CN1763065A - A kind of carbon-21 steroidal glycosides with immunosuppressive action - Google Patents

Abstract

The present invention relates to extract the separated a kind of new C with chemical structure of general formula (I), immunosuppressive activity and resisting rheumatoid arthritis effect obtaining from herbal medicine wax flower (Stephanotismucronata) ₂₁steroidal glycoside---SMA, R in its Chinese style ₁represent CH ₃cO, R ₂represent

, R ₃represent β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-oxygen methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyranoside [β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside].The invention still further relates to the preparation method of this compound, take the pharmaceutical composition that this compound is activeconstituents and the compounds of this invention and the purposes of medicinal compositions aspect immunosuppression and resisting rheumatoid arthritis.

Description

A kind of carbon-21 steroidal glycosides with immunosuppressive action

Technical field

The present invention relates to extract the separated a kind of new C with immunosuppressive activity and resisting rheumatoid arthritis effect obtaining from herbal medicine wax flower (Stephanotis mucronata) ₂₁steroidal glycoside---SMA and preparation method thereof and take the pharmaceutical composition that this compound is activeconstituents and their application aspect immunosuppression and resisting rheumatoid arthritis.

Background technology

Along with the development of basis and clinical immunology, many "X" diseases can be explained by autoimmune response.Autoimmune response can not only cause the generation of autoimmune disease, and itself has vicious cycle, can make disease protracted course of disease, prognosis mala.The formation of the autoimmune disorder such as lupus erythematosus, rheumatoid arthritis is a very complicated process, and the principle that people mainly apply immunological tolerance adopts immunosuppressor to treat, and has obtained certain effect improving aspect symptom.On the other hand, along with the improvement of surgery operating technology and the raising for the treatment of level, organ transplantation day by day universal, immunosuppressor plays an important role in the prevention of organ transplant rejection and treatment.

Wax flower is dry root and the rattan of asclepiadaceae plant wax flower Stephanotis mucronata, is distributed widely in south China area, and resource is very abundant.The effect with strengthening the bones and muscles, wind-damp dispelling, among the people for rheumatoid arthritis.

Summary of the invention

The object of this invention is to provide a kind of new C with immunosuppressive activity and resisting rheumatoid arthritis effect ₂₁steroidal glycoside---SMA, provides and from traditional herbal medicine wax flower, extracts separated preparation C ₂₁the method of steroidal glycoside---SMA, provides the pharmaceutical composition of immunosuppression and resisting rheumatoid arthritis and this compound thereof the purposes aspect preparation immunosuppressor and resisting rheumatoid arthritis medicine.

C of the present invention ₂₁steroidal glycoside comprises the separated SMA obtaining of extraction from herbal medicine wax flower, and this compound has the chemical structural formula (I) of following formula, R in formula ₁represent CH ₃cO, R ₂represent

r ₃represent β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-oxygen-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyranoside [β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside].

SMA provided by the invention, its chemical system is called 12-oxy-acetyl-20-oxygen-(N-methyl) o-amino benzoyl acyl group sarcostin 3-β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-oxygen-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyranoside [12-O-acetyl-20-O-(N-methyl) anthraniloyl sarcostin 3-O-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-cymaropy-ranosyl-(1 → 4)-β-D-cymaropyranoside], to extract from the dry root of asclepiadaceae plant wax flower Stephanotis mucronatum and rattan, preparation method comprises the following steps:

A. by after the root of wax flower (Stephanotis mucronata) or rattan pulverizing, with extraction using alcohol;

B. ethanol extract concentrated after with organic solvent extraction or wax flower root or rattan meal directly with organic solvent extraction, concentrated, obtain total steroid saponin;

C. total steroid saponin carries out chromatographic separation in silica gel or alumina column, and take methyl alcohol, ethanol, ethyl acetate, chloroform, methylene dichloride or their mixture is eluent;

D. silica gel thin-layer chromatography checks elutriant, and the elutriant merging with same blob is concentrated, obtains the stream part containing SMA;

E. the stream part that contains SMA, again through silica gel (comprising Rp-18, Rp-8) or HPLC chromatography, with eluent wash-out, separation, obtains C ₂₁steroidal glycoside---SMA.

In above-mentioned steps b, said organic solvent is chloroform, methylene dichloride, ethyl acetate, and in step e, said eluent is the mixture of first alcohol and water or acetonitrile and water or acetone and water.

