Post on 26-Feb-2020
transcript
54
Chapter-4
Antidiabetic activity of the selected plants by in vitro models
The selected 15 medicinal plants [Madhuca indica (bark), Ancardium occidentalis
(bark), Basella alba (aerial parts), Echinaps echinatus (aerial parts), Holoptetea integrifolia
(bark), Mangifera indica (kernels), Caesalpinia bounduc (Kernels), Limonia acidissima (bark),
Bauhinia varigata (bark), Spondias mangifera (bark), Erythrina variegate (bark), Amaranthus
viridis (areal parts), Pondanus odoratus (aerial parts) , Ichnocarpus frutescens (aerial parts)
and Cressa critica (aerial parts) ] for the screening of anti diabetic potential, these plants
are being used by the vidyas for the treatment of Diabetes mellitus in and around
Siddarabetta region. In the previous chapter some the extracts shown free radical
scavenging property. Hence, here an attempt made to know the in vitro anti diabetic
activity of the selected drug extracts by using -amylase and -glucosidase enzyme
inhibition assay models. These enzyme activities in the body are responsible for
postprandial hyperglycemia by break down of dietary carbohydrates in to glucose.
Hence, any of the selected plant extracts posses the inhibitory effect of these enzymes
may lead to reduction in post prandial hyperglycemia in diabetic condition.
Postprandial hyperglycemia has been proposed as an independent risk factor for
CVD (Cavarape et al., 2001). Therefore, control of postprandial hyperglycemia is
suggested to be important in the treatment of diabetes and prevention of cardiovascular
complications. Inhibitors of DPP IV, -amylase and -glucosidase, which are
particularly useful in postprandial glycemic control, are good candidates for prevention
of diabetic complications.
In previous chapters, we have discussed the in vitro antioxidant activity of all
the selected plant extracts. Some extracts exhibited potent free radical scavenging
property. It is well known that control of postprandial glacemia is very essential in
controlling long term deleterious effect of hyperoglycemia. Therefore, we thought it
55
worthwhile to elucidate the effect of all the selected plant extract in battery of targets
involved glucose homeostatic. In the present chapter, we propose to investigate the
effect of all the selected plant extracts in the following enzyme inhibition assay.
Alpha-amylase inhibition assay
Alpha-glucosidase inhibition assay
Importance Alpha-amylase enzyme in the body
In humans, the digestion of starch involves several stages. Initially, partial
digestion by the salivary amylase results in the degradation of polymeric substrates into
shorter oligomers. Later on in the gut these are further hydrolyzed by pancreatic -
amylases into maltose, maltotriose and small malto-oligosaccharides. The digestive
enzyme (-amylase) is responsible for hydrolyzing dietary starch (maltose), which
breaks down into glucose prior to absorption. Inhibition of -amylase can lead to
reduction in post prandial hyperglycemia in diabetic condition (Roux et al., 2001.,
Lonkisch et al., 1998) .
Importance of Alpha-glucosidase enzyme in the body
Alpha-glucosidase is a membrane bound enzyme located on the epithelium of
the small intestine, catalyzing the cleavage of disaccharides to form glucose. Inhibitors
can retard the uptake of dietary carbohydrates and suppress post-prandial
hyperglycemia. Therefore, inhibition of -glucosidase could be one of the most
effective approaches to control diabetes (Sheikh et al., 2008). Glucosidases are not only
essential to carbohydrate digestion, but also vital for the processing of glycoprotein and
glycolipids. This enzyme is a target for antiviral agents that interfere with the formation
of essential glycoproteins required in viral assembly, secretion and infection (Gruters et
56
al., 987). Glucosidases are also involved in a variety of metabolic disorders and
carcinogenesis (Dennis et al., 1987).
4.1. Materials and methods
The selected 15 plants were shade dried and coarsely powdered and extracted
with ethyl alcohol and distilled water as described in Chapter 3. These, extracts were
serially diluted to get a required concentration to perform both alpha amylase and alpha
glucosidase enzyme inhibition assays.
4.1.1. Alpha- amylase inhibition assay principle and procedure.
Alpha-amylase activity can be measured in-vitro by hydrolysis of starch in
presence of -amylase enzyme. This process was quantified by using iodine, which
gives blue colour with starch. The reduced intensity of blue colour indicates the
enzyme-induced hydrolysis of starch in to monosaccharides. If the substance/extract
possesses -amylase inhibitory activity, the intensity of blue colour will be more. In
other words, the intensity of blue colour in test sample is directly proportional to -
amylase inhibitory activity (Sheikh et al., 2008).
