{"id":51032,"date":"2023-09-30T11:32:52","date_gmt":"2023-09-30T11:32:52","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=51032"},"modified":"2023-10-07T08:50:16","modified_gmt":"2023-10-07T08:50:16","slug":"the-effect-of-administering-forest-honey-to-rats-exposed-to-physical-stress-on-corticosteroid-levels-folliculogenesis-and-the-number-of-corpus-luteum","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no3\/the-effect-of-administering-forest-honey-to-rats-exposed-to-physical-stress-on-corticosteroid-levels-folliculogenesis-and-the-number-of-corpus-luteum\/","title":{"rendered":"The Effect of Administering Forest Honey to Rats Exposed to Physical Stress on Corticosteroid Levels, Folliculogenesis and the Number of Corpus Luteum"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Livestock stress can cause several\nphysical health disorders and various mental and psychosomatic disorders.1<\/sup>\nSources of stress\nthat commonly occur in livestock originate from the environment and also maintenance\nmanagement such as temperature, weather and\nclimate change, light\nintensity, feed factors and over-exercise of livestock. Stress resulted from over-exercise leads to disruption of the homeostasis of the endocrine\nsystem in livestock and a decrease in livestock productivity.2<\/sup> <\/sup>Due to the changes in living environment, livestock is more likely to suffer from poor psychophysiological\nhealth. Repeated and prolonged stress induces\nhypothalamic-pituitary-adrenal (HPA) dysregulation, which disrupts homeostasis.3<\/sup> Activation of the HPA axis\nresults in the secretion of various stress hormones including glucocorticoids,\ncorticotropin-releasing factor (CRF), and cortisol.4<\/sup> Furthermore, excessive HPA\nactivity is reportedly associated with inadequate activation of the\nhypothalamus-pituitary-ovarian axis, which controls the growth and development\nof ovarian follicles and oocytes.5<\/sup><\/p>\n\n\n\n

Livestock that has stress releases glucocorticoids in response to it.2,3<\/sup> Glucocorticoids during stress are synthesized in the adrenal cortex with stimulation from adrenocorticotropic hormone (ACTH) to stimulate gluconeogenesis during the fight-or-flight response. The body responds to short-term and long-term stress in different ways which refer to a pattern known as the general adaptation syndrome (GAS). Early stage of GAS is called the alarm reaction. This is a short-term stress, the fight-or-flight response, mediated by epinephrine and norepinephrine hormones from the adrenal medulla. They function to prepare the body for extreme physical exertion. Once this stress is relieved, the body quickly returns to normal.6<\/sup> However, excessive secretion of corticosteroids has a negative effect on the reproductive system because of the allocation of energy to other organ system.7<\/sup> <\/p>\n\n\n\n

This suppression mechanism was explicitly described by Mahabadi8<\/sup> that activation of glucocorticoid receptors\non gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus induces\napoptosis of neuronal cells, causing hypogonadism due to loss of pulsatility of\nGnRH. GnRH neuronal cell death also affects the\nsynthesis of dehydroepiandrosterone (DHEA) which occurs in the mitochondria of\nthe reticular zone of the adrenal cortex which functions to synthesize estrogen\ndue to the loss of gonadotropin stimulus.9<\/sup> The disruption on GnRH release causes inhibition of the\nhypothalamus-pituitary-gonadal axis (HPG) signaling pathway and reduces\nreproductive efficiency in livestock.5,10<\/sup> <\/p>\n\n\n\n

The use of natural-based ingredients is a trend that starts to emerge\nglobally in the current globalization era, both from the points of view for humans and livestock.11,12<\/sup> Natural ingredients that positively affect\nthe reproductive system are forest honey.13<\/sup> Research conducted by Luqman14<\/sup> showed that the antioxidant content of\nforest honey also has a high range of phenolic and flavonoid compounds due to\nits multi-flora nature honey and can reduce cortisol levels\nin response to physical stress.15<\/sup><\/p>\n\n\n\n

