pubs.acs.

org/JAFC Article

Moringa oleifera Lam Seed Oil Augments Pentobarbital-Induced

Sleeping Behaviors in Mice via GABAergic Systems
Wei-Liang Liu, Bai-Fen Wu, Jian-Hua Shang, Yun-Li Zhao,* and Ai-Xiang Huang*
Cite This: J. Agric. Food Chem. 2020, 68, 3149−3162 Read Online

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ABSTRACT: Moringa oleifera Lam. (MO), which is widely consumed as both food and herbal medicine in tropical and subtropical
regions, has a wide spectrum of health benefits. Yet, whether the oil obtained from MO seeds could affect (improve) the sleep
activity remains unclear. Herein, we used the locomotor activity, pentobarbital-induced sleeping, and pentetrazol-induced
convulsions test to examine sedative-hypnotic effects (SHE) of MO oil (MOO) and explored the underlying mechanisms. Besides,
the main components of MOO like oleic acid, β-Sitosterol, and Stigmasterol were also evaluated. The results showed that they
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possessed good SHE. Except for oleic acid and Stigmasterol, they could significantly elevate γ-amino butyric acid (GABA) and
reduce glutamic acid (Glu) levels in the hypothalamus of mice. Moreover, SHE was blocked by picrotoxin, flumazenil, and
bicuculline, except for oleic acid, which could not be antagonized by picrotoxin. Molecular mechanisms showed that MOO and β-
Sitosterol significantly upregulated the amount of protein-level expression of Glu decarboxylase-65 (GAD65) and α1-subunit of
GABAA receptors in the hypothalamus of mice, not affecting GAD67, γ2 subunits. These data indicated that MOO modulates sleep
architectures via activation of the GABAA-ergic systems.
KEYWORDS: MOO, sedative-hypnotic, GABAA receptors, GABAA-ergic systems

■ INTRODUCTION
In mammals, sleep is a vital physiological need, which can
drumstick tree, horseradish tree, marango, and malunggay.8 It
is also commonly used as a medicinal plant, a vegetable, and a
maintain homeostasis, as well as healthy physical and mental source of cooking oil in the developing world.9 Due to its fast-
states. Thus, lack of sleep can result in fatigue, drowsiness, growing abilities, strong vitality, potential medicinal values, and
decreased immunity, anxiety, and depression.1 The increased socioeconomic advantages, it is the most widely cultivated
specie that has been naturalized by many subtropical and
work competition, rapid lifestyle pace, irregular daily habits,
tropical countries. In China, it dates back to the 1960s, when it
and similar other factors have led to an increased number of
was initially introduced to Yunnan Province of China from
sleep disorders among the general population. Globally,
India into local cultivation, spreading to Taiwan, Fujian,
approximately 10−15% of the adults have chronic insomnia,
Guangdong, Guangxi, and other regions.10 MO is a miracle
while an additional 25−35% suffer from transient occasional
and multipurpose tree, which has been identified as a native
sleep disorders.2 Besides affecting the quality of daily life and
source of highly digestible protein, potassium, calcium, iron,
work efficiency, these disorders can lead to various health and
vitamins, essential amino acids, carotenoids, and antioxidants
socioeconomic issues.3 Among the conventional approaches
that are suitable to fight malnutrition, particularly among
for insomnia treatment, pharmacological therapy is the most
infants and nursing mothers in many “developing” nations
commonly used treatment option. It has been reported that
where malnourishment is a major concern.11,12 Almost all parts
some prescription drugs, such as benzodiazepines (BDZ) and
of MO are potentially useful and store important nutrients.13
nonbenzodiazepines, could produce sedative-hypnotic effects
Some studies have shown that the seeds of the plant are
(SHE) to improve the patient’s condition; however, long-term
probably the most valuable part, which contains a series of
use of these compounds can cause adverse effects like drug
biologically active agents such as steroids, terpenoids, protease,
dependency, tolerance, muscle relaxation, addiction, amnesia,
flavonoids, alkaloids, and 4(α-L-rhamnosyloxy) benzylisothio-
and cognitive impairment.4,5 Thus, it is necessary to develop
cyanate, and produce a significant percentage of high-quality
more effective and safe drugs with fewer side effects.
oil yield, i.e., 30−40% by weight. This oil is also called “ben
It is well known that some naturally functional foods, such as
oil”, which is a nondrying and nonsticky oil, which can be used
whole grains, Lingzhi, maca, panax, kiwifruits, walnut, and milk,
can improve sleep in people.6,7 Although the relationship
between natural products and sleep is clear, their molecular Received: January 3, 2020
mechanisms should be further clarified. Revised: February 16, 2020
Moringa oleifera Lam. (MO), which belongs to the family of Accepted: February 16, 2020
Moringaceae, is a perennial, angiosperm specie, indigenous to Published: February 17, 2020
the sub-Himalayan tracts of India, Pakistan, Bangladesh, and
Afghanistan, where it is known by several alias names such as

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.jafc.0c00037

3149 J. Agric. Food Chem. 2020, 68, 3149−3162
Journal of Agricultural and Food Chemistry
against rancidity.14,15 Beyond its valued nutritional advantages,
modern pharmacological studies have indicated that different
parts of MO, such as seeds, pods, leaves, flowers, stem, and
roots, possess diverse biological properties, such as curd
effect,16,17 antispasmodic, antiulcer, anti-inflammatory, anti-
oxidant, antidiabetic, anticancer, as well as anticonvulsion.18
With the development of the MO industry, which has
become one of the special plant resources of Yunnan Province,
characterized by unique properties like the high quantity of
production, easy collection, and low cost. It also has been
identified as a homology of medicine and food plants with a
variety of health benefits. The seed oil of MO resembles olive
oil and is generally applied in lighting, cooking, perfume, and
cosmetics industries.19 A number of reports had demonstrated
that MO oil (MOO) is rich in some major bioactive agents
including the abundance of the unsaturated fatty acids (oleic
acids), β-Sitosterol, and Stigmasterol.20 The health benefits of
MOO and its bioactive compounds have a wide range of
pharmacological activities such as anticancer, antibacterial,
anti-inflammatory, antioxidants, antidyslipidemia, antidiabetic,
and neuroprotection effects.21 Moreover, numerous research-
ers are still investigating the health improvement of MOO and
its use in functional foods and pharmaceuticals.
Despite there are different studies that have elucidated the
involvement agents of MOO in a variety of physiological
functions, there are no studies that investigate the effect of
MOO on sleep. Interestingly, β-Sitosterol and Stigmasterol are
well-known triterpenoids, which have a preventive effect on
some diseases like atherosclerosis, hyperlipidemia, tumor, and
Alzheimer’s disease (AD).22 Notably, β-Sitosterol also has
sedative-hypnotic activities,23 thus indicating that MOO may
potentially have a depressant effect. Taking into consideration
the lack of combating insomnia reports of MOO, in this study,
we investigated the sleep improvement effects and the
mechanism of its major biologically active agents, such as
oleic acids, β-Sitosterol, and Stigmasterol.
■
MATERIALS AND METHODS
Plant Materials. The dried and cleaned seeds of MO were
purchased from Yunnan Tianyou Biotechnology, Inc. (Kunming,
Yunnan, China) in March 2019; the cultivated variety was PKm2.
Preparation of MOO Extraction and Phytochemical Anal-
ysis. The MO seeds (1000 g) were weighed, their coat was removed,
and then they were fed to an expeller. According to the previously
reported method24 that was somewhat adjusted, the oil was obtained
by continually pressing and crushing seeds by the expeller. During the
expeller pressing (cold-pressed), no external heat was applied. After
the extracted oil was received, the percentage of oil yield was
calculated, the crude oil was sent to a filter to remove the impurities,
and stored in sterile containers at 4−8 °C in a refrigerator for further
use.
Determination of percentage oil yield (%) was as follows: %
extraction yield = (weight of sample before extraction − weight of the
sample after extraction/weight of sample before extraction) × 100%.
Determination of Chemical Characteristics. Sterol Composi-
tion. The sterols of the MOO were determined by gas
chromatography (GC), following the Association of Official Analytical
Chemists method.25 Analyses were carried out on an Agilent gas
chromatograph (model 7890A-5975C) equipped with an SE-54
capillary column (50 m × 0.25 mm, 0.10 μm film thickness; Supelco,
Inc., Sigma) and a flame ionization detector (FID). Initial oven
temperature, 240 °C; ramp rate, 4 °C/min; and a subsequent increase
from 240 to 255 °C were applied. The injector and FID temperatures
were set at 320 °C. Extrapure H2 at a flow rate of 36 cm/s was used as
a carrier gas a; a split injector was used with a split ratio of 20:1.
Sample (1 μL) was injected into the GC system for measurement.
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The internal standard used was betulin, and unknown sterol
components were identified and quantified using a pure sterol
standard mixture. The relevant contents of the chemical components
of the sterols were calculated using the peak area normalization
method.
Fatty Acid (FA) Composition. The FA composition of the MOO
was extracted by Soxhlet and determined using GC after
derivatization to fatty acid methyl esters (FAME). FAME were
prepared according to the standard International Union of Pure and
Applied Chemistry (IUPAC) method 2.30126 and analyzed on a
PerkinElmer model 8700 gas chromatograph, fitted with BPX70 (70%
cyanopropyl poly(silphenylene-siloxane)) highly polar capillary
column (100 m × 0.25 mm, 0.20 μm film thickness; SGE Japan,
Inc., Yokohama, Japan) and a flame ionization detector (FID). The
oven temperature was programmed as follows: initial temperature was
100 °C that was maintained for 6 min from 100 to 180 °C at 10 °C/
min, from 180 to 200 °C at 1 °C/min for 20 min, from 200 to 230 °C
at 4 °C/min for 10.5 min; injector and FID temperatures were set at
270 and 280 °C, respectively. Extrapure N2 at a flow rate of 3 mL/min
was used as the carrier gas, and a split injector was used with a split
ratio of 100:1. Sample (1 μL) was injected into the GC system for
measurement. The internal standard used was triglyceride undeca-
noate, and unknown fatty acid components were identified and
quantified using a pure fatty acid standard mixture. The relevant
contents of the chemical components were calculated using the peak
area normalization method.
Animals. Healthy Adult Male Institute of Cancer Research (ICR)
mice, weighing 18−22 g, were purchased from the SJA Laboratory
Animal, Inc. (Hunan, China, License No. SYXK 2016-002). The
animals were kept under steerable circumstance conditions with a
constant temperature (24 ± 1 °C), relative humidity (50 ± 10%), and
12/12 h light−dark cycle. All of the mice were fed with standard
laboratory food (High-Quality Lab Animals Diets, ShooBree, Inc.,
Jiangsu, China) and water ad libitum. All of the mice were randomly
divided into different groups (n = 12). All animal studies (including
the mice euthanasia procedure) were done in compliance with the
regulations and guidelines of Yunnan Agricultural University institu-
tional animal care and conducted according to the Association for
Assessment and Accreditation of Laboratory Animal Care (AAALAC)
and the Institutional Animal Care and Use Committee (IACUC)
guidelines.
The behavioral evaluations were implemented during the day (8:00
a.m. to 5:00 p.m.) in a quiet place with the same conditions as
mentioned above. To ensure rapid adaptation to the new environ-
ment, the mice were acclimated for 3 days before testing.
Reagents and Drug Administration. The MOO was prepared
in the laboratory as mentioned above. Pentobarbital sodium (PENT),
flumazenil (FMZ), muscimol (MUS), bicuculline (BIC), sodium
carboxymethyl cellulose (CMC-Na), polyoxyethylene sorbitan
monooleate (Tween-80, TW), and picrotoxin (PIC) were purchased
from Sigma-Aldrich Chemical, Inc. (St. Louis, MO); pentylenete-
trazole (PTZ) was obtained from National Institutes for Food and
Drug Control (Beijing, China); and estazolam tablets (EST) were
bought from Huazhong Pharmaceutical, Inc. (Wuhan, Hubei, China).
Mice γ-amino butyric acid (GABA) and glutamic (Glu) ELISA kit
were purchased from Bio-Swamp Industrial, Inc. (Wuhan, Hubei,
China). Oleic acid (OA, ≥99%), Stigmasterol (Stigma, ≥95%), and β-
Sitosterol (β-Sito, ≥95%) were purchased from Shanghai Yuanye
Biotechnology, Inc. (Shanghai, China). Specific rabbit polyclonal
antibodies against GABAA receptors subunits (α1, γ2) or glutamate
decarboxylase (GAD65/67) and the corresponding conjugated anti-
rabbit immunoglobulin G-horseradish peroxidase were obtained from
Abcam, Inc. (Cambridge, U.K.). All other reagents and chemicals
were of the highest possible grade.
CMC-Na, which is used as a food additive, exerts emulsification
effect and is not harmful to human health. TW (a nonionic surfactant)
is commonly used as an intestinal absorption enhancer; it is generally
considered safe for the development of oral drug-delivery systems.60
In our recent study, the test samples, such as MOO, OA, β-Sito, and
Stigma, have shown to be hardly soluble in purified water. Therefore,
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Figure 1. Experimental procedure scheme. (A) Sedative-hypnotic effects evaluation of MOO. (B) Sleep improvement mechanism of MOO
investigated using the synergic and antagonistic test on GABAA receptor. (C) Molecular mechanism of MOO promoting sleep test. PENT =
pentobarbital sodium; PTZ = pentylenetetrazole; MUS = muscimol; EST = estazolam tablets; PIC = picrotoxin; BIC = bicuculline; FMZ =
flumazenil; ELISA = enzyme-linked immunosorbent assay; WB = western blot; GABA = γ-amino butyric acid; Glu = glutamic; GAD65/67 =
glutamate decarboxylase; GABAA = γ-amino butyric acid type A receptor.
for intragastric administration (20 mL/kg, ig), MOO (2, 1 g/kg), OA
(2, 1 g/kg), Stigma (50, 10 mg/kg), and β-Sito (100, 20 mg/kg) were
prepared in 0.5% CMC-Na containing 2% TW immediately before
use to obtain a uniform suspension and to improve absorption in the
mice body.27,28 The positive control, EST (2 mg/kg), was
administered in the same manner as the MOO mentioned above.
