CN101297828B - Uses of Petasites tricholobus franch extract in medicament preparation for preventing and controlling cardiovascular and cerebrovascular diseases - Google Patents
Uses of Petasites tricholobus franch extract in medicament preparation for preventing and controlling cardiovascular and cerebrovascular diseases Download PDFInfo
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- CN101297828B CN101297828B CN 200810039033 CN200810039033A CN101297828B CN 101297828 B CN101297828 B CN 101297828B CN 200810039033 CN200810039033 CN 200810039033 CN 200810039033 A CN200810039033 A CN 200810039033A CN 101297828 B CN101297828 B CN 101297828B
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- China
- Prior art keywords
- bakkenolide
- ococh
- rhizoma petasitis
- petasitis tricholobi
- tricholobi lactone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- HWJHWSBFPPPIPD-UHFFFAOYSA-N ethoxyethane;propan-2-one Chemical compound CC(C)=O.CCOCC HWJHWSBFPPPIPD-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the field of medical technology, which discloses a new usage of a petasites tricholobus franch extract and the contained bakkenolide compound in the preparation of drugs for prevention and treatment for cardiovascular and cerebrovascular diseases. The biological activity tests show that, the petasites tricholobus franch extract and the contained bakkenolide compound can significantly reduce the encephalic necrosis percentage of the rats with local cerebral ischemia, improve the behavior score of the ischemic rats and alleviate volume of brain edema and cerebral infarction of the rats with ischemia reperfusion injury, thus prompting that the petasites tricholobus franch extract and the contained bakkenolide compound have significant protective effect on the cerebral ischemia injury. The petasites tricholobus franch extract and the contained bakkenolide compound can further significantly reduce the J-point displacement caused by ISO and the LDH level in plasma, significantly reduce the scope of myocardial ischemic myocardial infarction of the rats caused by coronary artery ligation and lower the LDH level in the plasma of the rats with the myocardial ischemia. As the petasites tricholobus franch extract and the contained bakkenolide compound have good effects on the prevention and the treatment for heart and brain ischemic diseases aspects, the petasites tricholobus franch extract and the contained bakkenolide compound can be used in the preparation of drugs for prevention and treatment for cardiovascular and cerebrovascular diseases, including coronary heart disease, cerebral ischemia, cerebral infarction (stroke), myocardial infarction and so on.
Description
Technical field
The present invention relates to medical technical field, is that Petasites tricholobus franch extract and contained Rhizoma Petasitis tricholobi lactone compounds thereof are for the preparation of the new purposes of control cardiovascular and cerebrovascular diseases medicament.
Background technology
Rhizoma Petasitis tricholobi Petasites tricholobus Franch. belongs to the butterbur plant, and for detoxification and removing stasis, traumatic injury, fracture snakebite etc. are controlled in external application to its rhizome among the people.For developing this plant, the inventor has carried out chemical constituent and pharmacological research than system to this plant, once to four Rhizoma Petasitis tricholobi lactone noval chemical compounds in the Rhizoma Petasitis tricholobi and Rhizoma Petasitis tricholobi lactone compounds be used for the treatment of may be with the purposes application of histamine and/or leukotriene-related disease patent (patent publication No.: CN1844114), Rhizoma Petasitis tricholobi rhizome extract and preparation method thereof and this extract have also been applied for patent (number of patent application: 200710038961.1) for the preparation of the purposes for the treatment of and prophylaxis of tumours disease medicament.But up to the present, there is not yet the report that relevant Petasites tricholobus franch extract and contained active substance Rhizoma Petasitis tricholobi lactone compounds thereof have the cardiovascular and cerebrovascular vessel pharmacologically active.
Summary of the invention
The objective of the invention is provides a kind of new purposes for the preparation of the control cardiovascular and cerebrovascular diseases medicament for Petasites tricholobus franch extract and contained active substance Rhizoma Petasitis tricholobi lactone compounds thereof.
Show through zoopery, it is active that the Petasites tricholobus franch extract that the present invention is prepared and contained Rhizoma Petasitis tricholobi lactone compounds thereof have the cardiovascular and cerebrovascular vessel pharmacology, thereby can comprise for the preparation of control the medicine of the cardiovascular and cerebrovascular diseases such as coronary heart disease, cerebral blood supply insufficiency, cerebral infarction (apoplexy), myocardial infarction.
The preparation method of Petasites tricholobus franch extract of the present invention and contained active substance Rhizoma Petasitis tricholobi lactone compounds thereof is as follows:
(1) extracts
After the Rhizoma Petasitis tricholobi rhizome was pulverized, heat was carried routinely, merceration or percolation extract with the aquiferous ethanol of 20-95%, gets extracting solution, the extracting solution concentrating under reduced pressure, concentrated after centrifugal or filtration, get supernatant or filtrate;
(2) purification
Above-mentioned supernatant or filtrate are adsorbed by polystyrene type porous adsorbent resin, impurity is removed in the moisture lower alcohol drip washing of water-50%, doubly measure again the moisture lower alcohol eluting of the 60-95% of volume with the 4-12 of loading crude drug quality, collect eluent, said lower alcohol is selected from methanol, ethanol, propanol, butanols or amylalcohol;
(3) concentrated, dry
With eluent concentrating under reduced pressure, drying, namely get Petasites tricholobus franch extract routinely.
(see number of patent application for details: 200710038961.1).
(4) separation and purification of contained Rhizoma Petasitis tricholobi lactone compounds in the Petasites tricholobus franch extract:
With the water-soluble suspension of making of the above-mentioned Petasites tricholobus franch extract that makes, use ethyl acetate extraction, obtain ethyl acetate extraction part, through concentrating under reduced pressure, obtain acetic acid ethyl ester extract.Acetic acid ethyl ester extract is carried out repeatedly silica gel column chromatography, finally obtained 43 Rhizoma Petasitis tricholobi lactone compounds.
