In vitro ?-glucosidase inhibitory activity of isolated fractions from water extract of Qingzhuan dark tea
?-Glucosidase inhibitory activity of Qingzhuan tea extracts
?-Glucosidase plays a central role in modulating postprandial hyperglycemia, which
breaks down ?-1,4-glucosidic linkages of disaccharides, resulting in simpler sugars.
A previous study reported the established ?-glucosidase inhibitors and their effects
on delaying the expeditious generation of blood glucose after food uptake 21].
In the present study, the crude water extract of Qingzhuan tea was divided into five
fractions by polarity and the ?-glucosidase inhibitory activities of these fractions
were detected using pNPG as the reaction substrate. As shown in Table 1, the crude water extract of Qingzhuan tea exerted an obvious inhibitory effect on
?-glucosidase, with an IC
50
value of 2.47 mg/mL. A previous in vivo study 22] indicated the hypoglycemic effect of dark tea for diabetic mice. In our experiment,
no ?-glucosidase inhibition was observed in the chloroform fraction of Qingzhuan tea,
but the ethyl acetate, n-butanol, sediment and residual aqua fractions exhibited a dose-dependent inhibitory
effect on ?-glucosidase activity. The IC
50
values of the four fractions ranged from 0.26 to 5.24 mg/mL, and followed the sequence
of residual aqua fraction (5.24 mg/mL)??sediment fraction (3.02 mg/mL)??n-butanol fraction (2.94 mg/mL)??ethyl acetate fraction (0.26 mg/mL), indicating that
the ethyl acetate fraction had the greatest inhibitory activity. Meanwhile, apart
from the residual aqua fraction, the sediment, n-butanol and ethyl acetate fractions could exhibit significantly more potent inhibitory
effects than acarbose (an effective inhibitor of ?-glucosidase with the IC
50
value of 4.64 mg/mL, p??0.01), suggesting Qingzhuan tea extracts could be potential ?-glucosidase inhibitors.
Table 1. Inhibitory effects of Qingzhuan tea fractions on ?-glucosidase
In order to clarify the inhibitory mechanism of ?-glucosidase activity, the basic
active components in Qingzhuan tea extracts were determined (Table 2). The crude water extract contained 18.25 % polyphenols and 26.83 % tea-pigments
(including theaflavins, thearubigins and theabrownins), 14.74 % carbohydrates, and
5.82 % caffeine. Due to the special pile-fermenting process, Qingzhuan tea had a high
content of tea-pigments, especially thearubigins and theabrownins. Among the five
fractions, the ethyl acetate fraction had the highest total polyphenols (62.72 %)
and theaflavins (1.70 %), and the second highest amount of total thearubigins (24.21 %).
Caffeine was the dominant active compound (70.55 %) in the chloroform fraction. The
n-butanol fraction contained a certain amount of polyphenols (31.09 %), carbohydrates
(15.56 %), thearubigins (28.00 %), and theabrownins (24.82 %). For the sediment fraction,
16.65 % carbohydrates, 9.16 % polyphenols, and 27.18 % theabrownins were obtained.
The residual aqua fraction almost had the same components as the crude water extract.
Table 2. The main constituent of Qingzhuan tea extracts (%)
All the aforementioned results indicated that the ethyl acetate fraction had the highest
inhibitory effect on ?-glucosidase, and the highest contents of total polyphenols
and theaflavins. Correlation analysis between the levels of total polyphenols, theaflavins
and IC
50
values in ethyl acetate, n-butanol, sediment and residual aqua fractions showed that both polyphenols and theaflavins
were tightly correlated with ?-glucosidase inhibitory activities (R 2
?=??0.884 and ?0.878, respectively), suggesting that ?-glucosidase inhibitory activity
was likely attributed to the synergistic interaction of polyphenols and theaflavins
in the Qingzhuan tea extracts. It has been reported that plant phenolic compounds
could inhibit ?-glucosidase effectively 23]. Different teas (green, oolong and black teas) showed different ?-glucosidase inhibitory
profiles, which was associated with their major polyphenol contents 24]. Theaflavin was reported to have ?-glucosidase inhibitory activity in the following
order: theaflavin-3-O-gallate??theaflavin ?3,3?-di-O-Gal??theaflavin ?3?-O-Gal??theaflavin
25]. It seems that the presences of hydroxyl group and gallate group were closely associated
with ?-glucosidase inhibitory effects.
