the serum of rats with acute gouty arthritis, FIG. 6E is the effect of MSQ on the MCP-1 content in the serum of rats with acute gouty arthritis, and FIG. 6F is the effect of MSQ on the TNF-. alpha. content in the serum of rats with acute gouty arthritis.
Detailed Description
The present invention will be further described in detail with reference to the following examples; however, the following examples are merely illustrative, and the present invention is not limited to these examples.
A preparation method of an extract of Panax quinquefolium for treating gout is shown in a flow chart 1, and comprises the following steps:
step 1): collecting root, stem and leaf of Panax notoginseng as raw materials, pulverizing, extracting with 50% -100% ethanol aqueous solution with pH of 2-7 at a ratio of 1: 5-1: 20 at room temperature under ultrasonic for 2 hr, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell is produced;
step 2): adding 80-100% ethanol aqueous solution with volume 3-6 times of that of the solution obtained in the step 1) respectively, putting the mixture into a refrigerator at 4 ℃ for overnight alcohol precipitation, centrifuging the mixture to collect supernatant, and concentrating the supernatant under reduced pressure until no alcohol smell exists to obtain root, stem and leaf extracting solutions of the panax notoginseng respectively;
step 3): extracting the panax notoginseng root extract with an organic solvent for three times, combining the extracts, and concentrating under reduced pressure to obtain a panax notoginseng root extract; extracting stem extract of Panax notoginseng with organic solvent for three times, mixing extractive solutions, and concentrating under reduced pressure to obtain Panax notoginseng stem extract;
step 4): subjecting the obtained extract to macroporous resin column chromatography, collecting the same fractions, mixing, concentrating under reduced pressure, and drying to obtain leaf extract of Panax schinseng;
step 5): mixing root, stem and leaf extracts of Panax notoginseng according to the mass ratio of 1:1:2-1:1:3, and shaking uniformly to obtain Panax notoginseng extract, wherein the content of chlorogenic acid is not less than 40%, the content of isochlorogenic acid A, B and C is not less than 3%, and the content of cerebroside is not less than 0.15%.
Example 1
Taking 100g of panax notoginseng leaves, crushing the leaves by using a crusher, adding the crushed leaves into a conical flask, extracting 50% ethanol water solution with pH 2 by using an extraction solvent at the normal temperature for 2 hours under ultrasonic extraction at a liquid-material ratio of 1:20, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution 4 times the volume of the obtained solution, placing in a refrigerator at 4 deg.C overnight for alcohol precipitation, centrifuging, collecting supernatant, concentrating under reduced pressure until no alcohol smell is obtained, and purifying by column chromatography, wherein the macroporous resin D101 is washed with pure water, and then eluted with 30% ethanol water solution for collection, concentration under reduced pressure, and drying to obtain 5.36g of folium Notoginseng extract with yield of 5.36%, and the liquid chromatogram of folium Notoginseng extract is shown in FIG. 2.
Taking 100g of panax notoginseng roots, crushing the panax notoginseng roots by using a crusher, adding the crushed panax notoginseng roots into a conical flask, extracting 50% ethanol water solution with pH 7 by using an extraction solvent at a liquid-material ratio of 1:20 at normal temperature for 2 hours by ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution 4 times the volume of the obtained solution, placing in a refrigerator at 4 deg.C overnight for alcohol precipitation, centrifuging, collecting supernatant, concentrating under reduced pressure until no alcohol smell is obtained to obtain extract of root of Panax notoginseng, extracting with solvent (dichloromethane/methanol (3: 1)), mixing the extracts, concentrating under reduced pressure, and drying to obtain 3.78g extract of root of Panax notoginseng with yield of 3.78%, wherein the liquid chromatogram of the extract of root of Panax notoginseng is shown in FIG. 2.
Taking 100g of panax notoginseng stems, crushing the stems by using a crusher, adding the crushed stems into a conical flask, extracting 50% ethanol water solution with pH 7 by using an extraction solvent at the liquid-material ratio of 1:20 at normal temperature for 2 hours by ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution 4 times the volume of the obtained solution, placing in a refrigerator at 4 deg.C overnight for alcohol precipitation, centrifuging, collecting supernatant, concentrating under reduced pressure until no alcohol smell is obtained to obtain stem extract of Panax notoginseng, extracting with solvent, wherein the extraction solvent is ethyl acetate, mixing the extractive solutions, concentrating under reduced pressure, and drying to obtain 2.78g of Panax notoginseng stem extract with yield of 2.78%, wherein the liquid chromatogram and high-resolution mass spectrogram of the Panax notoginseng stem extract are shown in FIG. 3.
