CN105726554B - Dioscin is preparing the application in acute gastrointestinal damage protection drug - Google Patents
Dioscin is preparing the application in acute gastrointestinal damage protection drug Download PDFInfo
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Abstract
Present invention discover that Dioscin has acute gastrointestinal damage protective effect, it can be used for being made acute gastrointestinal damage protection drug.MTT and morphological observation prove that Dioscin has significantly protective effect to GES-1, Caco-2 and IEC-6 cell of hypoxia in vitro reoxygenation.Meanwhile Dioscin increases with apparent downward effect MDA caused by acute gastrointestinal damage and MPO, has obvious up-regulation effect to the reduction of T-SOD and GSH-PX.After Dioscin is intervened, gastric mucosal damage index after ischemical reperfusion injury increases, gastric mucosal erosion bleeding, phenomena such as local gland structure disorder, is obviously improved, and the villus shedding after ischemical reperfusion injury is obvious, phenomena such as telangiectasis, body of gland is impaired obvious, partially visible bleeding ulcer is obviously improved.According to clinical application demand, drug can be made using Dioscin as single active ingredient, can also be shared with other drugs and compound preparation is made.
Description
Technical field
The present invention relates to a kind of new application of effective component of chinese medicine, especially high-purity Dioscins to prepare acute stomach and intestine
Application in injury protection drug.
Background technique
Dioscin tool is illustrated in " Dioscin is preparing the application in injury of kidney protection drug " letters patent in the prior art
There is kidney protection effect, can be used for being made injury of kidney protection drug；Kidney organ belongs to a part of urinary system, is mainly responsible for
The balance of impurity, maintenance body fluid and electrolyte in filtering blood, finally generates urine and excretes via subsequent pipeline, simultaneously
Also has endocrine function to adjust blood pressure；However stomach and intestine are the important digestive organs of human body and toxin expelling organ, the master of stomach
Wanting function is receiving and digesting food, and major function emergenoy ration residue of intestines forms excrement, then is discharged by rectum per anum
In vitro.Acute gastrointestinal damage is the common illness of digestive system, makes therapeutic scheme appropriate to gastrointestinal damage, to mitigation patient
The condition of the injury, improve prognosis situation it is most important.Therefore, acute gastrointestinal damage protection drug is studied and is opened constantly in recent years
Among hair.Research finds that Chinese medicine has significant protective effect to acute gastrointestinal damage, and developed drug has following a few classes.1)
Flavonoids, such as curcumin；2) triterpene saponin, such as ginsenoside Rb1, notoginsenoside R；3) polyphenols, as tea is more
Phenol, resveratrol；4) alkaloids, such as anisodamine, ligustrazine.Dioscin (Dioscin) is that one kind is present in Chinese yam
Triterpene saponin componds in the plants such as section, Liliaceae, pulse family, wherein in dioscorea nipponica (Discorea nipponica
Makino content is higher in).Modern pharmacology research show Dioscin have adjust immune, platelet aggregation-against, it is antimycotic,
The multiple pharmacological effects such as antiviral and antitumor and lipid-loweringing, it is also the important source material of synthesizing steroid hormone medicine, and has made
For the main component of the fat-reducing medicaments such as Diaoxinxue Kang, Wei C Yinqiao Granules.In addition, " Dioscin is preparing injury of kidney protection medicine to patent
Application in object " it can only prove that Dioscin increases with bright urea nitrogen caused by injury of kidney (BUN) and urine creatinine (SCr)
Aobvious downward effect can be used for being made injury of kidney protection drug so having kidney protection effect.But it so far, has no
The relevant report that Dioscin is protected and applied in preparing acute stomach and intestine protection drug in acute gastrointestinal damage.
Summary of the invention
The present invention is that discovery Dioscin has protective effect to acute gastrointestinal damage, provides a kind of Dioscin and is preparing
Application in acute gastrointestinal damage protection drug.It specifically refers to Dioscin and is preparing Acute gastric injury protection drug or acute bowel
Application in injury protection drug.Especially to acute caused by Acute gastric injury caused by ischemia-reperfusion or ischemia-reperfusion
Damage of intestines has protective effect.
The Dioscin is preparing the application in acute gastrointestinal damage protection drug, Dioscin can be prepared into
The pharmaceutical preparation of single chemical component, or shared with other drugs and be prepared into compound medicinal formulation.It can also be according to pharmacy phase
Close require and clinic need to be made various dosage forms be applied to clinic in, such as: tablet, capsule, injection, oral liquid,
Granule etc..
