CN1569088A - 'Heiguteng' exract, its pharmaceutical composition and its use - Google Patents
'Heiguteng' exract, its pharmaceutical composition and its use Download PDFInfo
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- CN1569088A CN1569088A CN 03146799 CN03146799A CN1569088A CN 1569088 A CN1569088 A CN 1569088A CN 03146799 CN03146799 CN 03146799 CN 03146799 A CN03146799 A CN 03146799A CN 1569088 A CN1569088 A CN 1569088A
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Abstract
The invention provides a Heiguteng extract, its extraction method, the pharmaceutical composition containing the extract, and the use of the extract in preparing medicament for treating self-immunity diseases or inflammation resisting medicament, wherein the self-immunity diseases include rheumatoid arthritis, glomerulonephritis, nephropathy syndrome etc.
Description
Technical field
The present invention relates to the Heiguteng exract extract, its extracting method contains the pharmaceutical composition of this extract and is used to prepare the purposes of immunosuppressant or anti-inflammatory drug.
Background technology
The Heiguteng exract formal name used at school is southwestern P. sepium Bunge (Periploca forrestii Schltr.), is that asclepiadaceae (Asclepiadaceae) P. sepium Bunge belongs to (Periploca L.) plant, is referred to as Heiguteng exract in area, China west and south.According to medication experience among the people, its root is pharmaceutically acceptable, and function such as have the meridian dredging, wind-damp dispelling, Shujin, invigorate blood circulation can be used for treating diseases such as traumatic injury, arthralgia, menoxenia, stomatitis, mastitis.Heiguteng exract both can use separately, also can go into compound recipe.Draw in the rifle the Miao Ethnomedicine father, Heiguteng exract and Herba Anemones vitifoliae, Lysimachia trientaloides Hemsl. compatibility cure mainly anemofrigid-damp arthralgia, shoulder arm lumbago and skelalgia.
The inventor is through discovering extensively and profoundly, the Heiguteng exract extract that obtains by method described below can suppress the mouse boosting cell breeder reaction significantly, delayed hypersensitivity and various inflammatory reactions, have significant immunosuppressant and antiinflammatory action, and demonstrate the good curing autoimmune disease, as: rheumatoid arthritis, glomerulonephritis, the nephrotic syndrome, struma lymphomatosa, idiopathic thrombocytopenic purpura, systemic lupus erythematosus (sle), behcets disease, autoimmune hepatitis, the immunity angiopathy, and inflammatory diseases, as: the drug effect of rheumatic arthritis.Be accomplished based on above discovery the present invention.
Summary of the invention
An aspect of of the present present invention relates to the Heiguteng exract extract, this extract has tangible immunosuppressant and anti-inflammatory activity, to autoimmune disease, particularly rheumatoid arthritis, the nephrotic syndrome, behcets disease, autoimmune hepatitis, immunity angiopathy have therapeutical effect.
The invention further relates to the Heiguteng exract extract, it is characterized in that: this extract is by obtaining Heiguteng exract with organic solvent, water-containing organic solvent or water extraction.
Another aspect of the present invention relates to pharmaceutical composition, and it comprises Heiguteng exract extract of the present invention and one or more pharmaceutical carriers or excipient as active component.
The invention still further relates to the Heiguteng exract extract and be used for preparation treatment autoimmune disease as the purposes of the medicine of rheumatoid arthritis, the nephrotic syndrome, behcets disease, autoimmune hepatitis, immunity angiopathy and inflammatory diseases such as rheumatic arthritis.
The invention still further relates to the method for preparing the Heiguteng exract extract, it comprises Heiguteng exract is extracted with organic solvent, water-containing organic solvent or water.
