CN1144591C - Application of Sichuan champion bauhinia saponin in preparing anti-tumor medicine - Google Patents

Abstract

The present invention relates to an application of a Sichuan champion bauhinia saponin in preparing anti-tumor medicine, and a compound extracted from plants. Sichuan champion bauhinia is used as a raw material to which a series of extraction methods of alcohol extraction, pressure reducing concentration, extraction, low pressure chromatography, elution, etc. are carried out so that a crystal compound, namely the Sichuan champion bauhinia saponin is obtained. Experiments prove that the Sichuan champion bauhinia saponin has inhibiting effect on the gene replication of tumor cells, and the dose of oral administration LD50 is about 12 times of the least effective dose, which indicate that the saponin has high safety and is worth further developing.

Description

The application of ardipusilloside as the preparation antitumor drug produced in the river

Technical field

The chemical compound that the present invention relates to extract from plant is used to prepare the application of antitumor drug.

Background technology

It is the herb of Myrsinacea Ardisa plant that A.pusilla A.DC. (Ardisea pusilla A.DC) is produced in the river, and the effect of heat-clearing and toxic substances removing, reducing swelling and alleviating pain is arranged, the traumatic injury that is used among the people, and promoting blood circulation to remove obstruction in the collateral also can be used for venom.Producing the chemical compound-river product ardipusilloside that extracts the A.pusilla A.DC. from the river is used to prepare antitumor drug, does not appear in the newspapers as yet both at home and abroad.

Summary of the invention

The objective of the invention is to produce the chemical compound-river that extracts the A.pusilla A.DC. and produce ardipusilloside, be used to prepare anti-tumor drug from the river.

Technology implementation scheme of the present invention: with 95% ethanol extraction A.pusilla A.DC. herb coarse powder, concentrating under reduced pressure gets extractum, after adding water and stirring, leave standstill filtration, filtrate extracts in turn with petroleum ether, ether, ethyl acetate and n-butyl alcohol, with the extract acetone precipitation, through the silica gel low pressure column chromatography, solubilizer CHCl ₃-MeOH-H ₂O (65: 35: 10, lower floor) eluting gets crystalline compounds, and its molecular formula is:

3-O-[α-L-pyrans rhamnose (1 → 2)-β-D-Glucopyranose. (1 → 3)] [β-D-Glucopyranose. (1 → 2)] α-L-arabinose-quick A of Xi carat;

Its structural formula is:

The present invention can be used for preparing the medicine that suppresses hepatocarcinoma, pulmonary carcinoma, gastric cancer, ovarian cancer, cervical cancer, the esophageal carcinoma and renal carcinoma.

In detail, the present invention is the ethanol extraction A.pusilla A.DC. herb coarse powder with 95%, and concentrating under reduced pressure gets extractum, after adding the water stirring, leave standstill filtration, filtrate extracts in turn with petroleum ether, ether, ethyl acetate and n-butyl alcohol, with the extract acetone precipitation, through the silica gel low pressure column chromatography, solubilizer CHCL ₃-MeOH-H ₂O (65: 35: 10, lower floor) eluting gets crystalline compounds.Chemical compound is white needle, and mp239-241 ℃, infrared spectrum shows hydroxyl (3423cm ^-1), aldehyde radical (2700 and 1716cm ^-1) and cyclic ethers (1075 and 1044cm ^-1).Mass spectrum m/z:1097 (M+Na) ⁺, molecular weight 1074 is C in conjunction with elemental analysis molecular formula ₅₃H ₈₆O ₂₂.2H ₂O.Chemical compound ¹³C nuclear magnetic resoance spectrum δ: 101.7,104.4,105.3 and the 103.2ppm signal, its acetylate ¹The H nuclear magnetic resoance spectrum has 11 acetyl group signals; all show to have four glycosyls in the chemical compound, chemical compound is used the acid vapour hydrolysis on the high-efficient silica gel lamellae, detect D-glucose, L-arabinose and L-rhamnose; scan to such an extent that each sugared ratio is 2: 1: 1 through thin, chemical compound ¹³C nuclear magnetic resoance spectrum δ _C-389.1ppm move 11.0ppm, δ to low field than glycoside unit _C-226.6ppm move 1.7ppm (table 1) than glycoside unit to High-Field, the C of sugar chain and glycoside unit be described ₃-OH is connected.The important information m/:1097 (M+Na) that mass spectrum has pointed out sugar chain to connect ⁺, 951 (M+Na-146) ⁺, 935 (M+Na-162) ⁺, 626 (glycoside unit+Na+132) ⁺, show that sugar chain is branched.The mass spectrum m/z:273 of the acetylate of chemical compound (acetyl rhamnose), 331 (acetyl glucosamines), 561 (acetyl rhamnose-acetyl glucosamine).The fractional hydrolysis of chemical compound test detects each sucrose solution and separates order and be rhamnose, glucose and arabinose, more than show that all the sugared order of connection is that a glucose links to each other with arabinose with a rhamnose Fructus Vitis viniferae disaccharidase simultaneously.By chemical compound ¹³C- ¹Anomeric proton (1-H) signal of four glycosyls of the relevant spectrum of H appears at respectively: δ: 6.39,5.37,5.21 and 4.94ppm, the proton signal of each glycosyl overlaps each other in δ: 3.8-4.7 spectrum district, be difficult to determine its ownership with conventional method, then adopted the relevant spectrum of one-dimensional multi relay new method to do research, whole proton signals of each glycosyl can be separated and identify to this method from the spectrum peak that highly overlaps, each glycosyl proton chemical shifts and coupling constant are summarized in table 2.By each sugared end group carbon ¹³C nuclear magnetic resonance, NMR spectrum signal has been determined the configuration that each is sugared, and glucose is β-D-type, and rhamnose and arabinose are α-L-type, again in conjunction with the (irradiation of NOE difference spectrum ¹H nuclear makes with its coupling ¹³It is NOE effect or NOE difference spectrum that the effect that the signal intensity of C nuclear increases is called this effect of nuclear furan person of outstanding talent difficult to understand) 1-H of each glycosyl of selective exitation, observe the NOE gain signal, contrast with one-dimensional multi relay spectrum again, these MOE gain signals are belonged to (seeing Table 3).According to MOE effect and nuclear separation relation, thereby the structure that determines sugar chain is:

Table 1 glycoside unit molecule in deuterated pyridine solution ¹³The C chemical shift

C ardipusilloside cycalamin A C ardipusilloside cycalamin A

1 39.6 39.5 16 76.9 76.8

2 26.6 28.3 17 48.3 48.2

3 89.1 78.1 18 50.4 50.6

4 39.2 39.5 19 24.2 24.1

5 55.6 55.7 20 36.9 37.3

6 18.0 18.2 21 36.8 36.8

7 32.7 32.7 22 32.4 32.3

8 44.1 44.0 23 28.1 28.6

9 53.4 53.3 24 16.4 16.3

10 42.6 42.6 25 16.5 16.5

11 19.2 19.2 26 19.8 19.8

12 30.5 30.7 27 18.6 18.6

13 86.5 86.4 28 77.7 77.7

14 44.6 44.6 29 24.4 4.1

15 33.4 33.4 30 207.9 207.3

Table 2 is glycan molecule proton chemical shifts value and coupling constant in deuterated pyridine solution

Proton	Chemical displacement value δ (ppm)	Coupling constant J (Hz)	Proton	Chemical displacement value δ (ppm)	Coupling constant J (Hz)
						Rham rhamnose 123456 (CH 3) Glu-2 glucose-2 12345 6a 6b	6.39bs 4.75bs 4.71dd 4.32t 5.04m 1.79d 5.21d 4.27t 4.21t 4.12t 3.34m 4.50d 4.31dd	3.7，9.4 9.4，9.4 9.4，6.1 6.1 8.4 8.4，8.4 8.4，8.5 8.5，8.4 8.4，5.3 9.8 9.8，5.3	Glu-2 glucose-1 12345 6a 6b Ara arabinose 1234 5a 5b	5.37d 4.09t 4.36t 4.28t 4.05m 4.51d 4.40dd 4.94bd 4.59m 4.51m 4.40m 4.40m 3.82d	8.7 8.7，8.7 8.7，8.6 8.6，8.7 8.7，4.4 9.4 9.4，4.5

The NOE value of table 3 glycan molecule in deuterated pyridine solution

According to the time NOE value added
				Proton	Chemical displacement value	Belong to	NOE(％)
Arabinose rhamnose glucose 2 glucoses 1	3.12(dd) 4.27(t) 4.51(m) 4.59(m)	Glycoside unit-H 3Glucose 2-H 2Arabinose-H 3Arabinose-H 2	18.9 11.1 8.1 10.2

