CN107446014A - The Dulong Paris polyphylla ethanol extract and its application in pharmacy is separated with its chemical composition and authentication method - Google Patents

The Dulong Paris polyphylla ethanol extract and its application in pharmacy is separated with its chemical composition and authentication method Download PDF

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CN107446014A
CN107446014A CN201710661441.XA CN201710661441A CN107446014A CN 107446014 A CN107446014 A CN 107446014A CN 201710661441 A CN201710661441 A CN 201710661441A CN 107446014 A CN107446014 A CN 107446014A
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pde5
dulong
pde4
paris polyphylla
methanol
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王跃虎
汉秋燕
李晓艳
左何英
杨珺
夏梦媛
罗吉凤
李恒
杨永平
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Gongshan Yiyuan Chinese Herbal Medicine Planting Farmer Specialized Cooperatives
Kunming Institute of Botany of CAS
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Abstract

Dulong Paris polyphylla (Paris dulongensis H. Li ＆ Kurita) ethanol extract (PDE4) and total saposins (PDE5) with active anticancer and preparation method thereof and its application in the medicine for treating or preventing leukaemia, liver cancer, lung cancer, breast cancer, colon cancer is prepared are provided, and the separation of chemical composition and authentication method in PDE5 are provided.Using column chromatography and the separation method of half preparative high-performance liquid chromatographic, and the authentication method of mass spectrum and NMR spectrum, separated from PDE5, identify chonglou saponin V, chonglou saponin I, β moulting hormone, protodioscin, Parisaponin I and ginsenoside Rg1.The present invention illustrates PDE5 main chemical compositions first, by the method for offer, above-mentioned chemical composition can be simply and rapidly obtained from PDE5, for establishing the quality control standard of Dulong Paris polyphylla, Dulong Rhizoma Paridis extract PDE4 or Dulong Chinese paris rhizome total saponin PDE5.

Description

Dulong Paris polyphylla ethanol extract and its application in pharmacy separate with its chemical composition And authentication method
Technical field：
The invention belongs to pharmaceutical technology field and natural product extraction separation technology field, more particularly to Dulong Paris polyphylla ethanol Extract PDE4, Dulong Chinese paris rhizome total saponin PDE5 and preparation method thereof and its medicine for being used to prepare treating cancer, and Dulong Paris polyphylla active anticancer position PDE5 chemical composition separation and authentication method.
Background technology：
Melanthiaceae (Melanthiaceae) Paris (Paris) plant has very high medical value, wherein Yunnan Paris polyphylla [Paris polyphylla var.yunnanensis (Franch.) Hand.-Mazz] and paris polyphylla [Paris Polyphylla var.chinensis (Franch.) Hara] record in version in 2015《Pharmacopoeia of People's Republic of China》.Paris polyphylla The artificial growth of platymiscium has been made significant headway, for example in Yunnan Province's Nujiang Lisu Autonomous Prefecture, Paris is planted within 2014 Thing cultivated area just have about 200 hectares [Wang Yuehu, Niu Hongmei, Zhang Zhaoyun, face south recklessly, the medical value of Li Heng paris plants And its chemical substance basis CHINA JOURNAL OF CHINESE MATERIA MEDICAs, 2015,40 (5), 833-839].Planted in the Paris of Nujiang Prefecture artificial growth In thing, in addition to Yunnan Paris polyphylla, Rhizome of Forrest Paris [Paris forrestii (Takht.) H.Li], Dulong Paris polyphylla (Paris Dulongensis H.Li＆Kurita) also occupy certain proportion.At present, Dulong Paris polyphylla is there is no in the relevant report of medicinal aspect, The present invention on the basis of early stage screening active ingredients, find Dulong Paris polyphylla ethanol extract (PDE4) and its total saposins (PDE5) have it is aobvious The cytotoxic activity of work, therefore PDE4 and PDE5 chemical substance basis is illustrated, for establishing Dulong Paris polyphylla, PDE4 or PDE5 Quality control standard, and the new drug development based on Dulong Paris polyphylla, PDE4 or PDE5, it is significant.