Further, the Heiguteng exract extract can obtain as follows:
Heiguteng exract whole plant dried and crushed with fresh collection obtains the Heiguteng exract coarse powder.Heiguteng exract coarse powder organic solvent, water-containing organic solvent or water is diafiltration or heating and refluxing extraction repeatedly, obtains the Heiguteng exract extract behind the extracting solution concentrate drying.Used organic solvent is selected from alcohols such as methanol, ethanol, propanol, butanols etc.; Ketone such as acetone; Halo alkanes such as chloroform, dichloromethane etc.; Esters such as methyl acetate, ethyl acetate etc.; Ethers such as ether; Alkanes such as petroleum ether, gasoline etc.
According to the present invention, Heiguteng exract extract of the present invention can be differentiated as follows:
(1) acetic anhydride concentrated sulphuric acid test (Liebermann-Burchard reaction)
The Heiguteng exract extract leaches with an amount of 95% ethanol, gets leachate with the ethanol evaporate to dryness, adds 1 milliliter of glacial acetic acid in the residue and makes its dissolving, adds 1 milliliter of acetic anhydride again, splashes into 1 concentrated sulphuric acid at last.The test tube color is successively through Huang-red-purple-indigo plant-blackish green variation, and expression has Heiguteng exract sterol or steroidal saponin to exist.
(2) 3,5-dinitrobenzoic acids reactions (Kedde reaction):
The Heiguteng exract extract leaches with an amount of 95% ethanol, gets leachate 1ml in test tube, adds 3-4 and drips 3, (preparation by the following method: 1g 3, and the 5-dinitrobenzoic acid is dissolved in 50ml methanol, add 50ml 2N sodium hydroxide solution again for 5-dinitrobenzoic acid reagent, join with stylish), produce aubergine.
(3) the Heiguteng exract extract leaches with an amount of 95% ethanol, and leachate is a liquid to be measured.Get Heiguteng exract crude drug 5g, add 50ml 70% alcohol reflux 30 minutes after the pulverizing, reuse 5ml 95% dissolve with ethanol, liquid in contrast after the extracting solution evaporated under reduced pressure.Liquid to be measured and contrast liquid spotting launch with developing solvent A (chloroform/acetone, 95: 5) or developing solvent B (chloroform/ethyl acetate, 60: 40) in silica gel g thin-layer plate, and with the colour developing of 25% phosphomolybdic acid ethanol solution, liquid to be measured has identical colour developing speckle with contrast liquid.
According to the present invention, Heiguteng exract extract of the present invention can use separately or with pharmaceutical compositions, administering mode can be decided as the case may be, and can pass through oral, non-intestinal or topical, and form of administration can be for example tablet, capsule, unguentum, patch, injection etc.
The specific embodiment
The following examples and biological activity test are to further describe of the present invention, but do not mean that any limitation of the invention.
Embodiment 1
The preparation of Heiguteng exract ligroin extraction
After Heiguteng exract whole plant dry product 5kg pulverizing, use the petroleum ether heating and refluxing extraction, use petroleum ether 20L at every turn, extract three times, merge extractive liquid,, reduction vaporization obtains ligroin extraction 230g to doing, and it has aforesaid qualitative reaction feature.
Embodiment 2
The preparation of Heiguteng exract ethanol extraction
After Heiguteng exract whole plant dry product 5kg pulverizing, use the dehydrated alcohol heating and refluxing extraction, use ethanol 20L at every turn, extract three times, merge extractive liquid,, reduction vaporization obtains ethanol extraction 450g to doing, and it has aforesaid qualitative reaction feature.
Embodiment 3
The preparation of Heiguteng exract water extract
After Heiguteng exract whole plant dry product 5kg pulverizing, the water heating and refluxing extraction, each water 20L extracts three times, merge extractive liquid,, be evaporated to 5L, add dehydrated alcohol 35L, staticly settle, behind the centrifugal disgorging, the supernatant reduction vaporization obtains water extract 200g to doing, and it has aforesaid qualitative reaction feature.
Embodiment 4
The biological activity test of Heiguteng exract extract (HGTA)
Observation index: mouse boosting cell breeder reaction (external, body in); Dinitrochlorobenzene inducing mouse delayed hypersensitivity (immunosuppressive action); Rat anaphylactic type Arthus reacts (immune antiinflammatory action); Oleum Tiglii brings out mice ear test (anti-acute inflammation effect).
