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Rapid discrimination of three kinds of Radix Puerariae and their extracts by Fourier transform infrared spectroscopy and two-dimensional correlation infrared spectroscopy
Journal of Molecular Structure 1018 (2012) 88–95
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
Rapid discrimination of three kinds of Radix Puerariae and their extracts by Fourier transform infrared spectroscopy and two-dimensional correlation infrared spectroscopy Beilei Xu a, Guijun Zhang a,⇑, Suqin Sun b,⇑, Changhua Xu b, Jianbo Chen b, Ya Tu c, Qun Zhou b, Meng Cui a, Jingjuan Wang a, Chunxian Wen a a
School of Chinese Pharmacology, Beijing University of Chinese Medicine, Beijing 100102, China Department of Chemistry, Tsinghua University, Beijing 100084, China c Chinese Academy of Chinese Medical Sciences, Beijing 100700, China b
a r t i c l e
i n f o
Article history: Received 30 August 2011 Received in revised form 11 December 2011 Accepted 11 December 2011 Available online 17 December 2011 Keywords: Radix Puerariae Ethanol extracts Multi-level IR macroscopic fingerprint Fourier transform infrared spectroscopy (FT-IR) Two-dimensional correlation infrared spectroscopy (2D-IR) Rapid discrimination
a b s t r a c t In this study, a macroscopic IR fingerprint method, conventional Fourier transform infrared spectroscopy (FT-IR) combined with second derivative infrared spectroscopy and two-dimensional correlation infrared spectroscopy (2D-IR), was applied to quickly identify three kinds of Radix Puerariae and their ethanol extracts (AL extracts). They are authenticated as Pueraria thomsonii Benth (PTB), Pueraria lobata (Willd.) Ohwi (PLW) and simmered P. lobata (Willd.) Ohwi (PLS). By comparing the IR spectra and correlation coefficients, PTB, PLW and PLS all have comparable profiles to starch, however, PTB has relatively higher content starch comparing to PTB and PLW, and on the other hand PLW and PLS have relatively more puerarin than PTB. Though the IR spectra of PLW and PLS were almost the same, significant differences were found in 2D-IR spectra with much higher resolution. The strongest auto-peaks are at 1636 cm 1 and 1654 cm 1 in PLW and PLS, while the relative intensities are different. Moreover, the AL extracts of the three medicines were also perceivably distinguished step by step with FT-IR and 2D-IR. For example, cross-peaks at (1518, 1561) and (1518, 1568) with PLW are superior to PLS. The AL extracts had much higher correlation coefficients with puerarin than their corresponding raw materials. The contents of puerarin in the AL extracts followed the order: PLW > PLS > PTB. Therefore, it was demonstrated that the macroscopic fingerprint method of FT-IR and 2D-IR spectroscopy could be used to discriminate the three types of Radix Puerariae and their ethanol extracts rapidly and effectively. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Radix Puerariae (Gegen), a commonly used traditional Chinese medicine (TCM), is the dry root of Pueraria lobata (Willd.) Ohwi (PLW) and Pueraria thomsonii Benth (PTB) of leguminous plant, known as ‘‘YeGe’’ and ‘‘FenGe’’ in China, respectively. ‘‘WeiGe’’ is P. lobata (Willd.) Ohwi simmered by wheat bran or wet paper (PLS). The three kinds of Radix Puerariae are stipulated in the clinical prescription [1–3]. Although the three medicines have similar appearances and derive from same genera, their therapeutic functions are dramatically different in practical application of traditional medicine. PLW has stronger sweating and antipyretic effects than PTB, while the latter has stronger effect of enhancing production of body fluids to extinguish thirst. Divergent effect of PLS is reduced, and antidiarrheal effect is enhanced [1,4]. Modern pharmacological stud⇑ Corresponding authors. Address: Analysis Center, Tsinghua University, Beijing 100084, China. Tel.: +86 1062787661 (S. Sun), tel.: +86 13501027481 (G. Zhang). E-mail addresses: [email protected] (G. Zhang), [email protected]. edu.cn (S. Sun). 0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2011.12.017
ies of Radix Puerariae also have shown that its extract exhibits antioxidant effect [5], anti-cancer effect [6], and neuro-protective effect [7,8]. However, it is difficult to distinguish the three kinds of Radix Puerariae and their concentrated extracts rapidly by using the common analytical methods. The methods in previous studies [9–16] for determining the components in Radix Puerariae involved techniques such as HPLC [12–16], HPTLC [12,16] and near-infrared spectroscopy (FTNIR) [11], but all of them had limitations in differentiating similar complicated mixture system. For example, in term of qualitative discrimination, FTNIR (13330–4000 cm 1) could only provide limited structural information and has poor fingerprint, especially when studying complicated system, such as TCM and their extract products. TCM contains hundreds of components and produces curative effects through synergic reaction, thus the specific markers identified by chromatography cannot fully reflect the real qualities of TCM. Therefore, it is highly desirable to find a quick and effective identification and discrimination method to entirely monitor and capture the whole constituents of similar TCM and extract products. Characterized by direct and rapid nature, Fourier transform infrared spectroscopy (FT-IR) combined with two-dimensional
B. Xu et al. / Journal of Molecular Structure 1018 (2012) 88–95
Fig. 1. FT-IR spectra in the range of 4000–400 cm
89
1
: (a) starch; (b) PTB; (c) PLW; (d) PLS and (e) puerarin.