The contained Rhizoma Petasitis tricholobi lactone compounds of Petasites tricholobus franch extract of the present invention all contains the Rhizoma Petasitis tricholobi lactone female ring, and their chemical structure of general formula is:
R wherein
1, R
2Respectively representative:
R
1:-H ,-OCOC (CH
3)=CHCH
3,-OCOCH=C (CH
3)
2,-OCOCH=CHSCH
3Or cis-OCOCH=CHSCH
3,-OCOCH
3,-OCOC (CH
3)=CHCH
3, cis-OCOCH=CHSOCH
3Or (R)-cis-OCOCH=CHSOCH
3, (S)-cis-OCOCH=CHSOCH
3Or (S)-trans-OCOCH=CHSOCH
3,-OCOCH
2CH (CH
3)
2,-OCOCH (CH
3)
2,-OCOCH (CH
3) CH
2CH
3,-OH ,-OCOC (=CH
2) CH (OH) (CH
3);
R
2:-H ,-OCOCH=C (CH
3)
2,-OCOCH=CHSCH
3Or cis-OCOCH=CHSCH
3,-OCOC (CH
3)=CHCH
3,-OCOCH
3,-OCOCH
3, cis-OCOCH=CHSOCH
3Or (R)-cis-OCOCH=CHSOCH
3Or (S)-cis-OCOCH=CHSOCH
3Or (S)-trans-OCOCH=CHSOCH
3,-OCOCH
2CH (CH
3)
2,-OCOC (CH
3) CHCH
3,-OCOCH (CH
3)
2,-OH;
[annotate: (R) expression dextrorotation in the group, (S) expression is left-handed, and trans represents cis, and cis represents trans.] the present invention carried out pharmacology and the test of pesticide effectiveness to Petasites tricholobus franch extract and contained part Rhizoma Petasitis tricholobi lactone compounds thereof, title and the chemical constitution thereof of said part Rhizoma Petasitis tricholobi lactone compounds see Table 1.
Each R group on the table 1 Rhizoma Petasitis tricholobi lactone compounds female ring
| Numbering | The chemical compound title | R 1 | R 2 |
| 1 | Rhizoma Petasitis tricholobi lactone | H | H |
| 2 | Rhizoma Petasitis tricholobi lactone Ia | OCOC(CH 3)＝CHCH 3 | OCOCH＝C(CH 3) 2 |
| 3 | Rhizoma Petasitis tricholobi lactone IIa | OCOCH＝C(CH 3) 2 | OCOCH＝C(CH 3) 2 |
| 4 | Rhizoma Petasitis tricholobi lactone IIIa | OCOCH＝CHSCH 3 | OCOC(CH 3)＝CHCH 3 |
| 5 | Rhizoma Petasitis tricholobi lactone IVa | OCOCH＝CHSCH 3 | OCOCH＝C(CH 3) 2 |
| 6 | Rhizoma Petasitis tricholobi lactone-Va | OCOCH＝CHSCH 3 | OCOCH(CH 3) 2 |
| 7 | Bakkenolide B. | OCOC(CH 3)＝CHCH 3 | OCOCH3 |
| 8 | Bakkenolide D. | OCOC(CH 3)＝CHCH 3 | OCOCH 3 |
| 9 | Homofukinolide | OCOC(CH 3)＝CHCH 3 | OCOC(CH 3)＝CHCH 3 |
| 10 | Bakkenolide Db. | (R)-cis-OCOCH＝CHSOCH 3 | -OCOCH 3 |
| 11 | Bakkenolide Dc. | (S)-cis-OCOCH＝CHSOCH 3 | -OCOCH 3 |
| 12 | Bakkenolide Dd. | -OCOCH 3 | cis-OCOCH＝CHSCH 3 |
| 13 | Bakkenolide De. | -OCOCH 3 | (S)-trans-OCOCH＝CHSOCH 3 |
| 14 | Bakkenolide Df. | OCOCH＝CHSOCH 3 | -OCOCH 3 |
| 15 | Bakkenolide Dg. | -OCOCH 3 | (R)-cis-OCOCH＝CHSOCH 3 |
| 16 | Bakkenolide Dh. | -OCOCH 3 | (S)-cis-OCOCH＝CHSOCH 3 |
| 17 | Bakkenolide Fa. | -OCOC(CH 3)CHCH 3 | -OCOCH 2CH(CH 3) 2 |
| 18 | Bakkenolide Fb. | -OCOCH 2CH(CH 3) 2 | -OCOC(CH 3)＝CHCH 3 |
| 19 | Bakkenolide I. | H | -OCOCH(CH 3) 2 |
| 20 | Bakkenolide J. | H | -OCOCH 2CH(CH 3) 2 |
| 21 | Bakkenolide K. | -OCOCH(CH 3) 2 | -OCOC(CH 3)CHCH 3 |
| 22 | Bakkenolide L. | -OCOCH 3 | -OCOCH 3 |
| 23 | Bakkenolide M. | -OCOCH(CH 3)CH 2CH 3 | -OCOCH(CH 3) 2 |
| 24 | Bakkenolide Na. | -OCOCH 2CH(CH 3) 2 | -OCOCH(CH 3) 2 |
| 25 | Bakkenolide Nb. | -OCOCH(CH 3) 2 | -OCOCH 2CH(CH 3) 2 |
| 26 | Rhizoma Petasitis tricholobi lactone-O | -OCOCH(CH 3)CH 2CH 3 | -OCOCH 2CH(CH 3) 2 |
| 27 | Rhizoma Petasitis tricholobi lactone-P | -OCOCH(CH 3)CH 2CH 3 | -OCOC(CH 3)CHCH 3 |
| 28 | Rhizoma Petasitis tricholobi lactone-Q | -OCOCH 2CH(CH 3) 2 | -OCOCH 2CH(CH 3) 2 |
| 29 | Rhizoma Petasitis tricholobi lactone-R | -OH | OCOC(CH 3)＝CHCH 3 |
| 30 | Rhizoma Petasitis tricholobi lactone-S | H | -OH |
| 31 | Rhizoma Petasitis tricholobi lactone-T | (R)-cis-OCOCH＝CHSOCH 3 | -OCOCH 2CH(CH 3) 2 |
| 32 | Rhizoma Petasitis tricholobi lactone-Ua | -OH | -OCOCH(CH 3) 2 |
| 33 | Rhizoma Petasitis tricholobi lactone-Ub | -OCOCH(CH 3) 2 | -OH |
| 34 | Rhizoma Petasitis tricholobi lactone-V | -OCOCH 2CH(CH 3) 2 | -OH |
| 35 | Rhizoma Petasitis tricholobi lactone-W | -OCOC(＝CH 2)CH(OH)(CH 3) | -OCOCH 3 |
| 36 | Rhizoma Petasitis tricholobi lactone-X | -OCOCH 3 | -OH |
| 37 | Rhizoma Petasitis tricholobi lactone-Ya | -OCOCH(CH 3) 2 | cis-OCOCH＝CHSOCH 3 |
| 38 | Rhizoma Petasitis tricholobi lactone-Yb | cis-OCOCH＝CHSOCH 3 | -OH |
| 39 | Rhizoma Petasitis tricholobi lactone-Za | -OCOCH(CH 3) 2 | -OCOCH＝C(CH 3) 2 |
| 40 | Rhizoma Petasitis tricholobi lactone-Zb | -OCOCH＝C(CH 3) 2 | -OCOCH(CH 3) 2 |
| 41 | Bakkenolide I. II | -OH | -OH |
| 42 | Bakkenolide G. | -OCOCH 3 | -OCOCH 2CH(CH 3) 2 |
| 43 | Bakkenolide H. | -OCOCH(CH 3) 2 | -OCOCH(CH 3) 2 |
| 44 | Rhizoma Petasitis tricholobi lactone-Uc | H | -OCOCH(CH 3) 2 |
Wherein No. 6 chemical compound Rhizoma Petasitis tricholobi lactone-Va are noval chemical compound.Evaluation to Rhizoma Petasitis tricholobi lactone-Va is as follows:
Colourless needle, draws molecular weight 459.1814[M+Na by the HR-EIMS high resolution mass spectrum by fusing point: 124-126 ℃]
+(value of calculation: 459.1817), the molecular formula of determining chemical compound is C
23H
32O
6S.Calculating its degree of unsaturation is 8.
IR (KBr, cm
-1): 1776,1165,1046 are shown with the gamma lactone ring; 1721,1696,1211 are shown with ester bond; 3086,1570,927,799 are shown with two keys, and 1570,927 are shown with terminal double bond.
13C-NMR (CDCl
3, δ, ppm) and the DEPT data show, 5 CH contained in this chemical compound
3, 5 CH
2, 7 CH and 6 quaternary carbons.Contain 3 carbonyl C (δ 177.4,175.84,165.7) in the molecule, two two keys (δ 152.7,148.1,112.5 and 108.2), chemical shift includes two carbon signals at 70.43 places, and one is CH, and one is CH
2According to the degree of unsaturation of chemical compound, can infer to remove in the molecule and contain 3 carbonyls that 2 two keys outside 1 gamma lactone ring, may also contain two circuluses.
1H-NMR (CDCl
3, δ, ppm) and show 5 methyl signals: 0.93 (3H, d, J=7.0Hz), 1.146 (3H, s), 1.18 (3H, d, J=6.5Hz), 1.20 (3H, d, J=7.5Hz) and 2.41 (3H, s).Wherein 2.41 may be the methyl that connects S.Other shows the signal of 2 two key H: 5.21 (H, s) and 5.23 (H, s).
According to HMQC, this chemical compound hydrocarbon made further ownership, infer that the mother nucleus structure of this chemical compound is: Rhizoma Petasitis tricholobi lactone class skeleton.Remove outside C in the mother nucleus structure, the H signal, also have 2 substituent groups in the molecule.Find out that in HMBC two key C and C=O, sulfur-containing methyl exist relevant, carbon signal is δ 112.52,152.59,165.73,19.26.Therefore infer in the chemical compound and contain-OCOCH (CH
3)
2With-OCOCH=CHSCH
3, two fragments are replaced on the female ring structure.Can see that in HMBC spectrum there are distant relation in the carbon signal of δ 175.84 (C-1 ') and the hydrogen signal of δ 5.785 (H-9), prove-OCOCH (CH
3)
2Be positioned at the C-9. position, so on the C-1 position be-OCOCH=CHSCH
3
By analyzing the NOESY spectrum, determined two substituent stereochemical structures, because of H-1, there is NOE between H-10 and H-15, infer and be substituted in the α position on the C-1; NOE between H-9 and H-4 then points out the C-9 substituent group to be in the β position.
In sum, can determine that this chemical compound is noval chemical compound, its structural formula is as follows:
The physics and chemistry of this chemical compound and spectral data:
Colourless needle (MeOH), mp 124-126 ℃; [α]
D 10-213.0 (C 0.0115, EtOAc); HR-MS shows that molecular formula is: C
23H
32O
6S, m/z 459.1814[M+Na]
+(calcd.459.1817);
UV (MeOH) λ max (203,234,289nm); IRV
Max KBrCm
-1: 1776,1165,1046 (gamma lactones), 1721,1696,1211 (esters), 3086,1570,927,799 (two strong);
1H (500MHz) and
13C NMR (DEPT) (125MHz), data see Table 2.
Table 2
1H NMR (500MHz),
13C NMR (125MHz) and HMBC data (CDCl
3, δ ppm)
Through pharmacology and pharmacodynamics zoopery, it is active to show that Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds thereof all have the cardiovascular and cerebrovascular vessel pharmacology, Bakkenolide I. a particularly, Bakkenolide I. Ia, Bakkenolide I. IIa, Bakkenolide I. Va, Rhizoma Petasitis tricholobi lactone-Va, Homofukinolide, Bakkenolide B., Bakkenolide D., Bakkenolide G., Bakkenolide H., Bakkenolide Fa., Rhizoma Petasitis tricholobi lactone-P, Rhizoma Petasitis tricholobi lactone-R, Rhizoma Petasitis tricholobi lactone-W etc. have obtained good result, thereby can comprise coronary heart disease for the preparation of control, cerebral blood supply insufficiency, cerebral infarction (apoplexy), the medicine of the cardiovascular and cerebrovascular diseases such as myocardial infarction.
The specific embodiment
Now in conjunction with the embodiments and biological activity test, the present invention is described in detail.This is the present invention's usefulness as an illustration only, can not be used as limiting the scope of the invention.
Experiment is collected in respectively suburb, Chengdu, Sichuan and Huzhou City suburb, Zhejiang with Rhizoma Petasitis tricholobi Petasites tricholobus Franch. and Petasites japonicus Petasites japonicus (Sieb etZucc).
