CN101648937B - Isoflavones compound and preparation and application thereof - Google Patents

Isoflavones compound and preparation and application thereof Download PDF

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CN101648937B
CN101648937B CN200810041753A CN200810041753A CN101648937B CN 101648937 B CN101648937 B CN 101648937B CN 200810041753 A CN200810041753 A CN 200810041753A CN 200810041753 A CN200810041753 A CN 200810041753A CN 101648937 B CN101648937 B CN 101648937B
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isoflavonoid
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CN101648937A (en
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沈征武
寿清耀
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Jiangsu beihede Pharmaceutical Technology Co.,Ltd.
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Basilea Pharmaceutica China Ltd
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Abstract

The invention discloses a novel isoflavones compound as shown by a formula I. The results of animal experiments show that the isoflavones compound can significantly inhibit the proliferation of T cells and B cells, and can be used for preparing immunosuppressants or medicaments for treating immunologic diseases. The SI values (IC50/CC50) of the isoflavones compound are high, so the toxic side effects of the isoflavones compound are small, and the treatment interval of the isoflavones compound is wide. The isoflavones compound is obtained by extracting and separating clover shrubs, and the preparation method is simple and convenient.

Description

Isoflavonoid and preparation thereof and application
Technical field:
The present invention relates to Natural Medicine Chemistry, the isoflavonoid and preparation method thereof that is specifically related to extraction separation from the stem of Radix Campylotropis Hirtella (Herba Myrsines Africanae) (Myrsine Africana L.) with in the preparation immunosuppressor or treat the application aspect the various immunological disease medicines.
Background technology:
Known naturally occurring plant NOVASOY 400 performance wide biological activity, comprise anti-oxidant, to signal conduction and activity regulation of enzymes; Mitotic inhibition and cytotoxicity to human cancer cell; Increase perviousness capillaceous; Increase cell adhesion; Increase the reaction of vascular smooth muscle to the blood vessel relaxant; Excited ERs etc.
These biological activitys have produced certain therapeutic action, comprise treatment and prevention endometriosis, fibroma uteri, hyperlipidaemia, cardiovascular disorder, premenstrual syndrome, exhausted smart symptom such as osteoporosis and senile dementia, benign prostatauxe and cancer such as prostate gland, mammary gland and large bowel cancer etc.
But isoflavonoid also has other activity, needs further further investigation.
Radix Campylotropis Hirtella (Herba Myrsines Africanae) is the root and the cauline leaf of the young platymiscium iron of Myrsinacea iron young (Myrsine Africana L.), and " Guizhou folk medicine " record: " wind-damp dispelling is invigorated blood circulation.Control dysentery." " Yunnan herbal medicine " record: anti-inflammatory, pain relieving ends dysentery.Control toothache, enteritis, dysentery.Its function of record cures mainly and is wind-expelling pain-stopping, clearing away heat-damp and promoting diuresis, astringing to arrest bleeding in China's book on Chinese herbal medicine.Cure mainly: rheumatic arthralgia, toothache is had loose bowels, dysentery, metrorrhagia is had blood in stool, hemoptysis of pulmonary tuberculosis.
Radix Campylotropis Hirtella (Herba Myrsines Africanae) contains multiple isoflavonoid, but less to its chemical constitution study, the research of the chemical ingredients in the Radix Campylotropis Hirtella (Herba Myrsines Africanae) that especially do not appear in the newspapers aspect preparation immunosuppressor or treatment immunological disease.
Summary of the invention:
The technical problem that the present invention will solve is to study the multiple isoflavonoid of Radix Campylotropis Hirtella (Herba Myrsines Africanae) and preparation and application.
The present invention's extraction separation from Chinese medicine Radix Campylotropis Hirtella (Herba Myrsines Africanae) (Myrsine Africana L.) obtains 8 new isoflavonoids, and finds that they have immunosuppressive activity.
The invention provides a kind of isoflavonoid, general structure is suc as formula shown in the I
Figure G2008100417531D00021
In the formula, when being pair key between numbering 2 and 3 the carbon atom,
R1=H;
Figure G2008100417531D00022
R3=OH, R4=H or
R1=OH;
Figure G2008100417531D00023
R3=OH, R4=H or
R1=H;
Figure G2008100417531D00024
R3=OH, R4=OH or
R1=H, R2 and R3 form following six-ring:
or
Figure G2008100417531D00026
When being singly-bound between the carbon atom of numbering 2 and 3,
R1=OH;
Figure G2008100417531D00027
R3=OH, R4=H or
R1=OH;
Figure G2008100417531D00028
R3=OH, R4=H or
R1＝OH， R3＝OH，R4＝H
Concrete isoflavonoid is represented as follows:
Compound 1 (3 '-geranyl-5,7,2 ', 4 '-the tetrahydroxy NOVASOY 400), yellow oil.
UVλ max(MeOH)：262.0；
HREIMS：m/z?422.1743[M] +(Calc.422.1729?for?C 25H 26O 6)；
EIMS：m/z(rel.int.)：353(10)，299(38)，128(38)，86(100)，69(79)，59(61)，55(85)；
ESI-MS：m/z?423[M+H] +；
IRν maxcm -1(KBr)：3352，2924，2854，1649，1610，1500，1448，1364，1306，1259，1200，1167，1150，1094，1051，1038，820；
1HNMR(CDCI 3)：7.93(H-2)，6.36(H-6)，6.43(H-8)，6.51(H-5’)，6.89(H-6’)，3.56(H-1”)，5.31(H-1’)，2.08(H-4”)，2.10(H-5”)，5.06(H-6”)，1.59(H-8”)，1.84(H-3”Me)，1.67(H-7”Me)；
13CNMR(CDCI 3)：155.5(C-2)，123.9(C-3)，182.3(C-4)，162.7(C-5)，100.5(C-6)，163.5(C-7)，94.5(C-8)，158.2(C-9)，105.7(C-10)，112.7(C-1’)，154.5(C-2’)，117.4(C-3’)，157.5(C-4’)，109.5(C-5’)，128.2(C-6’)，26.6(C-1”)，121.9(C-2”)，139.1(C-3”)，39.6(C-4”)，26.0(C-5”)，124.1(C-6”)，132.3(C-7”)，16.6(C-8”)，15.4(C-3”Me)，23.1(C-7”Me)。
Figure G2008100417531D00031
Compound 1
Compound 2 (5,7,4 '-trihydroxy--3 '-[7-hydroxyl-3,7-dimethyl--2 (E)-octene] NOVASOY 400) the white solid thing, mp:179.3-180.9
UVλ max(MeOH)：262.0；
HREIMS：m/z?424.1890[M] +(Calc.424.1886?for?C 25H 28O 6)；
