Drug Testing and Analysis

Research article Received: 18 October 2013

Revised: 29 December 2013

Accepted: 30 December 2013

Published online in Wiley Online Library: 12 February 2014

(www.drugtestinganalysis.com) DOI 10.1002/dta.1612

Direct analysis of traditional Chinese medicines using surface-enhanced raman scattering (SERS) Jing Zhao,a,b Yang Liu,c,d Andrew M. Fales,b,c Janna Register,b,c Hsiangkuo Yuanb,c and Tuan Vo-Dinhb,c,d* Surface-Enhanced Raman Scattering (SERS) spectrometry provides an excellent tool to characterize chemical constituents in Traditional Chinese Medicines (TCMs) without requiring separation and extraction procedures. This study involved the use of SERS to analyze two TCMs, namely Coptis chinensis and Phellodendron amurense, and their main active constituent, berberine. Using silver nanospheres as SERS-active probes, the decoctions of two raw TCMs and their counterfeits were analyzed. Density functional theory (DFT) was used to calculate the expected Raman spectrum of berberine, and liquid chromatography- mass spectrometry (LC-MS) was used as a comparative technique to quantify the amount of berberine in the samples. The results of the SERS measurements were consistent with the results of DFT calculations and LCMS analyses. To our knowledge, this is the first time that the potential of SERS was demonstrated as a sensitive, rapid, and non-destructive method to qualitatively and quantitatively analyze the active constituents in raw TCM products. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: surface-enhanced Raman scattering (SERS); traditional Chinese medicines (TCMs); berberine; Coptis chinensis; Phellodendron amurense

Introduction

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* Correspondence to: Tuan Vo-Dinh, Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA. E-mail: [email protected] a College of Science, South China Agricultural University, Guang Zhou, China b Department of Biomedical Engineering, Duke University, Durham, USA c Fitzpatrick Institute for Photonics, Duke University, Durham, USA d Department of Chemistry, Duke University, Durham, USA

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Traditional Chinese Medicines (TCMs) have been used for centuries in the prevention and treatment of human diseases, such as malaria and cancer. To date, TCMs still play an important role in healthcare and are increasingly attracting interest worldwide because of their therapeutic effects, long historical clinical practice, and high availability.[1–5] Unfortunately, the progress in modernization and globalization of TCM usage remains slow due to the difficulty of understanding their complex characteristics, such as multi-target, multi-pathway effects, and multiple therapeutic chemical constituents, when compared to currently used modern medicines. Because little is known about how the chemical constituents of TCMs could treat or affect a disease’s progression, it is important to develop sensitive, selective, and practical techniques for the analysis of TCMs and their active constituents to better investigate these species and thus extend their medical applications. Traditionally, TCMs are only qualitatively identified according to morphology, such as shape, colour, or smell. However, this simple profiling method is highly subjective. Because some TCMs can look similar in appearance, misidentification is common. Moreover, it is impossible to identify the active constituents by this rather crude profiling method. In recent years, either thinlayer liquid chromatography (TLC) or high performance liquid chromatography (HPLC) is often employed to identify TCMs by separating and testing their main constituents. Although these methods are accurate and sensitive for analysis of TCMs, they are not practical for wider use in factories, drug stores, farms, or small clinics in remote areas because of the requirement of relatively sophisticated instrumentation, the stringent requirements of separation and the time-consuming analysis process.[6–8] In contrast, Raman scattering has been demonstrated as a rapid

and non-destructive tool to identify TCMs. For example, ginseng and its counterfeit products were successfully identified using Raman scattering techniques.[9] In previous studies, however, TCMs could only be identified qualitatively without any quantitative constituent information. This limitation was due to the fact that Raman scattering is intrinsically inefficient, and the content of active constituents in TCMs is relatively low (the content of active constituents is different with different TCMs, being usually less than 10%). To improve the detection sensitivity, surfaceenhanced Raman scattering (SERS) was used to identify TCMs in this study. Their active constituents were qualitatively and quantitatively analyzed using SERS without requiring sample separation and extraction. To our knowledge, this is the first report on the use of SERS to directly identify and quantify the active constituents in raw TCM products. SERS is a promising tool for identifying the molecular structure of chemical and biological species. Raman scattering involves an inelastic scattering process associated with an energy shift equal to the vibrational or rotational energy of the analyte molecule. Although Raman scattering is intrinsically inefficient (Raman crosssections are typically on the order of 10-28–10-30cm2), the energy shift is very specific to the characteristic vibrational structures of