Embodiment 1. preparation Petasites tricholobus franch extracts
The preparation method step of Petasites tricholobus franch extract of the present invention is as follows:
(1) extracts
After Rhizoma Petasitis tricholobi rhizome 10kg pulverized, the aquiferous ethanol 50L with 90% according to a conventional method percolation got extracting solution,
Centrifugal behind the extracting solution concentrating under reduced pressure, it is for subsequent use to get supernatant;
(2) purification
Above-mentioned supernatant is carried out polystyrene type porous adsorbent resin absorption, after impurity is removed in water and 50% aquiferous ethanol drip washing, with the aqueous alcohol eluting of 80L 80%, collect eluent.
(3) concentrated, dry
With eluent concentrating under reduced pressure routinely, drying namely gets Petasites tricholobus franch extract.
The contained part Rhizoma Petasitis tricholobi lactone compounds of embodiment 2. preparation Petasites tricholobus franch extracts
Get the Petasites tricholobus franch extract 20g of embodiment 1 preparation, be dissolved in petroleum ether, carry out routinely silica gel column chromatography, with petroleum ether-acetone 50-5: 1 chromatography, get successively Homofukinolide (7.9mg), Bakkenolide I. a (29mg), Bakkenolide I. Ia (65mg), Bakkenolide B. (1.6g), Bakkenolide I. IIa (2.8g), Bakkenolide I. Va (2.9g), Rhizoma Petasitis tricholobi lactone-Va (50mg), Bakkenolide D. (2.6g), wherein Rhizoma Petasitis tricholobi lactone-Va is noval chemical compound.The chemical constitution of these chemical compounds sees Table 1, they are Rhizoma Petasitis tricholobi lactone compounds, through the HPLC determination and analysis, wherein the content sum of Bakkenolide D., Bakkenolide B., Bakkenolide I. Va and 4 chemical compounds of Bakkenolide I. IIa accounts for more than 50% of total extract, so the main component of Petasites tricholobus franch extract is the Rhizoma Petasitis tricholobi lactone compounds.Detect through many experiments, the ratio of the content of Bakkenolide D., Bakkenolide B., Bakkenolide I. Va and Bakkenolide I. IIa is followed successively by: 0.1-5: 0.1-5: 1-9: 1-9.
Embodiment 3. usefulness Rhizoma Petasitis tricholobis prepare the Rhizoma Petasitis tricholobi lactone compounds
The dried leaves 35Kg of Rhizoma Petasitis tricholobi uses alcohol reflux three times, and the ethanol consumption is respectively 175L, 70L, 35L, and extraction time is respectively 2 hours, 1 hour, half an hour.Be dissolved in water behind the extracting solution concentrating under reduced pressure, make suspension, use successively petroleum ether and ethyl acetate extraction, get respectively petroleum ether extract, acetic acid ethyl ester extract.Acetic acid ethyl ester extract is utilized silica gel column chromatography (chromatographic silica gel 200-300 order, the yellow affair silica gel of Yantai, Shandong Zhifu development experiments factory), with normal hexane-ethyl acetate (8: 1-1: 1) gradient elution, according to the thin layer chromatography monitoring result, eluent is merged into 13 parts.Carry out silica gel column chromatography with the 4th part, take normal hexane: ethyl acetate was as 20: 1 eluting, proceed silica gel column chromatography after eluent is concentrated, ratio with normal hexane-ethyl acetate is 50: 1 eluting, get successively Homofukinolide (26.9mg), Bakkenolide I. a (65mg), Bakkenolide I. Ia (186.3mg), Bakkenolide Fa. (96.2mg), Bakkenolide Fb. (36.9mg), Bakkenolide I. (24.6mg), Bakkenolide J. (18.3mg), Bakkenolide K. (27.7mg), Bakkenolide M. (23.4mg), Bakkenolide Na. (13.0mg), Bakkenolide Nb. (34.8mg), Rhizoma Petasitis tricholobi lactone-O (21.7mg), Rhizoma Petasitis tricholobi lactone-P (32.9mg) and Rhizoma Petasitis tricholobi lactone-Q (11.0mg).With the 5th part through silica gel column chromatography, the ratio of normal hexane-ethyl acetate is 10: 1 eluting, get Bakkenolide De. (4.2mg), Bakkenolide Df. (1.9mg), Bakkenolide Dg. (29.0mg), Bakkenolide Dh. (4.2mg), Bakkenolide G. (536.1mg), Bakkenolide H. (843.2mg), Bakkenolide B. (3.1g), Bakkenolide I. IIa (6.2g), Rhizoma Petasitis tricholobi lactone-R (36.9mg), Rhizoma Petasitis tricholobi lactone-S (23.7mg), Rhizoma Petasitis tricholobi lactone-T (15.8mg), Rhizoma Petasitis tricholobi lactone-Ua (15.6mg), Rhizoma Petasitis tricholobi lactone-Ub (17.0mg), Rhizoma Petasitis tricholobi lactone-V (14.2mg), Bakkenolide I. II (52.3mg), Rhizoma Petasitis tricholobi lactone-W (35.5mg), Rhizoma Petasitis tricholobi lactone-X (38.7mg), Rhizoma Petasitis tricholobi lactone-Ya (11.0mg), Rhizoma Petasitis tricholobi lactone-Yb (11.0mg), Rhizoma Petasitis tricholobi lactone-Za (11.3mg), Rhizoma Petasitis tricholobi lactone-Zb (11.1mg).The 8th part is filtered to get Bakkenolide I. Va (4.8g), Rhizoma Petasitis tricholobi lactone-Va (50mg) through repeated crystallization.Session 9 is separated through silica gel column chromatography, obtain Rhizoma Petasitis tricholobi lactone-Uc (1.1g).Carrying out silica gel column chromatography with the 13 part separates, take the ratio of normal hexane-ethyl acetate as 5: 1 eluting, get Bakkenolide D. (5.6g), Bakkenolide Db. (60.0mg), Bakkenolide Dc. (25.0mg), Bakkenolide Dd. (15.4mg), Bakkenolide L. (10.8mg).