EIMSm/z(rel.int.)：424(3.5)，406(39)，338(16)，337(22)，335(22)，321(33)，284(100)，283(73)，153(60)；
ESI-MS：m/z?407[M-OH] +；
IRν maxcm -1(KBr)：3362，2968，2935，1653，1616，1506，1446，1364，1263，1198，1047，825，669；
1HNMR(CDCI 3)：8.12(H-2)，6.27(H-6)，6.40(H-8)，7.35(H-2’)，6.89(H-5’)，7.27(H-6’)，3.38(H-1”)，5.40(H-2”)，2.05(H-4”)，1.52(H-5”)，1.41(H-6”)，1.12(H-8”)，1.73(H-3”Me)，1.12(H-7”Me)；
13CNMR(CDCI 3)：153.5(C-2)，123.7(C-3)，181.0(C-4)，163.3(C-5)，99.2(C-6)，164.4(C-7)，93.8(C-8)，158.4(C-9)，105.5(C-10)，122.5(C-1’)，130.5(C-2’)，128.1(C-3’)，155.3(C-4’)，114.9(C-5’)，127.9(C-6’)，28.3(C-1”)，122.7(C-2”)，136.1(C-3”)，40.34(C-4”)，22.7(C-5”)，43.7(C-6”)，69.5(C-7”)，29.0(C-8”)，15.5(C-3”Me)，29.0(C-7”Me)。
Figure G2008100417531D00041
Compound 2
Compound 3 (5, the 7-dihydroxyl-[6 " methyl-6 "-(4-methyl-3-pentenyl) pyrans also (2 ", 3 ": 3 ', 4 ')] NOVASOY 400), yellow oil
UVλ max(MeOH)nm：290.0；
HREIMS：m/z?404.1635[M] +(Calc.404.1624?for?C 25H 24O 5)；
EIMS?m/z(rel.int.)：404(5)，322(23)，321(100)；
ESI-MS：m/z?405[M+H] +；
IRν maxcm -1(KBr)：3339，2924，1652，1618，1578，1491，1448，1360，1306，1261，1180，1045，831；
1HNMR(CDCI 3)：8.20(H-2)，6.29(H-6)，6.42(H-8)，7.31(H-2’)，6.85(H-5’)，7.33(H-6’)，6.49(H-4”)，5.75(H-5”)，1.71(H-7”)，2.13(H-8”)，5.13(H-9”)，1.57(H-1”)，1.40(H-6”Me)，1.64(H-10”Me)；
13CNMR(CDCI 3)：153.9(C-2)，123.1(C-3)，181.0(C-4)，163.2(C-5)，99.2(C-6)，164.4(C-7)，93.9(C-8)，158.9(C-9)，105.4(C-10)，123.6(C-1’)，127.4(C-2’)，121.1(C-3’)，153.6(C-4’)，115.9(C-5’)，130.3(C-6’)，122.7(C-4”)，130.0(C-5”)，99.0(C-6”)，41.4(C-7”)，22.8(C-8”)，124.3(C-9”)，131.4(C-10”)，17.0(C-11”)，26.3(C-6”Me)，25.2(C-10”Me)。
Figure G2008100417531D00051
Compound 3
Compound 4 (5,7,4 ", 5 " tetrahydroxys-[6 "-methyl-6 " (4-methyl-3-pentenyl) pyrans also (2 ", 3 ": 3 ', 4 ')] NOVASOY 400), yellow oil
UVλ max(MeOH)nm：290.0；
HREIMS：m/z?442.1989[M] +(Cal.442.1992?for?C 25H 30O 7)；
EIMS?m/z?rel.int.：442(5)，424(49)，300(42)，299(45)，153(100)；
ESI-MS?m/z：425[M-OH] +；
IRν maxcm -1(KBr)：3367，2966，2931，1699，1684，1635，1452， 1379，1269，1184，1465，835，800；
1HNMR(CDCI 3)：8.08(H-2)，6.22(H-6)，6.34(H-8)，7.61(H-2’)，6.81(H-5’)，7.34(H-6’)，4.58(H-4”)，3.65(H-5”)，1.85(H-7”)，2.17(H-8”)，5.16(H-9”)，1.65(H-1”)，1.19(H-6”Me)，1.69(H-10”Me)；
13CNMR(CDCI 3)：153.8(C-2)，123.2(C-3)，181.0(C-4)，162.7(C-5)，99.0(C-6)，164.9(C-7)，93.7(C-8)，158.5(C-9)，105.1(C-10)，123.5(C-1’)，128.7(C-2’)，124.6(C-3’)，152.9(C-4’)，116.5(C-5’)，129.7(C-6’)，69.1(C-4”)，73.2(C-5”)，80.3(C-6”)，38.7(C-7”)，21.1(C-8”)，124.3(C-9”)，131.2(C-10”)，16.5(C-11”)，17.1(C-6”Me)，24.7(C-10”Me)。
Figure G2008100417531D00052
Compound 4
Compound 5 (5,7,2 ', 4 '-tetrahydroxy-3 '-[7-hydroxyl-3,7-dimethyl--2 (E)-octene] isoflavanone) yellow oil;
UVλ max(MeOH)nm：290.0；
IRν maxcm -1(KBr)：3367，2966，2931，1699，1684，1635，1452，1379，1269，1184，1465，835，800；
HREIMS?m/z?442.1989[M +](Cal.442.1992?for?C 25H 30O 7)；
EIMS?m/z(rel.int.)：442(5)，424(49)，300(42)，299(45)，153(100)；
ESI-MS?m/z?425[M-OH] +；
1HNMR(CDCI 3)：4.60(H-2α)，4.70(H-2β)，4.17(H-3)，5.97(H-6)，5.97(H-8)，6.43(H-5’)，6.93(H-6’)，3.43(H-1”)，5.25(H-2”)，1.93(H-4”)，1.46(H-5”)，1.36(H-6”)，1.13(H-8”)，1.77(H-3”Me)，1.13(H-7”Me)；
13CNMR(CDCI 3)：70.4(C-2)，46.1(C-3)，197.9(C-4)，164.9(C-5)，96.2(C-6)，167.0(C-7)，95.1(C-8)，163.8(C-9)，102.2(C-10)，114.6(C-1’)，154.1(C-2’)，116.3(C-3’)，155.8(C-4’)，107.7(C-5’)，126.5(C-6’)，22.7(C-1”)，122.8(C-2”)，135.3(C-3”)，40.4(C-4”)，22.5(C-5”)，43.7(C-6”)，69.4(C-7”)，29.0(C-8”)，15.6(C-3”Me)，29.0(C-7”Me)。
Figure G2008100417531D00061
Compound 5
Compound 6 (3 '-geranyl-5,7,2 ', 4 '-the tetrahydroxy isoflavanone .), yellow oil
UVλ max(MeOH)nm：290.0，230.0；
IRν maxcm -1(KBr)：3304，2926，1639，1614，1450，1379，1263，1614，1450，1379，1263，1182，1163，1091，835；
HREIMS?m/z?424.1882[M+](Calc.424.1886?for?C 25H 28O 6)；
EIMSm/z(rel.int.)：424(12)，301(46)，201(26)，187(36)，179(31)，153(100)，123(60)，91(35)，69(80)；
ESI-MS?m/z?425[M+H] +；
1HNMR(CDCI 3)：4.66(H-2α)，4.76(H-2β)，4.00(H-3)，5.95(H-6)，5.95(H-8)，6.39(H-5’)，7.12(H-6’)，3.45(H-1”)，5.24(H-2”)，2.08(H-4”)，2.09(H-5”)，5.03(H-6”)，1.57(H-8”)，1.80(H-3”Me)，1.65(H-7”Me)；
13CNMR(CDCI 3)：70.0(C-2)，45.6(C-3)，197.5(C-4)，165.1(C-5)，97.2(C-6)，166.6(C-7)，95.8(C-8)，163.4(C-9)，102.0(C-10)，115.1(C-1’)，154.2(C-2’)，115.8(C-3’)，155.5(C-4’)，108.9(C-5’)，126.1(C-6’)，23.0(C-1”)，121.7(C-2”)，139.4(C-3”)，39.9(C-4”)，26.6(C-5”)，124.0(C-6”)，132.3(C-7”)，18.0(C-8”)，16.5(C-3”Me)，26.0(C-7”Me)。
Compound 6
Compound 7 (3 '-geranyl-4 '-methoxyl group-5,7,2 '-the trihydroxy-isoflavanone), yellow oil
UVλ max(MeOH)nm：288，226；
IRν maxcm -1(KBr)：3342，2966，2925，1643，1634，1495，1454，1385，1315，1275，1227，1182，1163，1094，835，756；
HREIMS?m/z?438.2057[M+](Calc.438.2042?for?C 26H 30O 6)；
EIMS?m/z(rel.int.)：438(28)，353(25)，327(15)，316(29)，315(100)，286(14)，215(22)，204(11)，203(31)，201(26)，189(30)，153(48)；
ESI-MS?m/z?439[M+H] +；
1HNMR(CDCI 3)：4.66(H-2α)，4.76(H-2β)，4.00(H-3)，5.95(H-6)，5.95(H-8)，6.39(H-5’)，7.12(H-6’)，3.45(H-1”)，5.24(H-2”)，2.08(H-4”)，2.09(H-5”)，5.03(H-6”)，1.57(H-8”)，1.80(H-3”Me)，1.65(H-7”Me)；
13CNMR(CDCI 3)：70.2(C-2)，46.1(C-3)，197.8(C-4)，164.9(C-5)，97.1(C-6)，166.5(C-7)，95.8(C-8)，163.6(C-9)，102.5(C-10)，115.3(C-1’)，154.2(C-2’)，117.2(C-3’)，158.1(C-4’)，103.9(C-5’)，126.8(C-6’)，22.8(C-1”)，122.0(C-2”)，138.3(C-3”)，40.0(C-4”)，26.7(C-5”)，124.2(C-6”)，132.0(C-7”)，17.9(C-8”)，16.4(C-3”Me)，25.9(C-7”Me)，56.0(C-4’OMe)。
Figure G2008100417531D00081
Compound 7
Compound 8 (3 '-geranyl-5,7,4 ', 5 '-the tetrahydroxy isoflavanone), yellow oil;
UVλ max(MeOH)nm：262.0；
IRν maxcm -1(KBr)：3371，2964，2922，2854，1650，1614，1574，1514，1441，1367，1306，1283，1177，1051，839，822，793；
HREIMS?m/z?422.1719[M] +(Calc.422.1729?for?C25H26O6)；
EIMS?m/z(rel.int.)：422(16)，353(27)，337(14)，300(67)，153(93)，123(49)，69(100)；
ESI-MS?m/z?423[M+1] +；
1HNMR(CDCI 3)：7.96(H-2)，6.20(H-6)，6.32(H-8)，6.32(H-2’)，6.86(H-6’)，3.34(H-1”)，5.32(H-2”)，2.08(H-4”)，?2.10(H-5”)，5.06(H-6”)，1.55(H-8”)，1.71(H-3”Me)，1.58(H-7”Me)；
13CNMR(CDCI 3)：153.5(C-2)，124.0(C-3)，181.1(C-4)，162.7(C-5)，98.9(C-6)，165.0(C-7)，93.6(C-8)，158.5(C-9)，105.2(C-10)，121.7(C-1’)，121.0(C-2’)，128.5(C-3’)，143.5(C-4’)，144.6(C-5’)，113.6(C-6’)，28.3(C-1”)，122.8(C-2”)，135.6(C-3”)，39.7(C-4”)，27.9(C-5”)，124.2(C-6”)，131.0(C-7”)，16.6(C-8”)，15.0(C-3”Me)，24.7(C-7”Me)。
Figure G2008100417531D00082
Compound 8
Another object of the present invention has provided the preparation method of isoflavonoid formula I, and this method comprises the following steps:
