Advancement of analytical techniques in some South African commercialized medicinal plants: Current and future perspectives

SAJB-02475; No of Pages 18 South African Journal of Botany xxx (2019) xxx

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Advancement of analytical techniques in some South African commercialized medicinal plants: Current and future perspectives N.A. Masondo, N.P. Makunga ⁎ Department of Botany and Zoology, Stellenbosch University, Private Bag X1, Matieland 7600, South Africa

a r t i c l e

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Article history: Received 1 April 2019 Received in revised form 12 June 2019 Accepted 16 June 2019 Available online xxxx Edited by JJ Nair Keywords: Biomarker compounds Chemometrics Chromatographic techniques DNA fingerprinting Hyperspectral imaging Quality control

a b s t r a c t As the use of medicinal plants continues to gain popularity worldwide, there is dire need for herbal medicines to be guaranteed in their safety and efficacy. South Africa has a largely under-explored medical flora scientifically due to the vast number of plant species that are consumed for medicinal purposes by the public, creating an urgent need to better define plants with therapeutic effects. To meet these aims, a combination of high-throughput analytical techniques that are sensitive and versatile are used for the standardization and authentication of commercialized natural products as part of quality control regimes. These methods also serve to highlight the role of analytical methods in providing accurate and reliable information pertaining to the biochemicals of medicinal plants in those species whose chemistry remains partially understood or poorly characterized. The review highlights the advancements made in analytical technology for the assessment of biochemical profiles, biomarker compounds and quality control in chosen commercialized products, produced from indigenous South African species. We also summarize studies on the phytochemistry, pharmacology, clinical trials and available patents associated with some of South Africa’s medicinal plants where commercialization has occurred or is imminent. This was achieved through a detailed literature search using web-based database searches including Google Scholar, Scopus and Web of Science (WoS) as well as ethnobotanical literature on South African medicinal plants. Bibliometric analysis was performed on the data mined from WoS. It is clear that future advancements and further development of the natural products industry in South Africa will benefit from a diverse range of technological approaches. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.

Abbreviations: CAD, Charged aerosol detector; nCRISPR, Clustered regularly interspaced short palindromic repeats; CoMPP, Comprehensive microarray polymer profiling; CID, Collision-induced dissociation; CZE, Capillary zone electrophoresis; DAD, Diode array detector; DLLME, Sail-aided liquid-liquid micro-extraction; DNA, Deoxyribonucleic acid; DART, Direct analysis in real time; ELSD, Light scattering detector; ESI, Electrospray ionization; FID, Flame-ionization detector; GC, Gas chromatography; HCA, Hierarchical cluster analysis; HPLC, High-performance liquid chromatography; HPTLC, High-performance thin-layer chromatography; IR, Infrared spectroscopy; IT, Ion trap; LC, Liquid chromatography; LLE, Liquid-liquid extraction; LPA, Local polynomial approximation; MAE, Microwave-assisted extraction; MALDI, Matrix-assisted laser desorption/ionization; MIR, Mid infrared spectroscopy; MS, Mass spectrometry; NACE, Non-aqueous capillary electrophoresis; NiO, Nickel oxide; NIRS, Near infrared spectroscopy; NGS, Next-generation sequencing; NMR, Nuclear magnetic resonance; NP-HPCCC, Normal phase high performance countercurrent chromatography; OPLS-DA, Orthogonal projections to latent structures-discriminant analysis; PCA, Principal component analysis; PDA, Photodiode array; PLS, Partial least squares; QqQ, Triple quadrupole; QTOF, Quadrupole-time-of-flight; RP-HPLC, Reversed-phase high-performance liquid chromatography; SFC-MS, Supercritical fluid chromatography- MS; SPE, Solid-phase extraction; SWIR, Short wave infrared; TLC, Thin layer chromatography; UHPLC, Ultra-high-performance liquid chromatography; UV, Ultraviolet detectors; WoS, Web of Science; ZnO, Zinc oxide. ⁎ Corresponding author. E-mail addresses: [email protected] (N.A. Masondo), [email protected] (N.P. Makunga).

1. Introduction Complexities of separation, identification and quantification of plant extracts remains a challenge in the commercialization of medicinal plant products. Accordingly, several separation techniques and detectors are developed to improve selectivity, sensitivity and speed during extract separation. These techniques include (i) titrimetric, (ii) chromatographic [thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), high-performance thin layer chromatography (HPLC), gas chromatography (GC)], (iii) spectroscopic [spectrophotometry, near infrared spectroscopy (NIRS), nuclear magnetic resonance spectroscopy (NMR), fluorimetry and phosphorimetry], (iv) electrochemical, (v) kinetic analysis, (vi) electrophoretic evaluation, (vii) flow injection and sequential injection analysis (Nováková and Vlčková, 2009; Siddiqui et al., 2017). Chromatographic approaches, particularly liquid chromatography (LC), are the preferred methods for bio-analytical monitoring of drugs in biological materials. Since the establishment of LC-MS [mass spectrometry (MS)] in the past 40 years, the technique has remained relevant and efficient, with continuous development of instrumentation [e.g. ultra-high-performance LC (UHPLC), ion mobility (MS), etc.], application of hyphenated techniques

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Please cite this article as: N.A. Masondo and N.P. Makunga, Advancement of analytical techniques in some South African commercialized medicinal plants: Current ..., South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.06.037

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[supercritical fluid chromatography-MS (SFC-MS), two-dimensional techniques, etc.] and a variety of analyzed compounds (Ganzera and Sturm, 2018). The combined effect of hyphenated techniques has led to highly effective systems for comprehensive metabolite profiling. These methods include the coupling of separation techniques together with online spectroscopic detection methods. However, the capacity of different approaches is greatly dependent on the applied detectors. Therefore, introduction of detectors since the 18th century has contributed broadly to the accurate characterization of complex compounds (Zhang et al., 2019). Detectors allow for advance peak annotation or de-replication as well as additional online structural data on the separated compounds (Wolfender et al., 2015). For instance, photodiode array (PDA), MS, nuclear magnetic resonance (NMR) and electrospray ionization (ESI) are generally used for liquid-based separation. On the other hand, GC is mainly paired with MS, infrared (IR) spectroscopy, flame-ionization detector (FID) and near infrared (NIR) detectors amongst others. Diode array detector (DAD), evaporative light scattering detector (ELSD) or MS are mostly applied in the SFC approach (Saito, 2013). Up to now, MS is the most preferred detector as a result of its high specificity and sensitivity plus it provides structural features of the analytes, regardless of the applied MS [single quadrupole (Q), triple quadrupole (QqQ), time-of-flight (TOF), ion trap (IT) or matrixassisted laser desorption/ionization (MALDI)] (Ganzera and Sturm, 2018). A recently published review on “universal detectors” details some of the hyphenated detectors that can be used based on the type of molecule and its uniformity (Zhang et al., 2019). The review created a timeline since the introduction of “universal” detectors [e.g. ultraviolet detectors (UV)] in the early 19th century up to the application of charged aerosol detectors (CADs) in the early 20th century. Some of the features of these hyphenated detectors include compound detection during the analysis of complex samples or a library of diverse compounds, quantification of drug metabolites without the need for internal standards, rapid evaluation of pure and concentrated samples, analysis of volatile compounds using GC method and the description of structurally similar compounds. Beside the widespread use of chromatographic techniques, hyperspectral imaging and vibrational spectroscopy combined with various chemometrics techniques have been well-received in herbal medicine authentication (Kiani et al., 2018; Rohman et al., 2014; Wolfender et al., 2015). Medicinal plants are known to contain chemically diverse compounds synthesized in very low quantities and are prone to variation due to environmental factors and manufacturing conditions. As the use of medicinal plants or herbal remedies continues to gain popularity worldwide, there is a dire need for the products’ guaranteed safety and efficacy. Therefore, a combination of advanced, sophisticated, sensitive and versatile analytical techniques is appropriate for the standardization and authentication of commercialized natural products (for formal or informal markets) for quality control purposes (Steinmann and Ganzera, 2011; Wolfender et al., 2015). In this review, we will focus on currently commercialized South African medicinal products that have been processed, standardized and are sold as teas, tinctures, tablets or capsules, both locally and internationally. The review highlights the advancements made in the assessment of biochemical profiles, especially using biomarker compounds, and the provision of quality control standards in commercialized products using analytical technologies. We also briefly summarize studies on the phytochemistry, pharmacology and clinical trials associated with some of South Africa’s medical plants and we specifically focus on those species that have been commercialized or where commercialization efforts are likely to follow in the near future. Our focus is mainly limited to ten South African medicinal plants species namely: Agathosma betulina Berg. (Buchu) Rutaceae, Aloe ferox Mill. (Bitter aloe) Asphodelaceae, Aspalathus linearis Burm.f. Dahlg. (Rooibos tea) Fabaceae, Harpagophytum procumbens DC. Ex Meisn. (Devil's claw) Pedaliaceae, Hypoxis hemerocallidea Fisch., C.A. Mey. & Avé-Lall. (African potato) Hypoxidaceae, Pelargonium sidoides DC. (African geranium, Umckaloabo) Geraniaceae, Sceletium tortuosum

(L.) N.E. Br. (syn. Mesembryanthemum tortuosum; Kanna), Sclerocarya birrea subsp. caffra, A.Rich. Hochst. (Marula) Anacardiaceae, Siphonochilus aethiopicus Schweinf. B. L. Burtt (African ginger) Zingiberaceae and Sutherlandia frutescens (L.) R.Br. (syn. Lessertia frutescens; Cancer bush) Fabaceae. These plants are part of this list because of their commercial value and/or interesting phytochemical makeup. For many of these species, there is also a considerable amount of work that is linked to their phytochemistry and pharmacology (discussed briefly in Section 5) that has served both commercial and scientific interest. They also serve to highlight the role of analytical methods in providing accurate and reliable information pertaining to the biochemicals of medicinal plants in those species whose chemistry remains partially understood or poorly characterized. 2. Materials and methods The main body of this review was constructed using online resources, e.g. Scopus, Google Scholar and Web of Science (WoS). By combining searches from these platforms, this has been shown to increase the robustness of gathering appropriate research articles, thus bolstering the coverage to access scientific papers in the primary literature (allowing access to journal articles, theses and dissertations, and books) (Lasda-Bergman, 2012). Access to theses, dissertations and books is easier with Google Scholar and through Google Patents, patent materials were downloaded. Keywords such as: 'analytical techniques, quality control, South African commercial medicinal plants' were used in the above-mentioned search engines (Table 1 refers). We identified several species that we used as target and/or species of interest, where we searched for literature pertaining directly to application of various methods such as: “metabolomics, DNA barcoding, in vitro bioassays and in vivo animal studies, pharmacokinetics and pharmacodynamics,” using these as keywords along with the species’ names. A bibliometric study of the different techniques applied in the success of commercialized plants is provided (based on Google Scholar and WoS (accessed from 9 October till March 31, 2019) and Scopus, (accessed between December 12, 2018 through to March 31, 2019)) (Table 1). For the purpose of constructing Fig. 1, documents published from the years of 1967 to date were used and a refined search using only the word “Sceletium” was used to identify the papers that have been published on this species during the respective time period (Fig. 2). For this purpose, Scopus was used because WoS may track fewer citation data than Scopus; and therefore, Scopus is regarded as being more informative to generate citations and this database also has the capacity to perform large-scale calculations of published scientific articles to generate graphics, a feature that is not possible with Google Scholar (Lasda-Bergman, 2012). Google Scholar has the disadvantage of providing more gray literature and lower quality documents, and we were thus more discerning with our criteria to eliminate such materials (e.g. from predatory journals) (refer to Lasda-Bergman, 2012 for details). 3. Biomarker compounds and authentication techniques Biomarkers or biological markers, as a term, is synonymous with the field of medicine where their use is a common practice and dates back centuries ago. In the context of medicine, a biological marker is thus defined as a naturally occurring molecule, gene, and/or characteristic which facilitates the identification of a specific physiological issue, pathology, and/or disease, as a means to enable accurate diagnosis (Fernandez et al., 2016). Its use in plant biology and particularly in the context of natural products, a biomarker is now regarded as a naturally occurring molecule, gene, or characteristic by which a particular pathological or physiological process, disease, etc. can be identified (Simoneit, 2005). With respect to medicinal plants, biomarkers could facilitate genotyping or phenotyping processes involving changes to the transcriptome, proteome, metabolome and phenome (Steinfath et al.,

