Establishment of the Determination Methods of Total Anthraquinone Content in Different Processed Products of Rhubarb

Jiangcun WEI, Xiumei MA, Chunli TANG, Shaofeng CHEN, Yusheng HUANG, Bihua NONG, Dongmei HUANG, Guangli LU

1.Guangxi International Zhuang Medicine Hospital, Nanning 530201, China; 2. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China

Abstract [Objectives] Ultraviolet spectrophotometry was established for the determination of total content of anthraquinone in different processed products of rhubarb. [Methods] Emodin was used as the control substance, and its color was developed by magnesium acetate methanol solution with the concentration of 1%, the absorbance was determined at the wavelength of 516 nm, the content of total anthraquinone in different processed products of rhubarb was determined to compare the content differences. [Results] The regression equation was y=0.045 2x+0.001 5, R2=0.999 1. Emodin had a good linear range in the concentration of 0.98-23.52 μg/mL, with high accuracy, good repeatability and stable detection results. This method revealed the content changes of total anthraquinone in rhubarb before and after processing. [Conclusions] This method could be used as the quality control method of different processed products of rhubarb, and provided reference for the comprehensive development and utilization of rhubarb resources in the future.

Key words Rhubarb, Total anthraquinone, Ultraviolet spectrophotometry

1 Introduction

Rhubarb is the rhizome ofRheumpalmatumL.,RheumtanguticumMaxim. ex Regel. orRheumofficinaleBaill. of Polygonaceae plants[1-2]. Rhubarb is used as monarch medicine in compound prescription, such as Dachengqi decoction, the safety of the compound is determined by the traditional Chinese medicine that makes up the compound, after the processing of traditional Chinese medicine, the drug properties change and the toxic side effects decrease[3]. Rhubarb mainly contains total anthraquinone, sennoside and tannin,etc., and total anthraquinone can be divided into free anthraquinone and binding anthraquinone[4]. The cathartic components of rhubarb are binding anthraquinone and dianthraquinone[5-6], free anthraquinone has antibacterial, anti-inflammatory and other effects[7-8]. The anthraquinones have quinone structure and strong absorption in visible area, ultraviolet spectrophotometry was established to measure the content of total anthraquinone in different processed products of rhubarb and compare the difference of the content of total anthraquinone. The 2015 edition ofChinesePharmacopoeiacontains only Radix et Rhizoma Rhei, prepared Radix et Rhizoma Rhei with wine, prepared Radix et Rhizoma Rhei and charred Radix et Rhizoma Rhei.

2 Materials

2.1InstrumentsUV-1780 Shimadzu UV-VIS Recording Spectrophotometer, pure water filter (France, Millipore), electronic analytical balance(Sartorius Scientific Instruments Co., Ltd.).

2.2ReagentsMethyl alcohol(chromatographic purity, Fisher), magnesium acetate and chloroform were purchased from.

Sinopharm Chemical Reagent Co., Ltd.; Emodin (batch No.170224) with the purity of >98% was purchased from Shanghai Winherb Medical Technology Co., Ltd.; Rhubarb (Sichuan, batch No.170307001) was purchased from Guangdong Kangmei Pharmaceutical Co., Ltd.

3 Methods and results

3.1Thepreparationofreferencesolution12.25 mg of emodin reference substance was obtained, weighed precisely, placed in a 25 mL volumetric flask, added 1% magnesium acetate methanol to the scale, and shaken up to 0.49 mg/mL.

3.2Thepreparationoftestarticles0.5 g of rhubarb was taken out, then 50 mL of water was added, refluxed for 20 min, and then cooled and filtered, 5 mL of the filtrate was precisely taken, 5 mL of 8% hydrochloric acid and 20 mL of chloroform were added for 15 min of ultrasound, chloroform was stratified, the acid solution was extracted with chloroform for 3 times, 10 mL each time, chloroform was volatilized, and the volume was fixed to 10 mL with 1% magnesium acetate methanol solution.

3.3Theselectionofwavelength0.45 mL of the reference solution was precisely obtained and placed in a 25 mL volumetric flask, 1% magnesium acetate methanol solution was added to the scale and shaken well. With 1% magnesium acetate and methanol solution as blank control, UV-VIS spectrophotometry was used to scan in the wavelength range of 200-700 nm, and the results showed that 1, 8-dihydroxy anthraquinone is highly absorbed at 516 nm, and the wavelength is not easily interfered by solvents and impurities, so the wavelength is chosen for the determination at 516 nm. The results are shown in Fig.1.

