J Sci Food Agric 1990,52, 527-536

Studies on Amylase Inhibitors in Dieffenbachia macdata

Sriram Padmanabhan and Narayan V Shastri"

Department of Biochemistry, Nagpur University, LIT Campus, Amravati Road, Nagpur-440 010, India

(Received 9 October 1989; accepted 24 February 1990)

ABSTRACT

Different parts of the Dieffenbachia maculata Schott plant have been found to possess amylase inhibitor activity. The inhibitor has been found to be non- dialysable and relatively heat-stable. Studies with crude extracts of D maculata plant indicate that the inhibitor is of the non-competitive type and is active against human salivary amylase, porcine pancreatic a-amylase, Bacillus subtilis a-amylase and sweet potato P-amylase.

Key words: Diffenbachia, ci-amylase, amylase inhibitors.

INTRODUCTION

Dieffenbachia, a popular indoor plant of the Araceae family, is well known for its toxicity. A large number of pets and children have been reported to be poisoned every year by this plant (Arditti and Rodriguez 1982). However, the compound(s) responsible for its toxic effects has not been fully identified. This plant has also been shown to possess some therapeutic properties such as antigout activity (Walter and Khanna 1972; Arditti and Rodriguez 1982).

The possible involvement of proteinases in the toxic effects suggested by some workers (Walter and Khanna 1972; Kuballa et a1 1981) remains controversial. The present workers have found that the proteinase activity in various parts of the plant is low. On the other hand, considerable trypsin inhibitor activity has been observed (Padmanabhan S and Shastri N V unpublished).

Experiments conducted in this laboratory also indicate the presence of amylase inhibitors in this plant. Amylase inhibitors are known to be autinutritional (Strumeyer 1972; Silano 1978). It was therefore thought worth while to conduct experiments on the amylase of D rnaculata.

* To whom correspondence should be addressed.

527

J Sci Food Agric 0022-5142/90/$03.50 0 1990 SCI. Printed in Great Britain

528 S Padmanabhan, N V Shastri

EXPERIMENTAL

Materials

Plant samples Dieffenbachia maculata Schott plants grown in the author's garden were used. Only fresh and mature parts of the plant were used, unless mentioned otherwise.

Chemicals and enzymes Pancreatin of porcine pancrease, Bacillus subtilis a-amylase (type XIA, EC 3.2.1 . l ) and polyvinyl polypyrrolidone (PVPP) were procured from Sigma Chemical Company, St Louis, MO, USA. Soluble starch was the product of Reanel, Budapest, Hungary, and 3,5-dinitrosalicylic acid (DNS) was the product of Loba Chemicals, Bombay, India. All other reagents were of analytical grade.

Methods

Preparation of Dieffenbachia maculata extract Homogenates of various parts of D maculata were prepared in cold 0.15 M NaCl containing 10 g litre-' PVPP, unless mentioned otherwise. The homogenate (250 g litre-') was centrifuged at 0-4°C and 5000 x g for 15 min. The supernate was taken as the inhibitor source.

Preparation of human salivary amylase (EC 3.2.1.1) Human saliva was collected and centrifuged at 5000 x g for 20 min at 0-4°C. The supernate was taken as the enzyme source.

P-Amylase (EC 3.2.1.2) 8-Amylase of sweet potato was prepared according to Bernfeld's method (Bernfeld 1955).

Preparation of substrate For a-amylase soluble starch was dissolved (10 g litre-') in 0.02 M sodium phosphate buffer, pH 6.9. For p-amylase soluble starch was dissolved (10 g litre-') in 0.016 M acetate buffer, pH 4.9.

Assay of a-amylase (EC 3.2.1.1) activity Amylase activity was measured by the method of Bernfeld with some modifications (Bernfeld 1955). A suitable aliquot (0.1 ml) of human salivary amylase was incubated with 0.15 M NaCl in a final volume of 1.5 ml for 15 min at 37°C. Starch solution (0.5 ml, l o g litre-') was added to initiate the reaction. After 5 min incubation at 37°C the reaction was terminated by adding 1 ml of DNS reagent. The solution was kept in a boiling water bath for lOmin, cooled and diluted to 13 ml with distilled water. The colour intensity was measured at 540 nm. A calibration curve established with maltose was used to calculate the amylase activity. One unit of amylase activity is defined as that amount of enzyme which liberates 1 mg maltose under the given assay conditions (5 min, 37°C).

