The following examples will describe the present invention in detail.
Embodiment 1: the extraction of stilbene analog derivative and structure are identified:
Get the rheum rhabarbarum decoction pieces, add 8 times of amount 95% alcohol reflux three times, each 2 hours.Merge extractive liquid,, concentrating under reduced pressure.Ethanol extract disperses with kieselguhr, drying.With chloroform flush away liposoluble constituent, continue with eluent ethyl acetate, obtain the ethyl acetate soluble fraction.This part row silica gel column chromatography (60-100 order) with eluent ethyl acetate, continues with ethyl acetate/methanol (4: 1~2) eluting, promptly gets crude product, and crude product through water/acetone recrystallization promptly.
Identify:
Compd E is white amorphous powder (rare acetone), mp.138-140 ℃.Be bluish violet fluorescence under the ultraviolet light, the Molisch reacting positive.
1HNMR (the δ ppm:7.14 (1H, d, J=8.5Hz, H-5 ') of acetone-d6), 7.06 (1H, d, J=2.1Hz, H-2 '), 6.97 (1H, d, J=16.3Hz, H-β), (6.94 1H, dd, J=2.1/8.5Hz, H-6 '), 6.89 (1H, d, H=16.3Hz, H-α), 6.52 (2H, d, J=2.1Hz, H-2,6), 6.24 (1H, t, J=2.1Hz, H-4), 4.79 (1H, d, J=7.5Hz, the end group isomery-H), 3.9-3.3 (sugar-H);
13CNMR (the δ ppm of acetone-d6): aglucon 159.5 (C-3,5), 148.5 (C-4 '), 146.0 (C-3 '), 140.3 (C-1), 134.2 (C-1 '), 128.6 (C-α, β), (119.3 C-5 '), 118.9 (C-6 '), 114.2 (C-2 '), (105.6 C-2,6), 104.0 (C-4), glucosyl group 102.9 (C-1 "), 77.8 (C-3 "), 77.1 (C-5 "); 74.4 (C-2 "), 70.9 (C-4 "), 62.2 (C-6 ").
1HNMR and
13(2 Yoshiki Kashiwada et al.Chem.Pharm.Bull.988 such as CNMR data and Yoshiki Kashiwada, 36 (4): 1545) Bao Dao data consistent, compd E be accredited as 3,3 ' 4 ' 5-tetrahydroxystilbene-4 '-the O-glucoside (Piceatannol-4 '-O-β-D-glucopyranoside).
Compd B
50Be white, needle-shaped crystals (rare acetone), mp.222-224 ℃.Be bluish violet fluorescence Molisch reacting positive under the ultraviolet light.
1HNMR (the δ ppm:7.46 (1H, d, J=2Hz, H-2 ') of acetone-d6), 7.10 (1H, dd, J=2/8.3Hz, H-6 '), 6.96 (1H, d, J=16.3Hz, H-β), (6.87 1H, d, H=16.3Hz, H-α), 6.82 (1H, d, J=8.2Hz, H-5 '), 6.50 (2H, d, J=2.1Hz, H-2,6), 6.22 (1H, t, J=2.1Hz, H-4), 4.82 (1H, d, J=7.5Hz, the end group isomery-H), 4.0-3.3 (sugar-H);
13CNMR (the δ ppm of acetone-d6): aglucon 159.5 (C-3,5), 148.1 (C-3 '), (146.5 C-4 '), 140.5 (C-1), 130.5 (C-1 '), (128.7 C-β), (127.5 C-α), 123.3 (C-6 '), 116.8 (C2 '), (116.5 C-5 '), (105.4 C-2,6), 104.1 (C-4); Glucosyl group 102.6 (C-1 "), 78.0 (C-3 "), 77.2 (C-"), 74.4 (C-2 "), 71.1 (C-4 "), 62.3 (C-6 ").
1HNMR and
13(2 Yoshiki Kashiwada etal.Chem.Pharm.Bull.1984,32 (9): 3501) Bao Dao data consistent, compd B such as CNMR data and Yoshiki Kashiwada
50Be accredited as 3,3 ' 4 ' 5-tetrahydroxystilbenes-3 '-O-glucoside (Piceatannol-3 '-O-β-D-glucopyranoside).
Compound F 17-hydroxy-corticosterone is white, needle-shaped crystals (dilute methanol), mp.228-230 ℃.Be bluish violet fluorescence under the ultraviolet light, the Molisch reacting positive.
