Quality assessment of Rhizoma et Radix Notopterygii by HPTLC and HPLC fingerprinting and HPLC quantitative analysis

doi:10.1016/j.jpba.2007.03.029

Journal of Pharmaceutical and Biomedical Analysis

Volume 44, Issue 3, 27 July 2007, Pages 812-817

https://doi.org/10.1016/j.jpba.2007.03.029 Get rights and content

Abstract

This paper describes an improved quality assessment method for Rhizoma et Radix Notopterygii (the rhizome and root of Notopterygium incisum Ting ex H.T. Chang or Notopterygium forbesii Boiss). The method was established by using fingerprinting and quantitation of marker compounds (isoimperatorin, notopterol and bergapten) in this herbal medicine. The authentication of Rhizoma et Radix Notopterygii using high performance thin-layer chromatography (HPTLC) fingerprinting was achieved by comparing the colors and R_f values of the bands in TLC fingerprints with those of the marker compounds. The HPLC fingerprints of 16 batches of herbal samples from different regions of China showed similar chromatographic patterns. Five peaks were selected as characteristic peaks, and three of these were identified by using LC–MS–MS techniques. The relative retention times of these characteristic peaks in the HPLC fingerprint were established as an important parameter for identification of Rhizoma et Radix Notopterygii. Finally, the pharmacologically active marker compounds isoimperatorin, notopterol and bergapten in this herb were quantitatively determined using a validated reverse-phase HPLC method.

Introduction

Rhizoma et Radix Notopterygii (Qianghuo), the dried rhizome and root of Notopterygium incisum Ting ex H.T. Chang or Notopterygium forbesii Boiss (family Umbelliferae), is a well-known traditional Chinese medicine that is used widely as an anti-rheumatic and pain-relieving herb for the treatment of colds and rheumatism [1]. Previous research on the chemical constituents of Rhizoma et Radix Notopterygii has shown that it contains a variety of coumarins, sugars, amino acids, organic acids, mono and sesquiterpenes, polyacetylenes and phenolic compounds [2], [3], [4], [5]. Pharmacological studies have indicated that coumarins such as isoimperatorin, notopterol and bergapten possess anti-inflammatory, analgesic, anti-cancer and anti-coagulant activities [6], [7], [8].

Because the clinical use of Rhizoma et Radix Notopterygii and its medicinal products is common in China, a reasonable assessment method for evaluating the quality of this crude drug is essential to ensure the efficiency and stability of its products. In Chinese Pharmacopoeia (2005 edition), the quantitation of total essential oils was set as the parameter for quality control of this herb [1]. Authentication of Rhizoma et Radix Notopterygii using chromatographic fingerprinting techniques such as TLC and HPLC have been rarely reported. Most of these previous reports focused on the quantitative analysis of one or two constituents using TLC and HPLC [9], [10], [11], [12]. Recently, an HPLC fingerprinting study of the water extract of “Qianghuo” was conducted, and ferulic acid, imperatorin and isoimperatorin were identified [13]. To establish an improved quality assessment method for Rhizoma et Radix Notopterygii, in the present paper high performance thin-layer chromatography (HPTLC) fingerprinting and HPLC fingerprinting of multiple components in this herb were conducted. These fingerprinting chromatograms can provide sufficient qualitative information for the identification and authentication of Rhizoma et Radix Notopterygii. Sixteen batches of authenticated herbal drugs, originating from two species of the genus Notopterygium, were collected from different regions of China for the analysis. In addition, three pharmacologically active components (isoimperatorin, notopterol and bergapten) in the herbal medicine were quantitatively determined by using reverse-phase HPLC for the purpose of quality assessment.

Section snippets

Herbal materials

Ten and six samples of Rhizoma et Radix Notopterygii derived from N. incisum and N. forbesii, respectively, were collected from different regions of China and were air-dried according to the procedure described in Chinese Pharmacopoeia (2005 edition). All of the samples were identified by one of the authors (Z. Zhao) and deposited in the Centre of Chinese Materia Medica, Hong Kong Baptist University.

Standards and reagents

Isoimperatorin and bergapten (purity > 98%) were purchased from the National Institute for the

HPTLC fingerprinting identification

The performance of TLC using a sorbent with a homogeneous particle size of ∼5 μm and with a narrow particle size distribution has been confirmed to be superior to that of the conventional TLC plate that was used in many pharmacopoeias for the identification of herbal materials. Therefore, in this study HPTLC plates were used to establish a TLC fingerprinting method. The chromatographic conditions, in particular the developing solvents (i.e., types of solvents and ratio), were carefully optimized

Conclusion

A comprehensive quality assessment method for Rhizoma et Radix Notopterygii was established. In contrast to the conventional quality assessment standard of Rhizoma et Radix Notopterygii [1], the present improved method adopts HPTLC and HPLC fingerprinting of total extracts of the herb. This method provides more chemical information that can be used for the identification of the crude drug as well as for the quantitation of three pharmacologically active marker compounds that are directly

Acknowledgments

This work is part of a project studying some common Chinese Materia Medica in Hong Kong that was commissioned by the Department of Health, Hong Kong Government of Special Administrative Region, PR China. The authors thank Mr. Chi Leung Chan for technical support in the LC–MS–MS experiments.

