*Corresponding Author: B. Lowkika, Department of Pharmacology, Chalapathi Institute of
Pharmaceutical Sciences (A), Guntur, A.P, Email: lowkikachowdary@gmail.com. 17
International Journal of Zoology and Applied Biosciences
ISSN: 2455-9571
Volume 11, Issue 1, pp: 17-22, 2026
http://www.ijzab.com
https://doi.org/10.55126/ijzab.2026.v11.i01.003
Research Article
MAZE LEARNING MEETS AI: SMART ANALYSIS OF MEMORY
ENHANCEMENT IN RODENTS USING 8 ARM RADIAL MAZE
*Lowkika B, Roshini P. Charanya S, Jyothika M, Divya P, Chandana S, Eswar Tony D,
Rama Rao Nadendla
Department of Pharmacology, Chalapathi Institute of Pharmaceutical Sciences (A), Guntur, Andhra Pradesh.
Article History: Received 11th October 2025; Accepted 17th December 2025; Published 1st January 2026
ABSTRACT
Memory and learning are crucial cognitive functions, often compromised in neurodegenerative disorders such as
Alzheimer's disease, dementia, and age-related cognitive decline. There is increasing interest in herbal nootropic agents
due to their safety and reduced side-effect profile compared to synthetic drugs. Ocimum sanctum (Tulasi) and
Trachyspermum ammi (Ajwain) are two medicinal plants traditionally used in Ayurveda for their neuroprotective and
adaptogenic properties. This study investigates the memory-enhancing effects of a combination of Ocimum sanctum and
Trachyspermum ammi powders at two different concentrations (100 mg/kg and 200 mg/kg), compared with the standard
nootropic drug Piracetam (1 mg/kg), using the 8-Arm Radial Maze model in mice. Male mice were randomly divided into
four groups: Group A (Control, 0.9% saline), Group B (Piracetam 1 mg/kg), Group C (Test 1: 100 mg/kg of Tulasi-Ajwain
combination in 1:1 ratio), and Group D (Test 2: 200 mg/kg of the same combination). Each group contained four animals
(n = 4). The 8-Arm Radial Maze was used to assess spatial memory by recording the number of correct arm entries (out of
8) at 2, 4, 6, 8, 12, and 24 hours post-administration. Higher numbers of correct entries indicate
better memory performance. The control group (Group A) showed a gradual increase in correct entries over time, reaching
an average of ~11 at 24 hours. Group B (Piracetam) consistently demonstrated improved performance, with an average of
15 correct entries at 24 hours, indicating strong nootropic activity. Test groups C and D, receiving the herbal combination
at 100 mg/kg and 200 mg/kg respectively, also showed significant improvement in correct entries over time. Group D (200
mg/kg) exhibited superior performance among the test groups, with correct entries reaching 12–17 by 24 hours, closely
approaching Piracetam’s efficacy. Group C (100 mg/kg) showed moderate but consistent improvement, with 11–15 correct
entries at 24 hours. These results suggest a dose-dependent enhancement of spatial memory by the herbal combinations.
The present study demonstrates that the combination of Ocimum sanctum (Tulasi) and Trachyspermum ammi (Ajwain)
exerts significant memory-enhancing effects in mice, as evidenced by improved performance in the 8-Arm Radial Maze
test. Both test doses (100 mg/kg and 200 mg/kg) showed progressive enhancement in spatial memory, with the higher dose
(200 mg/kg) producing results comparable to the standard nootropic drug Piracetam. These findings suggest a dose-
dependent nootropic potential of the Tulasi–Ajwain combination, supporting its traditional use in cognitive enhancement
and highlighting its promise as a safe, herbal alternative for managing cognitive decline and neurodegenerative disorders.
Keywords: Ocimum sanctum, Trachyspermum ammi, Nootropic, Memory enhancement, Spatial memory.
