{"id":62910,"date":"2024-12-30T10:20:02","date_gmt":"2024-12-30T10:20:02","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=62910"},"modified":"2025-01-07T04:14:08","modified_gmt":"2025-01-07T04:14:08","slug":"the-effect-of-ethanolic-extract-of-annona-muricata-l-leaves-on-cerebellum-neurons-in-noise-exposed-adult-wistar-rats","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no4\/the-effect-of-ethanolic-extract-of-annona-muricata-l-leaves-on-cerebellum-neurons-in-noise-exposed-adult-wistar-rats\/","title":{"rendered":"The Effect of Ethanolic Extract of Annona muricata L. Leaves on Cerebellum Neurons in Noise-Exposed Adult Wistar Rats"},"content":{"rendered":"\n

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

Soursop\nleaf extract is obtained from the soursop plant (Annona muricata L.<\/em>), this plant has\nbeen widely used for traditional medicine and is believed to have health\nbenefits. Several studies have previously been conducted on this extract,\nshowing its high antioxidant content and benefits such as anticancer and\nanti-inflammatory.<\/p>\n\n\n\n

This\nextract’s compounds such as acetogenin, alkaloids and flavonoids can\nsignificantly inhibit the growth of cancer cells 1<\/sup>. Soursop leaf\nextract also has significant antimicrobial activity against pathogens 2<\/sup>. Its benefits as an\nantioxidant and anti-inflammatory are associated with its potential to protect\nneurons from oxidative damage. Antioxidants are compounds that can protect body\ncells from damage caused by free radicals. Previous research has found that\nsoursop leaves contain various antioxidant compounds, such as alkaloids,\nflavonoids, tannins and phenolics 3<\/sup>.<\/p>\n\n\n\n

The\nhuman brain has a vital function in the body. The brain functions to control\nthe internal environment and external responses of the human body. One part of\nthe brain is the cerebellum which is responsible for motor coordination and\nbalance. Both the brain and cerebellum are susceptible to external disturbances\nsuch as noise.<\/p>\n\n\n\n

Noise\nis an environmental factor that is often considered trivial, but can have an\nimpact on brain health. Previous research has shown that prolonged exposure to\nnoise can cause various neurological problems. Noise can interfere with\ncognitive function, increase stress levels, and potentially increase the\nprogression towards neurodegenerative diseases 4<\/sup>. The cerebellum can\nalso be negatively impacted by noise. Disorders of the cerebellum can cause\nproblems with human motor coordination and balance. Previous research shows\nthat exposure to noise can disrupt the process of motor learning and\nadaptation, which is a function of the cerebellum 5<\/sup>. Protection of the\nbrain may include cerebellar function. Chronic inflammation and neuronal\ndegeneration can have a negative impact on the central nervous system including\nthe cerebellum 6<\/sup>,<\/sup>7<\/sup>.Compounds in this\nextract such as flavonoids and acetogenin have anti-inflammatory and\nanti-neurodegenerative activities. <\/p>\n\n\n\n

Most\nprevious studies tend to focus only on the effects of Annona\nmuricata L.<\/em> leaves extracts in the context of general\noxidative stress, or on other organs, and few have examined its protection of\ncerebellar neurons exposed to noise. This creates a significant research gap\nregarding the use of Annona muricata L.<\/em> leaves extracts for\nnervous system protection against adverse environmental exposures, such as\nnoise. To address this gap, this study aimed to evaluate the neuroprotective\neffects of Annona muricata L. <\/em>extract on cerebellar\nneurons of adult Wistar rats exposed to noise. This study is expected to\nprovide a deeper understanding of Annona muricata L.<\/em> protection against\nnoise-induced neuronal damage, as well as enrich the literature on\nantioxidant-based therapeutic approaches to reduce the impact of oxidative\nstress on the brain. <\/p>\n\n\n\n

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

Preparation and phytochemical analysis of soursop leaves\nethanol extract <\/strong><\/p>\n\n\n\n

Soursop leaf extract was obtained\nfrom locations that met organic farming standards, and the extraction process\nand phytochemical tests were carried out at the Integrated Services Laboratory,\nFaculty of Agricultural Technology, Universitas Udayana.  The selected leaves were then cleaned, dried,\nand ground into powder using a grinder. The simplicia powder is then subjected\nto an extraction process using the maceration method and 96% ethanol solvent. <\/p>\n\n\n\n

Ethanol extract of soursop leaves\nwere analyzed to identify the main phytochemical compounds, namely alkaloids,\nflavonoids, tannins, phenols and other related compounds. Phytochemical\nscreening methods used include testing with certain reagents, such as the\nWagner test for alkaloids, the Shinoda test for flavonoids, and the ferric\nchloride test for tannins.<\/p>\n\n\n\n

To evaluate the ability of\nsoursop leaf ethanol extract in reducing free radical compounds (antioxidant\ncapacity) IC50<\/sub> using the Ferric Reducing Antioxidant Power (FRAP).\nTo measure the phytochemical content of phenols, flavonoids, tannins, vitamin\nC, beta-carotene, tocopherol, and saponins using the Colorimetric Method.<\/p>\n\n\n\n

Extraction process of soursop<\/strong><\/p>\n\n\n\n

In this study, quantitative analysis of phytochemical compounds including phenols, flavonoids, tannins, vitamin C, tocopherols, and beta-carotene was carried out, as well as evaluation of the quality of saponins in Annona muricata L.<\/em> leaf extract. This procedure was carried out using a colorimetric method that is appropriate for each compound.<\/p>\n\n\n\n

Sample Preparation<\/strong><\/p>\n\n\n\n

Annona muricata L.<\/em> leaf simplicia to be analyzed was prepared by extracting it with ethanol solvent. This process is carried out to ensure that the desired phytochemical compounds are well dissolved.<\/p>\n\n\n\n