The control groups received an oral administration of 0.5% CMC-Na
consisting of 2% TW.
To the test agents such as MOO, OA, Stigma, and β-Sito, dosages
were prepared according to a previously described approach:29 briefly,
by assuming that human clinical dose = X mg/kg, is converted into
the rat’s dose = (X mg/kg × 70 kg × 0.018/200 g, i.e., 6.3X mg/kg;
the mice’s dose = (X mg/kg × 70 kg × 0.0026/20 g i.e., 9.1X mg/
kg)). World Health Organization (WHO) recommended a plant oil
intake of 25−30 g once daily for adults with moderate manual labor;30
thus, it gets converted into a mice’s dose of 3.6−4.3 g/kg. Rodrigues
et al. reported that oral ingestion OA (0.22 g/kg) could accelerate the
inflammatory phase of wound healing in rats. In addition, the Food
and Drug Administration (FDA) and European Commission for Food
Science (ECFC) recommended daily intake of 1.3−3.0 g phytosterols
for preventing cardiovascular disease (CAD);31 thus, it gets converted
into a mice’s dose of 200−400 mg/kg. Consequently, according to the
previous studies reported with slight modification, we select a
reasonable dose for animal study.
To test agent safety, and based on our preexperiment, all mice
received a 5 g/kg by gavage. Animal conditions were recorded for 14
days, and the results showed that no abnormality occurred. OA is one
of the healthy bioactive compounds present in olive oil, nuts, butter,
and cheese.32 Hepburn et al. reported a subchronic 90-day oral
toxicity study on phytosterol esters (up to 3.9 g/kg), a novel
functional food, where there have no significant adverse effects on
rats.33 Moreover, Weststrate et al. reported that oral phytosterols (8.6
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g/day) have no significant adverse effects on the normal metabolism
of the body as well as intestinal microflora structure and function in
adults.34 In addition, the FDA and Chinese Ministry of Health
recommended that intake of moderate phytosterols (1.3−2.4 g/day)
was safe for humans.35 Besides, Ajibade et al. reported that the extract
of seeds of MO is safe when used as medicinal and nutritional
supplement.36 Moreover, in 2014, Bolanle et al. reported that MOO
was a kind of edible oil with no toxicity, and it could be used as a new
functional food for people in the future.37 Taken together, considering
MOO might be developed as new resource food, its safety dosage
evaluation should be carefully evaluated in the future.
For intraperitoneal injection (10 mL/kg, ip), PENT and PTZ were
diluted in 0.9% saline. PIC (4 mg/kg), BIC (6 mg/kg), FMZ (10 mg/
kg), and MUS (0.05 mg/kg) were dissolved in saline containing 2%
TW.38 The dosages were determined according to previous studies
and our preliminary experimental results (unpublished data). All
samples were freshly prepared on the test day. The hypnotic dose of
PENT (50 mg/kg, ip) and subthreshold dose (30 mg/kg, ip) were
used during the experiment. MOO, OA, Stigma, β-Sito, and EST were
administered (ig) 30 min before pentobarbital or PTZ was injected
intraperitoneally (ip) into the mice. FMZ, MUS, BIC, and PIC were
prepared 15 min after the oral administration of test samples.
Measurement of Spontaneous Activity. The locomotion
activity of the mice was measured using an autonomous activity
instrumentZZ-6 locomotor activity tester (Taimeng Software, Inc.,
Chengdu, Sichuan, China). Each mouse was individually placed in the
activity cage 30 min after the last administration. The mice were
acclimated to the environment for 3 min, which was automatically
measured and recorded for 5 min by a microcomputer control system,
where the horizontal locomotor activity was converted to the total
ambulatory frequency.39 A schematic diagram of the treatment
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J. Agric. Food Chem. 2020, 68, 3149−3162
Journal of Agricultural and Food Chemistry
Figure 2. Major components of MOO GC chromatogram: sterol (A) and fatty acid (B).
schedule is shown in Figure 1A. Experimental details are provided in
the Supporting Information.
PENT-Induced Sleeping Test. Male ICR mice were randomly
divided into 10 groups. The test was initiated 30 min after the last oral
administration when the mice were treated ip with a hypnotic (50
mg/kg, 10 mL/kg) or subhypnotic dose of PENT (30 mg/kg, 10 mL/
kg). Then, the sleep number within 15 min was recorded and the
percentage of sleep onset was calculated.40 Moreover, sleep latency
and sleep duration were also recorded.41 A schematic diagram of the
treatment schedule is shown in Figure 1A.
PTZ-Induced Convulsions. In this experimental model of PTZ-
induced convulsions,42 male ICR mice were randomly grouped into
several groups; 30 min after the last oral administration, all of the
mice were treated with PTZ (85 mg/kg, 10 mL/kg) to induce
clonic−tonic convulsion. Then, parameters like death number and
mortality were measured for the anticonvulsant property. Moreover,
the mortality protection rate was recorded for each group. A
schematic diagram of the treatment schedule is shown in Figure 1A.
GABAA Receptor Synergic Test. Two specific GABAA receptor
agonists, MUS (0.05 mg/kg) and EST (0.25 mg/kg), were used to
explore whether GABAA receptors participate in the sedative-hypnotic
effect of MOO and its active constituents. MUS is a GABA site
agonist, and EST is a benzodiazepine site-specific agonist, which were
given 15 min after MOO, while their active constituents were
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individually administrated. A schematic diagram of the treatment
schedule is shown in Figure 1B.
GABAA Receptor Antagonistic Test. Three specific GABAA
receptor antagonists, PIC (4 mg/kg), BIC (6 mg/kg), and FMZ (10
mg/kg), were used to explore whether GABAA receptors participate in
the sedative-hypnotic effect of MOO and its active constituents. PIC
is a noncompetitive inhibitor, BIC is a light-sensitive competitive
antagonist, and FMZ is a benzodiazepine site-specific antagonist,
which were treated 15 min after MOO, after which their active
constituents were administrated respectively. A schematic diagram of
the treatment schedule is shown in Figure 1B.
Determination of Brain GABA and Glu Content. To
investigate the effects of MOO and its bioactive agents on the brain
GABA and Glu levels, mice used in the above spontaneous activity
test were sacrificed after accomplishing the test, after which their total
brain was rapidly taken on an ice pad, and the region of hypothalamus
was isolated, weighed, and stored at −80 °C until extraction.43 GABA
and Glu levels in the brain were measured according to the
manufacturer’s instructions. A schematic diagram of the treatment
schedule is shown in Figure 1C.
Western Blot (WB) Analysis. This test was performed as
previously described with a slight modification.44 Concisely, tissue
samples from the hypothalamus were removed from the brain at the
end of treatment and total protein was extracted by using
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radioimmunoprecipitation assay (RIPA) lysis buffer and protease
inhibitors, which were added immediately before use. The
immunoreactive proteins were detected using the enhanced
chemiluminescence WB detection system. A schematic diagram of
the treatment schedule is shown in Figure 1C.