Cancerous cell differentiation-inducing action-the mtt assay of ardipusilloside to In vitro culture produced in the river: its principle is that tetrazolium blue chemical compound MTT (Methylthiazolyltertrazolium) cleavable under the effect of living cells line liquid enzymes is the insoluble first chemical compound of purple, measure its optical density variation with microplate reader after adding dimethyl sulfoxide, can obtain the oncocyte number change of cancer therapy drug effect, obtain the survival rate of cell with this, according to the median lethal dose(LD 50) (ID of tumor cell ₅₀) judge the sensitivity of tumor cell to anticarcinogen.Hepatocarcinoma (SMMC-7721), hepatocarcinoma (HCC-9204), pulmonary carcinoma (A549), gastric cancer (SGC-7901), ovarian cancer (HO-8910), cervical cancer (Hela), the esophageal carcinoma (Eca-109), 8 kinds of human tumor cells of renal carcinoma (GRC-1) are made single cell suspension, in 96 empty plane culture plates, cell (5 * 10 ⁴/ ml) and after the river produces the ardipusilloside aqueous solution and cultivate 72h together, add MTT20ul, continue to cultivate 4h, centrifugal, abandon supernatant, add 150 μ l dimethyl sulphoxide solutions precipitation, measure OD490.Calculate the formula of cell survival rate:

The result: mtt assay is measured and is shown that river product ardipusilloside all is dosage-effect relation to the inhibitory action of 8 kinds of human tumor cells, increases with drug concentrations, and cell survival rate descends gradually.(seeing Table 4).

Table 4 mtt assay is measured 8 kinds of tumor cell survival rates (%)

Tumor cell	Ardipusilloside (μ g/ml)						ID 50
								1.5625	3.125	6.25	12.5	25	50	(μg/ml)
	A549 SGC-7901 Hela GRC-1 SMMC-7721 HCC-9204 HO-8910 Eca-109	84.66 100.8 91.32 94.25 91.91 111.4 85.83 98.26	77.53 90.54 91.32 91.88 79.97 93.63 60.39 92.07	37.36 49.75 15.11 58.26 25.61 33.19 47.19 60.59	34.34 30.81 11.9 43.51 3.98 4.47 10.68 25.52	155.33 28.58 3.22 36.88 2.29 2.13 4.67 16.18	9.25 25.64 3.86 24.34 0.82 1.68 2.9 14.66								4.94 5.80 3.95 8.89 4.12 4.94 4.94 7.57

Advantage of the present invention:

Produce the chemical compound-river of extracting the A.pusilla A.DC. from the river and produce ardipusilloside, it is newfound chemical compound with antitumor drug, experiment shows that this chemical compound all is dose-effect relationship to the inhibitory action of 8 kinds of human tumor cells, increases with drug concentrations, and cell survival rate descends gradually.

Experiment also shows river product ardipusilloside aqueous solution mice LD ₅₀Be respectively iV:10.29 ± 0.84mg/kg, the 95% credible 9.50～11.17mg/kg that is limited to; Ig:666.70 ± 55.82mg/kg, the 95% credible 613.30～724.82mg/kg that is limited to.Compound i v administration LD is described ₅₀Very little, should not be recommended as the clinical administration mode, and oral administration LD ₅₀Be about 12 times of least effective dose (LED), illustrate that the safety of this approach is bigger, be worth further exploitation.

The specific embodiment

A.pusilla A.DC. herb coarse powder 20kg uses 95% ethanol extraction, and concentrating under reduced pressure gets extractum 1785g, adds H ₂O leaves standstill filtration after stirring, and filtrate extracts in turn with petroleum ether, ether, ethyl acetate and n-butyl alcohol, and n-butyl alcohol extract gets total Saponin 61g with acetone precipitation, through silica gel low pressure column chromatography, solvent C HCL ₃-MeOH-H ₂O (65: 35: 10, lower floor) eluting gets crystalline compounds 20g.

Claims (1)

1, the application of ardipusilloside as the preparation antitumor drug produced in the river, it is characterized in that, ethanol extraction A.pusilla A.DC. herb coarse powder with 95%, concentrating under reduced pressure gets extractum, after adding water and stirring, leaves standstill filtration, filtrate extracts in turn with petroleum ether, ether, ethyl acetate and n-butyl alcohol, with the extract acetone precipitation, through the silica gel low pressure column chromatography, solubilizer CHCl ₃-MeOH-H ₂O, 65: 35: 10, lower floor, eluting gets crystalline compounds, and its molecular formula is:

3-O-[α-L-pyrans rhamnose (1 → 2)-β-D-Glucopyranose. (1 → 3)] [β-D-Glucopyranose. (1 → 2)] α-L-arabinose-quick A of Xi carat;

Its structural formula is:

Be used to prepare the medicine that suppresses hepatocarcinoma, pulmonary carcinoma, gastric cancer, ovarian cancer, cervical cancer, the esophageal carcinoma or renal carcinoma.