AnalyticalMethods

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aModern Research Center for Traditional Ch

92, Wucheng Road, Taiyuan 030006, Shan

lizhenyu@sxu.edu.cn; qinxm@sxu.edu.cn;

7018379bCollege of Chemistry and Chemical Eng

Wucheng Road, Taiyuan 030006, Shanxi, PcInstitute of Materia Medica, Chinese Acade

People's Republic of China

† Electronic supplementary informa10.1039/c3ay42119h

Cite this: Anal. Methods, 2014, 6, 2736

Received 28th November 2013Accepted 10th February 2014

DOI: 10.1039/c3ay42119h

www.rsc.org/methods

2736 | Anal. Methods, 2014, 6, 2736–27

1H NMR basedmetabolic profiling of the processingeffect on Rehmanniae Radix†

Pan He,ab Zhen-Yu Li,*a Jie Xing,a Xue-Mei Qin*a and Guan-Hua Duac

The roots of Rehmannia glutinosa Libosch, Rehmanniae Radix, also known as Dihuang in China, are an

important herbal drug in Traditional Chinese Medicine (TCM). According to the theory of TCM, the raw

and processed Rehmanniae Radix are used quite differently. In this study, the chemical change of

Rehmanniae Radix induced by processing is investigated by 1H NMR spectroscopy based fingerprinting

coupled with multivariate data analysis. The results indicated that big chemical change had occurred

after the processing of Rehmanniae Radix. Due to the high concentration of sugars, liquid–liquid

partition approach was used to eliminate the interfering effect from sugars, in which more metabolites

could be detected, and phenethylalcohol glycosides were concentrated in the EtOAc fractions, while the

iridoid glycoside were enriched in the n-BuOH part. In addition, various statistical methods were used to

find the differential metabolites between the groups. The results obtained in this study suggest that more

than one extraction and statistical method should be used for comprehensive metabolic profiling of

medical plants, which contained various metabolites in different concentrations.

1 Introduction

The roots of Rehmannia glutinosa Libosch, Rehmanniae Radix,also known as Dihuang in China, are an important herbal drug inTraditional Chinese Medicine (TCM). It was recorded in Chinesemedical classics “Shennong's Herba” and thought as a “topgrade” herb in China. According to the traditional uses,Rehmanniae Radix was used in TCM for nourishing yin andtonifying the kidney.1 Recent pharmacological studies haverevealed that Rehmanniae Radix shows a wide range of bioactiv-ities, including haemostatic, promoting blood coagulation,cardiotonic, diuretic, anti-inammatory, hypoglycemic, anti-tumor, immediating type allergic reaction inhibition, tumornecrosis factor-a (TNF-a) secretion inhibition, and stimulating theproliferation and activities of osteoblasts.2 Besides the pharma-cological research, extensive chemical studies have also beenconducted, suggesting the presence of iridoid glycosides,3 nor-carotenoid glycoside (ionone),4 phenethylalcohol glycosides,5

avonoid, amino acids, organic acids, monosaccharide, oligo-saccharide, and volatile essential oil in Rehmanniae Radix.6

inese Medicine of Shanxi University, No.

xi, People's Republic of China. E-mail:

Fax: +86-351-7011202; Tel: +86-351-

ineering of Shanxi University, No. 92,

eople's Republic of China

my of Medical Sciences, Beijing 100050,

tion (ESI) available. See DOI:

44

The processing of medicinal plant material has a longhistory in TCM, and the importance of processing has beendocumented in the Huang Di Nei Jing for 2000 years. Depend-ing on the therapeutic application, the same plant material canbe processed differently. For Rehmanniae Radix, the raw (driedroot) and processed drug are used in quite different ways, andthe choice is strictly dened in TCM theory and practice. Thedried, raw Rehmanniae Radix has a “cold” property, which isable to cure “heat” syndrome. Meanwhile, the processedRehmanniae Radix treated by steaming and drying possesses aslightly “warm” property, and is used for treatment of “cold”syndrome.2 For the chemical composition change in Rehman-niae Radix due to the processing, previous studies have revealedthat the contents of catalpol and stachyose decreased signi-cantly.7,8 However, synergistic effects of different componentsare vital for the therapeutic efficacy of herbal drugs. Thus, atechnique able to cover a wide range of metabolites is needed tostudy the processing effect on the Rehmanniae Radix.