It is described in the embodiment of the present invention, Dioscin is preparing the application in acute gastrointestinal damage protection drug, tool
Body administration mode is in the case of being administered orally, wherein Mouse oral Dioscin (20,40 and 80mg/kg) and Oral Administration in Rats
Dioscin (15,30 and 60mg/kg) there is significant protective effect to extrapolate using clinically acute gastrointestinal damage
One adult (60kg) daily oral about 140~550mg (i.e. 2.3~9.17mg/kg weight) Dioscin to there is acute stomach and intestine
Damage has significantly protective effect.
Present invention discover that Dioscin has acute gastrointestinal damage protective effect, it can be used for being made acute gastrointestinal damage protection
Drug.MTT and morphological observation prove that Dioscin has GES-1, Caco-2 and IEC-6 cell of hypoxia in vitro reoxygenation
There is significantly protective effect.Meanwhile Dioscin increases MDA caused by acute gastrointestinal damage and MPO under having significantly
Tune effect has obvious up-regulation effect compared with the reduction of model group to T-SOD and GSH-PX, all has dose dependent.Through potato
After Chinese yam saponin(e is intervened, the gastric mucosal damage index after ischemical reperfusion injury is increased, gastric mucosal erosion bleeding, local gland structure
Disorder, gap enlargement, and there is phenomena such as local Gastric Mucosal Cells fall off to be obviously improved；After Dioscin is intervened, Ischemia Reperfusion
Intestinal villi after note damage falls off obviously, telangiectasis, and body of gland is impaired obviously, partially visible bleeding ulcer and
Chui's is obviously improved phenomena such as scoring increases.It, can be using Dioscin as single active ingredient system according to clinical application demand
At drug, it can also be shared with other drugs and compound preparation is made.Dioscin have safely (having no toxic side effect), effectively, it is economical,
The advantages that practical.
Detailed description of the invention
Shadow of 1 Dioscin of Fig. 1 embodiment of the present invention to hypoxia in vitro reoxygenation GES-1 cell viability and cellular morphology
It rings.
The influence that 1 Dioscin of Fig. 2 embodiment of the present invention damages ischemia-reperfusion injury model gastric mucosa.Stomach lining damage
Hurt assessment of indices result to be confined to the length accumulation score of the dotted rotten to the corn, ulcer of Weishang skin and hemorrhagic focus: it is normal for 0 point,
Damage≤1mm counts 1 point, > 1mm simultaneously≤2mm counts 2 points, > 2mm simultaneously≤3mm counts 3 points, the rest may be inferred by analogy.Score when lesion width is more than 1mm
It doubles.The average value of every group of Damage of Rats score be damage index, as a result, it has been found that high dose group (Dioscin 60mg/kg) and
IPC processing group can significantly reduce gastric mucosal damage index, and compared with model group, the Dioscin of 30 and 15mg/kg is to stomach
Mucosa injury index does not have significant impact.
The influence that 1 Dioscin of Fig. 3 embodiment of the present invention dyes ischemia-reperfusion injury model rat stomach histopathology HE.
Rats in sham-operated group stomach trouble reason, which has no, as the result is shown substantially change；Model group stomach lining has gastric mucosal erosion bleeding, local body of gland
Structure disturbance, gap enlargement, and there are local Gastric Mucosal Cells to fall off；High dose Dioscin group epithelium is basic complete continuous, gland
Body arrangement is more neat, and gastric mucosa congestion is slighter, has clear improvement compared with model group gastric injury degree；Compared with model group, middle dose
Amount and low dosage Dioscin group can also improve these variations, but degree is all not so good as high dose group.
Influence of 1 Dioscin of Fig. 4 embodiment of the present invention to ischemia-reperfusion injury model rat stomach oxidative stress index.
Influence of 2 Dioscin of Fig. 5 embodiment of the present invention to hypoxia-reoxygenation Caco-2 cell viability and cellular morphology.
Influence of 2 Dioscin of Fig. 6 embodiment of the present invention to hypoxia-reoxygenation IEC-6 cell viability and cellular morphology.
2 Dioscin of Fig. 7 embodiment of the present invention is to Gut ischmia-reperfusion C57BL/6J mouse and SD rat model tissue disease
The influence of HE dyeing of science.The visible intestinal mucosa of sham-operation group is complete as the result is shown, no bleeding and inflammatory phenomena；C57BL/6J mouse
Villus shedding visible with the model group of SD rat is obvious, telangiectasis, and body of gland is impaired obvious, and partially visible bleeding, which is burst, swings；
And give Dioscin (80,40 and 20mg/kg) processing C57BL/6J mouse after and Dioscin (60,30 and 15mg/kg)
After handling SD rat, intestinal mucosal injury is substantially reduced.In addition, model group Chui's scoring be significantly higher than sham-operation group (P <
0.01), and compared with model group, high, middle dosage Dioscin group and the scoring of IPO+IPC group are substantially reduced, low dosage Chinese yam
The no significant changes of the scoring of saponin(e group.
