Research on the technology of refining flavonoidfrom lotus leaf by

macroporous adsorption resin

Abstract: Flavonoids were extracted from lotus leaves， and the HPD-100 was choosen by
comparing absorption effects of 11 kinds of macroporous resin． A new process for the
separation and purification of total flavonoids from lotus leaves was determined by studing the
effects of temperature and acidity on the adsorption capacity of HPD-100 and the desorption of
different volume fraction of ethanol on the resin． The results showed that HPD-100 had the
greatest absorption ability at pH 3° and 25 ℃ ． When the volume concentration of alcohol was
70 % ， the desorption was most effective and the purity of flavonoids could reach up to 55．
28 % ．

Key words: lotus leaf ; flavonoid; macroporous resin; absorption; desorption

1 Materials and methods

1. 1 Raw materials and reagents

1. 1. 1 raw material

Fresh lotus leaves were collected from Xihe Town, Xiaogan City.

1. 1. 2 main instruments

UV-2401PC UV-visible spectrophotometer, Shimadzu, Japan; constant temperature shaker,

Shanghai Shiping Experimental Equipment Co., Ltd.; adsorption column φ (300 mm × 30 mm),
Shanghai Dafeng Glass Instrument Factory.

1. 1. 3 main reagent adsorption resins (X-5, NCA-9, HPD-100, HPD-400, HPD-500, HPD-600, D-
3520, S-8, AB-8, 09A7, S-8), all Provided by the laboratory of the Institute of Biological Sciences,
Hubei Institute of Technology; other reagents are of analytical grade.

1. 2 Test methods

1. 2. 1 Preparation of crude extract of lotus leaf flavonoids

Wash and drain the fresh lotus leaves, dry them in a constant temperature drying oven at 50°C,
crush them through a 420 μm sieve, and seal them for later use. Accurately weigh 8 portions of
lotus leaf powder, each portion is 10 g, and put it into a 500 mL flat-bottomed flask. Add 60%
ethanol by volume according to the liquid-to-material ratio 1:40 (g/mL), and put it into a 70 ℃
digital display constant temperature water bath. Extraction in the pot 1. 5 h, filter under
reduced pressure, combine the extracts and centrifuge, and the supernatant is the crude lotus
leaf extract.
1. 2. 2 Analysis methods of lotus leaf flavonoids

Since rutin has a maximum absorption peak at a wavelength of 415 nm, and the absorbance of
the solution is positive to its concentration, quantitative analysis can be performed based on the
absorbance value of the solution. Use the absorbance A of the rutin (standard product) solution
at a wavelength of 415 nm as the ordinate and the concentration C of the rutin standard
solution as the abscissa to obtain a standard curve. A = 0. 027 2C + 0． 008 2( R2 = 0. 998 3),
the linear range is good.

1. 2. 3 Pretreatment of resin

Take the spare macroporous resin, soak it in anhydrous ethanol for 24 hours, rinse it repeatedly
with anhydrous ethanol until the eluate is no longer white turbid after adding an appropriate
amount of water, then wash it with distilled water until it is free of alcohol, filter and dry it for
later use.

1. 2. 4 Calculation of adsorption rate

Precisely measure 1 mL of each solution before and after the resin adsorbs lotus leaf flavonoids,
place it in a 25 mL volumetric flask, add an ethanol solution with a volume fraction of 70% to 10
mL, then add 3 mL of the color developer AlCl3 solution, and finally use a volume fraction of
70%. % ethanol solution to constant volume, shake well and let it stand for 15 minutes to
develop color and measure the absorbance value. Calculate the adsorption rate of lotus leaf
flavonoids according to formula (1).

Adsorption rate = A1 - A2 A1 × 100 % (1) In the formula: A1 is the absorbance of the solution
before adsorption; A2 is the absorbance of the solution after adsorption.

1. 2. 5 Calculation of resin elution rate

After using different eluents to elute the resin that has adsorbed a certain amount of lotus leaf
flavonoids, accurately measure 1 mL of the eluted solution and place it in a 25 mL volumetric
flask. Add an ethanol solution with a volume fraction of 70% to 10 mL, and then add significant
amounts. 3 mL of color agent AlCl3 solution was finally diluted to volume with 70% ethanol
solution, shaken well and allowed to stand, and the absorbance value was measured after 15
min of color development. Calculate the elution rate according to formula (2). Elution rate = A' 1
A' 2 × 100 % (2) In the formula: A' 1 is the absorbance of the solution after elution; A' 2 is the
absorbance of the lotus leaf flavonoids crude extract.

