CN1682874A - Use of acalypha copperleaf water extract in preparing medicine for treating chronic ulcerative colitis - Google Patents
Use of acalypha copperleaf water extract in preparing medicine for treating chronic ulcerative colitis Download PDFInfo
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Abstract
The present invention relates to medicine technology, and is the new use of water extract of acalypha copperleaf as a kind of euphorbiaceae plant in preparing medicine for treating chronic ulcer colonitis. Animal test shows that water extract of acalypha copperleaf can improve effectively the symptoms, including diarrhea, hemafecia, losing weight, colon tissue form change, etc. of ulcer colonitits rat model; and can eliminate neutral granulocyte. Biochemical detection and molecular biological test proves its antioxidant and anti-NO free radical effect, and the function of inhibiting the expression of inducing NO syntase in the RNA level. Therefore, water extract of acalypha copperleaf may be used in preparing medicine for treating chronic ulcer colonitis.
Description
Technical field:
The present invention relates to medical technical field, is the new purposes that the euphorbia plant acalypha copperleaf water extract is used to prepare treatment chronic ulcerative colitis medicine.
Background technology:
Ulcerative colitis (ulcerative colitis UC) is called chronic nonspecific colitis again, with the diarrhoea of outbreak repeatedly, to suffer from abdominal pain, have blood in stool etc. be cardinal symptom.Because this disease delay is difficult, very easily is converted into colon cancer again, and does not have specific Therapeutic Method at present, therefore is defined as by World Health Organization (WHO) one of " world's difficult treatment ".Chronic ulcerative colitis is very high at the sickness rate of American-European countries, also is the trend that rises year by year at the sickness rate of China.Because pathogenesis that clearly should disease also, so therapeutic effect is not fine.Clinical application at present mainly is aminosallcylic acid class, corticosteroid and immunosuppressant, and all there is the problem that curative effect is not good enough and side effect is bigger in these medicines, the for example clinical Salazosulfamide arsenic pyridine (SASP) that is usually used in treating light medium-sized UC, nearly 45% patient can not tolerate its intensive side effect.Corticosteroids comprises prednisone, hydrocortisone, budesonide etc., and this class medicine is not answered life-time service.Immunosuppressant mainly contains azathioprine, ammonia first dish purine etc., and this type of medicine is because toxicity is bigger, and its application is restricted.Have in addition report use the cyclosporin A treatment to corticosteroids have repellence serious symptom UC patient, because of must heavy dose of intravenous injection use, so toxicity is also bigger.Therefore, exploitation determined curative effect and the little medicament for resisting ulcerative colitis of toxic and side effects have become task anxious to be solved.Herba Acalyphae claims Acalypha australis again, is the herb of euphorbia plant Herba Acalyphae (Acalyphaaustralis L.), has the effect of heat clearing away, dampness removing, detumescence, detoxifcation.Dysentery, the disease such as hemorrhage of being usually used among the people.Toxicological experiment finds that Herba Acalyphae toxicity is very little, and its aqueous extract is through lumbar injection or press crude drug amount 10g/kg filling stomach, all do not cause dead mouse (Jin Jishu, etc. " research of the antibiotic effective ingredient of relievining asthma of Herba Acalyphae ", the Chinese herbal medicine communication, 1977,10:13).So far do not see that relevant acalypha copperleaf water extract is used for the treatment of the report of chronic ulcerative colitis.
Summary of the invention:
The invention provides a kind of new purposes that acalypha copperleaf water extract is used for the treatment of chronic ulcerative colitis.The preparation method of acalypha copperleaf water extract of the present invention is as follows: the dry herb of Herba Acalyphae is ground into coarse powder, decocts twice with the distilled water of 10 times of amounts respectively routinely, and each 1 hour, the merging decoction liquor, concentrating under reduced pressure is water extract of the present invention.
