Original Research Article                                                             DOI: 10.18231/2348-7682.2017.0005 

Panacea Journal of Medical Sciences, January-April,2017;7(1): 15-18                                                                   15 

Inhibitory effect of alternariol on nitric oxide synthase in different parts of rat brain 
 

Hassan MY Osman 

 
Assistant Professor, Dept. of Biochemistry, Medical Research Institute, Alexandria University, Alexandria, Egypt 

 
Email: hassanyousry12@yahoo.com 

 

Abstract 
Alternariol (3,4,5 trihydroxy 6-methyl dibenzoxy-a –pyrone) is a metabolite result of various strains of alternariatenuis 

organism. Its structure has closeness to cannabinol subordinates. The impact of the alternariol on nitric oxide snythase (NOS) 

extracted from distinct parts of rat brain; particularly: frontal cortex, basal ganglia, cerebellum, pons, medulla oblongata was 

considered. Kinetic studies were done to decide the sort of inhibition of NOS and the inhibitor dissociation constants (KI) by 

alternariol. The outcomes showed that alternariol inhibited NOS of the cortex, medulla oblongata and cerebellum more than that 

of alternate parts of the brain. These parts are in-charge of observation, tactile, psychic exercises and reflex focuses of breath. The 

inhibition of these enzymes increased with increasing the dose of alternariol added to the examined blend, i.e., the inhibition was 

dose dependent and of the competitive type. The estimations of KI for alternariol –NOS complexes shifted from 1.8 to 5.6 mM. 

The distinction in the level of inhibition of the extracts of these brain parts could be ascribed to the slight contrast in the structure, 

i.e. course of action of their amino acids (isozyme phenomenon) and to their particular gene loci. 

 

Keywords: Alternariol, Nitric oxide synthase, Brain parts, Inhibition. 

 

Introduction 
Alternariolis, a metabolite created by distinctive 

strains of alternariatenuis.(1) Its structure was built up as 

3,4,5 trihydroxy 6-methyl dibenzoxy -  - pyrone C14 

H10 O5 (I). It has striking likeness to cannabinol 

subordinates (II). 

 

 
 

 
 

Common events of Alternaria toxins have been 

accounted for in different natural products, handled 

organic product items, for example, squeezed apple, 

tomato items, wheat and different grains, sunflower 

seeds and pecans.(2-3) Pre-cancerous changes were 

observed in the esophageal mucosa of Alternariol fed 

mice for 10months.(4) 

Nitric oxide (NO) is an administrative organic 

substance and an essential intracellular errand person 

that goes about as a particular middle person of 

different neuropathological disorders.(5)NO is 

incorporated by nitric oxide synthase (NOS), and 

increased in an assortment of tumor cells. NO controls 

various cell reactions by S-nitrosylation.(6) Nitric oxide 

synthase (NOS) was initially recognized and depicted 

in 1989. The development of NO by NOS in vascular 

endothelial cells opened up what can be viewed as 

another territory of organic research. 

Brain NOS is a constitutive compound, the 

capacity of which is to deliver NO on interest for 

various neurophysiological exercises.(7) NOS intercedes 

the development of the neurotransmitter NO in addition 

to citrulline from L-arginine. NO plays an important 

role as a neurotransmitter in the nervous system, as a 

vasodilator in the cardiovascular system and as a 

cytotoxin in the host defence mechanism of 

macrophages.(8) 

The present review was led to research the 

inhibitory impact of alternariol on NOS extracted from 

entire and five sections of rat brain, to be specific; 

frontal cortex, basal ganglia, cerebellum, pons and 

medulla oblongata. Compound kinetic studies were 

done to decide the sort of inhibition and enzyme-

inhibitor dissociation constant (KI) of NOS by 

alternariol, and to know which of these parts was 

repressed by alternariol more than different parts. 

 

Material and Methods 
Alternariol was prepared by growing a strain of 

alternariatenuis, Catalogue number S.M.108, on 

Czepek-Doxmedium using either glucose or molasses, 

as a carbon source.(1)The metabolite was extracted from 

the dried defatted mycellian with ether, then purified by 

repeated crystallization from dioxane giving colorless 

needles m.p. 350° (decomp). 

Chemicals: Naphthyl ethelenediamine dihydrochloride, 

Sigma (Deisonhofen Germany), Sulfanylamide, Sigma 



Hassan MY Osman              Inhibitory effect of alternariol on nitric oxide synthase in different parts of rat brain 

Panacea Journal of Medical Sciences, January-April,2017;7(1): 15-18                                                                   16 

Deisonhofen Germany, Phosphoric acid (H3Po4): 

Deisonhofen Germany and Buffer: Phosphate 

(KH2PO4 and Na2HPO4) 0.1 M, pH 8.0 BDH. 

Animals: Thirty male albino rats (125-150 gram body 

weight) age 2 months were used in the experiments. 

