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Indian Journal of Natural Products and Resources
Vol. 12(4), December 2021, pp. 538-543
In vitro antidiabetic and anti-inflammatory activities of the bark of
Ehretia acuminata R.Br.
Amanpreet Kaur1*, Abha Shukla1 and Rishi Kumar Shukla2
1Department of Chemistry, Kanya Gurukula Campus, 2Department of Chemistry, Gurukula Kangri Vishwavidyalaya,
Haridwar 249404, Uttarakhand, India
Received 19 October 2019; Revised 24 August 2021
Ehretia acuminata is native to Southeast Asia, America, and Africa. It is commonly used as fodder and in quite a variety
of medicinal applications. E. acuminata is widely used in India, but there are few reports in the literature of studies on its
biological properties. In this study, the antidiabetic and anti-inflammatory activities of plant E. acuminata was evaluated.
The antidiabetic activity of the different extracts was measured by α-amylase and α-glycosidase inhibition method. The anti-
inflammatory activity was evaluated by the egg albumin of hen. Petroleum ether, chloroform, ethyl acetate, ethanol and
water extracts of bark of E. acuminata was evaluated for in-vitro anti-inflammatory and antidiabetic activity. Ethyl acetate
and ethanol extract showed significant anti-inflammatory effects (IC50 170 and 172 µg/mL respectively). Chloroform is the
only extract that showed significant inhibition against the antidiabetic effect (IC50 42-45 µg/mL). The findings suggest that
the bark of E. acuminata contains potential antidiabetic and anti-inflammatory compounds, which could be tested as drug
candidates against diabetic and inflammation-related pathological processes in medicinal chemistry studies.
Keywords: Antidiabetic, Anti-inflammatory, Ehretia acuminata R.Br., α-Amylase, α-Glucosidase.
IPC code; Int. cl. (2015.01)- A61K 36/00, A61K 129/00, A61P 3/00, A61P 3/10, A61P 29/00
Introduction
The use of secondary metabolites present in plants
represents a large source of traditional medicine that
could be serving as leads for the development of novel
drugs. Several anti-inflammatory, digestive,
antinecrotic, neuroprotective, and hepatoprotective
drugs have recently been shown to have radical
scavenging mechanism as part of their activity1.
Diabetes mellitus is a metabolic disease, characterized
by hyperglycemia together with impaired metabolism
of glucose and other energy-yielding fuels, such as
lipids and proteins2. This metabolic disorder is the
result of a deficiency in insulin secretion or a resistance
to insulin action or both3. More than 220 million people
worldwide have diabetes and by the year 2030, this
number might be more than double4. Diabetic patients
also exhibit oxidative stress, which leads to lipid
peroxidation and tissue damage including retinopathy,
nephropathy, and coronary heart disease5,6.
Dyslipidemia or hyperlipidemia is also involved in the
development of cardiovascular complications, which
are a major cause of morbidity and mortality7.
Inflammation and oxidative stress have an
important role in various diseases. Inflammation is an
immunological defence mechanism elicited in
response to mechanical injuries, burns, microbial
infections, allergens and other noxious stimulus8. The
use of anti-inflammatory agents may therefore be
helpful in the treatment of inflammatory disorders9.
Steroidal or non-steroidal anti-inflammatory drugs are
usually used to treat different inflammatory diseases.
The contrary effects of the presently usable anti-
inflammatory drugs however pose a major problem in
their clinical use, therefore naturally originated agents
with very minor side effects are desirable to replace
chemical therapeutics10. There has been a rising
interest in phenolic components of natural resources
like vegetables and fruits, which may promote human
health or decrease the risk of disease. Recent studies
have focused on the health functions of phenolics
including flavonoids from fruit and vegetables11. In
search of sources of natural antioxidants, some
medicinal plants have been extensively studied for
their antioxidant activity and radical scavenging
activity12,13. Regenerate attention to alternate
medicines and natural treatment has raised
researchers’ interest in traditional herbal medicine.
