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European Journal of Applied Sciences – Vol. 12, No. 6
Publication Date: December 25, 2024
DOI:10.14738/aivp.126.17865.
Ma, W., & Wang, C. (2024). Preparation of High-Purity Rutin from Sophora japonica. European Journal of Applied Sciences, Vol -
12(6). 135-147.
Services for Science and Education – United Kingdom
Preparation of High-Purity Rutin from Sophora japonica
Wenjing Ma
School of Chemistry and Chemical Engineering,
Nanjing University of Science and Technology, Nanjing 210094, China
Chuanjin Wang
School of Chemistry and Chemical Engineering,
Nanjing University of Science and Technology, Nanjing 210094, China
ABSTRACT
High-Purity Rutin (HPR) was extracted from Sophora japonica by ultrasonication,
decolorized using activated carbon, separated by polyamide chromatography, and
recrystallized. An orthogonal design of experiment was used to determine the
optimal ultrasonic extraction time, number of ultrasonic extractions, and
ultrasonic extraction temperature to maximize the extraction yield of rutin. The
optimal conditions were determined as follows: 2000 mL of CH3OH were used to
extract 20 g of the dried Sophora japonica flower bud powder for 30 minutes and
the extraction was repeated 2 times under ultrasonic conditions (ultrasound
frequency: 40 KHZ; water temperature: 40 °C). The decolorization of the CH3OH
extract of Sophora japonica using active carbon was studied. The optimal
processes are as follows: The filtrate of CH3OH extract was concentrated to 200 mL
by rotary evaporation; 7.2 g of activated carbon powder was added; and the
mixture was refluxed in an 80 °C water bath for 0.5 hours (twice). The polyamide
chromatography separation conditions for crude rutin were investigated. The
influence of particle size and amount of polyaminde used on extraction yield of
rutin was studied. It was found that best separation results were obtained when
50 g of polyamide with particle size of 0.170~0.210 mm was used to purify 3 g of
crude rutin. The purity of the HPR obtained was 99.8%, the extraction yield of HPR
was 18.6%, and the extraction rate of HPR was 70.8%. The structure of the final
product was identified by elemental analysis, IR, HPLC and 1H NMR. It was
experimentally demonstrated that the proposed process was a safe, mild, low-cost
and waste-free procedure.
Keywords: High-Purity of Rutin (HPR), Ultrasound extraction, Activated carbon
decolorization, Polyamide chromatography separation.
INTRODUCTION
Sophora japonica is the dried flower bud of Sophora japonica L., which is widely cultivated in
China for its favorable medicinal value. It has been reported that Sophora japonica, which has
many active substances, has various biological activities, including antibacterial capacity,
vasodilation, and antioxidation capacity, etc. The dried flower bud of Sophora japonica L. has
been widely applied in health and food products [1]. There is a large amount rutin (Fig. 1) in
the flower buds of Sophora japonica L. Rutin, a flavonoid compound, has many important
physiological activities in the treatment of inflammation, cerebrovascular diseases,
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cardiovascular disorders, cancer, etc., attributed to its anti-inflammatory [2], antiviral [3],
remarkable antioxidation [4], and antifree radical properties [5].
Fig. 1: Chemical structure of rutin
The extraction methods of rutin mainly include alkali extraction and acid precipitation [6, 7],
hot extraction and cold precipitation [8], and organic solvent extraction [9~12]. However,
these methods have some drawbacks, such as thermal degradation of rutin due to prolonged
heat at high temperature, potential effects on human health and the environment through the
use of large amounts of harmful organic solvents. In order to overcome these problems, the
use of environmentally friendly technologies has been highly valued by researchers, including
microwave-assisted extraction [13], ultrasound-assisted extraction [14], enzyme-assisted
extraction [15], supercritical fluid extraction [16], etc. Rutin purification methods mainly
include alkali solubilized acid precipitation method [17], cold water washing method [18],
dextran gel method [19], macroporous adsorption resin method [20], polyamide
chromatography method [21, 22], macroreticular resin method [23], high-pressure liquid
phase preparation method [24], two-step chromatographic process (MCI GEL® CHP20P and
Sephadex® LH-20 columns) method [25], etc. Among them, both cold water washing method
and alkali solubilized acid precipitation method have the problems of low purification rate
and incomplete impurity removal. The latter few methods have the problems of cumbersome
operations, low yields, and high costs. These shortcomings limit the wide application of rutin
in food, cosmetics, and medicine areas.