Statistical Analysis. Data from the experimental analyses are
expressed as mean ± standard error of the mean (S.E.M.). For
multiple comparisons, data were analyzed by one-way analysis of
variance (ANOVA) followed by the Student−Newman−Keuls test
with SPSS software and GraphPad Prism 5. For the test with a
subthreshold dose of PENT and the antiseizure test, the χ2 test was
used to compare the number of mice that fell asleep or died. p < 0.05
was considered to indicate a statistically significant difference between
groups.
■
RESULTS
MOO Components Analysis. The GC chromatogram
profiles of OA, β-Sito, and Stigma, the functional components
of MOO, are shown in Figure 2. The absolute concentrations
were 769, 9.5, and 5.4 g/kg of MOO, respectively, and the
retention time was 32.362, 18.451, and 17.608 min (Figure
2A,B). Data are provided in Tables S1 and S2.
Locomotor Activity. Compared to the control group
(0.5% CMC-Na consisting of 2% TW) (43.33 ± 15.47), all of
the animals from the drug treatment group showed different
degrees of reduced locomotion activity; the activity was
reduced in a dose-dependent manner: MOO-2 g/kg, 29.33 ±
8.09 (p < 0.01); MOO-1 g/kg, 34.58 ± 12.43 (p > 0.05); OA-2
g/kg, 30.83 ± 6.99 (p < 0.05), OA-1 g/kg, 33.75 ± 12.45 (p >
0.05); β-Sito-100 mg/kg, 29.75 ± 10.87 (p < 0.05), β-Sito-20
mg/kg, 36.58 ± 6.43 (p > 0.05); Stigma-50 mg/kg, 24.00 ±
10.48 (p < 0.01), Stigma-10 mg/kg, 35.25 ± 16.23 (p > 0.05)
(Figure 3). Moreover, EST-2 mg/kg significantly inhibited the
locomotion activity in a positive group compared to the
control group 11.08 ± 7.86 (p < 0.001; Figure 3). These
results indicated that MOO and its bioactive components
exerted an inhibitory effect on the central nervous system
(CNS) with dose−response curves.
Figure 3. Effects of oral administration of MOO on locomotor
activity test in mice. Each column represents mean ± S.E.M. (n = 12).
The mice were acclimated to the environment for 3 min in the
machine, 30 min after i.g. administration of EST (2 mg/kg), MOO (2,
1 g/kg), OA (2, 1 g/kg), β-Sito (100, 20 mg/kg), Stigma (50, 10 mg/
kg). The significance of the effects of the compounds was assessed
using analysis of variance (ANOVA) followed by the Student−
Newman−Keuls test. *p < 0.05, **p < 0.01, ***p < 0.001 compared
to control (0.5% CMC-Na consisting of 2% TW). S.E.M. = standard
error of the mean; Con = control; MOO = Moringa oleifera oil; EST =
estazolam tablets; OA = oleic acid; β-Sito = β-Sitosterol; Stigma =
Stigmasterol.
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Augmented of PENT-Induced Sleeping. To determine
the hypnotic activity of MOO, we first implemented a sleep
test to mice treated with the subthreshold dose of PENT
(Figure 4A). In comparison to the control group (0.5% CMC-
Na consisting of 2% TW), statistical analysis revealed increased
hypnotic activity in all treatment group: MOO (2 g/kg, p <
0.01), OA (2 g/kg, p < 0.05), β-Sito (100 mg/kg, p < 0.01),
Stigma (50 mg/kg, p < 0.05), which indicated that MOO and
its bioactive components have a hypnotic affect. Furthermore,
the hypnotic activity increased in the positive group EST (2
mg/kg, p < 0.001) compared to the control group (Figure 4A).
Second, we studied the sleep latency (Figure 4B) and sleep
duration (Figure 4C) in mice with a threshold dose of PENT.
Compared to the control group (5.53 ± 1.34 min), the sleep
latency occurred in a dose-dependent manner at MOO-2 g/kg,
4.01 ± 0.81 min (p < 0.01), MOO-1 g/kg, 4.65 ± 0.70 min (p
> 0.05); OA-2 g/kg, 4.00 ± 1.10 min (p < 0.01), OA-1 g/kg,
4.57 ± 1.21 min (p > 0.05); β-Sito-100 mg/kg, 3.93 ± 0.59
min (p < 0.001), β-Sito-20 mg/kg, 4.57 ± 1.12 min (p > 0.05);
Stigma-50 mg/kg, 3.95 ± 0.77 min (p < 0.01), Stigma-10 mg/
kg, 4.59 ± 0.90 min (p > 0.05). Moreover, the positive group
EST-2 mg/kg, 2.55 ± 0.93 min (p < 0.001) shortens the sleep
latency of the normal mice dramatically. Simultaneously, in
comparison to the control group (31.1 ± 21.2 min), the sleep
duration also occurred in a dose-dependent manner at MOO-2
g/kg, 62.85 ± 18.96 min (p < 0.001), MOO-1 g/kg, 44.96 ±
12.49 min (p > 0.05); OA-2 g/kg, 62.60 ± 23.55 min (p <
0.01), OA-1 g/kg, 41.17 ± 10.35 (p > 0.05); β-Sito-100 mg/
kg, 50.53 ± 13.77 min (p < 0.01), β-Sito-20 mg/kg, 42.99 ±
14.18 min (p > 0.05); Stigma-50 mg/kg, 49.15 ± 8.42 min (p
< 0.01), Stigma-10 mg/kg, 38.14 ± 10.68 min (p > 0.05).
Additionally, EST-2 mg/kg, 117.12 ± 1.52 min (p < 0.001)
significantly prolonged the sleep duration in the normal mice.
These results demonstrated that MOO and its major active
components exerted synergistic effects with PENT.
Effect of MOO on PTZ-Induced Convulsion. PTZ,
which is a noncompetitive antagonist of the GABAA receptor,
is generally used to induce a seizure in the animal model of
epilepsy.45 Mice treated with PTZ (85 mg/kg, ip) can produce
generalized tonic-clonic seizures. Therefore, we examined the
antiseizure effects of MOO and its activity components on the
series of characteristic behavioral symptoms such as tremor of
the vibrissae and muscles, which were observed after PTZ
injection. Besides, the mortality and the percentage of
mortality protection of each group were calculated. As depicted
in Table 1, compared to the control group (0.5% CMC-Na
consisting 2% TW), all of the animals administrated with EST
(2 mg/kg) were protected (100%) and these mice did not have
any convulsion after PTZ injection (p < 0.001); the other oral
treatment group animals showed different degrees effect
against the PTZ-induced convulsion and produced a dose-
dependent protection; especially, MOO (2 g/kg, p < 0.05), OA
(2 g/kg, p < 0.05), β-Sito (100 mg/kg, p < 0.01), and Stigma
(50 mg/kg, p < 0.01) groups had significantly reduced the
mortality rate, which was 81.8 and 27.3%. These results
indicated that MOO and its activity components have an
antiseizure role in the PTZ-induced model.
