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pISSN 2302-1616, eISSN 2580-2909
Vol 10, No. 1, June 2022, pp. 37-43
Available online http://journal.uin-alauddin.ac.id/index.php/biogenesis
DOI https://doi.org/10.24252/bio.v10i1.26488
Copyright © 2022. The authors. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
Purwoceng (Pimpinella pruatjan Molk.) nanosuspension repairs spatial white
albino wistar strains’ spatial memory degeneration after sleep deprivation
Rangga Wisnu Wardana1, Tuti Sri Suhesti2, Fitranto Arjadi3*
1Department of Biomedical Sciences, Faculty of Medicine, Universitas Muhadi Setiabudi
Jl. Pangeran Diponegoro No. KM2, Rw. 11, Wanasari, Brebes, Central Java, Indonesia. 52212
2Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Jenderal Soedirman
Jl. Profesor DR. HR Boenyamin No. 708, Grendeng, North Purwokerto, Banyumas, Central Java, Indonesia. 53122
3Department of Anatomy, Faculty of Medicine, Universitas Jenderal Soedirman
Jl. Profesor DR. HR Boenyamin No. 708, Grendeng, North Purwokerto, Banyumas, Central Java, Indonesia. 53122
*Email: fitranto.arjadi@unsoed.ac.id
ABSTRACT. Paradoxical sleep deprivation (PSD) induces oxidative stress and interferes permanent
memory consolidation in hippocampus. Purwoceng (Pimpinella pruatjan Molk.) is herbal medicine with
antioxidant effect. These effects will be more effective with nanosuspension technology. The research
purposes were to find effect of PSD to rats’ spatial memory reduction and to find Purwoceng
nanosuspension extract effect to improve rats’ spatial memory after PSD stress induction. The research
method is randomized controlled trial with posttest only with control group approach to 36 white male
albino rats Wistar strain which is induced by 96 h PSD stress model. Experiment groups is regular extract
25 mg/300g BW/day, Purwoceng nanosuspension extract 25 mg/300 gBW/day, 50 mg/300 gBW/day, and
75 mg/300 gBW/day. Statistical analysis found that there is no significant effect of Purwoceng extract to
memory traveling time (p=0.414) and memory track length (p=0.316) between pre-PSD and post-PSD
group. There is significant effect of PSD stress after Purwoceng administration to memory track length and
memory travelling time (p=0,005). The conclusion is that there is significant correlation between the
difference of memory latency and memory traveling time of morris water maze (MWM) test between the
various Purwoceng nanoemulsion dose. The 25-50 mg/300 gBW/day is effective dose of Purwoceng
nanoemulsion extract to repairs spatial memory degeneration after PSD stress.
Keywords: Antanan gunung; nanoemulsion; paradoxical sleep deprivation; spatial memory; sleep
deprivation
Article History: Received 13January 2022; Received in revised form 30 January 2022; Accepted 14 April 2022; Available online 30
June 2022. Ver: Pre-Press
How to Cite This Article: Wardana RW, Suhesti TS, Arjadi F. 2022. Purwoceng (Pimpinella pruatjan Molk.) nanosuspension repairs
spatial white albino wistar strains’ spatial memory degeneration after sleep deprivation. Biogenesis: Jurnal Ilmiah Biologi. vol 10(1):
37–43. doi: https://doi.org/10.24252/bio.v10i1.26488.
INTRODUCTION
Sleep deprivation (SD) or lack of sleep can disrupt circadian rhythm and physiology mechanism
of body (Ma et al., 2019). It induces oxidative stress which damages temporary and permanent
memory consolidation as we find in Alzheimer, Parkinson, and another brain degenerative disease
(Wu et al. 2019). Characteristics of these diseases are reduction of quality and quantity of post
synapses neuron and hippocampus cell (Havekes, 2017). Paradoxical sleep deprivation elevates
glucose metabolisme up to 30 % until there is excess of free radicals, disrupt sleep-and-wake cycle,
leads to antioxidant imbalance, oxidative stress in hippocampus and the other central nervous system
part (Owens et al., 2016). Sleep deprivation intensifies corticosterone level, induces CA3 neuron
atrophy and reduces arborization (dendrite branching) in hippocampus. Those effect generates spatial
memory problem and anxiety behavior to experimental animals (Konankanchi, 2022).The potential
of Purwoceng as an antioxidant is found in bioflavonoid compounds, the exogenous antioxidants that
can increase antioxidant levels and suppress free radical formation (Nurcahyanti et al., 2018).
