Determination of Anthraquinone Derivatives in Chang-QingTea by Using Cloud-Point Extraction and High-PerformanceLiquid Chromatography

Zhihong Shi & Huixian Jiang & Junjing Yan &

Hongyi Zhang

Received: 20 June 2011 /Accepted: 1 September 2011 /Published online: 10 September 2011# Springer Science+Business Media, LLC 2011

Abstract In this paper, a cloud-point extraction methodwas developed for the determination of five anthraquinonederivatives in Chang-Qing tea by high-performanceliquid chromatography. The optimum conditions formicelle extraction were obtained as follows—15% (w/v)Genapol X-080 as extractant, pH 3.5, liquid/solid ratio 80,and extraction time, 40 min. For cloud point preconcentration,20% (w/v) NaCl was added, and the solution was incubatedat 55 °C for 30 min. The detection limits for the fiveanthraquinone derivatives were in the range of 0.55–3.30 ng ml−1. Average recoveries for the anthraquinonederivatives at three spiked levels were in the range of 84.3–104.1%. Relative standard deviations for six replicatedeterminations of Chang-Qing tea sample were below 2.39.The established method has been successfully applied to thedetermination of anthraquinone derivatives in Chang-Qingtea products from three different manufacturers.

Keywords Cloud-point extraction . HPLC . Anthraquinonederivatives . Chang-Qing tea

Introduction

Nowadays, people pay more and more attention to health.As a health product which can improve bowel function withlaxative effect, Chang-Qing tea has become popular withChinese people. Although Chang-Qing tea products fromdifferent manufacturers have different formulas ranging

from five to seven ingredients, their indispensible compo-nent is semen cassiae. Semen cassiae is the seed of Cassiatora L and Cassia obtusifolia L (The PharmacopoeiaCommittee of China 2010). It has a long history as a foodand medicinal herb to treat dizziness and headache, and tobenefit the eyes by anchoring and nourishing the liver(Chen and Chen 2001). Semen cassiae can also be used tolower fat levels in blood and relax the bowels (Ou1992). Recently, semen cassiae has been reported to haveneuroprotective effects (Drever et al. 2008; Ju et al. 2010).A chemical composition study points out that semencassiae mainly contain anthraquinone compounds, whichhave been shown to possess various therapeutic propertiesincluding anti-bacterial, anti-viral, and anti-cancer activities(Jin et al. 2007; Wong et al. 1981). Commonly used methodsfor the determination of anthraquinone derivativesinclude high-performance liquid chromatography (HPLC; Liet al. 2009), micellar electrokinetic capillary chromatography(Zheng et al. 2004), and UV-spectrophotometry (Li et al.2007) coupled to refluxing extraction or ultrasonicextraction with ethanol, methanol, or ethanol/chloroform(1:1) as solvent.

These conventional extraction methods not onlyinvolve the use of large volumes of organic solventbut are also time-consuming. While cloud point extrac-tion (CPE) is a good alternative which employs theunique characters of surfactant: solubility for solutes ofdifferent nature and phase separation behavior. Thesmall volume of the surfactant-rich phase allows the pre-concentration of the analytes. The CPE process is simple,safe, less-toxic and economical and has been used forextraction and concentration of different compoundsfrom various solid matrix or aqueous samples (Trivediet al. 2011; Akinlua et al. 2011; Ortega et al. 2011; Zhanget al. 2011; Pourreza et al. 2011; Tang et al. 2010).

Z. Shi :H. Jiang : J. Yan :H. Zhang (*)Key Laboratory of Analytical Science and Technology of HebeiProvince, College of Chemistry and Environmental Science,Hebei University,Baoding, China 071002e-mail: hyzhang@hbu.edu.cn

Food Anal. Methods (2012) 5:659–663DOI 10.1007/s12161-011-9299-9

In this paper, we used CPE for the extraction ofanthraquinone derivatives in Chang-Qing tea, whichcomprises two steps: micelle extraction and cloud-pointconcentration. The effects of surfactant concentration,liquid/solid ratio, extraction time, equilibration temperature,and time and concentration of NaCl have been investi-gated in detail.

