TWI810657B - Use of a marigold extract for preparing a composition for reducing blood uric acid concentration - Google Patents

Abstract

The present invention provides a marigold extract with the effect of reducing uric acid in the blood. The preparation method comprises: taking a marigold plant ( Tagete sp.), carrying out reflux extraction with ethanol, and then removing the ethanol to obtain the marigold Chrysanthemum Extract. The marigold extract provided by the present invention and the quercetin tagetin contained therein can be used for preparing compositions for treating hyperuricemia or treating gout.

Description

Use of a marigold extract for preparing a composition for reducing blood uric acid concentration

The invention relates to the field of plant extracts, in particular to the technical field of using marigold extracts for reducing blood uric acid concentration.

Uric acid is a metabolite of purine. In the human body, purine can be metabolized by the liver to form uric acid, and then excreted with urine. However, when the amount of uric acid produced in the body is too high or the excretion is poor, it will accumulate in the body, resulting in too much uric acid in the blood. Clinically, when the fasting blood uric acid value is greater than 7.0 mg/dL in adults, regardless of gender, it is hyperuricemia.

Hyperuricemia is not only the most important risk factor for gout, but also has a clear correlation with cardiovascular disease, kidney disease, metabolic syndrome, diabetes and various complications, such as coronary heart disease, heart failure, atrial fibrillation, Erectile dysfunction, hypertension, chronic kidney disease, kidney stones, neuropathy, retinopathy, nephropathy, diabetic foot and vascular disease, etc.

The prevalence rate of hyperuricemia in western countries is about 2.3-17.6% in adult males, and the survey statistics in Taiwan are 17.3-25.8%; the prevalence rate of gout in foreign countries is 0.16-1.36%, and Taiwan is 0.16- 1.6%. Gout is more likely to occur in adult males aged 40-60, but it is currently increasing and younger.

At present, the treatment of hyperuricemia is still dominated by western medicine, and the therapeutic drugs can be divided into two categories: uric acid production inhibitors and uric acid excretion promoters. Uric acid production inhibitors are represented by Allopurinol, which is the most commonly used urate-lowering drug in clinical practice, while Febuxostat, which has recently been approved for marketing, is gradually widely accepted. Uric acid excretion accelerators include drugs such as probenecid and Benzbromarone.

Although these chemically synthesized western medicines have therapeutic effects, they may cause side effects such as allergies, skin rashes, gastrointestinal discomfort or stones in some patients. Therefore, the technical field of the present invention is in urgent need of a safe and effective ingredient for lowering uric acid from natural substances; it is also the problem to be solved by the present invention.

Purine is a metabolite of nucleic acid, nucleotide and nucleoside. Under the action of deaminase, purine is hydrolyzed and deaminated, such as adenine and guanine are hydrolyzed and deaminated to generate hypoxanthine respectively. (hypoxanthine) and xanthine (xanthine), hypoxanthine is oxidized to xanthine under the action of xanthine oxidase, and xanthine is further oxidized to uric acid under the action of xanthine oxidase. In addition, in the process of producing uric acid by xanthine oxidase, superoxide anion and hydrogen peroxide will be generated, which are the key enzymes that cause the increase of oxidative stress in the organism. Based on this, it can be seen that xanthine oxidase is the key to the synthesis of uric acid in the body, and inhibiting the activity of xanthine oxidase can reduce the production of uric acid.

Tagetes ( Tagetes sp.) is a perennial herb of Asteraceae, which contains polyphenolic compounds with special biological activities such as anti-oxidation and the ability to scavenge free radicals. However, whether tagetes can be used to reduce blood uric acid has not yet been investigated. It can be seen.

The object of the present invention is to provide a method for preparing a marigold extract with the effect of reducing uric acid in blood, which includes: step 1, take a marigold plant ( Tagete sp.), and carry out reflux extraction with ethanol, Then remove the ethanol to obtain a marigold ethanol extract; the marigold ethanol extract is the marigold extract.

