Research on Extraction and Purification Technology of Flavonoids from Quinoa (Cheuopodium quinoa) Le

Wei LYU　Huilong ZHANG　Hongmin LI　Haichuan WANG　Qiuhong LIU　Zhimin WEI

Abstract [Objectives] This study was conducted to investigate the extraction process of quiona （Cheuopodium quinoa） leaves.

[Methods] Firstly， single factor experiments were carried out with the volume fraction of ethanol， extraction time， extraction temperature and liquidtomaterial ratio as factors. Based on the single factor experiments， a response surface test was designed by the central composite design method of Design-Expert software to establish a mathematical model.

[Results] The optimal extraction conditions of quinoa flavonoids were obtained as follows： the ethanol volume fraction of 70%， the liquidtomaterial ratio at 30∶1， the extraction time of 100 min and the extraction temperature at 60 ℃.

[Conclusions] The results of the study are stable with small deviation， and can provide reference for related processes.

Key words Quinoa leaf; Flavonoid; Extraction; Response surface methodology

Quinoa （Cheuopodium quinoa）， belonging to Cheuopodium in Chenopodiaceae， has a planting history of more than 5 000 years. It is native to the Andes and is the main traditional food of the Inca indigenous people. Quinoa is recognized by the world for its high nutritional value and its various development and utilization values. National Aeronautics and Space Administration （NASA） lists quinoa as one of the ideal foods for astronauts who have long been engaged in space missions. Food and Agriculture Organization （FAO） recommends quinoa as the only monomeric plant that meets the basic nutritional needs of human body. It is thus known as "the nutritious gold"[1]. With the cultivation of quinoa， the edible value of quinoa seedlings has been more and more widely recognized. Quinoa leaves contain various nutritional ingredients such as amino acids， vitamins and microelements necessaryfor human body， and have high edible value， and certain medicinal value due to rich bioactive components such as flavonoids[2].

Flavonoids are polyphenol compounds that are widely distributed in the plant kingdom. Medical research has shown that flavonoids have high medicinal value with the effects of capturing free radicals， reducing fragility of blood vessels and improving vascular permeability， and can be used for treating coronary heart disease， angina， hypertension， cough， asthma and phlegm. They also have the effects of inhibiting platelet activating factor and promoting blood circulation and brain metabolism[3]. A diagram of response surface analysis is the threedimensional space composed of various factors corresponding to the specific response value， which intuitively reflects the influence of each factor on the response value. From the diagrams of response surface analysis obtained from experiments， the interaction between various factors can be analyzed[4]. In this study， with quinoa leaves as test materials， total flavonoids in the quinoa leaves as research objects and ethanol as an extraction solvent， based on single factor experiments， the response surface methodology was applied to optimize the extraction process of flavonoids from quinoa leaves.

Materials and Methods

Materials and Reagents

Quinoa seedlings were harvested on May 6， 2019 at the Qiema Experimental Station of the Hebei Academy of Agricultural Sciences. The collected plants had a height of 15 cm， and were subjected to freezedrying， pulverization， grinding and sieving with a 50 mesh sieve， obtaining the powder for later use.

The used reagents included rutin standard （Sinopharm Chemical Reagent Co.， Ltd.）， anhydrous ethanol and sodium hydroxide （Tianjin Damao Chemical Reagent Co.， Ltd.）， and sodium nitrite and aluminum nitrate （Shanghai Sinopharm Chemical Reagent Co.， Ltd.）， all of which were analytically pure.

Instruments and equipment

The instruments and equipment included 6202 highspeed pulverizer （Beijing Huanya Tianyuan Machinery）， UV3200PCS UVVis spectrophotometer （Shanghai Mapada Instrument Co.， Ltd.）， electronic balance （Ohaus International Trading （Shanghai） Co.， Ltd）， SHZD （III） circulating water vacuum pump， RE 522A rotary evaporator （Shanghai Yarong Biochemical Instrument Factory）， and DKS26 digital display constant temperature water bath （Suzhou Weier Experiment Equipments Co.， Ltd.）.

Drawing of standard curve

A certain amount of rutin standard （5.0 mg） was weighed accurately in a 25 ml volumetric flask， and added with 70% ethanol to dissolve it fully， followed by shaking well. The solution was then diluted to constant volume， obtaining 0.2 mg/ml rutin standard solution. Certain amounts of the rutin standard solution （0.0， 0.5， 1.0， 2.0， 3.0， 4.0 and 5.0 ml） were accurately absorbed in 25 ml test tubes， respectively， into each of which 0.3 ml of 5% NaNO2 solution was added， followed by standing for 6 min at room temperature. Then， 0.3 ml of 10% Al （NO3）3 solution was added into each of the test tube， obtaining the solution which was stood for 6 min. Each of the solutions was then added with 4.0 ml of 4% NaOH solution， diluted to constant weight with 70% ethanol， and shaken and finally stood for 15 min. With the solution in the first test tube as a blank control， the absorbance was measured by ultraviolet spectrophotometer at the wavelength of 510 nm. The obtained data were subjected to regression analysis with the absorbance as the vertical axis （Y） and the rutin concentration as the horizontal axis （X）， obtaining a linear regression equation： Y=0.007 1x-0.001 6， R2=0.999 6.