Nowadays, metabolic ngerprinting has been widely used asa state of the art technique in medicinal plant research.Compared with GC-MS and LC-MS, NMR has some uniqueadvantages, such as rapidity, non-selectiveness, reproducibility,and stability.9,10 In addition, detailed structural information ofmetabolites, including chemical shis and coupling constants,can be directly obtained, which makes NMR an ideal choice forthe proling of medicinal plants, such as Tussilago farfara L.,11

Magnoliae officinalis cortex,12 ginseng13 etc. A recent NMR basedngerprinting study by Chang et al. has demonstrated that thecontent of catalpol, raffinose and stachyose decreased, whereassome monosaccharide increased in the processed Rehmanniae

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Radix.2 However, due to the high concentration of sugar presentin the extracts, other types of compounds, which also contributeto its bioactivities, were not detected. Previous studies haveshown the potential benets of sample preparation methods,such as solid phase extraction (SPE), in plant metabolomicsstudies.14,15 In this study, direct extraction and liquid–liquidpartition, which can separate the sugar signals from the othermetabolites effectively, were used for metabolic ngerprintingof Rehmanniae Radix, and identifying the differential metabo-lites between the raw and processed Rehmanniae Radix.

2 Materials and methods2.1 Solvents and chemicals

Chloroform was bought from Fengchuan Chemical Co. Ltd.(Tianjin, China). Methanol and ethyl acetate (EtOAc) were fromBeijing Chemical works (Beijing, China). Petroleum ether wasacquired from Tianjin University Chemical experimental factory(Tianjin, China) and n-butanol (n-BuOH) was from DengfengChemical reagent factory (Tianjin, China). All chemical reagentsabove mentioned were of analytical grade. Deuterated chloro-form (CDCl3, 99.8%D) containing tetramethylsilane (TMS,0.03%, m/v) and methanol-d4 (CD3OD, 99.8%D) were obtainedfrom Merck (Darmstadt, Germany). D2O was bought fromNorell (Landisville, USA). Sodium 3-trimethlysilyl [2,2,3,3-d4]propionate (TSP) was from Cambridge Isotope Laboratories Inc.(Andover, MA), and NaOD was purchased from Armar (Dottin-gen, Switzerland).

2.2 Plant material

The raw roots (Fig. 1) of the plant materials Rehmannia glutinosawere collected in November 2012, from Linyi County, ShanxiProvince of China, and authenticated by Prof. Xue-Mei Qin ofShanxi University. The processed roots were prepared accordingto the procedures described in the Chinese Pharmacopeia.16

Voucher specimens (DH-201201 and DH-201202) were depos-ited in the herbarium of the Center of Modern Research forTraditional Chinese Medicine, Shanxi University, China. Allsamples were air-dried and ground into ne powder with pestleand mortar to be analyzed.

Fig. 1 The cross section pictures of processed (A), and raw (B) Rhe-mannia Radix samples.

This journal is © The Royal Society of Chemistry 2014

2.3 Sample preparation

Two different extraction procedures were used in this study. Inthe rst procedure (M1), the sample powders (200 mg) ofraw and processed Rehmanniae Radix were weighed into a 10mL glass centrifuge tube. 6 mL of CHCl3–MeOH–H2O (2 : 1 : 1,v/v/v) mixture was added to the tube followed by vortexing for 1min and ultrasonicating for 20 min. Then the material wascentrifuged for 25 min at 3500 rpm. The aqueous methanol(M1M) and chloroform (M1C) fractions were transferred sepa-rately into a 25mL round-bottomed ask and dried with a rotaryvacuum evaporator. The M1C was dissolved in CDCl3, and theM1M was dissolved in a mixture (1 : 1, v/v) of CD3OD andKH2PO4 buffer in D2O (adjusted to pH 6.0 by 1 N NaOD) con-taining 0.05% TSP. Then the samples were centrifuged for 10min at 13 000 rpm. The supernatants (600 ml) of all the sampleswere transferred into 5 mm NMR tube for NMR analysis.