INTRODUCTION
Memory and learning have led to increasing interest in
herbal and natural nootropic substances, which possess
Memory and learning are essential cognitive processes that
form the basis of intelligence and behavior. Impairment in
these functions is a characteristic feature of several
neurological disorders such as Alzheimer’s disease,
dementia, and age-related cognitive decline. The search for
safe and effective agents that can enhance fewer side
effects compared to synthetic drugs. The 8-Arm Radial
Maze is one of the most widely used behavioral models for
evaluating spatial learning and memory in laboratory
animals such as mice and rats. The maze consists of a
central platform from which eight arms radiate outward,
Lowkika et al. Int. J. Zool. Appl. Biosci., 11(1), 17-22, 2026
www.ijzab.com 18
each arm potentially containing a food reward. During the
training and testing sessions, animals learn to remember
which arms they have already visited and which ones
contain rewards. The number of errors made and the time
taken to complete the task serve as indicators of memory
and learning performance. Hence, this model is highly
reliable for screening potential memory-enhancing
(nootropic) agents. In the present study, Piracetam is used
as the standard drug, as it is a well-established nootropic
that improves memory and cognitive function. Piracetam
acts by enhancing neuronal communication, improving
cerebral blood flow, and modulating neurotransmitter
functions, particularly those related to acetylcholine, which
is vital for learning and memory processes.
Trachyspermum ammi L. (Ajwain / Carom Seeds),
belonging to the family Apiaceae, is an annual aromatic
herb native to Egypt and the Eastern Mediterranean
(Timalsina et al., 2023), now widely cultivated across
India. The plant grows up to 60–90 cm tall, with feathery
leaves, small white umbels of flowers, and oval brownish
fruits possessing a distinct thyme-like aroma due to thymol-
rich essential oil (Sharma et al., 2023). Its phytochemical
profile includes thymol, carvacrol, γ-terpinene, p-cymene,
flavonoids, saponins, and tannins, which contribute to its
diverse pharmacological activities such as (Timalsina et
al.,2023), antioxidant, antimicrobial, anti-inflammatory,
hepatoprotective, (Ghasemi et al.,2023), and
neuroprotective effects. Traditionally, Ajwain is used in
Ayurveda and Unani systems as a digestive aid, (Musa et
al.,2024) carminative, and remedy for colds, coughs, and
abdominal discomfort.
Ajwain exhibits notable memory-enhancing properties
attributed to its key compounds, (Hejazian et al.,2014),
thymol and carvacrol, which improve cholinergic
transmission and reduce oxidative stress in the brain. These
bioactives act as acetylcholinesterase inhibitors, increasing
acetylcholine levels and enhancing learning and memory.
They also provide neuroprotection by scavenging free
radicals, (Bairwa et al., 2012) suppressing
neuroinflammation (via NF-κB inhibition), and supporting
mitochondrial function. Flavonoids and other polyphenols
further protect neurons by preventing lipid peroxidation
and promoting synaptic plasticity. Collectively, these
mechanisms help maintain neuronal integrity, regulate
neurotransmitters like acetylcholine, dopamine, and
GABA, and enhance cognitive performance under
oxidative or inflammatory stress.
Ocimum sanctum L. (Holy Basil / Tulsi), belonging to the
family Lamiaceae, (Arya et al.,2024) is a sacred aromatic
herb widely cultivated across India and Southeast Asia. It is
a small, (Rodrigues et al.,2022) branched perennial plant
with fragrant leaves, purplish flowers, and oval fruits. The
plant contains essential oils rich in eugenol, ursolic acid,
carvacrol, linalool, and flavonoids such as orientin and
vicenin. Traditionally, (Pushpangadan et al.,2012) Tulsi has
been revered in Ayurveda for its adaptogenic,
antimicrobial, antidiabetic, (Joshi et al.,2006) and anti-
inflammatory properties and is used to treat respiratory
ailments, digestive disorders, and stress-related conditions.
Tulsi demonstrates strong neuroprotective (Stockburger et
al., 2016) and memory-enhancing effects due to its
antioxidant and cholinergic-modulating properties.
Compounds like eugenol and ursolic acid scavenge free
radicals, reduce lipid peroxidation, and protect neurons
from oxidative and inflammatory damage. Flavonoids such
as orientin and vicenin enhance cognitive performance by
improving synaptic transmission and reducing stress-
induced neurodegeneration. Tulsi also regulates
neurotransmitters like dopamine, serotonin, and
acetylcholine, improving learning, memory, and focus.
Through its combined antioxidant, anti-inflammatory, and
adaptogenic mechanisms, Ocimum sanctum supports brain
health and prevents cognitive decline associated with aging
and oxidative stress.