(1) Radix Campylotropis Hirtella (Herba Myrsines Africanae) pulverizing medicinal materials is doubly measured W/V 60%-90% alcohol solvent with medicinal material 5-10 and under 50-90 ℃, is carried out heating and extracting 1-3 time, and each 0.5-2h obtains extracting solution;
(2) extracting solution is at pressure 98Kpa, and temperature 50-90 ℃ of following concentrating under reduced pressure is dispersed in liquid concentrator in the water of identical weight and processes suspension;
(3) use and the isopyknic chloroform extraction of suspension three times, combining extraction liquid, chloroform extraction liquid be at pressure 98Kpa, under the 30-50 ℃ of temperature behind the concentrating under reduced pressure chloroform extract;
(4) separating compound
The i chloroform extract gets silica gel mixed sample with equivalent, separates with silica gel column chromatography, uses sherwood oil successively: ETHYLE ACETATE 5: 1-2: the 1V/V gradient elution obtains cut 1, cut 2, cut 3, cut 4, cut 5; Cut 1 usefulness anti-phase C18 post separates, methyl alcohol: water 8: the 2V/V wash-out gets compound 3; Use methyl alcohol: water 7: the 3V/V wash-out gets compound 7; Cut 2 usefulness sephadex lh-20s separate, and get compound 1 with methanol-eluted fractions; Cut 3 usefulness anti-phase C18 posts separate, methyl alcohol: water 7: 3V/V gets compound 2; Cut 4 usefulness anti-phase C18 posts separate, and methyl alcohol: water 6: 4V/V gets compound 5; Cut 5 usefulness anti-phase C18 posts separate, methyl alcohol: water 6: 4-7: the 3V/V wash-out gets compound 8;
The ii chloroform extract is used the equivalent silica gel mixed sample, separates with silica gel column chromatography, uses sherwood oil successively: acetone 4: 1-2: the 1V/V gradient elution, obtain cut 1 and cut 2, and cut 1 obtains compound 6 with the sephadex lh-20 separation again; Cut 2 usefulness anti-phase C18 posts separate, and methyl alcohol: water 6: 4V/V gets compound 4.
Radix Campylotropis Hirtella (Herba Myrsines Africanae) according to the invention is collected in the stem of the young platymiscium iron of Myrsinacea iron young (MyrsineAfricana L.) in Yunnan Province.Preparing method of the present invention is easy.
Another object of the present invention has provided the medicine of isoflavonoid formula I in preparation immunosuppressor or treatment immunological disease.
Immunosuppressive activity experiment of the present invention:
The inventor studies the immunosuppressive activity of above-mentioned eight kinds of neoflavonoids, has tested it to the influence of LPS inductive bone-marrow-derived lymphocyte propagation function and the influence of ConA inductive T lymphopoiesis function.And simultaneously its cytotoxicity is tested.Find that this compounds has significant inhibitory effect to bone-marrow-derived lymphocyte and the lymphocytic propagation of T; Can be used for the medicine for preparing immunosuppressor or treat various immunological diseases; Like rheumatoid arthritis, lupus erythematosus etc., and be used to prepare B cellular type chronic lymphocytic leukemia medicine.
Embodiment
Embodiment 1: the preparation of compound 1
At first use Radix Campylotropis Hirtella (Herba Myrsines Africanae) medicinal material 2kg, add 16L (adding 2L first) 80% ethanol 80 ℃ of heating and extracting 3 times, each 1 hour.Extracting solution is in decompression (pressure 98Kpa) then, temperature 50 ℃ concentrate after, be dispersed in liquid concentrator in the 2L water and process suspension.Add chloroform extraction three times, each 2L gets chloroform extraction liquid, and chloroform extraction liquid gets chloroform extract 91.3g at 40 ℃ of following concentrating under reduced pressure (pressure 98Kpa).
Get chloroform extract 15g and use the equivalent silica gel mixed sample, separate sherwood oil: ETHYLE ACETATE (3: 1) wash-out with silica gel column chromatography (300-400 order); Inspect with TLC; Merge elutriant, concentrate sephadex lh-20 on the eluate (methyl alcohol isocratic elution); Inspect the collection elutriant according to TLC, wash-out concentrates to such an extent that change
Compound 1
The physico-chemical property of compound 1 and wave spectrum characteristic
Compound 1 (3 '-geranyl-5,7,2 ', 4 '-the tetrahydroxy NOVASOY 400), yellow oil.
UVλ max(MeOH)：262.0；
HREIMS：m/z?422.1743[M] +(Calc.422.1729?for?C 25H 26O 6)；
EIMS：m/z(rel.int.)：353(10)，299(38)，128(38)，86(100)，69(79)，59(61)，55(85)；
ESI-MS：m/z?423[M+H] +；
IRν maxcm -1(KBr)：3352，2924，2854，1649，1610，1500，1448，1364，1306，1259，1200，1167，1150，1094，1051，1038，820；
1HNMR(CDCI 3)：7.93(H-2)，6.36(H-6)，6.43(H-8)，6.51(H-5’)，6.89(H-6’)，3.56(H-1”)，5.31(H-1’)，2.08(H-4”)，2.10(H-5”)，5.06(H-6”)，1.59(H-8”)，1.84(H-3”Me)，1.67(H-7”Me)；
13CNMR(CDCI 3)：155.5(C-2)，123.9(C-3)，182.3(C-4)，162.7(C-5)，100.5(C-6)，163.5(C-7)，94.5(C-8)，158.2(C-9)，105.7(C-10)，112.7(C-1’)，154.5(C-2’)，117.4(C-3’)，157.5(C-4’)，109.5(C-5’)，128.2(C-6’)，26.6(C-1”)，121.9(C-2”)，139.1(C-3”)，39.6(C-4”)，26.0(C-5”)，124.1(C-6”)，132.3(C-7”)，16.6(C-8”)，15.4(C-3”Me)，23.1(C-7”Me)。
Figure G2008100417531D00111
Compound 1
Embodiment 2: the preparation of compound 2
At first use Radix Campylotropis Hirtella (Herba Myrsines Africanae) medicinal material 2kg, add 12L (adding 2L first) 70% ethanol 90 ℃ of heating and extracting 2 times, each 1 hour, extracting solution was in decompression (pressure 98Kpa) then, temperature 50 ℃ concentrate after, be dispersed in liquid concentrator in the 2L water and process suspension.Add chloroform extraction three times, each 2L gets chloroform extraction liquid, and chloroform extraction liquid gets chloroform extract 82.5g at 40 ℃ of following concentrating under reduced pressure (pressure 98Kpa).
Get chloroform extract 20g, use the equivalent silica gel mixed sample, separate with silica gel column chromatography (300-400 order); Sherwood oil: ETHYLE ACETATE (2: 1) wash-out, inspect with TLC, merge elutriant; Concentrate, eluate separates with anti-phase C18 post again, methyl alcohol: water (7: 3) wash-out; Inspect the collection elutriant according to TLC, elutriant concentrate compound 2.