1HNMR (acetone-d6) δ H-β), 6.96 (1H, dd, J=2/8.3Hz, H-6 '), (6.90 1H, d, J=7.9Hz, H-5 '), 6.89 (1H, d, H=16.5Hz, H α), 6.77 (1H, br.s, H-2), 6.66 (1H, br.s, H-2), 6.48 (1H, t, J=1.8Hz, H-4), 4.90 (1H, d, J=7.7Hz, the end group isomery-H), 3.82 (3H, s ,-OCH
3), 4.0-3.3 (sugar-H);
13CNMR (the δ ppm of acetone-d6): aglucon 160.1 (C-5), 159.5 (C-3), 148.4 (C-4 '), (147.5 C-3 '), 140.5 (C-1), 131.5 (C-1 '), (129.5 C-β), 127.2 (C-α), 119.7 (C-6 '), (113.3 C-2 '), (112.5 C-5 '), 108.0 (C-2), 106.5 (C-6), 103.8 (C-4), 56.2 (OCH3); Glucosyl group 1101.9 (C-1 "), 77.7 (C-3 ", 5 "), 74.4 (C-2 "), 71.1 (C-4 "), 62.5 (C-6 ").
1HNMR and
13(2Yoshiki Kashiwada et al.Chem.Pharm.Bull.1984,32 (9): 3501) Bao Dao data consistent, compound F 17-hydroxy-corticosterone are accredited as ponticin (rhaponticin) for CNMR data and Yoshiki Kashiwada etc.
Embodiment 2 biological assessments
One, mainly observes the therapeutical effect of above-claimed cpd in the biotic experiment below, make positive control drug, its drug effect is estimated with the two croaks (to call metformin in the following text) of dimethyl to the hyperglycemia mice of alloxan modeling.
1 materials and methods
1.1 material
1.1.1 medicine and animal
Metformin, lot number 96064, Chinese-foreign joint Zhengan County pharmacy (Tianjin) company limited production.
Kunming mouse, body weight 22-24g, male, animal housing of Beijing Drug Control Inst. provides, the quality certification number: No. the 037th, the accurate word (1994) of the moving pipe in capital
1.1.2 instrument and reagent
The UV-265 spectrophotometer, day island proper Tianjin company produces; Alloxan, lot number 42011-3E, Hong Kong produces; Glucoseoxidase (GOD), the packing of Beijing hundred safe Biochem Technology, INC.; Peroxidase (POD), lot number 9712193, the beautiful pearl east wind in Shanghai Bioisystech Co., Ltd produces.
1.2 method
Above-mentioned healthy mice behind the fasting 20hr., with alloxan normal saline solution tail vein injection 80mg/Kg body weight, makes into hyperglycemia model.
With determination of glucose oxidase mice fasting blood sugar, select blood glucose value the mice of 7-25mmol/L be divided at random six groups (between group mean blood glucose differ<0.6mmol/L), each is organized dosage and sees Table.Every day, gastric infusion continuous 12 days, was surveyed the fasting blood sugar of respectively organizing mice in 30 minutes, with comparison between the blood glucose difference is organized before and after each group mouse blood sugar mean and the administration after the last administration.
2. result
Blood glucose value difference value after the blood glucose value administration before the group animals administer dosed administration
Number mg/kg mmol/L mmol/L mmol/L
Blank 11 ordinary water 20ml/Kg 7.08 ± 1.17 7.02 ± 1.31 0.22 ± 1.38
Model contrasts 12 ordinary water 20ml/Kg 22.70 ± 3.91 22.77 ± 2.92
Δ Δ Δ-0.76 ± 3.07
Metformin group 13 500 22.63 ± 4.85 17.35 ± 5.29
※5.85 ± 6.46
※
E is big by 13 1,000 22.47 ± and 3.52 16.41 ± 5.44
※6.41 ± 7.54
※
Among the E 13 500 22.58 ± 3.15 16.26 ± 5.27
※ ※6.93 ± 6.27
E is little by 13 250 22.51 ± and 4.60 20.93 ± 5.68 1.58 ± 6.73
Glyburide 13 6.6 22.25 ± 2.46 21.06.0 ± 4.29 0.06 ± 7.03
Annotate: compare Δ Δ Δ p<0.01 with the blank group; Compare ※ p<0.05, ※ ※ p<0.01 with model group;
Negative value is represented blood sugar increasing.
As seen from the above table, the positive control drug metformin demonstrates good blood sugar reducing function, and the illustrative experiment method is reliable.The big or middle dosage of compd E all can obviously reduce the blood glucose value of high sugared model mice, and (500mg/Kg) its effect is similar or slightly be better than the metformin group when equal dosage, illustrates that this medicine blood sugar reducing function is obvious.
Two, above-claimed cpd is to the influence of normal mouse carbohydrate tolerance
2. materials and methods
2.1 material
2.1.1 medicine and animal
Metformin, lot number 96064.Sino-foreign joint venture Zhengan County's pharmacy (Tianjin) company limited production.
Animal: Kunming mouse, body weight 20-23g, male and female are usefulness all, and the quality certification numbers 921103 is provided by Military Medical Science Institute's Experimental Animal Center.
2.1.2 instrument and reagent
Semi-automatic production analyser, IMPACT 400 types, U.S. GILFORD company produces.
Adopt the determination of glucose oxidase blood glucose value, provide the blood glucose test kit by Beijing Zhongsheng Biological Engineering High Technology Company.