2 Dioscin of Fig. 8 embodiment of the present invention refers to Gut ischmia-reperfusion C57BL/6J mouse and SD rat model biochemistry
Target influences.
Specific embodiment
Following non-limiting embodiments can with a person of ordinary skill in the art will more fully understand the present invention, but not with
Any mode limits the present invention.
The research that 1 Dioscin of embodiment acts on ischemia-reperfusion injury in treating
1. In vitro cell model is established and grouping
Vitro culture of human gastric epithelial cells system (GES-1) establishes stomach anoxia/reoxygenation (H/R) model, experiment point
Group: pretreated group (6.25,12.5,25,50,100,200,400 and of Dioscin is administered in control group, model group, various dose
800ng/mL).12h administration group starts to be administered before cell modeling, model group and administration group cell hypoxia 2h, respectively reoxygenation 12,24
And after 48h, cellular morphology is observed under inverted microscope, mtt assay detects cell viability.
Influence of the hypoxia-reoxygenation model to GES-1 cell:
GES-1 cell viability and its form after anoxia/reoxygenation injury are shown in Fig. 1, the results showed that, it is compared with the control group, multiple
Model group cell viability after oxygen is remarkably decreased, and Dioscin can significantly inhibit this variation, and it was found that Dioscin
Dosage be greater than 200ng/mL when have significant cytotoxicity, in addition, 200ng/mL Dioscin is to the cell after reoxygenation 12h
Model has maximum protective effect, therefore subsequent cell experiment uses the model and 200,100 and 50ng/ of reoxygenation 12h
The dosage of mL.White according to observing by the cell of inverted microscope, Hypoxia-reoxygenation model can result in the micro- knot of GES-1 cell
Structure is destroyed, and cell pyknosis eventually disengages from wooden partition；And it can after Dioscin (200,100 and 50ng/mL) pretreatment 12h
In dose-dependently obviously inhibiting this variation.
2. Animal Model and grouping in body
Male SD rat is randomly divided into 6 groups, every group 10: sham-operation group, model group, high dose administration group (Dioscin
60mg/kg), middle dosage administration group (Dioscin 30mg/kg), low dosage administration group (Dioscin 15mg/kg), Ischemia Reperfusion
It infuses pretreated group (Dioscin 60mg/kg+IPC group).
The preoperative 7 days administration groups of experimental animal and IPC group start to be administered, and once a day, preoperative fasting for 24 hours is freely intake, art
It is preceding with 10% chloraldurate solution (3.5mL/kg) intraperitoneal injection of anesthesia.Model group and administration group: supine is fixed on
On operating table, abdomen is opened in cropping, is separated and is pressed from both sides and closes the left and right artery of stomach and gastroepiploic artery, and artery clamp is removed after 30min and restores blood
Stream；IPC group: No. 3 folders repeatedly are carried out using blood vessel clip according to the method described above and close 15s, Song Jia 15s as pretreatment, then folder closes
Artery 30min；Sham-operation group: the left and right artery of stomach and gastroepiploic artery are not pressed from both sides and are closed, anesthesia and remaining operating procedure and model group
It is identical.1h after Reperfu- sion quickly takes stomach, observes gastric mucosal damage index and leaves and takes gastric tissue, is put into -80 DEG C of refrigerators and saves.
The variation of each group gastric mucosa damage:
Gastric mucosal damage index measurement result is shown in Fig. 2, to be confined to dotted rotten to the corn, ulcer and hemorrhagic focus the length of Weishang skin
Degree accumulation score: normal is 0 point, and damage≤1mm counts 1 point, > 1mm simultaneously≤2mm counts 2 points, > 2mm simultaneously≤3mm counts 3 points, the rest may be inferred by analogy.
Score doubles when lesion width is more than 1mm.The average value of every group of Damage of Rats score is damage index, as a result, it has been found that high dose group
(Dioscin 60mg/kg) and IPC processing group can significantly reduce gastric mucosal damage index, and compared with model group, 30 Hes
The Dioscin of 15mg/kg does not have significant impact to gastric mucosal damage index.
