Effects of Different Drying Methods on Content of Paederosidic Acid in Paederia scandens

Jingrong LU, Haisheng ZENG, Silu HE, Xiumei MA, Dongmei HUANG, Guilin YANG, Chengtong LIU, Qiji ZHOU

1. Guangxi International Zhuang Medicine Hospital, Nanning 530201, China; 2. The Affiliated Hospital of Youjiang Medical University for Nationalities, Baise 533000, China

Abstract [Objectives] To establish a method to simultaneously determine the content of paederosidic acid in Paederia scandens dried by four different methods. [Methods] Reversed-phase high performance liquid chromatography (RP-HPLC) was adopted. The chromatographic column: Thermo SCIENTIFIC Hypersil GOLD Dim. (mm); mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution; gradient elution; column temperature: 30 ℃; flow rate: 1 mL/min; detection wavelength: 236 nm; injection volume: 10 μL. [Results] The linear range of the detection injection volume of paederosidic acid was 0.64-9.60 μg (R=0.999 2); the limit of quantity (LOQ) was 5.10 ng, and the limit of detection (LOD) was 1.36 ng; the RSD of the precision, stability and reproducibility test was all less than 3%; the sample recovery rate was 98.87%, RSD<3.00, n=6. The results show that the content of paederosidic acid in the shade-dried P. scandens is the highest, and the order of the content is P. scandens (dry in shade)>P. scandens (dried at 50 ℃)>P. scandens (dried at 60 ℃)>P. scandens (dried at 70 ℃). [Conclusions] This method is sensitive, reliable and reproducible, and can be used to simultaneously determine the content of paederosidic acid in P. scandens dried by four different methods.

Key words Paederia scandens (Lour) Mreeill, Paederosidic acid, Reversed-phase high performance liquid chromatography (RP-HPLC), Content determination

1 Introduction

Paederiascandens(Lour) Mreeill belongs to the genus Paederia in family Rubiaceae, perennial herbaceous vine, and is a traditional Chinese herbal medicine in China[1-2]. Its roots, stems, leaves and fruits have obvious clinical therapeutic effects, and have curative effects on rheumatic pain, diarrhea, dysentery, abdominal pain, bruises andetc.[3-4].P.scandenscontains iridoids, glycosides, flavonoids, sterols, triterpenoids and volatile oils[5-7], and has anti-inflammatory, analgesic, anti-tumor, antibacterial, and affecting gastrointestinal function and other pharmacological effects[5-7].

Therefore, the current research on the extraction of active components of its drugs and their pharmacological effects has received greater attention. At present, the quality evaluation methods ofP.scandensinclude high performance liquid chromatography (HPLC), ultraviolet spectroscopy, thin layer chromatography (TLC), gas chromatography (GC),etc., of which the HPLC method is mainly used for the determination of effective components. Guangxi is the main producing area ofP.scandens. In order to further understand the content of paederosidic acid inP.scandensproduced in Guangxi, we optimized the chromatographic conditions, which can be used for the quality control analysis ofP.scandensmedicinal materials produced in Guangxi.

2 Instruments and medicinal materials

2.1 Experimental instrumentWaters 2695 UV high-performance liquid chromatography and chromatography workstation (American Waters Corporation); SQP electric balance (Sartorius Sartorius Scientific Instruments (Beijing) Co., Ltd.); LGL-16G high-speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory); 101A-3E electric air blowing dryer (Shanghai Laboratory Instrument Works Co., Ltd.); HWS-26 electroheating thermostatic water bath (Shanghai Bluepard Instruments Co., Ltd.).

2.2 Reference substance and reagentsPaederosidic acid reference substance (batch No.7392) was bought from Shanghai Shidande Biological Technology Co.,Ltd.), and silica gel G plate (batch No.20190610) was produced by Qingdao Haiyang Chemical Co., Ltd. Chromatographic acetonitrile was provided by Fisher Company of the United States. Methanol, ethanol, and phosphoric acid were of analytical grade. The experimental water was pure water.

