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 VOLUME Vol.05 Issue07 2025 

PAGE NO. 11-15 

DOI 10.37547/ajbspi/Volume05Issue07-03 

 
 
 
 

Study of The Effect of Polyphenol Plantagin On Calcium 

Transport in The Presence of NMDA Receptor Agonists 

and Antagonists 
 

Mamadaminov Rakhmatjon Rakhimjon oglu 

Namangan State University. Namangan, Uzbekistan 

 

Khoshimov Nozimjon Numonjonovich 

Institute of Biophysics and Biochemistry under the National University of Uzbekistan. Tashkent, Uzbekistan 

 

 

Received: 18 May 2025; Accepted: 14 June 2025; Published: 16 July 2025 

 

Abstract: This study investigated the effect of Plantagin polyphenol on NMDA receptor binding sites using glycine, 
Mg2+ and Zn2+ ions. A suspension of rat brain synaptosomes was isolated by differential centrifugation. Changes 
[Ca2+]in concentration in the suspension medium of rat brain synaptosomes were measured using the fluorescent 
probe Fluo-4 AM. It was noted that synaptosomal Ca2+ transport remained virtually unchanged under the 
influence of plantago polyphenols in Zn2+ and Mg2+ in rat brain synaptosomal suspensions incubated with L-
glutamate-Fluo-4 AM. The polyphenol Plantagin competes with glycine for binding to the glycine site of NMDA 
receptors, reducing the likelihood of full receptor activation even in the presence of glutamate, which leads to a 
limitation of Ca²⁺ influx, and therefore plays an important role in preventing excitotoxicity. 

 

Keywords: Plantagin polyphenol, NMDA receptor, Mg2+, Zn2+, synaptosome. 

 

Introduction: NMDA receptors are present in all parts 
of the central nervous system, located mainly in the 
postsynaptic and partly in the presynaptic membrane 
[1]. NMDA receptors have also been found to function 
in astrocytes in the cerebral cortex [2]. 

The structure of the NMDA receptor consists of a 
complex of 4 separate basic subunits that form an ion 
channel in the central part that is permeable to Ca2+ 
ions. NMDA receptor subunits differ in their 
physiological and pharmacological properties, and 
seven subunits have been identified within the 
complex, including NR1, NR2A–D, NR3A, and NR3B. The 
NR1 subunit, which binds to glycine, has 8 isomeric 
forms in the NMDA receptor structure, and the NR2A–
D subunit, which binds to 1 or 2 glutamic acid and 
provides the receptor with the function of an ion 
channel, are the functionally basic structures [3,4]. It is 
suggested that the NR3A and NR3B subunits expand 
the spectrum of functional properties of the NMDA 
receptor [5,6]. 

In clinical and experimental neurology, divalent cations 
such as Mg2+ and Zn2+ are widely used as NMDA 
receptor modulators [6,7]. 

Mg2+ ions, which are usually considered endogenous 
modulators, have been found to be more likely to bind 
to a specific site of the NMDA receptor at a highly 
negative membrane potential. Zn2+ ions bind to the 
GluN2A/GluN2B subunits [7]. 

It is known that magnesium ions play an important role 
in regulating the activity of NMDA receptors. They are 
potential-dependent receptor blockers, preventing 
excessive calcium influx and reducing excitotoxicity. 
Adequate amounts of Mg2+ are necessary for normal 
NMDA receptor function and synaptic plasticity. Zinc 
(Zn2+) ions can also modulate the activity of NMDA 
receptors. They act as positive allosteric modulators, 
enhancing the response of glutamate receptors. 
However, excessive levels of Zn2+ can lead to 
neurotoxicity and dysfunction of NMDA receptors. 

NMDA receptors are heteromeric tetrameric proteins 

 

https://doi.org/10.37547/ajbspi/Volume05Issue07-03
https://doi.org/10.37547/ajbspi/Volume05Issue07-03
https://doi.org/10.37547/ajbspi/Volume05Issue07-03
https://doi.org/10.37547/ajbspi/Volume05Issue07-03


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American Journal of Applied Science and Technology (ISSN: 2771-2745) 
 

 

composed of GluN1 and GluN2 subunits, which contain 
D-serine/glycine and glutamate binding sites, 
respectively. 

