Method Article

Extraction and Characterization of the Flavonoid Rutin from Traditional Chinese Medicine Flos sophorae immaturus

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DOI:

10.3791/67417

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October 31st, 2025

 ,  ,  ,  ,  ,  ,  ,  , 
1School of Chinese Materia Medica, Beijing University of Chinese Medicine

In This Article

Summary

Traditional Chinese medicine (TCM) represents a rich treasure trove of natural treatment options and plays an important role in addressing diverse health needs worldwide. This study presents a comprehensive experimental investigation on the extraction, separation, and physicochemical properties of rutin, the key active component of Flos sophorae immaturus.

Abstract

Traditional Chinese medicine (TCM) has long served as a rich repository of natural therapeutic solutions, playing a pivotal role in addressing diverse healthcare needs worldwide. Among the various herbs used in TCM, Sophora flavescens is known for its wide range of medicinal properties. Its application dates back centuries and continues to be highly valued in modern medicine, particularly for its therapeutic benefits in treating cardiovascular diseases. Rutin, a bioactive flavonoid compound, is widely distributed across the plant kingdom, with the highest concentration found in Flos sophorae immaturus. Rutin has been reported to reduce abnormal capillary permeability and fragility and acts as a protective agent for cardiovascular and cerebrovascular vessels. This study presents a detailed experimental protocol for the extraction, separation, and characterization of the physicochemical properties of rutin derived from Flos sophorae immaturus. Additionally, the protocol for the extraction of flavonoids from TCM using the alkali extraction and acid precipitation method is introduced and described.

Introduction

Flos sophorae immaturus (FSI), the dried flower bud of Sophora japonica L., has been a cornerstone of traditional Chinese medicine (TCM) for centuries. Its main active components include rutin, isoquercitrin, Sophorae Flos, and other flavonoids and saponins. These constituents collectively confer a variety of pharmacological effects, such as clearing heat and detoxifying, cooling blood and hemostasis, and lowering blood pressure. In clinical practice, FSI is primarily valued for its ability to protect capillary permeability1, maintain cardiovascular system function2, cool blood, arrest bleeding, and regulate liver fire3. Its therapeutic properties are attributed to a high concentration of rutin (quercetin-3-rutinoside), a bioactive flavonoid that constitutes up to 20%-30% of its dry weight4.

Rutin is a flavonoid widely distributed in plants. Its chemical name is Que-3-O-rutin (C27H30O16) (Figure 1). At room temperature, rutin appears as a light yellow to yellow-green crystalline powder. Its melting point varies with crystal form and purity, typically ranging between 125 °C and 195 °C. Rutin exhibits pronounced polar characteristics: it is slightly soluble in cold water, highly soluble in polar organic solvents such as ethanol and methanol, and nearly insoluble in non-polar solvents like ether and chloroform. In addition, its solubility is pH-sensitive and significantly increases under alkaline conditions due to the dissociation of phenolic hydroxyl groups forming sodium or potassium salts. These physical properties are closely associated with the polyhydroxyl and glycoside groups in its molecular structure5, directly influencing its extraction process and applications in medicine, food, and other industries6. Rutin, while ubiquitous in plants, is most abundant in FSI and exhibits multiple pharmacological activities, including capillary stabilization7, anti-inflammatory, antioxidant8, antimicrobial9, and metabolic regulatory effects10.

Despite its clinical potential11, rutin's poor aqueous solubility and stability pose challenges for extraction and formulation5. To address these limitations, the alkali extraction-acid precipitation (AEAP) method has emerged as an effective alternative, leveraging the pH-dependent behavior of its phenolic hydroxyl groups.

Based on rutin's physical properties, the AEAP method has become a standardized technique for isolating rutin from FSI, utilizing its solubility in alkaline solutions and subsequent crystallization under acidic conditions12. AEAP exploits the pH-switchable ionization of phenolic hydroxyl groups to improve yield and achieve high-purity rutin extraction from FSI. Under alkaline conditions (pH >10), phenolic -OH groups deprotonate to form hydrophilic -O- ions, enabling complete solubilization from plant tissues while minimizing oxidative degradation. Acidification to pH 2-3 reverses this ionization, precipitating the target phenolics with minimal co-precipitation of polar impurities. As a polyphenol with pH-dependent solubility, rutin dissolves readily in alkaline solutions (e.g., sodium hydroxide) but precipitates under acidic conditions due to hydroxyl group protonation. AEAP provides a cost-effective, scalable, and environmentally friendly alternative, aligning with green chemistry principles by avoiding toxic solvents13. It reflects a broader trend in natural product chemistry that favors pH-driven separation techniques14. Comparable methods are employed to isolate curcumin (from turmeric) and berberine (from Coptis chinensis), exploiting solubility changes at specific pH values for selective extraction. The AEAP method is ideal for high-yield rutin production from flavonoid-rich sources like FSI, requiring only basic laboratory equipment (e.g., pH meters, centrifuges). For plant matrices with low rutin content or complex interfering compounds, enzymatic hydrolysis pretreatment may be considered in the future to enhance extraction efficiency.