MATERIALS AND METHODS
Preparation of plant powder
The collected plants of Trachyspermum ammi (Ajwain),
(Rahman et al., 2018), and Ocimum sanctum (Tulsi), (Le et
al.,2019), were shade-dried and coarsely powdered. The
powders were mixed in an equal ratio (1:1 w/w) to prepare
the test formulation (Hening et al.,2018). For
administration, (Sarker et al.,2006), the mixed powder was
suspended in distilled water to achieve the desired
concentrations corresponding to two dose levels: Test I
(100 mg/kg) and Test II (200 mg/kg). The required dose for
each mouse was calculated based on its body weight, and
the suspension was freshly prepared each day to ensure
stability. The formulation was administered orally by
mixing with the animals’ drinking water, maintaining a
calculated intake volume of 4 mL per animal. The
suspension was continuously stirred to ensure uniform
dispersion of the powder throughout administration.
Experimental Groups
The experimental animals were divided into four groups,
each consisting of 6 number of mice: Group I – Control:
Received 0.9% v/v normal saline orally, serving as the
baseline control group. Group II – Standard: Administered
Piracetam at a dose of 150 mg/kg/day orally, serving as the
reference standard for cognitive enhancement. Group III –
Test I: Received the polyherbal formulation of
Trachyspermum ammi and Ocimum sanctum (1:1 ratio) at a
dose of 100 mg/kg/day orally. Group IV – Test II:
Received the same polyherbal formulation at a higher dose
of 200 mg/kg/day orally. All treatments were given once
daily, and the formulations were freshly prepared and
administered throughout the study period to ensure
consistency and accuracy.
Ethical Concerns
Healthy adult Swiss albino mice of either sex, weighing
between 20–30 g, were used for the study. The animals
were housed in clean, properly ventilated polypropylene
Lowkika et al. Int. J. Zool. Appl. Biosci., 11(1), 17-22, 2026
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cages under standard laboratory conditions, maintained at a
temperature of 22 ± 2°C, relative humidity of 50–60%, and
a 12-hour light/dark cycle. They were provided with a
standard pellet diet and water ad libitum throughout the
experimental period. The animals were allowed to
acclimatize to the laboratory environment for at least seven
days before the commencement of the experiment to
minimize stress and ensure uniform physiological
conditions. All animal handling and experimental
procedures were conducted in accordance with the
guidelines of the Committee for Control and Supervision of
Experiments on Animals (CCSEA), Government of India.
The study protocol was reviewed and approved by the
Institutional Animal Ethics Committee (IAEC) of the
respective institute, and due care was taken to minimize
pain or discomfort to the animals during the study (IAEC
No: 11/IAEC/CLPT/2024-25).
Equipment – 8 Arm Radial Maze
The 8-Arm Radial Maze is a behavioral apparatus designed
to assess learning and memory in rodents. It consists of a
central platform with eight arms radiating outward,
resembling the spokes of a wheel, with a food reward (such
as a pellet) placed at the end of each arm. The task requires
the animal to use spatial memory (Kohler et al.,2022) to
remember which arms have already been visited and which
still contain rewards. This model evaluates two types of
memory: working memory, which involves recalling the
arms (Waegemans et al., 2002) visited within a single trial
to avoid revisits, and reference memory, which involves
remembering across multiple trials which specific arms are
consistently baited or unbaited. The 8-Arm Radial Maze is
widely used in neuropharmacological research to assess
cognitive enhancers and memory-improving drugs,
including cholinergic agents and nootropics, and serves as a
valuable model for studying Alzheimer’s disease and
dementia. It offers advantages such as high sensitivity to
both working and reference memory, reliability,
reproducibility, and non-invasiveness, although it requires
slight food deprivation to maintain the animals’ motivation
for the task.
Procedure
The 8-arm radial maze test was conducted to assess spatial
learning (Dubreuil et al.,2003) and reference memory in
mice. After a 7-day acclimatization period, animals were
mildly food-restricted to 85–90% of their normal body
weight to enhance motivation for the food reward. The
maze consisted of a central platform with eight arms
radiating outward, and visual cues were placed around the
room for spatial orientation. During the habituation phase,
all arms were open, and food pellets were scattered to allow
free exploration for 5–10 minutes. In the training phase,
(Leuner et al.,2010), a fixed set of arms (two to four) were
consistently baited across sessions. Each animal was placed
on the central platform, and the session continued until all
baits were collected or 10 minutes elapsed. Entries into
unbaited arms were recorded as reference memory errors,
while re-entries into previously visited arms were noted as
working memory errors. The number of correct choices,
total errors, and latency to complete the task were recorded
daily. Trials were conducted for consecutive days until
performance stabilized, with a decrease in reference
memory errors indicating learning (Olney et al.,2015) and
memory retention. The maze was cleaned between trials to
eliminate scent cues, and data were analyzed statistically to
compare treatment effects.
