Flavonoids are ubiquitious in many edible plants. They consist of two aromatic rings linked through three carbons. Most flavonoids in plant are present as flavonoid glycosides, aglycone with sugar substitution (Figure 1.2) (Ross and Kasum, 2002).
A common method of flavonoid extraction is using ethyl acetate. In this study, EtOAc fraction was separated in a C18-HPLC column with a UV detector and a MS detector. HPLC is able to effectively separate various flavonoids under the specific conditions and mass spectrum and UV spectrum provides some structure information of each peak such as molecular weight, major stable ions and existence of phenol ring.
A combination of HPLC separation and MS are useful in identification of flavonoids from a mixture (Stobiecki, 2000). Since the EtOAc fraction was originally from a water extract, it was separated in a reverse phase column using a gradient solution, a combination of water and acetonitrile, after optimization. Figure 2.3 shows that there are 13 major peaks.
The structure of each of the 13 peaks was identified according to the information provided by MS and UV and made reference to available literature (Hu et al., 1994; Liu et al., 2001; Lee et al., 2003; Hu et al., 2004). Mass spectrum provides important information about the peak, in particular the molecular weight. Increasing the voltage of APCI will result in more fragments and provide additional information about its possible structure. Take peak 2 (retention time 20.23 min) as an example, the mass spectrum using low collision energy of APCI showed two major peaks, m/z 449 and m/z 287.5 (Figure 2.5A). Thus, m/z 449 is the molecular weight ion peak [M+H]+ and its molecular weight (MW) is 448. Higher collision energy of APCI caused the peak m/z 449 disappeared and only peak m/z 287.5 remained (Figure 2.5B). The loss
of 162 (from 449 to 287.5) is evidently due to the loss of a sugar residue (C6H12O6 - H2O = 162). Loss of 162 resulted in a stable structure m/z 287.5. According to literature (Hu et al., 2004), it is putatively considered as luteolin. UV spectrum confirmed the existence of a phenol ring (data not shown). According to the literature, the sugar is established as a glucose side chain (Hu et al., 2004; Hu and Kitts, 2004).
Thus, peak 2 is luteolin glucoside. Figure 2.5C shows the conversion from peak m/z 449 to m/z 287 under APCI.
Similarly, the putative structures of other peaks were identified based on their mass spectrum and earlier reports (Hu et al., 1994; Liu et al., 2001; Lee et al., 2003;
Hu et al., 2004) (Figures 2.4-2.16). Peak 8 appears to be a mixture of two flavonoids, one is a baicalein glucuronide and another is a hesperetin glycoside. However, the latter contains an unknown group attached to glucose. The structure of a small peak (peak 4) was not identified. In this investigation, a total of 13 flavonoids from 12 peaks were identified.
The 13 flavonoids of EtOAc fraction can be classified into five groups according to their aglycones (Figure 2.3). Four peaks are related to luteolin, including luteolin-rhamonosyl-glucoside (peak 1), luteolin-glucoside (peak 2), luteolin- glucuronide (peak 3) and luteolin-methoxyl-glucoside (peak 7). Two peaks are apigenin glycosides; including apigenin-glucoside (peak 6) and apigenin-methoxyl- glucoside (peak 10). There are three hesperetin glycosides, including hesperetin- rhamonosyl-glucoside (peak 5), heperetin-glucuronide (peak 9) and heperetin- methoxyl-glucoside (peak 8). Two peaks are baicalein glycosides, including baicalein-glucuronide (peak 8) and baicalein-methoxyl-glucoside (peak 12). The last group consists of two acacetin-glycosides, acacetin-rhmnosyl-glucoside (peak 11) and
RT:17.45 - 43.67
18 20 22 24 26 28 30 32 34 36 38 40 42
Tim e (min) 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Relative Abundance
NL:
7.48E8 TIC MS try3A
1 2 3
4 5
6 7
8 9
10
11
12 13
? Hesperetin glycosides
Acacetin glycosides Luteolin glycosides Apigenin glycosides
8
Baicalein glycosides
Figure 2.3 Flavonoids in the EtOAc fraction
The flavonoids in the EtOAc fraction are grouped into five groups, luteolin glycosides, apigenin glycosides, hesperetin glycosides, baicalein glycosides and acacetin glycosides.