Enzyme: (Type VI B: From porcine pancreas, 5,00,000 U) [15.8 U/mg solid at pH
6.9]- Stored at 2-8C- Sigma, USA (A3176)
Substrate: Starch 1%
Positive Control: Acarbose- Stored at RT-Glucobay (Bayer pharma, India)
Sodium dihydrogen orthophosphate (NaH2PO4.2H2O)- Himedia (RM-1255)- stored
at RT
Disodium hydrogen phosphate (Na2HPO4.2H2O)- Himedia (RM-257)- stored at RT
Indicator: Iodine solution 1%
Instrument- UV- Visible Spectrometer.
57
Preparation of working solution
Phosphate Buffer (40 mM, pH 7, 25C):
Solution A: 6.24 g of NaH2PO4.2H2O ----- 1L
Solution B: 7.12 g of Na2HPO4.2H2O ----- 1L
Enzyme (0.5128 U/ml)- 3.246 mg -amylase ----- 100 ml of 40 mM Phosphate
Buffer
NaCl solution (0.006 M)
Positive control:
Stock- 50 mg of Acarbose in 50 ml of 40 mM Phosphate buffer
Working stock: Take 25 l of stock, made upto 10 ml (2.5 g/ml) with 40 mM
Phosphate buffer
Procedure
Alpha-amylase activity was carried out by starch-iodine method. 10 L of α-
amylase solution (0.025 mg/mL) was mixed with 390 L 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 L 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/ (B-C),
where, A= absorbance of the sample, B= absorbance of blank (without α-amylase), and
C= absorbance of control (without starch).
58
Chart 1. Schematic flow chart of -amylase enzyme inhibition assay procedure
4.1.2. Alpha-glucosidase inhibition assay principle and procedure.
Alpha-glucosidase activity can be measured in-vitro by determination of the
reducing sugar (glucose) arising from hydrolysis of sucrose by -glucosidase enzyme,
isolated from small intestine of rat.
Materials
-glucosidase enzyme- isolated from rat intestine (stored at -20C)
Substrate: Sucrose- Himedia, India (RM 3063)- Stored at RT
Positive control: Acarbose- Stored at RT-Glucobay (Bayer Pharma, India)
Total protein estimation kit (Biuret method)- Span diagnostics (B-0211)- Stored at
2-8C.
Added 390 l of 0.02M Phosphate buffer pH 7/ Positive control/ Different concentration of test samples +10 µ L of
-amylase
Pre-incubated at 37 C for 10 mins
Re-Incubated at 37 C for 1h
1hhh1811mins
Measured OD at 565 nm
Added 10 l of Starch
Added 0.1 ml 1% Iodine
solution+5ml of distill water
59
Sodium dihydrogen orthophosphate (NaH2PO4.2H2O)- Himedia, India (RM-1255)-
stored at RT
Disodium hydrogen phosphate (Na2HPO4.2H2O)- Himedia (RM-257)- stored at RT
Glucose reagent- ErbaAGAPPE diagnostics, India (AFP 11208100)- Stored at 2-
8C.
Instrument- UV- Visible Spectrometer.
Preparation of working solution
Phosphate Buffer (80 mM, pH 7.0, 25C):
Solution A: 1.248 g of NaH2PO4.2H2O ----- 100 ml
Solution B: 1.242 g of Na2HPO4.2H2O ----- 100 ml
Mix 39 ml of solution A + 61 ml of solution B, made upto 200 ml with
deionized water.
Isolation of enzyme: Rats were sacrificed, intestine removed and chilled with ice
cold 80 mM phosphate buffer. The intestine was then cut open, the mucosa scraped
off with a piece of glass rod and homogenized with four parts (v/v) of cold buffer.
Nuclei and large cell debris were removed by centrifugation at 2000 to 4000 rpm
for 10 mins and supernatant aliquoted into 1.5 ml vials and stored at -20C.
[Protein content= 0.5g/dl by Biuret method]
Substrate (37 mM): 316 mg of sucrose dissolved in 25 ml 80 mM Buffer.
