Quality evaluation of a traditional Chinese medicine compound preparation containing Rhodiola rosea, Astragalus membranaceus, and Carthamus tinctorius

1 Introduction

Flavonoids are widely distributed in natural plants and constitute an important class of natural active constituents. Numerous studies have confirmed that flavonoids represented by isorhamnetin exhibit prominent pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, and cardioprotective effects. Accordingly, medicinal and edible Chinese herbal materials rich in flavonoids serve as high-quality natural raw materials for the development of functional health foods and possess excellent research and application potential (Elmehdi et al., 2026; Abdelhakim et al., 2026; Haytham et al., 2026). With the increasing prevalence of scenarios such as high-altitude exposure, intense physical exercise, and long-term mental work, health issues related to hypoxia and physical fatigue have become significant concerns affecting daily performance and wellbeing (Huang and Zhang, 2019). Rhodiola rosea, Astragalus membranaceus, and Carthamus tinctorius are three well-documented medicinal plants widely used in traditional herbal formulations for their complementary biological activities. R. rosea is recognized for its adaptogenic properties and the ability to enhance tolerance to hypoxia and stress (Panossian and Wikman, 2007); A. membranaceus exhibits immunomodulatory and anti-fatigue effects, largely attributed to its polysaccharides and saponins (Li and Qu, 2021); C. tinctorius is valued for its blood-circulation-promoting activity, primarily mediated by water-soluble quinone pigments such as hydroxysafflor yellow A (HSYA) and anhydrosafflor yellow B (ASYB) (Zhou and Zuo, 2006). Based on this functional synergy, a scientifically formulated combination—known as the R. rosea, A. membranaceus, and C. tinctorius capsule (RAC capsule)—was developed and registered as a national health food (Approval No. G20080473) in China (National Medical Products Administration NMPA, 2008). Preclinical studies have confirmed that RAC capsules effectively improve hypoxia tolerance and alleviate physical fatigue, supporting their use in conditions associated with oxygen deficiency and energy depletion (Wang and Chen, 2024; Wang and Wang, 2025).

The manufacturing process of RAC capsule integrates distinct extraction strategies tailored to the physicochemical properties of active constituents from each herb. R. rosea and A. membranaceus are first extracted with 75% ethanol (solid-to-liquid ratio 1:8, 1.5 h × 2) to recover medium-polarity compounds such as salidroside, tyrosol, and astragalosides. The residual marc is then subjected to aqueous extraction (1.5 h × 2) to capture polar components, including polysaccharides and glycosides (Li and Qu, 2021). The ethanolic and aqueous extracts are concentrated separately, combined, and dried to yield a uniform dry extract for later use. C. tinctorius, whose key pigments (e.g., HSYA) are heat-sensitive and highly polar, is processed without thermal extraction; the raw herb is milled into a fine powder (passing an 80-mesh sieve) and directly incorporated into the final blend (Zhou and Zuo, 2006). This hybrid formulation approach—combining solvent-based extraction with raw herb powdering—maximizes the retention of diverse bioactive compounds but introduces considerable complexity in quality control (Rao et al., 2025). Batch-to-batch variability in the extraction yield, powder homogeneity, or raw material composition can significantly affect the content of marker compounds, potentially compromising product consistency and efficacy (Zhang and Sun, 2024). Therefore, reliable analytical methods capable of both qualitative verification and precise multi-component quantification are critically needed to ensure the quality, safety, and functional reliability of RAC capsules.

2 Methodology

R. rosea, A. membranaceus, and C.s tinctorius are the main raw materials of RAC capsules. The quality standards of these three medicinal materials are all included in the Chinese Pharmacopoeia 2025 Edition.

For the analytical method of Rhodiola Radix et Rhizoma, salidroside is used as the reference substance in both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses, with a concentration of 0.5 mg/mL (dissolved in methanol). Approximately 0.5 g of the sample (passing a 50-mesh sieve) is weighed accurately, methanol is added at a solid–solvent ratio of 1:20 (W/V), the mixture is sonicated for 30 min and filtered, and the successive filtrate is used as the test solution (Wang and Wang, 2025).

