World Journal of Traditional Chinese Medicine

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 8  |  Issue : 3  |  Page : 395--401

Simultaneous determination of eleven bioactive constituents in honey-processed licorice by high-performance liquid chromatography-diode array detector and its application from the perspective of processing influence under orthogonal design


Li-Hong Chen1, Yuan Sun1, Hao Cai1, Shuang Guo2, Xia-Chang Wang3, Wei-Dong Li1, Chun-Qin Mao2, Xun-Hong Liu4, Lin-Yong Yan5, Heng-Li Jiang6, Tu-Lin Lu7,  
1 Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China
2 Teaching and Research Department of Traditional Chinese Medicine Processing, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China
3 Jiangsu Key Laboratory for Functional Substances of Chinese Medicine, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China
4 Teaching and Research Department of Traditional Chinese Medicine Identification, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China
5 Sales Department, China Medico Corporation, Tianjin 300301, China
6 Quality Management Department, China Medico Corporation, Tianjin 300301, China
7 Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing; Teaching and Research Department of Traditional Chinese Medicine Processing, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China

Correspondence Address:
Prof. Tu-Lin Lu
Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023
China

Abstract

Objective: The aim of this study was to develop a reliable approach to simultaneously quantify 11 markers and explore the quality variation in honey-processed licorice. Materials and Methods: A high-performance liquid chromatography-diode array detector method was developed for the simultaneous determination of 11 markers (nine flavonoids and two triterpenoid saponins) in honey-processed licorice. The changes to the 11 markers in honey-processed licorice were investigated using an orthogonal design with three input factors. Results: The established method was precise, accurate, and sensitive enough for the simultaneous quantitative evaluation of 11 markers in honey-processed licorice. Intuitive analysis and variance analysis revealed that (1) the soaking time of crude licorice, stir-frying temperature, and stir-frying time remarkably influenced the content of liquiritin apioside, signifying the decomposition of liquiritin apioside to liquiritin or transformation of liquiritin apioside to isoliquiritin apioside, (2) stir-frying temperature significantly influenced licorice-saponin G2, (3) stir-frying temperature was the most important factor of the three input factors, (4) in terms of composition, honey fried licorice had significant effects on two components, namely liquiritin apioside and licorice-saponin G2. Conclusions: Honey processing influenced the content of the 11 licorice analytes differently. This paper highlights the first report on how the quality of honey-processed licorice varies under different processing conditions and suggests the optimal levels of the investigated three factors as A2B2C3 according to the degrees of influence of these factors on the 11 components. Specifically, the soaking time of crude licorice with honey solution, stir-frying temperature, and stir-frying time were 40 min, 100°C, and 20 min, respectively.



How to cite this article:
Chen LH, Sun Y, Cai H, Guo S, Wang XC, Li WD, Mao CQ, Liu XH, Yan LY, Jiang HL, Lu TL. Simultaneous determination of eleven bioactive constituents in honey-processed licorice by high-performance liquid chromatography-diode array detector and its application from the perspective of processing influence under orthogonal design.World J Tradit Chin Med 2022;8:395-401


How to cite this URL:
Chen LH, Sun Y, Cai H, Guo S, Wang XC, Li WD, Mao CQ, Liu XH, Yan LY, Jiang HL, Lu TL. Simultaneous determination of eleven bioactive constituents in honey-processed licorice by high-performance liquid chromatography-diode array detector and its application from the perspective of processing influence under orthogonal design. World J Tradit Chin Med [serial online] 2022 [cited 2022 Dec 1 ];8:395-401
Available from: https://www.wjtcm.net/text.asp?2022/8/3/395/344543


Full Text



 Introduction



Licorice, one of the most popular herbal medicines, was originally described in Shennong Materia Medica, an ancient immortal masterpiece of clinical medicine in China.[1] At present, there are three common pharmaceutical forms of licorice (dried roots and rhizomes of licorice, crude licorice, and processed licorice with honey roasting) on the market. According to Chinese medical theory, crude and honey-processed licorice have different efficacies in clinical applications. Crude licorice is good at relieving cough, dissipating phlegm, and detoxication and is usually used to cure bronchitis, sore throat, and intoxication caused by drugs and food, while honey-processed licorice is known for invigorating vital energy, and is usually used to treat immune deficit, dyspepsia, and arrhythmia.[2],[3],[4] However, in the Chinese Pharmacopeia, the quality standards of raw licorice and honey-processed licorice stipulate the same index components.[5] Nonetheless, honey-processed licorice lacks specific index components, which may influence its therapeutic effects.

