Rhizome

As described previously (Lum et al., 2002 (link)), we divided ginseng into its main root (MR), lateral root (LR) and rhizome head (RH) (Figure 1). Different parts were weighed on an electronic scale (0.01 g), and the root mass ratio (RMR) was calculated (g·g⁻¹) (Gregory et al., 1995 (link)). We placed the plant parts on a glass board covered with graph paper to measure their length (0.1 mm) and calculated the specific root length (SRL) (cm·g⁻¹) (Ostonen et al., 2007 (link)). The total length and biomass were determined as the sum of every part. Average values were calculated from 25 samples per developmental stage of F. Ginseng. Relative growth rate (RGR) was measured as the increase in mass per biomass per year and was calculated using the following equation: RGR = (ln W2 − ln W1) / (t2 − t1); where ln = natural logarithm, t1 = time one (in years), t2 = time two (in years), W1 = weight of plant at time one (in grams), W2 = weight of plant at time two (in grams). The phenotypic plasticity index [PPI, (F. Ginseng mean − W. Ginseng mean)/W. Ginseng mean] was calculated for each trait (Caplan and Yeakley, 2013 (link)), which was used to evaluate the morphological difference between F. Ginsengs (in different growth time) and W. Ginseng.

GC–MS analyses of leaf and rhizome extracts were carried out using the Perkin-Elmer Clarus 680 system (Perkin-Elmer, Inc. U.S.A) equipped with a fused silica column, packed with Elite-5MS) capillary column (30 m in length × 250 μm in diameter × 0.25 μm in thickness). Pure helium gas (99.99%) was used as the carrier gas at a constant flow rate of 1 mL/min. For GC–MS spectral detection, an electron ionization energy method was adopted with high ionization energy of 70 eV (electron Volts) with 0.2 s of scan time and fragments ranging from 40 to 600 m/z. The injection quantity of 1 μL was used (split ratio 10:1), and the injector temperature was maintained at 250 °C (constant). The column oven temperature was set at 50 °C for 3 min, raised at 10 °C per min up to 280 °C, and final temperature was increased to 300 °C for 10 min. The contents of phytochemicals present in the test samples were identified based on comparison of their retention time (min), peak area, peak height and mass spectral patterns with those spectral database of authentic compounds stored in the National Institute of Standards and Technology (NIST) library⁶⁰ .

UV-Vis spectrophotometrical assays were used to determine the total content of flavonoids (as mg/g quercetin equivalents) [23 (link)], catechins (as mg/g (+)-catechin equivalents) [24 (link)], procyanidins (as mg/g procyanidin B₁ equivalents) [25 (link)], phenylpropanoids (as mg/g rosavin equivalents) [26 (link)], gallotannins (as mg/g gallic acid equivalents) [27 (link)], ellagitannins (as mg/g ellagic acid equivalents) [28 (link)], coumarins (as mg/g umbelliferon equivalents) [29 (link)], and anthocyanes (as mg/g cyanidin-3-O-glucoside equivalents) [30 (link)] in dry herbal samples of R. rosea (roots, rhizomes, leaves, flowers, stems). All the analyses were carried out in triplicate and the data were expressed as mean value ± standard deviation (SD).
Antioxidant activity of total extracts and selected compounds was determined using spectrophotometric assays. Trolox was used as a positive control (PC; 10 mg/mL), and water was used as a negative control (NC). Scavenging activity against 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH^•) was studies as the following assay: 500 μL DPPH^• (freshly prepared MeOH solution, 100 μg/mL) and 500 μL of Rhodiola rosea extract (freshly prepared 50% MeOH solution, 1–200 μg/mL) or pure compound (freshly prepared MeOH solution, 1–200 μg/mL). Absorbance (520 nm) was measured after 15 min. The DPPH^• scavenging capacity was calculated using equation: Scavenging capacity (%) = ((A₅₂₀^NC – A₅₂₀^PC) – (A₅₂₀^Sample – A₅₂₀^PC)/(A₅₂₀^NC – A₅₂₀^PC)) × 100, where A₅₂₀^NC is the absorbance of the negative control, A₅₂₀^PC is the absorbance of the positive control, and A₅₂₀^Sample is the absorbance of the sample solution. For studing 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radicals (ABTS^•+) scavenging capacity ABTS (water solution; 7 mM) reacted with potassium persulphate (water solution; final concentration 2.45 mM) in the dark at 20 °C (12–16 h before use). The ABTS^•+ solution was diluted with MeOH to an absorbance of 0.70 at 734 nm and equilibrated at 20 °C. Rhodiola rosea extract (500 μL; freshly prepared 50% MeOH solution, 1–200 μg/mL) was mixed with ABTS^•+ solution (500 μL) and the absorbance was measured at 734 nm after 20 min. The ABTS^•+ scavenging capacity was calculated using equation: Scavenging capacity (%) = ((A₇₃₄^NC – A₇₃₄^PC) – (A₇₃₄^Sample – A₇₃₄^PC)/(A₇₃₄^NC – A₇₃₄^PC)) × 100, where A₇₃₄^NC is the absorbance of the negative control, A₇₃₄^PC is the absorbance of the positive control, and A₇₃₄^Sample is the absorbance of the sample solution. Superoxide radicals (O₂^•−) scavenging capacity was determined using Rhodiola rosea extract (50 μL; freshly prepared solution in Tris-HCl buffer, 0.05 M, pH 8.2; 10–1000 μg/mL) mixed with pyrogallol (50 μL, 6 mM) and Tris-HCl buffer (1 mL). The absorbance was measured at 325 nm after 5 min. The O₂^•− scavenging capacity was calculated using equation: Scavenging capacity (%) = ((A₃₂₅^NC – A₃₂₅^PC) – (A₃₂₅^Sample – A₃₂₅^PC)/(A₃₂₅^NC – A₃₂₅^PC)) × 100, where A₃₂₅^NC is the absorbance of the negative control, A₃₂₅^PC is the absorbance of the positive control, and A₃₂₅^Sample is the absorbance of the sample solution. To determine hydroxyl radicals (^•OH) scavenging capacity Rhodiola rosea extract (100 μL; freshly prepared solution in 0.2 M phosphate buffer (pH 7.4; 1–500 μg/mL) mixed with deoxyribose solution in the same buffer (100 μL; 2.8 mM), H₂O₂ (10 μL; 3.6 mM), FeCl₃ (10 μL; 5.0 mM) and EDTANa₂ (100 μL; 100 μM). After addition of ascorbic acid (50 μL; 200 μM) the mixture was incubated at 55 °C for 20 min. Finally, 2-thiobarbituric acid (800 μL; 10 mg/mL) and trichloroacetic acid (800 μL; 50 mg/mL) were added and heated at 95 °C for 20 min. The absorbance was measured at 530 nm. The ^•OH scavenging capacity was calculated using equation: Scavenging capacity (%) = ((A₅₃₀^NC – A₅₃₀^PC) – (A₅₃₀^Sample – A₅₃₀^PC)/(A₅₃₀^NC – A₅₃₀^PC)) × 100, where A₅₃₀^NC is the absorbance of the negative control, A₅₃₀^PC is the absorbance of the positive control, and A₅₃₀^Sample is the absorbance of the sample solution. The IC₅₀ value is the effective concentration at which free radicals (DPPH^•, ABTS^•+, O₂^•−, ^•OH) was scavenged by 50%. Values are expressed as mean obtained from five independent experiments. Carotene bleaching assay was performed as described previously using β-carotene as a substrate (Sigma-Aldrich, St. Louis, MO, USA, cat. No. C9750) [31 (link)].

