Introduction
Mounting evidence that dietary polyphenols may modulate dysglycaemia, a major metabolic
aberration associated with the development of type 2 diabetes, is rooted in the demonstration
by Von Mering in 1886 that high doses of the dihydrochalcone phloridzin (2′-β -D-glucopyranosyloxyphloretin) reduced glucose reabsorption from the renal filtrate,
causing glucosuria in dogs and later also confirmed in humans [1 ]. This earned phloridzin the distinction as “the only compound known to have a definite
action in man” as stated in a book chapter on the economic importance of flavonoid
compounds in foodstuffs, published in 1962 [2 ]. Since the discovery of Von Mering, studies on phloridzin have progressed to the
development of C -glucosyl analogues, not only with longer pharmacokinetic half-lives and duration
of action than O -glucosides, but with high selectivity for SGLT2 over SGLT1 (reviewed by Idris and
Donelly [3 ], Jesus et al. [4 ], and Blaschek [5 ]). In silico modelling of the natural C -glucosyl dihydrochalcone aspalathin ([Table 1 ]) predicts that it may exhibit antidiabetic effects through inhibition of SGLT2 [6 ]. The present review provides a short discussion of the link between metabolic syndrome
and, respectively, glucose metabolism, lipid metabolism, oxidative stress, and inflammation,
as a background to a discussion of the potential of aspalathin in this context. Various
in vitro and in vivo studies on aspalathin are highlighted, demonstrating its potential to target underlying
metabolic dysfunction relative to the development or progression of serious metabolic
diseases such as obesity, type 2 diabetes, and cardiovascular diseases. Studies describing
the relevant biological activity of extracts prepared from green rooibos, containing
high levels of aspalathin, are included to indicate potential mechanisms of action
and provide perspective on the use of an aspalathin-rich extract versus pure compound.
Other aspects of aspalathin covered in this review are its natural source, the variation
in content and the impact of processing the plant material, physical and chemical
properties, chemical synthesis, bioavailability, and potential interference with the
metabolism of hypoglycaemic and hypocholesterolaemic drugs. To achieve this, the authors
independently and systematically searched major databases, including PubMed, EMBASE,
and Google Scholar, for available studies reporting on the chemical and physical properties
of aspalathin, its bioavailability profile, as well as its ameliorative potential
against various metabolic complications. The systematic search was conducted without
any language restrictions, while unpublished and ongoing studies, in addition to review
articles, were screened for primary findings.
Table 1 Physical properties of aspalathin.
Property (units)
Value
Reference
Structure
IUPAC name
3-(3,4-Dihydroxyphenyl)-1-(2,4,6-trihydroxy-3-[(2S ,3R ,4R ,5S ,6R )-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H -pyran-2-yl]phenyl)propan-1-one
[7 ]
Molecular formula
C21 H24 O11
[7 ], [8 ]
Molecular weight
452.412
[7 ], [8 ]
Melting point
152 – 154 °C
[9 ]
Polar surface area (Å2 )
208
[7 ]
Log P (predicted)
2.07
[8 ]
Log D (pH 7.4) (experimental)
0.13
[10 ]
Log D (pH 5.5) (experimental)
−0.347
[11 ]
H bond acceptors
11
[7 ], [8 ]
H bond donors
9
[7 ], [8 ]
Freely rotating bonds
6
[7 ], [8 ]
“Rule of 5′′ violations
2
[8 ]
Solubility at pH 2 (µM)
153
[10 ]
Solubility at pH 6.5 (µM)
123
[10 ]
Solubility in FaSSIF at pH 6.5 (µM)
119
[10 ]
Natural source
A recent review of the health effects of phloretin, the aglycone of phloridzin, stated
that to date, about 200 dihydrochalcones, isolated from more than 300 plant families,
have been identified [12 ]. Aspalathus linearis (Burm.f.) Dahlg., one of more than 270 species of the genus Aspalathus (Family Fabaceae, Tribe Crotalarieae) and endemic to the Cape Floristic Region [13 ], [14 ], is the natural and, until recently, only reported source of aspalathin. A closely
related species, Aspalathus pendula R. Dahlgren, was recently also shown to contain aspalathin [15 ]. A. linearis is an erect to spreading shrub up to 2 m high with green, needle-like leaves on straight,
slender branches. The leaves (15 – 60 mm long; up to 1 mm thick) are densely clustered
without stalks and stipules. The small, yellow flowers of the cultivated type appear
in spring to early summer and are solitary or arranged in groups at the tips of branches.
The fruit is a small lance-shaped pod usually containing one or two hard seeds. The
species is exceptionally polymorphic with ecotypes differing in morphology, fire survival
strategy (reseeding or resprouting), geographical distribution, and phenolic composition
[16 ], [17 ], [18 ], [19 ]. Van Heerden et al. [19 ], investigating the phenolic profile of ecotypes of A. linearis , as well as that of the closely related A. pendula , did not detect aspalathin in the collected A. pendula samples, but demonstrated it to be the major compound in most A. linearis populations, including the cultivated type. Recently, however, Stander et al. [15 ], using state-of-the-art mass spectrometric techniques, could detect aspalathin in
two populations of A. pendula . They concluded that A. linearis and A. pendula produce similar combinations of main compounds with no diagnostic patterns. Only
the selected and improved Nortier type (one of the Red Rocklands types) is used for
commercial cultivation of rooibos tea ([Fig. 1 ]) in the Cederberg region of the Western Cape.
Fig. 1 Commercial plantation of cultivated A. linearis (rooibos).
The aspalathin content of the plant material is known to vary substantially between
plants from the same plantation [20 ], [21 ] due to the fact that the plants are propagated from open-pollinated seeds [22 ]. [Fig. 2 ] depicts the variation in the aspalathin content of the leaves of individual plants,
harvested on the same date from the same plantation. The largest number of leaf samples
contained ca. 8% aspalathin (dry weight basis). [Table 2 ] summarises ranges and means for aspalathin content in various types of plant material,
extracts, and infusions. Aspalathin is present in substantially higher amounts in
the leaves (6 – 13%) [21 ] than the stems (0.16 – 0.78%) ([Fig. 3 ]) and thus also the whole dried shoots (4 – 10%) [20 ]. Rooibos produced in the traditional manner that includes the “fermentation” (oxidation)
step for development of the characteristic red-brown leaf colour and “woody”, “fynbos-floral”,
and “honey” flavour [23 ], [24 ], and contains very little aspalathin (0.02 – 1.2%) [20 ], while the “unfermented product”, green rooibos, has an aspalathin content between
2.5 and 4.5% [25 ]. Hot water extracts prepared from fermented and green rooibos are used as food and/or
cosmetic ingredients. If a high aspalathin content is required, a hot water extract
(aspalathin content > 8%) of green rooibos [25 ] is preferable. On the other hand, a hot water extract (aspalathin content < 2%)
of fermented rooibos [26 ] is more economical to produce and it is preferred in food products, such as rooibos
iced tea, when the flavour is important [27 ]. Similarly, green rooibos infusions at “cup-of-tea” strength have a much higher
aspalathin content (78 – 251 mg/L) [28 ] than fermented rooibos infusions (not detected-16 mg/L) [29 ]. The bulk of rooibos production is processed to supply the demand for the traditional
“fermented” herbal tea product [22 ].
Table 2 Aspalathin content and variation in various types of plant material (PM), extracts,
and infusions.
Sample type
N
Range (mean)
Units
Reference
Dried leaves
54
6.0 – 13.5 (9.7)
g/100 g PM
[21 ]
Stems
6
0.16 – 0.78 (0.33)
g/100 g PM
[Fig. 3 ]
Whole dried shoots
97
3.8 – 9.7 (6.6)
g/100 g PM
[20 ]
Green product
47
2.5 – 4.5 (3.6)
g/100 g PM
[25 ]
Fermented product
89
0.02 – 1.2 (0.3)
g/100 g PM
[20 ]
Green hot water extract
47
8.1 – 12.3 (10.5)
g/100 g extract
[25 ]
Fermented hot water extract
74
0.16 – 1.52 (0.58)
g/100 g extract
[26 ]
Green infusion
10
78 – 251 (158)
mg/L
[28 ]
Fermented infusion
114
nd-15.7 (5.8)
mg/L
[29 ]
Fig. 2 Normal distribution plots depicting the distribution of aspalathin content (g/100 g
PM) of leaves from 21 individual A. linearis plants harvested on the same date from the same plantation (unpublished data; HPLC
analysis as described in De Beer et al. [30 ]).