The content of the invention：
The problem of present invention exists for prior art, there is provided Dulong Paris polyphylla has the ethanol extract PDE4 of active anticancer With total saposins PDE5, and chemical composition in PDE5 extraction and authentication method.Because PDE4 and PDE5 has significant resist Cancer activity, but its chemical composition and active component are unclear, it is impossible to its quality control standard is established very well.So the present invention carries A kind of PDE5 isolation and identification method has been supplied to be used for solving this technical problem.
In order to realize the above-mentioned purpose of the present invention, the invention provides following technical scheme：
Dulong Paris polyphylla ethanol extract PDE4, it is characterised in that described PDE4 is prepared by following methods：Dulong weight After building dry rhizome crushes, with 90%~95% ethanol, 60~65 DEG C of 3~4h of extraction, filtrate and filter residue are obtained after filtering, filter residue is pressed Above-mentioned condition is extracted 2 times again, merges 3 extraction gained filtrates, Dulong Paris polyphylla ethanol extract PDE4 is obtained after concentration.
Dulong Paris polyphylla ethanol extract PDE5, it is characterised in that described PDE5 is prepared by following methods：By PDE4 Add water that suspension is made, extracted respectively 3 times with petroleum ether, chloroform, n-butanol successively, after extract concentration, respectively obtain oil Contain chonglou saponin V, Paris polyphylla soap in ether extraction part, chloroform extraction part and n-butanol portion PDE5, described PDE5 Glycosides I, β-moulting hormone, protodioscin, Parisaponin I and ginsenoside Rg1, total content are 10-95% weight.
Present invention also offers a kind of pharmaceutical composition, wherein the Dulong Paris polyphylla ethanol extract containing therapeutically effective amount PDE4 and pharmaceutically acceptable carrier.
And another pharmaceutical composition, wherein Dulong Paris polyphylla ethanol extract PDE5 and medicine containing therapeutically effective amount Acceptable carrier on.
The present invention additionally provides compound chonglou saponin V, Paris polyphylla in described Dulong Paris polyphylla ethanol extract PDE5 simultaneously Saponin I, β-moulting hormone, protodioscin, separation and the authentication method of Parisaponin I and ginsenoside Rg1, this method Comprise the steps：
(1) PDE5 is carried out with the silica gel column chromatography of 80~100 mesh drawing section, chloroform-methanol v/v5:1、3:1、2:1、1:1 With 0:1 carries out gradient elution successively, is divided into 6 part C1~C6 through TLC detections；
(2) C1 parts in RP-18 through pressing reversed-phase silica gel column chromatography, with methanol-water 5:95→100:0, v/v gradient elution, Again through TLC combining data detections, 10 part C-1-1~C-1-10 are obtained, C-1-2 parts are 50% methanol-eluted fractions, warp After Sephadex LH-20 gel column chromatographies, then through semi-preparative HPLC, Welch Materials Ultimate AQ-C18Color Spectrum post, 5.0 μm, 4.6 × 300mm of φ；Flow velocity v=1.0mL/min；Acetonitrile-water, 16:84 are prepared β-moulting hormone, tR= 23.918min, C-1-10 part are 90% methanol-eluted fractions, after Sephadex LH-20 gel column chromatographies, then by just Phase silica gel column chromatography methylene chloride-methanol, 12:1 respectively obtains chonglou saponin V and chonglou saponin I；
(3) C3 parts are through pressing reversed-phase silica gel column chromatography, methanol-water 30 in RP-18:70→100:0, v/v gradient elution, then Through TLC combining data detections, 10 part C-3-1~C-3-10 are obtained, C-3-4 is 50% methanol-eluted fractions, through Sephadex LH- After 20 gel column chromatographies, then through normal-phase silica gel column chromatography methylene chloride-methanol-water, 5:1:0.06 and semi-preparative HPLC, Welch Materials Ultimate AQ-C18Chromatographic column, 5.0 μm, 4.6 × 300mm of φ；Flow velocity v=1.0mL/min；Second Nitrile-water, 25:75 are prepared ginsenoside Rg1, tR=14.671min；
(4) C5 parts are through pressing reversed-phase silica gel column chromatography, methanol-water 5 in RP-18:95→100:0, v/v gradient elution, then Through TLC combining data detections into 17 part C-5-1~C-5-17, C-5-10 is 50% methanol-eluted fractions, through Sephadex LH- After 20 gel column chromatographies, then through semi-preparative HPLC, Zorbax SB-C18Post, 5.0 μm, 9.4 × 250mm of φ；Flow velocity v= 2.0mL/min；Acetonitrile-water, 30:70 preparations respectively obtain protodioscin, tR=24.174min and Parisaponin I, tR =29.064min；Then, by mass spectrum MS and NMR spectrum NMR to isolated compound from PDE5 Learn the determination of structure.
