3.1 FTIR spectra analysis of four types of R. glutinosa and their extracts
The chemical components of R. glutinosa mainly include polysaccharides, oligosaccharides, iridoid glycosides (catalpol was the main element of iridoid glycosides in R. glutinosa), amino acids, and etc. These were material basis for the pharmacological effects of R. glutinosa. Fig. 2 illustrated the FTIR spectra of four types of R. glutinosa and their extracts. Detailed peak positions and assignments of the samples were summarized in Tab. 1. As shown in Fig. 2 and tab 1, FTIR spectra of four types of R. glutinosa and their extracts showed higher similarities and typical absorption peaks of polysaccharides, oligosaccharides, catalpol, amino acids, and etc. There were 7 groups of typical common peaks. These absorption peaks were as follows. 1) -OH stretching vibration of the hydroxide radical from R. glutinosa polysaccharides, oligosaccharides, and catalpol molecules was near 3418 cm−1. 2) -CH stretching vibration of methyl and methylene from polysaccharides and oligosaccharides was near 2926 cm−1. 3) C=O stretching vibration of polysaccharides and amide I band produced by bending vibration of -NH group in amino acid molecules was near 1636 cm−1. 4) the peak of 1419 cm−1 was attributed by the variable angle vibration of C-H from polysaccharides and oligosaccharides. 5) C-O stretching vibration of glycoside molecules was near 1263 cm−1. 6) the region of 1145-1025cm−1 corresponded to ring vibrations overlapped with stretching vibrations of (C–OH) side groups and (C–O–C) glycosidic bend vibration of polysaccharides and oligosaccharides. 7) C-O stretching vibration of carbohydrates and glycosides was near 586 cm−1. In a nutshell, there were many common features in FTIR spectra of four types of R. glutinosa and their extracts. These indicated that the chemical components of four types of R. glutinosa and their extracts were basically the same.
Table 1
Peak positions of FTIR of four types of R. glutinosa and their extracts
sample
|
Wave number /cm−1
|
RR
|
3418
|
2926
|
1636
|
1518
|
1419
|
1263
|
1145
|
1070
|
-
|
-
|
-
|
598
|
RRP
|
3421
|
2929
|
1636
|
-
|
1419
|
1260
|
1145
|
1075
|
-
|
871
|
799
|
586
|
RRC
|
3431
|
2926
|
1636
|
-
|
1419
|
1238
|
1149
|
1072
|
-
|
-
|
-
|
529
|
RRPC
|
3426
|
2924
|
1636
|
-
|
1419
|
1259
|
1150
|
1075
|
-
|
-
|
-
|
586
|
RRE
|
3407
|
2926
|
1617
|
-
|
1419
|
1230
|
1150
|
1075
|
1026
|
899
|
772
|
578
|
RRPE
|
3384
|
2924
|
1636
|
-
|
1419
|
1239
|
1151
|
1077
|
1025
|
867
|
774
|
578
|
PPCE
|
3407
|
2926
|
1635
|
-
|
1418
|
1240
|
1150
|
1078
|
1025
|
868
|
770
|
578
|
RRPCE
|
3387
|
2927
|
1635
|
|
1413
|
1237
|
1151
|
1078
|
1025
|
867
|
766
|
578
|
As shown in Fig. 2a, the intensities of absorption peaks of RRP were stronger than those of RR at around 3418 cm-1 and 2926 cm-1, and the absorption peaks were observed at 871 cm−1 and 799 cm−1 for RRP. This was due to RRP was obtained from RR through processing, steaming, and drying. When RR was processed and processed into RRP, polysaccharides and oligosaccharides were converted to monosaccharides (galactose, fructose, and glucose). According to the literature, monosaccharides made RRP "sweet as sugar", fructose could react with amino acids to form melanin, which made RRP "black as lacquer", and the monosaccharide content of RRP was more than twice of RR 1, 22, which was the reason of RRP to turn black. Due to the presence of -OH and -CH in the galactose, fructose, and glucose molecules, the intensities of absorption peaks in 3418 cm-1 and 2926 cm-1 of RRP were stronger than those of RR. It could also be seen from Fig. 2a that the intensities of absorption peaks near 3418 cm−1, 2926 cm−1, 1636 cm−1, and 1263 cm−1 were significantly reduced in RRC and RRPC, which indicated lower levels of polysaccharides, oligosaccharides, catalpol, amino acids, and etc. The reason was that RRC and RRPC were obtained from RR and RRP, respectively. Partial carbonization occurred during processing, which reduced the intensities of the characteristic peaks of polysaccharides, oligosaccharides, and catalpol hydroxyl groups. The order of the intensities of absorption peaks of four types of R. glutinosa were RRP> RR> RRPC> RRC near 3418 cm−1 and 2926 cm−1.