Fig. 3 Aspalathin content (g/100 g PM) of leaves and stems of six individual A. linearis plants (unpublished data; HPLC analysis as described in De Beer et al. [30 ]).
Quantification of aspalathin
Several HPLC methods, based on UV-Vis detection, have been published through the years
since the first HPLC method was developed to quantify the change in aspalathin content
of the plant material with fermentation [31 ]. The need to quantify the phenolic content of a cup of fermented rooibos tea and
extracts used in biological studies resulted in the development of a variety of methods
[32 ], [33 ], [34 ], [35 ], [36 ]. The method developed by Beelders et al. [34 ] to characterise the phenolic content of infusions of fermented rooibos could, besides
aspalathin, quantify the content of the dihydrochalcone nothofagin, the phenolic acid
ferulic acid, and (Z )-2-(β -D-glucopyranosyloxy)-3-phenylpropenoic acid, an enolic glucoside of phenylpyruvic
acid, as well as the major flavones and flavonols. Quantitative data for rutin and
quercetin-3-O -robinobioside indicate that these compounds most likely co-eluted when using previous
methods. Comprehensive analysis of the phenolic composition of fermented rooibos are
problematic since a number of critical peak pairs need to be resolved. The most recent
method was shown to be suitable for quantification of the flavanone oxidation products
of aspalathin [37 ]. A need to screen large numbers of green rooibos samples led to the development
of a rapid HPLC method to quantify aspalathin, nothofagin, isoorientin, and orientin
[30 ]. Kazuno et al. [38 ] used a triple quadrupole MS detector in a selected reaction monitoring mode to quantify
a number of rooibos phenolic compounds, including aspalathin. The use of mass spectrometric
detection increases the sensitivity and specificity of the method. The suitability
of capillary zone electrophoresis for quantification of aspalathin and other phenolic
compounds in rooibos was investigated by Arries et al. [39 ]. It was deemed less sensitive and a smaller number of compounds could be quantified
with suitable reproducibility, but has potential as a rapid, inexpensive alternative
method, especially for quantification of the major compounds.
Physical and chemical properties
Aspalathin (PubChem CID: 11282394; Chemspider ID: 9457391) is a natural C -glucosyl dihydrochalcone (3′-β -D-glucopyranosyl-2′,3,4,4′,6′-pentahydroxydihydrochalcone) with the molecular formula
C21 H24 O11 (MW 452.412 g/mol) ([Table 1 ]) [7 ], [8 ]. The compound was first described by Koeppen [40 ], [41 ], originally designated “compound J”, and tentatively identified as a flavanone based
on its chromatographic properties, chromogenic reactions with a variety of reagents,
UV-Vis and infrared spectra, spectral shifts, and hydrolysis products. Aspalathin
was eventually identified as a dihydrochalcone based on its oxidation products [9 ] and NMR data acquired at 60 MHz [42 ]. Renewed interest in the phenolic composition of rooibos more than 30 years after
the first isolation of aspalathin resulted in full elucidation of its structure via
1 H NMR data acquired at 300 MHz [43 ].
Aspalathin is readily soluble in water and polar solvents, but insoluble in nonpolar
solvents. Other physical properties, relating specifically to bioavailability, are
discussed later. It is highly susceptible to oxidation in the presence of oxygen in
solution [30 ], [42 ], [44 ], [45 ], [46 ], [47 ] and rooibos plant material [31 ]. In the latter matrix, enzymes catalyse the reaction (unpublished data). Bruising
of the fresh leaves results in rapid browning. The oxidation products identified in
solution are shown in [Fig. 4 ]. Oxidation commences by cyclisation of aspalathin (1 ) to form the diastereomeric flavanone mixture (S )- and (R )-6-β -D-glucopyranosyleriodictyol (2 and 3 ) as major products and the diastereomeric mixture (S )- and (R )-8-β -D-glucopyranosyleriodictyol (4 and 5 ) as minor products. The flavanones 2 and 3 are oxidised to form the flavone isoorientin (6 ). The latter compound is susceptible to a Wessely-Moser-type rearrangement, i.e.,
hydrolysis of the enolic ether functionality and subsequent recyclisation of a 1,3-diketo
intermediate via the alternative o -hydroxy group, to form orientin (7 ) irreversibly, although the major oxidation products are unidentified brown material.
The flavanones 4 and 5 are not oxidised directly to orientin (7 ), but these compounds are postulated to be reversibly transformed into the thermodynamically
more stable flavanones 2 and 3 via the intermediate quinone methide. Further oxidation products of aspalathin include
8 and 9 atropo-diastereomeric phenols formed via phenol oxidative A- to B-ring coupling.
These dimers can undergo a second phenol oxidative coupling to form the dimer 10 . The 9H -fluorene 11 is postulated to form via a two-step oxidation process. The dimers are also susceptible
to further oxidation into unidentified brown products. A study to investigate the
formation of the brown colour upon oxidation identified aspalathin as the most important
substrate for the formation of coloured products [47 ].
Fig. 4 Oxidation of aspalathin in the presence of oxygen. [1 , aspalathin; 2 , (S )-6-β -D-glucopyranosyleriodictyol; 3 , (R )-6-β -D-glucopyranosyleriodictyol; 4 , (S )-8-β -D-glucopyranosyleriodictyol; 5 , (R )-8-β -D-glucopyranosyleriodictyol; 6 , isoorientin; 7 , orientin; 8, 9, 10, 11 , aspalathin dimers; R = β -D-glucopyranosyl]. Compiled from previous reports [45 ], [46 ], [47 ].
Degradation of aspalathin is pH dependent ([Fig. 5 ]). Aspalathin stability is highest at a low pH and substantial degradation (47%)
occurs over 24 h at pH 7 in a citric acid-phosphate buffer at room temperature ([Fig. 5 A ]) [30 ]. Other factors such as buffer type, ascorbic acid, and environment also have an
effect ([Fig. 5 B, C ]; unpublished results). A phosphate buffer (pH 7.4) without citric acid showed up
to 97% degradation over 24 h at room temperature ([Fig. 5 B ]), indicating that citric acid has a protective effect. The metal chelating properties
of citric acid may be a contributing factor. Ascorbic acid, on the other hand, was
able to completely prevent aspalathin degradation in a phosphate buffer (pH 7.4) over
24 h at room temperature ([Fig. 5 B ]). For a study on the permeability of aspalathin in a Caco-2 cell model [10 ], the stability of aspalathin as a pure compound and when present in an aspalathin-rich
green rooibos extract was determined under cell culture conditions (pH 7.4) over 2 h
in HBSS ([Fig. 5 C ]). Under these conditions, 14 and 27% degradation of the compound occurred when pure
aspalathin and aspalathin-rich extract, respectively, were tested. At pH 6 under cell
culture conditions, the aspalathin concentration slightly increased, which was attributed
to evaporation of the medium.
Fig. 5 Effect of pH on stability of pure aspalathin (ASP) and when present in green rooibos
extract. Conditions were as follows: A citric acid-phosphate buffer at room temperature (adapted from De Beer et al. [30 ]); B phosphate buffered saline at room temperature (AA = 1% ascorbic acid) (unpublished
data; HPLC analysis as described in Joubert et al. [48 ]); C HBSS under cell culture conditions (37 °C, 5% CO2 , 95% humidity) (unpublished data; HPLC analysis as described in Bowles et al. [10 ]).
Antioxidant and pro-oxidant properties
Research interest in natural phenolic antioxidants began to escalate from 1995 as
evidenced by the increase in the number of papers published [49 ]. Prior to this date, Japanese interest in rooibos produced a few papers relating
to the free radical scavenging ability of fermented rooibos (as reviewed by Joubert
et al. [50 ]). The latter research led to the association of rooibos with “anti-ageing” properties
[22 ]. These findings, together with the demonstration of high aspalathin levels in the
“unfermented” plant material and the susceptibility of aspalathin to oxidation [31 ], prompted research into the antioxidant properties of aspalathin. This research
focussed at first on aspalathin as a potential replacement for synthetic antioxidants
in food, but later the focus shifted to its potential role as an exogenous antioxidant,
due to the hypothesis that phenolic antioxidants in the diet could assist in maintaining
redox homeostasis in the cell gaining traction [51 ].