The physico-chemical property of compound 2 and wave spectrum characteristic
Compound 2 (5,7,4 '-trihydroxy--3 '-[7-hydroxyl-3,7-dimethyl--2 (E)-octene] NOVASOY 400) the white solid thing, mp:179.3-180.9;
UVλ max(MeOH)：262.0；
HREIMS：m/z?424.1890[M] +(Calc.424.1886?for?C 25H 28O 6)；
EIMSm/z(rel.int.)：424(3.5)，406(39)，338(16)，337(22)，335(22)，321(33)，284(100)，283(73)，153(60)；
ESI-MS：m/z?407[M-OH] +；
IRν maxcm -1(KBr)：3362，2968，2935，1653，1616，1506，1446，1364，1263，1198，1047，825，669；
1HNMR(CDCI 3)：8.12(H-2)，6.27(H-6)，6.40(H-8)，7.35(H-2’)，6.89(H-5’)，7.27(H-6’)，3.38(H-1”)，5.40(H-2”)，2.05(H-4”)，1.52(H-5”)，1.41(H-6”)，1.12(H-8”)，1.73(H-3”Me)，1.12(H-7”Me)；
13CNMR(CDCI 3)：153.5(C-2)，123.7(C-3)，181.0(C-4)，163.3(C-5)，99.2(C-6)，164.4(C-7)，93.8(C-8)，158.4(C-9)，105.5(C-10)，122.5(C-1’)，130.5(C-2’)，128.1(C-3’)，155.3(C-4’)，114.9(C-5’)，127.9(C-6’)，28.3(C-1”)，122.7(C-2”)，136.1(C-3”)，40.34(C-4”)，22.7(C-5”)，43.7(C-6”)，69.5(C-7”)，29.0(C-8”)，15.5(C-3”Me)，29.0(C-7”Me)。
Figure G2008100417531D00121
Compound 2
Embodiment 3: the preparation of compound 3
At first use Radix Campylotropis Hirtella (Herba Myrsines Africanae) medicinal material 2kg, add 16L (adding 2L first) 80% ethanol 80 ℃ of heating and extracting 3 times, each 1 hour.Extracting solution is in decompression (pressure 98Kpa) then, temperature 50 ℃ concentrate after, be dispersed in liquid concentrator in the 2L water and process suspension.Add chloroform extraction three times, each 2L gets chloroform extraction liquid, and chloroform extraction liquid gets chloroform extract 91.3g at 40 ℃ of following concentrating under reduced pressure (pressure 98Kpa).
Get chloroform extract 15g, use the equivalent silica gel mixed sample, with silicagel column (200-300 order) chromatographic separation; Sherwood oil: ETHYLE ACETATE (5: 1) wash-out, inspect with TLC, merge elutriant; Concentrate, eluate separates with anti-phase C18 post again, methyl alcohol: water (8: 2) wash-out; Inspect the collection elutriant according to TLC, elutriant concentrate compound 3.
The physico-chemical property of compound 3 and wave spectrum characteristic
Compound 3 (5, the 7-dihydroxyl-[6 " methyl-6 "-(4-methyl-3-pentenyl) pyrans also (2 ", 3 ": 3 ', 4 ')] NOVASOY 400), yellow oil;
UVλ max(MeOH)nm：290.0；
HREIMS：m/z?404.1635[M+](Calc.404.1624?for?C 25H 24O 5)；
EIMS?m/z(rel.int.)：404(5)，322(23)，321(100)；
IRν maxcm -1(KBr)：3339，2924，1652，1618，1578，1491，1448，1360，1306，1261，1180，1045，831；
1HNMR(CDCI 3)：8.20(H-2)，6.29(H-6)，6.42(H-8)，7.31(H-2’)，6.85(H-5’)，7.33(H-6’)，6.49(H-4”)，5.75(H-5”)，1.71(H-7”)，2.13(H-8”)，5.13(H-9”)，1.57(H-1”)，1.40(H-6”Me)，1.64(H-10”Me)；
13CNMR(CDCI 3)：153.9(C-2)，123.1(C-3)，181.0(C-4)，163.2(C-5)，99.2(C-6)，164.4(C-7)，93.9(C-8)，158.9(C-9)，105.4(C-10)，123.6(C-1’)，127.4(C-2’)，121.1(C-3’)，153.6(C-4’)，115.9(C-5’)，130.3(C-6’)，122.7(C-4”)，130.0(C-5”)，99.0(C-6”)，41.4(C-7”)，22.8(C-8”)，124.3(C-9”)，131.4(C-10”)，17.0(C-11”)，26.3(C-6”Me)，25.2(C-10”Me)。
Compound 3
Embodiment 4: the preparation of compound 4
At first use Radix Campylotropis Hirtella (Herba Myrsines Africanae) medicinal material 2kg, add 12L (adding 2L first) 70% ethanol 90 ℃ of heating and extracting 2 times, each 1 hour, extracting solution was in decompression (pressure 98Kpa) then, temperature 50 ℃ concentrate after, be dispersed in liquid concentrator in the 2L water and process suspension.Add chloroform extraction three times, each 2L gets chloroform extraction liquid, and chloroform extraction liquid gets chloroform extract 82.5g at 40 ℃ of following concentrating under reduced pressure (pressure 98Kpa).
Get chloroform extract 20g and use silica gel mixed sample, separate sherwood oil with silica gel column chromatography: acetone (2: 1) wash-out, inspect with TLC; Merge elutriant, concentrate, eluate separates with anti-phase C18 post again; Methyl alcohol: water (6: 4), inspect the collection elutriant according to TLC, elutriant concentrate compound 4.
The physico-chemical property of compound 4 and wave spectrum characteristic
Compound 4 (4,5,7,4 ", 5 " tetrahydroxys-[6 "-methyl-6 " (4-methyl-3-pentenyl) pyrans also (2 ", 3 ": 3 ', 4 ')] NOVASOY 400), yellow oil;
UVλ max(MeOH)nm：290.0；
HREIMS：m/z?442.1989[M] +(Cal.442.1992?for?C 25H 30O 7)；
EIMS?m/z?rel.int.：442(5)，424(49)，300(42)，299(45)，153(100)；
ESI-MS?m/z：425[M-OH] +；
IRν maxcm -1(KBr)：3367，2966，2931，1699，1684，1635，1452，1379，1269，1184，1465，835，800；
1HNMR(CDCI 3)：8.08(H-2)，6.22(H-6)，6.34(H-8)，7.61(H-2’)，6.81(H-5’)，7.34(H-6’)，4.58(H-4”)，3.65(H-5”)，1.85(H-7”)，2.17(H-8”)，5.16(H-9”)，1.65(H-1”)，1.19(H-6”Me)，1.69(H-10”Me)；
13CNMR(CDCI 3)：153.8(C-2)，123.2(C-3)，181.0(C-4)，162.7(C-5)，99.0(C-6)，164.9(C-7)，93.7(C-8)，158.5(C-9)，105.1(C-10)，123.5(C-1’)，128.7(C-2’)，124.6(C-3’)，152.9(C-4’)，116.5(C-5’)，129.7(C-6’)，69.1(C-4”)，73.2(C-5”)，80.3(C-6”)，38.7(C-7”)，21.1(C-8”)，124.3(C-9”)，131.2(C-10”)，16.5(C-11”)，17.1(C-6”Me)，24.7(C-10”Me)。
Figure G2008100417531D00141
Compound 4
Embodiment 5: the preparation of compound 5
At first use Radix Campylotropis Hirtella (Herba Myrsines Africanae) medicinal material 2kg, add 16L (adding 2L first) 80% ethanol 80 ℃ of heating and extracting 3 times, each 1 hour.Extracting solution is in decompression (pressure 98Kpa) then, temperature 50 ℃ concentrate after, be dispersed in liquid concentrator in the 2L water and process suspension.Add chloroform extraction three times, each 2L gets chloroform extraction liquid, and chloroform extraction liquid gets chloroform extract 91.3g at 40 ℃ of following concentrating under reduced pressure (pressure 98Kpa).
Get chloroform extract 20g and use the equivalent silica gel mixed sample, separate sherwood oil: ETHYLE ACETATE (2: 1) wash-out with silica gel column chromatography; Inspect with TLC, merge elutriant, concentrate; Eluate separates with anti-phase C18 post again; Methyl alcohol: water (6: 4) wash-out, inspect the collection elutriant according to TLC, elutriant concentrate compound 5.