In the second procedure (M2), 10 g sample powders of rawand processed Rehmanniae Radix were extracted by ultra-sonicating with 100 mL of 50% water-methanol mixture for 20min and then ltered at vacuum. The same procedure wasrepeated twice. The ltrates were combined and evaporated invacuum. The residue was suspended in 100 mL of water, andpartitioned by equal volume of petroleum ether, EtOAc, and n-BuOH in sequence. The soluble fractions in petroleum ether(M2P), EtOAc (M2E), n-BuOH (M2B), and the residual water(M2W) fractions were transferred separately into round-bottomed asks and taken to dryness with a rotary vacuumevaporator. The M2P and M2E were redissolved in CDCl3 andCD3OD, respectively, whereas the M2B and M2W were redis-solved in KH2PO4 buffer in D2O. All the samples were trans-ferred into 5 mm NMR tubes for NMR analysis.

2.4 NMR measurements and data analysis

All spectral data were obtained at a temperature of 298 K on aBruker Avance 600 NMR spectrometer (600.13 MHz protonfrequency, Bruker, Germany) equipped with a Bruker 5 mmdouble resonance BBI probe. TSP (0.05%, w/v) was used asinternal standard for D2O, while TMS (0.05%, w/v) was used forCDCl3. CD3OD and CDCl3 were used for internal lockpurposes. Each 1H NMR spectrum consisted of 64 scansrequiring 5 min acquisition time with the following parame-ters: spectral width ¼ 12 345.7 Hz, spectral size ¼ 65 536points, pulse width (PW) ¼ 30� (12.7 ms), and relaxation delay(RD) ¼ 5.0 s. A presaturation sequence was used to suppressthe residual HDO signal with low power selective irradiation atthe HDO frequency during the recycle delay. FIDs were Fouriertransformed with LB ¼ 0.3 Hz. The resulting spectra weremanually phased and baseline-corrected, and calibrated toTSP at 0.00 ppm for M1M and M2B, and to methanol at 3.30ppm for M2E.

The 1H NMR spectra were processed using MestReNova(version 8.0.1, Mestrelab Research, Santiago de Compostella,Spain). For M1M, spectral intensities were scaled to TSP anddivided into integrated regions of equal width (0.04 ppm) cor-responding to the region of d 0.54–10.02. The regions of d 4.70–5.02 and d 3.30–3.38 were excluded from the analysis because of

Anal. Methods, 2014, 6, 2736–2744 | 2737

Fig. 2 Representative 1HNMR spectrumof processed (A) and raw (B) Rhemannia Radix of M1M (the aqueousmethanol extracts in M1 procedure),M2E (the ethyl acetate extracts in M2 procedure), M2B (the butanol extracts in M2 procedure).

2738 | Anal. Methods, 2014, 6, 2736–2744 This journal is © The Royal Society of Chemistry 2014

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the residual signals of HDO and CHD2OD, respectively. ForM2E, the spectral 1H NMR region from d 0.52 to 10.00 wassegmented into regions with widths of 0.04 ppm, and the tworegions of d 4.64–4.88 and d 3.24–3.36 were excluded from theanalysis due to the residual signal of water and methanol. ForM2B, the spectral 1H NMR region from d 0.76 to 10.16 wasintegrated, and the region of d 4.68–4.80 was excluded from theanalysis. Multivariate data analysis (MVDA) by principalcomponent analysis (PCA) and orthogonal projection to latentstructure with discriminant analysis (OPLS-DA) were performedwith the SIMCA-P+ soware (v.13.0, Umetrics, Umea, Sweden)using Pareto scaling method. In addition, the SignicanceAnalysis of Microarray (SAM) and Random Forest Classication(RF) analysis were performed by MetaboAnalyst 2.0 (http://www.metaboanalyst.ca), a comprehensive tool suited formetabolomic data analysis.

3 Results and discussion3.1 Metabolites identication

Two-phase extraction method, composed of CHCl3–MeOH–

H2O in the ratio of 2 : 1 : 1 (M1), was rstly used to extract themetabolites of Rehmanniae Radix. Aer extraction, two frac-tions, M1C and M1M, were obtained and subjected to 1H NMRanalysis. The typical 1H NMR spectra of M1M of raw andprocessed Rehmanniae Radix are shown in Fig. 2, and both ofthem are dominated by the high concentration of sugars. Thesignals were assigned based on comparisons with the chem-ical shi of standard compounds using the Chenomx NMRsuite soware,17,18 Human Metabolomics Database (HMDB),19

and Biological Magnetic Resonance Data Bank (BMRB),20 aswell as reported literature data. The 1H NMR spectrum can be