Triterpene saponins and flavonoids in licorice have been reported as the main bioactive constituents, which are predominantly responsible for various bioactivities.[6],[7],[8],[9],[10] Previous pharmacological studies have demonstrated that honey-processed licorice extracts exhibit a significantly higher inducible effect on granulocyte colony-stimulating factor secretion in cells than unprocessed or processed licorice extracts without honey-roasting pretreatment.[11] Another study also revealed that total flavonoids offer a greater contribution than total triterpenoids toward medicinal efficacy of roasted licorice,[12] illustrating that licorice processing may possibly have a greater impact on flavonoids than triterpenoids, resulting in differences in quality between crude and honey-processed licorice. In addition, several analytical studies have shown that different processing technologies influence the contents of glycyrrhizin and several flavonoids in licorice.[4],[13] To explore the variation in quality and ensure its consistency in crude and honey-processed licorice from the perspective of processing influences, it is imperative and urgent to develop a practical, reliable, and comprehensive analytical method to monitor the changes in triterpenoids and multiple flavonoids in licorice during processing.

The aim of this study was to develop a reliable and economical approach using high-performance liquid chromatography-diode array detector (HPLC-DAD) to simultaneously quantify 11 marker substances and explore the quality variation of honey-processed licorice from the perspective of processing influences.

 Materials and Methods



Chemicals and materials

Glucoliquiritin and licorice-saponin G2 were purchased from Nanjing Senberga Biotechnology Co., Ltd. (Nanjing, China); liquiritin apioside, liquiritin, isoliquiritin apioside, isoliquiritin, liquiritigenin, licochalcone B, isoliquiritigenin, and glycyrrhizin were purchased from Chengdu Reifensis Biotechnology Co., Ltd. (Chengdu, China); ononin was acquired from Shanghai Shidande Biotechnology Co., Ltd. (Shanghai, China). The chromatographic solvents were HPLC grade, and all other chemicals were of analytical grade.

Crude licorice (No. 18110201) and honey were provided by the China Medico Corporation (Tianjin, China). Honey-processed licorice was prepared as follows: first, 25 g of honey was mixed with an appropriate amount of hot water; second, 100 g of crude licorice was soaked with the honey solution at room temperature (25°C) until the honey solution was completely absorbed; third, the mixture was exposed to mild heat on an induction cooker and the moistened crude licorice stir-fried to yellow or deep yellow at various stir-frying times; last, the processed licorice was allowed to cool to room temperature (25°C). The preparation method was followed as outlined in the Chinese Pharmacopeia.

Preparation of standard solutions

Each analytical standard was weighed and dissolved in methanol by ultrasonication for 5 min. The standard mixture was prepared by mixing 11 individual standard stock solutions to give concentrations of 171.2 μg/mL for glucoliquiritin, 658.8 μg/mL for liquiritin apioside, 732.4 μg/mL for liquiritin, 257.8 μg/mL for isoliquiritin apioside, 186.1 μg/mL for isoliquiritin, 34.93 μg/mL for ononin, 7.060 μg/mL for licochalcone B, 8.396 μg/mL for liquiritigenin, 8.012 μg/mL for isoliquiritigenin, 351.6 μg/mL for licorice-saponin G2, and 4324 μg/mL for glycyrrhizin. To prepare the working solutions, the stock solution was further diluted to obtain a series of concentrations for construction of the calibration curves.

Preparation of sample solutions

Each sample was powdered, and the powdered sample (0.5 g) constituents were extracted with 25 mL of 70% methanol under ultrasonication for 30 min then centrifuged at 3500 rpm for 10 min.

All the sample and standard solutions were stored away from light at 4°C and filtered through 0.22 μm Millipore filters before HPLC analysis.

Chromatographic conditions

The HPLC system was a Waters 2695 Alliance® chromatographic system (Waters Corporation, Milford, USA), which consisted of a quaternary pump, a temperature-controlled autosampler, and a column heater, coupled to a Waters® 2998 DAD. The Waters Empower 3 software was used for signal monitoring and processing.