Quantitative<\/strong> Analysis with Colorimetric Method<\/strong><\/p>\n\n\n\n

After the extract is obtained, the quantity of phytochemical compounds is measured using various colorimetric methods, namely the phenol content is measured using the Folin-Ciocalteu method. Folin-Ciocalteu reagent is added to the ethanol extract, then the absorbance is measured to determine the phenol concentration. To measure flavonoids, the aluminum chloride method is used, where the extract is mixed with the reagent and the color change is measured spectrophotometrically. Tannin levels were determined using FeCl\u2083 reagent, where the extracts were mixed and the absorbance was measured to determine the concentration of tannins based on the formation of a color complex. The DCPIP (dichlorophenolindophenol) method was used to measure vitamin C levels, where the extracts were tested with DCPIP reagent and the change in absorbance was measured. Tocopherol levels were measured using a colorimetric method that is appropriate for assessing the concentration of tocopherol in the extract. The spectrophotometric method was used to measure the concentration of beta-carotene by assessing the absorbance at a certain wavelength. For saponins, quality testing was carried out using the Schales method, namely the ethanol extract solution was mixed with water to check for foam formation. Saponins have the ability to form stable foam. Observation of the amount and stability of the foam will provide an indication of the presence of saponins in the sample. <\/p>\n\n\n\n

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

After all measurements were carried out, data from the colorimetric method was analyzed to calculate the concentration of each phytochemical compound. The results of the saponin quality test will also be recorded to provide an overview of the presence of saponins in the extract.<\/p>\n\n\n\n

The antioxidant potential (IC50<\/sub>) of Annona muricata L.<\/em> leaf extract was measured using the FRAP method, which is the concentration of extract required to reduce 50% of free radical activity.<\/p>\n\n\n\n

Sample Preparation<\/strong><\/p>\n\n\n\n

Preparation of ethanol extract of Annona muricata L.<\/em> leaves<\/p>\n\n\n\n

Preparation of FRAP Reagent<\/strong><\/p>\n\n\n\n

This reagent consists of a mixture of acetic acid solution (pH 3.6), FeCl\u2083 solution, and TPTZ solution (2,4,6-tris(2-pyridyl)-s-triazine) in certain proportions. This mixture is needed to assess the reduction ability of the antioxidant compounds in the extract.<\/p>\n\n\n\n

Determination of Extract Concentration<\/strong><\/p>\n\n\n\n

Next, several different extract concentrations were determined, namely 100, 200, 400, 600, 800, and 1000 \u03bcg\/mL. These various concentrations will be tested to determine the antioxidant activity shown by the extract.<\/p>\n\n\n\n

Reaction Process<\/strong><\/p>\n\n\n\n

For each concentration that has been prepared, a certain amount of extract (for example 0.1 mL) is mixed with 2.9 mL of FRAP reagent solution that has been prepared previously. The mixture is then incubated at room temperature for 30 minutes, allowing the reaction between the compounds in the extract and the FRAP reagent to occur optimally.<\/p>\n\n\n\n

Absorbance Measurement<\/strong><\/p>\n\n\n\n

After the incubation process, the absorbance of each mixture is measured using a spectrophotometer at a wavelength of 593 nm. This measurement is carried out to determine how effective the extract is in reducing Fe\u00b3\u207a to Fe\u00b2\u207a, which is indicated by changes in absorbance.<\/p>\n\n\n\n

Determination of Antioxidant Activity<\/strong><\/p>\n\n\n\n

Antioxidant activity is calculated based on the observed absorbance changes. The lower the absorbance value measured, the higher the reduction activity shown by the compounds in the extract. Data from this measurement are very important for further analysis.<\/p>\n\n\n\n

IC50 <\/sub>Calculation<\/strong><\/p>\n\n\n\n

The absorbance data obtained from each concentration are\nevaluated to create a concentration versus percent activity curve. The IC50<\/sub>\nvalue was determined using regression analysis, namely the concentration of\nextract required to reduce free radical activity by 50%.<\/p>\n\n\n\n

Animal Study Design and Setting<\/strong><\/p>\n\n\n\n

This research was conducted on\nadult Wistar rats (Rattus norvegicus<\/em>) as experimental animals. There\nwere 45 experimental animals, divided into 15 experimental animals in each of 2\ncontrol groups (K+ and K-) and 1 treatment group (P). Groups K+ and P were\ngiven noise exposure of 95dB for 4 hours for 14 days. Group P was given ethanol\nextract of soursop leaves at a dose of 100mg\/kg body weight before being\nexposed to noise for 14 days. Meanwhile, the K+ group was given distilled\nwater.<\/p>\n\n\n\n

Animal Care and Treatment<\/strong><\/p>\n\n\n\n

The research samples were all\nmale Wistar strain white rats (Rattus norvegicus<\/em>) aged 2-3 months,\nweighing 100-150 grams, kept at the Integrated Laboratory of Universitas\nUdayana. Animals are kept and cared for by giving them food and water and\nkeeping their cages clean. Experimental animals were fed ad libitum, both on a\nhigh-fat diet and a standard diet. Drinking is also provided ad libitum, in the\nform of bottled water. Experimental animals were kept in groups in cages\nmeasuring 30x40x40 cm, which were made of wire and had sufficient ventilation.\nEach cage contains 5 experimental animals, where each cage is given a partition\nfor each experimental animal. Every three days the cage is cleaned of dirt and\nfood waste. Lighting is regulated with a cycle of 12 hours of light and 12\nhours of darkness (light cycle from 6 am to 6 pm) with a room temperature of 30\n\u00b1 10C. For 1 week the animals are tried to acclimate so they get used to living\nin this environment. The physical health of the test animal can be seen to\nensure the animal can make adjustments. Healthy experimental animals can be\nseen from open and clean eyes, smooth and shiny fur, active activity and good\nappetite.<\/p>\n\n\n\n

