Comparison on FTIR Spectrum and Thermal Analysis for Four Types of Rehamnnia Glutinosa and Their Extracts


 Rehmannia glutinosa (R. glutinosa) is a plant material and widely used clinically in China. Due to different processing methods and various healing effects, there are four types of R. glutinosas (RR, RRP, RRC, and RRPC) and their corresponding extracts for convenient use. As complicated mixture systems, chemical components of R. glutinosa are very difficult to identify and discriminate. In order to effectively control its quality during processing, Fourier transform infrared spectroscopy (FTIR), second derivative spectrum, and thermal analysis were used in this study. It was found that the major active ingredients were retained in the extracts when extracted from their corresponded R. glutinosa and subtle differences could be distinguished by the second derivative spectrum. FTIR spectrum, second derivative spectrum, and thermal analysis are valid method to validate and identify plant material, traditional Chinese medicine material, as well as their corresponding extracts.

RRPE, RRCE, and RRPCE). Nowadays, there are considerable study reports on pharmacological effects of R. glutinosa [15][16][17][18][19][20][21][22][23] . However, there were relatively less researches on the similarities and differences of RR, RRP, RRC, RRPC and their extracts (RRE, RRPE, RRCE, RRPCE) by means of FTIR spectroscopy and thermal analysis. FTIR spectroscopy has ngerprint characteristics with strong features; it is the main method for drug identi cation in various pharmacopoeias 24 . Thermal analysis is also a primary method for quality control of drugs according to its record in pharmacopoeia [24][25][26][27][28] . In this study, the similarities and differences of four types of R. glutinosa and their extracts were investigated by FTIR spectroscopy, second derivative spectrum and thermal analysis. The aim of this study is to develop an effective analysis method for studying integrally the main constituents in the complicated mixture systems such as plant materials and their corresponding extracts.

FTIR spectral analysis
The sample of four types of R. glutinosa and their extracts were vacuum dried (110˚C, 2h), ground into powder, and sieved with a 80-mesh screen. The powder (0.002 g) was thoroughly mixed with the KBr powder (0.2 g) and pressed into a pellet for measurement by a Fourier transform infrared (FTIR) spectrophotometer (Nicolet iS-1063001, Thermo Fisher Scienti c (China) Co. Ltd.) in the range of 400-4000 cm −1 with a resolution of 4 cm −1 . An average of 16 scans was used for each sample. In this study, the second derivative spectra of FTIR spectrograms were analyzed by Omnic 8.3 software and SPSS 22.0 software.

Similarity:
Correlation coe cient is a common method evaluating the similarity for Infrared spectroscopy and other ngerprints of traditional Chinese medicine. In this study, the correlation coe cient was obtained using the calculation function of origin software, which indicates the similarity of two infrared spectra matching each other. The similarity was evaluated by the correlation coe cient of four types of R. glutinosa and their extracts. The correlation coe cient formula is as follows:

Thermal analysis
Thermal analysis experiments of the samples of four types of R. glutinosa and their extracts were carried out under nitrogen atmosphere in a thermal analyzer (NETZSCH STA449 F3 Jupiter, Hexico Scienti c Instruments (Shanghai) Co., Ltd.). The TG (Thermal Gravity Analysis) curves were conducted at heating rates in the range of 20 C°/min from room temperature to 800°C in a nitrogen atmosphere (20 ml/min). The sample amount used was between 10 and 15 mg per specimen, and the collected data were used for further analysis.
3 Results And Discussion 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. 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. 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  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 speci c 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 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.

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 re ected the absorption of C-O and C-OH vibration of polysaccharides, oligosaccharides, and monosaccharides. There manifested as multiple "sawtooth" peaks in the range of 1200 -500 cm −1 wave bands with signi cant 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 "sawtooth" 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.

The similarity of FTIR of four types of R. glutinosa and their extracts
The correlation coe cient could indicate the similarity of two infrared spectra matching each other. Tab 2 showed signi cant 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.  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 rst 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 rst 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 rst stage(≤190°C) due to the removal of water vapor molecules. RRCE and RRPCE have low water absorption due to processing into extracts.
Based on systematical analysis of four types R. glutinosa and their extracts using FTIR, second derivative spectrum, and thermal analysis, it can be concluded that these methods could support large numbers of microscope structure information and entire rules of chemical constituents in medicinal materials. By using microscope ngerprint characters of FTIR spectrum, second derivative spectrum, and thermal analysis, the constituents in the extracts can be tested accurately, instantly, and effectively and the quality of medicinal materials can be validated. Thus, FTIR spectrum, second derivative spectrum, and thermal analysis re ecting objectively the panorama of chemical constituents in complex system is the most credible method to validate and identify the mix-substance systems, such as traditional Chinese medicine, herbal medicine, as well as their corresponding extracts.

Declarations
Funding: This work was supported by the Natural Science Foundation Youth Fund of China (No. 81503299).
Statement: The materials used in this study comply with relevant institutional, national, and international guidelines and legislation. Second derivative spectra of four types of R. glutinosa and their extracts