Embodiment 4. usefulness Petasites japonicus prepare the Rhizoma Petasitis tricholobi lactone compounds
The dry rhizome 5Kg of Petasites japonicus extracts three times with methanol eddy, and methanol usage is respectively 25L, 10L, 5L, and extraction time is respectively 2 hours, 1 hour, half an hour.Be dissolved in water behind the extracting solution concentrating under reduced pressure, make suspension, use successively petroleum ether, ethyl acetate extraction, get acetic acid ethyl ester extract.Petroleum ether is extracted thing utilize silica gel column chromatography (chromatographic silica gel 200-300 order, the yellow affair silica gel of Yantai, Shandong Zhifu development experiments factory), with normal hexane-ethyl acetate (20: 1-1: 1) gradient elution, get successively Homofukinolide (6.9mg), Bakkenolide I. a (5mg), Bakkenolide I. Ia (6.3mg), Bakkenolide Fa. (6.2mg), Bakkenolide Fb. (26.9mg), Bakkenolide I. (14.3mg), Bakkenolide J. (7.2mg), Bakkenolide K. (17.3mg), Bakkenolide M. (13.5mg), Bakkenolide Na. (7.0mg), Bakkenolide B. (600.6mg).With acetic acid ethyl ester extract, use chloroform: the ratio of methanol is 40: 3 eluting, gets successively Bakkenolide Nb. (24.9mg), Rhizoma Petasitis tricholobi lactone-O (11.7mg), Rhizoma Petasitis tricholobi lactone-P (12.9mg), Rhizoma Petasitis tricholobi lactone-Q (11.0mg).Again with using chloroform: the ratio of methanol is 20: 3 eluting, get successively Rhizoma Petasitis tricholobi lactone-O (21.7mg), Rhizoma Petasitis tricholobi lactone-Uc (31.24mg), Bakkenolide D. (300.6mg), Bakkenolide Db. (10.0mg), Bakkenolide Dc. (15.0mg), Bakkenolide Dd. (5.4mg), Bakkenolide L. (2.8mg).
Biological activity test
One, Petasites tricholobus franch extract and contained Rhizoma Petasitis tricholobi lactone compounds thereof are to the therapeutical effect of focal cerebral ischemia in rats
1, test material
1.1, experimental apparatus
SW-30 radio frequency bipolar coagulator (production of detecting instrument factory of Shanghai Detecting Technology Inst.), 307-6 table electric dental engine car (Shanghai Dental Medical Apparatus and Instrument Factory's production), SXP-1B operating microscope (production of Shanghai medical optical instrument factory), water bath with thermostatic control.
1.2, test sample:
The Petasites tricholobus franch extract of embodiment 1 preparation, Rhizoma Petasitis tricholobi lactone compounds represent with the numbering in the table 2, i.e. chemical compound 4,5,7,8.
2, experimental animal
Strain: Wistar rat
Sex: male
Body weight: 260～290 grams
Source: Chinese Academy of Sciences's Shanghai animal center (together lower)
3, test method
(1) obtain solution: take edible peanut oil as solvent, preparation Petasites tricholobus franch extract solution, nimotop injection (Bayer A.G's production) is as positive control drug.
(2) preparation cerebral ischemic model: get healthy male Wistar rat, totally 80, be divided at random 8 groups: negative control group, low, the high dose group of Petasites tricholobus franch extract, lactone compound 4,5,7,8 groups and positive controls.Press method (Tumura A, et al:J Cereb Blood Flow and Metab, 1981,1:53) the preparation cerebral ischemic model of Tamura.
(3) administration: the 1st group of negative matched group (lumbar injection equal-volume solvent), 2nd, 3 groups is low, the high dose group of Petasites tricholobus franch extract, dosage is respectively the 4th, 5,6,7 group of lactone compound 4 of per kilogram of body weight 10mg, 30mg (following represent with mg/kg), 5,7,8 groups, the 8th group of positive matched group, the Nimotop dosage is 2mg/kg.Administration time is lumbar injection immediately behind the ischemia, and the administration volume is 1ml.
(4) scoring and check weighing: carried out behavioristics's scoring in 24 hours in postoperative, single blind method is adopted in scoring; Rat right side middle cerebral artery occlusion 24 hours, broken end is got brain, further proving conclusively the right side middle cerebral artery at microscopically has blown between tractus olfactorius and inferior cerebral vein and the harmless person of brain essence, it is carried out section statining, divide another name necrotic area and right hemisphere weight, calculate downright bad percentage rate.
4, statistical procedures
All data all use X ± SD to represent, statistical analysis is with t check (seeing the Jin Pihuan chief editor for details: medical statistical methodology, publishing house of Shanghai Medical Univ, February in 1993 the 1st edition, the 36th～40 page).
5, experimental result sees Table 3
Table 3 Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds are on histology and behavioristics's impact of rats with cerebral ischemia
Compare with negative control group
*P＜0.05,
*P＜0.01
As can be seen from Table 3, compare with negative control group, Petasites tricholobus franch extract high dose group and Rhizoma Petasitis tricholobi lactone compounds 4,5,7,8 all can obviously improve the neuroethology defective (P＜0.05) of focal cerebral ischemia rat, even are better than the Nimotop group; The cerebral infarction weight (P＜0.01) that Petasites tricholobus franch extract is low, high dose group and chemical compound 4,5,7,8 all can reduce the focal cerebral ischemia rat approaches even is better than the Nimotop group.
This experimental result shows, Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds intraperitoneal injection immediately behind ischemia can obviously improve the neuroethology defective of focal cerebral ischemia rat, reduces the cerebral infarction weight of focal cerebral ischemia rat; Petasites tricholobus franch extract of the present invention and Rhizoma Petasitis tricholobi lactone compounds can be brought into play good therapeutical effect to acute focal cerebral ischemia.
Two, Petasites tricholobus franch extract and contained Rhizoma Petasitis tricholobi lactone compounds thereof are to the protective effect of focal brain ischemia-reperfusion injury in rats
1, test material
(1) laser doppler flowmetry (the Periflux4001 type that Sweden Perimed company produces), SXP-1B
Operating microscope (Shanghai medical optical instrument factory), SW-30 radio frequency bipolar electrocoagulator (production of detecting instrument factory of Shanghai Detecting Technology Inst.).
Test specimen is the same.
(2) positive control: nimotop injection (Bayer A.G's production).
(3) 2, experimental animal
Strain: SD rat
Sex: male
Body weight: 230～270 grams
3, test method:
Take edible peanut oil as solvent, preparation Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds solution, the positive contrast medicine of Nimotop.