Groups K+ and P were exposed to a\nnoise stressor for 4 hours with a sound pressure level of 95 dB from Real-time\nanalyzer software version 5.2.0 (Yoshimasa Electronic Inc., Japan) connected to\na loudspeaker (Sony SRS XB30, Japan) for 14 days on a drum soundproof measuring\n100 x 100 cm. Group P was given Annona muricata<\/em> leaf extract 100mg\/kg\nbody weight orally once a day before noise exposure.  Several previous studies have shown that a\ndose of Annona muricata L.<\/em> extract of 100 mg\/kg body weight is effective\nin providing health benefits, such as increasing HDL, reducing various breast\ncancer markers, inhibiting the growth of prostate tumors, and reducing blood\nglucose and regenerating pancreatic beta cells 1<\/sup>,<\/sup>8<\/sup>,<\/sup>9<\/sup>,<\/sup>10<\/sup>. The acute toxicity of ethanol extract of Annona\nmuricata L.<\/em> leaves is more than 2000 mg\/kg. Previous studies with mice\nshowed this effect within 2 hours after administration and subsided after 4\nhours. The effects on the kidneys and liver were mild and insignificant,\nindicating that this extract is practically non-toxic 11<\/sup>,<\/sup>12<\/sup>. <\/p>\n\n\n\n

Rat Dissection and Brain Organ Removalal<\/strong><\/p>\n\n\n\n

On day 15, the rats were\nsacrificed by peritoneal injection of ketamine at a dose of 150 mg\/kgbb and the\nbrain organs were taken. Brain organs were fixed in 10% buffered formalin\nsolution in plastic containers that had been labeled according to the group and\nsample number of the experimental animal. After 8 hours (maximum 24 hours), the\nbrain organ is made into a histology preparation using the paraffin method.\nParaffin blocks were cut with a rotary microtome with a thickness of 4-5 \u03bcm and\nattached to glass objects, followed by hematoxylin and eosin staining.<\/p>\n\n\n\n

Measurement of Effects on Neurons in the Cerebellum<\/strong><\/p>\n\n\n\n

After Hematoxylin and Eosin (HE) staining, detailed cell morphology observations were carried out using a microscope (magnification 40x). Detailed morphological observations include identification of the layers of the cerebellum, namely the molecular layer, the Purkinje layer and the granular layer. In the molecular layer, observations were made on the density of nerve fibers (dendrites). Observations of Purkinje cells and granular layer were carried out for signs of degeneration in the cells. <\/p>\n\n\n\n

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

Phytochemical Analysis of Soursop Leaf Ethanol Extract<\/strong><\/p>\n\n\n\n

This extract is measured for its antioxidant content per\n100 grams. The contents measured were phenols, flavonoids, tannins,\nbeta-carotene, vitamin C, tocopherol and saponins (shown in Table 1).<\/p>\n\n\n\n

Table 1: Antioxidant content of soursop leaf ethanol extract<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Antioxidant content (per 100 gram)<\/strong><\/p>\n<\/td>\n

\n

Quantity\/Quality (mg)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Phenols<\/p>\n<\/td>\n

\n

4876,82<\/p>\n<\/td>\n<\/tr>\n

\n

Flavonoids<\/p>\n<\/td>\n

\n

847,23<\/p>\n<\/td>\n<\/tr>\n

\n

Tannins<\/p>\n<\/td>\n

\n

20822,09<\/p>\n<\/td>\n<\/tr>\n

\n

Beta-carotene<\/p>\n<\/td>\n

\n

4,1556<\/p>\n<\/td>\n<\/tr>\n

\n

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

\n

30402,1<\/p>\n<\/td>\n<\/tr>\n

\n

Tocopherol<\/p>\n<\/td>\n

\n

39850,7<\/p>\n<\/td>\n<\/tr>\n

\n

Saponins<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n

The results of measuring antioxidant capacity showed very\nstrong results, namely antioxidant capacity of 24,228.60 milligrams of gallic\nacid per liter of solution (24,228.60 mg\/L GAEAC). IC50<\/sub> measurement\nresults show moderate effectiveness in inhibiting certain targets, namely\n32.6230 ppm. In this study, various antioxidant contents were found in the\nethanol extract of soursop leaves, namely phenols, flavonoids, tannins,\nbeta-carotene, vitamin C, tocopherol and saponins.<\/p>\n\n\n\n

The ethanol extract of soursop leaves per 100 grams\nshowed a very high phenol content. The phenol content in other plants is\ngenerally 200-1500 mg 13<\/sup>. The ethanol extract of soursop leaves per 100 grams\nalso shows a very high flavonoid content. The flavonoid content in other plants\nis generally 50-300 mg 14<\/sup>,<\/sup>15<\/sup>. Tannins in the ethanol extract of soursop leaves in\nthis study were also found in very high levels. In general, the tannin content\nin plants is 50-3000 mg per 100 grams of plant 14<\/sup>,<\/sup>16<\/sup>,<\/sup>17<\/sup>. The beta-carotene content was found to be quite high in\nthis study. Beta-carotene in other vegetables and fruit is usually 0.4-10 mg\nper 100 grams 18<\/sup>,<\/sup>19<\/sup>. The results of this research show that the vitamin C\ncontent in the ethanol extract of soursop leaves shows extra-ordinary high\nresults. In other plants it is usually only 50-1677 mg per 100 grams 20<\/sup>. The tocopherol content in soursop leaf extract was also\nfound to be extraordinarily high. In other plants it is usually only 20-150 mg\nper 100 grams 21<\/sup>,<\/sup>22<\/sup>,<\/sup>23<\/sup>. In this study, qualitative saponin content was also\nfound in soursop leaf extract.<\/p>\n\n\n\n

