CN104804107B - A kind of Semen Herpetospermi extraction method of polysaccharides, Semen Herpetospermi polyoses extract and purposes that the extracting method is obtained - Google Patents
A kind of Semen Herpetospermi extraction method of polysaccharides, Semen Herpetospermi polyoses extract and purposes that the extracting method is obtained Download PDFInfo
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Abstract
The invention provides a kind of Semen Herpetospermi extraction method of polysaccharides, it comprises the following steps：A, degreasing；B, heating water bath refluxing extraction；C, Sevag method removing protein；D, precipitation.Semen Herpetospermi polyoses extract prepared present invention also offers extracting method and application thereof.Semen Herpetospermi polyoses extract of the present invention follows the traditional medication custom of the traditional Chinese medical science, meets clinical application feature, and extracting method is heating water bath refluxing extraction, and technique and extraction equipment are simple, with low cost and comparatively safe, are adapted to industrialized production.
Description
Technical field
The present invention relates to Semen Herpetospermi extraction method of polysaccharides, the Semen Herpetospermi polyoses extract that the extracting method is obtained, category
In drug field.
The content of the invention
Immunological liver injury is generally caused the activation of liver inner cell by biological and chemical irritation, and it is by a series of
The a large amount of inflammatory immune relevant cell factors of signal cascade reaction secretion, by various different cells and cell factor each other
Complexing action, produces immune/inflammatory response, causes the hepatic injury based on immune response.Immunological liver injury determines disease
Lapse to or develop.Long-term protracted course of disease can cause the last lesion eventually such as liver fibrosis, hepatic sclerosis or even liver neoplasm.It is immune
Property hepatic injury it is common the reason for be similar to pathology of viral hepatitis disease for virus infection, the hepatic injury pathology course of disease of its animal pattern
Journey.
At present, virus hepatitis is still public health problem main in world wide and the most common hepatopathy cause of disease.
China is also the district occurred frequently of virus hepatitis, especially most commonly seen with hepatitis type B virus (HBV) infection, seriously threatens people's
Health.At present, the specific medicament and method for the treatment of hepatitis B are there is no, the medicine for the treatment of hepatitis B is mainly that doctor trained in Western medicine is conventional
Interferon, thymosin extrasin, ucleosides, vaccine etc. and the conventional Radix Astragali crude preparation of the traditional Chinese medical science and compound, liver Kang Chong based on the Radix Astragali
The medicine such as agent and kuh-seng, but curative effect is not good enough.Therefore, exploitation can effectively treat the new drug of virus B hepatitis and clinically become
It is particularly urgent.
It is reported that Tibetan medicine and pharmacology achieves good achievement during virus hepatitis is prevented and treated, its treatment to hepatopathy
The extensive concern of domestic and foreign scholars is obtained.
Semen Herpetospermi is one of choice drug for the treatment of hepatopathy in Tibetan medicine, has obtained extensive during hepatopathy is treated
Using.Modern pharmacological research also shows that there is anti-hepatitis, anti-hepatitis virus, anti-liver to damage for Semen Herpetospermi total lignan and fat oil
Hinder, adjust the effect such as immune.It is reported that plant polyose also have it is antiviral, adjust the different physiological roles such as immune, anti-oxidant,
But have not yet to see the research report of Semen Herpetospermi polysaccharide liver protection effect.
The content of the invention
The technical scheme is that there is provided a kind of Semen Herpetospermi extraction method of polysaccharides, the ripple rib that the extracting method is obtained
Melon seeds polyoses extract.Another technical scheme of the present invention there is provided the purposes of the extract.
A kind of Semen Herpetospermi extraction method of polysaccharides of the present invention, it comprises the following steps：
A, degreasing：Take Semen Herpetospermi dry product to crush, add petroleum ether, water-bath backflow 4-6h, cold at 60-90 DEG C
But, centrifuge；
B, heating water bath refluxing extraction：After above-mentioned residue is volatilized, distilled water is added, 100 DEG C of heating water bath backflows are cooled down,
Extract solution is collected, and centrifugation takes supernatant liquor, again filtrate decompression concentrated after suction filtration；
C, Sevag method removing protein：Sevag reagent (chloroforms are added in above-mentioned concentrate：N-butanol=5:1), sample liquid
Than 1:4-4：1, supernatant is collected in acutely concussion, centrifugation；
D, precipitation：Absolute ethyl alcohol is added in above-mentioned supernatant, is stood overnight, suction filtration, precipitation vacuum drying obtains herpetospermum pedunculosum
Sub- polysaccharide.