Polysaccharides have also been reported to have hypoglycemic activities, partly due
to their ?-glucosidase or ?-amylase inhibitory activity 26], 27]. The present work also showed that the sediment fraction containing a substantial
portion of polysaccharides exerted an ability to inhibit ?-glucosidase. A similar
result was observed that polysaccharides obtained from leaves and flowers of Camellia sinensis by different extraction methods have been reported to have ?-glucosidase and ?-amylase
inhibitory activities 28].
The chemical components change greatly due to the special manufacturing process of
Qingzhuan tea. For instance, catechins, the predominant polyphenols in tea, accounted
for 70–80 % of total polyphenols 29] and could be oxidized and polymerized to generate theaflavins, thearubigins, theabrownins
and even unidentified polyphenols polymers under fungal, damp and hot conditions during
postfermentation 30]. Theabrownins were responsible for the characteristic color of Qingzhuan tea and
were enriched in the sediment, n-butanol and residual aqua fractions. Although the chemical composition of theabrownins
is still unknown, some biological activities have been demonstrated, such as serum
lipid-lowering effect, antioxidant property and nitric oxide scavenging ability 31], 32]. It is possible that theabrownins also contribute to the ?-glucosidase inhibition,
which needs further research. Thearubigins have been reported to have ?-glucosidase
inhibitory activity, while the inhibitory effect was weaker than catechins and theaflavins
33]. Under the present isolation conditions, the n-butanol fraction had the highest thearubigins, which might also contribute to the
?-glucosidase inhibitory activity.
Inhibitory effect of eight subfractions from ethyl acetate fraction on in vitro ?-glucosidase
activity
The aforementioned results showed the ethyl acetate fraction had the greatest inhibitory
effect on ?-glucosidase activity in vitro, and thus it was further separated into
eight subfractions on a Sephadex LH-20 column with different mixtures of methanol
or acetone with water or water alone as mobile phase. The inhibitory effects of those
subfractions on ?-glucosidase were investigated, and their IC
50
values were listed in Table 3. All subfractions showed ?-glucosidase inhibition capabilities, and their IC
50
values followed the sequence of QEF1 (12.31 mg/mL)??QEF2 (7.57 mg/mL)??QEF3 (6.22 mg/mL)??QEF4
(5.39 mg/mL)??QEF5 (3.33 mg/mL)??QEF6 (1.61 mg/mL)??QEF7 (0.95 mg/mL)??QEF8 (0.066 mg/mL),
indicating that the QEF8 had the greatest inhibitory activity. As shown in Table 3, there was significant difference in the ?-glucosidase inhibitory activities among
the eight subfractions compared to acarbose (p??0.01). The IC
50
values of QEF5, QEF6, QEF7 and QEF8 were significantly lower than that of acarbose,
especially QEF8, with IC
50
value being 70-fold lower than that of acarbose and 4-fold lower than that of the
ethyl acetate fraction.
Table 3. ?-Glucosidase inhibition of various subfractions obtained from the ethyl acetate fraction
The inhibitory mechanisms of QEF8 were further explored in this study. As shown in
Fig. 1, the Lineweaver-Burk plots for various concentrations of QEF8 showed the same intersection
on y-axis, indicating that the 1/V max
remained unchanged in the absence of the substrate. The QEF8 inhibited the enzymatic
activity in a competitive manner. According to Michaelis-Menten equations, the value
of the inhibition constant (K i
) was 77.10 ?g/mL (Table 4). Therefore, the mode of action of the inhibitory activity of QEF8 on ?-glucosidase
could be similar to that of acarbose. Acarbose has been shown in an animal study to
be a competitive inhibitor of intestinal brush-border ?-glucosidase enzymes through
the acarviosine moiety binding with high affinity to the active centers of the proteins
in this enzyme family 34].
Fig. 1. Lineweaver-Burk plot of QEF8 towards the substrate pNPG at different concentrations
Table 4. K m
and V max
values of ?-glucosidase in the presence of different concentration of QEF8
The components in QEF8 were analyzed by HPLC-MS method (Fig. 2). EGCG (m/z 459.2) and ECG (m/z 443.1) were found to be the predominant active compounds
in QEF8 (Table 5). To further confirm which components in QEF8 are the most effective as inhibitors,
EGCG and ECG were tested on the inhibition of ?-glucosidase. As shown in Figs. 3 and 4, EGCG showed greater inhibitory effect than ECG, with IC
50
values of 59 ?mol/L (27.02 ?g/mL) and 1626 ?mol/L (719.29 ?g/mL), respectively. Matsui
et al. 25] investigated the ?-glucosidase inhibitory activities of catechins (EC, ECG, EGC and
EGCG) and found EGCG exhibited greater inhibitory activity of maltase than ECG in
rat intestinal acetone powder. Kamiyama et al. 35] further compared the inhibitory activities of ten catechins toward maltase and sucrase
in rat brush border membrane vesicles prepared freshly and obtained a similar result.