In conclusion, AEAP represents a convenient approach for rutin extraction, balancing efficacy, safety, and scalability. Its role in TCM modernization highlights its potential to bridge traditional practices with contemporary pharmaceutical standards.

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Protocol

Rutin is a flavonoid containing multiple phenolic hydroxyl groups and is soluble in hot alkaline solutions, followed by acidification and precipitation. It can be extracted using the alkali extraction-acid precipitation process. The reagents and equipment used in this study are listed in the Table of Materials.

1. Experimental preparation

  1. Obtain the following materials: Flos sophorae immaturus (20 g), borax (0.8 g), several beakers, lime powder (1.5 g), concentrated hydrochloric acid, thermometer, and pH test strips. See Figure 2 for a larger version of this image.

2. Extraction of rutin

NOTE: Use a borax-to-water ratio of 0.8 g borax to 200 mL water. Use a lime milk ratio of 1.5 g lime powder to 10 mL water. Adjust the pH quickly and accurately. Replenish lost water as needed.

  1. Alkali extraction
    NOTE: Filter using 6 cm round filter paper.
    1. Add 250 mL of water and 0.8 g of borax to a 500 mL beaker. Heat to boiling, then add 20 g of Flos sophorae immaturus. Continue boiling for 2-3 min.
    2. After boiling, adjust the pH to 8-9 with lime milk under stirring. Continue heating for 20 min. Filter through gauze while hot to obtain a yellow-brown filtrate.
  2. Acid precipitation extraction
    1. Adjust the extract to pH 2-3 using concentrated hydrochloric acid and allow it to settle. A change from a clear yellow-brown solution to a turbid one, along with the gradual formation of orange-yellow flocculent precipitate at the bottom of the container, indicates effective rutin separation.
    2. After standing for 6 h, subject the precipitate to atmospheric pressure suction filtration. Collect and dry to obtain crude rutin.
      NOTE: Cut the filter film according to the Brinell funnel size used (150 mm diameter).
    3. Wash the precipitate with water until neutral during atmospheric pressure suction filtration.

3. Refining of rutin

  1. After coarse weighing, recrystallize rutin using hot water at a solubility ratio of 1:200. Boil the aqueous solution for 30 min and filter while hot. Ensure that the final volume of the rutin solution is 210 mL.
  2. Filter out the residue. Allow the filtrate to stand undisturbed, then filter again and dry under vacuum in an oven at 60-70 °C to obtain refined rutin (Figure 3).
    NOTE: Cut the filter film according to the Brinell funnel size used (150 mm diameter).

4. Physicochemical identification of rutin

  1. Hydrochloride-magnesium salt reaction - identification of flavonoid parent nuclei
    1. Dissolve 1-2 mL of the sample in methanol and add 50 mg of magnesium powder.
    2. Add a few drops of concentrated hydrochloric acid. Shake gently. The solution should turn red (Figure 4).
  2. Molisch reaction - identification of sugar compounds
    1. Add a 10% ethanol solution of α-naphthol and shake. Tilt the test tube and carefully add concentrated sulfuric acid dropwise along the inner wall.
    2. Observe the formation of a brown ring at the interface (Figure 5).
  3. Greening zirconia-citric acid reaction
    1. Add 1-2 mL of 10% α-naphthol solution to the ethanol solution of the sample. Shake well. Slowly add 10 drops of concentrated H2SO4 along the wall of the tube without shaking.
    2. Add 2% citric acid methanol solution. The solution color should lighten (Figure 6).
  4. Thin layer discrimination
    1. Use both the refined rutin and rutin standard.
    2. Use chloroform:methanol:formic acid (15:5:1) as the developing agent, and 1% ethanol solution of AlCl3 as the chromogenic agent. Observe fluorescence under a UV lamp at 365 nm.

5. TLC examination of rutin

NOTE: A thin-layer chromatography (TLC) method was employed to evaluate the purity of rutin. A silica gel plate was used as the stationary phase.

  1. Spot the refined rutin sample and rutin standard (1 mg/mL in methanol) onto the plate. Develop the plate in a mobile phase consisting of ethyl acetate:formic acid:water (8:1:1, v/v/v).
  2. After drying, visualize the plate under UV light at 254 nm.
  3. Calculate the retention factor (Rf) of the sample spot and compare it to the standard.
    NOTE: A single spot with an identical Rf value (approximately 0.35-0.50 under these conditions) indicates high purity, while additional spots suggest the presence of impurities.

