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

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.

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.

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 presence of flavone structures containing 3-OH or 5-OH groups. Upon subsequent addition of 2% citric acid in methanol, the solution color became lighter, indicating the presence of only 5-OH.

In the TLC experiments comparing rutin samples and rutin standards, the standard exhibited specific spot locations and characteristic coloration16. A comparison of the chromatograms showed that the number and position of the spots in the sample closely matched those of the standard, indicating the high purity of the rutin extracted via the alkali extraction and acid precipitation method17.

The combination of TLC, HPLC, and LC-MS/NMR established a comprehensive analytical system for rutin, spanning from preliminary screening to accurate qualitative analysis. TLC enabled rapid evaluation of sample purity, while HPLC quantitatively confirmed high purity. LC-MS and NMR provided final confirmation of the compound as rutin through multi-dimensional analysis of molecular weight, fragmentation patterns, and fine structural features.

The color reaction of rutin
The technique of alkaline extraction and acid precipitation plays a significant role in identifying the physicochemical properties of rutin. In this experiment, the hydrochloric acid-magnesium powder reaction, the Molisch reaction for sugar compound identification, and the citric acid reaction were carried out to evaluate rutin. The refined rutin sample extracted using this method was compared with a negative control group (methanol solvent control) and a positive control group (baicalin standard).

Hydrochloric acid-magnesium powder reaction
Rutin contains phenolic hydroxyl groups typical of flavonoids. In this reaction, hydrochloric acid reacts with the phenolic hydroxyl groups in rutin, followed by the complexation of the hydrolysate with magnesium powder. The resulting colored complexes typically appear red to purplish-red, depending on the specific flavonoid structure and reaction conditions. The color change observed in this reaction serves as a preliminary indicator of the flavonoid nature of rutin and helps distinguish it from non-flavonoid compounds (Figure 4).

Molisch reaction
Rutin is a flavonoid glycoside containing two sugar moieties -- glucose and rhamnose. These saccharides react with concentrated sulfuric acid and α-naphthol to form a purplish-red complex, confirming the presence of sugar components in the molecule (Figure 5).

Zirconia-citric acid greening reaction
The zirconia-citric acid greening reaction is commonly used to detect specific functional groups or structural features in flavonoids. In the case of rutin, phenolic hydroxyl groups or other reactive sites in the molecule interact with zirconia, leading to oxidation or complexation that results in a color change. This reaction provides additional confirmation of the flavonoid structure of rutin and aids in distinguishing it from other flavonoid compounds (Figure 6).

In addition to the comparative analysis of color reactions, a yield assessment was conducted based on three independent extraction experiments. Results showed that the yield of rutin remained stable at approximately 8%-9% (Table 1), demonstrating the consistency and reliability of the extraction method. The stability of the yield is likely influenced by the precision of key operational steps, such as filtration and sample transfer. Measures including the use of suitable filter paper, controlled filtration speed, and accurate weighing help reduce sample loss and enhance reproducibility. Furthermore, optimization of extraction parameters -- such as solvent type, concentration, temperature, and duration -- may further improve rutin yield.

TLC examination of rutin
Thin-layer chromatography analysis of the refined rutin samples (Figure 7) showed that the Rf values of the hydrolysate spots were consistent with those of reference rutin and mixed samples (Table 2), confirming the identity of the extracted compound as rutin. The chromatographic spots were well-separated, with no additional or unexpected spots observed, indicating that the refined rutin product possessed high purity.

Determination of rutin purity by LC-MS
In the negative ion mode, the quasimolecular ion peak [M−H]- appeared at m/z 609.1555, with a predicted molecular formula of C27H30O16. The [M−H]- peak at m/z 609.1561 was consistent with the rutin standard, also corresponding to C27H30O16. The refined rutin sample was identified as rutin by comparison with the quasimolecular ion peak of the rutin standard and confirmation using the SciFinder database.

Both the refined sample and the rutin standard (Figure 8) were identified as flavone-3-rutinoside, 3,3′,4′,5,7-pentahydroxy (CAS No. 153-18-4), or its isomers, based on mass spectral data. The LC-MS results for the refined rutin sample are presented in Figure 9. Analysis and comparison of the ¹H NMR spectra of the rutin standard and purified sample showed a high degree of consistency, further confirming the identity and purity of the extracted compound as rutin.