For TLC analysis:

A measure of 10 µL each of the reference solution and the test solution is added to the same silica gel G thin-layer plate. It is developed using the lower layer of a mixture of chloroform–methanol–acetone–water (6:3:1:1) as the mobile phase. The plate is removed, dried in air, and visualized by exposure to iodine vapor until distinct spots appear. In the chromatogram of the test solution, spots of the same color are observed at the positions corresponding to the principal spot in the chromatogram of the reference solution (Chinese Pharmacopoeia Commission, 2025).

For HPLC analysis:

A C18 column packed with octadecylsilane-bonded silica gel is used, with a methanol–water (15:85) mixture as the mobile phase. The detection wavelength is set at 275 nm. The content of salidroside in the sample is calculated by referring to the concentration and peak area of the salidroside reference standard. The chromatographic column is considered suitable if the number of theoretical plates calculated based on the salidroside peak is no less than 2,000. The medicinal material is deemed qualified when the salidroside content in the sample is not lower than 0.5% (Chinese Pharmacopoeia Commission, 2025).

Analytical methods for the quality standard of A. membranaceus.

TLC identification

Two identification methods are included in TLC analysis.

Method 1

Reference substance: astragaloside IV, concentration 0.5 mg/mL (dissolved in 80% methanol). Sample powder (passing a 65-mesh sieve) is extracted with 80% methanol containing 4% strong ammonia solution (prepared by taking 4 mL of strong ammonia solution, adding 80% methanol to 100 mL, and shaking well) at a solid–solvent ratio of 1:50 (W/V). It is followed by heating under reflux for 1 h, filtering, accurately measuring 25 mL of the successive filtrate, and evaporating to dryness. The residue is dissolved in 80% methanol, transferred to a 5-mL volumetric flask, diluted to volume with 80% methanol, shaken well, filtered, and used as the test solution (Chinese Pharmacopoeia Commission, 2025).

A measure of 5 µL–10 µL each of the reference solution and the test solution is added to the same silica gel G thin-layer plate. It is developed using the lower layer of a mixture of chloroform–methanol–water (13:7:2) as the mobile phase. The plate is removed, dried in air, sprayed with 10% sulfuric acid in ethanol, and heated at 105 °C until distinct spots appear. The sample is examined in daylight and under ultraviolet light (365 nm). In the chromatogram of the test solution, spots of the same brownish color are observed in daylight, and fluorescent spots of the same orange–yellow color are observed at 365 nm, at positions corresponding to the principal spot in the chromatogram of the reference solution (Chinese Pharmacopoeia Commission, 2025).

Method 2

Sample powder is extracted with ethanol at a solid–solvent ratio of 1:15 by heating under reflux for 20 min, filtered, and the filtrate is evaporated to dryness. The residue is dissolved in 15 mL of 0.3% sodium hydroxide solution and filtered. The pH of the filtrate is adjusted to 5–6 with dilute hydrochloric acid, and it is extracted with 15 mL of ethyl acetate by shaking. The ethyl acetate layer is separated, the sample is filtered through filter paper covered with appropriate anhydrous sodium sulfate, and the filtrate is evaporated to dryness. The residue is dissolved in 1 mL of ethyl acetate to obtain the test solution. Separately, 2 g of Astragali Radix reference drug is weighed, and the reference drug solution is prepared in the same manner (Chinese Pharmacopoeia Commission, 2025).

The sample is developed using a mixture of chloroform–methanol (10:1) as the mobile phase. The plate is removed, dried in air, exposed to ammonia vapor, and then examined under ultraviolet light (365 nm). In the chromatogram of the test solution, fluorescent principal spots of the same color are observed at positions corresponding to those in the chromatogram of the reference drug solution (Chinese Pharmacopoeia Commission, 2025).

HPLC Assay: two assay methods are included in HPLC analysis.

Method 1 (Astragaloside IV)

Reference substance: astragaloside IV. The concentrations of the reference solution and sample preparation are the same as those described in TLC Method 1 (Chinese Pharmacopoeia Commission, 2025).

Octadecylsilane chemically bonded silica is used as the stationary phase; a mixture of acetonitrile–water (32:68) is used as the mobile phase; the sample is detected with an evaporative light-scattering detector (ELSD). The number of theoretical plates calculated by the astragaloside IV peak is not less than 4,000 (Chinese Pharmacopoeia Commission, 2025).