1. 2. 6. Determination of purity of lotus leaf flavonoids. The purity of lotus leaf flavonoids was
determined according to the method of Zhou Taoying et al. [7].

2 Results and analysis

2. 1. Static adsorption experiment screening resin

Take 11 kinds of treated resins and shake them at a constant temperature for 24 hours to make
their adsorption saturated. Press 1． 2. 4 method was used to calculate the adsorption rate,
and the results are shown in Table 1.

Table 1 Static adsorption characteristics of lotus leaf flavonoids by different types of resins

Resin type adsorption rate/% Resin type adsorption rate/% X-5 94. 90 HPD-500 95. 13 NCA-9 94.
46 HPD-400 95. 34 HPD-600 95. 18 09A7 95. 04 D-3520 94. 90 HPD-100 95. 91 S-8 98. 25 03FC
95. 62 AB-8 94. 46

It can be seen from Table 1 that: S-8 and HPD-100 have higher adsorption rates for lotus leaf
flavonoids and strong adsorption capacity for lotus leaf flavonoids. Among them, the adsorption
rate of S-8 reaches 98. 25%, the best effect. However, in actual production, since S-8 is difficult
to elute, HPD-100 macroporous resin was selected as the adsorbent to systematically study its
adsorption and desorption of lotus leaf flavonoids.

2. 2 Effect of pH on the adsorption performance of HPD-100 Accurately weigh 9 parts of the

spare HPD-100 macroporous adsorption resin, each part is 1. 0 g, placed in a 250 mL Erlenmeyer
flask. Take 9 portions of the initial extract of lotus leaf flavonoids, each 100 mL, and adjust the
pH with citric acid-sodium hydrogen phosphate buffer. Then take 100 mL of the above solution
respectively, put it into an Erlenmeyer flask containing resin, and shake at a constant
temperature for 24 hours to reach adsorption equilibrium and make the adsorption saturated.
Press 1. 2. 4 method was used to calculate the adsorption rate, and the results are shown in
Table 2.

Table 2 Effect of pH on the adsorption performance of HPD-100 pH adsorption rate/% pH

adsorption rate/% 2. 0 94. 32 4. 5 94. 57 2. 5 94. 98 5. 0 94. 49 3. 0 96. 76 5. 5 94. 17 3. 5 95. 32
6. 0 93. 86 4. 0 94. 84

It can be seen from Table 2: When the pH is 3. At 0, HPD-100 macroporous resin has the highest
adsorption rate for lotus leaf flavonoids.

2. 3 Effect of temperature on adsorption performance of HPD-100

Weigh accurately respectively 1. 0 g of 7 parts of spare HPD-100 macroporous adsorption resin,

placed in a 250 mL Erlenmeyer flask, 100 mL of each, pH 3. 0 lotus leaf crude extract was
adsorbed at different temperatures with constant temperature shaking for 30 minutes. Press
1． 2. 4 method to calculate the adsorption rate, the results are shown in Table 3.

Table 3 Effect of temperature on the adsorption performance of HPD-100 Temperature/ ℃

Adsorption rate/% Temperature/℃ Adsorption rate/% 15 91． 44 35 43． 36 20 92. 77 40 43.
21 25 94． 39 45 42. 92 30 91. 15

It can be seen from Table 3 that when the temperature exceeds 25 °C, the adsorption rate
shows an obvious downward trend as the temperature increases. It shows that the adsorption
of HPD-100 macroporous resin is an exothermic process. As the temperature increases, the
adsorption capacity decreases. Therefore, the adsorption effect is best at 25 °C.

