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Development and application of a rapid HPLC method for simultaneous determination of hyperoside, isoquercitrin and eleutheroside E in Apocynum venetum L. and Eleutherococcus senticosus

Development and application of a rapid HPLC method for simultaneous determination of hyperoside,... Apocynum venetum L. and Eleutherococcus senticosus have been used for hundreds of years to treat hypertension in China. In previous research, there was not a suitable quality control of method for the formulas of Apocynum venetum L. and Eleutherococcus senticosus. It is urgent and essential to develop modern analytical methods for Apocynum vene- tum L. and Eleutherococcus senticosus to ensure the quality of the formulas. A rapid approach for simultaneous deter‑ mination of hyperoside, isoquercitrin and eleutheroside E in Apocynum venetum L. and Eleutherococcus senticosus by high‑performance liquid chromatography with a diode array detector was described and validated. The full method validation, including the linearity, limits of detection and quantification, precision, repeatability, stability and recovery, was examined. All target components, including isomers of hyperoside and isoquercitrin, were baseline separated in 35 min. The developed method was sensitive, reliable and feasible. With this method, the optimal decoction condi‑ tions of Apocynum venetum L. and Eleutherococcus senticosus were selected, and their quality analysis was carried out. Furthermore, an herbal compatibility study of Apocynum venetum L. and Eleutherococcus senticosus based on detect‑ ing variations in the content of their active ingredients was performed by the developed HPLC method. It could be an alternative for the quantitative analysis of herbs that contain hyperoside, isoquercitrin or (and) eleutheroside E in the future. Keywords: Hyperoside, Isoquercitrin, Eleutheroside E, Quality control Introduction Acanthopanax senticosus (Rupr. et Maxim.) Harms, have Botanical herbs are consumed globally not only as an been used for hundreds of years to treat hypertension in essential component of the diet but also as medicines or China. In addition to reducing blood pressure, the for- as functional food supplements. As herbs used in tradi- mulas can also be used to relieve stroke and other heart tional Chinese medicine (TCM), Apocynum venetum L. diseases [1]. Based on the multifold efficacy, an increas - (AV) and Eleutherococcus senticosus (ES), also named ing number of patients with hypertension take these herbs. Thus, it is urgent and essential to develop modern analytical methods to ensure the quality of the formulas. Apocynum venetum L. (Luobuma in Chinese) is a per- *Correspondence: slightsusu@163.com The First Affiliated Hospital of Zhejiang Chinese Medical University, No. ennial herbaceous or half-shrub plant that grows in 54 Youdian Road, Hangzhou 310 006, Zhejiang Province, China central to northwestern China [2]. Apocynum venetum Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Shen et al. BMC Chemistry (2020) 14:35 Page 2 of 10 L. has been used as an “antihypertensive tea” in China Eleutheroside E is known to reduce physical fatigue and and Japan. It appears to be a popular beverage all over to enhance endurance performance [21]. According to the world [3]. Previous studies reported AV has some previous research results, eleutheroside E is the major pharmacological activities, such as antioxidants, [3–5] component attributed to the pharmacological effects of anti-hypertensive [1, 6] and anti-depressant effects, [7, ES [20, 21]. Therefore, eleutheroside E should be detected 8] cholesterol lowering [1] and anti-diabetic activity [9]. in the formulas. It has been shown to lower blood pressure in  vivo and In previous research, hyperoside and isoquercitrin cause in vitro vasodilatation of rat aortic and mesenteric in AV were simultaneously separated and detected by arterial rings [6, 10]. AV is rich in minerals and flavo - mixed cloud point extraction (MCPE) combined with noids, and its main active fractions are found to be phe- HPLC [22]. A sensitive LC–MS-MS method for simulta- nolic acid, flavone and flavan-3-ol components [11, 12]. neous quantification of hyperoside and isoquercitrin was Among them, hyperoside and isoquercitrin are the main also reported [23]. Some studies have also reported ana- effective components [13]. New reports have demon - lytical methods for eleutheroside E detection by HPLC strated that hyperoside and isoquercitrin exhibit potent [24]. However, until now, no article has reported the antioxidant activities, anti-hypertensive and cardiovas- simultaneous detection of hyperoside, isoquercitrin and cular protection [14, 15]. Therefore, hyperoside and iso - eleutheroside E (Fig. 1). The quantification of hyperoside quercitrin must be measured in the formulas. and isoquercitrin by HPLC–MS or MCPE-HPLC is com- Eleutherococcus senticosus (Ciwujia in Chinese), a plicated and not suitable for quality control. Therefore, shrub native to the taiga of China, Korea, Russia and this study aims to develop an efficient method for simul - Hokkaido Island of Japan, has been used as an adaptogen taneous separation and determination of hyperoside, [16]. Currently, there are some ES products, including isoquercitrin and eleutheroside E and apply it in quality drugs and health food, on the market in many countries control of formulas of AV and ES. [16, 17]. In  vitro and in  vivo studies have demonstrated that ES possesses many pharmacological effects, such Materials and methods as antistress, antifatigue and antidepressive effects [18]. Chemicals and reagents It also has beneficial effects on hypertension, ischemic Standard substances for content determination, includ- heart disease, chronic bronchitis, autoimmune diseases, ing hyperoside (Batch No. 111521-201406), isoquer- gastric ulcers and allergic responses [19]. It has been citrin (Batch No. 111809-201403) and eleutheroside reported that ES has some active constituents, includ- E (Batch No. 111713-200502), were purchased from ing lignans (eleutheroside E), glycans (eleutheroside D), the National Institutes for Food and Drug Control triterpene saponins (eleutherosides I, K, L and M), ster- (Beijing, China). The Chinese herbs Apocynum vene - oid glycosides (eleutheroside A), hydroxycoumarins, tum L. and Eleutherococcus senticosus were obtained phenylacrylic acid derivatives and flavones [20, 21]. from Huadong Medicine Co., Ltd. (Hangzhou, China). Fig. 1 Chemical structures of hyperoside, isoquercitrin and eleutheroside E Shen  et al. BMC Chemistry (2020) 14:35 Page 3 of 10 Chinese medicine granules of Apocynum venetum L. Preparation process of electuary of AV and ES formulas (Batch No. 1512617) and Eleutherococcus senticosus Chinese herbs of 2 kg AV and 4 kg ES were mixed. Fifteen (Batch No. 1507077) were kindly supplied by Guang- volumes of water were added to the mixture. The mixture dong Yifang Pharmaceutical Co., Ltd. China (Foshan, was soaked for 0.5 h. It was decocted 3 times for 20 min China) and Jiangyin Tianjiang Pharmaceutical Co., Ltd. per time. The decoctions were mixed, filtered and con - (Jiangyin, China), respectively. HPLC-grade acetoni- centrated to a relative density of 1.2. Then, the decoction trile and phosphoric acid were purchased from Merck was processed to an electuary by marumerization. The (Darmstadt, Germany) and Sigma (Fairfield, USA), dosage of cyclodextrin was 400 g in the marumerization. respectively. The deionized water used throughout the experiments was produced using a Milli-Q water purifi - Samples prepared for HPLC analysis cation system (Milford, MA, USA). Ten milligrams of powder decoctions of AV and ES, 10 mg electuary of AV and ES, and 10 mg granules of