Single factor experiment design

Extraction of flavonoids with different ethanol volume fractions

Six parts of 1.00 g of quinoa seedling powder were accurately weighed and placed in 100 ml round bottom flasks， respectively. With the extraction temperature fixed at 60 ℃ and the liquidtomaterial ratio at 25∶1 （ml/g）， 25 ml of ethanol solutions with the volume fractions of 40%， 50%， 60%， 70%， 80% and 90% were added into the powder， which was refluxextracted for 100 min， obtaining the extracts which were concentrated to near dryness. The concentrates were dissolved with 70% ethanol and made up to 50 ml， and the contents of flavonoids extracted from quinoa seedlings with different ethanol volume fractions were determined finally.

Extraction of flavonoids at different temperatures

Six parts of 1.00 g of quinoa seedling powder were accurately weighed and placed in 100 ml round bottom flasks， respectively. With the liquidtomaterial ratio at 25∶1 （ml/g） and the ethanol volume fraction of 70%， the quinoa powder were refluxextracted at 30， 40， 50， 60， 70 and 80 ℃ for 100 min. The extracts were concentrated to near dryness with 70% ethanol， obtaining the concentrates which were then dissolved with 70% ethanol and made up to 50 ml. The contents of flavonoids extracted from quinoa seedlings at different temperatures were finally determined.

Extraction of flavonoids with different extraction time

Six parts of 1.00 g of quinoa seedling powder were accurately weighed and placed in 100 ml round bottom flasks， respectively. The weighed quinoa powder was then refluxextracted with 70% ethanol under the liquidtomaterial ratio of 25∶1 （ml/g） and extraction temperature of 60 ℃ for 60， 80， 100， 120， 140 and 160 min.The extracts were concentrated to near dryness with 70% ethanol， obtaining the concentrates which were then dissolved with 70% ethanol and made up to 50 ml. The contents of flavonoids extracted from quinoa seedlings for different time were finally determined.

Extraction of flavonoids under different liquidtomaterial ratios

Six parts of 1.00 g of quinoa seedling powder were accurately weighed and placed in 100 ml round bottom flasks， respectively. The weighed quinoa powder was then refluxextracted with 70% ethanol at the extraction temperature of 60 ℃ for 100 min under the liquidtomaterial ratios of 10∶1， 15∶1， 20∶1， 25∶1， 30∶1and 35∶1 （ml/g）， respectively. The extracts were concentrated to near dryness with 70% ethanol， obtaining the concentrates which were then dissolved with 70% ethanol and made up to 50 ml. The contents of flavonoids extracted from quinoa seedlings for different time were finally determined.

Results and Analysis

Effect of ethanol volume fraction on flavonoid extraction

It can be seen from Fig. 1 that as the volume fraction of ethanol increased， the amount of flavonoids extracted from quinoa leaves gradually increased. When the volume fraction of ethanol reached 70%， the extraction amount of flavonoids was the highest. When the volume fraction exceeded 70%， the amount of flavonoids started to decrease. Therefore， the optimal ethanol volume fraction was 70%.

Effect of extraction temperature on flavonoid extraction

It can be seen from Fig. 2 that under the ethanol volume fraction of 70%， the liquidtomaterial ratio of 25∶1 and the extraction time of 60 min， the extraction amount of flavonoids increased with the increase of the extraction temperature. When the extraction temperature was 60 ℃， the extraction amount of flavonoids was the highest. Over 60 ℃， the extraction amount decreased with the increase of temperature. Therefore， the extraction temperature at around 60 ℃ can be appropriate.

Effect of extraction time on flavonoid extraction

It can be seen from Fig. 3 that with the ethanol volume fraction of 70%， the liquidtomaterial ratio at 25∶1 and the extraction temperature at 60 ℃， the extraction amount of flavonoids was the largest when extracted for 100 min. After 100 min， the extraction amount decreased with time instead. Therefore， it was more appropriate to choose an extraction time of about 100 min.

Effect of liquidtomaterial on flavonoid extraction

It can be seen from Fig. 4 that under the ethanol volume fraction of 70%， the extraction temperature of 60 ℃ and the extraction time of 100 min， the flavonoid extraction amount increased with the increase of the liquidtomaterial ratio， and reached the peak value at the liquidtoliquid ratio of 30∶1， over which the flavonoid extraction amount of quinoa leaves decreased instead. Therefore， it was more suitable to choose a liquidtoliquid ratio of about 30∶1.

