Optimization of Processing Technology for Roasted Licorice with Water by Orthogonal Method

Yali XU, Xiaopeng LI, Liyang HA, Zhilong SHI, Jian GU

College of Pharmacy, Southwest Minzu University, Chengdu 610000, China

Abstract [Objectives] To optimize the processing technology for roasted licorice with water. [Methods] Through the orthogonal experimental design, taking the water added, moistening time, frying temperature and frying time as the factors, and the content of glycyrrhizin and glycyrrhizic acid as the evaluation index, the processing technology for roasted licorice with water was optimized. [Results] The best processing technology of licorice was as follows: Pure licorice slices were mixed with water and moistened for 3 h, and then fried at 160 ℃ for 6 min. 20 kg of water was added to every 100 kg of licorice. [Conclusions] The best processing technology for roasted licorice with water was established, laying a foundation for the research and application of roasted licorice with water and its preparation.

Key words Roasted licorice with water, Orthogonal test, Processing technology, High performance liquid chromatography

1 Introduction

Licorice is the dry root and rhizome of leguminous plantGlycyrrhizauralensisFisch.,GlycyrrhizainflateBat. orGlycyrrhizaglabraL.[1]. It is one of the most widely used traditional Chinese medicine in clinic. Licorice tastes sweet, neutral in nature, having the effects of clearing away heat and detoxification, eliminating phlegm and relieving cough, relieving pain and reconciling various medicines[2]. After being roasted with honey, the effect changes to tonifying the spleen and stomach, and it is mainly used for treating spleen and stomach weakness, fatigue, palpitation, regularly interrupted pulse and other diseases.

Ancient licorice processing methods are very rich, but the 2015 edition of theChinesePharmacopoeiacontains only licorice and roasted licorice with honey. By consulting the ancient books of traditional Chinese medicine, it is found that licorice can be simmered in addition to being roasted with honey. The excipients for production include wine, vinegar, salt, oil, ginger, bile and so on[3]. In theCompendiumofMateriaMedicain the Ming Dynasty, Li Shizhen said: "Licorice is dipped in running water and roasted and peeled, which can invigorate spleen-stomach; if it is directly used without processing, it can purge pathogenic fire[4]." It is recorded in theDrugProcessingMethodwithSupplementaryMaterialsin the Ming Dynasty that licorice can be dipped in water and roasted[5]. TheLife-savingManualofDiagnosisandTreatmentofExternalDiseasesin the Qing Dynasty recorded: "Licorice is cut into three-inch sections, and then they are soaked in water, simmered with charcoal on an iron sieve, peeled and sliced. Roasted licorice can strengthen the spleen and stomach, while raw licorice can dissolve all kinds of toxins, moisturize the lungs, relieve sore and gangrene, and benefit the throat."[6]. The "running water" and "pure water" mentioned in the three books can be understood as a kind of liquid excipients, and this processing method can be understood as "water-baking method". At the same time, it is mentioned that the emphasis of efficacy of licorice has changed, but the processing technology of licorice has not been described in detail, and the process parameters are not clear. Therefore, in this experiment, the orthogonal design L9(34) was used to optimize the processing technology for roasted licorice with water using the content of glycyrrhizin and glycyrrhizic acid as the evaluation index. In this experiment, the results of water-roasted products and varieties collected inPharmacopoeiawere compared through parallel experiments, and proved that the best technology was scientific and reasonable, which provided a basis for the utilization of multi-processed varieties of licorice.

2 Materials

2.1InstrumentsAgilent 1260 high performance liquid chromatograph, G7115A-1260DAD detector; TG-16 desktop high-speed centrifuge (Sichuan Shuke Instrument Co., Ltd.); KQ-250DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., frequency 40 kHz, power 250W); FA224 1/10 000 electronic balance (Shanghai Shunyu Hengping Scientific Instrument Co., Ltd.); HC-100T multi-function grinder (Hecheng Trading Co., Ltd.).