Materials and Methods

Apparatus

The chromatographic analysis was performed on ShimadzuLC-10AT liquid chromatograph with SPD-10A UVdetector (Shimadzu, Japan). A high-speed centrifugewas used to centrifuge the sample solutions (LG10-2.4,Beijing, China). A KQ2500E ultrasonic generator (10 L,40 KHz; Jiangsu, China) was used for the ultrasonic-assistedextraction of anthraquinone derivatives from Chang-Qing tea.A PHS-3 C Precision pH/mV Meter was used to measure thepH value (Shanghai, China). Incubation was performed on adigital thermostatic water bath (Jintan, China).

Materials and Reagents

All anthraquinone derivatives (aloe-emodin, rhein, emodin,chrysophanol, and physcion) were purchased from the Nation-al Institute for the Control of Pharmaceutical and BiologicalProducts (Beijing, China). The individual stock standardsolution was prepared in methanol at concentrations of 100,700, 900, 100, 200 μg ml−1 and stored at 4 °C. The standardworking solutions were prepared through diluting stockstandard solution with methanol to the required concentra-tions. Non-ionic surfactant Genapol X-080 was obtained fromFluka (USA) and used as received without further purifica-tion. Various concentrations (w/v) of aqueous surfactantsolutions were prepared by weighing appropriate amounts ofthe surfactant and directly dissolving the surfactant in double-distilled water. Phosphoric acid was of analytical grade, andmethanol was of HPLC grade. The water used was purifiedon a MYQ-sub-boiling distilled water purification system(Changsha, China). All the solvents were filtered through a0.45-μm membrane to eliminate particulate matter beforeanalysis. All the Chang-Qing tea products were purchasedfrom a local drugstore (Yushengtang Co., Beijing, batch no.091207; Tongrentang Co., Beijing, batch no. 52090506;Rongboshi Co., Beijing, batch no. 100305).

Sample Preparation

Chang-Qing tea sample was ground into powder. Some0.1 g of the powder (for Rongboshi product, 0.025 g is

enough) was precisely weighed and put into a 50-mlcentrifugal tube. Eight milliliters of 15% Genapol X-080was added, and the pH value was adjusted to 3.5 withphosphoric acid, then the tea powder was ultrasonicallyextracted (40 kHz, 25 °C) for 40 min. The solution wascentrifuged for 10 min at 3,500 rpm. Supernatant fluidwas transferred, and 20% (w/v) NaCl was added. AfterNaCl was completely dissolved by vortex mixing, thesolution was kept for 30 min in a 55 °C water bath forphase separation. Then, the aqueous phase was suckedout, and the surfactant-rich phase was left in thecentrifugal vial with a volume of 1.4 ml. The preconcen-tration factor is about 5.7. Then, methanol was added toreduce its viscosity, and the final volume of the phase wasdiluted to 3.0 ml. Finally, the solution was filtered througha 0.45-μm membrane, and a 5-μl aliquot was injected intoHPLC for analysis.

HPLC Conditions

The HPLC separation was performed on a Diamonsil-C18

column (150×4.6 mm ID, 5 μm; Dikma, Beijing, China).The mobile phase was prepared by mixing methanol and0.1% phosphoric acid at a ratio of 85:15 (v/v). The flow ratewas 1.0 ml min−1. The column temperature was room-temperature. The detection wavelength was set at 254 nm.The injection volume was 5 μl. Data acquisition wasperformed by using N-2000 software.