Mechanism of Hypnotic Effect of MOO. Effects of
Coadministration of MOO with MUS on PENT-Induced
Sleeping. We coadministrated MOO with MUS on PENT-
induced sleeping to investigate the direct relationship between
the hypnotic activity of MOO and the γ-amino butyric
acidergic (GABAergic) system. The pretreatment low doses of
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Figure 4. Effects of MOO on hypnotic response in PENT-treated mice. All values are mean ± S.E.M. (n = 12). (A) The number of mice falling
asleep, (B) sleep latency, and (C) sleep duration. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control (0.5% CMC-Na consisting 2% TW).

Table 1. Anticonvulsant Effect of MOO on PTZ-Induced of MUS (0.05 mg/kg) exhibited the synergistic effect of
Convulsions in ICR Micea decreasing sleep latency (p < 0.05, p < 0.01, p < 0.001, Figure
5A) and significantly prolonged the sleeping duration (p <
mortality
dose death mortality protection 0.05, p < 0.01, Figure 5B), in comparison to the self-control
treatment number (kg−1) number (%) (%) group. Moreover, the coadministration also remarkably
Control 11 20 mL 10 90.9 increased the number of sleeping mice (p < 0.05, p < 0.01,
EST 11 2 mg 0 0 100*** Figure 5C) in mice treated with subhypnotic dosages of
the low dose of 11 1g 8 72.7 20 PENT.
MOO Effects of Coadministration of MOO with EST on PENT-
the high dose 11 2g 5 45.4 50* Induced Sleeping. To explore the mechanisms underlying the
of MOO
depressant activity of MOO, the effect of coadministration of
the low dose of 11 1g 9 81.8 10
OA MOO and its bioactive components with EST (0.25 mg/kg), a
the high dose 11 2g 5 45.4 50* well-known agonist of the GABAA-BDZ receptor, was tested.
of OA The pretreatment low doses of MOO (1 g/kg), OA (1 g/kg),
the low dose of 11 20 mg 8 72.7 20 β-Sito (20 mg/kg), Stigma (10 mg/kg), or EST (0.25 mg/kg)
β-Sito
alone did not affect sleep latency and sleep duration induced
the high dose 11 100 mg 3 27.3 70**
of β-Sito by PENT. Surprisingly, the coadministration of EST (0.25 mg/
the low dose of 11 10 mg 7 63.6 30 kg) exhibited the synergistic effect of decreasing sleep latency
Stigma (p < 0.001, Figure 5D) and significantly prolonged sleeping
the high dose 11 50 mg 4 36.4 60** duration (p < 0.05, p < 0.01, p < 0.001, Figure 5E), in
of Stigma comparison to the self-control group. The coadministration
a
*p < 0.05, **p < 0.01, ***p < 0.001 significant compared to control. also dramatically increased the number of sleeping mice (p <
Abbreviations: MOO = Moringa oleifera Oil; EST = estazolam tablets; 0.05, p < 0.01, p < 0.001, Figure 5F) in mice treated with
OA = oleic acid; β-Sito = β-Sitosterol; Stigma = Stigmasterol; subhypnotic dosages of PENT.
mortality (100%) = (number of mice died after convulsion/total
number of mice used) × 100%; mortality protection (%) = (the death
Effects of Coadministration of MOO with PIC on PENT-
number of control group − the death number of experimental Induced Sleeping. To verify the mechanisms of the hypnotic
groups)/the death number of control group × 100%. effect of MOO and its bioactive components, we utilized
GABAA receptor noncompetitive inhibitor picrotoxin. As
shown in Figure 6, statistical analysis revealed that MOO (2
MOO (1 g/kg), OA (1 g/kg), β-Sito (20 mg/kg), Stigma (10 g/kg), OA (2 g/kg), β-Sito (100 mg/kg), or Stigma (50 mg/
mg/kg), or MUS (0.05 mg/kg) alone did not affect sleep kg) alone significantly shortened the sleep latency (2.91 ±
latency and sleep duration induced by PENT (50 mg/kg). The 0.28, 3.34 ± 1.04, 3.59 ± 0.91, and 3.06 ± 0.75 min,
rate of sleep onset induced by the subhypnotic dosages of respectively) and increased the sleep duration (46.61 ± 14.33,
PENT was not affected either. Interestingly, coadministration 57.98 ± 14.91, 50.81 ± 13.60, and 53.52 ± 16.17 min),
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Figure 5. Synergic effects of MOO with GABAA receptor agonist on hypnotic response in PENT-treated mice. Sleep latency (A), sleep duration
(B), and the number of mice falling asleep (C) combined with MUS. Sleep latency (D), sleep duration (E), and the number of mice falling asleep
(F) combined with EST. All data were presented as mean ± S.E.M. (n = 12). *p < 0.05, **p < 0.01, ***p < 0.001 compared to control; #p < 0.05,
##
p < 0.01, ###p < 0.001 compared to agonist plus each treatment group. NS = not significant.

individually (p < 0.05, p < 0.01, p < 0.001), in comparison to
the control group (4.95 ± 0.87 min and 35.31 ± 8.00 min).
The pretreatment of PIC (4 mg/kg) alone did not influence
sleep latency (Figure 6A) and sleep duration (Figure 6B)
induced by PENT (50 mg/kg). When the mice were
administrated MOO and its bioactive compounds with PIC,
the hypnotic effect was completely blocked by PIC (p < 0.05, p
< 0.01), compared to the self-control group, except for the OA
group, which showed no significant attenuation (p > 0.05) in
this research. These data indicated that MOO could partly
constitute exerted sedative effects via GABAergic neuro-
transmission.
Effects of Coadministration of MOO with BIC on PENT-
Induced Sleeping. Some reports have demonstrated that the
GABAA receptor possesses benzodiazepines, barbiturates, and
nerve sterol-binding sites that have been used to treat
insomnia; however, the hypnotic effect was competitively
inhibited by BIC. Consequently, we choose BIC to investigate
the relationship among MOO and its bioactive components
and GABAergic system. As shown in Figure 6, MOO (2 g/kg),
OA (2 g/kg), β-Sito (100 mg/kg), or Stigma (50 mg/kg)
alone significantly shortened the sleep latency (2.82 ± 0.42,
2.74 ± 0.31, 2.68 ± 0.35, and 2.87 ± 0.39 min, respectively)
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and increased the sleep duration (64.98 ± 19.65, 66.03 ±
15.87, 67.20 ± 21.75, and 78.38 ± 30.85 min, respectively) (p
< 0.01, p < 0.001), in comparison to the control group (4.06 ±
0.99 and 41.68 ± 15.24 min). The pretreatment of BIC (6 mg/
kg, ip) alone did not influence sleep latency (Figure 6C) and
sleep duration (Figure 6D) induced by PENT (50 mg/kg).
When the mice were administrated MOO and its bioactive
compounds with BIC, the tranquilizing effect was completely
antagonized by BIC (p < 0.05, p < 0.01), compared to the self-
control group. According to these results, we inferred that the
hypnotic effects of MOO and its bioactive compounds were
due to its GABAergic action.