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Because NRR and its medicinal products are widely used in clinical practice in China, a reasonable evaluation method for the quality control of NRR is essential to ensure the safety and efficiency of its products. So far, many methods have been reported for the determination of the active components in NRR, mainly including high performance thin layer chromatography (HPTLC)[10], gas chromatography-mass spectrometry(GC–MS)[11], high performance liquid chromatography-ultraviolet detector(HPLC-UV)[12], thin layer chromatography(TLC)[13], high performance liquid chromatography-fluorescence detector(HPLC-FLD)[14], liquid chromatography-electrospray ionization-multistage mass spectrometry(HPLC-ESI-MS/MS)[15], and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)[16], etc. Although HPLC-MS/MS or UHPLC-MS/MS has become the first choice due to their high sensitivity and high selectivity, they also have some disadvantages, such as high detection cost and technical requirements, consumption of a large number of organic solvents, etc.
This work has developed an isocratic micellar liquid chromatographic method for simultaneous determination of nodakenin, isoimperatorin, psoralen, ferulic acid, and chlorogenic acid in notopterygium and notopterygium processed products for the first time. Analytes were successfully separated within 15 min. The optimized chromatographic conditions were obtained by Box-Behnken design optimization, and the mobile phase consisted of 0.064 M sodium lauryl sulfate aqueous solution and 8.15% n-pentanol with a pH value of 3.04. The analytes were eluted isocratically through a C18 column at a flow rate of 1 mL/min. The linear ranges, limit of detection, limit of quantitation, recovery, intra-day and inter-day precision of the proposed method were validated according to ICH guidelines. The proposed method was successfully applied to the quantitative the 5 active components in notopterygium and its processed products. It is a simple, efficient, and low-cost analytical method that provides an alternative method for the determination of the 5 active ingredients in notopterygium.
The Natural Compound Notopterol Binds and Targets JAK2/3 to Ameliorate Inflammation and Arthritis
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Here, we described that notopterol, a natural active ingredient of the traditional Chinese medicinal herb Notopterygium incisum Ting ex H.T. Chang, may have significant therapeutic effects for the treatment of RA. Notopterol can be readily extracted from Notopterygium incisum Ting ex H.T. Chang and is less expensive in long-term treatment (Qian et al., 2007). In addition, notopterol is equally efficacious delivered orally or intraperitoneally, which can effectively treat individuals of RA mouse model without inducing detectable side effects at our tested dosages.
The traditional Chinese medicinal herb Notopterygium incisum Ting ex H.T. Chang has anti-rheumatism activity, and a mass spectrometry assay of patients’ serum after administration of the herb revealed that notopterol is the most abundant component enriched. However, the functions of notopterol and its molecular target in rheumatoid arthritis (RA) treatment remain unknown. Here, we show in different RA mouse strains that both oral and intraperitoneal administration of notopterol result in significant therapeutic effects. Mechanistically, notopterol directly binds Janus kinase (JAK)2 and JAK3 kinase domains to inhibit JAK/signal transducers and activators of transcription (JAK-STAT) activation, leading to reduced production of inflammatory cytokines and chemokines. Critically, combination therapy using both notopterol and tumor necrosis factor (TNF) blocker results in enhanced therapeutic effects compared to using TNF blocker alone. We demonstrate that notopterol ameliorates RA pathology by targeting JAK-STAT signaling, raising the possibility that notopterol could be effective in treating other diseases characterized by aberrant JAK-STAT signaling pathway.
Separation and quantitation of notopterol enantiomers in notopterygii rhizoma et radix using solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry
2020, Journal of Pharmaceutical and Biomedical Analysis
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Currently, commercial single notopterol enantiomer was unavailable, and there was no literature about the enantioseparation of notopterol enantiomers. Thus, the silica gel G plate with toluene-ethylacetate (70:30, v/v) as the developing solvent, was selected for the preparation of the (+)-notopterol, of which configuration confirmation was determined by circular dichroism spectrum [17]. As can be seen from Fig. 4, the Rf value of notopterol was 0.5, and notopterol was completely separated from other components.
