NMR Technique and Methodology in Botanical Health Product Analysis and Quality Control

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1 NMR technique and methodology in botanical health product analysis and quality control
2 Jianping Zhao a *, Mei Wang b, Seethapathy G. Saroja a, Ikhlas A. Khan a, c *

3
4
5 a National Center for Natural Products Research (NCNPR), School of Pharmacy, University of
6 Mississippi, University, MS 38677, USA
7 b Natural Products Utilization Research Unit, Agricultural Research Service, U.S. Department of
8 Agriculture, University, MS 38677, USA
9 c Division of Pharmacognosy, Department of BioMolecular Sciences, School of Pharmacy,
10 University of Mississippi, University, MS 38677, USA
11
12
13
14
15 * Corresponding authors:
16 Jianping Zhao (E-mail: jianping@olemiss.edu)
17 Ikhlas A. Khan (E-mail: ikhan@olemiss.edu)
18 National Center for Natural Products Research, School of Pharmacy, University of Mississippi,
19 University, MS 38677, USA
20 Phone: +1 662 915 7821, Fax: +1 662 915 7062;
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© 2021 published by Elsevier. This manuscript is made available under the Elsevier user license
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1 Abstract
2 Botanicals have played an important role in maintaining human health and well-being throughout
3 history. During the past few decades in particular, the use of botanical health products has gained
4 more popularity. Whereas, quality, safety and efficacy concerns have continuously been critical
5 issues due to the intrinsic chemical complexity of botanicals. Chemical analytical technologies
6 play an imperative role in addressing these issues. Nuclear magnetic resonance (NMR)
7 spectroscopy has proven to be a powerful and useful tool for the investigation of botanical health
8 products. In this review, NMR techniques and methodologies that have been successfully applied
9 to the research and development of botanical health products in all the stages, from plants to
10 products, are discussed and summarized. Furthermore, applications of NMR together with other
11 analytical techniques in a variety of domains of botanical health products investigation, such as
12 plant species differentiation, adulteration detection, and bio-activity evaluation, are discussed
13 and illustrated with typical examples. This article provides an overview of the potential uses of
14 NMR techniques and methodologies in an attempt to further promote their recognition and
15 utilization in the field of botanical health products analysis and quality control.
16
17 Keywords: Botanical health products; NMR; Methodology; Analytical technique; Quality
18 control; Application
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1 Contents:
2 1. Introduction
3 2. NMR techniques and methodologies used for botanical health products
4 2.1. Chemometrics-aided NMR

5 2.2. Quantitative NMR (qNMR)
6 2.3. Hyphenated NMR
7 2.4. Diffusion-ordered (DOSY) NMR
8 2.5. Computation-aided NMR
9 2.6. Site-specific natural isotope fractionation (SNIF) NMR
10 2.7. High-resolution magic angle spinning (HR-MAS) NMR
11 2.8. Low-field (LF) NMR
12 2.9. Other NMR techniques
13
14 3. Applications of NMR in botanical health product analysis and quality control
15 3.1. Quality control

