CN110343116B - Wild chrysanthemum extract, preparation method thereof and application thereof in preparation of medicine for treating nasopharyngeal carcinoma - Google Patents
Wild chrysanthemum extract, preparation method thereof and application thereof in preparation of medicine for treating nasopharyngeal carcinoma Download PDFInfo
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Abstract
The invention provides a wild chrysanthemum extract, a new application thereof in preparing a medicine for preventing and treating nasopharyngeal carcinoma, and a preparation method of the wild chrysanthemum extract. The wild chrysanthemum extract provided by the invention has obvious effect on inhibiting the growth of nasopharyngeal carcinoma cells, can block the nasopharyngeal carcinoma cells HONE-1 in the G2/M stage, and can induce the nasopharyngeal carcinoma cells to die.
Description
Technical Field
The invention relates to the technical field of Chinese herbal medicine active ingredients and medicines, and particularly relates to a wild chrysanthemum flower extract, a preparation method thereof and application thereof in preparing medicines for treating nasopharyngeal carcinoma.
Background
Nasopharyngeal carcinoma (NPC) is a high-grade malignant tumor disease in the south of china, accounting for about 18% of cancer population in China. Nasopharyngeal carcinoma formation may be associated with Epstein-Barr Virus (EBV) infection, environmental factors and genetic factors. The clinical manifestations are headache, hearing loss, nasal obstruction, blood in nasal discharge, etc. The treatment of nasopharyngeal carcinoma mainly comprises radiotherapy, usually combined with chemical drugs, and chemotherapy usually uses broad-spectrum antitumor drugs, such as 5-fluorouracil, cisplatin, gemcitabine, etc. However, radiotherapy and chemotherapy bring many side effects to patients, such as headache, dry mouth and fever, cardiac and renal toxicity, etc., which seriously affect the quality of life of people. Moreover, the treatment of nasopharyngeal carcinoma often shows local recurrence and the nasopharyngeal carcinoma cells are easy to be transferred to the far end.
Natural products are an important source of lead compounds in the process of drug development. Some natural compounds have been reported to have activity against nasopharyngeal carcinoma at home and abroad, including alkaloids, triterpenes, flavonoids, etc. The flos Chrysanthemi Indici is dried head-shaped inflorescence of perennial herb of Chrysanthemum indicum L of Compositae, and has abundant wild resource. Widely distributed in northeast, northwest, east and southwest of China. Modern researches show that the wild chrysanthemum flower mainly contains chemical components such as volatile oil, flavone, terpenes, organic acid and the like, and has the activities of broad-spectrum antibiosis, antivirus, blood pressure reduction and the like. The invention reports the nasopharyngeal cancer resisting activity of a wild chrysanthemum extract.
Disclosure of Invention
One of the purposes of the invention is to provide a wild chrysanthemum flower extract.
The invention also aims to provide a preparation method of the wild chrysanthemum flower extract.
The invention further aims to provide application of the wild chrysanthemum extract in preparation of medicines for treating nasopharyngeal carcinoma.
The invention adopts the following technical scheme to realize the technical purpose:
a wild chrysanthemum extract has a structural formula shown as formula (I) and formula (II):
wherein R is1Is hydrogen, hydroxyl or C1-C5 saturated or unsaturated alkyl, wherein R1Wherein hydrogen may be substituted by cyano, nitro, carboxyl, amide or ester, R2Is hydrogen, hydroxyl or C1-C5 saturated or unsaturated alkyl, wherein R2Wherein hydrogen may be substituted by cyano, nitro, carboxyl, amide or ester, R3Is hydrogen, hydroxyl or C1-C5 saturated or unsaturated alkyl.
As a preferred embodiment, R1Is hydrogen, hydroxyl or C1-C5 saturated alkyl, wherein R1The hydrogen in the hydrogen is substituted by-CO-R ', and R' is C1-C5 saturated alkyl or C1-C5 unsaturated alkyl; r2Is hydrogen, hydroxyl or C1-C5 saturated or unsaturated alkyl, wherein R2The hydrogen in the compound can be substituted by-CO-R ', and R' is C1-C5 saturated alkyl or C1-C5 unsaturated alkyl.
As a preferred embodiment, the unsaturated alkyl group is an alkenyl group.