Figure 2.4 Structure elucidation of peak 1, RT 18.31 min
EtOAc fraction was separated on reverse phase-HPLC and detected A
B
C
Figure 2.5 Structure elucidation of peak 2, RT 20.23 min
EtOAc fraction was separated on reverse phase-HPLC and detected by APCI. The Mass spectrums of the peak (RT20.23min) were shown in A and B. A, under low energy APCI; B, under high energy of APCI. C, Structure of peak 2 and its conversion under APCI A
B
C
Figure 2.6 Structure elucidation of peak 3, RT 21.22 min
EtOAc fraction was separated on reverse phase-HPLC and detected by APCI. The mass spectrum of peak RT21.22 were shown. A, under low energy APCI; B, under high energy of APCI. Structure of peak 3 A
B
C
Luteolin-Glucuronide RT21.22 min
m/z 463 m/z 287
-Glucuro (176)
Figure 2.7 Mass spectrum of peak 4, RT 22.79 min
EtOAc fraction was separated on reverse phase-HPLC and detected by APCI. The mass spectrum of peak RT22.79 were shown. A, under low energy APCI; B, under high energy of APCI.
A
B
Figure 2.8 Structure elucidation of peak 5, RT 23.5 min
EtOAc fraction was separated on reverse phase-HPLC and detected A
B
C Hesperdin (Hesperetin-Glucoside-Rhamnoside) RT 23.50 min
Figure 2.9 Structure elucidation of peak 6, RT 25.2 min
EtOAc fraction was separated on reverse phase-HPLC and detected by APCI. The mass spectrum of peak RT25.2 were shown. A, under low energy APCI; B, under high energy of APCI. Structure of peak 2 and its conversion under APCI
A
B
C
Figure 2.10 Structure elucidation of peak 7, RT 23.18 min EtOAc fraction was separated on reverse phase-HPLC and detected by APCI. The mass spectrum of peak RT23.18 min were shown. A, A
B
C
Luteolin-(methoxyl)-Glucoside RT23.18min
m/z 491.1 m/z 287.4
- Methoxyl-Glu (204)
Figure 2.11 Structure elucidation of peak 8, RT 21.22 min EtOAc fraction was separated on reverse phase-HPLC and detected by APCI. The mass spectrum of peak RT21.22 were shown. A, under low energy APCI; B, under high energy of APCI. Structure of peak 3
A B
C
m/z 447 Baicalin m/z 271 Baicalein
-Glucuronic (176)
Baicalin (Baicalein-7-O-D-Glucuronide) RT 27.22 min
Hesperetin-Glycoside RT 27.22 min D
Figure 2.12 Structure elucidation of peak 9, RT 28.70 min EtOAc fraction was separated on reverse phase-HPLC and detected by APCI. The mass spectrum of peak RT28.70 were shown. A, under low energy APCI; B, under high energy of APCI. Structure of peak 9 A
B
C
m/z 477
m/z 301 -Glucuronic (176)
Hesperetin-7-D-O-Glucuronide RT 28.70 min
Figure 2.13 Structure elucidation of peak 10, RT 33.20 min EtOAc fraction was separated on reverse phase-HPLC and detected by APCI. The mass spectrum of peak RT33.20 were shown. A, under low energy APCI; B, under high energy of APCI. Structure of peak 10 and its conversion under APCI.
A
B
C Apigenin-(Methoxyl)-Glucoside RT33.20min
-Methoxyl-Glu (204)
m/z 271.4 m/z 475.2
Figure 2.14 Structure elucidation of peak 11, RT 36.61 min EtOAc fraction was separated on reverse phase-HPLC and detected by APCI. The mass spectrum of peak RT36.61 were shown. A, under low energy APCI; B, under high energy of APCI. Structure of peak A
B
C
Figure 2.15 Structure elucidation of peak 12, RT 42.09min EtOAc fraction was separated on reverse phase-HPLC and detected by APCI. The mass spectrum of peak RT42.09 were shown. A, under low energy APCI; B, under high energy of APCI. Structure of peak 12 and its conversion under APCI.
A
B
C Baicalein-(Methoxyl)-Glucoside RT 42.09min
m/z 475.1 m/z 271.4
- Methoxyl-Glu(204)
Figure 2.16 Structure elucidation of peak 13, RT 43.08min EtOAc fraction was separated on reverse phase-HPLC and detected by APCI. The mass spectrum of peak RT43.08 were shown. A, under low energy APCI; B, under high energy of APCI. Structure of peak A
B
C