Get totally 90 of male SD rats, be divided at random 9 groups, 10 every group, the 1st group is sham operated rats, the 2nd group of negative matched group, that is group of solvents, injection equivalent solvent, 3rd, 4 groups is low, the high dose group of Petasites tricholobus franch extract, dosage is respectively 10mg/kg, 30mg/Kg, and the 5th, 6,7,8 group is Rhizoma Petasitis tricholobi lactone compounds 4,5,7,8, and dosage is 15mg/kg, the 9th group is the Nimotop group, dosage 2mg/kg.Administration time is lumbar injection immediately behind the ischemia, and the administration volume is 1ml.
Line bolt model with reference to Longa EZ report prepares models of cerebral ischemia-reperfusion injury (Longa EZ, et al:Reversable middle cerebral artery occlusion without craniectomy in rat.Stroke, 1989,20:84).Ischemia poured into laggard row behavioristics scoring in 24 hours in 2 hours again, broken end is got brain, TTC dyeing, leaving and taking sample carries out cerebral infarct volume and measures and (to see the Zhang Juntian chief editor for details: the modern pharmacology experimental methodology, the 1241st page, combined publication society of China Concord Medical Science University of Beijing Medical University, October in 1998 the 1st edition).
4, all data all adopt X ± SD to represent, statistical analysis adopts the t check.
5, experimental result sees Table 4:
Table 4 Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds are to the protective effect of focal brain ischemia-reperfusion injury in rats (n=10, X ± SD)
*P＜0.05,
*Compare with group of solvents P＜0.01
As can be seen from Table 4, Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds can significantly reduce the rat behavior scoring, dwindle brain infarction area, alleviate ischemia side brain hemisphere edema degree.Show that Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds have stronger protective effect to focal brain ischemia-reperfusion injury in rats.
Three, Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds to isoproterenol (ISO) institute to the myocardial ischemia in rats therapeutical effect
1, test sample: the same.
2, other medicines: chloral hydrate: China Medicine (Group) Shanghai Chemical Reagent Co.,, AR, lot number: 20030227, be made into 12% solution with distilled water; Isoproterenol: the solution that is made into before use 8mg/ml with normal saline; (bio-engineering research institute, lot number: 20060311) are built up in Nanjing to lactic acid dehydrogenase (LDH) testing cassete.
3, experimental animal: SD rat (Shanghai Si Laike laboratory animal responsibility company limited), female, 180-220 gram, animal feeding in positive pressure purification ventilation Animal House, 25 ± 2 ℃ of room temperatures, ad lib and drinking-water.
4, instrument: ECG-6511 type electrocardiograph (Japanese NIHON-KOHDEN produces).
5, dosage setting: Petasites tricholobus franch extract employing 10,2 dosage of 30mg/kg, the Rhizoma Petasitis tricholobi lactone compounds is 15mg/kg.
6, experimental technique
64 of Healthy female SD rats are divided into 8 groups at random, are respectively low, high 2 the dosage groups of blank group, model control group and Petasites tricholobus franch extract, Rhizoma Petasitis tricholobi lactone compounds 4,5,7,8 groups.Blank group and model control group be intraperitoneal injection of saline (with the capacity such as administration group) in advance respectively, and each dosage group of extract is intraperitoneal injection respectively, and every day 1 time, volume is the 2ml/kg body weight, successive administration three days.Subcutaneous injection gives ISO (0.15ml/100g body weight) behind model control group, Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds group second day and the 3rd day administration 0.5h, Normal group waits the normal saline of capacity, respectively record last give before the tested medicine and the 2nd injection ISO after the electrocardiogram (standard I I leads) of 30min, measure the J point value, respectively organize the absolute value of rat J point displacement behind the calculating injection ISO.The rat aorta blood sampling, centrifugal separation plasma behind the anticoagulant heparin, LDH active unit in the spectrophotometry blood plasma.Each administration group statistical result and model group are relatively carried out the t check.The lactic acid dehydrogenase computing formula is as follows:
7, experimental result
Each group causes myocardial ischemia in rats II lead electrocardiogram J point change in displacement to ISO and sees Table 5.Each group causes the variation of myocardial ischemia in rats LDH active unit to ISO and sees Table 6.
Table 5, Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds are on the impact of myocardial ischemia in rats J point displacement due to the ISO (n=8, x ± SD)
*P＜0.05,
*Compare with model control group p＜0.01
△P＜0.05,
△ △Compare with the blank group p＜0.01
Table 6, Petasites tricholobus franch extract are on the impact (x ± SD, n=8) of ISO rat LDH active unit
*P＜0.05,
*Compare with model control group p＜0.01
△P＜0.05,
△ △Compare with the blank group p＜0.01
Experimental result shows, behind rat skin lower injection ISO, the model group animal displacement of J point occurs and increases, the significance myocardial ischemia that the LDH unit of activity increases in the blood plasma changes, Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds intraperitoneal injection can reduce LDH level in J point displacement and the blood plasma by significance, compare with the feminine gender group, Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds group all present function of resisting myocardial ischemia, and the Petasites tricholobus franch extract group presents dose dependent.
This shows that the myocardial ischemia in rats that Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds cause ISO has significant inhibitory action, and can alleviate the damage of ischemic myocardium, therefore myocardial ischemia is had obvious protective effect.
Four, Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds are to the protective effect of rat coronary artery caused by ligature acute myocardial ischemia
1, test sample: Petasites tricholobus franch extract is mixed with suspension with 0.5% tragakanta.
2, medicine: control sample: Propranolol (Shanghai Xin Pasi pharmaceutical Co. Ltd), adding 0.5% tragakanta solution, to be made into content be the 2mg/ml suspension.Other medicines: chloral hydrate: China Medicine (Group) Shanghai Chemical Reagent Co.,, A is made into 12% solution with distilled water; NBT (N-BT) (Shanghai advance chemical reagent factory production) is with front 0.1% the fresh solution that is mixed with normal saline.Lactic acid dehydrogenase (LDH) testing cassete (bio-engineering research institute is built up in Nanjing).