As a preferred technical scheme, R' is-C (CH)3)＝CH(CH3) or-CH ═ C (CH)3)2。
As a preferable technical scheme, when the wild chrysanthemum extract is represented by the structural formula (I), R is1Is hydrogen, hydroxyl or C1-C3 saturated alkyl, wherein R1The hydrogen in the compound can be substituted by-CO-R ', R' is-C (CH)3)＝CH(CH3) or-CH ═ C (CH)3)2；R2Is hydrogen, hydroxyl or C1-C3 saturated alkyl, wherein R2The hydrogen in the compound can be substituted by-CO-R ', R' is-C (CH)3)＝CH(CH3) or-CH ═ C (CH)3)2，R3Is H, C1-C3 saturated alkyl or ═ CH2。
As a preferable technical scheme, when the wild chrysanthemum extract is represented by the structural formula (II), R is1Is hydrogen, hydroxyl or C1-C3 saturated alkyl, wherein R1The hydrogen in the compound can be substituted by-CO-R ', R' is-C (CH)3)＝CH(CH3) or-CH ═ C (CH)3)2；R2Is H, C1-C3 saturated alkyl or ═ CH2。
Preferably, the wild chrysanthemum extract is one of the following structural formulas:
the invention also provides a preparation method of the wild chrysanthemum extract, which comprises the following steps: drying the above-ground wild chrysanthemum flower, pulverizing, extracting with 95% ethanol water at room temperature for four times, each time for 72 hours, combining the concentrated solutions, extracting with ethyl acetate, and recovering ethyl acetate under reduced pressure to obtain ethyl acetate part extract. Passing the ethyl acetate part through normal phase silica gel column, passing petroleum ether/ethyl acetate column according to the percentage of 10:0, 9:1, 4:1, 2:1 and 0:1 respectively, and repeatedly separating the fraction to obtain the ethyl acetate.
The invention also protects the application of the wild chrysanthemum extract in preparing the medicine for preventing and treating nasopharyngeal carcinoma.
Further, the medicine also comprises a pharmaceutically acceptable carrier or auxiliary material.
When the compound of the present invention is used as a medicament, it may be used as it is or in the form of a pharmaceutical composition. The pharmaceutical composition preferably contains 0.1-99% by weight of active ingredients, namely the compounds of the invention, namely the formula (I) and the formula (II), most preferably 0.5-90% by weight of the compounds of the invention, and the balance of pharmaceutically acceptable carriers and/or excipients which are nontoxic and inert to human and animals.
The pharmaceutically acceptable carrier or excipient is one or more of solid, semi-solid and liquid diluents, fillers and pharmaceutical adjuvants. The pharmaceutical composition of the present invention is used in the form of a dose per unit body weight. The medicine of the present invention may be administrated through injection and oral taking.
The administration amount of the compound of the present invention may vary depending on the route of administration, age, body weight of the patient, type and severity of the disease to be treated, etc., and the daily dose thereof may be 0.01 to 10mg/kg body weight, preferably 0.1 to 5mg/kg body weight. Can be used once or more.
Compared with the prior art, the invention has the following advantages and effects:
the wild chrysanthemum extract provided by the invention has an obvious effect on inhibiting the growth of nasopharyngeal carcinoma cells, can block the nasopharyngeal carcinoma cells HONE-1 in the G2/M stage, can induce the nasopharyngeal carcinoma cells to die, has safe and reliable components, and can be applied to the preparation of medicines for treating the nasopharyngeal carcinoma.
Drawings
FIG. 1 shows the effect of Compound 5 on the apoptosis of HONE-1 cells in nasopharyngeal carcinoma.
FIG. 2 shows the results of experiment of cell cycle arrest of HONE-1 in nasopharyngeal carcinoma with Compound 5.
FIG. 3 is a structural diagram of a compound separated by the method of the present invention.
FIG. 4 is a key NMR spectrum of Compound 11H-1COSY of H
HMBC
And NOESY
And (5) a relational graph.
FIG. 5 is a single crystal structural diagram of Compound 1.
Figure 6 is an experimental and calculated ECD profile for compounds 2, 10 and 12.
FIG. 7 is a key NMR spectrum of compound 7,10-121H-1COSY of H
HMBC
And NOESY
And (5) a relational graph.
