Royal Jelly Induced Anxiolytic Effects and Prevent Hot Flushes in a Menopausal Model on Wistar Rat

Menopause also known as the climacteric at short term, is the time in women lives when menstrual period stops permanently [1]. At a physiological level, menopause happens because of a cessation in ovarian hormones [2]. This decrease in ovarian hormones is responsible for some physiological disorders which include vasomotor disorders (night sweats and hot flushes), mood changes [3], anxiety and irritability. Statistically, 10 % of the world population suffer from several forms of anxiety [4] and 51% of these are menopausal women [5]. In the same light, 80% of menopausal women suffer from hot flushes [6]. These menopausal disorders affect the social life, sexual life, and increase morbidity in women. The first approach of treatment of anxiety was the use of benzodiazepines; a group of anxiolytic drugs that modulate GABAA receptors but failed due to its secondary effects like muscle relaxation and sedation. However, these psychotropic agents seem not appropriate for the primary treatment of symptoms associated to a lack of estrogens [7]. Hormone Replacement Therapy (HRT) is also commonly used for the treatment of physiological disorders observed in menopausal women [8]. Despite the effectiveness of hormone replacement therapy to alleviate menopause symptoms, it is associated with high risk of cancer and cardiovascular diseases [9,10]. Following this, the needs for developing new alternative treatments for the management of physiological and/or psychological disorders related to menopause remain urgent 11]. Royal Jelly (RJ) is a yellowish white creamy liquid secreted by nurse bees (Apismellifera L) [12]. It serves as food for honey bee larva Received: July 09, 2019 Published: July 19, 2019


Introduction
Menopause also known as the climacteric at short term, is the time in women lives when menstrual period stops permanently [1]. At a physiological level, menopause happens because of a cessation in ovarian hormones [2]. This decrease in ovarian hormones is responsible for some physiological disorders which include vasomotor disorders (night sweats and hot flushes), mood changes [3], anxiety and irritability. Statistically, 10 % of the world population suffer from several forms of anxiety [4] and 51% of these are menopausal women [5]. In the same light, 80% of menopausal women suffer from hot flushes [6]. These menopausal disorders affect the social life, sexual life, and increase morbidity in women. The first approach of treatment of anxiety was the use of benzodiazepines; a group of anxiolytic drugs that modulate GABAA receptors but failed due to its secondary effects like muscle relaxation and sedation. However, these psychotropic agents seem not appropriate for the primary treatment of symptoms associated to a lack of estrogens [7]. Hormone Replacement Therapy (HRT) is also commonly used for the treatment of physiological disorders observed in menopausal women [8]. Despite the effectiveness of hormone replacement therapy to alleviate menopause symptoms, it is associated with high risk of cancer and cardiovascular diseases [9,10]. Following this, the needs for developing new alternative treatments for the management of physiological and/or psychological disorders related to menopause remain urgent 11].
Royal Jelly (RJ) is a yellowish white creamy liquid secreted by nurse bees (Apismellifera L) [12]. It serves as food for honey bee larva during their maturation process and it is the specific food given to the queen for her whole lifespan. Royal jelly is one of the most effective and beneficial remedy for human being, as it is widely used both in folk and in modern medicine and as a dietary supplement [12]. Previous work has shown that RJ can bind to oestrogen receptors and brings about the same effects as that of estrogens [13]. Royal Jelly shown many pharmacological activities such as antioxidant, neurotropic, hypoglycemiant, hypocholesterolaemiant, hepatoprotective and nephroprotective [12]. Nevertheless, there are no previous report on the effects of royal jelly on anxiety and hot flushes. Therefore, the aim of this study was to evaluate the effects of royal jelly on menopause related anxiety and on hot flushes in ovariectomised Wistar rat.

Animals
The experiments were carried out with juvenile female Wistar rats weighing between 150 ± 10 g and aged between 10

Substance Tested and Determination of Doses
The substance tested in this study was royal jelly ("GELEE ROYAL BIO") provided by "PLANETE AU NATUREL" / France. The doses of administration were prepared based on the prescription on the box (14.3 mg/kg BW for human); the equivalent doses in rat were extrapolated from this human dose, to afford 100 mg/kg BW.
To obtain a dose response curve of the substance, 2 other doses (200 and 300 mg/kg BW) were generated.

Setting of Data Loggers
The data loggers used were calibrated in a computer having the "ACR smart button". They were programmed to take temperatures every 2 minutes, 6 hours before the beginning of treatment till the end. These data loggers were robed with paraffin and inserted into the abdominal cavity of the rats during operation. This was done under anesthesia induced with Valium and ketamine respectively through the intraperitoneal pathway at the doses of 10 and 50 mg/kg BW respectively. After insertion, the abdominal cavity was sutured.