16 3.1.1. Quantification of markers
17 3.1.2. Quality assessment
18 3.2. Adulteration detection
19 3.2.1. Detection of synthetic pharmaceutical ingredients
20 3.2.2. Identification/recognition of substituents
21 3.3. Exploration of metabolite variability
22 3.3.1. Geographic origin
23 3.3.2. Aging or growing stage
24 3.3.3. Part or tissue region
25 3.3.4. Growing environment
26 3.4. Discrimination of botanical materials
27 3.4.1. Species
28 3.4.2. Cultivar/variety
29 3.5. Effect of processing
30 3.6. Biological activity evaluation
31 3.7. Other applications
32
33 4. Conclusions and prospects
34
35
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1 1. Introduction
2 Plants play a vital role in human life. In addition to contributing to the global ecosystem and
3 environment, plants provide farm produce, vegetable and fruit for our daily life, and serve as
4 renewable resources for energy, raw material, forage, etc. Yet, another important usage of plants
5 is as nutrient or therapeutic agents to maintain human health and well-being. The term
6 “botanicals” can be defined as plants, plant parts or products derived from plant sources valued
7 for its nutritional, medical or therapeutic properties in healthcare profession. Products made from
8 plants that are used to maintain or improve health condition are sometimes also known as
9 botanical health products, botanical supplements, herbal products, botanical products, herbal
10 medicines, or phytomedicines [1]. For centuries, the human race has accumulated and passed
11 down the knowledge of using botanicals for health purposes from generation to generation, and
12 developed many organized traditional medical systems including Ayurveda, Unani, Kampo and
13 traditional Chinese medicine (TCM), as well as apprenticeship systems such as herbalism,
14 folklore and shamanism. According to a World Health Organization report in 2002 [2], about 80%
15 of the population which lives in developing countries rely upon traditional and herbal medicine
16 as their primary source of health care. The herbal usage and sale of herbal products in the US
17 have increased dramatically since the last few decades. According to the Nutrition Business
18 Journal (NBJ), herbal supplement sales in the US increased 8.6% from 2018, reaching an
19 estimated total of $9.6 billion USD in 2019 [3]. The global market for herbal supplements and
20 remedies estimated at US$104.6 billion in the year 2020, and it is projected to reach a size
21 of US$166.2 billion by 2027 [4].
22 More and more people are accepting the concept that herbal products are a significant part of
23 modern medicine and pharmacy. It is undeniable that the prevalence of botanicals is making a
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1 far-reaching impact on human healthcare. At the same time, however, such widespread use of
2 herbal products throughout the world has raised serious concerns related to their quality, safety
3 and efficacy [5, 6]. The use of plant products is based primarily on empirical knowledge, and the
4 preliminary information regarding appropriate indication, formulation, and dosing are often
5 derived from the ways of traditional use that generally has not been scientifically evaluated [7].
6 Unlike conventional medicine, botanicals contain complex mixtures of chemical constituents,
7 and their compositions are often poorly characterized. Aside from the variation in the chemical
8 content of botanical preparations, as well as the instability of some constituents, the identities of
9 the true active constituent(s) in botanical supplements are often unknown [5, 8]. Furthermore, in
10 most cases, it is believed that there are multiple chemical components, sometimes from different
11 compound classes, that synergistically or antagonistically contribute to the promised
12 physiological activity [9]. All these complexity and uncertainty make it a very challenging task
13 to ensure the transparency that the formulating products are consistent, reliable and safe, and able
14 to deliver the expected physiological effect.
15 To reach the stage where the quality and effectiveness of botanical products are assured is an
16 ultimate goal for sufficient utilizing plants as a natural source and treasure for human healthcare.
17 Many practices (strategies, measures, or regulations) have been proposed and/or applied at each
18 stage, from farming and supplying of raw materials, to processing and manufacturing, up to
19 packaging and distributing finished products [10], including the guidelines for good agricultural,
20 good harvesting and good manufacturing practices. For instance, the U.S. Pharmacopoeia (USP)
21 began to develop monographs on major botanicals in commerce in 1995; the European
22 Directorate for the Quality of Medicines & Health Care (EDQM) has started to work on quality
23 control monographs for herbal drugs in European Pharmacopoeia (EP) since 1997; the World
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1 Health Organization (WHO) has set forth universal quality control methods for medicinal plant
2 materials and guidelines for good manufacturing practices since 1998. In the last decades, many
3 methods with use of markers (or reference compounds) and chemical profiles of botanicals have
4 been proposed to make the quality and safety control more practicable [7, 12]. Definitely,
5 chemical analyses play an imperative role in the practices. Analytical techniques, such as liquid
6 chromatography (LC), gas chromatography (GC), Fourier transferred infrared spectroscopy (FT-
7 IR), near-infrared (NIR) spectroscopy, thin-layer chromatography (TLC), mass spectrometry
8 (MS), nuclear magnetic resonance (NMR), and their serial or parallel combinations, have been
9 successfully employed for improving and ensuring the quality of botanical health products [11-
10 14].
11 NMR spectroscopy is one of the most powerful chemical analytical tools, and it is widely
12 used for characterization and structure determination of natural products. NMR spectrum can
13 provide a wealth of accurate qualitative and quantitative information even regarding the
14 components of a mixture. NMR has therefore become one of the most useful analytical
15 techniques which can contribute to the field of metabolomics. NMR spectroscopy combined with
16 multivariate analysis techniques have been successfully used to solve a number of problems
17 including plant species and cultivar discrimination, metabolite profiling, and quality assessment
18 of food or herbal medicines. In most cases, in contrast to GC-MS and LC-UV/MS techniques,
19 the NMR approach does not require separation or isolation of analytes from a mixture. Hence,
20 the loss of the whole metabolite information caused by the filtration of chromatographic column
21 can be avoided (such loss is very likely for botanicals since they have a wide range of
22 distributions both on molecular weight and polarity). Unlike most of other conventional methods
23 in which the detections are biased or limited owing to the physic-chemical properties of the
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1 analytes under investigation (e.g., lack of chromophores, poor ionizability, poor volatility, etc.),
2 NM
TLC/HPTLC GC/MS HPLC/UV/DAD HPLC/MS NMR
3 R
4 has
5 a
6 uni
7 versal detection capability. In principle, any component containing one or more atoms with a
8 non-zero magnetic moment (such as 1H, 13C, 14N, 15N, and 31P), is potentially detectable by NMR,
9 providing unbiased detections and covering compounds of all organic chemical classes in
10 botanicals. In addition, because a direct proportionality exists between the signal integral and the
11 number of nuclei giving rise to it, organic chemicals can be accurately quantified with integral
12 data. This feature essentially makes the quantitative NMR (qNMR) a standard-free quantification
13 method, that is, unlike most other analytical techniques, NMR quantification can be conducted
14 without the need of chemically identical standard compounds. In addition to the excellent
15 qualitative and quantitative capacities, NMR has other attractive advantages such as simplicity in
16 sample preparation, non-invasive/destructive measurement, ability to simultaneously detect and
17 quantify a large number of chemical compounds, and high reproducibility. Furthermore, solid
18 samples can be analyzed by using solid-state NMR spectroscopy. Pros and cons in different
19 analytical techniques used for botanical health product analysis with regards to different aspects
20 are highlighted in Table1.
21
22 Table 1. Comparison of pro (+) and con (-) in analytical techniques used for botanical health
23 product analysis with regards to different aspects
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Detection un-bias - -- -- - + +1
Less preparation + - - - +
2
Sensitivity - + + + -
Quantification - ++ ++ ++ +
3 D
Identification - + + + ++
Detection simultaneity + - - - + +4 urin
Information Content - ++ + ++ +++
5 g
Reproducibility - + + + ++
6 the
Ruggedness - + + + ++
7 past decades, tremendous advances in the NMR instrumentation (i.e., cryo- and high-sensitive
8 probe, high-strength magnet, hyphenation, low-field and benchtop NMR), automation (auto-
9 tuning, auto-shimming, and auto-sampling), data handling capabilities, spectral deconvolution,
10 and statistical analyses have boosted its applications in the analysis and quality control of
11 complex botanicals and herbal products. Fig. 1 illustrates the steady increase in the volume of
12 publication on the topics of NMR and botanical health products since the year 2000.
13
14 Figure 1. Yearly volume of publication on the topics of NMR and botanical health products
15 (found in the Scifinder database with the searching keywords: NMR and botanical/plant; access
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1 April 13th, 2021; publications related to using NMR as a tool for structural elucidation of isolates
2 from plants or plant-derived products are excluded)
3 This review intends to provide an overview of the potential applications of NMR
4 methodologies in botanical R & D and quality control of botanical health products, so that the
5 NMR techniques discussed may be further recognized by researchers, and the NMR application
6 examples demonstrated in this review may enlighten the updating path of further explorations in
7 this field. The review covers three main topics. Firstly, the NMR techniques and methodologies
8 commonly used in botanical analysis and quality control will be summarized. Those techniques
9 and methodologies are categorized and discussed based on their technical characteristics to
10 improve their recognition. To be concise, the most essential NMR concepts that are commonly
11 recognized by the majority of natural products chemists will not be covered or only be slightly
12 presented since an enormous amount of literature is available on them. Secondly, the
13 applications of NMR techniques and methodologies in different areas of botanical health product
14 analysis and quality control will be summarized, and selected application examples will be
15 presented for demonstration purposes. The NMR approaches and applications in structural
16 elucidations of isolates from botanicals are exclude because they are beyond the scope of this
17 review. Lastly, a brief summary including some perspectives on NMR and its use in botanical
18 health products will be presented. It is expected that this review may be helpful to the
19 developments of NMR techniques and applications in botanical product research and practice;
20 and it is certain that such further developments will improve and extend the utilization of
21 botanical products in health care, with regard to the promotion of quality, safety, and efficacy.
22
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1 2. NMR techniques and methodologies used in botanical health product analysis and
2 quality control
3 NMR spectroscopy is an analytical technique based on the measurement of resonance signals
4 of magnetic active nuclei. The intensity, resonance frequency, coupling constant, line shape, line
5 width and relaxation times (spin-lattice or longitudinal relaxation time and the spin–spin or
6 transverse relaxation time) of NMR signals are the main features of interest. They can be used to
7 reveal a great deal of information about the property of analyte, including molecular structure,
8 concentration, chemical composition, inter- and intra-molecular interaction, the physical and
9 chemical environment of magnetic nuclei, and to provide insights into the nature of the
10 intracellular medium [15-17]. A number of sophisticated NMR acquisition sequences and
11 techniques with various pulses and delays for spectral editing/filtering or for coherence transfer
12 and chemical shift encoding have been developed to generate various types of signals and spectra
13 [16, 18-20]. On the other hand, the technical advances in magnetic field and probe, signal
14 processing and data analysis, as well as hyphenation with other analytical techniques, have
15 greatly enhanced the capability of NMR, and at the same time, extended its applications in the
16 field of botanicals research and quality control [18, 19, 21, 22]. An overview of the NMR
17 techniques and methodologies commonly used in the analysis of botanical health products and
18 quality control, as summarized in Fig. 2, is presented below.
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1
2 Figure 2. Summary of NMR techniques and applications for the investigation of botanical health
3 products
5 2.1. Chemometrics-aided NMR
6 Chemometrics-aided NMR technique is a widely used tool for the investigation of botanicals,
7 and the applications of this technique have tremendously increased during the past two decades
8 [16, 23, 24]. Botanical samples are usually multicomponent mixtures. An NMR spectrum
9 obtained from one sample could be characterized by a large number of resonance signals. When
10 conducting the analysis of a large number of samples, the data exploration is very complex,
11 leading to the interpretation being very difficult. Chemometrics is a discipline which uses
12 mathematical, statistical and other methods to reduce the dimensions of chemical datasets
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1 obtained from non-targeted analyses and to decipher the relevant information. In addition, it can
2 be used to predict chemical data and to design optimal experimental procedures [25, 26].
3 Chemometrics has been successfully applied to simplify complex and massive NMR data, to
4 classify samples into groups based on the spectral similarity, to discover the possible intrinsic
5 relations between different groups, to reveal the characteristic chemical or signal markers, and to
6 predict the authenticities of samples. As a matter of fact, chemometrics-aided NMR technique is
7 the kernel of NMR-based metabolomics, which is currently widely used in food, agricultural,
8 biochemical, medical, and pharmaceutic areas [27, 28].
10 Figure 3. Workflow of chemometrics-aided NMR methodology
11
12 Fig. 3 illustrates the workflow of chemometrics-aided NMR methodology used in
13 botanicals study. Multiple botanical samples are usually investigated when using this
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1 methodology, and all samples are treated and measured under the same experimental conditions.
2 Both liquid-state and solid-state NMR analyses are suitable for use with chemometrics. In the
3 case of solid-state NMR analysis, plant materials can be directly put into the probe for
4 measurement. For liquid-state NMR analysis, a protocol for sample extraction should be
5 established to ensure the maximum recovery of the constituents in the samples, and to make the
6 procedure both reproducible and simple. Parameters such as homogenization, polarity and
7 selectivity of extraction solvent(s), pH, temperature, and the source/nature of botanical samples
8 (i.e., leaves, flowers, roots, or commercial products) should be considered. An internal reference
9 standard may be added for both chemical shift calibration and quantification purposes. The
10 prepared samples are usually dissolved in deuterated solvents prior to NMR measurement. If
11 necessary, a buffer can be used to control the pH of the solution. In the NMR data acquisition
12 portion of the workflow, the magnetic isotope of interest, most commonly 1H or 13C, can be
13 chosen for investigation. Either one-dimensional (1D) or two-dimensional (2D) NMR data,
14 which is acquired with the corresponding 1D or 2D pulse sequence and experimental settings
15 respectively, can be used for chemometric analysis [16, 17]. NMR data are acquired with
16 optimized apparatus settings (i.e., probe tune/match, shimming, lineshape, lock phase, pulse
17 length, and temperature control) and suitable acquisition parameters (i.e., acquisition time,
18 relaxation delay, spectral width, number of data-points, and number of repetition). A pre-
19 saturation sequence is usually used to suppress the undesired solvent/water signals. After
20 acquisition, the detected free induction decay (FID) is treated by apodization and zero filling,
21 followed by Fourier transformation (FT), then the NMR spectrum in the frequency domain is
22 obtained. Prior to conducting chemometrics analysis, phase and baseline corrections, as well as
23 noise elimination and spectral alignment are performed. NMR spectrum may contain several
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1 thousand digital points as variables. Binning (bucketing) is commonly used to reduce the data
2 dimensionality, and intensity normalization is often applied to make all sample spectra
3 correspond to the same overall concentration. Furthermore, in order to position all of the
4 variables on a comparable scale, the NMR dataset is generally pre-treated by using Pareto or
5 other scaling methods [25, 26].
6 Chemometric approaches for NMR data deciphering can be divided into two types:
7 unsupervised (also known as descriptive or undirected) and supervised (also known as predictive
8 or directed). The unsupervised approach makes no prior assumption about the sample
9 information, in contrast to the supervised approach in which the information about the sample’s
10 nature such as identity or class is utilized. Depending on the objective of the investigation, one of
11 the two approaches or both can be applied to generate chemometric models for data
12 interpretation. Principal components analysis (PCA), hierarchical cluster analysis (HCA),
13 canonical-correlation analysis (CCA), non-linear mapping (NLM), soft independent modelling of
14 class analogies (SIMCA), projection to latent structure discriminant analysis (PLS-DA), and
15 orthogonal projection to latent structure discriminant analysis (OPLS-DA) are the chemometric
16 approaches that are commonly used in botanical health product investigations. These methods
17 have been successively used for the detection of adulteration, identification of origin,
18 differentiation of species or varieties, and identification of diagnostic markers for botanical
19 authentication. Application examples using chemometrics-aided NMR technique for botanical
20 health product analysis can be seen in section 3 of this review.
21 2.2. Quantitative NMR
22 Quantitative NMR (qNMR) has been increasingly used for quantitation of characteristic (or
23 marker) compounds in various plant species, as well as for quality control of botanical products.
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1 qNMR is considered to be an unbiased analytical technique and a versatile tool for the analysis
2 of multi-component mixtures [29, 30]. The integrated intensities of NMR signals are directly
3 proportional to the number of nuclei of interest in the mixture. Therefore, the reference
4 compound that is used for calibration in quantitative analysis does not need to be identical or
5 even chemically related with the analyte, thus, providing a unique advantage for qNMR over
6 other analytical tools. In addition, for resolved NMR signals from multiple components in a
7 mixture, qNMR can potentially be used for quantification of multiple analytes simultaneously
8 [31]. The basic concept in qNMR technique in botanical analysis is to establish a linear
9 correlation between the intensities/integrals of NMR signals in frequency domain and the
10 quantity of specific component in the botanical mixtures. qNMR is rather uncomplicated with
11 regards to the practical aspects. Yet the intensities of NMR signals corresponding to the target
12 compound can be affected by the experimental parameters for data acquisition (i.e., relaxation
13 delay, digitization, and pulse sequence) and spectral processing (i.e. signal deconvolution and
14 peak integration), as well as the sample conditions (i.e., residual proteins, solvent, buffer, salt
15 concentration, and temperature) [20, 32, 33]. Therefore, in order to obtain accurate quantification
16 results, all of the parameters and experimental conditions need to be taken into consideration
17 when a qNMR experiment is designed. For instance, the interscan delay usually should be set
18 long enough for qNMR data acquisition to allow the magnetization to longitudinally relax to full
19 equilibrium in order to ensure that the measurement is truly quantitative. More detailed
20 discussions on qNMR experimental settings and method validations can be found in the related
21 literature [20, 29, 31].
22 1D 1H-qNMR is the most commonly used method for quantitative analysis of botanical
23 mixtures due to its sensitivity, universality, and precise nature. However, signal overlapping,
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1 arising from relatively narrow dispersion of 1H chemical shifts, a large amount of resonance
2 signals, and multiplet patterns due to homonuclear couplings, restricts its application in botanical
3 analysis. To address the signal overlap issue in 1D 1H-qNMR, 1D 13C and homonuclear or
4 heteronuclear 2D qNMR techniques have been increasingly recognized and exploited for
5 botanical quality assessment or concentration measurement. Several different 1D 13C-qNMR
6 techniques, such as Q-INEPT-CT (Quantitative Constant-Time Insensitive Nuclei Enhanced by
7 Polarization Transfer) and CHORAD (CHirped, ORdered Pulses and Adiabatic Decoupling),
8 have been proposed and applied for the quantitative measurements of mixtures [34-37]. Using
13
9 C NMR spectra for quantitative analysis may be beneficial because the chemical shift
10 dispersion of 13C is much wider than that of 1H, resulting in much less signal overlapping.
11 Homonuclear or heteronuclear 2D NMR creates an additional dimension to 1D 1H-NMR.
12 Overlapped signals may be resolved with the dispersion onto the second dimension. 2D NMR
13 spectra of mixtures could contain higher proportion of resolved peaks, but the signals are usually
14 more difficult to quantify because of the resonance-specific signal attenuation during the
15 coherence transfer periods that is intrinsically required for 2D NMR data acquisition. 2D cross-
16 peak intensities (or volumes) are influenced by more factors than those of 1D 1H-qNMR peaks,
17 including excitation profile, relaxation parameters, transfer efficiency, evolution times, mixing
18 times, etc. [20, 38, 39]. Therefore, for accurate quantifications, calibration using standards with
19 known concentrations are usually needed. The 2D qNMR technique has become increasingly