Detailed Description
The present invention will be further described with reference to the following specific examples and drawings, which are not intended to limit the invention in any manner. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
Example 1:
a method for preparing compounds of formula (I) and formula (II) from wild Chrysanthemum flower (Chrysanthemum indicum):
1. the method comprises the steps of drying and crushing overground parts (6kg) of wild Chrysanthemum (Chrysanthemum indicum), leaching four times with 95% ethanol water at room temperature for 72 hours each time, combining concentrated solutions to obtain a crude extract (700g), extracting with ethyl acetate, directly subjecting ethyl acetate (300g) parts to silica gel column chromatography (2500g, 200-mesh 300-mesh, 7.0-100 cm) and petroleum ether/ethyl acetate gradient elution (10:0, 9:1, 4:1, 2:1 and 0:1), collecting one part per 500mL, and combining the same parts through TLC detection to obtain 5 fractions Fr.A-E.
2. Fr.D (30g) fraction via C18The reverse column separation yielded Fr.D1-D3 three fractions. Wherein, the Fr.D1 component passes through a silica gel column and is eluted by petroleum ether-ethyl acetate (4:1-1:1) to obtain 5 fractions Fr.D1a-D1 e. Fraction fr.d1a gave 8(1g) of formula (II) by semipreparative liquid phase separation and fraction fr.d1a gave 17(162mg) of formula (II) by semipreparative liquid phase separation.
3. And Fr.D2 is eluted by petroleum ether-ethyl acetate (5:1-1:1, V/V) to obtain 4 fractions Fr.D2 a-D2D. D2d was separated by semipreparative liquid phase separation to give 16(5mg) of formula (II).
4. Fr.E (100g) fraction through C18Reverse phase column chromatography (MeOH/H)2O, 50% → 100%) to fr.e 1-E3. Fr.E1 was eluted with petroleum ether-ethyl acetate (4:1-1:2, V/V) to give 3 fractions Fr.E1a-E1 c. Fr.E1a is eluted with dichloromethane-methanol (1:1) through Sephadex LH-20 to obtain Fr.E1a-1-Fr.E1a-3. E1a-1 gave, by semi-preparative liquid phase separation, compounds of formula (II)10(29.5mg),11(24.3mg) and 12(15 mg). E1a-2 gave, by semi-preparative liquid phase separation, 7(15mg) and 15(34.3mg) of formula (II). E2 was eluted with petroleum ether-ethyl acetate (4:1-1:2, V/V) to give 5 fractions Fr.E2a-E2E. E2b on Sephadex LH-20, eluting with dichloromethane-methanol (1:1) to give fr e2 b', and by semi-preparative liquid phase separation to give compounds of formula (I)1(9mg),2(11mg),3(10.8mg),4(7mg),5(24.4mg),6(181mg),13(10mg), and 14(5 mg).
The structural formula is shown as formula (I) and formula (II).
Structural identification of the novel compounds:
table 1: nuclear magnetic data of novel compounds 1-4
TABLE 2 Nuclear magnetic data of novel Compounds 5-8
TABLE 3 Nuclear magnetic data of novel Compounds 9-12
Sodium ion peak 633.3046[ M + Na ] according to mass spectrum]+(calcd.for C35H46O9Na,633.3034) to give compound 2 of formula C35H46O9. Discovery of compounds by analysis of nuclear magnetic data2 has a structure similar to that of the compound 1, and the compound 2 has no exocyclic double bond and has one more bimodal methyl group and one more tegloyl side chain substituent [ delta ]H1.79,d(J＝7.2Hz),1.82,s,6.85,m；δC167.1,128.6,138.7,14.8 and 12.3]. By H-8' (delta) in HMBC spectraH5.13) with C-1' (delta)C167.1) and C-11' (delta)C42.4) correlation, indicating that the tigloyl unit is attached at the C-8' position.1H-1In the H COSY spectrum, H-11 'and H-7'/H313' correlation and H in HMBC spectra3-13′(δH1.14,d,J＝7.2Hz)/C-12′(δC178.8) and H3-13′(δH1.14)/C-7′(δC51.1) the relevant signals all indicate that the methyl group is attached at the C-11' position. By NOESY spectrum H-7/H-5, H-5/H-1, H-6/H-8, H-8/H2-13,H3-15′/H-6′,H-8′/H-6′,H313 '/H-7 ', H-11 '/H-8 ' and H-5 '/H-7 ', indicating that the relative configuration of Compound 2 is rel-1R,5R,6R,7R,8S,11R,1 ' R,4 ' R,5 ' S,6 ' R, -7 ' R, 8' S,11 ' S, and combining the experimental ECD and calculation of the ECD map, the absolute configuration of the compound is confirmed to be 1R,5R,6R,7R,8S,10R,11R,1 ' R,4 ' R,5 ' S,6 ' R,7 ' R, 8' S,10 ' R,11 ' S.