The Heiguteng exract extract that is adopted in the biological activity test is according to the method for embodiment 2 with ethanol as extracting the extract that solvent obtains.
1. the Heiguteng exract extract is to the influence of mouse boosting cell breeder reaction
(1) the external application of Heiguteng exract extract is to the influence of mouse boosting cell breeder reaction:
Put to death mice, the aseptic spleen of getting prepares splenocyte suspension, and adjusting cell concentration with complete RPMI-1640 culture fluid is 5 * 10
6Cell/ml, the Heiguteng exract extract that derives from embodiment 2,50 μ l Con A (final concentration is 0.5 μ g/ml) and the 100 μ l cell suspension of getting 50 μ l variable concentrations (concrete concentration see Table 1 listed) add in 96 orifice plates successively, matched group uses RPMI-1640 replaced C on A or/and medicine places 37 ℃, 5%CO
2Incubated incubation 56 hours, every hole adds 20 μ l
3H-TdR (10 μ Ci/ml) continues to be cultured to 72 hours,, after 80 ℃ of oven dry, glass fiber filter paper is placed in the scintillation solution on glass fiber filter paper with multiple tracks cell harvesting instrument collecting cell, surveys the cpm value with liquid scintillation counter.Each sample is made 4 parallel holes, and the result is with average cpm value representation.
Experimental result shows (table 1), and the Heiguteng exract extract is in 10 μ g/ml～500 μ g/ml concentration ranges, and breeder reaction has significant inhibitory effect to the Balb/c mouse boosting cell, and tangible dose-effect relationship is arranged.
Table 1, Heiguteng exract extract treated in vitro are to the influence of Balb/c mouse boosting cell breeder reaction
Group concentration (μ g/ml)
3The H-TdR value of mixing (cpm, x ± s)
1640 - 135±54
Con?A 0.5 13618±3272
Con?A+HGTA 10 7999±1704**
50 3240±1498**
100 2019±904**
200 1866±354**
300 567±292**
500 26±4**
One factor analysis of variance Dunnett multiple comparisons, n=4 are compared with Con A matched group in * P＜0.01.HGTA: Heiguteng exract extract
(2) oral Heiguteng exract extract is to the influence of mouse boosting cell breeder reaction:
The Balb/c mice, female, 6-8 week.Being divided into 3 groups at random is matched group, Heiguteng exract extract 0.5g/kg and two dosage groups of 1.0g/kg, 8 every group.The administration group is oral administration once a day, totally 5 days.The isopyknic physiologic saline for substitute of matched group.Put to death mice on the 6th day, the aseptic spleen of getting prepares splenocyte suspension, and adjusting cell concentration with RPMI-1640 (containing 10% hyclone) culture fluid is 5 * 10
6/ ml gets 100 μ l cell suspension and equivalent 1640 or Con A (final concentration 0.5 μ g/ml) and adds in 96 well culture plates, in 5%CO
2, 37 ℃ of incubations 54 hours, every hole adds 20 μ l
3H-TdR (10 μ Ci/ml) continues to be cultured to 72 hours, and on filter paper, the cpm value is surveyed with liquid scintillation counter in dry back with multiple tracks cell harvesting instrument collecting cell.Each sample is made 8 parallel holes, and the result is with average cpm value representation.
Experimental result shows (table 2), orally give 0.5g/kg or 1g/kg Heiguteng exract extract, and breeder reaction has significant inhibitory effect to the Balb/c mouse boosting cell, and dose-dependence is arranged.
Table 2, oral Heiguteng exract extract are to the influence of Balb/c mouse boosting cell breeder reaction
Dosage
3The H-TdR value of mixing (cpm, x ± s)
Group
(g/kg) Con?A(-) Con?A(0.5ug/ml)
Matched group 0 279 ± 98 37713 ± 11083
HGTA 0.5 489+271 27158±11116*
1.0 276±134 16532±3850**
HGTA: Heiguteng exract extract, continuous oral 5 days.* P＜0.05, * * P＜0.01, with matched group relatively, one factor analysis of variance Dunnett multiple comparisons, n=8.