20 recognized [40, 41], and more applications of this approach in the field of botanicals analysis are
21 expected in the near future. Application examples using qNMR for botanical health product
22 analysis can be seen in section 3.1.1 and Table 3 of this review.
23 2.3. Hyphenated NMR
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1 NMR could be the most powerful and versatile tool to provide structural information on
2 analyte. In addition, NMR has universal and unbiased detection capacities, making it a tool of
3 choice for the analysis of unknown compounds. However, as for the analysis of botanical
4 mixtures, its application potential is restricted due to the signal overlapping caused by the multi-
5 component nature of botanicals. Hyphenation of NMR with chromatographic techniques, such as
6 liquid chromatography (LC) or high-performance liquid chromatography (HPLC), can thus
7 provide an appropriate solution [42, 43]. HPLC-NMR can provide important complementary
8 structural information or complete structural assignments of analytes on-line. Since NMR is a
9 highly non-selective detection technique, it can provide a comprehensive detection of any
10 hydrogen containing compounds present in HPLC elute. Unfortunately, some contradictory
11 requirements in HPLC and NMR practice with respect to solvent and time-scale of the
12 experiment hamper the development of hyphenating these two techniques [44]. For example,
13 deuterated solvents are needed when conducting 1H-NMR measurements, but using deuterated
14 solvents for HPLC elution is expensive and the selection is limited. Particularly, the use of
15 buffers with high ionic strengths and gradient mobile phase system, which are necessary in most
16 HPLC separation, could change the magnetic susceptibility and field homogeneity in the NMR
17 probe. From NMR’s perspective, due to the inherently low sensitivity of NMR detection, the
18 analyte amount eluted from the HPLC column into the NMR flow-probe needs to be above the
19 limit of detection in order to be detectable by the NMR. Nevertheless, since the early 80’s,
20 progress towards the hyphenation of HPLC and NMR have been made to overcome the
21 impediments for coupling these two techniques [42, 45]. Strategies and technique improvements,
22 such as operations in stop-flow mode and loop-collection mode, use of solid-phase extraction
23 (SPE) for elute trapping, construction of highly sensitive probe and high-field magnet, use of
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1 reduced-diameter solenoid NMR coil (capillary NMR: CapNMR), application of pulse programs
2 for solvent suppression, etc., have enabled hyphenated NMR techniques to become a powerful
3 and useful tool to study complex mixtures [42, 43, 46, 47]. Furthermore, multiple hyphenated
4 systems, in which more than one separation or detection techniques are involved, have also been
5 proposed and implemented, such as LC–MS-NMR, HPLC-HRMS-SPE-NMR, and LC–PDA–
6 MS–NMR [42, 48, 49]. These modern hyphenated NMR techniques are expected to provide an
7 increasing number of applications in botanical health product analysis and quality assessment.
8 Application examples using hyphenated NMR for botanical health product analysis can be seen
9 in section 3 of this review.
10 2.4. Diffusion-ordered (DOSY) NMR
11 Diffusion-ordered spectroscopy (DOSY) is another useful NMR technique for the analysis of
12 multicomponent mixture [50]. DOSY is a 2D NMR experiment. For botanical analysis, one
13 dimension usually accounts for 1H NMR chemical shifts and another for the diffusion coefficient
14 of the component molecule in the detection mixture solution. The diffusion coefficient is a
15 molecular property related to the molecular weight and other hydrodynamic properties such as
16 molecule size, shape and charge [51]. In addition, the surrounding environmental conditions of
17 the molecule, such as temperature, solvent, viscosity, and concentration, have effects on the
18 diffusion coefficient. The 2D DOSY NMR technique enable the crowded signals to be dispersed
19 along the diffusion coefficient dimension based the differences of translational self-diffusion
20 coefficients for the different components in the solution. Thus, a complex mixture can be
21 virtually separated and the NMR spectra of different components can be extracted for qualitative
22 and quantitative analyses without the need of any prior physical separations. In DOSY NMR
23 approach, a series of pulsed field gradient (PFG) stimulated spin-echo (STE) experiments with
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1 systematic variations of the gradient pulse amplitude are applied, then the diffusions coefficient
2 of molecules can be measured by analysis of the exponential signal decay [51]. Signals from
3 molecules with large diffusion coefficients (D) (i.e., small molecules) decay faster than those
4 with small Ds (i.e., large molecules) as the PFG is incremented. The DOSY NMR spectrum can
5 be obtained after the Fourier transformation for the NMR signal and the inverse Laplace
6 transformation for the decaying signals are performed. All cross-peaks belonging to the same
7 molecule are aligned, and the diffusion coefficient can be measured [51]. Certain limitations may
8 exist when using DOSY to analyze very complex mixtures, especially for those containing
9 molecules with similar molar masses exhibiting similar diffusion coefficients, or those with a
10 large dynamic range of concentrations [50, 52, 53]. Nevertheless, DOSY NMR is a well-
11 established technique for mixture analysis because it can provide comprehensive compositional
12 information by means of a virtual separation of the components, and the technique has been
13 successfully used for adulteration detection in commercial botanical products [52, 54].
14 2.5. Computation-aided NMR and 1H iterative full spin analysis (HiFSA)
15 Structural information of compounds is embedded in NMR spectra through the signal
16 parameters including chemical shifts, intensities, coupling constants, and linewidths. The
17 quantum mechanical (QM) theory of NMR can represent complex spectra by these parameters
18 [55, 56]. The current well-established models based on density functional theory and ab initio
19 calculations have been used for computational predictions of the NMR parameters for complex
20 molecular structures [57]. A wide variety of computational approaches has emerged to predict
21 and analyze chemical shifts, spin–spin coupling constants, relaxation rates and other parameters
22 [57]. Advances of computational 1H NMR in the calculation of 1H NMR chemical shifts and 1H–
23 1H spin–spin coupling constants of natural products are very well documented, and the data set
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1 of 1H NMR chemical shifts calculated could be very consistent with the experimental results [56].
2 In other words, modern computational tools for spectral prediction, simulation, and iteration
3 have made it possible to decode resonance patterns and to portray molecular structures by using
4 1H NMR experimental data.
5 1H iterative full spin analysis (HiFSA) is a computation-aided NMR methodology performed
6 to understand and interpret the complex resonances found in 1H NMR spectra of molecules. This
7 method has been shown to be useful in analyzing and deconvoluting the complex “multiplets”
8 present in the NMR spectra of structurally diverse natural products [55]. HiFSA is performed via
9 iteration of the spin parameters until a best match of the calculated spectrum with the
10 experimental spectrum is achieved. The process can be performed manually with a spin
11 simulation tool, but the process is mostly (semi-) automated using a computational tool such as
12 the PERCH and Cosmic Truth software. In order to perform a HiFSA analysis, 1H NMR data of
13 reasonable quality is required, from which the chemical shifts and coupling constants are used as
14 the starting values to facilitate the QM-based calculation of the NMR spectrum [55]. QM-based
15 spin calculations and iteration can be performed on any magnetic field strength, making HiFSA a
16 powerful and useful tool for structure dereplication [58, 59]. Notably, HiFSA profile can be used
17 for the accurate quantification of marker compounds in mixtures such as botanical extracts. The
18 targeted analysis of all 1H resonances belonging to the selected markers using HiFSA profiles
19 can not only provide a reliable strategy for structure verification, but also produce a much more
20 accurate quantification result than any peak-fitting qNMR approaches do. In addition, once the
21 HiFSA profiles are generated, they can be used as surrogate standards for future qHNMR
22 analyses [55, 60].
23 2.6. SNIF-NMR (Site-Specific Natural Isotope Fractionation)
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1 Authenticity and adulteration are generally the primary issues related to the quality of
2 botanical health products. Determination of isotope ratios for the major stable isotopes (i.e.,
3 2H/1H, 13C/12C, and 15N/14N) found in natural processes is a well-recognized method for the
4 investigation of plant biology, geochemistry, and authentication [61]. The isotope ratio is well
5 established as a molecular marker, and can be used as a criterion for molecule traceability and
6 product authenticity. In 1981, Martin et al. initially proposed and developed an NMR method to
7 measure position-specific 2H isotope compositions, known as the site-specific natural isotope
8 fractionation by NMR (SNIF-NMR) [62]. SNIF-NMR can provide isotopic information on each
9 position of molecular structure without molecule being destroyed [63]. These site-specific
10 intramolecular isotope ratio variations define the isotopic fractionation of a molecule and
11 characterize its isotopic profile [63, 64]. The profile represents a unique molecular fingerprint
12 that can be related to the geographical or botanical origin of a compound [64].
13 2H-SNIF-NMR has proven to be a powerful technique to authenticate the origin of natural or
14 synthetic products. It can also be a useful tool for the investigation of counterfeiting (adulteration,
15 substitution, and imitation of premium products). The applications of 2H-SNIF-NMR methods
16 for authentication of the sugar origins added in wines and fruit juices, as well as for origin
17 determination of vanillin and acetic acid, are recognized by official authorities [65]. In the past
18 two decades, thanks to the advances of NMR techniques and methodologies (such as the use of
19 multi-pulse sequences to improve the sensitivity or resolution and the use of adiabatic pulse to
20 improve the uniformity of 1H decoupling), the approach has been extended from 2H to 13C-
21 SNIF-NMR [66]. This improvement has made it possible to trace and analyze more constituents
22 in food, pharmaceuticals, and botanicals, opening a wide range of new applications and
23 possibilities for the analyses of authenticity, origin, biosynthetic pathway, and adulteration.
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1 2.7. High-resolution magic angle spinning (HR-MAS) NMR
2 Botanical health products are usually solid or semisolid heterogeneous complex matrixes
3 containing many chemical components with different molecular weights and polarities. To obtain
4 the chemical composition information, the extraction of components from a sample is necessary
5 when using conventional analytical approaches. Some drawbacks, such as partial extraction or
6 insolubility of constituents which leads to a loss of qualitative and/or quantitative information,
7 along with the occurrence of chemical degradation for unstable components, may be associate
8 with the extraction process. High-resolution magic angle spinning (HR-MAS) NMR offers a
9 novel and useful way to eliminate the extraction procedure, which allows solid and intact tissue
10 samples to be directly investigated [67, 68]. In HR-MAS NMR, the sample is placed in a
11 zirconia rotor spinning at a high frequency (few kilohertz) around an axis tilted by ∼54.74° (the
12 “magic angle”) with respect to the static magnetic field of the NMR magnet. This can effectively
13 minimize or remove the line broadening of NMR signal arising from magnetic susceptibility
14 differences, anisotropic interactions, and dipolar couplings. As a result, a highly resolved NMR
15 spectra similar or comparable to liquid-state NMR spectra in terms of resolution [69] can be
16 obtained. Notably, the technical advancements in microcoil HR-MAS NMR probes (HR-μMAS)
17 has dramatically reduced the sample size requirement, making it possible for the measurement of
18 1H NMR spectra for a sample with small quantity (< 0.5 mg) [70]. Like liquid-state NMR, HR-
19 MAS NMR can perform both 1D and 2D experiments for the characterization and quantification
20 of constituents in mixture [22, 68]. A wide range of applications by using HR-MAS NMR
21 approach have been developed, including the investigation of geographical origin of botanicals
22 [71] and food products [72], identification of biomarkers for the quality control [73], and
23 metabolite profiling of the chemotypes/cultivars [74]. It is expected that HR-MAS NMR will
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1 become a popular, useful and important tool in botanical health product research and quality
2 control in the near future.
3 2.8. Low-field (LF) NMR
4 In the early stages of NMR development, field strengths up to 2.35 T (or 100 MHz 1H
5 frequency) were applied in the researches of physics, chemistry, and material science. With the
6 development of superconducting magnet technology, cutting-edge high-field analytical NMR
7 systems are available at proton frequencies up to 1 GHz (23.5 T) currently, which greatly
8 enhance NMR detection capability with regards to sensitivity, resolution and specificity, thus
9 tremendously expand the applicability of NMR technique in biology, biochemistry, and life
10 science [75]. However, modern NMR spectrometers are expensive, the maintenance costs (i.e.,
11 liquid helium and nitrogen, hardware and service, etc.) are high, and skilled personnel are needed
12 for operation. They are not suitable for small and medium size enterprises, quality control
13 agencies, and regular laboratories. In recent years, low-field (LF) NMR spectrometers (40–100
14 MHz) have gained more attention and acceptance with the innovation and commercialization of
15 compact benchtop NMR spectrometers from several manufactures. These instruments utilize
16 permanent rather than superconducting magnets, in which no cryogenic fluids are needed. They
17 combine a small footprint, lower price, no consumables, almost zero maintenance, and easy
18 operation [21]. One of the advantages of LF NMR is that it can be installed and operated in
19 locations other than research laboratories, and are capable of capturing as many data points per
20 frequency interval as their high-field counterparts. Modern LF instruments are capable of
21 performing almost the entire methodical portfolio of today’s high-field instruments (≥ 300 MHz
22 1H frequency), such as measuring multiple nuclei and recording two-dimensional (2D) NMR
23 spectra [76]. So far, applications of LF NMR have been developed in a wide range, with the
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1 prospects being promising for small benchtop instruments to be employed in areas ranging from
2 medicine and clinical science, to food and environmental sciences [21]. The two main limitations
3 of this technology are its insensitivity and poor spectral resolution. Appealing solutions to
4 overcome these limits include the use of 2D NMR experiments with ultrafast 2D NMR
5 acquisition approach and the treatment of LF 1H NMR spectral data with chemometric
6 methodologies [77], These solutions remarkably enhance the capability of LF NMR, making it a
7 useful tool for rapid screening and analyzing complex mixture. It is expected that LF NMR
8 technique will be widely utilized for the quality control of botanical health products in the
9 industry.
10 2.9. Other NMR techniques
11 NMR is a unique and powerful analytical tool. Unlike other analytical approaches in which
12 only a few or limited choices are available for experimental variations or manipulations, NMR
13 has great versatility, allowing data-acquisition modes or/and parameters to be manipulated or
14 modified to meet the needs of different analytic projects and to extend applications in specific
15 fields. Various NMR techniques and methods with the capability to deal with different analytic
16 issues have been developed. Except for those mentioned above, the other NMR techniques that
17 are also commonly used in botanical health product analysis and quality control are summarized
18 and presented in Table 2.
19
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Table 2. Other commonly used NMR techniques and methodologies for investigation of botanical health products
Technique/Method Mode Usage Reference
¹H-¹H correlation spectroscopy 2D homonuclear NMR; Revealing connection information of ¹H signals; Assisting [78]
(COSY) and variants correlation via spin-spin coupling structure elucidation and confirmation; Dispersing
overlapping signals, reducing congestion and increasing
specificity
Total correlation spectroscopy 1D or 2D homonuclear NMR; Revealing connection information of ¹H signals; Assisting [79]
(TOCSY) correlation via spin-spin coupling structure elucidation and confirmation; Dispersing
network overlapping signals, reducing congestion and increasing
specificity
Selective excitation ID or 2D NMR; selectively Extracting specific signals of interest from complex and [80]
exciting individual signals allows overlapping mixtures; Reducing congestion and increasing
their connectivity and interaction specificity
with other nuclei to be probed
Distortionless Enhancement by ID or 2D NMR; transferring Enhancing signal intensity and detection sensitivity; [36]
Polarization Transfer (DEPT) polarization from an excited Separating resonances by phase
and variants nucleus to another – most
commonly 1H → 13C
J-resolved spectroscopy (J-RES) 2D J-coupling correlation NMR Revealing coupling information for spin-spin splitting [81]
signals; Dispersing overlapping signals, reducing
congestion and increasing specificity
Carr-Purcell-Meiboom-Gill 1D relaxation-edited NMR Eliminating or attenuating macromolecule signals [82]
(CPMG)
Heteronuclear single quantum 2D heteronuclear NMR; one- Revealing one-bond connection information between the [19, 83, 84]
coherence (HSQC) and variants; bond connection hetero-nuclei signals (i.e., ¹H-13C or 1H-15N); Assisting
Heteronuclear multiple quantum structure elucidation and confirmation; Dispersing
coherence (HMQC) overlapping signals, reducing congestion, and increasing
specificity
Heteronuclear multiple bond 2D heteronuclear NMR; Revealing multiple-bond connection information between [85, 86]
coherence (HMBC) multiple-bond connection the hetero-nuclei signals (i.e., ¹H-13C or 1H-15N); Assisting
Page 25 of 98
structure elucidation and confirmation; Dispersing
overlapping signals, reducing congestion, and increasing
specificity
Pure shift yielded by chirp 1D homonuclear decoupling efficiently suppressing homonuclear couplings, producing [87]
excitation (PSYCHE) resolution-enhanced spectra
Nuclei other than 1H 1D or 2D NMR for 13C, 14N, 15N, Increasing analytical capability; Detecting chemical species [88-91]
19
F, or 31P selectively; Reducing congestion and increasing specificity
Page 26 of 98
1 3. Applications of NMR in botanical health product analysis and quality control
2 3.1. Quality control
3 Quality is of prime concern due to the increasing commercialization of botanical health
4 products and the lack of strict regulation norms for most of the products. The inherent
5 complexity and variations of chemical composition in plant raw materials pose challenges for the
6 quality control of botanicals. Various techniques and methodologies have been used to assure the
7 quality of botanicals [92]. As a powerful tool to provide both unbiased qualitative and
8 quantitative measurements, NMR has been widely and increasingly applied in the quality control
9 of botanicals and products. The applications can be classified as: 1. using qNMR techniques for
10 quantification of various marker constituents in botanicals as quality control parameters; 2. using
11 NMR spectral fingerprint and metabolite profile coupled with chemometric analyses for quality
12 assessments.
13 3.1.1. Quantification of markers
14 Quantitative estimation of characteristic marker constituents in plant materials and
15 botanical products has been a useful strategy to assure consistency, to minimize batch-to-batch
16 variation, to maintain quality, and to ensure safety and efficacy. NMR techniques have been
17 successfully applied to both qualitative and quantitative analyses of markers in botanical health
18 products. As examples, by using 1D 1H NMR, 1D TOCSY, 2D 1H−13C HSQC and HMBC
19 techniques, a total of 13 compounds including artemisinin and five of its analogs along with five
20 flavonoids, an aromatic ketone, and camphor in Artemisia annua (a folk medicine for the
21 treatment of fever and malaria) extract were simultaneously identified and quantified without the
22 need of laborious isolation of the individual analytes [93]. The method is very specific as
23 identification and quantification are based on the structure information of characteristic 1D
Page 27 of 98
1 TOCSY, 1H–13C HSQC, and HMBC cross peaks. The qNMR method was validated in terms of
2 precision, linearity, and limit of detection. In addition, the analytical results were found to be in
3 excellent agreement with those obtained with the use of the time consuming HPLC/DAD/ESI-
4 MSn approach for those compounds for which the reference standards were available. The
5 developed NMR method could be therefore useful for evaluating the quality of A. annua
6 preparations. A 13C qNMR method was developed for quantitative analysis of cannabinoids in
7 hemp (Cannabis sativa L.) inflorescences [34]. The method, which provided reliable results in a
8 competitive time with respect to the HPLC technique and gave sufficiently precise and sensitive
9 results, could be used as a promising and appropriate tool for comprehensive chemical analysis
10 of bioactive cannabinoids in hemp and related products to ensure their quality. A fast and precise
11 1H qNMR method was developed for quantification of principal bioactive constituents (esculin,
12 fraxin and (–)-epicatechin) in bark of horse chestnut (Aesculus hippocastanum L.) and its derived
13 products [94]. The obtained results were cross-validated by a HPLC-PDA method, with no
14 statistical differences being observed between the results obtained by the two methodologies,
15 however, the qNMR assay did have the advantage of having higher precision.
16 NMR has the unique ability to identify and quantify numerous metabolites
17 simultaneously without the need of reference standards. A 2D HSQC qNMR method was
18 developed for quantification of active principles (anthraquinones) in bark of alder buckthorn
19 (Frangula alnus), a plant medicine used to treat constipation and other complaints [40]. The
20 method used surrogate standards with similar chemical features rather than the identical
21 reference standards which are usually commercially unavailable or chemically unstable in pure
22 or isolated form. The validation in terms of accuracy, precision, specificity, linearity, and limit of