Positive control:
Stock- 50 mg of Acarbose in 50 ml of 80 mM Phosphate buffer
Working stock: 50 l of stock, made upto 10 ml (5 g/ml) with 80 mM
Phosphate buffer
60
Chart. 2. Schematic flow chart of -amylase enzyme inhibition assay procedure
(Matsui et al., 2007)
Pre-incubated at 37 C for 30 mins
Incubated at 37 C for 20 mins
Added 500 l of 37 mM Sucrose
Added 225 l of 80mM Phosphate buffer pH 7.0/ Positive control/ Different concentration of test samples
+ 75 l of -glucosidase
Pre-incubation mix (300l)
Kept in boiling water bath for 2 mins,
cooled and added 250 l of glucose reagent
Incubated at RT for 10 mins
Measured OD at 510 nm
61
4.2. Result
4.2.1. Alpha- amylase inhibition assay
Table 4.1 shows inhibition -amylase by 30 plants extracts. All the tested extracts
showed dose dependent inhibition of enzyme. The extracts exhibited IC50 less than 100
µg/mL will be considered active in comparison with other tested extracts. Pondanus
odoratus aqueous extract (POAE), Madhuca indica aqueous extract (MIAE),
Mangifera indica ethanolic extract (MAEE), Cressa critica ethanolic extract (CCEE ),
Erythrina variegate aqueous extract (EVAE), Limonia acidissima ethanolic extract
(LAEE), Ancardium occidentalis aqueous extract (ACAE), Erythrina variegate
ethanolic extract (EVEE), Holoptetea integrifolia aqueous extract (HIAE), Bauhinia
varigata ethanolic extract (BVEE), Ichnocarpus frutescens aqueous extract (IFAE),
Caesalpinia bounduc ethanolic extract (CBEE), Pondanus odoratus ethanolic extract
(POEE ), Ichnocarpus frutescens ethanolic extract (IFEE), Caesalpinia bounduc
aqoueous extract (CBAE), Madhuca indica ethanolic extract (MIEE), Cressa critica
aqueous extract (CCAE), Holoptetea integrifolia ethanolic extract (HIEE) &
Ancardium occidentalis ethanolic extract (ACEE) showed enzyme inhibition activity
with IC50 10.36, 19.00, 20.00, 24.66, 33.3, 42.33, 44.67, 48.00, 48.00, 52.00, 54.66,
55.53, 56.00, 63.33, 64.00, 64.00, 72.00 & 84.66 g/mL respectively. Among these 19
extracts, aqueous extract of Pandanus odoratissimus (POAE) was considered as most
potent -amylase enzyme inhibition activity with low IC50 10.36 μg/mL value.
Standard acarbose showed an IC50 of 0.32 μg/mL under similar experimental conditions
(Fig. 4.1).
4.2.2. Alpha-glucosidase inhibition assay
Table 4.2 shows the -glucosidase inhibitory activity of 30 tested plant extracts.
All the tested extracts exhibited dose dependent inhibition of enzyme. Among the
62
tested extracts only 10 plant extracts showed IC50 less than 100 g/mL. Limonia
acidissima aqueous extract (LAAE), Echinaps echinatus aqueous extract (ECAE),
Mangifera indica aqueous extract (MAAE), Amaranthus viridis ethanolic extract
(AVEE), Amaranthus viridis aqueous extract (AVAE), Bauhinia varigata ethanolic
extract (BVEE), Caesalpinia bounduc ethanolic extract (CBEE), Mangifera indica
ethanolic extract (MAEE), Cressa critica aqueous extract (CCAE), Ancardium
occidentalis ethanolic extract (ACEE) have exhibited enzyme inhibition activity with
IC50 values 64.17, 68.33, 73.33, 75.83, 78.90, 75.83, 81.67, 85.33, 86.66 and 90.83
μg/mL. Aacarbose showed an IC50 of 5.36±2.69 μg/mL under similar experimental
conditions (Fig. 4.2). The order of -glucosidase inhibitory activity as Acarbose >
LAAE > ECAE > MAAE > AVEE > AVAE > BVEE > CBEE > MAEE > CCAE >
ACEE.
Table : 4.1. Comparison of the IC50 in μg /ml of various extracts against α amylase enzyme inhibition.