Molecular Layer Morphology (Outermost Layer)<\/strong><\/p>\n\n\n\n

In the molecular layer, nerve fiber density was assessed\nvisually by comparing preparations in the control and treatment groups. The\nscoring system uses a value range of 0-3 with a value of 0 (normal) if the\nchange that occurs is <25%, a value of 1 (mild) if the change that occurs is\n25-50%, a value of 2 (moderate) if the change that occurs is 50-75%, and a\nvalue of 3 (severe) if the changes that occur are > 75%.  <\/p>\n\n\n\n

In this study, a significant difference was obtained in\nthe density of nerve fibers (dendrites) between the K- and K+ groups (p=0.000)\nand P (p=0.015). In addition, a significant difference was obtained between the\nK+ and P groups (p=0.015), as seen in Figure 1 and 2, and Table 2.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1:<\/strong>\u00a0Density of nerve fibers in the molecular layer<\/strong><\/p>\n

\u00a0<\/p>\n

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

\u00a0<\/p>\n\n\n

Table 2: Density of nerve fibers in the molecular layer<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Density of nerve fibers in the molecular layer<\/strong><\/p>\n<\/td>\n

\n

Frequency and percentage in each group<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

\u00a0<\/strong><\/p>\n<\/td>\n

\n

K-<\/strong><\/p>\n<\/td>\n

\n

K+<\/strong><\/p>\n<\/td>\n

\n

P<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

0%<\/p>\n<\/td>\n

\n

4 (26.7%)<\/p>\n<\/td>\n

\n

0 (0%)<\/p>\n<\/td>\n

\n

1 (6.6%)<\/p>\n<\/td>\n<\/tr>\n

\n

25%<\/p>\n<\/td>\n

\n

5 (33%)<\/p>\n<\/td>\n

\n

0 (0%)<\/p>\n<\/td>\n

\n

3 (20%)<\/p>\n<\/td>\n<\/tr>\n

\n

50%<\/p>\n<\/td>\n

\n

6 (40%)<\/p>\n<\/td>\n

\n

4 (26.7%)<\/p>\n<\/td>\n

\n

6 (40%)<\/p>\n<\/td>\n<\/tr>\n

\n

75%<\/p>\n<\/td>\n

\n

0 (0%)<\/p>\n<\/td>\n

\n

4 (26.7%)<\/p>\n<\/td>\n

\n

4 (26.7%)<\/p>\n<\/td>\n<\/tr>\n

\n

100%<\/strong><\/p>\n<\/td>\n

\n

0 (0%)<\/p>\n<\/td>\n

\n

7 (46.7%)<\/p>\n<\/td>\n

\n

1 (6.6%)<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n

In group K-, nerve fiber density was found to appear\nnormal to mild changes. In the K+ group, there was a change in nerve fiber\ndensity from mild to severe. While in the P group, the density of nerve fibers\nwas from normal to moderate, only 1 rat experienced a severe change in nerve\nfiber density. In the K- group preparation, the molecular layer of the\ncerebellum showed that the nerve fibers appeared dense and neatly structured.\nIn K+, there was a decrease in the number of nerve fibers, with areas that\nappeared emptier or “sparse”. In the P group, the decrease in nerve\nfibers was not clearly visible, the nerve fibers appeared denser and more\nneatly structured compared to the K+ group, as seen in Figure 2.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: The layers of the cerebellum, namely the molecular layer (M), the Purkinje layer (P) and the granular layer (G). a. Control group K-. b. Control group K+, nerve fiber density decreases (orange arrow). c. Treatment group P<\/strong><\/p>\n

\u00a0<\/p>\n

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

\u00a0<\/p>\n\n\n

Purkinje Layer Morphology (Middle Layer)<\/strong><\/p>\n\n\n\n

In the Purkinje layer, Purkinje cell degeneration were\nassessed. The scoring system uses a value range of 0-3 with a value of 0\n(normal) if the change that occurs is <25%, a value of 1 (mild) if the\nchange that occurs is 25-50%, a value of 2 (moderate) if the change that occurs\nis 50-75%, and a value of 3 (severe) if the changes that occur are > 75%.<\/p>\n\n\n\n

To assess Purkinje cell degeneration, an assessment is\nmade on nuclear changes such as karyorrhexis (nuclear fragmentation) or pyknosis\n(nuclear shrinkage). In addition, an assessment is made on signs of necrosis\nsuch as cell swelling, cell membrane disintegration, cell content leakage, and\ncells that appear blurry and less structured.<\/p>\n\n\n\n

In this study, a significant difference was obtained in\nPurkinje cell degeneration between the K- and K+ groups (p=0.000) and P\n(p=0.003). In addition, a significant difference was obtained between the K+\nand P groups (p=0.015), as seen in Figure\n3 and Table 3.<\/p>\n\n\n\n\n\n
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Figure 3: Purkinje cell degeneration<\/strong><\/p>\n

\u00a0<\/p>\n

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

\u00a0<\/p>\n\n\n

Table 3: Purkinje cell degeneration<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Purkinje cell degeneration<\/strong><\/p>\n<\/td>\n

\n

Frequency and percentage in each group<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

