CN1150204C - Cyclobornane triterpenoid saponin compound and its application in immunosuppresion and treating tumor - Google Patents

Abstract

The present invention relates to a new compound and a derivative thereof extracted and separated from whole herbs or roots of Marshmarigold-leaved beesia, which has a chemical structural formula in the formula (1) and has immunosuppressive activity, angiogenesis inhibiting activity and osteoplast proliferation inhibiting activity. The present invention also relates to a usage for preparing medicine from the compound and the derivative thereof, particularly to the fields of preparation of immunosuppressants of organ transplantation, angiogenesis inhibiting medicine, antitumor medicine, etc.

Description

A kind of cycloartane triterpenoid saponin compound and the application in immunosuppression and oncotherapy thereof

Technical field

The present invention relates to isolating a kind of compound from Beesia calthifolia herb or rhizome, its skeleton belongs to the cycloartane type triterpenoid saponin.The invention still further relates to the purposes of this compound aspect the preparation medicine, the particularly fields such as immunosuppressor, angiogenesis-inhibiting medicine and antitumor drug aspect the preparation organ transplantation.

Background technology

Beesia calthifolia [Beesia calthaefolia (Maxim.) Ulbr.] is that the Ranunculaceae Beesia calthifolia belongs to (Beesia) plant; Being a kind of herbal medicine commonly used among the people, also is the distinctive medicinal plant of China.Its rhizome or herb are medicinal, have clearing heat and detoxicatingly, and cool blood is invigorated blood circulation, detumescence, analgesia, the effect of dispersing wind and cold; Among the peoplely be used for treating common cold caused by wind-cold, rheumatic arthritis, red-white dysentery, swelling and pain in the throat, headache, diseases such as toothache, external application for curing sore furuncle and venomous snake bite.

Summary of the invention

This laboratory utilizes modern separation technology and structure determination means that Beesia calthifolia is carried out in the chemical constitution study process, therefrom separates the monomeric compound that obtains a series of novel structures, by screening active ingredients, finds that these monomeric compounds have very strong pharmacologically active.One of purpose of the present invention provides the compound of a kind of structural formula (1).Many physicians think, the success of organ transplantation should be given the credit to a kind of natural product one S-Neoral to a great extent clinically, surplus S-Neoral and other 10 the non-peptide medicament of kind entered effectively clinical or clinical before the development phase, therefore natural product becomes the important source of organ transplantation aspect immunosuppressor.Wherein compound 1 is tested in the mouse body and can obviously be suppressed the cell proliferation by ConA inductive T, has immunosuppressive action, is a kind of immunosuppressor.Find that by deep pharmacologically active screening this compound 1 also has activity (20 μ g/ml) that suppresses capillary blood vessel generation aspect and the activity (IC that suppresses osteoblastic proliferation in addition ₅₀=32.78 μ g/ml).

Formula 1 compound involved in the present invention up to now, still finds no relevant patent or bibliographical information.Two of purpose of the present invention provides the purposes of compound aspect the preparation medicine, the particularly fields such as immunosuppressor, angiogenesis-inhibiting medicine and antitumor drug aspect the preparation organ transplantation of a kind of structural formula (1).

Technical scheme of the present invention comprises following steps successively:

With Beesia calthifolia [Beesia calthaefolia (Maxim.) Ulbr.] rhizome (3.5Kg) is raw material, through with 95% alcohol reflux twice, each two hours, the dregs of a decoction are used 50% alcohol reflux twice again, and each two hours, behind the merging extracted twice liquid, reclaim solvent, get medicinal extract.With the medicinal extract water dissolution, use chloroform, n-butanol extraction successively, get chloroform extract 292g.The chloroform extract capable low pressure column chromatography of silica gel H post, with petroleum ether-ethyl acetate-methyl alcohol system gradient elution (9: 1: 0-8: 2: 0-7: 2.5: 0.5-6: 3: 1), every part of 2000ml, collect 16 parts altogether, 8-11 part (70g) wherein, use the capable low pressure column chromatography of silica gel H post once more, (10: 0-8: 2), every part of 2000ml collects 7 parts altogether with chloroform-methanol system gradient elution, wherein 1-2 part merges,, obtaining a white amorphous powder through preparation thin-layer chromatography (petroleum ether-ethyl acetate-methyl alcohol launches seven times at 5: 4: 0.4), TLC checks and is single spot, be compound 1,1.25g.

