INTRODUCTION:

Nutmeg, scientifically known as Myristica fragrans, is commonly found in Maluku, Indonesia. It belongs to the Myristicaceae family¹. Nutmeg is extensively utilized to treat diverse ailments such as diarrhea, antiinflammation, analgesic, antihyperlipidemia, nausea, kidney illness, sleeplessness, rheumatism, and cancer^2–5. Additionally, it has been historically employed as a condiment and fumigant. Nutmeg butter, a lipid obtained from the seed, finds application in fragrance, tobacco, and toothpaste^3,6.

M. fragrans comprises many secondary chemicals, including flavonoids, saponins, alkaloids, cardiac glycosides, anthraquinones, and terpenes^1,7–11. Nutmeg primarily consists of myristicin, a phenylpropanoid molecule derivative^12–14. The nutmeg pulp has the highest concentration of the myristicin chemical, with a percentage of 36.05%, compared to mace, which has a percentage of 17.54%, and seeds, which have a rate of 16.50%^15,16.

Myristicin is a bioactive molecule derived from nutmeg with diverse pharmacological properties. However, it is essential to note that excessive use of myristicin can lead to hazardous effects, albeit within specific thresholds¹². Between 1996 and 1998, there were multiple instances of myristicin poisoning in humans who ingested 14-80 grams of nutmeg powder. Research has also been conducted on the toxicity of nutmeg in laboratory animals. Specifically, nutmeg spices were given orally to test animals in 500 and 1,000mg/kg doses over 42 days. This resulted in organ degeneration and atrophy^17,18.

Toxic effects of Myristicin, a compound found in nutmeg, primarily affect the liver - one of the largest and heaviest organs in the body that functions in metabolic processes^19,20 The liver metabolizes Myristicin, producing toxic compounds that result in liver degradation^19.Moreover, excessive consumption of Myristicin causes hepatic steatosis and hepatic necrosis²⁰. The toxic dose of Myristicin is limited to 1-2 mg. If consumed in more significant amounts, it can lead to central nervous system disorders and, in severe cases, even death^21.However, research has yet to be conducted regarding the toxicity limits of consuming nutmeg pulp. Preclinical tests, including subchronic toxicity tests, must be carried out to determine the toxic limit of the compound. Toxicity is usually assessed based on the LD50 (Lethal Dose50), a dosage estimated to kill 50% of the test animal population²²

A subchronic toxicity test was carried out on white rats to ascertain the toxicity limit (LD50) of an ethanol extract obtained from nutmeg pulp (Myristica fragrans Houtt.). The test involved observing the rats' behavior, body weight, organ weight, and liver histopathology after administering doses of the extract at 125mg/kgBW, 250mg/kgBW, and 500mg/kgBW.

MATERIALS AND METHODS:

Materials:

Alcohol, distillate, sulfuric acid (H₂SO₄) (supelco®), acetic anhydride (Emsure®), iron III chloride (FeCl3) (supelco®), nutmeg pulp (Myristica fragrans Houtt), ethanol 96%, ethanol 70%, formalin 10%, hematoxylin-eosin, chloroform, formalin buffer solution, xylol solution, sodium chloride, sodium carboxymethyl cellulose, paraffin, and female white rats (Rattus norvegicus).

Animals Preparation:

Twenty female white rats of the Wistar strain, aged approximately 2 months and weighing between 150-200 grams, were acclimatized for a period of 2 weeks in a research room at a temperature of 27°C. During acclimatization, the rats were provided with 20% of their body weight in pellets and 45mL of water per day. The rats were housed in four square plastic cages, each with a wire cover and husks for bedding. The cages were cleaned twice a week, with the husks replaced and the cages disinfected.

Extract preparation:

The nutmeg pulp (Myristica fragrans Houtt.) were sourced from Ternate City, North Maluku, Indonesia. After washing and drying, the samples were weighed, with a total weight of 500grams. They were then macerated using 70% ethanol solvent with a ratio of (1:4) for 24hours. The liquid extract obtained was concentrated via a Rotary vacuum evaporator and then aired until all solvents were removed²³.

