An experimental study involving rats was performed in Department of Pharmacology, Santhiram Medical College, Nandyal, India, from December 2016 to February 2017. Before conducting this study, Institutional Animal Ethical Committee (IAEC) (897/PO/Re/S/05/CPCSEA) permission was taken. This study was strictly conducted according to Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines.

Fresh and dried Mace was purchased from wholesale grocery store for the preparation of extract. Authentication was done by Dr. Suryanarayana Moorthi, PhD, Professor of Botany. Its uses were explained by Dr. Gurunath Reddy. B.A.M.S. Nandyal, Andhra Pradesh.

Dried mace was ground to a fine powder. From the stock, 30 gm of powder was weighed and placed in the Soxhlet basket. The Soxhlet flask was filled with 300 mL of ethyl alcohol which acts as solvent. The Soxhlet connected with condenser and tap water was running through the inlet and outlet of the condenser. The solvent was heated to reflux. The solvent vapour travels up a distillation arm and floods into the Soxhlet basket which contains solid. The condenser ensures that any solvent vapour cools, and drips back down into the sac which was filled with mace powder in the basket. The Soxhlet basket was filled slowly with solvent. Some of the desired compounds in the powder dissolved in the warm solvent. When the basket was almost full, it was automatically emptied by a side arm, and the solvent running back down to the Soxhlet flask. This cycle was allowed to repeat many times in 24 hours. After many cycles the desired compounds were concentrated in the distillation flask. Finally, the extracted solvent was placed on the water bath for evaporation for yielding the extracted compound. The resultant solid extract was measured [17]. Out of 30 gm of dry powder, 5 gm of solid extract was obtained. The percentage of yield extract was 16. 6%. This extract was dissolved in 5 mL distilled water and administered orally by gastric intubation.

(It consisted of three variants): [18].

The different variants were fed on alternate days throughout the treatment period (10 weeks). First five weeks without treatment and last five weeks treatment with M. fragrans extract in Group-IV, and Group-V.

Acute toxicity study was done as per OPPTS guidelines in healthy Albino rats by up and down Procedure [19]. The rats were divided into six groups of six rats each. The groups were treated with ethanolic extract of M. fragrans in doses of 10 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg, 500 mg/kg and 1000 mg/kg respectively. The animals were observed for behavioural changes and death rate. No abnormal changes were found in the first five groups up to the dose of 500mg/kg. In the sixth group (1000 mg/kg), abnormal behavioural changes were observed and two rats died. Thus, M. fragrans extract was found to be safe below 500 mg/kg. The doses of 200 mg/kg and 400 mg/kg were chosen for the present study.

Thirty wistar Albino rats of both the sexes, weighing 150-180 gm, were selected and taken from the Central Animal House of Santhiram Medical College. They were fed with commercial standard pellet diet and water ad libitum and maintained under standard laboratory conditions with 12:12 hour light: dark cycle [20]. The rats were divided into five groups of six animals each:

Group-I-Control: Treated with Standard Pellet diet (SPD) + Normal saline.

Group-II-Test-1: Treated with Standard Pellet diet (SPD) + M. fragrans extract 200 mg/kg.

Group-III-Test-2: Treated with Standard Pellet diet (SPD) + M. fragrans extract 400 mg/kg.

Group-IV-Test-3: Treated with CD + M. fragrans extract 200 mg/kg.

Group-V-Test-4: Treated with CD + M. fragrans extract 400 mg/kg.

All the test compounds were administered orally once a day before food [16].

The BW of all the rats were recorded on day one in the first week and last day of the 10^th week. All the five groups were given standard pellet diet and water from day one to last day of the 10^th week. FI per day of each rat was assessed and the average FI capacity was calculated. From first day of sixth week to last day of 10^th week (five weeks of duration), the rats in Group I were given Normal saline. The rats in Group II and III were treated with SPD and M. fragrans extract in the doses of 200 mg/kg and 400 mg/kg respectively. These two groups were considered as weight Preventive models. Group IV and V were given CD along with pellet diet and treated with M. fragrans extract in the doses of 200 mg/kg and 400 mg/kg respectively. These two groups were considered as obesity treatment models. BW and FI of each rat was measured at the end of the 10^th week.