23 quantitation demonstrated that this 2D HSQC qNMR method had clear advantages over the
Page 28 of 98
1 method in the European Pharmacopeia. In another investigation, quantum-mechanics-driven
2 quantitative 1H NMR (QM-qHNMR) approach, in which 1H iterative full spin analysis (HiFSA)
3 was involved for spectral deconvolution, was employed and evaluated for simultaneous
4 determination of eleven markers in the extract of red clover (Trifolium pratense L.). The
5 outcomes were compared with HPLC-based standardization, it was found that the QM-qHNMR
6 method exhibited a significantly lower percent relative quantitation error [95]. This study
7 demonstrated that the QM-qHNMR method could be accepted as a highly reliable analytical
8 method for botanical standardization. A large number of applications utilizing NMR for
9 quantification of characteristic and biologically relevant markers in various botanical materials
10 and botanical health products have been published thus far; they are summarized in Table 3.
11
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Table 3. Applications of NMR for quantification of markers
Botanical / Product Methods/Techniques Remarks Reference
Licorice (Glycyrrhiza 1D 1H and 2D HMBC HMBC-qNMR enables the simultaneous identification and quantification [86]
uralensis) NMR for data mining; of many flavonoid chemotypes, without need for prior high-resolution
Peak fitting 1H qNMR separation and/or isolation of identical calibrants. Each absolute amount of
(PF-qHNMR) all 15 unambiguously identified metabolites was determined using
deconvoluted peak areas generated from the line fittings
Paederia foetida qNMR and UV–Vis Scopoletin was determined and quantified using qNMR in the different [96]
extracts of P. foetida twigs. qNMR method was deemed accurate and
reliable for quality control of P. foetida without extensive sample
preparation
1 1
Hawthorns (Crataegus H qNMR H qNMR was used in quantification of four flavonoids (naringenin, [97]
spp.) hyperoside, rutin, and vitexin-2″-O-rhamnoside) and chlorogenic acid in
leaf extracts of four Crataegus species
Artemisia annua crude 1D 1H qNMR, 1D Simultaneous identification and quantification of artemisinin and five of its [93]
extracts TOCSY, 2D 1H–13C analogs along with five flavonoids, an aromatic ketone, and camphor (in
HSQC and HMBC NMR total, 13 compounds) in crude diethyl ether A. annua extract without
techniques; LC-MS isolation of the individual analytes. The quantitative results provided by
1D 1H qNMR data are in excellent agreement with those obtained by
HPLC and LC-MS/MS
Sambucus williamsii 2D HSQC NMR method Comparison of the peak heights of the oxybenzyl-substituted carbon [98]
for quantitative analysis resonance signals of the lignans in the botanical extract was made against
those of a standard lignan pinoresinol. The application of this simple and
reliable NMR method can be used to estimate amounts of related
compounds and chemical families in complex mixtures or botanical
extracts and offers measurable scientific evidence in quality processes
1
Horse chestnut bark H qNMR Direct and simultaneous quantification of esculin, fraxin and (–)- [94]
(Hippocastani cortex) epicatechin in Hippocastani cortex.
The obtained results were cross-validated by an HPLC-PDA method, and
no statistical differences were found between the results, but with the
advantage of higher precision of the qNMR assay
Page 30 of 98
13
Fibre-type Cannabis C qNMR Multi-component analysis and determination of the main non-psychoactive [34]
sativa L. (Hemp) cannabinoids (cannabidiol, cannabidiolic acid, cannabigerol and
cannabigerolic acid) in female inflorescences of different hemp varieties
by means of 13C qNMR
1 1
Acorus rhizome herbal H qNMR H qNMR was used as an alternative reliable method to estimate β- [99]
drugs asarone, α-asarone, and asaronaldehyde levels in Acorus rhizomes
Goldenseal herbal 1D 1H qNMR and 2D 2D NMR approach called Q QUIPU (Quick QUantItative Perfected and [100]
supplements (Hydrastine Quick QUantItative pUre shifted) in combination with 1D 1H NMR capable to access the
canadensis) Perfected and pUre concentration of three major alkaloids, berberine, β-hydrastine and
shifted (Q QUIPU) canadine in the root extract of goldenseal
HSQC
1
Mucuna pruriens H qNMR The statistical analysis confirmed the accuracy and reproducibility of [101]
single point qNMR method (internal standard) for determining L‐Dopa,
as well as other commercial preparations of this species
1 1
Ferula ovina (Boiss.) H qNMR H qNMR method for simultaneous quantification of ferutinin, stylosin and [102]
Boiss roots tshimgine in the plant roots
1
Artocarpus lacucha H qNMR The validated 1H qNMR as a reliable analytical means to determine [103]
heartwood oxyresveratrol in A. lacucha heartwood
Bark of Frangula alnus 2D HSQC qNMR Using surrogate standards in 2D HSQC qNMR for the quantification of [40]
Mill. (syn. Rhamnus anthraquinones in the bark of alder buckthorn (F. alnus). Apart from being
frangula L.) a useful alternative in the quality control of alder buckthorn, the presented
approach demonstrated the versatility of sophisticated 2D measurements in
quantitative NMR
1
Garcinia fruits and food H qNMR Using 1H qNMR to quantify the content of (−)-hydroxycitric acid and (−)- [104]
supplements hydroxycitric acid lactone in raw herbal drugs and Garcinia food
supplements
1
Ginkgo biloba H qNMR Analyzing quantitatively the terpene trilactone components in Ginkgo [105]
biloba leaf extract with 1H qNMR and obtained almost identical results to
data reported using HPLC
1 1
Stephania epigeae H qNMR H qNMR as a rapid, accurate, and precise method for simultaneous [106]
quantitation of dicentrine (1) and sinomenine (2) in S. epigeae
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Copaiba oleoresin 2D HSQC qNMR; Quantification of eight diterpene acids in the oleoresin of C. reticulata [107]
(Copaifera reticulata UHPLC-ELSD Ducke by UHPLC-ELSD and quantitative HSQC. In terms of precision,
Ducke) the HSQC method was advantageous for the quantification of the three
main compounds, whereas UHPLC-ELSD was superior in the
determination of the minor components
1 1
Black cohosh (Actaea H qNMR H qNMR approach for determining the total amount of cycloartanoids in [108]
racemosa L.) and black cohosh (A. racemosa) rhizomes
dietary supplements
Red clover (Trifolium Quantum mechanical The study identified quantum-mechanics-driven quantitative 1H NMR [95]
pratense) extract (QM) 1H qNMR (QM- (QM-qHNMR) as a capable and flexible method to achieve the targeted
qHNMR) multi-marker standardization of a complex botanical extract
1
Salviae miltiorrhizae H qNMR The 1H qNMR method was validated and applied to simultaneously [109]
Radix product (Danshen determine eight main components in 10 sample batches
Dripping Pill)
1
Crude extracts of Annona H qNMR A simple 1H qNMR method was developed for the direct quantitation of [110]
muricata L. fruit pulp the majority of Annonaceous acetogenins (AAGs) (sub-types 1a and 1b) in
crude extracts
1
Radix et Rhizoma Rhei H qNMR A fast 1H qNMR method for determination and quantitation of five free [111]
(Rheum spp.) anthraquinones in Radix et Rhizoma Rhei was developed
1 1
Essential oil of Inula H qNMR H qNMR method was used for the quantitation of major sesquiterpene [112]
racemosa and Saussurea lactones in two important medicinal plants, I. racemosa and S. lappa
lappa
1
Compound Danshen H qNMR Some metabolites present in Compound Danshen extract including [113]
(Salviae miltiorrhizae) phenolic acids, saponins, saccharides, organic acids, and amino acids, were
extract detected by 1H qNMR method, and metabolites profiles were further
analyzed by selected chemometrics algorithms to define the threshold
values for product quality evaluation
1
TCM preparation H qNMR A highly reproducible, fast, accurate and simple 1H qNMR method without [114]
Salvianolate lyophilized the need for calibration curves and complex computation was established
injection (aqueous by optimizing the solvent system and acquisition parameters to
extracts of Salvia simultaneously determine nine salvianolic acids and mannitol
Page 32 of 98
miltiorrhiza)
Silybum marianum fruit uHPLC−DAD; 1H qNMR A head-to-head comparison of 1H qNMR to an uHPLC−DAD-based [115]
extracts quantitative analysis of six flavonolignan congeners (silychristin,
silydianin, silybin A, silybin B, isosilybin A, and isosilybin B) of the
silymarin complex is presented
1 1
Persicae semen, H qNMR H qNMR method was used to measure amygdalin content in Persicae [116]
Armeniacae semen, and semen, Armeniacae semen, and Mume fructus
Mume fructus
1 1
Gentianae radix and H qNMR H qNMR method was used to measure gentiopicroside content in [117]
Gentianae scabrae radix Gentianae radix and Gentianae scabrae radix. Gentiopicroside is a major
(Gentiana lutea Linne component of Gentianae radix and Gentianae scabrae radix
and G. scabra Bunge)
1
Peucedani Radix H qNMR The contents of praeruptorin A (PA) and proaeruptorin B (PB) were [118]
(Peucedanum simultaneously determined using 1H qNMR
praeruptorum Dunn)
1
Ginkgo biloba H qNMR A combination of 1H qNMR and adsorbent-free countercurrent separation [119]
preparations (CS) methodology was used to establish a quantification method for
ginkgotoxin (4′-O-methylpyridoxine) in Ginkgo preparations
1 1
Leonurus japonicus and H qNMR H qNMR was applied to quantify the pharmacologically active alkaloid [120]
L. cardiaca stachydrine
1 1
Green tea catechins H qNMR; HiFSA H qNMR approach utilizing computer-assisted HiFSA was developed for [60]
simultaneous identification and quantification of green tea constituents and
enabled rapid profiling of seven catechins in commercial green tea extracts
1 1
Paeoniae radix H qNMR H qNMR was used to measure paeoniflorin content in Paeoniae radix, of [121]
(dried root of Paeonia which paeoniflorin is a major component
lactiflora)
Leonurus cardiaca, HPTLC and 1H qNMR 1
H qNMR was used to measure stachydrine [122]
Leonurus japonicus,
Leonotis leonurus:
1 1
Andrographis paniculata H qNMR H qNMR was used to quantify andrographolide, dehydroandrographolide, [123]
Page 33 of 98
deoxyandrographolide, and neoandrographolide in Andrographis
paniculata, a commonly used important traditional Chinese medicine
1
Rhizoma coptidis H qNMR A rapid, simple and accurate 1H qNMR method was developed for [124]
(rhizomes of Coptis simultaneous determination of berberine, jatrorrhizine, epiberberine,
chinensis, C. deltoidea coptisine, palmatine, and columbamine in rhizomes of three Coptis species
and C. teeta)
1 1
Ligusticum porteri H qNMR H qNMR was developed for rapid and direct quantification of dimeric [125]
rhizome extracts phthalides and others constituents in fresh L. porteri rhizomes
Page 34 of 98
1 3.1.2. Quality assessment
2 Botanicals, in general, are mixtures containing diverse and complex chemical
3 constituents. The targeted analysis of one or multiple marker constituents may provide a measure
4 for quality control, but such analysis may be insufficient to reflect the quality reality and to
5 warrant the promising efficacy. The holistic untargeted analysis of botanical metabolite profile
6 can make it possible to gain a broader insight into the chemical composition of botanicals, thus
7 provide an alternative means for quality assessment. NMR fingerprinting and profiling in
8 combination with chemometrics may provide an untargeted analysis for assessing the overall
9 consistency of chemical composition as evidenced by a good number of applications reported in
10 the past years. For example, a 1H NMR profiling method was developed by Jiang et al. [126] for
11 quality evaluation of an herbal medicine injection – Danhong injection (DHI), which was made
12 from the extracts of Radix Salviae miltiorrhizae and Flos Carthami (Carthamus tinctorius L.).
13 Due to the complexity of chemical composition, a limited number of selected bioactive
14 constituents would not be able to fully represent the properties of DHI. With the developed
15 method, 23 primary metabolites together with seven polyphenolic acids were simultaneously
16 identified, of which 13 metabolites with fully separated proton signals were quantified and
17 employed for further multivariate quality control assays. The first two principal components
18 from the established independent principal component analysis (IPCA) model were used to
19 calculate the upper control limits of χ2 and Hotelling T2 control charts. Based on the constructed
20 upper control limits, the proposed method was successfully applied to 36 batches of DHI to
21 examine the outlier samples. In another study, 1H NMR together with UHPLC-QTOF/MS and
22 HPLC-ELSD were applied to assess the quality of a botanical drug candidate (DA-9801) used
23 for the treatment of diabetic neuropathy [127]. DA-9801 was a standardized 50% aqueous
Page 35 of 98
1 ethanolic extract of a mixture of Dioscorea japonica and D. nipponica. Using 1D and 2D NMR,
2 26 major metabolites were identified. The 1H NMR fingerprints were used to evaluate the batch-
3 to-batch consistency of DA-9801 preparations. The study demonstrated that the combined
4 method strategy could be an effective solution for the quality control of botanical drugs.
5 A comparative metabolomics approach via NMR and MS analyses was employed for
6 evaluating the quality and authenticity of Senna alexandrina in its various commercial
7 preparations [128]. S. alexandrina is used as an active ingredient in the laxative medicine, Senna
8 drug. The 1D and 2D 1H NMR analyses allowed 30 metabolites to be simultaneously identified
9 and quantified, while PCA was used to define relative metabolite differences among Senna
10 preparations. The established model can be used to test the phyto-equivalence of commercial
11 Senna preparations. 1H NMR-based metabolomics combined with HPLC-PDA-MS-SPE-NMR
12 was employed to characterize and evaluate the standardized Ginkgo biloba preparations [129].
13 The method allowed simultaneous assessment of terpene trilactones and flavonoid glycosides. At
14 the same time, fortification with quercetin and potentially harmful Δ8-15:1-anacardic acid in the
15 products could be detected. In addition, the non-conformity to the standard ratio between
16 ginkgolides and bilobalide, as well as the presence of constituents that are usually not considered
17 in the standardization protocols, could be revealed. 1H HR-MAS NMR combined with
18 chemometric analyses was proposed for authenticity and quality assessment of Baccharis
19 trimera samples [130]. B. trimera, popularly known as carqueja, has been traditionally used for
20 treating several diseases particularly associated with hepatic and gastric disorders. It is the only
21 species of the genus Baccharis recommended by the Brazilian Pharmacopoeia for its medicinal
22 properties. The developed HR-MAS NMR methodology allowed the discrimination of the B.
23 trimera and B. myriocephala samples from the other three investigated Baccharis species. The
Page 36 of 98
1 PLS-DA and SIMCA models were used to evaluate the authenticity of commercial samples with
2 the results showing that none of the samples were classified as B. trimera. In a recently published
3 article [131], the capability and limitation of low-field (LF) NMR (60 MHz) for quality control
4 of cinnamon samples (14 cooking spices and 14 dietary supplements) were evaluated. The results
5 indicated that the quantification based on the LF NMR data focused on the typical signals of (E)-
6 cinnamaldehyde or coumarin was predictable. The established PLS models could be used to
7 predict concentrations of these compounds in commercial samples.
8 There is no doubt that NMR in combination with other analytical techniques and
9 chemometric methodologies is a potential tool for the quality assessment of botanical health
10 products. More successful applications in this field could be expected in the future.
11 3.2. Adulteration detection
12 Adulteration has been a persistent issue in botanical health products. Many factors such as
13 increased demand, limited availabilities of plant resources, supply shortages, high market values
14 of products, unintentional misuses of wrong or misidentified plant species, intentional
15 substitutions or dilutions with low-cost materials, additions of active pharmaceutical ingredients
16 to improve efficiency, and insufficient oversights or inspections may lead to adulteration. The
17 risk associated with the adulterating and mislabeling botanical health products presents an
18 alarming public health concern. On the other hand, the chemical complexity of botanicals makes
19 it challenging to identify adulterants and unlabeled ingredients that present in botanical health
20 products. NMR can provide a non-selective and universal detection of components in a mixture,
21 while obtaining qualitative and quantitative information on chemical composition without the
22 need for chromatographic separation or pre-knowledge of the formulation. Such advantages
23 make NMR a useful and powerful tool in adulteration detection for botanicals.
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1 3.2.1. Detection of synthetic pharmaceutical ingredients
2 Addition of synthetic pharmaceutical ingredients to botanical health products is a very
3 common form of adulteration. Various NMR techniques have been applied to the detection of
4 this form of adulteration. As examples, 1H NMR and qNMR techniques were applied to detect
5 the presence of adulterants in botanical dietary supplements marketed for weight loss [132]. Six
6 synthetic active pharmaceutical ingredients (API), namely sibutramine, phenolphthalein,
7 sildenafil, fluoxetine, orlistat and lorcaserine were identified and quantified. Among the 164
8 samples analyzed, 56% were found to be tainted with at least one of the APIs. The study
9 successfully demonstrated the efficiency of 1H NMR for adulterant detection in botanical dietary
10 supplements. 1D 1H and 13C, as well as 2D homo- and heteronuclear correlation (gCOSY, 1H-
15
11 N CIGAR, HSQCAD, and gHMBCAD) NMR were used to identify the presence of
12 phenethylamines (PEAs) in sports dietary supplements sold in the US market (Fig. 4) [133].
13 From 32 investigated commercial supplement products, eight PEAs, namely phenethylamine,
14 synephrine, oxilofrine, hordenine, β-methylphenethylamine, N-methyltyramine, octopamine, and
15 deterenol, along with several other nitrogen-containing substances, were detected. Moreover, a
16 1H qNMR method was developed and validated for simultaneously determining the contents of
17 those PEAs in the products. 1D 1H and 2D homonuclear correlation (COSY and TOCSY) NMR
18 were applied to detect and quantify synthetic cannabinoids in herbal products [134]. Synthetic
19 cannabinoids, known by their street name “spice”, are potent agonists binding to the cannabinoid
20 receptors CB1 and CB2 distributed throughout the central nervous system (CNS) and immune
21 system. These compounds can produce potent psychoactive effects, leading to adverse effects
22 ranging from excited delirium to kidney damage. 1H NMR in conjunction with COSY and
23 TOCSY provided unambiguous structure elucidation, with the ability to differentiate the
Page 38 of 98
1 isomeric cannabinoids that are closely related in structure. The quantitative results obtained by
2 qNMR for the synthetic cannabinoids were comparable to those obtained by HPLC-DAD. In
3 another application study, 19F and 1H NMR qNMR methods were successfully employed to
4 quantify fluorinated third-generation synthetic cannabinoids in herbal incense packages [135].
5 The results demonstrated that the 19F qNMR method was fast and accurate with no background
6 interference from the plant-material matrix.
8 Figure 4. 2D NMR spectra of a sports dietary supplement sample. A. gCOSY; B.1H-15N CIGAR;
9 C. HSQCAD; D. gHMBCAD (Reprinted from [133], with permission from Elsevier).
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1 There are many other application articles published, in which different NMR techniques
2 and methodologies were employed. 1H NMR coupled with chemometric analysis was used for
3 the detection of adulteration of Iranian saffron samples [136]. A PLS-DA discrimination model
4 was established and validated for its prediction capability. The developed method could be used
5 for detecting a wide spectrum of adulterants, ranging from synthetic dyes to herbal materials.
6 Assemat et al. used high field 1H NMR (500 MHz) to screen for adulteration in the seized spice
7 herbal blends [137]. Nine synthetic cannabinoids (SC) were detected and quantified. In the
8 second step, a compact benchtop LF NMR spectrometer (60 MHz) was explored as a detection
9 method for SC contamination of herbal samples. The results indicated that LF NMR was a
10 suitable method to uncover added SC by rapidly screening of the herbal mixtures. However, the
11 limits of LF NMR would be reached for very complex mixtures in which several SC or a mixture
12 of SC and other illicit drugs of different classes were present. In such cases, the method would
13 permit the detection of unexpected substances but would not allow their precise identification.
14 The application potential of LF 1H NMR in adulteration detection was also demonstrated by the
15 use of a benchtop 60 MHz NMR spectrometer for uncovering adulteration of “100% natural”
16 sexual enhancement and weight loss dietary supplements [138]. Even if high-field (HF) NMR
17 spectroscopy had to be employed for an unambiguous structural identification of the adulterants,
18 LF NMR could provide valuable clues on their chemical structure so that the adulterants could
19 readily be detected after a very simple and rapid sample preparation. In a recently publication
20 [139], a LF NMR (60 MHz) method was explored for detection of sibutramine or
21 phenolphthalein adulterated slimming dietary supplements. A PLS-DA model constructed based
22 on the LF 1H NMR spectral data from forty weight-loss dietary supplements was used to predict
23 the presence of the adulterant (sibutramine or phenolphthalein) in the screened supplement
Page 40 of 98
1 samples. The study demonstrated that applying a chemometric treatment to LF 1H NMR data
2 could be a useful means to widen the application of LF NMR technique in the field of complex
3 mixture analysis.
4 Hyphenated NMR techniques are a useful tool for dereplication and discovery of
5 constituents of interest from complex mixtures. LC–MS–SPE-NMR [140] and HPLC–UV–SPE–
6 NMR [52] were successfully applied to identify undeclared synthetic drugs in herbal medicines.
7 Diffusion-ordered (DOSY) NMR is another useful technique in the detection of synthetic
8 pharmaceutical adulterants in botanical health products. 2D DOSY 1H NMR was applied to
9 analyze 17 herbal dietary supplements marketed as natural substances for the enhancement of
10 sexual function [53]. Adulteration with pharmaceutical drugs (PDE-5 inhibitors or analogues)
11 was detected in eight supplements. Meanwhile, the 3D DOSY–COSY method was used to
12 provide structural information of the adulterants. 2D DOSY and 3D DOSY–COSY 1H NMR
13 methodologies were also applied to adulterant detection in herbal medicines and dietary
14 supplements marketed for weight loss [54]. Among 20 investigated products, 13 were found to
15 be adulterated with sibutramine alone or in combination with phenolphthalein.
16 3.2.2. Identification/recognition of substituents
17 The quality of botanical health products is directly related to the proper use of authentic
18 plant materials. The traceability of both the identity and part of the source plant is important to
