Issue:
109
Page: 60-61
Botanical Integrity: Part 2
Traditional and Modern Analytical Approaches
by Charlotte Simmler, PhD, Shao-Nong Chen, PhD, Jeff Anderson, MS, David C. Lankin, PhD, Rasika Phansalkar, Elizabeth Krause, PharmD, Birgit Dietz, PhD, Judy L. Bolton, PhD, Dejan Nikolic, PhD, Richard B. van Breeman, PhD, Guido F. Pauli, PhD
HerbalGram.
2016; American Botanical Council
The concept of botanical integrity
(BI), introduced previously in HerbalGram issue 106, involves the
determination of identity, homogeneity, bioactivity, and safety of
plant-derived materials designated for human consumption.1It
goes beyond previously established quality control principles. The inaugural
article in this series described the three major domains of expertise that are
required to assess BI (as noted in Figure 1): botanical examination (botany),
phytochemical analysis (chemistry), and biological efficacy and safety
assessments (bioactivity, which encompasses the fields of pharmacology and
toxicology).
This article explores contemporary
and comprehensive analytical techniques, focusing on the fields of botany and
chemistry for the determination of BI. Recently, new approaches, such as
authentication assays, have become available to characterize plant-derived
material used in botanical dietary supplements (BDSs).
Botanical raw materials typically
undergo post-harvest examination prior to extraction, and this process is
fundamental to material authentication and the detection of adulteration. In
addition, phytochemical analyses, such as chemical profiling and fingerprinting
techniques, are required in order to study a plant’s metabolite composition.
These analyses often are performed on extracts, but they also can be performed
on botanical raw materials after sample preparation, which usually involves a
small-scale extraction made in a laboratory. Phytochemical analyses are used to
confirm the identity of the chosen plant and plant part. This can be accomplished
by characterizing metabolites qualitatively and quantitatively and by targeting
species-specific markers, bioactive constituents, and, sometimes, undesired
compounds (negative markers).
Collectively, botanical
identification and phytochemical characterization are techniques that are a
traditional part of pharmacognosy — the study of medicines of natural origin.
This scientific field has evolved considerably in terms of organisms covered
and methodologies used (e.g., see www.pharmacognosy.us).
Analyses Performed on Plant Raw Materials
Traditional Methods: Organoleptic,
Macroscopic, and Microscopic Examination
The first step in plant material
identification requires an understanding of taxonomic principles. If possible,
a reference herbarium voucher specimen or photograph of the source material
should be obtained to facilitate a definitive taxonomic determination. The
identification of whole or powdered botanical materials usually is achieved
through organoleptic, macroscopic, and microscopic analyses performed by
trained experts, such as botanists and pharmacognosists.
Organoleptic assessment of aroma,
taste, and appearance characteristics can provide important clues about the
identity, uniformity, and potential adulteration of the raw material. Macroscopic
analysis involves the observation of morphological keys and the description of
fruits, flowers, and vegetative parts (e.g., leaves and roots) obtained during
cultivation or at the time of harvest (Step 1 in Figure 1). At this stage, the
Latin binomial and plant part(s) used are documented.
Botanical authentication is
challenging when plant materials are powdered or extracted. Authentication of
dried plant powders is usually performed with microscopic techniques such as
normal light microscopy, scanning electron microscopy, or fluorescent
microscopy. These methods are used to detect characteristic plant tissues or
the presence of particular cell types such as hair, oil gland, secretory canal,
vascular tissue, seed, starch grain, and pollen, or crystals in cells.
For most traditional herbal products
(e.g., leaves of ginkgo [Ginkgo biloba, Ginkgoaceae] and aboveground
parts of St. John’s wort [Hypericum perforatum, Hypericaceae]), these
analyses are sufficient to identify the plant material. However, closely
related plant species and hybrids usually share macroscopic and microscopic
features. Thus, accurate botanical authentication requires the use of
orthogonal or more specific analyses. These may include DNA methods (Step 2 in
Figure 1), which test for underlying genetic differences between samples, and
an array of phytochemical analyses that are used to determine characteristic
metabolite profiles.2-5
Evolving Methodology: DNA Authentication
The use of DNA-based tools,
including the Sanger method of DNA sequencing and high-resolution melting
analysis, can be fundamental for the unambiguous identification of
botanical materials.6,7 This is especially true for materials
with a high risk of contamination or adulteration (e.g., due to
misidentification), or if macroscopic and microscopic analyses are not
sufficient to distinguish closely related species or hybrids.2
The roots of the three major species
of licorice (Glycyrrhiza spp., Fabaceae), for example, have nearly
identical macroscopic and microscopic features. In the absence of
distinguishable taxonomic features, accurate botanical authentication of
commercial licorice powder is possible through the use of DNA barcoding,
complementing phytochemical analysis. In a 2015 study, DNA authentication was
found to be important in the initial stages of building reliable BI assays for
licorice.5 Subsequent phytochemical tests may be useful as
well. However, since DNA degradation can occur during industrial processing,
genetic analyses are generally more appropriate for the identification of
unprocessed raw plant material, as detailed in the first article in this
series.