The use of forest honey as a natural ingredient that can reduce corticosteroid level as a stress response from physical stress induction is\nexpected to increase reproductive efficiency including folliculogenesis and the\nformation of the corpus luteum as parameters in ovulation. Based on this background, the study was conducted to determine the effect of administering\nforest honey to rats (Rattus noverg<\/em>icus<\/em>)\nexposed to physical stress on corticosteroid levels, folliculogenesis and the\nnumber of corpus luteum.<\/p>\n\n\n\n

Material and Methods\n<\/strong><\/p>\n\n\n\n

Ethical approval<\/strong><\/p>\n\n\n\n

This research is an\nexperimental animal study. This research has been declared ethically by the\nResearch Ethics Commission of the Faculty of Veterinary Medicine, Universitas\nAirlangga, Indonesia with the ethical eligibility number: No: 1.KEH.041.04.2022.<\/p>\n\n\n\n

This research is an experimental\nlaboratory study using a completely randomized design (CRD) at the Embryology\nLaboratory and Pathology Laboratory, Faculty of Veterinary Medicine, Universitas\nAirlangga. This study used 32 rats (Rattus novergicus<\/em>)\nwhich were divided into 4 treatment groups, and each group consisted of 8 rats. Before the\ntreatment, the experimental animals were first acclimatized by resting them and were given enough food and drink for 7 days.<\/p>\n\n\n\n

Materials<\/strong> <\/p>\n\n\n\n

The tools and materials used in this study were ZD\u00ae forest honey, aquadest, oral gavage,\nEppendorf tube,\ncentrifuge, Olympus\u00ae microscope, Neutraled Buffered\nFormaldehyde 10% (NBF 10%), tissue pot, Onemed\u00ae 5cc syringe, scalpel, surgical scissors, anatomical tweezers, ketamine (Ket-A-100\u00ae),\nand xylazine (Xyla\u00ae). The Feed used for mice was Hi-Pro-Vit Medicated\n593\u00ae.<\/p>\n\n\n\n

Methods<\/strong><\/p>\n\n\n\n

There were 4 treatments groups in this study, namely positive control (C)  treated with\nphysical stress, treatment 1 group (T1) treated with\nphysical stress + honey 2 g\/ rat \/day PO, treatment 2 group (T2) treated with physical stress + honey 4 g\/ rat \/day PO and\ntreatment 3 group (T3) treated with physical stress + honey 6 g\/ rat \/day PO. All\ntreatments\nwere carried out for 14 days.<\/p>\n\n\n\n

P<\/strong>hysical Stress Treatments in Rats<\/strong><\/p>\n\n\n\n

Physical stress using\nrat model was based on\na modified by placing the rats in a specific box measuring 50x30x25 cms and two\nthird of the box was filled with water at the temperature of 24-28o<\/sup>C. Next, the rats were forced to swim for 5 minutes  once a day and then\nthey were placed in a dark box. It was conducted daily at 09.00 AM. In T1, T2 and T3 groups after swimming\ntreatment, forest honey with doses of 2 g, 4 g, and 6 g was\ngiven orally (PO) to each rat daily for 14 days. Forest honey was dissolved in\ndistilled water and was given through the rat\u2019s mouth using a special nasogastric sonde to reach the gastrointestinal organ.<\/p>\n\n\n\n

Sample Collection and Histopathology<\/strong><\/p>\n\n\n\n

After 14 days of treatment, all rats were euthanized using anesthetic Ket-A-100\u00ae and Xyla\u00ae. After the rats\nwere anesthetized, the incision was made using a scalpel in the ventral midline\narea and then it was also made using scissors on the thorax and abdomen. Blood\nwas drawn using a 5 cc syringe intra-cardiac. The collected blood was allowed\nto freeze to take off the serum using a centrifuge at a speed of 2500rpm to examine corticosteroid level by ELISA. Ovarian organs were taken\nand then they were placed in a sample pot that was filled with 10% NBF. The organ samples were\nthen taken to the Pathology Laboratory Faculty of Veterinary Medicine, Universitas\nAirlangga to make histological preparations. <\/p>\n\n\n\n

