Content Determination of Total Flavonoids from Paulownia fortunei Flower

Xu LIU, Juan TAO, Qing LI, Tingting LU

School of Pharmacy and Chinese Medicine, Jiangsu College of Nursing, Huai’an 223005, China

Abstract [Objectives] The research aimed to explore the determination method for content of total flavonoids from Paulownia fortunei flower. [Methods] Rutin was taken as control. By comparing absorption characteristics of total flavonoids in NaNO2-Al(NO3)3-NaOH and AlCl3 chromogenic system, an appropriate method for the determination of total flavonoids from P. fortunei flower was screened, and the method was verified by methodology. On the basis of single factor experiment, the coloration time of the method was optimized by orthogonal experiment. [Results] NaNO2-Al(NO3)3-NaOH chromogenic system was more suitable for the determination of total flavonoids from P. fortunei flower, and the adding standard recovery was between 99.2% and 105.5%, and RSD was 2.18%, with better repeatability, precision, stability and accuracy. The optimized coloration time of NaNO2, Al(NO3)3 and NaOH was 6, 6, and 10 min. Under the condition, average content of total flavonoids from P. fortunei flower was 3.78%, and RSD was 2.05%. [Conclusions] The optimized NaNO2-Al(NO3)3-NaOH chromogenic method is simple, rapid, and accurate, and could be used as a method for the determination of total flavonoids from P. fortunei flower.

Key words Paulownia fortunei flower, Total flavonoids, Content determination, Methodology validation, Orthogonal experiment, Coloration time

1 Introduction

Paulowniafortuneiis perennial deciduous tree and belongs toPaulowniaof Scrophulariaceae.P.fortuneiresources are abundant and various in China, and it has been used as traditional medicine in the Yellow River basin for hundreds of years[1]. Due to easy reproduction, fast growth and good material,P.fortuneihas been introduced and cultivated in many countries, and it is mainly used in ecological greening and industrial and agricultural production.P.fortuneihas not only high economic value, but also important medicinal value. The roots, flowers, leaves, barks and fruits ofP.fortuneican be used as medicine, and have pharmacological activities such as antioxidation[2], antibiosis[3-4], and antiviral effect[5].P.fortuneiflower mainly contains flavonoids, phenylpropanoids, terpenes, sugars and other active ingredients, and has the effects of clearing heat and detoxification, dispersing wind and heat, drying dampness, stopping dysentery, clearing liver, and brightening eyes[6-7]. Total flavonoids fromP.fortuneiflower has the antibacterial[8], anti-inflammatory[9], antiviral[10-11], antioxidant[12], enhancing immunity[13]and other biological activities, and has attracted extensive attention. At present, the methods of determining the content of total flavonoids in plants mainly contain high performance liquid chromatography and colorimetry[14]. Among them, high performance liquid chromatography is cumbersome and mainly used for the determination of flavonoids, but is not suitable for the determination of total flavonoids, while NaNO2-Al(NO3)3-NaOH and AlCl3colorimetry are widely used for the content determination of total flavonoids due to simple operation and strong practicability[15-17].

In this paper, rutin was taken as control. By comparing absorption characteristics of total flavonoids in NaNO2-Al(NO3)3-NaOH and AlCl3chromogenic system, an appropriate method for the determination of total flavonoids fromP.fortuneiflower was screened, and the method was verified by methodology. On the basis of single factor experiment, the coloration time of the method was optimized by orthogonal experiment, which could provide reference for studying determination method of total flavonoids fromP.fortuneiflower.

2 Materials and methods

2.1 MaterialsP.fortuneiflower was bought from Lankao Chinese herbal medicine market of Henan, and it belonged toPaulowniaof Scrophulariaceae via identification. It was crushed into 300 meshes by multifunctional crusher for use.

Rutin control sample (purity≥98%), Hefei Bomei Biotechnology Co., Ltd.; NaNO2, Al(NO3)3, NaOH, and AlCl3were all AR, Wuxi Yasheng Chemical Co., Ltd.