Mixing root, stem and leaf extracts of Panax notoginseng with a ratio of 1:1:2, and shaking to obtain Panax notoginseng extract, wherein chlorogenic acid content is 41.23%, isochlorogenic acid A, B and C content are 3.12%, 3.25% and 3.42%, respectively, and cerebroside content is 0.21%.
Example 2
Taking 100g of panax notoginseng leaves, crushing the leaves by using a crusher, adding the crushed leaves into a conical flask, extracting 60% ethanol water solution with pH of 3 by using an extraction solvent at a liquid-material ratio of 1:15 at normal temperature for 2 hours by ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution with 5 times volume into the obtained solution, placing in a refrigerator at 4 ℃ overnight for alcohol precipitation, centrifuging, collecting supernatant, concentrating under reduced pressure until no alcohol smell is produced to obtain panax notoginseng leaf extract, purifying the panax notoginseng leaf extract by column chromatography, using macroporous resin D101, washing with pure water, eluting with 30% ethanol aqueous solution, collecting, concentrating under reduced pressure, and drying to obtain panax notoginseng leaf extract 5.67g, wherein the yield is 5.67%.
Taking 100g of panax notoginseng roots, crushing the panax notoginseng roots by using a crusher, adding the crushed panax notoginseng roots into a conical flask, extracting 70% ethanol water solution with pH 5 by using an extraction solvent at a liquid-material ratio of 1:20 at normal temperature for 2 hours by ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution with 4 times volume into the obtained solution, placing in a refrigerator at 4 deg.C overnight for alcohol precipitation, centrifuging, collecting supernatant, concentrating under reduced pressure until no alcohol smell is obtained to obtain radix Notoginseng root extract, extracting with solvent such as dichloromethane/methanol (3:1), mixing the extracts, concentrating under reduced pressure, and drying to obtain radix Notoginseng extract 3.45g with yield of 3.45%.
Taking 100g of panax notoginseng stems, crushing the stems by using a crusher, adding the crushed stems into a conical flask, extracting with 80% ethanol water solution with the pH value of 5 at the liquid-material ratio of 1:10 at normal temperature for 2 hours by ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution with 4 times volume of the obtained solution, putting into a refrigerator at 4 ℃ overnight for alcohol precipitation, centrifuging, collecting supernatant, concentrating under reduced pressure until no alcohol smell is produced to obtain stem extract of Panax notoginseng, extracting with solvent, wherein the extraction solvent is ethyl acetate, mixing the extracts, concentrating under reduced pressure, and drying to obtain 2.98g of Panax notoginseng stem extract with yield of 2.98%.
Mixing root, stem and leaf extracts of Panax notoginseng at a ratio of 1:1:3, and shaking to obtain Panax notoginseng extract, wherein chlorogenic acid content is 42.35%, isochlorogenic acid A, B and C content are 3.57%, 3.42% and 3.18%, respectively, and cerebroside content is 0.22%.
Example 3
Taking 100g of panax notoginseng leaves, crushing the leaves by using a crusher, adding the crushed leaves into a conical flask, extracting 80% ethanol water solution with pH 4 by using an extraction solvent at a liquid-material ratio of 1:20 at normal temperature for 2 hours by ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution with 5 times volume into the obtained solution, placing in a refrigerator at 4 ℃ overnight for alcohol precipitation, centrifuging, collecting supernatant, concentrating under reduced pressure until no alcohol smell is produced to obtain panax notoginseng leaf extract, purifying the panax notoginseng leaf extract by column chromatography, using macroporous resin D101, washing with pure water, eluting with 30% ethanol aqueous solution, collecting, concentrating under reduced pressure, and drying to obtain panax notoginseng leaf extract 5.44g, wherein the yield is 5.44%.
Taking 100g of panax notoginseng roots, crushing the panax notoginseng roots by using a crusher, adding the crushed panax notoginseng roots into a conical flask, extracting the panax notoginseng roots by using a 75% ethanol aqueous solution with pH 7 at a liquid-material ratio of 1:10 at normal temperature for 2 hours by ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution with 4 times volume into the obtained solution, placing in a refrigerator at 4 ℃ overnight for alcohol precipitation, centrifuging, collecting supernatant, concentrating under reduced pressure until no alcohol smell is produced to obtain Panax notoginseng root extract, extracting with solvent (dichloromethane/methanol (3: 1)) to obtain Panax notoginseng root extract, mixing the extracts, concentrating under reduced pressure, and drying to obtain 4.58g of Panax notoginseng root extract with yield of 4.58%.