Corticosteroid Level<\/strong><\/p>\n\n\n\n

Examination of Corticosteroid levels was carried out at\nthe Laboratory of Physiology, Faculty of Medicine, Universitas Brawijaya using the Colorimetric ELISA\nmethod with kit. Corticosteroid levels are expressed in ng\/ml<\/p>\n\n\n\n

Folliculogenesis Profile<\/strong><\/p>\n\n\n\n

Examination of the folliculogenesis profile was carried\nout on histological preparation of ovarian\ntissue that was previously prepared. The examination was carried out using an Olympus\u00ae microscope with 400x magnification and then\nthe number of primary follicles, secondary follicles, tertiary follicles, de\nGraff follicles, and corpus luteum were counted. The calculation was carried out in 5 fields of view and then in the average values of\nthe results.<\/p>\n\n\n\n

Statistical Analysis<\/strong><\/p>\n\n\n\n

Statistical Analysis was conducted using an application named IBM SPSS 23 using One-Way ANOVA with post hoc Duncan to find out\nthe differences between\ngroups on the variables of corticosteroid levels, number of primary follicles,\nsecondary follicles, tertiary follicles, de Graff follicles and corpus luteum\nto find out differences between groups of each variable.<\/p>\n\n\n\n

Results and\nDiscussion<\/strong><\/p>\n\n\n\n

The results\nof corticosteroid level examination using a colorimetric ELISA are\nshown in table 1. The results show that group C that was treated with the physical\nstress which is swimming has a corticosteroid level of 149.78\u00b132.67 ng\/ml. The treatment groups (T1,T2 dan T3) which also obtained\nphysical stress treatment and then were treated with forest honey at doses\nof  2 g, 4 g, dan 6 g\/ rat \/day have the following results : T1 shows\nthe lowest corticosteroid level of all treatment group which is at 57.03 \u00b1 3.21 ng\/ml and the corticosteroid level of this group is significantly\ndifferent (p<0.05) compared to that of group C. The corticosteroid\nlevel of T3 is significantly different (p<0.05) compared\nto that of group C, which is lower, however, it is still higher if compared to that\nof T1 (p<0.05).<\/p>\n\n\n\n

Overall,\nthe results of the folliculogenesis profile examination presented in table 2\nshow that the number of primary follicles, secondary follicles, tertiary\nfollicles and de Graff follicles of T1 \nis significantly different (p<0.05) when\ncompared to that of the group C and the other treatment groups (T2 dan T3). The lowest folliculogenesis profile is obtained by  T3 and in terms of several variables, such as\nthe number of primary follicles, tertiary follicles, and de Graff follicles,\nit  does not  have a significant difference with group C (p>0.05), while for secondary follicles it is significantly different from\ngroup  C (p<0.05).<\/p>\n\n\n\n

The calculation of the\ncorpus luteum in Table 3 shows that the T1 has the highest corpus luteum, which\nis 9.16\u00b11.72 and the number of corpus luteum in this group is significantly\ndifferent (p<0.05) from that of group C. The number of corpus luteum of group C , T2,\nand T3 is not significantly different (p>0.05) compared to T3 that has the lowest number of\ncorpus luteum.<\/p>\n\n\n\n

Table 1: Corticosteroid levels (ng\/ml) of each treatment group using ELISA examination (Mean \u00b1 SD)<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n
\n

Treatment Groups<\/strong><\/p>\n<\/td>\n

\n

Corticosteroid Levels (ng\/ml)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

C<\/p>\n<\/td>\n

\n

149.78c<\/sup> \u00b1 32.67<\/p>\n<\/td>\n<\/tr>\n

\n

T1<\/p>\n<\/td>\n

\n

57.03a<\/sup> \u00b1 3.21<\/p>\n<\/td>\n<\/tr>\n

\n

T2<\/p>\n<\/td>\n

\n

72.66ab<\/sup> \u00b1 4.87<\/p>\n<\/td>\n<\/tr>\n

\n

T3<\/p>\n<\/td>\n

\n

87.71b<\/sup> \u00b1 14.56<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Note: Different superscripts show significant difference (p<0.05).<\/p>\n\n\n

