23

5

Biomed. Chromatogr. 2010; 24: 235–244 Copyright © 2009 John Wiley & Sons, Ltd.

Research Article

Received 17 February 2009, Revised 23 April 2009, Accepted 24 April 2009, Published online in Wiley Interscience: 10 July 2009

(www.interscience.wiley.com) DOI 10.1002/bmc.1278

Application of an effi cient strategy based on MAE, HPLC-DAD-MS/MS and HSCCC for the rapid extraction, identifi cation, separation and purifi cation of fl avonoids from Fructus Aurantii Immaturus

Chen Wanga, b, Yaju Pana, b, Guorong Fana, b*, Yifeng Chaia, b and Yutian Wua, b

ABSTRACT: This study presents an effi cient strategy based on microwave-assisted extraction (MAE), HPLC-DAD-MS/MS and high-speed counter-current chromatography (HSCCC) for the rapid extraction, identifi cation, separation and purifi cation of active components from the traditional Chinese medicine Fructus Aurantii Immaturus. An LC-DAD-MS/MS method was applied for the screening and structural identifi cation of main components in crude extract, and fi ve components were preliminarily identifi ed as neoeriocitrin, narirutin, naringin, hesperidin and neohesperidin according to their UV and mass spectra. An effi cient MAE method for the extraction of the three most abundant components (narirutin, naringin and neohesperidin) was optimized by the combination of univariate and multivariate approaches. The crude extract was then separated and purifi ed by HSCCC and a total of 61.6 mg of narirutin, 207.3 mg of naringin and 159.5 mg of neohesperidin at high purities of 98.1, 97.2 and 99.5%, respectively, were obtained from 1.42 g of crude extract. The recoveries of these compounds were 86, 93 and 89%, respectively. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: LC-MS/MS; LC-DAD; Fructus Aurantii Immaturus; microwave-assisted extraction; counter-current chromatography.

* Correspondence to: Guorong Fan, Department of Pharmaceutical Analysis,

School of Pharmacy, Second Military Medical University, 325 Guohe Road,

Shanghai 200433, People’s Republic of China. E-mail: Guorfan@yahoo.com.cn

a Department of Pharmaceutical Analysis, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China

b Shanghai Key Laboratory for Pharmaceutical Metabolite Research, 325 Guohe Road, Shanghai 200433, People’s Republic of China

Abbreviations used: HRE, heat-refl ux extraction; HSCCC, high-speed coun-

ter-current chromatography; MAE, microwave-assisted extraction; TCM, tra-

ditional Chinese medicine.

Introduction

Traditional Chinese medicine (TCM) represents a rich resource for

new drug discovery, and lots of TCMs have been phytochemically

and pharmacologically investigated, from which a number of

bioactive principles have been developed into new drugs and

launched into the market (Xu, 2006; Balunas and Kinghorn, 2005).

However, in most cases, the compositions of TCMs are very com-

plicated and their biologically active compounds are not, or only

partially, known (Li et al., 2008). Therefore, two steps: (i) rapid

screening and analysis of bioactive components in TCMs; and (ii)

effi cient and selective extraction, separation and purifi cation of

these bioactive principles, are of great importance for the quality

control and standardization of TCMs.

Recently, a new approach based on LC-DAD-MS/MS has been

widely introduced for the screening and analysis of natural com-

pounds, making this method the most effi cient analytical tech-

nology in crude plant extracts (Li et al., 2008). Microwave-assisted

extraction (MAE) is a new technique to facilitate the extraction of

components in TCMs (Rostagno et al., 2007; Zhu et al., 2007; Hong

et al., 2001). This technique has advantages over traditional

Soxhlet and heat-refl ux extraction (HRE), which are rather time-

consuming and labor-intensive and use large amounts of organic

solvents. For the isolation and separation of natural products,

high-speed counter-current chromatography (HSCCC), a sup-

port-free all-liquid chromatographic technique that can elimi-

nate the irreversible adsorption of samples onto the solid

support, has been recently successfully used for the preparative

separation of many natural products (Sun et al., 2006; Ling et al.,

2007; Peng et al., 20051, b, 2006; Zhou et al., 2005; Assimopoulou

et al., 2009). Therefore, an effi cient strategy based on MAE, HPLC-

DAD-MS/MS and HSCCC may meet the demand of rapid extrac-

tion, identifi cation, separation and purifi cation of the bioactive

components of great interest from TCMs.

Fructus Aurantii Immaturus (Zhishi in Chinese) is well known

as a traditional Chinese medicine, prepared from the dried young

fruit of Citrus aurantium L. and its cultivars or Citrus sinensis

Osbeck (Fam. Rutaceae). It has gained ever-increasing popularity

because of its therapeutic eff ects for gastro-intestinal food reten-

tion, fullness and distending pain, stuffi ness in the stomach, pro-

lapse of the rectum and uterus, and other syndromes (China

C. Wang et al.

www.interscience.wiley.com/journal/bmc Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 235–244

23

6

Pharmacopoeia Committee, 2005). Previous phytochemical and

pharmacological studies have shown that fl avonoids are one

kind of active secondary metabolite of the plant (Tripoli et al.,

2007). Narirutin, naringin and neohesperidin are the most abun-

dant fl avonoids in Fructus Aurantii Immaturus. Recently, the

existing bibliography on biological and pharmacological of these

fl avonoids was reviewed (Tripoli et al., 2007). Several pharmaco-

logical studies have also shown that these fl avonoids possess

anti-allergic (Inoue et al., 2002), anti-oxidant (Wang et al., 2003;

Rajadurai et al., 2006), anti-infl uenza (Kim et al., 2001), anti-tumor

(Vanamala et al., 2006) and anti-infl ammatory (Panthong et al.,

2007) eff ects. Therefore, the screening and analysis of the fl avo-

noids, and extraction, separation and purifi cation of the abun-

dant fl avonoids (narirutin, naringin and neohesperidin), is very

important for the quality control and standardization of Fructus

Aurantii Immaturus.