Extract IC50 in μg/ml EVEE 48.00 ± 0.67
EVAE 33.3 ± 1.13
MIEE 64.00 ± 1.15
MIAE 19.00 ± 0.15
POEE 56.00 ± 1.15
POAE 10.36 ± 3.32
IFEE 56.66 ± 2.31
IFAE 54.66 ± 1.76
CBEE 55.53 ± 2.40
CBAE 63.33 ± 1.76
SMEE > 200
SAME > 200
HIEE 72.00± 0.33
HIAE 48.00 ± 1.20
BAEE > 200
63
All values reported as Mean ± S.E.M (n=3). Where as Madhuca indica ethanolic extract (MIEE), Madhuca indica aqueous extract (MIAE), Ancardium occidentalis ethanolic extract(ACEE), Ancardium occidentalis aqueous extract (ACAE),Basella alba ethanolic extract (BAEE), Basella alba aqueous extract (BAAE), Echinaps echinatus ethanolic extract (ECEE), Echinaps echinatus aqueous extract (ECAE), Holoptetea integrifolia ethanolic extract (HIEE), Holoptetea integrifolia aqueous extract (HIAE), Mangifera indica ethanolic extract (MAEE), Mangifera indica aqueous extract (MAAE), Caesalpinia bounduc ethanolic extract (CBEE), Caesalpinia bounduc aqoueous extract (CBAE), Limonia acidissima ethanolic extract (LAEE), Limonia acidissima aqueous extract (LAAE), Bauhinia varigata ethanolic extract (BVEE), Bauhinia varigata aqueous extract (BVAE), Spondias mangifera ethanolic extract (SMEE), Spondias mangifera aqueous extract (SMAE), Erythrina variegate ethanolic extract (EVEE ),Erythrina variegate aqueous extract (EVAE), Amaranthus viridis ethanolic extract (AVEE ), Amaranthus viridis aqueous extract (AVAE ), Pondanus odoratus ethanolic extract (POEE ), Pondanus odoratus aqueous extract (POAE ),Ichnocarpus frutescens ethanolic extract (IFEE ), Ichnocarpus frutescens aqueous extract (IFAE ), Cressa critica ethanolic extract (CCEE ), Cressa critica aqueous extract (CCAE ).
Table: 4.2. Comparison of the IC 50 in μg /ml of various extracts against α glucosidase enzyme inhibition.
Extract IC 50 in μg/ml
EVEE 150.83 ± 1.67
EVAE 170.80 ± 8.78
MIEE 131.67 ± 0.83
MIAE 135.83 ± 1.67
POEE 167.50 ± 0.63
POAE 165.83 ± 2.20
BAAE > 200
ECEE > 200
ECAE > 200
LAEE 42.33± 0.88
LAAE > 200
BVEE 52.00± 1.15
BVAE > 200
ACEE 84.66± 2.35
ACAE 44.67 ±1.33
MAEE 20.00±0.58
MAAE 74.67± 1.76
AVEE > 200
AVAE > 200
CCEE 24.66±2.32
CCAE 64.00±1.02
Acarbose 0.32 ± 3.2
64
IFEE 151.67 ± 0.83
IFAE 103.33 ± 2.20
CBEE 81.67 ±5.83
CBAE > 200
SMEE > 200
SAME > 200
HIEE > 200
HIAE > 200
BAEE 105.83 ± 1.59
BAAE > 200
ECEE 185.00 ± 4.09
ECAE 68.33 ± 3.00
LAEE 185.00 ± 5.00
LAAE 64.17 ± 0.83
BVEE 75.83 ± .025
BVAE > 200
ACEE 90.83 ± 0.58
ACAE 108.33 ± 0.83
MAEE 85.33 ± 0.83
MAAE 73.33 ± 0.83
AVEE 75.83 ± 0.23
AVAE 78.90 ± 0.43
CCEE 140.83±2.36
CCAE 86.66±3.54
Acarbose 5.36±2.69 All values reported as Mean ± S.E.M (n=3). Where as Madhuca indica ethanolic extract (MIEE), Madhuca indica aqueous extract (MIAE), Ancardium occidentalis ethanolic extract(ACEE), Ancardium occidentalis aqueous extract (ACAE),Basella alba ethanolic extract (BAEE), Basella alba aqueous extract (BAAE), Echinaps echinatus ethanolic extract (ECEE), Echinaps echinatus aqueous extract (ECAE), Holoptetea integrifolia ethanolic extract (HIEE), Holoptetea integrifolia aqueous extract (HIAE), Mangifera indica ethanolic extract (MAEE), Mangifera indica aqueous extract (MAAE), Caesalpinia bounduc ethanolic extract (CBEE), Caesalpinia bounduc aqoueous extract (CBAE), Limonia acidissima ethanolic extract (LAEE), Limonia acidissima aqueous extract (LAAE), Bauhinia varigata ethanolic extract (BVEE), Bauhinia varigata aqueous extract (BVAE), Spondias mangifera ethanolic extract (SMEE), Spondias mangifera aqueous extract (SMAE), Erythrina variegate ethanolic extract (EVEE ),Erythrina variegate aqueous extract (EVAE), Amaranthus viridis ethanolic extract (AVEE ), Amaranthus viridis aqueous extract (AVAE ), Pondanus odoratus ethanolic extract (POEE ), Pondanus odoratus aqueous extract (POAE ),Ichnocarpus frutescens ethanolic extract (IFEE ), Ichnocarpus frutescens aqueous extract (IFAE ), Cressa critica ethanolic extract (CCEE ), Cressa critica aqueous extract (CCAE ).