Compound results of animal of the present invention demonstrates remarkable immunosuppressive activity and resisting rheumatoid arthritis effect.This compound is active in clinical widely used immunosuppressor---cyclosporin A is suitable now to the inhibition of cellular immunization, and its humoral immunization restraining effect is better than cyclosporin A.This compound is to the preventive and therapeutic effect of rat assist agent arthritis and tripterygium glycosides (Tripterysium glucosides, TG) there was no significant difference.So compound of the present invention is expected to exploitation becomes a kind of new immunosuppressive drug.

Pharmaceutical composition of the present invention contains the C that treats significant quantity ₂₁steroidal glycoside---SMA is activeconstituents, and contains one or more pharmaceutically acceptable carriers.

Compound of the present invention and pharmaceutical composition can be used for preparing the medicine of immunosuppressor and preparation treatment resisting rheumatoid arthritis.

Pharmaceutically acceptable carrier mentioned above refers to the pharmaceutical carrier of pharmaceutical field, such as: thinner, vehicle are as water etc., and weighting agent is as starch, sucrose etc.; Tackiness agent is as derivatived cellulose, alginates, gelatin and polyvinylpyrrolidone; Wetting agent is as glycerine; Disintegrating agent is as calcium carbonate and sodium bicarbonate; Absorption enhancer is as quaternary ammonium compound; Tensio-active agent is as cetyl alcohol; Absorption carrier is as kaolin and soap clay; Lubricant is as talcum, calcium stearate, Magnesium Stearate and polyoxyethylene glycol etc.Can also in composition, add in addition other auxiliary material as flavouring agent, sweeting agent etc.

The compounds of this invention can composition the mode of form by oral, rectum, parenteral or percutaneous dosing be applied to the patient who needs this treatment.When oral, can be made into conventional solid preparation as tablet, pulvis, granule, capsule nationality, pill, sustained release pellet, solid dispersion, inclusion etc., the liquid preparation of making is as suspensoid, emulsion, sol, syrup, mixture, solution etc.; When the administered parenterally, can be made into solution, water or oiliness suspensoid, emulsion, liposome, micro-capsule, microballoon, the nanoparticle of injection, the ointment of external application etc.Preferential form is tablet, coated tablet, capsule, micropill, ointment and injection.

The various formulations of pharmaceutical composition of the present invention can be according to the conventional production method preparation of pharmaceutical field.For example make activeconstituents mix with one or more carriers, be then made into required formulation.

Pharmaceutical composition of the present invention preferably contains activeconstituents, and to account for gross weight ratio be 0.1%～99.5%.Most preferably containing activeconstituents, to account for gross weight ratio be 0.5%～95%.

The usage quantity of the compounds of this invention can be according to variations such as route of administration, patient age, body weight, the organ transplantation kind for the treatment of, rheumatoid arthritis severity, and its per daily dose can be 0.01～100mg/Kg body weight, preferably 0.1～10mg/Kg body weight.Can use by one or many, and Side effect is little.

Accompanying drawing explanation

Fig. 1: SMA's ¹³c NMR spectrogram

The DEPT spectrogram of Fig. 2: SMA

Fig. 3: SMA's ¹h NMR spectrogram

The HMBC spectrogram of Fig. 4: SMA

The HMQC spectrogram of Fig. 5: SMA

Fig. 6: SMA's ¹h- ¹h COSY spectrogram

Fig. 7: SMA is exempted from the impact of mouse lymphocyte proliferative response on the OVA of ConA induction

Fig. 8: SMA is exempted from the impact of mouse lymphocyte proliferative response on the OVA of OVA induction

Fig. 9: SMA is exempted from the impact of specific IgG antibodies level in mice serum on OVA

Embodiment

By example, further illustrate the present invention below.

Embodiment 1:C ₂₁steroidal glycoside SMA preparation

After wax flower dry root or rattan 20kg pulverize, with alcohol reflux 3 times, each 2 hours, united extraction liquid, decompression and solvent recovery, obtains ethanol extraction.For ethanol extraction, chloroform, methylene dichloride or ethyl acetate backflow are extracted, or wax flower root or rattan meal directly extract with chloroform, methylene dichloride or ethyl acetate backflow, chloroform, methylene dichloride or acetic acid ethyl acetate extract or extract, concentrated, obtain total steroid saponin.Total steroid saponin carries out silica gel or alumina column chromatography, first with chloroform, ethyl acetate or methylene dichloride and methyl alcohol (or ethanol), carry out gradient elution, with thin-layer chromatography, check elutriant, stream part of containing SMA spot merges, concentrated, medicinal extract is further through Rp-18 column chromatography or HPLC, and, purifying separated with water elution with water or acetone with first alcohol and water or acetonitrile, obtain C ₂₁steroidal glycoside---SMA.