The content of astragaloside IV (C₄₁H₆₈O₁₄) in the sample is calculated using the logarithmic equation of the two-point external standard method based on the concentration and peak area of the reference substance and the peak area of the sample. The content of astragaloside IV shall be not less than 0.08% (Chinese Pharmacopoeia Commission, 2025).

Method 2 (Calycosin-7-O-β-D-glucoside)

Reference substance: calycosin-7-O-β-D-glucoside, concentration 50 μg/mL (dissolved in methanol) (Chinese Pharmacopoeia Commission, 2025).

The sample powder (passing a 65-mesh sieve) is accurately weighed, extracted with methanol at a solid–solvent ratio of 1:50 (W/V) by heating under reflux for 4 h, and filtered. A measure of 25 mL of the successive filtrate is accurately measured, and the solvent is recovered to dryness. The residue is dissolved in methanol, transferred to a 5-mL volumetric flask, diluted to volume with methanol, shaken well, filtered, and used as the test solution (Chinese Pharmacopoeia Commission, 2025).

Octadecylsilane chemically bonded silica is used as the stationary phase; acetonitrile is used as mobile phase A, and 0.2% formic acid solution is used as mobile phase B. The gradient elution is as follows: 0 min–20 min: mobile phase A from 20% to 40%; 20 min–30 min: mobile phase A maintained at 40% (Chinese Pharmacopoeia Commission, 2025).

Detection wavelength: 260 nm. The number of theoretical plates calculated by the calycosin-7-O-β-D-glucoside peak is not less than 3,000 (Chinese Pharmacopoeia Commission, 2025).

The content of calycosin-7-O-β-D-glucoside (C₂₂H₂₂O₁₀) in the sample is calculated based on the concentration and peak area of the reference substance and the peak area of the sample. The content shall be not less than 0.020%.

Analytical methods for the quality standard of C. tinctorius

TLC identification

Approximately 0.5 g of the sample powder is accurately weighed, and 80% acetone is added at a solid–solvent ratio of 1:10 (W/V); the sample is then stoppered tightly, shaken for 15 min, and allowed to stand to obtain the test solution. Separately, 0.5 g of Carthami flos reference drug is weighed, and the reference drug solution is prepared in the same manner (Chinese Pharmacopoeia Commission, 2025).

A measure of 5 μL each of the reference drug solution and the test solution is applied to the same silica gel H thin-layer plate. It is developed using a mixture of ethyl acetate–formic acid–water–methanol (7:2:3:0.4) as the mobile phase. The plate is removed and dried in air. In the chromatogram of the test solution, spots of the same color are observed at the positions corresponding to those in the chromatogram of the reference drug solution (Chinese Pharmacopoeia Commission, 2025).

HPLC assay

Reference substance: hydroxysafflor yellow A with a concentration of 0.13 mg/mL (dissolved in 25% methanol) (Chinese Pharmacopoeia Commission, 2025).

The sample powder (passing a 50-mesh sieve) is accurately weighed, 25% methanol is added at a solid–solvent ratio of 1:125 (W/V), the mixture is sonicated for 40 min and filtered, and the successive filtrate is used as the test solution (Chinese Pharmacopoeia Commission, 2025).

Octadecylsilane chemically bonded silica is used as the stationary phase; a mixture of methanol–acetonitrile–0.7% phosphoric acid solution (26:2:72) is used as the mobile phase; the detection wavelength is 403 nm. The number of theoretical plates calculated by the hydroxysafflor yellow A peak is not less than 3,000 (Chinese Pharmacopoeia Commission, 2025).

The content of hydroxysafflor yellow A (C₂₇H₃₂O₁₆) in the sample is calculated based on the concentration and peak area of the reference substance and the peak area of the sample. The content of hydroxysafflor yellow A shall be not less than 1.0% (Chinese Pharmacopoeia Commission, 2025).