Bioflavonoids also activate ascorbic acid which is useful as an antioxidant and neuroprotectant
(Teleanu et al., 2019). Tests on the antioxidant activity of Purwoceng ethanol extract showed that
Purwoceng was able to capture free radicals (Qodri et al., 2013).
Indonesian unique herbal medicine (2019), Purwoceng (Pimpinella pruatjan Molk.) has
antioxidant effect because of phenol and bioflavonoid content). Nanosuspension technology makes
colloid particle can be dispersed into submicron size less than 1 μm, with mean sizes is 200 - 600 nm
Wardana et al. Biogenesis: Jurnal Ilmiah Biologi 38
(Ansari et al., 2012). The technology has been expected to increase absorption, bioavailability, onset,
peak level and penetration compared with regular extract. Bioflavonoid as exogen antioxidant can
escalate antioxidant body level and reduces ROS free radicals as product of oxidative stress. This
research purposes were to find sleep deprivation effect to spatial memory ability and antioxidant
ability of Purwoceng nanosuspension. This research uses nano preparation technology that can
increase the bioavailability and absorption of active ingredients so that the selectivity, effectiveness
and safety of herbs are more higher (Ansari et al., 2012). The release of active compounds can be
controlled to minimize side effects and with nano size can be given in high concentrations due to
their small size and high loading capacity (Dewandari et al., 2017). We expected that these results
can eliminate the disobedience of patient because the available formulations usually use large doses
and minimize the lack of drug target specificity for chronic disease.
MATERIALS AND METHODS
This is experimental, randomized controlled trial with posttest only, control group design
research. The subjects are 36 white male albino rats Wistar strain, divided into 6 groups: Goup A:
PSD with sleep recovery; group B: PSD without sleep recovery; group C: PSD and Purwoceng extract
25 mg/300 gBW/day, group D: PSD + Purwoceng nanosuspension extract 25 mg/300 gBW/day,
group E: 50 mg/300 gBW/day; group F: 75 mg/300 gBW/day. The chosen dose is based on recent
research by Pribadi, (2012) and Nuryadin dan Nabilla (2018). Their research doses are 25 mg/mL/day
for 300-gram body weight or 83,25 mg/kgBW. Nanoemulsion extract dose is 25 mg/3 mL by single
dose 3 mL nanoemulsion solution given by oral tube.
The Purwoceng has been obtained from Batur village, Dieng plateau, Banjarnegara. Plant
determination has been done in plant taxonomy laboratory of Biology Faculty, Universitas Jenderal
Soedirman, Purwokerto. Nanoemulsion process has been done in Pharmacy Laboratory of Pharmacy
Faculty, Jenderal Soedirman University, Purwokerto. Purwoceng nanoemulsion extract has been
made by mixing purwoceng ethanol extract with Tween 80, stirred with magnetic stirrer 500 rpm for
15 minutes, added by PEG 400 and Virgin coconut oil, and then stirred for 15 minutes. The solution
is added by a few aqudest slowly, and then stirred by magnetic stirrer 1000 rpm for 15 min, and then
sonicated in 25 – 40°C temperature for 30 min. The nanoemulsion phytochemicals are being screened
to measure alkaloid and flavonoid components, followed by nanoemulsion characteristics test, such
as organoleptic, transmittance percentage (%T), particle size, and particle size distribution and also
Zeta Potential.
Paradoxical Sleep Deprivation. Experimental animals were being placed in stainless steel cage
filled with water. It has 24x26x29 cm dimension, two platform with 6 cm diameter. The foothold has
muscle atonia shock device which automatically turns on every 10 min to give a shock effect for 1
second that causes a wake-up effect. This treatment is carried out for 96 h with a break between 07.00
– 11.00 WIB (Arjadi et al., 2021).
Morris Water Maze (MWM) test. Assessment of the spatial memory ability of rats using the
MWM test in a pool with a diameter of 180 cm and a height of 76 cm divided into 4 parts, one of
which is given a footing (on the shallow bottom of the water, not on the surface of the water) to stand
and stand on. The water filled with the pond consists of water and coconut milk/milk, which is useful
for clouding the water, so the rats are judged to find a foothold based on memory. The test takes 7
days, namely the first 5 days the experimental animals are swam 4 times a day within 60 s, until they
find a foothold (acquisition trial / acquisition test phase). On the 6th day, the stepping platform was
taken and the experimental animals were allowed to swim to find a foothold according to what was
remembered (probe trial/probe phase) and recorded the results of the trial probe as initial data for the
memory test of the experimental mice and on the 7th day a spatial ability test/sensorineural MWM
test/examination trial phase was conducted.
The treatment was documented by video camera and observed the spatial latency time ability test
the spatial memory capabilities of the rats by counting the time recorded from the time it was entered
Vol 10(1), June 2022 Biogenesis: Jurnal Ilmiah Biologi 39
into the pond until it was on a platform. The spatial ability test was tested for the length of the test
track to assess memory capabilities. We get spatial data of the rats by measuring the area and distance
of the path traveled in finding a foothold, and the results of the comparison of the distance measured
in the actual pond diameter with the diameter of the pond in the video, multiplied by the distance in
the video. The data is presented in tabular and graphical form, where the X-axis represents the day
session from the acquisition trial to the 7th day of testing and the Y-axis represents latency time (in
seconds) or track length (in cm) (Buccafusco, 2009).
Fig. 1. Test of spatial memory ability of experimental animals: A. Morris Water Maze pool; B. Acquisition test phase; C.
Test results between normal and impaired memory.
The MWM test was carried out at the end of each treatment phase as reference data for indicators of
spatial memory ability and became the basis for the analysis of the data used. The degree of degeneration of
spatial memory abilities is determined by the longer the path length and the longer the latency time required
to reach the target. Tests a few days before the sensorineural test (acquisition phase and probe phase) were
carried out as a means of practice and trials to increase the frequency of target recognition by footing. The data
displayed in the pre-PSD, post-PSD, and post-Purwoceng phases are descriptive data to see the pattern of
spatial memory development per experimental rat in the MWM pool and analyzed to get a conclusion.
Research ethics were obtained from the Health Research Ethics Commission, Faculty of Medicine, Universitas
Jenderal Soedirman, Number 097/KEPK/V/2021 dated 10 May 2021.
Data analysis. The research data was tested for the normality of the data distribution using the
Shapiro-Wilk and homogeneity test and data variance using Levene's Test. If the data transformation
is normal and heterogeneous, it is analyzed using One Way of Variance (ANOVA) as a parametric
test and if it is not normal, it is tested with Kruskal Wallis. The test was continued with the Tukey
Post-Hoc test to determine whether there was a significant difference between the control group and
the treatment group with a significance level of p < 0.05.
RESULTS AND DISCUSSION
The transmittance test showed that the size of the clarity and purity of the nanoemulsion formed
was 84.5% and although it did not reach 100%, it was sufficient as a marker of the validity of the
nanoemulsion solution structurally. The particle size distribution test of the Purwoceng nanoemulsion
solution was found to be 123.87±28.9 nm which indicates that the particle size distribution is ideal
Wardana et al. Biogenesis: Jurnal Ilmiah Biologi 40
because it is in the range of 20-200 nm, so that the stability and distribution of molecules in the
dissolution medium is optimal. The results of the polydispersity index of the Purwoceng
nanoemulsion solution obtained the number 0.37±0.11 which indicates that the intermediate disparity
is between homogeneous and heterogeneous. The results of the zeta potential of the Purwoceng
nanoemulsion solution were found to be -16.56±1.19 mV, which indicated good molecular stability
of the nanoemulsion solution from particle attraction aggregation, because it was in the range of -30
to 30 mV. The results of the phytochemical screening test showed that the Purwoceng extraction
solution was reactive in the alkaloid and flavonoid testing which indicated that the extraction solution
was positive for qualitatively qualitative alkaloids and flavonoids.