As shown in Fig. 2b, the intensities of absorption peaks of RRPE were slightly stronger than those of RRE at around 3407 cm−1 and 2926 cm−1, while RRCE and RRPCE were slightly lower. The peak intensities at around 3418 cm−1 and 2926 cm−1 of four types of extracts exhibited the same order as corresponding R. glutinosa, which were RRPE> RRE> RRPCE> RRCE. Therefore, it could be concluded that RRE and RR, RRPE and RRP, RRCE and RRC, and RRPCE and RRPC share similar chemical components, respectively. The absorption peaks were observed at around 1025 cm−1, 867 cm−1, and 774 cm−1 for four types of R. glutinosa extracts while no obvious corresponding peaks were seen for four types of R. glutinosa except RRP at 871 cm−1 and 799 cm−1. The reason might be that four types of extracts were extracted from corresponding R. glutinosa respectively, and a small amount of malt dextrin were added during extraction, which could cause absorption peaks at around 1025 cm−1, 867 cm−1, and 774 cm−1. It could also be seen from Tab. 1 that the absorption peaks moved towards low wavenumber near 3418 cm−1 and 1260 cm−1 for four types of R. glutinosa extracts, and these might be caused by malt dextrin. In addition, the absorption peaks disappeared at 1518 cm−1, which mainly corresponded to amide I band and amide II in RRE. Thus, it could be deduced that the relative content of protein and plant acid in RRE were lower than those in RR. The characteristic absorption peaks of excipient malt dextrin were at around 1025 cm−1, 867 cm−1, and 774 cm−1.
3.2 Comparative analysis of FTIR spectra of four types of R. glutinosa and their extracts
Figure 3 presented a comparison of FTIR spectra and relative intensity of absorption peaks of extracts with those of corresponding R. glutinosa (relative intensity of absorption peaks was the ratio of the intensities of 5 groups of characteristic peaks to those of the maximum peak at around 3400 cm−1 respectively). As shown in Figure 3, the main absorption peak positions and shapes of four types of extracts were quite similar with those of corresponding R. glutinosa. Comparison between two spectra in Figure 3a-3d revealed that the main specific peak positions and shapes were fairly similar to each other for RR and RRE, as well as for RRP and RRPE, RRC and RRCE, and RRPC and RRPCE, respectively, which demonstrated that four types of R. glutinosa extracts and corresponding R. glutinosa had similar chemical compositions. However, the differences were also observed that some absorption peak intensities and absorption peak width of the extracts were wider than those of R glutinosa at around 3418 cm−1. The reason was caused by adding malt dextrin.
It could also be seen from Fig. 3 that the relative intensity of peaks showed differences at around 1636 cm−1, 1419 cm−1, 1240 cm−1, 1150 cm−1 and 1075 cm−1 for four types of R. glutinosa and their extracts ( i.e. RR and RRE, RRP and RRPE, RRC and RRCE, RRPC and RRPCE ). For example, the relative intensity of peak near 1636 cm−1 of RR was higher than that of RRE, and slight differences near 1419 cm−1and 1240 cm−1, whereas the relative intensity of peaks near 1150 cm−1, and 1075 cm−1 of RR were lower than those of RRE. The peak of 1636 cm−1 was produced by C=O stretching vibration of polysaccharides and amide I band by bending vibration of -NH group in amino acid molecules, It showed that when RR was extracted into RRE, the content of polysaccharides and the amino acids were reduced. The peak of 1419 cm−1 was attributed by the variable angle vibration of C-H from polysaccharides and oligosaccharides and the peak of 1150 cm−1 was mainly generated by polysaccharide with hydroxide radical (C-OH), the region of 1145-1025cm−1 corresponded to ring vibrations overlapped with stretching vibrations of (C–OH) side groups and (C–O–C) glycosidic bend vibration of polysaccharides and oligosaccharides and C-O stretching absorption peaks were identified at 899 cm−1 and 772 cm−1 for glycosides and carbohydrates, respectively. Therefore, glycosides and carbohydrates characteristic peaks were more prominent for RRE, and Similarly, RRP and RPE, RRC and RCE, RRPC and RRPCE had the same characteristics as the above analysis. In short, the characteristic peaks of glycosides and carbohydrates were more obvious for four types of extracts.