AV and (or) ES were all diluted with 1  mL methanol. These Sample preparation samples were prepared by centrifugation (12000 rpm for Standard solution 15 min) at 4 °C. Then, the supernatants were acquired for The standard stock solution of hyperoside (2.0  mg/ HPLC analysis. mL), isoquercitrin (2.0  mg/mL) and eleutheroside E (2.0  mg/mL) was prepared in methanol and stored at Instrumentation and analytical conditions 4  °C. Working solutions with the lower concentrations HPLC analyses were performed on an Agilent 1260 were prepared by an appropriate dilution of the stock series system (Agilent Technologies, USA) consisting solution. of a quaternary pump, online vacuum degasser, autosa- mpler, thermostated column compartment and DAD full-wavelength scanning detector. An Agilent TC-C18 Decoctions of AV and ES column (150  mm × 4.6  mm i.d., 5.0  μm particle size) Herbs AV and ES were purchased from the Chinese from Agilent Technologies (USA) was used for all chro- Medicine Factory of Zhejiang University of Tradi- matographic separations. A linear gradient elution of tional Chinese Medicine (Hangzhou, China). The herbs Eluents A (0.1% (v/v) aqueous phosphoric acid) and B were obtained in September and identified by profes - (0.1% (v/v) phosphoric acid in acetonitrile) was used for sor Zheng. The leaves of AV and the roots of ES were the separation. The elution program was well optimized taken for preparation of the decoction. Five milligrams and conducted as follows: the first linear gradient was 5% of AV and 10 mg ES were soaked in water together and Eluent B in the range of 0 ~ 3  min, the second one was decocted in marmite. The decocting conditions (L 3 4 5% ~ 12% Eluent B in the range of 3 ~ 8 min, the third one orthogonal assay) are listed in Table  1. The decoctions was 12% ~ 15% Eluent B in the range of 8 ~ 11  min, the were filtered and metered to 250  mL with water. The fourth one was 15% Eluent B in the range of 11 ~ 25 min decoctions were concentrated to a powder by lyophili- and the last one was 15% ~ 100% Eluent B in the range of zation. It was stored at 4 °C until analysis. 25 ~ 35  min. Then, the system was restored to the initial conditions after 5 min. The solvent flow rate was 1.0 mL/ min, the detection wavelength was set at 210 nm, the col- Table 1 Decoctions of  Apocynum venetum L. umn temperature was maintained at 25 °C and the injec- and  Eleutherococcus senticosus prepared under  different tion volume was 20  μL. Chemstation software (Agilent decocting conditions Technology) was used for peak detection and peak area Decoctions Soaking Water Decoction Frequency calculation. time (h) addition duration of decoction (volumes) (min) (frequency) Method validation 1 0.5 10 10 1 Linearity and range 2 0.5 12 15 2 The linearity and range of the analytical assay were 3 0.5 15 20 3 determined by serial dilution of a standard stock solu- 4 1 10 15 3 tion. Standard calibration curves were generated by cal- 5 1 12 20 1 culating the ratios between the chromatographic peak 6 1 15 10 2 area of each standard substance and the corresponding 7 2 10 20 2 concentration. 8 2 12 10 3 9 2 15 15 1 Shen et al. BMC Chemistry (2020) 14:35 Page 4 of 10 Limit of quantification (LOQ) and limits of detection (LOD) Results and discussion The LOQ and LOD were both determined using a signal- Chromatographic separation to-noise approach. LOQ was defined as the lowest con - An aqueous acetonitrile solvent system was used to ana- centration level resulting in a peak height of 10 times the lyze of hyperoside and isoquercitrin by HPLC. Water baseline noise (the signal-to-noise ratio (S/N) is 10). LOD (0.1% (v/v) phosphoric acid) and acetonitrile were was defined as the minimum concentration that could be selected as the mobile phases, and 360  nm was selected calculated at S/N = 3. as the detection wavelength in Shi’s experiment [25]. Another study reported a microemulsion mobile phase consisting of 2.5% (v/v) n-butanol, 1.2% (v/v) Genapol Precision, repeatability and stability X-080, 0.5% (v/v) ethyl acetate and 95.8% (w/v) aque- The precision was determined by analyzing the same ous 20  mM phosphoric acid, and 360  nm was selected concentration solutions of standards five consecutive to detect six flavonoids of Apocynum venetum L. extract, times with the established HPLC method. The repeatabil - including hyperoside and isoquercitrin [26]. Fan [27] ity was determined using five duplicate samples from the used water (0.5% (v/v) trifluoroacetic acid) and acetoni - powder of the same batch decoction, treated according trile as the mobile phases and, 210  nm as the detec- to the sample preparation procedure (2.2.4) and analyzed tion wavelength to detect eleutheroside E. According with the established HPLC method. The stability was to the above literature, water (0.1% (v/v) phosphoric determined by testing the same powder decoction sam- acid) and acetonitrile (0.1% (v/v) phosphoric acid) were ple at six time points (0, 3, 6, 9, 12 and 24  h) over 24  h selected as the mobile phases in our assay, and 210  nm with the established HPLC method. and 360  nm were both investigated to determine which one was better for detecting hyperoside, isoquercitrin and eleutheroside E simultaneously. As seen in the chro- Recovery test matograms (Fig.  2), hyperoside and isoquercitrin had In the recovery test, three different quantities (low, larger responses at 210 nm than at 360 nm. Furthermore, medium and high) of standards were added to the same 210 nm was suitable wavelength for the determination of powder decoction sample. Then, the mixture was treated eleutheroside E, reported in the literature, as no response according to the sample preparation procedure (2.2.4) was observed at 360  nm for eleutheroside E. Therefore, and analyzed by the developed HPLC method. Then, the 210  nm was selected as the detection wavelength for quantity of each component was subsequently calculated simultaneous determination of hyperoside, isoquercitrin from the corresponding calibration curve. and eleutheroside E. The column temperature was set at 25  °C, and the flow rate was 1.0  mL/min for the recom - mended chromatographic column. To rapidly separate hyperoside, isoquercitrin and eleutheroside E, a gradient Fig. 2 HPLC chromatograms of 0.05 mg/mL standard solutions of hyperoside, isoquercitrin and eleutheroside E at 210 nm (blue chromatogram) and 360 nm (red chromatogram) separately. 1. Eleutheroside E, 2. Hyperoside, 3. Isoquercitrin Shen  et al. BMC Chemistry (2020) 14:35 Page 5 of 10 Precision, repeatability and stability elution procedure was carried out. Eleutheroside E was Various concentrations (1.0, 10.0 and 100.0  μg/mL) of separated first without interference from hyperoside or mixed standard solutions of hyperoside, isoquercitrin isoquercitrin. However, the chromatographic peaks of and eleutheroside E were injected and analyzed in quin- hyperoside and isoquercitrin were not cleanly separated tuplicate by HPLC to determine the precision of the because of isomerization. Thus, isocratic elution with method. The precision was evaluated by the RSD values 15% eluent B was performed until the baseline separation of the peak areas of the three components, which ranged of hyperoside and isoquercitrin was achieved. As shown from 0.61 to 1.19% (hyperoside), 0.37% to 0.87% (isoquer- in Fig. 3, when the running time was 23–25 min, hypero- citrin) and 0.69% to 1.81% (eleutheroside E). The results side and isoquercitrin were separated. The gradient elu - confirmed that the HPLC method had good precision. tion was then repeated. As seen from the above results, The repeatability was evaluated by the RSD values of adjusting the mobile phase gradient and the time allowed the contents of hyperoside, isoquercitrin and eleuthero- the