Smilax glabra Roxb. (SGB), which belongs to the Smilacaceae family, is the dry rhizome of the Sarsaparilla plant known as “tufuling” in China, and has been widely used as food and folk medicine in many countries for years. SGB has been extensively used worldwide for its marked pharmacological activities for treating syphilitic poisoned sores, morbid leucorrhea, eczema pruritus, carbuncle poisoning, and many other human ailments [1]. In the past 25 years, the studies of endophytes and their metabolites in plants, especially the medicinal plants, have become a vast and thriving field of natural product research [2].

The discovery of natural products has contributed greatly to the development of drugs for the treatment of various diseases [3]. However, as the discovery of natural products becomes increasingly challenging, the development of innovative strategies is essential for the further evolution of natural product chemistry. Studies combining genome sequencing and secondary metabolites has demonstrated that under laboratory conditions, the majority of genes in a strain are silent, with only a few being triggered to produce secondary metabolites [4], [5], [6]. Therefore, a promising approach to unlocking the biosynthetic potential of natural products is to activate silent gene clusters through genetic manipulation or environmental stimuli [7], [8]. In an attempt to activate these cryptic biosynthetic clusters, the researchers have developed multiple strategies including the mimic of the competitive microbial ecosystems of a natural environment by co-culturing two distinct fungi within the same medium. Recent researches into fungal co-cultures have confirmed the capacity to activate dormant biosynthetic gene clusters, substantially expanding the chemical diversity of natural products and making them a valuable resource for drug discovery and development, deserving of further exploration [9], [10], [11], [12], [13].

In the course of our ongoing research into metabolites generated by co-cultures, we analyzed the mixed culture of fungi C. virescens and X. grammica isolated from the rhizome of Smilax glabra Roxb. (Smilacaceae), respectively. Molecular networking analysis by the Global Natural Products Social (GNPS) Web-based platform can be used to effectively organize and visualize the cluster of structurally related compounds, providing guidance for the resolution of the dereplication issue in the co-cultivation of natural products [14], [15], [16]. In addition, the Network Annotation Propagation (NAP) can prioritize molecular families of interest, even in the absence of experimental library matches [17]. In this study, the MolNetEnhancer workflow was employed to analyze the generated molecular networking data, by combining outputs from molecular networking, MS2LDA, in silico structure annotation tools (such as NAP or DEREPLICATOR) and automated chemical classification through ClassyFire [18]. The molecular network analysis of the co-cultured EtOAc extract was conducted with the MolNetEnhancer, which classified the clusters of polyketide metabolites (Fig. 1). Analyzing the polyketide clusters, it was found that most structures of them possessed the chromone skeletons, which are variable and have extensive pharmacological activities. At the same time, there are also a large number of unknown nodes in the clusters, which are speculated to be novel compounds. Through a LC-MS guided isolation, twelve polyketides, including seven new chromone derivatives (1–7) and five known compounds (8–12), were obtained. After determining their structures, the clusters classified as polyketides by MolNetEnhancer were annotated (Fig. 2). Herein, the isolation, structural elucidation, and biological activities of these metabolites are discussed (Fig. 3).