Taking 100g of panax notoginseng stems, crushing the stems by using a crusher, adding the crushed stems into a conical flask, extracting with 80% ethanol water solution with pH of 6 at a liquid-material ratio of 1:10 at normal temperature for 2 hours by ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution 6 times the volume of the obtained solution, putting the obtained solution into a refrigerator at 4 ℃ overnight for alcohol precipitation, centrifuging to collect supernatant, concentrating under reduced pressure until no alcohol smell is produced to obtain panax notoginseng stem extract, extracting the panax notoginseng stem extract with solvent which is dichloromethane, mixing the extracts, concentrating under reduced pressure, and drying to obtain 2.98g of panax notoginseng stem extract, wherein the yield is 2.98%.
Mixing root, stem and leaf extracts of Panax notoginseng at a ratio of 1:1:3, and shaking to obtain Panax notoginseng extract, wherein chlorogenic acid content is 42.33%, isochlorogenic acid A, B and C content are 3.02%, 3.55% and 3.62%, respectively, and cerebroside content is 0.25%.
Example 4
Taking 100g of the leaf of the panax notoginseng, crushing the leaf of the panax notoginseng by using a crusher, adding the crushed leaf of the panax notoginseng into a conical flask, extracting the crushed leaf of the panax notoginseng into 70 percent ethanol aqueous solution with pH 4 at a liquid-material ratio of 1:20 at normal temperature for 2 hours by ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution with 4 times volume into the obtained solution, placing in a refrigerator at 4 ℃ overnight for alcohol precipitation, centrifuging, collecting supernatant, concentrating under reduced pressure until no alcohol smell is produced to obtain panax notoginseng leaf extract, purifying the panax notoginseng leaf extract by column chromatography, using macroporous resin D101, washing with pure water, eluting with 30% ethanol aqueous solution, collecting, concentrating under reduced pressure, and drying to obtain 6.35g of panax notoginseng leaf extract, wherein the yield is 6.35%.
Taking 100g of panax notoginseng roots, crushing the panax notoginseng roots by using a crusher, adding the crushed panax notoginseng roots into a conical flask, extracting 85% ethanol water solution with pH 5 by using an extraction solvent at a liquid-material ratio of 1:20 at normal temperature for 2 hours by ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution with 4 times volume into the obtained solution, placing in a refrigerator at 4 deg.C overnight for alcohol precipitation, centrifuging, collecting supernatant, concentrating under reduced pressure until no alcohol smell is obtained to obtain radix Notoginseng root extract, extracting with solvent (petroleum ether/ethyl acetate (3: 1)), mixing the extracts, concentrating under reduced pressure, and drying to obtain radix Notoginseng extract 4.85g with yield of 4.85%.
Taking 100g of panax notoginseng stems, crushing the stems by using a crusher, adding the crushed stems into a conical flask, extracting 90% ethanol water solution with pH 4 by using an extraction solvent at a liquid-material ratio of 1:15 at normal temperature for 2 hours by ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell exists. Adding 100% ethanol solution with 4 times volume of the obtained solution, putting the mixture into a refrigerator at 4 ℃ overnight for alcohol precipitation, centrifuging the mixture to collect supernatant, concentrating the supernatant under reduced pressure until no alcohol smell exists to obtain panax notoginseng stem extract, extracting the panax notoginseng stem extract by using a solvent which is n-butyl alcohol, combining the extracts, concentrating the extract under reduced pressure, and drying the extract to obtain 4.58g of panax notoginseng stem extract, wherein the yield is 4.58%.
Mixing root, stem and leaf extracts of Panax notoginseng with a ratio of 1:1:2, and shaking to obtain Panax notoginseng extract, wherein chlorogenic acid content is 43.65%, isochlorogenic acid A, B and C content are 2.98%, 3.86% and 3.78%, respectively, and cerebroside content is 0.28%.