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the molecule, thus providing a molecular ‘fingerprint’ of the analyte. The SERS effect can enhance the Raman signal of compounds adsorbed on nanostructured metallic substrates by several orders of magnitude. Our laboratory has been involved in the development and application of various SERS platforms ranging from nanoparticles, to nanopost arrays, nanowires, and nanochips.[10–15] An optical analytical technique such as SERS, which has been widely used in many fields such as food safety and biological detection, can provide a rapid, accurate, and non-destructive identification of TCMs. In this study, we applied the SERS technique for identification of two important species of TCMs, Coptis chinensis and Phellodendron amurense, and their main active constituent, berberine. Platycodon Grandiflorum Radix, which is often used as a counterfeit medicine that does not contain berberine, was also analyzed. Studies on both Coptis chinensis and Phellodendron amurense have demonstrated anti-inflammatory and anti-cancer functions from their multi-alkaloid constituents.[16,17] The main alkaloids of Coptis chinensis are berberine, palmatine, jatrorrhizine, and coptisine. The main alkaloids of Phellodendron amurense are berberine, phellodendrine, and magnoflorine. In both species of TCMs, berberine, the dominant alkaloid, is believed to be the most important contributor to therapeutic effects. Therefore, the berberine content is often used to represent the therapeutic value of Coptis chinensis and Phellodendron amurense.[18,19] Radix of Platycodon Grandiflorum, another kind of TCM, is also used as an anti-inflammatory due to its dominant active constituent platycodigenin.[20,21] In this study, TCMs with and without berberine were differentiated, and the content of berberine in raw TCMs were evaluated to illustrate the potential use of SERS in identification of TCMs and their active constituents.

TCMs sample preparation and SERS measurements Berberine (Sigma B3251; berberine chloride form) was purchased from Sigma-Aldrich, Inc (St. Louis, MO, USA). 18 berberine solutions ranging from 0.5μM to 9μM in increments of 0.5μM, were prepared with deionized water (DI, 18MΩ/cm). Coptis chinensis, Phellodendron amurense, and Radix of Platycodon Grandiflorum were purchased from Asia Natural Products, Inc (San Francisco, CA, USA). The TCMs decoctions were prepared by soaking the medicine in DI water for 10 min followed by boiling for another 20 min. Samples were cool down and stored at room temperature. TCMs samples of various dilutions were mixed with silver colloid with volume ratio of 1:10, and then placed into a 96-well plate for measurements. All spectra were collected using a Renishaw InVia Raman system (633nm HeNe laser, 8mW, 1800gr/mm grating; Renishaw Inc., IL, USA).

DFT calculations To determine the vibrational modes involved in Raman spectra, quantum chemical calculations were performed using density

Materials and methods SERS probe synthesis Silver (Ag) nanoparticle colloids were used as SERS nanoprobes, which were prepared using a previously reported method.[22,23] Silver nitrate (99.995%) was purchased from Alfa Aesar (Ward Hill, MA, USA). Hydroxylamine hydrochloride and sodium hydroxide (pellet) were purchased from Mallinckrodt Baker, Inc (Phillipsburg, NJ, USA). Silver nanoparticles were prepared using hydroxylamine as the reducing agent. Briefly, a silver nitrate solution (10 mL, 10 mM) was rapidly added to a hydroxylamine hydrochloride solution (90 mL, 1.67 mM) containing NaOH (3.3 mM) under vigorous stirring for 1 h. The colloidal solutions were then stored at 4°C. The synthesized Ag NPs were characterized under a transmission emission microscope (TEM). The size of the particles had an average diameter of 40 ± 6.3 nm.

Figure 2. Comparison of SERS and Raman spectra of berberine, Berberine concentration is 5μΜ in (a), Berberine concentration is 5μΜ for SERS and 1mM for traditional Raman in (b).

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Figure 1. Structure of berberine(a), structure of berberine with numbering scheme used in DFT calculations(b).

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Copyright © 2014 John Wiley & Sons, Ltd.