Sophora japonica L., which is also widely grown in China, and has become an important source
of rutin. The current upper limit purity of rutin available on the market is about 98%. To meet
higher needs arising in production and scientific research, as well as to control the quality of
rutin products, we have successfully prepared HPR using activated carbon decolorization
method polyamide chromatography method, and recrystallization method. The purity of HPR
was determined using the HPLC method. The structure of rutin was also characterized by IR,
elemental analysis, and 1H NMR. Compared with the extraction and purification processes
reported in the literature, the method for preparing HPR described in this paper has the
advantages of simple process, non-toxic and pollution-free solvents, safety, low cost, and
reusable solvents and polyamides, making it suitable for industrial production.
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Ma, W., & Wang, C. (2024). Preparation of High-Purity Rutin from Sophora japonica. European Journal of Applied Sciences, Vol - 12(6). 135-147.
URL: http://dx.doi.org/10.14738/aivp.126.17865
MATERIALS AND METHODS
Materials and Instruments
Chromatographic methanol and dimethyl sulfoxide (DMSO) were obtained from Sigma
Chemical Co., Ltd. (Shanghai, China). Polyamide (particle size: 0.140 mm-0.575 mm) was
obtained from Jiangsu Changfeng Chemical Co., Ltd. (Changzhou, China). Powdered activated
carbon (particle size: 1.0 μm-150 μm) was obtained from Shanghai Bilang Environmental
Protection Technology Co., Ltd. (Shanghai, China). Sophora japonica was obtained from
Chuzhou, Anhui Province, China.
A Waters e2695 instrument and a 2498UV/Vis detector were obtained from Waters
Corporation of Shanghai (Shanghai, China). A Bruker (500 MHz) NMR and Vector 22 type
infrared spectrometer were obtained from Brooke Company (Karlsruhe, Germany). A CHN-O- Rapid elemental analyzer was obtained from Heraeus Analytical Instruments (Hanau,
Germany). A chromatography column was obtained from Beijing Synthware Glass Instrument
Co., Ltd. (Beijing, China).
Content Determination and Extraction Rate Calculation of Rutin From Sophora japonica
Based on previous research methods [26], 55 mg of rutin standard was weighed and placed in
a 250 mL volumetric bottle; 60% ethanol was added to the scale; and rutin standard solution
was obtained at a concentration of approximately 0.22 mg/mL. A rutin standard solution of
0.22 mg/mL was placed in a 10 mL volumetric flask (0.0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL). Next,
0.5 mL of 5% NaNO2 solution was added and placed for 6 min; 0.5 mL of 10% Al (NO3)3
solution was added and calmly placed for 6 min. Next, added 4 mL of 4% NaOH solution was
added, and the distilled water was diluted to 10 mL, shaken evenly, and then calmly placed for
15 min. Rutin was not added to the blank control group. The absorbance value was measured
at 500 nm. The standard curve was plotted using the standard solution absorbance as the
ordinate Y and the mass concentration of rutin in the standard solution as the abscissa X. The
regressive equation of the curve was Y = 9.5182X-0.0072, R2 = 0.9973. The results showed
that its mass concentration and absorbance were quite linear in the range from 22 μg/mL to
110 μg/mL.
Based on a previous research method [27], Sophora japonica powder (1 g) and ether (120 mL)
were added to a Suo Shi extractor, heated, and refluxed to obtain a colorless extracted liquid.