The in vitro antioxidant activity of aspalathin ([Table 3 ]) has been assessed in a variety of assays including radical scavenging and lipid
peroxidation assays [52 ], [53 ], [54 ], [55 ], [56 ]. Aspalathin generally had lower antioxidant activity than the well-known radical
scavenger quercetin [52 ], [54 ], [55 ], [57 ], although similar activity was reported in the ABTS radical cation [54 ], [56 ], Fremyʼs radical [56 ] and superoxide radical anion [52 ] scavenging assays. Aspalathin also showed pro-oxidant activity in the deoxyribose
assay [53 ]. In this assay, hydroxy radicals are generated in a Fenton reaction model system
containing FeCl3 -EDTA and H2 O2 , where aspalathin was able to reduce Fe3+ to Fe2+ , thereby increasing formation of hydroxy radicals. In this system, potent antioxidants
act as potent pro-oxidants. In a recent paper, the dual antioxidant and/or pro-oxidant
role of rooibos polyphenol constituents, in particular aspalathin, was postulated
to play a role in the prevention of UVB-induced skin carcinogenesis [58 ]. With regard to lipid peroxidation, the activity of aspalathin was compared to that
of isoquercitrin, a quercetin glucoside, in the Rancimat and LDL oxidation assays.
Aspalathin showed higher and lower inhibitory activity than isoquercitrin in these
assays, respectively. In the β -carotene bleaching and Fe(II)-induced microsomal lipid peroxidation assays, aspalathin
showed lower activity than quercetin [54 ], [55 ]. The Fe(II)-induced microsomal lipid peroxidation assay was chosen as a model for
oxidation in a membrane system. For better comprehension of its interaction with the
membrane to protect against oxidation, in particular, in comparison to its 3-deoxy
and co-occurring analogue, nothofagin, the minimum energy conformations of these dihydrochalcones
were considered to explain their relative affinity to interact with the membrane [54 ]. According to their most likely conformers, aspalathin would have a more “open”
structure, postulated to improve accessibility of its catechol group to interact with
the polar heads of the lipid bilayers for radical scavenging at the membrane interphase
[54 ]. Aspalathin shares the 2′,6′-dihydroxyacetophenone antioxidant pharmacophore with
phloretin [59 ]. The oxidation products of aspalathin, namely (S )- and (R )-6-β -D-glucopyranosyleriodictyol, isoorientin and orientin, generally had lower antioxidant
activity than aspalathin [52 ], [54 ], [56 ]. The two exceptions are that aspalathin and orientin showed similar DPPH radical
scavenging activity [52 ], while isoorientin and orientin showed higher activity than aspalathin in the ORAC
assay [57 ].
Table 3 Summary of in vitro antioxidant and pro-oxidant activity of aspalathin.
Assay
Antioxidant mechanism
Antioxidant measure
Relative activity
Reference
DPPH radical scavenging
Synthetic radical scavenging
% Inhibition
Higher activity than BHT, BHA, α -tocopherol, luteolin, vitexin, rutin, and phenolic acids
[55 ]
DPPH radical scavenging
Synthetic radical scavenging
% Inhibition
Higher activity than isoorientin, catechin, epicatechin, rutin, Trolox, vitexin, and
chrysoeriol
[52 ]
ABTS radical cation scavenging
Synthetic radical scavenging
IC50
Higher activity than Trolox, catechin, orientin, isoorientin, luteolin, chrysoeriol,
rutin, isoquercitrin, hyperoside, nothofagin, vitexin, and isovitexin
[54 ]
ABTS radical cation scavenging
Synthetic radical scavenging
TEAC
Higher activity than nothofagin, isoorientin, orientin, isovitexin, vitexin, (S )- and (R )-6-β -D-glucopyranosyleriodictyol, rutin, isoquercitrin, and hyperoside
[56 ]
Fremyʼs radical scavenging
Synthetic radical scavenging
Mole radicals scavenged per mole compound
Higher activity than nothofagin, isoorientin, orientin, isovitexin, vitexin, (S )- and (R )-6-β -D-glucopyranosyleriodictyol, rutin, isoquercitrin, and hyperoside
[56 ]
Superoxide radical scavenging
Oxygen radical scavenging
% Inhibition
Higher activity than orientin, luteolin, isoquercitrin, isoorientin, catechin, epicatechin,
rutin, Trolox, vitexin, and chrysoeriol
[52 ]
ORAC with fluorescein
Peroxyl radical scavenging
ORAC index
Higher activity than nothofagin
[57 ]
ORAC with pyrogallol red
Peroxyl radical scavenging
ORAC index
Higher activity than nothofagin, orientin, isoorientin, vitexin, isoquercitrin, and
hyperoside
[57 ]
Deoxyribose degradation in Fenton system
Hydroxyl radical scavenging/
TBARS vs. control
Pro-oxidant activity in the absence of ascorbic acid
[53 ]
Rancimat lard oxidation
Lipid peroxidation
Induction period
Higher activity than p -hydroxybenzoic acid, ferulic acid, and p -coumaric acidα -tocopherol, catechin, luteolin, rutin, isoquercitrin, caffeic acid, and protocatechuic
acid
[55 ]
Coupled β -carotene bleaching and linoleic acid oxidation
Lipid peroxidation
AAC after 120 min reaction time
Higher activity than catechin, vitexin, rutin, isoquercitrin, and phenolic acidsα -tocopherol, and luteolin
[55 ]
LDL oxidation
Lipid peroxidation
Lag time
Higher activity than isoorientin, orientin, nothofagin, and (S )-6-β -D-glucopyranosyleriodictyol
[56 ]
Fe(II)-induced microsomal lipid peroxidation
Lipid peroxidation
IC50
Higher activity than Trolox, orientin, isoorientin, luteolin, chrysoeriol, rutin,
isoquercitrin, hyperoside, nothofagin, vitexin, and isovitexin
[54 ]
Chemical and potential biocatalytic syntheses
The emphasis to obtain a process for chemical synthesis of aspalathin stems from its
relatively low content in the bulk of rooibos produced (i.e., fermented rooibos tea),
the cost and challenge of isolation from the plant material, and the limited supply
of plant material, especially green rooibos. Synthesis of aspalathin would represent
a sustainable option if economically feasible. An eight-step process using tri-O -benzylglucal, tri-O -benzylphloroglucinol, and 3,4-bis(benzyloxy)phenylacetylene as starting materials
was reported by Yepremyan et al. [60 ]. This process involved a stereoselective Lewis acid-promoted coupling of 1,2-di-O -acyl-3,4,6-tri-O -benzylglucose with tri-O -benzylphloroglucinol, leading to the corresponding β -D-glucopyranosylphloroglucinol derivative that was subsequently transformed to aspalathin.
Nothofagin was also synthesised in a similar manner. Since the aforementioned synthetic
protocol resulted in a low yield (20%), an alternative sequence was developed by Van
der Westhuizen and coworkers ([Fig. 6 ]) [61 ]. Glucosylation of the di-O -benzylacetophenone 14 with the α -fluoroglucosyl derivative 13 under Lewis acid-catalysis afforded the α -glucopyranosyloxy analogue 15 in an 86% yield. Increasing the temperature from −40 to −15 °C permitted a facile
BF3 -catalysed rearrangement to the β -glucopyranosyl derivative 16 in an 84% yield. Aldol condensation of 16 with 3,4-dibenzyloxybenzaldehyde 17 afforded the protected chalcone 18 (96% yield), which upon hydrogenation gave aspalathin in an 80% overall yield. Based
on the excellent overall yield and its scalability potential, the process was subsequently
patented [62 ]. Recently, a number of C -glycosyl chalcone analogues of aspalathin were synthesised by a simple three-step
process [63 ]. These analogues displayed good DPPH radical scavenging activity and inhibited proliferation
of liver and breast cancer cells.
Fig. 6 Four-step synthesis of aspalathin. Adapted from Han et al. [61 ].
Another possibility for the synthesis of aspalathin may be the biotransformation by
enzymatic C -glycosylation of the aglycone, as was achieved for nothofagin [64 ], [65 ]. A C -glycosyltransferase from rice, which was perfectly selective, was used to glucosylate
phloretin using uridine diphosphate-glucose, giving an 80% yield of nothofagin [64 ]. In further work, the same group increased the efficiency of conversion by complexing
phloretin with β -cyclodextrin to improve its solubility [65 ].