Table 2: Number of Primary follicles, secondary follicles, tertiary follicles, and de Graff follicles of each treatment group (Mean \u00b1 SD)<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n
\n

Treatment Groups<\/strong><\/p>\n<\/td>\n

\n

Primary Follicles<\/strong><\/p>\n<\/td>\n

\n

Secondary Follicles<\/strong><\/p>\n<\/td>\n

\n

Tertiary Follicle<\/strong><\/p>\n<\/td>\n

\n

De Graff Follicles<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

C<\/p>\n<\/td>\n

\n

1.67 \u00b1 0.81a<\/sup><\/p>\n<\/td>\n

\n

1.67 \u00b1 0.81b<\/sup><\/p>\n<\/td>\n

\n

1.83 \u00b1 0.75a<\/sup><\/p>\n<\/td>\n

\n

0.67 \u00b1 0.13a<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

T1<\/p>\n<\/td>\n

\n

4.67 \u00b1 1.63b<\/sup><\/p>\n<\/td>\n

\n

3.00 \u00b1 1.09c<\/sup><\/p>\n<\/td>\n

\n

3.83 \u00b1 1.72b<\/sup><\/p>\n<\/td>\n

\n

4.33 \u00b1 1.75b<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

T2<\/p>\n<\/td>\n

\n

1.00 \u00b1 0.63a<\/sup><\/p>\n<\/td>\n

\n

0.67 \u00b1 0.12ab<\/sup><\/p>\n<\/td>\n

\n

1.00 \u00b1 0.63a<\/sup><\/p>\n<\/td>\n

\n

0 a<\/sup> \u00b1 0<\/p>\n<\/td>\n<\/tr>\n

\n

T3<\/p>\n<\/td>\n

\n

0.50 \u00b1 0.2a<\/sup><\/p>\n<\/td>\n

\n

0.17 \u00b1 0.04a<\/sup><\/p>\n<\/td>\n

\n

0.50 \u00b1 0.08a<\/sup><\/p>\n<\/td>\n

\n

0.17 \u00b1 0.04a<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Note: Different superscripts show significant difference (p<0.05)<\/p>\n\n\n

Table 3: Number of Corpus Luteum of each treatment group (Mean \u00b1 SD)<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n
\n

Treatment Groups<\/strong><\/p>\n<\/td>\n

\n

Corpus Luteum<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

C<\/p>\n<\/td>\n

\n

6.50 \u00b1 1.05a<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

T1<\/p>\n<\/td>\n

\n

9.16 \u00b1 1.72b<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

T2<\/p>\n<\/td>\n

\n

5.00 \u00b1 1.09a<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

T3<\/p>\n<\/td>\n

\n

5.83 \u00b1 1.16 a<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Note: Different superscripts show significant difference (p<0.05)<\/p>\n\n\n

In this study, there was a significant increase in corticosteroid levels in group C that was treated with physical stress in the form of swimming and without supplementing forest honey. Physical stress induced by swimming is Variability in Behavioral Phenotypes after Forced Swimming-Induced Stress is associated with acute stress and it increases the expression of several stress proteins such as glucocorticoid receptor(GR), Nurr1, and IL-1\u03b2. The increase in GR expression is caused by an increase in plasma corticosteroid levels in response to physical stress.16<\/sup> <\/p>\n\n\n\n