C ₂₁the unformed powder of steroidal glycoside SMA white, Lieberman-Buchard reacting positive.High resolution mass spectrum determines that its molecular formula is C ₅₈h ₈₉nO ₂₃(1168.5867[M+H] ⁺, Calcd 1168.5898). ¹³c NMR (Fig. 1) and DEPT spectrum (Fig. 2) (125MHz, C ₅d ₅n) show that SMA contains 58 carbon atoms, wherein 11 CH ₃, 10 CH ₂, 27 CH and 10 quaternary carbons.By ¹h NMR (Fig. 3) and ¹³c NMR data (table 1) are known: aglycon part spectroscopic data and the known compound prosapogenin basically identical [K.Yoshikawa, N.Okada, Y.Kann, S.Arihara, Chem.Pharm.Bull.1996,44,1790] of SMA.Comparative compound SMA aglycon and prosapogenin's ¹³cNMR data, can observe following glycosidation displacement: C2 (1.9ppm), and C4 (2.6ppm) and C3 (+5.9ppm) illustrate that sugar chain is partly connected on the C-3 position hydroxyl of aglycon prosapogenin. ¹³on the steroidal parent nucleus of C NMR data presentation compound S MA, there are two acyl substituents; be N-methyl o-amino benzoyl acyl group (N-methylanthraniloyl) (δ: 111.1; 152.7; 111.6; 135.2,114.8,132.7; 168.3,29.6) and ethanoyl (acetyl) (δ: 171.4 and 22.1).In HMBC collection of illustrative plates (Fig. 4), the carbon signal δ 168.3 of N-methylanthraniloyl and the signal δ 5.21 (q of parent nucleus C-20 (δ 74.9) methyne hydrogen, J=7.0Hz) relevant, illustrate that N-methylanthraniloyl is connected on C-20 hydroxyl; The carbon signal δ 171.4 of ethanoyl and the signal δ 5.19 (dd of parent nucleus C-12 (δ 74.2) methyne hydrogen; J=11.5; 4.5Hz) relevant; illustrate that ethanoyl is connected on C-12 hydroxyl; thereby further determine that its aglycon is prosapogenin, i.e. 12-O-ethanoyl-20-O-(N-methyl)-o-amino benzoyl acyl group sarcostin.Compound S MA's ¹in H NMR spectrum, show 3 methyl of desoxy sugar, the signal of 3 methoxyl group signals and 4 anomeric protons: δ 5.29 (1H, d, J=9.5Hz), 5.13 (1H, d, J=9.5Hz), 5.17 (1H, d, J=7.5Hz) and 4.73 (1H, d, J=8.0Hz), show to contain in molecule 4 sugar, and all with β-key, connect.Therefore compound S MA is 12-O-ethanoyl-20-O-(N-methyl)-o-amino benzoyl acyl group sarcostin 3-O-tetrose glycosides.

