Phytochemical Analysis and In-vitro Anti-Diabetic
and Anti- Inflammatory study of root extract of Apama siliquosa LAMK.
Manu Jose1*, Stephin Baby2, Dona
Mathew2, Naurin Muhammed2, Jayalakshmi P M3
1Assistant
Professor, Nirmala College of Pharmacy Muvattupuzha-686661, Dist-Ernakulam, Kerala,
India.
2Nirmala College
of Pharmacy Muvattupuzha-686661, Dist-Ernakulam, Kerala, India.
3College of
pharmaceutical Sciences, Gov medical college, thiruvanandapuram, Kerala.
*Corresponding Author E-mail: manujose09@gmail.com,
stephinbaby4@gmail.com, donamathew13@gmail.com, naurinmuhammed@gmail.com, pmjayalakshmi94@gmail.com
ABSTRACT:
The
demand for herbal medicines in many pharmaceutical sectors is growing at a drastic
rate due to their improved pharmacological actions, minimal side effects and cost-effectiveness.
Anti-inflammatory phytochemicals are found to be efficacious against the treatment
of inflammatory diseases like rheumatoid arthritis, glomerulonephritis, hepatitis,
inflammatory bowel disease, etc. Anti-diabetic phytochemicals are found to treat
the increasing incidence of diabetes prevalent globally. This work aims to perform
the phytochemical screening and to evaluate the antidiabetic and anti-inflammatory
activity of crude extract of Apama siliquosa Lamk. The method employed for
obtaining the active principles includes soxhlation technique with methanol as solvent.
The anti-inflammatory property was studied in vitro using inhibition of albumin
denaturation technique as well as heat-induced hemolysis and IC50 value
was found to be 39.5μg/ml and 36.30μg/ml respectively. The anti-diabetic
activity was estimated using the alpha-amylase inhibition assay and Glucose diffusion
inhibitory study. The IC50 value for alpha-amylase inhibition assay was
found to be 15.75μg/ml. It also shows a strong inhibition of glucose across
the dialysis membrane.
KEYWORDS: Invitro Antidiabetic, Invitro Anti-inflammatory,
Apama siliquosa Lamk, α-amylase.
1.
INTRODUCTION:
Over
the past twenty years, there has been an enormous rise in the use of herbal medicine;
however, there is still a lack of research data in this field. Therefore since 1999,
WHO has published three volumes of the WHO monographs on selected medicinal plants.
Modern searches for drugs utilize sophisticated bioassays and bioassay-guided fractionation
of medicinal plants used in traditional system of medicine. This has resulted in
the isolation of compounds with bio-potency and molecules with novel therapeutic
targets1.
Diabetes mellitus (DM) is a chronic metabolic disorder
characterized by hyperglycemia and impaired carbohydrates, lipids and proteins metabolism2, 3. Medicinal plants are being looked up once again
for the treatment of diabetes. To date, over
400 traditional plant treatments for diabetes have been reported, although only
a small number of these have received scientific and medical evaluation to assess
their efficacy4.
The
inflammation is the earliest organic response before tissue damage or infection.
Before a tissue injury, the local accumulation of prostaglandins, thromboxane's,
and other chemical mediators cause a change in the threshold nociceptors, resulting
in hyperalgesia. 5,6. The inflammatory process is part of a mechanism
of host defense against stimuli that cause injuries, but when this process is not
controlled, can damage the health of the individual7 The cardinal signs
that identify the inflammation are heat, flushing (redness), tumor (swelling), pain
and loss of function, of which the first four were described by Cornelius Celsus
8. A number of natural products are used in various traditional medical
systems to treat relief of symptoms from inflammation 9.The mature roots
of A. siliquosa are reportedly used by Ayurvedic physicians of Konkan and
Malnad districts of Karnataka for the treatment of dysentery10. The present
study aim to perform the phytochemical screening and to evaluate the antidiabetic
and anti-inflammatory activity of crude extract of Apama siliquosa Lamk by
various invitro screening methods.