The molecular formula of the compound 3, namely 8' -thioglycolyl chrysolide D, is determined to be C by mass spectrometry35H44O9. By comparing the nuclear magnetic data of the compound 3 and the compound 2, the compound 3 has one more exocyclic double bond (delta)H5.34,d,J＝3.2Hz；6.06,d,J＝3.2Hz；δC120.7) and one less bimodal methyl group (. delta.)H1.14,d,J＝7.2Hz；δC14.8), the rest being almost the same. By H in HMBC mapping2-13′(δH5.34,6.06) and C-7' (delta)C51.1) and C-12' (delta)C170.1) and determining that the exocyclic double bond is at C-11'. Similarly, the NOESY-related signals H-7/H-5, H-5/H-1, H-6/H-8, H-8/H2-13,H315 '/H-6 ', H-8 '/H-6 ', and H-5 '/H-7 ' define the relative configuration of the compound as rel-1R,5R,6R,7R,8S,10R,11R,1 ' R,4 ' R,5 ' S,6 ' R,7 ' R, 8' S,10 ' R.
The molecular formula of the compound 4(8-angeloyl-8' -hydroxy chrysinolide D) can be shown in the specificationMass spectrum fragment ion peak M/z 631.2890[ M + Na [ ]]+(calcd.for C35H44O9Na,631.2878) was determined to be C35H44O9The nuclear magnetic data are very similar to those of compound 3, except that the side chain substitution in compound 4 is an angeloyl unit, while in compound 3 is a tigloyl. The presence of angeloyl in which the two methyl groups are in trans position can be demonstrated by NOE correlation signals H-3 '/H-4' and H-3 '/H-5' and COSY patterns H-7 and H-8, in combination with the gamma bystander effect. Based on NOESY related signals H-7/H-5, H-5/H-1, H-6/H-8, H-8/H2-13,H3The relative configurations of compounds 4-15 '/H-6 ', H-8 '/H-6 ' and H-5 '/H-7 ' are identified as rel-1R,5R,6R,7R,8S,10R,11R,1 ' R,4 ' R,5 ' S,6 ' R,7 ' R, 8' S,10 ' R.
The molecular formula of compound 5(8,8' -diglyoyl chrysolide D) was determined to be C based on high resolution mass spectrometry40H50O10. Analysis by one-dimensional nuclear magnetic data (Table 2) revealed that this compound is similar to compound 3, except that a tigloyl unit (. delta.) is attached to the C-8 position of this compoundH1.72,d,J＝7.2Hz,1.79,s,6.85,overlap；δC166.6,129.0,139.8,14.8, and 12.2), HMBC correlation signal H-8(δ)H5.51)/C-1″(δC166.6) can prove this. NOESY for Compound 5 is the same as for Compound 3, so the relative configuration of this compound is rel-1R,5R,6R,7R,8S,10R,11R,1 ' R,4 ' R,5 ' S,6 ' R,7 ' R, 8' S,10 ' R.
The molecular formula of compound 6 is determined by adding sodium ion peak M/z 615.2939[ M + Na ]]+(calcd.for C35H44O8Na,615.2928) was determined to be C35H44O8. The structure of this compound was found to be similar to that of 1 by one-dimensional nuclear magnetic data analysis (Table 2), wherein the substitution of compound 6 with one more tigloyl fragment at position C-8 in 1 was demonstrated by the relative signals H-8/C-1'/C-11 in HMBC spectra. NOESY correlation indicates that compound 6 has the same relative configuration as compound 1. The structure of compound 6 was thus identified as rel-1R,5R,6R,7R,8S,10R,11R,1 'R, 4' R,5 'S, 6' S,7 'S, 10' R, under the name 8-tigloyl chrysanoide D.
Compound 7(chrysanolide F) is absentColored powder of the formula sodium ion peak M/z 289.1405[ M + Na ] given by high resolution mass spectrometry]+(calcd.for C15H22O4Na,289.1410) was determined to be C15H22O4A total of 5 unsaturations. The one-dimensional nuclear magnetic data (Table 2) indicates that compound 7 is the epimer of the known compound chrysanolide B. NOESY-RELATED SIGNAL H3-13/H-8,H-6/H 313, H-5/H-7 and H-5/H-1 indicate that H-1, H-5, H-7 and H-11 are coplanar and in the alpha orientation, then H-6, H-8 and H3-13 (fig. 7) is in the beta orientation. Thus, compound 7 is structurally designated rel-1R,5R,6R,7R,8S,10R, 11R.