Table 1 Comparison of assignment of main peaks of the three kinds of Radix Puerariae and their extracts. (See below-mentioned reference for further information.)
correlation infrared spectroscopy (2D-IR), has been used successfully for TCM, such as identification, quality control and stability prediction [17–21]. The combination of FT-IR, second derivative IR and 2D-IR, conceptually called as ‘‘IR macro-fingerprint method’’, cannot only present the whole features but also the macro-fingerprint characters of the sample [22–28]. In this paper, three different Radix Puerariae and their extracts are rapidly discriminated and the main components and the holistic variation rules of their chemical constituents are also revealed by the macro-fingerprint method. The aim of this study is to develop a method rapidly discriminating TCM, such as Radix Puerariae with simple procedure, good reproducibility and repeatability.
from the accumulation of a total of 32 scans in the range of 4000–400 cm 1 with a resolution of 4 cm 1. Temperature was controlled by a portable programmable temperature controller (Model 50-886, Love Control Corporation). 2.2. Materials P. lobata (Willd.) Ohwi (PLW), P. thomsonii Benth (PTB) and simmered P. lobata (Willd.) Ohwi (PLS) were provided and identified by Beijing University of TCM. Ethanol (AR grade) was purchased from Beijing Chemical Works (Beijing, China). Puerarin (provided by the National Institute on Drug Abuse of China with the batch number of 110752–200511).
2. Experimental 2.3. Procedure 2.1. Apparatus Spectrometer: spectrum GX FT-IR system (Perkin Elmer), equipped with a DTGS detector. Each spectrum was obtained
The three kinds of Radix Puerariae were pulverized and then were extracted with ethanol under ultrasonic condition for two cycles and 30 min each [29]. The extract liquids were combined.
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Fig. 2. Second derivative IR spectra in the range of 1200–500 cm
Fig. 3. Second derivative IR spectra in the range of 1750–1350 cm
Ethanol in extract liquids was then evaporated by using evaporator yielding the ethanol extracts (AL extracts). Each sample was ground into powder and was over 200 mesh. About 2–4 mg of the obtained powder was blended with 200 mg KBr powder and then was ground again and pressed into a tablet. Likewise, about 2–4 mg of puerarin and starch were blended with 200 mg KBr powder and then pressed them into tablets, respectively. After that, the IR spectra of all samples were collected. Second derivative spectra were obtained by using Savitzky Golay polynomial fitting method. For collecting the dynamic FT-IR spectra at different temperatures from 50 to 120 °C, the tablet was put into the pool of the temperature controller to record the IR spectra at interval of 10 °C. The sample pool was continuously heated with an increasing rate of 2 °C/min. 2D-IR correlation spectra were obtained by the treatment of the series of dynamic spectra with 2D-IR correlation analysis software developed by Tsinghua University, Beijing, China.
1
1
: (a) starch; (b) PTB; (c) PLW and (d) PLS.
and 920–760 cm
1
: (a) PTB; (b) PLW; (c) PLS and (d) puerarin.
3. Results and discussion 3.1. Analysis of three kinds of Radix Puerariae 3.1.1. FT-IR spectra analysis of three kinds of Radix Puerariae in the range of 4000–400 cm 1 Fig. 1 shows the IR spectra of three Radix Puerariae samples compared with starch and puerarin. Detailed peak positions and assignments of the samples are summarized in Table 1. As shown in Fig. 1 and Table 1, the peak around 1158 cm 1 is attributed to ACAOAC group of polymerized carbohydrates, and peaks around 1080 cm 1 and 1015 cm 1 are attributed to ACAO group [26]. In the range of 1200–1000 cm 1, the profiles of the three medicines are similar to starch. This is because Radix Puerariae is rich in starch [31]. Furthermore, the content of starch is different [32], which can be used for rapid discrimination. By
B. Xu et al. / Journal of Molecular Structure 1018 (2012) 88–95
Fig. 4. Synchronous 2D-IR correlation spectra (left) in the range of 1800–1400 cm
comparing the correlation coefficients with starch (not shown) in the range of 1200–1000 cm 1, PTB is more similar to starch than the PLW and PLS, suggesting that PTB has higher content of starch components than others. By comparing the IR spectra of PTB, PLW, PLS and pure puerarin, characteristic peaks of puerarin at 1633 cm 1, 1447 cm 1 and 1515 cm 1 can be found in PLW and PLS correspondingly, while there is no obvious corresponding peaks can be observed in PTB, it is suggesting that PLW and PLS could contain more puerarin than PTB. This finding is also consistent with the results from the correlation coefficients analysis. According to the spectra and the correlation coefficient (0.9685), however, no significant difference can be found between the IR spectra of PLW and PLS. 3.1.2. The second derivative IR spectra of three kinds of Radix Puerariae Figs. 2 and 3 are the second derivative IR spectra of the three kinds of Radix Puerariae and two pure materials of starch and puerarin in different range. The second derivative IR spectra can enhance the apparent resolution and amplify subtle differences
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1
: (a) PLW and (b) PLS with their curves of auto-peaks (right): (c) PLW and (d) PLS.
in ordinary IR spectra. As shown in Fig. 2, most identical profiles can be observed in PTB, PLW and PLS compared with pure starch in the range of 1200–500 cm 1. Hence, it is further proved that the three samples have a lot of starch. From Fig. 3, it is easy to discriminate PTB from PLW and PLS. The most obvious differences are in peaks around 1730 cm 1, 1627 cm 1 and 1461 cm 1. In addition, the peaks around 1627 cm 1 and 1514 cm 1, two typical characteristic peaks of puerarin [26], appear in the second derivative IR spectra of PTB with higher resolution. It is indicated that PTB has small content of puerarin. More importantly, PLW and PLS show some differences in the peak bands around 1730, 1691, 1650, 1633, 1541 and 1446 cm 1 though they are still similar. To present the differences more distinctly, we analyze the 2D-IR spectra in the range of 1800–1400 cm 1. 3.1.3. 2D-IR correlation spectra analysis of PLW and PLS 2D-IR spectrum can considerably enhance the resolution of spectrum and provide more information by showing the influences of the perturbation on each of molecules in the sample and then processing the data by a mathematical correlation analysis
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Fig. 5. FT-IR spectra in the range of 4000–400 cm
Fig. 6. Second derivative IR spectra in the range of 1800–1350 cm
1
1
: (a) AL extract of PTB, (b) AL extract of PLW, (c) AL extract of PLS and (d) puerarin.
