Determination of Total Anthraquinone and Free Anthraquinone in Different Processed Products of Rhubarb

Bing WU, Jinhua LIU, Meiyuan LU, Chao ZENG*, Tingting MO

1. Liuzhou Hospital of Traditional Chinese Medicine, Liuzhou 545001, China; 2. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China; 3. Guangxi Food and Drug Evaluation & Inspection Center, Nanning 530029, China

Abstract [Objectives] The content of total anthraquinone and 5 free anthraquinones in different processed products of rhubarb were determined. [Methods] Using emodin as the reference, the concentration of 1% magnesium acetate methanol solution was used for color development, and the absorbance was measured at 516 nm wavelength; the mobile phase was methanol-0.1% phosphoric acid (76∶24) aqueous solution, the detection wavelength was set at 256 nm, the flow rate was 1.0 mL/min, the column temperature was 30 ℃, and the sample size was 10 μL. [Results] The content of total anthraquinones in rhubarb was determined by UV spectrophotometry. The linear range was 0.98-23.52 μg/mL, R2=0.999 3, the average recoveries were 100.12%, 99.66% and 99.15%, with RSDs of 1.58%, 0.94% and 1.41%, respectively. The linear ranges of emodin, emodin, emodin, chrysophanol and emodin methyl ether were 0.020 7-0.414 (R2=0.999 9), 0.172 1-3.442 (R2=0.999 6), 0.020 3-0.406 (R2=0.999 9), 0.148-2.96 (R2=0.999 8) and 0.164-3.28 (R2=0.999 3), respectively. [Conclusions] The contents of total anthraquinones and free anthraquinones in different processed products of rhubarb were determined by UV spectrophotometry and HPLC. The method can be used for quality control of rhubarb and its different processed products.

Key words Rheum L., Total anthraquinone, Free anthraquinones, High-performance liquid chromatography (HPLC), Ultraviolet-visible spectrophotometry(UV-Vis)

1 Introduction

RheumL. belongs to the purgative medicine of traditional Chinese medicine, which was first recorded inShennong’sClassicofMateriaMedica. Rhubarb is the dried roots and rhizomes ofRheumpalmatumL. andRheumtanguticumMaxim. ex Balf. orRheumofficinaleBaill.[1]. The main effective components of rhubarb include anthraquinones, sennosides, rhubarb polysaccharides,etc[2], which have purgative, anti-inflammatory, antibacterial and other effects[3]. In the process of heating, steaming, frying and processing of rhubarb, the composition content changes greatly, and its pharmacology and efficacy change accordingly[4]. The medicinal properties of traditional Chinese medicine change before and after processing, and the pharmacology is different. According to the need of syndrome differentiation, different processed products can be reasonably selected to improve the curative effect of traditional Chinese medicine and avoid toxic and side effects[5]. The main components of rhubarb are anthraquinone compounds, in order to ensure the quality and clinical efficacy of rhubarb, the content of total anthraquinone and five free anthraquinones in different processed products of rhubarb will be determined by ultraviolet spectrophotometry and HPLC, which provides a scientific method for evaluating the quality of different processed products of rhubarb.

2 Instruments and reagents

2.1 InstrumentsWaters 2695 high-performance liquid chromatograph; Shimadzu UV1780; PRACTUM224-KN SOP electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); DHG-9203A electric constant temperature blow dry oven (Shanghai Qixin Scientific Instrument Co., Ltd.); TGL-16G High-speed table top centrifuge (Shanghai Anting Scientific Instrument Factory).

2.2 ReagentsMethanol (Chromatographic pure, Fisher Scientific World Corporation, USA), methanol and ethanol (Analytical Pure, Sinopharmaceutical Chemical Reagents Co., Ltd.), water was ultra-pure water, and other reagents were analytical pure. Aloe emodin (batch No.200523), rhein acid (batch No.200216), emodin (batch No.2002251), chrysophanol (batch No.200124), emodin methyl ether (batch No.200605), the purity was >98%, which were all purchased from Shanghai Ronhe Pharmaceutical Technology Co., Ltd.

2.3 Determination of total anthraquinone content in different processed products of rhubarb by UV-Vis spectrophotometry

2.3.1Preparation of reference solution. 12.25 mg of emodin reference substance was accurately weighed and placed in a 25 mL volumetric flask, 1% magnesium acetate and methanol were added to the scale and shaken evenly to get 0.49 mg/mL.

2.3.2Preparation of rhubarb test solution. 0.5 g of rhubarb was added with 50 mL of water for 20 min of reflux, then cooled and filtered for later use. Then 5 mL of filtrate was precisely taken, 5 mL of 8% hydrochloric acid was added and 20 mL of chloroform was added, ultrasonic was carried out for 15 min to separate the chloroform layer. The acid was extracted with chloroform for two times, 10 mL each time. Chloroform was dried by waving, and 1% magnesium acetate methanol solution was used for constant volume to 10 mL.