It is further preferred that it comprises the following steps：
A, degreasing：Take Semen Herpetospermi dry product to crush, add 8 times of amount petroleum ethers, the water-bath backflow 2h at 80 DEG C, cooling,
Centrifugation, is repeated twice；
B, heating water bath refluxing extraction：After above-mentioned residue is volatilized, sequentially add 10,8,6 times of amount distilled water, 100 DEG C of water-baths
Each extraction 2h, 1.5h, 1.5h are heated to reflux, cooling is collected and merges No. 3 extract solutions, 4000r/min centrifugation 5min take upper strata clear
Liquid, concentrates filtrate to every 1ml decoctions equivalent to 1g bulk drugs again after suction filtration；
C, Sevag method removing protein：Sevag reagent (chloroforms are added in above-mentioned concentrate：N-butanol=5:1), sample liquid
Than 3：1,25min, 4000r/min centrifugation 5min are acutely shaken, supernatant is collected, repeats 6 times, last gained supernatant；
D, precipitation：The absolute ethyl alcohol of 6 times of volumes is added in aforesaid liquid, is stood overnight, suction filtration, precipitation vacuum drying is obtained
Semen Herpetospermi polyoses extract.
Present invention also offers the Semen Herpetospermi polyoses extract that described extracting method is prepared.
Present invention also offers purposes of the described Semen Herpetospermi polyoses extract in the medicine of liver protecting is prepared.
Wherein, described medicine is the medicine for treating immunological liver injury.
Wherein, described medicine is the medicine of protecting liver, lowering enzymes.
Wherein, described medicine is with the immune medicine of removing free radical, anti-oxidant, anti-inflammatory, regulation.
The big constituents with hepatoprotective effect are lignanoid and fat oil in document report, Semen Herpetospermi, and fat oil exists
Almost eliminated in the first step (petroleum ether degreasing) of extraction, and oil does not dissolve in water for fat；Lignanoid is more with free in plant
State is present, and is liposoluble substance, is insoluble in water, and the final step prepared is polysaccharide precipitation volume fraction of ethanol up to 80%
More than, remnants lignanoid is dissolved in supernatant liquor ethanol.Therefore, in Semen Herpetospermi polyoses extract of the present invention not
Contain lignan component.
Semen Herpetospermi polyoses extract of the present invention follows the traditional medication custom of the traditional Chinese medical science, meets clinical application feature, extraction side
Method is heating water bath refluxing extraction, and technique and extraction equipment are simple, with low cost and comparatively safe, are adapted to industrialized production.
Brief description of the drawings
Fig. 1 each group murine liver tissue HE coloring pathological sections (A：Blank group B：Model group C：Positive controls D：Polysaccharide is low
Dosage group E：Polysaccharide middle dose group F：Polysaccharide high dose group)
Embodiment
The Semen Herpetospermi polyoses extract preparation method of the present invention of embodiment 1
1) degreasing：Semen Herpetospermi dry product is crushed, and adds 8 times of amount petroleum ethers, the water-bath backflow 2h at 80 DEG C, cooling, from
The heart, is repeated twice.
2) heating water bath refluxing extraction：After above-mentioned residue is volatilized, sequentially add 10,8,6 times of amount distilled water, 100 DEG C of water-baths
Each extraction 2h, 1.5h, 1.5h are heated to reflux, cooling is collected and merges No. 3 extract solutions, 4000r/min centrifugation 5min take upper strata clear
Liquid, concentrates filtrate to every 1ml decoctions equivalent to 1g bulk drugs again after suction filtration.
3) Sevag methods removing protein：Sevag reagent (chloroforms are added in above-mentioned concentrate：N-butanol=5:1), sample liquid
Than 3：1,25min, 4000r/min centrifugation 5min are acutely shaken, supernatant is collected, repeats 6 times, last gained supernatant.
4) precipitate：The absolute ethyl alcohol of 6 times of volumes is added in aforesaid liquid, is stood overnight, suction filtration, precipitation vacuum drying is obtained
Semen Herpetospermi polysaccharide.It is 3.54% to calculate recovery rate.
Beneficial effects of the present invention are proved below by way of specific pharmacodynamics test.
