Ultra performance liquid chromatography coupled with principal component and cluster analysis of Swertia chirayita for adulteration check

doi:10.1016/j.jpba.2018.10.054

Journal of Pharmaceutical and Biomedical Analysis

Volume 164, 5 February 2019, Pages 302-308

https://doi.org/10.1016/j.jpba.2018.10.054 Get rights and content

Highlights

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UPLC-PDA method for simultaneous analysis of four glycosides (iridoid and xanthone).
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Five Swertia chirayita ecotypes and five other related species were analysed.
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Chemometric tools like similarity analysis, PCA, and HCA used to distinguish the admixing of substitute/adulterants in the raw material.
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PCA analysis of fingerprint confirmed role of sweroside & swertiamarinin in Swertia species discrimination.

Abstract

This article describes the study to standardize phytochemically and distinguish Swertia chirayita from that of possible substitution/adulteration using ultra performance liquid chromatography (UPLC) with photodiode array detector (PDA) and chemometric tools viz. principal component analysis (PCA) and hierarchical clustering analysis (HCA). Five ecotypes of Swertia chirayita and five possible substitutions, e.g.,Swertia bimaculata (SB), Swertia chordata (SCH), Swertia ciliata (SCL), Swertia paniculata (SP), and Halenia elliptica (HE) collected from different Indian Himalayan region. Samples evaluated for 04 marker compounds- swertiamarin (SM), mangiferin (MF), gentiopicroside (GP), and sweroside (SW). Reverse phase column (Waters Acquity BEH C₁₈, 50 mm × 2.1 mm , 1.7 μm) provided high resolution for all target analytes with binary gradient elution. The detector response was linear (concentration 2.5–125 μg/mL, R² > 0.999). The limit of detection (LOD) and quantification (LOQ) of targeted compounds was in the range of 1.40–2.06 and 4.57–6.27 μg/mL respectively. The combined relative standard deviation (%RSD) for intra-day and inter-day precision values were less than 2%. The recoveries study comply the method suitability. Chromatogram similarity analysis based on congruence coefficient was higher than 0.925 for the chirayita ecotypes while much lower than 0.629 for possible substitutes. HCA showed that the samples could be clustered (all 5 clusters in two-level) reasonably into different ecotypes and substitutes. HCA together with loading plots has indicated different chemical properties of all five groups. PCA results showed that the discrimination of chirayita ecotypes is because of the presence of SW while SM may have more influence on the targeted substitutes to discriminate from chirayita ecotypes. Therefore, UPLC fingerprint in association with chemometric tools provides a reliable and accurate quality assessment and detection of possible adulteration.

Graphical abstract

Introduction

Swertia chirayita (Family: Gentianaceae) is considered an important medicinal plant for its broad spectrum medicinal properties [1]. The plant is mainly distributed in the temperate Himalayan region of India at an altitude of 1200–3000 m (4000 to 10,000 ft), from Kashmir to Bhutan, and in the Khasi hills at 1200–1500 m (4000–5000 ft) [2]. It is not only used in Indian systems of medicine such as Ayurveda, Unani, and Siddha but also in traditional Chinese medicines. Its demand in botanical or functional food intended for immunity enhancement is growing worldwide. Recently, the use of Swertia as a functional food additive to elevate the antioxidant capacity by suppressing enzymatic browning has been demonstrated successfully [3].

Research updates on Swertia have demonstrated its utility as antidyslipidemic, antidiabetic, anti-inflammatory, antihepatotoxic, and antiviral [4,5] in pharmacological studies and emphasized many promising prospects for modern medicine. However, herbal medicines need to address many challenges starting from raw material sustainability, uniform quality, standardize process and mechanism of action before its global acceptance as therapeutics. Variation in the content of pharmacologically active compounds is widespread due to G × E (genetic and environmental interaction), adulteration of similar plant species, non-standardized collection, and processing.

The therapeutic actions of the S. chirayita are primarily assigned to iridoid glycoside viz. SM, GP, SW, and xanthone glycoside-MF. However, their content is not only varied within S. chirayita but also in other Swertia species as SB, SCH, SCL, SP and species of another genus HE. Therefore, there are considerable challenges to identify the possible adulteration in the S. chirayita in raw material as well as their derived products. Some efforts made for the determination of single or multiple chemical compounds in a single Swertia species [[6], [7], [8], [9], [10]]. However, the information about contrasts chemical flux in different other mentioned Swertia species had not attempted so far for adulteration/substitution check.