Separation was achieved on a Waters® CORTECS C18 column (150 mm × 4.6 mm, 2.7 μm). The mobile phase was composed of 0.2% (v/v) formic acid (A) and acetonitrile (B) with gradient elution as follows: 0 min, 10% B; 5 min, 15% B; 5.2 min, 20% B; 20 min, 26% B; 30 min, 30% B; 65 min, 70% B; 68–90 min, 100% B; and 100–110 min, 10% B. The flow rate was 0.56 mL/min and the column temperature was set at 35°C. The injection volume of each sample was 5 μL, and detection wavelengths were set at 250 nm for ononin, licorice-saponin G2, and glycyrrhizin; 276 nm for glucoliquiritin, liquiritin apioside, liquiritin, and liquiritigenin, and 376 nm for isoliquiritin apioside, isoliquiritin, licochalcone B, and isoliquiritigenin. [Figure 1] shows the typical separation of the mixed standard (A) and honey-processed licorice (B) obtained at 250 nm, 276 nm, and 376 nm at the optimized chromatographic conditions.{Figure 1}

 Results



Method validation

The analytical method was validated in terms of linearity, limit of detection (LOD, S/N = 3), limit of quantitation (LOQ, S/N = 10), precision, repeatability, stability, and accuracy of the 11 analytes.

The results of linearity, LOD, LOQ, and precision are listed in [Table 1]. The precision was performed by injecting six replicates of the standard mixtures of glucoliquiritin (1) (85.60 μg/mL), liquiritin apioside (2) (329.4 μg/mL), liquiritin (3) (366.2 μg/mL), isoliquiritin apioside (4) (128.9 μg/mL), isoliquiritin (5) (93.04 μg/mL), ononin (6) (17.46 μg/mL), licochalcone B (7) (3.530 μg/mL), liquiritigenin (8) (4.198 μg/mL), isoliquiritigenin (9) (4.006 μg/mL), licorice-saponin G2 (10) (175.8 μg/mL), and glycyrrhizin (11) (2162 μg/mL). The calibration curves for the 11 bioactive components were linear for the investigated concentration ranges. The RSD values of the precision experiments were found to not exceed 1.5%.{Table 1}

Method validation results in terms of repeatability, stability, and recovery of the 11 analytes in honey-processed licorice are summarized in [Table 2]. Repeatability was examined by analyzing six different working solutions prepared from the same sample; stability was assessed by a single sample solution after 0, 2, 4, 8, 16, and 24 h. As shown in [Table 2], the RSD values of repeatability and stability did not exceed 3.0%. Accuracy was determined by the recovery test of the standard addition method. Sextuple experiments were performed at a specific concentration level for honey-processed licorice. The mean recoveries of the 11 markers ranged from 98.69% to 101.9%. Therefore, the established method was precise, accurate, and sensitive enough for the simultaneous quantitative evaluation of the 11 marker compounds in honey-processed licorice.{Table 2}

Investigating degrees of influence of various factors on 11 compounds using an orthogonal design.

An orthogonal design[14] was used to optimize the parameters for licorice honey processing. The soaking time of crude licorice with honey solution (a), stir-frying temperature (b), and stir-frying time (c) was the input factors. Eleven bioactive substances were considered as the evaluation indexes. L9 (34) was used for the orthogonal test [Table 3]. The control was crude licorice with 11 components, as shown in [Table 4]. According to “Preparation specification of Chinese herbal slices in Beijing (2008 Edition),” commonly used processing temperature (slices temperature) reference range values are as follows: 80°C–120°C for mild fire, 120°C–150°C for moderate fire, and 150°C–220°C for flaming fire. Therefore, the level range of the stir-frying temperature (b) was set to 80°C, 100°C, and 120°C. The parameter ranges of the honey solution (a) and stir-frying time (c) were determined following preexperiment or preliminary studies.{Table 3}{Table 4}

Nine batches of honey-processed licorice and their corresponding crude licorice were analyzed. Intuitive analysis and variance analysis (one-way ANOVA) (Minitab 19.2020.1.0 software package) were used for multigroup comparisons. The data obtained were statistically analyzed and P < 0.01 revealed that the difference was highly significant, while P < 0.05 revealed that the difference was statistically significant.

By max-min difference and variance analysis, the significant influencing factors of each evaluation index were determined. The results of the orthogonal design and intuitive analysis are presented in [Table 5]. [Table 6] and [Table 7] list the results of variance analysis of liquiritin apioside and licorice-saponin G2 performed during the orthogonal experiment.{Table 5}{Table 6}{Table 7}

Tukey's method and 95% confidence intervals were used to group the information. There was a significant difference between the mean values of nonshared letters [Table 8].{Table 8}

In [Table 8], taking factor A for liquiritin apioside as an example, Group A contained levels 2 and 3, Group B contained levels 3 and 1, with level 3 thus being in two groups. The difference between the mean values of sharing one letter was not statistically significant. Levels 2 and 1 did not share the same letter, which indicated that the average value of level 2 was much higher than that of level 1, and so on down the list.