2. MATERIALS AND METHODS:
2.1. Collection and Extraction
of Plant materials
Apama
siliquosa Lamk. obtained from the Thattekkad
forest area, Eranakulam district, Kerala identified and authenticated at the Department
of Botany Nirmala College Muvattupuzha, Kerala. The fresh roots were thoroughly
washed with water, allowed to dry under shade. The dried roots were finely powdered
and used for extraction. The phytoconstituents from Apama siliquosa
were extracted by soxhlation using methanol as solvent
2.2. Qualitative analysis
Test
for tannins: Two ml of plant extract
was taken in a test tube and two ml of water and few drops of ferric chloride solution
was added .blue-black precipitate formed indicates the presence of tannins.
Test
for alkaloids: Two ml of the plant
extract was taken in a test tube and 1%hydrochloric acid and 6 drops of Wagner’s
reagent was added. Brownish –red precipitate indicates the presence of alkaloids.
Test
for saponins: To 0.5 ml of plant extract
added 5 ml distilled water, the formation of frothing shows the presence of saponins.
Test
for steroids: To 2 ml of plant extract
added 2 ml acetic anhydride and conc. sulphuric acid. A blue-green ring indicated
the presence of terpenoids.
Test
for flavonoids: To the plant extract
added conc. hydrochloric acid and magnesium ribbon, pink –tomato red colour indicated
the presence of flavonoids11.
2.3. Pharmacological screening by in vitro methods
2.3.1. Antidiabetic activity of root extracts
A)
Alpha-amylase Inhibition Assay
12, 13, 14
The
alpha-amylase activity was carried out by starch-iodine method. 10 mL of αamylase
solution (0.025 mg/mL) was mixed with 390 mL of phosphate buffer (0.02 M containing
0.006 M NaCl, pH 7.0) containing different concentration of extracts. After incubation
at 37 °C for 10 min, 100 mL of starch solution (1%) was added, and the mixture was
re-incubated for 1 h. Next, 0.1 mL of 1% iodine solution was added, and after adding
5 mL distilled water, the absorbance was taken at 565 nm. Sample, substrate and
α-amylase blank determinations were carried out under the same reaction conditions.
Inhibition
of enzyme activity was calculated as (%) = (A-C) X100/ (BC)
Where,
A= absorbance of the sample, B= absorbance of blank (without α-amylase), and
C= absorbance of control (without sample).
B)
Glucose diffusion inhibitory
study15, 16
An
aqueous extract of all the plants was prepared, 1ml of the extract was then placed
in a dialysis membrane along with a glucose solution (0.22mM in 0.15 M sodium chloride).
It was then tied at both ends using thread and it was immersed in a beaker containing
40ml of 0.15 M sodium chloride and 10ml of distilled water. The control contained
1ml of 0.15M sodium chloride containing 22mM glucose and 1ml of distilled water.
The beakers were then placed on an orbital shaker and kept at room temperature.
The movement of glucose into the external solution was monitored every half hour.
Three replications of this were done for 3 hours
2.3.2. Anti-inflammatory activity of root extracts
A)
Inhibition of albumin denaturation
17, 18, 19
The
anti-inflammatory activity of Apama siliquosa was studied by using inhibition
of albumin denaturation technique which was studied according to Mizushima et
al and Sakat et al followed with minor modifications. The reaction mixture
consists of test extracts and 1% aqueous solution of bovine albumin fraction, pH
of the reaction mixture was adjusted using a small amount of 1N HCl. The sample
extracts were incubated at 37 °C for 20 min. and then heated to 51 °C for 20 min.,
after cooling the samples the turbidity was measured at 660nm. The experiment was
performed in triplicate. The percentage of inhibition of protein denaturation was
calculated as follows
Percentage
inhibition= (Ab Control-Ab Sample) 100/Ab control
B)
Membrane stabilization 20, 21, 22
1.
Preparation of red blood cells (RBCs) suspension
The
blood was collected from a healthy human volunteer who has not taken any NSAIDs
(Non-Steroidal Anti-Inflammatory Drugs) for 2 weeks prior to the experiment and
transferred to the centrifuge tubes. The tubes were centrifuged at 3000 rpm for
10 min and were washed three times with equal volume of normal saline.
2.
Heat-induced hemolysis:
The
reaction mixture (2ml) consisted of 1ml test sample of different concentrations
(100-500μg/ml) and 1ml of 10%RBCs suspensions, instead of the test sample the
only saline was added to the control tube. Aspirin was used as a standard drug.
