Extraction Process and Content Determination of Total Flavonoids in Lonicera japonica

Yao HUANG, Xiumei MA, Chao ZENG, Xiaoling CAI, Jie HE, Xi FENG, Jiangcun WEI, Hongxia CHEN

1. Affiliated Hospital of Guilin Medical University, Guilin 541001, China; 2. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China; 3. Guangxi University of Chinese Medicine, Nanning 530200, China

Abstract [Objectives] To optimize the extraction process of total flavonoids of Lonicerae japonica and establish a method for its extraction and content determination. [Methods] The total flavonoids of L. japonica were extracted by reflux extraction method. Through a single factor experiment, the effects of extraction method, extraction solvent concentration, extraction volume and extraction time on the total flavonoids content of medicinal materials were investigated to select the optimal extraction process of the total flavonoids of L. japonica. [Results] The optimal extraction process of the total flavonoids of L. japonica was 70% ethanol, 1∶30 of solid-to-liquid ratio, and 1.0 h of reflux extraction time. [Conclusions] This method can effectively determine the content of total flavonoids in L. japonica and is expected to provide a certain scientific basis for the study of the quality standard of L. japonica. This method has high reproducibility. It is stable and feasible in extraction of total flavonoids from L. japonica.

Key words Lonicera japonica Thunb, Total flavonoids, Extraction process, UV-visible spectrophotometry

1 Introduction

Honeysuckle has a long history of medication and is the dried bud or initial flower ofLonicerajaponicaThunb[1].L.japonicais cold in nature, sweet in taste, enters the lung, heart, and stomach meridians, and has functions of clearing away heat and detoxification, anti-inflammation, and replenishing deficiency and dispelling the wind, and is mainly used for treating distention and fullness, warm disease and fever, abscess and ulcer due to heat toxin, and erysipelas and tumors,etc.[2]. The main chemical components ofL.japonicaare flavonoids, triterpene saponins, iridoid glycosides, organic acids, and volatile oils[3-5]. Related pharmacological experiments have shown thatL.japonicaas a common Chinese medicine can not only dispel wind-heat, but also clear away heat and detoxify, broad-spectrum antibacterial, antiviral, antipyretic and anti-inflammatory, choleretic and liver protection, lipid-lowering. It is mainly used for various infections and inflammations, hyperlipidemia, tumor radiotherapy and chemotherapy, xerostomia, various febrile diseases[3,6].

Plant total flavonoids are natural resources with extremely high medicinal value. It is of great significance to determine the content of total flavonoids inL.japonica, so as to improve the quality standards ofL.japonicamedicinal materials. Taking the total flavonoids content ofL.japonicaas the indicator, we used the single factor experiment to examine the effects of extraction method, extraction solvent, extraction volume, extraction time and other factors on the total flavonoids content ofL.japonica. Then, we performed an orthogonal experiment to further optimize the optimal extraction process. This experiment established a method for determining the content of total flavonoids inL.japonicaand is expected to provide a scientific basis for the quality standard ofL.japonica.

2 Instruments and test drugs

2.1 InstrumentsGL Sciences UV1780 ultraviolet-visible spectrophotometer; 1/100000 electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); KQ5200B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); DFT-1000 high speed universal pulverizer (Wenling LINDA Machinery Co., Ltd.).

2.2 Reagents and medicinal materialsRutin (batch No.205125), purity > 98%, purchased from Shanghai Ronghe Medical Technology Development Co., Ltd.; the remaining reagents used in the experiment were all of analytical reagents. Honeysuckle was purchased from Guangxi Xianzhu Pharmaceutical Co., Ltd. with batch number of 20190701 and identified by Zeng Chao, deputy chief Chinese pharmacist from Department of Pharmacy, the First Affiliated Hospital of Guangxi University of Chinese Medicine, as the dried bud or initial flower ofL.japonicaThunb.

3 Methods and results

3.1 Preparation of reference solutionPrecisely weighed 6.13 mg of rutin reference substance, and dissolved with 70% ethanol solution and fixed to a 25-mL volumetric flask, shook well, to obtain the reference solution with concentration of 0.245 2 mg/mL.

3.2 Preparation of sample solutionTook about 1.0 g of coarse powder ofL.japonica, precisely weighed, placed in a conical flask, added 30 mL of ethanol with volume fraction of 70% and weighed, extracted by reflux method for 1.0 h, weighed again, and made up the lost weight with 70% ethanol, shook well, filtered, and accurately took 1 mL of the subsequent filtrate into a 25-mL measuring flask to obtain the sample solution.