The compound 8 is colorless oily compound with molecular formula C20H28O5. Compound 8 was found to be the epimer of the reported compound 8 α -angelyloxy-10 β -hydroxy-slov-3-en-6,12-olide by analysis of one-dimensional nuclear magnetic data (Table 2). Except that the C-8 position of the compound is linked to a tigloyl unit. Based on NOESY correlation signal H3-13/H-8,H-6/H 313, H-5/H-7 and H-5/H-1, Compound 8 was identified as rel-1R,5R,6R,7R,8S,10R,11R, and was designated 8-tigloyl chrysanolide F.
Fragment ion peak M/z 369.1656[ M + Na ] based on high resolution mass spectrometry]+(calcd.for C20H26O5Na,369.1672), Compound 9 has the formula C20H26O5. The hydrogen and carbon spectra data (Table 3) are similar to the reported compound angeloylcumambrin B (16), except that compound 9 has a tigloyl fragment at the C-8 position and compound 16 is angeloyl. The two-dimensional nuclear magnetic spectrum of the compound 9 shows that the compound is haploid with 1-6 and 13. Thus, compound 9 was identified as rel-1R,5R,6R,7R,8S,10R and designated chrysanolide G by NOESY-related signals H-5/H-7, and H-5/H-1.
The molecular formula of the compound 10(chrysanolide H) is C20H26O6. The hydrogen spectrum and carbon spectrum data show that the compound has 5 methyl groups, two oxygen-linked methines, one oxygen-linked tertiary carbon, one olefinic carbon proton, two methylene groups, three methines, one ketone group and two carbonyl groups. These data indicate the compounds10 is an analog of compound 8. The keto group at the C-3 position and the double bond between the C-4/C-5 positions of the compounds were confirmed by the relative signals H-15/C-3/C-5 in HMBC (FIG. 7). NOESY spectrum H3The signals associated with H-1/H-7 indicate that H-1 and H-7 are in alpha orientation, 13/H-7, H-11/H-8, H-11/H-6, H-8/H-6. H-6, H-8 and H-11 are in the beta orientation. And determining the absolute configuration of the compound to be 1R,6S,7R,8S,10R and 11S by calculating and comparing the ECD with an experimental ECD map.
The molecular formula of the compound 11 is C20H26O6. Analysis by nuclear magnetic data (Table 4) revealed that the structure of this compound was similar to that of 10, except that the C-8 position in compound 11 was an angeloyl unit. The same NOESY correlation (figure 7) indicates that the two compounds have the same relative configuration. Therefore, compound 11(8-angeloyl chrysanolide H) was identified as rel-1R,6S,7R,8S,10R, 11S.
Compound 12(chrysanolide I)1H spectrum and13the C spectrum data (Table 3) shows that the compound contains five methyl groups, three vicinal oxymethylene groups, one vicinal tertiary carbon, one olefinic carbon proton, two methylene groups, three methine groups and two carbonyl groups. The nuclear magnetic data of compound 12 is very similar to that of indoumolide a. Except that the angeloyl unit at the C-8 position of the compound indoliumolide A is substituted with tigloyl. This is confirmed by the correlated HMBC signal in fig. 7. Signal H in NOESY spectrum3-13/H-7,H-11/H-8,H-11/H-6,H-6/H-8,H 315/H-5, H-5/H-7 and H-3/H3-15 indicates H-3, H-5, H-7, H3-13 and H 315 is the same plane, defined as alpha orientation, then H-6, H-8 and H-11 are beta orientations. Based on the same biogenic synthesis pathway, compound 12 was identified as 3S,4R,5S,6S,7R,8S,11S, as confirmed by calculation and experimental ECD mapping (fig. 6).
Chrysanolide D (1) colorless crystals; [ alpha ] to]20 D+9.4(c0.1,MeOH)；UV(MeOH)λmax(logε)202(4.23)nm；ECD(MeOH)λmax(logε)222(-0.43)nm；IR(KBr)νmax3414,2963,2927,2858,1767,1736,1459,1378,1261,1088,1025,914,796,732,and 522cm-1；1H and13C NMR data,see Table 1；HRESIMS m/z 533.2507[M+Na]+(calcd.for C30H38O7Na,533.2510).