Drug Test. Analysis 2014, 6, 1063–1068

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Direct analysis of traditional Chinese medicines using Surface-Enhanced Raman Scattering (SERS) functional theory (DFT) at B3LYP level and 6-311++G(d,p) basis set by using Gaussian 03W program. The relative intensity was calculated from the absolute differential Raman cross-section for the vibration.

LC-MS conditions Liquid chromatography (LC) separation was performed using an Agilent 1200 Series LC with a Phenomenexnt Luna C-18 Column (2x100mm, 3μm particles). The effluent was quantitatively measured using an Agilent 6224 TOF-MS with ESI probe. These LC-MS measurements performed for comparison to SERS measurements were modelled after previously published experiments using LCMS to quantitatively investigate traditional Chinese medicines.[24]

Figure 3. DFT results compared with Raman experiment results(a), and SERS experiment results(b).

Results and discussion Raman and SERS spectrum of berberine Fig. 1(a) shows the chemical structure of berberine. The structure of beberine with numbering scheme used in DFT calculations is shown in Fig. 1(b). Raman and SERS spectra of berberine, shown in Fig. 2, were collected and analyzed. As shown in Fig. 2(a), there is only a very weak Raman signal detected with 5μM berberine. In contrast, the addition of Ag NPs into the sample solution induces the SERS effect and greatly enhances the Raman signal of the same berberine solution. The SERS signal intensity from 5μM berberine was significantly greater than the Raman intensity from 1mM berberine (Fig. 2(b)). Characteristic spectral peaks from both Raman and SERS were in good agreement. The 727–770cm-1 spectral region exhibits the most prominent enhancement with SERS. These results show that SERS is an effective technique to sensitively detect and specifically identify berberine. To further investigate the possible vibrational modes in the Raman spectrum, we performed DFT numerical calculations. The normalized results of DFT, normalized spectra of Raman and SERS spectra are shown in Fig. 3. In the DFT results, the peaks at 726 cm-1, 1203cm-1, 1276cm-1, 1394cm-1, 1509cm-1, and 1560cm-1 are the main peaks that exhibit the strongest Raman intensities. The results are similar to those previously reported by Leona and Lombardi.[25] The experimental Raman results obtained in our study

Figure 4. SERS spectra of berberine, Coptis chinensis, Phellodendron amurense and Platycodon Grandiflorum Radix.

Table 1. SERS main peaks used in qualitative and quantitative analysis -1

SERSexp /cm

731 751 773 --1274 1397 1423 1565

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-1

Ramancal /cm

726 750 772 1143 1276 1394 1423 1560

Description No. of vibration atoms

Type of vibration

5,1,26 (CCN) 6,5,1,26 (CCCN) 8,9,10 (CCC) 26,41 (NC) 16,9 (OC) --26,4 (NC) 6,5 (CC)

Bending Torsion Bending Stretching Stretching Aromatic Ring Breathing Stretching Stretching

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727 752 770 1142 1274 1394 1421 1566

-1

Ramanexp /cm

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J. Zhao et al. berberine, Coptis chinensis, Phellodendron amurense and Radix of Platycodon Grandiflorum. Both Coptis chinensis and Phellodendron amurense, two TCMs containing berberine, show the common peaks of berberine at 727cm-1, 752cm-1, 770cm-1, 1142cm-1, 1274cm-1, 1394cm-1, 1421cm-1, and 1566cm-1. The peaks in the spectra of Radix of Platycodon Grandiflorum, which are located at 730cm-1 and 1327cm-1, are quite different. These results are very useful in two aspects. First, they demonstrate the possibility for identifying the TCMs containing berberine from their counterfeit by SERS. Secondly, the results also underline the ability of SERS to directly detect the active constituents in raw TCMs without physical separation and extraction of the samples. Quantitative analysis

Figure 5. Fitting result of the relationship of intensity and berberine concentration.