2.3 Medicinal materialsThe medicinal materials were collected in Wuming District, Nanning City of Guangxi, and identified as the perennial herbaceous vinePaederiascandens(Lour) Mreeill (the genusPaederiain family Rubiaceae). The processing methods ofP.scandensincluded shade drying, drying at 50 ℃, drying at 60 ℃ and drying at 70 ℃. All were checked in accordance with provisions ofChinesePharmacopoeia2015(Volume I), and the results conformed to the related provisions.

3 Methods and results

3.1 Determination of the content of paederosidic acid inP.scandens

3.1.1Preparation of reference substance solution. Precisely weighed appropriate amount of paederosidic acid reference substance, added methanol to dissolve and placed in a 10-mL volumetric flask, and passed through a 0.22 μm microporous filter to obtain the reference substance solution (the concentration was 0.64 mg/mL).

3.1.2Preparation of test sample solution. Precisely weighed 1.0 g of coarse powder ofP.scandens, placed in a proper conical flask with stopper, precisely added 25 mL of methanol, sealed, weighed, ultrasonic treated (200 W, 42 kHz) for 1.0 h, cooled down, weighed again, made up the lost weight with proper volume of methanol, shook up and filtered, took the filtrate 13 000 r/min, centrifuged for 10 min, and took the supernatant, to obtain the test solution.

3.1.3Selection of detection wavelength. Precisely weighed the paederosidic acid reference solution, inject it, and scanned with a PDA detector in the range of 190-400 nm, and measured the absorption spectrum, the maximum absorption wavelength was 236 nm. At this wavelength, it is not easy to be interfered by solvents and impurities, so the detection wavelength was selected at 236 nm. Results are shown in Fig.1.

Fig.1 Absorption spectrum of paederosidic acid reference substance

3.1.4Chromatographic conditions. Chromatographic column was Thermo SCIENTIFIC Hypersil GOLD Dim.(mm) (4.6 mm×4.6 mm, 5 μm), mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution, elution procedure is shown in Table 1, detection wavelength: 236 nm, column temperature: 30 ℃, flow rate: 1.0 mL/min; injection volume: 10 μL. The active components of paederosidic acid inP.scandenscan achieve good separation, and other components in the sample have no interference with the measured components; the components have good resolution according to the above conditions.

3.1.5Negative interference test. Precisely pipetted 10 μL each of the reference solution, test solution, and negative solution and measured according to the above chromatographic conditions. According to the results, the negative solution showed no peak at the retention time corresponding to the reference substance. There is no interference in the determination of the paederosidic acid.

Table 1 Gradient elution

3.1.6Linear relationship test. Precisely pipetted 1, 3, 5, 7, 9, 12, and 15 μL of reference solution, and injected into the liquid chromatography system according to the above chromatographic conditions. Taking the peak area value (Y) as the ordinate, and the reference sample injection amount (X) as the coordinates, the linear regression was made, obtained the regression equation: paederosidic acidY=1.49×106X+32 658 (R2=0.999 2). Results indicated that paederosidic acid showed a good linear relationship within the range of 0.64 -9.60 μg. Chromatograms were shown in Fig.2-3.

Fig.2 Chromatogram of shade dried samples (1: paederosidic acid)

Fig. 3 Chromatogram for paederosidic acid reference substance (1: paederosidic acid)

Gradually diluted the reference solution to obtain a series of reference solutions, and then operated using the method in Section3.1.4, and measured the signal to noise ratio (in terms of peak height), set the concentration whenS/N=3 as the limit of detection (LOD), and set the concentration whenS/N=10 as the limit of quantity (LOQ). The measurement results indicate that the LOD ofP.scandenswas 5.10 ng and the LOQ was 1.36 ng. Besides, the linear relationship of components was good within the content range.

