Effect of Nutmeg Administration on the Anterior Cingulate Cortex (Area 24a) of Adult Male Albino Rats and the Protective Role of Vitamin C: A Histological and Immunohistochemical Study

Research Article

Austin J Anat. 2017; 4(3): 1071.

Effect of Nutmeg Administration on the Anterior Cingulate Cortex (Area 24a) of Adult Male Albino Rats and the Protective Role of Vitamin C: A Histological and Immunohistochemical Study

El-Kholy WB and M El-Sherif N*

Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Egypt

*Corresponding author: M El-Sherif N, Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Egypt

Received: April 03, 2017; Accepted: May 19, 2017; Published: May 26, 2017


Nutmeg is commonly used as a spice in various dishes, as components of teas and soft drinks or mixed in milk and alcohol. We investigated for the first time, the effect of chronic consumption of nutmeg on the anterior cingulate cortex of adult Wistar rats and the possible protective role of vitamin C. Adult male albino rats (n = 20), with average weight of 200 gms. Were assigned into three groups: Control, nutmeg treated and protected. The whole experimental period lasted for 6 weeks. Animals of nutmeg treated group showed cellular degenerative changes relative to those in the control group. It was concluded that, long term consumption of nutmeg has adverse effect on microanatomy of anterior cingulate cortex. In the vitamin C-supplemented (Protected) group, there was improvement in the histological and immunohistochemical changes. Further research, including human observational studies, aiming at corroborating these observations is recommended.

Keywords: Nutmeg; Anterior cingulate cortex; Histological effect; Rat


The nutmeg tree (Myristicafragrans Houtt), which yields an apricot-shaped fruit, is a tropical aromatic evergreen tree. The fruit contains a nutmeg seed which is covered by a protective aril. The aril can be processed giving a spice called mace and the seed is processed to make nutmeg [1]. Being frequently used in baking and cooking, nutmeg is considered a common household spice. The market available forms of nutmeg include the volatile oils, ground powder, and whole nut. Moreover, nutmeg essential oils and butter are also obtained from M.fragrans. These products are widely used in the medicine, food, and perfume industries [2]. Nutmeg is commonly used in variable ways and for many purposes. Beside its traditional use as a spice, it is also used for many purposes in traditional medicine. Nutmeg is used less commonly in Western medicine than in Oriental medicine [3]. Recently, with the gain in popularity of herbal medicine all around the world, the abuse the use of M.fragrans is also possible because of its addictive properties. The spice ingestion in large quantities can be toxic may cause convulsions and even death. The use of nutmeg as a spice and its medicinal use suggest the presence of some components which are responsible for the noticed biological activities. Some of these active principles, as reported previously, may possess some adverse effects at the same time [4]. The main constituents of nutmeg are volatile oil, fixed oil and starch. The volatile oil contents are sabinene, pinene, elemicin, camphene, myristicin, isoelemicin isoeugenol, eugenol, methoxyeugenol, dimericphenylpropanoids, neolignans and lignans. The fixed oil contents are myristic acid and myristin. Phytochemical screening of the seed of M. fragrans aqueous extract revealed the presence of saponins, alkaloids, anthraquinones, cardiac glycosides, phlobatanins and flavonoids [1]. Dietary phytochemicals including polyunsaturated fatty acids, flavonoids and others have been the subject of increasing numbers of research studies for their potential beneficial effects and also because there is apparently increasing evidence that these compounds possess deleterious effects at certain doses. Many researches were done to study the effects of nutmeg on different organs such as liver, kidney, heart, spleen, testes, etc [4].

The medial part of the prefrontal cortex is represented by the anterior part of cingulate cortex (area 24). On the basis of the cingulate cortex lamination pattern, it can be classified into isocortex (area 1) and proisocortex (area 2) [5]. Some researchers [6] define area 1 as the dorsal anterior cingulate cortex and area 2 as the ventral anterior cingulate cortex, but other researchers [7] consider them as areas 24b and 24a, respectively. The area 24a (ventral anterior cingulate cortex) represents a transitional zone that displays stepwise changes in its architectonic lamination pattern between the characteristic allocortical structure and the typical isocortical zone [8]. As it lacks the granular cell layer (lamina IV), the ventral part of the anterior cingulate cortex has a prominent architectonic feature. This makes the differentiation between this region and rest of the neocortex clear [9]. It has several connections with many limbic structures as the amygdala, hippocampus, hypothalamus and thalamus [10]. The area 24a (ventral anterior cingulate cortex) is strategically involved in both autonomic visceromotor and cognitive functions [11].

Vitamin C (ascorbic acid) is an important dietary antioxidant of non enzymatic nature that acts to overcome oxidative stress. It is considered as one of the major water-soluble, antioxidant reducing agents within the body that acts as an electron donor, and probably all of its roles both biochemical and molecular can be explained by this function [12]. However, relatively high levels of this vitamin must be maintained in the body to function as an effective antioxidant [13].

This study was conducted to demonstrate the effect of nutmeg on the structure of the ventral anterior cingulate cortex (area 24a) in adult male albino rats, and to evaluate the protective role of vitamin C against the possible induced changes.