target compounds to achieve baseline separation side E in five duplicate samples from powders of the same without interference with each other. Finally, chromato- batch decoction. The RSD values were 0.42% for hypero - grams of the standard solution and sample solution under side, 1.20% for isoquercitrin and 2.42% for eleutheroside the optimized chromatographic conditions are described E, which demonstrated the good repeatability of this in Fig.  4. Hyperoside, isoquercitrin and eleutheroside E method. were separated and detected rapidly and simultaneously The stability test of the powder decoction sample gave by the developed method. a good result. The RSD values of contents of the hypero - side, isoquercitrin and eleutheroside E were 0.73%, 0.97% Calibration curves, LOQ and LOD and 0.77%, respectively, showing that the method had Six concentrations (0.5, 1.0, 5.0, 10.0, 50.0, 100.0 μg/mL) good stability for the determination of hyperoside, iso- of standard eleutheroside E solutions and seven con- quercitrin and eleutheroside E in 24 h. centrations (0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 200  μg/mL) of mixed standard hyperoside and isoquercitrin solu- tions were prepared in quintuplicate to generate calibra- tion curves. The integrated chromatographic peak areas Recovery test (Y) were plotted against the corresponding concentra- A sample of decoction powder was detected by the pro- tions (X, μg/mL) of the three constituents in the stand- posed HPLC method. The contents of hyperoside, iso - ard solutions to obtain calibration curves based on linear quercitrin and eleutheroside E in this sample were 30.63, regression analysis. Satisfactory linearity was obtained, as 37.23 and 42.32  μg/mL, respectively. Then, three differ - shown by the correlation coefficients higher than 0.999 ent quantities (low, medium and high) of standards were in the investigated ranges (Table  2). The LOD (S/N = 3) added to this sample. The recovery was evaluated by the value for three target components was 0.1  μg/mL. The following formula: Recovery = (amount found- amount LOQ (S/N = 10) value for three target components was sample)/amount standard spiked × 100%. The results are 0.5  μg/mL, which met the sensitivity requirement for shown in Table  3. The recoveries ranged from 97.82 to quantitative analysis. 104.54%, which validated the accuracy of the method. Fig. 3 HPLC chromatogram of the electuary of Apocynum venetum L. and Eleutherococcus senticosus formulas under the optimized chromatographic conditions. 1. Eleutheroside E, 2. Hyperoside, 3. Isoquercitrin Shen et al. BMC Chemistry (2020) 14:35 Page 6 of 10 Fig. 4 HPLC chromatograms of the standard solution and sample solution under the optimized chromatographic conditions. A. mixed standard solution, B. decoction of Apocynum venetum L. and Eleutherococcus senticosus, C. granules of Apocynum venetum L. and Eleutherococcus senticosus, D. granules of Eleutherococcus senticosus, E. granules of Apocynum venetum L. 1. eleutheroside E, 2. hyperoside, 3. isoquercitrin Shen  et al. BMC Chemistry (2020) 14:35 Page 7 of 10 Table 2 Results of regression analysis on calibration curves and limits of detection (n = 5) Compound Test range (μg/mL) Calibration curves R LOQ (μg/mL) LOD (μg/mL) Hyperoside 0.50–200.00 Y = 52.995X − 86.148 0.9996 0.50 0.10 Isoquercitrin 0.50–200.00 Y = 40.714X − 90.563 0.9994 0.51 0.12 Eleutheroside E 0.50–100.00 Y = 24.193X +7.4352 0.9998 0.50 0.10 Table 3 Recovery test of the developed HPLC analysis method (n = 5) Compound Standard spiked (μg/ Found (μg/mL) Recovery (%) Average recovery (%) RSD (%) mL) Hyperoside 24.50 60.72 ± 0.32 122.82 104.54 16.13 30.63 61.62 ± 0.26 101.18 36.75 63.56 ± 0.56 89.61 Isoquercitrin 29.78 70.60 ± 0.86 112.06 104.53 8.06 37.23 76.73 ± 0.51 106.10 44.68 79.87 ± 0.56 95.43 Eleutheroside E 33.86 77.17 ± 0.68 102.91 97.82 6.21 42.32 84.41 ± 0.48 99.46 50.78 88.58 ± 0.49 91.10 Table 4 The four factors and  three-level orthogonal experiment and  ANOVA results for  the  determination of eleutheroside E in decoctions of the formulas of AV and ES Sum of squares df Mean square F P (Sig.) Calibration model 96.34 6 16.06 21.73 0.05 Intercept 11733.95 1 11733.95 15880.77 0 Water addition 22.35 2 11.18 15.13 0.06 Decoction duration 26.50 2 13.25 17.93 0.05 Frequency of decoction 47.49 2 23.75 32.14 0.03 Error (Soaking time) 1.48 2 0.74 Sample analysis 0.324 and 0.442 for the factors of soaking time, decoc- Analysis of decoction engineering of the formulas tion duration and frequency of decoction, respectively. of Apocynum venetum L. and Eleutherococcus senticosus There were no significant differences among these The addition of water, soaking time, decoction dura - factors when the hyperoside content was considered tion and frequency of decoction were the major factors the dependent variable. Similarly, the content of iso- in the herbal decoction procedure. These four fac - quercitrin was selected as the dependent variable, tors and three-level orthogonal experiments of decoc- and the mean square values of the four factors were tion engineering were analyzed by determining the 3.708 (soaking time), 1.969 (water addition), 9.693 contents of hyperoside, isoquercitrin and eleuthero- (decoction duration) and 2.450 (frequency of decoc- side E. The data acquired were analyzed by ANOVA tion), respectively. Water addition was selected as the to obtain the optimal decoction conditions of the for- blank control because it had the lowest mean square mulas of AV and ES. First, the content of hyperoside value among the four factors. Then, the P values were was selected as the dependent variable, and the mean obtained by ANOVA, 0.347 for soaking time, 0.169 for square values of the four factors were 9.456 (soaking decoction duration and 0.446 for frequency of decoc- time), 4.366 (water addition), 9.121 (decoction dura- tion. There were no significant differences among these tion) and 5.502 (frequency of decoction). Water addi- factors when the content of isoquercitrin was consid- tion was the factor with the least effect. Thus, it was ered the dependent variable. Finally, the content of regarded as the blank control to assess the other fac- eleutheroside E was selected as the dependent variable tors. The results showed that the P values were 0.316, in the assay. The results are shown in Table  4. Soaking Shen et al. BMC Chemistry (2020) 14:35 Page 8 of 10 time was selected as the blank control because it had the lowest mean square value among the four fac- tors. The frequency of decoction had a greater impact on the content of eleutheroside E than the other fac- tors. Moreover, the frequency of decoction obviously affected the content of eleutheroside E, with a P value of 0.030 (P < 0.05, significant difference). It was illus - trated that the frequency of decoction had a significant influence on the decoction engineering of the formulas of AV and ES. Therefore, three frequencies of decoction were recommended in decoction engineering. Consid- ering the production efficiency, a soaking time of 0.5 h was determined to be suitable. It was suggested that a soaking time of 0.5  h, water addition of 15 volumes, a decoction duration of 20 min and a frequency of decoc- tion of 3 would be the optimal decoction conditions, as evident from decoction engineering analysis based on the four factors and three-level orthogonal experiment Fig. 5 The contents of hyperoside, isoquercitrin and eleutheroside E and ANOVA results. in AV and ES granules, AV granules, and ES granules Study on the combination of Apocynum venetum L. Determination of the electuary of the formulas of Apocynum and Eleutherococcus senticosus venetum L. and Eleutherococcus senticosus The Chinese herbs AV and ES have been prescribed The optimal decoction