mellitus (DM) is a metabolic condition characterized by impaired glucose
homeostasis (chronic hyperglycemia) with disruptions of carbohydrates, fats,
and proteins metabolism resulting from defects in insulin secretion, insulin
action, or both1,2. Chronic hyperglycemia during diabetes is
interconnected to cardiovascular, kidney, nerve, and ocular dysfunctions.
Moreover, its effects are mostly related to dyslipidemia, increased oxidative
stress, and changes in the body's antioxidant defense mechanism3.
Over the past few decades, diabetes cases and prevalence have gradually risen
worldwide (over 690 million people by 2045)4, but it is a severe
health issue and societal burden in developing nations5.
countries like Jordan, medicinal plants account for around 20% of Jordan's
flora6,7. Due to the relatively high cost of allopathic medicines,
it is advantageous to use local plant remedies. For to their efficiency, lack
of clinically significant side effects, and affordable price, herbal medicines
are gaining popularity8,9.
Jordan, several studies reported using indigenous medicinal plants to manage
diabetes mellitus. Al-Aboudi and Afifi (2011) reviewed that Achillea santolina
L., Ajuga iva L., Allium sativum L., Aloe vera L., Artemisia
herba-alba Asso., and Capparis spinosa L. showed in vitro/ in
vivo hypoglycemic activities10.
al. (2019) also reviewed other plants with an antidiabetic effect11
such as Artemisia vulgari, Fucus vesicolosus, Lepidium sativum,
Olea europae, Teucrium polium, Urtic apilulifera, Salvia
triloba, and Cinnamomum ceylanicum.
in the review reported by Abu-Odeh and Talib. (2021) some medicinal plants
treat diabetes mellitus in the Middle East and Jordan. Achillea santolina,
Geranium graveolens, Eryngium creticum, Pistaci aatlantica,
and Varthemia iphionoides have an antidiabetic effect12.
orientalis L. was used by traditional healers to manage
diabetes mellitus in Jordan. A. orientalis is an annual herbaceous
flowering plant belonging to the family Lamiaceae, also known as the Eastern
bugle. Ajuga species has been reported to have antibacterial13,
antitumor14, antioxidant14, and anti-inflammatory
vitro studies focus on the inhibitory effects of medicinal plants on
α-glucosidase and α-amylase. Alpha-amylase digests dietary starch
into maltose, which is digested by glucose in the intestine with
alpha-glucosidase. Inhibition of these two enzymes delays the digestion of
carbohydrates, lowering postprandial blood glucose levels2,3. Most
of the plants studied inhibit these two enzymes that regulate carbohydrate
metabolism. However, different activity levels may be detected for a single
plant, depending on extract preparation and study conditions15.
On the other
hand, there are no prior investigations on A. orientalis antidiabetic
effects. Therefore, the present study desired to assess the in vitro antidiabetic
activity of aqueous and ethanolic extracts of A. orientalis aerial part.
MATERIALS AND METHODS:
Chemicals and Reagents:
All the chemicals and reagents were of AR grade and
were procured from Sigma Chemical Co, USA.
Material and Extract Preparation:
The fresh aerial parts of A. orientalis were
collected from Ajloun county-Northern of Jordan (31.9494964, 35.9342189) in
April 2022 and authentically identified by Prof. Dr. Sawsan Oran, University of
Jordan, Amman- Jordan. A. orientalis aerial parts were air-dried at room
temperature in the dark for about six weeks before being ground to a fine
powder. Ten grams of powder were soaked in 100ml of solvents (absolute ethanol,
distilled water) (1:10 w/v sample-to-solvent ratio) for 72 h at room
temperature with frequent agitation to make ethanolic and aqueous extracts. The
extracts were then filtered with Whitman filter paper (No.1), and the solvents
were then evaporated using a rotary evaporator under reduced pressure. The
crude extract was then collected and kept at 20°C14.
Alpha-amylase Inhibitory Activity:
The a-amylase inhibitory activity was conducted using
the method reported by Ibrahim et al. (2017) with slight modifications16.