BF-10 type of multifunctional crusher, Hebei Benchen Technology Co., Ltd.; UV1800 type of ultraviolet visible spectrophotometer, Shimadzu Corporation; HH-2 type of digital display constant-temperature water bath, Guohua Electric Appliance Co., Ltd; BSA type of electronic balance, Saidoris Scientific Instrument (Beijing) Co., Ltd.

2.2 Preparation of solution

2.2.1Preparation of coloration reagent. 5% NaNO2solution: 2.5 g of NaNO2was weighed and dissolved in 50 mL of distilled water. 10% Al(NO3)3solution: 5 g of Al(NO3)3was weighed and dissolved in 50 mL of distilled water. 1.0 mol/L of NaOH solution: 4.0 g of NaOH was weighed and dissolved in 100 mL of distilled water. 0.1 mol/L of AlCl3solution: 1.34 g of AlCl3was weighed and dissolved in 100 mL of distilled water.

2.2.2Preparation of control solution. 5.0 mg of rutin control sample was taken and set in 25 mL of volumetric flask, and was dissolved by distilled water. The volume was fixed to the graduate, and 200 μg/mL of control solution was obtained.

2.2.3Preparation of test sample solution. 2.0 g ofP.fortuneiflower powder was weighed and set in round-bottom flask. 50 mL of 60% ethanol was added, and 90 ℃ heating reflux extraction was conducted for 2.0 h. After cooling, the weight was supplemented, and it was centrifuged. 20 mL of supernatant was taken, and 60% of ethanol was used to dilute to 50 mL, and test sample solution was obtained.

2.3 Screening of determination methods for content of total flavonoids fromP.fortuneiflower

2.3.1NaNO2-Al(NO3)3-NaOH colorimetry[18-19]. 1.00 mL of test sample solution and 2.00 mL of control solution were measured precisely and set in beaker, and 0.3 mL of 5% NaNO2solution was added. After shaken evenly, it was set for 6 min.0.3 mL of 10% Al(NO3)3solution was added. After shaken evenly, it was set for 6 min. Finally, 3.0 mL of 1.0 mol/L NaOH solution was added, and distilled water was used to fix the volume to 10 mL, and it was set for 15 min. Taking blank reagent as the reference, ultraviolet visible spectrophotometer was used for scanning absorption curve in the wavelength of 400-600 nm.

2.3.2AlCl3colorimetry[20]. 1.00 mL of test sample solution and 2.00 mL of control solution were precisely measured and set in beaker. 2.0 mL of 0.1 mol/L AlCl3solution was added, and distilled water was used for fixing the volume to 10 mL, and it was set for 30 min. Taking blank reagent as the reference, ultraviolet visible spectrophotometer was used for scanning absorption curve in the wavelength of 300-500 nm.

2.4 Optimization of coloration time

2.4.1Single factor experiment[16,19,21]. In preliminary experiment, it was found that the adding proportion of three kinds of chromogenic reagents [NaNO2, Al(NO3)3and NaOH] was 1∶1∶10. So, when determining the content of total flavonoids fromP.fortuneiflower, it was only necessary to control the addition proportion of three coloration reagents, and the addition amount of reagents had little effect on the absorbance. Therefore, it was only necessary to investigate the influence of the standing time (coloration time) of the three coloration reagents on the absorbance. 2 mL of test sample solution was measured, and NaNO2-Al(NO3)3-NaOH colorimetry was used to determine the content of total flavonoids fromP.fortuneiflower, and the influence of NaNO2coloration time (2, 4, 6, 8, and 10 min), Al(NO3)3coloration time (2, 4, 6, 8, and 10 min), and NaOH coloration time (5, 10, 15, 20, and 25 min) on absorbance was inspected.