Results and Discussion

Optimization of Extraction Conditions

Effect of the Surfactant Concentration

Triton X series and Genapol X-080 were all tested asextracting solvents in this paper. However, Triton X seriessurfactants show high UV absorbance and give three largepeaks during 5.0–11.0 min in the HPLC chromatogram,which interfere with the determination of rhein (5.76 min)and emodin (8.68 min). Genapol X-080 is a polyoxy-ethylene glycol monoethertype surfactant, which has eightoxyethylene units and tridecyl alkyl moieties. Possessing noaromatic moiety, Genapol X-080 shows very low UVabsorbance and gives three small peaks before 3 min andone tiny peak at 6.5 min in the HPLC chromatogram. Thus,the elution of Genapol X-080 will not interfere with thedetermination of the anthraquinones. Figure 1 shows thetypical chromatograms of Genapol X-080, Genapol X-080spiked with authentic anthraquinone standards, and Chang-Qing tea sample. In this case, Genapol X-080 was chosenas the CPE surfactant for the study.

660 Food Anal. Methods (2012) 5:659–663

The effect of the surfactant concentration on theextraction efficiency was investigated from 1% to 20%. Itcan be seen from Fig. 2 that the amount of extractedanthraquinone derivatives increases when the surfactantconcentration increases from 1.0% to 15% (w/v). So, 15%was chosen as the optimum surfactant concentration for thefollowing experiments.

Effect of Liquid/Solid Ratio

The liquid/solid ratio is the proportion of the extractantvolume to the mass of herbal material. It is one of thefactors influencing the extraction efficiency. In this part,liquid/solid ratios of 20, 40, 60, 80, and 100 ml g−1

were investigated to optimize extraction procedure. Asshown in Fig. 3, the liquid/solid ratio of 80 has the bestextraction efficiency, so it was employed in thefollowing experiments.

Effect of Ultrasonic Extraction Time

The influence of ultrasonic extraction time was studied byvarying the extraction time between 10 and 50 min. Thepeak area reached the maximum value for all the fiveanthraquinone derivatives when the extraction time extendedfrom 10 to 40 min. So, 40 min was chosen to be the optimumultrasonic extraction time.

Effect of Equilibration Temperature and Time

Studies indicate that CPE should be performed at atemperature higher than the cloud-point temperature andthat two phases should be maintained for a given periodof time. The effect of equilibration temperature wasinvestigated between 50 °C and 70 °C. The resultsshowed that the two phases could not separate well at50 °C. From 55 °C on, good phase separation could beobtained and the peak areas of anthraquinones showedno obvious change. Therefore, 55 °C was selected asthe optimum equilibration temperature.

The optimum equilibration time was determined between10 and 50 min. The peak areas of anthraquinones increasewith the increase of the equilibration time, as the soluteswere given more time to transfer into the surfactant richphase. The highest peak area of anthraquinone wasobtained at 30 min. Therefore, 30 min of equilibration timewas finally selected.

Fig. 1 Typical chromatograms of Genapol X-080 (A), Chang-Qing teasample (B), and Genapol X-080 spiked with standards (C). (1)Aloe-emodin, (2) rhein, (3) emodin, (4) chrysophanol, (5) physcion

Fig. 2 Effect of surfactant concentration on the extraction efficiencyof the anthraquinone derivatives. Extraction conditions—0.1 g Chang-Qing tea; pH 3.5; liquid/solid ratio, 60 (milliliters per gram); extractiontime, 40 min; NaCl, 20%; equilibration temperature, 55 °C; equilibrationtime, 40 min

Fig. 3 Effect of liquid/solid ratio on the extraction efficiency of theanthraquinone derivatives. Extraction conditions—0.1 g Chang-Qingtea; pH 3.5; concentration of Genapol X-080, 15% (w/v); extractiontime, 40 min; NaCl, 20%; equilibration temperature, 55 °C;equilibration time, 40 min

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Effect of the Concentration of NaCl

The addition of electrolytes may facilitate the separation ofthe surfactant-rich phase and the aqueous phase for somenon-ionic surfactant systems (Ferrera et al. 2004). In thispaper, sodium chloride was chosen as the modifier. Theeffect of the concentration of NaCl on CPE behavior wasinvestigated in the range of 10–30%. It was found that,when the NaCl concentration was between 10% and 15%,the two phases could not separate well. When the NaClconcentration was above 30%, it could not be dissolvedcompletely. The CPE efficiency showed no difference whenthe NaCl concentration was between 20% and 25%. Thus,20% (w/v) NaCl was chosen as additive electrolyte.

