Jun TANG, Jianguang ZENG, Hao LI, Chenzhong JIN
Abstract With Pueraria edulis from different habitats in Yunnan Province， Guangdong Province and Guangxi Zhuang autonomous region as materials， the medicinal materials of P. edulis were extracted by ethanol condensation reflux extraction and ultrasound-assisted extraction， and the effects of different extraction methods the contents of total flavonoids in P. edulis from different areas were compared. The results showed that the best extraction time for P. edulis in Yunnan Province was 20 min， and that for Guangdong and Guangxi Zhuang Autonomous Region was 30 min. The yield of total flavonoids from P. edulis extracted by the condensation reflux extraction method was relatively better. The content of total flavonoids in P. edulis in Yunnan was the highest with an average content of 0.255 7%， while the average content of total flavonoids in P. edulis in Guangdong was the lowest with an average content of 0.210 8%.
Key words Pueraria edulis; Total flavonoids; Condensation reflux extraction method; Ultrasound-assisted extraction
Received：
December 26， 2022 Accepted：
February 28， 2023
Supported by Hunan Provincial University Innovation Platform Open Fund Project （19K049）.
Jun TANG （1999-）， male， P. R. China， master， devoted to research about resource utilization and environmental protection.
*Corresponding author. E-mail：
532479626@qq.com.
Pueraria edulis， also known as Geteng， Gemaye， and Fengeteng， is the tuberous roots of kudzu or kudzu vine， a leguminous plant. It is sweet and pungent in taste and mild in nature. It is used for externally induced fever and headache， strong pain in the neck and back， thirst， consumptive thirst， measles without adeqrate eruption， heat dysentery， diarrhea， dizziness， stroke hemiplegia， chest stuffiness and pains， and alcohol poisoning［1-2］. P. edulis is a kind of medicinal and edible homologous plant， which has always been known as "millennium ginseng"［3］， and has high edible and medicinal value. Pharmacological studies have shown that P. edulis has the effects of maintaining beauty and keeping young， stabilizing blood sugar and blood pressure， protecting the heart， and preventing osteoporosis［4］. The medicinal functions of P. edulis have been documented in Shen Nongs Herbal Classic. According to Supplementary Records of Famous Physicians， P. edulis powder can be directly swallowed or soaked， and can also be consumed in decoction， and it can be used to treat typhoid fever， stroke， headache， syndrome of external wind and cold deficiency， metal-inflicted wound， and hypochondriac pain. Its quality is directly related to the safety and effectiveness of public medicine and food［5-6］.
Currently， traditional extraction processes commonly used in the production and detection of industrial traditional Chinese medicine （such as Soxhlet extraction and reflux extraction） have long extraction time， which affects extraction efficiency， and some medicinal materials are also damaged due to long heating time［7-8］. It is necessary to overcome the shortcomings of traditional methods， search for extraction methods that require simple equipment and easy operation， and utilize modern science and technology to strive to develop the plant resources of the motherland［9］.
In this study， two extraction methods， ethanol condensation reflux and ultrasound-assisted extraction， were applied to compare and analyze the total flavonoids content of P. edulis samples from three different habitats， providing experimental basis for standardized planting and reasonable development and utilization of P. edulis in the later stage， as well as certain reference value for the establishment of quality standards for total flavonoids in P. edulis.
Materials and Methods
Instruments and reagents
KQ-300E ultrasonic cleaner （Kunshan Ultrasonic Instrument Co.， Ltd.）; HH-S2s thermostatic water bath （Jintan Dadi Automatic Instrument Factory）; BSA224S electronic balance （Sartorius Scientific Instruments （Beijing） Co.， Ltd.）; UV-2600 ultraviolet spectrophotometer （Shanghai Jinghua Technology Instrument Co.， Ltd.）.
Rutin （concentration ≥ 98%， Beijing Shiji Aoke Biotechnology Co.， Ltd.， biochemical reagent）; 95% ethanol solution， anhydrous ethanol solution （Hubei Xingdongcheng Chemical Co.， Ltd.， analytical pure）; sodium nitrite （NaNO2） （Xiaxian Yunli Chemical Co.， Ltd.， analytical pure）; aluminum nitrate ［Al（NO3）3］ （Jinan Kunfeng Chemical Co.， Ltd.， analytical pure）; sodium hydroxide （NaOH） （Cangzhou Zhanghua Chemical Products Co.， Ltd.， analytical pure）.