DHJSD comprised 6 g each of Radix glycyrrhizae, Panax ginseng, Radix achyranthis bidentatae, Eucommiae ulmoidis cortex, Poria cocos, Cortex cinnamomi, Radix paeoniae alba, Radix rehmanniae, Radix angelicae sinensis, Rhizoma chuanxiong, Herba Asari, Radix saposhnikoviae, Radix gentianae macrophyllae, Ramulus loranthi and 9 g of Radix angelicae pubescentis. The above-mentioned herbs were provided by the First Hospital of Wuhan. The specific preparation method of DHJSD has been previously reported [28 (link)]. It was formulated by the Pharmacy Department of the Wuhan First Hospital to contain 1 g/mL of crude drug. The stock solution was cooled at room temperature and stored at 4 °C prior to usage. In subsequent experiments, we diluted the DHJSD stock solution to ultimate contents of 500, 400, 300, 200 and 100 μg/mL, adopting DMEM/F12 medium with 15% fetal bovine serum (FBS).

Fresh Rhizomes of Rehmannia glutinosa were obtained from Wuzhi County, Henan Province, China (35° 1′23″ north latitude, 113° 18′76″ east longitude) in December 2021. Stachyose (NO. 112031-201701), sucrose (NO.1 11507-202105), raffinose (NO. 190225-201901), and verbascose (NO. 111530-201914) were purchased from China Institute for Food and Drug Control.
Dexamethasone tablets were purchased from Tangshan Longkang Pharmaceutical Co., Ltd.
The lipopolysaccharide (LPS) was purchased from Sigma Company in the United States. The detection kits for TNF-α, IL-6, IL-17, and IL-1β were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. Moreover, the detection kits for SOD, MDA, GSH-Px, and CAT were purchased from the Nanjing Jiancheng Bioengineering Research Institute. Additionally, the digestion enzyme test kits were purchased from Beijing SOLEBAR Technology Co., Ltd. All other chemicals, solvents, and reagents were of pure analytical grade.

Fresh Rhizomes of Rehmannia glutinosa were cut into small pieces of 5–10 mm after being washed, added four times the amount of water, and extracted twice at 90°C, at 1 h duration. The two extracts were combined, adding activated carbon (2 g/100 ml) and activated clay (2 g/100 ml) to the extract. It was stirred and decolored at 80°C for 30 min, then centrifuged. The supernatant was passed through 001 × 7 cation exchange resin column (diameter: high = 6:1), D201 type anion exchange resin column (diameter: high = 6:1), D101 macroporous adsorption resin column (diameter: high = 10:1) one by one, sample volume (mL): resin column volume = 1:1.5, flow rate was 500 mL/h. Finally, the macroporous adsorption resin effluent was collected and concentrated and dried at 60°C to get white powder, that is RGO.
The type and content of oligosaccharides in RGO were detected using high-performance liquid chromatography (HPLC) (Agilent1260), configured using a Refractive Index Detector (RID) (13 (link)). The standard reference substances of sucrose, stachyose, raffinose, and mulberry sugar were weighed precisely and prepared with 70% acetonitrile aqueous solution into the standard reference solution with a concentration of 0.5 mg/mL, respectively. The RGO powder was also weighed precisely, and prepared with 70% acetonitrile aqueous solution into the sample solution with a concentration of 1 mg/mL. The chromatographic column was Agilent ZORBOX NH2 (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile: water (7:3); the injection volume was 10 μL, the flow rate was 1.0 mL/min, and the temperature of column incubator was 40°C. The temperature of the detection was 50°C with RID. The types of oligosaccharides in RGO were determined by comparing the HPLC peaks of reference substance with those in RGO, and the content of oligosaccharides was calculated by external standard method.