Isolated 6 compounds, chemical constitution is carried out by mass spectrum MS and NMR spectrum NMR to it from PDE5 Be defined as chonglou saponin V (PDE10), chonglou saponin I (PDE11), β-moulting hormone (PDE12), protodioscin (PDE14), Parisaponin I (PDE15) and ginsenoside Rg1 (PDE22), these compounds can be used for establishing Dulong weight Building or Dulong Paris polyphylla ethanol extract PDE4 or Dulong Chinese paris rhizome total saponin PDE5 quality control standard.So the present invention provides Chonglou saponin V, chonglou saponin I, β-moulting hormone, protodioscin, Parisaponin I and ginsenoside Rg1 are establishing Application in Dulong Paris polyphylla or Dulong Paris polyphylla ethanol extract PDE4 or Dulong Chinese paris rhizome total saponin PDE5 quality control standard.
PDE4, PDE5, PDE10, PDE11, PDE14 and PDE15 are thin to leukemia HL-60 cell, liver cancer SMMC-7721 Born of the same parents, lung cancer cell A-549, MCF-7 Breast Cancer Cell and colon cancer SW480 cells have significant inhibitory activity (being shown in Table 1), and it is lived Property is suitable with positive control medicine cis-platinum.PDE4 or PDE5, which can be used for preparing, treats or prevents Cancerous disease such as leukaemia, liver Cancer, lung cancer, breast cancer, colon cancer medicine in application.
Dulong Paris polyphylla ethanol extract PDE4 and Dulong Chinese paris rhizome total saponin PDE5 is preparing treatment or prevention Cancerous disease Application in medicine.According to the application of described PDE4 and PDE5 in the medicine for treating or preventing Cancerous disease is prepared, wherein Described cancer is leukaemia, liver cancer, lung cancer, breast cancer, colon cancer.
Present invention also offers chonglou saponin V, chonglou saponin I, β-moulting hormone, protodioscin, Parisaponin I With application of the ginsenoside Rg1 in the medicine for treating or preventing leukaemia, liver cancer, lung cancer, breast cancer, colon cancer is prepared.
In addition, present invention also offers the method for the pharmaceutical composition for preparing treating cancer, this method presses following weight Percentage is mixed：PDE4 or PDE5 accounts for 20-80%, dispersant 2-20%, disintegrant 3-5%, emulsifying agent 3-8%, bonds Agent 0.2-2%, wetting agent 0.5-10%, remaining is filler.
Compared with prior art, the present invention has following excellent beneficial effects：Dulong Paris polyphylla ethanol extract PDE4 and Dulong Chinese paris rhizome total saponin PDE5 is thin to leukemia HL-60 cell, SMMC-7721 liver cancer cells, lung cancer cell A-549, breast cancer MCF-7 Born of the same parents and colon cancer SW480 cells have significant inhibitory activity, and its activity is suitable with according to drugs Cisplatin.The present invention illustrates solely first Imperial Paris polyphylla active anticancer position PDE5 main chemical compositions, these compositions can be used for working out Dulong Paris polyphylla, Dulong Paris polyphylla second Alcohol extracting thing PDE4 or Dulong Chinese paris rhizome total saponin PDE5 quality control standard.
Embodiment：
Further illustrate the essentiality content of the present invention with embodiments of the invention below, but this is not limited with this Invention.
Embodiment 1：
PDE4 and the PDE5 preparation and its separation of contained chemical composition of the present invention：
Dulong Paris polyphylla dry rhizome 1kg, after crushing, with 3~4 liters of 90%~95% ethanol, 60~65 DEG C are extracted 3~4h, Filtrate and filter residue are obtained after filtering, filter residue is extracted 2 times again by above-mentioned condition.Merge 3 extraction gained filtrates, Dulong is obtained after concentration Paris polyphylla ethanol extract 133g (being designated as PDE4).
Suspension is made in PDE4 plus 1 liter of water, is extracted respectively 3 times with 1 liter of petroleum ether, chloroform, n-butanol successively.Extraction After liquid concentration, petroleum ether portion (2g), chloroform extraction part (30g) and n-butanol portion (30g) are respectively obtained.Just Butanol, before immunoassay part is exactly Dulong Chinese paris rhizome total saponin, is designated as PDE5.
PDE5 (30g) is carried out with the silica gel column chromatography of 80~100 mesh to draw section, chloroform-methanol (v/v) 5:1、3:1、2:1、 1:1 and 0:1 carries out gradient elution successively, is divided into 6 parts (C1~C6) through TLC detections.
C1 parts (1.4g) in RP-18 through pressing reversed-phase silica gel column chromatography, with methanol-water (5:95→100:0, v/v) gradient Elution, then through TLC combining data detections, obtain 10 parts (C-1-1~C-1-10).C-1-2 parts (50% methanol-eluted fractions, 58.0mg), after Sephadex LH-20 gel column chromatographies, then through semi-preparative HPLC (Welch Materials Ultimate AQ-C18Chromatographic column, 5.0 μm, 4.6 × 300mm of φ；Flow velocity v=1.0mL/min；Acetonitrile-water, 16:84) it is prepared into To compound PDE12 (13.3mg, tR=23.918min).C-1-10 (90% methanol-eluted fractions) has 139.4mg, warp After Sephadex LH-20 gel column chromatographies, then by normal-phase silica gel column chromatography (methylene chloride-methanol, 12:1) respectively obtaining Compound PDE10 (2.3mg) and PDE11 (35.3mg).
C3 parts (14g) are through pressing reversed-phase silica gel column chromatography, methanol-water (30 in RP-18:70→100:0, v/v) gradient is washed It is de-, then through TLC combining data detections, obtain 10 parts (C-3-1~C-3-10).C-3-4 (50% methanol-eluted fractions) has 596.9mg, after Sephadex LH-20 gel column chromatographies, then through normal-phase silica gel column chromatography (methylene chloride-methanol-water, 5:1: And semi-preparative HPLC (Welch Materials Ultimate AQ-C 0.06)18Chromatographic column, 5.0 μm, 4.6 × 300mm of φ； Flow velocity v=1.0mL/min；Acetonitrile-water, 25:75) compound PDE22 (15.5mg, t is preparedR=14.671min).
C5 parts (3g) are through pressing reversed-phase silica gel column chromatography, methanol-water (5 in RP-18:95→100:0, v/v) gradient elution, Again through TLC combining data detections into 17 parts (C-5-1~C-5-17).C-5-10 (50% methanol-eluted fractions) has 322.0mg, warp After Sephadex LH-20 gel column chromatographies, then through semi-preparative HPLC (Zorbax SB-C18Post, 5.0 μm, φ 9.4 × 250mm；Flow velocity v=2.0mL/min；Acetonitrile-water, 30:70) prepare and respectively obtain compound PDE14 (4.8mg, tR= 24.174min) and PDE15 (5.3mg, tR=29.064min).
Embodiment 2
The Structural Identification of isolated chemical composition in the Dulong Chinese paris rhizome total saponin (PDE5) of the present invention：
Chonglou saponin V (PDE10), white solid, molecular formula C39H62O12, CAS registration numbers 19057-67-1.1H NMR (C5D5N, 500MHz) δ:6.40 (1H, br s), 5.29 (1H, br d, J=5.1Hz), 1.78 (3H, d, J=6.3Hz), 1.12 (3H, d, J=7.0Hz), 1.04 (3H, s), 0.81 (3H, s), 0.68 (3H, d, J=5.5Hz).13C NMR(C5D5N, 126MHz)δ:140.9,121.8,109.3,102.2,100.4,81.1,79.7,78.4,77.9,77.8,74.2,72.9, 72.7,71.8,69.6,66.9,62.9,62.7,56.6,50.3,42.0,40.5,39.9,39.0,37.5,37.2,32.3, 32.3,31.8,31.7,30.6,30.2,29.3,21.1,19.5,18.8,17.4,16.4,15.1。ESIMS m/z 721[M- H]-.Its NMR data with document, [grind by Li Zhongqiong, Lin Ruichao, Fu Wen, Wang Gangli, Wei Feng, Hangzhoupro too pretty asparagus lettuces flower chemical composition Study carefully Chinese herbal medicines, 2001,32 (2):101-104] in data it is basically identical.
