CN101978864B - Rape bee-pollen extract and use thereof - Google Patents
Rape bee-pollen extract and use thereof Download PDFInfo
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- CN101978864B CN101978864B CN2010102957917A CN201010295791A CN101978864B CN 101978864 B CN101978864 B CN 101978864B CN 2010102957917 A CN2010102957917 A CN 2010102957917A CN 201010295791 A CN201010295791 A CN 201010295791A CN 101978864 B CN101978864 B CN 101978864B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention provides an anti-inflammatory, antalgic and antibacterial extract which is extracted from rape bee-pollen according to lots of process researches. Strict tests prove that the extract has anti-inflammatory, antalgic and antibacterial functions and inhibition effect on human leukocyte elastase, excellent antalgic effect on visceral pain caused by ethylic acid and external pain caused by a hot plate in mice and excellent inhibition effect on Escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis and other bacterial. The extract can be used in the development of plant-source anti-inflammatory agents, antalgics and bacteriostatic agents. In addition, China's rape pollen productivity is huge, so the rape pollen can be used as the raw material for the plant-source anti-inflammatory agents, antalgics and bacteriostatic agents; and the extract has the advantages of low cost and simple process flow.
Description
Technical field
The present invention relates to a kind of bio-extract, particularly relate to a kind of Brassica campestris L pollen extract and application thereof.
Background technology
Rape (Brassica campestris L.) is the Cruciferae Brassica plants, bee pollen form cole is the rape male sex-cell that honeybee gathers, it not only contains many nutritional labelings, also contains many effective components relevant with life science, is traditional full-natural nutritive health products.Because it contains abundant nutrient numerous physiologically active ingredients is arranged again, causes bee pollen form cole to have diversified nutrition pharmacotoxicological effect.Confirm so far, bee pollen form cole has enhancing development, antifatigue, improves locomitivity, promotes hematopoiesis function, regulates immunologic function, anti-ageing, control agent intracellular metabolite and endocrine function, Suppress hyperplasia of prostate and anti-inflammatory, anti-liver injury, antiulcer, improves digestive tract function; In addition, promote the effects such as learning and memory function, antitumor, detoxifcation report is also arranged.The discovery of these effects directly provides foundation for developing extraordinary nutrition and health care and medicine.
Experiment showed, that pollen has preferably antiinflammatory action.Oral pollen extract T60 or GBX have inhibitory action to the swelling of rat toes and the filter paper granuloma due to croton oil or the ovalbumin.The corn pollen polysaccharide extract has bacteriostasis according to another report.And have no report about the dependent interaction of bee pollen form cole.
Summary of the invention
The purpose of this invention is to provide a kind of Brassica campestris L pollen extract with anti-inflammatory, analgesia, bacteriostatic activity, and its preparation method and methods for using them further is provided.
For achieving the above object, technical scheme of the present invention provides a kind of Brassica campestris L pollen extract, and its preparation process is:
(1) get bee pollen form cole, add 3～10 times of volume distilled water or deionized water, water extraction is carried out in ultrasonic auxiliary normal temperature or heating, the centrifugation supernatant, and precipitation repeats to extract, and merges supernatant, is evaporated to 0.5～1g pollen/ml;
(2) slowly add ethanol to no longer generation of precipitation in above-mentioned concentrate, the centrifugal precipitation of going is got the supernatant reduced pressure concentration and is obtained medicinal extract;
(3) get above-mentioned medicinal extract, add methyl alcohol to just fully dissolving, slowly add acetone to precipitation and no longer produce, the centrifugal precipitation of going is after supernatant reduced pressure concentration and the drying and get final product.
Can also be included in the front process of bee pollen form cole being carried out broken wall of water extraction in the above-mentioned steps (1), recovery rate is significantly improved.The method of broken wall can adopt any known Bee Pollen physical wall breaking method, such as overcritical broken wall method, machinery broken wall law, microwave broken wall method, comminution by gas stream etc.