For the assay of pancreatin (from porcine pancreas) and a-amylase from B subtilis,

Amylase inhibitors in Dieffenbachia maculata 5 29

25 pg protein of pancreatin a-amylase or 27 pg protein of B subtilis a-amylase was used in the assay mixture. For P-amylase activity, 0.1 ml of P-amylase was used.

Assay of amylase inhibitors A suitable amount of inhibitor from D maculata was preincubated with 0.1 ml of amylase and 0-15 M NaCl in a total volume of 1.5 ml for 15 min at 37°C. Controls without inhibitors were run simultaneously. The enzyme reaction was initiated by the addition of 0.5 ml of starch solution ( l o g litre-'). The decrease in amylase activity was a measure of the inhibitory activity. One unit of inhibitor activity is defined as the amount of inhibitor that reduces the amylase activity under the assay conditions by one unit. Specific activity of the inhibitor is expressed as units mg-' protein. Protein was determined by the method of Lowry et a1 (1951) using bovine serum albumin as standard.

Extraction of amylase inhibitors The inhibitors from various parts of D maculata were extracted in different media and were examined for inhibitory activity against human salivary amylase.

Effect of heat on a-amylase inhibitors Extracts of D maculata leaf and stem (middle section) in different extraction media were heated in a boiling water bath for various periods of time, cooled and assayed for residual inhibitory activity against human salivary amylase.

Effect of p H on a-amylase inhibitors Suitable aliquots of inhibitor were incubated with 0.1 M buffers of different pH values, acetate buffer pH 3*0,4.0,5.0,6.0, sodium phosphate buffer pH 6.0,7.0,8*0, glycine/NaOH buffer pH 8.0,9.0,10.2, and 0.1 M NaOH (pH 12.4) for 16 h at 4°C in a total volume of 0.8 ml. After addition of 0.4 ml of 0.2 M sodium phosphate buffer (pH 6.9), 0.1 ml of amylase and 0.2 ml of 0.15 M NaCl, the inhibitory activity was measured against that of human salivary amylase. Controls without inhibitors were run simultaneously.

Effect of preincubation with a-amylase and starch on amylase inhibitors

Human salivary amylase (0.1 ml) was incubated with suitable aliquots of inhibitor solution and 015 M NaCl in a final volume of 1.5 ml for various periods of time. The reaction was initiated by the addition of 0.5 ml of 10 g litre-' starch solution.

Starch solution (0.5 ml, 10 g litre-') was preincubated with the inhibitor for various periods of time, and the enzyme reaction was initiated by the addition of human salivary amylase and 0.15 M NaCl.

Effect of dialysis on a-amylase inhibitors Inhibitors of D maculata leaf and stem (middle section) were extracted in distilled water, 0.15 M NaCl and 0.005 M HCl (pH 5.1) separately and were dialysed against 50 volumes of the respective medium for 16 h at 04°C . The dialysates were centrifuged at 5000xg for 10min to remove the inert precipitate, and the supernates were examined for inhibitory activity against human salivary amylase.

TA

BL

E 1

E

xtra

ctio

n of

a-a

myl

ase

inhi

bito

rs fr

om d

iffer

ent p

arts

of D

ieffe

nbac

hia

mac

ulat

a

Plan

t par

t a-

Amyl

ase

inhi

bito

r ac

tivity

for

a g

iven

ext

ract

ion

med

ium

Dis

tille

d w

ater

So

dium

pho

spha

te b

uffe

r 0.

15 M

NaC

l 0.

02 M

pH

69

U g-'

FW

U

mg-

' pr

U

g-'

F

W

U m

g-'

pr

U g-

' F

W

U m

g-'

pr

Leaf

1.

31

0.23

1 *

60

0.37

2-

48

044

Petio

le

0,58

0.

54

0.43

0.

50

2.03

1.

41

Stem

(api

cal s

ectio

n)

3.93

1.

54

2.33

1.

33

2.76

1-

33

Stem

(mid

dle

sect

ion)

4.

50

2.06

3.

35

1.29

3.

34

1.70

St

em (b

asal

sec

tion)

1.

89

1 .oo

2.03

1.

04

1.75

1 -04

Amylase inhibitors in Dieffenbachia maculata 53 1

RESULTS AND DISCUSSION

The inhibitory activities in different parts of D maculata against human salivary amylase are shown in Table 1 and Figs 1-3. The stem, which is reported to be the most toxic part, shows the highest inhibitory activity followed by the petiole and leaf. Different parts of the stem show different inhibitory activities, the middle portion showing the highest specific activity and the basal portion showing minimal specific activity. Table 1 also indicates that the efficacy of different extraction media differs from tissue to tissue; overall, water was the best extractant. None of the parts tested had any detectable endogenous a-amylase activity under the assay conditions.

