**1. Introduction**

*Lepidium meyenii* (Maca), is a Brassicaceae *Lepidium* plant native to the Andes Mountains of South America. It has been traditionally used as a food and machine over 5000 years [1]. As is usual with many traditional folk medicines, many claims have been made regarding the efficacy of Maca in treating a wide range of illnesses and medical conditions [2,3]. However, in the 20th century most of the scientific attention has been focused in the areas where the pharmacological actions of Maca seem most strongly attested, these include, enhancement of sexual drive in humans, increasing overall vigour and

energy levels, and increasing sexual fertility in humans and domestic livestock [3]. *Lepidium meyenii* is rich in nutrients and secondary metabolites with a variety of biological activities. Its main chemical compositions are polysaccharide, flavone, saponin, microelement and amino acid [4]. Low polarity magamide is considered to be its unique iconic ingredient, at present, the method of solvent reflux, ultrasonic extraction, high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry were used to detect it [5].

The present studies abroad have been studying the pharmacological effects of *L. meyenii*, they focus mainly on the effect of sexual function in mice. However all of these studies are segmentary, and lack of a comprehensive and systemic assessment, as well as the effect mechanism of improving sexual function is not yet clear. Especially, there is no research on the *L. meyenii* active monomers in promoting the mechanism of sexual function [6–8].

Testosterone is a prerequisite for normal spermatogenesis. Leydig cells are the main cells responsible for the production and secretion of the testosterone hormone [9]. The raw material for testosterone synthesis is cholesterol. The rate-limiting enzyme steroidogenic acute regulatory protein (StAR) in testosterone synthesis is responsible for accelerating the transport of cholesterol to the mitochondria, which is the first step in testosterone biosynthesis. For the maintenance of the StAR function, the homeostasis of the mitochondrial function is indispensable. In the process of maintaining mitochondrial function homeostasis, CypD plays an important regulatory role. Activation of CypD leads to opening of the mitochondrial permeability transition pore (mPTP) on the outer membrane of mitochondria which causes mitochondrial damage [10,11]. Mitochondrial dysfunction results in the inhibition of StAR expression, hindering cholesterol from entering the mitochondrial stromal membrane and inhibiting testosterone secretion; the CypD inhibitor can effectively bind CypD and inhibit the *cis*-*trans* isomerase activity of CypD, making the StAR expression stable, ultimately promoting testosterone secretion. Although the complete mechanism of the mPTP opening remains unclear, cyclosporine A (CsA), a high-affinity cyclophilin inhibitor, blocks the mPTP opening by binding to the CypD [12–16].

Inspired by the applications mentioned above, in order to find out the bioactive markers reflecting the traditional efficacy, an effective strategy on the high-performance liquid chromatography-electrospray ionization/mass spectrometry (HPLC-ESI-MS/MS) coupling with multivariate statistical analysis was developed to screen and identify the bioactive ingredients in *L. meyenii* [17]. Molecular docking was used to investigate the mechanism of bioactive compounds for improving sexual function, as depicted in Figure 1. The present study illustrated and explained the underlying correlations between active constituents and mechanisms of action [18].

**Figure 1.** Strategy based on high-performance liquid chromatography-electrospray ionization/mass spectrometry (HPLC-ESI-MS/MS) coupling with the multivariate statistical analysis method to screen and identify the bioactive ingredients for the proliferation of mouse leydig cells (TM3) and promoting testosterone secretion in *Lepidium meyenii*. Molecular docking was used to investigate the mechanism of bioactive compounds. (**a**) The HPLC fingerprints of ten fractions. (**b**) Effects of the ten fractions on TM3 (a *p* < 0.01, b *p* < 0.05). (**c**) Model effect weights of the ten compounds on TM3. (**d**) Effects of the three compounds on TM3 and testosterone secretion (a *p* < 0.01). (**e**) The crystal structure of human cyclophilin D (PDB ID: 2Z6W). (**f**) The chemical structures of three bioactive markers. (**g1**) Molecular docking of compound (**9**) with CypD showed in three-dimensional (3D) and two-dimensional (2D). (**g2**) Molecular docking of compound (**6**) with CypD showed in 3D and 2D. (**g3**) Molecular docking of compound (**6**) with CypD showed in 3D and 2D.