2.2Medicinalmaterialsandreagents

2.2.1Medicinal materials. Licorice is produced in Longxi, Gansu Province (batch No.17111411). It was identified as the dry root and rhizome of leguminous plantG.uralensisFisch. by Professor Gu Jian from College of Pharmacy, Southwest Minzu University.

2.2.2Reagents. Glycyrrhizin reference substance (National Institute for Food and Drug Control, batch No.111610-201607, purity 93.1%, for content determination); ammonium glycyrrhizinate reference substance (National Institute for Food and Drug Control, batch No.110731-201619, purity 93.0%, for content determination); anhydrous ethanol and phosphoric acid (chromatographic purity, Tianjin Komeo Chemical Reagent Co., Ltd.); acetonitrile (chromatographic purity, Sigma Aldrich Shanghai Trading Co., Ltd.); pure water.

3 Methods and results

3.1RoastedlicoricewithwaterThe raw licorice was taken and mixed well with a certain amount of pure water, and moistened. After frying at the set temperature for a certain time, the processing experiment of licorice was arranged according to the design of L9(34) orthogonal test. After stir-frying, it was taken out, cooled, and bagged.

3.2Determinationofglycyrrhizinandglycyrrhizicacid

3.2.1Chromatographic conditions. Chromatographic column Kromasil 100-5-C18(4.6 mm×150 mm,5 μm); the mobile phase was acetonitrile (A)-0.05% phosphoric acid solution (B), gradient elution (0-8 min,19% A; 8-35 min,19%-50% A; 35-36 min，50%-100% A); detection wavelength 237 nm; volume flow 1 mL/min; column temperature 30 ℃; injection volume 10 μL. The number of theoretical plates should not be less than 5 000 according to the peak of glycyrrhizin.

3.2.2Preparation of reference solution. The right amount of reference substance of glycyrrhizin and ammonium glycyrrhizinate was accurately weighed.

Then 70% ethanol was added to prepare a reference solution containing 540 μg of glycyrrhizin and 610 μg of ammonium glycyrrhizinate per 1 mL (glycyrrhizic acid weight=ammonium glycyrrhizate weight/1.020 7).

500 μL of glycyrrhizin reference solution and ammonium glycyrrhizinate reference solution were precisely taken to prepare the mixed reference solution.

3.2.3Preparation of sample solution. The licorice sample was weighed, ground and sifted with 20-mesh sieve, and about 0.02 g of sample was taken and weighed precisely. 10 mL of 70% ethanol was precisely added, and weighed. After ultrasonic treatment (250 W, 40 kHz) for 30 min, it was cooled and then weighed. The weight loss was made up for with 70% ethanol, and it was shaken well and centrifuged (8 000 rpm, 5 min). The supernatant was filtered through a microporous membrane (0.45 μm), and the continuous filtrate was used as the sample solution[1]. Taking the content of glycyrrhizin and glycyrrhizic acid as indexes, the processing technology of licorice was optimized.

3.2.4Determination method. 10 μL of mixed reference solution and test solution were precisely taken respectively and injected into liquid chromatograph, and the results were obtained (Fig.1).

Note: 1. Glycyrrhizin; 2. Ammonium glycyrrhizinate.

Fig.1 HPLC chromatogram

3.2.5Investigation of linear relationship. 2, 4, 6, 8 and 10 μL of mixed reference solutions under Section3.2.2were precisely taken respectively and determined according to the chromatographic condition of Section3.2.1, and the peak area was recorded. Linear regression was carried out with glycyrrhizin and ammonium glycyrrhizinate (x, μg) as abscissa and peak area (y) as ordinate, respectively, and the regression equation and linear range were obtained (Table 1).

Table 1 Regression equation and linear range

3.2.6Precision test. An appropriate amount of mixed reference substance solution under Section3.2.2was taken and determined continuously for 6 times according to the chromatographic condition of Section3.2.1, and the peak area was recorded. As a result, theRSDof glycyrrhizin and glycyrrhizic acid was 0.32% and 0.32% (n=6), respectively, indicating that the precision of the instrument was good.