Increased plasma levels of corticosteroids have a\nnegative effect on the reproductive system as reported by Ackerman17<\/sup> that the increase of cortisol and\ncorticosteroid levels due to physical stress inhibits the\nhypothalamic-pituitary-gonadal (HPG) signaling pathway so that it becomes a factor that inhibits LH secretion. On the other hand, Mahabadi8<\/sup> explained that high plasma corticosteroid\nlevels activate GR on GnRH neurons in the hypothalamus and induce apoptosis of\nthe neurons, causing hypogonadism due to loss of pulsatility of GnRH. The\nresults of this study are in line with\nthe study in which in\ngroup C, there was a significant decrease in the number of primary, secondary,\ntertiary, de Graff follicles and corpus luteum when compared to that of T1. Furthermore, GnRH is an important hormone that stimulates FSH and LH as\nthe main factors for folliculogenesis and ovulation. Suppressing these hormones by corticosteroids disrupts these processes.18<\/sup><\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Histopathology of Ovaries. a) Group C: red arrow shows the preantral follicles (primary and secondary), black arrow shows antral follicles (tertiary and de Graff). b) Group T1: red arrow shows the preantral follicles, <\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) mRNAs in corpus luteum have a role to stimulate GnRH in order to\nrelease FSH and LH. SHBG binds and transports testosterone, estradiol, and other sex steroids in the plasma,\nreduces their metabolic clearance rate and affects their bioavailability. SHBG exhibits high affinity for testosterone\nand a low-capacity binding protein (estradiol), and other low-affinity but higher-capacity binding proteins including\nhuman serum albumin (HSA), corticosteroid-binding globulin (CBG) and\nOrosomucoid or \u03b11 acid glycoprotein. The free active testosterone concentrations\nin plasma are highly influenced by SHBG concentrations because\nonly 1\u20132% of testosterone in the circulation is free (unbound) and active; 65%\nis bound to SHBG and the rest is bound to albumin. So, the levels of SHBG have a correlation with serum steroid hormone levels.19,20<\/sup>  <\/p>\n\n\n\n

The mechanism of physical stress disorder in the\nreproductive system is also reported to be caused by low energy availability.\nThis causes a decrease in insulin levels and insulin-like growth\nfactor1 (IGF-1).21<\/sup> Impaired IGF-1 secretion, which is an essential factor for folliculogenesis process and oocyte maturation, causes apoptosis of\ngranulosa cells and failure of further follicular development into de Graff\nfollicles.22<\/sup> This is in line with the result of this study, in which in group C de Graff follicles that are produced are lower\ncompared to those of T1.<\/p>\n\n\n\n

Increased levels of corticosteroids consistently increase\napoptosis of neuron cells and are neurotoxic. Several neurotoxin mechanisms in\nneuronal cells include reducing the ability of neurons to use Ca2+<\/sup>, increasing Ca2+<\/sup> <\/sup>intracellular levels uncontrollably, changing\nmitochondria and endoplasmic reticulum function, and decreasing Bcl-2 expression due to translocations in the\nmitochondria of neuron cells.9<\/sup> Several studies show that the potential protective effects of forest honey\nagainst organic compounds that induce neurotoxicity in the reproductive system.<\/p>\n\n\n\n

Honey is known to be rich in enzymatic\nand non-enzymatic antioxidants,\nincluding catalase, ascorbic acid, flavonoids, alkaloids, glucose oxidase,\nphenolics acid, carotenoid derivatives, Maillard reaction products, amino acids\nand proteins. A unique flavonoid, known as\npinocembrin, is present in propolis and honey; other types of flavonoids,\nincluding quercetin, chrysin, galangin, luteolin and kaempferol, are also found\nin honey. Previous study found that\nquercetin and kaempferol contained\nin\nhoney exerted the free radical scavenging activity. In addition, estrogenic\nproperties of quercetin and kaempferol might compete with EDC to bind to\nestrogen receptors.23,24<\/sup>  <\/p>\n\n\n\n