The physico-chemical property of compound 5 and wave spectrum characteristic
Compound 5 (5,7,2 ', 4 '-tetrahydroxy-3 '-[7-hydroxyl-3,7-dimethyl--2 (E)-octene] isoflavanone) yellow oil
UVλ max(MeOH)nm：290.0；
IRν maxcm -1(KBr)：3367，2966，2931，1699，1684，1635，1452，1379，1269，1184，1465，835，800；
HREIMS?m/z?442.1989[M] +(Cal.442.1992?for?C 25H 30O 7)；
EIMS?m/z(rel.int.)：442(5)，424(49)，300(42)，299(45)，153(100)；
ESI-MS?m/z?425[M-OH] +；
1HNMR(CDCI 3)：4.60(H-2α)，4.70(H-2β)，4.17(H-3)，5.97(H-6)，5.97(H-8)，6.43(H-5’)，6.93(H-6’)，3.43(H-1”)，5.25(H-2”)，1.93(H-4”)，1.46(H-5”)，1.36(H-6”)，1.13(H-8”)，1.77(H-3”Me)，1.13(H-7”Me)；
13CNMR(CDCI 3)：70.4(C-2)，46.1(C-3)，197.9(C-4)，164.9(C-5)，96.2(C-6)，167.0(C-7)，95.1(C-8)，163.8(C-9)，102.2(C-10)，114.6(C-1’)，154.1(C-2’)，116.3(C-3’)，155.8(C-4’)，107.7(C-5’)，126.5(C-6’)，22.7(C-1”)，122.8(C-2”)，135.3(C-3”)，40.4(C-4”)，22.5(C-5”)，43.7(C-6”)，69.4(C-7”)，29.0(C-8”)，15.6(C-3”Me)，29.0(C-7”Me)；
Figure G2008100417531D00151
Compound 5
Embodiment 6: the separation preparation of compound 6
At first use Radix Campylotropis Hirtella (Herba Myrsines Africanae) medicinal material 2kg, add 16L (adding 2L first) 80% ethanol 80 ℃ of heating and extracting 3 times, each 1 hour.Extracting solution is in decompression (pressure 98Kpa) then, temperature 50 ℃ concentrate after, be dispersed in liquid concentrator in the 2L water and process suspension.Add chloroform extraction three times, each 2L gets chloroform extraction liquid, and chloroform extraction liquid gets chloroform extract 91.3g at 40 ℃ of following concentrating under reduced pressure (pressure 98Kpa).
Get chloroform extract 20g and use the equivalent silica gel mixed sample, separate sherwood oil: acetone (4: 1-2: 1 gradient elution) with silica gel column chromatography; Inspect with TLC; Merge elutriant, concentrate, eluate is used sephadex lh-20 (methanol-eluted fractions) again; Inspect the collection elutriant according to TLC, elutriant concentrate compound 6.
The physico-chemical property of compound 6 and wave spectrum characteristic
Compound 6 (3 '-geranyl-5,7,2 ', 4 '-the tetrahydroxy isoflavanone), yellow oil
UVλ max(MeOH)nm：290.0，230.0；
IRν maxcm -1(KBr)：3304，2926，1639，1614，1450，1379，1263，1614，1450，1379，1263，1182，1163，1091，835；
HREIMS?m/z?424.1882[M] +(Calc.424.1886?for?C 25H 28O 6)；
EIMSm/z(rel.int.)：424(12)，301(46)，201(26)，187(36)，179(31)，153(100)，123(60)，91(35)，69(80)；
ESI-MS?m/z?425[M+H] +；
1HNMR(CDCI 3)：4.66(H-2α)，4.76(H-2β)，4.00(H-3)，5.95(H-6)，5.95(H-8)，6.39(H-5’)，7.12(H-6’)，3.45(H-1”)，5.24(H-2”)，2.08(H-4”)，2.09(H-5”)，5.03(H-6”)，1.57(H-8”)，1.80(H-3”Me)，1.65(H-7”Me)；
13CNMR(CDCI 3)：70.0(C-2)，45.6(C-3)，197.5(C-4)，165.1(C-5)，97.2(C-6)，166.6(C-7)，95.8(C-8)，163.4(C-9)，102.0(C-10)，115.1(C-1’)，154.2(C-2’)，115.8(C-3’)，155.5(C-4’)，108.9(C-5’)，126.1(C-6’)，23.0(C-1”)，121.7(C-2”)，139.4(C-3”)，39.9(C-4”)，26.6(C-5”)，124.0(C-6”)，132.3(C-7”)，18.0(C-8”)，16.5(C-3”Me)，26.0(C-7”Me)。
Figure G2008100417531D00161
Compound 6
Embodiment 7: the preparation of compound 7
At first use Radix Campylotropis Hirtella (Herba Myrsines Africanae) medicinal material 2kg, add 16L (adding 2L first) 80% ethanol 80 ℃ of heating and extracting 3 times, each 1 hour.Extracting solution is in decompression (pressure 98Kpa) then, temperature 50 ℃ concentrate after, be dispersed in liquid concentrator in the 2L water and process suspension.Add chloroform extraction three times, each 2L gets chloroform extraction liquid, and chloroform extraction liquid gets chloroform extract 91.3g at 40 ℃ of following concentrating under reduced pressure (pressure 98Kpa).
Get chloroform extract 15g and use the equivalent silica gel mixed sample, separate sherwood oil: ETHYLE ACETATE (4: 1) wash-out with silica gel column chromatography; Inspect with TLC, merge elutriant, concentrate; Eluate separates with anti-phase C18 post again; Methyl alcohol: water (7: 3) wash-out, inspect the collection elutriant according to TLC, elutriant concentrate compound 7.The physico-chemical property of compound 7 and wave spectrum characteristic
Compound 7 (3 '-geranyl-4 '-methoxyl group-5,7,2 '-the trihydroxy-isoflavanone), yellow oil
UVλ max(MeOH)nm：288.0，226.0；
IRν maxcm -1(KBr)：3342，2966，2925，1643，1634，1495，1454，1385，1315，1275，1227，1182，1163，1094，835，756；
HREIMS?m/z?438.2057[M] +(Calc.438.2042?for?C 26H 30O 6)；
EIMS?m/z(rel.int.)：438(28)，353(25)，327(15)，316(29)，315(100)，286(14)，215(22)，204(11)，203(31)，201(26)，189(30)，153(48)；
ESI-MS?m/z?439[M+H] +；
1HNMR(CDCI 3)：4.66(H-2α)，4.76(H-2β)，4.00(H-3)，5.95(H-6)，5.95(H-8)，6.39(H-5’)，7.12(H-6’)，3.45(H-1”)，5.24(H-2”)，2.08(H-4”)，2.09(H-5”)，5.03(H-6”)，1.57(H-8”)，1.80(H-3”Me)，1.65(H-7”Me)；
13CNMR(CDCI 3)：70.2(C-2)，46.1(C-3)，197.8(C-4)，164.9(C-5)，97.1(C-6)，166.5(C-7)，95.8(C-8)，163.6(C-9)，102.5(C-10)，115.3(C-1’)，154.2(C-2’)，117.2(C-3’)，158.1(C-4’)，103.9(C-5’)，126.8(C-6’)，22.8(C-1”)，122.0(C-2”)，138.3(C-3”)，40.0(C-4”)，26.7(C-5”)，124.2(C-6”)，132.0(C-7”)，17.9(C-8”)，16.4(C-3”Me)，25.9(C-7”Me)，56.0(C-4’OMe)。
Figure G2008100417531D00171
Compound 7
Embodiment 8: the preparation of compound 8
At first use Radix Campylotropis Hirtella (Herba Myrsines Africanae) medicinal material 2kg, add 12L (adding 2L first) 70% ethanol 90 ℃ of heating and extracting 2 times, each 1 hour, extracting solution was in decompression (pressure 98Kpa) then, temperature 50 ℃ concentrate after, be dispersed in liquid concentrator in the 2L water and process suspension.Add chloroform extraction three times, each 2L gets chloroform extraction liquid, and chloroform extraction liquid gets chloroform extract 82.5g at 40 ℃ of following concentrating under reduced pressure (pressure 98Kpa).