—
—————
*Correspondent author
Email: amanpreet2225@gmail.com
KAUR et al.: ANTIDIABETIC AND ANTI-INFLAMMATORY POTENTIAL OF E. ACUMINATA
539
Because of their perceived effectiveness, with
minimal side effects in clinical experience and
relatively low costs, herbal drugs are prescribed
widely, even when the contents of the biologically
active constituents are unknown.
Ehretia acuminata is a wild medicinal plant found
in Asia, America, Australia, and Africa. Many
traditional claims are reported as medical treatments
for various diseases like some kind of fever, dysentery
and other health ailments14. The bark of E. acuminata
is useful in sores on the tongue, ripe fruit is edible and
unripe fruit is used as a pickle by tribal communities.
Ethno-botanical surveys on genus Ehretia also
suggest snake antivenom activity and anti-tubercular
activity of different parts of different species15,16,
antioxidant and antiarthritic activity17. An
antitrypanosomal18, antiprotozoal activity19, and anti-
inflammatory of leaves and root extract of Ehretia20,21
were also reported. Such activities can be due to the
presence of phytochemicals alkaloids22,23,
flavonoids24-26, and fatty acids27.
E. acuminata is a tree that belongs to the family
Boraginaceae. Previous literature provides meagre
information about therapeutic uses of this plant28.
Branchlets of E. acuminata are used traditionally in
Chinese herbal medicines. Antioxidant activities are
attributed to the fact that the bark of E. acuminata
contain a variety of secondary metabolites, such as
flavones, sterols, several hydrocarbons,
anthraquinones, glycosides, etc. Among these
flavones, glycosides, and sterols are considered to be
useful as anti-inflammatory and antidiabetic agents.
Materials and Methods
Plant materials
Bark material of plant E. acuminata was collected
from Pantnagar (in the daytime, at temperature
26–27.9 °C), Udham Singh Nagar district,
Uttarakhand in Kumaun region of India in the month
of April 2017 and authenticated from Botanical
Survey of India (BSI) Dehradun (Voucher specimen
number 117138 05/2017). Bark was dried for 10–20
days under shade until the bark material was ready for
grinding and stored at room temperature. It was
ground in a laboratory grinder and stored at 5 °C.
Chemicals
Nitrophenyl-α-D-glucopyranoside (SRL Pvt., Ltd),
tris buffer (Merck), α-amylase ex porcine pancreas
(SRL Pvt., Ltd), dimethyl superoxide (DMSO)
(Merck), 3,5-dinitrosalicylic acid (DNSA) (SRL Pvt.,
Ltd), α-glucosidase for biochemistry ex
microorganism (SRL Pvt Ltd); and acarbose (Bayer
India Limited), diclofenac sodium, sodium carbonate
(CDH) were purchased. All other chemicals,
solvents and reagents used were of analytical or
HPLC grade.
Preparation of crude plant extract
Collection and extraction of plant material were done
by the method used by the authors earlier29. Bark
material of E. acuminata was collected from the
Kumaun region Uttarakhand. Bark was dried under
shade and powdered using a grinder. The extraction was
done by soxhlet method at room temperature by taking
100 g of dried material. It was soaked in different
solvents (600 mL) according to increasing order of
polarity {petroleum ether (60-80 ºC), chloroform, ethyl
acetate, ethanol, water} for 6-8 days with stirring every
20 h by using a sterilized glass rod. The final extracts
were filtered through Whatman filter paper No.1. The
filtrates obtained were concentrated under vacuum on a
rotary evaporator at 42 oC and stored at low temperature
for further experiment.
Phytochemical screening
Phytochemical screening of solvent extracts of
E. acuminata bark was done to test for the presence
of alkaloids, glycosides, flavonoids, carbohydrate,
tannins, terpenoids, anthraquinones, fat and oil30.