De novo synthesis of nothofagin and a number of other related dihydrochalcones was recently
accomplished using metabolically engineered Saccharomyces cerevisiae
[66 ]. This was done by expressing the full-length biosynthetic pathways, consisting of
between four and nine genes, in the microbial host. Although the authors note that
the process is not yet economically viable, metabolically engineered microbes have
great potential for targeted sustainable production of high-value phenolic compounds
such as aspalathin.
Bioavailability
Aspalathin, normally ingested by drinking rooibos or consuming food supplemented with
rooibos extract, requires absorption through the gut into the blood stream to reach
the systemic site of action in adequate concentrations to be bioefficient, apart from
its beneficial gastrointestinal luminal effects. Considering that various physical
properties of aspalathin ([Table 2 ]) related to membrane permeability fail to meet various criteria, limited absorption
is to be expected. Whilst its molecular weight and log P value do not exceed the limits
of 500 and 5, respectively, the number of H-bond donors and acceptors violate Lipinskiʼs
“Rule of 5′′ [67 ]. The hydrophilic nature of aspalathin not only indicates poor permeability for gastrointestinal
penetration, but also susceptibility to renal clearance [68 ]. Additionally, the polar surface area of aspalathin exceeds 140 Å2 , indicating a high probability of poor oral bioavailability [69 ].
Low membrane permeability of aspalathin has been reported in Caco-2 monolayer cell
model studies. Relevant details of these studies are summarised in [Table 4 ]. Notably, Huang et al. [11 ] found that aspalathin absorption increased when present in green rooibos extract
as opposed to the pure compound, indicating that other plant components present in
the extract may assist in its transport across the membrane. In contrast, Bowles et
al. [10 ] found absorption of pure aspalathin to be similar to that when present in green
rooibos extract. Experimental differences may account for this disparity in results,
in particular the substantially higher aspalathin concentration, as well as the higher
permeability of the monolayer used by Huang et al. [11 ]. Courts and Williamson [70 ] postulated that the passive diffusion of aspalathin is most likely responsible for
its absorption across the intestinal epithelial monolayer. Insight into the mechanism
of aspalathin transport, using the Caco-2 monolayer cell model, was gained from the
study by Bowles et al. [10 ]. Following inhibition of aspalathin transport in the presence of a high glucose
concentration (20.5 mM), the role of active glucose transporters such as SGLT1 and
GLUT2 was investigated by performing experiments in the presence and absence of SGLT1
(phloridzin), GLUT2 (phloretin), and efflux (Pg) (verapamil) inhibitors. No effect
on aspalathin transport was observed, leading to the conclusion that aspalathin is
transported paracellularly, especially given its physical characteristics. Transport
of aspalathin across the intestinal epithelial monolayer occurred without evidence
of deglucosylation [70 ] or formation of other metabolites [10 ].
Table 4 Rate of transport of aspalathin from buffered solutions of pure aspalathin and green
rooibos extracts across the Caco-2 monolayer from apical to basolateral side.
Treatment
Concentration1
Papp × 10−6
% Passage
Reference
1 Extract concentration in mg/mL with molar concentration of aspalathin; 2 molar concentrations > 150 µM were cytotoxic to Caco-2 cells
Green rooibos extract in DPBS with calcium and magnesium (pH 7.4)
1 mg/mL (0.43 mM)
4 ± 0.42
[11 ]
5 mg/mL (2.15 mM)
3.49 ± 1.45
10 mg/mL (4.29 mM)
20.93 ± 3.61
~ 100%
Aspalathin in DPBS with calcium and magnesium (pH 7.4)
0.2 mg/mL (0.44 mM)
0.91 ± 0.37
1 mg/mL (2.21 mM)
2.48 ± 0.03
2 mg/mL (4.42 mM)
15.34 ± 1.66
79%
Aspalathin in HBSS (pH 6.0)2
1 µM
2.28 ± 0.09
[10 ]
150 µM
1.73 ± 0.97
5%
Aspalathin in HBSS (with high glucose; 20.5 mM; pH 6.0)
150 µM
0.29 ± 0.08
2%
Green rooibos extract in HBSS (pH 6.0)
0.38 mg/mL (153 µM)
2.00 ± 1.10
Enriched green rooibos fraction in HBSS (pH 6.0)
0.15 mg/mL (149 µM)
2.11 ± 0.20
In vitro phase II metabolism of aspalathin with microsomal and cytosolic subcellular rat liver
fractions and added cofactors produced two glucuronidated and one sulphated metabolite
[71 ]. Methylation of aspalathin was demonstrated when treated with human liver and intestinal
cytosolic fractions, following the addition of the cofactor [72 ]. Conjugates of aspalathin with glucuronic acid, sulphate, a methyl group, or a combination
were detected in animal and human urine [10 ], [33 ], [36 ], [72 ], [73 ]. Deglucosylation of aspalathin is thus not a prerequisite for its absorption. Traces
of methyl conjugates were found in the plasma of Vervet monkeys after being fed a
single bolus containing green rooibos extract delivering 25 mg aspalathin per kg body
weight [28 ]. Unconjugated aspalathin was detected in human plasma after ingesting a green rooibos
beverage containing 287 mg aspalathin ([Table 5 ]). In all of the in vivo studies, except the mouse study, aspalathin was ingested as part of a rooibos extract,
either mixed into the feed of the animals or consumed as a rooibos beverage by human
subjects ([Table 5 ]). Major outcomes of these studies were that aspalathin metabolites reached maximum
concentration in the plasma in 3 h or less [33 ] and that only a small quantity of the ingested amount was bioavailable [33 ], [36 ], [72 ].
Table 5 Summary of studies investigating oral bioavailability of aspalathin (ASP).
Model
ASP dose
Dosage form
ASP and metabolites in plasma
ASP and metabolites in urine
Excretion in urine
Refer-ence
Mouse
50 mg/kg BW, single dose
Pure ASP in PBS; orogastric gavage
nd
sulphated ASP (4); glucuronidated ASP (2); methylated ASP (2), methylated and glucoronidated
ASP (2), methylated and sulphated ASP; methylated and glucuronidated ASP aglycone;
a C -glucopyranosyl eriodictyol
nq
[10 ]
Pig
157 – 167 mg/kg BW daily for 11 days
ASP-rich GR extract (16.3%), mixed with feed
nd
ASP; glucuronidated ASP; methylated ASP; methylated and glucuronidated ASP; glucuronidated
ASP aglycone
0.1 to 0.9%
[73 ]
Vervet monkey
25 mg/kg BW, single dose
ASP-rich GR extract (18.4%) mixed with bolus
methylated ASP (2)
Methylated ASP; dimethylated ASP
nq
Unpub-lished
Human
91 mg/subject, single dose
300 mL of GR infusion
nd
Methylated ASP; methylated and glucuronidated ASP
Max. conc. reached < 2 h after ingestion; 0.74% excreted during 0 – 24 h
[72 ]
Human
41 mg/subject, single dose
500 mL GR “ready-to-drink” beverage
nd
Glucuronidated ASP (2); methylated and glucuronidated ASP (3); methylated and sulphated
ASP; sulphated ASP
Most excreted < 5 h after ingestion; 0.22% excreted during 0 – 24 h
[36 ]
Human
3.6 mg/subject, single dose
500 mL fermented rooibos “ready-to-drink” beverage
nd
methylated and glucuronidated ASP (3); methylated and sulphated ASP; sulphated ASP
0.09% excreted during 0 – 24 h
[36 ]
Human
287 mg/subject, single dose
GR beverage
ASP
ASP; glucuronidated ASP; methylated ASP; methylated and glucuronidated ASP (3); methylated
and sulphated ASP; sulphated ASP; glucuronidated ASP aglycone
0.2% excreted during 0 – 24 h
[33 ]
Of interest is the presence of the conjugated aspalathin aglycone, observed in mouse
[10 ] and human [33 ] urine. The resistance of the C-C bond to the action of lactase phloridzin hydrolase,
present in the brush-border of the small intestinal epithelial cells, as well as the
action of cytosolic β -glucosidase [74 ], requires liberation of the aglycone by colonic microflora. Human colonic bacteria
able to hydrolyse the C-C bond have been identified for a number of C -glucosyl compounds [75 ], [76 ], [77 ]. Eubacterium cellulosolvens , isolated from mice, are able to deglucosylate orientin and isoorientin [78 ]. Using oxidation products of aspalathin as a starting point and outcomes of studies
on the anaerobic catabolism of these flavones, Muller et al. [28 ] proposed a microbial biotransformation pathway for aspalathin, ultimately leading
to the formation of dihydrocaffeic acid and organic acids in the colon.