The extracted liquid of ether was discarded after being cooled. Methanol (90 mL) was added
to the Suo Shi extractor once again, heated, refluxed to colorless extracted liquid, and
transferred to a 100 mL volumetric flask. Methanol was added to the scale, and methanol
extract solution was obtained. Five milliliters of methanol extract solutions were placed in a
50 mL volumetric flask, and distilled water was added to the scale. Two milliliters were
removed from the previously prepared solution and placed in a 10 mL volumetric flask, and
the absorbance was measured according to the standard curve method. The mass fraction (c)
of rutin in Sophora japonica was calculated as 25.4% based on the concentration-absorbance
of the standard sample.
In the subsequent ultrasonic extraction and decolorization process, each extract or
decolorization liquid was transferred to a 100 mL volumetric bottle, methanol was added to
the scale, and a methanol extract solution was obtained. Five milliliters of methanol extract
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solutions were placed in a 50 mL volumetric flask, and distilled water was added to the scale
and used as the test solution. In the polyamide column layer separation process study, 100 mL
of eluate was taken each time. Two milliliters of eluate were transferred to a 50 mL
volumetric flask, and 50% ethanol was added to the scale and used as the test solution.
Two milliliters were removed from each test solution and placed in a 10 mL volumetric flask,
and the absorbance was measured according to the standard curve method. The mass fraction
(c') of rutin in the test solution was calculated according to the standard linear equation of
concentration-absorbance of the standard sample, and the extraction rate of rutin was
calculated according to the following formula:
Extraction rate of rutin (%) = (c'/c) × 100%
Polyamide Pretreatment and Packing
Polyamide (140 g) mixed with an appropriate amount of anhydrous ethanol was added to a
1000 mL round bottom flask, heated under reflux in a constant temperature water bath (90
°C), filtered and washed with distilled water more than three times after 2 h. The filter cake
was transferred to a 1000 mL beaker filled with distilled water (500 mL), stirred well, and
then soaked overnight. The next day, the polyamide powders that removed some bubbles in
the beaker were poured into the chromatography column filled with absorbent cotton at the
bottom of the column and naturally settled. Excess water is released from the
chromatography column and then stopped by closing the piston at the bottom of the column
until the water surface is slightly higher than the surface of the polyamide powder. The
chromatography column was installed and used for the separation and purification of rutin.
Extraction and Isolation
The dried flower bud powders of Sophora japonica (20.0 g) were loaded into a flask (5000
mL) and extracted with CH3OH (2000 mL × 2) under ultrasonic conditions (ultrasound
powder: 40 KHZ; water temperature: 40 °C). Each extraction time was 30 min. The solutions
were filtered and merged. The filtrate was concentrated to 200 mL by rotary evaporation; 7.2
g of activated carbon powder was added; and the mixture was refluxed in an 80 °C water bath
for 0.5 h (two times), filtered and merged to obtain a brown-yellow liquid. As previously
mentioned, the brown‒yellow liquid was concentrated and dried under vacuum to obtain a
crude extract of rutin (6.2 g). The crude rutin (3.0 g) was separated on a with polyamide
chromatographic column using elution solvent with H2O-C2H5OH (V/V, 1:1) to afford 30
fractions (F1-F30, 100 mL/fraction). Fractions F6–F20 were merged, concentrated, dried in
vacuum and further recrystallized from 400 mL H2O to obtain HPR (1.8 g) with a yield of
approximately 18.6% from Sophora japonica.
A High-performance liquid chromatography (HPLC) system of (Waters) was selected to
determine the purity of rutin. The analytical chromatography conditions are listed as follows:
rutin (5 mg) was dissolved in methanol in a 100 mL volumetric flask. After filtration with a
0.22 μm filter, purity analysis of rutin was carried out with a C18 column (4.6 mm i.d ×250
mm, particle size of 5 μm). The mobile phases were H2O and CH3OH (70:30, V/V), the flow
rate was 1.0 mL/min, and the sample volume was 10 μL. The temperature of the column was
maintained at 30 °C, and the UV detection wavelength was set to 254 nm. Identification of
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Ma, W., & Wang, C. (2024). Preparation of High-Purity Rutin from Sophora japonica. European Journal of Applied Sciences, Vol - 12(6). 135-147.
