Abstract

Objective To establish an HPLC method for the simultaneous determination of morroniside, loganin, cinnamic acid, paeonol, 7-hydroxycoumarin, and acteoside in Jinkui Shenqi Pills and conduct a comprehensive quality evaluation. Methods Separation was performed on an Agilent EC-C18 column (150 mm×2.1 mm, 1.9 μm) with a mobile phase of acetonitrile–0.3% phosphoric acid solution under gradient elution. The flow rate was 1.0 mL/min, detection wavelength 254 nm, column temperature 35 ℃, and injection volume 10 μL. Contents of the six components in different batches of Jinkui Shenqi Pills were determined, and quality was evaluated via principal component analysis (PCA). Results All six components showed excellent linear relationships (r≥0.9995) within their respective concentration ranges. The average spiked recoveries ranged from 98.69% to 102.54%. PCA extracted two principal components with a cumulative variance contribution rate of 88.75%. Conclusion The established HPLC method enables simultaneous quantification of multiple index components and reliable quality assessment of Jinkui Shenqi Pills, supporting standardized quality control of classic TCM formulations.

Keywords: Jinkui Shenqi Pills; HPLC; content determination; principal component analysis; TCM herb for kidney health; TCM herb for fatigue relief; TCM herb for immune support; Cistanche tubulosa extract; acteoside

 

 

Introduction

Jinkui Shenqi Pills are a classic TCM formula originally recorded in Synopsis of Prescriptions of the Golden Chamber. Composed of Rehmannia glutinosa, Dioscorea opposita, wine-processed Cornus officinalis, Poria cocos, Moutan cortex, Alisma orientale, Cinnamomum cassia, Achyranthes bidentata, salt-processed Plantago asiatica, processed Aconitum carmichaelii, and honey, this formula functions to warm and tonify kidney yang, promote qi transformation, and induce diuresis.

 

With the advancement of modern medicine, quality control of TCM has become increasingly stringent. The quality of TCM patent medicines directly affects clinical efficacy and safety. Although Jinkui Shenqi Pills are included in the Ministry of Health of the People's Republic of China Drug Standards: Traditional Chinese Medicine Preparations, current quality control systems are limited and cannot fully reflect intrinsic quality.

To improve quality evaluation standards, this study developed an HPLC method to simultaneously quantify six bioactive components: morroniside, loganin, paeonol, cinnamic acid, acteoside, and 7-hydroxycoumarin. These markers support kidney function, relieve fatigue, and enhance immunity-TCM herb for kidney deficiency, TCM herb for physical fatigue, TCM herb for low immunity.

Notably, acteoside (verbascoside) is also a signature active ingredient in Cistanche tubulosa extract, a premium TCM herb for anti-fatigue, kidney health, antioxidant defense, and immune enhancement. As a specialized manufacturer, we supply high-purity Cistanche tubulosa extract with superior acteoside content for functional foods, dietary supplements, and skincare.

 

1 Instruments and Materials

1.1 Instruments

Agilent 1260 High-Performance Liquid Chromatography (Agilent Technologies, USA)

EP225SM-DR Electronic Balance (Precisa, Switzerland)

MJ-300 Ultrasonic Cleaner (Wuxi Meiji Ultrasonic Equipment Co., Ltd.)

1.2 Materials

Reference substances: morroniside (batch 111998-202406, 97.8%), loganin (111640-202309, 99.2%), paeonol (110708-202309, 99.9%), cinnamic acid (110786-202305, 99.8%), acteoside (111530-202315, 97.6%), 7-hydroxycoumarin (111739-200501, 98.0%) (National Institutes for Food and Drug Control, China)

Acetonitrile (HPLC grade); phosphoric acid (analytical grade); ultrapure water

Herbal materials: authenticated genuine by Deputy Chief Pharmacist Ding Hongqing

Six batches of Jinkui Shenqi Pills (6 g/pill) from Tongrentang and Kuihua Pharmaceutical

 

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2 Methods and Results

2.1 Preparation of Test Solution

Finely cut ~2.0 g of Jinkui Shenqi Pills, accurately weigh, add 25 mL of 70% methanol, weigh again, and extract ultrasonically (300 W, 40 kHz) for 35 min. Cool to room temperature, replenish weight loss with 70% methanol, mix well, filter through a 0.45 μm membrane filter, and use the filtrate as the test solution.