In order to achieve the purpose of the aforementioned invention, the ratio of the marigold plant to the ethanol is 1 g: 1~30 mL; the preferred ratio of the marigold plant to the ethanol is 1 g: 2.5~25 mL; the The optimal ratio of marigold plants to this ethanol is 1 g: 5~20 mL.

In order to achieve the purpose of the aforementioned invention, the preparation method may further include: step 2, after suspending the marigold ethanol extract with water, perform liquid-liquid separation and extraction with n-hexane and water to obtain a n-hexane layer and a first A water layer; step 3, the first water layer is subjected to liquid-liquid separation and extraction with ethyl acetate to obtain an ethyl acetate layer and a second water layer, and the ethyl acetate in the ethyl acetate layer is removed Finally, a marigold ethyl acetate extract is obtained; the marigold ethyl acetate extract is the marigold extract.

In order to achieve the aforementioned purpose of the invention, the ratio of the ethanol extract of marigold to the n-hexane is 1 g: 1~30 mL; the preferred ratio of the ethanol extract of marigold to the n-hexane is 1 g: 2.5~ 20 mL; the optimal ratio of the marigold ethanol extract to the n-hexane is 1 g: 5~15 mL.

For the purpose of the aforementioned invention, wherein the ratio of the first water layer to the ethyl acetate is 1 g: 1~30 mL; the preferred ratio of the first water layer to the ethyl acetate is 1 g: 2.5~20 mL ; The optimal ratio of the first aqueous layer to the ethyl acetate is 1 g: 5-15 mL.

In order to achieve the purpose of the aforementioned invention, wherein the preparation method may further include: step 4, the second water layer is subjected to liquid-liquid separation and extraction with n-butanol to obtain a n-butanol layer and a third water layer, and the After the n-butanol in the n-butanol layer is removed, a marigold n-butanol extract is obtained; the marigold n-butanol extract is the marigold extract.

For the purpose of the aforementioned invention, wherein the ratio of the second water layer to the n-butanol is 1 g: 1-30 mL; the preferred ratio of the second water layer to the n-butanol is 1 g: 2.5-20 mL mL; the optimal ratio of the second aqueous layer to the n-butanol is 1 g: 5-15 mL.

In order to achieve the purpose of the aforementioned invention, the preparation method may further include: step 5, freeze-drying the third water layer to obtain a marigold water extract; the marigold water extract is the marigold extract.

The present invention further provides a marigold extract, which comprises the marigold extract prepared by the aforementioned preparation method.

The present invention further provides a marigold extract, which contains a main active ingredient, wherein the main active ingredient is quercetin.

The present invention further provides a use of a marigold extract for preparing a composition for treating hyperuricemia or gout, wherein the marigold extract is the aforementioned marigold extract.

The present invention also provides an application of quercetin tagetin in the preparation of a composition for treating hyperuricemia or gout.

In order to achieve the purpose of the aforementioned invention, wherein the treatment of hyperuricemia is to inhibit the activity of xanthine oxidase.

In order to achieve the purpose of the aforementioned invention, wherein the treatment of hyperuricemia is to reduce the uric acid value in blood.

The present invention further provides a composition comprising a marigold extract or quercetin as described above.

To achieve the purpose of the aforementioned invention, the composition may further include pharmaceutically acceptable carriers, excipients, and diluents.

To achieve the purpose of the aforementioned invention, wherein the composition comprises a pharmaceutical composition, a food composition or a composition thereof.

The present invention also provides a use of a marigold extract for preparing an anti-oxidation composition, wherein the marigold extract is the aforementioned marigold extract.

In conclusion, the marigold extract provided by the present invention has the characteristics of high antioxidant capacity, which can not only inhibit xanthine oxidase, but also exhibit the effect of significantly reducing the concentration of uric acid in blood. In addition, the marigold extract provided by the present invention is obtained from natural plants, has no significant toxicity, and has the advantages of both effectiveness and safety.

All technical and scientific terms used in this specification, unless otherwise defined, have meanings commonly understood by those skilled in the art.

The singular terms "a", "an" and "the" mentioned in this specification and claims may refer to more than one object unless otherwise specified.