Rats were supplied from Medical Research Institute 

Animal House, Alexandria University (Egypt) and were 

housed in group cages (five in each) and given free 

access of food and tap water (ad libitum). The brains of 

these rats were used as a source for NOS. The rats were 

profoundly anesthetized and slaughtered by beheading 

after an overnight fast, with free access of water. Each 

brain weighed about 1.6 gm. 

Assay of Nitric oxide synthase (NOS) activity:

 Brain Nitric oxide synthase (NOS) is a 

constitutive compound and the capacity of which is to 

create nitric oxide (NO):  

L-Arginine
NOS

  L-citrulline + NO 
NOS was controlled by evaluating nitrite (NO2) 

(being the most stable metabolite). It is utilized as a file 

for NOS.(9-10) 

Because of the transient and unpredictable nature 

of NO which makes it precarious, continually oxidized 

to the steady forms nitrite (NO2) and nitrate (NO3), the 

most advantageous discovery technique for NO was to 

be measured as far as NO2 which can be identified by 

photometric strategy utilizing Griess reagent(9)at 546 

nm. Griess reagent consists of one part of naphthyl 

ethelenediamine dihydrochloride (NED) in distilled 

water and one part of 1% sulfanilamide in 5% 

concentrated phosphoric acid (H3PO4). The two parts 

being mixed together within 12 hours of use and kept 

chilled. Each part may be stored separately refrigerated 

up to 2 months. The mixture of the two parts incubate 

with a NO2-continaing sample (ratio 1:1) to form purple 

azo dye and its absorbance is measured at a wave length 

of 546 nm.(9,10) 

To decide NO2 in brain tissue; 150 μl brain 

homogenate containing 20mg tissue were blended with 

1.5 ml of Griess reagent and the blend incubated for 5 

min at room temperature. The color formed was 

measured at absorbance 546 nm. Nitrite was computed 

from NO2 standard curve. 

For the assurance of the binding constant (KB), 

bimolecular rate constant (ka) and the inhibition rate 

(ki),
(11)alternariol was added to the previously 

mentioned blend in the following concentrations: 140, 

280, or 420 or 560 µM while keeping the substrate at a 

steady fixation (166 µM), then the catalyst was 

examined after various interims (0, 15, 30, 45 and 60 

sec). 

The experimental design and interpretation of the 

results were based on the following equation:(11) 

 

 
 

where [I] is the inhibitor fixation , and (△ t/2.3 △ log v) 
is the first order rate constant at steady [I]. 

For the assurance of the kind of inhibition and the 

enzyme-inhibitor dissociation constant (KI), the 

substrate fixation was differed: 111, 166, 222 and 333 

µM while the inhibitor (alternariol) was kept constant 

for every investigation: 140, 280 or 560µM.The 

inhibitor and substrate were added all the while 

simultaneously to the previously mentioned blend. 

 

Results 
Graphical representation of the inhibition of NOS 

by alternariol at various concentrations (140, 280, 420 

or 560 µM) got by plotting log v (the speed of reaction) 

against time (t) gave straight lines (Fig. 1a). The slopes 

were processed and gave the esteem 2.3∆ log v/∆t. 

These results were utilized to develop the chart of 

[I].∆t/2.3∆ log v plotted against [I] (Fig 1b). 

The slope of the straight line gave 1/ki(ki is the rate 

of inhibition), the catch on the [I] pivot gave KB 

(binding constant) and the capture on the ordinate gave 

1/ka(ka is the bimolecular rate constant).The estimations 

of ki, KB and ka are given in Table 1. 

As regards the kind of inhibition of NOS by 

alternariol; Figure 2 demonstrates that the double 

reciprocal curves of 1/v (v is the speed of the reaction) 

versus 1/[S] (S is the substrate concentration in 

µmol/L), keeping the inhibitor [I] constant for every 

analysis and changing the substrate concentration as 

mentioned by Dixon and Webb.(12) The 

slopereplots(inset of Fig.2) demonstrate that alternariol 

is a linear competitive inhibitor (after Cleland 

nomenclature).(13) 

The subsequent estimations ofKI (enzyme -

inhibitor dissociation constant), Km (Michaelis' 

constant) and KI/Km (affinity constant) for the 

distinctive parts under analysis, are recorded in Table 1. 

 

Table 1: Kinetic constants characterizing the inhibition of NOS of whole and different parts of rat brain by 

alternariol. Values represent mean of 3 repeated experiments. S.D. was 10% 

Part of the brain ki(a) 

(min-1) 

KB(b) 

(mM) 

ka (c) 

(µM.min)-1 

KI(d) 

(mM) 

Km(e) 

(µM) 

KI/Km(f) 

 

Whole brain  3.25 1.1 3.0 3.5 325 10.7 

Basal ganglia 1.7 0.8 2.2 1.8 333 5.4 

Frontal cortex  5.4 0.4 14 5.6 290 19.3 

Medulla oblongata 5.6 0.8 7.0 5 400 12.5 



Hassan MY Osman              Inhibitory effect of alternariol on nitric oxide synthase in different parts of rat brain 