Effects of Coadministration of MOO with FMZ on PENT-
Induced Sleeping. The GABAergic system has an important
role in sleep regulation, and the benzodiazepines (BDZ)
binding site on the GABAA receptor is the target for most
hypnotic drugs.46 FMZ, which is the GABAA-BDZ antagonist,
can inhibit the sedative-hypnotic activity of GABAA-BDZ
agonists such as diazepam (DZP), EST, by preventing them
from binding to GABAA-BDZ receptors.47 To validate that
MOO and its bioactive components affect the GABAergic
nervous system, we exploited GABAA-BDZ receptor inhibitor
FMZ. As shown in Figure 6, in comparison to the control
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Figure 6. Effects of GABAA receptor antagonists on the hypnotic activity of MOO. All data were presented as mean ± S.E.M. (n = 12). Sleep
latency and sleep duration with PIC (A, B), BIC (C, D), and FMZ (E, F). *p < 0.05, **p < 0.01, ***p < 0.001 significant compared to control; #p
< 0.05, ##p < 0.01, ###p < 0.001 significant between the antagonist treatment and nonantagonist treatment. GABAA = γ-amino butyric acid type A
receptor.

group (0.5% CMC-Na consisting 2% TW) (3.58 ± 0.74 and
27.86 ± 7.96 min), MOO (2 g/kg), OA (2 g/kg), β-Sito (100
mg/kg), Stigma (50 mg/kg), or EST (2 mg/kg) alone
significantly decreased the sleep latency (2.62 ± 0.27, 2.54 ±
0.39, 2.74 ± 0.39, 2.68 ± 0.45, and 2.28 ± 0.24 min,
respectively) and increased the sleep duration (51.46 ± 6.11,
62.34 ± 6.58, 47.21 ± 5.34, 55.85 ± 14.53, and 110.79 ± 23.92
min, respectively) (p < 0.01, p < 0.001). The preliminary
investigation of FMZ (10 mg/kg) alone did not influence sleep
latency (Figure 6E) and sleep duration induced by PENT
(Figure 6F). As expected, the hypnotic activity of EST was
found to be significantly inhibited by FMZ (p < 0.001). When
the mice were administrated MOO and its bioactive
constituents with FMZ, the hypnotic effect was also fully
reversed by FMZ (p < 0.05, p < 0.01, and p < 0.001),
compared to the self-control group. These results authenti-
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cated that MOO and its bioactive components can indeed act
on the GABAergic system that can be interrupted by FMZ.
Levels of Neurotransmitters in the Hypothalamus. GABA
is the main inhibitory neurotransmitter in the brain. It is well
established that activation of GABAA receptors favors sleep and
most of the sedative-hypnotics that are used in insomnia
treatment target the GABAA receptors.48 Glu, which is one of
the excitatory neurotransmitters in the central nervous system
(CNS), is also involved in the regulation of sleep. The brain
function is based on an exquisite balance between excitatory
and inhibitory neurotransmission, and when the balance
between the activity of these two amino acid neurotransmitters
is interrupted and pharmacologically shifted in favor of
GABAergic transmission, sedation can be induced.49 On the
contrary, it can result in arousal, anxiety, insomnia, and even
epileptic seizures. Accordingly, these two neurotransmitters
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Figure 7. Effects of MOO on the levels of GABA and Glu in the hypothalamus of mice. Each column represents mean ± S.E.M. (n = 12). **p <
0.01, ***p < 0.001 compared to control.

Figure 8. Effect of MOO on the expression of GABAA receptor subunits and of GAD65/67. GAPDH levels were needed for the normalization of the
protein expression. Each column represents the mean with S.E.M (n = 3). *p < 0.05 compared to control. Control = Con; oleic acid = OA;
Stigmasterol = Stigma; β-Sitosterol = β-Sito; Moringa oleifera oil = MOO; GABAA = γ-amino butyric acid type A receptor; GAD65/67 = glutamate
decarboxylase; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

have a key role in intercellular neural signal transmission and
many depressant active agents commonly exert their
pharmacological effects by regulating the concentration of
GABA and Glu. Some reports have demonstrated that in
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several sleep-related regions such as cerebral cortex,
diencephalon, midbrain, pons, and hypothalamus, these two
neurotransmitters are widely distributed, especially in the
hypothalamus.50 Accordingly, we examined the changes of
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GABA and Glu levels in the hypothalamus of the mice by the
ELISA kit. As depicted in Figure 7, compared to the control
group (0.5% CMC-Na consisting 2% TW), statistical analysis
revealed that MOO (2 g/kg, p < 0.01), β-Sito (100 mg/kg, p <
0.01), and EST (2 mg/kg, p < 0.001) significantly increased
GABA levels (Figure 7A) and decreased Glu in hypothalamus,
respectively (Figure 7B). However, OA (2 g/kg, p > 0.05) and
Stigma (50 mg/kg, p > 0.05) had no significant effect on the
levels of GABA and Glu in the hypothalamus (Figure 7).
Effect of MOO on the Expression of GAD65/67 and GABAA
Receptor Subunits. To confirm whether MOO and its active
constituents augment sleeping behaviors via biosynthesis of
GABA, the mice were treated with corresponding drugs for 14
days and they were sacrificed to examine the effect of these
drugs on the abundance of GAD65/67 and GABAA receptor
subunits in the hypothalamus (Figure 8). The findings revealed
that MOO (2 g/kg) and β-Sito (100 mg/kg) treatment
increased expression of GAD65 and α1-subunit (p < 0.05) but
did not influence the amounts of GAD67, γ2-subunits in the
GABAA receptors (p > 0.05); OA and Stigma did not affect
expression of these subunits, compared to the control group
(0.5% CMC-Na containing 2% TW). Moreover, EST (2 mg/
kg) significantly enhanced the amounts of the GAD65 and α1-
subunit (p < 0.05) but did not affect the abundance of GAD67,
γ2-subunits.
■
DISCUSSION
Sleep and wakefulness are regulated by a network of brain
nuclei that interact in a complex fashion, integrating
homeostatic and circadian regulations.51 Nowadays, due to
the rapid pace of modern life, sleep quality has a critical role in
keeping and improving the normal lifestyle, while it has also
been identified as essential in diagnosing the psychiatric
disturbance.52 Some reports have demonstrated that sleeping is
controlled by various neurotransmitters containing 5-hydrox-
ytryptamine (5-HT) and GABAergic neurotransmitter systems,
which are mostly related in the activation of sleep−wake
cycles.53 Among these neurotransmitters, GABA is a well-
known inhibitory neurotransmitter in the brain. Accordingly,
insomnia is a sophisticated nerve pathological state induced by
the dysfunction of a network of nervous systems. At present,
pharmacological treatments are considered as an easily
accessible, time-saving option. Still, the hypnotic agents
currently applied in clinic can produce undesirable adverse
effects such as drug addiction, tolerance, and dependence,
which can even occur at lower doses. In view of these side
effects, many researchers aim to develop more novel and safer
hypnotics. To date, three generations of hypnotic drugs have
been developed, including benzodiazepines and barbiturates,
which are based on the GABAA receptor exerted inhibitory
actions. Although the GABAA receptor agonists are still a drug
of choice, other new generations of hypnotic drugs are
currently being studied and developed. Accordingly, several
studies have reported the positive effects of natural products
for the treatment of sleep disorders.54
MO is an important tropical food plant with rich nutritional,
therapeutic, industrial, agricultural, and socioeconomic values.