2.3.3Wavelength selection. 0.45 mL of reference solution was precisely measured, and then put into a 25 mL volumetric flask, 1% magnesium acetate and methanol solution was added to the scale, and shaken well. 1% magnesium acetate methanol solution was taken as blank control, according to UV-Vis spectrophotometry test, it was scanned in the wavelength range of 200-700 nm, the results showed that there was a large absorption at 516 nm, and the wavelength was not easy to be interfered by solvents and impurities, so the determination wavelength was 516 nm.

2.3.4The drawing of a standard curve. 0.05, 0.1, 0.3, 0.5, 0.7, 0.9 and 1.2 mL of emodin reference solution were precisely measured and placed into 25 mL volumetric flask respectively, and 1% magnesium acetate solution was added to the scale. The absorbance was measured at the wavelength of 516 nm. Emodin concentration and absorbance were respectively taken as the horizontal and vertical coordinates, the standard curve was drawn, and the linear equationy=0.045 2x+0.001 5 andR2=0.999 3 were obtained, indicating that the emodin reference substance had a good linear range in the concentration of 0.98-23.52 μg/mL.

2.3.5Measurement. Using 1% magnesium acetate methanol solution as blank control, the absorbance was measured at the wavelength of 516 nm to calculate the content of total anthraquinone.

2.3.6Precision test. 0.5 g raw rhubarb was taken and accurately weighed to prepare the test product. The absorbance was measured at 516 nm for 6 consecutive times with 1% magnesium acetate and methanol solution as blank, and theRSDwas 0.43%. The precision of this method was good.

2.3.7Repeatability test. Six samples were prepared, and the absorbance was measured at 516 nm in parallel with 1% magnesium acetate methanol solution as the blank. The average contents of raw rhubarb, steamed rhubarb and cooked rhubarb were calculated as 2.19, 2.049 and 1.886 mg/g, respectively. TheRSDs were 1.31%, 1.09% and 1.17%, respectively, indicating that the method had good reproducibility.

2.3.8Stability test. 0.5 g of raw rhubarb was taken, accurately weighed and prepared. 1% magnesium acetate methanol solution was taken as blank, and the absorbance was measured at 516 nm wavelength at 0, 10, 20, 30, 40, 50, 60, 80, 100 and 120 min, respectively.RSDs of raw rhubarb, steamed rhubarb and cooked rhubarb were calculated to be 1.41%, 2.08% and 2.17%, indicating that the sample was stable within 2 h.

2.3.9Added sample recovery test. 0.25 g of rhubarb was accurately weighed to prepare 6 parts of test solution, the corresponding emodin reference solution was added, 1% magnesium acetate methanol solution was taken as the blank, its absorbance parallel at 516 nm wavelength was measured, and the content of total anthraquinone was calculated. It can be seen from Table 1-3 that this method has a good recovery rate.

Table 1 Results of sample recovery test of raw rhubarb (n=6)

Table 2 Results of steam rhubarb sampling recovery test (n=6)

Table 3 Results of added sample recovery test of cooked rhubarb (n=6)

2.3.10Determination of the sample. Three batches of different processed rhubarb products were respectively taken to prepare the test solution. The absorbance was determined at 516 nm wavelength and repeated for three times by the regression equation. The average contents of rhubarb, steamed rhubarb, cooked rhubarb, wine rhubarb, gooseberry rhubarb and rhubarb carbon were calculated as 2.190, 2.046 and 1.895 mg/g, withRSDs of 1.57%, 1.20% and 1.81%, respectively.

3 Methods and results

3.1 Chromatographic conditionsThe determination was performed on a Thermo BDS Hypersil C18column (4.6 mm×250 mm, 5 μm) with mobile phase methanol-0.1% phosphoric acid (76∶24). The detection wavelength was 256 nm, the flow rate was 1.0 mL/min, the column temperature was 30 ℃, and the sample size was 10 μL. According to the above conditions, the resolution of each component was good.

3.2 Preparation of reference solutionAloe emodin, emodin, emodin acid, chrysophanol and emodin methyl ether were accurately weighed and dissolved in methanol to prepare aloe emodin 20.7 μg/mL, chrysophanol 172.1 μg/mL, emodin 20.3 μg/mL, chrysophanol 148.0 μg/mL and emodin methyl ether 164.0 μg/mL, to obtain a mixed reference solution.

3.3 Preparation of rhubarb test sampleFree anthraquinone: About 0.5 g powder of different processed products of rhubarb were separately taken, accurately weighed, and put into conical flasks with covers, 25 mL of methanol was added accurately, weighed, refluxed in heat for 1 hour, cooled, then weighed again, methanol was used to make up the lost weight, shaken well, filtered, and the filtrate was added.