Extract of the present invention, wherein in the preparation process (1), the water extraction temperature is preferably 15～50 ℃, 1～4 hour extraction time, repeats extraction time 2～4 times; The reduced pressure concentration temperature is 40～60 ℃.
Extract of the present invention, wherein in the preparation process (2), the temperature when reduced pressure concentration is sloughed ethanol is preferably 35～50 ℃.
Extract of the present invention, wherein drying mode is constant pressure and dry, low temperature drying, vacuum drying or freeze drying in the preparation process (3).
The present invention finds that resulting extract has good anti-inflammatory, analgesia, bacteriostatic activity, but so can be with its various food, medicine or health products that are prepared into clinical practice or conveniently take according to conventional method, such as a kind of anti-inflammatory composition, or a kind of analgesic composition, or a kind of bacteria inhibiting composition.
The present invention has obtained a kind of extract with anti-inflammatory, analgesia, bacteriostatic activity of containing in the bee pollen form cole by a large amount of technical studies.By rigorous evidence, this extract has anti-inflammatory, analgesia, bacteriostasis, and the HLE is had inhibitory action; Mice viscera pain due to the Dichlorodiphenyl Acetate and the body surface pain due to the hot plate all have good analgesic activity; The bacteriums such as Escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis had good restraining and sterilizing bacteria effect.The present invention can be applicable to the exploitation of plant source anti-inflammatory, analgesia, bacteriostatic agent.And China's rape pollen output is huge, and it also has with low cost, the simple advantage of technological process as the raw material of producing plant source anti-inflammatory, analgesia, bacteriostatic agent.
Bee pollen form cole water extraction concentrate that the present invention obtains has been removed alcohol-insoluble substance through the ethanol precipitation, adds acetone in the methyl alcohol saturated solution, and extract has been carried out further removal of impurities and purifying, the extract enrichment correlation function composition, action effect is obvious.Preparation method of the present invention requires low to appointed condition, method is simple, and is easy to implement, and manufacturing cycle is short, and cost is low, is fit to produce.
Description of drawings
Fig. 1 is the anti-inflammatory experimental result of Brassica campestris L pollen extract of the present invention.
The specific embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
A kind of preparation method of Brassica campestris L pollen extract, this preparation method may further comprise the steps successively:
Take by weighing bee pollen form cole 100g, add 300ml distilled water, 50 ℃ of ultrasonic assisted extraction 1 hour, centrifugal acquisition extract, the precipitation part repeats to extract once, centrifugal, merge supernatant, 50 ℃ of rotary evaporations are concentrated into 1g pollen/ml, under magnetic agitation, slowly add 95% ethanol in the condensed water extract till no longer produce precipitation, leave standstill, the centrifugation supernatant, 50 ℃ of rotary evaporations are sloughed ethanol and are condensed into medicinal extract, add methyl alcohol to just fully dissolving, slowly add acetone and extremely no longer produce till the precipitation centrifuging and taking supernatant, 50 ℃ of rotary evaporated to dryness.Namely obtain this extract.
Embodiment 2
A kind of preparation method of Brassica campestris L pollen extract, this preparation method comprises following step successively:
Being 10.0% with fresh Bee Pollen vacuum drying to moisture, getting the 300g dry pollen 1L extractor of packing into, under the condition of extraction kettle temperature 45 C, is that 99% carbon dioxide boosts to extraction kettle pressure 45MPa with purity; Keep opening the rapid emptying carbon dioxide of extractor air bleeding valve behind the pressure 10min, collect pollen and get final product.Detect through Electronic Speculum, pollen cell is broken wall.