19 ensure quality, safety and efficacy of botanical products. Since botanical supplements do not
20 follow the same strict regulations as medicinal drugs, adulterations associated with using
21 misidentified plant species and substituting (or diluting) genuine plant material with low-cost
22 alternative have frequently occurred. NMR has proven to be a useful tool for detecting such
23 adulterations. For instance, the rhizomes of Rhodiola rosea L. are the principal raw material used
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1 for the production of R. rosea supplements, which are widely used in Europe, the US, and Asia
2 with the putative properties of mental, cognition and immunomodulation enhancement. The
3 ever-growing demand for the products, the scarcity of authentic raw material resource, and the
4 absence of a consistent worldwide program for quality assessment make it very likely that many
5 other Rhodiola species, which are similar and difficult to be discriminated morphologically, are
6 accidentally and/or deliberately used as substitutions in commercial R. rosea products. To deal
7 with this issue, Marchev et al. applied 1D and 2D NMR in combination with chemometric
8 analysis, together with HPLC, as an analytical platform for quality and quantity assessment of
9 the characteristic components in three Rhodiola species (R. rosea, R. kirilowii and R. crenulata)
10 [141]. The specific patterns in the metabolite profiles for different Rhodiola species were
11 revealed by PCA and OPLS-DA analyses. It was found that the coumarin crenulatin only existed
12 in R. crenulate. Thus, crenulatin could be used as a marker to detect potential adulteration of R.
13 crenulate in commercial products.
14 1H NMR coupled with chemometric analysis was employed to distinguish pure and
15 adulterated Curcuma longa with C. heyneana [142]. C. longa has many pharmacological
16 activities and is widely use in botanical health products. Other Curcuma species such as C.
17 heyneana have the potential to be used as adulterant due to their lower price and wide
18 availability. Based on the fingerprint differences in the aromatic regions of 1H NMR spectra, C.
19 longa and C. heyneana could be differentiated. The adulteration of C. longa by adding C.
20 heyneana in different concentrations from 5% to 100% could be revealed by PCA and OPLS-DA.
21 1H NMR coupled with chemometric analysis was also applied to assess the consistency and
22 quality of 47 St John's Wort (Hypericum perforatum) commercial products (granulated powders
23 and extracts) sourced from different suppliers [143]. The results demonstrated that there was
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1 significant compositional variation among commercial products. Adulteration with other
2 Hypericum species or use of chemically distinct H. perforatum cultivars or chemotypes was
3 detected in 36% of the products, and adulteration with food dyes (tartrazine, amaranth, brilliant
4 blue, sunset yellow) was detected in 19% of the products. Several other applications of NMR
5 methodology for the detection of substitutional adulteration in botanical products reported are
6 summarized in Table 4.
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Table 4. Applications of NMR for detection of adulteration substituents
1
Saposhnikoviae radix H NMR coupled with The cultivated Saposhnikoviae radix samples were discriminated from the [144]
(Saposhnikovia chemometric analysis wild samples; Saposhnikoviae radix was differentiated from the root of
divaricata) (PCA and PLS-DA) Peucedanum ledebourielloides
1
Java Turmeric H NMR coupled with Adulteration of C. xanthorrhiza with C. aeruginosa was investigated [145]
(Curcuma chemometric analysis
xanthorrhiza) (PCA and OPLS-DA)
Products of Rhodiola 1D and 2D NMR The developed method was used for investigations of the phytochemical [141]
rosea L. coupled with diversity of different Rhodiola species (R. rosea, R. kirilowii and R.
chemometric analysis crenulata), the recognition of unique metabolites between them and the
(PCA and OPLS-DA); identification of adulterated products
HPLC/UV
1
Curcuma longa L. 1D and 2D J-RES NMR; H NMR spectra-based metabolite fingerprinting and chemometric analyses [142]
chemometric analysis (PCA and OPLS-DA) were used to differentiate pure and adulterated C. longa
(PCA and OPLS-DA) with C. heyneana. Sugar compounds and curcuminoid were found to be the
important metabolites for discrimination of C. longa and adulterated C. longa
with C. heyneana
1
St John's wort H NMR coupled with Significant compositional variation among commercial finished products was [143]
(Hypericum chemometric analysis observed, and two main causative quality problems were identified as
perforatum L.) (PCA); HPTLC adulteration by incorrect species or with food dyes
products
Saraca asoca (Roxb.) 1D and 2D NMR The widespread adulteration of market samples of S. asoca was observed. The [146]
Willd (DOSY, COSY, HSQC, results showed that more than 80 % of the tested samples were spurious,
HMBC, and TOCSY); representing plant material from at least seven different families
chemometric analysis
(PLS-DA)
1
Rhodiola rosea L. H NMR coupled with The phytochemistry of different Rhodiola species was investigated and the [147]
products chemometric analysis potential of R. crenulata as an adulterant in R. rosea products was assessed.
(PCA); HPTLC; MS Approximately 80% of the commercial products were lower in rosavin
content than the registered products and appeared to be adulterated with other
Page 44 of 98
Rhodiola species
1
saffron (Crocus sativus H NMR coupled with OPLS-DA and O2PLS-DA models for the 1H NMR data was used for [148]
L.) chemometric analysis authentication and prediction of authentic and adulterated saffron. The
(PCA, OPLS-DA and developed method was reliable in assessing the type of adulteration and could
O2PLS-DA) be viable for dealing with extensive saffron frauds at a minimum level of 20%
(w/w)
1
Asian ginseng, and H NMR and multi-step The representative differences of Asian ginseng from American ginseng were [149]
cultivated and wild PCA found and the key metabolites responsible for differentiation were identified.
American ginseng A substitute cultivated American ginseng was noticed
1
lavender essential oil H NMR coupled with PCA was conducted for the 1H NMR data of 13 authentic [150]
(LEO) chemometric analysis LEOs and 27 unidentified samples. 12 out of the 27 unidentified samples were
detected as the adulterated
1 1
Panax ginseng H NMR-based H NMR and OPLS-DA were applied for distinguishing between samples [151]
metabolomics approach from two countries and seven primary metabolites were identified as
(OPLS-DA) discrimination markers
1
Polygoni Multiflori H NMR fingerprints The characteristic profiling of 1H NMR fingerprints was applied to identify [152]
Radix (root of coupled with Polygoni Multiflori Radix (PMR) from its common counterfeit Cynanchi
Polygonum chemometrics Auriculati Radix (CAR) using both the N3 and PLSDA models.
multiflorum Thunb)
1
Danggui (Angelica H NMR fingerprints Significant differences in secondary and primary metabolites between [153]
sinensis) and European coupled with Danggui and European Danggui were revealed by 1H NMR fingerprints
Danggui (Levisticum chemometrics coupled with chemometric analysis
officinale)
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1 3.3. Exploration of metabolite variability
2 Quality and efficacy of botanical products are directly related to the type and content of
3 chemical components contained in plant materials. The primary, and particularly, the secondary
4 metabolites accumulated in specimens of the same plant species could vary significantly due to
5 the influence of various factors such as growth location, cultivation condition, age, harvesting
6 time (season), and plant part (i.e., root, bark, or leaf). Such variations could lead to inconsistent
7 therapeutic and health promoting properties of botanical health products. NMR in combination
8 with chemometric analysis has proven to be a useful tool for investigating the variability of
9 metabolites effected by those factors.
10 3.3.1. Geographic origin
11 1H-NMR combined with chemometric analyses (PCA and OPLS-DA) was applied to
12 study the intraspecies chemotypic variation in Sceletium tortuosum (L.) samples collected from
13 two distinct regions of South Africa [154]. S. tortuosum is widely recognized for the treatment of
14 stress, anxiety and depression, as well as for obsessive compulsive disorder. The PCA model
15 built from the NMR dataset of 145 samples revealed two main clusters that were found to be
16 region-specific (Fig. 5). 2D NMR was used to identify the marker compounds that contributed to
17 the clustering as revealed by the S-plot from OPLS-DA. qNMR analysis indicated significant
18 variations in the contents of the marker compounds in samples of the two groups. Lamiophlomis
19 rotata (Benth.) samples from three different geographical regions were investigated by using 1H-
20 NMR for the variability of chemical composition [155]. L. rotata is an herbal folk medicine that
21 has traditionally been used to treat hemorrhage, as well as to relieve pain and tumescence. The
22 PCA and HCA analyses of NMR data allowed the classification of L. rotata samples into three
23 groups corresponding to their geographic origins. In this study, nine iridoid glycosides, one
Page 46 of 98
1 flavonoid, and three phenylpropanoid glycosides were profiled. Several other botanical
2 specimens that are commonly used in traditional medicines, including
3 Korean angelica (Angelica gigas) [156], rhizomes of Rhodiola rosea L. [157], roots of
4 Saposhnikovia divaricata (Turcz.) [158], Narcissus Bulbs (Leucojum aestivum) [159], Rhizoma
5 chuanxiong (Ligusticum chuanxiong Hort.) [160], ginseng roots (Panax ginseng) [161], Mexican
6 plant Galphimia glauca [162], and medicinal grass Eleusine indica [163], have also been
7 investigated using NMR coupled with chemometric analysis to better understand the variabilities
8 of their chemical compositions under the influence of geographic location.
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1
2 Figure 5. A) PCA scores plot and B) OPLS-DA scores plot revealing two main clusters, and C)
3 OPLS-DA S-plot indicating variables responsible for the differentiation between the groups
4 (Reprinted from [154], with permission from Elsevier).
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1 3.3.2. Aging or growing stage
2 The change of chemical composition in the flowers of Tussilago farfara L. collected at
3 different development stages was investigated by using NMR in combination with chemometric
4 analysis [164, 165]. The flower buds of T. farfara (Farfarae Flos), an important ingredient in
5 many TCM formulas, are widely used for the treatment of cough, bronchitis, tuberculosis, and
6 asthmatic disorders. Traditionally, only the flower buds can be used as the herbal drug. The buds
7 are usually collected in winter of the first year or spring of the next year before blooming. It is
8 believed that the bloomed flower cannot be used as it would possess “diminished medicinal
9 qualities”; however, the underlying scientific basis is not clear. The NMR fingerprints obtained
10 for the flower buds and the bloomed flowers indicated significant differences. Further
11 investigations using PCA and 2D NMR analyses, as well as bioactivity assays were conducted to
12 identify the endogenous metabolites correlated with the antitussive and expectorant effects of the
13 flower buds. Zheng et al. applied NMR coupled with chemometric techniques (PCA and OPLS-
14 DA) to investigate metabolite variation of different-age samples of Astragali Radix (the root of
15 Astragalus membranaceus Bunge or A. membranaceus var. mongholicus, one of the most well-
16 known traditional medicines and widely used in far-East Asia) collected in different locations
17 [166]. It was concluded that age had more influence on the sample discrimination than
18 geographical location. The quantity of individual flavonoids and some primary metabolites
19 contributed the greatest to the age differentiation. Isoflavonoids were found to be influenced both
20 by age and location but saponins were mostly associated with geographical origin. 1H NMR
21 metabolic fingerprinting in combination with principal component and hierarchical clustering
22 analyses was used to explore the metabolic differences of the mature and immature fruits, along
23 with the leaves of Sambucus ebulus, a traditional medicine used for the treatment of various
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1 disorders [167]. It was found that immature fruits and leaves of S. ebulus had a similar
2 metabolome, while significant changes occurred during the fruit ripening stage. A 1H qNMR
3 method was applied by Wang et al. [168] to reveal the composition changes of five
4 anthraquinones in the roots of Rheum palmatum (an important traditional medicinal herb listed in
5 Chinese Pharmacopoeia) harvested at both different ages and seasons. A distinct seasonal pattern
6 in the contents of individual and total anthraquinones across different growth years was observed.
7 The qNMR analysis was rapid, reference-free and convenient with less sample pre-treatment. In
8 addition, the results agreed with those obtained by the HPLC–UV. The relevance of harvesting
9 dates to the chemical profile of Isatis tinctoria, a well-known traditional medicine, was
10 investigated by using 1H NMR, and the spectral data were analyzed by a combination of different
11 statistical methods [169]. The results showed the metabolite composition varied significantly at
12 different harvesting time from June to September. In addition to the aforementioned applications,
13 NMR in combination with chemometric analyses used to compare differences of chemical
14 composition in mature and young leaves of Melicope ptelefolia (a healthy vegetable) [170] and
15 in different-age roots of ginseng (Panax ginseng) [171, 172] have also been reported.
16 3.3.3. Part or tissue region
17 A 1H NMR-based metabolomics approach was used to study the variability of
18 metabolites in different parts of Cistanches Herba (Cistanche deserticola), one of the most well-
19 known edible tonic and medicinal plants [173]. 1D and 2D NMR techniques in combination
20 with available databases and authentic compounds were utilized for the assignment of resonance
21 signals. The OPLS-DA analysis demonstrated that the chemical profile of upper parts of
22 Cistanches Herba significantly differed from the lower parts of the plant. In addition, some
23 metabolites such as oligosaccharides and fatty acids were screened out as the chemical markers
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1 for the discrimination. Ashwagandha (Withania somnifera), known as Indian ginseng, is a
2 popular medicinal plant and widely used as a dietary supplement. The metabolite variation in
3 leaves and roots of the plant was investigated using NMR and chromatographic (HPLC and GC–
4 MS) techniques [174]. Significant qualitative and quantitative differences were observed
5 between the leaf and root tissues, particularly with respect to the secondary metabolites. In
6 another investigation, HR-MAS NMR technique was recruited for the rapid metabolomic
7 analysis of leaf and root tissues of different chemotypes of W. somnifera [175]. A total of 41
8 metabolites were identified from both the leaf and root tissues of the chemotypes. HR-MAS
9 NMR is a useful analytical tool to obtain chemical information directly from samples in their
10 natural, unaltered states. Three parts (leaves, roots, and stems) of Berberis laurina Billb., a well-
11 known medicinal plant, were study by means of HR-MAS NMR analysis [176]. Berberine, a
12 characteristic alkaloid of the genus Berberis with manifold biological activities, was found in
13 high amounts in roots, when compared to stems and leaves. HR-MAS NMR, as a rapid, non-
14 destructive method which requires minimal sample preparation with no solvent extraction
15 procedure, has been increasingly used to provide a comprehensive localized metabolic profile of
16 large heterogeneous specimens. Notably, the advanced high-resolution micro-MAS probe (HR-
17 μMAS) enable the high-quality NMR data acquisition to be implemented for small-scale
18 specimens (at micro-gram level). This technique was successively applied to investigate the
19 metabolic profiles of four homogeneous anatomical garlic regions (skin, flesh, inner epidermis,
20 and sprout) [70]. Significant variations of different regions were identified by PCA and OPLS-
21 DA. The significant presence of fructose in both the skin and flesh, and the predominance of
22 sucrose and glucose in the core were revealed. Among the characteristic sulfur compounds of
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1 garlic, allicin was found to be dominant in the core, while allyl mercaptan was predominately
2 found in both the skin and flesh.
3 3.3.4. Growing environment
4 The effect of growing location on the chemical composition alteration of maca (Lepidium
5 meyenii Walpers) hypocotyls was investigated by using NMR combined with chemometric
6 analysis [177]. Maca, also called Peruvian ginseng, is widely used as a dietary supplement for
7 aphrodisiac purposes and to increase stamina. The PCA analysis of NMR spectral data
8 differentiated the maca accessions in terms of different cultivation conditions. The planting
9 location was found to be the major determining factor with regards to metabolite variations. The
10 1H NMR coupled with PCA was applied to evaluate the chemical composition of seeds from
11 different Chia (Salvia hispanica L.) populations [178]. With high nutritive value, Chia seeds are
12 used as both an ethnic medicine and a dietary supplement. The results obtained from NMR
13 analysis of the seeds cultivated with different fertilization conditions revealed significant
14 variations in the contents of carbohydrates, flavonoids, and aliphatic amino acids.
15 Pseudostellariae Radix (PR, root of Pseudostellaria heterophylla) is an important traditional
16 medicine and cultivated on a large scale in China. NMR coupled with PCA and OPLS-DA were
17 applied to gain insight on chemical composition differences of PR from two types of cultivation
18 fields [179]. A total of 34 metabolites were identified in PR based on 1H NMR analysis, while 14
19 of them were found to vary significantly in content due to the different cultivation conditions. 1H
20 NMR coupled with chemometric analysis and anti‐inflammatory bio-assays were also used to
21 evaluate the metabolite and bioactivity variabilities of Scurrula ferruginea (Jack) Danser
22 parasitizing on three different host plants [180]. S. ferruginea is a hemi‐parasitic shrub and
23 used as a traditional medicine in many Asian countries. The PCA analysis of NMR data resulted
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1 in a clear discrimination of S. ferruginea samples based on different host plants. Several
2 metabolites that might be responsible for the discrimination and activity were identified.
3 3.4. Discrimination of botanical materials
4 3.4.1. Species differentiation
5 To ensure that a botanical product is consistent, safe, and effective for its intended use, it
6 is crucial that the correct plant species is used at the beginning of botanical products
7 manufacturing. Generally, plant species are identified by macroscopic and microscopic
8 morphological features. For those species that have close taxonomic relationships or have subtle
9 differences in morphological features, it becomes very difficult to differentiate them on the basis
10 of morphological features alone, especially when they are distributed in the botanical material
11 supply chain in dried, partially processed, or even ground forms. Therefore, alternative strategies
12 such as DNA and chemical methods are usually needed in most cases. NMR is considered to be
13 the most promising analytical tool for plant species differentiation because the method allows the
14 simultaneous detection of primary and secondary metabolites in a single run, while producing a
15 non-biased holistic metabolic profile containing qualitative and quantitative information.
16 Combined with chemometrics, NMR has been increasingly used as a powerful tool for species
17 discrimination and for discriminator (marker metabolites that make significant contribution for
18 species discrimination) exploration. A large number of successful application cases, as presented
19 in Table 5, have demonstrated that NMR is a suitable and adequate tool to accomplish such tasks.
20
Page 53 of 98
Table 5. Applications NMR in plant species differentiation
1
Three different Rhodiola H NMR fingerprinting and Three Rhodiola species were clearly discriminated by 1H NMR [181]
species (R. crenulate, R. chemometric analyses (PCA, fingerprinting involved 22 resonance signals of chemical
kirilowii, and Rhodiola PLS-DA, and HCA) constituents. The chemometric analyses revealed that gallic acid,
fastigiate) salidroside, α-D-glucose, glycine, alanine, caffeic acid and tyrosol
and are the discriminators
1
Six different Polygonatum H NMR based metabolite Metabolite profiling was applied to discriminate between [182]
species profiling and chemometric Polygonatum species. 4-Aminobutyrate, alanine, asparagine,
analysis; UPLC/Q-TOF MS isoleucine, malate, O-phosphocholine and phenylalanine were
found to be the most influential metabolites for discriminating
Polygonatum species by 1H NMR
1
Leaves from 17 different H NMR fingerprinting and A total of 78 metabolites were tentatively identified, of which [183]
species within the genus profiling; chemometric several flavonoid conjugates were for the first time to be reported
Passiflora analyses; UPLC-MS in Passiflora spp. The study provided the most complete map for
secondary metabolite distribution within the genus
1
Pelargonium sidoides and P. H NMR and UHPLC–MS Nontargeted 1H NMR and UHPLC–MS metabolomic approaches [184]
reniforme metabolomic profiling and were applied to differentiate P. sidoides and P. reniforme extracts.
chemometric analysis qNMR was used for quantitative comparison of the marker
constituents occurring in both species
1
Licorice botanicals (three H NMR fingerprinting and The developed chemometric models based on 1H NMR data [185]
Glycyrrhiza species: G. profiling; chemometric enable the identification and classification of Glycyrrhiza species
glabra, G. uralensis, and G. analyses; UHPLC-UV according to their composition and species-specific phenolic
inflata) compounds
1
Seven Hypericum species H NMR and UPLC–MS NMR and MS coupled with multivariate data analysis were used [186]
metabolomic profiling and and compared to obtain the experimental results, and interesting
chemometric analysis and meaningful differences between the various species and
detection methods were identified
1
Swertia mussotii and Swertia H NMR-based A rapid, simple, and comprehensive 1H NMR-based metabolomics [187]
chirayita (traditional Tibetan metabolomics method was developed to differentiate the two species. A broad
medicine) range of metabolites, including iridoid glycosides, xanthones,
Page 54 of 98
triterpenoids, flavonoids, carbohydrates, and amino acids, were
identified
1
Pueraria lobata and Pueraria H NMR profiling and Thirteen isoflavones were tentatively identified based on 1D and [188]
thomsonii chemometric analyses (PCA 2D NMR analyses. Obvious chemical compositional difference