In this study, Fructus Aurantii Immaturus was selected as a

model, a HPLC-DAD-MS/MS method was developed for the pre-

liminary identifi cation and structural studies of the fl avonoids in

the crude extract, and fi ve main fl avonoids (structures in Fig. 1)

were putatively identifi ed according to their UV and mass spectra

(Anagnostopoulou et al., 2005, 2006). Then, the three most abun-

dant fl avonoids with high bioactivity were extracted by an opti-

mized MAE method, and subsequently separated and purifi ed by

two steps of HSCCC. The fi nal goal was to develop an effi cient

strategy based on MAE, HPLC-DAD-MS/MS and HSCCC for the

rapid extraction, identifi cation, separation and purifi cation of

active components from natural plants, ultimately contributing

to the TCM modernization.

Experimental

Apparatus

The preparative HSCCC instrument employed in the present study was a

model TBE-300A high-speed counter-current chromatography (Tauto

Biotechnological Company, Shanghai, China) with three preparative

polytetrafl uoroethylene coils (internal diameter of tube, 1.6 mm; total

volume, 300 mL) and a 20 mL sample loop. The revolution radius or the

distance between the holder axis and central axis of the centrifuge (R)

was 5 cm, and the b value varied from 0.5 at the internal terminal to 0.8

at the external terminal (b = r/R where r is the distance from the coil to

the holder shaft). The HSCCC system was equipped with a model S

constant-fl ow pump (Beijing Boyikang Laboratory Implements,

Beijing, China), a UV–vis detector (Shanghai Institute of Biochemistry,

Shanghai, China) and a model N2010 workstation (Zhejiang University,

Hangzhou, China).

A Varian HPLC-MS/MS system (Palo Alto, CA, USA) consisting of a

ProStar 410 autosampler, two ProStar 210 pumps, a ProStar 330 photodi-

ode array detector and a 1200 L triple quadrupole mass spectrometer

equipped with an electrospray ionization source was used. A Varian MS

workstation version 6.3 software was used for data acquisition and

processing.

MAE was performed on a CW-2000 open vessel microwave-extraction

system (Shanghai Xintuo Microwave Technology, Shanghai, China) with

a rated power of 800 W. The vessel was placed in a microwave irradiation

cavity and fi tted to the condenser.

Materials and Reagents

All organic solvents used for HSCCC were of analytical grade and pur-

chased from WuLian Chemical Factory, Shanghai, China. Methanol used

for HPLC was of chromatographic grade (Merck, Darmstadt, Germany).

Reverse osmosis Milli-Q water (18.2 MΩ; Millipore, Bedford, USA) was

used for all solutions and dilutions.

The Fructus Aurantii Immaturus (Provenance, Jiangxi, China; season,

June–July) was purchased from a local drug store and identifi ed by

Professor Luping Qin (Department of Pharmacognosy, College of

Pharmacy, the Second Military Medical University, Shanghai, China).

Preparation of Crude Extract

In the present study, crude extracts were prepared by diff erent proce-

dures (MAE and HRE), and the extraction yields were compared. The

yields of target compounds were defi ned as follows: extraction yield

(mg/g) = mass of target compound/mass of material.

MAE was performed on 10 g of shattered Fructus Aurantii Immaturus.

The main factors aff ecting the effi ciency (extraction solvent composition,

liquid/solid ratio, extraction time and extraction temperature) were opti-

mized by the combination of univariate and multivariate approaches.

HRE was performed according to the literature, in which the contributing

factors of the extraction yields were optimized by an orthogonal design

(Liu et al., 2006). In brief, 10 g of Fructus Aurantii Immaturus was extracted

twice with 80 mL of 60% ethanol for 2 h. After extraction, the extract was

fi ltered and evaporated to dryness by rotary vaporization at 60°C under

reduced pressure.

LC-DAD-MS/MS Conditions

The chromatographic separation was performed on a reversed-phase

DiamonsilTM C18 (200 × 4.6 mm i.d., 5 μm; Dikma Technologies Company,

China) with a C18 guard column. The mobile phase was methanol–water–

acetic acid (35 : 65 : 0.5, v/v). The fl ow rate was 1 mL/min, and the detec-

tion wavelength was set in the range 190–400 nm (monitoring

wavelength 280 nm).

The HPLC eluent was split, and approximately 0.2 mL/min was intro-

duced into the mass spectrometer. The ESI-MS spectrometer was oper-

ated in both positive and negative modes. The electrospray capillary

voltage was set to 30 V. Nitrogen was used as a drying gas for solvent

evaporation. The API housing and drying gas temperatures were kept at

50 and 380°C. The collision energy was 20 eV. The mass range measured

was 200–800 amu. The scan time was 1 s and the detector multiplier

voltage was set to 1300 V.

HSCCC Separation Procedure and Identifi cation of

Peak Fractions

The crude extract was separated and purifi ed by two steps of HSCCC. In

the fi rst step of separation, a two-phase solvent system composed of

Figure 1. The chemical structures of main fl avonoids in the crude

extract of Fructus Aurantii Immaturus.

Effi cient strategy for fl avonoids from Fructus Aurantii Immaturus

Biomed. Chromatogr. 2010; 24: 235–244 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/bmc

23

7

ethyl acetate–n-butanol–water (2 : 1 : 3, v/v) was used. The coil column

was fi rst entirely fi lled with the upper phase and then the apparatus was

rotated at 800 rpm, while the lower phase was pumped into the column

at a fl ow rate of 2.0 mL/min. After the mobile phase front emerged and

hydrodynamic equilibrium was established in the column, the sample

solution (1.42 g of crude extracts dissolved in the 60 mL lower phase of

the solvent system, 15 mL for each injection) was injected through the

injection value. The effl uent of the column was continuously monitored

with a UV–vis detector at 254 nm. Peak fractions were manually collected

according to the elution profi le.