Rising burden of metabolic syndrome and preventative potential of aspalathin
The metabolic syndrome describes a cluster of metabolic anomalies that underlie the
development of serious metabolic disease such as type 2 diabetes, obesity, and cardiovascular
disease, contributing to the rising burden of noncommunicable diseases [79 ]. Perturbations of glucose and lipid metabolism as a result of insulin resistance
play a major role in the development of the metabolic syndrome. Individuals with this
condition exhibit multiple risk factors including elevated fasting plasma glucose,
high serum triglycerides, and high blood pressure, which increase the probability
of developing cardiovascular complications [80 ]. Urbanisation with accompanying lifestyle changes, namely, excessive energy intake
and lack of physical activity, contributes to these metabolic diseases [79 ]. Current global estimates show that more than 2 billion children and adults are
overweight or obese, whereas a total of 107.7 million children and 603.7 million adults
were recorded to be obese in 2015 [81 ]. Furthermore, the International Diabetes Federation has reported that 415 million
adults have diabetes at present and this number is expected to rise to 642 million
by 2040 [82 ]. Cardiovascular diseases, major comorbidities of type 2 diabetes, greatly contribute
to global mortality [79 ]. In addition to cardiovascular diseases, the metabolic syndrome increases the risk
of organ damage to the liver (nonalcoholic fatty liver disease), muscle (muscle deterioration),
and pancreas (pancreatic β -cell dysfunction) [83 ], [84 ]. The generation of excessive oxidative stress, a consequence of depleted antioxidant
systems due to the overproduction of ROS, and accelerated inflammation driven by elevated
proinflammatory cytokine production, are both implicated in metabolic syndrome-induced
organ damage [85 ]. Nevertheless, there is an increase in evidence that dietary interventions may reduce
oxidative stress and inflammation associated with the metabolic syndrome, thereby
decreasing cardiovascular risk [86 ], [87 ]. For example, antioxidant transcriptional factors such as Nrf2, as well as its associated
downstream target genes including those coding for GSH, have emerged as essential
targets in the amelioration of oxidative stress-induced cardiovascular and liver damage
[88 ], [89 ], [90 ]. Similarly, effective modulation of protein kinases such as JNK, IKK, ERKs, and
AMPK by certain natural products has been correlated with a reduced inflammatory response
and cell damage [91 ], [92 ], [93 ]. Evidence demonstrating the ameliorative potential of aspalathin against the metabolic
syndrome and its associated complications is attracting a lot of interest. The following
sections provide a brief overview of the metabolic processes involved in altered glucose
and lipid metabolism, leading to exacerbated oxidative stress and inflammatory-induced
cell damage. A separate section deals specifically with the protective potential of
aspalathin and its possible interference with the metabolism of hypoglycaemic and
hypocholesterolaemic drugs.
Metabolic syndrome and glucose metabolism
Disturbances in glucose metabolism remain central to the aetiology of metabolic syndrome.
In both the fed and fasted state, glucose-induced insulin secretion is tightly controlled,
and disturbances in insulin action cause an imbalance between glucose uptake and consumption
[94 ]. In the liver, insulin action promotes lipogenesis and glycogen synthesis, while
lipolysis and glycogenolysis are inhibited [94 ]. Peripheral glucose absorption and usage are stimulated by insulin in adipose and
muscle tissue. Insulin resistance dysregulates these processes, resulting in increased
blood glucose levels with associated lipid accumulation [95 ].
Insulin action is mediated through the phosphorylation of various signalling cascades,
culminating in glucose uptake into the cell. Simply put, activation of PI3K/AKT by
IRS-1 phosphorylates GSK3-β and initiates GLUT4 translocation from the cytosol to the cell membrane [96 ]. Both PI3K and AKT are crucial kinases that regulate GLUT4 translocation and have
subsequently gathered significant interest as possible drug targets for treating the
metabolic syndrome [94 ], [96 ]. Conversely, in an obese or type 2 diabetic state, elevated DAG and ceramide levels
can activate PKC, leading to a dysregulated IRS/PI3K/AKT cascade that suppresses GLUT4-mediated
glucose uptake in muscle and adipose tissues, as is observed in AKT knockout mice
[97 ].
Although the PI3K/AKT signalling pathway is essential in the regulation of insulin-stimulated
glucose uptake, glucose can also be regulated by phosphorylated AMPK in an insulin-independent
manner [92 ]. AMPK is a major energy sensor that plays a critical role in cellular homeostasis
and has been widely studied as a possible target to improve insulin resistance [98 ]. Through its maintenance of energy consumption, AMPK has contributed significantly
to the modulation of various cellular processes, including cell growth, autophagy,
and glucose metabolism [98 ]. For the latter, it is postulated that upon activation, AMPK promotes GLUT4 translocation
to the cell membrane, resulting in increased glucose uptake in skeletal muscle and
adipose tissue [99 ], [100 ]. Metformin, a biguanide class of oral antidiabetic, mediates its antidiabetic actions
by indirectly activating AMPK [101 ]. However, contradicting evidence also shows that activation of AMPK in a diabetic
heart can contribute to the inhibition of glucose utilisation, while abnormally enhancing
fatty acid oxidation that can lead to accelerated myocardial apoptosis [102 ], [103 ], [104 ]. Thus, in addition to the modulation of PI3K/AKT signalling, optimal regulation
of AMPK remains essential in promoting glucose uptake, reversing insulin resistance,
and attenuating metabolic disease-associated complications such as oxidative stress
and inflammation [98 ], [103 ].
Metabolic syndrome and lipid metabolism
Lipid metabolism can be defined as the synthesis or break down of fats for energy,
a process that plays a major role in normal body function, as well as the aetiology
of metabolic syndrome [105 ]. Fatty acids are major components of triglycerides and can either be absorbed through
the ingestion of food or synthesised by adipocytes or hepatocytes from carbohydrate
precursors such as acetyl-CoA [105 ]. This process begins in the intestine where triglycerides are degraded by pancreatic
lipases and bile salts into FFAs, where they, together with cholesterol molecules,
are packed into chylomicrons, which are transported within the lymphatic and circulatory
system to be metabolised by cells or stored in the adipose tissue [106 ]. Although adipose tissue is the largest energy reserve in mammals, fats are also
stored in muscle and liver tissues [106 ]. In muscle, fat stores are used as a substrate for fatty acid β -oxidation, whereas in the liver, fats are utilised in the synthesis of triacylglycerol
for energy [106 ]. To obtain energy from adipocytes, fats are hydrolysed into FFAs and glycerol molecules
in a process called lipolysis. These FFAs are further oxidised during mitochondrial
fatty acid β -oxidation to produce acetyl-CoA, which is a major substrate required in the Krebs
cycle for the production of energy in the form of ATP [105 ].
Fatty acid β -oxidation produces twice the amount of energy compared to carbohydrate or glucose
metabolism and its dysregulation is implicated in the aetiology of the metabolic syndrome
[107 ]. Altered β -oxidation in the muscle and liver is associated with diabesity (co-occurrence of
type 2 diabetes and obesity) and an increased cardiovascular risk [108 ]. During the progression of obesity, increased lipogenesis and diminished β -oxidation account for augmented hepatic expression of lipogenic genes including SCD1,
PPARγ , and SREBP1/2. In muscle, increased levels of long-chain FFA acyl-CoAs together with
their lipid intermediate metabolites, DAG and ceramide, impede insulin action through
activation of PKC [109 ]. Activation of PKC initiates an increased inflammatory response that augments muscle
insulin resistance via activation of IRS (serine 307) (IRSSer307 ). As previously explained, this process attenuates peripheral glucose uptake and
translocation of GLUT4. Nevertheless, enhanced oxidative stress appears to be one
of the devastating factors associated with accelerated tissue injury as a result of
impaired glucose and lipid metabolism.
