四 of 555 endorf.Hamburg. cler ep Germ Spearman 到and P.value 50351 .36±0 cousnitz and clinical parameters 如 05 HD groups, Results 品e品 rom was observe in plas Spplmmlarha majo nts are shown in the ous erse events occurred uero noimg o in COD decoction ( bar-coded the structural ange9uor 1) as on sity curve altho ed with the eeks of treatment equencing. most of the diversityhac ients.The HD and MD group 1T2 eicrobiote 1.5 可i 1 GOD signif ontrol and HOMA-8 in T2D pa al Ch in FBG.(b)cha The ISME Journalfurther analysis. Standard and quantified samples were performed in triplicate. PCR reactions were performed using iQ SYBR Green SuperMix (Bio￾Rad, Richmond, CA, USA) on a MasterCycler ep Realplex 4s (Eppendorf, Hamburg, Germany). Spearman’s correlation coefficient (R) and P-value were used to compare the amounts of F. prausnitzii measured by q-PCR and pyrosequencing. This coefficient was also used to evaluate the relation￾ship between F. prausnitzii and clinical parameters using MATLAB 2010b. Results The major components of GQD decoction There were four major categories of compounds in the GQD decoction. Flavones (baicalin, puerarin, wogonoside, daidzin, liquiritin, baicalein and wogonin), alkaloids (berberine, coptisine, palmatine and jatrorrhizine) and triterpenoid sapnins (glycyrrhizin) were detected in the decoction, among which baicalin, puerarin and berberine were the major components (Supplementary Table 2). The chemical structures of these 12 components are shown in the Supplementary Figure 2c. Carbohydrates (starch, sucrose, reducing sugar and soluble dietary fiber) were also detected. Insoluble dietary fiber was undetectable in GQD decoction (Supplementary Table 3). GQD significantly improved glycemic control in T2D patients In our 12-week, randomized, double-blinded, placebo-controlled clinical trial (Supplementary Figure 1), the data of 187 participants were analyzed as shown in Supplementary Table 4. The baseline variables were not significantly different among the four groups. After 12 weeks of treatment, GQD significantly improved glycemic control in T2D patients. The HD and MD groups, when compared with the placebo and LD groups, showed significant reductions in adjusted mean changes from baseline of FBG ( 1.46±0.23 and 1.09±0.21 vs 0.16±0.22 and 0.24±0.24 mmol l 1 ; Po0.001 for HD vs LD and placebo; Po0.01 for MD vs LD and placebo). Similarly, the HD and MD groups showed significantly reduced HbA1c ( 0.88±0.14 and 0.75±0.13 vs 0.35±0.13 and 0.36±0.15%; Po0.01 for HD vs LD; Po0.05 for HD vs placebo; Po0.05, MD vs LD and placebo) (Figures 1a and b). A decrease in the mean change of 2h-PBG from baseline was also observed in the treated groups, although not reaching significant level. (Supplementary Figure 3). In addition, ANCOVA analysis showed that HOMA-b was significantly improved by HD GQD treatment compared with the placebo and LD groups (Figure 1c). Plasma orosomucoid was significantly reduced by HD GQD treatment (P ¼ 0.023) (Supplementary Figure 4a) and the HD group showed a significant reduction (P ¼ 0.034) in mean change from baseline of plasma orosomucoid compared with the LD group (Supplementary Figure 4b). No significant differ￾ence was observed in plasma adiponectin, tumor necrosis factor-a or serum amyloid A among the four groups (Supplementary Figures 5a–c). Finally, no drug-related serious adverse events occurred in this study. Overall structural modulation of gut microbiota after GQD treatment First, we used a bar-coded pyrosequencing run to analyze the structural changes of gut microbiota in the four groups before and after GQD treatment. In total, 483 304 usable raw sequences (34 753 unique sequences) and 3222 OTUs were obtained from 235 samples with an average of 2057±396 per sample. Rarefaction and Shannon diversity curves revealed that, although no rarefaction curves plateaued with the current sequencing, most of the diversity had already been captured (Supplementary Figure 6). Weighted and unweighted UniFrac PCoA analysis revealed that gut microbiota structure of the treated groups showed a dose-dependent deviation Figure 1 GQD significantly improved glycemic control and HOMA-b in T2D patients. (a) Change in FBG, (b) change in HbA1c and (c) change in HOMA-b. Placebo (n ¼ 41), LD (n ¼ 50), MD (n ¼ 52) and HD (n ¼ 44). Data are presented as mean±S.E.M. *Po0.05, **Po0.01 and ***Po0.001 vs placebo using ANCOVA; þ Po0.05, þ þ Po0.01 and þþþ Po0.001 vs LD using ANCOVA. Microbiota shift in alleviation of type 2 diabetes J Xu et al 555 The ISME Journal