Embodiment embodiment 1: the separation method of new diterpene-kind compound:
The raw materials used sponge of the present invention (Strongyphora strongylata) is picked up from table island, Japan west, and is frozen in-20 ° refrigerator.Take out sponge during application, at room temperature thaw, chopping, with 3 times of weight dehydrated alcohol extraction 3 times, united extraction liquid, drying under reduced pressure.Residue is scattered in isopyknic water, with isopyknic chloroform extraction 2 times, combined chloroform layer, vacuum decompression drying, chloroform extract.With carrying out chromatographic separation, the methanol-water gradient elution with the open post of anti-phase ODS filler.60% methanol-water wash-out part obtains compound 1,2 through reversed phase high efficiency liquid phase separation (65% methanol-water); 70% methanol-water wash-out part obtains compound 5 through reversed phase high efficiency liquid phase separation (73% methanol-water); The active stream part of 80% methanol-water wash-out part obtains compound 3,4,6 through reversed phase high efficiency liquid phase separation (78% methanol-water) again.
Compound 1 and 2: white powder, UV (MeOH) λ
MaxNm (ε): 203 (8450), 293 (3264); HRFABMS, m/z430.2706[M]
+(calcd for C
26H
38O
5, 430.2719);
13C and
1H NMR data see Table 2.
Compound 3 and 4: white powder, UV (MeOH) λ
MaxNm (ε): 204 (12882), 220 (s, 4097), 230 (s, 3074), 296 (2085); HRFABMS, m/z 412.2595[M]
+(calcd for C
26H
36O
4, 412.2614);
13C and
1H NMR data see Table 2.
Compound 5: white powder, UV (MeOH) λ
MaxNm (ε): 208 (10865), 220 (s, 6580), 228 (s, 5752), 250 (1592), 257 (1558), 263 (s, 1340), 296 (3396); HRFABMS, m/z 414.2774[M]
+(calcd for C
26H
38O
4, 414.2770);
13C and
1H NMR data sees Table 2.
Compound 6: white powder, white powder, UV (MeOH) λ
MaxNm (ε): 206 (9257), 220 (s, 4696), 229 (s, 4015), 295 (2089); HRFABMS, m/z 398.2792[M]
+(calcd for C
26H
38O
3, 398.2821);
13C and
1The HNMR data see Table 2.
Compound 7: white powder, UV (MeOH) λ
MaxNm (ε): 207 (21645), 229 (s, 5434), 257 (2492), 297 (1624); HRFABMS, m/z 427.2465[M+H]
+(calcd for C
26H
35O
5, 427.5609);
13C and
1H NMR data data see Table 2.
The 2 sea star egg parent cell cycles of embodiment are suppressed active testing method:
The sea star egg parent cell is the spontaneous early stage that is synchronized with initial meiosis before the ripening stage, i.e. the G2/M phase, in this stage, can observe complete nucleus.Ovum enters the M phase with maturation under the effect of hormone (1-methyladenines) but induce in maturation, and nucleus breaks and carries out reduction division.
It is littoral that female starfish (Asterina pectinifera) individuality picks up from Japan, gathers in annual 4-5 month and 8-10 month, cultures that (ASW, 10mM EPPS-NaOH (pH8.2) contains 0.46M NaCl, 10mM KCl, 35.9mMMgCl in artificial seawater
2, 1.75mM MgSO
4, and 9.18mM CaCl
2) in.Get one of female starfish Asterina pectinifera, cut off, take out red ovary, put into artificial seawater washing 2-3 time of calcium ions not, be suspended in the not calcareous artificial seawater of 30mL along each tentacle center.Ovary is shredded, remove floating follicular cell, three layers of filtered through gauze.Low-speed centrifugal is suspended in the precipitation that obtains in the 200ml artificial seawater then, and microscopic count transfers to 100/ml with cell count, promptly gets required starfish immature egg parent cell.