2.2 Preparation of Mixed Reference Solution

Accurately weigh appropriate amounts of morroniside, loganin, cinnamic acid, paeonol, 7-hydroxycoumarin, and acteoside, dissolve in 70% methanol, and prepare a mixed reference solution with concentrations of 0.2957, 0.2026, 0.2020, 0.2006, 0.2942, 0.2967 mg/mL, respectively.

2.3 Preparation of Negative Sample Solutions

Prepare placebo samples lacking Cornus officinalis, Cinnamomum cassia, Moutan cortex, or Rehmannia glutinosa following the standard manufacturing process, then process as in Section 2.1.

2.4 Chromatographic Conditions

Column: Agilent EC-C18 (150 mm×2.1 mm, 1.9 μm)

Mobile phase: acetonitrile (A) – 0.3% phosphoric acid (B), gradient elution

Gradient: 0–10 min 10%→20% A; 10–20 min 20%→30% A; 20–30 min 30%→50% A; 30–40 min 50%→90% A

Flow rate: 1.0 mL/min; detection wavelength: 254 nm; column temperature: 35 ℃; injection volume: 10 μL

2.5 Linearity

Calibration curves were constructed by plotting peak area vs. concentration. All components showed r≥0.9995, confirming excellent linearity.

2.6 Methodology Validation

Precision: RSDs = 0.47%–0.85% (n=6), indicating excellent instrument precision

Repeatability: RSDs = 0.46%–0.97% (n=6), demonstrating good method reproducibility

Stability: RSDs = 0.69%–1.01% within 24 h, confirming sample stability

Recovery: Average recoveries = 98.69%–102.54%, RSDs = 0.47%–0.92%, verifying accuracy

2.7 Sample Assay

Six batches were analyzed; contents of the six components were consistent across batches, reflecting stable manufacturing quality.

2.8 Principal Component Analysis

PCA extracted two principal components explaining 88.75% of total variance.

PC1: morroniside, cinnamic acid, paeonol (major contributors)

PC2: acteoside (major contributor)

Acteoside is a key quality marker for both Jinkui Shenqi Pills and Cistanche tubulosa extract, a TCM herb for kidney wellness, anti-oxidation, and cognitive support.

 

 

3 Discussion

3.1 Selection of Index Components

Morroniside: TCM herb for antioxidant, neuroprotection, and kidney function support

Loganin: TCM herb for immune regulation, anti-inflammation, and cardiovascular protection

Paeonol: TCM herb for anti-inflammatory, antimicrobial, and antioxidant effects

Cinnamic acid: TCM herb for immune enhancement, improved circulation, and antioxidant activity

Acteoside: TCM herb for potent antioxidant, anti-inflammatory, neuroprotective, and kidney-replenishing effects

7-Hydroxycoumarin: TCM herb for antimicrobial, anti-inflammatory, and immune-modulating activity

These components reflect the core efficacy of Jinkui Shenqi Pills and align with the bioactivity profile of Cistanche tubulosa extract, which is rich in acteoside and widely used in TCM herb for kidney deficiency, fatigue, and aging support.

3.2 Optimization of Chromatography

Acetonitrile–0.3% phosphoric acid provided sharp peaks and improved separation. 35 ℃ and 254 nm allowed simultaneous determination of all six components with stable response and low baseline noise.

3.3 Results and Application

The validated HPLC method is simple, robust, and accurate for multi-component quantification. Consistent component levels across batches confirm stable product quality. This approach supports the modernization of TCM quality control and can be extended to formulations containing Cistanche tubulosa extract-a premium TCM herb for kidney health, energy, and immune resilience with naturally high acteoside content.

 

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