Linearity and Detection Limits

The calibration curves of anthraquinone derivatives wereconstructed, and the linearity of the method was estimatedby regression analysis of their peak areas (y) against theconcentrations(x, in micrograms per milliliter). The regres-sion equations, correlation coefficients, linear ranges, andthe limits of detection for the analysis of the anthraquinonederivatives are shown in Table 1.

Precision

Chang-Qing tea produced by Rongboshi was used for theprecision test. Under the optimum conditions, the repeat-ability was determined by injecting the same sample for sixtimes. Relative standard deviation (RSD) for retention timewas less than 0.72%, and RSD for peak area was less than1.24%. The reproducibility was determined by analyzingsix replicate samples which were processed in the sameway. RSD for retention time was less than 0.56%, and RSDfor peak area was less than 2.39%.

Recovery Test

Rongboshi Chang-Qing tea at 0.025 g was spiked with theanthraquinone standards at low, medium, and high concen-trations, respectively, and aloe–emodin at 2.0, 3.0, and

4.0 μg ml−1; rhein, at 22.0, 33.0, and 47.5 μg ml−1; emodin,at 20.0, 30.0, and 44.5 μg ml−1; chrysophanol, at 2.0, 3.0,and 4.0 μg ml−1; physcion, at 4.0, 6.0, and 8.0 μg ml−1.The spiked samples were processed according to theestablished method. The recoveries at low, medium, andhigh spiked concentrations are as follows—91.3%, 84.3%,and 85.5% for aloe–emodin; 90.5%, 95.6%, and 95.3% forrhein; 85.8%, 87.8%, and 96.5% for emodin; 91.9%,94.4%, and 91.3% for chrysophanol; and 85.0%, 95.6%,and 104.1% for physcion, respectively.

Real Sample Analysis

The proposed analytical method was applied to thedetermination of anthraquinone derivatives in Chang-Qingtea produced by different manufacturers. The analysisresults are shown in Table 2. It could be seen from Table 2that contents of anthraquinones in Rongboshi Chang-Qingtea are much higher than those in the other two brands ofChang-Qing tea products. The results may be useful for thequality control of this kind of products.

Table 2 Analysis results of anthraquinone derivatives in Chang-Qingtea

Compound Manufacturer Found, mg g−1 RSD, %

Yushengtang 0.059 2.43

Aloe–emodin Tongrentang 0.058 1.59

Rongboshi 0.135 1.39

Yushengtang 0.529 1.85

Rhein Tongrentang 0.422 2.37

Rongboshi 1.036 1.02

Yushengtang 0.008 1.49

Emodin Tongrentang 0.112 1.98

Rongboshi 1.567 1.65

Yushengtang 0.023 2.15

Chrysophanol Tongrentang 0.022 1.28

Rongboshi 0.085 2.29

Yushengtang 0.005 2.77

Physcion Tongrentang 0.013 2.42

Rongboshi 0.251 1.91

Table 1 The regression equations, correlation coefficients, linear ranges, and the limits of detection (LOD) for the analysis of the anthraquinonederivatives

Linear equation R Linearity range, μg ml−1 LOD, ng ml−1

Aloe–emodin Y ¼ 3:57� 102 þ 3:00� 104X 0.99994 0.125–2.00 0.55

Rhein Y ¼ �1:27� 103 þ 2:55� 104X 0.99989 1.40–10.50 0.60

Emodin Y ¼ 8:43� 101 þ 1:76� 104X 0.99985 0.018–13.50 0.75

Chrysophanol Y ¼ 6:92� 102 þ 2:93� 104X 0.99994 0.100–1.00 0.93

Physcion Y ¼ 1:40� 102 þ 1:95� 104X 0.99993 0.050–2.00 3.30

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Conclusions

This work demonstrates an effective CPE coupled withHPLC method for extraction and simultaneous analysis ofanthraquinone derivatives in Chang-Qing tea. An importantaspect of the proposed method is the low organicsolvent consumption, which turns it into a low-costand environmentally friendly technique. So, this methodis promising and may be a good alternative to thetraditional techniques in the future for the quality control ofhealth products containing anthraquinone derivatives.

