INTRODUCTION:

Diabetes mellitus (DM) is the most significant metabolic condition of great concern to modern civilization. Both type 1 and type 2 diabetes are characterized by increased blood glucose levels, which can be attributed to either aberrant insulin production or impaired insulin sensitivity^1–3. Diabetes mellitus is caused by several conditions, such as damage to the function of insulin secretion and action or by carbohydrate, fat, and protein metabolism disorders, and impacts approximately 10% of the population^2,4–8.

The effects felt by people living with diabetes in the long term are retinopathy, nephropathy, and neuropathy, along with other complications. Diabetes patients also tend to be at increased risk of other diseases such as heart disease, obesity, cataracts, erectile dysfunction, liver damage, and some liver diseases^9,10.

Based on data from the International Diabetes Federation (IDF), in 2019, there were 463 million persons aged 20-79 who experienced DM (diabetes mellitus), and this figure is expected to increase to 700 million by 2045¹¹. In 2019, the Southeast Asia Region, the main area affected by DM, had 87.9 million individuals with DM. Among them, 34.3% resided in urban areas, and 49.4% lived in cities¹². Most individuals, precisely 99.2%, who are affected by DM reside in middle-income countries within South Asia. In Indonesia, the rate of diabetes is 8.2%, which is slightly higher than the average of 7.5% for all low- and middle-income countries (LMICs). They were ranked 4th after India, China, and the United States IDF, 2019. By 2030, it is estimated that there will be an increase to 21.3 million people who have diabetes. The increasing number of people living with diabetes in Indonesia will lead to a rise in the use of antidiabetic drugs^9,10.

Using synthetic medicines for an extended period can cause many disadvantages, including the risk of side effects, so currently, the use of natural ingredients as medicines can be a choice in therapy¹³. The use of plants for treatment has been widely chosen as an alternative because natural medicines are easy to obtain, cheaper, and have minimum side effects^14,15. Indonesia is a tropical country with diverse plant species, some of which have pharmacological activity but have not yet been identified. One of them is the nutmeg of Myristica fragrans^8,16–18.

Nutmeg (Myristica fragrans Houtt.) is a spice plant and is an essential ingredient in cooking in general. It is a native plant from Maluku and North Maluku, Indonesia¹³. Hasbullah et al. (2023) reviewed the pharmacological activities of Nutmeg as an antidiabetic. Almost all parts of nutmeg can reduce the risk of DM and reduce levels of blood glucose in animal tests, both rats and mice¹⁹. Leaves, pulp, mace, and seeds are the parts of nutmeg used since ancient times^20,21. However, most people take the seeds and nutmeg mace while discarding the pulp or using it as waste. Even though this one part contributes some significant components to nutmeg^13,22,23, apart from that, the pulp of nutmeg is also said to contain chemical compounds such as flavonoids, phenolics, alkaloids, and terpenoids. Various bioactive compounds these can provide pharmacological activity, for example, as research by Adeyeoluwa et al. (2020) demonstrated that there are the presence of phenolic and flavonoid compounds which act as antihyperglycemics in the pulp of nutmeg^24–27.

However, until now, it is unknown how the treatment effect will be diabetes mellitus or efforts to reduce blood sugar levels using the pulp of nutmeg (Myristica fragrans Houtt) and how it affects the histopathology of the pancreas. Thus, This research aims to see the antidiabetic effect of nutmeg pulp extract (Myristica fragrans Houtt), which is a spice commodity in Ternate City, North Maluku, on pancreatic histopathology in white rats (Rattus novergicus) diabetes.

MATERIALS AND METHODS:

Sample Preparation:

The nutmeg pulp used came from Ternate City, North Maluku. The samples obtained were then cleaned using running water, chopped into small pieces, and then dried until they contained water content below 10%. The dried samples were weighed at 500grams and extracted with 70% ethanol solvent using the maceration method; the solvent was added until all parts of the sample were submerged. Samples were left for 1x24h. Next, the process of filtering the maceration results is carried out and then evaporated using a rotary evaporator until a thick extract is obtained. The preparation of nutmeg pulp extract (NPE) is divided into three doses 100mg, 200mg, and 300mg.