All the centrifuge tubes containing reaction mixture were incubated in a water bath
at 56°C for 30min. At the end of the incubation, the tubes were cooled under running
tap water. The reaction mixture was centrifuged at 2500rpm for 5 min and the absorbance
of supernatants was taken at 560nm. The experiment was performed in triplicates
for all the test samples. The percentage inhibition of hemolysis was calculated
as follows
Ab control-Ab sample
Percentage inhibition= ––––––––––––––––––––––––––X
100
Ab control
2.4. Statistical analysis
All
data were expressed in Mean±SEM and calculations were done in Microsoft excel 2007.
3. RESULTS AND DISCUSSIONS:
The
extract was subjected to preliminary phytochemical screening and it revealed the
presence of various phytochemicals such as phenols, flavonoids, tannins, terpenes,
saponins, alkaloids and glycosides which are responsible for the anti-diabetic and
anti-inflammatory property.
Determination
of antidiabetic activity were performed by alpha-amylase inhibition and glucose
diffusion inhibitory study. In alpha amylase inhibition assay, it has been found
that the methanolic extract of the plant Apama siliquosa exhibited potent
inhibition on alpha-amylase. IC 50 of the plant extract was found to be 15.75 μg
/ml and that of standard drug was found to be 8.14mcg/ml. It is shown in Table no.1,
figure no.1 and 2.
Table
No.1 Determination of Antidiabetic activity by α-amylase enzyme inhibition
Blank
value=0.247±0.26
|
Concentration
(µg/ml)
|
Control
|
Control
(A.siliquosa)
|
Methanol Extract of A.siliquosa
|
Standard Drug Acarbose
|
Percentage inhibition of α-amylase
by Acarbose
|
Percentage inhibition of α-amylase
by A.siliquosa
|
|
100
|
0.066±0.07
|
0.068±0.21
|
0.205±0.19
|
0.224±0.26
|
87.29
|
76.53
|
|
75
|
0.057±0.13
|
0.053±0.04
|
0.191±0.11
|
0.212±0.08
|
81.57
|
71.13
|
|
50
|
0.051±0.09
|
0.051±0.15
|
0.172±0.11
|
0.183±0.14
|
67.34
|
61.73
|
|
25
|
0.047±0.10
|
0.044±0.12
|
0.150±0.16
|
0.160±0.09
|
56.50
|
52.21
|
Where,
A= absorbance of the sample, B= absorbance of blank (without α-amylase), and
C= absorbance of the control (without starch).
|
Figure No.1 Determination of Antidiabetic activity by
Inhibition of α-amylase enzyme by Acarbose IC 50 value of Acarbose=8.14
|
Figure No.2 Determination antidiabetic activity by inhibition
of α-amylase enzyme by A.siliquosa IC 50 value of A.siliquosa=15.75μg/ml
|
In the case of glucose diffusion inhibitory study, it has
been found that methanolic extract of the plant is effective in inhibiting the movement
of glucose across the membrane. The highest inhibitory effect of 72.72% is shown
at concentration of 150μg/ml.It is shown in the Table no 2, 3 and
figure no.3.
