Salvia miltiorrhiza subsp. roots

Chen, Haimin, Chen, Jialing, Qi, Yao, Chu, Siyuan, Ma, Yao, Xu, Linna, Lv, Shiyi, Zhang, Haihua, Yang, Dongfeng, Zhu, Yonghong, Mans, Dennis Ra. & Liang, Zongsuo, 2022, Endophytic fungus Cladosporium tenuissimum DF 11, an efficient inducer of tanshinone biosynthesis in Salvia miltiorrhiza roots, Phytochemistry (113021) 194, pp. 1-10 : 7

5.5. HPLC analysis of tanshinones in the S. miltiorrhiza roots

Tanshinones and salvianolic acids were analysed by a gradient elution program of HPLC, which established by xing et al. ( Xing et al., 2018; Zhang et al., 2020a). To dry the S. miltiorrhiza roots and crush them into powder and to accurately weigh 0.02 g, 70% methanol was added to the root powder and soaked overnight. After 45 min of ultrasonic extraction and centrifugation for 10 min at 10,000 rpm, the supernatant was filtered with a 0.22 μm filter membrane. Subsequently, the tanshinone contents of the samples were determined by a binary high-performance liquid chromatograph (Waters e2695, USA) with a Waters 2996 diode array detector and a Waters Sunfire C18 chromatographic column (250 mm × 4.6 mm, 5 μm), while the data acquisition software was Empower 3. Chromatographic conditions included a sample volume of 10 μl, column temperature of 30 ◦ C, flow rate of 1 mL min 1, and mobile phase of 0.026% phosphoric acid aqueous solution and acetonitrile.The detection wavelength of tanshinones was set at 270 nm, while that of salvianolic acids was set at 288 nm. The HPLC program was shown in Supplementary Table S1. The standard curves of tanshinones and salvianolic acid were shown in Supplementary Figure S1.

Figures

Fig. 1. The tanshinone biosynthesis pathway in S. miltiorrhiza. AACT: acetyl-CoA C-acetyltransferase, HMGS: 3-hydroxy-3- methylglutaryl-CoA synthase, HMGR: 3-hydroxy-3-methylglutaryl-CoA reductase, MK: mevalonate kinase, PMK: 5-phosphomevalonate kinase, MDC: mevalonate 5-diphosphate decarboxylase, DXS: 1-deoxy-Dxylulose-5- phosphate synthase, DXR: 1- deoxy-D-xylulose-5- phosphatereductoisomerase, MCT: 2-C-methyl-D- erythritol- 4-phosphate cytidylyltransferase, CMK: 4- (cytidine 5-diphospho) -2-C-methyl- Derythritolkinase, MECPS: 2-C-methyl- erythritol 2,4-cyclodiphosphatesynthase, HDS: 1-hydroxy-2- methyl-2-(E)- butenyl-4-diphosphate synthase, HDR: 1-hydroxy-2-methyl-2- (E)- butenyl-4- diphosphate reductase), IDI: isopentenyl diphosphate isomerase, GGPPS: geranylgeranyl diphosphate synthase, CPS: copalyl diphosphate synthase, KSL: kaurene synthase-like, CYP76AH1: cytochrome P450 enzyme (CYP) 76AH1.

Fig. 2. Morphological characteristics and phylogenetic analysis of strain DF11. (a)and(b) Frontal and backside morphology of DF11 colony respectively (Scale bar = 1.0 cm); (c)and(d) Microscopic morphology of spores and mycelium of DF11 respectively (40 × 10, Scale bar = 20 μm); (e)and(g) mycelium structure of DF11 showed by scanning electron microscopy (SEM); (e) Mycelium and conidiophore; (f) Conidiophore; (g) Conidium; (h) Neighbor-joining tree of DF11 based on the ITS gene sequences.

Fig. 3. Root phenotypes of S. miltiorrhiza seedlings 8 weeks after strain DF11 inocculation. (a)the control group inoculated with PDA liquid, (b) the treatment group inoculated with DF11 fungal suspension, (c) and (d) Red boxed areas of (a) and (b) are enlarged in (c) and (d) respectively. Scale bar = 1.0 cm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4. The colonization of DF11 in the root of aseptic seedling of S. miltiorrhiza after immunofluorescence staining (8 weeks). Magnify 400x; Green: ConA-FITC; Blue: DAPI. Control: PDA sterile liquid medium. Red arrow: DF11 is located within the root cell; Red triangle: DF11 is located in the root cell space. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5. DF11 effect on the accumulation of tanshinones and salvianolic acid in aseptic seedling roots of S. miltiorrhiza. Compared with the control group, *p <0.05, **p <0.01, ***p <0.001.