Fig. 3 Chemical structures of typical metabolites from Rhemannia Radi

This journal is © The Royal Society of Chemistry 2014

divided into three different regions, consisting of the aromaticregion (d 10.00–d 6.00), sugar region (d 6.00–d 3.50), andorganic and amino acid region (d 3.50–d 0.00). Organic acidssuch as succinic acid (d 2.45), acetic acid (d 1.93), citric acid(d 2.56, d 2.71), malic acid (d 2.70, d 2.41) and several aminoacids such as alanine (d 1.48), threonine (d 1.33), glutamic acid(d 2.15, d 2.47) and proline (d 2.08, d 2.38) were identied in theorganic acid and amino acid region. In the sugar region,sucrose (d 5.42, d 4.22), stachyose (d 5.41, d 4.97, d 4.13) andraffinose (d 5.43, d 4.96) were identied as the major sugars(Fig. 3). In addition, small amounts of monosaccharides, suchas glucose, galactose and xylose were identied in the pro-cessed Rehmanniae Radix. In the aromatic region, H-3 ofcatalpol (d 6.40) and leonuride (d 6.21), two iridoid glycosideswere clearly detected. In addition, formic acid (d 8.47) andphenylalanine (d 7.33, d 7.40) were also detected in this region.In the chloroform fraction (M1C), the dominant signals orig-inated from saturated and unsaturated fatty acids or theiresters (Fig. S1†), which were revealed by the terminal methyl (d0.98), a-CH2 (d 2.30), b-CH2 (d 1.60), allylic CH2 (d 2.05), andbis-allylic CH2 (d 2.77), all the other protons of hydrocarbonchain (d 1.20–1.30), and olenic protons (d 5.35).

Due to the high concentration of sugars contained in theM1M, only two secondary metabolites could be detected. Thus,another extraction method (M2) was used to eliminate thestrong sugar signals. In M2, the 50% MeOH extracts were sus-pended in water and partitioned sequentially by petroleumether, EtOAc, and n-BuOH, to give four fractions, M2P, M2E,M2B, and M2W. As seen in Fig. S2,† most of the sugars andother primary metabolites, such as amino and organic acidswere enriched in the M2W fraction, while fatty acids or theiresters were concentrated in M2P. For the M2E fraction (Fig. 2),

x.

Anal. Methods, 2014, 6, 2736–2744 | 2739

Table 1 Assignments of 1H NMR spectral peaks obtained from raw and processed Rehmanniae Radix

No. Compound dHa Method Sourcesb

1 Alanine 1.48 (d, J ¼ 7.2 Hz) M1 R&P2 Threonine 1.33 (d, J ¼ 6.5 Hz) M1 R&P3 Acetic acid 1.93 (s) M1 R&P4 Glutamic acid 2.15 (m), 2.47 (m) M1 R&P5 Proline 2.08 (m), 2.38 (m) M1 P6 Citric acid 2.56 (d, J ¼ 16.3 Hz), 2.71 (d,

J ¼ 16.3 Hz)M1 R

7 Succinic acid 2.45 (s) M1 R8 Malic acid 2.41 (dd, J¼ 15.3 Hz, 9.3 Hz),

2.70 (dd, J ¼ 15.3, 3.3 Hz),4.30 (dd, J ¼ 9.3, 3.3 Hz)

M1 R

9 Formic acid 8.47 (s) M1, M2B R&P10 Phenylalanine 7.33 (m), 7.40 (m) M1 R11 Choline 3.22 (s) M1 R&P12 b-Glucose 4.62 (d, J ¼ 8.0 Hz) M1 R&P13 b-Galactose 4.58 (d, J ¼ 8.0 Hz) M1 R&P14 b-Xylose 4.54 (d, J ¼ 8.0 Hz) M1 R&P15 a-Glucose 5.21 (d, J ¼ 3.7 Hz) M1 R&P16 a-Galactose 5.23 (d, J ¼ 3.7 Hz) M1 R&P17 a-Xylose 5.19 (d, J ¼ 3.7 Hz) M1 R&P18 Sucrose 5.42 (d, J ¼ 3.7 Hz), 4.22 (d, J