The reaction mixture contained 1ml of (sample) A. orientalis extracts at
concentration ranges of 100-500μg/ml and 1ml of a-amylase solution (0.5 mg/ml prepared in 0.20mM
phosphate buffer (pH 6.9). The mixture was pre-incubated for 30 min, and then 1
ml of starch solution (1%) in 0.02mol/L sodium phosphate buffer (pH 6.9) was
added to the reaction and incubated at 37°C for 10 min. The reaction mixture
was terminated by adding 1ml of 3, 5-dinitrosalicylic acid reagent (DNS), and
the mixture was boiled for 5 min. Acarbose(100-500μg/ml) was used as a
standard (positive control). The absorbance of the reaction mixture was
measured at 540nm using a UV- vis spectrophotometer. All assays were carried
out in triplicate. The percentage of inhibition was determined using the following
Inhibition = [(Absorbance Control - Absorbance Sample) /
Absorbance Control] x 100
Alpha-glucosidase Inhibitory Activity:
inhibitory activity of A. orientalis extracts was determined by
incubating 1ml of starch solution (2% w/v maltose) with 0.2M tris buffer (pH
8.0) and various concentrations of extracts (100-500 mg/ml). The reaction mixture was incubated at 37°C
for 10min. The reaction was started by adding 1ml of the α-glucosidase
enzyme (1U/ml) to the mixture and incubated at 35°C for 40 min. Then the
reaction was terminated by adding 2ml of 6 N HCl. Acarbose was used as a
positive control. All assays were done in triplicate. The absorbance of the
mixture was measured at 400 nm via a spectrophotometer17. The inhibitory
effect was calculated using the following equation.
Inhibition = [(Absorbance Control - Absorbance Sample) /
Absorbance Control] x 100
All assays were conducted in triplicate, and values
are expressed as the mean ± standard error of the mean (SEM) using GraphPad
Prism9.0.2 (GraphPad Software, San Diego, USA). Furthermore, the IC50
value was calculated to determine the concentration of the plant extract needed
to inhibit 50% of a-amylase or a-glucosidase activity under assayed
conditions. The differences between extracts were determined using one-way
ANOVA and Tukey's post hoc test, where a p-value < 0.05 was considered statistically
α- amylase and α- glucosidase inhibition
In the current study, A. orientalis extracts
were evaluated for their inhibitory effect on α-amylase and
α-glucosidase enzymes by the in-vitro method.
Both extracts significantly demonstrated inhibition to
α-amylase and α-glucosidase enzymes in a dose-dependent manner
(p-value< 0.05) (Figures 1 and 2). The aqueous and ethanolic extracts of A.
orientalis (at a concentration of 500 mg/ml) exhibited 67.21% and 77.9% α-amylase
inhibitory activity (Figure 1) and 72.23%, and 83.44% α-glucosidase
inhibitory activity (Figure 2), respectively. However, an ethanolic extract
inhibited α-amylase and α-glucosidase more than an aqueous extract.
Acarbose was used as a positive control (standard), which showed the best
α-amylase and α-glucosidase inhibitory activity of 89.18% and 93.35%,
respectively, at a concentration of 500 μg/ml.
Moreover, the ethanolic extract of A. orientalis
has shown higher enzyme inhibitory activity than the aqueous extract, with an
IC50 value of 226.79±3.22 and 209.81±1.33mg/ml (α-amylase and α-glucosidase) (Table 1)
compared with that of acarbose, which showed α-amylase inhibitory activity
with an IC50 value of 94.96 ± 4.25mg/ml and α-glucosidase inhibitory activity with
an IC50 value of 64.15±4.22mg/ml
Table 1. Alpha-amylase and alpha-glucosidase inhibitory
effects of aqueous and ethanolic extracts and of Ajuga orientalis, and
IC50 values of α-amylase (mg/ml)
IC50 values of α- glucosidase (mg/ml)
Aqueous extract of A. orientalis (AEAO)
Ethanolic extract of A. orientalis (EEAO)
Values were expressed as Mean ± SEM (N=3).
Figure 1. In vitro α-amylase inhibitory activity of
AEAO: Aqueous Extract of Ajuga orientalis and EEAO: Ethanolic Extract of
Ajuga orientalis. Acarbose was used as a standard. Values are
represented as Mean±SEM
(n=3). *p<0.05, **p<0.01, ***p<0.001 vs. Acarbose (ANOVA, Tukey post
Figure 2. In vitro α-glucosidase inhibitory
activity of AEAO: Aqueous Extract of Ajuga orientalis and EEAO:
Ethanolic Extract of Ajuga orientalis. Acarbose was used as a standard.