"Or", "and", "and" used in this manual, unless otherwise stated, all refer to "or/and". In addition, the terms "comprising" and "including" are not limited open conjunctions. The foregoing paragraphs are for systematic references only and should not be construed as limitations on the subject matter of the invention.

The term "extract" as used in this specification refers to a product prepared by extraction. The extract may be presented as a solution in a solvent, or the extract may be presented as a concentrate or essence free or substantially free of solvent. The extract can also be prepared in pharmaceutical composition or food. The term extract can be a single extract obtained from a particular extraction step or a series of extraction steps, or an extract can also be a combination of extracts obtained from separate extraction steps. Accordingly, such combined extracts are also encompassed by the term "extract".

The term "treatment" refers to methods of delaying, ameliorating, reducing or reversing the disorder or any symptoms associated with the disorder currently afflicting a patient, as well as methods of preventing the disorder or any emerging symptoms thereof.

The composition of the present invention can be further added with an edible material to be prepared as a food product or a health care product. The edible material includes, but is not limited to: Water, Fluid milk products, Milk, Concentrated milk, Yogurt, Sour milk, Frozen yogurt, Lactic acid bacteria-fermented beverages, Milk powder, Ice cream, Cream cheeses, Dry cheeses, Soybean milk , Fermented soybean milk, Vegetable-fruit juices, Juices, Sports drinks, Confectionery, Jellys, Candies, Infant formulas ), Health foods, Animal feeds, Chinese herbals or Dietary supplements.

The composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent.

The term "pharmaceutically acceptable carrier" means that the substance or composition must be compatible with the other ingredients of the formulation and not deleterious to the patient.

The pharmaceutically acceptable carrier may contain one or more agents selected from the following: Solvent, Emulsifier, Suspending agent, Decomposer, Binding agent ), excipient (Excipient), stabilizer (Stabilizing agent), chelating agent (Chelating agent), diluent (Diluent), gelling agent (Gelling agent), preservative (Preservative), lubricant (Lubricant), surface Active agent (Surfactant), and other carriers similar or applicable to the present invention.

The term "pharmaceutically acceptable excipient", as used herein, means any physiologically inert, pharmacologically inert substance known to those skilled in the art to be compatible with the physical and chemical properties of the marigold extract Inactive substances. Pharmaceutically acceptable excipients include, but are not limited to, polymers, resins, plasticizers, fillers, lubricants, diluents, binders, disintegrants, solvents, co-solvents, surfactants, preservatives , sweeteners, flavoring agents, pharmaceutical grade dyes or pigments or viscosity agents.

Appropriate routes of administration of the marigold extract or composition of the present invention include, but are not limited to, oral, intravenous, rectal, aerosol, gastrointestinal tract, eye, lung, mucosal penetration, skin penetration, vaginal, For ear, nasal or topical administration.

Furthermore, examples of gastrointestinal administration include, but are not limited to, intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intramyelinary, intraventricular, intraperitoneal, intralymphatic or intranasal injections.

The dosage form of the composition provided by the present invention can be appropriately adjusted according to the needs, and is not particularly limited, and the oral dosage form is preferred.

The human effective dose of marigold extract can be calculated based on the effective dose for mice provided in the embodiment of the present invention, based on the difference in body surface area (the conversion factor between mice and human is 12.3 times) and the formula proposed by the US Food and Drug Administration: Assuming that the effective dose for mice is 50 mg/kg/day, the effective dose for human body is 50 / 12.3= 4.065mg/kg /day; based on this calculation, the effective dose for 40kg human body is 50 / 12.3 x 40= 162.6 mg/day, 100kg The effective dose for human body is 50 / 12.3 x 100=406.5 mg.

The materials used in the present invention are all commercially available materials, unless otherwise specified. The sweet marigold ( Tagete lucida ) used in the examples of the present invention can be purchased from the market or collected in the wild . Tagete lucida ) , plants of the genus Tagetes ( Tagete sp.) should be included in the present invention.