When the fi rst step of separation procedure was fi nished, the two-

phase solvent in the separation column was blown out. The fractions

containing narirutin and neohesperidin were evaporated to dryness by

rotary vaporization, yielding 204.5 mg of residual (refi ned sample), and

was to be isolated and separated in the next run of HSCCC.

The second run of separation procedure was performed with the

two-phase solvent system chloroform–methanol–n-butanol–water

(4 : 3 : 0.5 : 2, v/v). Refi ned sample (204.5 mg of refi ned sample dissolved in

the 15 mL of lower phase of the solvent system) was further isolated and

purifi ed in a similar way to the fi rst run.

Identifi cation of the chemical structure was carried out by MS, 1H NMR

and 13C NMR spectra.

Results and Discussion

Preliminarily Structural Identifi cation of Flavonoids by

HPLC-DAD-MS/MS

In order to name fragment ions from fl avonoid conjugates, we

adopted the nomenclature proposed by Domon and Costello

(1988) and subsequently modifi ed by Claeys and coworkers (Ma

et al., 1997). Ions containing the aglycone were labeled k,lXj, Yj and

Zj, , where j is the number of the interglycosidic bond bronken,

counted from the aglycone, and the superscripts k and l indicate

the cleavages within the carbohydrate rings. The glycosidic bond

linking to the aglycone is number 0, Y0 and Z0 being the corre-

sponding fragments (Fig. 2).

The crude extract of Fructus Aurantii Immaturus was analyzed

by HPLC-DAD-MS/MS [Figs 3 and 4(a), Table 1]. Because of the

typical UV absorption pattern, compounds 1–5 can be rapidly

identifi ed as fl avonoids. MS spectra in positive-ion are mostly

often used in the structure analyses of fl avonoids, because these

positive-ion spectra were more informative than the correspond-

ing negative-ion spectra and contained extensive fragmenta-

tions that were diagnostic of structural features (Es-Safi et al.,

2005). Taking structural identifi cation of compound 3 [peak 3 in

Figs 3 and 4(a)] as an example, in the positive-ion mode, it gave

sodiated molecular ion [M + Na]+ (m/z 603) and protonated

molecular ion [M + H]+ (m/z 581). Fragment ion [Y1 + H]+ (m/z 435)

might due to the dissociation of terminal rhamnose (146 u) from

the protonated molecular ion. Fragment ions [Z1 + H]+ (m/z 419)

might be attributed to the dissociation of glucose (162 u) from

the protonated molecular ion, and further loss of H2O yielded

fragment ions [Z1 − H2O + H]+ (m/z 401) and [Z1 − 2H2O + H]+

(m/z 383). Fragment ion [0,2Y0 + H]+ (m/z 273) might be due to the

dissociation of rhamnosylglucose (308 u) from the protonated

molecular ion. Fragment ion [0,2X0 + H]+ (m/z 315) was also

observed. While in the negative-ion mode, fragment ions [0,2X1

− H]− (m/z 459) , [0,2X0 − H]− (m/z 313) and [Y0 − H]− (m/z 271) were

observed from the deprotonated molecular ion. According to the

typical UV absorption and fragmentation pattern proposed

above, compound 3 was preliminarily identifi ed as naringin,

which was in agreement with the literature (He et al., 1997; Zhou

et al., 2006, 2007; Shi et al., 2007; Anagnostopoulou et al., 2005).

In the same way, compounds 1–5 were preliminarily identifi ed as

neoeriocitrin, narirutin, naringin, hesperidin and neohesperidin,

respectively.

Extraction, separation and purifi cation of major fl avonoids

of Fructus Aurantii Immaturus extract

As we can see from the Figs 3 and 4(a), compounds 2, 3 and 5

(preliminarily identifi ed as narirutin, naringin and neohesperidin)

were the major components in the crude extract of Fructus

Aurantii Immaturus. Therefore, it is necessary to develop a rapid

method for the extraction and purifi cation of these compounds.

Such a method will not only facilitate quality control of existing

Fructus Aurantii Immaturus products, but also can provide high-

purity authentic standards to validate the structural identifi ca-

tion obtained by LC-MS/MS. Therefore an eff ective method based

on MAE and HSCCC was developed for the extraction, separation

and purifi cation of target compounds from Fructus Aurantii

Immaturus.

Optimization of MAE Conditions

Since various factors potentially aff ect the extraction yields, the

optimization of the factors was of great signifi cance. In this study,

the eff ects of these parameters (extraction solvent composition,

liquid–solid ratio, extraction time and extraction temperature)

were preliminarily studied using a univariate approach. As shown

in Fig. 5, the extraction yields reach a plateau with ethanol

Figure 2. Ion nomenclature followed for fl avonoids (illustrated on naringin).

C. Wang et al.

www.interscience.wiley.com/journal/bmc Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 235–244

23

8

Figure 3. HPLC-DAD chromatograms of crude extracts from Fructus Aurantii Immaturus by MAE.

Figure 4. TIC chromatograms: (a) of crude extracts from Fructus Aurantii Immaturus by MAE; (b) peak fraction II of fi rst-step HSCCC; (c) peak fraction

III of second-step HSCCC; (d) peak fraction IV of second-step HSCCC.