Ruslee24<\/sup> <\/sup>conducted a research that showed that Tualang honey had protective effects in\nreducing the ovarian toxicity induced by Cd. The protective effects observed were in the form of a reduction in\nmorphological abnormalities in the ovary, restoration of the gonadotropin\nhormones, reduction in the lipid peroxidation level and increase in the levels\nof enzymatic antioxidants. Many studies\nshow that flavonoids and phenolic acids\nare responsible for the antioxidant activity of honey as it has the ability to scavenge free radical formation.25,26,27<\/sup> Phenolic compounds work\nagainst oxidative stress as they have the properties of reducing agents (they\nhave hydrogen- or electron-donating capacity) with chemical structure of\nhydroxyl groups.28<\/sup> The more hydroxyl groups\nthere are in phenolic compounds, the more efficiently they can react as\nantioxidant agents due to their ability to donate hydrogen atoms to free\nradicals then free radical formation will\nbe reduced. Also, vitamin E and C contained in\nhoney also have a protective effect against\noxidative stress. Vitamin E helps to prevent lipid peroxidation reactions by\ninhibiting the production of lipid radicals in cellular membranes, whereas\nvitamin C is a water-soluble antioxidant that can interact directly against\nfree radicals in cytosol and extracellular fluids to reduce oxidative damage.29<\/sup><\/p>\n\n\n\n

The decrease in Bcl-2 levels causes the ability of buffer calcium in neuronal\ncells to decrease, therefore it increases ROS production, which will cause neuronal\ncell death.30<\/sup> On the other hand, GnRH secretion requires Ca2+ <\/sup>and ROS at normal levels, but the overload of Ca2+ <\/sup>and ROS production has an effect on GnRH secretion\n(hypogonadism) and interferes with normal reproductive function.8<\/sup> <\/p>\n\n\n\n

Forest honey has a high antioxidant content and consists\nof flavonoids, phenolic components, an enzymatic antioxidant such as catalase\nand glucose oxidase, carotenoids, several amino acids and vitamin C.14<\/sup> The potential of antioxidants contained in\nforest honey was measured by Apak31<\/sup> which\nused DPPH method\nto measure scavenging ability and obtained a relatively high IC50<\/sub> result of 5453.57 ppm. Usman32<\/sup> also explained in his research that administering honey was able\nto reduce cortisol\nlevels in plasma and to increase glutathione levels which counteracted the forced swimming stress\neffects.\nIncreased levels of glutathione can exert a protective effect on neurons\nagainst oxidative stress caused by overloaded ROS and maintain the secretory\nfunction of GnRH. Azman33<\/sup> said that forest honey (200 mg\/kg\/body weight) that was administered for 28 days successfully counteracted\nthe forced swimming stress effects in which the honey treated rats exhibited\nsignificant decrease in depressive-like behaviour and levels of ACTH,\ncorticosterone, and oxidative stress markers, with significant increase in\nantioxidant enzymes activities and total antioxidant status. The honey mediated\nantidepressant-like effects in stressed rats, possibly acting via restoration\nof hypothalamic-pituitary-adrenal axis through its antioxidant properties. Similarly, our current study\ndemonstrated that forest honey  was  able \nto  reverse  the \nincrease  of  corticosterone  levels \nin  the stressed rats. These\nfindings suggested that forest honey reduced the\nadverse effects of stress and may be beneficial for the nervous system and\nvasculature, and protect the brain and body from stress-induced damage. Azman33<\/sup> said\nthat honey may have modulated\ncorticosterone and ACTH levels either by suppressing HPA mobilization in\nresponse to stress or by facilitating elevated plasma corticosterone and ACTH\nlevels back to baseline following the termination of stress.<\/p>\n\n\n\n

Another mechanism of honey to prevent neuronal cell damage due to oxidative stress is by decreasing the\nexpression of pro-inflammatory cytokines, such as TNF-\u03b1, NF-kB, and MAPK and increasing anti-inflammatory cytokines\nsuch as IL-13.34<\/sup> Forest honey also provides energy and\nstimulates the secretion and stimulation of IGF-1, which is important for the folliculogenesis process and oocyte maturation.35<\/sup> The mechanism was proven in this study  in which group T1 with a\ndose of honey 2 g\/ rat \/day was the\nmost effective for providing protection to neuron cells as evidenced by the\nlowest corticosteroid levels, a good folliculogenesis profile and  high number of corpus luteum that indicated good ovulation process (enough LH). <\/p>\n\n\n\n