3.1.7System suitability test. (i) Precision test. Precisely pipetted the above reference solution, and continuously injected the sample for 6 times. According to the above chromatographic conditions, made a record of the respective peak areas. Calculated theRSD(%) of the paederosidic acid peak area, and theRSDwas 0.63%, indicating that the method has good precision. (ii) Stability test. Precisely weighed 1.0 g of shade dried sample, prepared the sample solution in accordance with the preparation method, and determined the peak area of the sample according to the chromatographic conditions at 0, 2, 4, 8, 12, and 24 h after the test solution was prepared. TheRSDvalue of paederosidic acid peak area inP.scandenswas 1.28%. AllRSDs were less than 3.0%, indicating that the test solution has high stability within 24 h. (iii) Reproducibility test. Precisely weighed 1.0 g of shaded driedP.scandenssample pieces in parallel, prepared the sample solution according to the test solution preparation method, determined the peak area of the sample component according to the chromatographic conditions, and calculated the average content of paederosidic acid inP.scandensand theRSDvalue of the content of the corresponding component. According to the results, the average content of paederosidic acid inP.scandenswas 153.48 μg/g and theRSDvalue of the content of the corresponding component was 2.46%.RSDvalues were less than 3.0%, indicating that this method has high reproducibility. (iv) Sample recovery rate test. Precisely weighed 6 pieces of 0.5 g shaded driedP.scandenssample (containing paederosidic acid 153.48 μg/g), each placed in a conical flask with a stopper, and precisely added the appropriate amount paederosidic acid reference substance, prepared according to the test solution preparation method, filtered, and centrifuged the filtrate at 13 000 r/min for 10 min at high speed. Took the supernatant and filtered through a 0.45 μm microporous membrane, and measured according to chromatographic conditions in Section3.1.4, and calculated the average recovery rate of paederosidic acid inP.scandensand the correspondingRSDvalues. The results are listed in Table 2, indicating that this method has high accuracy.

Table 2 Results of paederosidic acid sample recovery rate test (n=6)

3.2 Determination of sample contentTook powder ofP.scandensdried by 4 different methods, 1.0 g for each, preparedP.scandenstest solution in accordance with the method under Section3.1.2, and measured the content of test sample ofP.scandensin accordance with the chromatographic conditions in Section3.1.4, and measured three times in parallel, made a record of peak area of paederosidic acid inP.scandens, and calculated the sample content of paederosidic acid using one point external standard method. The results are shown in Table 3.

Table 3 Measurement results of sample content of Paederia scandens (n=3)

4 Discussions

In this experiment, we investigated the effects of methanol-water, acetonitrile-water, methanol-0.05% phosphoric acid, methanol-0.1% phosphoric acid, methanol-0.2% phosphoric acid, acetonitrile-0.05% phosphoric acid, acetonitrile-0.1% phosphoric acid and other systems on the separation effect of sample components. The results indicate that under the conditions of mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution; gradient elution; column temperature: 30 ℃; flow rate: 1 mL/min; detection wavelength: 236 nm; injection volume: 10 μL, the separation is good, so the mobile phase is determined to be acetonitrile-0.1% phosphoric acid aqueous solution with gradient elution.

Three different drying methods have affected the content of the medicinal components ofP.scandens. The results show that the order of the content isP.scandens(dry in shade)>P.scandens(dried at 50 ℃)>P.scandens(dried at 60 ℃)>P.scandens(dried at 70 ℃), possibly because paederosidic acid is not stable when heated. This method is sensitive, reliable and reproducible, and can be used to simultaneously determine the content of paederosidic acid inP.scandensdried by four different method, and also can be used for quality control of paederosidic acid inP.scandensproduced in Wuming District, Nanning City of Guangxi.

Taking content of active components paederosidic acid inP.scandensas the content determination index, we conducted the relevant experimental research and studied the content determination methodology. In the process of the experimental research, we also carried out relevant research and investigations according to chromatographic conditions and system adaptability experiments. Besides, we optimized the composition and ratio of different mobile phases; in the preparation of the sample solution, especially the preparation of the test solution, we optimized different solvents and dosages to determine the best preparation method of the test solu-

tion. The test results show that the method is fast, simple, accurate, and has high resolution, the recovery rate is high, and the results are reliable.