conditions of formulas of AV together to treat hypertension and cardiovascular dis- and ES were selected. Thus, the electuary of the formu - ease. To prove the advantage of the combination of las of AV and ES was prepared under optimal decoction AV and ES, the effective components of AV and ES conditions. Determination of hyperoside, isoquercitrin should be detected and analyzed by the developed and eleutheroside E was performed by the developed HPLC method. Granules of AV and ES both combined HPLC method to perform quality control of the electu- and separate were studied in the assay. The results are ary of the formulas of AV and ES. The results are shown shown in Fig.  5. The contents of hyperoside, isoquer - in Table  5. The RSD values of hyperoside, isoquerci - citrin and eleutheroside E increased when AV and ES trin and eleutheroside E from different batches were granules were dissolved together; in particular, the 3.285%, 3.695% and 3.749%, respectively. The contents isoquercitrin content increased by 10.63%. The combi - differed slightly among batches. It was demonstrated nation of AV and ES was beneficial for increasing the that the preparation of the electuary of the formulas content of effective components in AV and ES, and of AV and ES was stable and feasible. Furthermore, the thus, it improved the efficacy. The developed HPLC developed HPLC method was suitable for quality con- method was useful and rapid in the study of the combi- trol of the electuary of the formulas of AV and ES. nation of AV and ES. Conclusion Table 5 Determination of  hyperoside, isoquercitrin In this paper, an optimized HPLC analytical method and  eleutheroside E in  the  electuary of  the  formulas was established and validated for the simultane- of AV and ES by the developed HPLC method (n = 3) ous determination of hyperoside, isoquercitrin and Batch no. Content Content Content eleutheroside E in AV and ES. All target components of hyperoside of isoquercitrin of eleutheroside E including isomers of hyperoside and isoquercitrin were (μg/mL) (μg/mL) (μg/mL) baseline separated within 35  min by simple HPLC– 1,512,201 25.24 ± 0.55 26.98 ± 0.47 20.81 ± 0.23 DAD rather than HPLC–MS. The developed method 1,512,232 26.94 ± 0.43 28.89 ± 0.36 22.15 ± 0.15 was applied to optimize the decoction engineering of 1,512,291 25.26 ± 0.57 26.82 ± 0.46 20.69 ± 0.27 AV and ES, perform quality control of the electuary of 1,512,293 26.80 ± 0.48 28.77 ± 0.39 22.23 ± 0.19 AV and ES and research the herbal combination of AV and ES. The method was sensitive, rapid and reliable; 1,512,302 26.68 ± 0.42 28.68 ± 0.35 22.30 ± 0.18 Shen  et al. BMC Chemistry (2020) 14:35 Page 9 of 10 References thus, it could be an alternative for future quantitative 1. Xie W, Zhang X, Wang T, Hu J (2012) Botany, traditional uses, phytochem‑ analysis of herbs that contain hyperoside, isoquercitrin istry and pharmacology of Apocynum venetum L. (Luobuma): a review. J or (and) eleutheroside E. Ethnopharmacol 141:1–8. https ://doi.org/10.1016/j.jep.2012.02.003 2. Liang T, Yue W, Li Q (2010) Comparison of the phenolic content and antioxidant activities of Apocynum venetum L. (Luo‑Bu‑Ma) and two of its alternative species. Int J Mol Sci 11:4452–4464. https ://doi.org/10.3390/ Abbreviations ijms1 11144 52 AV: Apocynum venetum L; ES: Eleutherococcus senticosus; HPLC‑DAD: High‑ 3. Chan CO, Lau CC, Ng YF, Xu LJ, Chen SB, Chan SW, Mok DK (2015) performance liquid chromatographic with diode array detector; LOD: Limit Discrimination between leave of Apocynum venetum and its adulterant, of detection; LOQ: Limit of quantification; TCM: Traditional Chinese medicine; A. pictum based on antioxidant assay and chemical profiles combined MCPE: Mixed cloud point extraction; S/N: Signal‑to ‑noise ratio. with multivariate statistical analysis. Antioxidants 4:359–372. https ://doi. org/10.3390/antio x4020 359 Acknowledgements 4. Cao Y, Chu Q, Ye J (2003) Determination of hydroxyl radical by capillary We appreciate the work of the teams of central lab of Traditional Chinese electrophoresis and studies on hydroxyl radical scavenging activities of Medical Hospital of Zhejiang Province. We thanked Chiatai Qing Chun Bao Chinese herbs. Anal Bioanal Chem 376:691–695. https ://doi.org/10.1007/ Pharmaceutical Co., Ltd. for making granules of Apocynum venetum L. and s0021 6‑003‑1961‑7 Eleutherococcus senticosus. 5. Shirai M, Kawai Y, Yamanishi R, Terao J (2005) Approach to novel func‑ tional foods for stress control 5. antioxidant activity profiles of antidepres‑ Authors’ contributions sant herbs and their active components. J Med Invest 52:249–251. https JS contributed to design the assay, acquire of the data, analyze and interpret ://doi.org/10.2152/jmi.52.249 of the data, draft the article, critical revise of the article for important intel‑ 6. Kim D, Yokozawa T, Hattori M, Kadota S, Namba T (2000) Eec ff ts of aque ‑ lectual content and provide the study materials. KY contributed to critical ous extracts of Apocynum venetum leaves on spontaneously hyperten‑ revise of the article for important intellectual content and provide the study sive, renal hypertensive and NaCl‑fed‑hypertensive rats. J Ethnopharma‑ materials. CJ contributed to critical revise of the article for important intel‑ col 72:53–59. https ://doi.org/10.1016/S0378 ‑8741(00)00197 ‑5 lectual content. XM contributed to acquire of the data, critical revise of the 7. Butterweck V, Nishibe S, Sasaki T, Uchida M (2001) Antidepressant effects article for important intellectual content and provide the study materials. MZ of apocynum venetum leaves in a forced swimming test. Biol Pharm Bull contributed to critical revise of the article for important intellectual content 24:848–851. https ://doi.org/10.1007/s1070 5‑006‑6275‑7 and provide the study materials. C‑hS contributed to design the assay, critical 8. Zheng M, Liu C, Pan F, Shi D, Zhang Y (2012) Antidepressant‑like effect revise of the article for important intellectual content and provide study mate‑ of hyperoside isolated from Apocynum venetum leaves: possible cellular rials. All authors read and approved the final manuscript. mechanisms. Phytomedicine 19:145–149. https ://doi.org/10.1016/j. phyme d.2011.06.029 Funding 9. Yokozawa T, Nakagawa T (2004) Inhibitory effects of Luobuma tea and This work was financially supported by the Traditional Chinese Medical its components against glucose‑mediated protein damage. Food Chem Science and Technology Projects of Zhejiang Province [No. 2016ZQ017 and Toxicol 42:975–981. https ://doi.org/10.1016/j.fct.2004.02.010 No. 2015ZB044], the Clinical Pharmacy Research Fund of Chinese Integrative 10. Kwan C, Zhang W, Nishibe S, Seo S (2005) A novel in vitro endothe‑ Medicine Association of Zhejiang Province [No. 2013LYSX018], the projects lium‑ dependent vascular relaxant effect of Apocynum venetum leaf were responsible by the author Jie Shen who was in the design of the study; extract. Clin Exp Pharmacol Physiol 32:789–795. https ://doi.org/10.111 collection, analysis and interpretation of data; and in writing the manuscript. 