Drug effect of the Semen Herpetospermi polysaccharide of the present invention of test example 1 to the protective effect of ConA immunogenicity hepatic injury mouse livers
Learn experiment
1. experimental drug and reagent
Semen Herpetospermi polysaccharide:Prepared by embodiment 1, polysaccharide administration group dosage (being administered by mouse weight) is respectively 0.71g/
Kg, 0.99g/kg, 1.44g/kg, equivalent to 20g/kg, 28g/kg, 40.8g/kg of crude drug amount；
Above-mentioned dosage polysaccharide prepares standby with distilled water dissolving；
Bifendate, is provided by Zhejiang Medicine Co；
Con A (ConA), purchased from Sigma Co., USA；
ALT (ALT), aspartate amino transferase (AST), lactic dehydrogenase (LDH, lot number),
Superoxide dismutase (T-SOD), MDA (MDA), total protein (TP), nitric oxide (NO) kit, are purchased from Nanjing and build
There is provided into Bioengineering Research Institute；
Interleukin-6 (IL-6) ELASA kits, are provided by Suzhou Calvin bio tech ltd；
Sterile phosphate buffer (PBS), is provided by Bioroc companies.
2. experimental method
60 healthy male mice in kunming, 18-22g is randomly divided into blank group, model group, DDB (positive right
According to) group and the basic, normal, high dosage group of Semen Herpetospermi polysaccharide, every group 10.
The basic, normal, high dosage administration group mouse of Semen Herpetospermi polysaccharide presses body weight ig0.71,0.99,1.44g/kg respectively daily
Polysaccharide；
Positive controls mouse ig0.2g/kg DDBs；
DDB and polysaccharide are suspended and dissolved with distilled water；
Normal group and model group mouse ig20mL/kg distilled water；
Continuous ig8d；
Immunological liver injury in mice model is set up using disposable tail vein injection ConA：Last dose (fasting 9h) 1h
Afterwards, in addition to normal group, remaining mouse tail vein injection 30mg/kg ConA.
After 8h, mouse weight is weighed, eyeball is plucked and takes blood, serum is separated, detection ALT, AST, NO, LDH, IL-6 items refer to
Mark；
Eyeball takes puts to death mouse and takes liver, spleen immediately after blood, filter paper is wiped, and is weighed, and calculates liver, spleen internal organs
Index (organ weights/body weight × 100%)；
10% liver homogenate is made in clip partial liver tissue, ice bath of cold saline, centrifuges, takes supernatant, examines
Survey TP, SOD, MDA indices；
The liver same area hepatic tissue (left lobe of liver) of clip each group mouse, 10% formaldehyde (PBS preparations) is fixed, HE dyes
Color, light Microscopic observation liver tissue injury degree.
Statistical procedures：The data obtained is usedRepresent, variance analysis and independence are carried out with SPSS17.0 softwares
Sample T is examined, after the inflammatory necrosis degree of liver organization scores according to hierarchal grouping, is examined and is carried out at statistics using Riditu
Reason.P ＜ 0.05 or P<0.01 thinks there is significant difference.
3. experimental result
1) influence of the Semen Herpetospermi polysaccharide to organ index, experimental result is shown in Table 1.
The Semen Herpetospermi polysaccharide of table 1 to immunological liver injury in mice organ index influence (N=10)
Compared with model group**P ＜ 0.01；*P ＜ 0.05
The result of table 1 is shown, compared with blank control group, and the liver of model group mouse, index and spleen index significantly raise (P ＜
0.01), prompting modeling success；Compared with model group, high dose administration group can significantly reduce small in positive drug group and polysaccharide
Mouse liver and spleen index.
It can be obtained by result above：Positive drug and middle high dose polysaccharide can mitigate immunological liver injury in mice liver oedema journey
Degree.
2) influence of the Semen Herpetospermi polysaccharide to serum alt, AST, LDH content, the results are shown in Table 2.
The Semen Herpetospermi polysaccharide of table 2 to enzymatic activity in immunological liver injury in mice serum influence (N=10)
The * * P ＜ 0.01 compared with model group；* P ＜ 0.05
The result of table 2 shows, ALT, AST and LDH content (P ＜ significantly raised compared with normal group in model group mice serum
0.01) modeling success, is illustrated.In Semen Herpetospermi polysaccharide in high dose administration group and positive controls mice serum ALT, AST and
LDH contents equal conspicuousness reduction (P ＜ 0.01 or P ＜ 0.05) compared with model group.
It can be obtained by result above：Positive drug and middle high dose polysaccharide can be significantly reduced in immunological liver injury in mice serum
Transaminase and lactic acid dehydrogenase activity, the effect of protecting liver, lowering enzymes is served to mouse.
3) Semen Herpetospermi Polysaccharides on Mice serum NO, the influence of IL-6 contents, the results are shown in Table 3.
The Semen Herpetospermi polysaccharide of table 3 to immunological liver injury in mice serum NO, IL-6 contents influence (N=10)
Compared with model group**P ＜ 0.01；*P ＜ 0.05
The result of table 3 shows that NO, IL-6 content are significantly raised compared with normal group (P ＜ 0.01) in model group mice serum, explanation
Modeling success.NO, IL-6 content show compared with model group in Semen Herpetospermi polysaccharide administration group and positive controls mice serum
Work property reduction (P ＜ 0.01 or P ＜ 0.05).