Fig 1. Effect of substrate concentration on a-amylase activity in the presence (0) and absence (0) of the inhibitor from Dieffenbachia maculata leaf.

5-1

Fig 2. Effect of substrate concentration on a-amylase activity in the presence (0) and absence (0) of the inhibitor from Dieffenbachia maculata stem (middle section).

532

45t S Padmanabhan, N V Shastri

Fig 3. Effect of inhibitor concentration of Dieffenbachia maculata leaf and stem (middle section) on a-amylase activity; 1.418mg protein ml-' leaf inhibitor (a), 0.810mg protein ml-' stem inhibitor (0). Inhibitor (ml)

The data presented in Table 2 indicate that a-amylase inhibitors extracted in 0.15 M NaCl from various parts of D maculata were inhibitory to the different a- amylases used.

It is apparent from Table 3 that, although the a-amylase inhibitors of D maculata are relatively heat stable, their stability was highest in the presence of 0.15 M NaCl. It was also observed that a-amylase inhibitors of all parts of D maculata are more stable at the neutral pH although considerable activity was retained up to pH 3.0 on the acid side and 12.4 on the basic side.

The magnitude of a-amylase inhibitory activity was found to be dependent on the preincubation of the inhibitors with amylase. Maximal inhibition was observed after preincubation for 5 to 15 min. However, preincubation of the inhibitor with starch for various periods did not alter the inhibitor activity significantly. This probably indicates a minimal direct interaction involved between substrate and inhibitor during the course of the reaction. The nature of a-amylase inhibition by leaf and stem (middle section) inhibitor, as evaluated from the Lineweaver-Burke plot drawn using the method of least squares, was found to be noncompetitive.

Although the inhibitors appear to be non-dialysable (Table 4), the recovery after dialysis was rather low irrespective of the medium used. This was probably due to precipitation and inactivation of the inhibitors during dialysis. Gudiseva et a1 (1981)

TA

BL

E 2

E

ffect

of a-

amyl

ase i

nhib

itors

from

diff

eren

t par

ts o

f Die

ffenb

achi

a m

acul

ata o

n di

ffere

nt a-

amyl

ases

(inh

ibito

r ext

ract

ed in

0.1

5 M

NaC

l)

Plan

t par

t a-

Amyl

ase

inhi

bito

r ac

tivity

Hum

an s

aliv

ary

amyl

ase

Porc

ine

panc

reat

in

B su

btili

s u-

amyl

ase

U g

-'

FW

U

mg-

' pr

U

g-'

FW

U

mg-

I pr

U

g-'

FW

U

mg-'

pr

Leaf

2.

48

043

204

022

233

0.25

Pe

tiole

2.

03

1.41

3.

05

1.31

2.

62

1.13

St

em (

apic

al s

ectio

n)

2.76

1.

33

2.03

0.

67

3.20

1.

05

Ster

n (m

iddl

e se

ctio

n)

3.34

1.

70

2.9 1

1.

34

3.20

1.

47

Stem

(ba

sal s

ectio

n)

1.75

1.

04

2.04

1.

22

2.04

1.

22

534 S Padmanabhan. N V Shastri

TABLE 3 Effect of heat on a-amylase inhibitors of different parts of Dieffenbachia maculata

Plant part Extraction medium Amylase inhibitory activity remaining (%) afer treatment at 100°C

Omin Smin 10 min 30 min 60 rnin

Leaf Distilled water 100 100 77.2 77.2 33.3

Leaf 0.02 M, Na phosphate 100 91.3 91.3 91.3 54.6

Stem (middle section) 0.02 M, Na phosphate 100 100 59-7 33.0 33.0

Leaf 0.15 M, NaCl 100 94.2 64.8 64.8 64.8 Stem (middle section) 0.15 M, NaCl 100 100 87.7 87.7 87.7

Stem (middle section Distilled water 100 95 77.7 77.7 77.7

buffer, pH 6.9

buffer, pH 6.9

TABLE 4 Effect of dialysis on a-amylase inhibitors of Dieffenbachia maculata

Plant Extraction ~

a-Amylase inhibitor activity part medium

U ml-' s o h U mg- pr pr mg ml-' soh

Oh" I6h" O h 1 6 h O h 1 6 h

Leaf Distilled water 0.836 0.218 0.306 0.160 273 1.36 0.15 M NaCl 0.582 0.218 0.267 0.188 2.18 1.16 0-005 M HC1 0.764 0.364 0.362 0.293 211 1.24 (PH 5.1)

Stem (middle Distilled water 0.654 0.254 0.840 0.446 0.78 0.57 section) 0.15 M NaCl 0.474 0.218 0.585 0.436 0.81 0.50

0.005 M HCl 0.474 0.160 0.585 0.267 0.81 0.60 (PH 5.1)

~~ ___

Period of dialysis.

have reported that dialysis of amylase inhibitors of sorghum (IS-2059 variety) in the medium other than 0.005 M HC1, pH 5.0, resulted in complete loss of activity. In the present experiments, however, HCl was not found to improve recovery after dialysis.