and 920–750 cm
technique [22]. The 2D-IR correlation spectra, including synchronous and asynchronous spectrum, illustrate the sensitivity for each IR band or functional group and correlation between the functional groups, as well as the sequence of responses, when the investigated system is subjected to a given perturbation. Radix Puerariae has a complex constitution of various organic molecules with different functional groups, which have different susceptibilities of variations under thermal perturbation. Therefore, thermal perturbation is applied to the Radix Puerariae system to reflect more information about population changes of system constituents through the dynamic variation of the IR spectrum. Fig. 4 is the synchronous 2D-IR spectra of PLW and PLS in the range of 1800–1400 cm 1 from 50 to 120 °C. In PLW, the
1
: (a) AL extract of PTB, (b) AL extract of PLW, (c) AL extract of PLS and (d) puerarin.
strongest auto-peaks are at 1636 cm 1 and 1654 cm 1 with similar intensities. While in PLS, the strongest auto-peak is at 1653 cm 1 and the secondary one is at 1636 cm 1. The auto-peak of PLS at 1449 cm 1 is much weaker than 1560 cm 1 and 1596 cm 1, while the intensities of auto-peaks at 1449 cm 1, 1559 cm 1 and 1595 cm 1 are similar in PLW, which are attributed to aromatic ring. Additionally, a series of obvious auto-peaks at 1457, 1467, 1507 and 1540 cm 1 can be found in PLW, while only the auto-peak at 1502 cm 1 can be observed in PLS. The cross peak around (1400, 1500) is shown obvious negative correlation in PLS, while no obvious cross peak here in PLW. In the range of 1560–1700 cm 1, all positive cross peaks are much stronger in PLS than PLW.
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Fig. 7. Synchronous 2D-IR correlation spectra (left) in the range of 1800–1500 cm AL extract of PLW and (d) AL extract of PLS.
1
3.2. The multi-steps IR fingerprint analysis of Radix Puerariae extracts 3.2.1. FT-IR analysis of AL extracts of the three kinds of Radix Puerariae Fig. 5 is the IR spectra of the ethanol extracts (AL extracts). It is observed that the content of starch decreased about 8–15%, while the
: (a) AL extract of PLW and (b) AL extract of PLS with their curves of auto-peaks (right): (c)
puerarin increased about 36–65%. As shown in Fig. 5 and Table 1, the extracts have more and stronger characteristic peaks belonging to puerarin than their corresponding raw materials, such as 1627, 1515, 1444, 1397, 893, 836 and 797 cm 1. Especially, peak at 1516 cm 1 appears in the AL extract of PTB while the peak is missing
Table 2 The differences of the peaks intensities in 2D-IR correlation spectra of PLW and PLS extracts in the range of 1800–1500 cm Samples
Auto peaks (cm Intensity
Extract AL
PLW PLS
a
1
)
1
.
Cross-peaks (cm
a
1
)
a
Intensity 1518 +++ 1518 ++
1559 ++++ 1561 ++++
1568 +++ 1568 –
(–) Invisible, (+) weak, (++) middle, (+++) strong, (++++) very strong.
1634 ++ 1633 ++
1718 + 1720 ++
1744 + 1748 ++
(1518, 1559) ++++ (1518, 1561) +++
(1518, 1568) ++++ (1518, 1568) +++
(1744, others) ++ (1748, others) ++
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than PTB. The correlation coefficients with puerarin of raw materials are lower than their corresponding AL extracts. For instance, the AL extract of PLW is 0.7087 while its raw material is only 0.4226. It is suggested that the effective ingredients including puerarin are accumulated by extraction. In addition, the correlation coefficients with puerarin of extracts follow the order PLW > PLS > PTB, suggesting that the AL extract of PLW has more puerarin than PLS. 4. Conclusion
Fig. 8. The correlation coefficients of the three kinds of Radix Puerariae and their AL extracts with pure puerarin.