Chonglou saponin I (PDE11), white solid, molecular formula C44H70O16, CAS registration numbers 50773-41-6.1H NMR (C5D5N, 500MHz) δ:6.28 (1H, br s), 5.93 (1H, d, J=1.2Hz), 5.29 (1H, d, J=4.9Hz), 1.76 (3H, D, J=6.2Hz), 1.12 (3H, d, J=7.0Hz), 1.03 (3H, s), 0.81 (3H, s), 0.68 (3H, d, J=5.6Hz).13C NMR(C5D5N,126MHz)δ:140.8,121.9,109.6,109.3,102.0,100.2,86.7,82.7,81.1,78.1, 77.9,77.8,77.4,77.0,76.8,74.2,72.8,72.5,69.6,66.9,62.9,62.5,61.4,56.6,50.3, 42.0,40.5,39.9,39.0,37.5,37.2,32.3,32.2,31.8,31.7,30.6,30.2,29.3,21.1,19.4, 18.7,17.4,16.4,15.1.ESIMS m/z 853[M-H]-.Its NMR data is with document [Wang Yu, Gao Wenyuan, Yuan Lichun, Liu New bridge, Wang Shujun, the chemical constitution study Chinese herbal medicines of Chen Cui Yunnan Paris polyphylla, 2007,38 (1):17-20] in data basic one Cause.
β-moulting hormone (PDE12), white solid, molecular formula C27H44O7, CAS registration numbers 5289-74-7.1H NMR (C5D5N,500MHz)δ:6.26(1H,s),1.59(3H,s),1.36(6H,s),1.22(3H,s),1.06(3H,s)。13C NMR (C5D5N,126MHz)δ:203.6,166.2,121.7,84.2,77.6,76.9,69.6,68.2,68.1,51.4,50.1, 48.1,42.7,38.7,38.0,34.4,32.5,32.0,31.8,30.2,30.0,27.5,24.5,21.8,21.5,21.2, 17.9.ESIMS m/z 479[M-H]-.Its NMR data is with document [Zhao Wanshun, Gao Wenyuan, Huang Xian school, Huang Luqi, Xiao Peigen The chemical constitution study research and development of natural products of ball connective Paris polyphylla, 2011,23 (6), 1017-1020] in data one Cause.
Protodioscin (PDE14), white solid, molecular formula C51H84O22, CAS registration numbers 55056-80-9.1H NMR (C5D5N,800MHz)δ:6.38 (1H, s), 5.84 (1H, s), 5.27 (1H, br s), 1.74 (3H, d, J=6.2Hz), 1.60 (3H, d, J=6.2Hz), 1.30 (3H, d, J=6.8Hz), 1.02 (3H, s), 0.95 (3H, d, J=6.7Hz), 0.86 (3H, s)。13C NMR(C5D5N,201MHz)δ:140.8,121.9,110.7,105.0,102.9,102.1,100.3,81.1,78.6, 78.6,78.6,78.1,78.0,77.8,77.0,75.3,75.2,74.2,74.0,72.9,72.8,72.6,72.6,71.7, 70.4,69.6,63.9,62.8,61.3,56.6,50.4,40.8,40.7,40.0,39.0,37.5,37.2,37.2,34.3, 32.5,32.4,31.7,30.2,30.0,28.4,21.1,19.4,18.7,18.5,17.5,16.5。ESIMS m/z 1071[M+ Na]+.Its NMR data is with document [Kochkin DV, Khandy MT, Zaitsev GP, Tolkacheva NV, Shashkov AS,Titova MV,Chirva VY,Nosov AM.Protodioscin in Dioscorea deltoidea Suspension cell culture.Chem.Nat.Compd., 2016,52 (4), 664-668] in data it is consistent.