(3) discuss: above-mentioned experimental result shows that the Heiguteng exract extract all has the effect that suppresses the mouse boosting cell breeder reaction by external, vivo medicine-feeding, shows that it has the effect that suppresses the mice body's immunity.The above results prompting Heiguteng exract extract may have therapeutical effect preferably for the autoimmune disease that with immune hyperfunctioning is feature.
2. the influence of Heiguteng exract extract rat acute Arthus reaction that ovalbumin is brought out
(1) method and result: Wistar female rats, body weight 190 ± 10g.Every group 7-8, the ad lib water inlet.Model+Heiguteng exract extract group: intramuscular injection 0.5ml 1.7% ovalbumin, paraffin oil and 6.3% pertussis vaccine NaCl solution are made mixes the emulsion immune animal.Irritated stomach (ig) and give the Heiguteng exract extract the immune same day, every 1.0ml (0.5g/kg), once a day, totally 7 days.Model group: injections of antigens is the same, and the same day was irritated stomach equivalent distilled water once a day, totally 7 days in immunity.Non-immune matched group: intramuscular injection equal-volume solvent, irritated stomach equivalent distilled water the same day, once a day, totally 7 days.Each treated animal is 1h after the last administration all, with the sufficient mat thickness in a vernier caliper measurement left side, then at left foot palm injection 0.2ml 1.0% ovalbumin NaCl solution booster immunization.Measure left sufficient mat thickness once more in different time behind the booster immunization.Represent the inflammation degree with left sufficient mat thickness self difference before and after the booster immunization.The results are shown in Table 3.
The influence that the rat acute Arthus that table 3, Heiguteng exract extract bring out ovalbumin reacts
Left side foot self difference (mm, x ± SD)
Measuring Time
Non-immune matched group model group (n=7) model+HGTA (n=7)
(h)
(n＝8)
1 1.28±0.47 1.64±0.5 0.93±0.33
##
2 1.41±0.47 1.85±0.3
* 1.09±0.35
##
4 1.55±0.43 1.84±0.3 1.26±0.27
##
6 1.33±0.41 1.54±0.3 1.07±0.36
#
8 1.15±0.33 1.28±0.2 0.77±0.34
##
12 0.89±0.22 0.88±0.2 0.38±0.36
##
24 0.46±0.25 0.76±0.3 0.25±0.39
#
HGTA: the Heiguteng exract extract, 0.5g/kg, oral, once a day, totally 7 days.* compare with non-immune matched group P＜0.05, #P＜0.05, and compare with model group ##P＜0.01, the t check.
(2) discuss: above-mentioned experimental result shows that the administration of Heiguteng exract extract oral can suppress rat anaphylactic type Arthus reaction, shows that the Heiguteng exract extract may have immune antiinflammatory action.The above results prompting Heiguteng exract extract may have certain therapeutical effect to diseases such as autoimmune inflammation disease such as rheumatoid arthritis, glomerulonephritis, the nephrotic syndrome, struma lymphomatosa, idiopathic thrombocytopenic purpura, systemic lupus erythematosus (sle), behcets disease, autoimmune hepatitis, immunity angiopathy.
3. the Heiguteng exract extract is to the influence of Oleum Tiglii induced mice ear swelling
(1) method and result: Kunming mouse, male, 19-22g, with 2% Oleum Tiglii mixing proinflammatory agent coating auris dextra, left ear is a normal control, every 0.1ml, tow sides respectively are coated with 0.05ml.After causing inflammation, irritate stomach Heiguteng exract extract once, claim ear heavy behind the 8h.Or scorching that morning, dusk and morning next day respectively irritate stomach Heiguteng exract extract once in causing, administration is 3 times altogether, claims ear heavy behind the 32h.The results are shown in Table 4.