K-<\/strong><\/p>\n<\/td>\n

\n

K+<\/strong><\/p>\n<\/td>\n

\n

P<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

0%<\/p>\n<\/td>\n

\n

5 (33%)<\/p>\n<\/td>\n

\n

0 (0%)<\/p>\n<\/td>\n

\n

0 (0%)<\/p>\n<\/td>\n<\/tr>\n

\n

25%<\/p>\n<\/td>\n

\n

5 (33%)<\/p>\n<\/td>\n

\n

0 (0%)<\/p>\n<\/td>\n

\n

4 (26.7%)<\/p>\n<\/td>\n<\/tr>\n

\n

50%<\/p>\n<\/td>\n

\n

5 (33%)<\/p>\n<\/td>\n

\n

4 (26.7%)<\/p>\n<\/td>\n

\n

6 (40%)<\/p>\n<\/td>\n<\/tr>\n

\n

75%<\/p>\n<\/td>\n

\n

0 (0%)<\/p>\n<\/td>\n

\n

7 (46.7%)<\/p>\n<\/td>\n

\n

3 (20%)<\/p>\n<\/td>\n<\/tr>\n

\n

100%<\/p>\n<\/td>\n

\n

0 (0%)<\/p>\n<\/td>\n

\n

4 (26.7%)<\/p>\n<\/td>\n

\n

2 (13%)<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

In the K- group, only four out of 15 rats experienced\ndegeneration in Purkinje cells, from mild to moderate. In the K+ group, most\nsamples experienced moderate to severe Purkinje cell degeneration. In the P\ngroup, most samples experienced mild to moderate stage, only two rats\nexperienced severe degeneration. The differences in each group can be seen in Figure 2 and 4.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 4: In the K+ group, the degeneration process in Purkinje cells is visible (orange arrow).<\/strong><\/p>\n

\u00a0<\/p>\n

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

\u00a0<\/p>\n\n\n

Granular layer morphology (innermost\nlayer)<\/strong><\/p>\n\n\n\n

In the granular layer, granular cell degeneration were\nassessed. The scoring system uses a value range of 0-3 with a value of 0\n(normal) if the change that occurs is <25%, a value of 1 (mild) if the\nchange that occurs is 25-50%, a value of 2 (moderate) if the change that occurs\nis 50-75%, and a value of 3 (severe) if the changes that occur are > 75%.<\/p>\n\n\n\n

Granular cell degeneration is assessed by looking for\nsigns of degeneration such as karyorrhexis (nuclear fragmentation) or pyknosis\n(nuclear shrinkage). In addition, an assessment is made on signs of necrosis\nsuch as cell swelling, cell membrane disintegration, cell content leakage, and\ncells that appear blurry and less structured.<\/p>\n\n\n\n

In this study, there was no significant difference in\ngranular cell degeneration between the K- and K+ groups (p=0.061) and P\n(p=0.838), as well as between K+ and P (p=0.094) Figures 5 and Table 4.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 5. Granular cell degeneration<\/strong><\/p>\n

\u00a0<\/p>\n

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

\u00a0<\/p>\n\n\n

Table 4: Granular cell degeneration<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Granular cell degeneration<\/strong><\/p>\n<\/td>\n

\n

Frequency and percentage in each group<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

K-<\/strong><\/p>\n<\/td>\n

\n

K+<\/strong><\/p>\n<\/td>\n

\n

P<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

0%<\/p>\n<\/td>\n

\n

9 (60%)<\/p>\n<\/td>\n

\n

4 (26.7%)<\/p>\n<\/td>\n

\n

8 (53%)<\/p>\n<\/td>\n<\/tr>\n

\n

25%<\/p>\n<\/td>\n

\n

1 (6.6%)<\/p>\n<\/td>\n

\n

2 (13%)<\/p>\n<\/td>\n

\n

3 (20%)<\/p>\n<\/td>\n<\/tr>\n

\n

50%<\/p>\n<\/td>\n

\n

4 (26.7%)<\/p>\n<\/td>\n

\n

5 (33%)<\/p>\n<\/td>\n

\n

2 (13%)<\/p>\n<\/td>\n<\/tr>\n

\n

75%<\/p>\n<\/td>\n

\n

1 (6.6%)<\/p>\n<\/td>\n

\n

2 (13%)<\/p>\n<\/td>\n

\n

0 (0%)<\/p>\n<\/td>\n<\/tr>\n

\n

100%<\/p>\n<\/td>\n

\n

0 (0%)<\/p>\n<\/td>\n

\n

2 (13%)<\/p>\n<\/td>\n

\n

2 (13%)<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

In the K- group, only six out of 15 rats experienced\ngranular cell degeneration, from mild to moderate. In the K+ group, most\nsamples experienced granular cell degeneration from moderate to severe. In the\nP group, granular cell degeneration occurred at a mild to moderate stage, only\ntwo rats experienced severe degeneration. The differences in each group can be\nseen in Figures 5 and 6.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 6: In the K+ group, the degeneration process in granular cells is visible (orange arrow)<\/strong><\/p>\n

\u00a0<\/p>\n

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

\u00a0<\/p>\n\n\n

This study showed that noise exposure caused a\ndecrease in nerve fiber density in the K+ group. The group receiving soursop\nleaf extract (P) showed a non-significant decrease in nerve fiber density\ncompared to the group not receiving the extract, which was seen from the\nsignificant difference in the K+ and P groups in nerve fiber density. The\ndensity of nerve fibers in the molecular layer of the cerebellum indicates the\ndensity or number of synaptic connections in the layer, especially between\ngranular cell axons and Purkinje cell dendrites. The molecular layer serves as\na major signal integration area, where granular cell nerve fibers meet Purkinje\ncell dendrites and climbing fibers from the inferior olivary nucleus. Higher\ndensity indicates increased synaptic connectivity and neural activity in the\nprocess of motor coordination and motor learning. However, if the density of\nnerve fibers in this molecular layer decreases, it indicates nerve damage or\ndegeneration, which is likely caused by exposure to various factors such as\noxidative stress, noise, or other neurotoxic agents. This can lead to impaired\nmotor function and balance, because disrupted synaptic connectivity will affect\nsignal integration in the cerebellum 24<\/sup>,<\/sup>25<\/sup>.<\/p>\n\n\n\n