To address this need, the present study establishes a comprehensive analytical system for the quality evaluation of RAC capsules. A TLC method was developed for the specific identification of all three herbal ingredients. Additionally, a HPLC method with UV detection was validated for the simultaneous quantification of five major bioactive markers: salidroside and tyrosol from R. rosea, HSYA and ASYB from C. tinctorius, and rhodiolin—a characteristic phenolic constituent of R. rosea (Liu et al., 2023). The HPLC method was fully validated for linearity, precision, accuracy, stability, and specificity. By enabling multi-component quality control beyond the current single-marker standard (salidroside only), this work provides a robust scientific foundation for the standardization and quality assurance of RAC capsules (Lhaglham et al., 2024; Wu et al., 2025), facilitating their continued development as a reliable health product (Mu and Jin, 2023; Li et al., 2024).

There are two reasons for selecting salidroside, tyrosol, hydroxysafflor yellow A (HSYA), safflower yellow B (ASYB), and rhodiolin as the quantitative markers for RAC capsule.

First, these five components are derived from RAC capsules in the prescription and are all associated with the mechanisms of anti-hypoxia and anti-fatigue effects, consistent with the functional indications of the preparation. Among them, salidroside, tyrosol, and rhodiolin are the representative active constituents of R. rosea, while hydroxysafflor yellow A and safflower yellow B are the representative active constituents of C. tinctorius.

Second, in our preliminary studies, multiple components from the medicinal materials in the prescription were investigated; however, only the above five components could be well-separated and detected by HPLC with no interference from the negative samples. This study established an analytical method for the simultaneous determination of five active ingredients under identical chromatographic conditions. The proposed method is simple to operate and highly efficient. Given that HPLC is the most widely used quantitative detection technique in the industrial mass production of pharmaceutical products, the quantitative method developed in this work exhibits favorable practical value and promising application prospects.

Astragaloside IV was determined by evaporative light scattering detection, and astragalus polysaccharides were detected by ultraviolet spectrophotometry. Both methods showed relatively poor repeatability. In comparison, HPLC technology is more stable and precise. In recent years, numerous studies have been published on the simultaneous determination of multiple components in traditional Chinese medicines and their preparations using HPLC.

Although some literature have reported the extraction of components from R. using natural deep eutectic solvents (NADES), NADES have not yet been practically applied in the detection of Chinese medicinal materials. Furthermore, the present study focuses on the detection and analysis of all Chinese medicinal materials in the capsule, rather than targeting only R. rosea as a single medicinal material. Therefore, the extraction method using natural deep eutectic solvents is not suitable for RAC capsules.

This study is intended to construct and refine the quality control system of Hongjingtian Huangqi Honghua capsules and enhance the overall quality controllability of the preparation. Based on the quantitative determination of salidroside, four additional components were included in the assay. This allowed the indirect establishment of acceptable content ranges for the raw materials, thereby enhancing the quality control of raw materials and ensuring the final product quality.

3 Results and discussion

3.1 TLC identification

As a traditional Chinese multi-herb formulation, RAC capsules are dispensed in light-resistant bottles, with 45 capsules per container. Each capsule contains 0.4 g of a uniformly blended powder derived from R. rosea, A. membranaceus, and C. tinctorius, ensuring consistent dosing of active botanical ingredients (Figure 1).

The chemical matrix of RAC capsules is derived from the combination of a 75% ethanol extract (primarily targeting medium-polarity constituents such as salidroside and flavonoids), an aqueous extract (enriched in polar polysaccharides and saponins), and directly incorporated powdered C. tinctorius (which contributes both polar pigments such as HSYA and minor lipophilic compounds). Given this complex polarity profile, a developing system with moderate-to-high elution strength was required to achieve balanced migration of marker compounds from all three botanicals.

After the preliminary screening of various solvent systems, chloroform–methanol (15:1, v/v) was selected as the optimal mobile phase. This system provided sufficient polarity to mobilize key constituents while maintaining good resolution and compact spot morphology. Under the established TLC protocol, 2 µL of each reference drug’s solution (R. rosea, A. membranaceus, and C. tinctorius) and 4 µL of the RAC capsule test solution or corresponding negative control solutions were applied onto silica gel GF254 plates. Following development and air-drying, the plates were visualized under UV light at 365 nm without TLC developer.