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Table 1 Analytical methods currently applied in South African commercialized medicinal plants for the identification of novel biomarker compounds Species

Biomarkers compounds

Agathosma betulina (J.P Pseudo-diosphenol, diosphenol, menthone, l-pulegone, limonene, Bergius) Pillans 8-mercapto-p-menthan-3-one, (Buchu) isomenthone Aloe ferox Mill. (Bitter Aloesin, aloeresin A, aloeresin C, aloe) 5-hydroxyaloin, aloin A, aloin B, aloinoside B, Dihydrochalcones (aspalathin, Aspalathus linearis nothofagin), aspalalinin, (Burm. f.) R. flavones (isoorientin, orientin) Dahlgren (Rooibos tea)

Harpagophytum Harpagoside, harpagide, (E and Z) procumbens DC. Ex 8-O-p-coumaroylharpagide, (E and Z), Meisn. (Devil's claw) 6'-O-p-coumaroyl-harpagide, 8-O-cis-p-coumaroyl-harpagide, 6'-O-p-coumaroyl-procumbide, 8-feruloyol-harpagide, 8-cinnamoyl-myoporoside Hypoxis hemerocallidea Hypoxoside, rooperol, phytosterol (β-sitosterol, ergosterol, Fisch., C.A.Mey. & stigmasterol) Avé-Lall. (African potato) Pelargonium sidoides Umckalin, 5,6,7-methoxycoumarin DC. (Umckaloabo)

Analytical techniques

Detectors

References

GLC, GC, GC-MS, MIR, LC-MS, GCxGC-TOFMS

IR, UV (Katharometer), FID, MS, PDA

Fluck et al. (1961), Viljoen et al. (2006a), Sandasi et al. (2010), Mavimbela et al. (2014), Gorst-Allman and Naude (2016)

HPCCC, UHPLC-MS, RP-HPLC, GC-MS, LC-MS, HPLC-ESI-MS/MS,

MSD, UV, PDA, NMR, MS, DAD, MS UV, NMR, MS, UV, UV/Vis, PDA, DAD, germanium detector, MS/MS

Magwa et al. (2006), Zahn et al. (2008), Adhami and Viljoen (2015), Kanama et al. (2015), Zhao et al. (2016), Fan et al. (2018) Joubert (1996), Marais et al. (2000), Bramati et al. (2002), Schulz et al. (2003), Baranska et al. (2006), Shimamura et al. (2006), Joubert et al. (2008), Iswaldi et al. (2011), Beelders et al. (2012), De Beer et al. (2015), Walters et al. (2017a), Walters et al. (2017b), Malongane et al. (2018), Stander et al. (2019a) Boje et al. (2003), Baranska et al. (2005), Seger et al. (2005), Günther and Schmidt (2005), Clarkson et al. (2006), Qi et al. (2006), Qi et al. (2010), Karioti et al. (2011), Baghdikian et al. (2016), Tomassini et al. (2016), (Diuzheva et al., 2018), Rolland and Duval (2019) Albrecht et al. (1995), Nair and Kanfer (2006), Nair and Kanfer (2007), Boukes et al. (2008), Mkhize et al. (2013), Nsibande et al. (2018) White et al. (2008), Franco and de Oliveira (2010), Maree and Viljoen (2012), Viljoen et al. (2015) Smith et al. (1998), Patnala and Kanfer (2008), Shikanga et al. (2011), Shikanga et al. (2012a), Shikanga et al. (2012b), Roscher et al. (2012), Patnala and Kanfer (2013), Meyer et al. (2015), Lesiak et al. (2016), Sandasi et al. (2018) Braca et al. (2003), Viljoen et al. (2008), Njume et al. (2011), Kpoviessi et al. (2011), Russo et al. (2013), Jiménez-Sánchez et al. (2015), Komane et al. (2015), Shoko et al. (2018) Holzapfel et al. (2002), Viljoen et al. (2002), Lategan et al. (2009), Naudé et al. (2016)

FT-Raman, UV, HPCCC, HPLC-ESI-MS (TOF/IT), LC-UV-Vis/MS, NIR, RP-HPLC-UV, HPLC-DAD, LC-UV/DAD, RP-HPLC-DAD, LC-MS, LC-QTOF-MS

RP-HPLC, TLC, GC, NIR-FT-Raman, HPLC, UAE-HPLC, HPLC-SPE-NMR, LC-DAD-MS/SPE-NMR, HPLC-DAD, HPLC-ESI-MS, HPLC-NMR-MS, HPTLC, HPLC-PDA

NMR, UV, DAD, MS, germanium detector, FID, PDA

RP-HPLC-UV, CZE, TLC, HPLC-UV, GC, HPLC, LC-MS

UV, PDA, NMR, UV/Vis, MS, FID, DAD

TLC, HPLC, LC-MS, HPLC-DAD, UHPLC-MS

NMR, MS, UV, PDA, HgCdTe, DAD UV, MS, PDA, NPD, HgCdTe, NACE-MS, DAD

Sceletium tortuosum (L.) N.E. Br. (Syn. Mesembryanthemum tortuosum L.; Kanna)

Mesembrine, mesembrenone, mesembranol, epimesembranol

HPLC-UV, GC, GC-MS, TLC, CE, LC-HR-MSn, HPCCC, HPTLC, DART-HRTOFMS, RP-UHPLC, SWIR, CZE

Sclerocarya birrea Hochst. subsp. caffra (Sond.) Kokwaro (Marula)

β-caryophyllene, α-humulene, terpinen-4-ol, pyrrolidine, aromadendrene and gurjunene,

GC-MS, GC, TLC, HPLC-UV/PDA, LC-MS, RP-HPLC-ESI-QTOF/MS2,, GC/FID, UPLC-Q-TOF-MS, GC*GC-MS, HPLC-MS/MS

Siphonochilus aethiopicus Schweinf. B. L. Burtt (African ginger) Sutherlandia frutescens (L.) R.Br. ex W.T. Aiton (Syn. Lessertia frutescens; Cancer bush)

GC-MS, GC-TOFMS, HS-SPME, HPLC NMR, MS, DAD 4aαH-3,5α,8aβ-trimethyl-4,4a, 9-tetrahydro-naphtho[2,3-b]-furan-8-one; 2-hydroxy-4aαH-3,5α,8aβ-trimethyl-4,4a, 9-tetrahydro-naphtho[2,3-b]-furan-8-one Sutherlandin A-D, sutherlandiosides A-D LC-UV/ELSD, LC-ESI-TOF, HPLC, LC-MS, NMR, PDA, ELS, LC-MS/MS, UPLC-MS, UPLC-QTOF-MS, DAD, UV/Vis, MS HPLC-MS

UV, PDA, MS, FID, DAD

Avula et al. (2010), Albrecht et al. (2012), Faleschini et al. (2013), Acharya et al. (2014), Mbamalu et al. (2016), Mavimbela et al. (2018)

Gas chromatography (GC); capillary zone electrophoresis (CZE); diode array detection (DAD); electrospray ionization (ESI); evaporative light scattering detector (ELSD); lame ionization detector (FID); headspace solid phase micro-extraction (HS-SPME); high performance countercurrent chromatography (HPCCC); high performance thin layer chromatography (HPTLC); high performance liquid chromatography (HPLC); liquid chromatography (LC); mass spectrometer (MS); mercury-cadmium-telluride (HgCdTe); mid infrared spectroscopy (MIR); near infrared spectroscopy (NIR); nitrogen-phosphorous detector (NPD); nonaqueous capillary electrophoresis (NACE); nuclear magnetic detector (NMR); photodiode array (PDA); quadrupole-time-of-flight (QTOF); reversed-phase high-performance liquid chromatography (RP-HPLC); short wave infrared (SWIR); thin layer chromatography (TLC); ultra-performance liquid chromatography (UPLC); ultra-high performance liquid chromatography (UHPLC); ultraviolet (UV); ultraviolet/Visible (UV/Vis).

2010). It is said that for ease of use of biomarkers, to aid both basic scientific investigations and applied diagnosis, their application as a tool should be easy and relatively cheap, allowing both highly qualitative and quantitative predictions linked to a particular trait or problem. Chemical isolations followed by structural elucidation of plant metabolites has allowed for the assignment of some specialized chemicals to be confined to particular taxa, genera or even species, validating their status as biomarkers or metabolite markers. Secondary metabolites are the most frequently used biomarkers in medicinal plants for identification and distinction of morphologically similar genera. However, the use of biomarkers single-handedly proves to be a challenge in the identification of plants within an herbal mixture as well as in the authentication of commercial products. Authentication of plant species is an important task and requires a vast array of techniques. Over the years, established methodologies

continue to form ground-work for new and innovative tools that need to meet high demands brought about by the commercialization of natural products (Khan and Smillie, 2012; Techen et al., 2004). Some of these techniques include and are not limited to: (i) macroscopic and microscopic; (ii) analytical fingerprinting; (iii) DNA fingerprinting; (iv) hyperspectral imaging; and (v) chemometric methods. Macroscopic and microscopic techniques use traditional classification methods that involve the identification of plants as a whole, either as a fresh organ part or in a dried state. Generally, macroscopic techniques rely on distinctive identification of leaf morphology (shape, size, leaf margins), flowers (inflorescence, floral morphology, seeds per carpel, and so forth), characteristic color and roots (surface texture, type, e.g. corm, bulb, rhizome, tissue layering). On the other hand, the microscopic approach may be more effective since the method uses scanning electron microscopy, standard light microscopy and fluorescence microscopy.

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Fig. 1. Major research subjects (%) based on South African medicinal plants that have been targeted since 1967. This chart is based on keywords (South Africa + medicinal plants + pharmacology) used in Scopus. The graphic shows the areas of pharmacology, toxicology and pharmaceutics as contributing the highest number of documents.

However, both techniques have their shortcomings. They lack the ability to accurately ascertain multicomponent powdered samples and closely related genera with similar cellular morphologies. Thus, the use of chromatographic techniques provide the most reliable and applicable system. These methods, however, require the use of biomarker compounds which make up an analytical fingerprint distinct to each plant species. Accessibility of biomarkers is often a challenge because of their high costs, laborious work involving isolation methodologies (extraction, fractionation, isolation, and spectroscopic identification of unambiguous constituents), as well as the unavailability of plant material for compound isolation. Even worse, isolation systems differ from

species to species based on the phytochemical constituents inherent to a particular plant (Khan and Smillie, 2012). To counteract the inadequacies of chromatographic techniques, molecular authentication methods have several advantages suitable for herbal product identification relative to macroscopic, microscopic, and analytical fingerprinting. Molecular biological techniques thus become relevant for such cases. DNA-based molecular technology is a reliable and informative tool used for genetic composition of individual species extracted from fresh or dried organic tissues of botanical material (Heubl, 2010). Hyperspectral imaging is also a fast, sensitive, simple, non-destructive and non-invasive tool that provides valuable information on the

Fig. 2. A historical account of key research areas in Sceletium tortuosum that have dominated from 1970 based on the number of documents recorded in Scopus per year.