Take by weighing the bee pollen form cole 100g of above-mentioned broken wall, add 1000ml distilled water, 20 ℃ of ultrasonic assisted extraction 1 hour, centrifugal acquisition extract, the precipitation part repeats to extract once, centrifugal, merge supernatant, 60 ℃ of rotary evaporations are concentrated into 0.5g pollen/ml, slowly add 95% ethanol in the concentrate till no longer produce precipitation under magnetic agitation, leave standstill, the centrifugation supernatant, 35 ℃ of rotary evaporations are sloughed ethanol, heat up to slough moisture, be condensed into medicinal extract, add methyl alcohol to just fully dissolving, add acetone to not producing till the precipitation elimination precipitation, supernatant partly is evaporated to dried, namely obtains this extract.
Embodiment 3
A kind of preparation method of Brassica campestris L pollen extract, this preparation method comprises following step successively:
Take by weighing the broken wall bee pollen form cole 100g of embodiment 2, add 500ml distilled water, 50 ℃ of ultrasonic assisted extraction 1 hour, centrifugal acquisition extract, the precipitation part repeats to extract once, and is centrifugal, merges supernatant, 60 ℃ of rotary evaporations are to 0.8g pollen/ml, under magnetic agitation, slowly add absolute ethyl alcohol in the condensed water extract till no longer produce precipitation, leave standstill the centrifugation supernatant, 60 ℃ of reduced pressure concentrations become medicinal extract, add methyl alcohol to just fully dissolving, slowly add acetone to no longer producing precipitation, remove precipitation, supernatant partly is evaporated to dried, namely obtains this extract.
Embodiment 4
A kind of preparation method of Brassica campestris L pollen extract, this preparation method comprises following step successively:
Take by weighing bee pollen form cole 100g, add 500ml distilled water, 50 ℃ of ultrasonic assisted extraction 1 hour, centrifugal solid-liquid separates, and solid portion repeats to extract once, merges supernatant, and 50 ℃ of rotary evaporations are concentrated, obtain the condensed water extract.Slowly add 5 times of volume 95% ethanol in the condensed water extract and carry out alcohol precipitation (the ethanol final concentration is about 80%).Centrifugal, get 40 ℃ of rotary evaporations of supernatant and slough ethanol, 50 ℃ of rotary evaporations are condensed into medicinal extract again, add methyl alcohol to just fully dissolving, slowly add acetone to no longer producing precipitation, remove precipitation, supernatant partly are evaporated to dried, namely obtain this extract.
In order to prove the anti-inflammatory and antalgic fungistatic effect of said extracted thing, the Brassica campestris L pollen extract that the method that the inventor uses to be provided in above-described embodiment prepares has carried out pharmacodynamic experiment, result of study following (being the mean value of result of the test of the extract of each embodiment):
One, Brassica campestris L pollen extract anti-inflammatory experiment-human elastase enzyme (HLE) suppresses experiment
(250 μ l) successively adds PBS buffer solution 30 μ l, HLE enzyme liquid 50 μ l and sample liquid 20 μ l, 37 ℃ of balance 10min in the micro-sampling pipe.Add again substrate solution 50 μ l.Sample and blank be triplicate all.
Add 20% acetum 50 μ l cessation reactions behind 37 ℃ of reaction 120min, at last each reactant liquor is carried out HPLC and analyze under the 405nm wavelength, measure the PNA growing amount; Blank group replaces sample liquid with DMSO.Reaction volume 150 μ l.
Liquid-phase condition: chromatographic column C18 (VP-ODS, 150mm * 4.6mm, 5 μ m); Mobile phase: first alcohol and water; Gradient elution (0min～10min is 25% methyl alcohol, and 10～15min is 25%～90% methyl alcohol, and 15～20min is 90%～25% methyl alcohol, and 20～25min is 25% methyl alcohol); Flow velocity: 1mL/min; Detect wavelength: 405nm; Column temperature: 40 ℃; Sampling volume: 40 μ l.