Distinct fluorescent spots corresponding to the characteristic markers of all three herbs were clearly observed in the RAC capsule sample lanes with Rf values falling within the ideal range of 0.4–0.6, which is indicative of appropriate solvent strength and minimal tailing. Critically, the spot positions in the sample lanes matched those of the respective reference herbs, confirming the presence of R. rosea, A. membranaceus, and C. tinctorius in the final product. Moreover, when analyzed alongside negative controls (i.e., formulations prepared by omitting one herb at a time), no interfering or overlapping spots appeared at the same Rf regions, demonstrating excellent specificity. Notably, all three herbal components were successfully resolved on a single thin-layer plate using a unified developing system—a practical advantage for routine quality control of this complex formulation (Figure 2).

In the picture, the spot labeled R is the principal spot for the identification of Rhodiola; the spot labeled A is the principal spot for the identification of Astragalus. For the identification of Astragalus, there is one principal spot A1 and another identifiable spot A2; the spot labeled C is the principal spot for the identification of Carthamus. The Rf values of spots R, A1, A2, and C are 0.57, 0.63, 0.76, and 0.74, respectively.

For TLC identification, an Rf value within 0.4–0.6 is generally appropriate. However, traditional Chinese medicines contain multiple components, and the Rf value of the principal identification spot is usually controlled at approximately 0.5. For compound preparations with more complex components, more than one spot may be available for identification when reference medicinal materials are used as references. Therefore, according to the Chinese Pharmacopoeia, unless otherwise specified in the individual monograph, the Rf values of all spots should preferably be within 0.2–0.8. Accordingly, the present identification method is considered qualified.

3.2 HPLC method development

An HPLC method was systematically developed and optimized to support routine quality control and stability-indicating analysis of RAC capsules, with the aim of simultaneously quantifying multiple bioactive markers from its three constituent herbs: R. rosea, A. membranaceus, and C. tinctorius. The primary chromatographic goals were to achieve baseline separation, symmetrical peak shapes (asymmetry factor close to 1.0), high theoretical plate counts (>2,000 for all analytes), and adequate resolution (Rs > 1.5) between adjacent peaks.

Initial trials evaluated various column chemistries (C8, C18, and phenyl), mobile phase compositions (acetonitrile vs. methanol; phosphoric acid vs. Formic acid vs. ammonium acetate buffers), and gradient profiles. After comprehensive assessment, a Zafex Supperfex JX-C18 column (250 mm × 4.6 mm, 5 μm) coupled with a binary gradient system consisting of 0.1% aqueous phosphoric acid (A) and acetonitrile (B) was selected as optimal (Table 1). Phosphoric acid was chosen over volatile acids due to its superior peak-sharpening effect for phenolic and acidic constituents without compromising UV detection at 275 nm.

TABLE 1. Gradient elution program for HPLC analysis.

The gradient program was carefully designed to accommodate the wide polarity range of target analytes—from relatively polar compounds such as salidroside and tyrosol (eluting early) to more hydrophobic flavonoids such as rhodiolin (eluting later). This stepwise increase in organic phase ensured efficient elution while maintaining the resolution throughout the 100-min run. Representative chromatograms under the optimized conditions are shown in Figure 3. The retention time and relative retention time of the five characteristic chromatographic peaks in Figure 3 are listed in Table 2. Panel B shows the RAC capsule sample, where all five markers are clearly resolved and detectable. Panels C and D present negative control samples lacking R. rosea and C. tinctorius, respectively, confirming the absence of interfering peaks at the retention times of the target analytes.

TABLE 2. Results of retention time and relative retention time for five characteristic chromatographic peaks.

3.3 System suitability and method validation

To ensure the reliability and reproducibility of the developed HPLC method, comprehensive system suitability and validation parameters were evaluated in accordance with the guidelines of the Chinese Pharmacopoeia (CP), 2025 Edition. A series of standard solutions at varying concentrations were used to prepare the calibration curves by plotting peak areas against corresponding analyte concentrations. The calibration curves are illustrated in Figure 4.