The animal experiment in the embodiment of the present invention used 8-week-old, 30-35 g ICR mice, which were purchased from Lesco Biotechnology Co., Ltd. After purchase, the mice were quarantined and reared for one week to adapt to the environment. Sufficient drinking water and feed are provided throughout the feeding process, the cages are kept clean, and the feeding environment: IVC temperature 20.8±0.4 ℃, humidity 46.4±4.6%, pressure difference 2.1±0.3 Pa, air change rate 80 times/hour, noise level 51.5±0.6 dB , light and dark switching cycle.

The novel technical features of the present invention, including specific features, are disclosed in the scope of the patent application. For the technical features of the present invention, a better understanding is hereby combined with the description, the embodiment based on the principles of the present invention, and the drawings to describe the preferred embodiments of the present invention Detailed description.

The present invention is illustrated by the following examples, but the present invention is not limited by the following examples. Embodiment 1, the preparation of marigold extract

As shown in the process of Figure 1, take a stem or leaf of sweet marigold ( Tagete lucida ), dry it and grind it into a fine powder, then take 1g of the dried fine powder, and carry out reflux extraction with 20mL of 95% ethanol , followed by concentrating and drying under reduced pressure to remove the ethanol to obtain ethanol extracts of marigolds (ethanol extracts, EE); take 1 g of the ethanol extracts of marigolds and add water to suspend them, then carry out the extraction with 10 mL of n-hexane (hexane) and water Liquid-liquid separation and extraction to obtain a n-hexane layer (Hexane layer) and a first water layer, after the n-hexane in the n-hexane layer was concentrated and removed under reduced pressure, a marigold n-hexane extract (Hexane Extracts , HE); then, the first aqueous layer was subjected to liquid-liquid separation and extraction with 10 mL of ethyl acetate to obtain an ethyl acetate layer and a second aqueous layer, and the acetic acid After the ethyl acetate in the ethyl layer was concentrated and removed under reduced pressure, a marigold ethyl acetate extract (Ethyl Acetate Extracts, EAE) was obtained; then, the second aqueous layer was dissolved in 10 mL of n-butanol (butanol) Liquid-liquid separation and extraction were carried out to obtain an n-butanol layer (n-butanol layer) and a third water layer, and the n-butanol in the n-butanol layer was concentrated and removed under reduced pressure to obtain a marigold n-butanol layer. butanol extracts (Butanol Extracts, BE); then, the third water layer was freeze-dried to obtain a marigold water extract (Water Extracts, WE). Example 2, Xanthine Oxidase Inhibition Ability Test of Marigold Extract

The marigold ethanol extract (take 50 μL), marigold n-hexane extract (take 50 μL), marigold ethyl acetate extract (take 50 μL), marigold normal Butanol extract (take 50 μL) and marigold water extract (take 50 μL), add 35 μL of 50 mM PBS solution and 30 μL of 0.1 U/mL xanthine oxidase solution respectively, at room temperature After 30 minutes of continuous action, add 25 μL of 1 N HCl to terminate the reaction, then measure the absorbance value at a wavelength of 290 nm, and use the sample without extract as the blank control group (Negative Control), and then use the formula 1 to calculate Xanthine oxidase inhibition rate of these extracts: Xanthine oxidase inhibition rate (%) = (Formula 1) Among them, OD1 is the absorbance value of the extract experimental group added with xanthine oxidase; OD2 is the absorbance value of the extract experimental group without xanthine oxidase added; OD3 is the blank control group added with xanthine oxidase OD4 is the absorbance value of the blank control group without xanthine oxidase.

The results of the xanthine oxidase inhibition test are as shown in Figure 2, the marigold extract exhibits significant inhibitory properties to xanthine oxidase in the concentration range of 50-500 μg/mL; wherein, marigold ethyl acetate Extract (code: EAE) has the best inhibitory effect, marigold ethanol extract (code: EE), marigold n-butanol extract (code: BE) and marigold water extract (code: WE) The suppression effect is suboptimal. Among them, the IC ₅₀ (in the linear range) of the marigold ethyl acetate extract (code: EAE) is about 98 μg/mL. Example 3. The effect of marigold ethyl acetate extract (EAE) on the blood uric acid value of mice with hyperuricemia

Potassium oxonate (PO) is a uricase inhibitor for rodents. Intraperitoneal injection of potassium oxonate can inhibit the action of uricase, preventing uric acid from being metabolized into allantoin in animals. Induces an increase in uric acid levels in animal blood.