Experimental result as shown in Figure 1, from figure, can draw, Brassica campestris L pollen extract has inhibitory action to HLE, and when concentration was 0.25 μ g/ml, inhibiting rate (%) was 34.67 ± 1.58, the active increase with sample concentration of this inhibition increases, when concentration was 4 μ g/ml, inhibiting rate (%) was 64.76 ± 4.42, presents dose-effect relationship, calculate through returning, this extract is to the 503nhibiting concentration (IC of HLE
50) be 1.15 μ g/ml.
Two, Brassica campestris L pollen extract analgesic experiment
(1) hot-plate
Mouse is divided into 5 groups at random by body weight: blank group, aspirin group, extract group (840,420,210mgkg
-1).Successive administration 7 days places mouse on 55 ℃ the hot plate on test same day, is the threshold of pain index incubation period of licking metapedes take mouse.The same day was measured respectively the index of 30min, 60min after the administration, 120min, 240min in test, and carried out group difference relatively.The results are shown in Table 1.
Table 1
Compare * p＜0.05 * * p＜0.01 with the blank group
The as a result analgesic activity of the big or middle dosage group of Brassica campestris L pollen extract 30min onset after administration, wherein heavy dose of group relatively has utmost point significant difference (p＜0.01) with the blank group; Analgesic activity reaches the peak behind the 2h, and analgesic activity is still arranged behind the 4h; Small dose group demonstrates slow analgesia, and analgesic activity reaches the peak behind the 4h, with the blank group utmost point significant difference (p＜0.01) is arranged relatively.Experiment shows that Brassica campestris L pollen extract can improve the threshold of pain rate of thermic pain mouse, points out it can resist the thermic pain to the impact of mouse, and its pain is obviously prolonged incubation period, and low dose is slowly eased pain, and heavy dose of quickly easing pain has dose-effect relationship; Its analgesia intensity is suitable with aspirin with analgesia duration.
(2) writhing experiment
Mouse is divided into 5 groups at random by body weight: blank group, aspirin group, extract group (840,420,210mgkg
-1).Successive administration 8 days, test administration on the same day is after 1 hour, and each is organized mouse peritoneal and injects 0.7% glacial acetic acid normal saline solution.As observation index, and carry out group difference relatively with writhing number of times in 15 minutes and inhibiting rate.The results are shown in Table 2.
Table 2
| Group | Dosage (mg/kg) | N | Administration 1 hour (inferior) | Analgesia percentage (%) |
| Blank group | - | 7 | 50.57±12.19 | - |
| Aspirin | 500 | 8 | 8.00±5.95 ** | 84.18 |
| Heavy dose of | 840 | 10 | 38.20±13.97 | 24.46 |
| Middle dosage | 420 | 8 | 36.75±6.71 * | 27.33 |
| Low dose of | 210 | 8 | 30.38±8.18 ** | 39.92 |
Compare * p＜0.05 * * p＜0.01 with the blank group
The result shows, in the Brassica campestris L pollen extract, small dose group gavage Dichlorodiphenyl Acetate the mouse writhing number of times and the apparent in view minimizing of blank group that cause, statistical analysis, middle dosage group have significant difference (p＜0.05), and small dose group has utmost point significant difference (p＜0.01).The prompting Brassica campestris L pollen extract can suppress the pain reaction that acetic acid stimulates the abdominal cavity mucous membrane to cause, reduces the writhing number of times of mouse, illustrates to have analgesic activity, and low dose of (210mg/kg) can play analgesic effect.
Three, Brassica campestris L pollen extract bacteriostatic experiment
The culture medium preparation: by proportioning preparation culture medium, mixing is together sterilized with Oxford cup, pipette.Each dull and stereotyped 20ml sterilising medium that injects, drying is thoroughly stand-by.
Collecting cells: get a ring staphylococcus aureus and be dissolved in the 3ml sterilized water, use the liquid-transfering gun mixing.Make bacteria suspension, utilize blood counting chamber microscopically counting, adjust bacterial concentration and remain on 10
6-7About individual/ml.
