This is the first study to establish and present a discrimination method for PVV, PVF, and PVL, the three varieties of P. vietnamensis. Additionally, it is the first investigation of P. vietnamensis varieties that utilizes leaf samples for discrimination marker discovery. Given the morphological ambiguity observed among the varieties and the potential for unintentional misclassification during the cultivation process, it is imperative to develop a reliable and precise discrimination method that is also straightforward to implement. Furthermore, while the official market values of the three varieties remain undisclosed, the potential for adulteration among these varieties exists in the future. Here, we established a sequential discrimination algorithm by elucidating variety-specific phytochemicals as well as lipid enrichment characteristics, all while minimizing product damage.
As emphasized above in an earlier section, differentiation while preserving product integrity is crucial for the ginseng industry, where cultivation typically takes between 4 and 6 years. Considering that the TLC and MS analyses require less than 50 µL of sample extract (50 mg mL−1), it is estimated that approximately 2.5 mg of dried leaf material, which corresponds to less than one-tenth of a single ginseng leaf, is sufficient for analysis. In this regard, leaf sampling has the advantage of providing a sufficient amount of sample while minimizing the damage.
Already, a few studies have identified leaf-metabolite markers for differentiating various Panax species15,16. Notably, these studies all selected ginsenosides as potential discrimination markers. In the current study, we observed that the enrichments of four ginsenosides, compound K, ginsenosides F1, Rg1, and Rh3, were quite significantly different when we compared PVV versus PVL, as shown in Supplementary Fig. S8. However, the fold changes (FCs) of these compounds were less pronounced compared to the markers identified in current study. The individual log2FC values for compound K, ginsenosides F1, and Rh3 were below 2.00, while ginsenoside Rg1 showed a value of 2.83. In contrast, the log2FC values for ‘FA 18:3 + 2O_2’, ‘LPC 18:2’, ‘LPE 18:2’, and ‘LPE 16:0’ were significantly higher, at 3.26, 3.18, 3.16, and 3.11, respectively. Consequently, the predictability of the model trained with our markers (Fig. 5g) was notably superior to the model trained with the ginsenosides (Supplementary Fig. S9b). Regarding the role of ginsenosides in PVF differentiation, several ginsenosides exhibited significant differences compared to PVV or PVL. Nevertheless, none of the identified ginsenosides was unique to a specific variety. In contrast, panaxindole was exclusively present in PVF, demonstrating clear specificity.
Due to the relatively recent discovery and characterization of new varieties of P. vietnamensis, the chemical characteristics of individual varieties are less well understood. Nonetheless, one study attempted to establish a discrimination method among P. vietnamensis species10. The authors identified the unique expression patterns of ginsenosides in PVV and PVF. They applied chemometrics and machine learning modeling to assess the performance of these distinguishing factors. Our research strengthens the differentiation method of P. vietnamensis through the inclusion of an additional variety type, PVL, as well as the identification of a novel PVF-specific marker. As stated in the results section, this novel PVF-specific marker, panaxindole, was elucidated in a recent study but was initially misidentified as a PVV-derived compound14. This undoubtedly highlights the extreme difficulty in distinguishing among the varieties of P. vietnamensis, necessitating the implementation of a qualified differentiation method.
Although our study has not elucidated the biological function of panaxindole, several studies have investigated the function of indole alkaloid glycosides from various sources. As an example, it has been demonstrated that several indole alkaloid N-glycosides isolated from Ginkgo biloba possess anti-inflammatory and anti-aging properties17,18. Other indole alkaloid glycosides, including those identified in Gardneria nutans, Isatis indigotica, and I. tinctoria, exhibited consistent anti-inflammatory activity19,20,21. This suggests that PVF may possess an enhanced anti-inflammatory function, necessitating additional research in this area.
Intriguingly, the HFAs identified in this study are mostly mono- or polyhydroxylated forms of C18 fatty acids, which have been reported to correlate with reduced inflammatory processes. For instance, treatment of α-linolenic acid (FA 18:3) to THP-1 macrophages resulted in a decrease in the excretion of inflammatory cytokines such as IL-6, TNF-α, and IL-1β22. This effect was accompanied by increased levels of two hydroxy derivatives of FA 18:3 (FA 18:3 + 1O), namely 9-hydroxy-octadecatrienoic acid and 13-hydroxy-octadecatrienoic acid. Additionally, 13-hydroxy-octadecatrienoic acid has been shown to exhibit anti-inflammatory properties when applied to lipopolysaccharide-treated macrophages23. By inactivating NLRP3 inflammasome complex via the PPAR-γ pathway, it inhibits the expression of iNOS and TNF-α while upregulating IL-10 expression. HFAs can also conjugate with other fatty acids to form cross-esterified compounds known as FAHFAs (fatty acid esters of hydroxy fatty acids), and their potential health benefits have been actively studied. These include reverting metabolic dysregulations by inhibiting hepatic glucose production, enhancing insulin-stimulated glucose uptake, and reducing adipose tissue inflammation24,25. Moreover, FAHFAs have been implicated in calcium-dependent signaling pathways during myocardial ischemia and related pathological events26. Consistent with these findings, plasma levels of FAHFAs were found to be significantly lower in obese children and adults with coronary artery disease27. Given these observations, further studies are warranted to investigate the biological functions of P. vietnamensis in light of these potential benefits, particularly for PVV, which exhibits high levels of HFAs.
Numerous studies have applied mass spectrometry to identify root-based phytochemical markers for the discrimination of ginseng species and ages. Consistently, ginsenosides or related triterpenoid saponins were found to be the most predictive markers when it comes to the comparison of P. ginseng, P. quinquefolius, P. notoginseng, P. japonicus, and P. japonicus var. major28,29,30,31,32,33. In some cases, primary metabolites and lipids were incorporated to enhance the predictability31,32,33. Notably, several HFAs have been described as effective discriminating markers. For example, (15Z)-9,12,13-trihydroxy-15-octadecenoic acid (FA 18:1 + 3O) demonstrated noteworthy efficacy in the differentiation of P. quinquefolius cultivated in various provinces across China32. P. ginseng is reported to be enriched in 12-hydroxyoctadec-9-enoic acid (FA 18:1 + 1O) and hydroxyhexadecanoic acid (FA 16:0 + 1O), and the potential role of these HFAs as discriminant markers was discussed33. It is expected that the variation in HFA expression levels could serve as a discrimination feature among distinct Panax species. HFA was one of the most significant chemical classes capable of differentiating PVV from PVL, as shown in Fig. 5e. Conversely, lysophospholipid has rarely been described as a discriminant marker among the Panax genus in any other publications. However, in the case of other agricultural products, lysophospholipid was able to function as a robust and accurate discrimination marker for finding geographical origins or detecting adulterated admixtures34,35.
In summary, we established a benchmark of an intact sample distinguishing method for the three varieties of P. vietnamensis by profiling their phytochemical characteristics with multiple analytical platforms. It is our conviction that our research would contribute to the development of the Vietnamese ginseng industry through the provision of a framework for precise classification.