Compound 1, mp 196-200C ° (CHCl ₃-MeOH), [α] _D ²⁰-11.3 ° of (CHCl ₃: MeOH=1: 1, c0.12), and the Liebermann-Burchard reacting positive, the Molish reacting positive, thin layer hydrolysis inspection is known wood sugar.The FAB-MS (see figure 1) shows m/z685 (M+Na) ⁺, high resolution mass spectrum (HRFAB-MS) provides m/z663.408993[M+H] ⁺, [Calcd663.410824] determines that its molecular formula is C ₃₇H ₅₈O ₁₀, degree of unsaturation is 9.

IR spectrum (see figure 2) is at 3600-3100 and 1040,1090cm ^-1Strong absorption peak occurs, show glycoside compound; 1735,1249cm ^-1Show strong absorption band, show acetyl-containing in the molecular structure. ¹H NMR (see figure 3) and ¹³C NMR spectrum (see figure 4) shows that this compound is one 9,19-cycloartane type triterpene xyloside, and contain an ethanoyl and a ketal carbon (table 1).

¹H NMR spectrum high field region shows the signal [1H, d, J=4.0Hz, 19-H] of a pair of AB system proton, 0.43 (1H; d, J=4.0Hz, 19-H)], [δ 1.06,1.08 for the unimodal signal of seven uncle's methyl protons; 1.39,1.47,1.58,1.64] and an acetyl protons signal [δ 2.09].The terminal hydrogen signal of sugar appears at δ 4.86, and (1H, d J=7.5Hz), illustrate that the glucoside key is a beta comfiguration.Low place except the wood sugar proton signal, also show a pair of ABX system signal [δ 1.82 (and 1H, d, J=7.0Hz, H-17); 4.47 (1H, dd, J=7.0,2.0Hz, H-16); 5.67 (1H, d, J=2.0Hz, H-15)], ¹H- ¹(see figure 5) in the H COSY spectrum, H-15, H-16 is relevant with H-17 two protons; (see figure 6) in the HMQC spectrum, δ 86.23 (C-15) during H-15, H-16 and H-17 three proton signals compose with carbon respectively, 79.86 (C-16) are relevant with 51.19 (C-17).

¹³CNMR shows 37 carbon signals, and the end group carbon of sugar appears at δ 107.64, and low place also shows 3 even oxygen carbon signals [δ 72.03,82.44 and 110.70] except wood sugar carbon and C-3 signal; With beesioside II, IV relatively, δ 72.03 can belong to and is C-25.According to the degree of unsaturation of compound, deduction 9,19 cycloartane type tetracyclic triterpene parent nucleus, a sugar ring and an ethanoyl also should have 2 ring systems in the molecular structure.Connect oxygen quaternary carbon [δ 82.44] in the molecular structure, there are 16 β in ketal carbon [δ 110.70] and company's oxygen tertiary carbon [δ 79.86 (C-16)] prompting molecular structure, 24 and 20,24 bis-epoxy structures, therefore the two dimensional structure of this compound is determined, with beesioside IV relatively, difference is that 12 no hydroxyls replace; In conjunction with ¹H- ¹H COSY, ¹³C- ¹H COSY and with beesioside IV relatively, all hydrocarbon signals are all belonged to (table 1).

