CN108392509B

CN108392509B - Rape pollen extract and application thereof

Info

Publication number: CN108392509B
Application number: CN201810477607.7A
Authority: CN
Inventors: 张久亮; 王瑞丹; 张子程
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2018-05-18
Filing date: 2018-05-18
Publication date: 2021-07-02
Anticipated expiration: 2038-05-18
Also published as: CN108392509A

Abstract

The invention discloses an application of a rape pollen extract in preparing a medicine for reducing uric acid, and also discloses a rape pollen extract, which is prepared by the following method: (1) extracting the wall-broken rape pollen with 50-90% ethanol, and concentrating the extractive solution under reduced pressure until no ethanol exists; (2) extracting the extractive solution with water saturated n-butanol, concentrating n-butanol layer under reduced pressure, and vacuum freeze drying to obtain crude extract of rape pollen; (3) dissolving the crude extract with water, loading onto macroporous adsorbent resin column, washing with water, eluting with 30-70% ethanol, collecting ethanol eluate, concentrating under reduced pressure, and vacuum freeze drying. The results of in vitro cell experiments and in vivo animal experiments show that: the rape pollen extract can be used for reducing uric acid and treating gout.

Description

Rape pollen extract and application thereof

Technical Field

The invention belongs to the field of pharmacy, and relates to a rape pollen extract and application thereof, in particular to application in preparing a medicament for reducing uric acid.

Background

Gout is a group of metabolic diseases caused by purine metabolic disorder and/or uric acid excretion disorder, and urate deposition in joint parts can induce repeated attack of gouty acute arthritis, tophus deposition, joint deformity and the like. The kidney of a patient with severe gout is also damaged, such as uric acid kidney stones, chronic interstitial nephritis and the like. Hyperuricemia is the main biochemical basis for gout occurrence, is the 'fourth highest' after hypertension, hyperglycemia and hyperlipidemia, and seriously harms human health. Many studies indicate that hyperuricemia has close relationship with diabetes, hypertension, obesity, coronary heart disease, chronic kidney disease, etc. Recent epidemiological survey results show that the incidence of hyperuricemia and gout is on a continuous increase trend along with the improvement of living standard and the change of life style and dietary structure of people in recent years, and the incidence population tends to be younger, so that the search and development of medicines for preventing and treating gout and hyperuricemia are more and more important.

At present, the prevention of hyperuricemia and gout is generally to control the intake of high purine food and meat, increase the intake of fruits and vegetables, limit drinking, enhance exercise, and the like. The problem of uric acid metabolism abnormality cannot be fundamentally solved only by dietary intervention. For the treatment of hyperuricemia, most of the used medicines present toxic and side effects with different degrees. Therefore, the screening of high-efficiency, safe and nontoxic uric acid lowering drugs from natural products has gradually become a new direction of research.

The rape pollen contains various bioactive substances such as flavonoid compounds, carotenoid, sterol, spermidine compounds and the like, so that the rape pollen has wide biological effects such as oxidation resistance, inflammation resistance, radiation resistance, tumor inhibition, immunity regulation, bacteriostasis, blood fat regulation and the like. At present, no relevant literature report on intervention of the rape pollen extract in hyperuricemia exists at home and abroad.

Disclosure of Invention

The invention aims to provide a rape pollen extract and application thereof in preparing uric acid reducing medicines.

The results of in vitro cell experiments show that: the rape pollen extract has strong inhibition effect on xanthine oxidase and shows concentration dependence.

The results of in vivo animal experiments show that: (1) the rape pollen extract has no influence on the weight, the weight and the visceral index of the liver and the kidney of the mouse, which indicates that the invention can not damage the visceral index of the mouse;

(2) the rape pollen extract can reduce the serum UA, BUN and Cr of a hyperuricemia mouse, so that the kidney injury of the hyperuricemia mouse can be improved and relieved, the uric acid excretion is increased, and the rape pollen extract has mild effect and less side effect;

(3) the rape pollen extract can reduce XO activity in liver of mouse with hyperuricemia, and increase CAT activity and GSH content, thereby reducing uric acid production.

In conclusion, the cauliflower powder extract can be used for reducing uric acid and treating gout.

Preferably, the canola pollen extract is a canola pollen alcohol extract.

Further preferably, the alcohol is 50-90% ethanol, most preferably 75% ethanol.

Extracting rape pollen with ethanol, and concentrating the extractive solution to obtain rape pollen ethanol extract. Of course, in order to further improve the curative effect, reduce the side effect and reduce the dosage, the extract can be further subjected to impurity removal and refining treatment, for example, the impurity removal and refining treatment is carried out by adopting a polyamide column or a macroporous adsorption resin column.