Food intake: All the rats were fed with normal laboratory Pellet diet. Food was presented in the form of pellets in grams [21]. FI was measured manually. A known amount of food was given to the animal. After 24 hours, the food was weighed again and the amount consumed was calculated. FI in 24 hours by an individual rat was multiplied with 35 (for five weeks). FI was measured for first five weeks (before treatment), and the last five weeks (after treatment).

Body weight: Body weight was recorded on day one of first week and on last day of fifth week (without treatment) and at the end of the study on last day of 10^th week (after treatment with the extract).

The results obtained were expressed as mean±SEM and were analysed by the application of ANOVA, followed by Dunnett’s t-test. Results were considered to be statistically significant at p-value <0.05.

In Group-I, the BW and FI was almost the same on day one and day 70 [Table/Fig-2]. The average BW on day one, on day 35 and on day 70 was 162.83±4.81, 203.66±6.24, 225.00±5.11 respectively. FI capacity on day 35 and on day 70 was 835.83±10, 893.33±12 respectively. There was slight increase in body weight. This could be attributed to the fact that as the age (10 weeks) increases, BW and FI capacity also increases. There was not much difference observed in first five weeks (1-35 days) and last five weeks (36-70 days).

In Group-II, in the days 1-35, there was not much difference in the weight. In the days 36-70, there was a reduction in the BW. In the days 36-70, FI capacity was also reduced. The average BW on day one, on day 35 and on day 70 was 165.75±5.59, 194.5±5.92, 179.33±7.09 respectively. FI capacity on day 35 and on day 70 was 868.83±12, 808.66±10 respectively [Table/Fig-3].

In Group-III, the average BW on day one, on day 35 and on day 70 was 165.50±4.81, 190.00±6.23, 168.66±7.17 respectively. High dose of M. fragrans extract has shown more effect on BW gain than the low dose. FI capacity on day 35 and on day 70 was 900.00±10, 811.33±11respectively. From 36-70 days, food intake capacity also greatly reduced. Thus BW and FI significantly reduced in a dose dependent manner (p<0.01) when compared with control group [Table/Fig-4].

In group-IV, the average BW on day one, on day 35 and on day 70 was 169.50±2.36, 280.16±4.92, 250.83±5.63 respectively. From 1-35 days, there was great increase in BW due to high fat diet (diet induced obesity), but in the days 36-70, there was slight reduction of BW. FI capacity on day 35 and on day 70 was 895.50±0.6, 843.52±1.10 respectively. From 36-70 days, FI capacity also reduced but very less [Table/Fig-5].

In Group-V, the average BW on day one, on day 35 and on day 70 was 164.16±1.63, 287.33±6.11, 211.45±5.23 respectively. From 1-35 days, there was great increase in BW due to high fat diet (diet induced obesity), but in the days 36-70, BW was reduced greatly. High dose of M. fragrans extract has shown more effect on body growth than the low dose. FI capacity on day 35 and on day 70 was 866.23±13, 774.83±3.11 respectively. From 36-70 days, FI capacity also greatly reduced. Thus, BW and FI significantly reduced in a dose dependent manner (p<0.01) when compared with control group [Table/Fig-6].

From [Table/Fig-7] it can be concluded that high dose of M. fragrans extract (400 mg/kg) has shown more effect on both BW and FI capacity of the rats at the end of 70 days.

Studies have shown that medicinal extracts (obtained from traditional medicine) can be used to obtain new compounds with potential pharmacological activity [22]. The potential of M. fragrans to decrease FI and reduce BW was evaluated in rats. Rats were selected for the study because they enable us to obtain answers in a short time (10 days in the life of a rat accounts for approximately one year in the life of humans) when comparing changes in BW. Furthermore, there are similarities neuroanatomically in brain areas that play role in the control of FI. It is well established that a number of different neurotransmitters and peptides produce similar effects on FI and energy homeostasis in laboratory rodents and man. As the age increases in rats, its weight and FI capacity also increase rapidly within a short period [23].

Myristica fragrans (Mace) is a commonly used spice in various dishes and food preparations. It has been shown to possess hypolipidaemic properties [24]. However, studies have not been done so far to assess its impact on FI and BW. In the present study, the effect of M. fragrans on FI capacity and body growth in experimental animal models were studied as per standard guidelines. According to traditional system of medicine, different parts of M. fragrans have different medicinal properties.

