Extraction Process of Total Flavonoids from Eucommiae Cortex and Its in vitro Antioxidant Activity

Yunli TANG, Xiumei MA, Huazhen SU, Cheng PENG, Xiaofang XIE, Hongxia CHEN

1. Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China; 2. Affiliated Hospital of Guilin Medical University, Guilin 541001, China; 3. Guangxi University of Chinese Medicine, Nanning 530201, China

Abstract [Objectives] To optimize the extraction process of total flavonoids from Eucommiae Cortex, establish the extraction and content determination methods of its medicinal materials, and study its in vitro antioxidant activity. [Methods] The total flavonoids from Eucommiae Cortex were extracted by reflux extraction method, and the effects of extraction method, extraction solvent concentration, extraction volume, and extraction time on the total flavonoids content of the medicinal materials were explored through the single factor experiment. Orthogonal experiment was carried out to optimize the extraction process conditions, and the optimal extraction process for total flavonoids from Eucommiae Cortex was screened out. The total antioxidant capacity index was used to determine the free radical scavenging ability of the total flavonoids from Eucommiae Cortex. [Results] The optimal extraction process of total flavonoids from Eucommiae Cortex was 50% ethanol, 1∶40 solid-to-liquid-ratio, and 1.5 h reflux extraction time. Through the antioxidant experiment in vitro, it is found that the total flavonoids from Eucommiae Cortex have a higher scavenging ability for the total antioxidant capacity, and there is a certain positive correlation with the mass concentration of total flavonoids. [Conclusions] This method can effectively determine the total flavonoids content of Eucommiae Cortex medicinal material, and provide a certain scientific basis for the quality standard research of the medicinal material. The total flavonoids from Eucommiae Cortex have good in vitro antioxidant activity. And the method for extracting total flavonoids from Eucommiae Cortex medicinal material in this experiment has good repeatability, high stability and feasibility.

Key words Eucommia ulmoides Oliv., Total flavonoids, Extraction process, Ultraviolet-visible spectroscopy (UV-Vis), Antioxidant activity

1 Introduction

Eucommiae Cortex is the dried bark of Eucommiaceae plantEucommiaulmoidesOliv., and has functions of nourishing the liver and kidney, strengthening the bones and muscles, and protecting the fetus,etc[1]. There are records of various processing methods for bark ofE.ulmoides, including collecting barks and shade drying, removing coarse barks and sun drying, and removing coarse barks "sweating" and sun drying[1-2]. The total flavonoids of Eucommiae Cortex have blood pressure lowering, antioxidant and anti-tumor pharmacological effects[3], and are an important active component ofE.ulmoides. In this experiment, we took the total flavonoids content of Eucommiae Cortex as the evaluation indicator and explored the effects of extraction method, extraction solvent, extraction volume, extraction time and other factors on the total flavonoids content of Eucommiae Cortex through the single factor experiment. Then, we carried out the orthogonal experiment to further optimize the optimal extraction process, to provide a certain reference basis for further research on the quality standards of Eucommiae Cortex medicinal material. We established a method for the determination of total flavonoids in Eucommiae Cortex and the determination ofinvitroantioxidant capacity, to provide a scientific basis for the future development and utilization of this medicinal resource.

2 Materials and methods

2.1 Materials

2.1.1Instruments. Shimadzu UV1780 ultraviolet-visible spectrophotometer; 1/100000 electronic balance (XS105D; Mettler Toledo Equipment System Co., Ltd.); A-S082 multifunctional microplate reader (Switzerland Tecan company).

2.2.2Reagents. Total Antioxidant Capacity (T-AOC) Colorimetric Assay Kit was produced by Nanjing Jiancheng Institute of Bioengineering. Rutin (batch No.195126, purity > 98%) was purchased from Shanghai Winherb Medical Technology Co., Ltd.; the other reagents used in the experiment were of analytical grade. The medicinal materials were purchased from the Nanning LBX Pharmacy, and were identified as the dried bark ofEucommiaulmoidesOliv. by Zeng Chao, the director of the Department of Pharmacy of the First Affiliated Hospital of Guangxi University of Chinese Medicine.