Animals:

The test animals were male rats wistar, obtained from Murine Farm, Malang, Indonesia. The rats weighed 200-300grams and aged 3-4 months. Before being used for experimental, they adapted during one week in the laboratory. All experimental animals were acclimatized for two weeks under temperature conditions of room 25⁰±2⁰C and provided standard feed and boiled water²⁸. Acclimatization aims to enable the test animals to adapt to the new environment so that it does not affect the test results. Animals are declared healthy if, during the acclimatization process, the animal's weight does not show deviations of >10%. The entire research process has received ethical approval from the Animal Research Ethics Commission, Faculty of Agriculture, Khairun University with approval number 06/KEPH/PH/2023

Diabetes Induction:

The diabetes induction method is based on research conducted by Tri Yuliani et al. (2016) with modifications²⁹. Test animals were fasted for ±16 hours, baseline fasting blood sugar (T0) was measured, and blood was taken via the tail vein. Blood sugar measurement using Nesco® glucometer strips. Diabetes induction was carried out by administering white rice at a dose of 3.6g/200gbw for 14 days as a high-glucose feed (T14). Metformin 500mg was given as standard.

Antidiabetic Activity Test:

Test animals were divided into 5 groups (n=5), including negative control, Metformin, NPE 100mg, NPE 200mg, and NPE 300mg. Standards and extracts were given orally for 7 days, starting the 14th day after blood sugar measurement (T14). Blood sugar was measured on days 15, 17, 19 and 21. On day 21, surgery was carried out to remove the pancreas organ for histopathological observation.^1,28–30.

Statistical analysis:

The data are presented as the average value plus or minus the standard error of the mean for five rats in each experimental group. The data obtained from the study were analysed using One Way ANOVA followed by Tukey test. The statistical significance between the groups at a significance level of P<0.05.

RESULT:

Figure 1. Profile of blood sugar levels during a glucose tolerance test.

a b c

d e

Figure 2. Histopathology of the rats pancreas using a microscope with 40x magnification. (a) negative control; (b) standard (metformin 500mg); (c) NPE 100mg; (d) NPE 200mg; (e) NPE 300mg

DISCUSSION:

The extraction method used to obtain thick nutmeg pulp extract (NPE) is the maceration method. Maceration is a conventional method used to extract chemical compounds. This method is used because it has several advantages, including not requiring special equipment or special operators, an energy-saving process, not requiring high costs, and being able to attract chemical compounds stably without a heating process^31,32. The choice of 70% ethanol as a solvent aims to extract polar and nonpolar compounds. Phenolic compounds can be dissolved by solvents such as ethanol because they have hydroxyl groups. Ethanol 70% can extract more phenolic compounds than ethanol with high purity¹³.

Antidiabetic activity of nutmeg pulp ethanol extract in male Wistar rats induced by a high glucose diet. Male Wistar rats can metabolize drugs relatively quickly. Hence, they are more sensitive when used in research than female rats and can provide more stable research results because they are not influenced by pregnancy or hormonal influences³³, the estrogen hormone in female mice has receptors on pancreatic beta cells, stimulating insulin release³⁴. As a result, the measurement results using female rats are inaccurate because estrogen has a healing effect³⁵

Based on the results of blood sugar measurements before induction (see Table 1), all rats showed fasting blood sugar in a normal range and did not show significant differences between all groups. The normal rat's blood glucose levels in this measurement are 90-99 mg/dL. This value was published by Hidayaturrahmah et al. 2020. Typically, the blood glucose level of a white rat was recorded as 50-135mg/dL³⁶. Rats are said to have diabetes mellitus when blood sugar levels are >150mg/dL³⁷.

Before antidiabetic activity test, to create diabetic rats, the test animals were given 3,6g/200gbw of white rice induction, this amount is equivalent to the glucose content in 200g of white rice for adults can induce hyperglycemic^38,39. White rice was chosen as an inducer because it has a high glycemic index. The glycemic index is defined as the ranking of foods according to their effect on blood sugar levels. The glycemic index is a number that shows the potential for increasing blood glucose levels from carbohydrates available in a food. When consumed, foods with a high glycemic index will quickly increase blood glucose levels by a large amount. High glucose food was given daily for 14 days. All groups showed an increase in blood sugar (T14), and each group had no significant differences. So, it can be ascertained that giving high glucose feed can increase blood sugar (see table 1).