¹H NMR spectra of purified rutin samples
¹H NMR (600 MHz, DMSO) δ 12.61 (s, 1H), 10.83 (s, 1H), 9.67 (s, 2H), 9.18 (s, 2H), 7.55-7.54 (s, 2H), 6.85-6.84 (s, 2H), 6.39 (d, J = 2.0 Hz, 2H), 6.20 (d, J = 2.0 Hz, 2H), 5.36-5.28 (dd, J = 16.1, 5.5 Hz, 3H), 5.12-5.11 (dd, J = 12.1, 4.7 Hz, 2H), 4.53 (d, J = 5.2 Hz, 2H), 4.40-4.35 (m, 3H), 3.72-3.70 (d, J = 10.0 Hz, 2H), 3.09-3.04 (m, 3H), 1.00 (t, J = 7.0 Hz, 0H), 0.99 (d, J = 6.1 Hz, 6H).

Figure 1: Chemical structure of rutin. Please click here to view a larger version of this figure.

Figure 2: Materials used for decoction and extraction. (A) Flos sophorae immaturus; (B) Borax; (C) Beakers; (D) Lime powder; (E) Concentrated hydrochloric acid; (F) Thermometer; (G) pH test strips. Please click here to view a larger version of this figure.

Figure 3: Extraction and purification process of rutin. (A) Alkali extraction of Flos sophorae immaturus; (B) Filtration to obtain the filtrate; (C) Acid precipitation to obtain crude rutin; (D) Recrystallization to obtain purified rutin. Please click here to view a larger version of this figure.

Figure 4: Hydrochloric acid-magnesium salt color reaction used for flavonoid identification. Please click here to view a larger version of this figure.

Figure 5: Molisch test for the detection of sugar compounds. Please click here to view a larger version of this figure.

Figure 6: Zirconia-citrate greening reaction for the differentiation of flavonoids. Please click here to view a larger version of this figure.

Figure 7: Thin-layer chromatography (TLC) analysis of refined rutin samples. Left: Refined rutin sample; Middle: Mixed spot; Right: Rutin standard. Please click here to view a larger version of this figure.

Figure 8: Base-peak chromatograms of refined rutin standard and sample under negative ion detection mode by LC-MS. Please click here to view a larger version of this figure.

Figure 9: 1H NMR spectra of purified rutin samples. Please click here to view a larger version of this figure.

Table 1: Zirconia-citrate greening reaction for the differentiation of flavonoids.

Table 2: TLC analysis results of refined rutin samples compared to rutin standard.

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.

Studies have demonstrated that pH significantly influences both the solid extraction rate and the quercetin content in the precipitated solids. Therefore, precise control over the concentration of the alkaline solution and the extraction time is essential for maximizing rutin yield. Excess alkali can dissolve unwanted compounds, thereby compromising the purity of the extract¹⁸. Similarly, the type and concentration of acid used during precipitation play a critical role in the outcome; appropriate acidity ensures complete precipitation of rutin, while over-acidification may lead to its decomposition¹⁹. The use of boiling water during recrystallization is based on the differential solubility of rutin in hot versus cold water, allowing for further purification through selective crystallization.

While AEAP is effective and economical, it is sensitive to environmental contaminants and requires repeated washing and longer processing times to maintain product quality. Furthermore, this method may damage DNA and increase decomposition, compromising the integrity of samples, especially those with high DNA content, or requiring special handling²⁰.

AEAP yielded approximately 8% rutin within 2 h (including extraction and precipitation) from Flos sophorae immaturus (FSI), whereas UAE methods typically require 3-4 h to obtain a comparable yield (7%-9%) from FSI. Compared with UAE, the AEAP method stands out in terms of both efficiency and extract quality²¹. As research on Chinese herbal medicine advances, the demand for efficient extraction of bioactive compounds is increasing. Due to its cost-effectiveness and high yield, the AEAP technique holds promise for broader application in the field of herbal medicine extraction.

The AEAP method achieved a moderate rutin yield of around 8%, yet its practical benefits in scalability and operational simplicity are noteworthy. For example, challenges related to incomplete phenolic dissociation under suboptimal alkaline conditions (pH <10) can be mitigated by using 0.5-1 M NaOH (pH 10-12) with real-time pH monitoring. Additionally, employing a gradient acid precipitation strategy (pH 5 → 2-3) improves selectivity. The scalability of AEAP is evidenced by consistent yields of 7.5%-8.5% in 50 L pilot-scale extractions, along with 60%-70% lower solvent consumption compared to methanol reflux extraction. These features establish AEAP as a sustainable and scalable platform for phenolic compound production. When combined with waste valorization strategies, AEAP could evolve into a closed-loop system, bridging the gap between laboratory-scale efficacy and industrial feasibility. Although alternative methods such as UAE and MAE may deliver higher yields in tightly controlled laboratory settings, AEAP offers a uniquely adaptable, scalable, and cost-effective solution for routine phenolic extraction-particularly when integrated with green extraction technologies.