*2.2. Effects of FIR Treatment on Panasenoside and Kaempferol Contents of Ginseng Leaves*

Panasenoside (kaempferol 3-*O*-glucosyl-(1-2)-galactoside) is a kaempferol glycoside and has been found to be the main flavonoid in ginseng leaves [7]. Figure 2A,B shows the variation in the contents of panasenoside and its aglycone (kaempferol) before and after FIR treatment. Before FIR treatment (0), ginseng leaves had 10.53 mg/g d.w. of panasenoside and only 0.03 mg/g d.w. of kaempferol. Similarly, Yin et al. [7] reported contents of 15.84 mg/g d.w. and 0.43 mg/g d.w of panasenoside and kaempferol, respectively, in 4-year-old ginseng leaves. After FIR treatment, a significant decrease in the panasenoside content of ginseng leaves was observed with increasing FIR temperature, with only 2.29 mg/g d.w. remaining in the leaves after FIR-200 treatment. In contrast, our results showed a significant increase in kaempferol. Its content increased sharply to 0.42 mg/g d.w. until FIR-180, which was 14.3 times that of the untreated control (0). The kaempferol content did not significantly increase at temperatures greater than 180 ◦C, such as FIR-190 and FIR-200.

According to the structures of panasenoside and kaempferol (Figure 2C), it is assumed that FIR energy causes a significant increase in the kaempferol content due to the deglycosylation of panasenoside. It has been suggested that FIR has the capacity to transfer heat energy, cleave covalent bonds, and liberate low-molecular-weight compounds [12,22]. However, the content of kaempferol did not significantly vary when the temperature exceeded 180 ◦C, despite the continuous decrease in the panasenoside content with increasing FIR temperature (Figure 2A,B). Furthermore, the increased aglycone content was not as high as that of panasenoside. The effects of FIR heat energy on ginseng leaf flavonoids were assumed in two situations according to temperature variation: (1) The relatively fast deglycosylation of panasenoside accompanied by the slow degradation of kaempferol occurred when FIR temperatures were below 180 ◦C, which explains the relatively slow decrease in the panasenoside content and the sharp increase in kaempferol, and (2) the rapid deglycosylation of panasenoside accompanied with the faster degradation of kaempferol when the FIR temperature exceeded 180 ◦C, which explains the rapid decrease in panasenoside

and the lack of significant change in the kaempferol content. These assumptions were supported by Oliveira et al. [28]. They reported that the kaempferol content of *Poincianella pyramidalis* decreased significantly when the air inlet temperature was increased from 160 to 180 ◦C during spray-drying. Deng et al. [29] suggested that flavonol glycosides (kaempferol and quercetin derivatives) are degraded during the roasting process to correspond to an increase in aglycones in noni leaves (roasted at 175 ◦C from 10 to 60 min or roasted for 20 min from 100 to 250 ◦C, respectively). Moreover, FIR treatment usually provides higher energy during the material dry process compared to common dry processes under the same temperature control. In addition, it is expected that FIR treatment may lead to an intermediate compound by cleaving one glucoside of panasenoside rather than directly deglycosylating two glucosides to kaempferol. However, this intermediate compound was not detected in this study (data not shown). This result also supports our assumption. Previous studies on flavonoids in ginseng leaves have mainly focused on the types and contents of flavonoids in raw materials [7]. To the best of our knowledge, this is the first study to investigate flavonoid variation patterns in ginseng leaves during processing. According to our results, FIR treatment significantly increases the content of kaempferol via the deglycosylation of panasenoside (Figure 2), which has been found to possess higher bioactivities, such as antitumor, antioxidant, and anti-inflammatory activities, compared to its glycosides [30]. Thus, it can be concluded that FIR treatment (FIR-180) is a promising method for processing ginseng leaves to obtain more kaempferol. Here, it is also important to note that leaf burning was observed when the FIR temperature exceeded 200 ◦C during the 30 min treatment, suggesting that higher FIR temperatures are risky for ginseng leaf processing.

**Figure 2.** Changes in panasenoside (**A**) and kaempferol (**B**) contents and structural conversion pattern (**C**) from panasenoside to kaempferol in ginseng leaves treated by FIR at different temperatures. The values are expressed as the mean with standard error (*n* = 3). Different letters (a–d) above the bar graphs indicate significant differences at *p* < 0.05 on Tukey's HSD test.