3.3 Establishment of linear equationTook 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 mL of the reference solution under Section3.1into a 10-mL volumetric flask, added 0.4 mL of 5% sodium nitrite, and 6 min later, added 0.4 mL of 10% aluminum nitrate, placed for 6 min, then added 4 mL of 4% sodium hydroxide, added water to the scale, shook well, placed for 10 min, then measured at the wavelength of 510 nm. A linear equation was established based on absorbance versus concentration. The equation wasy=8.720 1x-0.006 2,R2=0.999 3. Within the range of 0.012 26-0.098 08 mg/mL, the concentration and absorbance had a good linear relationship.

4 Methodology examination

4.1 Precision testPrecisely weighed approximately 1.0 g of coarse powder ofL.japonica, prepared the test product according to the operation steps in Section3.2, and determined the color by the method in Section3.3. The absorbance was measured six times at a wavelength of 510 nm, and theRSDwas 0.58%, showing that this method had high precision.

4.2 Stability testPrecisely weighed approximately 1.0 g of coarse powder ofL.japonica, prepared the test product according to the operation steps in Section3.2, and determined the color by the method in Section3.3. Measured the absorbance of the same test solution at different times. Measured the absorbance at 0, 20, 40, 60, 80, 100, and 120 min at the wavelength of 510 nm, and calculated theRSDvalue of theL.japonica, the result was 1.53%, indicating that the sample was stable within 120 min.

4.3 Reproducibility testPrecisely weighed six pieces of approximately 1.0 g of coarse powder samples ofL.japonica, prepared the test product according to the operation steps in Section3.2, and determined the color by the method in Section3.3. Measured the absorbance in parallel at a wavelength of 510 nm, and calculated the mean value content of total flavonoids inL.japonica, the result was 48.72 mg/g, andRSDvalue was 2.04%, indicating that this method has high reproducibility.

4.4 Sample recovery rate testPrecisely weighed 6 pieces of approximately 0.5 g of coarse powder samples ofL.japonica, precisely added proper amount of the rutin reference substance, prepared the test product according to the operation steps in Section3.2, and determined the color by the method in Section3.3. Measured the absorbance in parallel at a wavelength of 510 nm, and calculated the content of total flavonoids. From the results, it can be seen that the average recovery rate was 99.20%, and theRSDvalue was 2.91% (n=6), indicating that the recovery rate of this method is high, as shown in Table 1.

5 Single factor and orthogonal experiments

5.1 Single factor experiments

5.1.1Determination of flavonoid maximum absorption wavelength. After color development, the sample solution and the rutin reference solution were scanned at the full wavelength in the wavelength range of 400-600 nm, as shown in Fig.1. Two maximum absorption wavelengths (510 nm and 512 nm) appeared in the range of 400-600 nm. The sample solution is a mixture with complex components and many impurities, and the maximum absorption wavelength is slightly different from that of rutin, so 512 nm was selected as the test wavelength.

Table 1 Sample recovery rate test (n=6)

Fig.1 Visible light absorption spectrum of rutin and sample solution

5.1.2Extraction method test. Took about 1.0 g of coarse powder ofL.japonica, precisely weighed, placed in a conical flask, took extraction time of 1 h, extraction volume of 30 mL, and solvent 70% ethanol as the extraction conditions, and extracted the total flavonoids fromL.japonicaby ultrasonic extraction method, reflux extraction method and soaking method, prepared the test product according to the operation steps in Section3.2, and determined the color by the method in Section3.3. The results indicate that the extraction rate of reflux extraction method was the highest, so reflux extraction was selected, as shown in Fig.2.

5.1.3Ethanol solvent concentration test. Took about 1.0 g of coarse powder ofL.japonica, precisely weighed, placed in a conical flask, took extraction time of 1 h, extraction volume of 30 mL different concentration ethanol as the extraction conditions, and extracted the total flavonoids fromL.japonicaby the reflux extraction method, performed single factor experiment on the total flavonoid yield ofL.japonicawith different concentrations of ethanol solvents. The designed ethanol concentrations were 30%, 40%, 50%, 60%, 70% and 80%, prepared the test product according to the operation steps in Section3.2, and determined the color by the method in Section3.3. The results indicate that when refluxing with 70% ethanol, the extraction rate of the total flavonoids ofL.japonicawas the highest, as shown in Fig.3.

5.1.4Extraction time test. Took about 1.0 g of coarse powder ofL.japonica, precisely weighed, placed in a conical flask, took extraction volume of 30 mL 70% ethanol as the extraction conditions, and extracted the total flavonoids fromL.japonicaby the reflux extraction method, performed single factor experiment on the total flavonoid yield ofL.japonicawith different extraction times. The designed extraction times were 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h, prepared the test product according to the operation steps in Section3.2, and determined the color by the method in Section3.3. The results indicate that when the extraction time was 2.0 h, the extraction rate of the total flavonoids ofL.japonicawas the highest, as shown in Fig.4.