3, experimental animal: SD rat (Shanghai Si Laike laboratory animal responsibility company limited), male and female half and half, body weight 190-210 gram, animal feeding in positive pressure purification ventilation Animal House, 25 ± 2 ℃ of room temperatures, ad lib and drinking-water.
4, instrument: ECG-6511 type electrocardiograph (Japanese NIHON-KOHDEN produces); 722 type digital display spectrophotometers, Shanghai the 3rd analytical tool factory produces.
5, dosage setting: low, high 2 the dosage groups of Petasites tricholobus franch extract, dosage is respectively 20,40mg/kg, Rhizoma Petasitis tricholobi lactone compounds 4,5,7,8 four group dosage are 20mg/kg, and positive control drug is beta receptor antagonist Propranolol, and dosage is 5mg/kg.
6, experimental technique
Get 80 of healthy SD rats about body weight 200g, male and female half and half are divided into 8 groups at random: model control group (normal saline 10ml/kg), Propranolol group (5mg/kg), low, the high dose group of Petasites tricholobus franch extract, Rhizoma Petasitis tricholobi lactone compounds 4,5,7,8 groups.All samples is through gastric infusion, every day 1 time, continuous 2 days.
1h after the last administration, lumbar injection 12% chloral hydrate 3ml/kg anaesthetizes, the record normal ECG, chest unhairing, sterilization, along the about 2cm of left mid-clavicular line longitudinal incision skin, at the 4th or the 5th intercostal blunt separation flesh layer, open the thoracic cavity, cut off pericardium, the light right side thorax of pressing is extruded heart, behind ligation left coronary artery in Coronary vein place between right side circular cone and the left auricle, heart is put back to the thoracic cavity, fully discharge air in the thoracic cavity, sew up and close thoracic cavity, respirator assisted respiartion, frequency 20 times/minutes, tidal volume 5ml.At once, 1h, 24h, 48h trace respectively one section electrocardiogram after ligation, and in 24h, 48h difference gastric infusion.
Behind coronary ligation 50h, rat is anaesthetized again, ventral aorta blood sampling, anticoagulant heparin, 3000 collect blood after leaving heart 10min, measure LDH, and after getting blood, take out rapidly heart, use normal saline flushing, remove blood stains, reject the non-cardiac muscular tissues such as blood vessel, fat, suck moisture with absorbent paper, claim whole-heartedly weight in wet base.Along coronary sulcus excision atrium, stay ventricle, weigh, along coronary sulcus from the apex of the heart to heart base portion 4 of parallel myocardium sheets with ventricular muscles crosscut Cheng Houyue 0.1cm, clean with normal saline flushing, myocardium sheet is placed in 0.1% the N-BT solution, the 15min that under 37 ℃ of water bath condition, dyes, unnecessary dyestuff is removed in immediately water flushing after the dyeing.Infarcted region is not painted, and non-infarcted region is dyed blueness by N-BT solution.Cut off infarcted myocardium, claim weight in wet base, accounting for whole-heartedly with the infarcted region weight in wet base, the percentage ratio of weight in wet base represents myocardial infarct size.Each administration group statistical result and model group are relatively carried out the t check.
7, result of the test
Extract of the present invention sees Table 7 to the impact that coronary artery ligation causes the variation of Acute Myocardial Ischemia in Rats LDH active unit, and the impact that coronary artery ligation is caused the myocardial ischemia in rats scope sees Table 8.
Table 7, Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds cause the impact (x ± SD, n=10, U/L) of Model Rats with Acute Myocardial Ischemia LDH activity on coronary artery ligation
*P＜0.05,
*Compare with model control group p＜0.01
Table 8, Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds cause the impact (x ± S, n=10, %) of Model Rats with Acute Myocardial Ischemia ischemia scope on coronary artery ligation
*P＜0.05,
*Compare with model control group p＜0.01
Experimental result shows, the displacement of rats underwent coronary artery ligation postoperative J point raises rapidly and peaked in 1 hour after ligation, ligation after 50 hours in the model treated animal blood plasma LDH activity have very significant to rise, myocardial ischemia-area also significantly increases.Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds can significantly reduce LDH activity (P＜0.01) and myocardial infarct size (P＜0.01).
This shows that Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds gastric infusion have significant protective effect to the myocardial ischemia in rats that coronary ligation causes, can significantly reduce the tissue necrosis that myocardial ischemia causes, ischemia group is woven with significant protective effect.
Five. the protective effect of the rat cerebral cortex neural cell injury that Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds cause hypoxia in vitro-reoxygenation
1. test material:
Test-compound title: Petasites tricholobus franch extract, Rhizoma Petasitis tricholobi lactone Ia (2), Rhizoma Petasitis tricholobi lactone IIa (3), Rhizoma Petasitis tricholobi lactone IIIa (4), Rhizoma Petasitis tricholobi lactone IVa (5), Rhizoma Petasitis tricholobi lactone-Va (6), Rhizoma Petasitis tricholobi lactone B (7), Rhizoma Petasitis tricholobi lactone D (8), Homofukinolide (9)
2 reagent: N-acetylcystein, H
2O
2, xanthine/xanthine oxidase, Fe
2+/ ascorbic acid, MTT solution, acidify isopropyl alcohol, Hank ' s balanced salt solution (HBSS liquid), MEM culture medium etc.
Laboratory animal: Wistar tire Mus (trimester of pregnancy 17-18 days, Charles River Japan, Kanagawn, Japan)
4 experiment equipments: incubator (Thermo Forma company), enzyme linked immunological monitor
2. test method:
Dosage arranges: Petasites tricholobus franch extract is 150 μ g/mL, the Rhizoma Petasitis tricholobi lactone compounds is the former culture of 100 μ g/mL cortical neurons: got Wistar tire Mus under the aseptic condition (trimester of pregnancy 17-18 days, Charles River Japan, the Kanagawn, Japan) cortical tissue cultivated 8-10 days.
The foundation of hypoxia/reoxygenation model:
Anoxia group: get the rat layer neurocyte of cultivating 8-10 days, matched group sucks original culture fluid, change Hank ' s balanced salt solution (pH 7.3 for HBSS, 10mM hydroxyethyl piperazine ethanesulfonic acid) into, put into 37 ℃, 5%CO2,95%N2, take out after cultivating 2h in the anaerobism culture systems of relative humidity 100% (Thermo Forma company), put 37 ℃, 5%CO2, continue to cultivate 24h in the 95%N2, relative humidity 100% incubator.