The results of this study also showed more\nsevere degeneration in Purkinje cells and granular cells in the group that did\nnot receive soursop leaf extract. Degeneration of cerebellar Purkinje cells\nindicates damage or loss of the main cells responsible for regulating\ncerebellar output and motor coordination. Purkinje cells are very large neurons\nwith extensively branched dendrites, which receive input from granular cell\nfibers and climbing fibers from the inferior olivary nucleus, which control\nsignals that exit the cerebellum to other brain structures. If Purkinje cells\ndegenerate, it can cause disturbances in motor coordination and balance,\nespecially fine and coordinated movements, decreased motor learning capacity\nthrough plasticity mechanisms, and dysfunction in signal processing and\nintegration, because Purkinje cells are the main signal receivers in the\ncerebellum. Degeneration of Purkinje cells can be caused by various conditions\nsuch as exposure to toxins, oxidative stress, infections and neurodegenerative\ndiseases, and genetics 26<\/sup>,<\/sup>27<\/sup>.<\/p>\n\n\n\n

Continuous noise exposure can trigger oxidative\nstress. Oxidative stress is a condition in which there is an imbalance between\nthe production of free radicals and the body’s ability to neutralize them. This\noxidative stress can damage cell membranes, proteins, and DNA, which can then\naffect nerve cell function. High noise can increase free radical production.\nThis contributes to nerve cell death through apoptosis (programmed cell death)\nand necrosis (unprogrammed cell death) 28<\/sup>. Cells in the cerebellum layer are particularly\nsusceptible to damage from oxidative stress. Previous research on Wistar rats\nshowed that exposure to high levels of noise can cause morphological damage to\nseveral brain areas, which can lead to impaired motor coordination and balance.\n29<\/sup>. <\/p>\n\n\n\n

Excessive exposure to noise will cause the body\nto respond by releasing stress hormones such as cortisol and adrenaline. This\nprocess can increase the production of free radicals, which are reactive\nmolecules that can damage cells. Free radicals are atoms or molecules that have\nunpaired electrons, making them highly reactive and can damage cellular\ncomponents. Increased free radicals can occur through several mechanisms, such\nas inflammation and mitochondrial dysfunction. Increased stress hormones such\nas cortisol can affect the immune system and increase the production of\npro-inflammatory cytokines. Immune cells that are activated in the inflammatory\nprocess can produce free radicals as part of the body’s defense response 30<\/sup>.<\/p>\n\n\n\n

Noise can also affect the function of\nmitochondria, which act as the main source of energy production in cells. When\nmitochondria are compromised, they produce more free radicals as a byproduct of\nenergy metabolism. Excessive ROS will disrupt mitochondrial function, including\nATP production and regulation of cell metabolism 31<\/sup>. Previous research in mice showed that chronic\ninflammation due to noise can also cause mitochondrial dysfunction.\nMitochondrial disorders can also reduce the activity of antioxidant enzymes,\nsuch as superoxide dismutase (SOD). With the reduction of these enzymes, the\nability of cells to neutralize ROS will decrease, thereby increasing the\naccumulation of free radicals 32<\/sup>. <\/p>\n\n\n\n

This study showed that rats that received\nsoursop leaf extract protection before being exposed to noise experienced a\nsmaller decrease in nerve fiber density compared to rats that did not receive\nextract protection. The degeneration process in Purkinje cells and granular\ncells was also lower in rats that received soursop leaf extract protection.\nThis protection can be mediated by phenols, flavonoids, tannins, beta-carotene,\nvitamin C, tocopherol, saponins contained in this soursop leaf extract.<\/p>\n\n\n\n

Soursop leaves are part of the soursop tree\nplant (Annona muricata L.<\/em>), growing in tropical regions such as\nSoutheast Asian countries. Soursop leaves are widely used in traditional\nmedicine by the community. The contents of soursop leaf extract such as\nalkaloids, phenols, flavonoids, tannins, saponins and acetogenin provide health\nbenefits such as anticancer, antimicrobial, antioxidant and anti-inflammatory 17<\/sup>,<\/sup>33<\/sup>,<\/sup>34<\/sup>,<\/sup>35<\/sup>,<\/sup>36<\/sup>. <\/p>\n\n\n\n

Phenol is a strong antioxidant and can\nneutralize free radicals thereby reducing oxidative stress in the body 13<\/sup>. Phenol can also reduce inflammation in the\nbody. Phenolic compounds are absorbed mainly in the small intestine, but their\nbioavailability varies due to metabolism by intestinal enzymes and gut\nmicrobiota. After metabolism, they are excreted in the urine, and to a lesser\nextent in the bile and feces 37<\/sup>. <\/p>\n\n\n\n

Like phenols, flavonoids are also compounds that\nhave strong antioxidant properties so they can neutralize free radicals and\nreduce oxidative stress in the body. Flavonoids can also inhibit enzymes and\nmolecular pathways that play a role in the inflammatory process 14<\/sup>,<\/sup>16<\/sup>,<\/sup>17<\/sup>. Flavonoids are metabolized in the liver and\nintestine, with limited absorption in the small intestine. Some flavonoids are\nfurther metabolized by the gut microbiota to produce more absorbable compounds.\nFlavonoids and their metabolites are excreted primarily in the urine. Some\nflavonoids are also excreted in the bile and excreted in the feces 38<\/sup>.<\/p>\n\n\n\n

The tannins contained in this extract also have\nthe same benefits as phenols and flavonoids 14<\/sup>,<\/sup>16<\/sup>,<\/sup>17<\/sup>.  Tannin\ncompounds are large and complex in size, so that little can be absorbed and\nmost of them undergo hydrolysis in the intestine by microbiota, then producing\nsimpler phenolic compounds that can be absorbed. Excretion of metabolite\nproducts from tannins is generally excreted through urine and feces 39<\/sup>.<\/p>\n\n\n\n