Chrysanolide E (2) as a colorless oil; [ alpha ] to]20 D+41.5(c0.1,MeOH)；UV(MeOH)λmax(logε)203(4.12)nm；ECD(MeOH)λmax(logε)214(-0.18)nm；IR(KBr)νmax3474,2968,2924,2854,1756,1699,1455,1377,1261,1082,1015,919,863,799,737,and 545cm-1；1H and13CNMR data,see Table 1；HRESIMS m/z 633.3046[M+Na]+(calcd.for C35H46O9Na,633.3034).
8' -Tigloyl chrysanolide D (3): colorless oil; [ alpha ] to]20 D+42.1(c0.1,MeOH)；UV(MeOH)λmax(logε)202(4.52)nm；ECD(MeOH)λmax(logε)210(-0.44),225(+0.13)nm；IR(KBr)νmax3474,2968,2931,2860,1756,1703,1458,1379,1262,1082,1021,913,797,731,and 545cm-1；1H and13C NMR data,see Table 1；HRESIMS m/z 631.2876[M+Na]+(calcd.for C35H44O9Na,631.2878).
8-Angeloyl-8' -hydroxy chrysanolide D (4): colorless oil; [ alpha ] to]20 D+24.1(c0.1,MeOH)；UV(MeOH)λmax(logε)202(4.25)nm；ECD(MeOH)λmax(logε)219(-0.78)nm；IR(KBr)νmax3482,2968,2927,2854,1748,1705,1457,1375,1354,1261,1157,1091,1022,969,908,797,732,667,and 548cm-1；1H and13C NMR data,see Table 1；HRESIMS m/z 631.2890[M+Na]+(calcd.for C35H44O9Na,631.2878).
8,8' -Ditigloyl chrysanolide D (5): colorless oil; [ alpha ] to]20 D+54.4(c0.1,MeOH)；UV(MeOH)λmax(logε)202(4.40)nm；ECD(MeOH)λmax(logε)210(-0.98),229(+0.25)nm；IR(KBr)νmax3489,2966,2927,2860,1766,1688,1641,1457,1415,1377,1261,1085,1015,866,797,736,and 699cm-1；1H and13C NMR data,see Table 2；HRESIMS m/z 713.3300[M+Na]+(calcd.for C40H50O10Na,713.3296).
8-Tigloyl chrysanolide D (6) as colorless oil; [ alpha ] to]20 D+4.0(c0.1,MeOH)；UV(MeOH)λmax(logε)202(4.37)nm；ECD(MeOH)λmax(logε)217(-0.99)nm；IR(KBr)νmax3499,2963,2933,1753,1699,1648,1454,1378,1345,1260,1146,1086,1030,908,804,727,and 521cm-1；1H and13C NMR data,see Table 2；HRESIMS m/z 615.2939[M+Na]+(calcd.for C35H44O8Na,615.2928).
Chrysanolide F (7) as a colorless solid; [ alpha ] to]20 D+92.4(c0.1,MeOH)；UV(MeOH)λmax(logε)201(3.76)nm；ECD(MeOH)λmax(logε)213(-0.12)nm；IR(KBr)νmax3258,2966,2921,2854,1772,1684,1454,1384,1261,1217,1146,1033,1000,924,798,721,and 539cm-1；1H and13C NMR data,see Table 2；HRESIMS m/z 289.1405[M+Na]+(calcd.for C15H22O4Na,289.1410).
8-Tigloyl chrysanolide F (8) as colorless oil; [ alpha ] to]20 D+86.3(c0.1,MeOH)；UV(MeOH)λmax(logε)203(4.07)nm；ECD(MeOH)λmax(logε)204(+0.25)nm；IR(KBr)νmax3455,3401,2962,2929,2913,2854,1772,1753,1702,1648,1446,1376,1267,1224,1182,1147,1116,1074,1024,1002,941,806,738,and 673cm-1；1H and13C NMR data,see Table 2；HRESIMS m/z 371.1804[M+Na]+(calcd.for C20H28O5Na,371.1829).
Chrysanolide G (9) as a colorless oil; [ alpha ] to]20 D+116.2(c 0.1,MeOH)；UV(MeOH)λmax(logε)203(4.37)nm；ECD(MeOH)λmax(logε)209(+0.42),222(+5.22),264(-0.46)nm；IR(KBr)νmax3434,2964,2921,2850,1760,1741,1702,1687,1646,1450,1382,1336,1263,1157,1133,1070,1010,941,912,815,740,and 674cm-1；1H and13C NMR data,see Table 3；HRESIMS m/z 369.1656[M+Na]+(calcd.for C20H26O5Na,369.1672).