5.1.5Extraction volume test. Took about 1.0 g of coarse powder ofL.japonica, precisely weighed, placed in a conical flask, took extraction time 1 h and 70% ethanol as the extraction conditions, and extracted the total flavonoids fromL.japonicaby the reflux extraction method, performed single factor experiment on the total flavonoid yield ofL.japonicawith different extraction volume of 70% ethanol. The designed extraction volumes were 10, 20, 30, 40, 50, and 60 mL, prepared the test product according to the operation steps in Section3.2, and determined the color by the method in Section3.3. The results indicate that when the extraction volume was 30 mL, the extraction rate of the total flavonoids ofL.japonicawas the highest, as shown in Fig.5.

Fig.2 Results of extraction method test

Fig.3 Results of ethanol concentration test

Fig.4 Results of extraction time test

Fig.5 Results of extraction volume test

5.2 Test of extraction process through orthogonal experiment

5.2.1Orthogonal experimental design[7-8]. According to related literature and with reference toChinesePharmacopoeia(2015), this experiment finally adopted the extraction method of different ethanol concentration as the solvent. Based on the analysis of the single factor experiment results, set the ethanol concentration (60% ethanol, 70% ethanol, 80% ethanol), the solid-to-liquid ratio (1∶20, 1∶ 30, 1∶40), extraction time (1.0, 1.5, 2.0 h) as extraction conditions. With the aid of the orthogonal design assistant software, selected L9(34) orthogonal experimental design, as shown in Table 3.

Table 2 Orthogonal factor levels of L9(34)

Precisely weighed about 1.0 g of coarse powder ofL.japonica, precisely weighed, prepared the test product according to the operation steps in Section3.2, and determined the color by the method in Section3.3, then performed the experiment according to L9(34) orthogonal design scheme, and determined the content of total flavonoids inL.japonicausing ultraviolet-visible spectrophotometer, as shown in Table 3-4.

5.2.2Orthogonal experiment and results. According to variance analysis results, ethanol concentration and extraction volume have a significant effect on the extraction process of total flavonoids; there are significant differences in ethanol concentration and extraction volume factors, but there is no significant difference in extraction time; the intensity of each influencing factor is solid-to-liquid ratio (B) > ethanol concentration (A) > extraction time (C), so the statistically significant factors for the extraction ofL.japonicaare ethanol concentration and extraction volume, but the extraction time has no significant significance; considering the efficiency of the experiment and the experiment time, the extraction time is taken at 1.0 h, so the most ideal extraction scheme for this experiment is A2B2C1, namely, the ethanol concentration is 70%, the solid-to-liquid ratio is 1∶30, the reflux extraction time is 1.0 h, under such conditions, the extraction effect is optimal.

Table 3 Results of L9(34) orthogonal experiment

Table 4 Variance analysis of orthogonal experiment

6 Determination of sample content

Precisely weighed three pieces of approximately 1.0 g of coarse powder samples ofL.japonica, prepared the test product according to the operation steps in Section3.2, and determined the content of total flavonoids by the color development method in Section3.3, and measured the total flavonoids of in parallel and the average value was taken. The results are shown in Table 5.

Table 5 Results of content of total flavonoids in samples of Lonicera Japonica produced in different areas

7 Conclusions

Taking the total flavonoids content ofL.Japonicaas the indicator, this experiment used the single factor experiment to examine the effects of extraction method, extraction solvent, extraction volume, extraction time and other factors on the total flavonoids content ofL.Japonica. The total flavonoids were extracted fromL.Japonicaby the reflux extraction method. Then, orthogonal experiment was performed to select the optimal extraction process. According to relevant literature, main factors affecting the extraction process of the total flavonoids inL.Japonicaare extraction volume, solvents concentration and extraction time,etc. According to selection of the influencing factors, L9(34) orthogonal experiment was carried out for many times. The experiment indicates that the most optimal extraction scheme for this experiment is as follows: the extraction method is reflux extraction, the ethanol concentration is 70%, the solid-to-liquid ratio is 1∶30, the reflux extraction time is 1.0 h, under such conditions, the extraction effect is optimal. The data of the optimal process extraction conditions obtained in this experiment is reliable.

L.japonicais rich in natural resources and widely used in clinical application. Through consulting related literature, it is found that there are few studies about the total flavonoids inL.japonica. In order to ensure its proper clinical application, safety, and efficacy, this experiment selected the optimal extraction process of total flavonoids ofL.japonicathrough orthogonal experiments. It is expected to provide a scientific basis for further research and development of medicinal resources ofL.japonica.