Although limited, the clinical evidence\ndescribed above suggests that honey has great potential in the management of asthma resistant of corticosteroid. The anti-asthmatic effect of raw Gelam honey (Apis\nmellifera<\/em>) which\nis originated from Malaysia showed that oral administration of Gelam honey (40%\nand 80% (v\/v)) exhibited a significant dose-dependent reduction in the airway\nepithelium thickening and infiltration of inflammatory cells (lymphocytes,\nneutrophils, and eosinophils) at peribronchiolar region and in the BALF of\nOVA-induced BALB\/c mice.36<\/sup> Another study investigated the\neffectiveness of aerosolized Tualang honey (25 and 50% (v\/v)) as both rescue\nand preventative agents in OVA-induced rabbits. Regardless of the dosage\nand treatment method (pretreatment or co-treatment), aerosolized\nTualang honey was able to significantly inhibit goblet cell hyperplasia, mucus\noverproduction, and infiltration of inflammatory cells (eosinophils,\nmononuclear, neutrophils, and macrophage) in the peribronchial region and BALF\nin OVA-induced rabbits.37<\/sup> <\/p>\n\n\n\n

In this study, group T1 which received the physical stress treatment of swimming and then was treated\nwith forest honey at dose of  2 g\/rat\/day showed that the number of primary follicles, secondary follicles, tertiary\nfollicles and de Graff follicles were significantly different (p<0.05) when compared with those of the other treatment groups and  the\nnumber of the corpus luteum of this group had the highest rate at 9.16\u00b11.72 and it\nwas significantly different (p<0.05) from\ngroup C. This showed significantly higher number of corpus luteum\nand antral follicles (primary follicles, secondary\nfollicles, tertiary follicles and de Graff follicles)\ncompared to those of the control\ngroup. The higher number of corpus luteum indicates an increase in ovulation\nrate, while the antral follicle shows that the normal folliculogenesis process\noccurs. Forest honey was reported to improve\noxidative stress status and normalize the hormonal and oestrus\ncycle disturbances. These hormonal and oestrus\ncycle corrections bring back physiological healthy folliculogenesis, which is\nmanifested by improvement in folliculogenesis-related factors and histological findings in this study. Oxidative\nstress and its enzymatic markers play roles to regulate the folliculogenesis\nprogress, oocyte development, and ovarian steroidogenesis.38,39 <\/sup><\/p>\n\n\n\n

In rat ovaries, Cyp17a1 is localized in theca cells of large\nantral and preovulatory follicles. Kamal40 <\/sup>study recorded a similar\nfinding in which the Cyp17a1 protein\ndistribution was significantly higher in theca cells of the large antral and\npreovulatory follicles of PCOS rats compared with that of the normal control rats. Similar\nwith that study, Kakuta  in his study showed that LH hypersecretion was one\nof the causes of Cyp17a1 overexpression.41 <\/sup> Tualang honey was reported to regulate the\nhypothalamic\u2013pituitary\u2013adrenal axis in ovariectomised rats.42<\/sup> In sexually mature\nanimals, aromatase is reported to be present in the granulosa cell layer of\nlarge antral healthy follicles, preovulatory follicles, and corpus luteum.43,44 <\/sup> In this study, group T1 showed that the number of primary follicles, secondary\nfollicles, tertiary follicles and de Graff follicles was higher.<\/p>\n\n\n\n

Ruslee24 <\/sup> study found that in BPA-exposed rats, Tualang honey,\nwhen consumed orally on a daily basis, could serve as an effective natural\nsupplement to reduce the toxic effects of BPA by restoring the level of FSH and\nLH hormones. This result reflected the\nnormalization of GnRH in the brain.\nIn BPA-exposed rats treated with Tualang honey, morphological abnormalities,\nsuch as the formation of large antral cystic-like follicles (anovulation\nfollicles), the insufficiency of the corpus luteum and preantral follicles and\nthe number of atretic follicles, were slightly reduced. In BPA-exposed rats, honey supplementation significantly improved\novarian and uterine morphological abnormalities, reduced lipid peroxidation and\nnormalized ER\u03b1, ER\u03b2 and C3 expression\nlevels and distribution. Quercetin and kaempferol are\nthe main naturally occurring flavonols in forest honey that share structural\nsimilarities with 17\u03b2-oestradiol. Therefore, the potential oestrogenic effects\nof these compounds are comparable with those of other xeno-oestrogens.45<\/sup><\/p>\n\n\n\n

