Quality Analysis of Chlorogenic Acid and Hyperoside in Crataegi fructus.

BACKGROUND: Crataegi fructus is a herbal medicine for strong stomach, sterilization, and alcohol detoxification. Chlorogenic acid and hyperoside are the major compounds in Crataegi fructus.
OBJECTIVE: In this study, we established novel high-performance liquid chromatography (HPLC)-diode array detection analysis method of chlorogenic acid and hyperoside for quality control of Crataegi fructus.
MATERIALS AND METHODS: HPLC analysis was achieved on a reverse-phase C18 column (5 μm, 4.6 mm × 250 mm) using water and acetonitrile as mobile phase with gradient system. The method was validated for linearity, precision, and accuracy. About 31 batches of Crataegi fructus samples collected from Korea and China were analyzed by using HPLC fingerprint of developed HPLC method. Then, the contents of chlorogenic acid and hyperoside were compared for quality evaluation of Crataegi fructus.
RESULTS: The results have shown that the average contents (w/w %) of chlorogenic acid and hyperoside in Crataegi fructus collected from Korea were 0.0438% and 0.0416%, respectively, and the average contents (w/w %) of 0.0399% and 0.0325%, respectively.
CONCLUSION: In conclusion, established HPLC analysis method was stable and could provide efficient quality evaluation for monitoring of commercial Crataegi fructus.
SUMMARY: Quantitative analysis method of chlorogenic acid and hyperoside in Crataegi fructus is developed by high.performance liquid chromatography.(HPLC).diode array detectionEstablished HPLC analysis method is validated with linearity, precision, and accuracyThe developed method was successfully applied for quantitative analysis of Crataegi fructus sample collected from Korea and China. Abbreviations used: HPLC: High-performance liquid chromatography, GC: Gas chromatography, MS: Mass spectrometer, LOD: Limits of detection, LOQ: Limits of quantification, RSD: Relative standard deviation, RRT: Relative retention time, RPA: Relation peak area.

INTRODUCTION

Herbal medicines have been widely used for the prevention and treatment of many diseases for 1000 of years. Herbal medicines had fewer side effects and were suitable for body. Various pharmacological effects of herbal medicines have been attributed to many bioactive compounds.[12]

Because of complex composition and variation by culture environment of the compounds in herbal medicines, quality consistency control of herbal medicines is difficult. Thus, accurate and stable analysis method of herbal medicine is required for quality evaluation. Analysis systems, such as high-performance liquid chromatography (HPLC), gas chromatography, and mass spectrometer have been widely used for qualitative and quantitative analysis of compounds in herbal medicines. Among systems, HPLC is the most common system for quality control of herbal medicines using characteristic fingerprint analysis.[34] HPLC chromatographic fingerprint technology is the comprehensive peak identification method and is applied to reveal bioactivity and chemical information of herbal medicines.[5] HPLC fingerprint is additionally used to approach a quality analysis of compounds in herbal medicine.[6]

Crataegi fructus is the fruit part of Crataegus pinnatifida Bunge var. typica Schneider (Rosaceae family) and is a well-known herbal medicine for strong stomach, analgesic effect, sterilization, and alcohol detoxification. Recent studies showed that Crataegi fructus prevents hyperlipidemia induced by alcohol and ameliorate arterial contraction via antioxidant effect.[7] Crataegi fructus also showed inhibition effect of sodium nitroprusside-induced cell lipid peroxidation and anti-inflammatory effect.[8]

Chlorogenic acid and hyperoside are major bioactive compounds in Crataegi fructus. Chlorogenic acid, a related family of hydroxycinnamic acid, showed anti-diabetic and anti-lipidemic effects.[9] Hyperoside is one of the phenolic compounds and showed antiviral activity, anti-inflammatory effect, and protective effect on PC12 cells against cytotoxicity.[101112]

Previous study reported simultaneous determination of eight polyphenols in the leaves of Crataegus pinnatifida by HPLC and HPLC analysis method of chlorogenic acid, hyperoside, and rutin for quality control of Crataegi fructus.[1314]

We determinate HPLC analysis method and improve the resolution of hyperoside using modified gradient system of mobile phase based on previous studies.

In this study, quantitative determination of chlorogenic acid and hyperoside in Crataegi fructus was developed and analyzed in 31 Crataegi fructus samples collected from Korea and China.

MATERIALS AND METHODS

Chemicals and plant materials

Water and acetonitrile as HPLC grade were purchased from J.T Baker. Standards of chlorogenic acid and hyperoside are both obtained from the Ministry of Food and Drug Safety (Korea) [Figure 1].