1/j.1440‑1681.2005.04255 .x The work was financially supported by the Public Service Technology Research 11. Han LW, Hou JJ, Zhao L, Liang TG, Li QS (2008) Establishment of Project of Zhejiang Province of China [No. 2016C33127] which was respon‑ HPLC fingerprint and its application in identification of Folium sible by the author Cheng Jiang. This work was also financially supported by Apocyni Veneti. Chin Tradit Herb Drugs 39:591–594. https ://doi. Zhejiang Province Traditional Chinese medicine (integration of Chinese and org/10.1111/j.1432‑1033.1982.tb069 93.x Western Medicine) key discipline identification and processing of traditional 12. An HJ, Wang H, Lan YX, Hashi Y, Chen SZ (2013) Simultaneous qualitative Chinese medicine (No. 2017‑xk ‑b01) which was responsible by the author and quantitative analysis of phenolic acids and flavonoids for the quality Min‑xia Zheng. control of Apocynum venetum L. leaves by HPLC‑DAD ‑ESI‑IT ‑ TOF‑MS and HPLC‑DAD. J Pharm Biomed Anal 85:295–304. https ://doi.org/10.1016/j. Availability of data and materials jpba.2013.07.005 We declared that materials described in the manuscript, including all relevant 13. Zhou C, Sun L, Bi K (2009) RP‑HPLC analysis of hyperoside and isoquerci‑ raw data, will be freely available to any scientist wishing to use them for non‑ trin in Apocynum venetum L. Chin J Pharm Anal 29:1001–1003. https ://doi. commercial purposes, without breaching participant confidentiality. org/10.1128/MCB.25.15.6760‑6771.2005 14. Piao X, Mi X, Tian Y, Wu Q, Piao H, Zeng Z, Wang D, Piao X (2009) Rapid Competing interests identification and characterization of antioxidants from Ligularia All the authors listed in the manuscript have declared that no competing fischeri. Arch Pharm Res 32:1689–1694. https ://doi.org/10.1007/s1227 interests exist. 2‑009‑2204‑z 15. Shibano M, Kakutani K, Taniguchi M, Yasuda M, Baba K (2008) Antioxi‑ Author details dant constituents in the dayflower (Commelina communis L.) and their The First Affiliated Hospital of Zhejiang Chinese Medical University, No. alpha‑ glucosidase‑inhibitory activity. J Nat Med 62:349–353. https ://doi. 54 Youdian Road, Hangzhou 310 006, Zhejiang Province, China. The First org/10.1007/s1141 8‑008‑0244‑1 Affiliated Hospital of Medical School of Zhejiang University, Hangzhou 310 16. Davydov M, Krikorian AD (2000) Eleutherococcus senticosus (Rupr. and 003, Zhejiang Province, China. Department of pharmacy, Tongde Hospital Maxim.) Maxim. (Araliaceae) as an adaptogen: a closer look. J Ethnophar‑ of Zhejiang Province, Hangzhou 310 012, Zhejiang Province, China. macol 72:345–393. https ://doi.org/10.1016/S0378 ‑8741(00)00181 ‑1 17. Weng S, Tang J, Wang G, Wang X, Wang H (2007) Comparison of the Received: 25 September 2019 Accepted: 29 February 2020 addition of siberian ginseng (Acanthopanax senticosus) versus fluoxetine to lithium for the treatment of bipolar disorder in adolescents: a rand‑ omized, double‑blind trial. Curr Ther Res Clin Exp 68:280–290. https ://doi. org/10.1016/j.curth eres.2007.08.004 18. Deyama T, Nishibe S, Nakazawa Y (2001) Constituents and pharmaco‑ logical effects of Eucommia and Siberian ginseng. Acta Pharmacol Sin 22:1057–1070. https ://doi.org/10.1007/s1167 0‑001‑0053‑5 Shen et al. BMC Chemistry (2020) 14:35 Page 10 of 10 19. Takahashi Y, Tanaka M, Murai R, Kuribayashi K, Kobayashi D, Yanagihara N, E analysis in Acanthopanax senticosus by ionic liquids‑ultrasound based Watanabe N (2014) Prophylactic and therapeutic effects of Acanthopanax extraction and high‑performance liquid chromatography detection. Food senticosus harms extract on murine collagen‑induced arthritis. Phytother Chem 141:2426–2433. https ://doi.org/10.1016/j.foodc hem.2013.05.094 Res 28:1513–1519. https ://doi.org/10.1002/ptr.5157 25. Shi Q, Deng F, Wu M (2014) Simultaneous determination of rutin, hyperin 20. Jung CH, Ahn J, Heo SH, Ha TY (2014) Eleutheroside E, an active com‑ and isoquercitrin in leaves of Apocynum venetum and Poacynum hender- pound from Eleutherococcus senticosus, regulates adipogenesis in 3T3‑L1 sonii located in Xinjiang by HPLC. Chin Tradit Herb Drugs 45:1326–1329. cells. Food Sci Biotechnol 23:889–893. https ://doi.org/10.1007/s1006 https ://doi.org/10.7501/j.issn.0253‑2670.2014.09.024 8‑014‑0119‑z 26. Song R, Zhou J (2015) Microemulsion liquid chromatographic method 21. Ahn J, Um MY, Lee H, Jung CH, Heo SH, Ha TY (2013) Eleutheroside E, an for simultaneous separation and determination of six flavonoids of active component of Eleutherococcus senticosus, ameliorates insulin resist‑ Apocynum venetum leaf extract. J Chromatogr B 995–996:8–14. https :// ance in type 2 diabetic db/db Mice. Evid Based Complement Alternat doi.org/10.1016/j.jchro mb.2015.05.019 Med 2013:934183. https ://doi.org/10.1155/2013/93418 3 27. Fan R, Fu H, Jin X, Wang J, Gao C, Gai C, Hu R (2014) Extraction technology 22. Zhou J, Sun JB, Xu XY, Cheng ZH, Zeng P, Wang FQ, Zhang Q (2015) Appli‑ for active constituents in Acanthopanax senticosus and comparison on cation of mixed cloud point extraction for the analysis of six flavonoids in its contents of A. senticosus from various habitats. Chin Tradit Herb Drugs Apocynum venetum leaf samples by high performance liquid chromatog‑ 45:260–264. https ://doi.org/10.7501/j.issn.0253‑2670.2014.02.020 raphy. Pharm Biomed Anal 107:273–279 23. Zhou C, Liu Y, Su D, Gao G, Zhou X, Sun L, Ba X, Chen X, Bi K (2011) A Publisher’s Note sensitive LC–MS–MS method for simultaneous quantification of two Springer Nature remains neutral with regard to jurisdictional claims in pub‑ structural isomers, hyperoside and isoquercitrin: application to pharma‑ lished maps and institutional affiliations. cokinetic studies. Chromatographia 73:353–359. https ://doi.org/10.1007/ s1033 7‑010‑1879‑0 24. Yang L, Ge H, Wang W, Zu Y, Yang F, Zhao C, Zhang L, Zhang Y (2013) Development of sample preparation method for eleutheroside B and Ready to submit your research ? 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Development and application of a rapid HPLC method for simultaneous determination of hyperoside, isoquercitrin and eleutheroside E in Apocynum venetum L. and Eleutherococcus senticosus

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Abstract

Apocynum venetum L. and Eleutherococcus senticosus have been used for hundreds of years to treat hypertension in China. In previous research, there was not a suitable quality control of method for the formulas of Apocynum venetum L. and Eleutherococcus senticosus. It is urgent and essential to develop modern analytical methods for Apocynum vene- tum L. and Eleutherococcus senticosus to ensure the quality of the formulas. A rapid approach for simultaneous deter‑ mination of hyperoside, isoquercitrin and eleutheroside E in Apocynum venetum L. and Eleutherococcus senticosus by high‑performance liquid chromatography with a diode array detector was described and validated. The full method validation, including the linearity, limits of detection and quantification, precision, repeatability, stability and recovery, was examined. All target components, including isomers of hyperoside and isoquercitrin, were baseline separated in 35 min. The developed method was sensitive, reliable and feasible. With this method, the optimal decoction condi‑ tions of Apocynum venetum L. and Eleutherococcus senticosus were selected, and their quality analysis was carried out. Furthermore, an herbal compatibility study of Apocynum venetum L. and Eleutherococcus senticosus based on detect‑ ing variations in the content of their active ingredients was performed by the developed HPLC method. It could be an alternative for the quantitative analysis of herbs that contain hyperoside, isoquercitrin or (and) eleutheroside E in the future. Keywords: Hyperoside, Isoquercitrin, Eleutheroside E, Quality control Introduction Acanthopanax senticosus (Rupr. et Maxim.) Harms, have Botanical herbs are consumed globally not only as an been used for hundreds of years to treat hypertension in essential component of the diet but also as medicines or China. In addition to reducing blood pressure, the for- as functional food supplements. As herbs used in tradi- mulas can also be used to relieve stroke and other heart tional Chinese medicine (TCM), Apocynum venetum L. diseases [1]. Based on the multifold efficacy, an increas - (AV) and Eleutherococcus senticosus (ES), also named ing number of patients with hypertension take these herbs. Thus, it is urgent and essential