*3.3. High-Performance Liquid Chromatography-Photodiode Array Detector-Electrospray Ionization*/*Mass Spectrometry Method*

An Agilent Technology 1100 Series HPLC system equipped with a quaternary pump, a degasser, a thermostatic auto-sampler and a photodiode array detector (DAD), was used for analysis (Agilent Technologies, Palo Alto, CA, USA). Chromatographic separations were carried out on a C18 analytical column Agilent Eclipse Plus-C18 (4.6 mm × 250 mm, 5μm) supplied by Agilent. The acetonitrile and water were used as the mobile phases (A) and (B), respectively, the optimized HPLC elution procedures were conducted as follows: 0–25 min, 80–90% (A); 25–70 min, 90–90% (A); 70–75 min, 90–100% (A). The flow-rate was 0.3 mL/min and the column temperature was maintained at 30 ◦C. The chromatogram was recorded at 210 nm. The injection volume of samples was 3.0 μL.

Agilent 1100 HPLC/MSD Trap mass spectrometer 6320 (Agilent) equipped with an electrospray ionization source was used in both positive and negative ion mode. An HPLC system coupled with DAD was controlled by an HPLC-MSD ChemStation software system. Auto MS2 mode of mass spectrometer was chosen to analyze the sample. The following operation parameters were used: capillary voltage: 4000 V; nebulizer pressure: 35 psi; drying gas: 9.0 L/min; gas temperature: 350 ◦C; skimmer voltage: 60 V. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) accurate mass spectra were recorded across the range from 50 to 1200 m/z. The data recorded was processed with the Applied HPLC-MSD ChemStation software system [26] (1200, Agilent Technologies).

#### *3.4. Cell Culture and Viability Assay*

Mouse leydig cell (TM3) line is a mouse epithelial Leydig cell line. The TM3 cell line were grown in Dulbecco's modified Eagle's medium/F-12 nutrient mixture (DMEM/F-12) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% penicillin (100 U/mL) and streptomycin (100 μg/mL) [27]. The human chorionic gonadotropin anhydrous (hCG) was obtained from Suolaibao Technology Co., Ltd. (Beijing, China).

Standard compounds *N*-benzyl hexadecanamide, *N*-benzyl-(9z,12z)-octadecadienamide and *N*-benzyl-(9z,12z,15z)-octadecatrienamide were dissolved in a culture medium containing a stock solution of 200 mg/L and further diluted to 62.5 μg/mL, 125 μg/mL, 250 μg/mL concentrations with culture medium containing 10% fetal bovine serum for 24 h. The DMEM/F12 concentrations (100 μL) was prepared as a control, and hCG concentrations (1 U/mL, 100 μL) were used as a positive control. Cells were cultured in a 37 ◦C incubator with 5% CO2 and 95% air [20]. The effects of fractions on Leydig cell viability were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Roche, Basle, Switzerland). The supernatant was collected to determine the testosterone levels using the Mouse Testosterone (T) ELISA kit (cat. no. JL10895; Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer's protocol.

### *3.5. Partial Least Squares Analysis and Statistical Analysis*

The multivariate analysis of the acquired data was carried out by PLS using the SIMCA 11 software (Umetrics, Umea, Sweden). All assays were performed at least in triplicate and the results were expressed as a mean ± standard deviation (SD). The significant difference analysis was evaluated by one-way analysis of variance (ANOVA) test completed by the software of IBM SPSS Statistics 19 (International Business Machines Corp., New York, NY, USA). Significance was accepted at *p* < 0.05 [28].

#### *3.6. Molecular Docking Studies*

To further study the probable mechanism of the bioactive compounds with CypD, a molecular docking study which could conjecture the interactions of ligands within the constraint of receptors binding sites was performed in silico.

In the prediction, The X-ray crystal structure of CypD in the complex with its inhibitor CsA, 0.96 Å, was obtained from the Protein Data Bank (PDB ID: 2Z6W). The three-dimensional (3D) structures of the ligands were drawn and converted using ChemBioDraw Ultra and ChemBio 3D Ultra [29] (Cambridgesoft Corp., Waltham, MA, USA). The ligands and water molecules were removed from the crystal structure and the polar hydrogen was added by using AutoDock [30] (4.2.6, Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA).

Each grid computation was set up covering all the active sites where CsA was bounded. The grid was then concentrated on the center (80 Å, 40 Å, 80 Å, 0.375 Å, central coordinates x = –20.347, y = 13.119, and z = 11.232), respectively. The calculation of the docking score was repeated three times for each ligand. Fifty ligand−receptor complex conformations were generated for each test compound, in which the least building energy was considered for further analysis. Finally, PyMOL and LigPlot were used to present the docking results [31,32].

#### **4. Conclusions**

This work used the multivariate analysis to reveal some potential components, which improved sexual function from *L. meyenii*. We established an effective strategy based on HPLC-ESI-MS/MS with the PLS analysis for screening and determining the bioactive compounds which promote leydig cells proliferation and testosterone secretion. The 10 fractions were fractionated and their promoting activities on TM3 were demonstrated. With the aid of HPLC-ESI-MS/MS and the multivariate statistical software, the three potential improving sexual function markers were identified. Molecular docking was employed for further illustration in the mechanism of action for bioactivity.

In this study, correlation analysis was studied to explore the internal relationship between chemical constituents and pharmacological effects and discover the bioactive markers reflecting the traditional efficacy of *L. meyenii*. The results specified the three compounds as potential bioactive markers could lay a foundation for the improvement of quality standard of *L. meyenii*.

**Author Contributions:** Conceptualization, J.-m.S. and X.-c.G.; methodology, J.-m.S.; software, J.-w.L.; validation, L.-l.T., C.-n.L. and N.Z.; formal analysis, X.-y.H.; investigation, L.-l.T.; resources, H.Z.; data curation, J.-w.L.; writing—original draft preparation, X.-c.G.; writing—review and editing, J.-m.S.; visualization, J.-w.L.; supervision, J.-m.S.; project administration, H.Z.; funding acquisition, J.-m.S.

**Funding:** This research was funded by the National Natural Science Foundation of China, grant number 31570347 and Science and Technology funds of Administration of Traditional Chinese Medicine of Jilin Province, grant number 2019050.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

1. Toledo, J.; Dehal, P.; Jarrin, F.; Hu, J.; Hermann, M.; Al-Shehbaz, I.; Quiros, C.F. Genetic Variability ofLepidium meyeniiand other AndeanLepidiumSpecies (Brassicaceae) Assessed by Molecular Markers. *Ann. Bot.* **1998**, *82*, 523–530. [CrossRef]


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