Terpene Glycosides from the Roots of Sanguisorba officinalis L. and Their Hemostatic Activities

Guided by a hemostasis bioassay, seven terpene glycosides were isolated from the roots of Sanguisorba officinalis L. by silica gel column chromatography and preparative HPLC. On the grounds of chemical and spectroscopic methods, their structures were identified as citronellol-1-O-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside (1), geraniol-1-O-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside (2), geraniol-1-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside (3), 3β-[(α-L-arabinopyranosyl)oxy]-19α-hydroxyolean-12-en-28-oic acid 28-β-D-glucopyranoside (4), 3β-[(α-L-arabinopyranosyl)-oxy]-19α-hydroxyurs-12-en-28-oic acid 28-β-D-glucopyranoside (ziyu-glycoside I, 5), 3β,19α-hydroxyolean-12-en-28-oic acid 28-β-D-glucopyranoside (6) and 3β,19α-di-hydroxyurs-12-en-28-oic acid 28-β-D-glucopyranoside (7). Compound 1 is a new mono-terpene glycoside and compounds 2, 3 and 5 were isolated from the Sanguisorba genus for the first time. Compounds 1–7 were assayed for their hemostatic activities with a Goat Anti-Human α2-plasmin inhibitor ELISA kit, and ziyu-glycoside I (5) showed the strongest hemostatic activity among the seven terpene glycosides. This is the first report that ziyu-glycoside I has strong hemostatic activity.


Introduction
Sanguisorba officinalis L. (Rosaceae) is a perennial plant widely distributed in China, and its roots have been used as a traditional Chinese medicine for the treatment of hemostasis and inflammation [1].

OPEN ACCESS
A variety of chemical constituents, including tannins, triterpenoids, flavonoids, anthraquinones, steroids were isolated from S. officinalis L., and pharmacological studies on its hemostatic and antiinflammatory properties have been reported [2,3], but the molecular level mechanisms of these activities have not been reported until now. In order to study the mechanism of hemostasis, we have carried out the bioassay-guided isolation and identification of hemostatic constituents of the roots of S. officinalis L. In the present study, we report the isolation and identification of a new monoterpene glycoside 1, together with two known monoterpene glycosides 2, 3 and four known triterpenoids 4-7, and their hemostatic activities.

Bioactivity Assay
The hemostasis assay was carried out by using a Goat Anti-Human 2-plasmin inhibitor kit which is a solid phase sandwich enzyme-linked quantitative immunoabsorbent assay (ELISA) with a purified antibody specific for α2-plasmin inhibitors. The above separated fractions and compounds were dissolved in dimethylsulfoxide (DMSO) (0.188 mg/μL for the fractions; 0.094 mg/μL for compounds), and were diluted with sample diluent (1:1). After an aliquot (50 μL) of the above samples or standards was added to each microplate well, a portion of biotinylated anti-α2-plasmin inhibitor (50 μL) was immediately added to each well, and the microplate was incubated for 1 h at 37 °C. After a wash with washing buffer, streptavidin-HRP was added, and the microplate was incubated for 30 min at 37 °C. Again, after a wash with washing buffer, substrate A and substrate B (50 μL each) were added to each well, and the microplate was incubated for 10 min at 37 °C. The enzyme-substrate reaction was stopped by quickly pipetting stopping solution (50 μL) into each well. The optical density absorbance (OD value) of each well was measured at 450 nm wavelength on a DG5033A enzyme immunoassay spectrophotometer. All the separated fractions and isolated compounds were tested for their hemostasis activities, and the results are summarized in Table 2.
The correlation of calibration curve test data is shown in Table 3. By using OD value as Y-axis(Y) and standards cocentration as X-axis (X), the linear regression equation, y = 0.0687x + 0.0317, r = 0.9997, was obtained. The results showed that the linear relation between OD value and concentration of standards were good and linearity domain of the measure is 0~40g/L. Therefore the OD value is directly proportional the concentration of α2-plasmin inhibitor present in the samples.

Conclusions
Compound 1 is a new monoterpene glycoside and compounds 2, 3 and 5 were isolated from the Sanguisorba genus for the first time. Compounds 1-7 were assayed for their hemostatic activities with a Goat Anti-Human α2-plasmin inhibitor ELISA kit, and the hemostatic constituent of S. officinalis wasidentified as ziyu-glycoside I (5). This is the first report that ziyu-glycoside I has strong hemostatic activity.