URL: http://dx.doi.org/10.14738/aivp.126.17865
rutin was performed by IR, elemental analysis and 1H NMR spectroscopy. IR was performed
using a Vevtor 22 infrared spectrometer. The 1H NMR experiment was carried out using a
Bruker DR×500 NMR spectrometer. Elemental analysis was performed by a CHN-O-Rapid
elemental analyzer.
RESULTS AND DISCUSSION
Identification of rutin Isolated from Sophora japonica
The structure of the final product separated was identified by IR, elemental analysis, HPLC
and NMR spectroscopy as follows: Purity (HPLC): 99.8% (Fig. 2); appearance: light yellow
powder; mp: 195-196 °C. In the infrared spectrum (Fig. 3), the strong and wide absorption
peak at 3427.3 cm-1 resulted from the -OH stretching vibration.
Fig. 2: HPLC spectrogram of rutin isolated methanol solution
Fig. 3: Infrared spectrum of rutin
The weak absorption peak of 2923.7 cm-1 resulted from CH3- and -CH2- stretching vibrations.
The strong and wide absorption peak of 1652.8 cm-1 indicated the presence of a conjugated
carbonyl group. The peaks at 1604.6 cm-1, 1506.2 cm-1, 937.3 cm-1, 877.5 cm-1 and 811.9 cm-1
were caused by aromatic ring skeletons and aromatic ring substitutions. The absorption peak
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of 1361.5 cm-1 showed the presence of methyl group in this compound. The absorption peak
of 1456.1 cm-1 was caused by C-H bending vibration of methylene. 1297.9 cm-1, 1243.9 cm-1,
1205.3 cm-1, 1164.8 cm-1 and 1062.3 cm-1 were attributed to various hydroxyl groups in
flavonoid glycosides.
The results of element analysis according to C27H30O16 were the measured values (calculated
values)/%: C, 53.10 (53.07); H, 4.88 (4.91). The structure of the isolated compound and rutin
are identical.
The chemical shifts of different types of protons are shown in the 1H NMR spectra (DMSO, 500
MHz) (Fig. 4). Apart from the sharp peak of δ 12.58 (1H, S, 5-OH), the remaining three peaks
of 10.83 (1H, S, 7-OH), 9.67 (1H, S, 4′-OH), and 9.18 (1H, S, 3′-OH) hydroxyl protons on the
aromatic ring are wide and flat, and all four hydroxyl protons appear in the low field (δ>9.0).
The five protons on the aromatic ring are 7.52 (1H, S, 6′-H), 7.51 (1H, S, 2′-H), 6.83 (1H, S, 5′-
H), 6.37 (1H, S, H-8), and 6.17 (1H, S, H-6). These protons appear in the field (9.0>δ>6.0).
The six –OH proton glycosides are 5.28 (1H, S, 2′′-OH), 5.11 (1H, S, 3′′-OH), 5.08 (1H, S, 4′′-
OH), 5.07 (1H, S, 2′′′-OH), 4.53 (1H, S, 3′′′-OH), and 4.35 (1H, S, 4′′′-OH). The three hydroxyl
glucose group appear in the lower field because the glucose groups is near the mother nucleus
of the flavonoid. The chemical shifts of two hydrogen atoms on the first carbon of
glucosylrhamnose are 5.31 (1H, S, H-1′) and 4.39 (1H, S, H-1′′′). The peaks of 0.97, and 0.96
are typical proton-induced chemical shift of rhamnose methyl. The chemical shifts of ten
hydrogen atoms on the glucose and pyran ring of rhamnose are located between 3.0 and 4.0,
forming overlapping multiple peaks. All data were identical to those of rutin [28].
