Ratanhiaphenol III from Ratanhiae radix is a PTP1B inhibitor.

Treatment and prevention of type 2 diabetes mellitus (T2DM) with its severe mostly hyperglycemia-induced micro- and macrovascular complications are major challenges of the present age [1]. Aberrantly high levels of visceral fat and subsequently acquired insulin resistance often precede the onset of T2DM. Currently used insulin sensitizers still have many side effects including weight gain or bone loss [2]. An optimal treatment for T2DM would consist of an increase in insulin sensitivity and weight loss. Protein tyrosine phosphatase 1B (PTP1B) [3] is a promising drug target since it negatively regulates insulin and leptin signalling, and its inhibition therefore leads to increased insulin sensitivity as well as higher energy expenditure, less food intake, and less weight gain [4-6]. In the course of a previous project focussing on constituents of Ratanhiae radix with antiphlogistic properties we have isolated and identified several benzofuran derivatives with marked anti-inflammatory activity in vitro and in vivo and provided a molecular explanation for the traditional use of this herbal drug against oropharyngeal inflammation [7]. Since chronic inflammation contributes to and, directly or indirectly, shares common molecular hubs with obesity and diabetes, we were prompted to investigate whether a lipophilic CH2Cl2 (DCM) extract of Ratanhiae radix (RR_ex) as well as the previously isolated lignan derivatives [7] also exert a beneficial impact on metabolism. Of note, several benzofuran derivatives have already been reported as PTP1B inhibitors (e.g., [8]). We therefore examined the PTP1B inhibiting potential of RR_ex and the lignans in vitro and whether they would enhance insulin signalling and glucose uptake in a cell-based model. In Fig. 1A we show that RR_ex (please see Fig. 1S in Supporting Information for an HPL chromatogram of the extract) concentration-dependently (3-30 μg/mL) reduced human recombinant PTP1B activity. A known natural product-derived PTP1B inhibitor, ursolic acid (UA, 10 μM) [9], was used as a positive control in this colorimetric in vitro enzyme assay. The same extract at a concentration of 30 μg/mL was able to significantly increase glucose uptake into murine C2C12 myocytes both in the absence and presence of insulin (Fig. 1B). Fig. 1 A DCM extract of Ratanhiae radix inhibits PTP1B in vitro and elevates glucose uptake into C2C12 myocytes. A Different concentrations of a DCM extract of Ratanhiae radix (RR_ex) were subjected to a PTP1B in vitro enzyme assay. Ursolic acid (UA, 10 μM) ... Prompted by these promising findings, we next examined eleven constituents recently isolated from RR_ex (benzofurans 1, 4-11, and 7,7′-epoxylignans 2 and 3; for structures, see Fig. 2S) [8] with regard to their capability of inhibiting PTP1B. At a concentration of 30 μM, only one compound, namely ratanhiaphenol III [compound (cpd) 9 in Fig. 2S], significantly and markedly suppressed PTP1B activity, as shown in Fig. 2A. This is surprising since several benzofuran derivatives (viz. cpds 1, 4–8, 10, 11) isolated from RR_ex just differ marginally from ratanhiaphenol III. Apparently, the isolated dihydrofurans (cpds 2 and 3) do not contribute to any PTP1B inhibiting activity. The prop-1-enyl moiety on position 5 of the 2-phenylbenzofuran skeleton seems to be one essential feature, whereas the corresponding hydroxypropyl derivatives are inactive (cpds 1 and 5). The substitution pattern of the phenyl ring obviously has a tremendous impact on the activity. A para substitution of a hydroxyl-group alone (cpd 8) does not reveal any activity; however, in combination with a methoxy-group in the ortho position (cpd 9), a distinct PTP1B inhibition could be observed. The impact of further variations on the 2-phenylbenzofuran skeleton with respect to the substitution pattern of the phenyl ring and the degree of saturation in the furan moiety remains to be clarified due to the limited number of available natural derivatives. Fig. 2 Ratanhiaphenol III from RR_ex inhibits PTP1B in vitro with an IC50 of 20 μM and enhances insulin-mediated glucose uptake and insulin receptor phosphorylation in C2C12 cells. A Compounds 1–11 (for structures, see Fig. 2S) from RR_ex were ... Testing of ratanhiaphenol III in different concentrations in the in vitro PTP1B assay revealed an apparent IC50 value of 20.2 μM ( Fig. 2B). Ratanhiaphenol III was furthermore administered to C2C12 myocytes and examined concerning its influence on glucose disposal ( Fig. 2C). In concentrations of 10 and 30 μM, ratanhiaphenol III significantly boosted the insulin-stimulated glucose uptake whereas the basal glucose uptake rate was not affected. This finding is in agreement with the inhibition of PTP1B by ratanhiaphenol III and subsequent sensitization of cells for the action of insulin. The increased basal glucose uptake observed upon exposure to the extract (see Fig. 1B) apparently cannot be assigned to the action of ratanhiaphenol III and seems to be, at least partly, dependent on activation of AMP-activated kinase presumably mediated by other compounds in the extract (Fig. 3S). Inhibition of PTP1B by ratanhiaphenol III in the cell-based model is further underpinned by the increased insulin-triggered phosphorylation of the insulin receptor at tyrosines Y1150 and Y1151, sites normally dephosphorylated by PTP1B [4]. Total levels of PTP1B remain, hereby, unaffected ( Fig. 2D). These findings demonstrate that ratanhiaphenol III is one PTP1B-inhibiting principle of RR_ex in vitro and consistently enhances the insulin response in relevant cell-based models. Our data demonstrate for the first time the beneficial potential of constituents in Ratanhiae radix, a traditionally anti-inflammatory herb, in the context of metabolic dysfunction. Its DCM extract elevates glucose disposal partly via inhibition of PTP1B by ratanhiaphenol III. Our findings encourage the examination of other herbal remedies for indications different from their traditional use in folk medicine. Such repurposing of herbal drugs is highly facilitated by the knowledge of the active compounds and/or their mode of action, as well as by the identification of common druggable molecular master switches involved in the etiology of seemingly unrelated disorders. It can be achieved by the synergy between phytochemistry and molecular pharmacology.