Effi cient strategy for fl avonoids from Fructus Aurantii Immaturus

Biomed. Chromatogr. 2010; 24: 235–244 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/bmc

23

9

Table 1. Retention times, MS data and UV lmax values for the main fl avonoids present in Fructus Aurantii Immaturus

No. RT UV lmax (nm) [M − H]− ESI-MS/MS m/z [M + H]+ [M + Na]+ (+ )ESI-MS/MS m/z Identifi cation

1 14.27 224, 282 595 287 597 619 451, 435, 417, 399, 289 Neoeriocitrin2 20.85 224, 282 579 270 581 603 435, 419, 401, 315, 273 Narirutin3 24.72 224, 282 579 459, 313, 271 581 603 435, 419, 401, 383, 315, 273 Naringin4 27.82 224, 282 609 301 611 633 465, 449, 431, 413, 303 Hesperidin5 33.29 224, 282 609 489, 343, 301 611 633 465, 449, 431, 413, 303 Neohesperidin

concentration at levels of 70, 80 and 90%; liquid–solid ratio at

10 : 1, 15 : 1 and 20 : 1; extraction time at 5, 10 and 15 min; and

extraction temperature at 60, 70 and 80°C. Based on these results,

an orthogonal design L9 (3)4 was applied for the further optimiza-

tion for the contributing factors producing interrelated infl uence,

as shown in Table 2. The extract obtained from each test of MAE

was weighed and analyzed by HPLC, and result is shown in Table

3. The results in Table 4 indicate that the factor ratio of liquid–

solid was the most important. To sum up, the optimum extraction

condition was ethanol concentration, liquid–solid ratio, extrac-

tion time and extraction temperature at 90%, 15 : 1, 5 min and

80°C, respectively. Under these conditions, a total of 1.42 g crude

extract was obtained from 10 g of Fructus Aurantii Immaturus,

and the extraction yields of narirutin, naringin and neohesperi-

din were 7.0, 21.8 and 17.6 mg/g, respectively, according to the

HPLC analysis (chromatogram shown in Fig. 3).

Comparison of MAE and HRE

The resulting recoveries using MAE method were compared with

those obtained using HRE (n = 3; Fig. 6). MAE was selected to

extract narirutin, naringin and neohesperidin from Fructus

Aurantii Immaturus when the extraction time and extraction

yields were taken into consideration. This is mainly due to the

factor that microwave energy is delivered effi ciently to materials

through molecular interaction with the electromagnetic fi eld

and off ers a rapid transfer to the extraction solvent and raw plant

materials (Thostenson and Chou, 1999).

Separation and Purifi cation of Flavonoids by HSCCC

A successful separation of the target compounds using HSCCC

requires a careful search for a suitable two-phase solvent system

to provide an ideal range of partition coeffi cients (K) (Ito, 2005).

In order to achieve effi cient separation of target compounds,

various two-phase systems were tested (Table 5). Preliminary

selections of the two-phase solvent systems were based on good

K values. The two-phase solvent systems chloroform–methanol–

water (4 : 3 : 2, v/v), ethyl acetate–n-butanol–water (3 : 2 : 5, 2 : 3 : 5,

v/v), n-butanol–water (1 : 1, v/v) and n-butanol–acetic acid–water

(4 : 1 : 5, v/v) were excluded because in which the target com-

pounds’ K values were large, and the two-phase solvent systems

chloroform–methanol–n-butanol–water (4 : 3 : 1 : 2, 4 : 3 : 0.5 : 2,

v/v) and ethyl acetate–n-butanol–water (4 : 1 : 5, 2 : 1 : 3, v/v) were

further evaluated. When the two-phase solvent system chloro-

form–methanol–n-butanol–water (4 : 3 : 0.5 : 2, v/v) was used,

narirutin was eluted with naringin, and neohesperidin was eluted

with other compounds. Narirutin, naringin and neohesperidin

were all eluted together when the two-phase solvent system

chloroform–methanol–n-butanol–water (4 : 3 : 1 : 2, v/v) was used,

and so was the two-phase solvent system ethyl acetate–n-butanol–

water (4 : 1 : 5, v/v). Finally, the two-phase solvent system ethyl

acetate–n-butanol–water (2 : 1 : 3, v/v) was applied for the fi rst run

of HSCCC. Under this condition, naringin was separated from the

other compounds, and narirutin was eluted with neohesperidin.

As for the second run of separation, the two-phase solvent

system chloroform–methanol–n-butanol–water (4 : 3 : 0.5 : 2, v/v)

could provide baseline separation for narirutin and neohesperi-

din, and was selected.

Although the selection of the two-phase systems is critical, the

infl uence of the fl ow rate of the mobile phase, separation tem-

perature and the revolution speed was also investigated. A low

fl ow rate can increase the retention of stationary phase and result

in an improved peak resolution. For the fi rst run of HSCCC from

the crude sample, preliminary tests were conducted at two fl ow

rates (1.0 and 2.0 mL/min), and it was not found that the former

(1.0 mL/min) gave a better resolution. In order to reduce the

separation time, the latter (2.0 mL/min) was employed. The sepa-

ration temperature also has signifi cant eff ects on K values and

the retention of the stationary phase. In this study, changing the

separation temperature to 20, 30 and 40°C did not aff ect the

resolution signifi cantly. The revolution speed is a factor aff ecting

the retention of the stationary phase and various revolution

speeds (700, 800, 900 and 1000 rpm) were tested. The retention

of the stationary phase was poor, resulting in low resolution for

target compounds when the revolution speed was set at 700 rpm.

Compared with the revolution speed of 800 rpm, higher speeds

(900 and 1000 rpm) increase the retention of stationary phase,

but also increase the separation time. Finally, a fl ow rate of

2.0 mL/min, revolution speed of 800 rpm and temperature of

30°C were applied in both separation runs.

In addition, in the previous study, the single injection mode

was applied. A total of 300 mg of crude extracts dissolved in the

15 mL lower phase of solvent system was injected through the

injection valve and the total separation time was about 10 h [Fig.