With the animal model TNBS (2 that is widely used at present the anti-UC medicine of screening both at home and abroad, 4, the 6-trinitrobenzene sulfonate) inductive rat chronic UC model has carried out zoopery, and pass through molecular biology test, confirmed acalypha copperleaf water extract of the present invention to the UC rat model, can significantly improve diarrhoea, have blood in stool, lose weight, symptom such as colon pathology morphological change; Can remove neutrophilic granulocyte, reduce the level of myeloperoxidase (MPO) (MPO); Antioxidation with rising superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reduction malonaldehyde (MDA) level; And the expression of inhibition inductivity nitricoxide synthase (iNOs) mRNA, thereby the generation of inhibition NO free radical.Illustrate that thus acalypha copperleaf water extract has the effect of the definite chronic UC of treatment, therefore can be used for preparing the medicine for the treatment of chronic UC.
Description of drawings
Fig. 1 is the curve chart of water extract of the present invention to the influence of UC rat model body weight change.
Fig. 2 is a rats in normal control group colon slice map.
Fig. 3 is UC model control group rat colon tissue slice figure.
Fig. 4 is dosage treatment group rat colon tissue slice figure in the water extract of the present invention.
The agarose gel electrophoretogram that Fig. 5 organizes iNOs mRNA to express for UC model control group rat colon.
The agarose gel electrophoretogram that Fig. 6 organizes iNOs mRNA to express for dosage treatment group rat colon among the present invention.
The specific embodiment:
Embodiment 1 is described in detail the present invention now in conjunction with the embodiments
The preparation acalypha copperleaf water extract: the dry decoction pieces of Herba Acalyphae herb is ground into coarse powder, adds the distilled water of 10 times of amounts,
Decoct twice, each 1 hour, merge decoction liquor, in the Rotary Evaporators concentrating under reduced pressure, finally obtain the concentrated solution of 1g crude drug/ml.
Embodiment 2 acalypha copperleaf water extracts are suffered from diarrhoea, are had blood in stool, lose weight the UC rat model and the therapeutical effect of colon morphological change.
1. animal: 60 cleaning levels, healthy male SD rat, body weight 180g-200g purchases the Shanghai Experimental Animal Center in the Chinese Academy of Sciences, the animal quality certification number: [SYXK (Shanghai) 2002-0026].60 rats are divided into 6 groups at random, and 10 every group, wherein 1 group as the normal control group, and all the other 5 groups are used for modeling.
2.UC the preparation of animal model: adopt Morris method (Morris G P, Beck PL, Herridge MS, et al.Hapten-induced model of chronic inflammation and ulceration in the rat colon.Gastroenterology1989,96 (3), 795-803.) carry out modeling, promptly get above-mentioned healthy male SD rat, fasting be can't help water 24 hours, after the slight anesthesia of the sodium pentobarbital of lumbar injection 2%, the latex tubing of diameter 2mm is inserted about 8cm place gently through the rat anus, by the 125mg/kg body weight with 50% alcoholic solution of TNBS with in the disposable enteric cavity that is injected into rat of syringe, the afterbody of rat is mentioned, be inverted 30 seconds, in case the modeling agent leaks, the UC rat model.
3. medicine: 1. sulfasalazine (SASP): be Shanghai Sunve Pharmaceutical Co., Ltd.'s product, this is clinical medicine with the chronic UC of treatment, is mixed with the suspension of 0.1g/ml with distilled water, as treatment contrast medicine.
2. acalypha copperleaf water extract: prepared by embodiment 1, concentration is 1g crude drug/ml.
4. experimental technique: experiment is divided into 6 groups: get 10 of above-mentioned healthy male SD rats as the normal control group for the 1st group; Get 10 UC rat models as model control group for the 2nd group; 3rd, 4,5 groups are respectively the basic, normal, high dosage treatment group of extract of the present invention, and every group of 10 UC rat models are irritated stomach acalypha copperleaf water extract 2.5,6.5,9.5g crude drug/kg body weight every day respectively; The 6th group of positive treatment matched group got 10 of UC rat models, and irritate stomach SASP suspension according to the 0.3g/kg body weight every day.Each organizes all beginning administrations in 6 hours after modeling of rat, and normal control group and model control group then give the normal saline of equal volume amounts respectively and irritate stomach, continuous irrigation stomach 10 days.