All five target compounds—salidroside, tyrosol, HSYA, ASYB, and rhodiolin—exhibited excellent linearity over their respective concentration ranges, with correlation coefficients (R ²) ranging from 0.9991 to 0.9998 (Table 3). The linear ranges were sufficiently wide to cover expected concentrations in both quality control and stability testing scenarios (e.g., 53.29 μg/mL–399.68 μg/mL for salidroside; 52.40 μg/mL–393.00μg/mL for tyrosol; 39.08 μg/mL–293.10 μg/mL for HSYA; 22.40 μg/mL–168.00 μg/mL for ASYB; 10.73 μg/mL–80.47 μg/mL for rhodiolin).

TABLE 3. Results of calibration curves.

System suitability was assessed by six consecutive injections of a mixed standard solution. The relative standard deviation (RSD) of the peak areas for all five compounds was within the range of 0.67%–1.02%, confirming excellent instrumental precision and system stability under the chromatographic conditions utilized (Table 4). Method repeatability was determined by preparing and analyzing six independent sample solutions from a single batch of RAC capsules. The RSD values for the measured contents of salidroside, tyrosol, HSYA, ASYB, and rhodiolin were 0.36%, 0.38%, 1.15%, 1.80%, and 1.79%, respectively—all below 2.0%, demonstrating high repeatability of the sample preparation and analytical procedure. Stability was evaluated by analyzing the HPLC sample solution at 0, 2, 4, 8, 12, 24, and 36 h after preparation. The RSD values of the peak areas over 36 h were calculated to be 0.28%, 0.33%, 0.89%, 1.67%, and 0.92%, respectively (Table 5).

TABLE 4. Results of system suitability related data (A).

TABLE 5. Stability RSD data of sample solution (36 h).

The theoretical plate numbers of salidroside, tyrosol, HSYA, ASYB, and rhodiolin in the batch sample (Lot No. 250101) were 79,962, 62,411, 127,813, 331,474, and 522,888, respectively; the resolution values were 2.2, 1.7, 2.3, 2.8, and 1.7, respectively, indicating that the method achieved satisfactory separation for the above five compounds. The tailing factors were 0.95, 1.05, 0.98, 0.84, and 1.2, respectively. Among them, the tailing factor of salidroside was outside the optimal range of 0.95–1.2, which represents the optimal result obtained under the current laboratory conditions (Table 6).

TABLE 6. Results of system suitability related data (B).

Accuracy was assessed via recovery experiments using the standard addition method. Known amounts of each reference standard were spiked into pre-analyzed RAC capsule powder at approximately 100% of their native concentrations, followed by sample preparation and analysis (n = 6 for each analyte). As summarized in Table 7, the average recoveries ranged from 95.07% to 105.61%, with RSD values between 0.53% and 0.83%.

TABLE 7. Recovery results for the five marker compounds (n = 6).

This method has not yet investigated factors such as flow rate, column temperature, and slight adjustments to the mobile phase ratio. Minor changes in the mobile phase or the testing environment may affect the determination of the sample, and further optimization will be performed in subsequent studies.

3.4 Application to real sample analysis

The validated HPLC method was subsequently applied to the quantitative analysis of six commercial batches of RAC capsules (labeled 250101 to 250901). The contents of the five marker compounds—salidroside, tyrosol, HSYA, ASYB, and rhodiolin—were determined in triplicate for each batch, and the results are presented in Table 8 (expressed in mg per gram of capsule content).

TABLE 8. Content of the five marker compounds in six commercial batches of RAC capsules (n = 3, mg/g).

The measured concentrations of the five marker compounds across all six batches of RAC capsules demonstrated excellent batch-to-batch consistency: salidroside ranged from 3.31 to 3.34 mg/g, tyrosol from 3.22 to 3.25 mg/g, HSYA from 2.08 to 2.14 mg/g, ASYB from 1.04 to 1.22 mg/g, and rhodiolin from 0.70 to 0.72 mg/g. The narrow variation in content underscores the robustness of both the manufacturing process and the analytical method.

The low inter-batch variability (RSD <2.5% for all analytes) demonstrates the robustness of both the manufacturing process and the analytical method. Notably, the consistent levels of C. tinctorius-derived markers (HSYA and ASYB) and R. rosea-derived markers (salidroside, tyrosol, and rhodiolin) confirm the uniformity of herbal composition across production lots. These findings underscore the suitability of the developed method for routine quality control and batch-to-batch consistency evaluation of RAC capsules.

