C₂₁ Steroidal Glycosides From Two Species Of Asclepiadaceae And Their Immunological Activities

Immunosuppressive agents are the mainstay treatment for patients that have received organ grafts and are becoming increasingly important in the treatment of autoimmune diseases. The development of high-powered with less side effects immunosuppressive agents is the hotspot of pharmaceutical chemists at all times. Cynanchum chekiangense M. Cheng ex Tsiang et PT Li. (Cynanchum Linn.) and Stephanotis mucronata (Blanco) Merr. (Stephanotis Thou.) are used to treat rheumatoid arthritis and rheumatic aches in Chinese folk medicine. In order to obtain new immunoactive compounds, chemical and immunological studies on the main active components, C-21 steroidal glycoside, of these two species were undertaken by screening with immunological tests in vitro and led to:Two new C₂₁ steroidal glycosides were isolated from the CHCl₃ soluble extract of the roots of Cynanchum chekiangense, named chekiangensosides A (K-1) and B (K-3), together with two known compounds. On the basis of chemical evidence and extensive spectroscopic methods, including one-dimensional and two-dimensional NMR, the structures of two new compounds were identified as cynajapogenin A 3-O-β-D-gIucopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranoside (chekiagensoside A, K-1), and glaucogenin A 3-O-β-D-glucopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4) -α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranoside (chekiagensoside B, K-3), respectively. The two known steroidal glycosides, K-2 and K-4, were determined as cynascyroside C and glaucoside H. On the basis of ¹³C-NMR, their structures were revised as cynajapogenin A 3-O-β-D-glucopyranosyl-(1→4)-α-L-cymaropyranosyl-(1→4)-β-D-digitoxopyranosyl-(1 →4)-β-D-cymaropyranoside (cynascyroside C), and glaucogenin A 3-O-β-D-gIucopyranosyl-(1 →4)-α-L-cymaropyranosyl-( 1 →4)-β-D-digitoxopyranosyl-( 1 →4)-β-D-cymaropyranoside(glaucoside H), respectively. These isolated compounds were tested for their immunological activities in vitro against concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced proliferation of mice splenocytes. Compounds K-1-K-4 showed immunosuppressive activities in vitro in a dose-dependent manner.Thirteen new C2i steroidal glycosides (M-1-M-13) were isolated from the CHC13 soluble extract of the stems of Stephanotis mucronata, together with four known compounds (M-14— M-17). Their structures were determined on the basis of chemical evidence and extensive spectroscopic methods including one-dimensional and two-dimensional NMR. Compounds M-1-M-13 were elucidated as 12-O-acetyl-20-O-tigloyl-5,6-dihydrosarcostin 3-0-/?-D-glucopy-ranosyl-( 1 —>4)-6-deoxy-3 -O-methyl-/?-D-allopyranosyl-( 1 —?4)-/?-D-cymaropyranosyl-(l —*4)-(3-D-cymaropyranoside (stemucronatoside H, M-1), 20-Otigloyl-5,6-dihydrosarcostin 3-O-/3-D-glucopyranosyl-(l—>4)-6-deoxy-3-0-methyl-y5-D-allopyranosyl-(l-+4)-y5-D-cymaropyranosyl-(1—>4)-/?-D-cymaropyranoside (stemucronatoside I, M-2), 12-O-deacetylmetaplexigenin 3-0-/?-D-glucopyranosyl-(l—>4)-6-deoxy-3-O-methyl-/?-D-allopyranosyl-(l—*4)-y5-D-cymaropyranosyl -(1—>4)-y#-D-cymaropyranoside (mucronatosides E, M-3), sarcostin 3-0-/?-D-glucopyranosyl-(l ->4)-6-deoxy-3-O-methyl-y9-D-allopyranosyl-(l->4)-/?-D-cymaropyranosyl-(l->4)-/?-D-cyma-ropyranoside (mucronatosides F, M-4), 12-O-cinnamoyl-20-O-tigloyl(205)-pregn-6-ene-3/S,5a, 8/?, 12/3,14p, 17/?,20-heptanol 3-0-/?-D-glucopyranosyl-(l -+4)-6-deoxy-3-O-methyl-y?-D-allopy-ranosyl-(l-^4)-y5-D-cymaropyranosyl-(l-^4)-/?-D-cymaropyranoside (mucronatoside G, M-5), 12-O-cinnamoyl-20-O-acetyl(20S)-pregn-6-ene-3 p,5 a,8/3,12/?, 14(3,17/?,20-heptanol 3-0-/?-D-thevetopyranosyl-(l—>4)-/?-D-cymaropyranosyl-(l-^4)-/?-D-cymaropyranoside (mucronatoside Hs M-6), 12-0-N-methylanthraniloyl sarcostin 3-0-y3-D-thevetopyranosyl-(l-*4)-^-D-cymarop-yranosyl-(l-^-4)-/?-D-cymaropyranoside (stemucronatoside J, M-7), deacetylkidjoladinin 3-0-/?-D-thevetopyranosyl-( 1 —>4)-/?-D-cymaropyranosyl-( 1 -+4)-/?-D-cymaropyranoside (mucronatoside I, M-8), 12-O-cinnamoyl-20-<9-acetyl(205)-pregn-6-ene-3^,5a,8/?,12/?,14y9,17/?,20-heptanol 3-O-/?-D-glucopyranosyl-(l —4)-6-deoxy-3-(9-methyl-/?-D-allopyranosyl-(l ->4)-/MD -cymaropyranosyl-(l—>4)-y#-D-cymaropyranoside (mucronatoside J, M-9), deacetylkidjoladinin 3-O-/?-D-glucopyranosyl-(l-+4)-6-deoxy-3-O-methyl-y5-D-allopyranosyl-(l—>4)-/?-D-cymarop-yranosyl-(l-^4)-/^-D-cymaropyranoside (mucronatoside K, M-10), 12-0-cinammoyl-2O-0-tigloyl-5,6-dihydrosarcostin 3-0-/?-D-glucopyranosyl-(1^4)-6-deoxy-3-0-methyl-/?-D-allopy-ranosyl -(1—?4)-/?-D-cymaropyranosyl-(l—>4)-/?-D-cymaropyranoside (mucronatoside L, M-11), 12-O-tigloyl-20-O-N-methylanthraniloyl sarcostin 3-0-/?-D-glucopyranosyl-(l —>4)-6-deoxy -3-0-methyl-/?-D-allopyranosyl-( 1 —>4)-/?-D-cymaropyranosyl-( 1 ^4)-/?-D-cymaropyranoside (mucronatoside M, M-12), and 12-O-cinnamoyl-20-<9-nicotinoyl(205)-pregn-6-ene-3y9,5o:,8/?, 12/?,14^,17/?,20-heptanol 3-0-y9-D-glucopyranosyl-(l-*4)-6-deoxy-3-0-methyl-/?-D-allopyrano-syl-(l-*4)-y^-D-cymaropyranosyl-(1^4)-/?-D-cymaropyranoside (mucronatoside N, M-13), respectively. The four known compounds M-14-M-17 were determined as sinomarinoside A, sinomarinoside E, stephanoside H, and stephanoside M, respectively. The results of splenocyte proliferation assay in vitro showed that M-1 > M-2> M-3s M-5 and M-15 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, M-4-. M-6x M-7x M-14 and M-16 significantly inhibited the Con A- and LPS-induced mice splenocyte proliferation in a concentration-dependent manner.