#### *2.3. Effects of FIR Treatment on Ginsenoside Contents of Ginseng Leaves*

Ginsenosides can be roughly divided into two types according to the position and amount of glycol groups in the glycosides: (1) protopanaxadiol (PPD) type and (2) protopanaxatriol (PPT) type [8]. Here, the variations in nine PPD-type and eight PPT-type ginsenosides in ginseng leaves during FIR treatment were qualified and quantified using HPLC, respectively (Figure 3 and Figure S1, and Table S1).

**Figure 3.** Flow schemes of the conversion patterns of PPD type (**A**) and PPT type (**B**) of ginsenosides in ginseng leaves treated by FIR at different temperatures. A heat map (**C**) of 17 kinds of ginsenoside changes in ginseng leaves treated by FIR at different temperatures. The unit of ginsenoside content is mg/g dry weight. GN indicates the glycosyl number attached on ginsenoisde. The values in bar graphs are expressed as the mean with standard error (*n* = 3). Different letters (a–f) above the bar graphs indicate significant differences at *p* < 0.05 on Tukey's HSD test.

#### 2.3.1. PPD-Type Ginsenosides

The effects of FIR treatment on the transformation of PPD-type ginsenosides in ginseng leaves are shown in Figure 3A,C, and Table S1. Overall, FIR treatment reduced the major ginsenoside content and increased the minor ginsenoside content. In detail, the ginsenosides Rb1 (4.25 mg/g d.w.), Rb2 (9.41 mg/g d.w.), Rb3 (1.42 mg/g d.w.), Rc (5.72 mg/g d.w.), and Rd (23.65 mg/g d.w.) were the predominant compounds in untreated ginseng leaves, whereas their contents gradually decreased after FIR treatment (Figure 3, light yellow). Each of the aforementioned ginsenosides showed a relatively low amount at FIR-200, with 0.58, 1.45, 0.13, 0.90, and 3.11 mg/g d.w. of Rb1, Rb2, Rb3, Rc, and Rd being observed, respectively. In contrast, non-FIR-treated ginseng leaves only had small amounts of minor ginsenosides (Figure 3, blue column), such as Rg3 (0.07 mg/g d.w.), Rk1 (0.04 mg/g d.w.), Rg5 (0.10 mg/g d.w.), and Rh2 (0.12 mg/g d.w.), whereas they significantly increased after FIR treatment. The ginsenoside Rg3 increased to its highest amount at FIR-180, presenting a 22-fold (1.49 mg/g d.w.) higher content than the untreated control. The highest amounts of Rk1, Rg5, and Rh2 were observed at FIR-190, which increased 77-fold (3.30 mg/g d.w. Rk1), 100-fold (10.01 mg/g d.w. Rg5), and 3-fold (0.41 mg/g d.w. Rh2), respectively, compared to the non-FIR-treated control.

FIR treatment significantly increased the content of minor PPD-type ginsenosides (Rg3, Rk1, Rg5, and Rh2) in ginseng leaves by achieving the deglycosylation and dehydration of the major ginsenosides. The major ginsenosides Rb1, Rb2, Rb3, Rc, and Rd had their glycosyl residue(s) at C-20 removed, causing a significant increase in the content of the minor ginsenoside Rg3. Furthermore, Rg3 was then converted to Rk1 and Rg5 via dehydration at C-20 and C-21/C-22 or to Rh2 by deglycosylation at C-3. Similar results have been reported for ginseng roots and observed in the ginsenoside variation patterns after different thermal processing methods, such as microwave heating, puffing, and steaming [9,31,32]. However, in our results, it is important to point out that most of the Rg3 was further converted to the dehydrated type (Rk1 and Rg5) instead of the deglycosylated type (Rh2). According to the carbenium ion mechanism [33], the deglycosylation of ginsenosides at C-20 is easy than at other positions because it can generate tertiary carbenium ions with more stability. Then, the active tertiary alcohol is further eliminated during thermal processing. In addition, Zaitsev's rule can potentially give the reason why more Rg5 is enhanced than Rk1 during FIR treatment [34,35]. Our previous study suggested that 10 min of FIR treatment (between 60 and 120 ◦C) significantly increases the major ginsenoside (Rb1, Rb2, Rc, Rd) content of ginseng leaves, whereas it has no effect on minor ginsenosides (Rg3, Rh2) [13]. These results indicate that both the temperature and time of FIR treatment are important factors affecting ginsenoside variation in ginseng leaves. In addition, Chen et al. [36] reported that polar PPD-type ginsenosides (Rb1, Rb2, Rb3, Rc, and Rd) can be converted to Rg3, F2, Rh2, C-K, PPD, and Rk2 during the creation of black ginseng leaves. Thus, it is important to select suitable processing methods in consideration of the final products before processing ginseng leaves. Compared to FIR-190, a decreased pattern of each PPD-type minor ginsenoside was observed in FIR-200-treated ginseng leaves, which can be explained by the destruction of ginsenoside structures by high FIR energy.