Sample treatment
Wild P. edulis samples produced in Yunnan， Guangdong， and Guangxi Zhuang Autonomous Region were used as test samples. The sample numbers are shown in Table 1. After washing the samples thoroughly， they were taken back and rinsed repeatedly with deionized water for several times. The samples were pre-frozen at -40.5 ℃ for 2 h， kept at low temperature （-15 ℃） for 5 h， dried （35 ℃） for 16.5 h， and sublimated （15 ℃） for 8 h. The freeze-dried P. edulis samples were pulverized with a pulverizer， sieved through a No. 3 sieve， and added into wide-mouth bottles with different numbers for later use.
Preparation of reference solution
Two milligram of rutin reference substance was accurately weighed， and 80% ethanol solution was slowly added to dissolve the reference substance. The obtained solution was diluted to constant volume in a 50 ml volumetric flask with a stopper， and a 40 μg/ml reference solution was prepared after shaking well.
Selection of wavelength
A 5 ml pipette was used to transfer 5 ml of rutin reference solution to a 25 ml colorimetric tube with a stopper， and a 0.5 ml pipette was used to accurately pipette 0.3 ml of 5% sodium nitrite solution to the colorimetric tube to obtain a solution， which was shaken to mix well， and stood for 6 min. A 0.5 ml pipette was used to accurately transfer 0.3 ml of 10% aluminum nitrate solution into the colorimetric tube， which was shaken to mix well， and then stood for 6 min. Next， 4.0 ml of 4% sodium hydroxide solution was added. Finally， the solution was diluted to 10 ml with 80% ethanol solution， mixed well after plugging with a stopper， and stood for 15 min. With the mixed solution without the addition of rutin as the blank control， the maximum absorption wavelength was determined to be 476 nm by scanning within the wavelength range of 200-900 nm［10］.
Ethanol condensation reflux extraction
First， 3 g of dried P. edulis powder was accurately weighed and added into a conical flask with a stopper that had undergone dry heat sterilization. A 25 ml pipette was employed to accurately add 50 ml of 80% ethanol solution， and the total weight of the solvent and the conical flask was weighed. The prepared samples were extracted under condensation reflux in a water bath at constant temperature for 30， 20 and 10 min， respectively. After cooling and weighing， the weight losses were supplemented， and the mixtures were filtered to obtain filtrates for later use.
A 1 ml of filtrate was added into a 50 ml volumetric flask with a stopper， and finally diluted to constant volume with 80% ethanol solution. Each volumetric flask was covered with its stopper and shaken to mix well. Finally， 5 ml of each test solution was added into a 10 ml colorimetric tube with a stopper， and measured for absorbance in turn with an ultraviolet spectrophotometer.
Ultrasonic extraction
First， 3 g of dried P. edulis powder was accurately weighed and added into a conical flask with a stopper. A 25 ml pipette was used to accurately add 50 ml of 80% ethanol solution， and the total weight of the solvent and the conical flask was weighed. The prepared samples were ultrasonically extracted for 10， 20， and 30 min， respectively. After cooling and weighing， the weight losses were supplemented， and the mixtures were filtered to obtain filtrates for later use.
A 1 ml of filtrate was added into a 50 ml volumetric flask with a stopper， and finally diluted to constant volume with 80% ethanol solution. Each volumetric flask was covered with its stopper and shaken to mix well. Finally， 5 ml of each test solution was added into a 10 ml colorimetric tube with a stopper［11］， and measured for absorbance in turn with an ultraviolet spectrophotometer.
Results and Analysis
Linearity test results
The absorbance values of reference solutions were measured at a wavelength of 476 nm， and the results are shown in Table 2. Using the absorbance of the reference substance as the ordinate and the concentration as the abscissa， a standard curve was drawn as shown in Fig. 1. The regression equation for rutin was A=0.352 7C+0.005 4 （R2=0.998 9）， and the concentration of total flavonoids in P. edulis in the range of 0.4-2.4 μg/ml conformed to Beers Law［12］， and there was a good linear relationship between absorbance and concentration.
Methodological investigation
Precision test
A total flavonoid reference solution was accurately measured to prepare a color developing solution， which was repeatedly determined for 6 times. The relative standard deviation result is shown in Table 3. From Table 3， it can be seen that the RSD was 0.02%， indicating a good test precision.