The invention provides a rape pollen extract which is prepared by the following method, comprising the following steps:

(1) extracting the wall-broken rape pollen with 50-90% ethanol, and concentrating the extractive solution under reduced pressure until no ethanol exists;

(2) extracting the extractive solution with water saturated n-butanol until the n-butanol layer is colorless, concentrating the n-butanol layer under reduced pressure, and vacuum freeze drying to obtain crude extract of rape pollen;

(3) dissolving the crude extract with water, loading onto macroporous adsorbent resin column, washing with water until the eluate is not turbid, eluting with 30-70% ethanol, collecting ethanol eluate, concentrating under reduced pressure, and vacuum freeze drying to obtain rape pollen extract.

Preferably, the ethanol in step (1) is 75% ethanol.

Preferably, the macroporous adsorption resin column in the step (3) is an AB-8 type macroporous adsorption resin column.

Preferably, the ethanol in step (3) is 50% ethanol.

The extract prepared by the method has the advantages of highest active ingredient content, least impurities, strongest inhibitory activity on xanthine oxidase and best uric acid reducing effect.

The ethanol concentration referred to in the present invention is a volume concentration, such as 50% ethanol, and means that 50ml of ethanol is contained per 100ml of the ethanol aqueous solution.

Drawings

FIG. 1 shows XO inhibitory activity of canola pollen extract.

FIG. 2 is a graph showing the effect of canola pollen extract on serum uric acid levels in hyperuricemic mice.

FIG. 3 is a graph showing the effect of canola pollen extract on serum urea nitrogen levels in hyperuricemic mice.

FIG. 4 is a graph of the effect of canola pollen extract on serum creatinine levels in hyperuricemic mice.

FIG. 5 is the effect of canola pollen extract on XO activity in the liver of hyperuricemia mice.

FIG. 6 is a graph showing the effect of canola pollen extract on CAT activity in the liver of hyperuricemia mice.

FIG. 7 is a graph of the effect of canola pollen extract on GSH activity in the liver of hyperuricemia mice.

FIG. 8 is an HPLC-DAD chart of crude extract (A) and fragment 5(B) of canola bee pollen.

Detailed Description

The invention is further described with reference to the following figures and specific embodiments.

Example 1 preparation of extract

(1) Taking the rape pollen after wall breaking treatment, taking 75% ethanol as a solvent, and carrying out the following steps of 1: extracting with material-liquid ratio of 10 (m: V), filtering, collecting extractive solution, extracting the residue twice, mixing extractive solutions, and concentrating under reduced pressure until no ethanol exists.

(3) dissolving the crude extract with water, loading onto polyamide column, washing with distilled water until the eluate is not turbid, sequentially eluting with 30%, 50% and 75% ethanol, mixing eluates of different concentrations, concentrating under reduced pressure, and vacuum freeze drying to obtain fragment 1, fragment 2 and fragment 3; dissolving the crude extract with water, loading onto AB-8 type macroporous adsorbent resin column, washing with distilled water until the eluate is not turbid, sequentially eluting with 30% and 50% ethanol, mixing eluates of different concentrations, concentrating under reduced pressure, and vacuum freeze drying to obtain fragments 4 and 5.

EXAMPLE 2 XO inhibition by extracts

(1) Test method

Xanthine Oxidase (XO) catalyzes xanthine (Xan) to generate uric acid and has a characteristic absorption peak at 290nm, the absorbance is measured every 15s by adopting dynamics/time software of an ultraviolet spectrophotometer for 20 times, the absorbance is linearly increased along with the time in the period, and the slope is the reaction rate of the enzyme. The larger the slope, the more active the enzyme.

First, 5 fragments prepared in example 1 were dissolved in 1% DMSO to prepare sample solutions with different concentrations, and 100. mu.L of 7.5X 10 fragment was removed^-8The mol/L XO solution was mixed with 50. mu.L of the sample solution, incubated at 37 ℃ for 5min and then 100. mu.L of substrate solution Xan (5X 10)^-5mol/L), measuring the absorbance change at 290nm and calculating the slope R. The sample was replaced by the same volume of 0.05mol/L phosphate buffer solution (pH 7.5) as a blank, the change in absorbance was recorded and the slope R was calculated₀. Relative Activity (%) of xanthine oxidase ═ R/R₀X 100%. At the same time, the IC of 5 purified fragments were calculated by SPSS 13.0 software₅₀The most active fragment was evaluated and screened.