in its raw material. Peak at 1516 cm 1 is the characteristic peak of puerarin. Because PTB contains a lot of starch but only a little of puerarin, the peak is covered up in the IR spectrum of PTB. However, puerarin is enriched after extraction, leading to the emergence of the peak at 1516 cm 1. Additionally, the peak around 1730 cm 1 becomes stronger in the extracts than the raw materials (Fig. 1), particularly the AL extract of PTB. It can also be used for rapid discrimination of the AL extracts of the three kinds of Radix Puerariae. Except for the peak around 1730 cm 1, most peaks of ethanol extracts of PLW and PLS are similar. Hence, second derivative IR spectroscopy are applied. 3.2.2. The second derivative IR spectra of AL extracts of the three kinds of Radix Puerariae In Fig. 6, it is easy to find more differences between the AL extract of PTB and the other extracts, such as 1787, 1720, 1627 and 1469 cm 1. Furthermore, small differences are observed at the peak including those around 1730 cm 1 and 1632 cm 1 in the spectra of PLW and PLS AL extracts. To further discriminate extracts of PLW and PLS, we analyze the 2D-IR spectra in the range of 1800–1500 cm 1. 3.2.3. 2D-IR correlation spectra analysis of AL extracts of the three kinds of Radix Puerariae Fig. 7 is the 2D-IR correlation synchronous spectra of PLW and PLS ethanol extracts in the range of 1800–1500 cm 1. The PLW and PLS extracts’ 2D-IR synchronous spectra main auto-peaks and cross-peaks are summarized in Table 2. The number, position and intensity of auto-peaks and cross-peaks are different in the two extracts. The strongest and the secondary auto-peaks are labeled in the red and blue frames, respectively. Both AL extracts have strong auto-peaks around 1518 cm 1, 1561 cm 1 and 1568 cm 1 and cross-peaks at (1518, 1561) and (1518, 1568) with PLW are superior to PLS. These peaks are attributed to stretching vibration of aromatic ring of puerarin. It is suggested that more puerarin is in the PLW AL extract than PLS AL extract. However, the auto-peaks around 1720 cm 1 and 1748 cm 1 and cross-peaks around (1748, others) in PLS AL extract are much stronger than PLW AL extract, indicating that the PLS AL extract could have higher content of phenolic glycoside. 3.3. The content of puerarin estimated by spectral correlation coefficient Through the spectral correlation coefficients with puerarin in Fig. 8, the contents of puerarin in the three medicines and their extracts can be estimated. The correlation coefficient of PTB is only 0.1334, while those of PLS and PLW are 0.4363 and 0.4226, respectively. This demonstrates that PLS and PLW have more puerarin
The three kinds of Radix Puerariae, PTB, PLW and PLS, and their ethanol extracts can be quickly and effectively distinguished by the multi-level IR macro-fingerprint method, although they are from the same genera and have similar shape and chemical compositions. According to the fingerprint characters and the spectral correlation coefficients, the chemical compositions and their relative contents have been rapidly estimated. Although, the PTB, PLW and PLS all have comparable profiles to starch, however PTB has relatively higher content starch comparing to PTB and PLW, and on the other hand PLW and PLS have relatively more puerarin than the PTB. It has also been proved that their AL extracts have more purearin than the corresponding raw materials. More importantly, the AL extract of PLW has higher content of purearin than PLS. It has been demonstrated that the multi-level IR macro-fingerprint analysis is a quick and effective method for identifying and discriminating confused TCM and their extracts which consist of hundreds of chemical compositions. Acknowledgements This work is sponsored by the State Administration of Traditional Chinese Medicine of the People’s Republic of China (2001ZDZX01), the Grand support of Ministry of Science and Technology of the People’s Republic of China (2002BA906A29-4), and the National Natural Science Foundation of China (30860391). References [1] Q.F. Gong, Science of Chinese Drug Processing, China Publishing House of Traditional Chinese Medicine, Beijing, 2003. pp. 360–361. [2] The State of Pharmacopoeia Commission of the People’s Republic of China, Pharmacopoeia of the People’s Republic of China, vol. 1, Chemical Industry Press, Beijing, 2005, pp. 203–233. [3] The State of Pharmacopoeia Commission of the People’s Republic of China, Pharmacopoeia of the People’s Republic of China, vol. 1, China Chemical Industry Press, Beijing, 2010, pp. 272–312. [4] L.X. Zhu, J. Fujian Coll. Chin. Med. 3 (1993) 25. [5] F.L. Xiong, X.H. Sun, L. Gan, et al., Eur. J. Pharmacol. 529 (2006) 1. [6] Z.L. Yu, W.J. Li, Cancer Lett. 238 (2006) 53. [7] H.Y. Zhang, Y.H. Liu, H.Q. Wang, J.H. Xu, H.T. Hu, Cell Biol. Int. 32 (2008) 1230– 1237. [8] H.Y. Lou, X.B. Wei, B. Zhang, X. Sun, X.M. Zhang, Acta Pharm. Sin. 42 (2007) 710–715. [9] L.M. Gong, J. Hunan Univ. Chin. Med. 27 (2007) 93–95. [10] E.L. Sun, B. Chen, B.J. Shi, J. Shandong Pharm. Indust. 16 (1997) 43–44. [11] C.C. Lau, C.O. Chan, F.T. Chau, D.K.W. Mok, J. Chromatogr. A 1216 (2009) 2130– 2135. [12] H. Wangner, R. Bauer, P.G. Xiao, P.G. Xiao, J.M. Chen, H. Michler, S. Bächer, Chinese Drug Monographs and Analysis, vol. 4, no. 20, Verlag für Ganzheitliche Medizin, Wald, 2003. [13] W. Cherdshewasart, S. Subtang, W. Dahlan, J. Pharm. Biomed. Anal. 43 (2007) 428. [14] S.L. Chen, S.B. Chen, D.J. Yang, A.S.C. Chan, H.X. Xu, Chin. Tradit. Herbal Drugs 23 (2003) 661. [15] C.B. Fang, X.C. Wan, C.J. Jiang, H.R. Tan, Y.H. Hu, H.Q. Cao, J. Planar. Chromatogr. Mod. TLC 19 (2006) 348. [16] S.B. Chen, H.P. Liu, R.T. Tian, D.J. Yang, S.L. Chen, H.X. Xu, A.S.C. Chan, P.S. Xie, J.Chromatogr. A 1121 (2006) 114. [17] S.Q. Sun, Q. Zhou, J. Liu, H. Huang, Spectrosc. Spect. Anal. 24 (2004) 427. [18] Y.W. Wu, S.Q. Sun, J. Zhao, L. Yi, Q. Zhou, J. Mol. Struct. 833–834 (2008) 48–54. [19] C.H. Xu, S.Q. Sun, C.Q. Guo, Q. Zhou, J.X. Tao, I. Noda, Vib. Spectrosc. 41 (2006) 118–125.