Arulmozhi DK et al., observed that hydroalcoholic extract of fruits of M. fragrans effectively attenuate the raised TG (47% reduction) and CH (66.7%), and at 450 mg/kg, significantly inhibited the hepatic lipoprotein secretion in high cholesterol fed rats. These results proposed that M. fragrans extract can be used to improve hyperglycaemia and hyperlipidaemia [25].

Nguyen PH et al., isolated the seven active compounds from the total extract of M. fragrans and have shown anti-obesity property [14]. The active compounds were 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignans, tetrahydrofuroguaiacin B (1), saucernetindiol (2), verrucosin (3), nectandrin B (4), nectandrin A (5), fragransin C(1) (6), and galbacin (7). In differentiated C2, C12 cells, the sole target area for the treatment of obesity and type-2 diabetes is AMP-Activated Protein Kinase (AMPK) enzyme. The active compounds from M. fragrans can stimulate the AMPK. THF is one of the important chemical compounds, and its preventive effect was tested on body weight in a diet-induced animal model. According to outcome of the animal study, the active compounds of the M. fragrans can be used for the evolution of agents to treat metabolic disorders [13].

Al-Shammary Hayfaa A et al., demonstrated the analgesic activity of M. fragrans seed extract in mouse model of acetic acid-induced visceral pain. It was concluded that the analgesic activity of seed extract was due to presence of various alkaloids and inhibit phospholipase A2, thus lowering the availability of arachidonic acid precursor for prostaglandin synthesis [26].

Olaleye MT et al., explained the antioxidant property of aqueous extract of M. fragrans in animal models of rat. The results showed that alkaloids present in M. fragrans, were saponins, anthraquinones, cardiac glycosides, flavonoids and phlobatanins have antioxidant property [27].

In another study, Kartika Dewi et al., demonstrated the anti-inflammatory effect of M. fragrans ethanolic extract by in vitro method -cell viability assay. They concluded that, anti-inflammatory effect was due to antioxidant property of Quercetin, the most commonly occurring flavonoid in M. fragrans seeds. Anti-inflammatory potential may be due to inhibition of TNF-α, IL-6, IL-1β and no production [28].

Jaiswal P et al., explained the biological effects of M. fragrans in a review article. The chemical constituents of M. fragrans was found to exhibit hypolipidaemic and hypocholesterolaemic effects, antimicrobial, antidepressant, aphrodisiac, memory enhancing, antioxidant and hepatoprotective properties. Recent studies revealed strong insecticidal and molluscicidal activities of M. fragrans [29].

In the previous animal studies, the whole fruit and seed (nutmeg) extracts have shown antioxidant, antidiabetic and hypolipidaemic properties [30,31]. The effect of Mace (fleshy covering of nutmeg) extract was not evaluated on body weight and FI capacity. Hence, mace was chosen for the present study.

The results of this study revealed that the M. fragrans extract can reduce FI capacity and prevent the increase in BW in a dose dependent manner. High dose (400 mg/kg) has shown maximum effect in reducing FI capacity and preventing the increase in BW in Group-III, and V (p<0.01). Low dose (200 mg/kg) has less effect on FI, BW in Group-II, and IV when compared with the high dose. The exact mechanism of action of the mace extract was not well established. Based on the observations and previous research studies, the possible mechanism for weight reducing property may be due to presence of saponins in mace extract. Saponins can inhibit the pancreatic lipase enzyme; thereby reduce the cholesterol levels [32]. Tetrahydrofuran is one of the active chemical compounds present in the Mace, also contributes to prevent weight gain by stimulating AMPK enzyme in differentiated C2, C12 cells. Its steroidal compounds reduced the FI capacity for its inhibitory effect on hunger sensory mechanism in hypothalamus [33]. This could be beneficial to the obese persons and to treat obesity associated complications. Acute toxicity study revealed that this plant extract was found to be safe for a short period. Prolonged use of M. fragrans extract can lead to toxic effects on various organs [27,34]. Further studies are needed for the establishment of safety.

The present study was carried out in the rat models but histological study, and plasma lipid profile was not conducted to establish its weight reducing effect by observing adipose tissue mass and cholesterol levels. Its delayed effects were not studied. Further evaluation is needed on the separation of the individual compounds present in the M. fragrans and their effects at the molecular level.

The present experimental study indicates that the commonly used spice M. fragrans extract has potential inhibitory effect on body weight and FI. Hence, this extract can be used as an anti-obesity preparation in obese and overweight individuals. However, it needs further studies to prove its weight reducing property in humans.