(2) Results of the experiment

FIG. 1 shows the inhibition of XO by 5 purified fragments, and the results show that 5 purified fragments all have inhibition of XO enzyme and show a certain concentration dependence relationship, i.e., the relative activity of XO is continuously reduced with the increase of the sample concentration. Compared with other 4 purified fragments, the fragment 5 has the lowest relative activity of XO at the concentration of 0-0.32 mg/mL, which indicates that the fragment 5 has stronger inhibition effect on XO. Table 1 shows the IC of 5 purified fragments for XO₅₀IC of value, fragment 5 vs XO₅₀Lowest value, followed by

fragment

43, 2 and 1. Thus, fragment 5 was the most potent fragment inhibiting XO activity.

TABLE 15 IC of different purified fragments for XO₅₀Value (

n＝4)

Sample (I)	IC₅₀Value (mg/mL)
		Fragment 1	0.34±0.04
Fragment 2	0.33±0.03
		Fragment 3	0.31±0.06
Fragment 4	0.23±0.02
		Fragment 5	0.21±0.02

EXAMPLE 3 evaluation of uric acid-lowering Effect of extract

(1) Test method

50 male Kunming mice of 18-22g are acclimatized for one week, animals are fed with common feed and drinking water freely, and after one week, the mice are randomly divided into 5 groups, namely a blank group, a model group, a positive group, a rape pollen crude extract group and a fragment 5 group, wherein each group comprises 10 mice. Mixing sodium carboxymethylcellulose with distilled water to obtain 0.5% solution, mixing potassium oxonate with 0.5% CMC-Na with distilled water, stirring to obtain potassium oxonate suspension, and mixing allopurinol as positive drug with 0.5% CMC-Na with distilled water to obtain allopurinol solution. Similarly, the crude extract and fraction 5 used in the sample group were mixed with 0.5% CMC-Na and distilled water, respectively, and stirred to prepare respective suspensions.

All mice were given a diet at 9 am each day and gavage started at 10 am. And (3) intragastric administration of 0.5% CMC-Na solution to the mice in the blank group, sequentially intragastric administration of the potassium oxonate suspension to the other four groups of mice according to the dose of 250mg/kg, and continuous intragastric administration for 7 days to construct a hyperuricemia mouse model. After 1 hour of intragastric Potassium Oxonate suspension and CMC-Na solution every day, the positive group of mice were intragastric allopurinol solution according to the dose of 5mg/kg, the sample group of mice were intragastric rape pollen crude extract and fragment 5 suspension respectively according to the dose of 200mg/kg, and the blank group and model group of mice were intragastric distilled water.

Measurement of organ index

All mice were given a diet 12 hours prior to gavage on day 7. The mice were weighed, and after the gavage was completed on day 7, the eyeballs were removed to obtain blood, and the mice were sacrificed by cervical dislocation. The liver and kidney tissues are taken out, rinsed in pre-cooled physiological saline at 4 ℃ and placed on filter paper. Weighing liver and kidney in sequence, freezing at 20 deg.C, and storing at-80 deg.C. Organ indices were calculated according to the following formula.

② measurement of UA, BUN and Cr contents in mouse serum

After continuous gavage for 7 days, the eyeball was removed and blood was taken, and the mouse was sacrificed by cervical dislocation. Naturally coagulating blood at room temperature for 1 hr, centrifuging at 4 deg.C and 3500rpm for 10min, collecting supernatant to obtain serum, subpackaging the serum, and freezing at-20 deg.C for storage. The contents of UA, BUN and Cr in the serum of mice were determined using Uric Acid (UA) test cell, urea nitrogen (BUN) test cell and creatinine (Cr) test cell.

Measuring the activity of XO and CAT and the content of GSH in the homogenate of mouse liver

Taking liver samples of mice of each group, adding pre-cooled physiological saline at 4 ℃, and mixing the pre-cooled physiological saline with the mixture according to a mass ratio of 1: 9, preparing a 10 percent liver tissue homogenate, centrifuging for 10min at 4 ℃ under 4000r/min, gently sucking off surface adipose tissues, slowly sucking supernatant, subpackaging and storing at-20 ℃ for later use. Total Protein (TP) test box is used to test total protein content in liver homogenate, and Xanthine Oxidase (XO) test box, Catalase (CAT) test box and reduced Glutathione (GSH) test box are used to test the activity of XO and CAT and GSH content in mouse liver homogenate.

(2) Results of the experiment

Influence of rape pollen extract on weight, liver and kidney weight and organ index of hyperuricemia mice

The body weight, liver and kidney weight and liver and kidney organ index of the mice after 7 days of continuous gavage are shown in table 2. The difference analysis is carried out on the weight and the index of the visceral organs of each group to obtain: the differences of the weight, the weight of the liver and the kidney and the organ indexes of the liver and the kidney of the mice among the groups are all not obvious (p is more than 0.05), which indicates that the rape pollen extract does not cause great damage to the organs of the mice.