3.4 Investigation of linear relationship1, 2, 5, 8, 12, 15, 20 μL of mixed reference solution of rhubarb were accurately absorbed and injected into the liquid chromatograph, each sample was injected twice, and the peak area was measured. The peak area and injection volume (μg) were used as ordinate and abscissor coordinates to draw the standard curve and calculate the regression equation. The linear relationship of each component within its respective content range was good, and the results were shown in Table 4.

Table 4 Linear equation and linear range of different processed products of rhubarb

3.5 Precision testA proper amount of the mixed reference solution was accurately measured and injected continuously for 6 times, and the peak areas were recorded respectively, and theRSDs of peak areas of aloe emodin, rhein, emodin, chrysophanol, emodin methyl ether were calculated. Results showed that theRSDs of peak areas of aloe emodin, chrysophanic acid, emodin, chrysophanol and emodin methyl ether in the mixed control substance were 0.7%, 0.7%, 0.37%, 0.35% and 0.42%, respectively. TheRSDs were all less than 3.0%, indicating that the precision of the liquid chromatograph was good.

3.6 Stability test0.5 g rhubarb powder was precisely weighed to prepare the test solution, the peak areas of each test sample were determined at 0, 2, 4, 8, 12 and 24 h after the preparation of the test solution. The results showed that theRSDs of aloe emodin, rhein, emodin, chrysophanol and physcion in raw rhubarb, steamed rhubarb and cooked rhubarb were all less than 1.52%, and theRSDs were all less than 3.0%, indicating that the stability of the test solution was good within 24 h.

3.7 Repeatability test0.5 g powder of different processed rhubarb products was precisely weighed, six parts of each processed rhubarb product were weighed to prepare the test solution, the peak area of each sample was determined. The results showed that theRSDs of aloe emodin, rhein, emodin, chrysophanol and physcion in raw rhubarb, steamed rhubarb and cooked rhubarb were less than 2.12%, and theRSDs were less than 3.0%, indicating that the method had good repeatability.

3.8 Added sample recovery test0.25 g rhubarb powder was precisely weighed, and 9 samples were divided into three groups, namely low, medium and high sample addition groups, each group was added with mixed reference solution to prepare test solution, the average recovery andRSDs of aloe emodin, rhein, emodin, chrysophanol and physcion in raw rhubarb low, medium and high sample addition groups were calculated. The results showed that the average recoveries of aloe emodin, rhein, emodin, chrysophanol and physcion in raw rhubarb, steamed rhubarb and cooked rhubarb were among 96.90%-101.60%,RSDs values were among 1.30%-3.09%,RSDs values were less than 4.0%, indicating that the method was accurate.

3.9 Sample determination0.5 g powder of different processed rhubarb products was weighed respectively in 3 parts, and then precisely weighed to prepare the test solution. The contents of emodin, rhein, aloe emodin, chrysophanol and physcion were calculated by one point external standard method. The results were shown in Table 5.

Table 5 Determination of different contents of rhubarb

4 Conclusions and discussion

4.1 Selection of mobile phaseIn this experiment, HPLC was used to determine the free anthraquinones in rhubarb, and the mobile phase was selected. It was found that the addition of different kinds of acids in the mobile phase had a great effect on the separation of five free anthraquinones. In this experiment, methanol-0.1% phosphoric acid solution, methanol-0.2% phosphoric acid solution, methanol-0.1% glacial acetic acid solution, methanol-0.2% glacial acetic acid solution and methanol-0.1% glacial acetic acid solution were selected and compared. It was concluded that the separation effect of five free anthraquinones in rhubarb was better when the methanol-0.1% phosphoric acid solution (76∶24) was used as the mobile phase, and the separation from the hybrid peak reached the baseline.

4.2 Correlation analysis of processed products and their corresponding pharmacologyIn the process of steaming, the steam heats up and the temperature is high, which destroys the combined rhubaric acid, the main reducing component, and reduces its reducing effect. The increase of emodin, chrysophanol and emodin methyl ether inhibits platelet agglutination, decreases platelet activity, improves the agglutination state, and the osmotic pressure tends to be normal. Therefore, compared with raw rhubarb, cooked rhubarb has a better effect of activating blood circulation and removing blood stasis. After the processing of raw rhubarb, its efficacy has changed, and the therapeutic scope has been expanded[6]. At the same time, after processing, cooked rhubarb has less gastrointestinal stimulation than raw rhubarb. Different processing methods of traditional Chinese medicine can produce different processed products of rhubarb, and the treatment of diseases will also have differences. Therefore, clinical treatment should be based on syndrome differentiation, and rhubarb and its processed products should be properly used.