CN108392509A - A kind of Pollen Brassicae campestris extract and application thereof - Google Patents
A kind of Pollen Brassicae campestris extract and application thereof Download PDFInfo
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Abstract
The invention discloses purposes of the Pollen Brassicae campestris extract in preparing anti-trioxypurine drug, and the invention also discloses a kind of Pollen Brassicae campestris extracts, it is prepared as follows：(1) rape pollen after broken wall treatment of learning from else's experience is extracted with 50 90% ethyl alcohol, when extracting solution is concentrated under reduced pressure into no ethyl alcohol until；(2) by the water saturated extracting n-butyl alcohol of extracting solution, n-butanol layer and vacuum freeze drying is concentrated under reduced pressure, obtains rape pollen crude extract；(3) it by upper large pore resin absorption column after crude extract water dissolution, is first rinsed with water, then with 30 70% ethanol elutions, collects ethanol eluate, through being concentrated under reduced pressure, after vacuum freeze drying to obtain the final product.In vitro cell experiment and interior animal experiment result show：Pollen Brassicae campestris extract can be used for anti-trioxypurine and treatment gout.
Description
Technical field
The invention belongs to pharmaceutical fields, are related to Pollen Brassicae campestris extract and application thereof, are especially preparing anti-trioxypurine drug
In purposes.
Background technology
Gout is one group of metabolic disease caused by purine metabolic disturbance and (or) uric acid excretion disorder, lithate
Gouty acute arthritis recurrent exerbation, tophaceous deposition and joint deformity etc. can be induced by being deposited on joint part.Serious pain
Its kidney of wind patient can also be damaged, such as cause uric acid kidney stones and arteriosclerotic kidney.Hyperuricemia is gout
The main biochemical basis of generation, is " the 4th is high " after hypertension, hyperglycemia, hyperlipidemia, seriously endangers the health of the mankind.
Many studies have shown that hyperuricemia and diabetes, hypertension, obesity, coronary heart disease, chronic renal disease etc. have close pass
System.Newest epidemiological survey is the results show that recently as the improvement of people's living standards, life style and dietary structure
Change, the trend continued to increase is presented in the incidence of hyperuricemia and gout, and morbidity crowd tends to rejuvenation, therefore,
It finds and the drug of research and development prevention gout and hyperuricemia is more and more important.
Currently, being usually the intake for controlling high purine food and meat for the prevention of hyperuricemia and gout, increasing
The intake of fruits and vegetables limits and drinks and enhance movement etc..Only by Dietary frequency, uric acid metabolism can't be fundamentally solved
Abnormal problem.Treatment for hyperuricemia, the drug big city used show different degrees of toxic side effect.Therefore,
The new direction that efficient, safe and non-toxic anti-trioxypurine drug has been increasingly becoming research is screened from natural products.
Contain a variety of biologies such as flavone compound, carotenoid, sterol and spermidine class compound in rape pollen
Active material, thus have extensive biological effect, such as anti-oxidant, anti-inflammatory, radioresistance, inhibit tumour, adjust it is immune, antibacterial,
Adjust blood fat etc..The pertinent literature for not intervening Pollen Brassicae campestris extract hyperuricemia both at home and abroad at present is reported.
Invention content
The object of the present invention is to provide a kind of Pollen Brassicae campestris extract and its purposes in preparing anti-trioxypurine drug.
In vitro cell experiment the result shows that：Pollen Brassicae campestris extract has stronger inhibiting effect to xanthine oxidase and is in
Existing concentration-dependent relation.
Interior animal experiment the result shows that：(1) Pollen Brassicae campestris extract is to the dirty of the weight of mouse, liver kidney weight and liver kidney
Device index does not influence, and illustrates that the present invention will not damage mice organs；
(2) Pollen Brassicae campestris extract can reduce hyperuricemia mice serum UA, BUN and Cr, thus can improve and
Alleviate the kidney injury of hyperuricemia mouse, increases uric acid excretion, and action temperature and few side effects；
(3) Pollen Brassicae campestris extract can reduce the activity of the XO in hyperuricemia mouse liver, improve CAT activity and GSH
Content, to reduce the generation of uric acid.