¼ 8.8 Hz)M1 R&P

19 Stachyose 5.41 (d, J ¼ 3.7 Hz), 4.97 (d, J¼ 2.8 Hz), 4.13 (m)

M1 R&P

20 Raffinose 5.43 (d, J ¼ 3.7 Hz), 4.96 (d, J¼ 3.8 Hz)

M1 R&P

21 Catalpol 2.30 (m), 2.6 (dd, J ¼ 9.6, 7.7Hz), 5.03 (d, J ¼ 9.8 Hz), 5.13(d, J¼ 5.9 Hz), 5.14 (d, J¼ 5.9Hz), 6.40 (dd, J ¼ 6.0, 1.8 Hz)

M1, M2B R&P

22 Leonuride 1.33 (s), 2.14 (dd, J ¼ 13.7,5.6 Hz), 2.62 (d, J ¼ 10.0 Hz),2.76 (d, J ¼ 7.6 Hz), 5.54 (d, J¼ 1.5 Hz), 6.21 (dd, J ¼ 6.0,2.0 Hz)

M1, M2B R&P

23 5-HMF 9.56 (s), 7.41 (d, J ¼ 3.6 Hz),6.62 (d, J ¼ 3.6 Hz), 4.65 (s)

M2E P

24 HMF analogue-1 9.46 (s) M2E P25 HMF analogue-2 9.40 (s) M2E P26 Acteoside 7.63 (d, J ¼ 15.8 Hz), 6.31 (d,

J ¼ 15.8 Hz), 7.09 (d, J ¼ 2.1Hz), 6.99 (dd, J¼ 2.1, 8.3 Hz),6.82 (d, J ¼ 8.2 Hz), 6.72 (d, J¼ 8.0 Hz), 6.61 (dd, J ¼ 8.0,2.0 Hz), 4.35 (d, J ¼ 7.5 Hz),2.76 (t, J ¼ 7.2 Hz), 1.13 (d, J¼ 6.0 Hz)

M2E R

27 Acteoside analogue-1 7.70 (d, J ¼ 15.8 Hz), 6.42 (d,J ¼ 15.8 Hz), 6.78 (d, J ¼ 2.1Hz), 6.68 (d, J ¼ 8.0 Hz), 6.57(dd, J¼ 2.1, 8.0 Hz), 1.03 (d, J¼ 6.0 Hz)

M2E R

28 Acteoside analogue-2 7.46 (d, J ¼ 16.3 Hz), 6.38 (d,J ¼ 16.1 Hz), 0.94 (d, J ¼ 6.0Hz)

M2E R

29 Ligustrazine 2.62 (s) M2B P30 Unknown-1 7.71 (d, J ¼ 15.48 Hz), 6.46

(d, J ¼ 15.6 Hz)M2B R

31 Unknown-2 7.68 (d, J ¼ 15.72 Hz), 6.38(d, J ¼ 15.84 Hz)

M2B R

32 Unknown-3 7.77 (d, J ¼ 15.9 Hz), 6.16 (d,J ¼ 15.12 Hz)

M2B R

2740 | Anal. Methods, 2014, 6, 2736–2744 This journal is © The Royal Society of Chemistry 2014

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Fig. 4 PCA score plots based on 1H NMR data of raw (square) and processed (dot) Rhemannia Radix. A, B, C represent data for M1M, M2E, M2B.

Table 1 (Contd. )

No. Compound dHa Method Sourcesb

33 Unknown-4 6.76 (t, J ¼ 6.3 Hz), 6.87 (m),7.21 (m), 7.36 (m), 7.39 (m)

M2B R

a s: singlet, d: doublet, t: triplet, m: multiplet, dd: doublet of doublet. b ‘R’ represents raw Rehmanniae Radix and ‘P’ represents processedRehmanniae Radix.

Fig. 5 OPLS-DA score plots and loading plots based on 1H NMR spectra of raw (square) and processed (dot) Rhemannia Radix. A, B, C representdata for M1M, M2E, M2B.