2.4.2Orthogonal experiment. On the basis of single factor experiment, NaNO2coloration time (A), Al(NO3)3coloration time (B) and NaOH color development time (C) were taken as investigation factors, and the content of total flavonoids fromP.fortuneiflower was taken as assessment index for L9(34) orthogonal experiment, to optimize determination conditions of NaNO2-Al(NO3)3-NaOH colorimetry for the content of total flavonoids fromP.fortuneiflower. The factor and level of orthogonal experiment were shown in Table 1.

Table 1 Factor and level of orthogonal experiment for the content of total flavonoids from P. fortunei flower min

3 Results and analyses

3.1 Definition of determination method for the content of total flavonoidsNaNO2-Al(NO3)3-NaOH and Al(NO3)3chromogenic system were used for color rendering of test sample solution and control solution, and ultraviolet visible spectrophotometer was used for scanning absorption curve in certain wavelength range, and the results were shown in Fig.1.

Fig.1 UV-Vis absorption spectra of solutions colored by NaNO2-Al(NO3)3-NaOH chromogenic system (a) and Al(NO3)3 chromogenic system (b)

Seen from Fig.1, when using NaNO2-Al(NO3)3-NaOH chromogenic system, the absorption peaks of the control solution and the test sample solution almost coincided, and the maximum absorption wavelength was 500 nm. When using Al(NO3)3chromogenic system, there was greater difference in the maximum absorption wavelength of control solution and test sample solution. Therefore, NaNO2-Al(NO3)3-NaOH colorimetry was selected to measure the content of total flavonoids fromP.fortuneiflower, and the detection wavelength was 500 nm.

3.2 Methodological investigation

3.2.1Standard curve and linear range. 1.0, 2.0, 3.0, 4.0 and 5.0 mL of control solution was measured precisely, and NaNO2-Al(NO3)3-NaOH chromogenic system was used, and ultraviolet visible spectrophotometer was used for determination at 500 nm. Taking rutin solution concentration as horizontal ordinate, and absorbance as longitudinal coordinate, standard curve was drawn (Fig.2), and linear equation was obtained:y=9.315x-0.035 3,R2=0.999 8. It showed that rutin concentration had a good linear relationship with absorbance in the range of 0.02-0.10 mg/mL.

Fig.2 Standard curve of rutin

3.2.2Repeatability. 6 copies ofP.fortuneiflower sample (2.0 g) were weighed, and test sample solution was prepared according to the method of Section2.2.3. NaNO2-Al(NO3)3-NaOH chromogenic system was used, and ultraviolet visible spectrophotometer was used for determination at 500 nm. Each sample was determined parallely for three times, and mean was obtained. The content of total flavonoids fromP.fortuneiflower was respectively 3.66%, 3.71%, 3.76%, 3.64%, 3.68%, and 3.72%, and average content was 3.70%.RSDwas 1.08%, and was less than 3.0%. It showed that NaNO2-Al(NO3)3-NaOH chromogenic system had good repeatability.

3.2.3Precision. 2.0 g ofP.fortuneiflower sample was weighed, and test sample solution was prepared according to the method of Section2.2.3. NaNO2-Al(NO3)3-NaOH chromogenic system was used, and ultraviolet visible spectrophotometer was used for determination at 500 nm, and it was continuously determined for 6 times. The content of total flavonoids fromP.fortuneiflower was respectively 3.52%, 3.63%, 3.67%, 3.53%, 3.64%, 3.66%, and average content was 3.61%.RSDwas 1.67%, and was less than 3.0%. It showed that NaNO2-Al(NO3)3-NaOH chromogenic system had good precision.

3.2.4Stability. 2.0 g ofP.fortuneiflower sample was weighed, and test sample solution was prepared according to the method of Section2.2.3. NaNO2-Al(NO3)3-NaOH chromogenic system was used, and sampling was conducted at 0, 10, 20, 30, 40, 50, and 60 min, and ultraviolet visible spectrophotometer was used for determination at 500 nm. Each sample was measured parallely for 3 times, and the mean was obtained, and they were 0.528 0, 0.527 2, 0.526 8, 0.526 1, 0.521 2, 0.518 1, and 0.512 0. It showed that absorbance had good stability within 0-30 min afterP.fortuneiflower sample solution was colored by NaNO2-Al(NO3)3-NaOH chromogenic system.