5. result of the test:
(1) acalypha copperleaf water extract is to the influence of UC rat model body weight change.
After each organizes rat modeling and administration, write down the body weight of rat every day, the results are shown in Figure 1.As seen from Figure 1, body weight begins rapid drawdown after the rat modeling, significantly is lower than normal rats body weight (p＜0.05) in 1-9 days, the 2nd day body weight after modeling drops to minimum, have only beginning in 80%, the 3 day of former body weight to recover gradually, returned to original weight on the 10th day.Extract 6.5g of the present invention, 9.5g crude drug/kg treatment group and SASP0.3g/kg positive treatment matched group can significantly promote the weight recovery of UC rat model, and rat the 2nd day body weight after modeling just begins to rise, and return to original weight in 5-6 days gradually.
(2) Herba Acalyphae is to the UC rat model diarrhoea and the influence of having blood in stool
Diarrhoea does not take place and has blood in stool in normal rats, and it is matt in a jumble that the rat after the modeling shows as hair color, and the look asthenia is dispirited, diarrhoea in various degree all occurred, has had blood in stool, and the results are shown in Table 1.By table 1 as seen, the number that middle and high dosage group of extract of the present invention and SASP positive treatment control rats are suffered from diarrhoea, had blood in stool obviously is less than model control group, illustrate that extract of the present invention has therapeutical effect to the diarrhoea of UC rat model with having blood in stool, the therapeutical effect that acalypha copperleaf water extract is had blood in stool to the UC rat model is particularly evident.
Table 1 acalypha copperleaf water extract is to UC rat model diarrhoea and the influence of having blood in stool
| Group | Rat number (n) | Diarrheal number (n) takes place in rat | The number that rat is had blood in stool (n) | ||||||||
| 1 day | 3 days | 5 days | 7 days | 10 days | 1 day | 3 days | 5 days | 7 days | 10 days | ||
| ????1 ????2 ????3 ????4 ????5 ????6 | ???10 ???10 ???10 ???10 ???10 ???10 | ???0 ???10 ???10 ???10 ???10 ???10 | ???0 ???9 ???8 ???7 ???7 ???7 | ???0 ???8 ???8 ???4 ???4 ???3 | ???0 ???6 ???6 ???2 ???0 ???1 | ???0 ???4 ???3 ???1 ???0 ???1 | ???0 ???10 ???10 ???10 ???10 ???10 | ???0 ???10 ???9 ???7 ???6 ???7 | ???0 ???7 ???6 ???3 ???2 ???6 | ???0 ???4 ???3 ???2 ???0 ???4 | ???0 ???3 ???2 ???0 ???0 ???2 |
Annotate: 1: the normal control group; The 2:UC model control group; 3: acalypha copperleaf water extract low dosage administration group; 4: dosed administration group in the acalypha copperleaf water extract; 5: acalypha copperleaf water extract high dose administration group; 6:SASP positive treatment matched group.
(3) acalypha copperleaf water extract is to the influence of UC rat model colon damage scoring and colon weight in wet base
Each organized the rat administration after 10 days, and excessive routinely injection pentobarbital sodium causes death, and cuts rat abdomen open, observed intestinal and changed substantially.To take out apart from the long latter end colon of anus 10cm,,, and weigh with the ice normal saline flushing along the mesentery longitudinal incision.Mucosa towards last expansion, is observed mucosa injury and marked according to the standards of grading of Wallace and Keenan1990: 0 minute, NIP and ulcer; 1 minute, contrafluxion but do not have ulcer; 2 minutes, ulcer but do not have hyperemia; 3 minutes, a place's ulcer and an inflammation; 4 minutes, above ulcer in two places or two places and inflammation; 5 minutes, ulcer extended beyond 2cm.The results are shown in Table 2.Rat is dissected the back observe colon outward appearance and mucous membrane surface discovery, the colon of model control group rat is most to have slight adhesion with adjacent internal organs, the colon congestion and edema, at the anus mouth 8cm place that makes progress bigger ulcer sexually transmitted disease (STD) kitchen range is arranged, the affected area colon wall thickens, curing ulcer erosion takes place and becomes pasty state in the intestinal mucosa surface, and colon obviously shortens, and colon weight significantly increases.Middle and high dosage group of acalypha copperleaf water extract and positive treatment matched group can improve inflammatory symptoms such as colon congestion and edema, reduce the ulcer area, promote ulcer healing, alleviate colon weight.