To sum up, cauliflower powder extracts can be used for anti-trioxypurine and treatment gout.
Preferably, the Pollen Brassicae campestris extract is rape pollen alcohol extracting thing.
It is further preferred that the alcohol is 50-90% ethyl alcohol, most preferably it is 75% ethyl alcohol.
Rape pollen is extracted with ethyl alcohol, rape pollen alcohol extracting thing is can be obtained after extracting solution is concentrated.Certainly,
In order to further increase curative effect, side effect is reduced, taking dose is reduced, extract can also be continued at impurity removal and purification
Reason carries out impurity removal and purification processing for example, by using polyamide column or large pore resin absorption column.
A kind of Pollen Brassicae campestris extract provided by the invention, it is prepared as follows, and this method includes following step
Suddenly：
(1) rape pollen after broken wall treatment of learning from else's experience is extracted with 50-90% ethyl alcohol, extracting solution is concentrated under reduced pressure into
Until when without ethyl alcohol；
(2) by the water saturated extracting n-butyl alcohol of extracting solution, until n-butanol layer is colourless, n-butanol layer is concentrated under reduced pressure
And vacuum freeze drying, obtain rape pollen crude extract；
(3) it by upper large pore resin absorption column after crude extract water dissolution, is first rinsed with water until eluent is not muddy,
Then 30-70% ethanol elutions are used, ethanol eluate is collected, obtaining rape pollen through being concentrated under reduced pressure, after vacuum freeze drying carries
Take object.
Preferably, the ethyl alcohol in step (1) is 75% ethyl alcohol.
Preferably, the large pore resin absorption column in step (3) is AB-8 type large pore resin absorption columns.
Preferably, the ethyl alcohol in step (3) is 50% ethyl alcohol
Minimum, the inhibition to xanthine oxidase using the extract active component content highest of this method preparation, impurity
Activity is most strong, and anti-trioxypurine effect is best.
Involved concentration of alcohol, is volumetric concentration in the present invention, such as 50% ethyl alcohol, refers to every 100ml ethyl alcohol
In aqueous solution, contain ethyl alcohol 50ml.
Description of the drawings
Fig. 1 is inhibitory activity of the Pollen Brassicae campestris extract to XO.
Fig. 2 is influence of the Pollen Brassicae campestris extract to hyperuricemia mice serum uric acid level.
Fig. 3 is influence of the Pollen Brassicae campestris extract to hyperuricemia mice serum urea nitrogen levels.
Fig. 4 is influence of the Pollen Brassicae campestris extract to hyperuricemia mice serum creatinine level.
Fig. 5 is Pollen Brassicae campestris extract on the active influences of XO in hyperuricemia mouse liver.
Fig. 6 is Pollen Brassicae campestris extract on the active influences of CAT in hyperuricemia mouse liver.
Fig. 7 is Pollen Brassicae campestris extract on the active influences of GSH in hyperuricemia mouse liver.
Fig. 8 is the HPLC-DAD figures of bee pollen form cole crude extract (A) and segment 5 (B).
Specific implementation mode
The present invention will be further described in the following with reference to the drawings and specific embodiments.
The preparation of 1 extract of embodiment
(1) rape pollen after broken wall treatment of learning from else's experience, using 75% ethyl alcohol as solvent, 1：10(m：V solid-liquid ratio) is carried
Take, filter and collect extracting solution, filter residue repeats extraction twice, when merging extracting solution and being concentrated under reduced pressure into no ethyl alcohol until.