Compound I

Molecular model shows that if C-20 is the R configuration, C-24 also should be R; If C-20 is the S configuration, C-24 also should be S.Sakurai N etc. are by with observed ¹H- ¹H coupling constant (J _15,16, J _16,17) and relatively determined C-15 among the compound b eesiosideIV according to Karplus formula calculated value, 20 and 24 stereochemistry.If C-20 and 24 is the R configuration, the coupling constant between H-16 and the H-17 should be 4.3Hz (boat form) or 7.2Hz (chair form) so; If C-20 and 24 is the S configuration, the coupling constant between H-16 and the H-17 should be 7.5Hz (boat form) or 7.0Hz (chair form) so.20 R, 24 RUnder the situation, if H-15 is the α configuration, the coupling constant between H-15 and the H-16 should be 7.2Hz (boat form) or 4.7Hz (chair form) so; If H-15 is a beta comfiguration, the coupling constant between H-15 and the H-16 should be 0.4Hz (boat form) or 7.2Hz (chair form) so.20 S, 24 SUnder the situation, if H-15 is the α configuration, the coupling constant between H-15 and the H-16 should be 5.4Hz (boat form) or 8.2Hz (chair form) so; If H-15 is a beta comfiguration, the coupling constant between H-15 and the H-16 should be so 7.5Hz (boat form) or 1.7Hz (chair form).Among the compound gbc-18, J _15,16=2.0, J _16,17Therefore=7.0Hz determines C-15, and 20 and 24 stereochemistry is 20 S, 24 SWith 15 α-OAc.

¹3 hydrogen appears at δ 3.50 (J=11.5 3.5Hz), splits the branch mode and the big I of coupling constant judges that 3 hydrogen are the α configuration according to it for 1H, dd in the HNMR spectrum.Compare it with the glucoside unit of beesioside IV ¹³C-3 position glucoside shift value in the CNMR spectrum (low field displacement 10.59ppm) determines that wood sugar is connected in the C-3 position.

Comprehensive above-mentioned spectroscopic analysis, the structure of measuring Compound I be (20S, 24S)-15 α-acetoxy-16 β, 24; 20,24-diepoxy-9,19-cyclolanostane-3 β, 25-diol-3-O-β-D-xylopyranoside.

The experimental data of Compound I structure determination:

IR(KBr)cm ^-1：3700-3100，2965，2930，2870，1735，1460，1380，1360，1240，1090，1040，965。FAB-MS m/z：685(M+Na) ⁺，665(M+H) ⁺，535，453，115(100)，59，43。 ¹H NMR and ¹³C NMR data see Table 1.

Table 1 Compound I ¹H NMR and ¹³C NMR data

positi δ ¹H(J in Hz) δ ¹³C posit δ ¹H(J in Hz) δ ¹³C

on ion

1 1.22m；1.58m 32.40 19 0.25d(4.0) 30.11

0.43d(4.0)

2 1.93m；2.35m 30.11 20 82.44

3 3.50dd(11.5， 88.46 21 1.33s 25.75

3.5)

4 41.33 22 2.00m；1.65m 40.07

5 1.31m 47.50 23 2.60m；1.98m 28.57

6 0.67q(12.5) 20.96 24 110.70

-

7 1.02m；1.30m 26.02* 25 72.03

8 1.76dd(12.8， 47.77 26 1.64s 25.47

5.0)

9 19.65 27 1.47s 25.21

10 26.42 28 1.39s 24.64

11 2.10m；1.05m 26.08* 29 1.06s 15.36

12 1.66m；1.05m 33.13 30 1.08s 13.66

13 46.54 CO CH ₃ 2.09s 21.47

14 48.95 COCH ₃ 107.28

15 5.67d(2.0) 86.23 1’ 4.86d(7.5) 107.64

16 4.47dd(7.0，2.0) 79.86 2’ 4.03t(8.5) 75.53

17 1.82d(7.0) 51.19 3’ 4.16t(8.5) 78.63

18 1.58s 21.55 4’ 4.23td(8.5，5.0) 71.21

19 0.25d(4.0) 30.11 5’ 3.73t(9.5) 67.13

0.43d(4.0) 4.36dd(11.0，5.0)

Through pharmaceutical research, the Compound I in vivo test has stronger immunosuppressive activity, and in vitro tests has stronger inhibition angiogenic activity and suppresses activity of osteoblast proliferation.