Another potential limitation for the
universal application of DNA-based botanical authentication is the reliability
and overall comprehensiveness of available DNA barcode databases. Ideally, such
databases should include reference sequences of plants that have been
taxonomically identified and unambiguously vouchered. But, as stated by
Coutinho Moraes et al., “at the present time, the number of DNA sequences
for herbal and botanical products is insufficient.”7
Analyses Performed on Crude Extracts of Raw Materials
Traditional Methods: Targeted
Chromatographic-based Analyses
Differences in cultivation methods,
geographic origin, drying processes, and extraction methods yield botanical
products with varying metabolic compositions. The documentation of these
variables and development of appropriate and reproducible analytical methods
for standardization are essential for the authentication and characterization
of botanical extracts.4 The most widely used phytochemical
methods for the characterization of extracts involve chromatographic separation
of constituents by high-performance liquid chromatography (HPLC),
ultra-high-performance liquid chromatography (UHPLC), gas chromatography (GC),
and regular or high-performance thin-layer chromatography (TLC or HPTLC).
Chromatographic systems often are
coupled (“hyphenated”)
with suitable detection methods, such as ultraviolet (UV), fluorescence,
refractive index (RI), evaporative light scattering detection (ELSD), charged
aerosol detection (CAD), and mass spectrometry (MS). The resulting metabolite
profiles depend on the type of separation and mode of detection. These
conditions determine which kind of metabolic snapshot the chromatogram
captures, and they should be chosen according to the physicochemical properties
of the compounds expected to be present in the extract. Depending on the
selectivity of the detection method, such choices can introduce a certain
amount of “chemical
bias” into the analysis.
Challenges in the phytochemical
characterization of extracts are associated with various factors including the
chemical diversity of plant metabolites, varying physicochemical properties,
and their extended dynamic ranges, in terms of concentrations of the extracted
compounds. Therefore, a combination of multiple analytical methods typically is
required to detect different sets of compounds on the basis of their
physicochemical properties (e.g., boiling point, volatility, presence of
chromophores, and ionizability). (HP)TLC systems allow for the parallel
comparison of multiple samples on a single plate, which then can undergo multiple
detections with UV, MS, and/or various chemical or biological reagents. (HP)TLC
systems generally are considered to be more practical, informative, economical,
and easier to perform than other chromatographic systems. Thus, these systems
are used frequently for routine quality control (QC) of plant material and as
identification assays in pharmacopeias.
In general, traditional
chromatographic analyses focus on the identification and quantification of
select compounds for which the method is considered sufficiently specific.
These designated marker compounds are then used to characterize the chemical
composition of plant extracts. Such approaches are considered targeted phytochemical
analyses and represent the gold standard in the QC of plant materials. When
assessing herbs to be used in BDSs, the selected marker compounds should
include specific bioactive phytochemicals. The frequent lack of commercially
available reference standards required for this process is a significant
practical challenge.3
Comprehensive Approaches: Techniques for Metabolomic
Analyses
It is widely believed that BDSs and
herbal medicines exert their health effects as a whole rather than by virtue of
a few selected, potentially bioactive phytochemicals.8-13 Because
of this, analytical approaches should have the capability to cover and
characterize a much wider (ideally the entire) chemical composition of a given
plant extract. This represents another major analytical challenge, which can be
approached by the use of metabolomics.
Metabolomics is defined as the study
of all chemicals (metabolites) produced by, and present in, a living organism.10,11 Hence,
for the analysis of plant extracts, the primary goal of metabolomics is to gain
insight into the chemical composition of the plant material at a specific time
point. This leads to a better understanding of the molecular signature produced
by diverse environmental influences, such as geographic origin, cultivation
conditions, harvest time, storage methods, and industrial processing.12,14 As
such, metabolomic analysis can aid in the development of standardization
methods for cultivation, harvest, and extraction, among other processes.
Three main types of techniques are
employed for metabolomic analysis of plant materials: LC- or GC-MS; nuclear
magnetic resonance (NMR) spectroscopy; and vibrational spectroscopy, such as IR
and Raman spectroscopy.