Metabolic syndrome and oxidative stress
It is generally accepted that oxidative stress plays a major role in the development
and exacerbation of the metabolic syndrome. The role of persistent exposure to high
glucose (chronic hyperglycaemia), linked to the increased production of ROS within
the disease state, is well established [110 ]. In diabetes, raised blood glucose levels induce various signalling pathways associated
with the aggravation of oxidative stress. For example, activation of the sorbitol-aldose
reductase pathway, a key process in the control of excess blood glucose, can cause
a decrease in the reduced form of NADP, i.e., NADPH. It is well known that the NADPH
cofactor plays a major role in the synthesis of GSH, an essential antioxidant that
detoxifies intracellular ROS [111 ]. Moreover, intracellular reduction of NADPH and GSH may promote the production of
nitric oxide as well as chain activation of other free radicals leading to accelerated
oxidative damage [112 ].
Some of the well-known sources responsible for exacerbated generation of oxidative
stress in many cell types include glucose autoxidation (normally identified with elevated
glucose levels in a diabetic state), abnormally enhanced activity of NADPH and xanthine
oxidases, and the overactivity of the mitochondrial electron transport chain [113 ], [114 ], [115 ]. Lipid peroxidation, which may arise as a result of oxidative degradation of lipids
by increased free radical production, is a widely-reported phenomenon identified in
obese individuals [116 ], [117 ]. Avci et al. [117 ] recently showed that enhanced lipid peroxidation inversely correlates with GSH content
in individuals with metabolic syndrome. This was supported by others demonstrating
that systemic markers of lipid peroxidation such as oxidised LDL and TBARS are elevated
in individuals with metabolic syndrome compared to control subjects [118 ], [119 ]. Such findings have also been supported by data from animal models of metabolic
syndrome with increased liver dysfunction, muscle insulin resistance, and subsequent
cardiomyocyte remodelling and apoptosis [83 ], [84 ], [88 ].
Metabolic syndrome and inflammation
Another devastating consequence interrelated with oxidative stress that normally arises
due to prolonged exposure to elevated blood glucose (hyperglycaemia) and circulating
lipids (hyperlipidaemia) is the activation of inflammation. Although an inflammatory
response is necessary for debridement after injury, tenacious inflammation is thought
to exacerbate cell damage under various disease conditions [85 ]. In an obese state, a proinflammatory response results in the infiltration of macrophages
into peripheral tissues, including adipose tissue and the liver as well as skeletal
and heart muscle [120 ], [121 ]. In adipose tissue, the macrophage infiltration stems from excessive lipid overload
of the various adipose tissue deposits, caused by adipocyte hypertrophy and hyperplasia,
culminating in ischaemic tissue congestion and dysfunction [122 ]. In the liver, the nutrient overload causes nonalcoholic fatty liver disease [123 ]. Furthermore, increased lipid stores, particularly visceral adiposity, promote a
low-grade systemic proinflammatory response, activating M1 macrophages (Th1 response)
to secrete elevated levels of MCP1, IL-6, IL-1β , TNF-α , and leptin, while repressing adiponectin levels [124 ], [125 ]. Adiponectin is an important adipokine that is secreted by adipocytes and is known
to play a significant role in obesity-induced insulin resistance. Numerous animal
studies have shown that increased adiponectin levels are inversely proportional to
the concentration of ceramides, an important factor in the modulation of obesity-induced
insulin resistance [126 ], [127 ]. Additional evidence shows that adiponectin levels are negatively correlated with
increased TNF-α levels [121 ], [124 ], [126 ]. Likewise, growing evidence suggests that decreased adiponectin levels with concomitant
increased TNF-α levels not only perpetuate obesity-induced insulin resistance, but are key cytokines
that aggravate the metabolic syndrome [127 ], [128 ]. Increased adiponectin levels are associated with diminished systemic inflammation
and lipid accumulation, leading to reduced vascular dysfunction that is linked to
the metabolic syndrome [129 ]. Indeed, reduced adiponectin levels negatively correlate with the degree of adiposity
and inflammation as indicated by decreased IL-6 and TNF-α production, both key mediators of diabesity-induced cardiac dysfunction [130 ], [131 ]. Thus, understanding the process that regulates TNF-α -induced inflammation and subsequent insulin resistance is key to unravelling possible
drug targets for therapeutic intervention.
According to Kwon and Pessin [124 ], TNF-α can induce insulin resistance through two possible mechanisms, i.e., the initiation
and propagation of lipolysis or by directly blunting insulin signalling. In the obese
state, TNF-α -induced lipolysis leads to an escalation in the availability of circulating FFAs,
and this exacerbates insulin resistance and ectopic fat accumulation in organs such
as the liver and muscle [132 ]. In the liver, this enhanced FFA delivery together with an increase in endoplasmic
reticulum stress can lead to the activation of several kinases, including JNK and
IKK/NF-κ B, which further contribute to the impairment of insulin resistance through the phosphorylation
of IRSSer307
[120 ], [133 ]. In addition, excessive de novo hepatic lipogenesis causes an increase in FFA flux, which inhibits β -oxidation through increased expression of long-chain acyl-CoA. This in turn augments
the hepatic triglyceride pool, which is exacerbated through an alternative mechanism
that implicates the upregulated expression of SREBP-1c [134 ], [135 ].
The escalation of lipid accumulation further triggers increased dyslipidaemia, in
the form of VLDL and LDL concomitant to raised proinflammatory markers such as macrophages
and leukocytes within the vascular wall [124 ], [136 ]. This process, together with elevated TNF-α levels increase the expression of CAM, P-selectin, and E-selectin, to the vascular
endothelium, resulting in an acute localised cellular inflammatory response that triggers
plaque development and cardiovascular dysfunction [121 ], [123 ], [137 ]. Similarly, in skeletal muscle of obese insulin-resistant individuals, TNF-α enhances the expression of proinflammatory cytokines in response to increased lipid
storage with a concomitant reduction of β -oxidation, leading to abnormally elevated ceramide levels that augment muscle insulin
resistance through PKC activation [94 ].
Protective Potential of Aspalathin against Metabolic Disease-Associated Complications
Effect of aspalathin on glucose and lipid metabolism
A growing body of evidence demonstrates that dietary supplements such as polyphenols,
displaying antioxidant and anti-inflammatory properties, can exert beneficial effects
on essential signalling molecules involved in carbohydrate and lipid metabolism [138 ]. Rooibos and its phenolic constituents, including aspalathin, are progressively
explored for their ameliorative effects on metabolic syndrome and associated complications,
including obesity and diabetes mellitus ([Table 6 ]). For example, in type 2 diabetic (db/db ) mice and cultured pancreatic β -cells, aspalathin was shown to be effective at improving glucose tolerance and stimulating
insulin secretion, respectively [139 ]. Consistent with these findings, Muller et al. [140 ] showed that an aspalathin-rich green rooibos extract and pure aspalathin are able
to modulate glucose metabolism by inhibiting α -glucosidase enzyme activity and promoting glucose uptake in pancreatic and skeletal
muscle cells as well as improving glucose tolerance in streptozotocin-induced diabetic
Wistar rats. Mikami et al. [141 ] demonstrated that green rooibos extract and aspalathin were effective at reducing
blood glucose levels of nondiabetic mice following ingestion of glucose, sucrose,
and starch solutions, while suppressing the activities of α -glucosidase and α -amylase, key enzymes involved in carbohydrate hydrolysis. Mazibuko et al. [92 ], [142 ] further investigated the molecular mechanisms associated with the ameliorative effect
of pure aspalathin and an aspalathin-rich green rooibos extract on palmitate-induced
insulin resistance in C2C12 myotubes and 3T3-L1 adipocytes. Both the extract and compound
were able to reverse palmitate-induced insulin resistance by increasing levels of
GLUT4 through the suppression of PKC and NF-κ B, while activating AMPK. Son et al. [143 ] supported these findings showing that aspalathin treatment improved glucose tolerance
in obese (ob/ob ) mice, while similarly enhancing glucose uptake by promoting AMPK phosphorylation
and GLUT4 translocation in L6 myotubes. Smit et al. [144 ] showed that aspalathin promotes insulin sensitivity in cardiomyocytes from young
and aged rats, but not in high-caloric diet animals, through a PI3K-dependent mechanism.
Aspalathin can protect cardiomyocytes from doxorubicin-induced cardiotoxicity by increasing
autophagy, while simultaneously decreasing apoptosis [102 ], [145 ].