Fig.1 UV scanning of emodin reference solution

3.4DeterminationmethodWith 1% magnesium acetate and methanol solution as blank control, the absorbance was determined at 513 nm, and the content of total anthraquinone was calculated.

3.5Methodologyinvestigation[6]

3.5.1Standard curve drawing. 0.05, 0.1, 0.3, 0.5, 0.7, 0.9 and 1.2 mL of emodin reference solution were precisely taken and placed in a 25 mL volumetric flask respectively, and 1% magnesium acetate solution was added to the scale. The absorbance was determined at the wavelength of 516 nm. The linear equationy=0.045 2x+0.001 5,R2=0.999 1 was obtained with emodin concentration and absorbance as horizontal and vertical coordinates respectively, which meant that emodin had a good linear range in the concentration range of 0.98-23.52 μg/mL (Fig.2).

Fig.2 Standard curve of emodin reference substance

3.5.2Precision test. 0.5 g Radix et Rhizoma Rhei was taken and weighed precisely, the test product was prepared according to the method under item Section3.2, the absorbance was measured at the wavelength of 516 nm for 6 times with 1% magnesium acetate and methanol solution as blank, and theRSDwas 0.43%. The method has good precision.

3.5.3Repeatability test. According to the method of Section3.2, 6 samples were prepared. The absorbance was measured at the wavelength of 516 nm with 1% magnesium acetate and methanol solution as the blank, the average contents of total anthraquinones in Radix et Rhizoma Rhei, prepared Radix et Rhizoma Rhei, prepared Radix et Rhizoma Rhei with wine and charred Radix et Rhizoma Rhei were 2.19, 1.886, 1.985 and 0.835 mg/g, respectively; theRSDs were 1.31%, 1.17%, 1.40% and 2.54%, respectively, indicating that the method had good repeatability.

3.5.4Stability test. 0.5 g of Radix et Rhizoma Rhei was obtained and weighed precisely, the test product was prepared according to the method under Section3.2, with 1% magnesium acetate and methanol solution as blank, the absorbance was determined at the wavelength of 516 nm at 0, 10, 20, 30, 40, 50, 60, 80, 100 and 120 min, respectively; theRSDs of Radix et Rhizoma Rhei, prepared Radix et Rhizoma Rhei, prepared Radix et Rhizoma Rhei with wine and charred Radix et Rhizoma Rhei were 1.41%, 2.17%, 1.41% and 1.65%, respectively. It can be seen that the samples were stable within 2 h.

3.5.5Sample recovery test. 0.25 g rhubarb was taken and weighed precisely, 6 samples of test solution were prepared according to the method under item Section3.2, appropriate amount of emodin reference solution was added, and the absorbance was measured at the wavelength of 516 nm with 1% magnesium acetate and methanol solution as blank, then total anthraquinone content was calculated. The results were shown in Table 1-4, indicating that the recovery rate of this method was good.

Table 1 Results of recovery test of Radix et Rhizoma Rhei with sample addition (n=6)

Table 2 Results of recovery test of prepared Radix et Rhizoma Rhei with sample addition (n=6)

Table 3 Results of recovery test of prepared Radix et Rhizoma Rhei with wine with sample addition (n=6)

Table 4 Results of recovery test of charred Radix et Rhizoma Rhei with sample addition (n=6)

4 Sample determination

Three batches of different processed rhubarb products were used to prepare the test solution according to the method under Section3.2, and the absorbance was repeatedly measured at the wavelength of 516 nm for three times. According to the linear equation, the average contents of Radix et Rhizoma Rhei, prepared Radix et Rhizoma Rhei, prepared Radix et Rhizoma Rhei with wine and charred Radix et Rhizoma Rhei were 2.190, 1.895, 1.987 and 0.849 mg/g, respectively, withRSDsof 1.57%, 1.81%, 1.89% and 2.68%, respectively (Table 5).

Table 5 Determination results of different processed rhubarb products

5 Discussion

In the process of processing rhubarb, due to the influence of heat and compatibility of different excipients and other factors, the content of rhubarb will change. The total anthraquinones of prepared Radix et Rhizoma Rhei decreased with the increase of steam temperature for a long time; the ratio of active components of Radix et Rhizoma Rhei could be effectively changed after being fried with high heat wine; and the content of total anthraquinones in charred Radix et Rhizoma Rhei was greatly destroyed at high temperature for a long time. In sum, the effective components of rhubarb changed after different methods of processing, different processing methods had different effects on the total anthraquinone content of rhubarb.

Acknowledgements:

In the process of research, the project was supported and helped by Zhuang Yao Medical Research Laboratory of Guangxi International Zhuang Medical Hospital.