and PLS-DA) between the two species was revealed by PCA and PLS-DA.
Puerarin was identified as the marker for the discrimination of the
two species
1 1
Two Bupleurum species (B. H NMR metabolic H NMR and multivariate data analysis were applied to 67 Radix [189]
chinense and B. fingerprinting and Bupleuri samples for discrimination of the two species. The
scorzonerifolium) used as chemometric analyses influences of habitat and culture method on the quality of Radix
Radix Bupleuri Bupleuri were explored based on their metabolomics profiles
1 1
Two Curcuma species (C. H NMR profiling and H NMR metabolite profiling coupled with multivariate analysis [190]
aromatica and C. longa) chemometric analysis was applied to characterize the differences between species or
origins. PCA showed significant differences between species than
origins, and the metabolites responsible for the differences were
identified
Glycyrrhiza species (G. Metabolite profiling and An unbiased multiplex approach integrated NMR and MS coupled [191]
glabra, G. uralensis, G. fingerprinting with a with multivariate data analyses was adopted to reveal
inflata and G. echinata) for multiplex approach of GC– compositional differences in primary and secondary metabolites
licorice roots MS, LC–MS, and 1H NMR among Glycyrrhiza species. Differences between various samples
and detection methods were identified
1 1
Five Verbascum species (V. H NMR fingerprinting in H NMR fingerprinting in combination with PCA and HCA [192]
nigrum, V. densiflorum, V. combination with allowed classification of Verbascum species in two groups. Two
phoeniceum, chemometric analyses species were found to accumulate high amounts of phenylethanoid
V. phlomoides, and V. glycosides
xanthophoeniceum)
1 1
Polygonum cuspidatum and P. H NMR fingerprinting in H NMR and multivariate analysis were applied to almost 60 [193]
multiflorum combination with samples collected in different places for discrimination of the two
chemometric analysIs Polygonum species. A clear distinction between the two species
was observed and the discriminant metabolites were identified
1 1
Cistus species and subspecies H NMR metabolic H NMR fingerprinting in combination with PCA was applied to [194]
fingerprinting in combination 678 samples from wild-growing Cistus populations, including
Page 55 of 98
with chemometric analysis seven species and six subspecies/varieties, to identify
discriminators and specific markers for the differentiation between
Cistus (sub-) species
1 1
Roots/rhizomes and aerial H NMR fingerprinting in H NMR fingerprinting in combination with PCA was applied to [195]
parts of three Actaea species combination with 33 Actaea plant samples for differentiation of three species.
(A. racemosa, A. podocarpa chemometric analysis Several characteristic peaks and peak patterns were identified for
and A. cordifolia) each group of samples
1
Four Crataegus species (C. H NMR fingerprinting in Differentiation of Crataegus spp. was best achieved by focusing [196]
laevigata, C. douglasii, C. combination with on the NMR spectral region containing signals unique to phenolic
okanaganensis, and C. chemometric analysis compounds. Identification of potentially significant metabolites for
monogyna) differentiation between species was approached
1
Five Actaea species (A. H NMR fingerprinting in Differences were observed between A. racemosa samples from [197]
racemosa, A. rubra, A. combination with four different sources, although the variance within species was
cimicifuga, A. pachypoda, A. chemometric analysis still significantly less than the variance between species. A model
podocarpa) based on combined A. racemosa samples from the four sources
permitted distinction between species
1
Rhodiola Species (R. rosea, R. H NMR fingerprinting in The utilization of a combined platform based on 1H NMR and [198]
crenulata, R. fastigiate, R. combination with HPTLC methods resulted in an integrated analysis of different
sachalinensis, and R. chemometric analysis; Rhodiola species.
quadrifida) HPTLC
1
Harpagophytum procumbens H NMR fingerprinting in UHPLC-MS and 1H NMR techniques were used for identifying [199]
and its close taxonomical ally combination with metabolites, and differentiating the metabolite patterns of two
H. zeyheri chemometric analysis and Harpagophytum species. Quantification of the biomarker,
UHPLC-MS harpagoside, showed that it was highly variable, and greater in H.
procumbens and detected in low concentrations in some
populations of H. zeyheri
1
Vaccinium Species (V. H NMR fingerprinting in The three Vaccinium species can be distinguished based on leaf [200]
angustifolium, V. ovalifolium, combination with extracts using 1H NMR. Spectra acquired at five different sites
and V. macrocarpon) chemometric analysis with differences in field strength, console, probe, sample-tube
diameter, and software were classified into the expected group in
the PCA
1
Two ginseng species (P. H NMR fingerprinting in The multivariate analysis indicated distinct patterns in different [201]
Page 56 of 98
quinquefolius and P. ginseng) combination with metabolites, which distinguish Ontario ginseng landraces from one
chemometric analyses another. In addition, Ontario ginseng (P. quinquefolius) was
compared with Asian ginseng (P. ginseng) to determine metabolite
differences
1
Five Phyllanthus species (P. H HR-MAS NMR and 1H Taxonomic distinction among the Phyllanthus species was [202]
amarus, P. caroliniensis, P. NMR in solution, combined successfully attained by PCA and HCA analyses of the data from
niruri, P. tenellus and P. with chemometric analysis; the three techniques evaluated: FT-IR, 1H HR-MAS NMR and 1H
urinaria) FT-IR NMR of aqueous and ethanolic extracts. The phyllanthin signals
were related to discrimination of the P. amarus sample
1
Rhizoma Coptidis from H NMR combined with NMR-based metabolic fingerprinting provided global metabolite [203]
different Species (C. chemometric analysis information; Chlorogenic acid, sucrose, and fatty acids were found
chinensis, C. deltoidea, and C. to be responsible for the discrimination of different Coptidis
teeta) species
1
Hoodia species (H. gordonii, H NMR fingerprinting in NMR fingerprinting and multivariate analysis were applied for [204]
H. currorii, H. parviflora, and combination with identification, discrimination, and quality analysis of Hoodia plant
H. rushii) chemometric analysis materials and commercial products
1
Eleven Leontopodium species H NMR fingerprinting in Metabolic patterns of 11 Leontopodium species were revealed by [205]
1
(Asteraceae) combination with H NMR and LC–MS metabolic fingerprinting. The insights
chemometric analysis; concerning species relationships within the genus could be
HPLC–ESI-MS acquired with both fingerprinting approaches
1 1
11 South American Ilex H NMR fingerprinting in H NMR combined with PCA, PLS-DA and HCA showed a clear [206]
species combination with separation between species and resulted in four groups based on
chemometric analysis metabolite similarities.
1
Echinacea species (E. H NMR fingerprinting in A clear distinction between three pharmaceutical species was [207]
purpurea, E. pallida, and E. combination with observed and useful metabolites were identified
angustifolia) chemometric analysis
1
Ephedra Species (E. sinica, E. H NMR fingerprinting in Major differences in three Ephedra species were found to be due [208]
intermedia and E. distachya combination with to benzoic acid analogues in the aqueous fraction and ephedrine-
var. distachya) chemometric analysis type alkaloids in the organic fraction
11 Ilex species NMR spectroscopy and Species could be discriminated by multivariate analysis of their [209]
multivariate data analysis metabolite fingerprints obtained by 1H NMR spectra for the crude
Page 57 of 98
extracts. The major compounds contributing to the discrimination
were found to be several phenylpropanoids and arbutin
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1 As examples, NMR combined with chemometrics was applied to investigate the
2 roots/rhizomes and aerial parts of three Actaea species, A. racemosa (AR), A. podocarpa (AP)
3 and A. cordifolia (AC) [195]. Roots and rhizomes of AR, known as black cohosh, are
4 traditionally used for indications of premenstrual syndrome (PMS), menstrual pain and cramping.
5 Currently, AR is widely used as a ingredient in dietary supplements. AP and AC were found to
6 be misused as AR due to misidentification and/or economic reasons. Based on the NMR spectral
7 and chemometric PCA analysis, the samples from the three Actaea species could be
8 discriminated. Several characteristic peaks and peak patterns were identified for each group of
9 samples. An investigation of metabolite distinction between Pelargonium sidoides and P.
10 reniforme was conducted by Viljoen and co-workers [184]. These two species are closely
11 related taxonomically. They can only be differentiated based on subtle morphological features.
12 Although both species are acceptable in some herbal pharmacopeias as ingredients to produce
13 phytomedicines, the use of P. sidoides is preferred according to the results of several clinical
14 studies on the efficacy of patented formulations containing different species. 1H-NMR in
15 combination with PCA and OPLS-DA approaches illustrated a chemical distinction between the
16 two species, which was in accordance with the parallel UHPLC–MS analytical result. 2D NMR
17 analyses (i.e., HSQC and HMBC) revealed that coumarines were the marker constituents
18 responsible for the discrimination of the two species, and the results were confirmed by UHPLC-
19 MS analysis. qNMR was also used for the quantitative comparison of coumarines occurring in
20 both species in this study.
21 3.4.2. Cultivar/variety discrimination
22 In addition to being used for plant species discrimination, NMR can also be used for
23 differentiation and characterization of cultivars (varieties) of the same species. Most of the
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1 popular and useful botanicals usually have long cultivation histories, resulting in various
2 cultivars. There is great interest in developing accurate methods for cultivar characterization that
3 could be used to compare cultivars reliably for differences and similarities in metabolic profiles,
4 to trace variability of nutritionally or biologically important components, and to support
5 germplasm preservation and reproduction of the best cultivars. NMR in combination with
6 chemometric analysis has been successfully applied in this area. As examples, Wang et al. used
7 NMR combined with chemometrics as a fast screening method for discrimination of Cannabis
8 cultivar samples [210]. Cannabis from different cultivars may be very different with respect to
9 their pharmacological properties and chemical compositions, and be marketed for different uses.
10 Thirty-two Cannabis samples were investigated by using 1D 1H and 2D 1H-1H correlation
11 (COSY) NMR. Six classification methods—fuzzy rule-building expert system, linear
12 discriminant analysis (LDA), super partial least-squares discriminant analysis, support vector
13 machine (SVM), and SVM classification trees (SVMTreeG and SVMTreeH) were used to
14 construct discriminatory models that could separate the spectra profiles into known classes. The
15 LDA method was found to have the best prediction accuracy of 99.8 ± 0.4%. The metabolite
16 profiles and classification of commercial hop (Humulus lupulus L.) cultivars were investigated
17 by Farag and co-workers using NMR and chemometric analysis [211, 212]. Cultivar segregation
18 in PCA plots generated from NMR data was found comparable to that from LC-MS data, and
19 mostly influenced by differences in the composition of bitter acids among cultivars. 2D HMBC
20 NMR based PCA analysis was successfully used for hop cultivar classification. 1H high
21 resolution MAS-NMR was used to analyze garlic (Allium sativum L.) belonging to red and white
22 varieties [71]. Using PLS-DA model, the varieties were discriminated, and the metabolites
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1 responsible for the discrimination were identified. A number of other applications of NMR in
2 discriminating cultivars/varieties have been reported. A summary is presented in Table 6.
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Table 6. Applications of NMR in cultivar/variety discrimination
Botanical / Product Methods / Techniques Remarks Reference
1
Chemotypes of H qNMR methods coupled to NMR analysis unveiled that distinct non-volatile compositions [213]
gastroprotective herb Egletes chemometrics were present in different chemotypes. The gastroprotective
viscosa compounds tanabalin and centipedic acid were higher in chemotype
A
1
Ten cultivars of Capsicum H NMR based metabolite Forty-eight metabolites were identified and quantified by 2D NMR [214]
annuum cv. Serrano profiling and chemometric and qNMR, respectively. PCA and PLS-DA separated the ten races
analysis into two clusters, from which citric acid, formic acid, fumaric acid,
malic acid, glucose, fructose, sucrose and galactose were found as
differential metabolites
Hancornia speciosa varieties HR-MAS NMR allied to Leaves of four varieties of H. speciosa was studied and [74]
chemometric analysis demonstrated that H. speciosa var. speciosa differed from the
others due to its specific metabolic profile, and var. pubescens was
discriminated based on its high phenolic compound content
Cannabis sativa cultivars 1D 1H and 2D COSY NMR A high throughput deuterated-solvent direct-extraction method was [210]
coupled with pattern applied to Cannabis samples and, coupled with NMR detection,
recognition classification yielded a facile approach to authenticating Cannabis botanicals
methods
1
Globe artichoke (Cynara H NMR based metabolite The detailed metabolite profiles of artichoke and cardoon varieties [215]
cardunculus L. var. profiling and chemometric were obtained. Relevant differences in the content of the
scolymus) and cardoon analysis corresponding metabolites were observed
(Cynara cardunculus L. var.
altilis)
Cultivars of Humulus lupulus An approach in coupling 2D 2D HMBC NMR profile maps of hop resins (H. lupulus) were [212]
L. (hop) NMR datasets with PCA generated for a comparative study of 13 hop cultivars
PGI (Protected Geographical HR MAS-NMR coupled with The established statistical models could provide clear [216]
Indication) chicory chemometric analyses discrimination among the two varieties and classification according
(Cichorium intybus L.) to geographical origins
Cultivars of Humulus lupulus MS and NMR methods in Resins and extracts prepared from 13 hop cultivars were analyzed [211]
Page 62 of 98
L. (hop) combination with PCA using NMR, LC-MS, and FTICR-MS in parallel and subjected to
PCA. Forty-six metabolites including 18 bitter acids, 12 flavonoids,
3 terpenes, 3 fatty acids and 2 sugars were identified and quantified
1
Italian garlic (Allium sativum HR MAS-NMR and H HRMAS-NMR was used to analyze garlic belonging to red and [71]
L.) multivariate data analysis white varieties and collected in different Italian regions in order to
address the traceability issue. 1D and 2D NMR spectra allowed the
assignment of organic acids, sugars, fatty acids, amino acids, the
nutritionally important fructo-oligosaccharides, and allyl-
organosulphur compounds
1
Silk extracts from seven H NMR based metabolite PCA of 1H NMR data set showed clear discrimination among the [217]
maize landraces (Zea mays profiling and chemometric maize varieties, pointing out three distinct metabolic profiles.
L.) cultivated in southern analysis Target compounds analysis showed significant differences in the
Brazil contents of protocatechuic acid, gallic acid, t-cinnamic acid, and
anthocyanins, corroborating the discrimination of the genotypes as
revealed by PCA
1
Catharanthus roseus H NMR fingerprinting and PCA of 1H NMR total, aliphatic, carbohydrate, and aromatic region [218]
cultivars PCA data, revealed possible relationship between eight cultivars.
Secologanin and polyphenols were contributed most profoundly to
the discrimination between cultivars
1
Cannabis sativa cultivars H NMR fingerprinting and The discrimination of the cultivars could also be obtained from a [219]
PCA water extract containing carbohydrates and amino acids. The level
of sucrose, glucose, asparagine, and glutamic acid are found to be
major discriminating metabolites of different cultivars
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1 3.5. Effect of processing
2 While converting raw plant materials into final commercial botanical health products, series
3 of processing operations is usually implemented. The processes may include drying, grinding,
4 extracting, decocting, fractionating, precipitating, concentrating, mixing, packaging, etc. In some
5 cases, fermenting, frying, steaming, or other forms of treatment may be used during processing.
6 To ensure the consistency and controllability of product quality, it is necessary to understand the
7 effect of processing on the change of chemical composition in botanicals. NMR is gaining
8 greater attention as a powerful tool for the investigation of chemical changes during botanical
9 processing. For examples, 1H NMR combined with chemometric analysis was applied to
10 investigate the effect of processing factors (heating temperature and duration) on the chemical
11 composition during the processing of Flos Lonicerae (flowers of Lonicera japonica) by stir-
12 frying [220]. The heat map derived from NMR spectral data was used to pinpoint the significant
13 intensity changes of signals in different stages of processing. PCA on the NMR dataset provided
14 a holistic view for the evaluation of the chemical composition change during processing. Five
15 chemical components, whose concentration changed significantly during the processing, were
16 identified and quantified. In another investigation, qNMR was used to quantitatively determine
17 the differences of ellagitannins and related polyphenols in Geranium thunbergii in both short-
18 time and long-time decoctions [221]. G. thunbergii is an important source of ellagitannins with
19 up to 10% content in dried leaves. Geraniin is the major ellagitannin in G. thunbergii, which has
20 long been used as a remedy for intestinal disorders. The qNMR results indicated that geraniin
21 was the major component in short-time decoction, while corilagin was the major compound in
22 long-time decoction.
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1 LF NMR approach was employed for monitoring the drying process of Gastrodia elata
2 Blume, an important traditional herb used in many Asian countries [222]. The variation and
3 distribution of three water states (bound, immobilized, and free) in G. elata samples during
4 drying were measured through multiexponential fitting and inversion of the NMR data. The
5 result demonstrated that LF-NMR was a feasible and convenient tool for nondestructively
6 monitoring the moisture content during the drying process of botanicals. The effect of two
7 different post-harvest drying methods (shade drying and tray drying) on the chemical
8 composition of Hibiscus sabdariffa calyces was investigated by using NMR and PCA [223]. H.
9 sabdariffa calyx is traditionally used as both a nutritious food and herbal medicine. The 1D and
10 2D NMR spectral analyses allowed 18 polar metabolites to be identified. The PCA result showed
11 that the metabolite composition significantly differed between shade-dried and tray-dried
12 samples. It was found that tray drying was more efficient than shade drying in reducing moisture
13 and maintaining both the metabolite and ash content of the Hibiscus biomass.
14 With the aid of 1H NMR combined with chemometric analysis, the influence of ethanol
15 concentration in the extraction solvent on the chemical composition of Salviae miltiorrhizae
16 Radix extract was investigated [224]. S. miltiorrhizae Radix extracts and derived products are
17 widely used for the treatment of various disorders, particularly cardiovascular diseases. Twenty-
18 eight metabolites including 6 sugars, 11 amino acids, 3 organic acids, 4 salvianolic acids, and 4
19 tanshinones were identified by NMR. The variation of chemical composition in extracts obtained
20 using different aqueous ethanol solutions ranging from 0 % to 90 % ethanol was revealed by
21 PCA, OPLS-DA and HCA. qNMR analysis revealed that an increase in tanshinones
22 concentration tended to be present in the extracts obtained with greater ethanol concentrations. In
23 another study, the effects of multi-step processing (seven steps) on the chemical changes in the
Page 65 of 98
1 process intermediates of S. miltiorrhizae Radix were evaluated by using 1H NMR combined with
2 chemometric analysis [225]. Thirty metabolites were identified by NMR spectral analysis. PCA
3 and OPLS-DA were applied to distinguish the major metabolite differences between the
4 intermediates. It was found that the second step, in which alkali-isolation and acid dissolution
5 methods were employed for intermediate processing, had the greatest influence on the quality of
6 intermediates.
7 HR-MAS NMR combined with chemometric analysis was used to reveal the chemical
8 composition diversification of four differently processed ginsengs [226], namely white ginseng
9 produced by dehydrating raw ginseng in sunlight, tae-geuk ginseng produced by boiling raw
10 ginseng in hot-water followed by drying, red ginseng produced by steaming followed by
11 dehydration, and black ginseng produced by steaming at followed by nine drying cycles. The
12 PCA and OPLS-DA analyses illustrated the clear discriminations of the four differently
13 processed ginseng samples, and significant variations in chemical profile were observed. In
14 another application reported last year, HR-MAS NMR was employed for the precise assessment
15 of the mixing degree between Ginkgo biloba extract (GB) and soy-lecithin-phosphatidylserine
16 (SLPS) in a product formulation. The processing outcomes obtained by using two different
17 mixing methods (mechanical and Phytosome®) were evaluated and compared [227]. The proton
18 relaxation information obtained from the solid-state NMR analysis highlighted a clear difference
19 between samples processed with the two different mixing methods.
20 3.6. Bio-activity evaluation and underlying mechanism exploration
21 The promotive health functions or therapeutic properties of botanical health products are
22 usually only substantiated with evidence based upon traditional use. In general, for most
23 products sold on the market, the active compounds responsible for the claimed properties are
Page 66 of 98
1 remain indefinite. Evaluating the purported function for a botanical health product is a great
2 challenge due to the complexity of chemical composition and possible synergistic effects
3 amongst different compounds contained in botanical products. Even so, in order to ensure the
4 quality, safety and efficacy of botanical health products, particularly for herbal medicine
5 products, it is of great significance to evaluate the biological (or pharmacological) activities, to
6 discover biomarker(s) and to expound the underlying mechanism of action. Metabolomics, based
7 on the comprehensive analysis of the metabolic composition in biological systems, is an effective
8 approach not only to evaluate systemic responses to interventions such as diet and drugs, but also
9 to provide complementary information for understanding of multifactorial mechanisms related to