Table 6 Summary of studies related to the antioxidant, antidyslipidaemic, antidiabetic, and
anti-inflammatory activities of aspalathin (ASP) and aspalathin-rich green rooibos
extracts.
Experimental model
Treatment and dose
Experimental outcome
Reference
L6 Myoblasts, RIN-5F cells and type 2 diabetic (db/db ) mice
ASP: Cell culture dose (1 – 100 µM)
Improved glucose uptake, insulin secretion, and glucose tolerance.
[139 ]
Online HPLC-biochemical assay for α -glucosidase inhibitory activity, C2C12 myotubules, Chang (CCL-13) cells, and streptozotocin-induced
diabetic rats
ASP: Cell culture dose (1, 10, and 100 µM)−5 -5 µg/mL)In vivo dose (3, 25, 30, or 300 mg/kg BW)
Displayed α -glucosidase inhibitory activity, improved glucose tolerance, and dyslipidaemia.
[140 ], [146 ]
Nondiabetic ddY mice; in vitro α -glucosidase and α -amylase inhibitory assays
ASP: In vitro dose (0, 0.5, 1, 2, 4, 8 mg/mL)In vivo dose (20 mg/mL/100 g BW)In vitro dose (0, 2.5, 5, 10, 20, 40 mg/mL)In vivo dose (80 mg/mL/100 g BW)
Reduced blood glucose levels following ingestion of glucose, sucrose, and starch solutions.
Also inhibited the activities of α -glucosidase and α-amylase.
[141 ]
L6 Myotubes, RIN-5F cells and obese (ob/ob ) mice
ASP: Cell culture dose (0 – 100 µM)
Dose-dependently increased glucose uptake, enhanced GLUT4 translocation to plasma
membrane, and promoted AMPK phosphorylation in L6 myotubes. Reduced oxidative stress
in RIN-5F cells and improved fasting plasma glucose levels in mice.
[143 ]
Caenorhabditis elegans
ASP: In vitro dose (0, 10, 20, 50 µM)
Promoted longevity by targeting stress and ageing-related genes, reducing the endogenous
intracellular level of reactive oxygen species.
[152 ]
HUVECs, human blood samples, and C57BL/6 mice (ex vivo model)
ASP: Cell culture dose (1 – 50 µM)Ex vivo dose (4.5, 9.1, 18.1, 27.1, and 45.2 µg/mouse)
Demonstrated antithrombotic activities by prolonging activated partial thromboplastin
time and blocking platelet aggregation and activities of thrombin and activated factor
X.
[156 ]
HUVECs and mice
ASP: Cell culture dose (1 – 50 µM)In vivo dose (4.5, 9.1, 27.1, or 45.2 µg/mouse)
Prevented high-glucose-mediated vascular hyperpermeability, adhesion of monocytes,
and expression of cell adhesion molecules. Inhibited generation of ROS and activation
of NF-κ B or ERKs.
[157 ], [162 ], [163 ]
HUVECs
ASP: Cell culture dose (1 – 50 µM)
Ameliorated HMGB1-induced septic responses.
[159 ]
3T3-L1 Adipocytes
ASP: Cell culture dose (10 µM)
Reversed palmitate-induced insulin resistance by repressing NF-κ B, IRS1 and AMPK phosphorylation, and increased AKT activation; only the extract upregulated
GLUT4 protein expression.
[92 ]
In vitro xanthine oxidase inhibitory activity assay and mice
ASP and extract: Effective dose used (4.5 µg/mL)
Competitively inhibited xanthine oxidase. In hyperuricaemic mice, markedly suppressed
increased plasma uric acid levels in a dose-dependent manner.
[153 ]
H9c2 cells, db/db mice and cardiomyocytes from rats
ASP: Cell culture dose (1 and 1000 µM)In vivo dose (13 and 130 mg/kg BW/day)
Improved diabetes associated cardiac deregulations, including enhanced glucose uptake,
reversed impaired myocardial substrate metabolism, inhibited inflammation, lipid storage,
oxidative stress, and cardiac remodelling. Reversed doxorubicin-induced cardiotoxicity
by activating AMPK and reducing tumour protein p53 expression.
[88 ], [91 ], [102 ], [144 ], [145 ]
Rats
Extract: In vivo dose of 29.5 mg/kg BW/day
Lowered serum total cholesterol and iron levels, whilst increasing alkaline phosphatase
enzyme activity and liver GSH levels.
[147 ], [151 ]
Diabetic nonhuman primates
Extract: In vivo dose (90 mg/kg BW)
Improved glucose tolerance, reduced total cholesterol, and LDL levels. Increased plasma
coenzyme Q10 and decreased oxidative status.
[154 ]
Furthermore, both in vitro and in vivo studies have indicated that aspalathin can reduce hyperlipidaemia [91 ], [146 ]. The effect of aspalathin to diminish cholesterol, triglycerides, and VLDL and LDL
cholesterol levels, while increasing high-density lipoprotein cholesterol levels,
has been investigated in a few studies. Najafian et al. [146 ] showed that streptozotocin-induced diabetic male Wistar rats receiving 5, 10, and
40 mg/kg of aspalathin for 21 days displayed a dose-dependent decrease in lipid levels
in conjunction with reduced blood glucose concentrations. Similarly, in a study by
Johnson et al. [91 ], a 6-week treatment with aspalathin modulated lipoprotein clearance in a dose-dependent
manner in db/db mice, with a higher dose (130 mg/kg) of the compound being more effective than a
lower dose (13 mg/kg). Additional evidence by Van der Merwe et al. [147 ] demonstrated that an aspalathin-rich green rooibos extract significantly reduced
serum total cholesterol of male Fischer rats after 90 days of treatment. Collectively,
these results indicate that aspalathin may beneficially modulate glucose and lipid
metabolism, thereby ameliorating the complications associated with metabolic syndrome.
The proposed cellular mechanisms by which aspalathin improves glucose and lipid metabolism
The cellular mechanism by which aspalathin targets metabolic syndrome and improves
glucose and lipid clearance remains to be fully elucidated. However, it has been proposed
that by supressing fatty acid synthesis, aspalathin enhances CPT1 expression and subsequently
increases β -oxidation in muscle tissue [92 ]. The latter process is under the control of essential lipid metabolism genes, including
ACC, FAS, and SCD1, that play an integral role in the development of insulin resistance
in fat, liver, muscle, and heart tissue. Furthermore, PPARγ , SREBP-1/2, and ChREBP are transcriptional factors that regulate and control the
expression of these enzymes involved in the lipogenic process [148 ]. However, this process can be repressed through AMPK phosphorylation [149 ]. Aspalathin activates AMPK and reduces the expression of hepatic enzymes and transcriptional
regulators that are associated with either gluconeogenesis and/or lipogenesis [91 ], [92 ], [102 ], [139 ], [143 ], [144 ], [150 ]. These studies proved that aspalathin controlled the balance between anabolism and
catabolism through increased AMPK expression, while decreasing the expression of ACC,
FAS, SCD1 and SREBP1 [91 ], [92 ], [143 ]. For example, Johnson et al. [91 ] showed that aspalathin protected vulnerable cardiomyocytes from diabetes-induced
lipotoxicity by modulating adiponectin, ApoB, CD36, CPT1, PPARγ , SREBP1/2, and SCD1, key regulators of lipid metabolism. Collectively, these results
suggest that aspalathin acts on multiple targets associated with fatty acid synthesis
and fatty acid oxidation, resulting in improved glucose and lipid metabolism.
Effect of aspalathin on oxidative stress and inflammatory markers
In addition to the strong antioxidant properties demonstrated by aspalathin in non-cell-based
assays ([Table 3 ]), its capacity to enhance endogenous antioxidants and prevent oxidative stress was
also shown in various experimental models ([Table 6 ]). Administration of an aspalathin-enriched green rooibos extract to nondiabetic
Fischer rats for 28 days increased GSH reductase activity (an important enzyme in
the maintenance of the reduced form of GSH), however, a longer treatment period (90
days) reduced the GSH content in the liver, suggesting an altered GSH redox cycle
[147 ], [151 ]. Alternatively, Chen et al. [152 ] showed that a green rooibos extract improved the survival rate of Caenorhabditis elegans by reducing acute oxidative damage caused by the superoxide anion radical generator
juglone. In RIN-5F pancreatic β -cells [143 ], aspalathin displayed an increased potential to prevent oxidative damage by suppressing
ROS induced by advanced glycation end products. The robust antioxidant properties
of aspalathin to reduce oxidative stress could be attributed to its inhibitory effect
on xanthine oxidase, a known superoxide radical-producing enzyme [153 ]. Recently, Orlando et al. [154 ] showed that an aspalathin-rich green rooibos extract (12.8% aspalathin content)
administered at 90 mg/kg three times daily with meals to high-fat fed diabetic vervet
monkeys protected against LDL oxidation and preserved endogenous coenzyme Q10 levels.