4 Materials and methods

4.1 Chemicals and materials

Chromatographic-grade acetonitrile was obtained from Tianjin Concord Technology Co., Ltd. (China), and phosphoric acid was purchased from Tianjin Damao Chemical Reagent Factory (China). Chloroform, analytical-grade methanol, and ethyl acetate were purchased from Chengdu Kelong Chemicals Co., Ltd. (China). Silica gel plates for TLC (TLC silica gel plates) were obtained from Qingdao Ocean Chemical Co., Ltd. (China). Purified water was prepared using a Milli-Q purification system.

Reference compounds were purchased from the Chinese Food and Drug Accreditation Institute (China), including R. rosea reference drug (Lot No.: 121412-202203), A. membranaceus reference drug (Lot No.: 120974-201813), C. tinctorius reference drug (Lot No.: 120907-201713), salidroside (Lot No.: 110818–202210, purity: >99.7%), tyrosol (Lot No.: 111676-200602), and hydroxysafflor yellow A (Lot No.: 111637–202412, purity: >97.7%). Anhydrosafflor yellow B (Lot No.: KB378750, purity: >98.9%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (China). Rhodiolin (Lot No.: MUST-24062901, purity: >99.4%) was obtained from Chendu MUST Biotechnology, Co. Ltd. (China). The RAC capsules were obtained from Shijiazhuang Zangnuo Pharmaceutical Co., Ltd. (China).

4.2 Preparation of standard TLC solutions

Standard TLC solutions were prepared individually for R. rosea, A. membranaceus, and C. tinctorius reference herbal materials as follows.

For R. rosea: 1 g of the reference drug is accurately weighed, 10 mL of 50% ethanol is added, and ultrasonic extraction is performed for 30 min. The mixture is filtered, and the filtrate is evaporated to dryness under reduced pressure at 40 °C. The residue is dissolved in 20 mL of water and then extracted twice with ethyl acetate (20 mL each time). The organic layers are combined, evaporated to dryness, and the residue is dissolved in 1 mL of methanol to obtain the R. rosea standard solution.

For A. membranaceus: 0.5 g of the reference drug is correctly weighed and processed identically to the above procedure to yield the A. membranaceus standard solution.

For C. tinctorius: 0.1 g of the reference drug is correctly weighed, 10 mL of 50% ethanol is added, and ultrasonic extraction is performed for 30 min. The mixture is filtered, and the filtrate is directly diluted to 1.0 mL with methanol. After filtration, the filtrate is concentrated and finally diluted to 1.0 mL with methanol to obtain the C. tinctorius standard solution.

4.3 Preparation of the standard HPLC solutions

Accurately weighed reference standards of salidroside, tyrosol, HSYA, ASYB, and rhodiolin are individually dissolved in methanol to prepare stock solutions with mass concentrations of 1.07 mg/mL for salidroside, 1.05 mg/mL for tyrosol, 0.98 mg/mL for HSYA, 1.12 mg/mL for ASYB, and 1.07 mg/mL for rhodiolin.

Aliquots of each stock solution are then diluted with 50% ethanol to yield a mixed working solution containing 399.7 μg/mL of salidroside, 393.0 μg/mL of tyrosol, 293.1 μg/mL of HSYA, 168.0 μg/mL of ASYB, and 80.5 μg/mL of rhodiolin. The final solution is vortex-mixed until homogeneous and used for HPLC analysis.

4.4 Preparation of TLC sample solutions

A measure of 10 g of the powdered contents of RAC capsule is accurately weighed into a conical flask, 50 mL of 50% ethanol is added, and the mixture is sonicated for 30 min. It is then filtered through qualitative filter paper. The filtrate is evaporated to dryness under reduced pressure at 40 °C. The residue is dissolved in 30 mL of water and extracted twice with ethyl acetate (30 mL each time). The organic layers are combined, evaporated to dryness, and the residue is dissolved in 2 mL of methanol to obtain the test solution.

Negative control solutions are prepared similarly using RAC formulations lacking R. rosea, A. membranaceus, or C. tinctorius, respectively, to assess the specificity of the TLC identification.