SMA is hydrolyzed under acidic conditions, obtains aglycon, cymarose, glucose (contrasting with standard cymarose, standard glucose on TLC) and a unknown sugar (showing on TLC).This aglycon ¹³the Prosapogenin data consistent of C NMR data and bibliographical information [K.Yoshikawa, N.Okada, Y.Kann, S.Arihara, Chem.Pharm.BulL.1996,44,1790].At HMBC, HMQC (Fig. 5) and ¹h, ¹in H COSY (Fig. 6) collection of illustrative plates, from end group carbon δ 104.9 and proton δ 5.17 (d thereof, J=7.5Hz) set out and can find out all the other protons of unknown sugar and the chemical displacement value (in Table 1) of carbon atom, and contrast [K.Yoshikawa with data in literature, N.Okada, Y.Kann, S.Arihara, Chem.Pharm.Bull.1996,44,1790.], result shows that this unknown sugar is 6-deoxidation-3-O-methyl-β-D-allose (6-deoxy-3-O-methyl-β-D-allose, referred to as AllMe).By comparing compound data presentation in document: in pyridine solution, the anomeric proton signal of AllMe appears at low of δ > 5.00, and β-D-chrysanthemum first bamboo peach sugar (β-D-thevetose, referred to as Thv) anomeric proton signal appear at the High-Field [K.Yoshikawa of δ < 5.00, N.Okada, Y.Kann, S.Arihara, Chem.Pharm.Bull.1996, 44, 1790.K.Yoshikawa, K.Matsuchika, S.Arihara, H.C.Chang, J.D.Wang, Chem.Pharm.Bull.1998, 46, 1239.], further determine the anomeric proton signal that δ 5.17 is AllMe.Compound S MA's ¹in H NMR, δ 5.29 and 5.13 should belong to respectively the anomeric proton signal on two Apocynum cannabinum pools, and is judged as beta comfiguration from coupling constant.At it ¹³in C NMR, δ 37.3 and 37.1 belongs to respectively the C-2 signal of two cymarose, further determines that they are β-D configuration [R.Vleggaar, F.R.van Heerden, L.A.P.Anderson, G.L.Erasmus, J.Chem.Soc., Perkin Trans.11993,483.].The C-3 (δ 77.7) of visible significantly relevant peaks: Sa-H1 (δ 5.29, d, J=9.5Hz) and aglycon in HMBC collection of illustrative plates; Sb-Hl (δ 5.13, d, J=9.5Hz) and Sa-C4 (δ 83.2); Sc-H1 (δ 5.13, d, J=9.5Hz, 1H) and Sb-C4 (δ 83.1); (δ 5.17 for Sd-H1, d, J=7.5Hz, 1H), with Sc-C4 (δ 83.1), (δ 4.73, d for Sd-H1, J=8.0Hz, 1H) with Sc-C4 (δ 83.5), show that the order of connection of sugar chain part is (β-D-Glucose)-(6-deoxidation-3-O-methyl-β-D-allose)-(β-D-cymarose)-(β-D-cymarose)-(C-3 position of prosapogenin), and be 1 → 4 connection.

In sum, the Structure Deduction of compound S MA is 12-O-ethanoyl-20-O-(N-methyl) o-amino benzoyl acyl group sarcostin 3-O-β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyranoside.

C ₂₁the experimental data white amorphous powder of steroidal glycoside SMA.HR-EI-MS：1168.5867(C ₅₈H ₈₉NO ₂₃，Cal.1168.5898[M+1] ⁺)。EI-MS m/z：1190.6[M+Na] ⁺，1005.2[M-glc] ⁺。 ¹³c NMR, ¹h NMR and HMBC data are in Table 1.

Get C ₂₁steroidal glycoside SMA 50mg is dissolved in 9% hydrochloric acid methanol of 10mL, and reflux 2h, lets cool, and adds Ag ₂cO ₃powder is neutralized to pH7, filters, and removes precipitation, and filtrate decompression is concentrated into dry, then uses column chromatography (silica gel 10g, CHCl ₃: MeOH=100: 1 → 50: 1V/V) separation, obtains aglycon (21mg) and sugared mixture.In TLC: hydrolysate shows and Cymarose, the consistent spot of glucose standard substance Rf value.Aglycon ¹³c NMR data are in Table 1.

Embodiment 2

Tablet: activeconstituents 1.0g

Lactose 18.7g

W-Gum 5.0g

Magnesium Stearate 0.3g

Preparation method: activeconstituents, newborn sugar and starch are mixed, and water is evenly moistening, the mixture after moistening sieves and be dried, after sieve, adds Magnesium Stearate, then by mixture compressing tablet, every weight 250mg, active component content 10mg.

Table 1 C ₂₁steroidal glycoside SMA's ¹³cNMR, ¹hNMR and HMBC data (C ₅d ₅n)

position	13C NMR	1H NMR	HMBC	13C NMR of aglycone
					1	38.9(t)			38.6
2	29.9(t)	1.70，m；2.03，m		30.8
					3	77.7(d)	3.84，m		71.8
4	39.3(t)			41.9
					5	139.3(s)			139.7
6	119.4(d)	5.33，br s		118.2
					7	35.0(t)			34.3
8	74.4(s)			74.1
					9	44.1(d)	1.70，m		43.1
10	37.3(s)			36.7
					11	25.7(t)	1.97，m；2.26，m		24.7
12	74.6(d)	5.19，dd(11.5，4.5)	C-1′，C-18	73.5
					13	57.0(s)			56.0
14	89.0(s)			87.8
					15	34.0(t)			32.2
16	33.8(t)			32.9
					17	87.7(s)			87.8
18	11.4(q)	2.05，s	C-12，C-14，C-17	10.3
					19	18.1(q)	1.32，s	C-5，C-9，C-10	18.2
20	74.9(d)	5.21，q(7.0)	C-7″，C-21	73.9
					21	15.7(q)	1.55，d(6.0)	C-17，C-20	15.0

