Table 2. Effect of the studied compounds on the level of anxiety in rats in the “Elevated Plus Maze” test

Behavioural response indicators	Groups
Behavioural response indicators	Intact rats	Control (no treatment)	Comparison group amitriptyline	1	2	3	4
Time spent in open arm, (sec.)	9.7± 16.0	8.0± 9.8	16.0± 4.5	18.8± 8.5	41.8± 17.3*	16.8± 8.5	12.0± 20.2
Number of entries into open arms, (times)	0.8±0.4	0.5± 0.5	0.8± 1.0	1.3± 0.5	2.8± 1.7*	0.8± 0.4	0.5± 0.5
Time spent in closed arm, (sec.)	113.8± 23.9	131.2± 35.3	133.8± 16.9	120.0± 18.5	103.8± 16.0	122.7± 19.1	152.0± 24.1
Number of entries into closed arms, (times)	4.0± 2.4	4.2± 1.7	3.8±1.5	2.2± 0.4*	5.5± 0.5	3.0± 0.6	2.0± 0.6*
Number of hangings, (times)	3.8±4.7	5.0±1.6	2.8±1.2*	10.0± 2.3*	11.2± 3.0*	3.3± 2.3	1.7± 3.1*
Number of stands, (times)	0.3±0.5	0	0.3±0.5	0	0.3± 0,8	0	0
Time spent on central platform, (sec.)	56.5± 34.7	40.8± 29.5	46.8± 17.6	28.7± 8.5	34.2± 6.3	40.5± 12.6	24.3± 5.7
Number of defecations	0.3±0.5	0	0	0	0	0	0
Number of urinations	1.3±2.1	0.8±1.0	0.2±0.4	0.3± 0.5	0	0.2± 0.4	0.3± 0.5

Note: *– p<0.05 compared to the values in animals of the control group; n – number of animals in the group

In the group treated with 20-ethyl-16-methoxy-4-(methoxymethyl)aconitane-1,6,7,8,14-pentol (2), a slight reduction in passive swimming time was observed, while active swimming time increased compared to the control group. No antidepressant effect was detected in the groups treated with 1-methyl-4-quinolone (3) and (20R)-21-ethyl-1α,15β-dihydroxy-4-methyl-16-methylene-7α,20-cycloveatchan-12-one (4).

Thus, in the Porsolt’s test, the compound 7-methoxy-1-methyl-9H-pyrido[3,4-b]indole (1) demonstrated a pronounced antidepressant effect. At a dose of 10 mg/kg, it significantly increased the duration of active swimming by 1.2 times and reduced immobility time by 2.1 times compared to the control group.

It is known that certain biological activities are determined by the presence of specific pharmacophores in the structure of alkaloid molecules. The presence of an aryl group in the molecules of 7-methoxy-1-methyl-9H-pyrido[3,4-b]indole (1), 20-ethyl-16-methoxy-4-(methoxymethyl)aconitane-1,6,7,8,14-pentol (2), 1-methyl-4-quinolone (3), and (20R)-21-ethyl-1α,15β-dihydroxy-4-methyl-16-methylene-7α,20-cycloveatchan-12-one (4) is of great importance for their neurotropic activity. The aryl group is part of the structure of many medicinal compounds and is, in particular, one of the pharmacophores of the anti-Parkinsonian drug levodopa.

7-Methoxy-1-methyl-9H-pyrido[3,4-b]indole (1) enhances the proliferation of neurons (hNPC), which explains its effect on proliferation in vitro and its antidepressant effects in vivo. Thus, the neurotropic activity of 7-methoxy-1-methyl-9H-pyrido[3,4-b]indole (1) observed in the experiment is supported by previous studies ¹².

The “structure-activity” relationship analysis indicated that the neurotropic activity of 20-ethyl-16-methoxy-4-(methoxymethyl)aconitane-1,6,7,8,14-pentol (2) and (20R)-21-ethyl-1α,15β-dihydroxy-4-methyl-16-methylene-7α,20-cycloveatchan-12-one (4) is associated with the presence of an N-ethyl group in their molecular structures. In 1-methyl-4-quinolone (3), it is linked to the N-methyl group in the pyridine moiety, while in 7-methoxy-1-methyl-9H-pyrido[3,4-b]indole (1), it is related to the NH group in the indole ring, which interacts with corresponding receptors.

The studied plant-derived alkaloids hold promise for the development of new pharmaceutical substances for the treatment of central nervous system disorders.