Table 4, Heiguteng exract extract are to the influence of Oleum Tiglii induced mice ear swelling
Group ear swelling rate (%) (n=5)
Dosage (g/kg)
8h 32h
Normal CON/7.2 ± 5.2 3.2 ± 1.7
MODEL C ON/95.1 ± 21.7**, 64.7 ± 19.3**
Prednisolone acetate 0.045 44.0 ± 11.7## 25.8 ± 25.0#
HGTA 0.5 59.3±24.0# 26.6±18.9#
1.0 65.7±14.0 29.1±9.4#
Compare with the normal control group * P＜0.05; #P＜0.05, compare with model control group ##P＜0.01, the t check.
HGTA: Heiguteng exract extract
(2) discuss: above-mentioned experimental result shows that the Heiguteng exract extract is inhibited to Oleum Tiglii induced mice ear swelling, points out it that inflammatory diseases such as rheumatic arthritis etc. is had therapeutical effect.
4. the Heiguteng exract extract is to the influence of delayed hypersensitivity
(1) method and result: Kunming mouse, 4-5 week, male.Mouse web portion loses hair or feathers with 12% sodium sulfide solution, and the 24h postabdomen is coated with 5% dinitro-chlorine propiophenone (DNBC) solution, and every 0.02ml makes it sensitization.The 6th day oral administration after the sensitization, once a day, totally 5 days.The 9th day every mice is coated with auricular concha (single face) with the DNBC of 0.02ml 2.5% and attacks after the sensitization, surveys the mice ear rate behind the 48h.The results are shown in Table 5.
Table 5, Heiguteng exract extract are to the influence of mice delayed hypersensitivity
Grouping dosage (g/kg) number of animals (n) ear swelling rate (%)
Normal group-8 2.6 ± 3.4
Attack group-8 9.16 ± 8.11
Model group-8 40.6 ± 16.2**
Prednisone 0.045 8 12.7 ± 9.7##
HGTA 0.25 6 22.4±12.5#
0.50 8 20.2±12.4#
Compare with the normal control group * P＜0.05, #P＜0.05, and compare with model control group ##P＜0.01, the t check.
HGTA: Heiguteng exract extract
(2) discuss: above-mentioned experimental result shows, the Heiguteng exract extract has the obvious suppression effect to the mice delayed hypersensitivity, pointing out it is a kind of immunosuppressant, may have certain therapeutical effect to autoimmune inflammation disease such as rheumatoid arthritis, glomerulonephritis, the nephrotic syndrome, struma lymphomatosa, idiopathic thrombocytopenic purpura, systemic lupus erythematosus (sle), behcets disease, autoimmune hepatitis, immunity angiopathy and inflammatory diseases such as rheumatic arthritis etc.
Claims (9)
1. Heiguteng exract extract, wherein Heiguteng exract is the whole plant of Asclepiadaceae P. sepium Bunge platymiscium southwest P. sepium Bunge.
2. Heiguteng exract extract, it is characterized in that: this extract is by obtaining Heiguteng exract with organic solvent, water-containing organic solvent or water extraction.
3. the method for preparing the described Heiguteng exract extract of claim 1 comprises with organic solvent, water-containing organic solvent or water extraction Heiguteng exract.
4. the described method of claim 3, organic solvent wherein is selected from methanol, ethanol, propanol, butanols, acetone, chloroform, dichloromethane, methyl acetate, ethyl acetate, ether, petroleum ether and gasoline.
5. pharmaceutical composition, it comprises as the Heiguteng exract extract of the claim 1 of active component and one or more pharmaceutical carriers or excipient.
6. the Heiguteng exract extract of claim 1 is used to prepare the purposes of the medicine for the treatment of autoimmune disease.