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molecular and structural composition of herbal remedies or plants, without the need for biomarker compounds (Gowen et al., 2007). Chemometric tools that allow for statistical deductions and mathematical applications where chemical data are reduced in dimensionality to reveal relationships through application of statistical models [i.e. hierarchical cluster analysis (HCA), principal component analysis (PCA), partial least squares (PLS)] for phytochemical fingerprinting have also proven resourceful. The approach applies pattern recognition parameters to analyze different components within a cluster and determine correlation efficiency within a population of authenticated material. The technique is a non-selective system that examines diverse phytochemicals within the sample and eliminates partiality, inherent in classical fingerprinting techniques. 3.1. Biomarker isolation in ten commercially important South African medicinal plants Isolation of biomarkers in natural products is linked to three categories (i) structure-directed isolation; (ii) bioactivity-guided isolation and (iii) chemotaxonomy-oriented isolation (Rasoanaivo et al., 2018). The first classification is based on a random search for new or novel structures; the second classification focuses on finding bioactive compounds; and lastly, the use of botanical taxonomy to search for available chemical constituents or quantitatively searching for active compounds or concentrated constituents. As part of bioprospection for new value added plant-based products, biomarker identification is a critical stage with intentions of commercialization. It is thus clear that research in South Africa on medicinal plants has largely focused on the areas of pharmacology, pharmaceutics and toxicology, specifically aimed at meeting both scientific and commercial interests. Chemistry has also over the years been an important research area for study (Fig. 1), assisting with the discovery of novel chemicals that now serve as biomarkers. Better detail in the characterization of plants assists with the development of appropriate chemical markers. Biomarker compounds of the 10 medicinal plants of interest have been mainly isolated based on available ethnobotanical literature (Hutchings et al., 1996; Street and Prinsloo, 2012; Van Wyk, 2011; Watt and Breyer-Brandwijk, 1962) detailed in Table 1. 3.1.1. Agathosma betulina (P.J. Bergius) Pillans (Buchu) The earliest report on Agathosma betulina revealed several biomarker compounds including limonene, menthone, diosphenol, lpulegone, 8-mercapto-p-menthan-3-one and isomenthone isolated using different chromatographic techniques (Collins et al., 1996; Fluck et al., 1961; Kaiser et al., 1975; Klein and Rojahn, 1967; Lamparsky and Schudel, 1971). As a follow up to the established reports, researchers have developed and validated different systems for biomarker quantification in A. betulina essential oils including chromatography and spectroscopy approaches (Mavimbela et al., 2014; Sandasi et al., 2010; Viljoen et al., 2006b). In these studies, the authors have either used the most frequently applied system in essential oils, GC and GC-MS analysis (Viljoen et al., 2006b) or a combination of chromatography (GC-MS, HPTLC, LC-MS), spectroscopy (NIR, MIR, Raman), and chemometric analysis software (Mavimbela et al., 2014; Sandasi et al., 2010). Comprehensive GC methods coupled with time of flight mass spectrometry (GC × GC with TOF-MS), applied in Rozendal Fynbos Vinegar® products, separated and differentiated complex mixtures of plants (including A. betulina), based on the biomarkers of individual species (Gorst-Allman and Naude, 2016). The method could quantitatively characterize several chemical constituents through high-resolution and accurate mass measurements of GC-HRT. Recently, a nanoparticle (NiO, ZnO) approach, a green novel and an environmentally friendly pathway has been successfully implemented using the extract of A. betulina, an effective chelating chemical agent (Thema et al., 2016; Thema et al., 2015). The natural products from A. betulina act as stabilizing and reducing agents for nanoparticle synthesis and their application

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in research involving pharmaceuticals and cosmetics continues to gain interest (Agarwal et al., 2017). 3.1.2. Aloe ferox Mill. (Bitter aloe) Established reports have confirmed that 5-methylchromone C-glucosides form the major constituents in Aloe ferox using HPLC methods. These are made up of aloeresin A (Gramatica et al., 1982), aloesin (formerly aloeresin B) (Haynes et al., 1970), aloeresin C (Speranza et al., 1985), aloeresin D (Speranza et al., 1986) and isoaloeresin (Speranza et al., 1988). Concentrations of aloeresin A, aloesin and aloin are approximately 4:3:2, making up the major compounds in fresh aloe bitters, which is about 70–97% of the total dry weight (Chen et al., 2012). A reversed-phase HPLC technique was developed and validated by Zahn et al. (2008) and they described the detection of aloesin, aloeresin and anthraquinone (barbaloin) in A. ferox. The method was reported to have a shorter run time, simpler standard preparation, and simultaneous detection of compounds of interest. Adhami and Viljoen (2015) found that in just one run, HPCCC could easily separate and isolate eight A. ferox compounds such as aloesin, aloeresin C, aloeresin A, 5-hydroxyaloin, aloin B, aloinoside B, aloin A and aloinoside A. The coupling of UHPLC-MS also resulted in an ultrafast, accurate and sensitive quantification of chromones (aloeresin A, aloesin) and anthrones (aloin A and B) in A. ferox exudates (Kanama et al., 2015). Apart from these chemicals, monosaccharides that constitute the polysaccharide layer are being developed as diagnostic tools to differentiate between different aloe-based products, allowing for those products with A. ferox to be separated from those that may be composed of A. vera gel, using techniques such as comprehensive microarray polymer profiling (CoMPP) (Ahl et al., 2018). This technology artistically combines the power of microarray analysis with various molecular probes that can be used as a fast and reliable diagnostic approach, for those plants which may hold their healing properties linked to leaf succulence, where a unique combination of mono-, di-, tri- and polysaccharide accumulates. 3.1.3. Aspalathus linearis (Burm. f.) R.Dahlgren (Rooibos tea) Aspalathus linearis constituents are very unique, and were identified as aspalathin, a C–C linked dihydrochalcone glucoside and aspalalinin, a cyclic dihydrochalcone (Koeppen and Roux, 1965; Rabe et al., 1994; Shimamura et al., 2006). In addition to the aforementioned biomarkers, analysis of processed leaves and stems in A. linearis using NMR, MS and UV discovered a new diastereomeric pair of flavanones; (S)- and (R)eriodictyol-6-C-ß-D-glucopyranoside, formed in the process of aspalathin oxidative cyclization (Marais et al., 2000). Due to the plant’s reputable health-promoting properties, numerous approaches have been established and re-modelled for the isolation and quantification of A. linearis biomarkers. These include and not limited to LC-MS, with the use of different detectors (Bramati et al., 2002; Schulz et al., 2003), i.e., RP HPLC (Joubert, 1996), HPLC-ESI-TOF-MS and HPLC-ESI-IT-MS2 (Iswaldi et al., 2011), HPLC-DAD coupled with MS and tandem MS (Beelders et al., 2012) and HPLC-DAD (De Beer et al., 2015). Aspalathin in A. linearis has been identified using FT-Raman spectroscopy, and compared to spectral data and reference HPLC values (Baranska et al., 2006). A new improved HPLC method utilizing a 2.7 mm superficially porous stationary phase in the analysis of A. linearis phenolic compounds was recently developed by Walters et al. (2017a). The method is able to detect changes in the phenolic compounds, which decreased during the fermentation process, while eriodictyol glucopyranoside isomers and orientin increased as a result of aspalathin oxidation during fermentation. The authors also demonstrated that normal phase high performance countercurrent chromatography (NP-HPCCC) and reversed phase ultra-high pressure LC (RP-UHPLC) are a highly orthogonal (∼80%) offline comprehensive two-dimensional separation system that allows for separation of A. linearis phenolics in just 17 hours (Walters et al., 2017b). High performance countercurrent chromatography enhanced separation efficiency of low phenolic content that could

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only be detected when 1D mobile phase evaporates. At the same time, UHPLC analysis enhanced separation of early eluting polar compounds in the 2D mobile phase. A high-resolution chromatography and MS system, in combination with a collisional cross-section data system in A. linearis populations, confirmed previously detected biomarkers as well as new compounds (Stander et al., 2017). Recently, the question of the caffeine-free status in A. linearis has been revisited (Jeszka-Skowron et al., 2018; Malongane et al., 2018; Stander et al., 2019b). Although Malongane et al. (2018) reported on the presence of caffeine in A. linearis using 1H NMR spectroscopic analysis and a non-targeted RP UPLC-Q-TOF/MS approach, Jeszka-Skowron et al. (2018) only found traces of caffeine (0.31–0.97 μg/g) in A. linearis with an LC-MS/MS system. Stander et al. (2019b) again confirmed the absence of caffeine in A. linearis after applying specific MRM and high resolution LC-MS methods at a relatively low detection limit (0.2 μg/L). These contradictory reports often indicate different data interpretations especially when putative chemical assignments are not followed with confirmation using purified chemical standards (Stander, personal communication). It is important to emphasize that rooibos is conclusively established as being caffeine-free, using the method of Stander et al. (2019b). Additional constituents in A. linearis that make up its flavonoid fraction are of interest from a metabolomics point of view, so as to differentiate chemotypes that may naturally be occurring, that are becoming important chemical signatures for comprehensive profiling of this particular species (Stander et al., 2019a) (discussed later in this paper).

3.1.4. Harpagophytum procumbens DC. Ex Meisn. (Devil's claw) Iridoid glycosides from Harpagophytum procumbens are characteristic of the species. The most active constituent was established to be harpagoside followed by harpagide and procumbide. Kikuchi et al. (1983) later reported on the isolation of three new iridoid glycosides, 8-O-(p-coumaroyl)-harpagide, 6′-O-(p-coumaroyl)-procumbide, and procumboside as well as the previously identified compounds, harpagide, harpagoside and lastly procumbide, confirmed by 1H-NMR spectrum. 8-Feruloylharpagide was identified for the first time amongst other formerly documented compounds as well as three new natural products with a RP-HPLC approach, elucidated with spectroscopic data (Boje et al., 2003). Minor constituents, isobaric iridoid glycoside regioisomers were characterized in H. procumbens, and identified as (E/Z) pairs of 6′-O-(p-coumaroyl)harpagide (6′- PCHG) and 8-O-(pcoumaroyl)-harpagide (8-PCHG) (Seger et al., 2005). Application of a hyphenated LC-DAD-MS/SPE-NMR system aided in the identification of a new constituent, 6′-(Z)- and 6′-(E)-PCHG, in root extracts of H. procumbens. Clarkson et al. (2006) used HPLC-SPE-NMR to confirm complex compound structures from extracts without the need for isolation: however, the technique had its own limitations. Shortcomings of the method included lack of sensitivity, inability to analyze crude extracts, indeterminate chirality of compounds, however, the latter could be compensated with extended hyphenation involving circular dichroism. Over the years, the search for new compounds continue to yield good results in H. procumbens aided by the application of different analytical techniques (Karioti et al., 2011; Qi et al., 2006, 2010; Tomassini et al., 2016). In addition to the pursuit for new compounds, advancement in rapid, easy and convenient techniques for qualitative and quantitative recovery of harpagoside remains a focal point (Baghdikian et al., 2016; Baranska et al., 2005; Karioti et al., 2011; Schmidt, 2005). To add to this, different protocols have been proposed for H. procumbens extraction. A more recent one, used a rapid and an environmentally friendly approach, sail-aided liquid–liquid microextraction (DLLME) and microwave-assisted extraction (MAE) in combination with an HPLC-PDA for the quantitative analysis of harpagoside content (Diuzheva et al., 2018). In 2019, a patent was published by Rolland and Duval on preparatory methods of purified H. procumbens extracts (Rolland and Duval, 2019).