Subchronic Toxicity Test:

In subchronic toxicity, the test animals were divided into four groups: the control group, NPE 125mg/kgBW, 250mg/kgBW and 500mg/kgBW. The treatment was given continuously for 50days²⁴. Before administering the extract, the weight is weighed by the body of each test animal. Behavioural observations were carried out every two days before administering the extract; behavioural observations were carried out in the form of toxic symptoms in animals such as Straub, pyrexia, pineal reflex, grooming, tremor, ptosis and lacrimation. Test animals are dissected on the 50th day, but if an animal dies during the sample period, it is immediately dissected to remove the liver. Additional parameters include determination of liver organ weight, macroscopic appearance and histopathology of the liver^25,26. All research procedures were ethically approved by the Animal Research Ethics Commission at Khairun University's Faculty of Agriculture (Approval No: 07/KEPH/PH/2023).

RESULT:

The research was conducted at the Laboratory of Pharmacology and Toxicology, Pharmacy Department, Faculty of Medicine, Khairun University. The results obtained are body weight, the weight of the liver organ, as well as macroscopic and microscopic observations of the rat liver organ have met the inclusion and exclusion criteria presented in the table and diagram following:

Results of LD50 Value (Lethal Dose 50):

Observation results regarding the death of experimental animals after the first 24 hours of administration of 50 days of extract can be seen in Table 1. The results show no experimental animal died starting from the lowest dose of 125mg/kg.BW to the highest dose of 500mg/kg BW.

Table 1. Observation of rat's death during treatment

Groups	Number of Rats
Groups	1^st day	50^th day
Control	0	0
NPE 125mg	0	0
NPE 250mg	0	0
NPE 500mg	0	0

NPE: Nutmeg Pulp Extract

Result of Behavioral Observation:

During the subchronic toxicity test of NPE, the rats' behavior was observed before administering the extract every two days to see any symptoms of toxicity that the treatment may cause, as seen in Table 2.

Table 2. Changes in Behaviour

Groups	Changes in Behaviour
Groups	GR	SR	PR	TR	RP	PT	LM
Control	N	N	N	N	N	N	N
NPE 125mg	N	N	N	N	N	N	N
NPE 250mg	N	N	N	N	N	N	N
NPE 500mg	N	N	N	N	N	N	N

GR (groming), SR (Straub), PR (pyloerection), RP (pineal reflex), PT (ptosis), LM (lacrimation), N (normal)

Result of Body weight weighing:

The body weight of the test animals was observed during testing at the time of administration of the extract for 50 days. The following are the results of observing the body weight of rats seen in Table 3 and the collected data was converted into a diagram, displayed in Figure 1.

Table 3. Rats' body weight during NPE administration.

Groups	Body weight (mean ± SD)
Groups	Initial Weight	Final Weight
Control	138,80 ± SD 27,20	148,20 ± SD 22,01
NPE 125mg	138,40 ± SD 17,99*	152,20 ± SD 15,23*
NPE 250mg	146,80 ± SD 32,22*	155,20 ± SD 33,44*
NPE 500mg	128,00 ± SD 33,08*	136,80 ± SD 29,79*

*Significant P>0.05, no difference with the control group.

Figure 1. Comparison of rats body weights

Result of liver organ index:

Determining the organ index is a key parameter in subchronic toxicity tests. The liver weight in rats typically ranges from 2.4% to 5% of their body weight. The test results are presented in Table 4.

Table 4. Liver organ weight

Groups	Liver organ weight (mean ± SD)
Kontrol	5,04 ± SD 0,82
NPE 125 mg	4,75 ± SD 0,07^*
NPE 125 mg	4,98 ± SD 0,95^*
NPE 500 mg	4.40 ± SD 0,51^*

*(Significant P>0.05, no difference with the control group).

Result of Macroscopic and Microscopic Liver:

After 50 days of administering the extract, the liver of all rats was dissected and observed both macroscopically and microscopically. Macroscopic observations involved examining any changes in the color and texture of the organ, which can be seen in table 5. On the other hand, microscopic observations were focused on the hepatocyte cells, which can be observed in Figure 2.

Table 5. Observation of the liver at a macroscopic level.

Groups	Observation result	Figure
Control	The object in question appears to have a dark brownish-red hue with a smooth and glossy surface.
NPE 125mg	The object in question appears to have a dark brownish-red hue with a smooth and glossy surface.
NPE 250mg	The object in question appears to have a dark brownish-red hue with a smooth and glossy surface.
NPE 500mg	The object in question appears to have a dark brownish-red hue with a smooth and glossy surface.

Microscopic observations were conducted using histopathological images of liver cells. The images were analyzed to determine the number of normal hepatocytes and necrotic cells, as well as the widening of the sinusoids. The findings are shown in Figure 2.