According to the literature, the β-carboline alkaloids used in our study are known for their interaction with the dopaminergic system, demonstrating their ability to modulate D1 and D2 dopaminergic receptors in the striatum. In particular, it has been shown that beta-carboline alkaloids can act as monoamine oxidase inhibitors, which also contributes to their interaction with dopamine systems ¹³. These data confirm the neurochemical activity of β-carbolines and their potential as drugs for the treatment of neurodegenerative diseases and depression. These alkaloids also interact with serotonin and dopamine receptors, affecting the psychoemotional state. This interaction may explain its moderate neurotropic activity, as shown in our results.

In terms of toxicological analysis, plant alkaloids, like other biologically active substances, can cause side effects such as gastrointestinal disorders or liver and kidney dysfunction. We suggest continuing toxicological studies to fully characterize their safety ¹⁴.

Future research will focus on testing alkaloids in models of neurodegenerative diseases, such as Alzheimer's and Parkinson's, as well as in models of chronic stress, which will allow us to evaluate the potential of these compounds as preventive agents in the treatment of disorders associated with neurodegenerative processes.

MATERIALS AND METHODS:

To study psychotropic activity, pharmacological screening models were used, including the “Open Field” Test, the “Elevated Plus Maze” Test ¹⁵, and the “Forced Swim” Test ¹⁶.

The research objects were alkaloid samples: 7-methoxy-1-methyl-9H-pyrido[3,4-b]indole (1), 20-ethyl-16-methoxy-4-(methoxymethyl)aconitane-1,6,7,8,14-pentol (2), 1-methyl-4-quinolone (3), and (20R)-21-ethyl-1α,15β-dihydroxy-4-methyl-16-methylene-7α,20-cycloveatchan-12-one (4), which were extracted from plant sources Peganum harmala L. ^17-26, Aconitum delphinifolium DC. ^27-30, Echinops albicaulis Kar & Kir. ^31-34, and Aconitum pendulum ^35-⁴⁰ (Table 4). Neurotropic effects were studied using experimental models of emotional stress.

Table 4. Numbering and structural formulae of the studied compounds 1–4.

Compound	IUPAC Name	Structural Formula
1	7-Methoxy-1-methyl-9H-pyrido[3,4-b]indole
2	20-Ethyl-16-methoxy-4-(methoxymethyl)aconitane-1,6,7,8,14-pentol
3	1-Methyl-4-quinolone
4	(20R)-21-Ethyl-1α,15β-dihydroxy-4-methyl-16-methylene-7α,20-cycloveatchan-12-one

Experiments were carried out on 84 white outbred male rats weighing 200-220 g.

Ethical Compliance:

All animal experiments were carried out in the Laboratory of Experimental and Clinical Pharmacology of Karaganda Medical University with the approval of the Local Ethics Committee of Karaganda Medical University (Reg. No. 8 dated 08.09.2020). The research was carried out in accordance with the “Rules of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes” and in compliance with the requirements for studying new pharmacological substances in sexually mature outbred laboratory rats.

The animals were kept in standard vivarium conditions with a normal diet and free access to food and water. Additionally, observations were conducted on their general condition, including body weight changes, motor activity, appetite, and response to external stimuli.

Emotional Stress Model:

Emotional stress was induced by placing the rats into confined plastic cylinders and subsequently immersing them in water (20-22°C) up to neck level for 2 hours daily over four days ⁴⁰.

The tested substances were administered intragastrically at a dose of 10 mg/kg via a probe for seven days prior to emotional stress modeling and then daily, one hour before placing the animals in plastic cylinders. The antidepressant drug “Amitriptyline” (Saneca Pharmaceuticals a.s., Slovak Republic) was used as a reference drug at a dose of 10 mg/kg, administered according to the same schedule. Both the test substances and the reference drug were administered orally as an aqueous solution at a volume of 1 ml/kg ⁴¹.

Behavioral Assessment:

On the fourth day after emotional stress modeling, the effects of the studied compounds on behavioral responses were assessed using standard methods in the following tests:

· Open Field Test

· Elevated Plus Maze Test

The antidepressant activity of the tested compounds was evaluated in vivo using the Porsolt’s Forced Swim Test (behavioral despair) ¹⁵.