In this study, a specific, sensitive, and reliable high performance liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantitative determination of notopterol enantiomers in Notopterygii Rhizoma et Radix. Solid-phase extraction was used for the extraction of notopterol enantiomers from Notopterygii Rhizoma et Radix. Enantiomeric separation of notopterol was achieved on a Chiralpak IA column with the mobile phase consisting of acetonitrile-water (50:50, v/v) at a flow rate of 0.6 mL/min. Quantification was performed on a triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source in the positive ionization and multiple reaction monitoring mode. The lower limit of quantification and lower limit of detection were 0.09 and 0.04 mg/g for each enantiomer in NRR samples. And each enantiomer showed good linearity (R²≥0.999) in the range of 0.09–9.55 mg/g. The precision, repeatability, and stability were all within satisfaction. The average recoveries of (-)-notopterol and (+)-notopterol were demonstrated to be 99.3 % and 101.1 %, respectively, with the relative standard deviations less than 5.0 %. Finally, the validated method was successfully applied to the quantification of notopterol enantiomers in Notopterygii Rhizoma et Radix from different sources.
Simultaneous quantification of 33 active components in Notopterygii Rhizoma et Radix using ultra high performance liquid chromatography with tandem mass spectrometry
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Through further analysis of chemical composition, dozens of phenolic acid esters, such as isopropylferulate, benzylsalicylate, p-hydroxyphenethyl anisate, (−)-bornylferulate, phenethylferulate, 4-methoxyphenethylferulate, and 4-methyl-3-trans-hexenylferulate, have been isolated from NRR and also demonstrated to be its bioactive components [11–13]. In view of the current situation, a variety of methods, including high-performance thin layer chromatography (HPTLC) [14], gas chromatography-mass spectrometer (GC–MS) [15], and high performance liquid chromatography (HPLC) coupled with a range of techniques, such as fluorescence detection (FLD) [16], diode array detection (DAD) [14, 15] and MS [17], have been employed for the determination of NRR. However, all these methods had a number of different shortcomings, such as, low sensitivity and selectivity for HPTLC and HPLC–DAD, narrow application scope for GC–MS and HPLC–FLD, and long assay time for HPLC–FLD, HPLC–DAD and HPLC–MS. Additionally, these methods only determinated a few analytes and were mainly focused on the quantification analysis of coumarins, including notopterol, isoimperatorin, nodakenin, bergaptol, bergapten, and bergamottin [18, 19].
A method of ultra high performance liquid chromatography with tandem mass spectrometry was developed for the simultaneous quantification of 33 active components including 26 coumarins and 7 phenolic acid esters in Notopterygii Rhizoma et Radix (NRR). Chromatographic separation was achieved on a Kinetex® C₁₈ column (100 mm × 2.1 mm i.d.; 2.6 μm) with a gradient elution in 18 min. Electrospray ionization tandem mass spectrometric detection was conducted in the positive and negative ionization modes with multiple reaction monitoring. All of 33 analytes showed good linearity (R² ≥ 0.9919) within the test range. The relative standard deviations of the precision, repeatability and stability were not exceeding 4.97%, and the recoveries were in the range of 85.37%–115.00%. The matrix effect on the response of target analyte was not obvious. The validated method was successfully applied to quantify the 33 components in ten batches of NRR samples. Quantitative results showed the coumarins and phenolic acid esters with large difference in level of content in the herb samples. Furthermore, hierarchical cluster analysis and principal component analysis were applied to classify and discriminate these samples. The variations of isoimperatorin, notopterol, bergamottin, nodakenin, phenethylferulate were suggested as important indicators of NRR quality. This work may serve for quality identification and comprehensive evaluation of NRR.
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The developed fingerprint pattern of components can then be used to determine not only the absence or presence of markers of interest but the ratio of all detectable analytes as well. Although high performance thin layer chromatography (HPTLC) has a few limitations, such as the limited developing distance and lower plate efficiency by comparison with HPLC and GC, it is still an effective tool for quality evaluation of herbal drugs due to its simplicity, low cost, and requirement, and it has been successfully utilized to develop the chromatographic fingerprint for botanical drugs (Chen et al., 2006; Anandjiwala et al., 2007; Qian et al., 2007). Moreover, the above mentioned shortcomings can be overcome by separately developing fractions of different polarity on two or several thin layer plates.
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Short communicationQuality assessment of Rhizoma et Radix Notopterygii by HPTLC and HPLC fingerprinting and HPLC quantitative analysis

Abstract

Introduction

Section snippets

Herbal materials

Standards and reagents

HPTLC fingerprinting identification

Conclusion

Acknowledgments

J. Ethnopharmacol.

Chem. Pharm. Bull.

Chin. Tradit. Herbal Drugs (Zhong Cao Yao).

Acta Pharmaceutica Sinica

Analytical Handbook of Active Component in Chinese Medicine

Chem. Pharm. Bull.

Short communication
Quality assessment of Rhizoma et Radix Notopterygii by HPTLC and HPLC fingerprinting and HPLC quantitative analysis