3.3 Second derivative spectra of four types of R. glutinosa and their extracts
Generally, the second derivative infrared spectrum could greatly enhance the spectral resolution and amplify tiny differences in FTIR spectrum. Since R. glutinosa and their extracts were a mixture of various active components, some absorption peaks were overlapped in FTIR spectra. The second derivative infrared spectroscopy with higher resolution could be applied to further analyze tiny differences of R. glutinosa and their extracts. As illustrated in Fig. 4, a number of differences invisible in FTIR spectra became clearer, especially in the range from 1200 to 500 cm−1, which mainly reflected the absorption of C-O and C-OH vibration of polysaccharides, oligosaccharides, and monosaccharides. There manifested as multiple “saw-tooth” peaks in the range of 1200 - 500 cm−1 wave bands with significant differences in peak number and peak intensities for four types of and their extracts. The peak intensities in the range of 900 - 770 cm−1 were higher in RRP and RRPE (the absorption peak at 830 cm−1 and 778 cm−1 were caused by α-glycosidic bond). Since RRP was processed by RR, polysaccharides and oligosaccharides of RR were converted into monosaccharides (galactose, fructose, and glucose). RRPE were extracts from RRP. Therefore, RRP and RRPE had greater contents of monosaccharide (fructose and glucose) and their characteristic peaks of monosaccharides were more obvious, by which they could be easily distinguished from others. RRC and RRPC were obtained from RR and RRP, respectively, and with partial carbonization, lower contents of polysaccharides, oligosaccharides, and monosaccharide were in RRC and RRPC, lower numbers of “saw-tooth” absorption peaks were in RRC and RRPC, and fewer characteristic peaks of polysaccharides, oligosaccharides, and monosaccharides could distinguish RRC and RRPC with other R. glutinosa and extracts. There were slightly higher contents of glycosides and carbohydrates in four types of extracts due to the addition of malt dextrin excipients during extraction. Thus, slightly higher numbers of “saw-tooth” absorption peaks were in four types of extracts. For example, some peak intensities of RRCE and RRPCE in the range of 1200 - 700 cm−1 were slightly higher than those of RRC and RRPC. Absorption peaks could also been seen at around 1150cm−1 and peak intensities were different and conformed to the above analyses in four types of R. glutinosa and their extracts. It could be seen that second derivative spectra amplify the differences and reveal the potentially characteristic FTIR absorption bands, as well as enhance the spectral resolution and obtain more new information for distinguishing similar complicated samples. Therefore, it could be concluded that the second derivative infrared spectrum could distinguish four types of R. glutinosa and their extracts.
3.4 The similarity of FTIR of four types of R. glutinosa and their extracts
The correlation coefficient could indicate the similarity of two infrared spectra matching each other. Tab 2 showed significant similarity of FTIR spectra between the extracts and corresponding R. glutinosa, and the similarities were 0.951, 0.963, 0.960, and 0.954 for RRE and RR, RRPE and RRP, PPCE and RRC, and RRPCE and RRPC, respectively. Among them, RRPE and RRP exhibited the highest similarity (0.963). In addition, the chemical structures of the extracts were similar to those of the corresponding R. glutinosa, which further proved that four types of extracts had multiple identical chemical components with their corresponding R. glutinosa and retained active ingredients from raw medicinal materials. The minor differences observed between the extracts and corresponding R. glutinosa may be caused by small amounts of components that were not completely extracted and the addition of malt dextrin excipients during extraction.
Table 2
The similarity of FTIR of four types of R. glutinosa and their extracts
|
RR
|
RRP
|
RRC
|
RRCP
|
RRE
RRPE
PPCE
RRPCE
|
0.951
|
0.963
|
0.960
|
0.954
|
3.5 Comparison of thermal analysis of four types of R. glutinosa and their extracts
Figure 5 showed a comparison of TG and DTG curves of four types of R. glutinosa and their extracts. It could be seen that TG and DTG curves of four types of extracts were very similar to those of corresponding R. glutinosa, and the peak positions and shapes of characteristic temperature in DTG curve of four types of extracts were also very similar to those of corresponding R. glutinosa, and the same time the TG and DTG curves of RR and RR were similar, while RRC and RRPC were more similar. This proved that four types of R. glutinosa extracts retained the effective ingredients of their raw materials. This also proved that the active ingredients of RR and RRP were similar, while PPC and RRPC were more similar, which was consistent with the above spectral analysis. It could be seen from DTG curve in Figure 5 that peak 1 and 3 of RR, RRP and their extracts were not obvious while peak 2 was sharp and larger, indicating a lower rate of mass loss in the first and third stage and a higher rate of mass loss in the second stage(about 190°C – 350°C). The reason could be that RR and its extract were rich in polysaccharides, catalpol and a variety of amino acids, and these organic biomasses, especially polysaccharides would be decomposed into monosaccharides when heated. Monosaccharides would be dehydrated to caramel at 190°C to 220°C. Caramel was subjected to further heating to form carbon dioxide and carbon monoxide at high temperature, resulting in a large mass loss. Thus, RR and its extract had a greater mass loss in the second stage. The same reason was for RRP and its extract. Peak 1 in DTG curves of RRC and RRPC were larger than those of RR and RRP, which indicated that RRC, RRPC had higher mass loss rates in the second stage, at the same time, the mass loss rate of RRC and RRPC were slightly higher than those of RR and RRP in the first stage. The reasons were that RRC and RRPC processed by RR and RRP respectively and they were partially carbonized, and these might caucused them to absorbed water easily, RRC and RRPCs had slightly higher mass loss rate in the first stage(≤190°C) due to the removal of water vapor molecules. RRCE and RRPCE have low water absorption due to processing into extracts.