In addition to D-serine/glycine and glutamate binding 
sites, they have several regulatory sites sensitive to 
polyamines, Zn2+, protons and glutathione [2,7,8,9]. 

It has been found that Mg2+ and Zn2+ ions have an 
inhibitory effect on NMDA receptors. These ions can 
block the NMDA receptor channel and reduce its 
activation by the neurotransmitter glutamate. Studies 
show that the inhibitory effects of Mg2+ and Zn2+ on 
NMDA receptors are not the same. It has been found 
that the blocking of the channel by Zn2+ is faster than 
by Mg2+. 

In addition, the effect of Mg2+ and Zn2+ ions on NMDA 
receptors may depend on changes in pH. Therapeutic 
approaches identified through studies of the effects of 
Mg2+ and Zn2+ on NMDA receptors suggest that by 
regulating Mg2+ and Zn2+ levels, NMDA receptor 
activity can be modulated and neurological diseases 
associated with NMDA receptor dysfunction, such as 
Alzheimer's disease and epilepsy, can be prevented and 
treated. 

The aim of the study was to determine the effect of the 
polyphenol Plantagin on NMDA receptor sites using 
glycine, Mg2+ and Zn2+ ions. 

METHODS 

Method for isolating rat brain synaptosome 

In the studies, rat brain synaptosome suspensions were 
isolated by differential centrifugation using the method 
developed and modified by C.W. Cotman [10] [11]. 

The rat was sacrificed by dislocation, the cranial cavity 
was opened surgically, and the brain was removed. The 
brain preparation was homogenized in an incubation 
medium (pH=7.4) containing sucrose (0.32 M), Tris-HCl 
(0.01 M) and EDTA (0.5 mM) in a ratio of 1:10 under ice 
water conditions. In experiments, rat brain 
synaptosomes were isolated using 2/4-step 
centrifugation [12]. The first centrifugation was carried 
out at a speed of 4500 rpm and a duration of 10 
minutes, and the obtained supernatant was 
centrifuged at a second stage at a speed of 14000 rpm 
and a duration of 20 minutes. 

In the experiments, a KCl solution (35 mM) was used as 
a plasma membrane depolarizing agent. It is known 
that under incubation conditions with KCl (35 mM), 
membrane depolarization occurs and, in turn, 
activation of Ca2+ channels is observed [11]. In the 
synaptosome, the process of neurotransmitter 
secretion from the vesicle occurs due to an increase in 
the concentration of [Ca2+]in due to the entry or exit 
of Ca2+ ions from the SR through Ca2+ channels 

located in the presynaptic membrane [13]. 

Method for studying changes [Ca2+]in concentration 
in rat brain synaptosomes. 

The change [Ca2+]in concentration in the suspension 
medium of rat brain synaptosomes was calculated 
using the method developed by Grynkiewicz et al. [14]. 

To determine the intracellular calcium concentration 
(1×108 cl/ml) in synaptosomes, the highly sensitive 
fluorescent probe Fluo-4 AM was used. 

In our experiments, 1 mg of Fluo-4 AM powdered 
fluorescent probe was dissolved in 135 μL of DMSO to 
obtain 1 mM Fluo-4 AM reagent solution. Before the 
experiment, the Fluo-4 AM solution in DMSO was kept 
at room temperature [15] and 80 μl synaptosomes and 
12 μl Fluo-4 AM were added to 2 ml Krebs-Ringer buffer 
and incubated for 30 min at 37°C. Fluo-4 AM is a 
fluorescent Ca2+ chelator with high affinity for calcium. 
Fluo-4 AM can specifically detect intracellular calcium 
ions with high sensitivity, low cytotoxicity and high 
content of acetyl methyl ester AM, which has good 
intracellular penetrating ability. After cleavage by 
intracellular esterase, it remains in the cell, binding to 
calcium ions and causing strong fluorescence. 