In order to test the effect of marigold ethyl acetate extract (EAE) on the blood uric acid value of mice with hyperuricemia, take 375 mg of potassium oxonate and add 10 mg of sodium carboxymethylcellulose (Sodium carboxymethyl cellulose, NaCMC) and 2 drops of Tween-80 (Tween-80), then add an appropriate amount of water and mix well to form a 10 ml PO suspension.

The test system first administered 250 mg/kg body weight of oxonate potassium to mice by intraperitoneal injection to induce hyperuricemia. During the experiment, the mice were only supplied with drinking water, so as not to affect the detection of uric acid value due to diet. After the mice were induced by potassium oxonate, they were then tube-fed. After 2 hours, the uric acid value was monitored for 3 hours, and at intervals of 90 minutes, venous blood was taken to monitor the change of uric acid in the blood. The machine used for monitoring the uric acid level is the Yilisi blood glucose/uric acid dual-function tester (Model ET-201, Yuxin Biotechnology Co., Ltd., Taiwan). The specific test process is as follows: (1) Weigh the body weight of the mice. (2) Calculate the volume of intraperitoneal injection of potassium oxonate and the volume of tube feeding target. (3) Test the uric acid value of mice at T1 (0 hours induced by potassium oxonate). (4) Inject potassium oxonate (250 mg/kg) intraperitoneally to induce hyperuricemia in mice. (5) Immediately feed the target objects, grouped as follows: Negative control group: the target is saline (Saline); Positive control group: the standard substance is 10 mg/kg of allopurinol (Allopurinol); EAE 50 experimental group: the standard substance is 50 mg/kg ethyl acetate extract of marigold; EAE 100 experimental group: the standard substance is 100 mg/kg ethyl acetate extract of marigold; EAE 250 experimental group: the standard substance is 250 mg/kg ethyl acetate extract of marigold. (6) Test the uric acid value of mice at T2 (2 hours induced by potassium oxonate). (7) Test the uric acid value of mice at T3 (3.5 hours induced by potassium oxonate). (8) Test the uric acid value of mice at T4 (5 hours induced by potassium oxonate). (9) ANOVA two-factor variance analysis was used to detect the change of uric acid value between T3 and T2 of mice in each group.

The results are shown in Figure 3. In terms of the change of uric acid value between T3 (3.5 hours) and T2 (2 hours) in each group, the uric acid level of the mice fed allopurinol (the main drug for hyperuricemia) was The uric acid value of mice fed 50 mg/kg, 100 mg/kg and 250 mg/kg of marigold ethyl acetate extract decreased by 46%, 50% and 58% respectively; , the uric acid value of the mice fed 250 mg/kg of marigold ethyl acetate extract (EAE 250 mg/kg experimental group) was the most significant. From the above test results, it can be seen that marigold extract has the effect of significantly reducing the concentration of uric acid in the blood, which can achieve similar or better efficacy than the clinical drug (allopurinol) for treating hyperuricemia. (The marked ^** in Figure 3 represents a very significant difference (p <0.01); the marked ^* represents a significant difference (0.01 < p < 0.05).) Example 4, Tagetes ethyl acetate extract (EAE) Determination of antioxidant capacity and analysis of total phenols and total flavonoids

Determination of 1,1-diphenyl-2-trinitrophenylhydrazine (1,1-diphenyl-2-picrylhydrazyl, DPPH) free radical scavenging ability: the marigold ethyl acetate extract (EAE) described in Example 1 was dissolved After methanol, take 0.8 mL and 0.2 ml of 10 mM DPPH and mix evenly for 30 minutes, then detect the absorbance value at 517 nm, and replace the extract with methanol as a blank (Negative Control), and then calculate DPPH according to formula 2 Free radical scavenging ability: DPPH free radical scavenging rate (%) = (Formula 2) Among them, OD1 is the absorbance value of the experimental group of ethyl acetate extract of marigold added with DPPH; OD2 is the absorbance value of the experimental group of ethyl acetate extract of marigold without DPPH added; The absorbance value of the blank control group; OD4 is the absorbance value of the blank control group without DPPH.