Chemical compound 2-9 group: 30min adds chemical compound 2-9 (be dissolved in the HBSS liquid, concentration is respectively 25,50,100 μ g/mL) before anoxic treatment, and all the other are processed with the anoxia group.
The positive drug group: 30min adds N-acetylcystein (be dissolved among the HBSS, concentration is 2000 μ g/mL) before anoxic treatment, and all the other are processed with the anoxia group.
Cell viability is analyzed: the MTT method is the most classical cell viability detection method.Cortical cell is inoculated in 96 well culture plates, every hole 200 μ L, 3000-10000/hole.Put 37 ℃, 5%CO
2, incubator is cultivated 24h under the saturated humidity condition.After cultivating 24h, every hole adds MTT solution (5mg/mL, Sigma) 20 μ L, hatches 3 hours for 37 ℃, stops cultivating, and careful the suction abandoned culture supernatant in the hole, and every hole adds acidify isopropyl alcohol hydrotropy, shakes 10 minutes, and crystal is fully dissolved.Select the 540nm wavelength, measure each hole absorbance value (Abs) at the enzyme linked immunological monitor.
Cell viability (%)=100 * (Abs
Damage+administration-Abs
Damage)/(Abs
Contrast-Abs
Damage)
Lactic acid dehydrogenase (LDH) is analyzed: in the hypoxia/reoxygenation experiment, cell injury can discharge percentage rate by LDH and quantize.Through after the reoxygenation of 24h, it is active to measure LDH.The interior remaining LDH of cellular layer dissolves with 0.1M phosphate-buffered salt (containing 0.5% Triton X-100) and measures.The LDH that measures in culture fluid and the cellular layer is active, accounts for culture fluid with LDH active unit in the culture fluid and adds the percentage ratio of LDH gross activity unit in the cellular layer as the percentage rate of LDH release, and estimate the cell injury degree with this.Then these numerical value are converted into the percentage ratio that accounts for oxygen content normal cell group LDH active (100%).
Experimental result sees Table 9.
Table 9 Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds discharge percentile impact to cell viability and the LDH of the injured neurons that hypoxia/reoxygenation causes
*) P＜0.01 and the comparison of damage group
Can find out that from experimental result the Rhizoma Petasitis tricholobi lactone compounds has significant protective effect to the neuronal damage that hypoxia in vitro-reoxygenation causes, can obviously improve the cell viability of injured neurons, reduce lactic acid dehydrogenase (LDH) and discharge.
Above-mentioned biological activity test result shows that Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds can obviously reduce locality brain rats with cerebral ischemia brain necrosis percentage rate, improves behavioristics's scoring of cerebral ischemic rats; Can make the ischemical reperfusion injury rat obviously alleviate cerebral edema and cerebral infarction volume, prompting Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds have obvious protective effect to cerebral ischemia.In addition, Petasites tricholobus franch extract and Rhizoma Petasitis tricholobi lactone compounds can significance reduce LDH level in J point displacement due to the ISO and the blood plasma; Can obviously dwindle the myocardial ischemia in rats myocardial infarct size that coronary ligation causes, reduce LDH level in the rats with myocardial ischemia blood plasma.Biological activity test also shows, the neuronal damage due to Petasites tricholobus franch extract and the Rhizoma Petasitis tricholobi lactone compounds hypoxia/reoxygenation has obvious protective effect.
In view of Petasites tricholobus franch extract and contained Rhizoma Petasitis tricholobi lactone compounds thereof have good effect aspect the control heart, the cerebral ischemia diseases, therefore can be used for preparing the medicine for the treatment of and prevention cardiovascular and cerebrovascular disease.
Claims (3)
1. the application of Rhizoma Petasitis tricholobi lactone compounds in the neuronal damage medicine due to preparation control cerebral ischemia, myocardial ischemia or the hypoxia/reoxygenation is characterized in that said Rhizoma Petasitis tricholobi lactone compounds chemical structural formula is as follows:
R wherein
1, R
2Respectively representative:
R
1：-OCOC(CH
3)＝CHCH
3，-OCOCH＝C(CH
3)
2，-OCOCH＝CHSCH
3，(S)-trans-OCOCH＝CHSOCH；
R
2：-OCOCH＝C(CH
3)
2，-OCOCH
3，-OCOC(CH
3)＝CHCH
3，-OCOCH(CH
3)
2。
2. by the application of Rhizoma Petasitis tricholobi lactone compounds claimed in claim 1 in the neuronal damage medicine due to preparation control cerebral ischemia, myocardial ischemia or the hypoxia/reoxygenation, it is characterized in that said Rhizoma Petasitis tricholobi lactone compounds is: Bakkenolide I. a, Bakkenolide I. Ia, Bakkenolide I. IIa, Rhizoma Petasitis tricholobi lactone-Va, Homofukinolide, Bakkenolide D., Bakkenolide I. Va, Bakkenolide B..
3. Rhizoma Petasitis tricholobi lactone compounds is characterized in that Rhizoma Petasitis tricholobi lactone compounds chemical structural formula is as follows:
R wherein
1For-OCOCH=CHSCH
3, R
2For-OCOCH (CH
3)
2
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| CN102086182B (en) * | 2011-01-05 | 2013-04-03 | 中国人民解放军第二军医大学 | Petasites tatewakianus Kitam. extract as well as preparation method and application thereof |
| CN102846484B (en) * | 2012-04-20 | 2014-04-16 | 朱丹 | Application of Petasites tatewakianus Kitam lactone in preparing anti-ultraviolet compositions |
| CN110585255A (en) * | 2016-12-12 | 2019-12-20 | 中国人民解放军第二军医大学 | Application of petasites tricholobus franch extract in preparation of medicines or health-care food for preventing and treating anoxia diseases |
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| CN1844114A (en) * | 2006-04-27 | 2006-10-11 | 中国人民解放军第二军医大学 | Fukinanolide compounds and pharmaceutical application thereof |
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| CN1844114A (en) * | 2006-04-27 | 2006-10-11 | 中国人民解放军第二军医大学 | Fukinanolide compounds and pharmaceutical application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 方志先等.地葫芦.土家族药物志.中国医药科技出版社,2007,436-437. * |
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