Repeatability test
Six portions of 3.000 0 g of P. edulis powder from Yunnan Province were accurately weighed to prepare test solutions， which were measured for the absorbance after color development at a wavelength of 467 nm， and the average value of total flavonoid contents and RSD were calculated according to the standard curve. The results are shown in Table 4.
Extraction of total flavonoids from P. edulis from different habitats for different time by condensation reflux extraction
First， 3.000 00 g of P. edulis powder from different habitats in Yunnan Province， Guangdong Province and Guangxi Zhuang Autonomous Region， were weighed and added in conical flasks， respectively. An appropriate amount of ethanol was added to dissolve the samples and perform condensation reflux extraction. The results are shown in Table 5. The total flavonoid contents of samples from different habitats were compared， and the results are shown in Table 6 and Fig. 2. The optimal extraction time for total flavonoids in P. edulis from Yunnan Province was 20 min， and the content of flavonoids was 0.255 7%. The optimal extraction time for total flavonoids in P. edulis from Guangdong Province was 30 min， and the content of flavonoids was 0.210 8%. The optimal extraction time for P. edulis in Guangxi Zhuang Autonomous Region was 10 min， and the total flavonoid content was 0.213 2%. The total flavonoid contents in P. edulis from different regions ranked as Yunnan Province>Guangxi Zhuang Autonomous Region>Guangdong Province.
Jun TANG et al. Effects of Ethanol Condensation Reflux Extraction and Ultrasound-assisted Extraction on the Content of Total Flavonoids of Pueraria edulis
Ultrasound-assisted extraction of total flavonoids from P. edulis
First， 3.000 00 g of P. edulis powder from different habitats in Yunnan Province， Guangdong Province and Guangxi Zhuang Autonomous Region were， respectively， weighed into conical flasks， each of which was added with an appropriate amount of ethanol to perform ultrasound-assisted extraction （Table 7）. The total flavonoid contents of P. edulis samples from different habitats were compared， and the total flavonoid contents of various samples are shown in Table 8 and Fig. 3. The optimal extraction time for total flavonoids in P. edulis from Yunnan Province was 20 min， and the content of flavonoids was 0.171 5%. The optimal extraction time for total flavonoids in P. edulis from Guangdong Province was 30 min， and the content of flavonoids was 0.155 9%. The optimal extraction time for P. edulis from Guangxi Zhuang Autonomous Region was 30 min， and the total flavonoids content was 0.204 6%. The total flavonoid contents in P. edulis from different regions were in order of Yunnan Province>Guangxi Zhuang Autonomous Region>Guangdong Province.
Conclusions and Discussion
In recent years， the quantities of components in tested medicinal materials has been determined by detecting the contents of components in the medicinal materials， and the quality of medicinal materials has been evaluated by comparing the contents of components extracted by different instruments［13］. The condensation reflux extraction process refers to the extraction of components from medicinal materials using organic solvents［14］. For example， the crude powder of P. edulis was added into a container， added with an appropriate amount of solvent， and extracted with a condensing device in a thermostatic water bath， and after refluxing and soaking for a specified time， the refluxing liquid was filtered to obtain its filtrate， which was then subjected to corresponding testing. Compared with ultrasonic extraction， the condensation reflux extraction method has the advantages of being less affected by external temperature and pressure and not affected by the gas-to-liquid ratio， stable recovery effect， direct recovery of organic liquids， no secondary pollution， and convenient operation［15］.
The contents of total flavonoids in P. edulis from different habitats in Yunnan， Guangdong， and Guangxi Zhuang Autonomous Region were determined by UV spectrophotometry. The average content of total flavonoids in P. edulis from Yunnan Province was 0.257 7%; the average content of total flavonoids in P. edulis from Guangdong Province was 0.210 8%; and the average content of total flavonoids in P. edulis from Guangxi Zhuang Autonomous Region was 0.213 2%. The contents of total flavonoids in P. edulis from different habitats ranked as Yunnan Province>Guangxi Zhuang Autonomous Region>Guangdong Province.
In summary， a more scientific and simple extraction method was obtained by comparing ultrasound-assisted extraction and condensation reflux extraction of total flavonoids from P. edulis， providing a reference method basis for extracting total flavonoids from P. edulis. Meanwhile， the determination results of total flavonoids in P. edulis from three different places in Yunnan Province， Guangdong Province， and Guangxi Zhuang Autonomous Region were compared and analyzed， providing a basis for standardized planting and reasonable development and utilization of P. edulis in the later stage， as well as a certain reference value for the establishment of quality standards for total flavonoids in P. edulis.
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