7(A)]. Obviously, it is time-consuming for such a small sample

load. Therefore, continuous separation mode was applied in the

present study. The noisy signal in chromatogram [Fig. 7(B)] indi-

cated small quantities of stationary phase bleedings (about

15 mL), which might aff ect the resolution of the target com-

pounds. In the earlier study, the fractions I and II of each injec-

tion were collected separately and analyzed by HPLC

(chromatogram not given), and the results indicate no signifi cant

diff erence on resolution of target compounds between the four

injections. Therefore, the continuous separation mode was avail-

able in the present study and its advantages were obvious. This

mode can reduce the cycle time and solvent consumption

C. Wang et al.

www.interscience.wiley.com/journal/bmc Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 235–244

24

0

Figure 5. Optimization of extraction parameters by a univariate approach.

Table 2. L9 (3)4 orthogonal test design

Level Factors(A) Extraction

solvent (ethanol %)

(B) extraction

time (min)

(C) Liquid–solid ratio

(volume/mass, mL/g)

(D) Extraction

temperature (°C)

1 70 2 10 602 80 5 15 703 90 10 20 80

Table 3. L9 (3)4 orthogonal test results

Test no. Factors Yield (mg/g)a

A B C D Compound 1 Compound 2 Compound 3

1 1 1 1 1 4.21 12.94 12.252 1 2 2 2 6.93 21.02 17.873 1 3 3 3 6.62 20.01 16.774 2 1 2 3 6.88 20.96 17.685 2 2 3 1 6.63 19.89 16.716 2 3 1 2 5.04 13.21 12.487 3 1 3 2 6.42 19.25 17.478 3 2 1 3 5.32 15.05 13.429 3 3 2 1 6.94 20.45 17.13

a Extraction yield (mg/g) = the amount of the target compounds/sample mass.

Effi cient strategy for fl avonoids from Fructus Aurantii Immaturus

Biomed. Chromatogr. 2010; 24: 235–244 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/bmc

24

1

Table 4. Analysis of L9 (3)4 orthogonal test

Compound 1 (mg/g) Compound 2 (mg/g) Compound 3 (mg/g)A B C D A B C D A B C D

K1 17.76a 17.51 14.57 17.78 53.97 53.15 41.20 53.28 46.89 47.40 38.15 46.09K2 18.55 18.88 20.75 18.39 54.06 55.96 62.43 53.48 46.87 48.00 52.68 47.82K3 18.68 18.60 18.57 18.82 54.75 53.67 59.15 56.02 48.02 46.38 50.95 47.87k1 5.92b 5.84 4.86 5.93 17.99 17.72 13.73 17.76 15.63 15.80 12.72 15.36k2 6.18 6.29 6.92 6.13 18.02 18.65 20.81 17.83 15.62 16.00 17.56 15.94k3 6.23 6.20 6.19 6.27 18.25 17.89 19.72 18.67 16.01 15.46 16.98 15.96R 0.31c 0.46 2.06 0.35 0.26 0.94 7.08 0.91 0.38 0.54 4.84 0.59Optimal level A3 B2 C2 D3 A3 B2 C2 D3 A3 B2 C2 D3

a kiA = ∑ extraction yield at Ai.

b kiA = Ki

A/3.c Ri

A = max{kiA} − min{ki

A}.

Figure 6. Comparison of MAE with HRE on the yields of narirutin, nar-

ingin and neohesperidin.

Table 5. The partition coeffi cients (k) of narirutin, naringin and neohesperidin in diff erent solvent systems

Solvent systems Narirutin Naringin Neohesperidin

n-Butanol–acetic acid–water (4 : 1 : 5) 3.86 4.75 5.79n-Butanol–water (1 : 1) 3.14 5.96 6.93Ethyl acetate–n-butanol–water (4 : 1 : 5) 1.02 1.07 0.71Ethyl acetate–n-butanol–water (2 : 1 : 3) 2.90 3.80 3.18Ethyl acetate–n-butanol–water (3 : 2 : 5) 4.42 5.75 4.71Ethyl acetate–n-butanol–water (2 : 3 : 5) 6.67 9.07 6.80Chloroform–methanol–water (4 : 3 : 2) 14.48 13.29 7.84Chloroform–methanol–n-butanol–water (4 : 3 : 0.5 : 2) 3.86 3.73 2.30Chloroform–methanol–n-butanol–water (4 : 3 : 1 : 2) 2.26 1.99 1.46

The K value was defi ned as the peak area of the compound in the upper phase divided by that in the lower phase.

(Table 6). These advantages were of great importance when

HSCCC was applied to industrial processing of narirutin, naringin

and neohesperidin.

Under the optimum HSCCC conditions, the crude extracts

were separated and purifi ed. The typical HSCCC chromatogram

is shown in Fig. 7(C). A yield of 207.3 mg of naringin (II) was

obtained from 1.42 g of crude extract in a one-step separation

with good resolution, and the retention of the stationary phase

was 38%. The peak fractions (I) were evaporated to dryness and

204.5 mg of refi ned sample was obtained. The refi ned sample

was then further separated by HSCCC, yielding 61.6 mg of nariru-

tin (IV) and 159.5 mg of neohesperidin (III), with the retention of

stationary phase at 50%. The typical HSCCC chromatogram is

shown in Fig. 7(B). The purity of narirutin, naringin and neohes-

peridin was 98.1, 97.2 and 99.5%, respectively, and the chromato-

grams are shown in Fig. 4(b–d). The recoveries of these

compounds were 86, 93 and 89%, respectively.