In this study, group T2 and T3 showed poor results\ncompared to group T1. Researchers suspect that forest honey with a dose 4 g\/rat\/day and 6 g\/rat\/day has a very high viscosity. This is in accordance\nwith the results of Bakier\u2019s research46 <\/sup>that honey in liquid form has a high\nviscosity which is influenced by temperature and water concentration.  Administering honey with those doses causes stress in experimental animals\nbecause viscosity is quite high.47<\/sup> Physical stress coupled with stress due to honey administration makes the therapy with honey ineffective. <\/p>\n\n\n\n

Another hypothesis to consider is the antioxidant\nparadox. Antioxidants become effective to prevent cell damage if used with the right dose. Forest honey that has  protective effects is observed in the form of  reduction in morphological abnormalities in\nthe ovary, restoration of the gonadotropin hormones, reduction in the lipid\nperoxidation level and increase in the levels of enzymatic antioxidants. Forest\nhoney contains flavonoids and phenolic acids that are responsible for antioxidant activity as\nit has the ability to scavenge free radical formation.27<\/sup> Phenolic compounds work\nagainst oxidative stress as they have properties of\nreducing agents (they have\nhydrogen- or electron-donating capacity) with chemical structure of hydroxyl\ngroups. The more hydroxyl groups\nthere are in phenolic compounds, the more efficiently they can react as\nantioxidant agents due to their ability to donate hydrogen atoms to free\nradicals then free radical formation will\nbe reduced. 28<\/sup><\/p>\n\n\n\n

Then, vitamin E and C contained in\nhoney also have  protective effects against oxidative stress. Vitamin E\nhelps to prevent lipid peroxidation reactions by inhibiting the production of\nlipid radicals and vitamin C is a\nwater-soluble antioxidant\nthat reduces oxidative damage in cytosol and extracellular\nfluids.29,48<\/sup> Excessive dose can cause reductive stress that increases ROS levels and pro-inflammatory cytokines and\nactivates the inflammatory cascade with increased expression of NF-kB and MAPK. Group T2 and T3 which used forest honey at\ndoses of 4 g\/rat\/day and 6 g\/rat\/day caused reductive stress and increased GnRH neuronal damage which was\ncaused by physical stress and interfered with the process of folliculogenesis\nand ovulation<\/p>\n\n\n\n

Conclusion <\/strong><\/strong><\/p>\n\n\n\n

Administration of forest honey\nat a dose of <\/strong>2 g\/rat\/day to rats treated with physical stress for 14 days decreases corticosteroid levels with reductive stress which increases ROS levels and pro-inflammatory cytokines and\nactivates  inflammatory cascade with increased expression\nof NF-kB and MAPK because forest honey contains flavonoids and phenolic acids: antioxidants that have the ability to scavenge free radical formation,\nincrease folliculogenesis profile, and the number of corpus luteum because Cyp17a1 is localized in theca cells of large\nantral and preovulatory follicles as well as LH hyper-secretion and increase corpus luteum development. <\/p>\n\n\n\n

The\nuse of forest honey could reduce corticosteroid levels\nas a stress response from physical stress induction which was expected to increase reproductive efficiency.<\/p>\n\n\n\n

Conflict of interest<\/strong><\/p>\n\n\n\n

All\nauthors declare that there is no conflict of interest.<\/p>\n\n\n\n

Funding<\/strong><\/p>\n\n\n\n

The\nauthors received financial support for the Implementation of Internal Research\nUniversitas Airlangga Number 978\/UN3\/2022 and publication of this article.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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