3.2.7Stability test. An appropriate amount of the same sample solution under Section3.2.3was taken and placed at room temperature for 0, 2, 4, 8, 12 and 24 h, respectively, and it was determined and the peak area was recorded under the chromatographic condition of Section3.2.1. As a result, the content of glycyrrhizin and glycyrrhizic acid was 0.130 6% and 4.803 9% respectively, and theRSDwas 6.82% and 1.95% (n=6), respectively. This showed that the solution of the sample was basically stable within 24 h at room temperature.

3.2.8Replication test. Six samples from the same batch were determined according to the chromatographic conditions of Section3.2.1. The peak area was recorded and the content of glycyrrhizin and glycyrrhizic acid was calculated according to the standard curve method. As a result, the content of glycyrrhizin and glycyrrhizic acid was 0.563 2% and 6.028 9%, respectively, and theRSDwas 4.12% and 3.81% (n=6), respectively, indicating that the method had good repeatability.

3.2.9Sample recovery test. The sample powder of roasted licorice with water was precisely weighed, a total of 6 groups, each about 0.02 g. An appropriate amount of reference solution (adding 0.010 8 mg of glycyrrhizin, 0.012 2 mg of ammonium glycyrrhizate) under Section3.2.2was precisely added to prepare the sample solution according to the method of Section3.2.3. Then the sample was injected and determined according to the chromatographic condition of Section3.2.1, the peak area was recorded and the recovery rate was calculated. The results showed that theRSDof glycyrrhizin and glycyrrhizic acid was 97.39% and 99.19%, respectively, indicating that the method conformed to the relevant regulations.

3.3SinglefactortestAccording to the results of the pre-experiment, the single factor was investigated with four factors: water added, moistening time, frying temperature and frying time. After processing, the sample solution was prepared according to the method of Section3.2.3. The content of glycyrrhizin and glycyrrhizic acid was determined under the chromatographic condition of Section3.2.1. The maximum content was 100 points, and the weighting coefficient was 0.5 according to the proportion of other content values to the maximum value. The calculation formula of comprehensive score is as follows:

Y=(Xi/maxi×0.5+Xj/maxj×0.5)×100

whereXirepresents valueiof glycyrrhizin;Xjrepresents valuejof glycyrrhizic acid;maxirepresents the maximum value of glycyrrhizin; maxjrepresents the maximum value of glycyrrhizic acid.

3.3.1Investigation on the water added. 20 g of licorice was taken, and mixed with proper amount of pure water and moistened for 2 h. The licorice was roasted for 9 min at 120 ℃ with different amount of water added. The content of glycyrrhizin and glycyrrhizic acid was determined. The results are shown in Table 2.

Table 2 Investigation on the water added to licorice

The results showed that the content of glycyrrhizin and glycyrrhizic acid changed obviously with different water added, and it could be seen that the water added was the factor affecting the change of their content.

3.3.2Investigation on moistening time. 20 g of licorice was taken for test, and mixed with 12 mL of pure water (60%), moistened, fried at 120 ℃ for 9 min, to get licorice roasted with water at different moistening time, and determine the content of glycyrrhizin and glycyrrhizic acid. The results are shown in Table 3.

Table 3 Investigation on the moistening time of roasted licorice with water

The results showed that the content of glycyrrhizin and glycyrrhizic acid changed obviously with different moistening time, and it could be seen that the moistening time was the factor affecting the change of their content.

3.3.3Investigation on frying temperature. 20 g of licorice was taken for test, and mixed with 12 mL of pure water (60%), and moistened for 9 min. It was fried for 9 min at different temperatures. The content of glycyrrhizin and glycyrrhizic acid in licorice roasted with water at different temperatures was determined. The results are shown in Table 4.

Table 4 Investigation on the frying temperature of roasted licorice with water

The results showed that the content of glycyrrhizin and glycyrrhizic acid changed obviously with different frying temperature. It could be seen that the frying temperature was the factor affecting the content change of both.