Figure 1

Chemical structures of chlorogenic acid and hyperoside

The 4 batches of Crataegi fructus samples (collected from Korea, 11D2001, 2002, 2024, 2027) were obtained from the Ministry of Food and Drug Safety (Korea). 11 batches of Crataegi fructus samples (collected from Korea, 12D3017-3027) and 16 batches of Crataegi fructus samples (collected from China, 12D3001-3016) were purchased from Kyungdong mart (Seoul, Korea).

Preparation of samples and standard solution

Samples of Crataegi fructus powder were accurately weighed and extracted by sonication with 70% methanol for 60 min. Before HPLC analysis, extract solutions were filtered with 0.45 µm membrane filter.

Stock standard solution of chlorogenic acid and hyperoside were prepared in methanol at a concentration of 1000 µg/mL. Stock standard solutions appropriately diluted to obtain work standard solutions by methanol.

High-performance liquid chromatography-diode array detection condition

HPLC was performed on Dionex Ultimate 3000 HPLC system (Dionex, Germany) equipped with a pump (LPG 3×00), an auto sampler (ACC-3000), a column oven (TCC-3000SD), and diode array UV/VIS detector (DAD-3000(RS)). HPLC chromatogram data were processed using Dionex Chromeleon™ Chromatography Data System.

Chromatographic separation was conducted on Agilent eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm) at column temperature 30°C The mobile phase consisted of aqueous with 0.1% trifluoroacetic acid solution (A) acetonitrile (B) using gradient elution system of 5% (B) at 0–5 min and 5–50% (B) at 5-40 min and flow rate was 1.0 mL/min. The ultraviolet (UV) wavelength was selected and monitored at 254 nm according to the wavelength of chlorogenic acid and hyperoside.

RESULTS AND DISCUSSION

Optimization of chromatographic conditions

In the previous study, resolution of hyperoside and rutin is low. Therefore, we evaluated HPLC condition for improvement of resolution of hyperoside. Mobile phase system (acetonitrile-water, methanol-water, and acetonitrile and aqueous with 0.1% trifluoroacetic acid), gradient elution system, and column temperature (25°C, 30°C and 35°C) were tested to obtain good resolution.[1516]

Acetonitrile and water were found to be effective for chromatographic separation, and trifluoroacetic acid was used for the inhibition of compounds and improvement of peak shape. Gradient elution system was proposed to achieve good separation. Overall, gradient elution system was varied as follows: 0–5 min, 5% acetonitrile; and 5–40 min, 5–50% acetonitrile based on previous study. Flow rate of 1.0 ml/min and column temperature of 30°C were selected. The detection UV wavelength of 254 nm was selected according to the wavelength of two compounds.

We evaluated resolution value (R) of hyperoside using the retention time (tR1 and tR2) and the widths (W1 and W2) of two peaks. R ≥1.5 represents good peak resolution in many cases. Resolution value of hyperoside was 1.76.

HPLC chromatograms of standard compounds and Crataegi fructus sample were obtained without interfering peaks on optimized separation condition [Figure 2].

Figure 2

High-performance liquid chromatography-diode array detection chromatograms for chlorogenic acid and hyperoside in Crataegi fructus. (a) Standards, (b)Crataegi fructus sample

Method validation

Linearity, limits of detection, and limits of quantification

The calibration curves of chlorogenic acid and hyperoside were constructed by plotting the peak area versus six different concentrations of standard solutions. Limits of detection (LOD) and limits of quantification (LOQ) of two compounds were investigated at noise ratio of 3 and 10, respectively. The linear regression equations were present as Y = ax + b (a: The slope of the calibration curve, b: The intercept of calibration curve, x: Concentration and Y: Peak area of compound). Regression equations and correlation coefficients of chlorogenic acid and hyperoside were Y = 0.0857x + 0.1784 (R2 = 0.9995) and Y = 0.1669x + 0.349 (R2 = 0.9986), respectively. The LOD of chlorogenic acid and hyperoside were 0.26 µg/ml and 0.04 µg/ml, respectively, and the LOQ were found to be 0.78 µg/ml and 0.12 µg/ml for chlorogenic acid and hyperoside, respectively [Table 1].

Table 1

Regression equation, correlation coefficient (R2), limits of detection, and limits of quantitation

Results showed that this HPLC method has good linearity and high detection sensitivity for chlorogenic acid and hyperoside.