3.2.5Accuracy. 6 copies ofP.fortuneiflower sample (2.0 g) were weighed, and 0.076 2 g of rutin control sample was added, and test sample solution was prepared according to the method of Section2.2.3. NaNO2-Al(NO3)3-NaOH chromogenic system was used, and ultraviolet visible spectrophotometer was used for determination at 500 nm. Each sample was determined parallely for 3 times, and the mean was obtained. The recovery rate of standard addition andRSDof total flavonoids fromP.fortuneiflower were calculated, and the results were shown in Table 2.

Seen from Table 2, the recovery rate of standard addition of total flavonoids fromP.fortuneiflower was between 99.2%-105.5%, andRSDwas 2.18%, which was less than 3.0%. It showed that NaNO2-Al(NO3)3-NaOH chromogenic system had good accuracy.

Table 2 Recovery rate of standard addition of total flavonoids from Paulownia fortunei flower

3.3 Optimization of color development time

3.3.1Single factor experimental results. NaNO2-Al(NO3)3-NaOH colorimetry was used to measure total flavonoids fromP.fortuneiflower, and effect of coloration time of three chromogenic reagents on absorbance was shown in Table 3.

Table 3 Effect of coloration time of three chromogenic agents on absorbance

Seen from Table 3, with the prolonging of coloration time, the absorbance decreased gradually and tended to be stable after adding different chromogenic reagents. After adding chromogenic reagents NaNO2, Al(NO3)3, and NaOH, it was set for 6, 6 and 15 min, and the absorbance was stable.

3.3.2Orthogonal experimental results. According to single factor experimental results, 6, 6 and 15 min were taken as center points of NaNO2coloration time (A), Al(NO3)3coloration time (B), and NaOH coloration time (C), and L9(34) orthogonal experiment was used to optimize chromogenic conditions of NaNO2-Al(NO3)3-NaOH colorimetry, and the results and analyses were shown in Table 4.

Seen from Table 4, the factor A had the maximum influence on the content of total flavonoids, followed by the factor B, and the factor C had the least influence. The optimal chromogenic condition for determining the content of total flavonoids fromP.fortuneiflower was A2B2C1, namely 6 min of NaNO2coloration time, 6 min of Al(NO3)3coloration time, and 10 min of NaOH coloration time.

Table 4 Results of L9 (34) orthogonal experiment for the determination of total flavonoids from Paulownia fortunei flower

3.4 Determination for the content of total flavonoids fromP.fortuneiflower6 copies ofP.fortuneiflower sample (2.0 g) was weighed, and sample solution was prepared according to the method of Section2.2.3. NaNO2-Al(NO3)3-NaOH chromogenic system was used for coloration reaction under the optimized condition, and ultraviolet visible spectrophotometer was used to measure absorbance at 500 nm. Each sample was measured parallely for three times, and mean was obtained. Average content of total flavonoids fromP.fortuneiflower was 3.78%, andRSDwas 2.05%. It illustrated that the method was stable and feasible for determining total flavonoids fromP.fortuneiflower. The optimized NaNO2-Al(NO3)3-NaOH colorimetry is simple, fast and accurate, which could be taken as the determination method of total flavonoids fromP.fortuneiflower.

4 Conclusions

Via single factor experiment and orthogonal experiment, it was determined that NaNO2-Al(NO3)3-NaOH colorimetry was suitable for determining total flavonoids fromP.fortuneiflower. The method had better repeatability, precision, stability and accuracy, and the optimal coloration time of NaNO2, Al(NO3)3and NaOH was respectively 6, 6 and 10 min. Under the condition, average content of total flavonoids fromP.fortuneiflower was 3.78%, andRSDwas 2.05%.