Table
No.2. Determination of percentage inhibition of Glucose diffusion of methanol extract
A.siliquosa
|
Time(min)
|
Control
|
Methanol extract of A.siliquosa
(400µg/ml)
|
S.E.M
|
Methanol extract of A.siliquosa
(200µg/ml)
|
S.E.M
|
Percentage Inhibition of glucose
diffusion
|
Percentage Inhibition of glucose
diffusion
|
|
30
|
0.492±0.057
|
0.191±0.164
|
0.0947
|
0.213±0.29
|
0.1676
|
56.70
|
61.17
|
|
60
|
0.569±0.121
|
0.187±0.093
|
0.0537
|
0.226±0.012
|
0.0069
|
60.28
|
67.13
|
|
90
|
0.582±0.098
|
0.171±0.348
|
0.2011
|
0.231±0.086
|
0.0497
|
60.30
|
70.61
|
|
120
|
0.601±0.136
|
0.169±0.046
|
0.0265
|
0.233±0.103
|
0.0595
|
61.23
|
71.88
|
|
150
|
0.616±0.124
|
0.168±0.206
|
0.1190
|
0.238±0.218
|
0.1260
|
61.36
|
72.72
|
Table
No.3. Determination of percentage inhibition of Glucose diffusion of Acarbose
|
Time(min)
|
Control
|
Acarbose
|
S.E.D
|
Percentage Inhibition of glucose
diffusion
|
|
30
|
0.492±0.057
|
0.157±0.267
|
0.1543
|
68.08
|
|
60
|
0.569 0.121
|
0.153±0.069
|
0.0398
|
73.11
|
|
90
|
0.582±0.098
|
0.149±0.043
|
0.0248
|
74.39
|
|
120
|
0.601±0.136
|
0.140±0.136
|
0.0786
|
76.70
|
|
150
|
0.616±0.124
|
0.138±0.084
|
0.0485
|
77.59
|
Figure No. 3. Determination of Glucose diffusion
To determine the anti-inflammatory activity protein denaturation
study and heat induced hemolysis were performed. In protein denaturation study,
ability of plant extract to inhibit protein denaturation was studied. The extract
was found to be effective in inhibiting heat induced albumin denaturation and IC50
value calculated as 39.5mcg/ml. Maximum inhibition of 74.76% was observed
at 500μg/ml. Aspirin is used as the standard anti-inflammatory drug and the
extract is less potent than the standard drug. It is shown in the Table no. 4,
figure no.4 and 5.
Table No. 4 Determination of percentage inhibition of Albumin
denaturation
|
Concentration
(µg/ml)
|
Control
|
Absorbance at 660nm
|
Percentage Inhibition
|
|
100
|
0.42±0.19
|
0.187±0.03
|
55.47
|
|
200
|
0.166±0.14
|
60.47
|
|
300
|
0.129±0.07
|
69.28
|
|
400
|
0.119±0.16
|
71.66
|
|
500
|
0.106±0.11
|
74.76
|
Figure No. 4. Determination of Inhibition of albumin denaturation
IC50 VALUE=39.5μg/ml
The extract was also effective in inhibiting heat induced
hemolysis at different concentrations such as 100,200,300,400 and 500μg/ml.
At concentration of 500μg/ml the plant extract showed maximum percentage inhibition
of 78.65%. IC 50 of the plant extract was found to be 36.30mcg/ml. It is shown in
the table no.5, figure no.5 and 6.
Table No. 5. Determination of Percentage Inhibition of Heat
induced hemolysis
|
Concentration
(µg/ml)
|
Control
|
Absorbance at 660nm
|
Percentage Inhibition
|
|
100
|
0.52±0.06
|
0.252±0.23
|
51.53
|
|
200
|
0.189±0.08
|
63.65
|
|
300
|
0.148±0.12
|
71.53
|
|
400
|
0.135±0.26
|
74.03
|
|
500
|
0.111±0.12
|
78.65
|
Figure No. 5. Determination of Inhibition of Heat induced
hemolysis IC50 value = 36.30 μg/ml
4. CONCLUSION:
The research was carried out to evaluate the anti-diabetic and
anti-inflammatory activity of the methanolic extract of the plant Apama siliquosa
Lamk. Anti-diabetic property of the extract was evaluated in-vitro by glucose
diffusion inhibitory assay and alpha-amylase inhibition assay. The present findings
in both tests, showed that the root extract of Apama siliquosa Lamk is capable
of exhibiting significant antidiabetic property. Anti-inflammatory property of the
extract was evaluated by inhibition of albumin denaturation as well as heat induced
hemolysis. The result of anti-inflammatory studies indicated that the root extracts
of Apama siliquosa possess significant anti-inflammatory properties. Further
studies to be conducted to find out the bioactive compounds responsible for these
effects are necessary.
5. ACKNOWLEDGEMENT:
Our heartful thanks to the Principal and Administrator, Nirmala
College of Pharmacy Muvattupuzha, for granting permission to utilize the facilities
to carry out this work.
6. CONFLICT OF INTEREST:
The authors have no conflicts of interest to declare that
they are directly relevant to the content of this manuscript
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