The pharmacodynamics test method:

One, immunological experiment

1. experiment material and instrument:

Mouse: the Balb/c mouse, male, the 18-20g body weight;

MTT: tetrazole, purchase company in Sigma;

Concanavalin A: purchase company in Sigma;

C-RPMI-1640 cell culture fluid: in every 1000ml distilled water, contain RPMI-1640 dry powder 10.4 grams, NaHCO ₃2 grams, L-glutaminate 0.3 gram, penicillin G 100U/ml, Streptomycin sulphate 100ug/ml, 10% calf serum, sterile filtration;

Cell work liquid: Hank ' the s liquid of 0.01M pH7.2;

SRBC: get defiber SRBC, be washed till not haemolysis with physiological saline, the long-pending SRBC of last pressure is made into 5%, 10% SRBC suspension;

Sample liquid: accurately take by weighing sample compound, be made into 1mg/ml solution, be diluted to sterile filtration behind the 10 μ g/ml with physiological saline again with dissolved in distilled water (DMSO hydrotropy).Be mixed with the soup to be measured of different concns during experiment.

Model 550 microplate reader: Microplate Reader Instruction ManualCatalog Number 170-6750

CO ₂Incubator: NAPCO Model 5410

2. experimental technique:

Sample thief liquid is given abdominal injection in the mouse body, and control group is set simultaneously, and successive administration is after seven days, and the aseptic respectively spleen of getting prepares splenocyte suspension, does mtt assay lymphocyte transformation experiment, record OD value.

2.1 the preparation of mouse boosting cell suspension: draw neck to put to death mouse, with 75% alcohol immersion 1-2 minute, the aseptic spleen of getting in super clean bench was put to shred with nook closing member on steel mesh in the plate that fills Hank ' s liquid and is ground gently, single cell suspension.Centrifugal 10 minutes (1000rpm) washes twice after abandoning supernatant liquor, carries out the cell numeration, and cell precipitation transfers to 1 * 10 with the c-RPMI1640 nutrient solution ⁷/ ml concentration.

2.2MTT method lymphproliferation response experiment

1 * 10 of fresh separated ⁷/ ml mouse boosting cell suspension adds in the 96 porocyte culture plates, every empty 100 μ l that add, add stimulant (ConA) 100 μ l/ holes according to experimental design again, each sample is established three parallel holes and is established control wells (only adding c-1640), cumulative volume 200 μ l in every hole place culture plate 37 ℃ 5%CO then ₂Cultivated 48 hours in the incubator.Take out culture plate, every hole is inhaled and is abandoned supernatant 100 μ l, and each hole adds 5mg/ml MTT 10 μ l, continues to cultivate 4 hours.Take out culture plate, each hole adds 10%SDS lysate 100 μ l, puts in the incubator and spends the night, and in 570nm place microplate reader colorimetric estimation OD value, and calculates conversion values OD value.

3. experimental result

Mtt assay lymphproliferation response experimental result sees Table 2.

Table 2 Compound I is lymphopoietic to ConA inductive mouse boosting cell

Influence (the OD value ± SD)

Group dosage (ng) conversion values OD ± SD Δ OD
	Blank
0 0.167-Compound I 10 0.285+0.008-0.368 Compound I 50 0.323=0.033-0.294

The T lymphocyte external be subjected to non-specific mitogen (ConA) and stimulate after, can be converted into that volume is big, metabolism is vigorous and can carry out the splitted lymphocyte, in this course, add flaxen MTT and can be reduced into hepatic MTT first the part between the ribs and the hips, the first the part between the ribs and the hips amount and the metaboilic level of generation are proportional.So, be the lymphocytic propagation degree of decidable according to the OD value by colorimetric to bluish voilet liquid transparence behind the dissolving first the part between the ribs and the hips.