Chrysanolide H (10) as a colorless oil; [ alpha ] to]20 D+143.9(c 0.1,MeOH)；UV(MeOH)λmax(logε)201(3.88),230(3.95)nm；ECD(MeOH)λmax(logε)236(+0.62),323(-0.30)nm；IR(KBr)νmax3493,2969,2936,1787,1693,1643,1459,1373,1253,1192,1095,1060,1009,973,797,734,and 545cm-1；1H and13C NMRdata,see Table 3；HRESIMS m/z 385.1613[M+Na]+(calcd.for C20H26O6Na,385.1622).
8-Angeloyl chrysanolide H (11) as a colorless oil; [ alpha ] to]20 D+114.3(c0.1,MeOH)；UV(MeOH)λmax(logε)201(3.95),231(4.16)nm；ECD(MeOH)λmax(logε)208(-0.36),238(+0.95),323(-0.06)nm；IR(KBr)νmax3482,2963,2937,2880,1784,1681,1639,1448,1373,1302,1207,1160,1091,1004,949,798,737,and 556cm-1；1H and13C NMRdata,see Table 3；HRESIMS m/z 385.1624[M+Na]+(calcd.for C20H26O6Na,385.1622).
Chrysanolide I (12) as a colorless oil; [ alpha ] to]20 D+9.3(c0.1,MeOH)；UV(MeOH)λmax(logε)202(4.11)nm；ECD(MeOH)λmax(logε)206(-0.94),222(+0.75)nm；IR(KBr)νmax3570,2968,2942,2856,1756,1700,1505,1446,1383,1259,1171,1083,1048,1014,971,925,801,733,627,and 522cm-1；1H and13C NMR data,see Table 3；HRESIMS m/z 387.1790[M+Na]+(calcd.for C20H28O6Na,387.1778).
Compound 1X-ray single crystal diffraction analysis: the crystal structure of compound 1 and its absolute configuration were determined by single crystal diffraction analysis. The crystals were subjected to a Xcalibur, Onyx, Nova diffractometer to collect data at 100K. The crystals were processed and optimized by XS and olex2.refine software. The single crystal pattern was drawn using ORTEP. Single crystal data for compound 1 are as follows: c30H38O7(M-510.60); colorless block-like, space group P2 12121(no.0),
Z＝2,T＝100K,μ(CuKα)＝0.755mm-1,Dcalc＝1.320g/cm324724 reflection measurement, 5045unique (Rint 0.0512). Applicable to all calculations. The final R1 value was 0.0326 and wR2 was 0.0849 (all data). -0.10 (7).
Example 2
Preparing formula (I) and formula (II) according to the method of example 1, adding water for injection, fine filtering, encapsulating and sterilizing to prepare injection according to the conventional method.
Example 3
Formula (I) and formula (II) were prepared as described in example 1, dissolved in sterile water for injection, stirred to dissolve, and filtered through a sterile suction funnel. Then sterile fine filtering, subpackaging in 2 ampoules, freeze drying at low temperature, and aseptically sealing by melting to obtain powder for injection.
Example 4
The formula (I) and formula (II) were prepared according to the method of example 1, and the excipients were added in a weight ratio of 5:1 to the excipients, and granulated and tabletted.
Example 5
The formula (I) and formula (II) were prepared according to the method of example 1, and the excipients were added in a weight ratio of 5:1 to the excipients, and capsules were prepared.
Example 6
Preparing formula (I) and formula (II) according to the method of the embodiment 1, adding excipient according to the weight ratio of 3:1 of the formula (I) and the formula (II) and preparing the capsule.
Application test example:
the compounds of the invention, formula (I) and formula (II), are tested for their inhibitory activity against nasopharyngeal carcinoma cells.
(1) And (5) culturing the cells. In vitro culture of nasopharyngeal carcinoma cell lines CNE1, CNE2, HONE-1 and SUNE 1. RPMI1640 medium containing 10% fetal bovine serum, streptomycin (100 μ g/ml), penicillin (100 units/ml) was used for routine maintenance culture and passage at 37 ℃ under 5% carbon dioxide concentration.