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Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
Rapid discrimination of three kinds of Radix Puerariae and their extracts by Fourier transform infrared spectroscopy and two-dimensional correlation infrared spectroscopy Beilei Xu a, Guijun Zhang a,⇑, Suqin Sun b,⇑, Changhua Xu b, Jianbo Chen b, Ya Tu c, Qun Zhou b, Meng Cui a, Jingjuan Wang a, Chunxian Wen a a
School of Chinese Pharmacology, Beijing University of Chinese Medicine, Beijing 100102, China Department of Chemistry, Tsinghua University, Beijing 100084, China c Chinese Academy of Chinese Medical Sciences, Beijing 100700, China b
a r t i c l e
i n f o
Article history: Received 30 August 2011 Received in revised form 11 December 2011 Accepted 11 December 2011 Available online 17 December 2011 Keywords: Radix Puerariae Ethanol extracts Multi-level IR macroscopic fingerprint Fourier transform infrared spectroscopy (FT-IR) Two-dimensional correlation infrared spectroscopy (2D-IR) Rapid discrimination
a b s t r a c t In this study, a macroscopic IR fingerprint method, conventional Fourier transform infrared spectroscopy (FT-IR) combined with second derivative infrared spectroscopy and two-dimensional correlation infrared spectroscopy (2D-IR), was applied to quickly identify three kinds of Radix Puerariae and their ethanol extracts (AL extracts). They are authenticated as Pueraria thomsonii Benth (PTB), Pueraria lobata (Willd.) Ohwi (PLW) and simmered P. lobata (Willd.) Ohwi (PLS). By comparing the IR spectra and correlation coefficients, PTB, PLW and PLS all have comparable profiles to starch, however, PTB has relatively higher content starch comparing to PTB and PLW, and on the other hand PLW and PLS have relatively more puerarin than PTB. Though the IR spectra of PLW and PLS were almost the same, significant differences were found in 2D-IR spectra with much higher resolution. The strongest auto-peaks are at 1636 cm 1 and 1654 cm 1 in PLW and PLS, while the relative intensities are different. Moreover, the AL extracts of the three medicines were also perceivably distinguished step by step with FT-IR and 2D-IR. For example, cross-peaks at (1518, 1561) and (1518, 1568) with PLW are superior to PLS. The AL extracts had much higher correlation coefficients with puerarin than their corresponding raw materials. The contents of puerarin in the AL extracts followed the order: PLW > PLS > PTB. Therefore, it was demonstrated that the macroscopic fingerprint method of FT-IR and 2D-IR spectroscopy could be used to discriminate the three types of Radix Puerariae and their ethanol extracts rapidly and effectively. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Radix Puerariae (Gegen), a commonly used traditional Chinese medicine (TCM), is the dry root of Pueraria lobata (Willd.) Ohwi (PLW) and Pueraria thomsonii Benth (PTB) of leguminous plant, known as ‘‘YeGe’’ and ‘‘FenGe’’ in China, respectively. ‘‘WeiGe’’ is P. lobata (Willd.) Ohwi simmered by wheat bran or wet paper (PLS). The three kinds of Radix Puerariae are stipulated in the clinical prescription [1–3]. Although the three medicines have similar appearances and derive from same genera, their therapeutic functions are dramatically different in practical application of traditional medicine. PLW has stronger sweating and antipyretic effects than PTB, while the latter has stronger effect of enhancing production of body fluids to extinguish thirst. Divergent effect of PLS is reduced, and antidiarrheal effect is enhanced [1,4]. Modern pharmacological stud⇑ Corresponding authors. Address: Analysis Center, Tsinghua University, Beijing 100084, China. Tel.: +86 1062787661 (S. Sun), tel.: +86 13501027481 (G. Zhang). E-mail addresses: [email protected] (G. Zhang), [email protected]. edu.cn (S. Sun). 0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2011.12.017
ies of Radix Puerariae also have shown that its extract exhibits antioxidant effect [5], anti-cancer effect [6], and neuro-protective effect [7,8]. However, it is difficult to distinguish the three kinds of Radix Puerariae and their concentrated extracts rapidly by using the common analytical methods. The methods in previous studies [9–16] for determining the components in Radix Puerariae involved techniques such as HPLC [12–16], HPTLC [12,16] and near-infrared spectroscopy (FTNIR) [11], but all of them had limitations in differentiating similar complicated mixture system. For example, in term of qualitative discrimination, FTNIR (13330–4000 cm 1) could only provide limited structural information and has poor fingerprint, especially when studying complicated system, such as TCM and their extract products. TCM contains hundreds of components and produces curative effects through synergic reaction, thus the specific markers identified by chromatography cannot fully reflect the real qualities of TCM. Therefore, it is highly desirable to find a quick and effective identification and discrimination method to entirely monitor and capture the whole constituents of similar TCM and extract products. Characterized by direct and rapid nature, Fourier transform infrared spectroscopy (FT-IR) combined with two-dimensional
B. Xu et al. / Journal of Molecular Structure 1018 (2012) 88–95
Fig. 1. FT-IR spectra in the range of 4000–400 cm
89
1
: (a) starch; (b) PTB; (c) PLW; (d) PLS and (e) puerarin.