Determination of reducing ability: Dissolve the marigold ethyl acetate extract (EAE) described in Example 1 in methanol and take 0.6 mL and 0.6 ml of 0.2 M sodium phosphate buffer (sodium phosphate buffer, pH 6.6) and 0.6 mL 1% K ₃ Fe(CN) ₆ aqueous solution was uniformly mixed, placed in a water bath at 50°C for 20 minutes, cooled rapidly, and then 0.6 mL of 10% trichloroacetic acid (trichloroacetic acid, TCA) was added at 3000 rpm After centrifugation for 10 minutes, take 1 mL of the supernatant, add 1 mL of H ₂ O and 0.2 mL of 0.1% FeCl ₃ and mix evenly, react at room temperature for 10 minutes, detect the absorbance at 700 nm, and use Different concentrations of butylated hydroxytoluene (BHT) were used as standard products, and then the reducing ability was calculated according to formula 3: Reducing ability (%) = (Formula 3) Among them, the absorbance value of the experimental group is the absorbance value of the experimental group with the addition of marigold ethyl acetate extract; the absorbance value of the control group is the absorbance value of the control group with methanol replacing the marigold ethyl acetate extract ; The absorbance value of the standard substance is the absorbance value of the standard substance when the ethyl acetate extract of marigold is replaced by BHT, and the reducing ability reaches saturation.

Analysis of total phenol content: using the Folin-Ciocalteu method, with gallic acid (gallic acid, GA) as the standard for determination.

Analysis of total flavonoids content: using the aluminum chloride chromogenic method, rutin (a flavonol quercetin and disaccharide rutinobiose (α-L-rhamnopyranosyl-(1→6)) -β-D-glucopyranose) formed between glycosides) as a reference standard.

According to the results in Table 1, the EC ₅₀ of the free radical scavenging ability of DPPH of marigold ethyl acetate extract (EAE) is 21.1 μg/mL; the reducing power of marigold ethyl acetate extract (EAE) is 687 mg BHT eq./g. The total phenolic compounds per gram of marigold ethyl acetate extract (EAE) is equivalent to 300 mg gallic acid equivalent; the total flavonoids per gram of marigold ethyl acetate extract (EAE) is equivalent to 700 mg of rutin equivalents. From the above results, we can know that marigold ethyl acetate extract (EAE) not only has high antioxidant capacity, but also is rich in polyphenols and flavonoids.

Table 1. Antioxidant capacity and total phenolic and total flavonoid contents of marigold ethyl acetate extract (EAE). DPPH radical scavenging capacity (EC ₅₀ ) ^* Restorative ability Total phenol content Total flavonoid content Marigold Ethyl Acetate Extract (EAE) 21.1 μg/mL 687 mg BHT eq./g 300 mg GA eq./g 700 mg Rutin eq./g *The EC50 of DPPH free radical scavenging ability of standard BHT is 32.4μg/mL. Example 5. Analysis of Active Index Components of Marigold Ethyl Acetate Extract (EAE)

Using column chromatography and high performance liquid chromatography (HPLC), further separate and purify the marigold ethyl acetate extract (EAE) described in Example 1, and use nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography in series Mass spectrometer (LC-MS/MS) was used to analyze its active index components. The specific method is as follows:

Take 1g of marigold ethyl acetate extract (EAE) and 30g of Sephadex, and carry out gradient extraction of 100-200 mL with 10 gradients of H ₂ O/MeOH (each gradient is: 90% H ₂ O/MeOH, 80% H ₂ O/MeOH, 70% H ₂ O/MeOH, 60% H ₂ O/MeOH, 50% H ₂ O/MeOH, 40% H ₂ O/MeOH, 30 % of H ₂ O/MeOH, 20% of H ₂ O/MeOH, 10% of H ₂ O/MeOH and 100% of MeOH), and collect the eluate at a fixed time, and combine the eluate from each tube into each Separation section, obtain 13 sub-separation sections altogether, as shown in Table 2:

Table 2 separation part Number of collection tubes separation part Number of collection tubes EA1 #2-6 EA8 #81-90 EA2 #7-10 EA9 #99-106 EA3 #11-30 EA10 #107-112 EA4 #31-44 EA11 #113-121 EA5 #45-57 EA12 #122-126 EA6 #58-68/#76-80/#91-98 EA13 #127-184 EA7 #69-75

Then, the equal fractions were prepared to 100 μg/mL, and the xanthine oxidase inhibitory ability test was carried out by the method described in Example 2. The results are shown in Table 3. The main active index component of marigold ethyl acetate extract (EAE) inhibiting xanthine oxidase is located in EA12, and the inhibition rate of xanthine oxidase of EA12 is 85.53%.

Table 3. Recovered weight and xanthine oxidase inhibitory activity of each fraction of sweet marigold EAE extract. EA1 EA2 EA3 EA4 EA5 EA6 EA7 Weight (mg) 62.1 54.6 203.8 144.3 68.2 92 26.7 Inhibition rate(%) 9.79 12.25 27.34 23.65 26.42 50.74 55.36 EA8 EA9 EA10 EA11 EA12 EA13 Weight (mg) 61.9 69.5 76.9 64.2 25.9 10.6 Inhibition rate(%) 55.97 34.11 58.13 69.83 85.53 31.96

Next, EA12 was further separated and purified by high-performance liquid chromatography (HPLC) and its components were analyzed. The specific method was: EA12 was collected by HPLC for isocratic extraction for 8 minutes, and the conditions were as follows: Pump: Hitachi L-2140; UV detector: Hitachi L-2420; reverse phase chromatography tube: Discovery®HS-C18 (250 x 10mm, 5 μm, Supelco); flow rate: 5 mL/min; injection volume: 400 mL; detection wavelength: 240 nm; mobile phase: acetonitrile (ACN).

Then, HPLC was used for isocratic eluting to collect the separation part with a retention time of 9.73 min. The conditions were as follows: Pump: Hitachi L-2140; UV detector: Hitachi L-2420; reversed-phase chromatography tube: Discovery®HS-C18 (250 x 10mm, 5 μm, Supelco); flow rate: 2.5 mL/min; injection volume: 100 μL; detection wavelength: 240 nm; mobile phase: methanol: acetonitrile: 0.1% trifluoroacetic acid (Trifluoroacetic acid) /H ₂ O = 50:10:40.

Then the fraction was concentrated to dryness, then dissolved in CH ₂ Cl ₂ -acetone (1:1), centrifuged, concentrated, and dried to obtain a yellow powder; the yellow powder was identified as quercetagetin (Quercetagetin, 3 , 3', 4', 5, 6, 7-Hexahydroxyflavone), the quercetin oxidase was tested for its ability to inhibit xanthine oxidase by the method described in Example 2, and it was found that it inhibited xanthine oxidase The IC ₅₀ is 60.5 μM. Among them, the identification data of the yellow powder are as follows: ¹ H NMR: 7.73 (1H, d, J = 2.0 Hz), 7.63 (1H, dd, J = 8.4 Hz), 6.88 (1H, d, J = 8.4Hz), 6.51 (1H, d, J = 2.5 Hz). ¹³ C NMR: 177.2, 154.5, 151.0, 148.6, 148.1, 146.7, 146.1, 136.8, 129.6, 124.3, 121.7, 116.2, 116.0, 104.7, 94.3. HRESIMS m/z 319.0443 [M+H] ⁺ (calcd for C ₁₅ H ₁₁ O ₈ ), m/z 317.0298 [MH] ^- (calcd for C ₁₅ H ₉ O ₈ ). IR (ν max 3379, 2978, 2839, 1659, 1612, 1570, 1493, 1420, 1381, 1319, 1281, 1254, 1204, 1126, 1092, 1057, and 1015 cm ^-1 .

