## 3 Results  
### Quantification of Caffe, Catechins, and Theantine in NGBTs  
HPLC was employed to quantify caffeine, total catechins, and theanine in the five NGBTs (Figure 2).  
Significant compositional differences were observed among the cultivars. Total catechin content was significantly higher in DXY and QTZ compared to JSH, DXE, and YHJ, though no significant differences were detected between DXY and QTZ or among JSH, DXE, and YHJ. Theobromine levels were significantly elevated in JSH and YHJ relative to DXY, DXE, and QTZ, while no significant differences existed between JSH and YHJ or among DXY, DXE, and QTZ. Theanine content differed significantly across all five teas, with JSH displaying the highest concentration, followed sequentially by YHJ, QTZ, DXY, and DXE. For caffeine, JSH and QTZ exhibited the highest concentrations, followed by YHJ, DXY, and DXE; however, no significant differences were observed between JSH and QTZ, between DXY and YHJ, or between DXY and DXE.  
Theanine is widely recognized as a key contributor to the refreshing umami taste (freshness) in tea [31], while catechins are primarily responsible for imparting bitterness and astringency [32]. The ratio of amino acids to catechins serves as a critical biochemical indicator for evaluating the quality attributes of mellowness (thickness) and briskness (freshness) in tea liquors [33]. Notably, JSH and YHJ exhibit significantly higher theanine-to-total catechin (T/C) ratios compared to DXY, DXE, and QTZ (_p_ < 0.05).  
These pronounced variations in total catechins, theobromine, theanine, caffeine, and gallic acid highlight their potential role as key discriminating non-volatile compounds contributing to the compositional distinctness of the five NGBTs.  
Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were employed to investigate the multivariate relationships among the five NGBT samples based on their non-volatile compound profiles. Both PCA score plots and OPLS-DA score plots demonstrated clear separation among all five NGBTs (Figure 3A,B), confirming significant compositional differences in their non-volatile constituents. The validity and absence of overfitting in the OPLS-DA model were confirmed by permutation testing (Figure 3C). Variable importance in projection (VIP) scores quantified each compound's contribution to the OPLS-DA model discrimination, with VIP \(>\) 1 indicating statistically significant contributions. This analysis identified total catechins, simple catechins, CG, EGCG, esterified catechins, and EGC as possessing VIP scores \(>\) 1 (Figure 3D), highlighting their potential as key markers differentiating the non-volatile profiles of the five NGBTs.  
Figure 2: Content of non-volatile compounds in 5 NGBTs. Different letters indicate statistically significant differences according to one-way ANOVA. (**A1–A5**) Content of total catechin, theobromine, theanine, caffeine, and GA in 5 NGBTs. (**B**) Content of ester catechin (including CG, GCG, ECG, and EGCG) in 5 NGBTs. (**C**) Content of simple catechin (including C, EC, and EGC) in 5 NGBTs. (**D**) Theanine-to-total catechin ratio in 5 NGBTs.