Parisaponin I (PDE15), white solid, molecular formula C50H82O22, CAS registration numbers 561007-63-4.1H NMR(C5D5N, 500MHz) δ:6.28 (1H, s), 5.92 (1H, s), 5.28 (1H, br s), 1.76 (3H, d, J=6.2Hz), 1.32 (3H, d, J=6.9Hz), 1.03 (3H, s), 0.97 (3H, d, J=6.7Hz), 0.88 (3H, s).13C NMR(C5D5N, 126MHz)δ:140.8,121.9,110.7,109.6,105.0,102.0,100.2,86.7,82.8,81.1,78.7,78.6, 78.2,77.9,77.7,77.4,77.0,76.8,75.3,75.2,74.2,72.8,72.5,71.7,69.6,63.9,62.8, 62.5,61.4,56.6,50.4,40.8,40.7,40.0,39.0,37.5,37.2,37.2,34.3,32.5,32.4,31.7, 30.2,30.0,28.4,21.2,19.4,18.7,17.5,16.5。ESIMS m/z 1057[M+Na]+.Its NMR data is with document [Matsuda H,Pongpiriyadacha Y,Morikawa T,Kishi A,Kataoka S,Yoshikawa M.Protective effects of steroid saponins from Paris polyphylla var.yunnanensis on ethanol-or indomethacin-induced gastric mucosal lesions in rats:structural requirement for activity and mode of Action.Bioorg.Med.Chem.Lett., 2003,13 (6), 1101-1106] in data it is consistent.
Ginsenoside Rg1 (PDE22), white solid, molecular formula C42H72O14, CAS registration numbers 22427-39-0.1H NMR (C5D5N, 500MHz) δ:5.23 (1H, m), 5.16 (1H, d, J=7.8Hz), 5.01 (1H, d, J=7.8Hz), 2.05 (3H, s), 1.60(3H,s),1.57(9H,s),1.13(3H,s),1.01(3H,s),0.78(3H,s)。13C NMR(C5D5N,126MHz)δ: 131.0,126.0,106.0,98.3,83.3,80.2,79.7,79.4,78.7,78.4,78.2,75.5,75.2,71.8, 71.6,70.2,63.1,62.9,61.4,51.6,51.4,50.0,49.2,45.1,41.1,40.4,39.7,39.4,36.1, 31.8,31.0,30.7,28.0,26.6,25.8,23.3,22.4,17.8,17.6,17.6,17.2,16.4。ESIMS m/z 823[M+Na]+.Its NMR data is with document [Kim DS, Chang YJ, Zedk U, Zhao P, Liu YQ, Yang CR.Dammarane saponinsfrom Panax ginseng.Phytochemistry,1995,40(5),1493-1497] In data it is consistent.
Embodiment 3：
Dulong Paris polyphylla ethanol extract (PDE4), extracting n-butyl alcohol position (PDE5) and isolated chemical combination from PDE5 Inhibitory action of the thing to cancer cell growth：
By PDE4, PDE5 and compound PDE10, PDE11, PDE12, PDE14, PDE15 isolated from PDE5 Cytotoxic activity test is carried out with PDE22, the results are shown in Table 1.As a result show, PDE4, PDE5, PDE10, PDE11, PDE14 and PDE15 is to leukemia HL-60 cell, SMMC-7721 liver cancer cells, lung cancer cell A-549, MCF-7 Breast Cancer Cell and colon Cancer SW480 cells have significant inhibitory activity, and its activity is suitable with positive control medicine cis-platinum.
The Dulong Rhizoma Paridis extract of table 1 and compound are to cancer cell growth inhibitory action
Embodiment 4：
Dulong Rhizoma Paridis extracts (PDE4), sodium carboxymethyl starch, appropriate starch are added, gelatinized corn starch granulation, is dried, whole grain, It is appropriate to add talcum powder, mixes, tabletting (every 0.5g, equivalent to Dulong rhizoma paris rhizome 3g), 2 tablets once, 3 times a day.