Comparative example
Pulverizing root, stem and leaf of Panax quinquefolium L with pulverizer, adding into conical flask, extracting with 90% ethanol water solution with pH of 4 at a liquid-material ratio of 1:15 at room temperature under ultrasonic extraction for 2 hr, filtering, and concentrating the filtrate under reduced pressure until no alcohol smell is produced. Adding 100% ethanol solution with 4 times volume into the obtained solution, placing in a refrigerator at 4 ℃ overnight for alcohol precipitation, centrifuging to collect supernatant, concentrating under reduced pressure until no alcohol smell is produced to obtain a panax notoginseng mixed extract, extracting the panax notoginseng mixed extract with a solvent which is n-butyl alcohol, combining the extracts, concentrating under reduced pressure, and drying to obtain 1.45g of panax notoginseng mixed extract, wherein the content of chlorogenic acid in the panax notoginseng mixed extract is 35.45%, the content of isochlorogenic acid A, B and C are respectively 0.12%, 0.14% and 0.03%, and the content of cerebroside is not detected.
Effect of Panax quinquefolium extract on hyperuricemia of mice caused by Potassium Oxonate
132 male BALB/c mice were randomly divided into 11 groups (number n: 12/group), including a normal control group (CTRL) and a model control group (MC group), and were administered with saline at a dose of 10mL/kg by gavage, and the positive control group received 10mL/kg of febuxostat tablets (FBT) at a concentration of 0.6 mg/mL. The panax notoginseng root extract group (namely the MSQ administration group, obtained in example 1 and example 2, and the root, stem and leaf extracts of panax notoginseng are mixed according to the ratio of 1:1:2 to obtain the panax notoginseng extract, the root, stem and leaf extracts of panax notoginseng are mixed according to the ratio of 1:1:3 to obtain the panax notoginseng extract, the two ratios are respectively set as a low dose group and a high dose group, 75mg (low dose group) and 300mg (high dose group) of panax notoginseng extract powder are dissolved in 10mL of normal saline for intragastric administration, the dosages are 75mg/kg and 300mg/kg, and the panax notoginseng root extract group, the panax notoginseng stem extract group and the panax notoginseng leaf extract group are dissolved in 10mL of normal saline for intragastric administration by adopting 300mg (high dose) of powder. The mixed extract of panax notoginseng obtained in the comparative example is used for gastric lavage administration by dissolving 300mg (high dose) of powder in 10mL of normal saline. On days 1 to 8 of the experiment, 20g/kg of yeast extract powder was administered by gavage administration 12 hours before oral administration, except for normal control group mice. Potassium oxonate (OXO) was intraperitoneally injected into mice at a dose of 300mg/kg 1 hour prior to gavage from day 6 to day 8. According to the related method, the kit detects the content of mouse blood uric acid, the activity of Xanthine Oxidase (XO), and the content determination of oxidative stress related factors such as Superoxide Dismutase (SOD), Glutathione peroxidase (GSH-Px), and Reactive Oxygen Species (ROS).
The results are shown in fig. 5, and compared with the normal control group, the increase of UA level in serum of experimental mice in the model control group is more than 20%, and has obvious difference (P <0.05), which indicates that we have successfully constructed a hyperuricemia mouse model. Two doses of MSQ administration groups (fig. 5, administration group of panax notoginseng extract obtained by mixing root, stem and leaf extracts of panax notoginseng according to 1:1:2) respectively reduced UA levels in serum of mice with hyperuricemia, indicating that MSQ administration group has activity of treating hyperuricemia; has inhibitory effect on XO activity in serum of mouse with hyperuricemia; no effect on the activity of XO in the liver; can obviously improve the activity of SOD and GSH-Px in mouse serum; can reduce the level of ROS in serum.
The effect of the panax notoginseng root, stem and leaf extract on the increase of mouse UA caused by oteracil potassium is shown in table 1 (statistical analysis is performed by applying SPSS22.0 software, all detection results are expressed by mean ± standard deviation, and variance analysis is performed by comparing multiple groups), the results show that the individual panax notoginseng root, stem and leaf administration groups and the panax notoginseng mixed extract group have no effect on the UA level in the serum of hyperuricemia mice, the panax notoginseng root, stem and leaf extract is mixed according to 1:1:2 to obtain the panax notoginseng extract and the panax notoginseng root, stem and leaf extract, the low dose group of the panax notoginseng extract is significant compared with the model control group (P <0.05), the panax notoginseng root, stem and leaf extract is mixed according to 1:1:3 to obtain the panax notoginseng extract and the root, stem and leaf extract, the high dose group of the panax notoginseng extract is significant compared with the model control group (P <0.01), the root, stem and leaf of the panax notoginseng are extracted, and the extract of the root, stem and leaf of the panax notoginseng obtained by mixing the extracts in proportion reduces the UA level in the serum of a mouse with hyperuricemia, so that the MSQ administration group has the activity of treating hyperuricemia, and the root, stem and leaf administration group and the mixed extract of the panax notoginseng have no obvious activity of treating hyperuricemia.