2.2 Methods

2.2.1Preparation of reference solution. Precisely weighed an appropriate amount of the rutin reference substance that has been dried to the constant weight at 105 ℃, added 70% ethanol to dissolve it, diluted to a 25-mL volumetric flask, shook well, and prepared the reference substance solution with the concentration of 0.52 mg/mL.

2.2.2Preparation of test solution. Precisely weighed 1.0 g of Eucommiae Cortex powder, placed in a conical flask, added 25 mL of 60% ethanol and weighed, ultrasonically extracted for 60 min, and then weighed again, and made up the lost weight with the 60% ethanol. Shook well and filtered and precisely weighed 2.0 mL of the filtrate and placed in a 50-mL measuring flask.

2.2.3Establishment of linear equation. Separately took 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 mL of the reference solution and placed in a 25-mL volumetric flask, added 1 mL of 5% sodium nitrite, and placed for 7 min, then added 1 mL of 10% aluminum nitrate and placed for 7 min. Added 10 mL of 4% sodium hydroxide, added water to the desired scale, shook well, placed for 12 min, and then measured at the wavelength of 510 nm[4]. Established the linear equation of absorbance vs concentration. The equation wasy=12.198x-0.028 4,R2=0.999 2. In the range of 0.010 4-0.083 2 mg/mL, there is a good linear relationship between concentration and absorbance.

2.2.4Methodological test. (i) Precision test. Precisely weighed 1.0 g of Eucommiae Cortex powder, prepared the test article, developed color, and measured the absorbance for 6 consecutive times at the wavelength of 510 nm, and the result was that theRSDwas 2.04%, which was less than 3, showing that the precision of this method is high. (ii) Stability test. Precisely weighed 1.0 g of Eucommiae Cortex powder, prepared the test article, developed color, and measured the absorbance of the same test solution in 0, 10, 20, 30, 40, 50, and 60 min at the wavelength of 510 nm. Calculated theRSDof the medicinal material was 1.91%, which was less than 3, showing that the absorbance is stable within 60 min, so the samples are stable within 60 min. (iii) Repeatability test. Precisely weighed 6 pieces of Eucommiae Cortex powder with each piece about 1.0 g, prepared the test article, developed color, and measured the absorbance at the wavelength of 510 nm in a parallel manner. Calculated the average content of Eucommiae Cortex medicinal materials and the result was 15.08 mg/g; theRSDwas 2.36%, which was less than 3%, showing that the method has good repeatability. (iv) Sample recovery test. Precisely weighed 9 pieces of 0.5 g of Eucommiae Cortex powder of the same batch with known total flavonoids content, divided them into 3 groups, namely, high, medium, and low content groups, prepared the test article, developed color, measured the absorbance at the wavelength of 510 nm in a parallel manner, calculated the total flavonoids content. The results show that the method has high accuracy (Table 1).

2.2.5Design of orthogonal experiment[5-6]. With reference to the related literature, we finally adopted the extraction method with different ethanol concentration as the solvent, set three conditions as follows: ethanol concentration (40%, 50%, and 60% ethanol), the solid-to-liquid ratio (1∶30, 1∶40, and 1∶50), extraction time (1.0, 1.5, and 2.0 h) in this experiment. According to the orthogonal design assistant software, we selected the L9(34) orthogonal experiment design (Table 2).

Table 1 Sample recovery test of extracting total flavonoids from Eucommiae Cortex (n=9)

Table 2 Factors and levels of L9(34) orthogonal experiment for extraction of total flavonoids from Eucommiae Cortex

2.2.6Invitroantioxidant activity test of total flavonoids. The total antioxidant capacity of the total flavonoids of Eucommiae Cortex was determined using a total Antioxidant Capacity (T-AOC) Colorimetric Assay Kit.

3 Results and analysis

3.1 Results of single factor experiment

3.1.1Extraction method test. Precisely weighed 1.0 g of Eucommiae Cortex powder, placed in a conical flask, took extraction time 1 h, 25 mL, 50% ethanol as the extraction conditions, extracted the total flavonoids from the medicinal materials separately using the soaking method, ultrasonic extraction method and reflux extraction method. The results showed that the extraction rate of reflux extraction method was the highest, so we selected the reflux extraction method (Fig.1).