The quality assessment of raw herbs by qualitative and quantitative analysis of several components using chemical fingerprint was proposed by the World Health Organization [11]. Later this approach has been accepted worldwide and adopted in the pharmacopeia of China [12], India [13], and USA [14]. HPLC chromatogram used for information like presence or absence of targeted compounds which are subjective. Such approach missed the minor difference between chromatogram of samples. Fingerprint pattern is a complex multivariate data set due to the complex sample matrix of herbs. Therefore, such chemical patterns are processed by chemometric methods viz. similarity analysis, hierarchical cluster analysis (HCA), and principal component analysis (PCA) to define the classification based on oxidant potential [15], ecotypes [16,17], species [18], and processing [19]. Acquisition of fingerprint for quantitative analysis requires a rigorous operation. Nowadays, ultra-high performance liquid chromatography UHPLC based fingerprint is emerging as a preferred technique over HPLC due to better resolution, separation capacity, shorter run time, and less consumption of solvents [20].

Similarity information related to bioactive chemical flux viz. swertiamarin (SM), mangiferin (MF), gentiopicroside (GP), and sweroside (SW) in inter- and intra-genus of the same Gentianaceae family are not available so far. Therefore, to evaluate the interspecific similarity of the genus Swertia, and closeness to the genus Halenia for bioprospecting and raw material authenticity are still needed to be studied. Therefore, we have developed a validated method for simultaneous determination of targeted compounds by ultra-performance liquid chromatography (UPLC) in Swertia species. Chromatographic fingerprint data analyzed using PCA and cluster analysis for inter- and intra-species similarities and diagnosis of possible adulteration in raw and powdered herb.

Section snippets

Chemicals and materials

Targeted phytochemicals viz. SM, GP, SW, and MF (Fig. 1S) of designated purity (94.3–98.6%) procured from Chromadex, USA, stored following instruction and used as such without further purification. Chromatographic grade solvents, e.g., acetonitrile, methanol, and water purchased from Merck (Darmstadt, Germany). Acetic acid, formic acid, and trifluoroacetic acid of analytical grade purchased from Merck (Mumbai, India). Plant materials of targeted Swertia and Halenia species were collected from

Optimization of the extraction procedure

Based on our previous experience and other research, ultrasonic assisted extraction (UAE) is having advantages over other extraction techniques like cold percolation, hot and Soxhlet extraction. Although few studies performed the extraction of targeted compounds either with methanol, ethanol or varying concentration of alcohol in water (60–80%) [23,24] using UAE but lacking to demonstrate the role of time and temperature. Therefore, we have optimized extraction efficiency of UAE for solvent

Conclusions

The first time UPLC-PDA method was validated for the simultaneous determination of four markers viz. SM, GP, SW, and MF in different ecotypes of S. chirayita, with good precision, accuracy and linearity. The method was capable of giving rapid analysis with acceptable resolution. The method validation data has been showing satisfactory performance for all critical parameters. Shorter runtime and less consumption of organic solvents as compared to the other analytical techniques could be the best

Conflict of interest

Authors have no conflict of interest.

Acknowledgments

The authors acknowledge the financial support provided by National Medicinal Plant Board (NMPB), New Delhi as well as CSIR-CIMAP, Lucknow for Central Instrumentation Facility. One of the Author (SM) is also acknowledging DST, New Delhi for INSPIRE Fellowship.

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Ultra performance liquid chromatography coupled with principal component and cluster analysis of Swertia chirayita for adulteration check

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals and materials

Optimization of the extraction procedure

Conclusions

Conflict of interest

Acknowledgments

Food Chem.

Nat. Prod. Res.

Phytomedicine

Food Sci. Technol.

Ind. Crops Prod.

J. Ethnopharmacol.

S. Afr. J. Bot.

J. Chromatogr. B

A review of Swertia chirayita (Gentianaceae) as a traditional medicinal plant

Front. Pharmacol.

Swertia chirayita—an overview

Curr. Sci.