Embodiment 5：
The medicine for the treatment of cancer is prepared, is mixed by following percentage by weight：Dulong Rhizoma Paridis extract PDE4 accounts for 20- 80%, dispersant 2-20%, disintegrant 3-5%, emulsifying agent 3-8%, binding agent 0.2-2%, wetting agent 0.5-10%, remaining is Filler.Then routinely the anticarcinogen that active ingredient is PDE4 is made in process for preparing medicine.
Embodiment 6：
The extracting n-butyl alcohol position (PDE5) of Dulong Rhizoma Paridis extract, add sodium carboxymethyl starch, appropriate starch, gelatinized corn starch Granulation, dry, whole grain, it is appropriate to add talcum powder, mixes, tabletting (every 0.5g, equivalent to Dulong rhizoma paris rhizome 3g), every time 2 Piece, 3 times a day.
Embodiment 7：
The medicine for the treatment of cancer is prepared, is mixed by following percentage by weight：Dulong Paris polyphylla extracting n-butyl alcohol position PDE5 accounts for 20-80%, dispersant 2-20%, disintegrant 3-5%, emulsifying agent 3-8%, binding agent 0.2-2%, wetting agent 0.5- 10%, remaining is filler.Then routinely the anticarcinogen that active ingredient is PDE5 is made in process for preparing medicine.

Claims (10)

1. Dulong Paris polyphylla ethanol extract PDE4, it is characterised in that described PDE4 is prepared by following methods：Dulong Paris polyphylla After dry rhizome crushes, with 90%~95% ethanol, 60~65 DEG C of 3~4h of extraction, filtrate and filter residue are obtained after filtering, filter residue is by upper State condition to extract again 2 times, merge 3 extraction gained filtrates, Dulong Paris polyphylla ethanol extract PDE4 is obtained after concentration.
2. Dulong Paris polyphylla ethanol extract PDE5, it is characterised in that described PDE5 is prepared by following methods：By PDE4 plus Suspension is made in water, is extracted respectively 3 times with petroleum ether, chloroform, n-butanol successively, after extract concentration, respectively obtains petroleum ether Contain chonglou saponin V, chonglou saponin in extraction part, chloroform extraction part and n-butanol portion PDE5, described PDE5 I, β-moulting hormone, protodioscin, Parisaponin I and ginsenoside Rg1, total content are 10-95% weight.
3. pharmaceutical composition, wherein Dulong Paris polyphylla ethanol extract PDE4 described in the claim 1 containing therapeutically effective amount and Pharmaceutically acceptable carrier.
4. pharmaceutical composition, wherein Dulong Paris polyphylla ethanol extract PDE5 described in the claim 2 containing therapeutically effective amount and Pharmaceutically acceptable carrier.
5. compound chonglou saponin V, chonglou saponin I, β-husking in the Dulong Paris polyphylla ethanol extract PDE5 described in claim 2 Hormone, protodioscin, separation and the authentication method of Parisaponin I and ginsenoside Rg1, it is characterised in that this method bag Include following step：
(1) PDE5 is carried out with the silica gel column chromatography of 80~100 mesh drawing section, chloroform-methanol v/v5:1、3:1、2:1、1:1 and 0:1 Gradient elution is carried out successively, is divided into 6 part C1~C6 through TLC detections；
(2) C1 parts in RP-18 through pressing reversed-phase silica gel column chromatography, with methanol-water 5:95→100:0, v/v gradient elution, then pass through TLC combining data detections, 10 part C-1-1~C-1-10 are obtained, C-1-2 parts are 50% methanol-eluted fractions, through Sephadex After LH-20 gel column chromatographies, then through semi-preparative HPLC, Welch Materials Ultimate AQ-C18Chromatographic column, 5.0 μ m,φ4.6×300mm；Flow velocity v=1.0mL/min；Acetonitrile-water, 16:84 are prepared β-moulting hormone, tR= 23.918min, C-1-10 part are 90% methanol-eluted fractions, after Sephadex LH-20 gel column chromatographies, then by just Phase silica gel column chromatography methylene chloride-methanol, 12:1 respectively obtains chonglou saponin V and chonglou saponin I；
(3) C3 parts are through pressing reversed-phase silica gel column chromatography, methanol-water 30 in RP-18:70→100:0, v/v gradient elution, then pass through TLC combining data detections, 10 part C-3-1~C-3-10 are obtained, C-3-4 is 50% methanol-eluted fractions, through Sephadex LH-20 After gel column chromatography, then through normal-phase silica gel column chromatography methylene chloride-methanol-water, 5:1:0.06 and semi-preparative HPLC, Welch Materials Ultimate AQ-C18Chromatographic column, 5.0 μm, 4.6 × 300mm of φ；Flow velocity v=1.0mL/min；Acetonitrile-water, 25:75 are prepared ginsenoside Rg1, tR=14.671min；
(4) C5 parts are through pressing reversed-phase silica gel column chromatography, methanol-water 5 in RP-18:95→100:0, v/v gradient elution, then through TLC Combining data detection is into 17 part C-5-1~C-5-17, and C-5-10 is 50% methanol-eluted fractions, through Sephadex LH-20 gels After column chromatography, then through semi-preparative HPLC, Zorbax SB-C18Post, 5.0 μm, 9.4 × 250mm of φ；Flow velocity v=2.0mL/min； Acetonitrile-water, 30:70 preparations respectively obtain protodioscin, tR=24.174min and Parisaponin I, tR= 29.064min；Then, chemistry is carried out to isolated compound from PDE5 by mass spectrum MS and NMR spectrum NMR The determination of structure.