Fig.1 Yield rate of total flavonoids from Eucommiae Cortex by different extraction methods

3.1.2Ethanol solvent concentration test. Precisely weighed 1.0 g of Eucommiae Cortex powder, placed in a conical flask, took extraction time 1 h, 25 mL ethanol with different concentration as the extraction conditions, extracted the total flavonoids from Eucommiae Cortex using the reflux extraction method. We carried out a single factor experiment on the yield rate of total flavonoids from Eucommiae Cortex with different concentrations of ethanol solvent. The designed ethanol concentrations were 30%, 50%, 60%, 70%, 80% and 95%. The results showed that 50% ethanol reflux extraction has the high yield rate of total flavonoids from Eucommiae Cortex (Fig.2).

Fig.2 Yield rate of total flavonoids from Eucommiae Cortex at different ethanol concentrations

3.1.3Extraction time test. Precisely weighed 1.0 g of Eucommiae Cortex powder, placed in a conical flask, took 25 mL of 50% ethanol as the extraction condition, adopted the reflux extraction method, carried out the single factor experiment for the yield rate of total flavonoids at extraction time of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h. The results showed that the yield rate of total flavonoids is the highest when the extraction time is 1.5 h (Fig.3).

Fig.3 Yield rate of total flavonoids from Eucommiae Cortex in different extraction time

3.1.4Extraction volume test. Precisely weighed 1.0 g of Eucommiae Cortex powder, placed in a conical flask, took extraction time of 1.5 h and 25 mL of 50% ethanol as the extraction conditions, adopted the reflux extraction method, carried out the single factor experiment for the yield rate of total flavonoids at the volume of 20, 30, 40, 50, 60, and 70 mL of 50% ethanol. The results showed that the yield rate of total flavonoids is the highest at the 40 mL ethanol volume (Fig.4).

Fig.4 Yield rate of total flavonoids from Eucommiae Cortex at different extraction volume

3.2 Results of orthogonal experimentAccording to the orthogonal experiment analysis (Table 3-4), it can be seen that the ethanol concentration and extraction time have a significant effect on the extraction process of total flavonoids from Eucommiae Cortex, while the solid-to-liquid ratio has no significant effect on the extraction process of total flavonoids from Eucommiae Cortex. The influencing factors are ethanol concentration (A) > extraction time (C) > solid-to-liquid ratio (B), so the factors that are statistically significant for the extraction effect of Eucommiae Cortex are ethanol concentration and extraction time, while the solid-to-liquid ratio is not statistically significant. Therefore, the most ideal extraction scheme obtained in this experiment is A2B2C2, that is, the ethanol concentration is 50%, the solid-to-liquid ratio is 1∶40, and the reflux extraction time is 1.5 h. Under these conditions, the extraction effect is optimal.

Table 3 Results of L9(34) orthogonal experiment for extraction of total flavonoids from Eucommiae Cortex

Table 4 Analysis of variance of orthogonal experiment

3.3 Determination of sample contentPrecisely weighed 6 pieces of Eucommiae Cortex powder with each piece about 1.0 g, placed in a conical flask, prepared the test article in accordance with the optimal extraction process, developed color, and determined the content of total flavonoids. The results are listed in Table 5.

Table 5 Results of content of total flavonoids from Eucommiae Cortex

3.4 Results of antioxidant activity testFrom Fig.5, it can be seen that the total flavonoids from Eucommiae Cortex and the Vc reference substance have a strong reducing ability to iron ions, and they increase with the increase in concentration. The total antioxidant capacity of total flavonoids from Eucommiae Cortex is stronger than Vc at the same concentration.

Fig.5 Total antioxidant capacity of total flavonoids from Eucommiae Cortex

4 Discussion and conclusions

In this experiment, we adopted L9(34) orthogonal experiment and carried out many times of experiments. The experimental results show that the optimal extraction process for Eucommiae Cortex, namely reflux extraction, with an ethanol concentration of 50%, a solid-to-liquid ratio of 1∶40, and an extraction time of 1.5 h. Under these conditions, the extraction effect is optimal.

Using the ultraviolet spectrophotometry, we measured the total antioxidant capacity of the total flavonoids from Eucommiae Cortex. The experiment showed that the total flavonoids from Eucommiae Cortex have a stronginvitroantioxidant effect. When the concentration of total flavonoids in Eucommiae Cortex was 3 mg/mL, its total antioxidant capacity was 132.20 U/mL, and its scavenging capacity increased with the increase of concentration.