Acknowledgments Financial support from the National NaturalScience Foundation of China (20875020, 20575016) and the NaturalScience Foundation of Hebei Province China (B2006000953) aregratefully acknowledged. The authors also thank the financial supportfrom “The Introduction of Talents” program of Hebei University.

References

Akinlua A, Jochmann MA, Laaks J, Ewert A, Schmidt TC (2011)Microwave-assisted nonionic surfactant extraction of aliphatichydrocarbons from petroleum source rock. Anal Chim Acta691:48

Chen JK, Chen TT (2001) Liver-calming and wind-extinguishingherbs, Chinese medical herbology and pharmacology. Art ofMedicine Press Inc, City of Industry, p 803

Drever BD, AndersonWG, Riedel G, KimDH, Ryu JH, Choi DY, Platt B(2008) The seed extract ofCassia obtusifolia offers neuroprotectionto mouse hippocampal cultures. J Pharmacol Sci 107:380

Ferrera ZS, Sanz CP, Santana CM, Rodriguez JJS (2004) The use ofmicellar systems in the extraction and pre-concentration of organicpollutants in environmental samples. Trends Anal Chem 23:469

Jin DH, Liang FH, Li JY (2007) Microwave assisted and liquidchromatography for simultaneous determination of anthraqui-none derivatives in semen cassiae. J Mol Sci 23:429

Ju MS, Kim HG, Choi JG, Ryu JH, Hur J, Kim YJ, Oh MS (2010)Cassiae semen, a seed of Cassia obtusifolia, has neuroprotectiveeffects in Parkinson’s disease models. Food Chem Toxicol48:2037

Li HB, Fang KY, Li XE (2007) Study on determination methodsof anthraquinones component of Cassia tora L. China FoodSci 28:427

Li GL, Xiao YQ, Zhang C, Li L, Pang Z (2009) Comparison study oftwo types of constituents in seeds of Cassia obtusifolia beforeand after roasted. China J Chin Materia Medica 34:1364

Ortega AC, da Silva DC, Visentainer JV, de Souza NE, Almeida VD,Oliveira CC (2011) Determination of vitamins A and E exploitingcloud point extraction and micellar liquid chromatography. AnalLett 44:778

Ou M (1992) Chinese–English manual of common-used intraditional Chinese medicine. Guangdong Science and TechnologyPublishing House, Guangdong, p 243

Pourreza N, Rastegarzadeh S, Larki A (2011) Determination of allurared in food samples after cloud point extraction using mixedmicelles. Food Chem 126:1465

Tang T, Qian K, Shi TY, Wang F, Li JQ, Cao YS (2010) Determinationof triazole fungicides in environmental water samples by highperformance liquid chromatography with cloud point extractionusing polyethylene glycol 600 monooleate. Anal Chim Acta680:26

The Pharmacopoeia Committee of China (2010) The Chinesepharmacopoeia, part I. The Chemical Industry Publishing House,Beijing, p 135

Trivedi P, Kumar JK, Negi AS, Shanker K (2011) HPLC methoddevelopment and validation of cytotoxic agent phenyl-heptatriyne in Bidens pilosa with ultrasonic-assisted cloud pointextraction and preconcentration. Biomed Chromatogr 25:697

Wong SM, Wong MM, Seligmann O (1981) Anthraquinone glycosidesfrom the seeds of Cassia tora. Phytochem 20:1951

Zhang WJ, Duan CM, Wang ML (2011) Analysis of sevensulphonamides in milk by cloud point extraction and highperformance liquid chromatography. Food Chem 126:779

Zheng WJ, Wang SF, Chen XG, Hu ZD (2004) Identification anddetermination of active anthraquinones in Chinese teas bymicellar electrokinetic capillary chromatography. BiomedChromatogr 18:167

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