Table 1 Comparison of assignment of main peaks of the three kinds of Radix Puerariae and their extracts. (See below-mentioned reference for further information.)
correlation infrared spectroscopy (2D-IR), has been used successfully for TCM, such as identification, quality control and stability prediction [17–21]. The combination of FT-IR, second derivative IR and 2D-IR, conceptually called as ‘‘IR macro-fingerprint method’’, cannot only present the whole features but also the macro-fingerprint characters of the sample [22–28]. In this paper, three different Radix Puerariae and their extracts are rapidly discriminated and the main components and the holistic variation rules of their chemical constituents are also revealed by the macro-fingerprint method. The aim of this study is to develop a method rapidly discriminating TCM, such as Radix Puerariae with simple procedure, good reproducibility and repeatability.
from the accumulation of a total of 32 scans in the range of 4000–400 cm 1 with a resolution of 4 cm 1. Temperature was controlled by a portable programmable temperature controller (Model 50-886, Love Control Corporation). 2.2. Materials P. lobata (Willd.) Ohwi (PLW), P. thomsonii Benth (PTB) and simmered P. lobata (Willd.) Ohwi (PLS) were provided and identified by Beijing University of TCM. Ethanol (AR grade) was purchased from Beijing Chemical Works (Beijing, China). Puerarin (provided by the National Institute on Drug Abuse of China with the batch number of 110752–200511).
2. Experimental 2.3. Procedure 2.1. Apparatus Spectrometer: spectrum GX FT-IR system (Perkin Elmer), equipped with a DTGS detector. Each spectrum was obtained
The three kinds of Radix Puerariae were pulverized and then were extracted with ethanol under ultrasonic condition for two cycles and 30 min each [29]. The extract liquids were combined.
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Fig. 2. Second derivative IR spectra in the range of 1200–500 cm
Fig. 3. Second derivative IR spectra in the range of 1750–1350 cm
Ethanol in extract liquids was then evaporated by using evaporator yielding the ethanol extracts (AL extracts). Each sample was ground into powder and was over 200 mesh. About 2–4 mg of the obtained powder was blended with 200 mg KBr powder and then was ground again and pressed into a tablet. Likewise, about 2–4 mg of puerarin and starch were blended with 200 mg KBr powder and then pressed them into tablets, respectively. After that, the IR spectra of all samples were collected. Second derivative spectra were obtained by using Savitzky Golay polynomial fitting method. For collecting the dynamic FT-IR spectra at different temperatures from 50 to 120 °C, the tablet was put into the pool of the temperature controller to record the IR spectra at interval of 10 °C. The sample pool was continuously heated with an increasing rate of 2 °C/min. 2D-IR correlation spectra were obtained by the treatment of the series of dynamic spectra with 2D-IR correlation analysis software developed by Tsinghua University, Beijing, China.
1
1
: (a) starch; (b) PTB; (c) PLW and (d) PLS.
and 920–760 cm
1
: (a) PTB; (b) PLW; (c) PLS and (d) puerarin.
3. Results and discussion 3.1. Analysis of three kinds of Radix Puerariae 3.1.1. FT-IR spectra analysis of three kinds of Radix Puerariae in the range of 4000–400 cm 1 Fig. 1 shows the IR spectra of three Radix Puerariae samples compared with starch and puerarin. Detailed peak positions and assignments of the samples are summarized in Table 1. As shown in Fig. 1 and Table 1, the peak around 1158 cm 1 is attributed to ACAOAC group of polymerized carbohydrates, and peaks around 1080 cm 1 and 1015 cm 1 are attributed to ACAO group [26]. In the range of 1200–1000 cm 1, the profiles of the three medicines are similar to starch. This is because Radix Puerariae is rich in starch [31]. Furthermore, the content of starch is different [32], which can be used for rapid discrimination. By
B. Xu et al. / Journal of Molecular Structure 1018 (2012) 88–95
Fig. 4. Synchronous 2D-IR correlation spectra (left) in the range of 1800–1400 cm
comparing the correlation coefficients with starch (not shown) in the range of 1200–1000 cm 1, PTB is more similar to starch than the PLW and PLS, suggesting that PTB has higher content of starch components than others. By comparing the IR spectra of PTB, PLW, PLS and pure puerarin, characteristic peaks of puerarin at 1633 cm 1, 1447 cm 1 and 1515 cm 1 can be found in PLW and PLS correspondingly, while there is no obvious corresponding peaks can be observed in PTB, it is suggesting that PLW and PLS could contain more puerarin than PTB. This finding is also consistent with the results from the correlation coefficients analysis. According to the spectra and the correlation coefficient (0.9685), however, no significant difference can be found between the IR spectra of PLW and PLS. 3.1.2. The second derivative IR spectra of three kinds of Radix Puerariae Figs. 2 and 3 are the second derivative IR spectra of the three kinds of Radix Puerariae and two pure materials of starch and puerarin in different range. The second derivative IR spectra can enhance the apparent resolution and amplify subtle differences
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1
: (a) PLW and (b) PLS with their curves of auto-peaks (right): (c) PLW and (d) PLS.
in ordinary IR spectra. As shown in Fig. 2, most identical profiles can be observed in PTB, PLW and PLS compared with pure starch in the range of 1200–500 cm 1. Hence, it is further proved that the three samples have a lot of starch. From Fig. 3, it is easy to discriminate PTB from PLW and PLS. The most obvious differences are in peaks around 1730 cm 1, 1627 cm 1 and 1461 cm 1. In addition, the peaks around 1627 cm 1 and 1514 cm 1, two typical characteristic peaks of puerarin [26], appear in the second derivative IR spectra of PTB with higher resolution. It is indicated that PTB has small content of puerarin. More importantly, PLW and PLS show some differences in the peak bands around 1730, 1691, 1650, 1633, 1541 and 1446 cm 1 though they are still similar. To present the differences more distinctly, we analyze the 2D-IR spectra in the range of 1800–1400 cm 1. 3.1.3. 2D-IR correlation spectra analysis of PLW and PLS 2D-IR spectrum can considerably enhance the resolution of spectrum and provide more information by showing the influences of the perturbation on each of molecules in the sample and then processing the data by a mathematical correlation analysis
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Fig. 5. FT-IR spectra in the range of 4000–400 cm
Fig. 6. Second derivative IR spectra in the range of 1800–1350 cm
1
1
: (a) AL extract of PTB, (b) AL extract of PLW, (c) AL extract of PLS and (d) puerarin.