Panacea Journal of Medical Sciences, January-April,2017;7(1): 15-18                                                                   17 

Pons  1.75 0.4 4.5 4.5 460 9.8 

Cerebellum 3.67 0.6 6.1 4.7 415 11.3 

(a) Inhibition constant   (d) enzyme-inhibitor dissociation constant  

(b) Binding constant   (e) Michaelis’ constant  

(c) Bimolecular rate constant  (f) Affinity constant  

 

Fig. 1(a): The time course of inhibition of cerebellum Fig 1(b): Main plot of the data of the inhibition of NOS 

by alternariol in vitro at constant substrate cerebellum NOS by alternariol. The slope of the concentration 

(0.3mM), concentrations of straight line gave 1/ki (ki: rate of inhibition), the alternariol: 140(

(∆), 560 (○) µM. intercept on [I] axis gave KB (binding constant) and the intercept on the ordinate gave 

1/ka(ka: bimolecular rate constant) 

 

 
Inset: Cleland replot of the slopes obtained from Fig. 2 against the inhibitor concentration [I]. 

Fig. 2: Competitive inhibition obtained from Line weaver-Burk plot of 1/V versus 1/S, under the effect of 

alternariol on activity of basal ganglia NOS. () Control, () 4.0, () 7.0 & () 10 mmol/L 

 

Discussion 
The preliminary experiments showed considerable 

difference in NOS activity in the various parts of rat 

brain. The highest activity was detected in the medulla 

oblongata, frontal cortex and cerebellum, lower in basal 

ganglia and pons. The high NOS activity in the 

cerebellum is in agreement with the results described by 

other authors(14) who found that NOS activity was 

significantly decreased by 75% in the cerebellum. 

Decreased NOS activity in the nervous tissue was 

associated with decreased motor activities and 

spontaneous behavior of rats. 

In the present work, the in vitro study of the effect 

of alternariolon NOS activity showed that alternariol 

had an inhibitory power on NOS of the cerebellum, 

pons and medulla oblongata more than that on the other 

parts which was in agreement with other authors(15) who 

found that the Vmax and Km values of the enzyme in 

cortex and cerebellum of rats were higher than in other 

brain parts. These studies show differential distribution 

and higher activity of NOS in rat brain regions and 

spinal cord compared to mouse tissues. 

Alternariol possessed higher affinity and binding to 

the enzyme extracts of medulla oblongata (responsible 

for neural control on heart rate, respiration and blood 

pressure) than to the extracts of the other parts. This 

indicated that it resembled serine(16) that was introduced 

as a NOS inhibitor which was time and dose-dependent. 

It also resembles carbaryl (sevin)(a carbamate analogue 

of eserine which was introduced as a cholinesterase and 

NOS inhibitor).(17) 

 

1.4

1.6

1.8

2

0 20 40 60

Time (sec)

lo
g

 a
c
t
iv

it
y

 %

0.0 
5.0 

10.0 

1/ka 

 

20.0 
25.0 
30.0 
35.0 

-0.6 

 

 

-0.4 -0.2 0 0.2 0.4 0.6 
[I] mM 

[I].  t / (2.3   log v) µM.min 1/ki 

 

KB 



Hassan MY Osman              Inhibitory effect of alternariol on nitric oxide synthase in different parts of rat brain 

Panacea Journal of Medical Sciences, January-April,2017;7(1): 15-18                                                                   18 

 
Carbaryl (Sevin) 

 

Alternariol was introduced in this work as a NOS 

inhibitor as it was previously postulated(18)that NOS 

inhibitor agents play crucial roles in neurodegeneration 

and neuropathic pain, as it is known that NO levels rise 

after neurological insults causing the previously – 

mentioned malfunctions. 

Alternariol was previously introduced as having a 

dual inhibitory effect on cholinesterase (ChE) and 

monoamine oxidase (MAO).(19) 

Finally; it is note-worthy to mention that the 

difference in the rate of NOS inhibition by alternariolin 

different parts of rat brain could be due to:  

a. The inhibition of NOS in the various regions of the 
brain is dependent on the concentration or the form 

of the enzyme i.e., isoenzyme phenomenon.(20) 

b. The high effect of alternariolon NOS activity may be 
due to the solubility of this compound in the lipid 

layer in the brain exactly as eserine behaved and also 

affecting the parts containing white matter (e.g. 

medulla oblongata) more than the parts containing 

gray matter, as previously proved by Osman et al.(21) 

c. The difference in isozyme inhibition could be due 
to distinctly different gene loci(22) and the amino 

acid sequence(23) leading to substrate specificity of 

the isozymes. 

d. The difference in inhibition could be due to NOS 
content in each part of the brain. 

So, it is recommended that further studies are to be 

done to produce medications from alternariol as a triple 

inhibitory agent on ChE, MAO and NOS to overcome 

neurodegenerative disorders and accompanying pain. 

 

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