TABLE 2 Effect of rape pollen extract on the body weight, liver and kidney weight and organ index of hyperuricemia mice

Group of	Body weight (g)	Liver weight (g)	Weight of kidney (g)	Liver organ index (%)	Kidney organ index (%)
						Blank group	29.76±1.84	1.47±0.10	0.47±0.05	4.96±0.50	1.57±0.15
Model set	29.44±1.60	1.44±0.12	0.48±0.03	4.84±0.27	1.60±0.12
						Positive group	29.89±1.24	1.45±0.07	0.44±0.04	5.13±0.23	1.56±0.12
Crude extract group	29.80±1.26	1.47±0.14	0.46±0.03	4.92±0.33	1.54±0.11
						Fragment 5 group	29.03±1.65	1.44±0.12	0.44±0.04	4.96±0.27	1.52±0.12

② the influence of rape pollen extract on the serum UA, BUN and Cr of hyperuricemia mouse

The serum UA value of each group of mice is shown in figure 2, compared with the blank group of mice, the serum UA value of the model group of mice is greatly increased (p is less than 0.001), and the animal model modeling success of the hyperuricemia mice is shown. Compared with a model group, the positive group of allopurinol can remarkably reduce the serum uric acid level of mice with hyperuricemia (p is less than 0.001), and the allopurinol has strong capability of reducing the serum UA and possibly generates certain side effects. The mouse serum UA value of the rape pollen crude extract group is 56.51 mu mol/L, which is reduced by about 39 percent compared with the model group (p <0.01), and the mouse serum UA value of the fragment 5 group is 52.24 mu mol/L, which is reduced by about 43 percent compared with the model group (p < 0.01). Therefore, the uric acid reducing capability of the fragment 5 is stronger than that of a crude rape pollen extract, the effect is milder than that of allopurinol, and the side effect is less.

Serum urea nitrogen (BUN) in each group of mice as shown in fig. 3, the serum BUN levels in the model group mice were significantly elevated (p <0.01) compared to the blank group, indicating a decrease in kidney function. Compared with the model group, the crude extract group and the fragment 5 group of the rape pollen both can remarkably reduce the serum BUN level of the hyperuricemia mice (p is less than 0.01), and the rape pollen can improve and relieve the kidney damage of the hyperuricemia mice. Wherein, after the crude extract is gavaged, the BUN value of the serum of the mouse is reduced by about 15 percent, and after the fragment 5 is purified by the gavage, the BUN value of the serum of the mouse is reduced by about 25 percent, so the fragment 5 can more effectively reduce the BUN level of the serum of the hyperuricemia mouse compared with the crude extract (p is less than 0.01).

The change in creatinine (Cr) levels in the mice of each group is shown in fig. 4, and the serum Cr levels in the mice of the model group are significantly increased (p <0.01) compared to the blank group, indicating that the renal function may be decreased. Compared with the model group, the rape pollen crude extract group and the fragment 5 group can obviously reduce the serum Cr level of the hyperuricemia mice (p is less than 0.01) by 23 percent and 36 percent respectively, which indicates that the rape pollen can improve the kidney injury caused by the hyperuricemia mice, and the fragment 5 can more effectively reduce the serum Cr level of the hyperuricemia mice compared with the crude extract.

③ the influence of the bee pollen extract on the activities of XO and CAT and the GSH content in the liver of the mouse with hyperuricemia

The change in the activity of XO in the liver of each group of mice is shown in fig. 5, and the activity of XO in the liver of the model group of mice induced by Potassium Oxonate was increased very significantly (p <0.001) compared to the blank group. Compared with the model group, allopurinol can reduce the activity of XO in the liver of hyperuricemia mice (p is less than 0.001) remarkably, and is even lower than the activity of XO in the liver of blank mice. The crude extract group and the fragment 5 group can both reduce the activity of XO in the liver of a hyperuricemia mouse (p is less than 0.01) by 25 percent and 34 percent respectively, and the fragment 5 can more effectively reduce the activity of XO in the liver of the hyperuricemia mouse (p is less than 0.01) compared with the crude extract, thereby reducing the generation of uric acid.

The change in CAT activity in XO livers of each group of mice is shown in fig. 6, with a very significant decrease in CAT activity in livers of model group mice compared to the blank group (p < 0.001). Compared with the model group, the crude extract group and the fragment 5 group can both remarkably improve the activity of CAT in the liver of the hyperuricemia mouse (p is less than 0.001) by about 37 percent and 53 percent respectively. Fragment 5 was more effective than the crude extract in increasing the activity of CAT in the liver of hyperuricemia mice (p < 0.01).

Changes in GSH activity in the XO livers of the groups of mice as shown in fig. 7, the GSH content in the livers of the model group of mice was very significantly reduced compared to the blank group (p < 0.001). Compared with the model group, the crude extract group and the fragment 5 group can both remarkably improve the content of GSH (p is less than 0.001) in the liver of the hyperuricemia mouse by about 17 percent and 27 percent respectively. Fragment 5 was more effective in increasing GSH content in the liver of hyperuricemia mice (p <0.01) than the crude extract.

Example 4 identification of uric acid-lowering effective ingredients in canola pollen extract

(1) Test method

Early in vivo animal experiment results show that the overall effect of the purified fragment 5 in reducing uric acid is better than that of the crude extract. Therefore, the invention mainly adopts HPLC-ESI-QTOF-MS/MS to carry out structural identification on the active ingredients in the rape pollen crude extract and the fragment 5.

Accurately dissolving 1mg of each of the crude extract and the fragment 5 of the rape pollen in chromatographic methanol to prepare a solution with the concentration of 1mg/mL, filtering the solution by using a filter membrane with the thickness of 0.45 mu m, collecting filtrate, and marking the filtrate for later use.

Chromatographic conditions are as follows: adopting an Accurate-Mass Q-TOF LC/MS 6520 system, taking a Diamonsil Plus C18 column (250mm multiplied by 4.6mm, 5 μm) as an analysis column, taking a sample amount of 5 μ L, taking 1% acetic acid solution (A) and acetonitrile (B) as mobile phases, and taking a flow rate of 0.5mL/min and gradient elution conditions of 0min and 5% B; 10min, 12% B; 15min, 16% B; 30min, 20% B; 40min, 30% B; 50min, 35% B; 60min, 50% B; 70min, 95% B; 75min, 5% B, 80min, 5% B. The column temperature was 30 ℃ and the DAD detector was used for detection analysis at 280 nm.

Mass spectrum conditions: the ESI ion source adopts a positive ion mode to capture molecular fragments, the flow rate of a drying gas is 10L/min, the temperature of the drying gas is 325 ℃, the pressure of an atomizing gas is 35psi, the voltage of a capillary is 3500V, and the scanning range of the mass-to-charge ratio is 50-1200 m/z.

(2) Results of the experiment

The liquid phase diagram of crude extract of rape pollen and fragment 5 is shown in FIG. 8. The crude extract of rape pollen mainly contains 8 compounds (peaks 1-8), and the fragment 5 mainly contains 3 compounds (peaks 4-6), which indicates that the material components of the crude extract and the purified fragment 5 are different. And the response values of the peaks 4, 5 and 6 in the fragment 5 are obviously higher than the response values of the peaks 4, 5 and 6 in the crude extract, which indicates that the content of the compounds 4, 5 and 6 in the fragment 5 is higher, so that the fragment is inferred to be an effective substance for reducing the uric acid of the rape pollen. The structures of the

compounds

1, 2 and 3 are identified and analyzed by HPLC-ESI-QTOF-MS/MSSequentially comprises quercetin-glucopyranoside, quercetin-rhamnopyranoside and kaempferol-glucopyranoside, and the structures of compounds 4, 5 and 6 are sequentially N¹,N⁵-di- (p-coumaroyl) -spermidine, N⁵,N¹⁰-di- (p-coumaroyl) -spermidine and N¹,N¹⁰-di- (p-coumaroyl) -spermidine. Therefore, the spermidine compound is judged to be the main effective component for reducing uric acid in the rape pollen extract.

Claims

1. The application of the rape pollen extract in the preparation of the uric acid reducing medicine is characterized in that: the rape pollen extract has xanthine oxidase inhibiting effect; can reduce serum uric acid, urea nitrogen and creatinine, thereby improving and relieving kidney injury of hyperuricemia and increasing uric acid excretion; can reduce xanthine oxidase activity in liver, improve catalase activity and reduced glutathione content, thus reduce the production of uric acid, the said rape pollen extract is prepared according to the following method, said method comprises the following steps:

(1) extracting the wall-broken rape pollen with 75% ethanol, and concentrating the extractive solution under reduced pressure until no ethanol exists;

(3) dissolving the crude extract with water, loading onto AB-8 type macroporous adsorbent resin column, washing with water until the eluate is not turbid, sequentially eluting with 30% and 50% ethanol, collecting 50% ethanol eluate, concentrating under reduced pressure, and vacuum freeze drying to obtain rape pollen extract.