Table 1. Comparison of observation results of mwm of latency time test data.
Groups
Latency time (seconds)
Pre-PSD
Post-PSD
After Purwoceng Treatment
A (PSD Non-Sleep)
20.25
42.25
-
B (PSD + Sleep)
5.25
9.25
-
C (PSD + 25 mg extract)
13
21.25
46
D (PSD + 25 mg nanoemulsion)
15
39.25
27.25
E (PSD + 50 mg nanoemulsion)
7.5
12.5
12.5
F (PSD + 75 mg nanoemulsion)
11
7
12.25
Table 2. Data comparison of observation results long path of mwm examination trial per phase between groups of
experimental wistar rats.
Groups
Track length (cm)
Pre-PSD
Post-PSD
After Purwoceng Treatment
A (PSD Non-Sleep)
562.5
900.25
-
B (PSD + Sleep)
112
205
-
C (PSD + 25 mg extract)
255
511.25
1013.25
D (PSD + 25 mg nanoemulsion)
321.5
837
487.5
E (PSD + 50 mg nanoemulsion)
180
271.75
190.75
F (PSD + 75 mg nanoemulsion)
197.5
167
2715
Based on the figures in Tables 1 and 2, it can be seen that there is a decreasing trend of spatial
memory ability between the average rats after PSD treatment but statistically there is no significant
difference between PSD to the decrease in spatial memory ability in rats and sleep recovery does not
provide a difference to spatial memory ability. The paired t-test between pre-PSD and post-PSD in
group A showed travel time p = 0.168> 0.05 and path length p = 0.145> 0.05 and Wilcoxon test in
group B obtained travel time p = 0.180> 0.05 and path length p = 0.068>0.05. These results differ
from the findings of several studies on memory disorders.
Recent research (Heba et al., 2021) stated that 72 hours of PSD treatment through reduced
catalase activity and glutathione levels, inhibition of Na/K ATPase in the hippocampus and cerebral
cortex, increased acetylcholinesterase and lipid peroxidation which resulted in the induction of
oxidative stress and increased brain excitability can reduce memory performance and increase anxiety
levels in rats. Recent research by Kamphuis et al., (2017) states that the disruptive consequences of
degeneration of the prefrontal cortex in experimental rats treated with sleep disturbances for 7 days
caused degeneration of the prefrontal cortex with symptoms of slower motor responses and impulsive
reflexes. Increased anxiety in post-PSD rats resulted in increased brain excitability and overactivity
of motor behavior that made the rat's instincts move faster which increased the length of the trajectory
in the sensorineural MWM test.
Atrooz et al. (2019) stated that prolonged sleep disturbances increase the incidence of forgetting
and cause memory fragmentation such as short and medium-term memory loss due to weakened
associative pathways characterized by a decrease in the number of pyramidal and dendritic cells in
the CA1 hippocampus area, the main area of memory pathway transmission. According to Wu et al.
Vol 10(1), June 2022 Biogenesis: Jurnal Ilmiah Biologi 41
(2019), the pathophysiological mechanism of memory degeneration due to sleep disturbances is
related to glymphatic-vascular-lymphatic clearance in brain macromolecular structures, increased
oxidative stress in the brain and decreased levels of melatonin in blood circulation.
Hunter's research (2019) states that there is no evidence of a detrimental effect of REM sleep
disturbances on the performance of experimental mice in the acquisition phase of the MWM test,
both during retention and reversal and REM is only useful in helping improve learning performance
and general memory. Research by Karabulut et al. (2019) states that the post-MWM sleep phase,
especially during REM sleep, plays a very important role in modulating the consolidation and rapid
repair of cells that play a role in memory which can improve the transcription of mRNA genes from
pyramidal cells and lead to hippocampal neuroplasticity.