Fig. 4:
1H NMR spectrogram of rutin
Factors Influencing the Separation and Purification of Rutin
Factors Influencing Ultrasonic Extraction:
The ultrasonic extraction process has shown many advantages, including a faster extraction
rate, lower energy input, lower solvent consumption, and lower temperature in the extraction
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Ma, W., & Wang, C. (2024). Preparation of High-Purity Rutin from Sophora japonica. European Journal of Applied Sciences, Vol - 12(6). 135-147.
URL: http://dx.doi.org/10.14738/aivp.126.17865
of natural products [29]. This extraction technology is widely utilized in industry. Ultrasound
waves cause cell destruction and ultrasonic jets and reduce the particle size, improving the
contact area of the solid-liquid phase and producing more active substances in the extraction
solvent. The increasing extraction yield of natural products is devoted to acoustic cavitation
phenomena under ultrasound conditions. The expansion cycle could produce numerous
microbubbles or cavities in the liquid phase at sufficient ultrasound intensity. These formed
bubbles could absorb the energy from ultrasonic waves and increase during the expansion
cycles. The pressure and temperature increased due to compression, making the bubbles
break up, which produces a shock wave that traversed the solvent, improving the mass
transfer effect within the ultrasound extraction system [30].
Rutin is an alcohol-soluble component that is extracted with methanol as the solvent. A three- factor and three-level orthogonal experiment was established with ultrasonic extraction times
(A) of 20 min, 30 min and 40 min, ultrasonic extraction times (B) of 1 time, 2 times and 3
times, and ultrasonic extraction temperatures (C) of 20 °C, 40 °C and 60 °C (Table 1). The
ultrasonic extraction technology of rutin from Sophora japonica was investigated. The
extraction rate of rutin was used as a marker. The dried flower bud powders of Sophora
japonica (1.0 g) were loaded into a flask (50 mL) and extracted with CH3OH (10 mL) under
ultrasonic conditions (ultrasound powder: 40 KHZ) every time. The experimental results are
shown in Table 2.
Table 1: Factors and levels of orthogonal experiments for ultrasonic extraction
Levels Factors
Time/A(min) Times/B(Times) Temperature/C (°C)
1 20 1 20
2 30 2 40
3 40 3 60
Table 2: Results of ultrasonic extraction orthogonal experiment
No
Factors and levels The extraction rate of rutin (%)
A B C D(Blank)
1 1 1 1 1 64.5
2 1 2 2 2 72.4
3 1 3 3 3 77.1
4 2 1 2 3 84.6
5 2 2 3 1 85.1
6 2 3 1 2 81.2
7 3 1 3 2 79.3
8 3 2 1 3 86.2
9 3 3 2 1 85.7
I 214.0 228.4 231.9 235.3
II 250.9 243.7 242.7 232.9
III 251.2 244.0 241.5 247.9
R 37.2 15.6 10.8 15.0
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As shown in Table 2, the A3B3C2 method (ultrasonic extraction time of 40 minutes, 3 ultrasonic
extraction times, and ultrasonic extraction temperature of 40 °C) was the optimal ultrasonic
extraction method for rutin from Sophora japonica.
With the extended ultrasound time, the extraction rate of rutin gradually increased. When the
time exceeded 30 minutes, the effect of time on the extraction rate of rutin was not obvious.
As shown in Table 2, the rutin extraction rate was nearly unchanged by ultrasound time at 30
minutes and 40 minutes. The concentration gradient of rutin inside and outside the plant cells
tended to 0 in a short time, and the rutin extraction rate exhibited minimal change with
increasing extraction time. Under the conditions of constant extraction time and extraction
temperature, the ultrasonic extraction time had a significant effect on the extraction rate of
rutin. If the number of extractions is too small, rutin leaching will be incomplete. With an
increase in extraction times, rutin leaching becomes relatively more complete; however, the
production cost is increased. Comprehensive consideration shows that the 2 extraction times
yields better results. The increase in ultrasonic extraction temperature can improve the
solubility and diffusion coefficient of rutin in the solvent and enhance the extraction rate.