Optimization of Extraction of Flavonoids from Quinoa Leaves by Response Surface Methodology and Its Results

Test design

In this study， the data were processed by Design-Exper10 software. According to the results of the single factor experiments， the four factors including ethanol volume fraction， extraction temperature， extraction time and liquidtomaterial ratio were selected as the response surface optimization combination. The designed factor levels are shown in Table 1.

Model establishment and significance test

According to the response surface methodology， such four test factors as the ethanol volume fraction， extraction temperature， extraction time and liquidtomaterial ratio were analyzed. The design and results are shown in Table 2.

The experimental results were analyzed by the response surface analysis method， and the regression equation with the flavonoid extraction amount as the response value was obtained as follows：

Extraction amount of flavonoids=+11.951 81+0.915 00*A+0.511 667*B+0.186 67*C+0.868 57*D+0.040 00*AB-0.220 00*AC+0.430 00*AD+0.255 00*BC-0.880 00*BD+0.095 00*CD-1.846 14*A2-1.996 14*B2-1.473 64*C2-2.267 27*D2It can be seen from Table 3 that with the flavonoid extraction amount as the response value， the model had P=0.000 4<0.005， indicating that the model or each factor was significant. Meanwhile， the lack of fit showed P=0.217 6>0.100 0， which meant that the orthogonal test results can be well fitted to the mathematical model， that is， the mathematical model can be used to infer the test results. The effects of various factors on the extraction amount of flavonoids from low to high ranked as the extraction time<extraction temperature<ethanol volume<liquidtomaterial ratio， in which the ethanol volume and liquidtomaterial ratio were extremely significant.

The response surface diagrams were the surface diagrams of the threedimensional space formed by the response values to each test factor A， B， C， and D. The optimal parameters and the inter action between the parameters can be visually seen from the response surface diagrams. The response surface analysis results of different factors according to the regression equation are shown in Fig. 1. From Fig. 1， the effects of interaction of various factors on the yield of total flavonoids in quinoa leaves can be seen more intuitively. If the curve is steeper， it indicates that the effect of this factor on the total flavonoid extraction is greater， which will be reflected on the corresponding change in the response value. The optimal flavonoid extraction parameters were determined by software analysis as follows： the ethanol volume fraction of 70%， the liquidtomaterial ratio at 30∶1， the extraction time of 100 min， and the extraction temperature at 60 ℃， with which the flavonoid extraction amount reached the maximum value of 12.41mg/g. The determination results were stable with large deviation， which proved that the results were reasonable and reliable.

Conclusions

In this study， the effects of ethanol volume fraction， extraction time， extraction temperature and liquidtomaterial ratio on the extraction of flavonoids from quinoa leaves were investigated by single factor experiments. Based on this， a response surface test was designed according to the central composite design method of Design-Expert software. Based on the response surface test， a mathematical model was established and the optimal extraction conditions were obtained. Combined with the mathematical model obtained by the response surface analysis， better process conditions were predicted and the verification experiment was carried out. Finally， the optimal extraction conditions of quinoa flavonoids were obtained as follows： the ethanol volume fraction of 70%， the liquidtomaterial ratio at 30∶1， the extraction time of 100 min and the extraction temperature at 60 ℃.

References

[1] WEI ZM， LI SG， XIA XY， et al. Discussion on the characteristics and development strategies of Cheuopodium quinoa[J]. Journal of Hebei Agricultural Sciences， 2016， 20（5）： 14-17. （in Chinese）

[2] WEI ZM， SONG SJ， ZHAO Y， et al. Introduction test of five quinoa varieties in central and Southern Hebei[J]. Journal of Hebei Agricultural Sciences， 2018， 22（5）： 1-3， 7. （in Chinese）

[3] WANG JN， HUANG YH， MU ZM， et al. Research progress on flavonoid of the plant secondary metabolites[J]. Science of Sericulture， 2007， 33（3）： 499-505. （in Chinese）

[4] HUANG YS， XIE MY， ZHANG ZW， et al. Determination for total flavonoids in horthorn[J]. Food Science， 2006，30 （5）： 473-477. （in Chinese）

[5] LI JX， CHEN X， DENG JQ， et al. Extraction and antioxidant activity in vitro of okra flavonoids[J]. Food Science， 2014， 35（10）： 121-125. （in Chinese）

[6] qIN WW， LIU Y， LI MM， et al. Optimization of extracting technology for total flavonoids in tea tree with response surface methodology[J]. Food Research and Development， 2018（2）： 67-71. （in Chinese）

[7] WANG LM， MA N， LI S， et al. The nutritional value of Cheuopodium quinoa and its application prospects[J]. Science and Technology of Food Industry， 2014， 35（1）： 381-384. （in Chinese）

[8] SHI ZX， ZHU YY， YANG XS， et al. Development of nearinfrared reflectance spectroscopy （NIRS） predication model for the quality components of flour in quinoa[J]. Cereals & Oils， 2017， 30（12）： 55- 57. （in Chinese）

Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU