Coffee processing generates a huge amount of waste that contains many natural products. Here, we report the discovery of a panel of novel cell-penetrating and metal ion-binding microproteins designated coffeetide cC1a–c and cL1–6 from the husk of two popular coffee plants, Coffea canephora and Coffea liberica, respectively. Combining sequence determination and a database search, we show that the prototypic coffeetide cC1a is a 37-residue, eight-cysteine microprotein with a hevein-like cysteine motif, but without a chitin-binding domain. NMR determination of cC1a reveals a compact structure that confers its resistance to heat and proteolytic degradation. Disulfide mapping together with chemical synthesis reveals that cC1a has a ginsentide-like, and not a hevein-like, disulfide connectivity. In addition, transcriptomic analysis showed that the 98-residue micrcoproten-like coffeetide precursor contains a three-domain arrangement, like ginsentide precursors. Molecular modeling, together with experimental validation, revealed a Mg²⁺ and Fe³⁺ binding pocket at the N-terminus formed by three glutamic acids. Importantly, cC1a is amphipathic with a continuous stretch of 19 apolar amino acids, which enables its cell penetration to target intracellular proteins, despite being highly negatively charged. Our findings suggest that coffee by-products could provide a source of ginsentide-like bioactive peptides that have the potential to target intracellular proteins.

Coffee processing generates a huge amount of waste which contains many natural products. Here, we report the discovery of a panel novel cell-penetrating and metal-ion binding microproteins designated coffeetide cC1a-c and cL1-6 from the husk of two popular coffee plants, Coffea canephora and Coffea liberica, respectively. Combining sequence determination and database search, we showed that the prototypic coffeetide cC1a is a 37-residue, eight-cysteine microprotein with a hevein-like cysteine motif but without a chitin-binding domain. NMR determination of cC1a revealed a compact structure that confers it resistance to heat and proteolytic degradation. Disulfide mapping together with chemical synthesis revealed that cC1a has a ginsentide-like, and not a hevein-like, disulfide connectivity. In addition, transcriptomic analysis showed that the 98-residue micrcoproten-like coffeetide precursor contains a three-domain arrangement like ginsentide precursors. Molecular modeling together with experimental validation revealed a Mg2+ and Fe3+ binding pocket at the N-terminus formed by three glutamic acids. Importantly, cC1a is amphipathic with a continuous stretch of 19 apolar amino acids, and which enables its cell penetration to target intracellular proteins, despite being highly negatively charged. Our findings suggest that coffee by-products could provide a source of ginsentide-like bioactive peptides that have potential to target intracellular proteins.

In China, Pyracantha fortuneana has been consumed as a nutritious plant to improve indigestion. In the current study, the main chemical composition of P. fortuneana fruits was extracted and analysed for composition. Free fatty acids (FFA)-induced normal human hepatic L02 cells were used to construct a high-fat cell model, and lipid deposition in Caenorhabditis elegans was induced by a high concentration of glucose to study the anti-hyperlipidemic effects of the main components. The results showed that the flavonoid content of PFF (P. fortuneana Flavonoid Fractions) was 80.28%, and it contained various flavonoids such as epicatechin, isoquercetin, rutin, quercetin, and myricitrin, while the saponin content of PFS (P. fortuneana Saponin Fractions) was 74.4%, and it contained saponins such as shionone, crategolic acid, and ursolic acid. PFF and PFS significantly reduced the content of lipid droplets in high-fat L02 cells, inhibited mitochondrial membrane potential decline, regulated the fat accumulation by up-regulating the relative mRNA expression levels in the Nrf2/ARE signaling pathway, as well as the CPT-1 and SIRT1 genes in lipid metabolism. Meanwhile, both PFF and PFS significantly reduced lipid deposition, reactive oxygen species (ROS) levels, malondialdehyde (MDA) content, and catalase activity in C. elegans. In summary, our results indicated that the flavonoids and saponins of P. fortuneana are potential natural products in antihyperlipidemic effect.

This study characterized the basic structure of partridge tea leaves polysaccharides and comparatively analyzed the in vitro lipid-lowering activity of total partridge tea polysaccharide (PTPS) and its two purified homogeneous fractions, namely PTPS-I (13,560 Da) and PTPS-III (30,935 Da). In terms of structural composition, PTPS-I and PTPS-III share identical monosaccharide types but differ significantly in monosaccharide proportions, glycosidic linkages and backbone structures. In vitro experiments demonstrated that PTPS, PTPS-I, and PTPS-III could effectively reduce intracellular lipid levels and oxidative stress in free fatty acids (FFA)-injured L02 cells and alleviate the decline of mitochondrial membrane potential in damaged hepatocytes. At the high concentration of 400 μg/mL, PTPS-III showed a superior effect in reducing triglyceride (TG) content compared with the other two samples, with the value reaching 0.31 ± 0.024 mmol/mg prot. Additionally, 400 μg/mL PTPS markedly decreased total cholesterol (TCHO) content and enhanced superoxide dismutase (SOD) activity, which were 0.55 ± 0.039 mmol/mg prot and 29.92 ± 0.22 μmol/mg prot, respectively. PTPS-I of 400 μg/mL significantly reduced malondialdehyde (MDA) content to 1.31 ± 0.288 μmol/mg prot and inhibited the decline of mitochondrial membrane potential (MMP) by 9.67%. The three polysaccharide fractions could elevate the mRNA expression of Nrf2, NQO1 and HO-1 in the Nrf2/HO-1 signaling pathway and the gene expression of PPARα, CPT-1 and ACOX1 in the lipid metabolism pathway, and ultimately regulate lipid accumulation in L02 cells. This study validated the in vitro antilipid activities of partridge tea leaves polysaccharide and provided fundamental data for research on its bioactivity and functional components. Further in vivo assays and mechanism exploration will be conducted to evaluate its potential application in fatty liver intervention product development.