Evaluation of in vitro antidiabetic activity
Inhibition assay of α-amylase enzyme
The enzymes inhibition procedure of α-amylase was
according to the method of Kazeem et al. with some
modification31. The α-amylase activity can be measured
by the determination of the reducing group arising from
hydrolysis of soluble starch by isolated pancreatic
α-amylase. Reduction of 3,5- dinitrosalicylic acid to
nitroaminosalicylic acid produces a colour shift which is
followed photometrically by a change in the absorbance
at 540 nm. Inhibition of starch hydrolysis by the
α-amylase inhibitor results in a diminished absorbance at
540 nm in the comparison with control. Acarbose (AC)
was used as a standard drug in α-amylas antidiabetic
assay. The percentage (%) of inhibition for α-amylase
was also expressed as the half-maximal inhibitory
concentration (IC50). The formula for % of inhibition is
as given below:
%ofinhibition
=Absorbanceofcontrol–(Absorbanceofextract)
Absorbanceofcontrol × 100
INDIAN J NAT PROD RESOUR, DECEMBER 2021
540
Inhibition assay of α-glucosidase enzyme
The enzymes inhibition procedure of α-glucosidase
was according to the method of Kim et al. with slight
modification32. The activity is based on the fact that
when the enzyme α-glucosidase is incubated with
the substrate p- nitrophenyl-α-D-glucopyranoside
(p-NPG), it hydrolyses the substrate to p- nitrophenol
and D- glucose respectively. Acarbose (AC) was used
as a standard drug for α-glucosidase inhibition assay.
The absorbance of the released p-nitrophenol was
measured at 410 nm. Each experiment was performed
in triplicates. The percentage (%) of inhibition for
all the enzymes except for α-glucosidase was
also expressed as the half-maximal inhibitory
concentration (IC50). The formula for % of inhibition
is as given above.
Evaluation of in vitro anti-inflammatory activity
Inhibition by albumin method
The ability of plant extract against protein
denaturation was determined according to the
method described by Chandra et al. with some
modification33. The reaction mixture contained 2 mL
of varying concentrations of different extract, 0.2
mL of egg albumin (fresh hen’s egg) and 2.8 mL of
phosphate-buffered saline (pH 6.4) and the final
concentrations are 25, 50, 100, 250, and 500 µg/mL.
Then the reaction mixtures were incubated at room
temperature in the BOD incubator for 15 min and
then, heated at 72 ºC for 5 min. Many kinds of
biological substances are observed in visible light
(400-700 nm) in spectrophotometry. Due to the
appearance of viscosity in the tested samples,
absorbance was taken at the high visible range 660
nm (Systronic 118, UV-VIS) by using a blank and
their viscosity was determined by using Ostwald
viscometer. Sodium Diclofenac was used as the
reference drug and treated similarly as plant extracts
for determination of absorbance. The following
formula was used to calculate the percentage
inhibition of protein denaturation:
%inhibition= 100×
Vt(absorbanceoftestsample)
Vc(absorbanceofcontrol) −1
Statistical analyses
All the experiments were done in triplicates and the
results were expressed as Mean±SD. The data were
statistically analyzed using one way ANOVA
followed by Duncan’s test. Mean values were
considered statistically significant when P >0.05.
Results and Discussion
Extracts of E. acuminata bark in different solvents
gave different colours and different yields. The lowest
yield was found in chloroform (0.467%) and the
highest yield was found in water (2.96%). Table 1
gives the total yield and colour of all extracts.
The qualitative phytochemical screening of all the
extracts of E. acuminata revealed the presence of
alkaloids, terpenoids, and glycosides in chloroform
extract and tannins, saponins, flavanoids in polar
solvents ethyl acetate and ethanol extract.