In the experiments, the fluorescence intensity was 
recorded using a USB 2000 spectrofluorimeter (Ocean 
Optics, USA, 2010). Statistical processing of the results 
was carried out using the specialized software package 
OriginPro 7.5 (OriginLab Corporation, USA). The results 
were obtained in n-fold repetition. Also, the statistical 
reliability of the values between the experimental 
results and the control group was calculated based on 
Student's t-test and was assessed as statistically 
significant at values of p<0.05, p<0.01. 

RESULTS OBTAINED AND THEIR ANALYSIS 

In our experiments, the effect of Plantagin polyphenol 
on the NMDA receptor was studied using Zn2+ (5 μM), 
Mg2+ (5 μM) ions and glycine (50 μM). It was found 
that Ca2+ transport in synaptosomes remained 
virtually unchanged under the influence of Plantagin 
polyphenol under conditions of incubation of L-
glutamate-Fluo-4 AM in a suspension of rat brain 
synaptosomes with Zn2+ and Mg2+ (5 μM). However, it 
was found that the activating effect of glycine was 
relatively suppressed in the presence of glycine (50 μM) 
(Figure 1). 

The results of this experiment allow us to conclude that 
the polyphenol Plantagin used has both a stimulating 
and competitive/blocking effect on antagonists and 
agonists that directly affect the functional activity of 
the ionotropic NMDA receptor. 

In experiments, glycine binds to the active site of the 
NMDA receptor, activating the calcium channel 



American Journal of Applied Science and Technology 13 https://theusajournals.com/index.php/ajast 

American Journal of Applied Science and Technology (ISSN: 2771-2745) 
 

 

through the receptor. In subsequent experiments, 
studies were conducted to determine the complex 
effect of 50 μM glycine incubated with Fluo-4AM in the 
presence of 50 μM concentration of the polyphenol 
Plantagin (Figure 1). 

It turns out that glycine binds to the glycine-binding site 

of NMDA receptors, promoting activation of the Ca2+ 
channel. The obtained results show that the 
polyphenol Plantagin inhibits the increase in calcium 
dynamics caused by glycine. 

 

 
Figure 1. Effect of polyphenol plantagin (50 μM) on fluorescence intensity in synaptosomal 

suspensions of rat brain incubated with glycine (50 μM), Zn2+ (5 μM) and Mg2+ (5 μM). Confidence 

level. *- P<0.05; **- P<0.01. 

This suggests that the polyphenol Plantagin may also 
affect glycine binding sites, suggesting that this 
polyphenol may be effective in receptor modulation. 

In terms of discussion of the results, the polyphenol 
Plantagin competes with glycine for binding to the 
glycine site of NMDA receptors, reducing the likelihood 
of full receptor activation even in the presence of 
glutamate. This partial inhibition results in a limitation 
of Ca2+ influx and is therefore important for preventing 
excitotoxicity. Competition with glycine reduces NMDA 
receptor activation and the subsequent increase in 
calcium influx. 

Normally, activation of NMDA receptors opens an ion 
channel, allowing Ca2+ to enter the postsynaptic 
neuron. In neurodegenerative diseases, disruption of 
this process leads to excessive calcium influx, which can 
trigger a cascade of harmful processes, including 
mitochondrial dysfunction, generation of reactive 
oxygen species (ROS), and activation of enzymes that 
destroy calcium-dependent cellular structures. The 
polyphenol Plantagin inhibits the entry of Ca2+ into the 

postsynaptic neuron by blocking the glycine site and 
partially inhibiting the activation of the NMDA 
receptor. This process reduces intracellular calcium 
concentration and stabilizes calcium homeostasis, 
preventing the harmful effects of excess calcium. 

Plantagin polyphenols reduce the harmful effects of 
receptor hyperactivation by competitively blocking 
postsynaptic NMDA receptors at the glycine site. This 
blockade limits calcium influx, reduces excitotoxicity 
and protects neurons from damage and death. 