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Table 2 Assessment parameters of the model quality

OPLS-DA model

N R2X (cum) R2Y (cum) Q2 (cum)

M1 (raw vs. processed) 1P + 1O 0.929 0.996 0.991M2E (raw vs. processed) 1P + 1O 0.85 0.999 0.997M2B (raw vs. processed) 1P + 1O 0.88 0.997 0.993

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no sugar signals were observed either in raw or processedRehmanniae Radix. The spectra of raw and processedRehmanniae Radix were quite different, as a phenethylalcoholglycoside, acteoside, was detected as the major compound inthe raw Rehmanniae Radix. In addition, two of its analogueswere also detected. On the other hand, in the processedRehmanniae Radix, higher levels of 5-hydroxymethylfurfural(5-HMF) and two of its analogues (compound no. 24 and 25)were obvious, and almost no phenethylalcohol glycosides weredetected. In the M2B (Fig. 2), carbohydrate signals were stillpresent, but those of iridoid glycosides were increased incomparison to the M1M. In addition, some unidentiedcompounds (unknown-1, unknown-2, unknown-3 andunknown-4) were detected in the aromatic region of rawRehmanniae Radix, while these signals were not detectable in

Fig. 7 Random Forest determined features ranked by their contributionsdata for M1M, M2E, M2B.

Fig. 6 SAM plots of M1M (A), M2E (B), M2B (C).

2742 | Anal. Methods, 2014, 6, 2736–2744

the processed samples. The chemical shis and couplingconstants of all the identied metabolites are summarized inTable 1.

3.2 Multivariate data analysis

Visual inspection of the M1M, M2E and M2B between the rawand processed Rehmanniae Radix revealed that raw Rehman-niae Radix contained more catalpol, stachyose, succinic acid,acteoside and less monosaccharides, 5-HMF, and ligustrazine.Then multivariate analysis was applied to any subtle differencebetween them. The M1M part was analyzed by PCA rstly, and aclear separation can be seen between the raw and processedRehmanniae Radix in the score plot (Fig. 4A) of the rst two PCs(PC1: 60.9%; PC2: 12.3%). Marked chemical alterations inducedby the processing were identied by orthogonal projection tolatent structure with discriminant analysis (OPLS-DA). Theparameters indicating the model quality are listed in Table 2. Inorder to evaluate the validity of the model, permutation tests(permutation number: 200) were also performed. All Q2 and R2

values were higher in the permutation tests, revealing greatpredictability and goodness of t. The corresponding OPLS-DAscore plot and loading plot (Fig. 5A) indicate that the rawRehmanniae Radix contained more catalpol, raffinose, phenyl-alanine, malic acid, succinic acid, acetic acid, alanine and less

to classification accuracy (mean decrease accuracy). A, B, C represent

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monosaccharides such as glucose, xylose and galactose, ascompared with processed Rehmanniae Radix.

For the ethyl acetate extracts (M2E), the PCA score plot alsoshowed clear separation between the raw and processedRehmanniae Radix (Fig. 4B). PC1 accounted for 80.5% of thetotal variance, while PC2 accounted for 9.4% of the total vari-ance. The OPLS-DA loading plot (Fig. 5B) showed that acteosideand its analogues (compound no. 27 and 28) were higher in theraw Rehmanniae Radix, and 5-HMF and its analogues(compound no. 24 and 25) were higher in the processedRehmanniae Radix.

For the butanol extracts (M2B), the separation between theraw and processed Rehmanniae Radix was also obvious(Fig. 4C), and increase of monosaccharide and ligustrazine,together with the decrease of catalpol, leonuride, unknown-1,unknown-2, unknown-3 and unknown-4, were observed aerthe processing (Fig. 5C).

In addition, MetaboAnalyst (http://www.metaboanalyst.ca),a web based tool for metabolomic data analysis, was used tond the differential metabolites between the raw and pro-cessed Rehmanniae Radix. Signicance Analysis of Microarray(SAM) was designed to address the issue in high-dimensiondata analysis, especially for the small sample size (3 to 8).21

This result suggested succinic acid, alanine, acetic acid, malicacid, choline, proline, phenylalanine, b-galactose, b-glucose,a-glucose, raffinose, catalpol, acteoside, acteoside analogue-1,

Table 3 Three different methods to identify significant metabolitesbetween raw and processed Rehmanniae Radix