3. gastric tissue pathological observation
According to histopathology conventional method, the gastric tissue being fixed in 10% formalin is embedded into paraffin, is cut
At 5 μm of tissue slice, slice is made, carries out histopathology sight under 200 times of mirrors behind Yihong and hematoxylin (HE) dyeing
It examines.
Each group rat stomach histopathological examination result:
As a result optical microphotograph microscopic observation each group rat HE stained slice is shown in Fig. 3, HE dyeing display rats in sham-operated group stomach
Pathology, which has no, to be substantially change；Model group stomach lining has gastric mucosal erosion bleeding, local gland structure disorder, gap enlargement, and has
Local Gastric Mucosal Cells fall off；High dose Dioscin group epithelium is basic complete continuous, and body of gland arranges more neat, gastric mucosa congestion
It is relatively slight, it has clear improvement compared with model group gastric injury degree；Compared with model group, middle dosage and low dosage Dioscin group also can
Improve these variations, but degree is all not so good as high dose group.
4. gastric tissue biochemical indicator detects
With the malonaldehyde (MDA) and glutathione peroxidase (GSH-PX) of kit measurement each group rat gastric tissue.
The investigation of each group rat stomach biochemical index:
The rat stomach biochemical index of ischemical reperfusion injury is investigated, the result is shown in Fig. 4.The MDA of model group
It is apparently higher than sham-operation group, and the MDA of administration group (60,30 and 15mg/kg) is in dose-dependently to decline compared with model group.Separately
Outside, compared with sham-operation group, the GSH-PX of model group is decreased obviously, and the GSH-PX of administration group (60,30 and 15mg/kg) is compared with mould
Type group is in dose-dependently to increase.
This research by hypoxia in vitro reoxygenation model GES-1 cell, Ischemia-Reperfusion Injury Model SD rat
Gastric mucosal damage index, the detection of histopathology and stomach functional biochemistry index, it was demonstrated that Dioscin can significantly mitigate
Ischemia-reperfusion damage has significant protective effect to Acute gastric injury.
Research of 2 Dioscin of embodiment to Intestine ischemia and reperfusion therapeutic effect
1. In vitro cell model is established and grouping
In vitro culture source of people colon adenocarcinoma cell strain (Caco-2) and crypts of small intestine cell strain (IEC-6) are established lack respectively
Oxygen reoxygenation (H/R) model, experimental group: control group, model group, various dose administration pretreated group (Dioscin 12.5,
25,50,100,200,400 and 800ng/mL).12h administration group starts to be administered before cell modeling, and model group and administration group cell lack
Oxygen 4h, respectively reoxygenation 6,12 and for 24 hours after, microscopically observation cellular morphology, MTT detect cell viability.
Influence of the Hypoxia-reoxygenation model to cell:
Caco-2 and IEC-6 cell viability and its form after anoxia/reoxygenation are shown in Fig. 5-6, the results showed that, and it compares
Group is compared, and the cell viability of model group is remarkably decreased, and Dioscin can significantly inhibit this variation, and it was found that Chinese yam soap
The dosage of glycosides has significant cytotoxicity when being greater than 200ng/mL, in addition, 200ng/mL Dioscin is to thin after reoxygenation 6h
Born of the same parents' model has maximum protective effect, therefore subsequent cell experiment uses the model and 200,100 and 50ng/ of reoxygenation 6h
The dosage of mL.White according to observing by the cell of inverted microscope, Hypoxia-reoxygenation model can result in Caco-2 and IEC-6 cell
Microstructure destroyed, cell pyknosis eventually disengages from wooden partition；And Dioscin (200,100 and 50ng/mL) pre-processes 6h
After can be in dose-dependently obviously inhibiting this variation.
2. Animal Model and grouping in body
Male C 57 BL/6 J mouse is randomly divided into 6 groups, every group 10: sham-operation group, model group, high dose administration group (potato
Chinese yam saponin(e 80mg/kg), middle dosage administration group (Dioscin 40mg/kg), low dosage administration group (Dioscin 20mg/kg), lack
Blood Reperfu- sion processing group (Dioscin 80mg/kg+IPC+IPO group).Male SD rat is randomly divided into 6 groups, every group 10: artificial hand
Art group, model group, high dose administration group (Dioscin 60mg/kg), middle dosage administration group (Dioscin 30mg/kg), low dose
Measure administration group (Dioscin 15mg/kg), ischemic preconditioning group (Dioscin 60mg/kg+IPC+IPO group).