7. the described purposes of claim 6, wherein said autoimmune disease is rheumatoid arthritis, glomerulonephritis, the nephrotic syndrome, struma lymphomatosa, systemic lupus erythematosus (sle), idiopathic thrombocytopenic purpura, behcets disease, autoimmune hepatitis or immunity angiopathy.
8. the Heiguteng exract extract of claim 1 is used to prepare the purposes of the medicine for the treatment of inflammatory diseases.
9. the described purposes of claim 8, inflammatory diseases wherein is a rheumatic arthritis.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1305480C (en) * | 2005-06-03 | 2007-03-21 | 中国科学院昆明植物研究所 | Antineoplastic medicinal composition, its preparation method and application |
| CN1939324B (en) * | 2005-09-29 | 2011-03-16 | 中国科学院上海药物研究所 | Medical usage of periplocin A and E |
| CN102565046A (en) * | 2011-12-29 | 2012-07-11 | 北京市药品检验所 | Method for rapidly detecting mifepristone |
| CN102579601A (en) * | 2011-04-06 | 2012-07-18 | 四川师范大学 | Medicinal composition for treating rheumatoid arthritis, and preparation method and application thereof |
| CN102631390A (en) * | 2012-04-12 | 2012-08-15 | 贵州师范大学 | Preparation method of periploca forrestii schltr extract as well as product and application of periploca forrestii schltr extract |
| CN101480419B (en) * | 2007-11-23 | 2013-07-03 | 中国人民解放军军事医学科学院毒物药物研究所 | Polysaccharide extract of Periploca forrestii Schltr, and preparation method thereof |
| CN104666461A (en) * | 2015-02-12 | 2015-06-03 | 贵阳中医学院 | Medicament for treating rheumatoid arthritis and preparation method of medicament |
| CN107875182A (en) * | 2017-11-17 | 2018-04-06 | 贵州医科大学 | Heiguteng exract active principle and its preparation method and application |
| CN108785321A (en) * | 2017-04-28 | 2018-11-13 | 南京葆赫生物技术有限公司 | Purposes of the Heiguteng exract C21 steroids in preparing IDO inhibitor |
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2003
- 2003-07-11 CN CN 03146799 patent/CN1569088A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1305480C (en) * | 2005-06-03 | 2007-03-21 | 中国科学院昆明植物研究所 | Antineoplastic medicinal composition, its preparation method and application |
| CN1939324B (en) * | 2005-09-29 | 2011-03-16 | 中国科学院上海药物研究所 | Medical usage of periplocin A and E |
| CN101480419B (en) * | 2007-11-23 | 2013-07-03 | 中国人民解放军军事医学科学院毒物药物研究所 | Polysaccharide extract of Periploca forrestii Schltr, and preparation method thereof |
| CN102579601A (en) * | 2011-04-06 | 2012-07-18 | 四川师范大学 | Medicinal composition for treating rheumatoid arthritis, and preparation method and application thereof |
| CN102565046A (en) * | 2011-12-29 | 2012-07-11 | 北京市药品检验所 | Method for rapidly detecting mifepristone |
| CN102565046B (en) * | 2011-12-29 | 2013-12-11 | 北京市药品检验所 | Method for rapidly detecting mifepristone |
| CN102631390A (en) * | 2012-04-12 | 2012-08-15 | 贵州师范大学 | Preparation method of periploca forrestii schltr extract as well as product and application of periploca forrestii schltr extract |
| CN102631390B (en) * | 2012-04-12 | 2013-10-09 | 贵州师范大学 | Preparation method, product and application of black bone vine extract |
| CN104666461A (en) * | 2015-02-12 | 2015-06-03 | 贵阳中医学院 | Medicament for treating rheumatoid arthritis and preparation method of medicament |
| CN108785321A (en) * | 2017-04-28 | 2018-11-13 | 南京葆赫生物技术有限公司 | Purposes of the Heiguteng exract C21 steroids in preparing IDO inhibitor |
| CN107875182A (en) * | 2017-11-17 | 2018-04-06 | 贵州医科大学 | Heiguteng exract active principle and its preparation method and application |
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