6. Determination of rutin purity by HPLC

NOTE: The purity of rutin was quantitatively analyzed by high-performance liquid chromatography (HPLC). Analysis conditions were as follows: Chromatographic column- (250 mm × 4.6 mm, 5 µm); mobile phase-acetonitrile (A) and 0.2% acetic acid aqueous solution (B). Linear gradient elution program: 0-15 min, 20%-30% A; 15-30 min, 30%-60% A. Flow rate-1 mL/min. Injection volume-10 µL. Detection wavelength-355 nm. Column temperature-40 °C.

  1. Precisely weigh 0.1 g of rutin sample powder and transfer it to a 10 mL centrifuge tube. Add 5.0 mL of 60% ethanol solution and shake thoroughly.
  2. Set the constant temperature water bath or column oven to 30 °C and ultrasonicate the sample for 30 min.
  3. Centrifuge the sample at 55,900 × g for 10 min at room temperature. Use the supernatant as the test solution for HPLC analysis.

7. Determination of rutin by LC-MS

NOTE: The isolated compounds were identified by liquid chromatography-mass spectrometry (LC-MS). LC conditions matched the HPLC method. Mass spectrometry was performed using electrospray ionization (ESI) in negative ion mode with a capillary voltage of 3.5 kV. Desolvation temperature-350 °C; mass scan range-m/z 100-1000.

  1. Weigh 10 mg of purified rutin sample and transfer it to a 2 mL microcentrifuge tube. Add 0.6 mL of deuterated DMSO using a pipette until the sample is fully dissolved. Prepare rutin standard solutions using the same procedure.
  2. Analyze the treated samples and standards using proton nuclear magnetic resonance (1H NMR) spectroscopy.
  3. Process the spectral data using compatible software15.
  4. Compare the spectral data with literature or database references such as NIST or PubChem for verification.

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Results

In the physicochemical identification experiment of rutin, the solution turned red after the addition of magnesium powder and a small amount of concentrated hydrochloric acid, followed by shaking, indicating the presence of a flavonoid parent nucleus. In the Molisch reaction, a purple ring formed at the interface, indicating a positive result and the presence of sugar moieties in the molecular structure. The sample solution turned bright yellow upon the addition of 2% chloro-oxidation reagent in methanol, suggesting the ...

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Discussion

The extraction of rutin is primarily based on the alkali extraction-acid precipitation (AEAP) method6, which offers the advantages of simple equipment requirements, ease of operation, high efficiency, and low cost. This technique is often supplemented by microwave-assisted extraction (MAE)7 and ultrasound-assisted extraction (UAE)8, which reduce reagent consumption, accelerate extraction time, and improve overall yield.

St...

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Disclosures

The authors of this paper hereby declare that they have disclosed any and all conflicts of interest that may have arisen in the course of conducting this research and writing this article. We confirm that we have no financial, personal, or professional relationships that could potentially bias our interpretation of the results or conclusions presented in this work. Specifically, we have no ties to any pharmaceutical companies, research institutions, or other entities that could benefit from the findings reported here. Finally, we affirm that our findings and conclusions are solely based on the data and analysis presented in this paper and are not influenced by any external factors. We remain committed to the highest standards of academic integrity and ethical conduct in all our research activities.

Acknowledgements

We would like to express our profound gratitude to the Pharmaceutical Chemistry Laboratory at the Beijing University of Chinese Medicine for providing the experimental platform. Their support and resources have been invaluable in the conduct of our research and experiments. We deeply appreciate their dedication to scientific excellence and their commitment to fostering the development of young researchers.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1-NaphtholMACKLINC16245764
Acetic acidMACKLINC12662811
AcetonitrileMACKLINC16129374
Aluminium ChlorideMACKLINC16485922
Citric acidMACKLINC16429480
DMSOFUCHEN (Tianjin) Chemical Reagent Co., LTD20230306
EthanolFUCHEN (Tianjin) Chemical Reagent Co., LTD20250102
Ethyl acetateFUCHEN (Tianjin) Chemical Reagent Co., LTD20240318
Flos sophorae immaturusBeijing Tongrentang10092026329782
Formic acidFUCHEN (Tianjin) Chemical Reagent Co., LTD20150402
‌MestReNovaMestrelab Research/
Methanol innochemC16365684
Silica gel 60F254STEEMA20210222
XcaliburThermo Fisher Scientific/
Zirconyl chloride octahydrateMACKLINC15350627

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Rutin ExtractionFlavonoid CharacterizationAlkali ExtractionAcid PrecipitationCardiovascular ProtectionPhysicochemical PropertiesFlavonoid SeparationBioactive Flavonoids
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