3.1.5. Hypoxis hemerocallidea Fisch., C.A.Mey. & Avé-Lall. (African potato) Hypoxoside, a new glycoside, was isolated in Hypoxis obtusa rhizome by a research group in Rome (Marini Bettolo et al., 1982). They confirmed the structure using an NMR and a UV spectrum as 1-(3′,4′dihydroxyphenyl)-5- (3″,4″-dihydroxyphenyl)-l-penten-4-yne and also based on its physico-chemical determinations and chemical behavior. At the same time, a group of South African researchers used column chromatography to isolate hypoxoside, (E)-1,5-bis(3′,4′dimethoxyphenyl)pent- 4-en-1-yne, in H. hemerocallidea (formerly known as H. rooperi). The history leading to the isolation of hypoxoside from Hypoxis plants is well-detailed in a review published by Drewes and Khan (2004). Advancement in the isolation of hypoxoside in Hypoxis species was achieved with an HPLC system (Betto et al., 1992; Kruger et al., 1994). Later work conducted by Laporta et al. (2007) showed that hypoxoside could be readily transformed into rooperol in corm extracts of H. hemerocallidea. Transformation of hypoxoside, a naturally occurring product with glucose substituents, occurs through an enzymic removal of the substituents which results in an end-product of rooperol, considered as a remarkable compound based on its active biological agents (Drewes et al., 2008). Nair and Kanfer (2006) employed a simple, accurate, and precise RP-HPLC-UV method to quantitatively detect hypoxoside in raw material and commercial products. The method proved to be effective in isolating hypoxoside and rooperol in H. hemerocallidea. In 2007, the authors validated a method that applies capillary zone electrophoresis to quantitatively identify hypoxoside in commercial products in just 12 min (Nair and Kanfer, 2007). Hypoxoside discovery in H. stellipilis and H. sobolifera species was achieved with an HPLC system. Both species contained higher levels (10 mg/ml) of the bioactive compound as compared to the frequently used H. hemerocallidea (5 mg/ml) (Boukes et al., 2008). Recently, Nsibande et al. (2018) used a UPLC-DAD-MS(-) to analyze hypoxoside in seven Hypoxis species. The biomarker compound was reported to occur in relatively minor levels in H. hemerocallidea when compared to H. gerrardii, H. argentea and H. filiformis plant species. 3.1.6. Pelargonium sidoides DC. (Umckaloabo) Earliest research on the chemical constituents of P. sidoides has been outlined by Bladt and Wagner (2007), and details seven coumarin derivatives, including umckalin (5, 6-dimethoxy-7-hydroxycoumarin and 7-O-glucoside. Reviews by Kolodziej (2007) and Brendler and van Wyk (2008) also highlight a cocktail of secondary metabolites, such as coumarins, coumarin glycosides, coumarin sulphates, flavonoids, proanthocyanidins, phenolic acids and phenylpropanoid derivatives constituted in P. sidoides and P. reniforme. Using umckalin as a biomarker, RP-HPLC method was developed and validated together with two extraction protocols, liquid–liquid extraction (LLE) and solidphase extraction (SPE) (Franco and de Oliveira, 2010). Viljoen et al. (2015) used 1H-NMR and UHPLC-MS to quantify metabolites in the popular Pelargonium species. Umckalin, identified as a marker compound, was very low (appearing as trace amounts) in P. reniforme, with high scopoletin, isofraxoside and a scopoletin isomer content. A combination of analytical techniques (HPTLC and LC-MS) was effective in detecting umckalin concentrations in Pelargonium plants from different localities in South Africa (Maree and Viljoen, 2012). The results revealed an absence of the biomarker compound in P. reniforme species. Other studies have also reported on different phenolic acid derivatives, coumarins and flavonoids detected with LC-MS, GC-MS and UPLC–MS/ MS (Colling et al., 2010a; Kumar et al., 2015). 3.1.7. Sceletium tortuosum (L.) N.E. Br. (Syn. Mesembryanthemum tortuosum L.; Kanna) Mesembrine-type alkaloids are considered to be the primary active constituents in Sceletium genus. Research on the isolation and characterization of mesembrine alkaloids has been ongoing. There is a long history documented on the discovery and biosynthesis of Sceletium alkaloids, such as mesembrenone, mesembranol, mesembrine, and

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mesembrenol (Arndt and Kruger, 1970; Herbert and Kattah, 1990; Jeffs et al., 1974a, 1974b; Jeffs et al., 1971; Jeffs et al., 1970; Stevens et al., 1975) (Fig. 2). Traditional and modern preparatory methods applied during S. tortuosum extraction yielded three mesembrine alkaloids (4'O-demethylmesembrenol, mesembrine and mesembrenone) with GC and GC-MS (Smith et al., 1998). Findings from this study, highlight the importance of extract preparation on alkaloid levels and ratios, with the traditional approach (involving crushed and bruised raw material before oven drying) yielding high alkaloid levels. Similar results have been recently confirmed by Chen and Viljoen (2018). In this case, fermentation increased the mesembrine concentration with an immediate decrease in mesembrenone levels, validated with the use of UPLC-MS. Five biomarker compounds (mesembrenone, epimesembranol, mesembrine, mesembranol, and Δ7 mesembrenone) were quantitatively identified using capillary zone electrophoresis (CZE) (Patnala and Kanfer, 2008). The method was said to be an accurate, precise and rapid approach for fingerprinting of mesembrine alkaloids in Sceletium products. Capillary electrophoresis was later coupled with MS to create a new system, non-aqueous capillary electrophoresis coupled to mass spectrometry (NACE-MS), that could separate complex alkaloid mixtures in S. tortuosum samples and drug products (Roscher et al., 2012). Application of high-speed countercurrent chromatography (HSCCC) for the isolation of S. tortuosum bioactive compounds rapidly improved isolation and alkaloid yields (Shikanga et al., 2011). In comparison to GC-MS methods, the RP-UHPLC-PDA technique made it possible to determine low levels of mesembrine alkaloids (Shikanga et al., 2012a). However, both chromatographic techniques were reported to be repeatable, precise and appropriate for quality control of mesembrinetype alkaloids. Recently, Zhao et al. (2018) used UPLC-MS and 1HNMR in S. tortuosum to identify and quantify alkaloid concentrations in plants. In addition, a non-destructive method using hyperspectral imaging (SWIR) was employed in honeybush-sceletium (Cyclopia genistoides–S. tortuosum) tea blends, and the procedure could easily detect major constituents of both species without the need for destructive sample preparation, as in chromatography-based approach (Sandasi et al., 2018). Additional research on the isolation of mesembrine-type alkaloids including Sceletium A4, tortuosamine and joubertiamine has been recently reviewed (Patnala and Kanfer, 2017). 3.1.8. Sclerocarya birrea Hochst. subsp. caffra (Sond.) Kokwaro (Marula) The chemical profile of Sclerocarya birrea subsp. caffra showed an abundance of sesquiterpene hydrocarbons and β-caryophyllene in samples using GC and GC-MS (Pretorius et al., 1985). Braca et al. (2003) later isolated a new flavonol glycoside, quercetin 3-O-α-L-(5”-galloyl)arabinofuranoside, in S. birrea wild plants using HPLC-UV/PDA and LCMS, together with other eight known phenolic acid compounds (Braca et al., 2003). Viljoen et al. (2008) confirmed the two major constituents of S. birrea, β-caryophyllene (91.3%) and heptadecene (16.1%) in fruit pulp and intact fruits, respectively, using SPME and GC-MS. From these findings, it is clear that fruit pulp extracts are mainly abundant in β-caryophyllene, with minor contents of α-humulene (8.3%) and germacrene D (0.1%), as compared to intact fruits, that are composed of diverse compounds and relatively high levels of heptadecene. In a study by Kpoviessi et al. (2011), steam distillation was applied in S. birrea (leaves) essential oils and compounds were measured using GC/ FID and GC-MS system. Essential oils comprised of sesquiterpenes, 7epi-α-selinene, α-muurolene, valencene, β-selenene and α-selinene as major chemical compounds with relatively low β-caryophyllene content. Ethyl acetate fractions from S. birrea essential oils constituted of high terpinen-4-ol, pyrrolidine, aromadendrene and α-gurjunene achieved through GC-MS analysis (Njume et al., 2011). The authors also found relatively low β-caryophyllene in S. birrea essential oils extracted from stem bark. A number of analytical methods continue to be applied in the search for S. birrea compounds together with efficient and faster approaches, i.e. HPLC (Ndhlala et al., 2007), HPLC-MS/MS (Russo et al., 2013), GCxGC-MS (Komane et al., 2015), RP-HPLC-ESI-

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QTOF/MS2 (Jiménez-Sánchez et al., 2015), UPLC-Q-TOF-MS (Shoko et al., 2018), and HPLC-ESI-TOF-MS (Cádiz-Gurrea et al., 2019). 3.1.9. Siphonochilus aethiopicus Schweinf. B. L. Burtt (African ginger) Furanoterpenoid derivatives isolated from Siphonochilus aethiopicus, (i) 4aαH-3,5α,8aβ-trimethyl-4,4a,9-tetrahydro-naphtho[2,3-b]-furan8-one and (ii) 2-hydroxy-4aαH-3,5α,8aβ-trimethyl-4,4a,9-tetrahydronaphtho[2,3-b]-furan-8-one were identified using NRM (Holzapfel et al., 2002). Essential oil composition in S. aethiopicus roots and rhizomes comprised of more than 60 compounds, with 4aαH-3,5α,8aβtrimethyl-4,4a,9-tetrahydro-naphtho[2,3-b]-furan-8-one (furanoterpenoid/siphonochilone) representing 20–30% of the detected compounds (Viljoen et al., 2002). Chemical composition analysis using GC-MS in S. aethiopicus roots and rhizomes was relatively similar, prompting a question of organ substitution in plants. Three new furanoterpenoids were isolated via solid phase extraction (SPE) with the application of HPLC in S. aethiopicus (Lategan et al., 2009). Compounds were identified as; (i) 9aβ-hydroxy 4aαH-3,5α,8aβ-trimethyl4,4a,8a,9-tetrahydronaphtho-([2,3-b]-dihydrofuran-2-one)-8-one, (ii) 4aαH-3,5α,8aβ-trimethyl-4,4a,8a,9-tetrahydronaphtho-([2,3-b]dihydrofuran-2-one)-8-one, and (iii) 4aαH-3,5α,8aβ-trimethyl4,4a,8a-trihydronaphtho-([2,3-b]-dihydrofuran-2-one)-8-one. The importance of preparation methods has been raised again in the research conducted by Naudé et al. (2016) during the quantification of eucalyptol (1,8-cineole). Results confirmed the abundance of eucalyptol in freshly prepared extracts using headspace micro-extraction (HS-SPME) when compared to dried S. aethiopicus extracts, analyzed with GC-TOF-MS. Although a study conducted by Malaka et al. (2017) was not intended for compound isolation in S. aethiopicus extracts, the study showed that steam distillation in essential oil extraction (6.37%) was better achieved in dry samples, with a fixed extraction time of 270 min when compared to extracts with a 10–30% moisture content, known to affect oil yields. The authors concluded that factors such as moisture content (10–30%), particle size (2.4–4 mm) and temperature (100 °C) influence oil yields in plant samples. 3.1.10. Sutherlandia frutescens (L.) R.Br. ex W.T.Aiton (syn. Lessertia frutescens; Cancer bush) Initially, canavanine (a non-protein amino acid with a limited distribution in plant families) was regarded as being an important biomarker for quality assessment of S. frutescens products before a set of secondary metabolites, that are uniquely produced by this species were isolated and their structural features resolved (Colling et al., 2010b). There has long been an interest in the chemical composition of the species and this dates back to the 1960s where triterpene saponins were shown to occur in the plant. The isolation and characterization of cycloartane triterpene glycosides, which at the time were named SU1 by Gabrielse (1996) and SU3 (24,25-O-β-D- diglucopyranosyl-6α-hydroxycycloart3-one by Olivier et al. (2009) were later termed sutherlandioside by Fu et al. (2008), providing for pioneer research that led to several biomarkers being identified in S. frutescens. These cycloartane glycoside compounds were established with spectroscopy and X-ray crystallography as sutherlandiosides A–D, and were chemically designated as (i) 1S,3R,24S,25-tetrahydroxy-7S,10S-epoxy-9,10-seco-9,19cyclolanost-9(11)-ene 25-O-β-D-glucopyranoside, (ii) 3R,7S,24S,25tetrahydroxycycloartan-1-one 25-O-β-D-glucopyranoside, (iii) 3R, 24S,25-trihydroxycycloartane-1,11-dione 25-O-β-D-glucopyranoside, and (iv) 7S,24S,25-trihydroxycycloart-2-en-1-one 25-O-β-Dglucoyranoside. Following these sutherlandiosides, four new 3hydroxy-3-methylglutaro-yl containing flavonol glycosides were isolated and identified as sutherlandins A–D (Fu et al., 2010). These were recognized as (i) 3-O-β-D-xylopyranosyl(1 → 2)-[6-O-(3-hydroxy-3methylglutaroyl)]-β-D-glucopyranoside, (ii) quercetin 3-O-β-Dapiofuranosyl(1 → 2)-[6-O-(3-hydroxy-3-methylglutaro-yl)]-β-Dglucopyranoside, (iii) kaempferol 3-O-β-D-xylopyranosyl (1→2)-[6-O(3-hydroxy-3-methylglutaroyl)]-β-D-glucopyranoside and (iv) and