12-O-acyl	Ac			Ac
						1′	171.4(s)			171.4
2′	22.1(q)	2.14，s	C-1′	21.7
					20-O-acyl	Anth			Anth
1″	111.1(s)			109.6
					2″	152.7(s)			152.2
3″	111.6(d)	6.74，d(8.5)	C-1″，C-5″	110.9
					4″	135.2(d)	7.43，ddd(8.5，8.0，1.5)	C-2″，C-6″	134.8
5″	114.8(d)	6.60，t(7.5)	C-3″	114.4
					6″	132.7(d)	8.36，d(8.0)	C-2″，C-4″，C-7″	131.4
7″	168.3(s)			167.2
					CH 3	29.6(q)	2.80，d(5.0)	C-2″	29.5
Cym
					Sa-C1	96.5(d)	5.29，d(9.5)	C-3
Sa-C2	37.3(t)	1.78，m；2.54，m
					Sa-C3	78.1(d)	4.05，m
Sa-C4	83.2(d)	3.50，dd(9.5，2.5)	Sb-C1，Sa-C6
					Sa-C5	69.4(d)	4.22，m
Sa-C6	18.7(q)	1.40，d(6.5)	Sa-C4；Sa-C5
					Sa-3-OCH 3	59.1(q)	3.63，s
Cym
					Sb-C1	100.5(d)	5.13，d(9.5)	Sa-C4
Sb-C2	37.1(t)	1.78，m；2.46，m
					Sb-C3	78.1(d)	4.00，m
Sb-C4	83.1(d)	3.88，d(9.0)	Sc-C1，Sb-C6
					Sb-C5	69.1(d)	4.23.m
Sb-C6	18.6(q)	1.59，d(6.0)	Sb-C4，Sb-C5
					Sb-3-OCH 3	59.0(q)	3.60，s
Allm
					Sc-C1	104.9(d)	5.17，d(7.5)	Sb-C4
Sc-C2	75.0(d)	4.05，m
					Sc-C3	86.0(d)	3.74，t(9.0)	Sc-C2，Sc-C4，Sc-3-OCH 3
Sc-C4	83.5(d)	3.54，dd(9.5，2.5)	Sd-C1

Sc-C5	72.1(d)	4.25，m
					Sc-C6	18.8(q)	1.78，d(6.0)	Sc-C4，Sc-C5
Sc-3-OCH 3	60.7(q)	3.97，s
					Glc
Sd-C1	106.1(d)	4.73，d(8.0)	Sc-C4
					Sd-C2	75.9(d)	4.00，m
Sd-C3	78.8(d)	4.26，m	Sd-C4
					Sd-C4	72.1(d)	3.91，m
Sd-C5	78.2(d)	4.09，m
					Sd-C6	63.2(t)	4.72，dd(12，5)；4.55，dd(12，3)	Sd-C4，Sd-C5

Embodiment 3

Tablet: activeconstituents 1.0g

Lactose 18.8g

Magnesium Stearate 0.2g

Preparation method: activeconstituents is mixed with lactose, Magnesium Stearate, sieve, pack hard capsule into, the heavy 200mg of each capsule, active component content 10mg.

Embodiment 4:C ₂₁immunosuppressive action in steroidal glycoside SMA body

Experimental technique

(1) immune programme for children ICR mouse is divided into 6 groups at random, take oralbumin (OVA) as antigen (200 μ g/ only), the aluminium hydroxide (1mg/ only) of take is adjuvant, respectively at the 1st and 15 days subcutaneous injection immune mouses, sets up physiological saline control group simultaneously.OVA immune mouse in the 1st day, respectively abdominal injection SMA (25 μ g/ only, 50 μ g/ only, 100 μ g/ only), cyclosporin A (CsA, 50 μ g/ only) and physiological saline, interval administration in 7 days 1 time, successive administration 5 times.Two exempt from latter 14 days, and separated splenic lymphocyte supplies lymphocyte proliferation assay, and prepares serum and carry out antibody titers mensuration.

(2) splenocyte suspension is prepared mouse sacrificed by exsanguination, and 70% alcohol immersion 1-2 minute, gets the spleen of mouse under aseptic condition, adds appropriate Hanks liquid and grinds, and with 200 order stainless (steel) wires, filters, and centrifugal 5 minutes of 1500rpm, abandons supernatant, adds Hanks liquid repeated washing 2 times.Collect splenocyte, add appropriate RPMI RPMI-1640 suspendible, with platform, expect that orchid refuses to dye method counting, viable count is not less than 95%, adds RPMI 1640 complete culture solutions dilutions, and adjusts cell concn to 1 * 10 ⁷individual/ml.