(2) Cytotoxic activity assay methods. Before 96-well plate, count the cells (2000-. After 48 hours, 20. mu.L of MTT at 5mg/mL in PBS was added and the mixture was treated for 4 hours. The medium was discarded again, 120. mu.L of DMSO was added to each well, the mixture was shaken for 10min, and the OD at 492nm was measured using a microplate reader. Calculating the inhibition rate of each drug concentration on cells according to OD value, and calculating the IC of each compound by nonlinear regression through GraphPad Prism software50The value is obtained.
(3) An apoptosis test method. HONE1 cells in logarithmic growth phase at 2X 105Perwell, 10% FBS medium per well, in 6-well plates, placed at 37 ℃ in 5% CO2The incubator is incubated for 24 hours; changing the solution and adding compounds with different concentrations to incubate the cells for 48 h; removing supernatant by suction (storing in 5mL centrifuge tube), washing with 200 μ L of PBS at 4 deg.C twice per well, adding 200 μ L of pancreatin without EDTA into each well for digestion, centrifuging, and collecting cells; adding 1mL Binding buffer into each tube for suspension; taking 400 mu L of cell suspension, adding 5 mu L of FITC and PI respectively; add 400. mu.L Binding buffer Beckmann flow cytometer to each tube for analysis, 10000 cells per sample were collected. The results of the experiment were analyzed using Flowjo software.
(4) Cell cycle experimental method. 2X 105HONE1 cells were seeded in 6-well plates and cultured for 24 h. HONE1 cells were treated with the compound at various concentrations for 48h, followed by trypsinization without EDTA, centrifugation, and cell collection. Fixation with frozen 70% ethanol overnight at 4 ℃ followed by washing with cold PBS followed by staining with cell cycle and apoptosis detection kit for 30 min at 37 ℃ in the dark. The treated samples were analyzed by Beckmann flow cytometry, and 10000 cells were collected per sample. The experimental results were analyzed with Modfit software. The activity is tabulated in Table 4.
TABLE 4 Compounds nasopharyngeal carcinoma cell proliferation inhibiting Activity of formula (I) and formula (II)
As shown in Table 4, FIG. 1 and FIG. 2, the compounds provided by the present invention have very good inhibitory activity against nasopharyngeal carcinoma cells, especially some compounds have better effect than cisplatin.
As shown in figure 1, after being treated by 0,1, 3, 5, 10 and 20 mu M of compound 5 for 48 hours, the apoptosis rates of HONE1 cells are respectively 3.61%, 3.34%, 3.46%, 17.61%, 33.99% and 37.18%, the apoptosis rates are obviously higher than those of blank groups, and the compound shows dose dependence, and has the function of promoting the apoptosis of nasopharyngeal carcinoma cells.
As shown in FIG. 2, after 48h of treatment with 0,1, 3, 5, 10 and 20 μ M compound 5, the proportion of cells in G0/G1 phase was 55%, 44.75%, 50.22%, 40.89% and 14.01%, respectively, the overall proportion was decreased, the proportion of cells in S phase was substantially unchanged, and the percentage of cells in G2/M phase was 0.3%, 5.93%, 7.52%, 16.52% and 38.22%, respectively, showing a dose-dependent increase, suggesting that compound 5 can induce blocking of nasopharyngeal carcinoma cells in G2/M phase.
Claims (4)
1. The wild chrysanthemum extract is characterized by being one of the following structural formulas:
2. a preparation method of the wild chrysanthemum extract as claimed in claim 1, characterized in that the wild chrysanthemum extract is prepared by drying the above-ground part of wild chrysanthemum, crushing, leaching four times with 95% ethanol water at room temperature for 72 hours each time, combining the concentrated solutions, extracting with ethyl acetate, recovering ethyl acetate under reduced pressure to obtain an ethyl acetate part extract, passing the ethyl acetate part through a normal phase silica gel column, passing petroleum ether/ethyl acetate through columns according to the percentage of 10:0, 9:1, 4:1, 2:1 and 0:1 respectively, and repeatedly separating the fractions.
3. The application of the wild chrysanthemum extract in preparing the medicine for preventing and treating nasopharyngeal carcinoma is characterized in that the wild chrysanthemum extract is one of the following structural formulas:
4. the use of claim 3, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.
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| CN113788840B (en) * | 2021-09-15 | 2023-08-11 | 中国科学院昆明植物研究所 | Guaiane sesquiterpene polymer and medicine thereof, and preparation method and application thereof |
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| CN115010720B (en) * | 2022-06-02 | 2023-08-11 | 中国科学院昆明植物研究所 | Chinese mugwort sesquiterpene dimer and pharmaceutical composition thereof, and preparation method and application thereof |
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