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kaempferol 3-O-β-D-apiofuranosyl(1 → 2)-[6-O-(3-hydroxy-3methylglutaroyl)]-β-D-glucopyranoside. A suite of both flavonoids (derived from quercetin and kaempferol metabolism), aptly named sutherlandins, and sutherlandiosides are now routinely used as features to monitor quality aspects in the species. Using an LC-UV/ELSD and LCMS approach, sutherlandins A–D and sutherlandiosides A–D were quantified in leaf and stems of S. frutescens (Avula et al., 2010). Although some of the detected compounds showed slight variation within the quantified constituents, sutherlandioside B (major compound) varied significantly, ranging from 2.75% in plant samples and 1.099–5.224 mg/average in capsules or tablets. Studies continue to focus on the isolation and quantification of sutherlandins and sutherlandiosides in S. frutescens, with the application of different techniques, such as HPLCDAD (Whisgary, 2011), HPLC-MS (Faleschini et al., 2013), LC-MS (Albrecht et al., 2012; Grobbelaar et al., 2014), RP-HPLC (Mbamalu et al., 2016) and CCC that applies spiral tubing support rotors (Chen et al., 2017). Vibrational spectroscopy (NIR, mid-IR and Raman) in combination with chemometric techniques allows for an authentic analysis of herbal medicine as a whole matrix, including species identification, more accurate prediction of geographic distribution and quality control measurement (Wang and Yu, 2015). In a recent study, Mavimbela et al. (2018) successfully developed a model to rapidly quantify sutherlandioside B (or SU1) using vibrational spectroscopy (NIR and MIR) in combination with a UHPLC-MS reference dataset as well as chemometric data analysis. These techniques allow for rapid screening of metabolites, circumventing steps of extraction, purification and separation linked to phytochemical analysis of plants. 4. Quality control in 10 commercially important South African medicinal plants A majority of natural/herbal products (e.g. tablets, capsules, tinctures, tea, to name a few) commercialized from South African medicinal plants are available in formal and informal markets for therapeutic purposes. However, majority of these products (especially from informal markets) are poorly defined, bringing about huge concerns on the safety, efficacy, standardization, and quality control (Khan and Smillie, 2012). Furthermore, plant substitution is a common practice in herbal markets, particularly when plants become rare due to excessive decline of wild populations. Therefore, the use of biomarker compounds as general standards for quality control in commercialized products is crucial. Comprehensive analysis of natural/herbal products can be obtained via chemical profile of biomarker compounds using analytical techniques such as chromatography and vibrational spectroscopy, with chemometric tools used for processing data, especially in metabolomics. “Targeted” metabolomics is reliant on biomarker compounds where a set of defined metabolites is actually being investigated whereas with “untargeted” metabolomic approaches a global perspective of understanding the plant cell's biology is enabled without a prior knowledge of metabolite targets (Cox et al., 2014). Both these types of metabolite profiling approaches have become increasingly important in defining metabolic similarities and pinpointing differences in a more holistic fashion, thus aiding with chemical phenotyping of key medicinal species. These types of analyses can add value in the assessment of chemotypic variation in metabolite profiles of species from the same genus, harvested from the wild or cultivated, and in commercialized products. 4.1. Quality assessment of biomarker compounds within a genus Increasing demands for Agathosma essential oils has brought about the need for proper validation of the two commonly used species (A. betulina and A. crenulata). The leaf structure of A. betulina and A. crenulata is often used as a basis for differentiating these two species, and so, rapid techniques to confirm these plants at the species level are highly relevant. Authentication of these species arises from the

widespread variation of pulegone composition in A. betulina (2.4– 4.5%) and in A. crenulata (31.6–73.2%) (Collins et al., 1996), with high concentrations of pulegone reported to cause hepatoxicity (Thorup et al., 1983). Similarly, pulegone content has been reported to be concentrated in high levels in A. betulina (8.4%) and A. crenulata (34.9%) essential oils when analyzed with GC and GC-MS method (Viljoen et al., 2006b). Since the species have variable morphotypes, characterization of these hybrids can be achieved by examining their pulegone and 8mercapto-p-menthan-3-one isomer ratio (Collins et al., 1996). The chemical profile in Agathosma species was unmistakable when chromatographic techniques (namely, GC-MS, HPTLC, LC-MS) or vibrational spectroscopy (Raman, FT-IR) (Mavimbela et al., 2014; Sandasi et al., 2010) were employed in the determination of essential oils. For Raman, FT-IR system, chemometric data analysis revealed two distinct clusters based on the species evaluated, authenticating the variability of Agathosma hybrids. Using hyperspectral imaging, local polynomial approximation (LPA) was more effective in generating accurate data as compared to PCA (Abe and Jordaan, 2016) to delineate samples. Biochemical genetic markers of Aloe arborescens × A. ferox hybrids can be distinguished at the DDH-2 and MNR-2 enzyme coding loci when analyzed using starch gel-electrophoresis (van der Bank and Van Wyk, 1996). Aloe arborescens and A. ferox seem to possess unique alleles, which suggest that different populations (hybrid or pure) can be easily identified using enzyme coding loci rather than their morphological features. An effective method developed by Zhao et al. (2016) uses HPLC-MS/MS and HPLC-DAD to qualitatively and quantitatively analyze three Aloe species (A. arborescens, A. barbadensis and A. ferox). The method simultaneously verifies biomarkers present within Aloe species and their compound levels in order to differentiate between species. High performance liquid chromatography and LC-ESI-MS/MS applied in Aloe species (A. barbadensis, A. chinensis, A. ferox, A. arborescens) separated aloin A, aloin B and aloe emodin in plants (Fan et al., 2018). Aloe arborescens and A. barbadensis accumulated high levels of aloin A, aloin B compared to A. chinensis, A. ferox, yet both species contained different concentrations of aloin. Aspalathus linearis and A. pendula LC-MS sample analysis showed a mixture of main phenolic compounds, with no characteristic differences between the two species harvested from different populations (Stander et al., 2017). Botanically and chemically similar Harpagophytum species were distinguished with the presence of 6-acetylacteoside in H. procumbens and its consequent absence in H. zeyheri (Boje et al., 2003). Mncwangi et al. (2014b) used a UHPLC-MS and 1H-NMR together with chemometric modelling to distinguish chemical disparity in harpagoside content between H. procumbens (0.17–4.37%) and H. zeyheri (0.00–3.07%) species. The chemical profile in Harpagophytum species showed a distinct separation between the most common species; H. procumbens and H. zeyheri, with putative hybrids sometimes being intermediate and transgressive (Muzila et al., 2018). Within the evaluated species, H. procumbens contained double the content of harpagoside than in H. zeyheri and putative hybrids, analyzed using HPLC-ESI-MS. However, isoverbascoside, verbascoside, acetylacteoside and pagoside composition was greater in pulp extracts of putative hybrids as compared to H. procumbens and H. zeyheri. Successful separation between species was displayed in the data set obtained from PCA, OPLS-DA and S-plot which characterized compounds based on their retention time–mass/ charge ratio (Rt_m/z). To further confirm the variation amongst Harpagophytum species, Mncwangi et al. (2014a) used MIR single point spectroscopy and SWIR hyperspectral imaging coupled with chemometric modelling. Even though morphologically similar and used interchangeably in traditional medicine, species are made up of different harpagoside content, with the compound not always present in H. zeyheri. Quantitative analysis of phytosterols in Hypoxis species was evaluated using HPLC-UV. Stigmasterol and ergosterol constituents were abundant in H. rigidula as compared to H. hemerocallidea (Mkhize et al., 2013). Fourier transform near- and mid-infrared spectroscopy

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were used in the differentiation of P. sidoides and P. reniforme, and both systems in combination with PCA and OPLS-DA could separate plants based on their phytochemistry (Maree and Viljoen, 2011). The metabolomic profiles of Pelargonium plants (P. sidoides and P. reniforme), assessed with an untargeted 1H-NMR and UHPLC-MS approach, showed high concentrations of umckalin (0.0012–0.2760% (w/ w)) in P. sidoides and traces of this biomarker compound (occurring at 0–0.0016% (w/w)) in P. reniforme (Viljoen et al., 2015). An untargeted LC-MS approach in Sutherlandia frutescens and S. microphylla detected sutherlandins (A–D) and sutherlandiosides (A–D) that played a key role in the differentiation of the two species (Acharya et al., 2014). During an S-plot analysis, S. frutescens was found to have five markers, whereas S. microphylla constituted of seven markers. Sutherlandia frutescens was made up of sutherlandin B, a new derivative of sutherlandin B {(sutherlandin B − pentose) + hexose}, new derivatives of sutherlandioside A and D and a new molecule with an unknown aglycone. Sutherlandia microphylla compounds were documented as, sutherlandin A, sutherlandioside B and D, new derivatives of sutherlandioside B, C and D and a new molecule with an unknown aglycone. Based on these results, it is evident that sutherlandioside B (still referred to as SU1) does not constitute the main active ingredient, therefore the compound cannot be used as the only quality control marker for Sutherlandia products. We emphasize this because sutherlandioside B is often the biomarker that is more rigorously tested with respect to commercial products. Hyperspectral imaging together with chemometrics proved to be a reliable set of tools in the identification of S. tortuosum and S. crassicaule species relative to the commonly used UPLC fingerprinting (Shikanga et al., 2013). 4.2. Quality assessment of biomarker compounds in wild and cultivated plant material Plant materials from various biogeographical localities are prone to differ in their phytochemical profiles. These differences are often ascribed to phenotypical plastic responses as part of adaptive mechanisms to cope with various environments and/or evolutionary traits that are genetically inherent in the population, impacts of different environmental conditions on genetically clonal individuals, fluctuations due to seasons and plant phenology, etc (Field and Lake, 2011; Zonyane et al., 2019). Such variations, associated with ecological adaptations relate to the plant–environment interplay that may lead to unpredictable bioactivity of plant-derived extracts, requiring for specialized standardization of active pharmaceutical ingredients during the manufacture of commercial products, when especially, wild collections (and even cultivated plant materials) are used as raw materials (Chen et al., 2018; Zonyane et al., 2019). With these challenges in mind, several papers have thus focused on quality monitoring to better understand chemical variability within different geographical localities in key medicinal plants. Much of this work has been the recent focus in studies, for example, GC analysis of Agathosma betulina harvested in different areas of Cederberg Mountains had a slight variation in the biomarker compounds assessed (Ntwana et al., 2011). The constituents were made up of low pulegone, high diosphenol and the absence of quantifiable levels of cis- and trans-acetylthio-p-menthan-3-one isomers. Subsequent to the aforementioned study, Ntwana et al. (2013) reported on high quantities of diosphenol and absence of any measurable amounts of cis- and transacetylthio-p-menthan-3-one isomers in Agathosma betulina cultivated at different pH levels. Based on these results, the authors indicated that the different plant material must have originated from pure A. betulina genetic material. Seasonal variation (monitored on a monthly basis) in Agathosma ovata caused an enormous change in oil and compound yield in wild populations (Viljoen et al., 2006a). Essential oil composition was highest in May, with a significant drop in oil content in September. Malgas et al. (2010) used PCA analysis to confirm three growth forms of A. linearis obtained from the northern Cederberg and on the Bokkeveld Plateau. The findings displayed a strong genetic