and 920–750 cm
technique [22]. The 2D-IR correlation spectra, including synchronous and asynchronous spectrum, illustrate the sensitivity for each IR band or functional group and correlation between the functional groups, as well as the sequence of responses, when the investigated system is subjected to a given perturbation. Radix Puerariae has a complex constitution of various organic molecules with different functional groups, which have different susceptibilities of variations under thermal perturbation. Therefore, thermal perturbation is applied to the Radix Puerariae system to reflect more information about population changes of system constituents through the dynamic variation of the IR spectrum. Fig. 4 is the synchronous 2D-IR spectra of PLW and PLS in the range of 1800–1400 cm 1 from 50 to 120 °C. In PLW, the
1
: (a) AL extract of PTB, (b) AL extract of PLW, (c) AL extract of PLS and (d) puerarin.
strongest auto-peaks are at 1636 cm 1 and 1654 cm 1 with similar intensities. While in PLS, the strongest auto-peak is at 1653 cm 1 and the secondary one is at 1636 cm 1. The auto-peak of PLS at 1449 cm 1 is much weaker than 1560 cm 1 and 1596 cm 1, while the intensities of auto-peaks at 1449 cm 1, 1559 cm 1 and 1595 cm 1 are similar in PLW, which are attributed to aromatic ring. Additionally, a series of obvious auto-peaks at 1457, 1467, 1507 and 1540 cm 1 can be found in PLW, while only the auto-peak at 1502 cm 1 can be observed in PLS. The cross peak around (1400, 1500) is shown obvious negative correlation in PLS, while no obvious cross peak here in PLW. In the range of 1560–1700 cm 1, all positive cross peaks are much stronger in PLS than PLW.
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Fig. 7. Synchronous 2D-IR correlation spectra (left) in the range of 1800–1500 cm AL extract of PLW and (d) AL extract of PLS.
1
3.2. The multi-steps IR fingerprint analysis of Radix Puerariae extracts 3.2.1. FT-IR analysis of AL extracts of the three kinds of Radix Puerariae Fig. 5 is the IR spectra of the ethanol extracts (AL extracts). It is observed that the content of starch decreased about 8–15%, while the
: (a) AL extract of PLW and (b) AL extract of PLS with their curves of auto-peaks (right): (c)
puerarin increased about 36–65%. As shown in Fig. 5 and Table 1, the extracts have more and stronger characteristic peaks belonging to puerarin than their corresponding raw materials, such as 1627, 1515, 1444, 1397, 893, 836 and 797 cm 1. Especially, peak at 1516 cm 1 appears in the AL extract of PTB while the peak is missing
Table 2 The differences of the peaks intensities in 2D-IR correlation spectra of PLW and PLS extracts in the range of 1800–1500 cm Samples
Auto peaks (cm Intensity
Extract AL
PLW PLS
a
1
)
1
.
Cross-peaks (cm
a
1
)
a
Intensity 1518 +++ 1518 ++
1559 ++++ 1561 ++++
1568 +++ 1568 –
(–) Invisible, (+) weak, (++) middle, (+++) strong, (++++) very strong.
1634 ++ 1633 ++
1718 + 1720 ++
1744 + 1748 ++
(1518, 1559) ++++ (1518, 1561) +++
(1518, 1568) ++++ (1518, 1568) +++
(1744, others) ++ (1748, others) ++
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than PTB. The correlation coefficients with puerarin of raw materials are lower than their corresponding AL extracts. For instance, the AL extract of PLW is 0.7087 while its raw material is only 0.4226. It is suggested that the effective ingredients including puerarin are accumulated by extraction. In addition, the correlation coefficients with puerarin of extracts follow the order PLW > PLS > PTB, suggesting that the AL extract of PLW has more puerarin than PLS. 4. Conclusion
Fig. 8. The correlation coefficients of the three kinds of Radix Puerariae and their AL extracts with pure puerarin.