It possesses many biological functions, including antiulcer,
anti-inflammatory, antioxidant, antidiabetic, anticonvulsion,
anticancer as well as antianxiety response. Furthermore, in
the folk medicine of Yunnan, India, Pakistan, and Bangladesh,
it is applied for the treatment of epilepsy and anxiety.8,55
Although MO has been investigated for its antianxiety activity,
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scarce information can be found on the sedative-hypnotic
activity of its seeds oil. Therefore, based on our preliminary
data and available literature, we assumed that MOO and its
active compounds would affect sleep, which we further
investigated in the present study. To the best of our
knowledge, this is the first study to investigate the effects of
MOO on sleep and the mechanisms through which its
hypnotic effect is generated. In several studies, OA, β-Sito, and
Stigma have been considered as major biologically active
components of MOO. Some of them revealed that β-Sito and
Stigma had some depressant effects, whereas these specific
substances responsible for the hypnotic effects of MOO had
not yet been identified. First, we determined the contents by
gas chromatography, which were 769, 9.5, and 5.4 g/kg of
MOO, respectively. Additionally, we were interested to learn
whether prolongation of PENT induced sleep behaviors after
administration of MOO and whether three bioactive
constituents could elucidate the possible underlying mecha-
nisms of sleep, possibly via GABAergic transmission.
As is well known, sleep is a sophisticated physiological
process affected by a network of neuronal systems, which is
hardly reproducible in vitro.56 Hence, to evaluate the SHE of
MOO and its major bioactive constituents, the locomotor
activity and PENT-induced sleep test, which are the two classic
behavioral pharmacology methods for assessing sedative-
hypnotic activity, were implemented.57 First, the results of
locomotor activity indicated that the movement frequencies
were reduced dependently by the treatment of MOO and its
three components (OA, β-Sito, Stigma). According to our
unpublished data, they reduced the excitability of the CNS as
well as MOO, which may be attributed to the GABAA receptor,
like that of EST. Second, we performed a PENT-induced sleep
test in mice. Previous reports have demonstrated that the
threshold dose of PENT could enhance the inhibitory effect of
drugs of the CNS by interfering with the polysynaptic
transmission of the brainstem system. It has also been shown
that the change of PENT-induced sleep time can be a useful
tool for detecting the stimulatory or inhibitory effects on CNS,
and particularly, for investigating the influences on GABAergic
systems in CNS.58 Nevertheless, the subthreshold hypnotic
dose of PENT cannot directly induce sleep, but can only
synergize weak hypnotic drugs into showing the effect.
Experimental data showed that MOO and three components
(OA, β-Sito, Stigma) also decreased sleep latency and
increased total sleep time with a dose-dependent effect.
Additionally, all of the groups with the higher doses of drug
had a similar increase in the number of mice falling asleep
compared to after being induced by a subhypnotic dose of
PENT. It is the powerful evidence to explain that MOO and its
major bioactive constituents interact with pentobarbital on the
CNS via GABAergic mechanisms in the brain. β-Sito, which is
isolated from Mallotus peltatus (Geist) Muell Arg. var
acuminatus (Euphorbiaceae) leaves and Tilia americana var.
mexicana, has been found to possess central nerve depressant
activities; nonetheless, its mechanism of action remained
unclear.59,60 Um et al.61 reported that Stigma, which is the
bioactivity constituent of rice bran supplement (RBS),
augmented PENT-induced sleep in mice, thus verifying that
its hypnotic effect may be due to an effect in the CNS via a
histaminergic pathway. Moreover, no evidence would suggest
that OA, which is the majority in MOO, possesses SHE. The
purpose of our present study was to confirm the SHE of MOO
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and to elucidate the mechanisms underlying these effects,
which are conceivably involved in the GABAergic system.
The antiseizure action was widely evaluated to investigate
sedation-hypnotic drugs.62 As PTZ has been shown to interact
with the GABA neurotransmitter, the protection against PTZ-
induced seizures probably indicates that it interferes with
GABAergic mechanisms when exerting the anticonvulsant
effect.63 The present study showed that MOO and its major
bioactive components significantly reduced the rate of PTZ-
induced convulsion death in a dose-dependent manner, which
might be mediated via the chloride channel of the GABAA−
BDZ receptor complex. Further studies are necessary to verify
the specific mechanisms of convulsion at the anticonvulsive
dose.
Most sedative-hypnotics used in the treatment of insomnia
target the BDZ−GABAA receptors.64 Thus, we evaluated
whether the SHE was blocked by BIC, FMZ, PIC, which is the
GABAA receptor antagonist or the BDZ receptor antagonist.
The synergistic effects of coadministration of MOO and its
major bioactive compounds with MUS or EST, which is the
GABAA receptor agonist or the BDZ receptor agonist, were
individually investigated. According to the findings, we
observed that the SHE could be blocked by BIC and FMZ.
In addition, it could be antagonized by picrotoxin, except for
OA. The results showed synergistic effects with MUS and EST
in PENT-treated mice. Thus, the above-reported test data
indicated that MOO has an important role in the involvement
of the BDZ−GABAA receptor complex.
The neuropharmacological actions such as sedative,
hypnotic, and anxiolytic and mutual regulations of central
nervous cells were mediated by changing the levels of the
corresponding neurotransmitter in mice.65 GABA, which acts
as one of the major inhibitory neurotransmitters in the brain,
not only regulates fast synaptic inhibition via GABAA receptors
but is also involved in mediating excitability of distinct
neuronal networks via extrasynaptic GABAA receptors.66 Glu is
an excitatory neurotransmitter, which mainly acts on the
cerebral cortex, hippocampus, and hypothalamus to regulate
neuron functions like autonomic activities, memory, and
sleeping in the CNS.67 The balance between GABA and Glu
levels is generally used as an objective index to evaluate the
excitation and inhibition of CNS function.68 Therefore, it is
not surprising that neurotransmitters have a critical role in
insomnia. Taken together, we examined GABA and Glu
concentrations in the hypothalamusone of the major
regulatory regions of the sleep−wake circulationto elucidate
these positive effects of MOO and its major bioactive
components. In our current experiment, the results showed
that MOO and β-Sito improved the levels of GABA and
obviously decreased the Glu levels in brain tissue (hypothal-
amus), thus suggesting that it exerts the SHE by upregulating
the levels of GABA. Additionally, this increased GABA level
might accumulate and affect various physiological functions. As
expected, this induced increase in GABA levels may affect
spontaneous locomotor behavior and anticonvulsant actions in
mice. Nevertheless, OA and Stigma did not have an apparent
action on Glu and GABA concentrations in the hypothalamus.
The reason for this might be that these two components do
not influence the synthesis and metabolism of the neuro-
transmitters through another pathway to exert SHE.