Values are represented as Mean±SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 vs. Acarbose (ANOVA,
Tukey post hoc test)
hyperglycemia in diabetes is associated with long-term damage and failure of
various organ systems, most notably the eyes, nerves, kidneys, and heart, while
macrovascular complications refer to increased atherosclerosis-related events
such as myocardial infarction and stroke18.
diabetes medications are effective, but they also have unavoidable side
effects. On the other hand, medicinal plants may serve as an alternative source
of anti-diabetic agents19,20. Many plants have been considered a
primary source of powerful anti-diabetic drugs for centuries. Medicinal plants
are used to treat diabetes in developing countries, mainly to alleviate the
financial burden of conventional medicines on the population21,22.
Currently, medicinal plants treat diseases such as diabetes23; because
they contain diverse bioactive constituents such as flavonoids, saponins,
terpenoids, carotenoids, alkaloids, and glycosides that may have anti-diabetic
hyperglycemia is primarily caused by two carbohydrate hydrolyzing enzymes
(a-amylase and a -glucosidase). Alpha-amylase initiates carbohydrate digestion
by hydrolyzing 1, 4-glycosidic linkages of polysaccharides (starch, glycogen)
to disaccharides, and a-glucosidase catalyzes disaccharides to monosaccharides, resulting
in postprandial hyperglycemia28. As a result, a-amylase and a-glucosidase inhibitors help manage hyperglycemia
because they delay carbohydrate digestion, lowering postprandial plasma glucose
bioactive compounds derived from various plants may be responsible for enzyme
inhibition (hypoglycemic effect). Most phenolics and triterpenoids positively
correlate as anti-diabetic agents31. These bioactive constituents
had a hypoglycemic effect via a variety of mechanisms. Polyphenolic compounds
may interact with or inhibit specific enzyme positions, reducing the potency of
α-amylase and α-glucosidase32. Flavonoid compounds may
protect against diabetes by preventing glucose absorption or improving glucose
tolerance through competitive inhibition of sodium-dependent glucose
transporter-133. Moreover, flavonoid compounds such as luteolin,
kaempferol, chrysin, and galangin reduced blood glucose levels by inhibiting
α-amylase and α-glucosidase activity in the intestine34.
study on the aqueous and ethanolic extracts of Ajuga remota, the results
showed that the aqueous extract caused more reduction in blood glucose levels
than ethanolic extract in diabetic mice 35.
the methanolic and aqueous extracts of Ajuga iva showed inhibition of
the vital digestive enzymes linked to type 2 diabetes, with a more potent inhibitory
effect against a-glucosidase
and a significant inhibition against a-amylase36.
2020, Alene and his colleagues conducted an in vivo and in vitro
study. They revealed that hydro-methanolic crude extract and its aqueous
fraction of Ajuga integrifolia root possess significant anti-diabetic
Jordan, there was no available data about the in-vitro (α-amylase
and α-glucosidase inhibitory activity) anti-diabetic studies of A.
orientalis. As a result, the current study sought to determine the
inhibitory activity of ethanolic and aqueous extracts of A. orientalis
against α -amylase and α -glucosidase.
the phytochemical constituents of methanolic extract of A. orientalis
were determined by Oran et al. (2022), where the major components were
9-octadecenoic acid, methyl ester, (E)- (27.2%), hexadecanoic acid, methyl
ester (12.8%), and methyl stearate (9.6%). Also, Oran et al. (2022)
reported that the ethanolic and aqueous extracts of A. orientalis have
antioxidant properties and anti-cancer activity14.
current study showed that both extracts significantly inhibited the α-amylase
and α-glucosidase (p-value<0.05). Ethanolic extract of A. orientalis
had more inhibitory activity to α-amylase and α-glucosidase than
aqueous extract. Hence, it may be helpful in the management of diabetes
results of this study show that A. orientalis ethanolic and aqueous
extracts are effective α-amylase and α-glucosidase inhibitors, which
may help reduce of postprandial glucose levels. However, the main compounds
responsible for α-amylase and α-glucosidase inhibitory action must be
identified and characterized further. Moreover, in-vivo studies are
needed to confirm the obtained results further, and it could help develop new
anti-diabetic agents based on native plant resources.
CONFLICT OF INTEREST:
author has no conflicts of interest regarding this investigation.
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