(3) lymphproliferation response is in 96 hole micro plates, and every hole adds 100 μ l splenocyte suspensions (1 * 10 ⁷cell/ml), each sample repeats 12 holes, and to add respectively Con A liquid (final concentration is 5 μ g/ml), LPS liquid (final concentration is 10 μ g/ml), OVA liquid (final concentration is 20 μ g/ml) or RPMI RPMI-1640 to cumulative volume be 200 μ l.Separately establish blank group.37 ℃, 5%CO ₂cultivate after 44h, each hole adds 50 μ l MTT solution (2mg/ml), continues to cultivate 4h.Centrifugal 5 minutes of 1000rpm, discards each hole supernatant, adds respectively the acid DMSO solution of 150 μ l, vibration, in room temperature dark place, place 15min, with microplate reader in the mensuration OD of wavelength 578nm place value, and be calculated as follows stimulation index (SI): SI (stimulation index)=

(4) in serum, OVA specific antibody titres is measured and is adopted OVA specific IgG antibodies titre in indirect elisa method detection serum.Method is as follows: on 96 hole enzyme plates, every hole adds 100 μ l coating buffers (containing the 0.01mol/L carbonate buffer solution of 50 μ g/ml OVA), puts 4 ℃ and hatches 24h, with washings washing 3 times, each 3 minutes.Every hole added confining liquid 200 μ l, hatches 2h in 37 ℃, with washings washing 3 times, each 3 minutes.Add 100 μ l test serum diluents, repeat 3 holes, doubling dilution to six extent of dilution.Hatch 2h for 37 ℃, with washings washing 3 times, each 3 minutes.Add the anti-mouse IgG antibody diluent of horseradish peroxidase-labeled rabbit of the working concentration of 100 μ l/ hole by specification regulations, hatch 2h for 37 ℃, wash each 3 minutes 3 times.Every hole adds 100 μ l substrate buffer solutions, and 10min is placed in 37 ℃ of dark places, adds 50 μ l/ hole 2N H ₂sO ₄solution termination reaction.Put microplate reader and in wavelength 492nm place, measure OD value.

Experimental result

(1) that the OVA of ConA induction is exempted to affecting in SMA body of mouse lymphocyte proliferative response exempted from the OVA of ConA induction that mouse T lymphocyte proliferative response affects in SMA body the results are shown in Figure 8.As seen from Figure 8, the OVA that SMA all can extremely significantly suppress ConA induction under 25 μ g, 50 μ g and 100 μ g dosage is exempted from mouse T lymphocyte proliferative response (P < 0.001).

(2) that the OVA of OVA induction is exempted to affecting in SMA body of mouse lymphocyte proliferative response exempted from OVA induction OVA that the reaction of mouse specificity lymphocyte proliferation affects in SMA body the results are shown in Figure 9.As seen from Figure 9, SMA all can be significantly under 25 μ g, 50 μ g and 100 μ g dosage or the OVA that extremely significantly suppresses OVA induction exempted from mouse bone-marrow-derived lymphocyte proliferative response (P < 0.01 or P < 0.001), and be dose-dependently relation.

(3) SMA is exempted from mice serum on OVA that OVA specific IgG antibodies is tired affects SMA and OVA is exempted to the impact that in mice serum, OVA specific IgG antibodies is tired sees Figure 10.As seen from Figure 10, SMA 25 μ g, 50 μ g and 100 μ g dosage all extremely significantly suppress OVA exempted from mice serum in OVA specific IgG antibodies tire (P < 0.05 or P < 0.01), and be dose-dependently relation.

Above-mentioned results of animal shows: compound S MA of the present invention has significant immunosuppressive activity, and its inhibition to cellular immunization is active suitable with cyclosporin A, and the restraining effect of humoral immunization is better than to cyclosporin A.