Laboratory sample is dissolved in DMSO, and concentration is 2mg/ml, as test liquid.The starfish immature egg parent cell of getting 1ml adds in 24 orifice plates, the test liquid 10ul that adds different concns simultaneously, cultivate after 10 minutes on the ice bath, add 1.25uM1-methyladenines 100ul, act on after 50 minutes, the form of observation of cell nuclear under inverted microscope, if compare with negative blank group, nucleus is kept perfectly and shows that promptly the sea star egg cell mature process is had the activity of inhibition.Starfish oocyte maturation inhibiting rate is calculated as follows.
Table 1: compound 1-7 is active to the sophisticated inhibition of sea star egg parent cell
Compound | Active (IC
50)
|
????1 ????2 ????3 ????4 ????5 ????6 ????7 | ????35.0μM ????35.0μM ????36.4μM ????36.4μM ????1.2μM ????35.2μM ????36.4μM |
Table 2: compound 1-7's
13C-NMR spectrum data (pyridine-d
5, 500MHz)
C#???????1??????????2???????????3??????????4???????????5???????????6???????????7
1????????40.4???????40.4????????40.5???????40.5????????35.6????????35.0????????39.9
2????????22.8???????22.9????????22.9???????22.7????????19.2????????19.0????????20.1
3????????35.3???????40.9????????35.1???????40.7????????36.4????????42.1????????40.3
4????????37.3???????37.1????????37.4???????37.1????????39.1????????33.2????????43.2
5????????51.1???????48.7????????51.5???????49.1????????57.3????????57.0????????49.7
6????????19.8???????20.0????????21.0???????20.4????????18.9????????18.4????????21.2
7????????40.1???????40.6????????38.9???????39.3????????42.9????????41.9????????31.9
8????????39.4???????39.7????????36.4???????36.6????????37.5????????37.5????????43.5
9????????56.9???????57.5????????56.9???????57.6????????62.4????????62.1????????46.3
10???????37.8???????37.6????????37.9???????37.7????????42.4????????42.8????????36.7
11???????17.6???????17.6????????19.0???????18.0????????22.1????????22.5????????19.1
12???????44.7???????44.7????????42.1???????42.1????????43.0????????43.1????????39.9
13???????73.8???????73.8????????76.3???????76.3????????76.7????????76.7????????74.0
14???????63.0???????63.0????????53.0???????53.0????????53.4????????53.4????????99.6
15???????27.6???????27.6????????22.9???????22.9????????23.1????????23.0????????31.9
16???????131.9??????131.9???????123.5??????123.5???????123.5???????123.5???????130.1
17???????149.9??????149.9???????146.8??????146.8???????146.9???????146.8???????154.0
18???????117.7??????117.7???????118.0??????118.0???????118.0???????118.0???????108.7
19???????114.6??????114.6???????115.3??????115.3???????115.3???????115.3???????114.4
20???????151.3??????151.3???????152.2??????152.2???????152.0???????152.0???????152.6
21???????119.4??????119.4???????116.9??????116.9???????116.9???????116.8???????112.4
22???????24.7???????24.7????????21.0???????21.0????????20.5????????20.5????????24.9
23???????16.4???????16.5????????15.7???????15.7????????15.5????????15.6????????17.5
24???????67.9???????62.0????????67.9???????62.0????????62.2????????61.5????????73.8
25???????24.3???????24.1????????24.0???????24.2????????28.6????????34.2????????23.5
26???????98.9???????99.1????????98.9???????99.1????????64.9????????22.0????????176.1
Table 3: compound 1-6,8
1H-NMR spectrum data (pyridine-d
5, 500MHz)
C#????1?????????????2???????????????3??????????????4???????????????5???????????????????????6??????????????????????7