Table 2 acalypha copperleaf water extract damages UC rat model colon
And the influence (n=10) of colon weight in wet base
| Group | Colon damage scoring | Colon weight in wet base (mg/100g body weight) |
| ????1 ????2 ????3 ????4 ????5 ????6 | ????0±0 ????3.84±1.21 ????3.17±1.1 ????2.56±0.77 ????2.09±0.68 ????2.12±0.78 | ??????0.54±0.05 ??????1.03±0.22 ??????1.00±0.29 ??????0.82±0.20 ??????0.81±0.17 ??????0.76±0.25 |
Annotate: each organizes note with table 1.
(4) acalypha copperleaf water extract is to UC rat model colon pathological effect
Tissue pathological slice is seen Fig. 2,3,4, finds out by Fig. 2, and normal rat colon clear in structure, mucosa is complete, and enteraden is abundant in the lamina propria, arranges closely; Find out that by Fig. 3 the necrosis of model control group rat colon mucosa comes off, form ulcer, a large amount of neutrophilic granulocytes, lymphocytic infiltration, remaining body of gland goblet cell disappears, fibrohistiocytic's hypertrophy, interstitial edema is obvious; Find out by Fig. 4, dosage treatment group in the acalypha copperleaf water extract, between matter though Mild edema and a small amount of lymphocytic infiltration are arranged, but rat colon mucosa active proliferation, surface mucous is recovered substantially, and ulcer healing illustrates that water extract of the present invention has the promotion ulcer healing, eliminate inflammatory cell infiltration, improve the effect of UC rat model colon pathological characters.
(5) acalypha copperleaf water extract is to the influence of myeloperoxidase (MPO) (MPO) vigor in the UC rat model colon
The MPO enzyme is the higher a kind of enzyme of content in neutrophilic granulocyte, and increasing of its content can reflect neutrophilic granulocyte increasing in a certain tissue, reflects the existence of inflammation in tissue indirectly.Therefore, the level of MPO can be used to weigh the degree of tissue inflammation.By table 3 as seen, the model control group rat colon is organized the specific activity normal rat of MPO enzyme significantly raise (p＜0.01), acalypha copperleaf water extract height, middle dosage group can significantly reduce the activity (p＜0.01) of MPO in the colon, illustrate that acalypha copperleaf water extract has certain antiinflammatory action, compare with the positive treatment group, do not have significant difference (p＞0.05).
Table 3. acalypha copperleaf water extract is to the influence of the UC rat model MPO of colon enzyme activity
| Group | The number of rat | MPO activity (U/g) |
| ????1 ????2 ????3 ????4 ????5 ????6 | ??????10 ??????10 ??????10 ??????10 ??????10 ??????10 | ????0.15±0.21 ????14.58±5.28 ????12.83±6.58 ????5.58±3.06 ????4.09±2.80 ????7.89±4.75 |
Annotate: each organizes note with table 1.
In sum, the middle dosage of acalypha copperleaf water extract (6.5g crude drug/kg body weight), high dose (9.5g crude drug/kg body weight) treatment can significantly improvement UC rat model symptoms such as diarrhoea that lose weight, have blood in stool, and have promote the colon mucosa ulcer healing, reduce the MPO enzyme activity, the effect of removing neutrophilic granulocyte.This shows that acalypha copperleaf water extract has therapeutical effect to ulcerative colitis.