The preventive and therapeutic effect test of example 5:C21 steroidal glycoside SMA to rat assist agent arthritis

Get 56 of SD rats, male, body weight 200～250g, by weight average, be divided into 5 groups, every group of 10-16 only, starts administration for 1 day from causing scorching, respectively by 20,40 and 80mg/kg dosage gavage give SMA, administration volume is 10ml/kg body weight, every day 1 time, continuous 21 days, positive group gave tripterygium glycosides (Tripterysium glucosides, TG) 80mg/kg, model control group gives isometric(al) ordinary water; Cause the scorching same day, with self-control measuring apparatus, measure rats with bilateral hind leg ankle joint girth as causing scorching front data, after administration 30 minutes, all rats all cause inflammation at left side toes subcutaneous injection Freund ' Freund's complete adjuvant 100 μ l, cause scorching rear 1d, 3d, 5d, 8d, 11d, 14d, 17d, 20,23d, with self-control measuring apparatus, measure left side ankle joint girth, by following method calculate swelling (swelling=(cause scorching after girth-cause scorching after girth)/cause scorching before girth * 100%), observe the impact of SMA on primary affection.Since the 8th day, measure right side ankle joint girth simultaneously, with method, calculate swelling, and the appearance situation of observing the secondary affections such as rat ear erythema, afterbody tubercle, front foot redness, end of record tubercle number, by the red and swollen degree of Pyatyi scoring system in post-therapeutic evaluation front foot, (0 minute without redness, 1 minute little toe redness and swelling of joints, 2 minutes toe joints and toes swelling, the following foot swelling of ankle joint in 3 minutes, comprises the whole sufficient pawl swelling of ankle joint for 4 minutes), with this, evaluate the impact of SMA on adjuvant arthritis rats secondary affection.The results are shown in Table 2～table 5.

Table 2 SMA is on the impact of rat assist agent arthritis primary pathology (X ± SD)

Group	Dosage (mg/kg)	Cause scorching front girth (mm)	Cause scorching rear different time and cause scorching paw swelling (%)
											1d	3d	5d	8d	11d	14d	17d	20d
			Control TG SMA SMA SMA	-- 80 80 40 20	22.1±0.6 21.9±0.7 21.7±0.8 22.1±0.6 21.4±0.6	31.8±8.8 28.8±9.7 27.5±7.2 33.2±5.4 37.0±7.0	28.5±10.5 23.1±8.1 25.7±8.8 29.9±10.6 34.9±8.8	24.1±7.0 18.1±7.2 17.4±6.9 22.4±7.7 29.0±9.2	21.8±12.0 17.2±8.1 15.8±8.8 22.7±13.8 32.0±10.4	15.4±8.5 15.1±7.8 9.9±9.0 16.0±11.3 17.0±9.8									18.3±9.4 11.0±4.6 * 8.1±6.8 * 14.3±12.0 16.6±10.3	12.5±9.5 4.0±6.6 * 4.7±5.7 * 5.9±11.8 16.6±12.3	10.6±10.8 7.4±7.1 7.2±5.4 11.7±11.9 17.4±10.3

Note: n=10 (control group n=16), ^*p < 0.05 (with model control group comparison)

Table 3 SMA is on the impact of rat assist agent arthritis secondary arthritis (offside paw swelling, X ± SD)

Group	Dosage (mg/kg)	Cause scorching front girth (mm)	Cause scorching rear different time offside paw swelling (%)
								8d	11d	14d	17d	20d
			Control TG SMA SMA SMA	-- 80 80 40 20	21.7±0.8 21.4±0.6 21.4±0.3 21.5±0.6 21.6±0.7	10.6±4.5 10.9±4.2 11.3±5.7 9.6±4.6 10.1±4.9	8.9±4.3 8.4±5.0 4.9±4.8 3.2±4.9 * 4.9±4.1						5.3±4.9 3.7±3.8 5.2±3.9 3.5±4.4 3.9±4.8	0.3±3.6 -0.3±2.4 -0.7±3.6 0.2±3.1 2.2±3.7	0.4±4.7 -0.2±5.1 2.7±4.7 1.0±5.7 5.4±4.7

Note: n=10 (control group n=16), ^*p < 0.05 (with control group comparison).

Table 4 SMA is on the impact of rat assist agent arthritis secondary affection (afterbody tubercle, X ± SD)

Group	Dosage (mg/kg)	Afterbody tubercle number
								8d	11d	14d	17d	20d	23d
		Control TG SMA SMA SMA	-- 80 80 40 20	3.1±0.6 2.2±0.4 * 2.3±0.5 * 2.4±0.5 * 2.2±0.7 *	3.1±0.5 2.5±0.5 * 2.6±0.5 * 2.8±0.4 2.9±0.3	3.6±0.5 2.9±0.3 * 2.8±0.4 * 3.0±0.5 * 3.0±0.0 *	3.6±0.5 2.7±0.5 * 2.6±0.5 * 2.9±0.3 * 3.0±0.0 *							3.4±0.5 2.7±0.5 * 2.7±0.5 * 3.0±0.5 3.1±0.6	4.0±0.4 2.8±0.4 * 2.7±0.5 * 3.0±0.0 * 2.8±0.4 *

