| C21 steroidal glycosides are one kind of important biological active compounds widely in the plants of Asclepiadaceae family and have been shown to possess extensive pharmacological effects such as inhibition against proliferation and invasion of tumor cells, immunomodulating, antifertility, antiepilepsy, and anti-acetylcholinesterase (AChE) activities. Therefore, C21 steroidal glycosides have attracted much attention from researchers in the world for their isolation, elucidation of their structures and pharmacological effect. The dried roots of Stephanotis mucronata (Asclepiadaceae) are used for the treatment of rheumatoid arthritis and rheumatic aches in folk medicine of southern China. However, the principle of Stephanotis mucronata used for the treatment of these diseases is still unknown. The study on isolation of total C21 steroidal glycosides from the roots of Stephanotis mucronata was undertaken, leading to 10 new C21 steroidal glycosides, named as stemucronatosides A-G(6-12) and K-M(13-15) respectively. The effects of these 10 compounds on mice splenocyte proliferation were determined in vitro by MTT method. Stemucronatoside K(13), the most main constituent, shown to have significant immunosuppressive activity in vitro, was further selected for evaluation of its immunosuppressive effect in vivo, effect on serum agglutinin in mice and prevention and treatment of adjuvant-induced arthritis in rats.The soluble fraction in CHCl3 of ethanol extract from the roots of Stephanotis mucronata was isolated and purified with column chromatography (CC, normal silica gel), Rp-C18 CC, Sephadex LH-20 CC or HPLC respectively to afford 10 new compounds. Their structures were determined on the basis of chemical evidence and extensive spectroscopic methods including ESI-MS, HRESI-MS, 1H NMR, 13C NMR, 1H-1H COSY, DEPT, HMQC and HMBC techniques.Their structures were elucidated as 12-0-deacetylmetaplexigenin 3-0-6-deoxy-3-0-methyl-/￡-D-allopyranosyl-(l —>4)-/?-D-cymaropyranosyl-(l —>4)-/?-D-cymaropyranoside (stemucronatoside A, 6), 12-0-deacetylmetaplexigenin 3-0-/^D-thevetopyranosyl-(l—*4)-/?-D- cymaropyranosyl-(l—> 4)-/?-D-cymaropyranoside (stemucronatoside B, 7), metaplexigenin 3-0-ft- D-glucopyranosyl-(1—>4)-6-deoxy-3-6>-methyl-/^-D-allopyranosyl-(l-*4)-^-D-cymaropyranosyl-(l—>4)-/?-D-cymaropyranoside (stemucronatoside C, 8), 20-0-tigloylsarcostin 3-0-/?-D-thevetopyranosyl-(l —>4)-/?-D-cymaropyranosyl-(l—>4)-/￡-D-cymaropyranoside (stemucronatoside D, 9), 20-0-tigloyl-5,6-dihydrosarcostin 3-0-/?-D-thevetopyranosyl-(l —>4)-/?-D-cymaropyranosyl-(l —*4)-J3-D-cymaropyranoside (stemucronatoside E, 10), rostratamine 3-0-/?-D-thevetopyranosyl-(l —*4) -/?-D-cymaropyranosyl-( 1 —>4)-/?-D-cymaropyranoside (stemucronatoside F, 11), 12-0-acetyl -20-0-tigloylsarcostin 3-0-y9-D-glucopyranosyl-(l—>4)-6-deoxy-3-0-methyl-/?-D-allopyranosyl-(1—*4)-y5-D-cymaropyranosyl-(1^4)-/0-D-cymaropyranoside (stemucronatoside G, 12), 12-0-acetyl-2O-0-(iV-methyl) anthraniloylsarcostin 3-0-/?-D-glucopyranosyl-(l —>4)-6-deoxy-3-0-methyl-^-D-allopyranosyl-(l —>4)-yff-D-cymaropyranosyl-(l ^4)-/?-D-cymaropyranoside (stemucronatoside K, 13), 12-0-acetyl-2O-0-(jV-methyl) anthraniloyl-5,6-dihydrosarcostin 3-0-/5-D-glucopyranosyl-(l—>4)-6-deoxy-3-0-methyl-/?-D-allopyranosyl-(l-^4)-/?-D-cymaropyranosyl-(l —^-/^-D-cymaropyranoside (stemucronatoside L, 14), 12-0-nicotinoyl-2O-0-cinnamoylsarco-stin 3-0-y9-D-glucopyranosyl-(l-^4)-6-deoxy-3-0-methyl-y0-D-allopyranosyl-(l—>4)-fi-D-cymaropyranosyl-(l-^-4)-/?-D-cymaropyranoside (stemucronatoside M, 15), respectively.The results of splenocyte proliferation assay in vitro showed that stemucronatosides A, B, C and F significantly enhanced the concanavalin A (Con-A)- and lipopolysaccharide (LPS)-induced mice splenocyte proliferation. The concentration-effect curves showed their immunomodulating effect to be bidirectional. However, stemucronatosides D, E, G, K, L, and M significantlyinhibited the Con A- and LPS-induced mice splenocyte proliferation in a concentration-dependent manner. The results of stemucronatoside K on the cellular and humoral immune responses of ovalbumin (OVA)- immunized mice showed that it not only significantly inhibited T splenocyte proliferation stimulated by Con A and B splenocyte proliferation stimulated by OVA, but also significantly decreased the serum OVA-specific IgG level in mice immunized with OVA in a dose-dependent manner. Stemucronatoside K significantly inhibited producing antibody in the experiment of serum agglutinin in mice. Stemucronatoside K showed significant inhibition against early local primary inflammation and subsequent inflammation after 11 days of adjuvant-induced arthritis in rats, and significant prevention and treatment of subsequent pathological changes such as red swelling in the front feet and nodes in the tails of rats.
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