MS determines the
mass-to-charge (m/z) ratio of the molecular ions, their fragmentation
patterns, and the relative ion intensities of the compounds present in a plant
extract (generally after chromatographic separation). With their high
sensitivity (pM-nM) and inherent specificity, LC- and GC-based MS approaches
are widely used for the identification and quantification of selected marker
compounds, notably those found in trace amounts. With the improvement of MS
instrumentation and data systems, LC/GC-MS techniques are also used for the
profiling and metabolomic analysis of plant extracts, which requires only
a small amount of the sample to be analyzed.
Historically, NMR and IR have been
employed predominantly for the structural elucidation of individual isolated
compounds. However, with the development of advanced software and the
accessibility of statistical analyses of chemical information (chemometrics),
the use of NMR and IR-based metabolomics has increased recently. Compared to
chromatographic techniques, both NMR and IR are non-destructive, allowing
a full recovery of the analyzed sample, and can readily accommodate crude
plant extracts without physicochemical separation of their constituents.
NMR measures the resonance frequency of
various nuclei, such as 1H,13C, and 31P, under
the influence of a magnetic field. NMR-based metabolomics of botanicals favor
the measurement of protons (1H), which are characterized by a
relatively high sensitivity, natural abundance, and nearly ubiquitous presence
in phytochemicals. The 1H-NMR spectrum of a complex plant
extract can be considered a metabolite fingerprint representing the
superposition of spectra from all compounds present in the extract, at their
relative abundance. Careful interpretation of the 1H
spectrum may enable the simultaneous identification of the most abundant
phytochemicals (low µM-mM) present, and, importantly, their quantification
without the need for structurally identical reference standards. Currently, NMR
and its quantitative form, qNMR, are considered robust and versatile tools for
the direct unbiased, untargeted metabolomic analysis of botanical extracts.12,15,16
IR measures the stretching,
wagging, and bending actions that occur within all molecules in an herbal
material. There are three different types of IR analyses: near-IR (NIR; ~
4,000-12,500 cm-1; vibrations and overtones), mid-IR (MIR; ~ 4,000-400
cm-1; bending and stretching vibrations), and far-IR (FIR; ~ 400-10 cm-1; lattice vibrations). Raman
spectroscopy can operate between 4,000 and 400 cm-1 and below. MIR and Raman have been
used for the QC of food products and, together with NIR, these techniques have
become an alternative analytical tool for metabolomic analysis of raw
botanicals (e.g., Panax ginseng, Araliaceae17; Digitalis purpurea,
Plantaginaceae18), plant powders, and extracts. The IR spectrum
offers an overall molecular fingerprint, representing characteristic chemical
features of the most abundant botanical compounds. The implementation of
chemometric analyses is required in order to compare spectra from different
plant materials, calibrate classification models, and obtain meaningful
qualitative/quantitative information.12,19 Following the
validation of the classification models, IR methods allow a rapid
identification of botanicals without extraction and sample destruction. Using
these methods, the detection of adulterants in tested materials requires
relatively large amounts of the contaminants (e.g., other plant species and
undesired chemicals).
Metabolite Profiling versus Metabolite
Fingerprinting
Since the introduction
of metabolome research, there have been two main groups of metabolomic
analyses: (1) metabolite profiling, which encompasses the identification and
quantification of selected metabolites; and (2) metabolite/metabolic
fingerprinting, which is dedicated to the comparison of chemical patterns in
samples without the need for metabolite identification. Metabolite
fingerprinting may be preferable when spectroscopic methods such as IR and NMR
techniques have been used. Metabolite profiling, on the other hand, is
associated more with LC-based methods. In the scientific literature, the terms
metabolite/metabolic fingerprinting, metabolite profiling, and metabolomics are
used interchangeably.10
Chemometrics
Metabolomics generally involves a
large number of samples, a significant amount of numerical data from the
chromatographic results, and spectra that have to be statistically processed
and analyzed. Data mining resources, including statistics and multivariate
analysis, are required to reduce both the dimension and complexity of datasets
to determine the most relevant information. Chemometrics comprises multivariate
data analyses of complex chemistry data sets and defines the use of these
statistical methods to measure, simplify, and interpret a large amount of
chemical information. Hence, the use of chemometrics is necessary to describe
spectral differences and to evaluate global changes in large sets of MS
profiles, or NMR spectra, so as to eventually achieve group classification.20 The
most widely applied chemometric analyses that enable the distinction and
classification of botanical extracts are principal component analysis (PCA);
soft independent modeling of class analogy (SIMCA), a classification model
based on PCA; hierarchical cluster analysis (HCA); and partial least squares
discriminant analysis (PLS-DA). Such analyses can be performed with various
software tools such as SIMCA-P (Umetrics; Umeå, Sweden), Pirouette (Infometrix;
Bothell, Washington), AMIX-TOOLS (Bruker; Billerica,
Massachusetts), and Matlab with Statistics Toolbox (MathWorks; Natick,
Massachusetts), among others.9,14,21
Metabolomics and the Determination of Botanical Integrity
The phytochemical composition of a
plant material is, in part, the result of its genomic expression and
environmental factors, such as cultivation conditions, herbivory/microbial
interactions, and the time of harvest. Hence, the metabolomic profiles of plant
materials are inevitably subject to fluctuation even when created from what is
considered the “exact
same” authenticated plant specimen or species.