4. experiment conclusion:

Experiment shows that monomeric compound I can obviously suppress the cell proliferation by ConA inductive T when the mouse vivo medicine-feeding, have immunosuppressive action, is a kind of immunosuppressor in the body.

One, chick chorioallantoic membrane (CAM) test

1. experiment purpose: observe the influence that monomeric compound I generates new vessel

2. experiment material and experimental technique

A. sample source: from Beesia calthifolia, separate the monomeric compound I that obtains.

B. plant the egg source: available from China's National Defense University.

C. medicament preparation and contrast: 0.5mg monomeric compound I is dissolved in 75% ethanol, 37.5 μ l, adds physiological saline again to cumulative volume 100 μ l, promptly gets the 5mg/ml soup, and the equal-volume mixed solvent is done negative control, and 1: 3 hydrogen is examined the heparin mixed solution and done positive control.

D. egg hatching: will plant egg and put in constant temperature, constant humidity (37 ℃, the 60%) incubator and hatch, and break eggshell after 72 hours and go into to continue in the 100mm plate dosing (as follows) after the hatching 72 hours.

E. dosing: above-mentioned 5mg/ml concentration liquid is diluted to 20 μ g/ml, solvent is added in from restricting on the 2mm glass fiber filter paper sheet after complying same dilution, every adds soup 3 μ l (tablet), tablet is placed on chick chorioallantoic membrane vascular arborization place respectively, and dissecting microscope is observed this place's blood vessel variation down after 24 hours.

3. experimental result:

This experimental result: add the tablet place of Compound I, blood vessel autolyze phenomenon, no new vessel forms; Solvent contrast tablet place blood vessel no change; Hydrogen is examined-heparin tablet place blood vessel appearance blank, and no new vessel forms.

4. experiment conclusion:

The tumour cell division hyperplasia is rapid, and growth of tumor is accompanied by the blood vessel hyperplasia of microcirculation aspect, and its nutritive ingredient is transported by capillary vessel; Chick chorioallantoic membrane (CAM) test is intended to check the influence of medicine to the microcirculation aspect, estimates and screening antineoplastic drugs from this angle.This experimental result shows that monomeric compound I has the activity that suppresses capillary blood vessel generation aspect.

Three, scleroblast in vitro tests and to the influence of alkaline phosphatase:

1. experiment purpose:

Monomeric compound I is to the influence of Wistar rat skull bone osteoblastic proliferation and right

The influence of alkaline phosphatase.

2. scleroblast in vitro tests method and step:

Get the Wistar rat skull bone of new birth under the aseptic condition, isolate parietal bone and frontal bone, in D-HankS liquid, peel off the fibering periosteum after, chopping, through 0.25% tryptic digestion 15min, vibration in per 3 minutes is once, discard Digestive system, at 37 ℃, digest 2 times with 0.1%I Collagen Type VI enzyme 10ml, each 60min, collect Digestive system, the centrifugal 5min of 1000rpm removes supernatant, with cell inoculation in the culturing bottle that contains 20% calf serum F12 nutrient solution, 37 ℃ of 5%CO ₂Cultivate in the incubator, 24h changes liquid, and 2-3 days when treating cell confluent culture bottle, the cultivation of going down to posterity, used experimental cell is 3-5 for cell, is 30,000 cells/ml with cell concn during experiment.

Step is flat middle 0.1ml cell, the 37 ℃ of 5%CO of adding in hole a.96 ₂Cultivate 24h.

B. every hole adds different concns and contains and be subjected to reagent liquid, solvent or blank 20 μ l, and it is intact that every hole adds F12

The basic 80 μ l of full training, whole reaction system is 200 μ l, cultivates 72h for 37 ℃.