The experiment of embodiment 3 acalypha copperleaf water extract antioxidation and the effect of anti-NO free radical
1. animal: healthy male SD rat, body weight 180--200g
2. medicine: acalypha copperleaf water extract is a trial drug; SASP is treatment contrast medicine
3. animal grouping and administration: animal is divided into four groups at random, 10 every group.Get wherein one group at random as the normal control group, its excess-three group is all used TNBS modeling (method is the same).
The 1st group is the normal control group: irritate the stomach normal saline every day.
The 2nd group is model control group: irritate the stomach normal saline every day.
The 3rd group is dosage group in the acalypha copperleaf water extract: irritate stomach acalypha copperleaf water extract 6.5g crude drug/kg body weight every day.
The 4th group of positive matched group: irritate stomach SASP suspension according to the 0.3g/kg body weight every day.Each organizes all administrations in 6 hours after modeling of rat, successive administration 10 days.
4. index is investigated: administration is after 10 days, excessive routinely injection pentobarbital sodium causes death, cut rat abdomen open, the colon of getting diseased region adds the ice normal saline, homogenate is made into colon's homogenate of 10% under the ice bath, measures according to superoxide dismutase (SOD), malonaldehyde (MDA), glutathione peroxidase (GSH-Px) and nitric oxide (NO), inducible nitric oxide synthase (iNOs) detection kit description respectively; Determining the protein quantity is operated (SOD, MDA, GSH-Px, NO, iNOs and Coomassie brilliant blue protein determination kit all build up the biological engineering company limited available from Nanjing) according to Coomassie brilliant blue protein determination kit description.The results are shown in Table 4 and table 5.
Table 4. is respectively organized rat colon tissue SOD, MDA, GSH-Px enzyme activity
| Group | The rat number | ???SOD(NU/mgprot) | ??MDA(nmol/mgprot) | ???GSH-Px(U/ ???mgprot) |
| ???1 ???2 ???3 ???4 | ????8 ????10 ????10 ????10 | ????95.96±7.74 ????80.67±7.78 ????158.68±10.23 ????115.16±14.07 | ????4.31±0.32 ????12.18±0.85 ????9.38±0.77 ????8.87±0.83 | ??15.32±1.76 ??8.66±1.07 ??13.09±1.21 ??12.07±2.12 |
Table 5. acalypha copperleaf water extract is to the influence of the UC rat model NO of colon content and iNOs vigor
| Group | The rat number | ??NO(μmol/gprot) | ????iNOs(U/mgprot) |
| ????1 ????2 ????3 | ?????8 ?????10 ?????10 | ????0.79±0.29 ????2.71±0.83 ????0.98±0.25 | ?????0.015±0.012 ?????0.84±0.11 ?????0.54±0.27 |
By table 4, table 5 as can be seen, model control group is compared with normal group, active significantly reduce (P＜0.05) of SOD enzyme and GSH-Px enzyme in the colon, and the MDA level significantly raises (P＜0.05), dosed administration group and SASP administration group are compared with model control group among the present invention, the remarkable vigor of increased SOD enzyme and GSH-Px enzyme (p＜0.05), and the dosed administration group is compared with the positive treatment matched group among the present invention, does not have significant difference (p＞0.05).The vigor of the content of NO and iNOs is very low in the normal rat colon, and significantly raises (p＜0.05) after the modeling, and behind the dosed administration, the vigor of the content of NO and iNOs significantly reduces (p＜0.05) in the colon in the Herba Acalyphae.Illustrate that thus acalypha copperleaf water extract has the effect that reduces UC rat model NO content and iNOs vigor.
Embodiment 4 RT-PCR detect the inhibition test material of acalypha copperleaf water extract to the expression of iNOs mRNA: RNA extraction agent box (purchasing in Shanghai China Shun biological engineering company limited); reverse transcription test kit and RNase inhibitor (purchasing in precious biological engineering Dalian company limited), iNOs, GAPDH primer synthesize by the rich inferior biological engineering company limited in Shanghai.