The first day after administration standard and NPE (T15), Metformin, NPE 200mg, and NPE 300 groups showed a significant decrease in blood sugar compared to the negative control (p<0.05). The results of blood sugar measurements on days 17, 19, and 21 showed that all extract groups showed significant differences (p<0.05) compared to the negative control group.

Figure 2 shows the pancreatic histopathology of each group. Figure 2a is the pancreas from the negative control group. Based on the picture, it can be seen that the Langerhans islets suffer damage in the form of degeneration followed by necrosis. According to Hermawati (2020), the degeneration and necrosis found were response cells after exposure to toxicants in the form of administration of high doses of glucose, which can damage metabolic pathways in rats so that when observed microscopically can be seen changes in the shape of endocrine cells in rats islets of Langerhans. Figure 2b shows that the proportion of endocrine cell structures that experience degeneration and necrosis in standard-group rats was less than in rats in the negative control group who were not given therapy. Cell degeneration occurs due to disturbances that affect structures within cells that can interfere with cell metabolism processes. This damage is reversible.

In Figure 2c, the NPE 100mg shows slightly more degeneration and necrosis of Langerhans's islets than the negative group but not better than the standard group. In Figure 2d, the NPE 200mg group shows degeneration and necrosis of the islets of Langerhans in rats with less diabetes than the negative group characterized by a reduced area of necrosis on the islets of Langerhans which provides a regenerative effect on pancreatic β cells and is effective in lowering blood glucose levels. In picture 2e, the NPE 300mg shows the proportion of endocrine cells that experienced less degeneration and necrosis than negative control test animals and showed improvement in islet cells that resembled the standard group given metformin.

The improvement in islets of Langerhans in the histopathological picture is due to the ability of secondary metabolite compounds, flavonoids which is found in the ethanol extract of nutmeg pulp. Flavonoids are known to have antioxidant activity, which is believed to protect the body against damage caused by reactive oxygen species so that it can prevent degenerative diseases such as diabetes mellitus^5,40,41. Flavonoids can lower blood glucose levels with their ability as an antioxidant. Flavonoids are protective against damage to β cells, which produce insulin. Well, it can restore the sensitivity of insulin receptors on cells and even increase insulin sensitivity^5,42.

From the research results above, it is suspected that the higher the dose of NPE given, the greater and faster the reduction in blood sugar. The smaller the dose, the slower it lowers blood sugar, so it is more effective as an antidiabetic.

CONCLUSION:

Nutmeg pulp extract showed activity in reducing blood sugar levels at doses of 200mg and 300mg. Also, it showed improvements in the histopathological appearance of the rat pancreas induced by high glucose feed.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We would like to express our gratitude to Khairun University for their financial support during the study.

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Received on 06.03.2024 Revised on 03.06.2024

Accepted on 09.08.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):89-93.

DOI: 10.52711/0974-360X.2025.00014

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Groups	Dose	Blood Sugar Level (mg/dL) ± SEM
Groups	Dose	T0	T14	T15	T17	T19	T21
Negative Control	-	95.80±12.85	167.40±3.96	167.80±4.72	164.40±7.13	157.60±8.63	154.00±9.88
Metformin	500mg	91.00±95.80	171.20±8.02	133.60±8.07^a	114.40±9.56^a	100.00±6.19^a	85.20±9.87^a
NPE	100mg	90.80±11.08	159.80±10.06	151.00±8.97	142.00±12.10^a	123.00±6.71^a	97.40±3.51^a
NPE	200mg	98.40±8.50	165.20±6.30	140.60±14.31^a	122.00±10.86^a	99.80±4.82^a	88.40±3.05^a
NPE	300mg	99.60±8.26	168.80±11.08	136.80±11.54^a	121.40±10.38^a	101.40±5.18^a	86.00±6.67^a