From result above：Semen Herpetospermi polysaccharide can adjust immune rapidly when lesion occurs for liver, reduce inflammatory mediator
Deng the release of harmful toxic matter, so as to mitigate the infiltration of liver inflammatory cell.
4) influence of the Semen Herpetospermi polysaccharide to SOD, MDA content in liver homogenate, the results are shown in Table 4.
The Pedunculate herpetospermum seed extract of table 4 to SOD, MDA content in immunological liver injury in mice liver homogenate influence (
N=10)
Compared with model group**P ＜ 0.01；*P ＜ 0.05
SOD activity is significantly reduced (P ＜ 0.01) compared with normal group in the homogenate of model group mouse liver；MDA contents are compared with normal group
Significantly rise (P ＜ 0.01), illustrates modeling success.High dose administration group mouse liver homogenate SOD activity in Semen Herpetospermi polysaccharide
The more significant rise (P ＜ 0.01 or P ＜ 0.05) compared with model group；Semen Herpetospermi polysaccharide high dose group and positive controls mouse
MDA contents significantly reduce (P ＜ 0.05) compared with model group in liver homogenate, and Semen Herpetospermi polysaccharide is low, middle dose group is without aobvious
Sex differernce is write, but MDA contents still have certain downward trend.
It can be obtained by result above：Semen Herpetospermi polysaccharide high dose can significantly improve SOD water in immunological liver injury in mice body
It is flat, and MDA contents are reduced, so as to reach scavenging activated oxygen, the enhancing oxidation resistant ability of body, liver cell biomembrane is protected,
Liver cell nuclear pyknosis or disintegration are avoided or reduce, to protect liver.
5) influence that Semen Herpetospermi polysaccharide changes to pathology of hepar
Normal group：Mouse lobuli hepatis structure is complete under light microscopic, has no that hyperplasia and pseudolobuli are formed, centrilobular vein has no
Extravasated blood, liver cell has no denaturation and necrosis；Also liver cell proliferation, fibrosis are had no, acholia in liver cell and in tiny bile duct
Alluvial, has no that various types of cells infiltrates, and portal area expands and fibrous connective tissue hyperplasia without obvious, and interlobular bile duct is without expansion and increases
Raw, envelope is complete, has no and thickens and ooze out.
Model group：Mouse lobuli hepatis structure is substantially clear, and sinus hepaticus is slightly expanded, and Di Shi gaps are broadening, and liver cell is visible not
With the focal shape oedema of degree, the visible large stretch of liver cell pyknosis in leaflet periphery is distributed in disperse shape.
Positive drug group：Mouse lobuli hepatis structure is clear, and lesion is mainly based on Mild edema and necrosis, and degree is substantially compared with mould
Type group lesion mitigates.High dose group：Mouse lesion is basic with positive drug group, but liver cell hydropic degeneration is compared with positive drug group weight.
In, low dose group：Mouse lobuli hepatis is clear, and lesion is mainly based on Mild edema and necrosis, and degree is significantly higher
Level dosage group weight.Liver organization pathological section is shown in accompanying drawing 1.
After the inflammatory necrosis degree of 6 groups of mouse liver tissues scores according to hierarchal grouping, examined and united using Riditu
Count processing, the hepatic tissue inflammatory necrosis degree of model group and high dose in blank group, positive controls and Semen Herpetospermi polysaccharide
The comparison of administration group, there were significant differences.It the results are shown in Table 5.
Influence that the Semen Herpetospermi polysaccharide of table 5 is classified to immunological liver injury in mice Hepatic fibrosis ( N=
8)
| Group | Dosage (g/kg) | Scoring |
| Blank control group | — | 0.00±0.00** |
| Model control group | — | 2.13±0.84 |
| Positive controls | 0.20 | 0.75±0.71** |
| Low dose group | 0.71 | 1.38±0.74 |
| Middle dose group | 0.99 | 1.13±0.64* |
| High dose group | 1.44 | 0.88±0.64** |
Compared with model group**P ＜ 0.01；*P ＜ 0.05
It can be obtained by result above：Model group hepatic disease compares in liver cell oedema and downright bad two classes lesion with blank group
There is significant difference, point out modeling success, the improvement immunological liver injury in mice that each dosage of Semen Herpetospermi polysaccharide can be different degrees of
Liver histopathology changes, wherein, high dose group mouse liver degree of necrosis is minimum, suitable with positive drug group mouse.
