*2.4. Activity Evaluation of Active Components*

The proliferation activity of the three compounds (**9**), (**6**), (**2**), were assessed using the MTT assay in TM3. All the compounds were found to possess proliferation activity.

To better evaluate the improving sexual function of the three compounds (**9**), (**6**), (**2**), the testosterone secretion assay was tested, and the results were presented in a strong correlation between the values determined by the HPLC-DAD-MS2 method and that predicted by the testosterone secretion tested data was observed [20] (Figure 6).

**Figure 6.** Effects of the three compounds on TM3 and testosterone secretion (a *p* < 0.01).

#### *2.5. Analysis of Molecular Docking*

The molecular docking study further elucidated the binding mode of the three compounds at the active site of CypD. The binding pocket of CypD was large and shallow, consisting of residues Arg55, Ile57, Phe60, Met61, Gln63, Gly72, Thr73, Gly74, Ala101, Asn102, Phe113, Trp121, Leu122, and His126 etc [21]. In which it was known, four specific residues (Arg55, Gln63, Asn102 and Trp121) were involved in hydrogen bond interactions with CsA [12]. Molecular docking simulation revealed that ligands interacted with important amino acid residues surrounding the active site through plenty of interactions including hydrogen bond acceptor, hydrogen bond donor, hydrophobic interactions. The docked molecules interacted with essential amino acid forming proteins' binding site. Unlike the case of full occupation by CSA, CypD- macamide complexes occupied only part of the binding pocket and might swing in the pocket [21]. (Figures 7a–c and 8a–c). The lowest binding energy were found: −4.79 kcal/mol for (**9**), −4.55 kcal/mol for (**6**) and −4.18 kcal/mol for (**2**). The negative binding energy (G < 0) indicated that there were good binding affinity between the three compounds and CypD.

Normally, the interactions between CypD and the macamide were dependent on the structures of the macamides, as the number of hydrogen bonds and hydrophobic interactions increased, the affinity degree might increase, it was shown between (**6**) and (**2**). It was interesting that, a hydrogen bond was formed between residual Arg55 and N atoms of macamide, causing the electrons of the N atom to form a regular tetrahedron of Sp3 hybrid, with single-button rotation. A mutant CypD with a single amino acid substitution (Arg to Ala at position 55) that was predicted to produce a 1000-fold attenuation in isomerase activity failed to reverse the CsA effect [22]. Therefore, the lowest binding energy were found in (**9**).

The results showed there was a specific ligand-binding ability of macamide for CypD, which could be used in the inhibition of MPT pore opening, which caused mitochondrial damage. The homeostasis of the mitochondrial function ensured the maintenance of the StAR function, which was the first step in testosterone biosynthesis. CypD inhibitor could effectively bind CypD and inhibit the cis-trans isomerase activity of CypD, making the StAR expression stable, ultimately promoting testosterone secretion [23,24]. Therefore, one of the possible mechanisms of promoting testosterone secretion for thee compounds, which could be the bioactive markers of *L. meyenii*.

**Figure 7.** (**a**–**c**) Molecular docking of the three bioactive ligands with CypD, respectively. Ligands were shown in stick form and gray dashed lines were hydrogen bonds. The figure was prepared with PyMol. The interactions between bioactive ligands and binding sites were detailed in the article. (Figure 7a-(**9**), Figure 7b-(**6**), Figure 7c-(**2**)).

**Figure 8.** (**a**–**c**) Two-dimensional (2D) representation of hydrogen bond and hydrophobic interactions. Dashed lines represent hydrogen bonds, and spiked residues form hydrophobic interactions. (Figure 8a-(**9**), Figure 8b-(**6**), Figure 8c-(**2**))**.**