2.2 method
Above-mentioned healthy mice is according to body weight size random packet.Each is organized dosage and sees the following form.Mice gastric infusion every day was taken 12 days continuously.Before the carbohydrate tolerance test, each treated animal fasting blood sugar is measured in animal fasting 12 hours, administration then, 1 hour pneumoretroperitoneum injectable dextrose monohydrate 2g/kg.Measure after the administration 0,0.5,1.0 and 2.0 hours blood glucose value at once, and calculate area under the blood glucose curve, between organizing relatively.
2. result
E is to the influence of normal mouse carbohydrate tolerance
Group | Number of animals | Dosage (mg/kg) | Blood glucose value (mmol/L) | Area AUC under the blood glucose curve |
Before the administration | After the administration 0 ' | 30’ | 60’ | 120’ |
Among the blank metformin E | 7 7 7 | Water 500 500 | 2.7±1.0 2.4±0.9 1.8±0.8 | 5.2±1.1 4.0±0.9 4.0±0.6
* | 13.5±3.0 9.6±2.0
* 6.4±1.0
# | 7.2±1.1 4.1±2.0
# 3.7±0.6
# | 5.0±0.7 0.6±0.6
# 0.8±1.1
# | 953.2±135.4 552.6±100.3
#40.6±69.6
#@ |
E is little for the blank metformin | 5 5 5 | Water 250 250 | 4.3±1.1 4.1±0.6 4.9±0.5 | 6.7±1.5 4.5±1.0 5.2±0.9 | 17.4±2.2 13.2±2.6 14.6±2.5 | 11.1±3.3 7.2±1.6 9.4±1.8 | 6.8±1.0 4.2±1.1 5.5±0.8 | 1327.6±227.6 917.0±143.5 1076.4±135.7 |
Annotate: compare * P<0.01 #P<0.001 with the blank group, compare @P<0.05 with metformin
As seen from the above table, normal mouse gavages E 500mg/kg or metformin 500mg/kg, continuous 12 days.Behind glucose load in 30-120 minute, blood glucose value is starkly lower than the blank group, and (P<0.005-0.001): area is also significantly less than blank group (P<0.001) under the blood glucose curve.Illustrate that metformin and E all have good blood sugar reducing function.With equal drug dose (500mg/kg), area is than metformin smaller (P<0.05) under the blood glucose curve of E.The hypoglycemic effect that E is described slightly is better than metformin.When dosage is reduced to 250mg/kg, also show certain blood sugar reducing function, but its effect weakens.
3 toxicity relatively
Drug toxicity
Mice, the Kunming kind, body weight 19-21g Military Medical Science Institute Animal Experimental Study central supply,
The quality certification numbers 921103
1. acute toxicity test
Lumbar injection
LD
50♀ 1242.5mg/kg MTD 641.3mg/kg
LD
50♂ 1139.2mg/kg MTD 641.3mg/kg
Oral
LD
50♀ 9007.5mg/kg MTD 5625mg/kg
LD
50♂ 8578.9mg/kg MTD 4219mg/kg
2. cumulative toxicity test
Mouse peritoneal is injected 1/10 LD
50Dosage (123.5mg/kg) once a day, totally 30 days
Mouse peritoneal is injected 1/2 LD
10Dosage (336.5mg/kg) once a day, totally 30 days
The above-mentioned two groups of mices of result (10 every group) all do not have dead, show no obvious depot action.But its influence for mice body weight and some index is as follows:
E is to the influence of body weight
Before the medicine behind (g) medicine 30 days (g)
Normal control 20.0 ± 0.4 32.0 ± 3.8
1/2 LD
10Dosage 20.0 ± 0.4 30.4 ± 1.6
1/10 LD
50Dosage 20.2 ± 0.6 29.6 ± 1.3
E is to the influence of some serum biochemistry index
GOT GPT Glu
Normal control 3004.8 ± 884.7 679.3 ± 114.2 6.96 ± 0.80
1/2 LD
10Dosage 3397.3 ± 690.8 994.5 ± 324.5 7.48 ± 0.48
1/10 LD
50Dosage 3097.3 ± 545.8 735.5 ± 23.6 6.86 ± 0.67
For some index such as body weight, GOT, GPT.1/2 LD
10Dosage (336.5mg/kg) connects was relatively had the trend that increases with the normal control group after 30 days, but did not have the statistics difference.1/10LD
50Dosage connects gives 30 days, and only body weight is low slightly like comparative control person, does not have the statistics difference.
It seems that by above-mentioned mouse test this toxicity of compound is quite low, in general the oral LD of mice
50Dosage should belong to low toxicity or non-toxic type material, and this chemical compound does not also have accumulative effect greater than 5g/kg person.
Metformin LD
50Be 247mg/Kg (ip)
Compd E LD
50About 1100mg/Kg (ip)
Therefore it seems that compd E is low to the toxicity of mice than metformin.