Get chloroform extract 20g and use the equivalent silica gel mixed sample, separate sherwood oil: ETHYLE ACETATE (2: 1-1: 1) gradient elution with silica gel column chromatography; Inspect with TLC, merge elutriant, concentrate; Eluate separates with anti-phase C18 post again; Methyl alcohol: water (6: 4-7: 3), inspect the collection elutriant according to TLC, elutriant concentrate compound 8.
The physico-chemical property of compound 8 and wave spectrum characteristic
Compound 8 (3 '-geranyl-5,7,4 ', 5 '-the tetrahydroxy isoflavanone), yellow oil;
UVλ max(MeOH)nm：262.0；
IRν maxcm -1(KBr)：3371，2964，2922，2854，1650，1614，1574，1514，1441，1367，1306，1283，1177，1051，839，822，793；
HREIMS?m/z?422.1719[M] +(Calc.422.1729?for?C 25H 26O 6)；
EIMS?m/z(rel.int.)：422(16)，353(27)，337(14)，300(67)，153(93)，123(49)，69(100)；
ESI-MS?m/z?423[M+1] +；
1HNMR(CDCI 3)：7.96(H-2)，6.20(H-6)，6.32(H-8)，6.32(H-2’)，6.86(H-6’)，3.34(H-1”)，5.32(H-2”)，2.08(H-4”)，2.10(H-5”)，5.06(H-6”)，1.55(H-8”)，1.71(H-3”Me)，1.58(H-7”Me)；
13CNMR(CDCI 3)：153.5(C-2)，124.0(C-3)，181.1(C-4)，162.7(C-5)，98.9(C-6)，165.0(C-7)，93.6(C-8)，158.5(C-9)，105.2(C-10)，121.7(C-1’)，121.0(C-2’)，128.5(C-3’)，143.5(C-4’)，144.6(C-5’)，113.6(C-6’)，28.3(C-1”)，122.8(C-2”)，135.6(C-3”)，39.7(C-4”)，27.9(C-5”)，124.2(C-6”)，131.0(C-7”)，16.6(C-8”)，15.0(C-3”Me)，24.7(C-7”Me)。
Figure G2008100417531D00181
Compound 8
Embodiment 9: the immunosuppressive activity test:
(1) TP:
1. experimental animal
The BALB/c mouse inbred lines, female, the 18-20 gram, available from Chinese Academy of Sciences's Shanghai Experimental Animal Center, animal production licence number: SYXK (Shanghai) 2002-0033 number.
2. the preparation of mouse spleen lymphocyte:
Mouse takes off vertebra puts to death, and aseptic its spleen of getting prepares the individual cells suspension, and erythrocyte cracked liquid is removed red corpuscle, washes 3 after this with containing 2%FBS, the RPMI-1640 nutrient solution that usefulness contains 10%FBS wash 1 this and cell concn transferred to 4 * 10 6/ ml.
3. the mtt assay detection compound is to the active influence of mouse spleen lymphocyte:
Mouse spleen lymphocyte suspension 100 μ l (4 * 10 5/ hole) be inoculated in 96 orifice plates, add the compound of different concns simultaneously, other establishes corresponding solvent contrast and the contrast of nutrient solution background, and TV is 200 μ l.37 ℃, 5%CO 2Culture supernatant in the incubator adds 150 μ lDMSO dissolved cells again, measures the OD value in the 570nm place with ELIASA.
4. 3H-TdR mixes the influence of method detection compound to mouse spleen lymphocyte propagation function:
Mouse spleen lymphocyte suspension 100 μ l (4 * 10 5/ hole) be inoculated in 96 orifice plates, add 50 μ l ConA (final concentration 5 μ g/ml), or add 50 μ l LPS (final concentration 10 μ g/ml), different concns compound 50 μ l, TV are 200 μ l.Each concentration three multiple holes, and establish corresponding no ConA or do not have the LPS control wells and do not have the medicine control wells.37 ℃, 5%CO 2Cultivate 48h in the incubator.Cultivate and finish preceding 8 hours, every hole adds 25 μ l 3H-thymidylic acid (10 μ Ci/ml).Continuing to be cultured to experiment finishes.Cell is collected on the glass fibre membrane with the cell harvesting appearance, adds behind the scintillation solution that (MicroBeta Trilux PerkinElmer) reads and mixes cell DNA in Beta numeration appearance 3H-TdR measures, and represents the situation of cell proliferation with the cpm value.
(2) sample is used in test
Compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8
(3) test-results
Table1 compound 1-8 is to the active influence of mouse spleen lymphocyte
Compound concentration (μ g/ml) necrocytosis (%) CC50 (μ g/ml)
100 85.27
10 15.66
35.94
Compound 1 1-9.19
0.1 -2.18
100 88.69
10 56.53
14.50
Compound 21 2.36
0.1 -1.61
100 65.69
10 16.06
Compound 3 1-17.07 49.05
0.1 -2.38
100 86.92
10 28.85
Compound 41 3.96 19.42
0.1 1.58
100 76.86
Compound 5 10 11.70 40.27
1 5.19
0.1 2.61
100 7.58
10 15.87
6.81
Compound 61 5.71
0.1 5.31
100 72.48
10 8.22
49.9
Compound 7 1-17.18
0.1 -11.68
100 64.02
10 16.46
Compound 81 19.48 31.7
0.1 -0.82
Table2 compound 1-8 is to the influence of LPS inductive bone-marrow-derived lymphocyte propagation function
Concentration (μ g/ml) B cell suppresses IC50 SI
(％) (μg/ml) (IC50/CC50)
100 98.92
Compound 1 10 54.11
1.61 22.32
1 25.15
0.1 20.16
100 99.97
Compound 2 10 99.03
0.60 24.2
1 43.93
0.1 10.34
100 99.97
Compound 3 10 95.55
1.22 40.2
1 15.73
0.1 11.39
100 99.78
Compound 4 10 90.90 16.2
1.20
1 26.56
0.1 14.62
100 99.92
Compound 5 10 44.38
1 20.92 1.63 ?24.7
0.1 9.77
100 99.19
Compound 6 10 86.56
1 33.77 1.11 ?6.13
0.1 13.30
100 98.77
Compound 7 10 77.15
15.03 3.31
1 -2.31
0.1 -17.80
100 97.18
Compound 8 10 21.47
24.86 1.27
1 11.58
0.1 -2.23
Table3 compound 1-8 is to the influence of ConA inductive T lymphopoiesis function
Concentration (μ g/ml) T cell suppresses IC50 SI
(％) (μg/ml) (IC50/CC50)
100 98.91
Compound 1 10 58.59
1 26.53 3.44 10.5
0.1 21.94
100 99.96
Compound 2 10 99.03 0.70 20.7
1 43.93
0.1 10.34
100 99.97
Compound 3 10 95.55
1 15.73 1.22 40.2
0.1 11.39
100 99.78
Compound 4 10 90.90
1 26.56 2.17 9.0
0.1 14.62
100 99.22
Compound 5 10 40.63
3.55 11.3
1 15.00
0.1 3.72
100 99.29
Compound 6 10 86.71
0.63 10.8
1 48.54
0.1 24.85
100 99.60
Compound 7 10 82.74
3.83 13.0
1 9.62
0.1 2.08
100 98.53
Compound 8 10 39.99
6.74 4.71
1 10.31
0.1 -12.52
(4) conclusion (of pressure testing)
Test-results shows that the propagation of significant suppressor T cell of such flavonoid compound ability and B cell shows that this compounds can be used in the preparation immunosuppressor; And to autoimmune disorder (like rheumatoid arthritis; Lupus erythematosus) has therapeutic action, simultaneously because such compound S I value (IC50/CC50) is all higher, therefore; Its toxic side effect is less, have broad treatment region between.

Claims (12)

1. the isoflavonoid of a general formula I:
Figure RE-RE-FSB00000761009600011
In the formula, when being pair key between numbering 2 and 3 the carbon atom,
R1=H;
Figure RE-RE-FSB00000761009600012
R3=OH, R4=H or
R1=OH;
Figure RE-RE-FSB00000761009600013
R3=OH, R4=H or
R1=H;
Figure RE-RE-FSB00000761009600014
R3=OH, R4=OH or
R1=H, R4=H, R2 and R3 form following six-ring:
Figure RE-RE-FSB00000761009600015
When being singly-bound between the carbon atom of numbering 2 and 3,
R1=OH; R3=OH, R4=H or
R1=OH;
Figure RE-RE-FSB00000761009600017
R3=OH, R4=H or
R1＝OH，?