to develop modern analytical methods to ensure the quality of the formulas. Apocynum venetum L. (Luobuma in Chinese) is a per- *Correspondence: slightsusu@163.com The First Affiliated Hospital of Zhejiang Chinese Medical University, No. ennial herbaceous or half-shrub plant that grows in 54 Youdian Road, Hangzhou 310 006, Zhejiang Province, China central to northwestern China [2]. Apocynum venetum Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Shen et al. BMC Chemistry (2020) 14:35 Page 2 of 10 L. has been used as an “antihypertensive tea” in China Eleutheroside E is known to reduce physical fatigue and and Japan. It appears to be a popular beverage all over to enhance endurance performance [21]. According to the world [3]. Previous studies reported AV has some previous research results, eleutheroside E is the major pharmacological activities, such as antioxidants, [3–5] component attributed to the pharmacological effects of anti-hypertensive [1, 6] and anti-depressant effects, [7, ES [20, 21]. Therefore, eleutheroside E should be detected 8] cholesterol lowering [1] and anti-diabetic activity [9]. in the formulas. It has been shown to lower blood pressure in  vivo and In previous research, hyperoside and isoquercitrin cause in vitro vasodilatation of rat aortic and mesenteric in AV were simultaneously separated and detected by arterial rings [6, 10]. AV is rich in minerals and flavo - mixed cloud point extraction (MCPE) combined with noids, and its main active fractions are found to be phe- HPLC [22]. A sensitive LC–MS-MS method for simulta- nolic acid, flavone and flavan-3-ol components [11, 12]. neous quantification of hyperoside and isoquercitrin was Among them, hyperoside and isoquercitrin are the main also reported [23]. Some studies have also reported ana- effective components [13]. New reports have demon - lytical methods for eleutheroside E detection by HPLC strated that hyperoside and isoquercitrin exhibit potent [24]. However, until now, no article has reported the antioxidant activities, anti-hypertensive and cardiovas- simultaneous detection of hyperoside, isoquercitrin and cular protection [14, 15]. Therefore, hyperoside and iso - eleutheroside E (Fig. 1). The quantification of hyperoside quercitrin must be measured in the formulas. and isoquercitrin by HPLC–MS or MCPE-HPLC is com- Eleutherococcus senticosus (Ciwujia in Chinese), a plicated and not suitable for quality control. Therefore, shrub native to the taiga of China, Korea, Russia and this study aims to develop an efficient method for simul - Hokkaido Island of Japan, has been used as an adaptogen taneous separation and determination of hyperoside, [16]. Currently, there are some ES products, including isoquercitrin and eleutheroside E and apply it in quality drugs and health food, on the market in many countries control of formulas of AV and ES. [16, 17]. In  vitro and in  vivo studies have demonstrated that ES possesses many pharmacological effects, such Materials and methods as antistress, antifatigue and antidepressive effects [18]. Chemicals and reagents It also has beneficial effects on hypertension, ischemic Standard substances for content determination, includ- heart disease, chronic bronchitis, autoimmune diseases, ing hyperoside (Batch No. 111521-201406), isoquer- gastric ulcers and allergic responses [19]. It has been citrin (Batch No. 111809-201403) and eleutheroside reported that ES has some active constituents, includ- E (Batch No. 111713-200502), were purchased from ing lignans (eleutheroside E), glycans (eleutheroside D), the National Institutes for Food and Drug Control triterpene saponins (eleutherosides I, K, L and M), ster- (Beijing, China). The Chinese herbs Apocynum vene - oid glycosides (eleutheroside A), hydroxycoumarins, tum L. and Eleutherococcus senticosus were obtained phenylacrylic acid derivatives and flavones [20, 21]. from Huadong Medicine Co., Ltd. (Hangzhou, China). Fig. 1 Chemical structures of hyperoside, isoquercitrin and eleutheroside E Shen  et al. BMC Chemistry (2020) 14:35 Page 3 of 10 Chinese medicine granules of Apocynum venetum L. Preparation process of electuary of AV and ES formulas (Batch No. 1512617) and Eleutherococcus senticosus Chinese herbs of 2 kg AV and 4 kg ES were mixed. Fifteen (Batch No. 1507077) were kindly supplied by Guang- volumes of water were added to the mixture. The mixture dong Yifang Pharmaceutical Co., Ltd. China (Foshan, was soaked for 0.5 h. It was decocted 3 times for 20 min China) and Jiangyin Tianjiang Pharmaceutical Co., Ltd. per time. The decoctions were mixed, filtered and con - (Jiangyin, China), respectively. HPLC-grade acetoni- centrated to a relative density of 1.2. Then, the decoction trile and phosphoric acid were purchased from Merck was processed to an electuary by marumerization. The (Darmstadt, Germany) and Sigma (Fairfield, USA), dosage of cyclodextrin was 400 g in the marumerization. respectively. The deionized water used throughout the experiments was produced using a Milli-Q water purifi - Samples prepared for HPLC analysis cation system (Milford, MA, USA). Ten milligrams of powder decoctions of AV and ES, 10 mg electuary of AV and ES, and 10 mg granules of AV and (or) ES were all diluted with 1  mL methanol. These Sample preparation samples were prepared by centrifugation (12000 rpm for Standard solution 15 min) at 4 °C. Then, the supernatants were acquired for The standard stock solution of hyperoside (2.0  mg/ HPLC analysis. mL), isoquercitrin (2.0  mg/mL) and eleutheroside E (2.0  mg/mL) was prepared in methanol and stored at Instrumentation and analytical conditions 4  °C. Working solutions with the lower concentrations HPLC analyses were performed on an Agilent 1260 were prepared by an appropriate dilution of the stock series system (Agilent Technologies, USA) consisting solution. of a quaternary pump, online vacuum degasser, autosa- mpler, thermostated column compartment and DAD full-wavelength scanning detector. An Agilent TC-C18 Decoctions of AV and ES column (150  mm × 4.6  mm i.d., 5.0  μm particle size) Herbs AV and ES were purchased from the Chinese from Agilent Technologies (USA) was used for all chro- Medicine Factory of Zhejiang University of Tradi- matographic separations. A linear gradient elution of tional Chinese Medicine (Hangzhou, China). The herbs Eluents A (0.1% (v/v) aqueous phosphoric acid) and B were obtained in September and identified by profes - (0.1% (v/v) phosphoric acid in acetonitrile) was used for sor Zheng. The leaves of AV and the roots of ES were the separation. The elution program was well optimized taken for preparation of the decoction. Five milligrams and conducted as follows: the first linear gradient was 5% of AV and 10 mg ES were soaked in water together and Eluent B in the range of 0 ~ 3  min, the second one was decocted in marmite. The decocting conditions (L 3 4 5% ~ 12% Eluent B in the range of 3 ~ 8 min, the third one orthogonal assay) are listed in Table  1. The decoctions was 12% ~ 15% Eluent B in the range of 8 ~ 11  min, the were filtered and metered to 250  mL with water. The fourth one was 15% Eluent B in the range of 11 ~ 25 min decoctions were concentrated to a powder by lyophili- and the last one was 15% ~ 100% Eluent B in the range of zation. It was stored at 4 °C until analysis. 