1.00,m???????1.00,m?????????1.00,m????????1.00,m?????????0.77,m?????????????????0.67,m????????????????0.86,m
1
2.17,m???????2.17,m?????????2.17,m????????2.17,m?????????2.51,m?????????????????2.53,m????????????????1.95,m
1.55,m???????1.56,m?????????1.56,m????????1.56,m?????????1.45,m?????????????????1.43,m????????????????1.15,m
2
2.84,m???????2.50,m?????????2.83,m????????2.56,m?????????1.88,m?????????????????1.74,m????????????????1.50,m
1.22,m???????1.34,m?????????1.20,m????????1.34,m?????????1.09,m?????????????????1.16,m????????????????1.30,m
3
2.50,m???????1.56,m?????????2.52,m????????1.56,m?????????2.14,m?????????????????1.37,m????????????????1.74,m
5?????0.88,m???????0.80,m?????????1.12,m????????1.04,m?????????1.13,m?????????????????0.90,m????????????????1.15,m
1.25,m???????1.50,m?????????1.06,m????????1.72,m????????????????????????????????????????????????????????1.15,m
6?????????????????????????????????????????????????????????????????1.73,2H,m?????????????1.44,2H,m
1.60,m???????2.50,m?????????1.65,m????????2.60,m????????????????????????????????????????????????????????1.50,m
0.88,m???????0.88,m?????????0.88,m????????0.88,m?????????0.95,m?????????????????1.00,m????????????????1.25,m
7
2.06,m???????2.06,m?????????1.65,m????????1.65,m?????????1.76,m?????????????????1.72,m????????????????1.58,m
9?????0.85,m???????0.85,m?????????0.94,m????????0.94,m?????????1.10,m?????????????????1.03,d(11.0)b?????????2.05,d(12.5)
1.28,m???????1.28,m?????????1.12,m????????1.55,m?????????2.03,m?????????????????2.05,m????????????????1.17,m
11
1.55,m???????1.55,m?????????1.74,m????????1.72,m?????????2.16,m?????????????????2.20,m????????????????1.70,m
1.75,m???????1.75,m?????????1.74,m????????1.74,m?????????1.76,m?????????????????1.74,m????????????????1.93,m
12
2.03,m???????2.03,m?????????2.08,m????????2.08,m?????????2.14,m?????????????????2.12,m????????????????2.22,m
14????1.95,m???????1.95,m?????????1.65,m????????1.65,m?????????1.74,m?????????????????1.72,m
2.65,m???????2.65,m??????????????????????????????????????????????????????????????????????????????????????3.02,d(15.0)
15
2.60,2H,m????2.60,2H,m?????2.66,2H,d,(9.0)??????2.68,2H,m
3.24,m???????3.24,m???????????????????????????????????????????????????????????????????????????????????????3.51,d(15.0)
18????7.18,m???????7.18,m?????????6.98,m????????6.98,m?????????6.99,d(9.0)????????????6.99,d(9.0)???????????6.80,d(9.0)
19????7.10,m???????7.10,m?????????7.04,m????????7.04,m?????????7.04,dd(3.0,9.0)??????7.04,dd(3.0,9.0)?????6.94,d(9.0)
21????7.34,m???????7.34,m?????????7.05,m????????7.05,m?????????7.09,d(3.0)????????????7.09,d(3.0)???????????7.12,br.s
22????1.36,3H,s???1.36,3H,s?????1.17,3H,s????1.16,3H,s?????1.25,3H,s?????????????1.25,3H,s????????????1.38,3H,s
23????0.87,3H,s???0.96,3H,s?????0.75,3H,s????0.86,3H,s?????1.15,3H,s?????????????1.17,3H,s????????????0.96,3H,s
3.65,d(12.5)?3.40,d(12.5)???3.67,d(11.0)??3.42,d(11.0)???4.09,dd(5.5.12.0)??????4.03,dd(5.5,12.0)????4.01,d(12.5)
24
4.17,d(12.5)?4.57,d(12.5)???4.20,d(11.0)??4.61,d(11.0)???4.23,dd(5.5,12.0)?????4.20,dd?(5.5,12.0)???4.76,d?(12.5)
25????1.01,3H,s???1.08,3H,s?????1.03,3H,s????1.11,3H,s?????1.25,3H,s?????????????0.87,3H,s????????????1.18,3H,s
3.75,d(10.0)
26????5.18,br.s????5.06,d(3.3)????5.20,br.s?????5.08,d(3.3)????????????????????????????0.80,3H,s
3.99,d(10.0)
OH????8.02,br.s????7.56,d(3.3)????8.10,br.s?????7.65,d(3.3)