Figure 1: Design of 8 Arm Radial Maze
Figure 1. Design of 8 Arm Radial Maze.
Lowkika et al. Int. J. Zool. Appl. Biosci., 11(1), 17-22, 2026
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RESULTS AND DISCUSSION
The results of the 8-Arm Radial Maze test demonstrated a
clear improvement in learning and memory performance in
mice treated with the standard drug (Piracetam), (Kurz et
al.,2010), and the polyherbal formulations (Trachyspermum
ammi and Ocimum sanctum) compared to the control
group. The average number of correct arm entries
progressively increased over time across all groups,
indicating learning with repeated exposure to the maze.
However, the rate and extent of improvement varied
notably among the groups. The control group (0.9% saline)
showed a gradual but limited increase in correct choices
from 2.5 at 2 hours to 11.0 at 24 hours, reflecting normal
learning through repetition without pharmacological
enhancement. In contrast, the standard Piracetam group
exhibited a substantial improvement, (Costa et al., 2013),
with correct entries rising from 5.75 at 2 hours to 16.0 at 24
hours, confirming its well-established nootropic effect
through enhancement of cholinergic transmission and
facilitation of synaptic plasticity (Table 1).
Both Test I (100 mg/kg) and Test II (200 mg/kg) groups
treated with the polyherbal formulation also showed
marked improvements compared to the control. Test I
animals increased from 4.75 at 2 hours to 14.0 at 24 hours,
while Test II animals rose from 4.75 at 2 hours to 15.5 at
24 hours. This dose-dependent enhancement suggests that
the combination of Trachyspermum ammi and Ocimum
sanctum effectively improved spatial reference memory
and learning ability. The higher dose (200 mg/kg)
demonstrated slightly better performance, likely due to
synergistic effects of active constituents such as thymol,
(Timalsina et al.,2023) carvacrol, eugenol, and flavonoids,
which are known for their antioxidant, anti-inflammatory,
and cholinergic-modulating properties. Overall, the
findings indicate that the polyherbal combination exhibits
significant cognitive-enhancing (Malik et al.,2022), effects
comparable to Piracetam. The observed improvements in
maze performance may be attributed to reduced oxidative
stress, inhibition of acetylcholinesterase, and
neuroprotective actions within the hippocampus. Thus, the
formulation shows potential as a natural nootropic agent for
improving learning and memory functions.
Table 1. Evaluation of memory enhancement activity for various treatment groups.
Time
Group A
(Control)
Group B (Piracetam 150
mg/kg)
Group C
(Test I 100 mg/kg)
2 Hours
2.50
5.75
4.75
4 Hours
3.50
7.50
5.00
6 Hours
4.75
9.50
6.50
8 Hours
6.50
12.50
9.50
12 Hours
8.50
14.50
13.00
24 Hours
11.00
16.00
14.00
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www.ijzab.com 21
Figure 2. Graphical representation memory enhancement activity for various treatment groups.
CONCLUSION
The present study demonstrates that the polyherbal
formulation containing Trachyspermum ammi (Ajwain) and
Ocimum sanctum (Tulsi) exerts significant cognitive-
enhancing effects in mice, as evidenced by improved
performance in the 8-Arm Radial Maze test. Both test doses
(100 mg/kg and 200 mg/kg) produced a dose-dependent
increase in correct arm entries, indicating enhanced spatial
learning and reference memory compared to the control
group. The higher dose (200 mg/kg) showed performance
comparable to the standard nootropic drug, Piracetam (150
mg/kg), suggesting potent memory-facilitating activity.
These effects may be attributed to the synergistic action of
bioactive compounds such as thymol, carvacrol, and
eugenol, which enhance cholinergic transmission and
protect neuronal cells from oxidative and inflammatory
damage. Overall, the results support the potential of this
polyherbal combination as a safe and effective natural
alternative for improving memory and cognitive function.
ACKNOWLEDGMENT
The authors express sincere thanks to the head of the
Department of Pharmacology, Chalapathi Institute of
Pharmaceutical Sciences (A), Guntur, Andhra Pradesh for
the facilities provided to carry out this research work.
CONFLICT OF INTERESTS
The authors declare no conflict of interest
ETHICS APPROVAL
Not applicable
FUNDING
This study received no specific funding from public,
commercial, or not-for-profit funding agencies.
AI TOOL DECLARATION
The authors declares that no AI and related tools are used to
write the scientific content of this manuscript.
DATA AVAILABILITY
Data will be available on request
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