10 the biochemical and physiological changes in the organism. NMR can provide stable, unbiased,
11 qualitative and quantitative results with less sample pretreatment requirements, making it widely
12 used in metabolomics research. In recent years, NMR-based metabolomics has demonstrated its
13 potential for evaluation of biological activity of botanical products. The approach is often
14 supported with the complementary use of in vivo or in vitro bio-assays.
15 Huiyang Shengji formula (HSF), a compounded Chinese herbal medicine prescription, is
16 used for treating chronic non-healing wounds. NMR-based metabolomics was applied to assess
17 the healing effects of HSF treatment [228]. A rat diabetic skin ulcer (DSU) model was
18 established for the study, with four different kinds of HSF treatments on DSUs being
19 investigated. A total of 30 metabolites were identified in the aqueous extracts derived from six
20 groups of rat skin tissues based on 1D and 2D NMR spectral analyses. The distinct metabolic
21 profiles of the six groups were analyzed and distinguished by PCA and PLS-DA statistical
22 models. The study found that two kinds of HSF treatments could partly reverse the metabolically
23 disordered pathological state of DSUs to the normal state. With the aid of metabolomic data
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1 analyses, the authors proposed that the wound healing effect of HSF might be in association with
2 the promotion of glucose and branched-chain amino acids (BCAAs) metabolisms and the
3 enhancement of antioxidant capacity and angiogenesis in DSU tissues.
4 Gastrodiae Rhizoma (GR), the dry tuber of Gastrodia elata Blume, is a well-known herbal
5 medicine used for treating headache, dizziness, epilepsy, and chronic atrophic gastritis. NMR-
6 based metabolomics was employed to assess the effects of different RG extracts (water, n-
7 butanol, ethyl acetate and petroleum ether extracts) on autoimmune chronic atrophic gastritis
8 (CAG) [229]. A rat CAG model was established for the study, stomach and serum samples were
9 collected and subjected to histopathologic, biochemical and metabolomic analyses. The results
10 showed that the water extract had the best therapeutic effect, suggesting that the polar
11 components might be more active. Base on the integrative data analysis, the authors proposed
12 that the therapeutic effects of GR extract on autoimmune CAG could be mainly related to the
13 reduction of inflammation and oxidative stress, as well as the amelioration of energy and amino
14 acid metabolisms.
15 The extract of leaves from Clinacanthus nutans (CN) is a well-established therapeutic
16 alternative for inflammation and used in Asian communities. The metabolic mechanism of the
17 effects of a 14 day oral treatment with CN aqueous extract administered to the
18 lipopolysaccharides (LPS) induced rats was explored by NMR-based metabolomics [230]. The
19 water-soluble components extracted from the homogenized rat brain tissue samples were profiled
20 by NMR approach, and the cytokine expression levels of the brain protein lysate samples were
21 measured using the G-series rat inflammation array. Based on the PCA of the NMR spectral data,
22 21 metabolites in the brain tissue were profiled as biomarkers for the LPS-induced
23 neuroinflammation following intracerebroventricular injection. It was found that CN treatment
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1 successfully altered lactate, pyruvate, phosphorylcholine, glutamine, and α-ketoglutarate when
2 compared to the negative control. The mechanistic role of CN in controlling the
3 neuroinflammatory conditions through the modulation of complex metabolite interactions in the
4 rat brain was discussed in this study.
5 To date, a number of investigations using NMR-based metabolomics approaches for
6 evaluating the bio-activities (effects) of botanical products have been published, a brief summary
7 is presented in Table 7.
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Table 7. Applications of NMR-based metabolomic technique in bio-activity evaluation and underlying mechanism exploration
Botanical / Product Methods / Techniques Remarks Reference
Huiyang Shengji NMR-based metabolomic The healing effects of four kinds of HSF treatments on DSUs were [228]
formula (HSF, an analysis for rat diabetic skin evaluated using NMR-based metabolomic analyses on skin tissues of the
herbal medicine) ulcer (DSU) model HSE-treated rats. It was found that the treatments up-regulated
expressions of two angiogenesis-related factors VEGF and CD31, and two
kinds of HSF treatments exhibited the most significant effects.
Shenling Baizhu San NMR-based metabolomic Significant dyspeptic symptoms such as weight loss, poor appetite, [231]
(SBS, a classical analysis was used to profile reduced gastrointestinal motility, and decreased absorptive capacity were
prescription of the fecal and urinary observed in the FD rats, which were subsequently improved by SBS. The
traditional Chinese metabolome in the functional metabolic disorders were amended with SBS intervention mainly by
medicine) dyspepsia (FD) rats during modulating the metabolic pathways involved in energy metabolism,
SBS intervention amino acid metabolism, and gut microbiota and host co-metabolism.
Gastrodiae Rhizoma NMR-based metabolomic The integrated metabolomics approach proved the validity of the [229]
(GR, tuber of analysis on the stomach and therapeutic effect of extract from different polar parts of Gastrodiae
Gastrodia elata serum samples collected Rhizoma on autoimmune CAG, providing new insights into the
Blume) from the rat model of chronic underlying mechanisms, and demonstrating the feasibility of
atrophic gastritis (CAG) metabolomics to evaluate efficacy of herbal drug, which is often difficult
by traditional means.
Toxicological risks of NMR-based metabolomic The study revealed that the RQCJ significantly altered the concentrations [232]
Renqingchangjue analysis on the serum and of 14 serum metabolites and 14 urine metabolites, which implied
(RQCJ, a traditional urine samples collected from disturbances in energy metabolism, amino acid metabolism, intestinal
Tibetan herbal the rat model flora environment, and membrane damage. Besides, the biochemical
medicine) analysis of serum samples was consistent with the histopathological
results, which indicated slight hepatotoxicity and nephrotoxicity.
Varieties of Ficus NMR-based metabolomic Metabolomics results showed the varieties were able to manage the [233]
deltoidea analysis; diabetic rat assay altered metabolites of diabetic rats by shifting some of the metabolites
back to their normal state. The most potential variety was recommended,
which may be useful for further pharmacological studies and herbal
authentication processes.
Huangqi Jianzhong NMR-based metabolomic The metabolomic approach was applied to reveal the efficacy of HQJZ on [234]
Tang (HQJZ, a famous analysis on the constructed the constructed CAG rats. The results revealed that the effect of HQJZ
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TCM formula consists chronic atrophic gastritis might be due to the balance of energy consumption, inflammatory
of seven herbs) (CAG) rats; UPLC-Q/TOF inhibition, improvement of the immune system and oxidative stress on the
MS constructed CAG rats.
Polysaccharides from NMR-based metabolomic NMR-based metabolomic analysis was used to investigate the metabolic [235]
polygonatum sibiricum analysis on the tissue and regulation effects of the polysaccharides on the mouse models. The
blood of mouse models. metabolite maps of the control group and the drug group in the liver had
significant changes. The main differential metabolites were identified.
Stem bark of Berberis NMR-based metabolomic B. vernae exerts a significant anti-diabetic effect and has potential as a [236]
vernae analysis on type 2 diabetic drug candidate for the treatment of type 2 diabetes. The NMR-based
rats; biochemistry assay; metabolomic analysis revealed that the anti-diabetic mechanisms of B.
molecular docking; network vernae might be related to its regulation of butanoate metabolism pathway
analysis and disease-related adipocytokine signaling pathway.
Defatted Dabai (fruit NMR-based metabolomic Assessment of 1H NMR urinary metabolomics revealed that [237]
of Canarium analysis on urine of supplementation of 2% of DDP could partially recover the dysfunction in
odontophyllum Miq.) hypercholesterolemic rats the metabolism induced by hypercholesterolemia via choline metabolism.
pulp extract (DDP)
Dried stem bark of NMR-based metabolomic A total of 28 metabolites were identified in rat serum by 1H NMR-based [238]
Berberids kansuensis analysis on serum of rat metabolomics method, 16 of which were significantly different in the
model normal group compared with the model group, and eight of them were
significantly reversed after B. kansuensis intervention.
Clinacanthus nutans NMR-based metabolomic Based on the PCA of the NMR spectral data, twenty-one metabolites in [230]
leaf extract analysis on the brain tissue of the brain tissue were profiled as biomarkers for the LPS-induced
rat model neuroinflammation following intracerebroventricular injection. A
moderate correlation, in the OPLS regression model, was found between
the spectral metabolite profile and the cytokine levels.
1
Astragali Radix (root H NMR-based metabolomic The results indicated that the optimal antifatigue effect of Astragali Radix [239]
of Astragalus approach on the serum and was observed at a dose of 3 g/kg body weight. Potential biomarkers in
membranaceus) urine metabolome of rats serum and urine were identified to elucidate the mechanism of Astragali
Radix in swimming rats to prolong exercise endurance. The antifatigue
effect of Astragali Radix is exerted by regulating pathways of
glycometabolism, lipid metabolism, energy metabolism, and changes of
other metabolites.
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1 3.7. Other applications
2 3.7.1. Dereplication
3 Botanical health products as complex mixtures contain a wide range of chemically
4 diverse constituents occurring in varying concentrations. It is important to obtain detailed
5 identity and quantity information about the constituents in botanical mixtures in regard to the R
6 & D of botanical products and the quality control. In doing so, constituent isolation is typically
7 indispensable. Repetitive isolation of known, unwanted or even readily available constituents is
8 one of the main factors limiting productivity of botanical products research. Dereplication, i.e.,
9 exclusion from further isolation efforts of constituents that have already been studied or are
10 otherwise unwanted, is of the utmost importance with respect to the outcome, as well as time,
11 cost, and effort spent on discovery of unknown or interesting constituents. Hyphenated NMR
12 techniques have been successfully used as a powerful tool for achieving dereplication in
13 botanical research. For instance, HPLC-PDA-HRMS-SPE-NMR was applied to the strategic
14 isolation of a series of targeted spiro compounds from Carthamus oxyacantha (wild safflower)
15 extract [240]. The extract was first analyzed by HPLC-PDA-HRMS-SPE-NMR, and selected
16 components were fully identified. These components were subsequently purified using a targeted
17 isolation procedure. Compared to the classical approach, in which as many extract components
18 as possible were isolated by preparative HPLC for final identification by spectroscopic methods
19 at a purified stage, the hyphenated NMR approach was considerably faster, while requiring less
20 solvent and other consumables. In another application, HPLC–PDA– MS–SPE–NMR was
21 employed to the rapid dereplication of an extract of aerial parts of Haplophyllum acutifolium
22 [241]. H. acutifolium is a rich source of quinoline alkaloids which are used in traditional
23 medicine as analgesics, antispasmodics, diuretics, and sedatives, as well as topical agents for
Page 72 of 98
1 skin diseases. The on-line analysis of nine peaks led to the identification of six new and two
2 known 4-quinolinone alkaloids as well as a hemiterpenoid 2-quinolinone alkaloid and a lignan.
3 The study demonstrated the fast and dependable dereplication for the isolation of alkaloid
4 constituents from a crude extract.
5 3.7.2. Discovery of bio-marker(s)
6 Discovering or determining bio-active components in botanical health products is often
7 hampered by difficulties, including the complexity of chemical composition and time-consuming
8 deconvolution. Conventional methodologies to discover bio-active constituents from natural
9 products require tedious and costly procedures including fractionation, isolation, purification,
10 bio-activity measurement, and other steps before an active compound can finally emerge from
11 the crude mixture, that is also the case in which bio-assay guided isolation strategies are applied.
12 NMR techniques have been used to facilitate and ameliorate the discovery of bio-active markers
13 from botanical medicines in recent years. As an example, 1H NMR in combination with
14 chemometric analysis was applied to investigate the correlation between the bioactivity of polar
15 extracts prepared from the leaves of Cissampelos sympodialis and the chemical composition
16 [242]. C. sympodialis is used for the treatment of respiratory diseases such as bronchitis and
17 asthma. Despite many pre-clinical pharmacological studies, the constituents mediating the anti-
18 asthma activity of polar extracts of C. sympodialis leaves have not been definitively identified. In
19 the reported study, the metabolic profiles of the polar extracts obtained from the leaves harvested
20 at different phenological stages were investigated through 1D and 2D NMR spectral analyses,
21 while spasmolytic activity was simultaneously screened using a guinea-pig tracheal assay.
22 Meanwhile, the content of the alkaloids previously implicated in the bioactivity of C.
23 sympodialis was determined by HPLC. The PCA analysis of the 1H NMR data revealed the
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1 discrimination of the extracts from different plant phenological stages, and the PLS regression
2 analysis demonstrated that the spasmolytic activity was better correlated with signals for flavonol
3 derivatives rather than the alkaloids. The findings shed light on the contribution of phenolic
4 compounds to the spasmolytic activity of the plant's polar extracts. In another application, the
5 use of NMR techniques for discovering active substances from Polygoni multiflori Radix was
6 demonstrated [243]. The radix of P. multiflori is a well-known herbal medicine used for the
7 treatment of hypercholesterolemia, atherosclerotic, diabetes, and other diseases. Unfortunately,
8 the molecular mechanisms or molecular targets of these therapeutic actions are not clear. In this
9 study, an NMR-based drug screening strategy was used for discovering the active constituents
10 from P. multiflori Radix using human fatty acid binding protein 4 (FABP4) as target protein.
11 FABP4 is a potential therapeutic target for metabolic diseases such as diabetes and
12 atherosclerosis. The radix extract was first subjected to moderate separation to generate pre-
13 purified subfractions, the target protein was then added to each subfraction. After that, the
14 ligand-protein interactions were investigated using NMR techniques through the measurements
15 of the line-broadening, the chemical shift perturbation and the saturation transfer difference-
16 spectrum. As a result, 2,4-dihydroxy-6-[(1E)-2-(4-hydroxyphenyl)ethenyl]phenyl-ß-D-
17 glucopyranoside was found to be an active constituent, for which the obvious interaction with
18 target protein FABP4 was observed. In addition, the interaction information at the atom level
19 revealed by NMR ligand-protein observation would be valuable in further stages of lead
20 optimization. This study, as an example, illustrated the promising potential of NMR techniques
21 in bio-marker discovery for botanical health products.
22
23 4. Conclusions and prospects
Page 74 of 98
1 botanicals or herbal medicines and consider them as alternatives to prescription drugs for
2 preventing illness, treating chronic diseases, cancer, and alleviating the stress and pressure of
3 daily life. Still, more investigations on botanical products are definitely needed to provide
4 scientific evidence, gain insights on the underlying mechanisms, and ensure their benefits to
5 human health. NMR has proven to be a powerful and useful tool for the investigation of
6 botanical health products. As illustrated in the above review, various NMR techniques and
7 methodologies have been successfully applied to the research and development of botanical
8 health products in all stages, from plants to products (from field to shelf). NMR techniques in
9 combination with chemometrics and other analytical tools have not only shown great potential in
10 differentiation of plant species, discrimination of cultivars (varieties), authentication of species,
11 and characterization of marker compounds, but have also been widely used for the exploration of
12 plant metabolite variabilities that are associated with different growing stages, geographic
13 locations, cultural environments, etc. In addition, NMR has been successfully utilized for
14 investigating chemical and compositional changes during the processing of botanicals and
15 products. Quality, safety and efficacy are the most important issues for the utilization and
16 regulation of botanical health products. qNMR has been increasingly accepted and used as a
17 routine analytical tool for quantification of various marker constituents for achieving quality
18 control of botanical health products, while NMR fingerprinting and profiling in combination
19 with chemometric analyses have been employed for quality assessments. Since NMR can
20 provide a non-selective and universal detection of components in a mixture, while obtaining both
21 qualitative and quantitative information on chemical composition without the need for
22 chromatographic separation or pre-knowledge of formulation, it has been used as a powerful tool
23 for detecting adulteration of commercial products. The potential of NMR to evaluate biological
Page 75 of 98
1 activity, discover biomarker(s), and explore the underlying mechanism of action has gained
2 considerable attention. A large number of applications by using NMR-based metabolomic
3 approaches have been reported in recent years. We expect that with the aid of other techniques,
4 NMR will be employed further as a holistic and multifaceted means to facilitate investigations in
5 the field so that more information on how multicomponent botanical products function
6 systematically in the body will be obtained.
7 It is obvious that NMR currently holds a firm position in the field of botanical health product
8 analysis and quality control. Nevertheless, NMR is still an underutilized methodology in the area,
9 mainly due to the high cost of the instrument and maintenance, relative low sensitivity, and the
10 need for skillful professionals. Additionally, the overlapping of signals and the difficulty
11 associated with the thorough interpretation of 1H NMR spectra of botanical complex mixtures
12 hamper the application to a much greater extent. In order to overcome those difficulties, both
13 technological and methodological progress has been made during the past decades. Compact LF
14 NMR spectrometers that rely upon permanent magnets without the need of any specific
15 operation have been developed and commercialized by several manufacturers since 2013 [21],
16 making NMR increasingly accessible. We expect that the application of LF NMR for quality
17 control of botanical health products will expand in the near future, and more LF NMR methods
18 suitable for industrial routine use as the protocols for the analyses of various botanical products
19 will be developed and established. Numerous developments have been implemented to improve
20 NMR sensitivity, such as increasing magnetic field strength and developing probes with greater
21 sensitivity. A remarkable advancement in NMR microcoil construction is the development of the
22 high-resolution micro-MAS probe (HR-μMAS) [70], which can greatly reduce the required
23 sample quantity, making HR-MAS a promising tool for the investigation of botanicals. Notably,
Page 76 of 98
1 alternative technological approaches for improving NMR sensitivity by applying
2 hyperpolarization methods, such as the dissolution dynamic nuclear polarization (D-DNP) and
3 the signal amplification by reversible exchange (SABRE) techniques, have been proposed and
4 adopted in practice [244, 245]. These techniques can drastically enhance NMR response by up to
5 four orders of magnitude. We expect these contemporary techniques will find applications in the
6 R & D of botanical health products in the near future. 1H NMR signal overlapping in
7 multicomponent mixtures, which often lead the identification and/or quantification of
8 metabolites to be difficult and ambiguous, is the predominant obstacle to utilizing NMR for the
9 investigation of botanical products. As demonstrated and reviewed in Sections 2 and 3 of this
10 review, various multi-dimensional NMR techniques and methodologies have been developed to
11 disentangle overlapping signals. It is worth mentioning that many of those techniques and
12 methodologies have not been fully used on a routine basis in the botanical health product field,
13 thus, it is still necessary to make end-users aware of recent methodology advances. Using
14 heteronuclear NMR other than 1H NMR is another useful strategy to overcome 1H signal overlap,
15 yet the applications have not been fully explored. With the advances of NMR instrumentation
16 and technology, we anticipate that 13C NMR will become a useful tool for investigating botanical
17 health products. Hyphenated NMR techniques have shown great potential in dealing with
18 complex mixture samples. It is expected that more state-of-the-art hyphenated NMR techniques,
19 particularly high-throughput concerted LC-MS-NMR techniques and methodologies, will be
20 innovated and commercialized; this would greatly promote the development and utilization of
21 botanical health products. We also anticipate that the development of NMR computational signal
22 processing to deconvolute overlapping signals and the establishment of dedicated NMR profile
23 databases to assist the identification of metabolites in botanical mixtures will be two important
Page 77 of 98
1 directions of development. A few initiative works have started in these two areas, yet more
2 explorations are still needed to be carried out in the future.
4 Declaration of competing interest
5 The authors declare no conflict of interest. The founding sponsor had no role in design of the
6 study, analyses, or interpretation of data, writing the manuscript, and decision to publish the
7 results.
9 Acknowledgements
10 This research is supported in part by “Science-Based Authentication of Dietary
11 Supplements” funded by the Food and Drug Administration grant number 2U01FD004246-06,
12 and the United States Department of Agriculture, Discovery & Development of Natural Products
13 for Pharmaceutical & Agricultural Applications No. 58-6060-6-015. The authors would like to
14 thank Dr. Joseph Lee for proofreading and editing of the manuscript.
15
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NMR Technique and Methodology in Botanical Health Product Analysis and Quality Control