3.3.4Investigation on frying time. 20 g of licorice was taken for test, and mixed with 12 mL of pure water (60%), and moistened for 2 h. The roasted licorice with water was prepared at 120 ℃ at different frying time. The content of glycyrrhizin and glycyrrhizic acid was determined. The results are shown in Table 5.

Table 5 Investigation on the frying time of roasted licorice with water

The results showed that the content of glycyrrhizin and glycyrrhizic acid changed obviously with different frying time. The frying time was the factor that affected the content change of both.

3.4OrthogonaltestdesignandprocessoptimizationAccording to the results of the single factor test, the experiment was carried out according to the L9(34) orthogonal table with the water added (A), moistening time (B), frying temperature (C), frying time (D) as factors. The content of glycyrrhizin and glycyrrhizic acid was determined according to the method of Section3.2.3, which was used as the evaluation index to optimize the process. The level of factors is shown in Table 6. The content and results of glycyrrhizin and glycyrrhizic acid in each sample of orthogonal test are shown in Table 7. The results of analysis of variance are shown in Table 8.

Table 6 Orthogonal test factors and level design of roasted licorice with water

Table 7 Results of orthogonal test of roasted licorice with water

Table 8 Results of variance analysis of orthogonal test of roasted licorice with water

The results of variance analysis showed that the water added (A), moistening time (B), frying temperature (C), frying time (D) had no significant effect on the test results. By comparing the rangeRvalue, we can see that the influence degree is B> C>D>A. The best technology of roasted licorice with water was A1B3C3D1, that is, the water added 20%, moistening time 3 h, frying temperature 160 ℃ and frying time 6 min.

3.5ProcessverificationtestWith reference to Section3.1, three groups of roasted licorice samples with water were prepared in parallel with the best process of A1B3C3D1. The color of water-roasted products was darker than that of raw products, yellowish brown, with a slight aroma. Under the chromatographic condition of Section3.2.1, glycyrrhizin and glycyrrhizic acid in roasted licorice with water were determined with reference to Section3.2.3. The results showed that the content of glycyrrhizin was 0.460 5%, 0.522 4% and 0.406 1%, respectively. The content of glycyrrhizic acid was 5.021 5%, 5.734 5% and 5.277 0%, respectively. The scores were 87.858 6, 100.004 3 and 84.882 7, respectively. The results showed that the best water-roasting process of licorice was stable and feasible.

3.6Comparativeexperimentofrawlicorice,honey-roastedandwater-roastedlicoriceIn order to explore the similarities and differences in the effects of roasted licorice with water and unprocessed licorice and honey-roasted licorice, and to better prove the rationality of the best water roasting technology, this experiment took glycyrrhizin and glycyrrhizic acid as the index, a comparative experiment was carried out on three different licorice slices: water-roasted licorice (The best technology in this experiment was A1B3C3D1: 20 g of licorice was mixed well with water for 4 mL, moistened for 3 h, and fried at 160 ℃ for 6 min), unprocessed licorice and honey-roasted licorice. The experimental results are shown in Table 9.

Table 9 Comparative test of three kinds of licorice processed products

4 Results and discussions

In the orthogonal test, the content of glycyrrhizin and glycyrrhizic acid was comprehensively scored, to objectively reflect the relationship between various factors and the effect of factor level on the quality of roasted licorice with water. Three batches of roasted licorice slices with water were prepared by the optimized processing technology. The verification test results showed that the optimized processing technology of roasted licorice with water was stable and feasible. The best technology of roasted licorice with water was as follows: pure licorice slices were mixed well with water, moistened for 3 h, fried at 160 ℃ for 6 min, cooled and sifted. 20 kg of pure water was added to every 100 kg of licorice.

The best processing technology of roasted licorice with water was optimized by orthogonal design. The specific parameters were determined in the ratio of licorice slices to water, moistening time, frying temperature and frying time. This provided a theoretical basis for rational selection of licorice varieties in clinic and further development and utilization of corresponding medicinal resources.