The in vitro α-amylase inhibitory activity of
E. acuminata bark extracts compared with acarbose
(Table 1 & Fig. 1) represents the alpha-amylase
Table 1 — Yield, colour of extracts and IC50 of antidiabetic & anti-inflammatory activity of Ehretia acuminata bark
Plant extract Bark extraction Results of α-amylase
antidiabetic activity
Results of α-glucosidase
antidiabetic activity
Results of anti-inflammatory
activity by egg albumin
yield % Colour IC50 (µg/mL) IC50 (µg/mL) IC50 (µg/mL) Viscosity (cp)
Petroleum ether 0.508 Light yellow 270 270 680 0.75
Chloroform 0.467 Pale yellow 45.35 42.90 1150 0.71
Ethyl acetate 1.785 Yellow 580 550 170 0.88
Ethanol 2.735 Brown 475 480 180 0.80
Water 2.96 Dark brown 242.90 250.80 185 0.79
Acarbose 40.25 38.45
Sodium diclofenac 120 0.84
Fig. 1 — Correlation between extract concentration an
d
percentage of α-amylase enzyme inhibition.
KAUR et al.: ANTIDIABETIC AND ANTI-INFLAMMATORY POTENTIAL OF E. ACUMINATA
541
inhibition on changing the concentration of each
extract and helps in the estimation of IC50 value of
each extract as well as standard acarbose. The IC50
value is the concentration of each extract or standard
drug which is required to inhibit 50 per cent of the
enzyme in reaction. Acarbose showed percentage
α-amylase inhibition of 32.24-98.45% on varying
concentration from 25-500 μg/mL with an IC50 value
40.25 μg/mL. A lower IC50 value corresponds to
greater potency and better therapeutic efficacy.
Chloroform extract reflects the highest α-amylase
inhibitory activity (IC50 = 45.35 μg/mL) followed by
water extract (IC50 = 242.90 μg/mL). The IC50 value
of chloroform extract is nearly comparable with
acarbose and thus can be regarded as an excellent
α-amylase inhibitor. The α-amylase inhibitory
activity shown by all extracts might be due to
various phytoconstituents present in each extract.
Furthermore, the potent activity of chloroform extract
must be attributed to its high potential and the
majority of phytoconstituents present which are
responsible for antidiabetic effect. The available
literature depicts that alkaloids, terpenoids, and
glycosides are known to show α-amylase inhibitory
activity34.
The in vitro α-glucosidase inhibitory activity of
E. acuminata bark extracts compared with acarbose
(Table 1 & Fig. 2) represents the α-glucosidase
inhibition on changing the concentration of each
extract and helps in the estimation of IC50 value of
each extract as well as standard acarbose. Again the
highest α-glucosidase inhibitory activity was
demonstrated by chloroform extract (IC50 42.90
μg/mL) followed by aqueous extract (IC50 250.80
μg/mL). The IC50 value of chloroform extract is even
close to acarbose (IC50 38.45 μg/mL) indicating its
extremely potent nature. Previous literature evidenced
alkaloids and terpenoids as potent α-glucosidase
inhibitors and these phytoconstituents could be well
cited in chloroform extract. These active extracts
could be used as antidiabetic agents in comparison to
synthetic drugs with numerous side effects like
abdominal discomfort, bloating, flatulence, and
diarrhoea35.
Polar solvents have been proved as effective
solvents to extract phenolic compounds. Considering
the ethyl acetate and ethanol extracts, ethanol extract
is more polar than ethyl acetate. Both extracts are also
a good source for obtaining polyphenolic compounds
and are safe for human consumption. Anti-
inflammatory potential of E. acuminata may be due to
the extractability of some bioactive compounds in
ethyl acetate and ethanol extracts. Previous work of
the authors on total phenolic content revealed that
E. acuminata bark can scavenge free radicals and that
the ethyl acetate and ethanol extracts showed good
antioxidant activity28. Therefore, the different extracts
were selected for further in vitro anti-inflammatory
studies. In the present investigation, the in vitro anti-
inflammatory effect of E. acuminata was evaluated
against the denaturation of egg albumin. Results are
summarized in Table 1. This study shows a
concentration-dependent inhibition of protein
(albumin) denaturation by E. acuminata throughout
the concentration range of 25 to 500 µg/mL.
