FIGURE 1 A. trifoliata growing in different habitats
2.3  | Determination of biomass
Separate the plant into three parts: root, stem and leaf. Rinse with water to remove impurities adhering to the plant sample, then rinse with deionized water 2 to 3 times and absorb moisture with absorbent paper. The sample was then treated at 105 °C for 30 minutes and then dried at 70 °C to constant weight.
2.4  | Determination of basic secondary metabolites and enzyme of phenylpropane pathway
The content of tannin was determined by vanillin method; The content of total phenols was determined by Folin ciocalteus method with gallic acid as standard; The content of flavonoids was determined by aluminum nitrate colorimetry, and the standard curve was established with rutin as the standard (Alhaithloul et al., 2019).
The PAL activity was assessed following the method as described by Koukol and Conn (1961). The C4H activity was determined by the method as described by Lamb and Rubery (1975). The 4CL activity was measured using the method as described by Knobloch and Hahlbrock (1975).
2.5  | Determination of medicinal ingredients
The content of oleanolic acid was determined by spectrophotometry (Fu et al., 2019): 5 g dry powder of roots, stems and leaves parts of A. trifoliata was precisely weighed and placed in 15 mL of 90% ethanol. Ultrasonic extraction was conducted at 70 ℃ for 90 min, and then the filtrate was filtered. Sufficient saturated clarified lime water was added to the filtrate, and the filtrate was stirred and precipitated at 60 ℃ for 30 min, and then the filtrate was filtered again. The filter cake was decomposed with 3% dilute sulfuric acid and filtered by suction. The filtrate was extracted twice with 40 mL trichloromethane, and the extraction solution was combined to a constant volume of 50 mL. 2 mL of the extract was added with 5% vanillin glacial acetic acid and 0.8 mL perchloric acid. The absorbance at 545 nm was determined by SPECTROMAX 250 (Molecular devices, USA) at room temperature. The standard curve of oleanolic acid concentration and absorbance was drawn with different concentrations of oleanolic acid as standard samples, and the content of oleanolic acid was calculated, expressed in mg·g-1.
Α-hederagenins were measured by High Performance Liquid Chromatography (HPLC) (Commission, 2010): (1) Reagent: α-hederagenin reference substance (CFDA, China); Acetonitrile (Tedia, USA); Phosphoric acid (Chengdu Jinshan Chemical Co., Ltd. China); Purified water (Wahaha Group, China). (2) Chromatographic conditions: Water 2695 HPLC instrument; The column was Agilent extend-c18 (4 × 250 mm, 5 μm); The mobile phase was acetonitrile: water: phosphoric acid (45:55:0.1); The flow rate was 1.0 mL·min-1; Injection volume 10 μL; The column temperature was 25 ℃; The detection wavelength was 203 nm. (3) Preparation of test solution: 1 g dry powder of roots, stems and leaves parts of A. trifoliata was precisely weighed, placed in 100 mL 75% ethanol, weighed, ultrasonicated for 30 min (330W, 50KHz), weighed in cold, added with 75% methanol, shaken and centrifuged. Using microporous organic filter membrane (0.22 μm) the supernatant was filtered twice to determine the content of α-hederagenins. Repeat 3 times.
2.6  | Data analysis
All data were analyzed with Excel 2010 and SPSS 19.0, and plotted with origin 2019 b. One-way ANOVA and Duncan multiple comparison were used to test the differences between the treatments. In order to comprehensively and systematically evaluate the growth physiological response ofA. trifoliata in rocky desertification habitat, principal component analysis was conducted on the average values of growth and physiological and biochemical indexes of various organs and parts in the experiment. In order to eliminate the differences between materials, the stress resistance coefficient method was used to evaluate the main response indexes (stress resistance coefficient = measured value under stress / measured value under control).
3   | RESULTS
3.1  | Biomass of A. trifoliata in different habitats
As shown in Figure 2, with the growth and development of A. trifoliata , the biomass of roots, stems, and leaves will gradually increase. Regardless of the habitat, the root biomass was the highest in the late growth period. In comparison, the biomass of roots, stems and leaf organs in rocky desertification habitats was less than that in normal habitats.