NMDA receptors are a subtype of glutamate receptors 
involved in synaptic plasticity and learning. NMDA 
receptor antagonists prevent calcium ions from 
entering the cell by blocking the NMDA receptor 
channel. This process helps regulate glutamate-
mediated excitatory signaling and prevents excessive 
calcium influx that can lead to neuronal damage. 
Examples of NMDA receptor antagonists include 
ketamine, memantine, and dextromethorphan. 

Memantine binds to NMDA receptors more efficiently 
than Mg+ ions. It counteracts the long-term influx of 

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 Control

 Plantagin (50 mkM)

 Glycine (50 mkM)

 Zn
2+

 (5 mkM)

 Mg
2+

 (5 mkM)

 Plantagin (50 mkM) + Glycine (50 mkM)

 Plantagin (50 mkM) + Zn
2+

 (5 mkM)

 Plantagin (50 mkM) + Mg
2+

 (5 mkM)



American Journal of Applied Science and Technology 14 https://theusajournals.com/index.php/ajast 

American Journal of Applied Science and Technology (ISSN: 2771-2745) 
 

 

Ca2+ ions through the NMDA calcium channel (which 
counteracts the voltage-dependent blockade of NMDA 
receptors by Mg+ ions due to excess glutamate levels) 
by temporarily maintaining the physiological activity of 
the channel due to the high concentration of glutamate 
released from the synaptic terminal. Control of 
intracellular Ca2+ concentration is critical for neurons 
because it determines their survival and physiological 
function. Because memantine is voltage dependent, it 
dissociates the average physiological signal from its 
context and mediates average signal transmission. 

Based on the above data, in our experiments the 
polyphenol Plantagin was compared with the drug 

Memantine, a non-competitive NMDA receptor 
blocker. The effect of the polyphenol plantagin used in 
these studies on the activity of synaptosomal Ca2+ 
channel suspensions in the presence of memantine was 
investigated (Figure 2). 

In our experiments, we found that psyllium 
polyphenols (50 μM) at memantine concentrations (50 
μM) had little effect on the calcium content of 
synaptosome suspension compared to memantine, 
indicating that this polyphenol does not affect the 
memantine binding site. 

 

 
Figure 2. Effect of polyphenol plantagin and memantine (50 μM) on the fluorescence 

intensity of Fluo4-AM in synaptosomal suspensions of rat brain. Level of significance. *- P<0.05; 

**- P<0.01; ***- P<0.001. (n=6). 

The results of the study indicate that the polyphenol 
Plantagin used does not affect the Mg2+ binding sites 
of the NMDA receptor. 

CONCLUSIONS 

It was found that synaptosomal Ca2+ transport was 
virtually unchanged in the presence of plantago 
polyphenols under Zn2+ and Mg2+ incubation 
conditions of L-glutamate-Fluo-4 AM in rat brain 
synaptosomal suspensions. The polyphenol Plantagin 
competes with glycine for binding to the glycine site of 
NMDA receptors, reducing the likelihood of full 
activation of the receptor even in the presence of 
glutamate. This partial inhibition results in a limitation 

of Ca2+ influx and is therefore important for preventing 
excitotoxicity. The results of this experiment allow us to 
conclude that the polyphenol Plantagin used has both 
a stimulating and competitive/blocking effect on 
antagonists and agonists that directly affect the 
functional activity of the ionotropic NMDA receptor. 

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Hirasawa M, Pittman QJ. Nifedipine facilitates 
neurotransmitter release independently of calcium 
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Lee M.–C., Ting K.K., Adams S., Brew B.J., Chung R., 
Guillemin G.J. Characterisation of the expression of 

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ty
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%
) 

(F
3
4
0/F

4
9
0)

X Axis Title

 Control

 Plantagin (50 mkM)

 Memantine (50 mkM)

 Plantagin (50 mkM) + Memantine (50 mkM)



American Journal of Applied Science and Technology 15 https://theusajournals.com/index.php/ajast 

American Journal of Applied Science and Technology (ISSN: 2771-2745) 
 

 

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https://doi.org/10.3390/encyclopedia3020027
https://doi.org/10.3390/encyclopedia3020027