Structural Identifi cation

The structural identifi cation was carried out by ESI-MS, 1H NMR

and 13C NMR. Narirutin: ESI-MS m/z: 579 [M − H]−; 13C NMR

(600 MHz, CD3OD), 198.56 (C-4), 166.88 (C-7), 164.84 (C-5), 164.43

(C-4′), 159.09 (C-9), 130.88 (C-1′), 129.15 (C-2′, 6′), 116.38 (C-3′, 5′), 104.3 (C-10), 97.96 (C-6), 97.08 (C-8), 80.59 (C-2), 44.08 (C-3),101.16

(Glc C-1), 77.88 (Glc C-3), 77.15 (Glc C-5), 74.67 (Glc C-2), 71.31 (Glc

C-4), 67.39 (Glc C-6), 102.14 (Rha C-1), 74.11 (Rha C-4), 72.38 (Rha

C-3), 72.06 (Rha C-2), 69.78 (Rha C-5), 17.91 (Rha C-6); 1H NMR

(600 MHz, CD3OD): 7.324 (2H, d, J = 8.4 Hz, H-2′, 6′), 6.809 (2H, d,

J = 8.4 Hz, H-3′, 5′), 6.193 (1H, d, J = 1.8 Hz, H-8), 6.169 (1H, d, J =

1.8 Hz, H-6), 5.396 (1H, d.d, J = 5, 11 Hz, H-2), 3.162 (1H, d.d,

J = 11, 17 Hz, H-3), 2.764 (1H, d.d, J = 5, 17 Hz, H-3), 4.932 (1H, d,

J = 7.2 Hz, Glc H-1), 4.682 (1H, d, J = 2.0 Hz, Rha H-1), 1.183 (3H,

d, J = 6.6 Hz, Rha H-6); Naringin: ESI-MS m/z: 579[M − H]−; 13C NMR

(600 MHz, DMSO-d6), 197.16 (C-4), 164.86 (C-7), 162.88 (C-5),

162.75 (C-9), 157.84 (C-4′), 128.62 (C-1′), 128.51 (C-2′), 128.41

(C-6′), 115.21 (C-3′, 5′), 103.33 (C-10), 96.32 (C-6), 95.09 (C-8),

C. Wang et al.

www.interscience.wiley.com/journal/bmc Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 235–244

24

2

A

B

C

Figure 7. HSCCC chromatograms of crude extracts: single injection mode (A) and continuous injection mode (B), and HSCCC chromatograms of

refi ned sample (C) from Fructus Aurantii Immaturus. (A) Two-phase solvent system: ethyl acetate–n-butanol–water (2 : 1 : 3, v/v); mobile phase, the lower

phase; revolution speed, 800 rpm; fl ow rate, 2.0 mL/min; retention of stationary phase, 42%; detection wavelength, 254 nm; separation temperature,

30°C; sample solution, 300 mg of crude extracts dissolved in the 15 mL lower phase of solvent system (B) Two-phase solvent system: ethyl acetate–n-

butanol–water (2 : 1 : 3, v/v); mobile phase, the lower phase; revolution speed, 800 rpm; fl ow rate, 2.0 mL/min; retention of stationary phase, 38%;

detection wavelength, 254 nm; separation temperature, 30°C; sample solution, 1.42 g of crude extracts dissolved in the 60 mL lower phase of solvent

system (15 mL for each injection). (C) Two-phase solvent system: chloroform–methanol–n-butanol–water (4 : 3 : 0.5 : 2, v/v); mobile phase, the lower

phase; revolution speed, 800 rpm; fl ow rate, 2.0 mL/min; retention of stationary phase, 50%; detection wavelength, 254 nm; separation temperature,

30°C; sample solution, 204.5 mg of refi ned sample dissolved in the 15 mL of lower phase of solvent system.

Effi cient strategy for fl avonoids from Fructus Aurantii Immaturus

Biomed. Chromatogr. 2010; 24: 235–244 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/bmc

24

3

Table 6. Comparison of continuous injection mode (CIM)

and single injection mode (SIM) in the fi rst step of HSCCC

Injection mode tsp (min) tehe (min) tsp (min) Δt (min)

SIM 30 90 440 560CIM 30 90 1400 380

tfsp, time for fi lling the coil column with stational phase; tehe,

time for establishing hydrodynamic equilibrium; tsp, time for

separation process; Δt, cycle time.

Table 7. Retention times, MS data and UV λmax values for the authentic compounds obtained by HSCCC

Compound RT UV lmax (nm) [M − H]− (−)ESI-MS/MS m/z [M + H]+ [M + Na]+ (+)ESI-MS/MS m/z

Narirutin 20.83 224, 282 579 271 581 603 435, 419, 401, 315, 273Naringin 24.79 224, 282 579 459, 313, 271 581 603 435, 419, 401, 383, 315, 273Neohesperidin 33.23 224, 282 609 489, 343, 301 611 633 465, 449, 431, 413, 303

78.61 (C-2), 42.10 (C-3), 100.38 (Glu C-1), 77.13 (Glc C-2), 76.89 (Glc

C-3), 76.10 (Glc C-5), 69.59 (Glc C-4), 60.44 (Glc C-6), 100.31 (Rha

C-1), 71.83 (Rha C-4), 70.48 (Rha C-3), 70.37 (Rha C-2), 68.26 (Rha

C-5), 18.02 (Rha C-6); 1H NMR (600 MHz, DMSO-d6): 7.317 (2H, d,

J = 8.4 Hz, H-2′, 6′), 6.792 (2H, d, J = 8.4 Hz, H-3′, 5′), 6.099 (1H, d,

J = 2.4 Hz, H-8), 6.073 (1H, d, J = 2.4 Hz, H-6), 5.492 (1H, d.d, J =

3.0 Hz, H-2), 2.715 (2H, q, H-3), 5.118 (1H, d, J = 5.4 Hz, Glc H-1),

5.093 (1H, d, J = 2.0 Hz, Rha H-1), 1.141 (3H, d, J = 6 Hz, Rha H-6);