Beta-carotene also has strong antioxidant\ncapabilities so it can also protect the body from oxidative stress 40<\/sup>. Beta-carotene is a precursor of vitamin A and\nis absorbed in the small intestine with the help of dietary fat. After being\nconverted to vitamin A in the liver, some beta-carotene is stored in fat\ntissue. Beta-carotene is excreted mainly in the feces, mainly in unabsorbed\nform, while vitamin A is excreted in the urine 41<\/sup>.<\/p>\n\n\n\n

Vitamin C is also a strong antioxidant so it can\nprotect cells from damage caused by free radicals 20<\/sup>. Vitamin C can be absorbed in the small\nintestine through active transport and passive diffusion. Its bioavailability\nis quite high until it reaches saturation, after which its excretion increases.\nUnused vitamin C is excreted in the urine. Excretion of this vitamin can\nincrease as the concentration in plasma increases beyond the body’s needs 42<\/sup>.<\/p>\n\n\n\n

Tocopherol provides health benefits because it\nis also a strong antioxidant 21<\/sup>,<\/sup>22<\/sup>,<\/sup>23<\/sup>. Tocopherol (vitamin E) is fat-soluble and can\nbe absorbed in the small intestine with dietary fat, then transported in the\nlymphatic system. The absorption process involves micelles and lipoproteins.\nThis vitamin is excreted mainly through bile and then into the feces. A small\nportion of the metabolite products of this vitamin can also be excreted through\nurine 43,44<\/sup>.<\/p>\n\n\n\n

Saponins are also beneficial for health. Saponin\nalso acts as an antioxidant and can inhibit cancer proliferation and trigger\napoptosis 45<\/sup>. Lastly, saponins can reduce inflammation\nbecause they can inhibit inflammatory mediators such as prostaglandins and\ninflammatory cytokines 46<\/sup>. Saponins are generally poorly absorbed due to\ntheir large and complex molecules, but some types of saponins can be hydrolyzed\nby intestinal enzymes or microbiota into more easily absorbed aglycones.\nUnabsorbed saponins and their metabolites are excreted primarily through feces 47<\/sup>.<\/p>\n\n\n\n

Based on previous research, it was found that\nsoursop leaf extract can help protect body cells from oxidative damage caused\nby free radicals due to the high phenolic content of this extract 17<\/sup>. Previous in vitro research using\nlipopolysaccharide-induced cells showed that soursop leaf extract also had\nanti-inflammatory effects on macrophages stimulated by lipopolysaccharide. This\nis supported by the results of other literature studies 16<\/sup>,<\/sup>35<\/sup>,<\/sup>48<\/sup>.<\/p>\n\n\n\n

The antioxidant capacity of the ethanol extract\nof soursop leaves is also very high. In other plants, several hundred to 20,000\nmg\/L GAEAC is usually obtained. This of course supports high protection against\nfree radicals which contribute to aging and the development of various\nchronic  diseases. High antioxidant\ncapacity can also reduce the risk of heart disease, prevent cancer, and\nstrengthen immunity 49<\/sup>,<\/sup>50<\/sup>,<\/sup>51<\/sup>,<\/sup>52<\/sup>,<\/sup>53<\/sup>.<\/p>\n\n\n\n

This research shows that the ethanol extract of\nsoursop leaves has a fairly good IC50<\/sub>. IC50<\/sub> (Inhibitory\nConcentration 50) is a measurement used as an indicator of the effectiveness of\na compound in inhibiting certain biological or biochemical processes. Herbal\nstudies show that if a compound has an IC50<\/sub> between 20-50 ppm (parts\nper million) it is categorized as having moderate to high antioxidant\neffectiveness. With an IC50<\/sub> of 32.6230 ppm, it shows that the\ncompounds in this extract have a good ability to inhibit free radicals and help\nprotect cells from oxidative damage. Apart from that, it can help improve cell\nhealth by reducing oxidative stress and inflammation which contribute to aging\nand various degenerative diseases. It can be concluded that the compounds in\nthis extract have good antioxidant potential for use in the formulation of\nantioxidant supplements, skin care products, and drug development for diseases\nrelated to oxidative stress 46<\/sup>,<\/sup>53<\/sup>,<\/sup>54<\/sup>,<\/sup>55<\/sup>.<\/p>\n\n\n\n

Previous research shows that soursop leaf\nextract has a protective effect against oxidative damage to neurons 56<\/sup>. Another study on rats that experienced nerve\ninjury due to radiation, also found that soursop leaf extract could reduce\ninflammation in the brain, which is the main risk factor for neuron damage.\nThis shows that soursop leaf extract can maintain the integrity of neurons in\nthe brain, including the cerebellum 57<\/sup>.<\/p>\n\n\n\n

Soursop leaf extract has neuroprotective\npotential, because this extract contains bioactive compounds that can protect\nneurons from oxidative stress, including inflammation 56<\/sup>. Soursop leaf extract has the ability to\nneutralize free radicals and reduce inflammation at the cellular level, thereby\nhaving a positive effect on the health of neurons, including the cerebellum 58<\/sup>.<\/p>\n\n\n\n

The content of anonaceous acetogenins can\ninfluence various cellular signaling pathways, such as activation of the\nPI3K\/Akt pathway which plays an important role in the regulation of cell\nsurvival, cell growth and metabolism. Activation of this pathway will protect\nneurons from apoptosis and oxidative stress 59<\/sup>. Another signaling pathway that can be\ninfluenced is by modulating the NF-\u03baB pathway involved in inflammatory\nresponses and cell survival. Apart from that, the content of acetogenin,\nalkaloids and annonaceous flavonoids contained in this extract can also\ninfluence the Mitogen-Activated Protein Kinase\/Extracellular Signal-Regulated\nKinase (MAPK\/ERK) pathway which plays a role in cell proliferation,\ndifferentiation and response to stress, so that contributes to neuroprotective\neffects by reducing apoptosis and increasing neuronal survival 56<\/sup>,<\/sup>60<\/sup>.<\/p>\n\n\n\n

