Transcriptome Analysis Provides Insights into Catalpol Biosynthesis in the Medicinal Plant Rehmannia glutinosa and the Functional Characterization of RgGES Genes.

Rehmannia glutinosa, a member of the Scrophulariaceae family, has been widely used in traditional Chinese medicine since ancient times. The main bioactive component of R. glutinosa is catalpol. However, the biogenesis of catalpol, especially its downstream pathway, remains unclear. To identify candidate genes involved in the biosynthesis of catalpol, transcriptomes were constructed from R. glutinosa using the young leaves of three cultivars, Beijing No. 3, Huaifeng, and Jin No. 9, as well as the tuberous roots and adventitious roots of the Jin No. 9 cultivar. As a result, 71,142 unigenes with functional annotations were generated. A comparative analysis of the R. glutinosa transcriptomes identified over 200 unigenes of 13 enzymes potentially involved in the downstream steps of catalpol formation, including 9 genes encoding UGTs, 13 for aldehyde dehydrogenases, 70 for oxidoreductases, 44 for CYP450s, 22 for dehydratases, 30 for decarboxylases, 19 for hydroxylases, and 10 for epoxidases. Moreover, two novel genes encoding geraniol synthase (RgGES), which is the first committed enzyme in catalpol production, were cloned from R. glutinosa. The purified recombinant proteins of RgGESs effectively converted GPP to geraniol. This study is the first to discover putative genes coding the tailoring enzymes mentioned above in catalpol biosynthesis, and functionally characterize the enzyme-coding gene in this pathway in R. glutinosa. The results enrich genetic resources for engineering the biosynthetic pathway of catalpol and iridoids.

Comparison of Inhibitory Effects of Safflower Decoction and Safflower Injection on Protein and mRNA Expressions of iNOS and IL-1ß in LPS-Activated RAW264.7 Cells.

OBJECTIVE: Safflower has antioxidant and anti-inflammatory activities. The two forms of preparations for safflower which are widely used in China are injection and decoction. The first step of the process for preparing an injection involves extracting safflower with water, which actually yields a decoction. This study is intended to investigate how the preparation process influences the anti-inflammatory activity of safflower in vitro. METHODS: Five samples, including a decoction (sample 1) and an injection (sample 5) of safflower, were prepared according to the national standard WS3-B-3825-98-2012 and were analyzed by the oxygen radical absorbance capacity (ORAC) method and the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) method for comparison. Sample 1 and sample 5 were further tested by the Griess assay and ELISA for their effects on nitric oxide (NO) production and interleukin- (IL-) 1ß content in lipopolysaccharide- (LPS-) activated RAW264.7 cells. The protein and mRNA levels of inducible nitric oxide synthase (iNOS) and IL-1ß were measured by Western blotting and real-time quantitative PCR. RESULTS: Sample 5 showed a significantly higher ORAC value and a lower half inhibitory concentration (IC50) for DPPH scavenging activity as compared to the other four samples (p < 0.05). LPS significantly upregulated the mRNA and protein expressions of iNOS and IL-1ß as compared to the solvent control (p < 0.01). As compared to sample 1, sample 5 significantly decreased NO production, iNOS protein expression, and the contents of IL-1ß mRNA and IL-1ß protein at both 100 µg/ml and 200 µg/ml (all: p < 0.05) and significantly downregulated iNOS mRNA expression at 100 µg/ml (p < 0.05). CONCLUSIONS: Results of this study demonstrate that the safflower injection prepared according to the national standard has a significant effect of suppressing protein and mRNA expressions of iNOS and IL-1ß as compared to its traditional decoction.