4.5 Preparation of the HPLC sample solutions

Approximately 1.0 g of the powdered contents of RAC capsules is accurately weighed into a 25-mL volumetric flask, 50% ethanol is added to volume, and the sample is sonicated in a water bath (40 KHZ, 500 W) at 25 °C for 30 min. The solution is filtered through a 0.45-μm membrane filter prior to HPLC analysis.

4.6 TLC chromatographic conditions

A volume of 2 μL of each reference herbal solutions (R. rosea, A. membranaceus, and C. tinctorius), 4 μL of the RAC capsule test solution, and the corresponding negative sample solution are applied as bands onto a silica gel GF254 plate (20 cm × 10 cm). The plate is developed in a twin-trough chamber saturated with chloroform–methanol as the mobile phase to a distance of approximately 8 cm–10 cm. After development, the plate is air-dried at room temperature and examined under ultraviolet light at 365 nm without TLC developer.

4.7 HPLC chromatographic conditions

Chromatographic separation is carried out on a Zafex Supperfex JX-C18 column (250 mm × 4.6 mm, 5 μm) with column and autosampler temperatures maintained at 30 °C. The mobile phase consists of 0.1% aqueous phosphoric acid (A) and acetonitrile (B). A gradient elution program is utilized. The flow rate is set at 1.0 mL/min. The samples are detected by UV at 275 nm. The injection volume is 10.0 µL, and the total runtime is 100 min.

5 Conclusion

The RAC capsule is a registered health food subject to pharmaceutical-like regulatory standards, requiring a national health food registration certificate (Guo Shi Jian Zhu Wen Hao) valid for 5 years, with renewal upon expiration (Panossian and Wikman, 2007). In line with evolving regulatory expectations for traditional Chinese medicine products, recent renewal guidelines mandate supplementary quality controls, including TLC identification of constituent herbs, which is now incorporated into technical specifications.

In comparison with existing standards, the present study established TLC identification methods for R. rosea, A. membranaceus, and C. tinctorius in RAC capsules. These methods provide a reliable basis for evaluating the authenticity and quality of the capsule. Notably, simultaneous identification of the three medicinal materials can be achieved on a single silica gel plate, thereby significantly improving analytical efficiency.

Chinese medicinal materials contain a wide range of chemical constituents, and the composition of compound preparations is even more complex. With the continuous progress of research on traditional Chinese medicine, comprehensive evaluation of the quality of medicinal materials and preparations using multiple components has become an inevitable trend.

In the present study on the HPLC assay method of RAC capsules, compared with existing standards, quantitative determination methods for four components, namely, tyrosol, HSYA, ASYB, and rhodiolin, were newly established in addition to salidroside. The analysis of six batches of large-scale production samples showed that the average contents of salidroside, tyrosol, HSYA, ASYB, and rhodiolin were 3.33 mg/g, 3.24 mg/g, 2.12 mg/g, 1.12 mg/g, and 0.71 mg/g, respectively. These results enable a more comprehensive and scientific quality evaluation of RAC capsules.

In this study, TLC and HPLC methods were developed to establish a comprehensive quality standard for RAC capsules—a formulation based on medicinal and edible homologous herbs. TLC enabled simultaneous identification of R. rosea, A. membranaceus, and C. tinctorius under unified conditions, ensuring the presence of all three botanicals. Meanwhile, a validated HPLC method allowed simultaneous quantification of five bioactive markers: salidroside, tyrosol, HSYA, ASYB, and rhodiolin. Together, these approaches enhance the quality control of RAC capsules, support standardization of multi-herb health foods, and provide a practical framework for quality assessment of similar products.

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Footnotes

Edited by: Bruno Alves Rocha, University of São Paulo, Brazil

Reviewed by: Kumar Katragunta, University of Mississippi, United States

Abhishek Kumar Mishra, Tergus Pharma, United States

Rachid Touzani, Mohamed Premier University, Morocco

Footnotes

RAC capsule, Rhodiola rosea, Astragalus membranaceus, and Carthamus tinctorius capsule; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography; HSYA, hydroxysafflor yellow A; ASYB, anhydrosafflor yellow B; NADES, natural deep eutectic solvents.