According to the results, soaking time of crude licorice (a) remarkably affected one component (liquiritin apioside) influenced but had no statistical significance on five components (liquiritin, isoliquiritin apioside, isoliquiritin, liquiritigenin, and licorice-saponin G2) and had minimal impact on five components (glucoliquiritin, ononin, licochalcone B, isoliquiritigenin, and glycyrrhizin); stir-frying temperature (b) remarkably affected two chemicals (liquiritin apioside and licorice-saponin G2), influenced but had no statistical significance on six chemicals (glucoliquiritin, liquiritin, isoliquiritin apioside, isoliquiritin, ononin, and glycyrrhizin), and had a slight impact on three chemicals (licochalcone B, liquiritigenin, and isoliquiritigenin); stir-frying time (c) remarkably affected one constituent (liquiritin apioside), influenced but had no statistical significance on four constituents (isoliquiritin apioside, isoliquiritin, licorice-saponin G2, and glycyrrhizin), and had a slight impact on six constituents (glucoliquiritin, liquiritin, ononin, licochalcone B, liquiritigenin, and isoliquiritigenin). From comparison of the effects of the three factors, the stir-frying temperature (B) was the most important/significant factor. In terms of composition, honey fried licorice had significant effects on two components, namely liquiritin apioside and licorice-saponin G2.

The degrees of influence of the three factors on the eleven components were determined according to grouping results for factors that had a significant impact on chemicals [Table 8], and the K values for factors that affected chemicals but had no statistical difference [Table 5]. The optimal levels of these three factors are listed in [Table 9].{Table 9}

According to the degrees of influence of the three factors on the eleven components, the best levels of the three factors were chosen, and A2B2C3 was taken as the optimal combination. Specifically, the soaking time of crude licorice with honey solution, stir-frying temperature, and stir-frying time were 40 min, 100°C, and 20 min, respectively.

The range values reflected the degrees of influence of various factors on eleven compounds. The greater the degree of influence is, the greater the value is.

 Discussion



The calibration curves for the 11 bioactive components were linear for the investigated concentration ranges. The RSD values for precision, repeatability, and stability were found to not exceed 3.0%. The recoveries of the 11 markers were found to be in the range of 98.69% to 101.9%. Therefore, the established method was precise, accurate, and sensitive enough for the simultaneous quantitative evaluation of the 11 marker compounds in honey-processed licorice.

It has been reported that honey-roasting promotes the transformation between liquiritin and isoliquiritin or between liquiritigenin and isoliquiritigenin[13] and the degradation of glycyrrhizin.[15] In the present study, the following findings are presented (1) the soaking time of crude licorice remarkably affected one component (liquiritin apioside), influenced five components but with no statistical significance, (2) stir-frying temperature remarkably affected two chemicals (liquiritin apioside and licorice-saponin G2) and influenced six chemicals with no statistical significance, (3) stir-frying time remarkably affected one constituent (liquiritin apioside) and influenced four constituents with no statistical significance, (4) from comparing the effects of the three input factors, stir-frying temperature (B) was the most important factor; (5) in terms of composition, honey fried licorice had significant effects on two components, namely liquiritin apioside and licorice-saponin G2. The results revealed that honey processing can influence the contents of the 11 analytes differently, which supports the selection of optimal processing conditions for honey roasting.

According to the degrees of influence of the three factors on the eleven components, the optimal levels of the three factors were chosen, and A2B2C3 was considered as the optimal combination. Specifically, the soaking time of crude licorice with honey solution, stir-frying temperature, and stir-frying time were 40 min, 100°C, and 20 min, respectively.

 Conclusions



The reported validated HPLC-DAD method, with acceptable linearity, precision, repeatability, and accuracy, was applied to investigate the changes in 11 constituents of honey-processed licorice. By comparing the effects of three input factors, the stir-frying temperature was the factor with the greatest impact. In terms of composition, honey fried licorice had significant effects on two components, namely liquiritin apioside and licorice-saponin G2.

This paper highlights the first report on how the quality of honey-processed licorice varies under different processing conditions and suggests the optimal levels (A2B2C3) of the three investigated factors according to their degrees of influence on the eleven components. Specifically, the soaking time of crude licorice with honey solution, stir-frying temperature, and stir-frying time were 40 min, 100°C, and 20 min, respectively.

Financial support and sponsorship

This work was financially supported by the National Key R and D Program of China (2018YFC1706500 and 2018YFC1707000).

Conflicts of interest

There are no conflicts of interest.

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