Those results showed that galloylated catechins had higher ?-glucosidase inhibitory
activities than non-galloylated catechins. The galloy group bonding at the 3 position
of catechins played an important role in the ?-glucosidase inhibitory activity. Among
galloylated catechins, the number of the hydroxyl group on the B ring was favorable
to the inhibitory activity. Galloylated catechins also had higher inhibitory effects
on ?-amylase that was another important digestive enzyme, while catechol catechins
(CG and ECG) were 2 times more inhibitory than pyrogallol catechins (GCG and EGCG)
36]. Intestinal glucose transporters were responsible for subsequent glucose uptake,
and ECG was also more effective against intestinal glucose transport than EGCG 37], 38]. Xu et al. 39] assessed the contribution of seven catechins to the inhibition of carbohydrate digestive
enzyme (?-glucosidase and ?-amylase) and intestinal glucose transport, and found the
inhibitory potency of seven catechins was ranked in a similar order. The inhibitory
kinetics of catechins on ?-glucosidase has been reported and still remain controversial.
Li et al. 40] reported that both EGCG and ECG inhibited Saccharomyces cerevisiae ?-glucosidase in non-competitive manners. While the mode of rat intestinal ?-glucosidase
of EGCG was a mix-competitive manner and the K i
value was 87.8 ?g/mL in the study of Xu et al. 39].
Fig. 2. HPLC chromatogram of individual phenolic compounds in the QEF8. peak 1 GA: [M?+?H]
+
171.0, MS
2
152.8, 126.9; peak 2 EGC: [M?+?H]
+
307.1, MS
2
288.9, 138.9; peak 3 C: [M?+?H]
+
291.0, MS
2
273.4, 139.4; peak 4 EGCG: [M?+?H]
+
459.2, MS
2
288.9, 138.9; peak 5 GCG: [M?+?H]
+
459.2, MS
2
288.9, 138.9; peak 6 EC: [M?+?H]
+
291.0, MS
2
273.4, 139.4; peak 7 ECG: [M?+?H]
+
443.1, MS
2
272.9, 150.9; and peak 8 CG: [M?+?H]
+
443.1, MS
2
272.9, 150.9
Table 5. Summary of data used to quantify major components of QEF8 by HPLC method
Fig. 3. Inhibitory effects of ECG on ?-glucosidase
Fig. 4. Inhibitory effects of EGCG on ?-glucosidase
Tea has been associated with reducing the risk of diabetes, while the ability of various
teas on antidiabetic effects was different. Koh et al. 41] investigated the ability of different teas (green, oolong and black teas) in inhibiting
?-glucosidase and ?-amylase, and found black tea was most potent in inhibiting ?-glucosidase
and ?-amylase. Theaflavins in black tea were associated with their potent inhibitory
effects, and catechins were weaker enzyme inhibitors in contrast to theaflavins. In
the study of Yang et al. 24], the inhibitory effect of oolong tea extract was significantly higher than that of
green tea and black tea extracts. The difference might be due to different sources
of tea, which correlated to their major polyphenol content (theaflavins and catechins).
Gomes et al. 42] revealed that black tea extract was more effective in reducing the blood glucose
concentration in streptozotocin-induced diabetic rats comparing with green tea extract,
while green tea extract was more effective in preventive. Tang et al. 43] performed the comparison between green tea extract and black tea extract in a type
II diabetic mouse model, suggesting that different tea extracts might exert hypoglycaemic
effects through different pathways. Qingzhuan tea, as a post-fermentation tea, might
reduce blood glucose in a different way. Our previous investigation showed that Qingzhuan
tea was more effective than green tea in losing weight, decreasing serum lipids, antioxidation
and protecting liver cells of hypolipidemic rats 14]. Cheng et al. 15] tried to identify bioactive components from Qingzhuan tea by successively isolating
the water extract and found QEF8 was the most active subfraction based on the in vitro
antioxidant and ?-amylase inhibitory activity, which is consistent with the present
results. HPLC chromatographic separation of QEF8 revealed that almost half of the
subfraction was catechins. It can concluded that catechins were an important factor
in contributing to the antioxidant and digestive enzymes inhibitory activity of Qingzhuan
tea. While during the process of Qingzhuan tea, the post-fermentation stage process
decreased the contents of catechins and formed some novel catechins derivatives. These
unidentified derivatives probably contributed to the anti-hyperglycemic potential,
which still requires further research.