Note: n=10 (control group n=16), ^*p < 0.05 (with model control group comparison)

Table 5 SMA is on the impact of rat assist agent arthritis secondary affection (the red and swollen score of front foot, X ± SD)

Group	Dosage (mg/kg)	The red and swollen score of front foot
								8d	11d	14d	17d	20d	23d
		Control TG SMA SMA SMA	-- 80 80 40 20	2.0±0.5 1.6±0.5 1.5±0.5 * 1.5±0.5 1.7±0.9	2.7±0.5 1.7±0.5 * 2.0±0.7 * 1.8±0.4 * 2.2±0.4 *	2.8±0.4 1.6±0.5 * 1.8±0.6 * 2.0±0.5 * 2.2±0.4 *	2.8±0.4 2.0±0.5 * 1.7±0.5 * 1.6±0.5 * 2.1±0.8 *							2.8±0.4 2.0±0.5 * 1.6±0.5 * 1.6±0.5 * 2.4±0.5	2.8±0.4 1.9±0.3 * 1.6±0.5 * 2.0±0.8 * 2.0±0.0 *

Note: n=10 (control group n=16), ^*p < 0.05 (with model control group comparison)

From table 2～table 5, SMA group has certain preventive and therapeutic effect to the early stage local primary inflammation of the rat arthritis due to adjuvant, and action intensity and tripterygium glycosides are suitable.Also can obviously suppress the swelling once again after 12 days, the generation of the secondary arthritis such as front foot redness and afterbody tubercle is also had to significant preventive and therapeutic effect.

In sum, C of the present invention ₂₁steroidal glycoside SMA all has significant inhibition activity to mouse cell immunity and humoral immunization, and rat assist agent arthritis is had to significant preventive and therapeutic effect examination.These pharmacodynamic experiment results show: C of the present invention ₂₁steroidal glycoside SMA can be used as immunosuppressor, can be used for treatment and comprises the various immunological diseases such as the autoimmune disorders such as organ transplantation, rheumatoid arthritis, systemic lupus erythematosis, chronic nephritis and allergic disorder.

Claims (8)

1, a kind of new C with immunosuppressive action ₂₁steroidal glycoside---SMA, this compound has the chemical structural formula (I) of following formula, R in formula ₁represent CH ₃cO, R ₂represent r ₃represent β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-oxygen-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyranoside [β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosy1-(1 → 4)-β-D-cymaropyranosy1-(1 → 4)-β-D-cymaropy-ranoside].

2, a kind of new C with immunosuppressive action ₂₁the preparation method of steroidal glycoside---SMA, the method comprises the following steps:

A. by after the root of wax flower (Stephanotis mucronata) or rattan pulverizing, with extraction using alcohol;

B. ethanol extract concentrated after with organic solvent extraction or wax flower root or rattan meal directly with organic solvent extraction, concentrated, obtain total steroid saponin;

C. total steroid saponin carries out chromatographic separation in silica gel or alumina column, and take methyl alcohol, ethanol, ethyl acetate, chloroform, methylene dichloride or their mixture is eluent;

D. silica gel thin-layer chromatography checks elutriant, and the elutriant merging with same blob is concentrated, obtains the stream part containing SMA;

E. the stream part that contains SMA, again through silica gel (comprising Rp-18, Rp-8) or HPLC chromatography, with eluent wash-out, separation, obtains C ₂₁steroidal glycoside---SMA.

3. preparation method according to claim 2, is characterized in that in step b, said organic solvent is chloroform, methylene dichloride, ethyl acetate.

4. preparation method according to claim 2, is characterized in that in step e, said eluent is the mixture of first alcohol and water or acetonitrile and water or acetone and water.

5. new C according to claim 1 ₂₁steroidal glycoside---SMA, is characterized in that having the pharmaceutical composition of immunosuppression and resisting rheumatoid arthritis.

6. new C according to claim 1 ₂₁steroidal glycoside---SMA, is characterized in that wherein comprising SMA and the pharmaceutically acceptable carrier for the treatment of significant quantity.

7. new C according to claim 5 ₂₁steroidal glycoside---SMA, is characterized in that it is 0.1%～99.5% that SMA in pharmaceutical composition accounts for gross weight ratio.

8. new C according to claim 1 ₂₁steroidal glycoside---SMA, is characterized in that this compound has the application in the medicine of preparation immunosuppressor and preparation treatment rheumatoid arthritis.

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