2. 4 Effect of ethanol concentration on desorption under static conditions

Take 7 portions of HPD-100 macroporous resin that has been saturated with lotus leaf
flavonoids, 1. 0 g, and add them to 250 mL Erlenmeyer flasks respectively, and prepare ethanol
solutions with different volume fractions at the same time. Measure 100 mL of the above
ethanol solutions and add them to the Erlenmeyer flask containing the resin, and place them in
a shaker at 25°C for constant temperature oscillation. 24 h to allow complete elution. Press 1．
2. 5 method to calculate the elution rate, the results are shown in Table 4.

Table 4 Elution effect of different ethanol aqueous solutions on lotus leaf flavonoids /% ethanol
volume fraction elution rate ethanol volume fraction elution rate 30 46． 26 70 97. 33 40 86.
67 80 98. 00 50 93. 06 90 98. 67 60 93. 33

It can be seen from Table 4 that the eluent with an ethanol volume fraction of 70%, 80%, and
90% has a higher elution rate, and the analysis of lotus leaf flavonoids is very strong, but a high
concentration of ethanol can remove the flavonoids adsorbed on the resin column. Some
impurities other than lotus leaf flavonoids are eluted out, affecting the purity of lotus leaf
flavonoids. Therefore, this study used 70% ethanol as the elution solution.

2. 5 Desorption of lotus leaf flavonoids under dynamic conditions

The HPD-100 macroporous resin was packed into the column by wet method, and after
adsorbing a certain amount of lotus leaf flavonoids, the glass column was first eluted with 3
times the column volume of distilled water, and then eluted with a volume fraction of 70%
ethanol solution, followed by 1. Elute the lotus leaf flavonoids on the resin column at a speed of
0 mL/min, use a small test tube to collect the effluent, and measure the absorbance of the
effluent at 415 nm every 6 minutes until the content of lotus leaf flavonoids in the effluent is
quite small, and obtain The elution curve is shown in Figure 1.

Figure 1 Desorption curve of lotus leaf flavonoids

It can be seen from Figure 1 that the ethanol solution with a volume fraction of 70% can better
elute the lotus leaf flavonoids adsorbed by the resin HPD-100. In the 9th tube of eluate, the
elution reached the peak (a total of 135 mL), 95% of the pigments were eluted, indicating that
the eluted lotus leaf flavonoids were relatively concentrated. The test found that cross-elution
with distilled water and ethanol with a volume fraction of 70% in the later stage of elution can
significantly improve the tailing phenomenon, which also has certain significance for industrial
production.
 2. 6 Comparison of lotus leaf flavonoids before and after purification

Lotus leaves are rich in lotus leaf flavonoids. After extraction with 70% ethanol, the purity of the
flavonoids in the crude extract is 7. 53%; after purification by HPD-100 resin, the content of lotus
leaf flavonoids in the product is greatly increased, and the purity reaches 55. 28%, 7. higher than
before purification. 34 times, and its properties are stable and the overall quality is significantly
improved, indicating that the macroporous adsorption resin HPD-100 is suitable for the
purification of lotus leaf flavonoids.

3 Summary

This article tested 11 kinds of macroporous resins, screened out the HPD-100 macroporous resin
with the best adsorption performance, and discussed the effects of temperature, acidity and
ethanol volume fraction on its performance. Finally, it was concluded that at a temperature of
25°C and a pH of 3. Under the condition of 0. ethanol volume fraction 70%, the macroporous
resin HPD-100 has the best adsorption and desorption effect.

Oil mass ratio 3:2 (mixed), 15% whole egg liquid, and about 40% water (need to increase or
decrease according to the softness and hardness of the dough); the black rice tough biscuits
made under these conditions are gray-black, uniform in color, and have a smooth surface.
Smooth without cracks, the internal structure has even pores, crisp texture, moderate
sweetness, and a certain black rice aroma.

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(Continued from page 69) Studies have shown that lotus leaf flavonoids can scavenge most
oxygen free radicals, increase the activity of SOD (superoxide dismutase), and reduce
peroxidized lipid malondialdehyde (MDA) and oxidized low-density lipids. The production of
protein (OX LDL) [8] shows that the purified lotus leaf flavonoids will become a new type of
natural antioxidant and play a wide range of roles in food, medicine, cosmetics and other
industries. This study provides a certain theoretical reference for the industrial production of
lotus leaf flavonoids.

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广泛的作用。本研究为荷叶黄酮的工业化生产提 供了一定的理论参考。

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