(2) by the water saturated extracting n-butyl alcohol of extracting solution, until n-butanol layer is colourless, n-butanol layer is concentrated under reduced pressure
And vacuum freeze drying, obtain rape pollen crude extract；
(3) will upper polyamide column after crude extract water dissolution, first with distilled water flushing to eluent it is not muddy until, so
Afterwards successively with 30%, 50% and 75% ethanol elution, merge the eluent of each concentration, be concentrated under reduced pressure, vacuum freeze drying,
It is respectively labeled as segment 1, segment 2 and segment 3；Another upper AB-8 type large pore resin absorption columns after taking crude extract water dissolution, first
With distilled water flushing to eluent it is not muddy until, then successively with 30% and 50% ethanol elution, merge each concentration
Eluent, is concentrated under reduced pressure, and vacuum freeze drying is respectively labeled as segment 4 and segment 5.
Inhibiting effect of 2 extract of embodiment to XO
(1) test method
Xanthine oxidase (XO) catalysis xanthine (Xan) generates uric acid and has characteristic absorption peak at 290nm, using purple
The dynamics of outer spectrophotometer/time software measures an absorbance every 15s, measures 20 times, during this period of time inhales altogether
Luminosity linearly increases at any time, and slope is the reaction rate of enzyme.Slope is bigger, illustrates that the vigor of enzyme is stronger.
5 segments prepared by embodiment 1 are dissolved separately in first be configured in 1%DMSO various concentration sample it is molten
Then liquid pipettes 100 μ L 7.5 × 10-8Mol/L XO solution and 50 μ L sample solution mix, and 100 are added after 37 DEG C of incubation 5min
The substrate solution Xan (5 × 10 of μ L-5Mol/L) start reaction, measure the absorbance change under 290nm wavelength and calculate slope R.
It replaces sample as blank control using the 0.05mol/L phosphate buffer solutions (pH=7.5) of same volume, records its absorbance
Variation and calculate slope R0.Relative activity (%)=R/R of xanthine oxidase0× 100%.It is soft by SPSS 13.0 simultaneously
Part calculates separately the IC of 5 purified fragments50It is worth and screens the most strong segment of inhibitory activity.
(2) experimental result
Fig. 1 shows inhibiting effect of 5 purified fragments to XO, as a result shows that 5 purified fragments have suppression to XO enzymes
It makes and uses and certain concentration-dependent relation is presented, i.e., with the increase of sample concentration, the relative activity of XO constantly reduces.Segment
5 have the relative activity of minimum XO compared to other 4 purified fragments in 0~0.32mg/mL of concentration, illustrate segment 5 to XO
With stronger inhibiting effect.Table 1 shows IC of 5 purified fragments to XO50Value, IC of the segment 5 to XO50Be worth it is minimum, secondly
For segment 4,3,2 and 1.Thus illustrate that segment 5 is to inhibit the strongest segment of XO activity.
IC of the different purified fragments of 15, table to XO50Value (N=4)
| Sample | IC50It is worth (mg/mL) |
| Segment 1 | 0.34±0.04 |
| Segment 2 | 0.33±0.03 |
| Segment 3 | 0.31±0.06 |
| Segment 4 | 0.23±0.02 |
| Segment 5 | 0.21±0.02 |
3 extract anti-trioxypurine evaluation of effect of embodiment
(1) test method
50 18-22g male mouse of kunming progress one week are adapted to raise, animal ad lib normal diet and drink
Mouse is randomly divided into 5 groups by water after a week, respectively blank group, model group, positive group, rape pollen crude extract group and
5 groups of segment, every group 10.Sodium carboxymethylcellulose plus distilled water are mixed be configured to 0.5% solution, Oteracil Potassium with
0.5%CMC-Na adds distilled water mixing to stir evenly, and is configured to the suspension of Oteracil Potassium, positive drug allopurinol with
0.5%CMC-Na adds distilled water mixing to be configured to allopurinol solution.Similarly, the crude extract used in sample sets and segment 5
Add distilled water to mix stirring with 0.5%CMC-Na respectively and is configured to corresponding suspension.
Run out of grain to all mouse for 9 points in every morning, 10 points start gavage.Naive mice gavage 0.5%CMC-
Na solution, by the suspension of Oteracil Potassium according to the dosage of 250mg/kg to remaining four groups of mouse successively gavage, continuous gavage 7
It, builds antihyperuricemic disease mouse model.After the suspension and CMC-Na solution 1 hour of daily gavage Oteracil Potassium, sun
Property group mouse according to 5mg/kg dosage gavage allopurinol solution, sample sets mouse according to 200mg/kg dosage distinguish gavage
The suspension of rape pollen crude extract and segment 5, blank group and model group intragastric administration on mice distilled water.
1. the measurement of organ index
Ran out of grain to all mouse within 12 hours before gavage at the 7th day.The weight for weighing mouse, terminated in the 7th day gavage
Afterwards, it plucks eyeball and takes blood, the neck that breaks puts to death mouse.Its liver, renal tissue are taken out, rinsed clean in the physiological saline being pre-chilled at 4 DEG C
It is placed on filter paper.It weighs successively to liver and kidney, being placed on -80 DEG C in 20 DEG C of freezings saves backup.According to following formula meter
Calculate organ index.
2. the measurement of mice serum UA, BUN, Cr content
Continuous gavage plucks eyeball after 7 days and takes blood, and the neck that breaks puts to death mouse.Blood is at ambient temperature after natural blood coagulation 1h in 4
3500rpm centrifuges 10min at a temperature of DEG C, takes its supernatant up to serum, -20 DEG C of freezen protectives are positioned over after serum is dispensed,
It is spare.Mice serum UA, BUN are measured using uric acid (UA) testing cassete, urea nitrogen (BUN) testing cassete and creatinine (Cr) testing cassete
And the content of Cr.
3. the vigor of XO, CAT and the measurement of GSH contents in mouse liver homogenate
The liver samples of each group mouse are taken, 4 DEG C of precooled physiological saline are added, according to mass ratio 1：9 ratio is made
10% hepatic homogenate after centrifuging 10min under the conditions of 4 DEG C of 4000r/min, after gently sopping up surface fat tissue, delays
Slow Aspirate supernatant, and dispensed, -20 DEG C are stored in spare.It is even that liver organization is measured using total protein (TP) testing cassete
Total protein content in slurry, and using xanthine oxidase (XO) testing cassete, catalase (CAT) testing cassete, reduced form paddy
The sweet peptide of Guang (GSH) testing cassete come measure mouse liver tissue homogenate in the vigor of XO, CAT and the content of GSH.
(2) experimental result
1. influence of the Pollen Brassicae campestris extract to hyperuricemia mouse weight, liver kidney weight and organ index
The organ index of mouse weight, liver kidney weight and liver kidney is as shown in table 2 after continuous gavage 7 days.To each group internal organs weight
Amount and organ index obtain after carrying out difference analysis：The organ index of the weight of mouse between each group, liver kidney weight and liver kidney
Equal unobvious (the p of difference>0.05), illustrate that Pollen Brassicae campestris extract does not cause prodigious damage to mice organs.
Influence of 2 Pollen Brassicae campestris extract of table to hyperuricemia mouse weight, liver kidney weight and organ index
| Group | Weight (g) | Liver weight (g) | Kidney weight (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 group | 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 |
| 5 groups of segment | 29.03±1.65 | 1.44±0.12 | 0.44±0.04 | 4.96±0.27 | 1.52±0.12 |
2. influence of the Pollen Brassicae campestris extract to hyperuricemia mice serum UA, BUN and Cr
For the serum UA values of each group mouse as shown in Fig. 2, compared with naive mice, the serum UA values of model group mouse are extremely aobvious
It writes and rises (p<0.001), show hyperuricemia mouse model modeling success.Compared with model group, positive group purine
Alcohol can pole significant decrease hyperuricemia mice serum uric acid level (p<0.001), show that allopurinol reduces serum UA abilities
It is too strong, certain side effect may be will produce.The mice serum UA values of rape pollen crude extract group are 56.51 μm of ol/L, compare mould
Type group has dropped about 39% (p<0.01), the mice serum UA values of 5 groups of segment are 52.24 μm of ol/L, are had dropped about than model group
43% (p<0.01).It can be seen that the anti-trioxypurine ability of segment 5 is better than rape pollen crude extract, and act on than other purine
Alcohol is milder, and side effect is less.