It can obtain based on the above results：Semen Herpetospermi polysaccharide have remove free radical, anti-oxidant, anti-inflammatory, regulation it is immune and
The activity of protecting liver, lowering enzymes, there is significant curative effect to immunological liver injury in mice, wherein, high dose polysaccharide drug effect is optimal.
Claims (4)
1. purposes of the Semen Herpetospermi polyoses extract in the medicine for preparing treatment immunological liver injury；Wherein, the Semen Herpetospermi
Extraction method of polysaccharides is prepared by following step：
A, degreasing：Take Semen Herpetospermi dry product to crush, add petroleum ether, water-bath backflow 4-6h, centrifugation at 60-90 DEG C；
B, heating water bath refluxing extraction：After above-mentioned residue is volatilized, distilled water is added, 100 DEG C of heating water bath backflows are cooled down, collected
Extract solution, centrifugation, takes supernatant liquor, again concentrates filtrate decompression after suction filtration；
C, Sevag method removing protein：Sevag reagents are added in above-mentioned concentrate, described Sevag reagents are chloroform：Positive fourth
Alcohol=5:1, sample liquid compares 1：4 - 4：1, supernatant is collected in acutely concussion, centrifugation；
D, precipitation：Absolute ethyl alcohol is added in above-mentioned supernatant, is stood overnight, suction filtration, it is many that precipitation vacuum drying obtains Semen Herpetospermi
Sugar.
2. purposes according to claim 1, it is characterised in that：Described Semen Herpetospermi extraction method of polysaccharides is by following steps
Prepare：
A, degreasing：Take Semen Herpetospermi dry product to crush, add 8 times of amount petroleum ethers, 2 h of water-bath backflow at 80 DEG C, cooling, from
The heart, is repeated twice；
B, heating water bath refluxing extraction：After above-mentioned residue is volatilized, sequentially add 10,8,6 times of amount distilled water, 100 DEG C of heating water baths
Flow back 2 h of each extraction, 1.5 h, 1.5 h, cooling, collects and merges No. 3 extract solutions, and 4000 r/min centrifuge 5 min, take upper strata clear
Liquid, concentrates filtrate to every 1 ml decoctions equivalent to 1g bulk drugs again after suction filtration；
C, Sevag method removing protein：Sevag reagents are added in above-mentioned concentrate, described Sevag reagents are chloroform：Positive fourth
Alcohol=5:1, sample liquid compares 3：1, violent 25 min of concussion, 4000 r/min centrifuge 5 min, collect supernatant, repeat 6 times, most
After obtain supernatant；
D, precipitation：The absolute ethyl alcohol of 6 times of volumes is added in above-mentioned supernatant, is stood overnight, suction filtration, precipitation vacuum drying obtains ripple
Rib melon seeds polyoses extract.
3. purposes according to claim 1 or 2, it is characterised in that：Described medicine is the medicine of protecting liver, lowering enzymes.
4. purposes according to claim 1 or 2, it is characterised in that：Described medicine is that have to remove free radical, antioxygen
Change, anti-inflammatory, the medicine for adjusting immunization.
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| CN112778432B (en) * | 2021-02-01 | 2022-04-22 | 西藏天虹科技股份有限责任公司 | Method for extracting herpetospermum pedunculosum seed polysaccharide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4229570A (en) * | 1976-07-07 | 1980-10-21 | Kureha Kagaku Kogyo Kabushiki Kaisha | Method of producing nitrogen-containing polysaccharides |
| CN1037714A (en) * | 1988-05-19 | 1989-12-06 | 中国科学院上海有机化学研究所 | From the Chinese medicine root of bidentate achyranthes, extract the method for Achyranthan |
-
2015
- 2015-04-24 CN CN201510201883.7A patent/CN104804107B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4229570A (en) * | 1976-07-07 | 1980-10-21 | Kureha Kagaku Kogyo Kabushiki Kaisha | Method of producing nitrogen-containing polysaccharides |
| CN1037714A (en) * | 1988-05-19 | 1989-12-06 | 中国科学院上海有机化学研究所 | From the Chinese medicine root of bidentate achyranthes, extract the method for Achyranthan |
Non-Patent Citations (2)
| Title |
|---|
| 藏药波棱瓜子提取液对四氯化碳致小鼠急性肝损伤保护作用的研究;姜汹;《中国优秀硕士学位论文全文数据库 农业科技辑》;20110615(第6期);D050-170 * |
| 超声波法提取藏药波棱瓜子中多糖的实验研究;哈文秀等;《青海师范大学民族师范学院学报》;20110525;第22卷(第1期);第88-89页 * |
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