65
Graphical representation of -amylase inhibition activity of selected medicinal plants
66
Graphical representation of alpha amylase enzyme inhibitory activity of ethanolic and aqueous extracts of the selected plants Erythrina variegate, Madhuca indica , Pondanus odoratus, Ichnocarpus frutescens, Limonia acidissima, Ancardium occidentalis, Mangifera indica, Cressa critica
67
Graphical representation of - glucosidase inhibition activity of selected medicinal plants
68
Graphical representation of alpha glucosidase enzyme inhibitory activity of ethanolic and aqueous extracts of the selected plants Pondanus odoratus, Mangifera indica, Ichnocarpus frutescens, Madhuca indica, Limonia acidissima, Erythrina variegate, Echinaps echinatus, Caesalpinia bounduc, Ancardium occidentalis, Amaranthus viridis , Basella alba.
4.3. Discussion
The treatment goal of diabetic patients is to maintain near normal levels of
glycemic control, in both fasting and post-prandial conditions. Many natural sources
have been investigated with respect to suppression of glucose production from the
carbohydrates in the gut or glucose absorption from the intestine (Mastsui et al .,
69
2001). Alpha-amylase catalyses the hydrolysis of alpha-1,4-glycosidic linkages of
starch, glycogen and various oligosaccharides. Alpha-glucosidase further breaks
down the disaccharides to simple sugars, readily available for intestinal absorption.
The inhibition of their activity in the digestive tract of humans is considered to be
effective tool to control diabetes. In addition, these effects may leads to diminished
absorption of monosaccharides (Hara and Honda., 1990). Therefore, effective and
nontoxic inhibitors of alpha-amylase and alpha-glucosidase have long been sought.
In this study, 30 extracts prepared from 15 selected traditionally claimed herbs
available around Sidderabetta region, Tumkur. The major outcome of this study
reveals that both the extracts of Erythryna varigata, Madhuca indica, Pandanus
odoratissimus, Ichnocarpus frutescens, Ancardia occidentalis, Mangifera indica,
Cressa critica have exhibited potent inhibition of alpha-amylase and alpha-
glucosidase enzyme activity. In addition, only aqueous extract of Caesalpinia
boundac was able to inhibit both the enzymes. However, both extracts of Holoptetea
integrifolia showed only alpha-amylase inhibition, whereas Amaranthus viridis
exhibited only alpha-glucosidase inhibition property.
In the present study, we selected 15 traditionally claimed herbs of
Sidderabetta region of Tumkur for the treatment of diabetes. Out of 30 extracts
prepared from 15 plants (ethanolic and aqueous of each) seven plants such as
Erythryna varigata, Madhuca indica, Pandanus odoratissimus, Ichnocarpus
frutescens, Ancardia occidentalis, Mangifera indica, Cressa critica exhibit potent
alpha-amylase and alpha glucosidase inhibitory activity. Similar trend of activity (in
vitro antioxidant) was observed for these plants (chapter 3).
70
The present study provided results to justify the traditional claim of herbs for
antidiabetic activity. Hence, the author further extended the work to confirm
antidiabetic activity by acute toxicity studies and on in vivo models.