25 ~ 35  min. Then, the system was restored to the initial conditions after 5 min. The solvent flow rate was 1.0 mL/ min, the detection wavelength was set at 210 nm, the col- Table 1 Decoctions of  Apocynum venetum L. umn temperature was maintained at 25 °C and the injec- and  Eleutherococcus senticosus prepared under  different tion volume was 20  μL. Chemstation software (Agilent decocting conditions Technology) was used for peak detection and peak area Decoctions Soaking Water Decoction Frequency calculation. time (h) addition duration of decoction (volumes) (min) (frequency) Method validation 1 0.5 10 10 1 Linearity and range 2 0.5 12 15 2 The linearity and range of the analytical assay were 3 0.5 15 20 3 determined by serial dilution of a standard stock solu- 4 1 10 15 3 tion. Standard calibration curves were generated by cal- 5 1 12 20 1 culating the ratios between the chromatographic peak 6 1 15 10 2 area of each standard substance and the corresponding 7 2 10 20 2 concentration. 8 2 12 10 3 9 2 15 15 1 Shen et al. BMC Chemistry (2020) 14:35 Page 4 of 10 Limit of quantification (LOQ) and limits of detection (LOD) Results and discussion The LOQ and LOD were both determined using a signal- Chromatographic separation to-noise approach. LOQ was defined as the lowest con - An aqueous acetonitrile solvent system was used to ana- centration level resulting in a peak height of 10 times the lyze of hyperoside and isoquercitrin by HPLC. Water baseline noise (the signal-to-noise ratio (S/N) is 10). LOD (0.1% (v/v) phosphoric acid) and acetonitrile were was defined as the minimum concentration that could be selected as the mobile phases, and 360  nm was selected calculated at S/N = 3. as the detection wavelength in Shi’s experiment [25]. Another study reported a microemulsion mobile phase consisting of 2.5% (v/v) n-butanol, 1.2% (v/v) Genapol Precision, repeatability and stability X-080, 0.5% (v/v) ethyl acetate and 95.8% (w/v) aque- The precision was determined by analyzing the same ous 20  mM phosphoric acid, and 360  nm was selected concentration solutions of standards five consecutive to detect six flavonoids of Apocynum venetum L. extract, times with the established HPLC method. The repeatabil - including hyperoside and isoquercitrin [26]. Fan [27] ity was determined using five duplicate samples from the used water (0.5% (v/v) trifluoroacetic acid) and acetoni - powder of the same batch decoction, treated according trile as the mobile phases and, 210  nm as the detec- to the sample preparation procedure (2.2.4) and analyzed tion wavelength to detect eleutheroside E. According with the established HPLC method. The stability was to the above literature, water (0.1% (v/v) phosphoric determined by testing the same powder decoction sam- acid) and acetonitrile (0.1% (v/v) phosphoric acid) were ple at six time points (0, 3, 6, 9, 12 and 24  h) over 24  h selected as the mobile phases in our assay, and 210  nm with the established HPLC method. and 360  nm were both investigated to determine which one was better for detecting hyperoside, isoquercitrin and eleutheroside E simultaneously. As seen in the chro- Recovery test matograms (Fig.  2), hyperoside and isoquercitrin had In the recovery test, three different quantities (low, larger responses at 210 nm than at 360 nm. Furthermore, medium and high) of standards were added to the same 210 nm was suitable wavelength for the determination of powder decoction sample. Then, the mixture was treated eleutheroside E, reported in the literature, as no response according to the sample preparation procedure (2.2.4) was observed at 360  nm for eleutheroside E. Therefore, and analyzed by the developed HPLC method. Then, the 210  nm was selected as the detection wavelength for quantity of each component was subsequently calculated simultaneous determination of hyperoside, isoquercitrin from the corresponding calibration curve. and eleutheroside E. The column temperature was set at 25  °C, and the flow rate was 1.0  mL/min for the recom - mended chromatographic column. To rapidly separate hyperoside, isoquercitrin and eleutheroside E, a gradient Fig. 2 HPLC chromatograms of 0.05 mg/mL standard solutions of hyperoside, isoquercitrin and eleutheroside E at 210 nm (blue chromatogram) and 360 nm (red chromatogram) separately. 1. Eleutheroside E, 2. Hyperoside, 3. Isoquercitrin Shen  et al. BMC Chemistry (2020) 14:35 Page 5 of 10 Precision, repeatability and stability elution procedure was carried out. Eleutheroside E was Various concentrations (1.0, 10.0 and 100.0  μg/mL) of separated first without interference from hyperoside or mixed standard solutions of hyperoside, isoquercitrin isoquercitrin. However, the chromatographic peaks of and eleutheroside E were injected and analyzed in quin- hyperoside and isoquercitrin were not cleanly separated tuplicate by HPLC to determine the precision of the because of isomerization. Thus, isocratic elution with method. The precision was evaluated by the RSD values 15% eluent B was performed until the baseline separation of the peak areas of the three components, which ranged of hyperoside and isoquercitrin was achieved. As shown from 0.61 to 1.19% (hyperoside), 0.37% to 0.87% (isoquer- in Fig. 3, when the running time was 23–25 min, hypero- citrin) and 0.69% to 1.81% (eleutheroside E). The results side and isoquercitrin were separated. The gradient elu - confirmed that the HPLC method had good precision. tion was then repeated. As seen from the above results, The repeatability was evaluated by the RSD values of adjusting the mobile phase gradient and the time allowed the contents of hyperoside, isoquercitrin and eleuthero- the target compounds to achieve baseline separation side E in five duplicate samples from powders of the same without interference with each other. Finally, chromato- batch decoction. The RSD values were 0.42% for hypero - grams of the standard solution and sample solution under side, 1.20% for isoquercitrin and 2.42% for eleutheroside the optimized chromatographic conditions are described E, which demonstrated the good repeatability of this in Fig.  4. Hyperoside, isoquercitrin and eleutheroside E method. were separated and detected rapidly and simultaneously The stability test of the powder decoction sample gave by the developed method. a good result. The RSD values of contents of the hypero - side, isoquercitrin and eleutheroside E were 0.73%, 0.97% Calibration curves, LOQ and LOD and 0.77%, respectively, showing that the method had Six concentrations (0.5, 1.0, 5.0, 10.0, 50.0, 100.0 μg/mL) good stability for the determination of hyperoside, iso- of standard eleutheroside E solutions and seven con- quercitrin and eleutheroside E in 24 h. centrations (0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 200  μg/mL) of mixed standard hyperoside and isoquercitrin solu- tions were prepared in quintuplicate to generate calibra- tion curves. The integrated chromatographic peak areas Recovery test (Y) were plotted against the corresponding concentra- A sample of decoction powder was detected by the pro- tions (X, μg/mL) of the three constituents in the stand- posed HPLC method. The contents of hyperoside, iso - ard solutions to obtain calibration curves based on linear quercitrin and eleutheroside E in this sample were 30.63, regression analysis. Satisfactory linearity was obtained, as 37.23 and 42.32  μg/mL, respectively. Then, three differ - shown by the correlation coefficients higher than 0.999 ent quantities (low, medium and high) of standards were in the investigated ranges (Table  2). The LOD (S/N = 3) added to this sample. The recovery was evaluated by the value for three target components was 0.1  μg/mL. The following formula: Recovery = (amount found- amount LOQ (S/N = 10) value for three target components was sample)/amount standard spiked × 100%. The results are 0.5  μg/mL, which met the sensitivity requirement for shown in Table  3. The recoveries ranged from 97.82 to quantitative analysis. 104.54%, which validated the accuracy of the method. Fig. 3 HPLC chromatogram of the electuary of Apocynum venetum L. and Eleutherococcus senticosus formulas under the optimized chromatographic conditions. 