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variation within the different growth forms when Aspalathus linearis was evaluated using DNA sequencing from a single chloroplast region. However, due to low sample size and different organ parts, different growth forms were suggested to have diverse genome evolutionary histories, and also, by only using the chloroplast genome, this can be a misrepresentation of their genetic variability. Different production batches of fermented A. linearis chosen from quality grades, A, B and C were analyzed during 2011, 2012 and 2013 harvest seasons (Joubert et al., 2016). It turned out that aspalathin and nothofagin constituents accumulated in higher amounts in samples collected from the Western Cape than those produced in the Northern Cape region after analysis with a RP-HPLC-DAD. De Beer et al. (2017) reported on metabolite variation; flavonoids, aspalathin, nothofagin, orientin and iso-orientin in different batches of samples originating from the same plantation at different harvest periods. Harvest from summer growth stages contained high amounts of aspalathin and nothofagin with a gradual decline in biomarker compounds after the winter harvest. During oxidation processes (fermentation), distinction between green and fermented rooibos tea samples can be achieved by using robust pair-wise logratios which allows for fast computation and identification of original variables (or peaks) (Tobin et al., 2017). For quality control purposes, Joubert et al. (2005) reported on the detrimental effect of sun drying on harpagoside retention, whereas tunnel drying provided the most proficient retention method in Harpagophytum procumbens when elucidated with an NIR method. During in vitro propagation, callus material sustained for three years were able to maintain iridoid glycoside levels (harpagoside, harpagide, verbascoside, isoverbascoside) in H. procumbens cultures (Grąbkowska et al., 2016). Umckalin levels in wild population (P. sidoides) was comparable to concentration yields in cultivated plants (White et al., 2008). Relatively similar compound content in cultivated P. sidoides together with high growth rates offer a good promising aspect for plant conservation and the international market supply. Transgenic culture clones of P. sidoides accumulated higher levels of metabolites, such as coumarins, flavonoids and phenolic acids, in comparison to non-transgenic control cultures (Colling et al., 2010a). Such gene modification techniques have an advantage as they allow for a better understanding of molecular regulation of secondary metabolism in plants. A series of studies utilizing metabolomic approaches was critical in pinpointing different chemotypes of Sceletium tortuosum (Fig. 2). Investigation of S. tortuosum plants (151 specimens) harvested from 31 localities in the south-western region of South Africa had varying degrees of mesembrine-type of alkaloid concentrations, ranging from 0.11 to 1.99% DW, as quantified using GC-MS (Shikanga et al., 2012b). Five chemotypes (A, B, C, D and E) were distinguished using hierarchical cluster analysis and PCA analysis. These consisted of chemotype A, which lacked mesembrine-type alkaloids, chemotype B (mesembrenol; 64.9–95.5%,) chemotype C (mesembrine; 51.2–92.5%), chemotype E (mesembrenone; 50.8–72.5%) and chemotype D comprising of all four alkaloids in moderate quantities. Chemotypic variation of S. tortuosum in populations of Northern Cape and Western Cape regions showed two distinct clusters in the S-plot (2D NMR), with the sugar alcohol, pinitol, and two alkylamines alkaloids distinguishing the two groups (Zhao et al., 2018). To confirm the high quantity of alkaloid content in Northern Cape species based on qNMR results, the UPLC method corroborated these findings with 4938.0–9376.8 mg/kg mesembrine alkaloids concentrated in Northern Cape samples and low levels of 16.4– 4143.2 mg/kg found in Western Cape samples. Furthermore, Northern Cape samples formed one cluster, whereas in the Western Cape, species grouped into two branches. Preparatory methods of S. tortuosum played an essential role in the chemistry and quantity of mesembrine alkaloids. A study by Patnala and Kanfer (2009) on the fermentation of S. tortuosum revealed a transformation of mesembrine to Δ7 mesembrenone, moreover, mesembrine content decreased while Δ7 mesembrenone increased. Sample fermentation in S. tortuosum significantly enhances mesembrine and total alkaloid content as had been

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previously indicated by the KhoiSan based on traditional knowledge (Chen and Viljoen, 2018). Chen and Viljoen (2018) thus also confirmed these claims that have a long history. Online analysis (via HPLC-UV/PDA and LC-MS) showed comparable flavonol glycosides in wild and cultivated Sclerocarya birrea extracts (Braca et al., 2003). Nevertheless, flavonol content accumulated more in the wild population (ca. 3 times) as compared to cultivated plants. Seasonal variation greatly influenced the chemical profile in S. birrea plants in Benin (Kpoviessi et al., 2011). Essential oil yield in samples collected in summer (February) was 0.10% and 0.24% in winter (August). On the contrary, essential oil analysis in plants harvested in winter had reduced chemical content in leaf extracts, with β-caryophyllene decreasing from 1.8 to 0.02%. Albrecht et al. (2012), using populations from the Western Cape and Northern Cape, showed that sutherlandioside B in S. frutescens is exclusive to the Northern Cape Karoo ecotypes and coastal plants, whilst the coastal plants of Gansbaai lack this particular chemical, and instead, synthesize a sutherlandioside D isomer. Untargeted metabolite profiling using LCMS as a discriminatory tool of S. frutescens showed chemical differences between geographical localities of analyzed plants (Acharya et al., 2014). Principal component analysis score plots separated wild and cultivated varieties based on their geographical localities, with less variation reported from cultivated plants harvested from Kruishof farm and wild samples collected from Darling and Melkbosstrand localities. This work was extended to plants growing in other regions of South Africa where populations from the Eastern Cape, and Free State were studied comprehensively for the first time. Geographic based metabolomic patterns were reliant on whether the sutherlandins or sutherlandiosides were the target compounds incorporated into the chemometric models that were used to display chemical associations between different groupings (Zonyane et al., 2019). 4.3. Quality assessment of biomarker compounds in commercialized products The global consumption of herbal medicinal products sold as overcounter-preparations is estimated at approximately US$107 billion and there is thus a lot at stake and this may be one reason which leads to fraud and adulterated products (Posadzki et al., 2013). This has been said to be common in both Chinese Traditional and Ayurvedic medicines. In the recent past, adulteration and contamination has thus become a growing concern, lowering trust in herbal-based medicines thus placing the complementary and alternative industries at a potential financial risk. Street et al. (2008) reported that there were no obvious cases of deliberate adulterations of medicinal plant products in South Africa in their review which was based on work between 1994 and 2007. As the commodification of medicinal plants continues to rise in South Africa, researchers are becoming more weary of the potential for cases of fraudulent adulterations to increase in their frequency and studies linked to authentication and detection of contaminants and adulterants are now appearing in the literature. Quality control and the standardization of natural/herbal products is very important and protects the integrity of the product in terms of its pharmaceutical quality. It also forms part of a prerequisite for the reproducibility of active ingredients from batch to batch, when they are tested. From issues such as spiked pharmaceutical additives, economic adulterants, and accidental/intentional substitution by commonly misidentified or improperly characterized plant material, adulterations and modifications do occur during commercial manufacture. Therefore, methodical systems that factor in inherent attributes of a given botanical, from seed to shelf, are probably the most reasonable approaches to dealing with quality control concerns. A systematic designed approach that includes botanical characterization (genetic, morphological, phytochemical, etc.) of natural/herbal products can add to safety standards (Khan and Smillie, 2012). Adulteration of essential oils including Agathosma betulina (synthetic compounds) has been reviewed by Do et al. (2015). Raman et al. (2015) also confirmed the issue of

adulteration in commercialized sample products (e.g. capsules, tea bags) of A. betulina with senna, grass and foreign materials using microscopy and HPTLC fingerprinting. Using NMR spectroscopy, Rasmussen et al. (2006) found discrepancies in pharmaceutical products prepared from H. procumbens, either from different suppliers or different batches supplied by the same company. Constituents of H. procumbens tinctures from different batches examined during accelerated thermal stability test with the coupling of HPLC-DAD and HPLCESI-MS revealed high stability of iridoids (8-E-p-coumaroyl-harpagide, pagoside, harpagoside), with no more than 90% degradation detected from the initial concentration up to 6 months (Karioti et al., 2011). Commercialized products of H. hemerocallidea using HPLC-UV showed inconsistencies in hypoxoside content (Nair and Kanfer, 2006). Some of the products contained hypoxoside while others showed no traces of the constituent even though the labels claimed otherwise. Capillary zone electrophoresis method detected lower levels of hypoxoside content in capsules of product A (18.83 mg) and B (11.24 mg) as compared to 250 mg Hypoxis rooperi/0.69 g unit weight (product A) and 200 mg Hypoxis powder/1.17 g unit weight (product B), as per product label (Nair and Kanfer, 2007). Pelargonium sidoides commercialized products purchased from Finzelberg (Berlin, Germany) and African Bush (San Antonio, USA) were composed of similar levels of umckalin (Franco and de Oliveira, 2010). Sceletium tortuosum capsule products (purchased from health shops and local manufacturers, South Africa) contained varying concentrations of alkaloid profiles (Shikanga et al., 2012b). For instance, mesembrenol varied from 0.20 to 59.91%, mesembrine (42.26–82.94%) was the main alkaloid constituent composed in all the analyzed products, whereas the other alkaloids varied based on the product tested. High quality in-source CID DARTHRTOFMS in Sceletium tortuosum exposed the presence of a stimulant known as ephedrine, a banned substance in herbal products and supplements (Lesiak et al., 2016). 4.4. Other approaches applied for quality control purposes in commercialized plants Genome fingerprinting generally applies in sample authentication and detects sample homogeneity based on individual plant, species and population (Sucher and Carles, 2008). Single chloroplast or nuclear gene sequences often need to be supplemented with DNA sequenced data from more than one gene or genomic region for an accurate species identification, powerful genetic markers that provide precision and accuracy are needed for DNA-based quality assessments. Therefore, DNA-based systems offer an alternative tool for rapid and robust characterization of species. The method incorporates the use of gel electrophoresis, sequencing, or hybridization with species-specific analyses through nuclear and chloroplast DNA. The use of DNA barcoding for picking up biological contaminants or adulterants is an important application and advantages and disadvantages of using this technique in plants are outlined in detail by Hollingsworth et al. (2016). These authors emphasize that for plants, a universal barcode with high resolving power, such as that used for animals, cytochrome oxidase, CO1, is not yet available but popular markers such as rbcL, matK, trnH-psbA, offer possibilities for species authentication. The ITS nuclear markers and especially trn-L for short sequence amplification can be used with some success in plants. Further advancement of DNA barcoding coupled with next-generation sequencing and high-resolution melting curve analysis has successfully led to species-level resolution recovered from commercialized herbal products (Mishra et al., 2016). The use of DNA barcoding has to be met with metabolomics, transcriptomics and proteomics for effective authentication of herbal products. The latter two applications are glaringly lacking for the interest plants that we discuss. Recent work by Raclariu et al. (2017) showed that Hypericum perforatum was detected in 68% of the products with the use of metabarcoding. There were also some discrepancies detected with metabarcoding between constituent species and those listed on the