This supports outcomes of the study by Marnewick et al. [155 ], showing that the consumption of six cups of rooibos tea improved plasma lipid and
oxidative stress levels in adults at risk for developing cardiovascular disease.
Recent studies from our laboratory have also reported on the potential of aspalathin
to protect cardiac cells against oxidative stress-associated damage ([Table 6 ]). In cardiomyocytes isolated from diabetic rats, a fermented rooibos extract (0.36%
aspalathin) prevented ROS-induced apoptosis by increasing intracellular GSH levels
[161 ]. Furthermore, in cardiomyocytes exposed to a high glucose concentration, the capacity
of aspalathin to prevent oxidative damage was associated with its modulatory effect
on glucose and lipid metabolism, specifically by reducing abnormally increased FFA
uptake and oxidation through the reduced phosphorylation of AMPK [102 ]. AMPK is a major energy regulator that plays a role in the reversal of peripheral
insulin resistance through the modulation of β -oxidation [92 ], [142 ], [143 ]. This kinase increases β -oxidation and improves peripheral insulin sensitivity through phosphorylation and
inactivation of ACC, a rate-limiting enzyme in fatty acid synthesis and subsequent
β -oxidation. However, in the diabetic heart, an abnormal increase in fatty acid oxidation,
as opposed to glucose oxidation, has been linked to reduced cardiac efficiency. In
a recent study [102 ], we showed that in the diabetic heart, aspalathin modulates AMPK hyperactivation
and improves glucose oxidation. This favourable shift in cardiac energy substrate,
in favour of glucose oxidation, is believed to be important to protect a diabetic
heart at risk of developing heart failure. The ameliorative effects of aspalathin
were confirmed in the hearts of db/db mice and cardiomyocytes exposed to high glucose concentrations [88 ], showing that an increased expression of Nrf2, an essential transcriptional factor
that is upregulated in response to oxidative stress and other stresses associated
with the metabolic syndrome [89 ], plays a partial role in the protective effect of aspalathin. This study further
showed that the upregulated expression of Nrf2 enhanced the endogenous antioxidant
systems such as GSH and superoxide dismutase, as well as UCP2, resulting in improved
cardiac ultrastructure. Although these findings need to be confirmed in other models,
the results infer that aspalathin might be a useful therapeutic against endogenous
oxidative stress and protect cardiovascular cells from diabetes-associated complications.
In addition to the ability of aspalathin to reduce oxidative stress, anti-inflammatory
properties have also been demonstrated for this dihydrochalcone. Lipopolysaccharide
is a known means of inducing vascular inflammation, both in in vitro and in vivo models. Aspalathin treatment suppressed lipopolysaccharide-induced membrane permeability
and CAM in both human endothelial cells and in mice [162 ]. Furthermore, aspalathin ablated this effect by downregulating the expression of
TNF-α , IL-6, and NF-κ B. In a follow-up study, the authors demonstrated that 10 – 30 µM of aspalathin averted
HMGB1-mediated vascular inflammation and hyperpermeability by inhibiting the expression
of CAM in both an in vitro (HUVECs) and in vivo (C57BL/6 mouse) model [158 ]. Although additional evidence is required to confirm these findings, a study by
Ku et al. [157 ] showed aspalathin suppressed ROS as well as activated NF-κ B and monocyte adhesion in both an in vitro cell and in vivo mouse model. Elevated plasma levels of sEPCR have been found to increase vascular
inflammation and subsequent thrombotic risk [159 ]. Kwak et al. [163 ] showed that aspalathin treatment inhibited phorbol 12-myristate 13-acetate-induced
TNF-α , IL-1β , and CLP-induced EPCR shedding by inhibiting the phosphorylation of several kinases
known to increase thrombin generation. These results present strong evidence in support
of aspalathin as a nutraceutical to protect against metabolic syndrome and associated
complications such as glucose and lipid intolerance, as well as oxidative stress and
inflammation that may result in accelerated cell injury. The ameliorative properties
of aspalathin against glucose and lipid metabolic perturbations in various tissue
targets are summarised in [Fig. 7 ].
Fig. 7 Summary of the ameliorative properties of aspalathin (ASP) and/or aspalathin-enriched
green rooibos extract (GRE) against glucose and lipid metabolic perturbations as observed
in in vitro and in vivo models. An obesogenic environment, characterised by over-nutrition and lack of physical
activity, promotes excess lipid accumulation, development of insulin resistance, and
metabolic syndrome. In adipose tissue, increased hypertrophy and hyperplasia of adipocytes
result in the activation of NF-κ B, an inflammatory kinase known to suppress insulin signalling and exacerbate inflammation.
Aspalathin effectively ameliorated these metabolic complications by improving insulin
response, the result of enhanced GLUT4 expression, and inhibition of NF-κ B-induced inflammation. In the liver, aspalathin suppressed cholesterol synthesis
by decreasing SREBP1-C, a transcriptional factor involved in fat synthesis, and by
decreasing glucose release from the liver, facilitated by gluconeogenesis and glycogenolytic
enzymes. In the pancreas, aspalathin exerted its effects by suppressing ROS induced
by advanced glycation end products, and by stimulating insulin secretion. In the heart,
aspalathin suppressed abnormally increased FFA oxidation resulting in improved cardiac
energy metabolism, and prevented oxidative damage by upregulating Nrf2. In the skeletal
muscle, aspalathin improved insulin signalling by reversing the inhibitory effect
of PKC on IRS1/2, thereby increasing glucose uptake and β -oxidation and reducing ROS.
Herb-drug interactions
The increasing custom of the health conscious public to supplement their diets with
natural products to enhance health and well-being is of concern due to possible herb-drug
interactions in the growing population of patients on chronic medications [164 ]. Natural products are generally considered to be safe with little regard for potential
adverse effects. Patients are drawn to using these products as adjunctive supplements
to enhance the therapeutic efficacy of their medication, mostly without informing
their health practitioner. In most countries, natural products are sold over-the-counter
or are freely available in supermarkets with sparse information about their health
risk benefits, either as a monotherapy or in combination with other chronic medications.
It is reasonable to assume that the concurrent use of aspalathin-based nutraceuticals
with chronic blood glucose-lowering medication by type 2 diabetic patients will escalate.
Although anecdotal evidence suggests that consumption of rooibos is generally regarded
as safe, recently, two case studies have suggested the potential for herb-drug hepatotoxicity.
In the first case [165 ], a 42-year-old woman treated for a low-grade B-cell malignancy with rituximab and
maintained on prednisolone and co-trimoxazole daily, presented with elevated liver
enzymes. She was advised to stop drinking rooibos flavoured with small amounts of
strawberry, chamomile, and petals of daisy and discontinue her prophylactic antibiotic
(co-trimoxazole). One week later, her liver enzymes returned to normal and she resumed
her prophylactic co-trimoxazole treatment without further adverse effects. A second
case study [166 ], [167 ] involved a 52-year-old hyperlipidaemic patient on atorvastatin who developed clinical
symptoms of hepatotoxicity following increased consumption of a rooibos-buchu herbal
tea. In the latter case, the presence of buchu, an indigenous South African medicinal
plant shown to inhibit CYP3A4 [160 ], a major phase I metabolising enzyme of atorvastatin, confounded results. Although
these two case studies do not conclusively infer causality, the sparsity of information
relating to the potential of rooibos- and aspalathin-drug interactions prompted us
to investigate their potential interaction with chronic medications such as statins
or oral hypoglycaemic drugs. Using an in vitro recombinant CYP450 enzyme assay, both an aspalathin-rich green rooibos extract and
aspalathin dose- and time-dependently inhibited CYP3A4, cautioning against the potential
of a herb-drug interaction with hypoglycaemic drugs such as sulfonylureas and statins,
including atorvastatin [168 ]. However, these findings still need to be confirmed by in vivo pharmacokinetic and pharmacodynamic studies.