1. Eleutheroside E, 2. Hyperoside, 3. Isoquercitrin Shen et al. BMC Chemistry (2020) 14:35 Page 6 of 10 Fig. 4 HPLC chromatograms of the standard solution and sample solution under the optimized chromatographic conditions. A. mixed standard solution, B. decoction of Apocynum venetum L. and Eleutherococcus senticosus, C. granules of Apocynum venetum L. and Eleutherococcus senticosus, D. granules of Eleutherococcus senticosus, E. granules of Apocynum venetum L. 1. eleutheroside E, 2. hyperoside, 3. isoquercitrin Shen  et al. BMC Chemistry (2020) 14:35 Page 7 of 10 Table 2 Results of regression analysis on calibration curves and limits of detection (n = 5) Compound Test range (μg/mL) Calibration curves R LOQ (μg/mL) LOD (μg/mL) Hyperoside 0.50–200.00 Y = 52.995X − 86.148 0.9996 0.50 0.10 Isoquercitrin 0.50–200.00 Y = 40.714X − 90.563 0.9994 0.51 0.12 Eleutheroside E 0.50–100.00 Y = 24.193X +7.4352 0.9998 0.50 0.10 Table 3 Recovery test of the developed HPLC analysis method (n = 5) Compound Standard spiked (μg/ Found (μg/mL) Recovery (%) Average recovery (%) RSD (%) mL) Hyperoside 24.50 60.72 ± 0.32 122.82 104.54 16.13 30.63 61.62 ± 0.26 101.18 36.75 63.56 ± 0.56 89.61 Isoquercitrin 29.78 70.60 ± 0.86 112.06 104.53 8.06 37.23 76.73 ± 0.51 106.10 44.68 79.87 ± 0.56 95.43 Eleutheroside E 33.86 77.17 ± 0.68 102.91 97.82 6.21 42.32 84.41 ± 0.48 99.46 50.78 88.58 ± 0.49 91.10 Table 4 The four factors and  three-level orthogonal experiment and  ANOVA results for  the  determination of eleutheroside E in decoctions of the formulas of AV and ES Sum of squares df Mean square F P (Sig.) Calibration model 96.34 6 16.06 21.73 0.05 Intercept 11733.95 1 11733.95 15880.77 0 Water addition 22.35 2 11.18 15.13 0.06 Decoction duration 26.50 2 13.25 17.93 0.05 Frequency of decoction 47.49 2 23.75 32.14 0.03 Error (Soaking time) 1.48 2 0.74 Sample analysis 0.324 and 0.442 for the factors of soaking time, decoc- Analysis of decoction engineering of the formulas tion duration and frequency of decoction, respectively. of Apocynum venetum L. and Eleutherococcus senticosus There were no significant differences among these The addition of water, soaking time, decoction dura - factors when the hyperoside content was considered tion and frequency of decoction were the major factors the dependent variable. Similarly, the content of iso- in the herbal decoction procedure. These four fac - quercitrin was selected as the dependent variable, tors and three-level orthogonal experiments of decoc- and the mean square values of the four factors were tion engineering were analyzed by determining the 3.708 (soaking time), 1.969 (water addition), 9.693 contents of hyperoside, isoquercitrin and eleuthero- (decoction duration) and 2.450 (frequency of decoc- side E. The data acquired were analyzed by ANOVA tion), respectively. Water addition was selected as the to obtain the optimal decoction conditions of the for- blank control because it had the lowest mean square mulas of AV and ES. First, the content of hyperoside value among the four factors. Then, the P values were was selected as the dependent variable, and the mean obtained by ANOVA, 0.347 for soaking time, 0.169 for square values of the four factors were 9.456 (soaking decoction duration and 0.446 for frequency of decoc- time), 4.366 (water addition), 9.121 (decoction dura- tion. There were no significant differences among these tion) and 5.502 (frequency of decoction). Water addi- factors when the content of isoquercitrin was consid- tion was the factor with the least effect. Thus, it was ered the dependent variable. Finally, the content of regarded as the blank control to assess the other fac- eleutheroside E was selected as the dependent variable tors. The results showed that the P values were 0.316, in the assay. The results are shown in Table  4. Soaking Shen et al. BMC Chemistry (2020) 14:35 Page 8 of 10 time was selected as the blank control because it had the lowest mean square value among the four fac- tors. The frequency of decoction had a greater impact on the content of eleutheroside E than the other fac- tors. Moreover, the frequency of decoction obviously affected the content of eleutheroside E, with a P value of 0.030 (P < 0.05, significant difference). It was illus - trated that the frequency of decoction had a significant influence on the decoction engineering of the formulas of AV and ES. Therefore, three frequencies of decoction were recommended in decoction engineering. Consid- ering the production efficiency, a soaking time of 0.5 h was determined to be suitable. It was suggested that a soaking time of 0.5  h, water addition of 15 volumes, a decoction duration of 20 min and a frequency of decoc- tion of 3 would be the optimal decoction conditions, as evident from decoction engineering analysis based on the four factors and three-level orthogonal experiment Fig. 5 The contents of hyperoside, isoquercitrin and eleutheroside E and ANOVA results. in AV and ES granules, AV granules, and ES granules Study on the combination of Apocynum venetum L. Determination of the electuary of the formulas of Apocynum and Eleutherococcus senticosus venetum L. and Eleutherococcus senticosus The Chinese herbs AV and ES have been prescribed The optimal decoction conditions of formulas of AV together to treat hypertension and cardiovascular dis- and ES were selected. Thus, the electuary of the formu - ease. To prove the advantage of the combination of las of AV and ES was prepared under optimal decoction AV and ES, the effective components of AV and ES conditions. Determination of hyperoside, isoquercitrin should be detected and analyzed by the developed and eleutheroside E was performed by the developed HPLC method. Granules of AV and ES both combined HPLC method to perform quality control of the electu- and separate were studied in the assay. The results are ary of the formulas of AV and ES. The results are shown shown in Fig.  5. The contents of hyperoside, isoquer - in Table  5. The RSD values of hyperoside, isoquerci - citrin and eleutheroside E increased when AV and ES trin and eleutheroside E from different batches were granules were dissolved together; in particular, the 3.285%, 3.695% and 3.749%, respectively. The contents isoquercitrin content increased by 10.63%. The combi - differed slightly among batches. It was demonstrated nation of AV and ES was beneficial for increasing the that the preparation of the electuary of the formulas content of effective components in AV and ES, and of AV and ES was stable and feasible. Furthermore, the thus, it improved the efficacy. The developed HPLC developed HPLC method was suitable for quality con- method was useful and rapid in the study of the combi- trol of the electuary of the formulas of AV and ES. nation of AV and ES. Conclusion Table 5 Determination of  hyperoside, isoquercitrin In this paper, an optimized HPLC analytical method and  eleutheroside E in  the  electuary of  the  formulas was established and validated for the simultane- of AV and ES by the developed HPLC method (n = 3) ous determination of hyperoside, isoquercitrin and Batch no. Content Content Content eleutheroside E in AV and ES. All target components of hyperoside of isoquercitrin of eleutheroside E including isomers of hyperoside and isoquercitrin were (μg/mL) (μg/mL) (μg/mL) baseline separated within 35  min by simple HPLC– 1,512,201 25.24 ± 0.55 26.98 ± 0.47 20.81 ± 0.23 DAD rather than HPLC–MS. The developed method 1,512,232 26.94 ± 0.43 28.89 ± 0.36 22.15 ± 0.15 was applied to optimize the decoction engineering of 1,512,291 25.26 ± 0.57 26.82 ± 0.46 20.69 ± 0.27 AV and ES, perform quality control of the electuary of 1,512,293 26.80 ± 0.48 28.77 ± 0.39 22.23 ± 0.19 AV and ES and research the herbal combination of AV and ES. 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