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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

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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.

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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).

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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

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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

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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

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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

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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

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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

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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 ).

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Graphical representation of -amylase inhibition activity of selected medicinal plants

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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

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Graphical representation of - glucosidase inhibition activity of selected medicinal plants

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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 .,

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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).

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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.