in its raw material. Peak at 1516 cm 1 is the characteristic peak of puerarin. Because PTB contains a lot of starch but only a little of puerarin, the peak is covered up in the IR spectrum of PTB. However, puerarin is enriched after extraction, leading to the emergence of the peak at 1516 cm 1. Additionally, the peak around 1730 cm 1 becomes stronger in the extracts than the raw materials (Fig. 1), particularly the AL extract of PTB. It can also be used for rapid discrimination of the AL extracts of the three kinds of Radix Puerariae. Except for the peak around 1730 cm 1, most peaks of ethanol extracts of PLW and PLS are similar. Hence, second derivative IR spectroscopy are applied. 3.2.2. The second derivative IR spectra of AL extracts of the three kinds of Radix Puerariae In Fig. 6, it is easy to find more differences between the AL extract of PTB and the other extracts, such as 1787, 1720, 1627 and 1469 cm 1. Furthermore, small differences are observed at the peak including those around 1730 cm 1 and 1632 cm 1 in the spectra of PLW and PLS AL extracts. To further discriminate extracts of PLW and PLS, we analyze the 2D-IR spectra in the range of 1800–1500 cm 1. 3.2.3. 2D-IR correlation spectra analysis of AL extracts of the three kinds of Radix Puerariae Fig. 7 is the 2D-IR correlation synchronous spectra of PLW and PLS ethanol extracts in the range of 1800–1500 cm 1. The PLW and PLS extracts’ 2D-IR synchronous spectra main auto-peaks and cross-peaks are summarized in Table 2. The number, position and intensity of auto-peaks and cross-peaks are different in the two extracts. The strongest and the secondary auto-peaks are labeled in the red and blue frames, respectively. Both AL extracts have strong auto-peaks around 1518 cm 1, 1561 cm 1 and 1568 cm 1 and cross-peaks at (1518, 1561) and (1518, 1568) with PLW are superior to PLS. These peaks are attributed to stretching vibration of aromatic ring of puerarin. It is suggested that more puerarin is in the PLW AL extract than PLS AL extract. However, the auto-peaks around 1720 cm 1 and 1748 cm 1 and cross-peaks around (1748, others) in PLS AL extract are much stronger than PLW AL extract, indicating that the PLS AL extract could have higher content of phenolic glycoside. 3.3. The content of puerarin estimated by spectral correlation coefficient Through the spectral correlation coefficients with puerarin in Fig. 8, the contents of puerarin in the three medicines and their extracts can be estimated. The correlation coefficient of PTB is only 0.1334, while those of PLS and PLW are 0.4363 and 0.4226, respectively. This demonstrates that PLS and PLW have more puerarin
The three kinds of Radix Puerariae, PTB, PLW and PLS, and their ethanol extracts can be quickly and effectively distinguished by the multi-level IR macro-fingerprint method, although they are from the same genera and have similar shape and chemical compositions. According to the fingerprint characters and the spectral correlation coefficients, the chemical compositions and their relative contents have been rapidly estimated. Although, the PTB, PLW and PLS all have comparable profiles to starch, however PTB has relatively higher content starch comparing to PTB and PLW, and on the other hand PLW and PLS have relatively more puerarin than the PTB. It has also been proved that their AL extracts have more purearin than the corresponding raw materials. More importantly, the AL extract of PLW has higher content of purearin than PLS. It has been demonstrated that the multi-level IR macro-fingerprint analysis is a quick and effective method for identifying and discriminating confused TCM and their extracts which consist of hundreds of chemical compositions. Acknowledgements This work is sponsored by the State Administration of Traditional Chinese Medicine of the People’s Republic of China (2001ZDZX01), the Grand support of Ministry of Science and Technology of the People’s Republic of China (2002BA906A29-4), and the National Natural Science Foundation of China (30860391). References [1] Q.F. Gong, Science of Chinese Drug Processing, China Publishing House of Traditional Chinese Medicine, Beijing, 2003. pp. 360–361. [2] The State of Pharmacopoeia Commission of the People’s Republic of China, Pharmacopoeia of the People’s Republic of China, vol. 1, Chemical Industry Press, Beijing, 2005, pp. 203–233. [3] The State of Pharmacopoeia Commission of the People’s Republic of China, Pharmacopoeia of the People’s Republic of China, vol. 1, China Chemical Industry Press, Beijing, 2010, pp. 272–312. [4] L.X. Zhu, J. Fujian Coll. Chin. Med. 3 (1993) 25. [5] F.L. Xiong, X.H. Sun, L. Gan, et al., Eur. J. Pharmacol. 529 (2006) 1. [6] Z.L. Yu, W.J. Li, Cancer Lett. 238 (2006) 53. [7] H.Y. Zhang, Y.H. Liu, H.Q. Wang, J.H. Xu, H.T. Hu, Cell Biol. Int. 32 (2008) 1230– 1237. [8] H.Y. Lou, X.B. Wei, B. Zhang, X. Sun, X.M. Zhang, Acta Pharm. Sin. 42 (2007) 710–715. [9] L.M. Gong, J. Hunan Univ. Chin. Med. 27 (2007) 93–95. [10] E.L. Sun, B. Chen, B.J. Shi, J. Shandong Pharm. Indust. 16 (1997) 43–44. [11] C.C. Lau, C.O. Chan, F.T. Chau, D.K.W. Mok, J. Chromatogr. A 1216 (2009) 2130– 2135. [12] H. Wangner, R. Bauer, P.G. Xiao, P.G. Xiao, J.M. Chen, H. Michler, S. Bächer, Chinese Drug Monographs and Analysis, vol. 4, no. 20, Verlag für Ganzheitliche Medizin, Wald, 2003. [13] W. Cherdshewasart, S. Subtang, W. Dahlan, J. Pharm. Biomed. Anal. 43 (2007) 428. [14] S.L. Chen, S.B. Chen, D.J. Yang, A.S.C. Chan, H.X. Xu, Chin. Tradit. Herbal Drugs 23 (2003) 661. [15] C.B. Fang, X.C. Wan, C.J. Jiang, H.R. Tan, Y.H. Hu, H.Q. Cao, J. Planar. Chromatogr. Mod. TLC 19 (2006) 348. [16] S.B. Chen, H.P. Liu, R.T. Tian, D.J. Yang, S.L. Chen, H.X. Xu, A.S.C. Chan, P.S. Xie, J.Chromatogr. A 1121 (2006) 114. [17] S.Q. Sun, Q. Zhou, J. Liu, H. Huang, Spectrosc. Spect. Anal. 24 (2004) 427. [18] Y.W. Wu, S.Q. Sun, J. Zhao, L. Yi, Q. Zhou, J. Mol. Struct. 833–834 (2008) 48–54. [19] C.H. Xu, S.Q. Sun, C.Q. Guo, Q. Zhou, J.X. Tao, I. Noda, Vib. Spectrosc. 41 (2006) 118–125.
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