The primer 1 of iNOs (5 '-TGGCTTGCCCTTGGAAGTTTCTC-3 ')
Primer 2 (5 '-TCCAGGCCATCTTGGTGGCAAGA-3 '),
Test method: respectively dosage group rat colon tissue in embodiment 2 described model control group and the acalypha copperleaf water extract is carried out the RNA extracting, get each sample RNA1.5 μ l, with Oligo (dT) is primer, the AMV reverse transcription is synthesized cDNA, with this cDNA is template, with GAPDH is contrast, the DNA of amplification iNOs and GAPDH in PCR reaction instrument, and response parameter is: behind 94 ℃ of pre-degeneration 2min; 94 ℃ of degeneration 30sec, 51.6 ℃ of annealing 30sec, 72 ℃ of C extend 1min, carry out 31 circulations after, 72 ℃ are extended 10min again, amplified production carries out electrophoresis with 1% agarose gel, and places biological electrophoresis imaging analysis system to carry out photographic analysis.Electrophoresis pattern sees 5,6.Use CMIAS-II true color magic magiscan two groups of PCR results' integral optical density is analyzed, the results are shown in Table 6.
Table 6.PCR is electrophoresis integral optical density value as a result
| Group | Integral optical density | ????GAPDH | Integral optical density/GAPDH |
| Dosage group in the model control group acalypha copperleaf water extract | ????3.895±1.010 ????2.418±0.847 | ????1.787±0.171 ????1.854±0.0503 | ????2.216±0.688 ????1.301±0.446 |
As seen from Table 6, model control group is compared with dosage treatment group among the present invention, the mRNA of iNOs expresses and has significant difference (p＜0.05), and the expression of the former iNOs mRNA is significantly higher than the latter, shows that water extract of the present invention has certain inhibitory action to iNOs.Because NO produces by iNOs catalysis L-arginine, illustrate that acalypha copperleaf water extract of the present invention is the generation of expression inhibiting NO by inhibition iNOs mRNA.
Claims (1)
1. the application of acalypha copperleaf water extract in preparation treatment chronic ulcerative colitis medicine, the preparation method of said acalypha copperleaf water extract is as follows: after the dry herb of Herba Acalyphae is pulverized, distilled water gradation with 10 times of amounts decocts, and merges decoction liquor, concentrating under reduced pressure.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103444995A (en) * | 2013-08-08 | 2013-12-18 | 镇江天和生物技术有限公司 | Preparation method of acalypha australis extractive grains |
| CN105770101A (en) * | 2016-04-06 | 2016-07-20 | 梁海月 | Medicine for treating ulcerative colitis |
| WO2019100602A1 (en) * | 2017-11-27 | 2019-05-31 | 大理大学 | Method for constructing animal model for ulcerative colitis, and application thereof |
| CN112057491A (en) * | 2020-10-15 | 2020-12-11 | 西南科技大学 | Application of copperleaf herb water extract in preparation of weight-losing and lipid-lowering medicines or weight-losing and lipid-lowering health-care products |
-
2005
- 2005-03-15 CN CNB2005100243926A patent/CN1316984C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103444995A (en) * | 2013-08-08 | 2013-12-18 | 镇江天和生物技术有限公司 | Preparation method of acalypha australis extractive grains |
| CN105770101A (en) * | 2016-04-06 | 2016-07-20 | 梁海月 | Medicine for treating ulcerative colitis |
| WO2019100602A1 (en) * | 2017-11-27 | 2019-05-31 | 大理大学 | Method for constructing animal model for ulcerative colitis, and application thereof |
| CN112057491A (en) * | 2020-10-15 | 2020-12-11 | 西南科技大学 | Application of copperleaf herb water extract in preparation of weight-losing and lipid-lowering medicines or weight-losing and lipid-lowering health-care products |
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