Experimental Design
All female Wistar rats were ovariectomized before each test except the SHAM group. The bilateral operation was done using the dorsal approach [14] under diazepam and ketamin anaesthesia (10 and 50 mg/kg BW respectively; i.p.). After 14 days of endogenous hormonal decline [15], animals were randomly distributed into different groups: SHAM operated and negative control (OVX) groups received distilled water; E2V and DZP groups received estradiol valerate and diazepam respectively at the dose of 1 mg/kg BW each; and three others groups received royal jelly at the doses of 100, 200 and 300 mg/kg BW (RJ100, RJ200 and RJ300). All substances were dissolved in distilled water (vehicle) and were administered per os (1 mL/100 g BW for oral administration), except diazepam (i.p.).
For behavioral assessments, the experiment was carried out in a calm room supply with day light after an acclimation period of three days in this room. The parameters were registered with a video-camera system. After each test, the apparatus was cleaned with 70% ethanol solution and dry cloth before placing the next animal, to avoid perturbation by faeces or urine of the previous animal. In this experiment, 49 female Wistar rats were used and there were distributed into seven groups (as listed above) of seven rats each. One hour after the administration of the different substances, the animals were placed in the centre of the EPM and observed for five minutes each. For each rat, the following parameters were taken: Time spent and number of entries into the open and closed arms, number of rearing, number of Grooming, number of head dipping [16] and weight of faeces produced. The rectal temperature was taken using a thermometer, immediately after the animal was removed from the EPM apparatus. The percentage of time spent in the different arms was calculated.

Open Field Test
Developed by Calvin S. Hall, the Open Field (OF) test is an experiment used to assay general locomotory activity levels, anxiety and willingness to explore in rodents in scientific research [17]. It is based on the fact that animals such as rats and mice display a natural aversion to brightly lighted areas. They also have a drive to explore a perceived threatening stimulus. The open field apparatus used in this study was a wooden square box 60 x 60 x 40 cm; the floor was divided into 16 smaller squares of equal dimensions (15 x15 cm).
To carry out this experiment, 49 female rats were used and there were distributed into seven groups (as previously describe) of seven rats each. One hour after the administration of the different substances, the animals were placed one after the other in the centre of the open field and observed for five minutes. For each rat, the following parameters were taken center square duration, number of crossing, number of grooming and number of rearing.

Hot Flushes Protocol
This experiment was carried out with 30 female rats distributed into six groups of five rats each (SHAM, OVX, E2V, RJ100, RJ200 and RJ300). Fourteen days after the introduction of the calibrated data loggers, the animals were treated for three consecutive days, after which they were weighed and dissected. The data loggers were removed, and results printed to a computer. Parameters taken were total number, average duration and frequency of hot flushes.

Statistical Analysis
The data from each experimental group was expressed as the mean ± standard error on the mean (S.E.M.). The significance of the difference between OVX group and NOVX group was determined using the unpaired t-test and the significance of the difference between treated groups and OVX group was determined using oneway ANOVA followed by Dunnett's test (Graph Pad Prism, version 5.03). The p-value < 0.05 was considered significant.

Anxiolytic Effects of Royal Jelly on Rats Subjected to the Elevated Plus Maze Test
Effects of Royal Jelly on the number of Entries into the Open and Closed Arms of the Elevated Plus Maze: Analysis of the results presented in Figure 1A shows that compared to the   and 300 mg/kg BW. ## p < 0,01 vs. SHAM (unpaired t-test); * p < 0.05 vs. OVX (one-way anova followed by dunnett's test).

Effects of Royal Jelly on the Number of Grooming, Weight of Faeces Produced and on Rectal Temperature
As shown in Figure 4A, estrogen depletion induced a significant increase (p< 0.05) of the number of grooming in the EPM, compared to SHAM group. As well as E2V and DZP, royal jelly at the dose of 300 mg/kg BW induced a non-significant decrease of the number of grooming compared to OVX group ( Figure 4B). This number of grooming passed from 5.00 ± 0.81 for the OVX group to 3.33 ± 0.52 for RJ at the dose of 300 mg/kg BW.
Analysis of the results presented in Figure 4B, show that compared to SHAM group, ovariectomy induced a significant increase (p < 0.05) of the weight of faeces. Same as E2V and DZP, royal jelly at all the tested doses induced a significant decrease (p< 0.01, p< 0.001) of the weight of faeces produced in the EPM compared to the OVX group ( Figure 4B).