TABLE 1 Effect of Panax notoginseng root, stem and leaf extracts on the increase of UA in mice by Potassium Oxonate
Compared with the normal control group, the composition has the advantages that,##P<0.01; compared with model group<0.05,**P<0.01 and P<0.001。
Panax notoginseng extract activity against acute gouty arthritis of rats
132 male Wistar rats were randomly divided into 11 groups (number n of each group was 12) including a normal control group (CTRL group) and a model control group (MC group), and the rats were gazed with 5mL/kg of physiological saline. The positive control group was Colchicine (COL) and COL was dissolved in physiological saline at a dose of 0.4mg/kg and at a volume of 5 mL/kg. The MSQ administration group (two groups of the root, stem and leaf extracts of the panax notoginseng mixed according to the ratio of 1:1:2 and the root, stem and leaf extracts of the panax notoginseng mixed according to the ratio of 1:1: 3) is subjected to gastric lavage administration by using panax notoginseng extract powder physiological saline, the administration dosage is 0.05g/kg (low dosage group) and 0.2g/kg (high dosage group), the administration dosage of the root extract group of the panax notoginseng, the stem extract group of the panax notoginseng and the leaf extract group of the panax notoginseng is 0.2g/kg, and the administration dosage of the mixed extract of the panax notoginseng (extracted according to the comparative example) is 0.2 g/kg. All administration groups were gavaged once a day for 8 days. On day 6 of dosing, rats were injected with 0.1mL of 30mg/m L sodium urate crystals (MSU) in the synovial space of the right ankle joint at 4:00 in the afternoon for 3 days. Normal control rats were treated with an equal volume of saline. The ankle joint circumference swelling rate on the right side of the rat is measured by using a vernier caliper, and biochemical factors related to inflammation in serum of the rat are measured by using an enzyme-linked immunosorbent assay (ELISA): IL-1 beta, IL-6, IL-10, MCP-1 and TNF-alpha.
The results are shown in FIG. 6 at 24h, 48h after injection of the sodium urate crystals. Compared with a normal control group, the swelling degrees of the right ankle joints of the rats with the acute gouty arthritis in the model group are obviously increased, and the result shows that the rat acute gouty arthritis model is successfully established by injecting sodium urate crystals into the right ankle joints of the rats. At the 48 th hour after the injection of the sodium urate crystals, the MSQ administration group (root: stem: leaf: 1:2) can obviously inhibit ankle swelling caused by the sodium urate crystals, and has obvious inhibition effect; the MSQ administration group can reduce the content of IL-1 beta, IL-6 and TNF-alpha in the serum of a rat with acute gouty arthritis; can increase the IL-10 content in the serum of the rat with acute gouty arthritis; can reduce the level of MCP-1 in the serum of rats.
Statistical analysis is carried out by using SPSS22.0 software, all detection results are expressed by mean +/-standard deviation, variance analysis is adopted for comparison among multiple groups, and as can be seen from table 2, the single root, stem and leaf of Panax notoginseng and the administration group of the mixed extract of Panax notoginseng have no obvious effect on inhibiting ankle swelling caused by sodium urate crystals, while the MSQ administration group (the root: stem: leaf: 1:2 group and the root: stem: leaf: 1:3 group) can obviously inhibit ankle swelling caused by sodium urate crystals, and has obvious inhibition effect.