Figure RE-RE-FSB00000761009600018
R3＝OH，R4＝H。
An isoflavonoid 13 '-geranyl-5,7,2 ', 4 '-the tetrahydroxy NOVASOY 400, it is characterized in that it has following structural and physico-chemical property:
Figure FSB00000507002300021
Compound 1
Yellow oil;
UVλ max(MeOH)：262.0；
HREIMS：m/z?422.1743[M] +(Calc.422.1729for?C 25H 26O 6)；
EIMS：m/z(rel.int.)：353(10)，299(38)，128(38)，86(100)，69(79)，59(61)，55(85)；
ESI-MS：m/z?423[M+H] +；
IR?v? maxcm -1(KBr)：3352，2924，2854，1649，1610，1500，1448，1364，1306，1259，1200，1167，1150，1094，1051，1038，820；
1HNMR(CDCl 3)：7.93(H-2)，6.36(H-6)，6.43(H-8)，6.51(H-5’)，6.89(H-6’)，3.56(H-1”)，5.31(H-1’)，2.08(H-4”)，2.10(H-5”)，5.06(H-6”)，1.59(H-8”)，1.84(H-3”Me)，1.67(H-7”Me)；
13CNMR(CDCl 3)：155.5(C-2)，123.9(C-3)，182.3(C-4)，162.7(C-5)，100.5(C-6)，163.5(C-7)，94.5(C-8)，158.2(C-9)，105.7(C-10)，112.7(C-1’)，154.5(C-2’)，117.4(C-3’)，157.5(C-4’)，109.5(C-5’)，128.2(C-6’)，26.6(C-1”)，121.9(C-2”)，139.1(C-3”)，39.6(C-4”)，26.0(C-5”)，124.1(C-6”)，132.3(C-7”)，16.6(C-8”)，15.4(C-3”Me)，23.1(C-7”Me)。
An isoflavonoid 25,7,4 '-trihydroxy--3 '-[7-hydroxyl-3,7-dimethyl--2 (E)-octene] NOVASOY 400, it is characterized in that it has following structural and physico-chemical property:
Figure FSB00000507002300022
Compound 2
The white solid thing, mp:179.3-180.9;
UVλ max(MeOH)：262.0；
HREIMS：m/z?424.1890[M] +(Calc.424.1886?for?C 25H 28O 6)；
EIMSm/z(rel.int.)：424(3.5)，406(39)，338(16)，337(22)，335(22)，321(33)，284(100)，283(73)，153(60)；
ESI-MS：m/z?407[M+H] +；
IR?v? maxcm -1(KBr)：3362，2968，2935，1653，1616，1506，1446，1364，1263，1198，1047，825，669；
1HNMR(CDCl 3)：8.12(H-2)，6.27(H-6)，6.40(H-8)，7.35(H-2’)，6.89(H-5’)，7.27(H-6’)，3.38(H-1”)，5.40(H-2”)，2.05(H-4”)，1.52(H-5”)，1.41(H-6”)，1.12(H-8”)，1.73(H-3”Me)，1.12(H-7”Me)；
13CNMR(CDCl 3)：153.5(C-2)，123.7(C-3)，181.0(C-4)，163.3(C-5)，99.2(C-6)，164.4(C-7)，93.8(C-8)，158.4(C-9)，105.5(C-10)，122.5(C-1’)，130.5(C-2’)，128.1(C-3’)，155.3(C-4’)，114.9(C-5’)，127.9(C-6’)，28.3(C-1”)，122.7(C-2”)，136.1(C-3”)，40.34(C-4”)，22.7(C-5”)，43.7(C-6”)，69.5(C-7”)，29.0(C-8”)，15.5(C-3”Me)，29.0(C-7”Me)。
An isoflavonoid 3 (5,7 dihydroxyl-[6 " methyl-6 "-(4-methyl-3-pentenyl) pyrans also (2 ", 3 ": 3 ', 4 ')] NOVASOY 400), it is characterized in that having following structural and physico-chemical property:
Figure FSB00000507002300031
Compound 3
Yellow oil;
UVλ max(MeOH)nm：290.0；
HREIMS：m/z?404.1635[M] +(Calc.404.1624?for?C 25H 24O 5)；
EIMS?m/z(rel.int.)：404(5)，322(23)，321(100)；
ESI-MS：m/z?405[M+H] +；
IR?v? maxcm -1(KBr)：3339，2924，1652，1618，1578，1491，1448，1360，1306，1261，1180，1045，831；
1HNMR(CDCl 3)：8.20(H-2)，6.29(H-6)，6.42(H-8)，7.31(H-2’)，6.85(H-5’)，7.33(H-6’)，6.49(H-4”)，5.75(H-5”)，1.71(H-7”)，2.13(H-8”)，5.13(H-9”)，1.57(H-1”)，1.40(H-6”Me)，1.64(H-10”Me)；
13CNMR(CDCl 3)：153.9(C-2)，123.1(C-3)，181.0(C-4)，163.2(C-5)，99.2(C-6)，164.4(C-7)，93.9(C-8)，158.9(C-9)，105.4(C-10)，123.6(C-1’)，127.4(C-2’)，121.1(C-3’)，153.6(C-4’)，115.9(C-5’)，130.3(C-6’)，122.7(C-4”)，130.0(C-5”)，99.0(C-6”)，41.4(C-7”)，22.8(C-8”)，124.3(C-9”)，131.4(C-10”)，17.0(C-11”)，26.3(C-6”Me)，25.2(C-10”Me)。
5. isoflavonoid 44,5,7,4 ", 5 " tetrahydroxys-[6 "-methyl-6 " (4-methyl-3-pentenyl) pyrans also (2 ", 3 ": 3 ', 4 ')] NOVASOY 400, it is characterized in that it has following structural and physico-chemical property:
Figure FSB00000507002300041
Compound 4
Yellow oil
UVλ max(MeOH)nm：290.0；
1HREIMS：m/z442.1989[M] +(Cal.442.1992?for?C 25H 30O 7)；
EIMS?m/z?rel.int.：442(5)，424(49)，300(42)，299(45)，153(100)；
ESI-MS?m/z：425[M-OH] +；
IR?v? maxcm -1(KBr)：3367，2966，2931，1699，1684，1635，1452，1379，1269，1184，1465，835，800；
1HNMR(CDCl 3)：8.08(H-2)，6.22(H-6)，6.34(H-8)，7.61(H-2’)，6.81(H-5’)，7.34(H-6’)，4.58(H-4”)，3.65(H-5”)，1.85(H-7”)，2.17(H-8”)，5.16(H-9”)，1.65(H-1”)，1.19(H-6”Me)，1.69(H-10”Me)；
13CNMR(CDCl 3)：153.8(C-2)，123.2(C-3)，181.0(C-4)，162.7(C-5)，99.0(C-6)，164.9(C-7)，93.7(C-8)，158.5(C-9)，105.1(C-10)，123.5(C-1’)，128.7(C-2’)，?124.6(C-3’)，152.9(C-4’)，116.5(C-5’)，129.7(C-6’)，69.1(C-4”)，73.2(C-5”)，80.3(C-6”)，38.7(C-7”)，21.1(C-8”)，124.3(C-9”)，131.2(C-10”)，16.5(C-11”)，17.1(C-6”Me)，24.7(C-10”Me)。
An isoflavonoid 55,7,2 ', 4 '-tetrahydroxy-3 '-[7-hydroxyl-3,7-dimethyl--2 (E)-octene] isoflavanone, it is characterized in that it has following structural and physico-chemical property:
Figure FSB00000507002300051
Compound 5
Yellow oil;
UVλ max(MeOH)nm：290.0；
IR?v? maxcm -1(KBr)：3367，2966，2931，1699，1684，1635，1452，1379，1269，1184，1465，835，800；
HREIMS?m/z?442.1989[M +](Cal.442.1992for?C 25H 30O 7)；
EIMS?m/z(rel.int.)：442(5)，424(49)，300(42)，299(45)，153(100)；
ESI-MS?m/z?425[M-OH] +；
1HNMR(CDCl 3)：4.60(H-2α)，4.70(H-2β)，4.17(H-3)，5.97(H-6)，5.97(H-8)，6.43(H-5’)，6.93(H-6’)，3.43(H-1”)，5.25(H-2”)，1.93(H-4”)，1.46(H-5”)，1.36(H-6”)，1.13(H-8”)，1.77(H-3”Me)，1.13(H-7”Me)；
13CNMR(CDCl 3)：70.4(C-2)，46.1(C-3)，197.9(C-4)，164.9(C-5)，96.2(C-6)，167.0(C-7)，95.1(C-8)，163.8(C-9)，102.2(C-10)，114.6(C-1’)，154.1(C-2’)，116.3(C-3’)，155.8(C-4’)，107.7(C-5’)，126.5(C-6’)，22.7(C-1”)，122.8(C-2”)，135.3(C-3”)，40.4(C-4”)，22.5(C-5”)，43.7(C-6”)，69.4(C-7”)，29.0(C-8”)，15.6(C-3”Me)，29.0(C-7”Me)。
An isoflavonoid 63 '-geranyl-5,7,2 ', 4 '-tetrahydroxy NOVASOY 400 alcohol, it is characterized in that it has following structural and physico-chemical property:
Figure FSB00000507002300061
Compound 6
Yellow oil
UVλ max(MeOH)nm：290.0，230.0；