The serum urea nitrogen (BUN) of each group mouse is as shown in figure 3, compared with blank group, the serum BUN water of model group mouse
HUD, which writes, increases (p<0.01), show that its renal function is declined.Compared with model group, rape pollen crude extract group and piece
Section 5 groups can pole significantly reduce hyperuricemia mouse serum BUN levels (p<0.01), show rape pollen can improve and
Alleviate the kidney injury of hyperuricemia mouse.Wherein, after gavage crude extract, mice serum BUN values have dropped about 15%, gavage
After purified fragments 5, mice serum BUN values have dropped about 25%, therefore segment 5 can more effectively reduce height compared to crude extract
The serum BUN levels (p of uricacidemia mouse<0.01).
For the horizontal variation of the creatinine (Cr) of each group mouse as shown in figure 4, compared with blank group, model group mice serum Cr is horizontal
Significantly increase (p<0.01), show that its renal function may be declined.Compared with model group, rape pollen crude extract group and
5 groups of segment can significantly reduce the change of serum C r levels (p of hyperuricemia mouse<0.01) 23% and 36%, table, are reduced respectively
Clear rape pollen can improve the kidney injury caused by hyperuricemia mouse, and segment 5 can be with compared to crude extract
The change of serum C r for more effectively reducing hyperuricemia mouse is horizontal.
3. influence of the bee pollen extract to the activity and GSH contents of XO, CAT in hyperuricemia mouse liver
In the liver of each group mouse the activity change of XO as shown in Figure 5, compared with blank group, Oteracil Potassium induction model
The activity of XO significantly increases (p by pole in group mouse liver<0.001).Compared with model group, allopurinol can pole significantly drop
Activity (the p of XO in low hyperuricemia mouse liver<0.001), even lower than in naive mice liver XO activity.Slightly carry
5 groups of object group and segment can pole significantly decrease the activity (p of XO in hyperuricemia mouse liver<0.01) it, reduces respectively
25% and 34%, segment 5 can more effectively reduce the activity (p of XO in hyperuricemia mouse liver compared to crude extract<
0.01), to reduce the generation of uric acid.
The activity change of CAT is as shown in fig. 6, compared with blank group in the XO livers of each group mouse, model group mouse liver
The active pole of middle CAT significantly reduces (p<0.001).Compared with model group, 5 groups of crude extract group and segment can pole significantly increase
Activity (the p of CAT in hyperuricemia mouse liver<0.001), it has been respectively increased about 37% and 53%.Segment 5 is carried compared to slightly
Object can more effectively improve the activity (p of CAT in hyperuricemia mouse liver<0.01).
The activity change of GSH is as shown in fig. 7, compared with blank group in the XO livers of each group mouse, model group mouse liver
The content pole of middle GSH significantly reduces (p<0.001).Compared with model group, 5 groups of crude extract group and segment can pole significantly increase
Content (the p of GSH in hyperuricemia mouse liver<0.001), it has been respectively increased about 17% and 27%.Segment 5 is carried compared to slightly
Object can more effectively improve the content (p of GSH in hyperuricemia mouse liver<0.01).
The identification of anti-trioxypurine active ingredient in 4 Pollen Brassicae campestris extract of embodiment
(1) test method
Early period, interior animal experiment was the results show that the anti-trioxypurine overall effect of purified fragments 5 is better than crude extract.Therefore this
Invention mainly carries out structure using HPLC-ESI-QTOF-MS/MS to the active ingredient in rape pollen crude extract and segment 5
Identification.
Accurate rape pollen crude extract and 5 each 1mg of segment are dissolved in chromatography methanol, are configured to the molten of a concentration of 1mg/mL
Liquid is collected filtrate, is marked with 0.45 μm of membrane filtration, spare.