C. change serum-free medium, every hole 0.1ml.

D. add 20 μ l, 5mg/ml MTT solution

E.37 ℃ hatch 4h,

F. sucking-off 120 μ l, (outwelling)

G. add 200 μ l dimethyl sulfoxide (DMSO), shake up

H. on microplate reader, measure the absorbance in each hole in 570nm.

3. scleroblast experiment in vitro result: see Table 3 and Fig. 7.

4. conclusion (of pressure testing): this experimental result shows that monomeric compound I is inhibited to scleroblast, inhibiting rate IC ₅₀=32.78 μ g/ml, experiment finds that also monomeric compound I has significant inhibitory effect to alkaline phosphatase to alkaline phosphatase activities in addition, the inhibition degree is "+"

Description of drawings:

Fig. 1: the FAB-MS spectrum of Compound I;

Fig. 2: the IR spectrum of Compound I;

Fig. 3: Compound I ¹The H-NMR spectrum;

Fig. 4: Compound I ¹³The C-NMR spectrum;

Fig. 5: Compound I ¹H- ¹H COSY spectrum;

Fig. 6: the HMQC spectrum of Compound I;

Fig. 7: the scleroblast inhibiting rate of Compound I.

The Gegenbaur's cell of table 3 Compound I suppresses experimental result

The experiment title	Gegenbaur's cell (mtt assay)											2000.11.20			490nm
																			Tested medicine	Monomer medicine				Compound number: Compound I
The cell title	Gegenbaur's cell						23 ℃ of indoor temperatures							Indoor humidity 22%
																				Serum		External dosage														Inhibiting rate	Average inhibiting rate
Medicine name	Numbering		ul/ml		1		2		3		4	5		6	Average	Standard deviation	％	％
																			Ethanol
			2		0.119										0.1190	######		0.122
																			Ethanol			1		0.119										0.1190	######
Ethanol			.5		0.132										0.1320	######
																			Ethanol			.25		0.119										0.1190	######
Compound	I		50		0.052		0.054		0.049						0.0530	0.00141	55.462	56.646
																			Compound	I		25		0.065		0.062		0.065						0.0640	0.00173	46.218	47.648
Compound	I		12.5		0.074		0.082		0.095						0.0837	0.01059	36.616	31.561
																			Compound	I		6.25		0.134		0.112		0.104						0.1167	0.01553	1.961	4.567
																			External dosage	Log10 dose	Inhibiting rate %		Average inhibiting rate %		Inhibiting rate=(solvent-medicine)/solvent * 100
50	1.6989	55.46218		56.6462
																			25	1.3979	46.21848		47.6482
12.5	1.0969	36.61616		31.5610		Inhibiting rate IC50=32.78							95% fiducial limit			26.41-40.68
																			6.25	0.7958	1.960784		4.56714		Regression equation				Y＝2.4128+1.72013X					r＝0.867698		sb＝0.214490
						Average rate IC50=24.89							95% fiducial limit			19.79-31.32
																									Regression equation				Y＝3.06978+1.382487X					r＝0.9633		sb＝0.20633

Claims (4)

1, a kind of cycloartane triterpenoid saponin compound (1), its chemical structure is as follows:

Compound (1)

2, the preparation method of cycloartane triterpenoid saponin compound (1), it is characterized in that with Beesia calthifolia (Beesia calthaefolia) rhizome be raw material, use extraction using alcohol, ethanol extraction is dissolved in the water, use chloroform extraction again, chloroform extract separates through silica gel column chromatography, obtains said compound.

3, the application of ring Artocarpus heterophyllus Lam alkane triterpenoid saponin compound (1) in the preparation immunosuppressor.

4, the application of ring Artocarpus heterophyllus Lam alkane triterpenoid saponin compound (1) in preparation angiogenesis inhibitor or antitumor drug.

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