Metabolites dH Loadingab d(i) Random Forest

Succinic acid 2.45 (s) Y*** �3.17 3

Alanine 1.48 (d) Y*** �3.51 3

Acetic acid 1.93 (s) Y*** �3.21 3

Glutamic acid 2.47 (m) [*** — 3

Malic acid 2.41 (dd) Y*** �3.11 3

Choline 3.22 (s) [*** 2.86 3

Proline 2.38 (m) [*** 3.24 3

Phenylalanine 7.33 (m) Y*** �3.37 3

b-Xylose 4.54 (d) [** — 3

b-Galactose 4.58 (d) [*** 3.07 3

b-Glucose 4.62 (d) [*** 2.94 —a-Glucose 5.21 (d) [*** 3.46 3

Raffinose 5.43 (d) Y*** �2.66 3

Catalpol 6.40 (dd) Y*** �3.41 3

Acteoside 7.08 (d) Y*** �17.98 3

Acteoside analogue-1 6.68 (d) Y*** �14.22 3

Acteoside analogue-2 7.46 (d) Y** — 3

5-HMF 9.56 (s) [*** 10.52 3

HMF analogue-1 9.46 (s) [** — 3

HMF analogue-2 9.40 (s) [** — 3

Leonuride 6.21 (dd) Y* — —Ligustrazine 2.62 (s) [** — 3

Unknown-1 7.71 (d) Y*** �13.82 3

Unknown-2 6.38 (d) Y*** �10.37 3

Unknown-3 7.77 (d) Y*** — 3

Unknown-4 7.39 (m) Y*** �26.76 3

a *P < 0.05, **P < 0.01, ***P < 0.001. b [-relatively higher levels in rawRehmanniae Radix; Y-relatively lower levels in processed RehmanniaeRadix.

This journal is © The Royal Society of Chemistry 2014

5-HMF, unknown-1, unknown-2 and unknown-4 are signi-cantly altered metabolites aer processing (Fig. 6). In addi-tion, the Random Forest (RF) function implemented inMetaboAnalyst was also used to nd the compounds alteredaer the processing. RF is a powerful nonparametric classi-cation method and can be used for both classication andimportant variable selection.22 As can be seen in Fig. 7, the rawRehmanniae Radix contained more succinic acid, alanine,acetic acid, malic acid, phenylalanine, raffinose, catalpol,acteoside and its analogues, unknown-1, unknown-2,unknown-3, unknown-4 and less glutamic acid, choline,proline, b-xylose, b-galactose, a-glucose, 5-HMF and itsanalogues, ligustrazine, if compared with the processedRehmanniae Radix. The differential metabolites determinedby the above three different methods are listed in Table 3,and relatively quantied using bucket data of 1H NMRspectroscopy.

4 Conclusion

In this study, the chemical composition of raw and processedRehmanniae Radix were systematically compared by 1H NMRspectrometry coupled with multivariate analysis. 20 primarymetabolites, including amino acids, organic acids, andsugars, as well as 13 secondary metabolites, were detectedwithout chromatographic separation. Clear differences can beseen between the raw and processed Rehmanniae Radix,indicating that big chemical change occurred aer theprocessing. By liquid–liquid partition, more secondarymetabolites could be detected, and acteoside, ligustrazine,unknown-1, unknown-2, unknown-3 and unknown-4 wereidentied for the rst time as the differential metabolitesrelated with the effect of processing. According to the theoryof TCM, the purpose of herb processing is to increase potency,reduce toxicity and side effects, and alter the properties orfunctions. Thus, further studies should be conducted to linkchemical differences to the biological change, to furtherexplain the processing mechanism of Rehmanniae Radix. Theunknown compounds which also related with the processingshould be also further investigated. In addition, the NMRbased ngerprint of Rehmanniae Radix can be used formonitoring the processing of Rehmanniae Radix for thepurpose of quality control.

In the samples preparation method M2, phenethylalcoholglycosides were concentrated in the EtOAc fractions, while theiridoid glycosides were enriched in the n-BuOH part. Comparedwith direct extraction, liquid–liquid extraction is relatively time-consuming, and could affect the reproducibility of samplepreparation. However, for samples containing high amounts ofprimary metabolites, such as Rehmanniae Radix, it is themethod of choice since it allows the detection of secondarymetabolites of lower content. Furthermore, three statisticalmethods were used to nd the differential metabolites betweenthe raw and processed Rehmanniae Radix. The results obtainedindicate that for comprehensive metabolic proling of medicalplants, various extraction and statistical methods should beused to obtain accurate results.

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