Precious and accuracy

The inter- and intra-day test with chlorogenic acid and hyperoside at three different concentrations were performed to investigate the precision of this HPLC method. Previously repeatability of this method was indicated and determined as relative standard deviation (RSD) values of inter- and intra-day. The intra-day test was continuously analyzed by injection standard solution 5 times on the same day and the inter-day test was analyzed for 3 consecutive days (1, 3, and 5 days).

The intra- and inter-day RSD values of chlorogenic acid and hyperoside were in the range from 0.05–1.02% to 0.62–2.49%, respectively [Table 2].

Table 2

Analytical results of intra- and inter-day variability

Accuracy of analysis method was determined as recovery test. Recovery was investigated by spike test. Three different concentrations of chlorogenic acid and hyperoside were added into the previously analyzed Crataegi fructus sample, and then the sample was analyzed in triplicate. Accuracy (%) was calculated by difference of spiked and unspiked sample.

The recovery value of two compounds ranged from 97.02% to 109.39% with RSD <1.17% [Table 3].

Table 3

Results of recovery test

Quantitative analysis of chlorogenic acid and hyperoside in Crataegi fructus samples

To obtain the HPLC fingerprint of Crataegi fructus, 31 batches of Crataegi fructus from different areas were analyzed by optimized HPLC method. Peaks of chlorogenic acid and hyperoside are identified in all the samples. Among different peaks, chlorogenic acid and hyperoside have relatively high intensity peak in Crataegi fructus [Figure 3].

Figure 3

Chromatographic fingerprints for 31 Crataegi fructus samples

Relative retention time and relation peak area of chlorogenic acid and hyperoside peak were calculated [Table 4].

Table 4

Relative retention time and relation peak area of the chlorogenic acid and hyperoside in Crataegi Fructus

This HPLC method was applied for the quantitative determination of chlorogenic acid and hyperoside in 31 Crataegi fructus samples using calibration curve of two compounds. Among Crataegi fructus sample, 15 species were collected from Korea and 16 species were collected from China. The content results of chlorogenic acid and hyperoside were shown in Table 5.

Table 5

Contents (μg/mg) of the chlorogenic acid and hyperoside in the 30 Crataegi Fructus

The content (w/w %) of chlorogenic acid in Crataegi fructus ranges were as follows: 0.0129–0.1065% (Korea) and 0.0121–0.1125% (China). The contents of hyperoside ranged from 0.0150–0.0660% (Korea) to 0.0204–0.0560% (China). The average content of chlorogenic acid in Crataegi fructus collected from Korea (0.0477%) higher than that in China sample (0.0399%). Moreover, the average content of hyperoside in Crataegi fructus collected from Korea (0.0450%) was higher than that in China sample (0.0325%).

The difference of contents in Crataegi fructus was due to cultivation environment including the year of the plant cultivation, harvest time, plant origins, climate, and cultivated location.

Principal components analysis (PCA) was conducted by scatterplot for quantitative evaluation. Results of PCA showed that the contents of Crataegi fructus collected from Korea and China were different by two developed clusters [Figure 4].

Figure 4

Principal components analysis projection of 31 Crataegi fructus samples. ■: Crataegi Fructus sample collected from China, ○: Crataegi fructus sample collected from Korea

We evaluated reproducibility of this HPLC method by cross-monitoring with other researchers. Cross-monitor was performed using some Crataegi fructus samples and compared to the content results of chlorogenic acid and hyperoside of other researchers. Reproducibility is determined from the difference between two test results obtained from same HPLC conditions. However, production of HPLC system and column is different. The results are listed in Table 6 and there was no difference between the contents of chlorogenic acid and hyperoside in other researchers [Figure 5].

Table 6

Results of cross-monitoring

Figure 5

Principal components analysis projection of cross-monitoring of Crataegi fructus samples

The results of cross-monitor indicated that our HPLC method had a good reproducibility.

CONCLUSION

In this study, we developed quantitative analysis method of chlorogenic acid and hyperoside in Crataegi fructus and validated with linearity, precision, and accuracy. Results of validation indicated that this HPLC method was accurate and stable for the analysis of Crataegi fructus. Moreover, the developed method was successfully applied for quantitative analysis of Crataegi fructus sample collected from Korea and China.

The results of validation and Crataegi fructus sample analysis could be used as a reference for monitoring and quality control of Crataegi fructus.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors declare no conflicts of interest.

Choong Je Ma

ABOUT AUTHOR

Choong Je Ma, has completed his PhD at the age of 32 years from Seoul National University and postdoctoral studies from the University of Michigan. He is the professor of Department of Medical Biomaterials Engineering, College of Biomedical science, Kangwon National University, Korea. He has published more than 20 papers in reputed journals.