#### 2.3.2. PPT-Type Ginsenosides

Re (47.71 mg/g d.w.) and Rg1 (28.47 mg/g d.w.) were the major PPT-type ginsenosides (Figure 3B,C, and Table S1) found in non-FIR-treated ginseng leaves. During FIR treatment, the Re and Rg1 contents gradually decreased as the FIR temperature increased, exhibiting 85% and 83% reductions at FIR-200, respectively. However, the content of minor ginsenosides, Rg2, Rh1, F4, Rg6, Rh4, and Rk3, significantly increased as the FIR temperature increased, presenting their highest amounts at FIR-190. Hundred-fold increases in most minor ginsenosides described above were found at FIR-190, namely 319-fold (5.34 mg/g d.w.) in F4, 169-fold (3.48 mg/g d.w.) in Rg6, 187-fold (3.14 mg/g d.w.) in Rh4, and 314-fold (1.21 mg/g d.w.) in Rk3 compared to the untreated control (less than 0.03 mg/g d.w. of

each ginsenoside). The increases in Rg2 and Rh1 at FIR-190 were relatively small, exhibiting 8.07- and 3.11-fold increases compared to the untreated control, respectively.

The conversion patterns of the PPT-type ginsenosides are shown in Figure 3B. The increase in the minor ginsenoside Rg2 was as a result of the glycosyl residue at C-20 of the major ginsenoside Re being detached due to high FIR energy [8,22]. As we mentioned in Section 2.3.1, the fact that the glycosyl residue at C-20 was easily deglycosylated than at C-6 can be explained by the carbenium ion mechanism [33]. Rg2 was then converted to F4 via dehydration between C-20 and C-22 positions or to Rg6 via dehydration between C-20 and C-21 positions. Similar results were reported by Kim et al. [11] using standard ginsenosides. The standard ginsenoside Re was deglycosylated and transformed to the less polar ginsenosides Rg2, F4, and Rg6 after heat processing at 120 ◦C. Likewise, the deglycosylation of Rg1 at C-20 caused an increase in the Rh1 content, followed by an increase in Rk3 and Rh4, resulting from the dehydration of Rh1 between C-20 and C-21/C-22. A similar result was reported by Hwang et al. [31]. They showed the variation in Rg1, Rh1, Rh4, and Rk3 in white ginseng heated at different temperatures at 20 MPa for 2 h. In our results, relatively higher amounts of F4 and Rg6 were transformed compared to Rh4 and Rk3. Differently, Chen et al. [36] investigated ginsenoside changes in black ginseng leaf products and suggested that the degradation of Re and Rg1 to minor ginsenosides, such as Rk3, Rh4, and PPT, is the main transforming PPT-type pattern. This can be explained by the different progressions of thermal processing (methods, time, temperature, etc.) affecting different substances [13,37–41], as Rk3 and Rh4 are the deglycosylated products of Rg6 and F4 at C-6, respectively. Ginsenoside mutations in ginseng leaves during heat treatment have not been widely studied in the literature, especially in terms of dry-heat treatment. In our results, it is important to point out that FIR treatment (especially FIR-190) significantly increased the content of each dehydrated minor ginsenoside compared to unprocessed ginseng leaves (Figure 3C). The moderate FIR energy that showed high dehydration efficiency at C-20 instead of deglycosylation at C-6 was considered a response to the significant increased minor ginsenosides F4, Rg6, Rh4, and Rk3. A decreased pattern of each minor ginsenoside at FIR-200 compared to those at FIR-190 was also observed in our results, which may be due to ginsenoside degradation caused by excessive FIR energy.

These results suggest that a higher FIR temperature is more advantageous for ginseng leaf processing and indicate that 190 ◦C is a suitable temperature. It is also recommended for obtaining more dehydrated minor ginsenosides based on the processing conditions. Compared to major ginsenosides, minor ginsenosides show stronger or even new biological activities [42], so numerous studies have been devoted to promote the transformation of major ginsenosides into profitable ginsenosides through thermal processing [31,43]. Although our previous results have shown that FIR treatment can promote the conversion of major ginsenosides to minor ginsenosides in ginseng roots and leaves [10,17], this study optimized the processing technology and significantly improved the conversion efficiency in ginseng leaves.