However, when the temperature is too high, ultrasonic cavitation may be weakened, which is
not conducive to the extraction of rutin [31]. Overall, an ultrasound temperature of 40 °C was
selected for subsequent experiments. The method of A2B2C2 (ultrasonic extraction time: 30
minutes; number of ultrasonic extractions: 2 times; ultrasonic extraction temperature: 40 °C)
was determined as the method of ultrasound extraction. The test was repeated three times
according to the optimized process conditions, and the average extraction rate of rutin was
88.6%. The result was better than that of any group in the orthogonal trials. The results
showed that the optimal extraction method that was selected was convenient and reliable,
had a high extraction rate and was suitable for extracting rutin from Sophora japonica.
Factors Influencing Activated Carbon Decolorization:
The methanol ultrasonic extract of Sophora japonica contains more chlorophyll, which should
be decolorized with activated carbon. Systematic research on the method of decolorization of
methanol ultrasonic extract of Sophora japonica was carried out. The dried flower bud
powders of Sophora japonica (9.0 g) were loaded into a flask (500 mL) and extracted with
CH3OH (90 mL) under ultrasonic conditions (ultrasound powder: 40 KHZ) according to the
optimal methanol ultrasonic extraction process A2B2C2 (ultrasonic extraction time: 30
minutes; ultrasonic extraction: 2 times; ultrasonic extraction temperature: 40 °C). The two
extracts were filtered, merged, concentrated to 90 mL by rotary evaporation, evenly divided
into nine aliquots, and decolorized according to the orthogonal experiment method (Table 3).
The extraction rates of rutin, chlorophyll A (at 663 nm) and chlorophyll B (at 645 nm)
absorption in decoloring solution were selected as the comprehensive marker, and the L9 (34)
orthogonal table was arranged for the experiment. The experimental results are shown in
Table 4.
Comprehensive marker (CM) = 25% (X̅/X) + 25% (Y̅/Y) + 50% (Z̅/Z)
X is the absorption of chlorophyll A in 9 parts of the decolorization solution; X̅ is the mean of
chlorophyll A absorption in 9 decolorization solutions; Y is the absorption of chlorophyll B in
9 parts of the decolorization solution; Y̅ is the mean of chlorophyll B absorption in 9
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Ma, W., & Wang, C. (2024). Preparation of High-Purity Rutin from Sophora japonica. European Journal of Applied Sciences, Vol - 12(6). 135-147.
URL: http://dx.doi.org/10.14738/aivp.126.17865
Table 6: Influence of the sample amount to the polyamide amount
M sample /M polyamide (g/g) Extracting rate of rutin/%
1:40 55.7
1:45 65.3
1:50 76.6
1:55 76.8
1:60 77.5
As show in Table 6, when the mass ratio of the crude rutin to polyamide used was 1:50, the
extraction rate of rutin did not increase. Therefore, the ratio of 1:50 is selected.
CONCLUSIONS
In recent years, natural products have been increasingly investigated as anticancer drugs. In
this study, HPR (HPLC: 99.8%) was successfully separated from Sophora japonica by
ultrasonic extraction, activated carbon decolorization, polyamide column chromatography,
and recrystallization. The extraction yield of HPR was 18.6%. The chemical structure of the
final product separated was identified by IR, element analysis and 1H NMR. Compared with
the traditional separation process, optimized extraction process of HPR in this paper has the
advantages of simple process, environmentally friendly solvent, safe and cheap, and
polyamide can be reused. so optimized extraction process is suitable for industrial
production. The extraction yield and purity of rutin increased significantly. The research
results have provided a strong foundation for the further development and utilization of
Sophora japonica resources.
ACKNOWLEDGMENTS
The author appreciates Lei Sun, Fu Da Testing Center, for the help with FT-IR and HNMR
characterizations.
Conflict of Interest
The authors declare no conflict of interest.
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