Number of Crossing:
The results presented in Figure 5A shows that ovariectomy induced a non-significant decrease of the time spent at the center of the open field compared to the SHAM group (passed from 2.00 ± 0.69 s for the SHAM group to 1.14± 0.26 s for the OVX group). Royal jelly, as well as E2V and DZP induced an increase on the time spent at the centre of the OF compared to OVX. This effect was significant (p<0.05) with the RJ treatment at the doses of 100 and 300 mg/kg BW.  As presented in Figure 6A, ovariectomy induced a significant increase of the number of grooming (p< 0.05), compared to the SHAM group. Royal jelly, as well as E2V and DZP, induced a decrease of the number of grooming, compared to OVX group ( Figure 6A). This effect was significant (p< 0.05) with RJ treatment at the dose of 200 mg/kg BW. The results presented in Figure 6B shows that estrogen depletion induced a significant decrease (p<0.05) of the number of rearing in the OF, compared to the SHAM group. # p < 0.05 vs. SHAM (unpaired t-test); *p < 0.05, **p < 0.01 vs. OVX (one-way anova followed by dunnett's test).

Duration of Hot Flushes
The analysis of the results in Figure 7A shows that ovariectomy induced a non-significant increase in the total number of hot flushes compared to SHAM group (passed from 42.  The results presented in Figure 7B show that ovariectomy  (unpaired t-test); * p < 0.05, ** p < 0.01 vs. OVX (one-way anova followed by dunnett's test).

Discussion
According to the World Health Organization [4], anxiety is the  [20,21] substance that increases locomotory activity, in stress condition as in the case of open field test has anxiolytic effects. These results confirm the anxiolytic effects of RJ observed in the EPM test. According to Anuradha et al. [22], drugs of natural origin can be useful in stress-induced anxiety.
The anxiolytic properties of royal jelly could result from its action as selective ER modulators [23]. It is known that anxiety behaviour increase the deficiency of ERβ in female mice [24]. Royal jelly could act through the ERβ because according to Lund et al. [25], estrogens and oestrogenic substances carry out their anxiolytic properties through this partway. Such as many other anxiolytic substances, RJ could act as agonist of Gamma Amino-Butyric Acid (GABA) on GABA receptors [26]. Royal jelly can also act as antagonist of N-Methyl-D-Aspartic Acid (NMDA) [27] and 5-Hydroxytryptamine (5-HT) by binding to their receptors [28].
The results obtained on evaluation of hot flushes show that ovariectomy induced an increase on the total number and average duration of hot flushes compared to SHAM group. The administration of E2V for three consecutive days, compared to OVX group, led to a decrease in the total number, average duration and frequency of hot flushes. These results confirm previous studies which explained that estrogen depletion causes hot flushes which can be reversed by hormone replacement therapies [29,30]. After three days treatment with royal jelly at all the tested doses, as well as E2V, there was a decrease on the total number, average duration and frequency of hot flushes compared to OVX group.
These effects could be due to the action of royal jelly on estrogen receptors in the brain. As suggested by Zingue et al. [31] substance with estrogenic effect like Ficus umbellate can act on estrogen receptor to reverse the thermoregulatory disorder caused by post ovariectomy depletion of endogenous estrogen. Given the fact that hot flushes at menopause are directly linked to anxiety, it will be of great importance to compare the results obtained from hot flushes and those from anxiety behavior.
According to these results, the untreated ovariectomised animals apart from being too anxious, showed increase in the total number, average duration and frequency of hot flushes. This is because the lack of oestrogens modifies the activity of neurotransmitters especially that of serotonin and noradrenalin, which are neurotransmitters involved in the regulation of central temperature and behavior [30]. The administration of estradiol valerate reduced at the same time anxiety and hot flushes, confirming the reports that suggest that hormone replacement suppresses hot flushes and menopause induced anxiety. Just like estradiol, royal jelly at all the tested doses and most at the dose of 200 mg/kg BW reduced hot flushes and menopause related anxiety, in nearly all the parameters in the two behavioral test. These results show that royal jelly is capable of regulating temperature and behavioral systems.

Conclusion
The aim of this study was to evaluate the effects of royal jelly on anxiety and hot flushes in ovariectomised rats. Our findings have shown that RJ exhibits anxiolytic properties and reduces the total number average duration and frequency of hot flushes.
These results suggest that royal jelly is capable of reversing some disorders related to estrogen depletion induced by ovariectomy.
The dose of 200 mg/kg BW showed the most beneficial effects on anxiety and hot flushes. Royal jelly could be considered as a safer health alternative product to relief anxiolytic menopausal symptoms. These anxiolytic effects and hot flushes prevention by royal jelly could justify it traditional use by older women.