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label in all products, which cannot be easily detected with TLC or HPLCMS methods. Overall, the study confirmed the application of metabarcoding in the detection of H. perforatum, plus the substitution, adulteration and/or mixture of other species in purchased herbal products. Electrophoresis was used to determine genetic variation in Siphonochilus aethiopicus wild and cultivated population, using a technique that was popular in the 90s. Allozyme results showed that genetic fingerprinting in S. aethiopicus clones was accomplished by using 11 polymorphic loci, and that clonal polymorphism may have resulted from interactions between vegetative and sexual reproduction (Makhuvha et al., 1997). Even though electrophoretic-based analyses for genetic profiling may now be out of favor, and viewed by some as being more archaic due to molecular biological advancements having a rapid evolution as a technology, they still hold value as they may be cheaper than current technologies such as next-generation sequencing and more readily accessible in some laboratories. Sclerocarya birrea micropropagated plantlets ensured an intraclonal genetic stability with explant parent when genetic fingerprinting was done using randomly amplified polymorphic (RAPD) (Mollel and Goyvaerts, 2012). Genome organization in S. birrea was characterized using a classical karyological approach, with physical mapping of heterochromatin and rRNA genes, and through assessing genome size (Bationo-Kando et al., 2016). From the results, S. birrea was found to be a diploid species (2n = 28 chromosome number), with a very small genome size of 2C = 0.81 pg within the Anacardiaceae, based on a flow cytometry. In situ hybridization (fluorescence) confirmed the detection of two ribosomal gene families, 5S and 35S, located in different chromosome pairs. Sclerocarya birrea populations collected from Malawi and Tanzania (ICRAF field genebank) contained a relatively rich gene pool (Fridah et al., 2017). Out of the 6 ISSR markers, 76 polymorphic bands across the 257 accessions studied were identified, with a polymorphic loci and heterozygosity that ranged from 75 to 78.9% as well as H = 0.362 to 0.043, respectively. Using AFLP-, sequence characterized amplified regions (SCARs-) and single nucleotide polymorphosmis (SNP)-based analyses, populations of Agathosma species were studied to provide a genetic platform that would easily be used to distinguish hybrids of A. betulina. The detection of hybridization is important in this species as hybridization would alter essential oils derived from A. betulina, generating oil that is of low to no economic value in the market (Husselmann, 2006). Populations were assigned to three different groups: (1) A. serratifolia and A. crenulata populations; (2) the putative hybrid, A. betulina X A. crenulata populations and (3) A. betulina plants. The data provided important evidence confirming that analyzed hybrids were more similar to A. betulina populations than those of A. crenulata. Authentication and identification of plants used in herbal remedies using molecular biology technologies, such as DNA barcoding, [and recently next-generation sequencing (NGS)] is increasingly useful for plant species discrimination, assisting with genotyping and identification of different taxa especially when the medicinal remedies are made of chopped up plant materials assembled into a polyherbal mixture (Techen et al., 2014). The review of Techen et al. (2014) provides a summary of techniques such as hybridization and microarray methods, sequencing of genetic regions and various PCR-based methods (e.g. RAPD and ISSR) that have been applied in medicinal plants. It is clear from this review that at present many species that have been assessed are those used in Chinese traditional medicines. Literature is thus currently lacking in the application of DNA barcodes as an authentication tool for commercialized South African medicinal plants (Williamson et al., 2016) but the establishment of the unit called the 'African Centre for DNA Barcoding' in 2010 in South Africa is meant to fill this particular gap, providing a scientific service to support regulatory enforcements in the natural products industry plus tracing the identities of species that may be sold at medicinal plant markets in South Africa, and, Africa as a whole (http://www.acdb.co.za). At this unit, a DNA barcode database has been established for medicinal South African plants and this will open further opportunities in genomic

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studies of many other species. There are thus, at present, only few examples to describe and discuss. Although the study of Williamson et al. (2016) is not on the previously mentioned interest species, it provides a good example of how barcoding can trace origins of species exposing illicit trade networks in traditional medicines markets in South Africa, especially of those species such as cycads (Encephalartos species) that are critically, high-to-nearly endangered. Using Sanger sequencing, with amplifications achieved with rbcLa, matK, nrITS and trnH-psbA primers, a sequencing success rate of 89–100% was achieved and 40 species representing 87% of test samples could be identified using BRONX software. The cycads were determined to be E. ferox, E. lebomboensis, E. natalensis, E. senticosus and E. villosus that were being traded at the medicinal markets. Molecular-based identifications are useful in picking out different plants that may comprise a polyherbal mixture, which is often the way in which plants are used in traditional medicines in South Africa. Other interesting works worthy of mention include the identification of endophytic fungi using ITS1, ITS4, EF1 and EF2, associated with Sceletium tortuosum. Aspergillus, Penicillium and Fusarium were dominant fungal associates but three new species were also confirmed (Manganyi et al., 2018). This is significant as the microbial biome is now accepted to cause major fluctuations in quality and quantitative profiles of plant phytochemicals, contributing to de novo changes in metabolite flux and chemical pools. This means that different microbial variants in plant tissues culminate in intraspecific chemical differentiation (Manganyi et al., 2018). Otherwise, next generation sequencing is gaining considerable popularity for application in medicinal plants for the very purpose of establishing plant taxonomic identities in macerated tissues where morphological characters cannot be applied as a means to distinguish species (Hollingsworth et al., 2017). 5. Phytochemistry, pharmacology and clinical studies in commercialized South African medicinal plants In South Africa, similarly to other parts of the world, those that are prolific consumers of natural products and plant-derived medicines have a culture of utilizing a variety of herbal medicinal systems when self-medicating, paying little attention to possible adverse drug–herb interactions that may occur or even those linked to herb–herb combinations. Adverse reactions become even more difficult to pinpoint when constituents of complex active pharmaceutical ingredients (APIs) are not well characterized and defined. We conducted a literature survey to obtain experimental data associated with the plants of interest in this paper. It is clear that testing of both herbal mixtures, single herb ingredient products and purified chemicals from isolated ethnobotanical medicines is limited and large gaps in our knowledge exist despite the commercialization of certain species. For species such as Aspalathus and Sceletium (as examples), there is a considerable growing body of information on potential changes to efficacy and safety when products based on these plants are taken together with other herbal products or mixtures and even allopathic medicines but for many other South African species information is limited. The main concern here is attached to: (1) unsafe herb-to-herb drug interactions; (2) combining purified chemical drugs with botanical pharmaceutics and (3) adulterations of plant-based medicines with pure chemicals that may cause toxicities that have previously not been detected or studied when consumed by humans. Refer to some of the main research areas linked to medicinal plants compiled from Scopus into a chart (Fig. 1), which highlight subject fields that have been the basis of scientific studies in terms of plant medicines based on South African plant taxa. It is obvious from Fig. 1 that recent attention has been paid to in vivobased analysis. Dosage is often critical in determining whether medicinal herbs and even pharmaceutical drugs have therapeutic and combinations may lead to unprecedented decreases or increases in toxicity that go without being clinically monitored (Zonyane et al., 2019). The use of herbal medicines is often hidden by patients, complicating diagnosis linked to them being unsafe for users (Street et al., 2008). Many

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medicinal herbs and pharmaceutical drugs are therapeutic at one dose and toxic at another. Interactions between herbs and drugs may increase or decrease the pharmacological or toxicological effects of either individual component and those medications that are used on a longterm basis for chronic diseases may be particularly difficult to provide correct dosing for users (Chen et al., 2018). Provision of quality chemical standards and plant materials in pharmacovigilance tests generated from important South African plants that are of sociocultural relevance and commercial interest is urgently required for a larger fraction of plants that, at present, have been tested. Combination of conventional (orthodox) pharmaceutical drugs together with herbal products is a common practice but the scientific monitoring of possible interactions remains poorly defined. Recently, a report on the combination of Lippia scaberrima and an unfermented A. linearis extract was conducted by Kok et al. (2018) and these authors indicated that this combination reduced in vitro hepatotoxicity in the presence of the drug, acetaminophen, which is commonly used to reduce fever and inflammation through the inhibition of CYP1B1. A study that focused on tablets made with H. procumbens used an in vitro technique to the effect of an artificial gastric fluid on the physico-chemical properties of the key constituents of these tables and little to no alterations to harpagoside were observed, with this chemical being reduced in its content by only 10% once the experiment was complete (Chrubasik et al., 2000). These studies have been followed by several others in a clinical trial setting (Chrubasik et al., 2003; Chrubasik et al., 2004), concluding that dosages of 50 mg harpagoside in the extract are important for pain relief in patients that were involved in a double-blind clinical trial. There has been some work with South African plants that are often used for HIV/AIDS-related disorders that have been evaluated for their pharmacokinetic effects such as H. hemerocallidea (e.g. Azu et al., 2016); Sutherlandia frutescens (Minocha et al., 2011) using in vivo models. For the latter, various pharmacokinetic measurements monitoring the maximum plasma concentration (Cmax), time needed to reach maximum concentration (Tmax) and area under the plasma concentration curve (AUC), in fact, formed the basis of the measurements. The chemical complexity of herbal medicines makes them suitable candidates to study interactions with conventional drugs as they contain a wide spectrum of pharmacologically active components beyond those that are identified as key biomarkers. Even so, it is only recently that the South African Health Products Regulatory Authority (also known as the South African Medicines Control Council) is calling for better regulation of herbal products and thus driving more research into herb–drug interactions. Ingested herbals and even those with a dermal application that become absorbed into the bloodstream, are more than likely to act on the cytochrome P450 (CYP450) system and illicit the efflux drug transporter mechanisms involving the P-glycoprotein (P-gp). A wide range of tissues need to be monitored such as liver cells, adrenal gland and kidney proximal tubules, colon and intestinal mucosa, blood-testis capillary epithelial cells, etc in order to better define effects on mammalian system (Li and Bluth, 2011). Interactions between the nevarapine and CYP3A4 gene expression in LS-180 cells became apparent, validating need for more intensive research as S. frutescens was at some stage encouraged for lowering symptoms linked to HIV/AIDS infections. Another study conducted by Faleschini et al. (2013) that focused on in vitro modulation of HL60 cell lines, showed production of TNF and IL8 cytokines especially when phorbol 12-myristate 13-acetate (PMA) was incorporated into the bioassay leading to values of 229.45 ± 13.89 for TNF and 5967.93 ± 226.86 pg/ml for IL8 cytokines. This study showed that both the whole extract and fractions derived from S. frutescens had anti-inflammatory activity (Africa and Smith, 2015). Recently, Chen et al. (2018) and Zonyane et al. (2019) reported on the use of the zebrafish embryo bioassay to study teratogenic effects of S. frutescens and high concentrations of an ethanolic extract (N50 mg/ml) caused cardiotoxicity in developing embryos. Although the water extracts were less toxic, all doses above 100 mg/ml caused reduced heart beats in zebrafish embryos and leading to a 100% mortality

rate. Ultimately, production of cardiac cysts and internal bleeding was obvious under the light microscope at high concentrations. The zebrafish study adds to the body of knowledge linked to this species where concerted effort to better define the cytotoxic effects have led to several studies that focus on its effects on the main cytochrome P450 isozymes by utilizing the liver microsomal in vitro plate assay (Sergeant et al., 2017), and monitoring ATP-binding cassette transporters (P-gp and BCRP) and human organic anion transporting polypeptides (OATP1B1 and OATP1B3) (Fang, 2011). With aims to pilot a future large-scale phase II human clinical trial, the study of Swead (2018) first developed a protocol using Chlorocebus aethiops (vervet monkey) as an animal model and provided preclinical evidence that could be ultimately used to set standards for testing Sutherlandia frutescens in a clinical setting with adult human patients who have type 2 diabetes at the South African Medical Research Council. The vervet monkey study showed that there were no abnormalities that could be detected in the hematological, biochemical, and physiological parameters that were measured in the monkeys. In a previous study by Johnson et al. (2007) that used a small sample size and a test that spanned three months, capsules made from pooled Sutherlandia frutescens cultivated from four different farms underwent a clinical trial using healthy human adults. In that clinical trial, the authors concluded that no ill-effects were induced by the consumption of the test capsules. Essentially, human patients taking the capsules for the threemonth period had similar physical to biochemical properties as those that were given the placebo treatment (Johnson et al., 2007). Unfortunately, pharmacokinetic information was glaringly lacking. Apart from this, in reality those that consume S. frutescens products for diabetes generally take it as a long-term chronic medication, and so, longer trial periods would be necessary to provide convinving data about the safety of these products. To follow on from this work, asymptotic patients with HIV between 21 and 64 years were given S. frutescens and their viral loads monitored after 24 weeks (Wilson et al., 2015). Some of the data presented seem to be inconclusive. There were no obvious changes in the HIV viral load and CD4 T lymphocytes with the exception of those on other medications such as isoniazid preventative therapies in the study of Wilson et al. (2015). Many of the biochemical and hematological measurements for individual asymptomatic female patients were the same at 12 and 24 weeks of the study period plus none of them developed vasculitis, however, the effect of combining isoniazid (a tuberculosis preventative drug), together with S. frutescens was unclear for the authors and they recommended a further study to better understand possible drug–herb interactions in a clinical setting. Recently, Muller et al. (2018) studied the pharmacokinetic profiles of healthy male adults to understand the effect of S. frutescens tables that were taken together with the antiretrovial protease inhibitor drug, atazanavir. The data showed a negative interaction where reduced bioavailability of the atazanavir drug was observed in study participants. Due to the popularity of Aspalanthus linearis worldwide, several studies providing preclinical and clinical analysis of extracts, fractions and even those extracts enriched with aspalathin, are on the increase. Some examples of such studies are those by (1) Sasaki et al., 2018 investigating the role of rooibos in diabetic rodents using an extract with high levels of PPAG; (2) histological and immunohistological studies focusing on modulatory effects linked to neurological inflammation; (3) using various neurochemical assays, are the interest areas explored by Akinrinmade et al. (2017); (4) Marnewick et al. (2011) analyzing the effects on those patients at higher risk of cardiovascular diseases and the oxidized glutathione (GSSG) measurements were taken together with blood pressure measurements, amongst the top evaluations conducted; (5) previous work determining lipid peroxidation, blood biomarker analysis for oxidative stress management and comprehensive analysis of clinical pathological markers (Marnewick et al., 2011; Marnewick et al., 2009) and (6) protective antioxidant effects of rooibos when used with other products such as red palm oil in terms of liver function in rats (Ajuwon et al., 2013).