C57BL/6J mouse and the preoperative 7 days administration groups of SD rat start to be administered, once a day, preoperative fasting for 24 hours, freely into
Water, it is preoperative with 10% chloraldurate solution (3.5mL/kg) intraperitoneal injection of anesthesia.I/R group and administration group: supine is consolidated
Due on operating table, abdomen is opened in cropping, softly separates surrounding tissue, and the mesentery root above exposure inferior caval vein and right kidney is looked for
To superior mesenteric artery, and folder closes removal artery clamp recovery blood flow after 45min；IPC+IPO group: blood vessel is used according to the method described above
Folder carries out No. 3 folders repeatedly and closes 15s, Song Jia 15s as pre-treatment (IPC), and then folder closes artery 45min, then carries out No. 1 folder and closes
10min, Song Jia 10min are as post-processing (IPO)；Sham-operation group: superior mesenteric artery is not pressed from both sides and is closed, anesthesia and remaining operation step
Suddenly identical as model group.6h after Reperfu- sion quickly takes small intestine, and leaves and takes small intestine, is put into -80 DEG C of refrigerators and saves.
3. intestinal tissue pathological observation
According to histopathology conventional method, by the C57BL/6J mouse being fixed in 10% formalin and SD rat
Small intestine is embedded into paraffin, is cut into 5 μm of tissue slice, and slice is made, after h and E dyes (HE dyeing)
Pathological study, and statistical damage degree are carried out under 200 times of mirrors.
C57BL/6J mouse and SD rat intestine histopathological examination result:
Optical microphotograph microscopic observation groups of animals HE stained slice result is shown in Fig. 7, the visible intestines of HE dyeing display sham-operation group
Mucous membrane is complete, no bleeding and inflammatory phenomena；The visible villus shedding of the model group of C57BL/6J mouse and SD rat is obvious, blood capillary
Enlargement of pipe, body of gland is impaired obvious, and partially visible bleeding, which is burst, swings；And give Dioscin (80,40 and 20mg/kg) processing C57BL/
After 6J mouse and after Dioscin (60,30 and 15mg/kg) processing SD rat, intestinal mucosal injury is substantially reduced.In addition, model
Group Chui's scoring is significantly higher than sham-operation group (P < 0.01), and compared with model group, high, middle dosage Dioscin group and
The scoring of IPO+IPC group is substantially reduced, the no significant changes of scoring of low dosage Dioscin group.
4. intestinal tissue biochemical indicator detects
With the myeloperoxidase (MPO) of kit measurement each group C57BL/6J mouse and SD rat intestinal, total super
Superoxide dismutase (T-SOD) and malonaldehyde (MDA).
The investigation of C57BL/6J mouse and SD rat intestine biochemical index:
The intestinal tissue biochemical indicator of ischemical reperfusion injury is investigated, the result is shown in Fig. 8.C57BL/6J mouse and SD
Model group MDA and the MPO level of rat is apparently higher than sham-operation group, and gives Dioscin (80,40 and 20mg/kg) processing
After C57BL/6J mouse and after Dioscin (60,30 and 15mg/kg) processing SD rat, serum MDA and MPO are compared with model group
In dose-dependently declining.In addition, the T-SOD of model group is decreased obviously, and Dioscin (80,40 compared with sham-operation group
And 20mg/kg) processing C57BL/6J mouse after and Dioscin (60,30 and 15mg/kg) processing SD rat after, serum T-
SOD is larger, mouse model group is in dose-dependently to increase.
This research by Caco-2 the and IEC-6 cell after hypoxia in vitro reoxygenation, ischemical reperfusion injury
The detection of C57BL/6J mouse and SD rat intestine histopathology and intestines biochemical indicator, it was demonstrated that Dioscin can significantly subtract
Light Intestine ischemia and reperfusion has significant protective effect to acute damage of intestines.
Claims (1)
1. application of the Dioscin as sole active agent in the drug that acute damage of intestines is treated in preparation.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101450144A (en) * | 2007-12-07 | 2009-06-10 | 卡南吉医药科技（上海）有限公司 | Solanum dulcamara total-saponin extract and preparation method and use thereof |
-
2016
- 2016-02-02 CN CN201610074517.4A patent/CN105726554B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101450144A (en) * | 2007-12-07 | 2009-06-10 | 卡南吉医药科技（上海）有限公司 | Solanum dulcamara total-saponin extract and preparation method and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 天然产物薯蓣皂苷的研究进展;许丽娜等;《中国中药杂志》;20150101;第40卷(第1期);第36-41页 |
| 薯蓣皂苷生物活性研究新进展;王晓鹏等;《国外医学中医中药分册》;20040530;第26卷(第3期);第140页右栏第3-13行 |
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