Neohesperidin: ESI-MS m/z: 609 [M − H]−; 13C NMR (600 MHz,

DMSO-d6), 196.96 (C-4), 164.28 (C-7), 162.88 (C-5), 162.55 (C-9),

147.95(C-4′), 146.47 (C-3′), 130.88 (C-1′), 117.75 (C-6′), 114.09

(C-2′), 112.02 (C-5′), 103.30 (C-10), 96.24 (C-6), 95.13 (C-8), 77.10

(C-2), 55.66 (-OCH3), 42.12 (C-3), 97.39 (Glc C-1), 78.37 (Glc C-2),

76.87 (Glc C-3), 76.04 (Glc C-5), 69.56 (Glc C-4), 60.39 (Glc C-6),

100.34 (Rha C-1), 71.79 (Rha C-4), 70.43 (Rha C-3), 70.34 (Rha C-2),

68.23 (Rha C-5), 17.99 (Rha C-6); 1H NMR (600 MHz, DMSO-d6):

9.087 (1H, s, 3′-OH), 6.926 (3H, m, H-2′, 5′, 6′), 6.103 (1H, d, J =

2.4 Hz, H-8), 6.077 (1H, d, J = 2.4 Hz, H-6), 5.284 (1H, d.d, J = 5,

11 Hz, H-2), 3.764 (3H, s, 4′-OCH3), 3.185 (1H, d.d, J = 11, 15 Hz,

H-3), 2.757 (1H, d.d, J = 5, 15 Hz, H-3), 5.106 (1H, d, J = 6.0 Hz, Glc

H-1), 5.093 (1H, d, J = 2.0 Hz, Rha H-1), 1.145 (3H, d, J = 6 Hz, Rha

H-6). Comparing with the reported data, the MS, 1H NMR and 13C

NMR data agree with those of narirutin, naringin and neohesperi-

din (Ya et al., 2007; Guo et al., 2000; Kim et al., 2007;

Anagnostopoulou et al., 2005).

The obtained high-purity narirutin, naringin and neohesperi-

din were analyzed by HPLC-DAD-MS/MS as authentic standards

analyses and their RT, UV spectra and MS data are listed in Table

7. By comparing the RT, UV spectra, molecular masses and their

fragmentation patterns obtained by CID-MS of the protonated

and deprotonated molecules of authentic standards (Table 7)

with that of compounds in crude extract of Fructus Aurantii

Immaturus (Table 1), three components [peaks 2, 3 and 5 in Figs

3 and 4(a)] can be unequivocally identifi ed as narirutin, naringin

and neohesperidin, respectively.

Conclusion

In conclusion, an effi cient strategy based on MAE, HPLC-DAD-MS/

MS and HSCCC was successfully applied for the extraction,

identifi cation, separation and purifi cation of fl avonoids from the

traditional Chinese medicine Fructus Aurantii Immaturus. Five

fl avonoids in the crude extract were preliminary identifi ed by

LC-DAD-MS/MS. MAE was applied for the rapid and effi cient

extraction of the fl avonoids from Fructus Aurantii Immaturus.

Through two steps of HSCCC separation, 61.6 mg of narirutin,

207.3 mg of naringin and 159.5 mg of neohesperidin were

obtained with high purity and recovery directly from the crude

extract. The obtained high-purity narirutin, naringin and neohes-

peridin were analyzed by HPLC-DAD-MS/MS as authentic stan-

dards to validate the preliminary structural identifi cation. The

present study indicates that the effi cient strategy based on MAE,

HPLC-DAD-MS/MS and HSCCC can meet the demand of rapid

extraction, identifi cation, separation and purifi cation of the bio-

active compounds of great interest from TCMs.

Acknowledgement

This work was supported by the Fundamental Research Key

Project founded by Science and Technology Department of

Shanghai, P. R. China, grant nos 036505016 and 06DZ19702.

References

Anagnostopoulou MA, Kefalas P, Kokkalou E, Assimopoulou AN and Papageorgiou VP. Analysis of antioxidant compounds in sweet orange peel by HPLC–diode array detection–electrospray ionization mass spectrometry. Biomedical Chromatography 2005; 19: 138–148.

Anagnostopoulou MA, Kefalas P, Papageorgiou VP, Assimopoulou AN and Boskou D. Radical scavenging activity of various extracts and frac-tions of sweet orange peel (Citrus sinensis). Food Chemistry 2006; 94: 19–25.

Assimopoulou ΑΝ, Sturm S, Stuppner Η and Papageorgiou VP. Preparative isolation and purifi cation of alkannin/shikonin derivatives from natural products by high-speed counter-current chromatography. Biomedical Chromatography 2009; 23: 182–198.

Balunas MJ, Kinghorn AD. Drug discovery from medicinal plants. Life Sciences 2005; 78: 431–441.

China Pharmacopoeia Committee. Pharmacopoeia of the People’s Republic of China. The First Division of 2005, English edn. China Chemical Industry Press: Beijing, 2005; 66.

Domon B and Costello CE. A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Gly-coconjugate Journal 1988; 5: 397–403.

Es-Safi N, Kerhoas L, Einhorn J and Ducrot P. Application of ESI/MS, CID/MS and tandem MS/MS to the fragmentation study of eriodictyol 7-O-glucosyl-(1→2)-glucoside and luteolin 7-O-glucosyl-(1→2)-glucoside. International Journal of Mass Spectrometry 2005; 247: 93–100.

Guo XL, Wang TJ, Guo MJ and Chen Y. Studies on chemical constituents of processed green tangerine peel. China Journal of Chinese Materia Medica 2000; 25: 146–148.

He XG, Lian LZ, Lin LZ and Bernart MW. High-performance liquid chroma-tography–electrospray mass spectrometry in phytochemical analysis of sour orange (Citrus aurantium L.). Journal of Chromatography A 1997; 791: 127–134.

Hong N, Yaylayan VA, Raghavan GS, Pare JR and Belanger JM. Microwave-assisted extraction of phenolic compounds from grape seed. Natural Product Letters 2001; 15: 197–204.

C. Wang et al.

www.interscience.wiley.com/journal/bmc Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 235–244

24

4

Inoue T, Sugimoto Y, Masuda H and Kamei C. Antiallergic eff ect of fl avo-noid glycosides obtained from Mentha piperita L. Biological and Pharmaceutical Bulletin 2002; 25: 256–259.