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NMR Technique and Methodology in Botanical Health Product Analysis and Quality Control

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2 Jianping Zhao a *, Mei Wang b, Seethapathy G. Saroja a, Ikhlas A. Khan a, c *

6 Mississippi, University, MS 38677, USA

8 Agriculture, University, MS 38677, USA

9 c Division of Pharmacognosy, Department of BioMolecular Sciences, School of Pharmacy,

10 University of Mississippi, University, MS 38677, USA

16 Jianping Zhao (E-mail: jianping@olemiss.edu)

17 Ikhlas A. Khan (E-mail: ikhan@olemiss.edu)

19 University, MS 38677, USA

20 Phone: +1 662 915 7821, Fax: +1 662 915 7062;

11 analytical techniques in a variety of domains of botanical health products investigation, such as

17 Keywords: Botanical health products; NMR; Methodology; Analytical technique; Quality

3 2. NMR techniques and methodologies used for botanical health products

4 2.1. Chemometrics-aided NMR

14 3. Applications of NMR in botanical health product analysis and quality control

15 3.1. Quality control

33 4. Conclusions and prospects

13 traditional Chinese medicine (TCM), as well as apprenticeship systems such as herbalism,

21 of US$166.2 billion by 2027 [4].

6 Unlike conventional medicine, botanicals contain complex mixtures of chemical constituents,

11 compound classes, that synergistically or antagonistically contribute to the promised

14 to deliver the expected physiological effect.

21 began to develop monographs on major botanicals in commerce in 1995; the European

7 IR), near-infrared (NIR) spectroscopy, thin-layer chromatography (TLC), mass spectrometry

16 sample preparation, non-invasive/destructive measurement, ability to simultaneously detect and

20 are highlighted in Table1.

23 product analysis with regards to different aspects

9 tuning, auto-shimming, and auto-sampling), data handling capabilities, spectral deconvolution,

2 from plants or plant-derived products are excluded)

3 This review intends to provide an overview of the potential applications of NMR

12 presented since an enormous amount of literature is available on them. Secondly, the

3 NMR spectroscopy is an analytical technique based on the measurement of resonance signals

10 intracellular medium [15-17]. A number of sophisticated NMR acquisition sequences and

18 quality control, as summarized in Fig. 2, is presented below.

5 2.1. Chemometrics-aided NMR

6 predict the authenticities of samples. As a matter of fact, chemometrics-aided NMR technique is

8 biochemical, medical, and pharmaceutic areas [27, 28].

10 Figure 3. Workflow of chemometrics-aided NMR methodology

12 Fig. 3 illustrates the workflow of chemometrics-aided NMR methodology used in

5 other scaling methods [25, 26].

12 interpretation. Principal components analysis (PCA), hierarchical cluster analysis (HCA),

13 canonical-correlation analysis (CCA), non-linear mapping (NLM), soft independent modelling of

18 differentiation of species or varieties, and identification of diagnostic markers for botanical

19 authentication. Application examples using chemometrics-aided NMR technique for botanical

20 health product analysis can be seen in section 3 of this review.

21 2.2. Quantitative NMR

21 literature [20, 29, 31].

5 botanical quality assessment or concentration measurement. Several different 1D 13C-qNMR

6 techniques, such as Q-INEPT-CT (Quantitative Constant-Time Insensitive Nuclei Enhanced by

11 Homonuclear or heteronuclear 2D NMR creates an additional dimension to 1D 1H-NMR.

22 analysis can be seen in section 3.1.1 and Table 3 of this review.

23 2.3. Hyphenated NMR

5 component nature of botanicals. Hyphenation of NMR with chromatographic techniques, such as

6 liquid chromatography (LC) or high-performance liquid chromatography (HPLC), can thus

8 structural information or complete structural assignments of analytes on-line. Since NMR is a

9 highly non-selective detection technique, it can provide a comprehensive detection of any

10 hydrogen containing compounds present in HPLC elute. Unfortunately, some contradictory

5 proposed and implemented, such as LC–MS-NMR, HPLC-HRMS-SPE-NMR, and LC–PDA–

9 in section 3 of this review.

10 2.4. Diffusion-ordered (DOSY) NMR

14 2.5. Computation-aided NMR and 1H iterative full spin analysis (HiFSA)

15 Structural information of compounds is embedded in NMR spectra through the signal

2 Jianping Zhao a , Mei Wang b, Seethapathy G. Saroja a, Ikhlas A. Khan a, c