To quantitatively determine the berberine concentration, the peak intensity at 727cm-1 was used, as its intensity changed proportionally with that of the other three common peaks. The peak intensities of different berberine dilutions are shown in Fig. 5. There is a good linear relationship between intensity and concentration when the concentration is below 5 μΜ. The fitting result within this range, which can be used to roughly evaluate concentration, is shown below: I ¼ 29211C  3943:8

(1) -1

Figure 6. Calculated berberine concentration in diluted Coptis decoction using fitting formula.

are consistent with the DFT calculations. There are only some small peak position shifts, such as a 1cm-1 shift at 726cm-1, 2 cm-1 shift at 1276cm-1, and 3cm-1 shift at 1394cm-1, because the calculated values are harmonic frequencies while the experimental values include anharmonic corrections.[26] The most intense peak in the SERS spectrum is at 727cm-1, which is an out-of-plane ring system bending deformation caused by No. 5, 1, 26 atoms in Fig. 1(b) based on the DFT results. Combining the results of numerical calculations and experiments, we assign the SERS main peaks for use in qualitative and quantitative analyses as shown in Table 1. Qualitative characterization Qualitative characterization is usually performed by the analysis of common peak positions. Fig. 4 shows the SERS spectra of

where I is intensity of SERS signal at 727cm , and C is concentration of berberine in solution. R2 = 0.9826, which demonstrates that the linear Eqn (1) predicts 98.26% of the variance in the variable I. When the concentration is higher than 6μΜ, the colour of the colloidal sample is visibly changed, as shown in Fig. 5, and the intensity data were not used for quantitative analysis. The berberine concentrations in the three vials in Fig. 5 are 2μΜ, 4μΜ, and 6μΜ from left to right. There is no obvious difference in colour between the berberine concentrations of 2μΜ and 4μΜ, but the colour of colloid with 6μΜ berberine changed to greenish grey. Two blind experiments were performed to evaluate how well the SERS method performs for quantitative analysis of raw medicine. Coptis decoction was prepared and divided into two parts for the SERS and LCMS measurements. For SERS identification, the decoction was diluted 1:200, 1:250, and 1:500, and then three decoctions were measured using SERS three times. The peak at 727cm-1 was selected to determine the concentration of berberine in the diluted decoction using the fitting formula. The results are shown in Fig. 6 and Table 2. The LC-MS data revealed that the decoction sample extract contains at least four other components. Although berberine and palmatine co-elute, the use of extracted ion chromatograms can distinguish between these two components quite well, as shown in Fig. 7. In Fig. 7, peak of berberine is at 336.1243 m/z. The concentration of berberine in the decoction sample is 1.15 ± 0.10mM. The LC-MS results were consistent with those obtained using SERS.

Table 2. Calculated berberine concentration in Coptis decoction using a fitting formula

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Diluted Factors

Intensity of -1 Peak 727/cm

1:500 1:250 1:200

2264 ± 177 8042 ± 476 10671 ± 1018

Calculated berberine concentration Calculated berberine concentration Average berberine concentration in diluted decoction(μm) in original decoction(mM) in original decoction(mM)

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2.13 ± 0.06 4.04 ± 0.16 5.00 ± 0.34

1.07 ± 0.03 1.01 ± 0.04 1.00 ± 0.07

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1.03 ± 0.05

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Figure 7. Positive ion electrospray mass scan spectrum of berberine.

Conclusion The results of this study show that SERS is a useful technique for direct identification of TCMs and their active constituents. Without requiring strict separation and purification like conventional analytical methods, SERS can be used to rapidly analyze TCMs in raw samples, and identify their main active constituents. Compared with other analytical methods, SERS is a highly sensitive, specific, rapid and non-destructive technique. In this study, Coptis chinensis and Phellodendron amurence are the TCMs that can be identified by their main constituent. For TCMs that need to be identified by several constituents, we can use the SERS spectra from the constituent mixtures to assess the authenticity of the TCMs. Since accurate identification of TCMs is an important quality control problem, the use of modern analytical tools to enhance the quality control of TCMs has become an important task. The SERS technique used for identification of basic TCM elements will offer small markets, stores, clinics and factories a simple but efficient analytical tool to rapidly identify and quantify TCMs and their active elements. Acknowledgements The authors appreciate the assistance of Dr George Dubay for his LC-MS analysis. This work was sponsored by the Duke University Exploratory Research Project, the National Science Foundation of China (Grant 60908038), and Guangdong Scientific and Technological Project (No. 2012B040302002).

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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.

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Drug Test. Analysis 2014, 6, 1063–1068

Direct analysis of traditional Chinese medicines using Surface-Enhanced Raman Scattering (SERS).

Surface-Enhanced Raman Scattering (SERS) spectrometry provides an excellent tool to characterize chemical constituents in Traditional Chinese Medicine...
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