The explanation for the absence of a significant difference between PSD and the role of sleep
recovery on spatial memory abilities in this study is still unknown but it is suspected that adequate
sleep compensation after PSD and before the MWM acquisition phase to sensorineural testing. The
short sleep compensation was able to provide a better consolidation effect on brain and hippocampal
performance so that it could reduce the effect of decreasing spatial memory ability which is expected
to exist in the rat group. Sleep compensation in PSD was more accommodating than when it was
replaced with total sleep deprivation (TSD) treatment where mice were not allowed to sleep at all for
several days. According to Tufik et al. (2009), adequate compensation from regular sleep-wake cycles
can improve neurophysiological and behavioral changes significantly despite having experienced
sleep disturbances, indicating that there is no significant difference in spatial memory ability between
the groups of rats with sleep recovery and without sleep recovery because all groups received sleep
compensation even with different duration.
Based on statistical tests, there was no significant difference between the administration of
Purwoceng on spatial memory abilities in rats based on the length of the track and the latency time.
The administration of purwoceng non-nanosuspension and nanosuspension extraction at a dose of 25
mg, 50 mg, or 75 mg did not give a significant difference in spatial memory ability. Data analysis
using Paired t-test group C (non-nanoemulsion Purwoceng extract) showed travel time p = 0.271(>
0.05) and path length p = 0.224 (>0.05), group D (Purwoceng nanoemulsion 25 mg/gBW) obtained
travel time p = 0.21> 0.05 and path length p = 0.249 (>0.05), Wilcoxon test in group E (Purwoceng
nanoemulsion 50 mg/gBW) obtained travel time p = 0.465(>0.05) and path length p = 0.465(>0.05),
and group F (Purwoceng nanoemulsion 75 mg/gBW) obtained travel time p = 0.651(>0.05) and path
length p = 0.647 (>0.05). Bivariate analysis testing the spatial memory ability of rats after
administration of Purwoceng through the One-Way ANOVA test showed a significant relationship
between travel time (p = 0.000 <0.05) and track length (p = 0.001 <0.05) in groups C, D, E, and C.
and F. The post hoc test showed that the lowest travel time was group D, followed by groups F and
E, while the highest travel time was found in group C and in the section on track length, the lowest
track length was group E, but not much different with groups D and F.
The explanation of the absence of significant differences between Purwoceng on post-PSD
spatial memory ability is not certainly known. It is suspected that the active flavonoid substances in
Purwaceng are able to reduce the level of cell aging and the level of tissue inflammation but cannot
provide instantaneous improvement in the quality of spatial memory. Flavonoids can modulate
neuronal signaling pathways that are important in memory processing, synaptic plasticity, and long-
term potentiation mechanisms as the basis for the formation of a memory (Rendeiro et al., 2009) and
improve cognitive memory through activation of kinases in the MAPK and P13 kinase pathways and
accelerate the process of regulation of the cAMP response element-binding protein (CREB) which is
important in the memory storage process in the hippocampus and cerebral cortex (Krishnaveni, 2012),
Nanoparticle preparation technology by emulsification was proven to produce significant
pharmacological effects which was indicated by the difference in the level of decline in spatial
memory ability at different doses of Purwoceng and non-nanoemulsion extract Purwoceng had a
Wardana et al. Biogenesis: Jurnal Ilmiah Biologi 42
higher rate of degeneration of spatial memory ability compared to the group that received Purwoceng
nanoemulsion.
CONCLUSION
There was no effect between the decrease in spatial memory ability and the number of pyramidal
cells and hippocampal volume in albino male wistar rats treated with PSD. There was no significant
effect of giving Purwoceng nanoemulsion on improving spatial memory ability of albino male wistar
rats treated with PSD. There is a significant effect on the difference in latency and path length of the
MWM test between doses of Purwoceng nanoemulsion. The dose of 25 - 50 mg is the effective dose
of the Purwoceng nanoemulsion which results in a shorter path length and latency time indicating an
improvement in post-PSD spatial memory degeneration.
ACKNOWLEDGEMENTS
The author would like to thank to Head of Research and Community Service, Universitas
Jenderal Sudirman, Head of the Department of Anatomy, Faculty of Medicine, Universitas Jenderal
Sudirman, Dzicky Rifqi Fuady as proofreader and other parties have assisted in the research.
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