Control NPE 125mg

NPE 250mg NPE 500mg

Figure 2. Histopathology of rat liver after administration of nutmeg pulp extract, 400x magnification. S (sinusoidal); H (hepatocytes/normal cells); N (necrosis); BD (node channel).

DISCUSSION:

Samples of nutmeg pulp were extracted using the maceration method because it is cheap, simple, and easy to do. The principle was breakdown of cell walls and membranes is due to pressure differences, resulting in secondary metabolites dissolved in solvents. Solvent selection 70% ethanol is used because it is non-toxic, cheap, suitable for various extraction methods, easy to obtain, efficient, environmentally friendly, and has a high extraction rate, Moreover, according to the solubility, myristicin is slightly soluble in Ethanol¹⁷

Subchronic toxicity is assessed through several evaluation parameters, including the LD50 value (Lethal Dose 50), changes in behaviour, body weight, and liver weight, as well as macroscopic and microscopic observations of the liver. Weighing Test animals aim to detect changes in body weight directly due to Changes in body weight, which are early symptoms that are easily detected/observed. Toxicity causes a decrease in appetite in test animals.

In this study, the LD50 (Lethal Dose 50) value was calculated to determine the level of toxicity of the test preparation. The calculation was based on the number of deaths of experimental animals 24hours after administering the test preparation for 50days. The results showed that there were no deaths of experimental animals during the test. This indicates that EDBP at doses of 125mg, 250mg, and 500mg is safe to consume, with an LD50 value of 246.036mg/kg, which is categorized as safe or non-toxic²⁷.

During the testing, the behavior of the test animals was closely monitored as it is an early indicator of any toxic symptoms that might arise after treatment. It was observed that the animals did not display any behavioral changes such as grooming, Straub, piloerection, pineal reflex tremor, ptosis, or lacrimation as shown in Table 2. This indicates that the doses of EDBP at 125mg, 250 mg, and 500mg are safe to consume and do not cause any toxic symptoms in the behavior of test animals.

The results of weighing the rat's body weight can be seen in Table 3, where There was an increase in the overall weight of the group by 8.4 - 13.8grams; the percentage increase in body weight was 5.72% - 9.97% according to the growth of the mice's normal average is 1.5 - 3% per day of initial weight or around 7.05 - 21% per week²⁸. The One Way Anova test results showed no significant differences between the test and control groups (P>0.05). So, there is no influence in giving EDBP doses of 125mg, 250mg, and 500mg to the body weight of rats.

Surgery was performed on test animals that had died, and their livers were examined. This is because the liver is responsible for metabolizing all foreign substances that enter the body²⁹. If a substance is toxic, it can directly damage the liver or cause metabolic changes that lead to liver damage³⁰. Hence, the presence of liver damage can indicate whether a substance is toxic or not. The liver weight observation results are presented in Table 5.6. The results indicate that all groups comply with the relative weight requirement for an average rat's liver, which is 2.3% - 5% of the rat's body weight^28,31. Additionally, the SPSS One Way test Anova results showed no significant differences between the control groups and test groups (P>0.05).

The macroscopic observations indicated that the livers of all rats in the group had a normal brownish-red color with a smooth and shiny surface, without any indications of toxicity. The results showed that the liver conditions were within the normal range, with a soft and even surface^32,33. The results of the histopathological image of liver cells can be observed in picture 5.2. In general, the liver is still in a normal condition, where the dose of 125 mg is similar to the control group. However, the groups that received doses of 250mg and 500mg experienced slight changes in the number of normal hepatocyte cells, cell necrosis, and sinusoial dilation. This demonstrates that the ethanol extract of Nutmeg pulp has a certain effect on liver histopathology, but it is still within safe limits. The average shows that the dose used is still considered safe.

CONCLUSION:

The toxicity of nutmeg pulp's ethanol extract has been studied at doses of 125mg, 250mg, and 2500mg. The results showed that the extract is safe with an LD50 value of 246.036mg/kg. Moreover, the nutmeg pulp extract did not have any impact on the body weight, organ weight, macroscopic and microscopic appearance of the liver in all groups. Additionally, there were no changes in the behavior of the test animals during the 50 days of administering the extract.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The author would like to express gratitude to the Research and Community Service Khairun University for their support during the research process.

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Received on 12.05.2024 Revised on 10.09.2024

Accepted on 22.11.2024 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2256-2260.

DOI: 10.52711/0974-360X.2025.00323

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