Amylase inhibitors of D maculuta have also been found to inhibit P-amylase of sweet potato to a large extent (Table 5) . No endogenous P-amylase activity was observed under the assay conditions.

Some trypsin inhibitors are also known to inhibit a-amylase (Manjunath et ul 1983). The amylase inhibitor reported in this paper seems to be different from the trypsin inhibitor of D maculata (reported by the present authors) as trypsin inhibitor has been found to be heat labile, being destroyed completely at 100°C within 15 min. On the other hand, the amylase inhibitor is comparatively heat stable.

Amylase inhibitors in Dieffenbachia maculata 535

536 S Padmanabhan, N V Shastri

As amylase inhibitors are known to be antinutritional, the known toxic effects of Dieffenbachia plants may be attributed, at least in part, to the presence of such inhibitors.

It has been reported that ingestion of Dieffenbachia extracts by humans causes excess salivation, swelling of salivary ducts and temporary speechlessness (Arditti and Rodriguez 1982). It is not known whether the functional properties of saliva such as amylase activity are affected in such a condition. Our observation that D rnaculata extracts are capable of inhibiting salivary amylase in vitro suggests the possibility that a similar effect is also produced in vivo.

It is known that defective digestion generally results from pancreatic diseases and consequent upon deficiency of pancreatic digestive enzymes (Kao and Scheer 1984). As human pancreatic amylase is similar to human salivary amylase, and as D rnaculata extracts inhibit the latter as well as porcine pancreatic a-amylase, the toxic effects of Dieffenbachia ingestion may be related to the presence of amylase inhibitors in these plants.

Recent studies in this laboratory on albino rats given D maculata extract indicate significantly lowered activity of liver and serum amylases (Padmanabhan S and Shastri N V unpublished).

ACKNOWLEDGEMENTS

Financial support by the Council of Scientific and Industrial Research, New Delhi, to one of the authors (SP) is gratefully acknowledged. The authors are indebted to Prof H F Daginawala, Head of the Department of Biochemistry, Nagpur University, Nagpur, for his keen interest.

REFERENCES

Arditti J, Rodriguez E 1982 Diefenbachia; uses, abuses and toxic constituents; a review. J Ethnopharmacol5 293-302.

Bernfeld P 1955 Amylases, a and 8. In: Methods in Enzymology, Vol 1, eds Colowick S P & Kaplan N 0. Academic Press, New York, pp 149-158.

Gudiseva C, Suryaprasad R D, Pattabiram T N 1981 Natural plant enzyme inhibitors, a- amylase inhibitors in millets. J Sci Food Agric 32 9-16.

Kao Y S, Scheer W D 1984 Todd-Sandford Clinical Diagnosis and Management by Laboratory Methods (15th edn), eds Davidson I & Henry B J. W B Saunders, Philadelphia.

Kuballa B, Lugnier A A J, Anton R 1981 Study of Dieffenbachia-induced edema in mouse and rat hindpaw; respective role of oxalate needles and trypsin-like protease. Toxic01 Appl Pharmacol58 444-451.

Lowry 0 H, Rosebrough N J, Farr A L, Randall R J 1951 Protein measurement with the folin phenol reagent. J Biot Chem 193 265-275.

Manjunath N H, Veerabhadrappa P S, Virupaksha T K 1983 Isolation and characterisation of a trypsin inhibitor from finger millet. Phytochemistry 22(11) 2349-2357.

Silano V 1978 Biochemical and nutritional significance of wheat albumin inhibitors of a- amylase. Cereal Chem 55 723-731.

Strumeyer D H 1972 Protein amylase inhibitors in the gliadin fraction of wheat and rye flour. Possible factors in celiac disease. Nutr Rep Int 6 45-52.

Walter W G, Khanna P N 1972 Chemistry of the aroids I. Dieffenbachia seguine, amoena and picta. Econ Bot 26 364372.