The production of representative
metabolomic profiles and fingerprints requires the analysis of a significant
number of botanically authenticated samples. In this regard, metabolomic and
chemometric approaches enable a simultaneous comparison of multiple samples and
integrate the information to produce representative classification
(chemometric) models. Results obtained from such analytical platforms
enable the assessment of the overall phytochemical composition of botanical
material, the verification of botanical identity and distinction of plant
species, the determination of parts used, the evaluation of geographic origin,
and the objective comparison of botanical samples from various batches.
It is important to note that the
building of robust chemometric models for metabolomic authentication requires
an appropriate sample size. This is defined by the overall objective of the
classification model, as determined by the investigators, and should ideally be
representative of the geographic distribution of the botanicals. The more
samples that are considered and included in the model, the more accurate (i.e.,
representative of the phytochemical variability) this model will be. Acquiring
a representative amount of botanically verified reference materials to build
accurate chemometric models remains a challenge. To the best of our knowledge,
there is no consensus about the amount of samples per class or species required
to build such classification models.
The chemometric models built through
metabolomic analyses favor the selection of samples that display unusual
chemical features and facilitate the detection of potential botanical or
chemical adulteration. Using metabolomic approaches to assess the compositional
quality of plant material, and comparing the results to representative
classification models, can be implemented as QC measures and for the
determination of BI.
Conclusion
Unambiguous identification of
botanicals represents the first critical step for the determination of
botanical integrity. DNA authentication is increasingly
considered to be a helpful tool accompanying traditional macro- and microscopic
examinations to identify botanicals accurately.2,6,7 Recently,
work conducted at the authors’ Botanical Center at the College of Pharmacy at
the University of Illinois at Chicago (UIC) has demonstrated that DNA authentication
is a necessary tool for the identification of Glycyrrhiza hybrids
(licorice), as well as for the detection of mixtures among Glycyrrhiza species.5 Furthermore, DNA identification has
been shown to enable the selection of suitable botanical samples for the
production of representative chemical fingerprints to build accurate
chemometric classification models.
Chromatographic-based analyses,
e.g., (HP)TLC and (U)HPLC-UV/ELSD/RI/MS, remain valuable techniques and
industry gold standards for the phytochemical authentication and
characterization of botanicals. These analyses focus on the generation of
representative metabolite profiles with the identification and quantitation of
selected marker compounds.
Metabolomic approaches can
better address the phytochemical complexity and variability of plant materials,
while satisfying the general need for a more holistic analysis. Currently, the
most widely applied techniques for metabolomic analysis of botanicals are
LC/GC-MS, (1H) NMR, and, to a lesser extent, IR. Notably, the
excellent reproducibility and simultaneous quantification capability of NMR
makes it of increasing interest as a QC tool. The building of chemometric
models, based on any of these data, can foster the selection of plant material
with desired chemical and biological features. Therefore, techniques for
metabolomic analysis occupy a crucial place in the toolset for the holistic
assessment of botanical integrity, aimed at ensuring quality and safety of
botanical products.
Acknowledgments
The authors acknowledge support by
grants P50 AT000155 and U41 AT 008706 from the National Center for
Complementary and Integrative Health and the Office of Dietary Supplements of
the US National Institutes of Health. The contents are solely the responsibility
of the authors and do not necessarily represent the views of the funding
agencies.
About the UIC/NIH Center for Botanical Dietary Supplements
Research
Established in 1999, the UIC/NIH
Center for Botanical Dietary Supplements Research addresses issues of
standardization, quality, safety, and efficacy of botanical dietary supplements
used for women’s health. Founded by the late Norman Farnsworth, PhD, and
directed by Richard van Breemen, PhD, the Center’s leaders, including Judy
Bolton, PhD, and Guido Pauli, PhD, take an integrated, collaborative approach
to botanical research. Based on extensive preclinical studies involving botany,
chemistry, and biology, the Center has completed clinical studies with black
cohosh (Actaea racemosa, Ranunculaceae), red clover (Trifolium pratense,
Fabaceae), and hops (Humulus lupulus, Cannabaceae), specifically
addressing botanical safety and efficacy. Educating the next generation of
pharmacognosists remains a primary goal of the Center, which has trained more
than 60 pre- and post-doctoral researchers.
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