Ito Y. Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. Journal of Chromatography A 2005, 1065: 145–468.

Kim HK, Jeon WK and Ko BS. Flavanone glycosides from Citrus junos and their anti-infl uenza virus activity. Planta Medica 2001; 67: 548–549.

Kim WC, Lee DY, Lee CH and Kim CW. Optimization of narirutin extraction during washing step of the pectin production from citrus peels. Journal of Food Engineering 2004; 63: 191–197.

Li P, Qi LW, Liu EH, Zhou JL and Wen XD. Analysis of Chinese herbal medi-cines with holistic approaches and integrated evaluation models. Trends in Analytical Chemistry 2008; 27: 66–77.

Ling JY, Zhang GY, Cui ZJ and Zhang CK. Supercritical fl uid extraction of quinolizidine alkaloids from Sophora fl avescens Ait. and purifi cation by high-speed counter-current chromatography. Journal of Chromatography A 2007; 1145: 123–127.

Liu WJ, Geng QJ, Cai R and Yao DL. Optimization of the extracting method of total fl avonoids from Frutus aurantii immaturus. Lishizhen Medicine and Materia Medica Research 2006; 17: 2233–2234.

Ma YL, Li QM, Van den Heuvel H and Claeys M. Characterization of 3-methoxyfl avones using fast-atom bombardment and collision-induced dissociation tandem mass spectrometry. Rapid Communications in Mass Spectrometry 1997; 11: 1357–1364.

Panthong A, Supraditaporn W, Kanjanapothi D, Taesotikul T and Reutrakul V. Analgesic, anti-infl ammatory and venotonic eff ects of Cissus qua-drangularis Linn. Journal of Ethnopharmacology 2007; 110: 264–270.

Peng JY, Fan GR and Wu YT. Isolation and purifi cation of clemastanin B and indigoticoside A from Radix Isatidis by high-speed counter-current chromatography. Journal of Chromatography A 2005a; 1091: 89–93.

Peng JY, Yang GJ, Fan GR and Wu YT. Preparative isolation and separation of a novel and two known fl avonoids from Patrinia villosa Juss by high-speed counter-current chromatography. Journal of Chromatography A 2005b; 1092: 235–240.

Peng JY, Fan GR, Chai YF and Wu YT, Effi cient new method for extraction and isolation of three fl avonoids from Patrinia villosa Juss. by super-critical fl uid extraction and high-speed counter-current chromatogra-phy. Journal of Chromatography A 2006; 1102: 44–50.

Rajadurai M and Prince PSM. Preventive eff ect of naringin on lipid peroxides and antioxidants in isoproterenol-induced cardiotoxicity in Wistar rats: biochemical and histopathological evidences. Toxicology 2006; 28: 259–268.

Rostagno MA, Palma M and Barroso CG. Microwave assisted extraction of soy isofl avones. Analytica Chimica Acta 2007; 588: 274–282.

Shi P, He Q, Song Y, Qu H and Cheng Y. Characterization and identifi cation of isomeric fl avonoid O-diglycosides from genus Citrus in negative electrospray ionization by ion trap mass spectrometry and time-of-fl ight mass spectrometry. Analytica Chimica Acta 2007; 598: 110–118.

Sun Q, Sun A and Liu R. Preparative isolation and purifi cation of four compounds from the Chinese medicinal herb rhizoma Anemarrhenae by high-speed counter-current chromatography. Journal of Chromatography A 2006; 1104: 69–74.

Thostenson ET and Chou TW. Microwave processing: fundamentals and applications. Composites Part A: Applied Science and Manufacturing 1999; 30: 1055–1071.

Tripoli E, Guardia ML, Giammanco S, Majo DD and Giammanco M. Citrus fl avonoids: molecular structure, biological activity and nutritional properties: a review. Food Chemistry 2007; 104: 466–479.

Vanamala J, Leonardi T, Patil BS, Taddeo SS, Murphy ME, Pike LM, Chapkin RS, Lupton JR and Turner ND. Suppression of colon carcinogenesis by bioactive compounds in grapefruit. Carcinogenesis 2006; 27: 1257–1265.

Wang M, Simon JE, Aviles IF, He K, Zheng QY and Tadmor Y. Analysis of antioxidative phenolic compounds in artichoke (Cynara scolymus L.). Journal of Agricultural and Food Chemistry 2003; 51: 601–608.

Xu XJ. New concepts and approaches for drug discovery based on tradi-tional Chinese medicine. Drug Discovery Today: Technologies 2006; 3: 247–253.

Ya J, Zhang XQ, Ye WC, Xu YJ and Shi Y. Chemical constituents from the seeds of Morus atropurpurea. Journal of China Pharmaceutical University 2006; 37: 301–303.

Zhou DY, Chen DL, Xu Q, Xue XY, Zhang FF and Liang XM. Characterization of polymethoxylated fl avones in Fructus aurantii by liquid chroma-tography with atmospheric pressure chemical ionization combined with tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2007; 43: 1692–1699.

Zhou DY, Xu Q, Xue XY, Zhang FF and Liang XM. Analysis of fl avonoid glycosides in Fructus Aurantii by high performance liquid chromatog-raphy–electrospray ionization mass spectrometry. Chinese Journal of Analytical Chemistry 2006; 34: S31–S35.

Zhou TT, Fan GR, Hong ZY, Chai YF and Wu YT. Large-scale isolation and purifi cation of geniposide from the fruit of Gardenia jasminoides Ellis by high-speed counter-current chromatography. Journal of Chromatography A 2005; 1100: 76–80.

Zhu M, Cao Y and Fan G. Microwave-assisted extraction and fi ngerprint studies of Schisandra chinensis (Turcz.) by high performance liquid chromatography and gas chromatography. Journal of Separation Science 2007; 30: 67–73.