Get dry flat board, get bacteria suspension 0.2ml spread plate, rear four Oxford cups of on flat board, evenly placing to be dried.Getting concentration is 200mg/ml sample, methyl alcohol, injects respectively the Oxford cup, and correspondence position is consistent, mark.Cultivate for 37 ℃ and observe, observed in 24 hours, the record experimental result.The results are shown in Table 3.
Table 3
Annotate: Oxford cup external diameter 8mm
Experimental result shows, Brassica campestris L pollen extract has good fungistatic effect, under same concentration (200mg/ml), different strain is suppressed to some extent difference of ability, bacteriostasis is followed successively by pseudomonas aeruginosa＞bacillus subtilis＞staphylococcus aureus＞Escherichia coli, wherein to pseudomonas aeruginosa inhibition the strongest (antibacterial circle diameter 21.75mm), to Escherichia coli inhibition the most weak (antibacterial circle diameter 17.89mm).Solvent (100% methyl alcohol) does not show bacteriostasis in experiment, and positive control (Sodium Benzoate) shows good fungistatic effect, but the sensitiveness for pseudomonas aeruginosa is inferior to other three bacterial classifications, and this point and Brassica campestris L pollen extract are antithesis.
The above only is preferred embodiment of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. a Brassica campestris L pollen extract is characterized in that, its preparation process is:
(1) get bee pollen form cole, add 3～10 times of volume distilled water or deionized water, water extraction is carried out in ultrasonic auxiliary normal temperature or heating, the centrifugation supernatant, and precipitation repeats to extract, and merges supernatant, is evaporated to 0.5～1g pollen/ml;
(2) slowly add ethanol to no longer generation of precipitation in above-mentioned concentrate, the centrifugal precipitation of going is got the supernatant reduced pressure concentration and is obtained medicinal extract;
(3) get above-mentioned medicinal extract, add methyl alcohol to just fully dissolving, slowly add acetone to precipitation and no longer produce, the centrifugal precipitation of going is after supernatant reduced pressure concentration and the drying and get final product.
2. extract as claimed in claim 1 is characterized in that, described step (1) also comprises the process of bee pollen form cole being carried out broken wall before water extraction.
3. extract as claimed in claim 1 is characterized in that, in the described step (1), 15～50 ℃ of water extraction temperature, repeat extraction time 2～4 times at 1～4 hour extraction time; The reduced pressure concentration temperature is 40～60 ℃.
4. such as each described extract of claim 1-3, it is characterized in that, in the described step (2), the temperature when reduced pressure concentration is sloughed ethanol is 35～50 ℃.
5. such as each described extract of claim 1-3, it is characterized in that, drying mode is constant pressure and dry, low temperature drying, vacuum drying or freeze drying in the described step (3).
6. an anti-inflammatory composition is characterized in that, contains each described extract of claim 1-5.
7. an analgesic composition is characterized in that, contains each described extract of claim 1-5.
8. a bacteria inhibiting composition is characterized in that, contains each described extract of claim 1-5.
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| CN102188350B (en) * | 2011-05-06 | 2012-08-22 | 湖南明园蜂业科技有限公司 | Rape bee pollen extract as well as preparation method and application thereof in anti-aging skincare products |
| CN102688178B (en) * | 2011-05-06 | 2013-08-21 | 湖南明园蜂业科技有限公司 | Application of rape bee pollen extract in anti-aging skin care products |
| CN103091406B (en) * | 2011-11-04 | 2015-06-24 | 上海医药工业研究院 | Detection method of fatty acid compounds and/or sterol compounds in rape bee pollen |
| CN103689308B (en) * | 2013-12-13 | 2015-07-08 | 广西科技大学 | Dissociation additive special for ultrasonically dissociating pollen pini tissue |
| CN112022881B (en) * | 2020-09-08 | 2022-04-19 | 中国农业科学院蜜蜂研究所 | Bee pollen fat extract and extraction method and application thereof |
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