Diclofenac sodium (25 to 500 µg/mL) which is used
as the standard drug, also exhibited concentration-
dependent inhibition of protein denaturation.
However, diclofenac sodium was highly effective
when compared with E. acuminata and more polar
solvent extracts ethyl acetate and ethanol show high
inhibition as comparison to other solvents. This is
confirmed by comparing their IC50 values in Table 1
and inhibition in Fig. 3. From antioxidant and
anti-inflammatory activities of E. acuminata bark, it is
proved that extracts have a high content of phenolic
and flavonoid compounds.
Antidiabetic activity of E. acuminata was based on
two biochemical assays conducted in vitro. There
could be some problems in using animals in
experimental in vivo research, such as ethical issues and
the lack of rationale for their use when other suitable
methods are available. The maltose standard curve for
α-amylase inhibitory assay was plotted using various
concentrations of maltose. The extracts were tested at
different concentrations for inhibition of a-amylase. In
Fig. 2 — Correlation between extract concentration an
d
percentage of α-glucosidase enzyme inhibition
INDIAN J NAT PROD RESOUR, DECEMBER 2021
542
this assay, the positive control, acarbose gave the IC50
value as 40.25 µg/mL. In the α-amylase method, the
inhibition effect by phytoconstituents responsible for
antidiabetic potential of each extract based on the
change in colour intensity was monitored at 540 nm.
IC50 values obtained for chloroform extract was lower
than the other extracts. Antidiabetic potential by
α-glucosidase was measured with the help of
P-nitrophenyl-α-D-glucopyranoside (NPG), tris buffer
and 50% inhibition of chloroform extract was reached
at a concentration much less than ethyl acetate extract.
E. acuminata was also evaluated for
anti-inflammatory activity using the egg albumin
method and the protein denaturation bioassay was
selected for the in vitro assessment. Reason for protein
denaturaton are diseases like inflammatory and arthritic.
The formation of antibodies in some arthritic diseases
may be due to the denaturation of proteins36. Factors that
can forbid protein denaturation therefore would be
favourable for anti-inflammatory drug development.
The increments in absorbances of test samples with
respect to control, indicated stabilization of protein
i.e., inhibition of heat-induced protein (albumin)
denaturation by different extracts and reference drug
sodium diclofenac. From the IC50values, it becomes
evident that ethyl acetate extract was most active after
sodium diclofenac, being effective in lower
concentrations. Changes in viscosities also indicate
denaturation. It is documented that the viscosities of
protein increase on denaturation37. The presence of
different extracts of the plant depicts the inhibition of
protein denaturation. Here, the viscosities decreased
when compared with blank where no test extract/drug
was added. However, the viscosities were found to
decrease with a co-occurrence decrease in the
concentration of plant extract and standard drug as
well. Viscosities will decrease with the decrease in the
concentration of plant extracts and standard drug in
reaction. It is reported that at pH 6.2-6.5, many non-
steroidal anti-inflammatory drugs can stabilize (forbid
denaturation) heat-treated egg albumin38. Hence, from
this experiment, it can be concluded that E. acuminata
bark showed a significant in vitro anti-inflammatory
effect against the denaturation of protein.
Conclusion
The present study revealed that different types of
extracts had a big influence on the antidiabetic and
anti-inflammatory properties. The results showed that
chloroform extract of E. acuminata could be a natural
source of antidiabetic and ethyl acetate and ethanol
extracts could have greater importance as therapeutic
agents in preventing or slowing oxidative stress and
inflammation-related disorders. Further studies are
currently underway to assess the in vivo biological
activities and to identify the active component
responsible for their antidiabetic and anti-
inflammatory properties.
Conflict of interest
The authors declare no conflict of interest.
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