The flavonoid content such as quercetin and\nkaempferol in soursop leaf extract has a strong antioxidant effect so it can\nreduce oxidative stress. Kaempferol can also inhibit NF-\u03baB which plays a role\nin inflammatory responses and apoptosis. By inhibiting NF-\u03baB activity, it will\nreduce the expression of pro-inflammatory genes and cytokines, thereby reducing\ninflammation and protecting neurons from damage due to inflammation 61<\/sup>. The flavonoid mechanism for reducing oxidative\nstress is by activating the Nrf2 pathway which is able to regulate the\nexpression of antioxidant enzymes 62<\/sup>. Research in mice shows that flavonoids have\nneuroprotective abilities from neurotoxins by increasing dopamine and serotonin\nlevels in the striatum, inhibiting oxidative stress, and the response of\nastroglia 63<\/sup>.<\/p>\n\n\n\n

Soursop leaf extract also contains saponins\nwhich have anti-inflammatory and antioxidant effects. Saponins can neutralize\nfree radicals, thereby helping protect neurons from damage and apoptosis.\nSaponins are also able to inhibit inflammatory pathways such as NF-\u03baB and\nreduce the production of pro-inflammatory cytokines, thereby reducing\ninflammation in neurons 64<\/sup>. Previous in vitro studies have shown that\nsaponins have neuroprotective effects on spinal cord neurons 65<\/sup>. <\/p>\n\n\n\n

Beta-carotene is a type of carotenoid, also\ncontained in this extract. Beta-carotene can neutralize free radicals and\noxidative stress in neurons, thereby preventing damage to cell membranes,\nproteins and DNA in neurons. Beta-carotene is also a precursor of vitamin A\nwhich is important for the normal function of the nervous system, where this\nvitamin plays a role in the regulation of gene expression related to the growth\nand development of neurons 66<\/sup>. Previous studies in rats have shown that\nbeta-carotene reduces the progression of nerve injury through inhibition of\nNF-\u03baB activation 67<\/sup>.<\/p>\n\n\n\n

The ability of soursop leaf extract to\nprovide neuroprotective effects can be linked to its ability to penetrate the\nblood brain barrier (BBB). However, research on the ability of soursop leaf\nextract to penetrate the BBB is still limited. There are several factors in\nthis extract that can affect its ability to penetrate the BBB, namely bioactive\ncomponents and molecular size. Smaller and lipophilic compounds contained in\nthe extract such as acetogenins, flavonoids, tannins, and phenols, have a more\nactive transport mechanism, so they are more likely to penetrate the BBB. In\naddition, other factors such as compounds require metabolism first to become\nsmaller molecules to be able to penetrate the BBB. This shows that not all\ncompounds in this extract are able to penetrate the BBB, but there is a\nmechanism of compounds in this extract that can produce neuroprotective effects\nwithout having to penetrate directly into the BBB, namely its systemic effects\nthrough reducing oxidative stress and inflammation throughout the body,\nstimulation of signaling pathways in the periphery that can affect brain\nfunction, and through metabolites that can penetrate the BBB 68<\/sup>,<\/sup>69<\/sup>,<\/sup>70<\/sup>.<\/p>\n\n\n\n

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

Ethanol\nextract of soursop leaves can provide neuroprotection against chronic noise\nexposure. In this study, it was proven that the content of ethanol extract of\nsoursop leaves in the form of phenols, flavonoids, tannins, beta-carotene,\nvitamin C, tocopherol, and saponins with moderate to high antioxidant\neffectiveness can provide protection for neurons in the cerebellum of rats\nexposed to noise. <\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

The author would like to thank the laboratory\nteam who were very persistent in helping the author during the implementation\nat the integrated laboratory at Universitas Udayana, as well as to all\nparticipants involved in completing this research. <\/p>\n\n\n\n

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

The author(s) received no financial support for the\nresearch, authorship, and\/or publication of this article <\/p>\n\n\n\n

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

The author(s) do not have any conflict of interest <\/p>\n\n\n\n

Data Availability Statement<\/strong><\/p>\n\n\n\n

This statement does not apply to this article.<\/p>\n\n\n\n

Ethics<\/strong> Statement<\/strong> <\/p>\n\n\n\n

The research\nprotocol has been approved by the Research Ethics Committee, Faculty of\nMedicine, Udayana University, Denpasar, Indonesia with approval number\n1072\/UN14.2.2.VII.14\/LT\/2024 for phytochemical testing of soursop leaf extract,\nand approval number 1397\/UN14.2.2.VII.14\/LT\/2024 for research with experimental\nanimals.<\/p>\n\n\n\n

Informed\nConsent Statement<\/strong><\/p>\n\n\n\n

This study did not involve human participants, and\ntherefore, informed consent was not required<\/p>\n\n\n\n

Clinical\nTrial Registration <\/strong><\/p>\n\n\n\n

This research does not involve any clinical trials<\/p>\n\n\n\n

Author Contributions<\/strong><\/p>\n\n\n\n

Dewa Ayu Agung Alit Suka Astini: Conceptualization, Methodology, Writing \u2013 Original Draft.<\/p>\n\n\n\n

I Wayan Putu Sutirta Yasa: Acquisition, Resources, Supervision.<\/p>\n\n\n\n

I Made Jawi: Acquisition, Resources, Supervision.<\/p>\n\n\n\n

I Nyoman Wande: Writing \u2013 Review & Editing.<\/p>\n\n\n\n

Putu Indah Budi Apsari: Writing \u2013 Review & Editing.<\/p>\n\n\n\n

Luh Gde Evayanti: Data Collection, Analysis.<\/p>\n\n\n\n

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

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