Forced Swim Test in Mice:

Stress conditions in mice were induced through forced swimming. The animals were placed in a cylindrical container with a diameter of 10 cm and a height of 25 cm, filled to one-third with water (27°C). After unsuccessful attempts to escape, the mice adopted a characteristic immobile posture, which was interpreted as the onset of behavioral despair. All active escape attempts within the first 6 minutes after immersion were recorded. Behavioral parameters included:

· Duration of the first act of motor activity

· Active swimming time

· Immobility time

The test substances, at a dose of 10 mg/kg, were administered orally as a suspension in 1% starch mucus intragastrically one hour before testing. Control animals received an equivalent volume of starch mucus. The reference drug, Amitriptyline, at a dose of 10 mg/kg, was also administered orally as a suspension in 1% starch mucus one hour before testing.

The experimental tests were conducted using a blind method. All experiments were carried out in such a way that the assessment of the animals' behavioral responses was performed without prior knowledge of which compound was used. The evaluation and analysis of the data were carried out independently of the therapeutic intervention, which ensures a high degree of objectivity of the results obtained.

Statistical Analysis:

Statistical analysis of the results was performed using the “Statistica 8.0” software package. Data were presented as “mean ± standard error of the mean”. Intergroup differences were assessed using the nonparametric Mann-Whitney’s U-test.

CONCLUSIONS:

Thus, the experimental study demonstrated that 20-ethyl-16-methoxy-4-(methoxymethyl)aconitane-1,6,7,8,14-pentol (2) at a dose of 10 mg/kg exhibited pronounced neurotropic activity under conditions of emotional stress, normalizing the emotional state of animals and reducing fear and anxiety. Moreover, in terms of effectiveness, 20-ethyl-16-methoxy-4-(methoxymethyl)aconitane-1,6,7,8,14-pentol (2) was not inferior to, and in some parameters surpasses, the neurotropic effects of the reference drug “Amitriptyline”.

The alkaloids 7-methoxy-1-methyl-9H-pyrido[3,4-b]indole (1), 1-methyl-4-quinolone (3) and (20R)-21-ethyl-1α,15β-dihydroxy-4-methyl-16-methylene-7α,20-cycloveatchan-12-one (4) at a dose of 10 mg/kg exhibited moderate neurotropic activity compared to the control group.

A pronounced antidepressant activity was identified for the alkaloid 7-methoxy-1-methyl-9H-pyrido[3,4-b]indole (1), comparable to the effect of the reference drug “Amitriptyline”.

The “structure-activity” analysis revealed pharmacophore centers within the molecular structures of the studied plant alkaloids.

The findings indicate the potential for developing original neurotropic agents based on 7-methoxy-1-methyl-9H-pyrido[3,4-b]indole (1), 1-methyl-4-quinolone (3) and (20R)-21-ethyl-1α,15β-dihydroxy-4-methyl-16-methylene-7α,20-cycloveatchan-12-one (4).

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 27.06.2025 Revised on 03.12.2025

Accepted on 24.02.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):2992-2998.

DOI: 10.52711/0974-360X.2026.00426

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Group	Spectrum of orientation-exploratory activity		Spectrum of anxiety manifestations
Group	Number of horizontal movements	Vertical motor activity	Grooming	Number of defecations	Number of urinations
Intact rats	23±4.4	7.7±1.4	2.2±0.8	1.0±0.6	0.6±0.8
Control (no treatment)	9.8±3.1	6.0±1.7	3.2±3.3	2.3±1.9	0.8±0.4
Comparison group (amitriptyline)	12.8±4.4	5.2±2.3	0.6±1.0	3.3±0.5*	0.6±1.0
1	13.8±5.4	6.2±1.5	4.7±1.0	1.3±0.5	0.5±0.8
2	33.6±6.0*	9.0±1.5*	0.5±0.8	0.7±1.6	0.3±0.5
3	7.3±5.5	4.2±3.8	0.3±0.5	1.2±0.6	0.8±0.8
4	10.6±4.2	4.8±1.7	9.3±2.7*	1.3±1.0	0.7±1.2

Name of substance, dose	Duration of the first act of motor activity (sec.)	Active swimming time (sec.)	Immobilization time (sec.)
Control	50.5±22.5	235.1±62.6	124.9±62.6
Comparison group (amitriptyline), at a dose of 10 mg/kg	57.5±15.6	303.9±36.6	56.1±36.6*
1	60.4±12.7	308.8±34.2*	63.2±24.7*
2	48.6 ± 15.9	257.6±71.4	101.4±71.2
3	59.8±18.8	236.3±78.0	115.6±65.0
4	56.7±24.2	215.5±81.4	136.5±80.6