TABLE 2 Effect of Panax quinquefolium root, Stem and leaf extract on swelling degree of right ankle joint of rat caused by sodium urate
Compared with the normal control group, the composition has the advantages that,##P<0.01，###P<0.001; compared with model group<0.05。
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. The panax notoginseng extract for treating gout and hyperuricemia is characterized by comprising the following components in parts by weight: respectively taking roots, stems and leaves of panax notoginseng as raw materials, respectively extracting an extract of panax notoginseng roots, an extract of panax notoginseng stems and an extract of panax notoginseng leaves, and then mixing the extract of panax notoginseng roots, the extract of panax notoginseng stems and the extract of panax notoginseng leaves according to the proportion of 1:1:2-1:1:3 to obtain an extract of panax notoginseng; the content of chlorogenic acid in the panax notoginseng extract is not less than 40%, the content of isochlorogenic acid A, B and C is not less than 3%, and the content of cerebroside is not less than 0.15%; the preparation method of the panax notoginseng extract comprises the following steps
Step 1): collecting root, stem and leaf of Panax notoginseng respectively, pulverizing, extracting with ethanol water solution at room temperature under ultrasonic condition, filtering, and concentrating under reduced pressure until no alcohol smell exists;
step 2): adding ethanol aqueous solution into the solutions obtained in the step 1), precipitating with ethanol, centrifuging, collecting supernatant, and concentrating under reduced pressure until no ethanol smell exists to obtain root, stem and leaf extractive solutions of Panax notoginseng;
step 3): extracting the panax notoginseng root extract with an organic solvent for three times, combining the extracts, and concentrating under reduced pressure to obtain a panax notoginseng root extract; extracting stem extract of Panax notoginseng with organic solvent for three times, mixing extractive solutions, and concentrating under reduced pressure to obtain Panax notoginseng stem extract; the organic solvent is any one of petroleum ether, n-hexane, dichloromethane, ethyl acetate, n-butanol, dichloromethane/methanol and petroleum ether/ethyl acetate;
step 4): subjecting the obtained extract to macroporous resin column chromatography, wherein the macroporous resin is D101, washing with pure water, eluting with 30% ethanol water solution, collecting eluate, mixing collected fractions, concentrating under reduced pressure, and drying to obtain folium Notoginseng extract;
step 5): mixing root, stem and leaf extracts of panax notoginseng according to the mass ratio of 1:1:2-1:1:3, and shaking uniformly to prepare the panax notoginseng extract.
2. A preparation method of a panax notoginseng extract for treating gout and hyperuricemia is characterized by comprising the following steps:
step 1): collecting root, stem and leaf of Panax notoginseng respectively, pulverizing, extracting with ethanol water solution at room temperature under ultrasonic condition, filtering, and concentrating under reduced pressure until no alcohol smell exists;
step 2): adding ethanol aqueous solution into the solutions obtained in the step 1), precipitating with ethanol, centrifuging, collecting supernatant, and concentrating under reduced pressure until no ethanol smell exists to obtain root, stem and leaf extractive solutions of Panax notoginseng;
step 3): extracting the panax notoginseng root extract with an organic solvent for three times, combining the extracts, and concentrating under reduced pressure to obtain a panax notoginseng root extract; extracting stem extract of Panax notoginseng with organic solvent for three times, mixing extractive solutions, and concentrating under reduced pressure to obtain Panax notoginseng stem extract; the organic solvent is any one of petroleum ether, n-hexane, dichloromethane, ethyl acetate, n-butanol, dichloromethane/methanol and petroleum ether/ethyl acetate;
step 4): subjecting the obtained extract to macroporous resin column chromatography, wherein the macroporous resin is D101, washing with pure water, eluting with 30% ethanol water solution, collecting eluate, mixing collected fractions, concentrating under reduced pressure, and drying to obtain folium Notoginseng extract;
step 5): mixing root, stem and leaf extracts of panax notoginseng according to the mass ratio of 1:1:2-1:1:3, and shaking uniformly to prepare panax notoginseng extracts; the content of chlorogenic acid in the extract of the panax notoginseng is not less than 40%, the content of isochlorogenic acid A, B and C is not less than 3%, and the content of cerebroside is not less than 0.15%.
3. The method for preparing the panax notoginseng extract for treating gout and hyperuricemia according to claim 2, wherein in the step 1), the extraction solvent is 50-90% ethanol water solution with pH =2-7, the liquid-material ratio is 1:10-20, the extraction temperature is normal temperature, ultrasonic extraction is performed for 2 hours, and the filtrate is concentrated under reduced pressure until no alcohol smell exists.
4. The method for preparing an extract of panax notoginseng for treating gout and hyperuricemia according to claim 2, wherein the step 2) comprises adding 80 to 100% ethanol aqueous solution in an amount of 3 to 6 times the volume of the solution obtained in the step 1), and performing alcohol precipitation in a refrigerator at 4 ℃ overnight.
5. Use of the extract of panax notoginseng as claimed in any one of claims 1 to 4 for the preparation of a medicament for the treatment of gout.
6. Use of an extract of Panax notoginseng as claimed in any one of claims 1 to 4 for the preparation of a medicament for the treatment of hyperuricemia.
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