IR?v? maxcm -1(KBr)：3304，2926，1639，1614，1450，1379，1263，1614，1450，1379，1263，1182，1163，1091，835；
HREIMS?m/z?424.1882[M] +(Calc.424.1886for?C 25H 28O 6)；
EIMSm/z(rel.int.)：424(12)，301(46)，201(26)，187(36)，179(31)，153(100)，123(60)，91(35)，69(80)；
ESI-MS?m/z?425[M+H] +；
1HNMR(CDCl 3)：4.66(H-2α)，4.76(H-2β)，4.00(H-3)，5.95(H-6)，5.95(H-8)，6.39(H-5’)，7.12(H-6’)，3.45(H-1”)，5.24(H-2”)，2.08(H-4”)，2.09(H-5”)，5.03(H-6”)，1.57(H-8”)，1.80(H-3”Me)，1.65(H-7”Me)；
13CNMR(CDCl 3)：70.0(C-2)，45.6(C-3)，197.5(C-4)，165.1(C-5)，97.2(C-6)，166.6(C-7)，95.8(C-8)，163.4(C-9)，102.0(C-10)，115.1(C-1’)，154.2(C-2’)，115.8(C-3’)，155.5(C-4’)，108.9(C-5’)，126.1(C-6’)，23.0(C-1”)，121.7(C-2”)，139.4(C-3”)，39.9(C-4”)，26.6(C-5”)，124.0(C-6”)，132.3(C-7”)，18.0(C-8”)，16.5(C-3”Me)，26.0(C-7”Me)。
An isoflavonoid 73 '-geranyl-4 '-methoxyl group-5,7,2 '-the trihydroxy-isoflavanone, it is characterized in that it has following structural and physico-chemical property:
Figure FSB00000507002300071
Compound 7
Yellow oil
UVλ max(MeOH)nm：288.0，226.0；
IR?v? maxcm -1(KBr)：3342，2966，2925，1643，1634，1495，1454，1385，1315，1275，1227，1182，1163，1094，835，756；
HREIMS?m/z?438.2057[M] +(Calc.438.2042for?C 26H 30O 6)；
EIMS?m/z(rel.int.)：438(28)，353(25)，327(15)，316(29)，315(100)，286(14)，215(22)，204(11)，203(31)，201(26)，189(30)，153(48)；
ESI-MS?m/z?439[M+H] +；
1HNMR(CDCl 3)：4.66(H-2α)，4.76(H-2β)，4.00(H-3)，5.95(H-6)，5.95(H-8)，6.39(H-5’)，7.12(H-6’)，3.45(H-1”)，5.24(H-2”)，2.08(H-4”)，2.09(H-5”)，5.03(H-6”)，1.57(H-8”)，1.80(H-3”Me)，1.65(H-7”Me)；
13CNMR(CDCl 3)：70.2(C-2)，46.1(C-3)，197.8(C-4)，164.9(C-5)，97.1(C-6)，166.5(C-7)，95.8(C-8)，163.6(C-9)，102.5(C-10)，115.3(C-1’)，154.2(C-2’)，117.2(C-3’)，158.1(C-4’)，103.9(C-5’)，126.8(C-6’)，22.8(C-1”)，122.0(C-2”)，138.3(C-3”)，40.0(C-4”)，26.7(C-5”)，124.2(C-6”)，132.0(C-7”)，17.9(C-8”)，16.4(C-3”Me)，25.9(C-7”Me)，56.0(C-4’OMe)。
An isoflavonoid 83 '-geranyl-5,7,4 ', 5 '-the tetrahydroxy isoflavanone, it is characterized in that it has following structural and physico-chemical property:
Figure FSB00000507002300072
Compound 8
Yellow oil
UVλ max(MeOH)nm：262.0；
IR?v? maxcm -1(KBr)：3371，2964，2922，2854，1650，1614，1574，1514，1441，1367，1306，1283，1177，1051，839，822，793；
HREIMS?m/z?422.1719[M] +(Calc.422.1729for?C 25H 26O 6)；
EIMS?m/z(rel.int.)：422(16)，353(27)，337(14)，300(67)，153(93)，123(49)，69(100)；
ESI-MS?m/z?423[M+1] +；
1HNMR(CDCl 3)：7.96(H-2)，6.20(H-6)，6.32(H-8)，6.32(H-2’)，6.86(H-6’)，3.34(H-1”)，5.32(H-2”)，2.08(H-4”)，2.10(H-5”)，5.06(H-6”)，1.55(H-8”)，1.71(H-3”Me)，1.58(H-7”Me)；
13CNMR(CDCl 3)：153.5(C-2)，124.0(C-3)，181.1(C-4)，162.7(C-5)，98.9(C-6)，165.0(C-7)，93.6(C-8)，158.5(C-9)，105.2(C-10)，121.7(C-1’)，121.0(C-2’)，128.5(C-3’)，143.5(C-4’)，144.6(C-5’)，113.6(C-6’)，28.3(C-1”)，122.8(C-2”)，135.6(C-3”)，39.7(C-4”)，27.9(C-5”)，124.2(C-6”)，131.0(C-7”)，16.6(C-8”)，15.0(C-3”Me)，24.7(C-7”Me)。
10. like the preparation method of each described isoflavonoid of claim 1-9, it is characterized in that this method comprises the following steps:
(1) Radix Campylotropis Hirtella (Herba Myrsines Africanae) pulverizing medicinal materials is doubly measured kg/L 60%-90% alcohol solvent with medicinal material 5-10 and under 50-90 ℃, is carried out heating and extracting 1-3 time, and each 0.5-2h obtains extracting solution;
(2) extracting solution is at pressure 98Kpa, and temperature 50-90 ℃ of following concentrating under reduced pressure is dispersed in liquid concentrator in the water of identical weight and processes suspension;
(3) use and the isopyknic chloroform extraction of suspension three times, combining extraction liquid, chloroform extraction liquid be at pressure 98Kpa, under the 30-50 ℃ of temperature behind the concentrating under reduced pressure chloroform extract;
(4) separating compound
The i chloroform extract gets silica gel mixed sample with equivalent, separates with silica gel column chromatography, uses sherwood oil successively: ETHYLE ACETATE 5: 1-2: the 1V/V gradient elution obtains cut 1, cut 2, cut 3, cut 4, cut 5; Cut 1 usefulness anti-phase C18 post separates, methyl alcohol: water 8: the 2V/V wash-out gets compound 3; Use methyl alcohol: water 7: the 3V/V wash-out gets compound 7; Cut 2 usefulness sephadex lh-20s separate, and get compound 1 with methanol-eluted fractions; Cut 3 usefulness anti-phase C18 posts separate, methyl alcohol: water 7: 3V/V gets compound 2; Cut 4 usefulness anti-phase C18 posts separate, and methyl alcohol: water 6: 4V/V gets compound 5; Cut 5 usefulness anti-phase C18 posts separate, methyl alcohol: water 6: 4-7: the 3V/V wash-out gets compound 8;
The ii chloroform extract is used the equivalent silica gel mixed sample, separates with silica gel column chromatography, uses sherwood oil successively: acetone 4: 1-2: the 1V/V gradient elution, obtain cut 1 and cut 2, and cut 1 obtains compound 6 with the sephadex lh-20 separation again; Cut 2 usefulness anti-phase C18 posts separate, and methyl alcohol: water 6: 4V/V gets compound 4.
11. like the application of each described isoflavonoid of claim 1-9 in preparation immunosuppressor or treatment immunological disease medicine.
12. like the application of each described isoflavonoid of claim 1-9 in preparation B cellular type chronic lymphocytic leukemia medicine.
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