Materials and Methods

Experimental protocol

This study was carried out on 20 adult male Wistar albino rats weighing 180–200 gms. Animals were housed in clean properly ventilated cages, fed on a standard laboratory diet, and maintained on a 12-h light/dark photoperiod in the animal house of the Faculty of Medicine, Menoufiya University. The animals were treated in accordance with the guidelines approved by the Animal care and Use Committee of Faculty of Medicine, Menoufiya University. Animals were randomly assigned into three groups:

(1) Group I (Control): It included 10 animals that were equally subdivided into two subgroups:

• Subgroup Ia (Negative control group) in which animals were kept without any treatment all over the experimental period.

• Subgroup Ib (Positive control group) in which animals received vitamin C at a dose of 500 mg /kg/day orally for 6 weeks. Vitamin C was obtained from Kahira Pharmaceuticals and Chemical Industry (Cairo, Egypt) (Cevilene drops in concentration of 100 mg/ ml).

2) Group II (Nutmeg-treated group): This group included 5 animals that were treated daily with nutmeg. Animals were given 1 ml of nutmeg extract at a dose of 500 mg/kg/day orally for 6 weeks.

(3) Group III (Protected group): This group included 5 animals that received vitamin C 30 minutes before oral administration of nutmeg at the same dose and duration as the previous two groups.

At the end of the experiment, the animals were sacrificed by an overdose of ether and perfused intracardially with saline and 10% neutral-buffered formalin. The brains were extracted from the skulls. The anterior cingulate cortex was dissected by a coronal cut rostral to the corpus callosum.

Preparation of the nutmeg seed’s extract

Nutmeg seeds were obtained from local nearby markets. To remove fungal spores, dust and/or other undesired particles; the dry seeds were washed thoroughly then left to dry, overnight, under room temperature. Using a mortar and pestle; the seeds were then macerated into a fine flour-like paste to pass through 0.2mm mesh. Then we used aliquot weight of nutmeg powder (1gm) which, then, was soaked in 2 ml hot distilled water and left to stand for 72 hrs and then the extract was filtered. That extract was kept a frozen form until used [4].

Histological procedures

Brains in each group were fixed in 10% neutral formalin for 48 h. Tissues were dehydrated in ascending concentrations of alcohol, cleared in xylene, and embedded in paraffin. Five-micrometer-thick sections were prepared. Sections were stained with Hematoxylin and Eosin (H&E) stain [14].

For immunohistological staining, paraffin sections (5 μm thick) were deparaffinized in xylene for 1–2 min and then rehydrated in descending grades of ethanol (100%, 95%, and 70% ethanol) two changes 5 min each, then brought to distilled water for another 5 min. Sections were rinsed with PBS, blocked for 30 min in 0.1% H2O2 as inhibitor for endogenous peroxidase activity. After rinsing in PBS, sections were incubated for 60 min in blocking solution (10% normal goat serum) at room temperature. The sections were then incubated with the primary antibody (iNOS 1:500; Bax-protein 1:500; Bcl-2 protein 1:500; TNF-a 1:1000 and GFAP 1:300) at room temperature for an hour. Sections were rinsed with PBS, followed by 20 min of incubation at room temperature with secondary biotinylated antibody. After rinsing the sections in PBS, enzyme conjugate “Streptavidin-Horseradish peroxidase” solution was applied to the sections for 10 min. Secondary antibody binding was visualized using 3,3'- Diaminobenzoic Acid (DAB) dissolved in PBS with the addition of H2O2 to a concentration of 0.03% immediately before use. Finally, sections were PBS rinsed and counterstaining of slides was done using two drops of hemotoxylin. Slides were washed in distilled water until the sections turned blue. Finally, slides were dehydrated in ascending grades of ethanol (70%, 95%, and 100%) for 5 min each and were cleared in xylene and finally cover slipped using histomount mounting solution.

Morphometric procedure and statistical analysis

In this work, an estimation of the number of immunopositive cells per area (3000 μm2) in the second layer (external granular layer) and third layer (pyramidal layer) of the cingulate cortex was made for all the studied groups. The number of immunopositive cells in the fields was counted using image J software and averaged per field for each animal. The numbers calculated for five animals/experimental group were considered for comparison and statistical analyses. The statistical analysis for each experimental parameter was performed using the arithmetic mean, standard deviation (X±SD), and analysis of variance (ANOVA). This was followed by Tukey’s test for multiple comparisons. P values less than 0.05 were considered significant.


There was no significant difference between the animals of the two subgroups of Group I in all the outcomes used in the study; therefore, these two subgroups were pooled in one group (control).

Group I (control)

The ventral anterior cingulate cortex (area 24a) is located just above the corpus callosum. It represents the medial part of the prefrontal cortex (Figure 1) and is composed of the following layers: layer I (molecular layer) – it contained sparsely scattered cells and nerve processes; layer II (external granular layer) – it contained packed rounded cells with rounded nuclei and distinct nucleoli; layer III (pyramidal layer) – this layer showed the presence of dispersed pyramidal cells of various sizes; layer IV – this layer was absent. Layer V (ganglionic layer), consisted of many pyramidal cells. Many nerve processes could be observed extending from the cells. Layer VI (multiform layer) contained cells of different sizes and shapes (Figures 2, 3a and b).