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The phytopharmaceutical, EPs 7630 has undergone numerous clinical evaluations in randomized, double-blind, placebo-controlled trials. Incidentally, it has been clinically tested against respiratory-related conditions, namely acute bronchitis, acute rhinosinusitis, common cold, chronic obstructive pulmonary disease and asthma, and some of the most recent work is shown by an investigation of Roth et al. (2019). The clinical trials conducted for EPs 7630 have included both older and younger patients (Moyo and Van Staden, 2014). Much of this work is driven by the interest of the phytopharmaceutical industry to provide efficacy of this extract on patients, funding such studies. Gericke (2001) in a review of those plants that at the time had undergone clinical analysis, i.e. P. sidoides, Harpagophytum procumbens, Hoodia gordonii (Masson) Sweet ex Decne, Sceletium tortuosum, and Sutherlandia frutescens also concludes that clinical studies are undertaken by commercial producers within the natural products industry to provide efficacy of their products so as to comply to regulatory bodies that govern the phytopharmaceutical sector in different countries. Regulatory barriers to the approval of novel plants extracts, preventing them from moving from research laboratories to commercial markets, may be more for those items that are earmarked as botanical pharmaceutics rather than those that are likely to enter the food industries. This is due to novel foods and functional foods facing lower regulatory standards than those products that claim pharmaceutical function in humans. However, preclinical and clinical data provide empirical evidence for research and development efforts, and provision of such information is absolutely crucial, as it may fast-track commercialization of natural products (Gericke, 2011). Protocols are thus required to assess the safety, tolerability and effectiveness using double blind randomized trials where placebo-controls are firmly in place especially for those formulations that have standardized extracts (one of the constraints for South African plants is limited standardization). Other clinical studies worthy of mention are those conducted on Sceletium as it becomes more popular as a potential treatment for depression. The studies discussed in this species would have not been possible without a major effort in understanding the ethnobotany, phytochemistry and pharmacology of S. tortuosum (for reviews refer to Gericke and Viljoen, 2008; Krstenansky, 2017; Smith et al., 1996; Van Wyk, 2011). Refer to Fig. 2 for a graphic summary of the timeline of research areas in S. tortuosum, showing seminal works that provided a foundation that led to pharmacological tests, using both in vitro and in vivo models, of various commercial and nowadays, also in a clinical set up. The first papers in the 1970s focused mainly on isolation and purification of compounds but it is the papers published in the mid-90s (Smith et al., 1996) that were a prerequisite to the in vivo studies that appeared from 2010 onwards (Fig. 2). Some of these papers are highlighted here. Using rat urine and human liver preparations, mesembrine or mesembrenone after the exposure to 1 mg/kg BW and cytochrome P450 expression of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, or CYP3A5 as key markers were assayed to facilitate forensic detection of Sceletium alkaloids in samples. In a study by Meyer et al. (2015), metabolized urine screening for mesembrenone revealed presence of acetylated demethyl-dihydro and hydroxy metabolites whereas N-demethylation, mono- or bis-Odemethylation, hydroxylation, N-oxidation, reduction of the keto group, and various combinations including the presence of Odemethyl metabolites that were recreated as glucuronides or sulphates were linked to the consumption of mesembrine. The study that used LCMS, GC-MS and NMR metabolomics provides a valuable pharmacokinetic platform towards the possible detection of these alkaloids in human urine (Meyer et al., 2015). The Sceletium extract (Zembrin) has been subjected to different tests using clinical settings where the central nervous systems vital signs and Hamilton depression rating scale (HAM-D) tests were used (Chiu et al., 2014) and in this study, the action of this extract is thought to function via the PDE-4/cAMP/CREB cascade. There has been a concerted effort to better define the effects of Zembrin within a clinical context in humans following work that has a

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foundation from ethnobotanical leads, ethnobotanical, in vitro and in vivo assays, that are congruent in the effects of this particular extract on serotonin function. Using electopharmocogram technology, Zembrin® was compared to several other herbal extracts on brain function. It is clear from the study this extract offers similar CNS properties as Gingko biloba and Cimicifuga racemosa extracts that are popular medicinal plants for enhancing cognition and relieving symptoms associated with depression. Recent data, generated by Coetzee et al. (2016) have provided new evidence of the mechanism(s) that may be important in the pharmacological effectiveness of the commercial extract Trimesemine™ generated from Sceletium. This extract is thought to function through cortical steroid enzymes that are implicated in inflammation and stress. It appears that the mode of action of this particular extract is therefore slightly different from Zembrin®, eliciting monoamine-releasing activity rather than solely functioning as a serotonin reuptake inhibitor in vitro (Coetzee et al. (2016). The Trimesimine™ extract, that is defined by having high levels of mesembrine, has also been tested using a purified monocyte in vitro model and stimulation by application of an Escherichia coli lipopolysaccharide and this work showed that extracts with different quantities of mesembrine alkaloids may vary in their antioxidant and anti-inflammation pharmacology (Bennett and Smith, 2018). These studies have shown that when the extract has high levels of mesembrine, it exhibits higher cytoprotective and anti-inflammation properties, whereas those extracts that contain a superior amount of δ-7-mesembrenone together with other polyphenolic compounds, show good antioxidant activity in vitro when using human astrocytes in a mitotoxicity and cytokine test (Bennett et al., 2018). There is clearly value in doing further clinical studies that are well aligned with the manner in which plants are being used using human subjects. The major challenge of this type of work is the lack of funding as clinical studies are extremely expensive and often they are time consuming, one of the reasons it is easier for researchers to focus more of their attention and funding on in vitro-based studies (much of this has been reviewed elsewhere before). It is noteworthy that these evaluations may often not translate directly to a clinical setting. Several other authors have emphasized on this, including Masondo et al. (2019) where they conclude that in order to advance plant based research in disease areas of the central nervous system, more studies on clinical trials are urgently needed (see the work of Van Wyk (2011) and Mahomoodally (2013)). It cannot be said enough that, existing natural products research which has seen a rapid accumulation of scientific information in the areas of ethnobotany and pharmacology (Fig. 1) plus artistic and innovative interrogations in local indigenous plants that are discussed in detail in this review, has been a major impetus that has driven commercialization of some medicinal plants from South Africa in world markets. For South African medicinal plants to continue to enter new sectors of the complementary and alternative industries, exploitation of cutting-edge analytical techniques that will ensure quality, safe and scientifically validated herbal therapies will play a greater role in years to come. 6. Conclusions and future perspectives The current position of natural product research in South Africa is at a point where the analysis of only few biomarkers causes a bottleneck that limits our understanding of the possible side effects, which may result from different active constituents in both standardized extracts and those earmarked for future development into APIs. This in turn decreases the reliability and repeatability of pharmacological and clinical studies, as well as the credibility of the medicinal plant or herbal product as an effective health-benefiting agent. With this being said, the value of efforts to better define extracts of various medicinal species using a battery of different methods such as metabolomics and genomics is enhancing our current knowledge and has potential to generate new insights into the phytochemistry of South African medicinal plants

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as analytical technologies develop to become even more robust. In the past decades, comprehensive quantitative and qualitative analysis of the different metabolites gained considerable momentum, and henceforth, it is hoped that in the future this knowledge will move towards a better understanding of metabolic and molecular controls that are occurring at the cell, tissue and organ level of those plants that are of commercial interest. Applications of new technologies such as CRISPR-Cas9 genome editing to non-model plant species of medicinal value are only possible if background genetic-to-metabolite information is available. Sequencing using next-generation technologies is thus urgently needed to provide basic genetic information which can be used to study the molecular physiology of medicinal plants, where applications such as transcriptomics and proteomics can provide basic information on plant function. We view this as being highly important, especially in the context of climate change, which has possibilities to alter not only the chemistry of plants but also their general phenology and epigenetics. Therefore, multi-hyphenated chromatographic techniques in combination with hyperspectral imaging and numerous chemometric systems represent the future direction for comprehensive and robust methods for the rapid metabolite profiling of natural product extracts, and have largely been developed by biotechnology companies, and now start to be applied in academic drug discovery programs, as evidenced in the current review. Other research avenues that require rapid development for key medicinal species in South Africa include those related to pharmacodynamics, pharmacokinetics and bioavailability of plant extracts with larger research inputs being urgently needed to study multiple drug–herb and/or herb–herb interactions particularly using animal models. Such studies will not only lead to new insights into the functions of extracts in living organisms but will require an integration of expertise and approaches. This in turn may lead to generation of new intellectual property patents and opportunities for further commercialization. Authors' contributions NPM conceived the idea for this paper and coordinated the writing of the review article. NAM and NPM sourced the online publications used for the manuscript. NAM and NPM drafted and proofread the final version of the manuscript. Acknowledgements NAM is a recipient of the National Research Foundation Postdoctoral Fellowship (NRF, Grant UID: 106493). NAM and NPM appreciate the financial support from the South African National Research Foundation (NRF, Grant UID: 109385) and Stellenbosch University Division of Research Development. We are grateful to the reviewers for their comments and editorial improvements made to this manuscript. References Abe, B.T., Jordaan, J.A., 2016. Identifying Agathosma leaves using hyperspectral imagery and classification techniques. Proceedings of the World Congress on Engineering and Computer Science. San Francisco, USA. Acharya, D., Enslin, G., Chen, W., Sandasi, M., Mavimbela, T., Viljoen, A., 2014. A chemometric approach to the quality control of Sutherlandia (cancer bush). Biochem. Syst. Ecol. 56, 221–230. Adhami, H.-R., Viljoen, A.M., 2015. Preparative isolation of bio-markers from the leaf exudate of Aloe ferox (“aloe bitters”) by high performance counter-current chromatography. Phytochem. Lett. 11, 321–325. Africa, L.D., Smith, C., 2015. Sutherlandia frutescens may exacerbate HIV-associated neuroinflammation. J. Neg. Res. BioMed., 14 https://doi.org/10.1186/s12952-015-0031-y. Agarwal, H., Kumar, S.V., Rajeshkumar, S., 2017. A review on green synthesis of zinc oxide nanoparticles – An eco-friendly approach. Resour. Effic. Technol. 3, 406–413. Ahl, L.I., Grace, O.M., Pedersen, H.L., Willats, W.G.T., Jørgensen, B., Rønsted, N., 2018. Analyses of Aloe polysaccharides using carbohydrate microarray profiling. J. AOAC Int. 101, 1720–1728. Ajuwon, O.R., Katengua-Thamahane, E., Van Rooyen, J., Oguntibeju, O.O., Marnewick, J.L., 2013. Protective effects of rooibos (Aspalathus linearis) and/or red palm oil (Elaeis guineensis) supplementation on tert-butyl hydroperoxide-induced oxidative

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