Based on the above results, it can be seen that the marigold extract provided by the present invention is rich in polyphenols and flavonoids, and has a high antioxidant capacity. The marigold extract provided by the present invention can not only inhibit xanthine oxidase, but also has the effect of significantly reducing the concentration of uric acid in blood as confirmed by animal experiments; among them, the marigold ethyl acetate extract (EAE) The efficacy was the best, and the efficacy of marigold ethanol extract (EE), marigold n-butanol extract (BE) and marigold water extract (WE) was suboptimal. And it can be seen from the analysis that the active index component of the marigold ethyl acetate extract (EAE) provided by the present invention is quercetin, and its IC ₅₀ for inhibiting xanthine oxidase is 60.5 μM.

In addition to the above effects, the marigold extract provided by the present invention also has the characteristics of less obvious toxicity than natural extracts. Not only is it safer to use, but it can also be environmentally friendly and healthy when made into health food.

In the content disclosed in the preferred embodiments of this specification, those with ordinary knowledge in the field of the present invention can clearly understand that the foregoing embodiments are only examples; those with ordinary knowledge in the technical field of the present invention can implement them through many transformations and substitutions, and It is not different from the technical features of the present invention. According to the embodiments of the description, the present invention can have various transformations without hindering the implementation. The claims provided in this specification define the scope of the invention, which covers the aforementioned methods and structures and inventions equivalent thereto.

The multiple functions mentioned above are fully in line with the statutory patent requirements of novelty and advancement. Please file an application in accordance with the law and urge your office to approve this invention patent application to encourage inventions.

Fig. 1 is the preparation flowchart of the marigold extract provided by the present invention; Fig. 2 is the test result diagram of the inhibitory ability of the marigold extract of the present invention to xanthine oxidase; Fig. 3 is a graph showing the test results of the influence of the marigold extract of the present invention on the blood uric acid level of mice with hyperuricemia.

Claims (6)

A method for preparing a marigold extract with the effect of reducing uric acid in blood, which comprises: step 1, taking a marigold plant ( Tagete sp.), carrying out reflux extraction with ethanol, and then removing the ethanol to obtain 1. Marigold ethanol extract; step 2, after suspending the marigold ethanol extract with water, perform liquid-liquid separation and extraction with n-hexane and water to obtain a n-hexane layer and a first water layer; step 3 , carrying out liquid-liquid separation and extraction of the first water layer with ethyl acetate to obtain an ethyl acetate layer and a second water layer, and after removing the ethyl acetate in the ethyl acetate layer, a Wanshou Ethyl chrysanthemum acetate extract; step 4, carry out liquid-liquid separation and extraction of the second water layer with a n-butanol to obtain a n-butanol layer and a third water layer, and the n-butanol layer in the n-butanol layer After butanol is removed, a marigold n-butanol extract is obtained; and step 5, the third water layer is freeze-dried to obtain a marigold water extract; wherein the marigold extract contains the marigold Ethanol extract, the marigold ethyl acetate extract, the marigold n-butanol extract, and the marigold water extract; the ratio of the marigold plant to the ethanol is 1g: 1~30mL; The ratio of the marigold ethanol extract to the n-hexane is 1g: 1~30mL; the ratio of the first water layer to the ethyl acetate is 1g: 1~30mL; the ratio of the second water layer to the n-butanol The ratio is 1g: 1~30mL. A marigold extract, which comprises the marigold extract prepared by the preparation method described in Claim 1. A use of a marigold extract for preparing a composition for treating hyperuricemia or treating gout, wherein the marigold extract is the marigold extract as described in Claim 2. The use as described in claim 3, wherein the treatment of hyperuricemia involves inhibiting xanthine oxidase activity. The use as described in claim 3, wherein the treatment of hyperuricemia is to reduce the uric acid level in blood. A composition comprising a marigold extract as described in Claim 2.