6. chonglou saponin V, chonglou saponin I, β-moulting hormone, protodioscin, Parisaponin I and ginsenoside Rg1 exist Establish in Dulong Paris polyphylla or Dulong Paris polyphylla ethanol extract PDE4 or Dulong Chinese paris rhizome total saponin PDE5 quality control standard should With.
7. the Dulong Chinese paris rhizome total saponin described in Dulong Paris polyphylla ethanol extract PDE4 and claim 2 described in claim 1 Applications of the PDE5 in the medicine for treating or preventing Cancerous disease is prepared.
8. applications of the PDE4 according to claim 7 and PDE5 in the medicine for treating or preventing Cancerous disease is prepared, its It is leukaemia, liver cancer, lung cancer, breast cancer, colon cancer to be characterised by described cancer.
9. chonglou saponin V, chonglou saponin I, β-moulting hormone, protodioscin, Parisaponin I and ginsenoside Rg1 exist Prepare the application in the medicine for treating or preventing leukaemia, liver cancer, lung cancer, breast cancer, colon cancer.
10. prepare the method for the pharmaceutical composition described in claim 3 or 4, it is characterised in that this method presses following weight hundred Ratio is divided to be mixed：PDE4 or PDE5 accounts for 20-80%, dispersant 2-20%, disintegrant 3-5%, emulsifying agent 3-8%, binding agent 0.2-2%, wetting agent 0.5-10%, remaining is filler.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111610271A (en) * 2020-06-04 2020-09-01 成都中医药大学 Identification method of authentic rhizoma paridis
CN114668773A (en) * 2022-04-08 2022-06-28 山东中医药大学 Application of rhizoma paridis extract in resisting Climiya Congo hemorrhagic fever virus

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CN104706896A (en) * 2015-03-16 2015-06-17 中国科学院昆明植物研究所 Vegetable drug for curing cancers and preparation method and application thereof
CN105106556A (en) * 2015-09-11 2015-12-02 中国科学院昆明植物研究所 Chemical component separation and identification method for Parisforrestii (Takht.) H. Li antitumor activity part PFE-PT3

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CN104706896A (en) * 2015-03-16 2015-06-17 中国科学院昆明植物研究所 Vegetable drug for curing cancers and preparation method and application thereof
CN105106556A (en) * 2015-09-11 2015-12-02 中国科学院昆明植物研究所 Chemical component separation and identification method for Parisforrestii (Takht.) H. Li antitumor activity part PFE-PT3

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111610271A (en) * 2020-06-04 2020-09-01 成都中医药大学 Identification method of authentic rhizoma paridis
CN114668773A (en) * 2022-04-08 2022-06-28 山东中医药大学 Application of rhizoma paridis extract in resisting Climiya Congo hemorrhagic fever virus
CN114668773B (en) * 2022-04-08 2023-10-03 山东中医药大学 Application of paris polyphylla extract in resisting Crimedes congo hemorrhagic fever virus

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