The classical view of GAD is that it is a rate-limiting enzyme
in GABA biosynthesis that has a crucial role in maintaining
GABA levels in the brain.69 Therefore, regulation of expression
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levels of this enzyme may change GABA transmission in the
CNS. We investigated the expression levels of GAD65/67 in vivo
experiment, which is responsible for GABA production. Our
results revealed that MOO and β-Sito significantly increased
the content of GAD65 protein in the hypothalamus of the
brain, thus suggesting that it might activate GAD65 and
increase GABA transmission.
As we know, GABAA receptors are important therapeutic
targets for the treatment of insomnia. It is noteworthy to
mention that the analysis of subunit expression furthers the
understanding of the potential basic pharmacology and
functionality of GABAA receptors subunits in various brain
regions.70 In the mammalian brain, GABAA receptors with a
classic high-affinity BDZ binding site contain α-, β-, and γ-
subunit combinations; meanwhile, some benzodiazepines
could be facilitators of fast inhibitory neurotransmission
mediated through GABAA receptors.71 GABAA R α1γ272 is
the most abundant subunit composition that is closely related
to the hypnotic-sedative effects of GABAA receptors agonist.
Our qualitative results from WB analysis denoted that MOO,
β-Sito, and EST increased the levels of α1-subunit and GAD65,
thus indicating that MOO and β-Sito have the same binding
site with EST exerted effects on GABAA receptor, which is
different from PENT. Based on this reason, the synergistic
effect on the PENT-induced mice sleeping model was
developed.
In conclusion, MOO is a functional oil, which is known
commercially as “ben oil” in Roman times.73 In the present
study, we found that MOO has sedative, hypnotic, and
anticonvulsive actions. Compared to other plant oils (rapeseed
oil, soya bean oil, and peanut oil), MOO has good stability and
high nutritional value, thereby useful for deep frying.74
Kiranawati et al. found that fried noodles with MOO could
improve the milk production of rats; phytosterol, tocopherols
(α, γ, and δ), and monounsaturated fatty acids (MUFA) in
MOO have been identified as the main regulators.75 Moreover,
Mansour et al. reported that MOO possesses potent
hepatoprotective action against carbon tetrachloride (CCl4)-
induced hepatic damage in rats; the mechanism might be
attributed to the antioxidant properties of the tochopherols
and phytosterols along with fatty acids.32 In addition, Scheim
and colleagues reported that unsaturated fatty acids (OA,
linoleic acid, eicosapentaenoic acid) of MOO could regulate
antitumor in vitro.76 Additionally, a large number of articles
verified that MOO contains high levels of unsaturated fatty
acids, especially omega-9-fatty acids (oleic, up to 76.29%),
sterols, flavonoids (quercetin, myricetin), alkaloids, terpenoids,
tocopherols (α, γ, and δ), and polyphenolic compounds.77 The
sterol fractions of the MOO consist mainly of β-Sitosterol,
Stigmasterol, campesterol, and Δ5-avenasterol, which accounts
for 92% of the total sterols in MOO.78 The present fatty acid
composition shows that MOO falls in the category of high
oleic oils and contains a high MUFA-to-saturated fatty acids
(SFA) ratio (MUFA/SFA); this ratio is a common character-
istic of oils, particularly olive oil, and has been associated with a
reduced risk of all-cause mortality, cardiovascular events, and
stroke.79 Therefore, MOO could be an acceptable substitute
for olive oil as the main dietary fat in countries where the tree
grows. Recently, MOO had been applied in the medical field,
cosmetics, biofuel production, and production of lubricating oil
and health products.80
Collectively, these results suggest that MOO possesses
multifunctional active ingredients and, through the activation
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of the GABAergic neurotransmitter system, influences CNS to
produce the depressant properties. Furthermore, MOO could
be a promising therapeutic agent for developing new functional
food to help treat insomnia. Finally, our findings make an
essential contribution to the understanding of the biological
effects of MOO and its active compounds. Yet, further studies
are necessary to verify whether the 5-hydroxytryptamine-ergic
(5-HTergic) system is involved in mediating the SHE. Besides,
other active agents that possess depressant actions present in
MOO should be investigated further.
■sı
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c00037.
Determination of chemical characteristics; experiments;
MOO components analysis; FA compositions and
relative amounts of MOO (Table S1); sterols
compositions and relative amounts of MOO (Table
S2) (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
Yun-Li Zhao − Key Laboratory of Medicinal Chemistry for
Natural Resource, Ministry of Education and Yunnan Province,
School of Chemical Science and Technology, Yunnan University,
Kunming 650091, People’s Republic of China; State Key
Laboratory of Phytochemistry and Plant Resources in West
China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650201, P. R. China; Email: zhaoyunli@
mail.kib.ac.cn
Ai-Xiang Huang − College of Food Science & Technology,
Yunnan Agricultural University, Kunming 650201, Yunnan,
China; Phone: +86 871 65227843; Email: aixianghuang@
126.com; Fax: +86 871 65227843
Authors
Wei-Liang Liu − College of Food Science & Technology, Yunnan
Agricultural University, Kunming 650201, Yunnan, China;
orcid.org/0000-0001-9997-8321
Bai-Fen Wu − Yunnan University of Business Management,
Kunming 650106, People’s Republic of China
Jian-Hua Shang − State Key Laboratory of Phytochemistry and
Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650201, P. R. China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c00037
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The National Science Foundation of China (grant no.
31560431) and the Yun-Ling Industrial Technology Leading
Talent program (grant no. 2014-1782) supported this study.
■
ABBREVIATIONS
MO, Moringa oleifera Lam; PTZ, pentylenetetrazole; MOO,
Moringa oleifera oil; GABA, γ-amino butyric acid; Glu,
glutamic; GAD65, glutamic acid decarboxylase-65; GAD67,
glutamic acid decarboxylase-67; GABAA, γ-amino butyric acid
type A receptor; FID, flame ionization detector; GC, gas
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chromatography; FAME, fatty acid methyl esters; FA, fatty
acid; IUPAC, International Union of Pure and Applied
Chemistry; AD, Alzheimer’s disease; ICR, Institute of Cancer
Research; AAALAC, Association for Assessment and Accred-
itation of Laboratory Animal Care; IACUC, Institutional
Animal Care and Use Committee; SHE, sedative-hypnotic
effects; TW, Tween-80; WHO, World Health Organization;
FDA, Food and Drug Administration; ECFC, European
Commission for Food Science; CAD, cardiovascular disease;
MUFA, monounsaturated fatty acids; SFA, saturated fatty
acids; CCl4, carbon tetrachloride; OA, oleic acid; Stigma,
Stigmasterol; β-Sito, β-Sitosterol; PENT, pentobarbital so-
dium; MUS, muscimol; EST, estazolam tablets; BIC, bicucul-
line; PIC, picrotoxin; FMZ, flumazenil; CMC-Na, sodium
carboxymethyl cellulose; GABAergic, γ-amino butyric acid-
ergic; 5-HTergic, 5-hydroxytryptamine-ergic; CNS, central
nervous system; BDZ, benzodiazepines; GAPDH, glyceralde-
hyde-3-phosphate dehydrogenase; WB, western blot; RIPA,
radioimmunoprecipitation assay; ig, intragastric; ip, intra-
peritoneal; S.E.M, standard error of the mean
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