Chromatographic condition：Using 6520 systems of Accurate-Mass Q-TOF LC/MS, with Diamonsil Plus C18
Column (250mm × 4.6mm, 5 μm) is analytical column, and 5 μ L of sample size, 1% acetic acid solution (A) and acetonitrile (B) are used as mobile phase, flow velocity
For 0.5mL/min, condition of gradient elution 0min, 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.Column temperature 30
DEG C, DAD detectors are detected analysis at 280nm.
Mass Spectrometry Conditions：ESI ion sources capture molecular fragment using positive ion mode, and dry gas stream speed is 10L/min, is done
Pathogenic dryness temperature is 325 DEG C, atomization pressure 35psi, capillary voltage 3500V, and mass-to-charge ratio scanning range is 50-
1200m/z。
(2) experimental result
Liquid phase figure in rape pollen crude extract and segment 5 is as shown in Figure 8.Rape pollen crude extract mainly contains 8 changes
Object (peak 1-8) is closed, segment 5 mainly contains 3 compounds (peak 4-6), has illustrated the material composition of crude extract and purified fragments 5
Difference.And the response at peak 4,5,6 is apparently higher than the response at peak 4,5,6 in crude extract in segment 5, shows in segment 5 and changes
The content higher of object 4,5,6 is closed, it is inferred that it is the active principle of rape pollen anti-trioxypurine.Through HPLC-ESI-QTOF-MS/
The structure of MS identification and analysis, compound 1,2,3 is followed successively by Quercetin -- glucopyranoside, Quercetin-rhamnopyranosyloxyhy glucosides, mountain
How phenol-glucopyranoside, the structure of compound 4,5,6 is followed successively by N1,N5- two-(p-Coumaric Acid acyl)-spermidines, N5,N10-
Two-(p-Coumaric Acid acyl)-spermidines and N1,N10- two-(p-Coumaric Acid acyl)-spermidines.Therefore judge that spermidine class compound is
The principle active component of anti-trioxypurine in Pollen Brassicae campestris extract.
Claims (7)
1. purposes of the Pollen Brassicae campestris extract in preparing anti-trioxypurine drug.
2. purposes as described in claim 1, it is characterised in that：The Pollen Brassicae campestris extract is rape pollen alcohol extracting thing.
3. purposes as claimed in claim 2, it is characterised in that：The alcohol is 50-90% ethyl alcohol.
4. a kind of Pollen Brassicae campestris extract, it is characterised in that be prepared as follows, this approach includes the following steps：
(1) rape pollen after broken wall treatment of learning from else's experience is extracted with 50-90% ethyl alcohol, and extracting solution is concentrated under reduced pressure into no second
Until when alcohol；
(2) by the water saturated extracting n-butyl alcohol of extracting solution, until n-butanol layer is colourless, reduced pressure n-butanol layer is simultaneously true
Vacuum freecing-dry obtains rape pollen crude extract；
(3) it by upper large pore resin absorption column after crude extract water dissolution, is first rinsed with water until eluent is not muddy, then
With 30-70% ethanol elutions, ethanol eluate is collected, through being concentrated under reduced pressure, Pollen Brassicae campestris extract is obtained after vacuum freeze drying.
5. Pollen Brassicae campestris extract as claimed in claim 4, it is characterised in that：Ethyl alcohol in step (1) is 75% ethyl alcohol.
6. Pollen Brassicae campestris extract as claimed in claim 4, it is characterised in that：Large pore resin absorption column in step (3) is
AB-8 type large pore resin absorption columns.
7. Pollen Brassicae campestris extract as claimed in claim 4, it is characterised in that：Ethyl alcohol in step (3) is 50% ethyl alcohol.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110101733A (en) * | 2019-03-27 | 2019-08-09 | 淮阴师范学院 | The purification process of rape flower general flavone |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110101733A (en) * | 2019-03-27 | 2019-08-09 | 淮阴师范学院 | The purification process of rape flower general flavone |
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