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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39294162
66781
10.1038/s41598-024-66781-9
Article
Unveiling Lobophytum sp. the neuroprotective potential of Parkinson's disease through multifaceted mechanisms, supported by metabolomic analysis and network pharmacology
Bakhsh Hussain T. 1
Abu-Baih Dalia H. 23
Abu-Baih Rania H. 4
Saber Entesar A. 5
Altemani Faisal H. 6
Algehainy Naseh A. 6
Alanazi Mohammad A. 6
Mokhtar Fatma Alzahraa 78
Bringmann Gerhard gerhard.bringman@uni-wuerzburg.de

9
Abdelmohsen Usama Ramadan usama.ramadan@mu.edu.eg

310
http://orcid.org/0000-0002-5554-2064
El-Mordy Fatma Mohamed Abd fatmamohammed.pharmg@azhar.edu.eg

11
1 https://ror.org/02ma4wv74 grid.412125.1 0000 0001 0619 1117 Department of Pharmacy Practice, Faculty of Pharmacy, King Abdulaziz University, 21589 Jeddah, Saudi Arabia
2 https://ror.org/05252fg05 Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Deraya University, New Minia City, Minia 61111 Egypt
3 https://ror.org/05252fg05 Deraya Center for Scientific Research, Deraya University, New Minia City, Minia 61111 Egypt
4 https://ror.org/02hcv4z63 grid.411806.a 0000 0000 8999 4945 Drug Information Center, Faculty of Pharmacy, Minia University, Minia, Egypt
5 https://ror.org/05252fg05 Department of Medical Science, Histology and Cell Biology, Faculty of Pharmacy, Deraya University, New Minia City, Minia 61111 Egypt
6 https://ror.org/04yej8x59 grid.440760.1 0000 0004 0419 5685 Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, University of Tabuk, 71491 Tabuk, Saudi Arabia
7 Fujairah Research Centre, Sakamkam Road, Fujairah, United Arab Emirates
8 Department of Pharmacognosy, Faculty of Pharmacy, El Saleheya El Gadida University, El Saleheya El Gadida, Sharkia 44813 Egypt
9 https://ror.org/00fbnyb24 grid.8379.5 0000 0001 1958 8658 Institute of Organic Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
10 https://ror.org/02hcv4z63 grid.411806.a 0000 0000 8999 4945 Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia, 61519 Egypt
11 https://ror.org/05fnp1145 grid.411303.4 0000 0001 2155 6022 Department of Pharmacognosy and Medicinal Plants, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, 11754 Egypt
18 9 2024
18 9 2024
2024
14 218109 1 2024
3 7 2024
© The Author(s) 2024
2024
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A main feature of neurodegenerative diseases is the loss of neurons. One of the most prevalent neurodegenerative illnesses is Parkinson disease (PD). Although several medications are already approved to treat neurodegenerative disorders, most of them only address associated symptoms. The main aim of the current study was to examine the neuroprotective efficacy and underlying mechanism of Lobophytum sp. crude extract in a rotenone-induced rat model of neurodegeneration mimicking PD in humans. The influence of the treatment on antioxidant, inflammatory, and apoptotic markers was assessed in addition to the investigation of TH (tyrosine hydroxylase) immunochemistry, histopathological changes, and α-synuclein. Metabolomic profiling of Lobophytum sp. crude extract was done by using High-Resolution Liquid Chromatography coupled with Mass Spectrometry (HR-LC–ESI–MS), which revealed the presence of 20 compounds (1–20) belonging to several classes of secondary metabolites including diterpenoids, sesquiterpenoids, steroids, and steroid glycosides. From our experimental results, we report that Lobophytum sp. extract conferred neuroprotection against rotenone-induced PD by inhibiting ROS formation, apoptosis, and inflammatory mediators including IL-6, IL-1β, and TNF-α, NF-кB, and subsequent neurodegeneration as evidenced by decreased α-synuclein deposition and enhanced tyrosine hydroxylase immunoreactivity. Moreover, a computational network pharmacology study was performed for the dereplicated compounds from Lobophytum sp. using PubChem, SwissTarget Prediction, STRING, DisGeNET, and ShinyGO databases. Among the studied genes, CYP19A1 was the top gene related to Parkinson’s disease. Dendrinolide compounds annotated a high number of parkinsonism genes. The vascular endothelial growth factor (VEGF) pathway was the top signaling pathway related to the studied genes. Therefore, we speculate that Lobophytum sp. extract, owing to its pleiotropic mechanisms, could be further developed as a possible therapeutic drug for treating Parkinson's disease.

Keywords

Lobophytum sp.
Neurodegenerative diseases
Parkinson's disease
Rotenone (ROT)
Network pharmacology
VEGF pathway
Subject terms

Biochemistry
Computational biology and bioinformatics
Drug discovery
Neuroscience
Plant sciences
Chemistry
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Parkinsonism is the general word used to describe a variety of neurological issues. It is also known as atypical Parkinson's disease or Parkinson's plus. Many factors can contribute to parkinsonism, such as drug side effects, persistent head injuries, metabolic disorders, pollutants, and neurological conditions1. Nevertheless, there are more than a dozen distinct types of parkinsonism, the most typical forms of Parkinson's disease are parkinsonism brought on by drugs (DIP), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and vascular parkinsonism2. Movement stiffness, sluggishness, trembling, and difficulty of moving are the most obvious symptoms3. For more than a century, a variety of medications have been used to treat parkinsonism, but their meager therapeutic success heavily depends on their central parasympatholytic actions4.

Oceanic biodiversity makes marine species an amazing source of secondary metabolites which exhibit a wide range of pharmacological activities. The soft corals of the genus Lobophytum (phylum Cnidaria, class Anthoza, subclass Octocorallia, order Alcyonacea, family Alcyoniidae) are a significant group of marine invertebrates that are found in coral reefs all over the world and are a good source of several secondary metabolites with distinctive structures and potential bioactivity5,6. Lobophytum sp. are distinguished by a wide range of colors, shapes, and sizes. These organisms are well recognized for producing a variety of terpenoid derivatives (mostly diterpenoids), some of which have distinctive skeletal structures and powerful biological activities7. In addition to diterpenes, triterpenoids, steroids, tocopherols, lipids, and zoanthamine-type alkaloids are all abundant in this soft coral, and they exhibit a wide range of biological functions and structural diversity6. The genus Lobophytum shows a range of pharmacological properties, including anti-inflammatory, neutrophil elastase-release activity, antiviral, HIV-inhibitory, antibacterial, cytotoxicity, antiarthritic, antidiabetic, and antiosteoporosis potential6,8,9.

The current study aimed to examine the metabolomic profiling of Lobophytum sp. soft corals using LC-HR-ESI-MS and determine whether it might be used to treat parkinsonism by examining the neuroprotective efficacy and underlying mechanism of Lobophytum sp. in a rotenone (ROT) induced rat model of neurodegeneration mimicking PD in humans. The influence of the treatment on antioxidant, inflammatory, and apoptotic markers was assessed in addition to the investigation of tyrosine hydroxylase immunochemistry and histopathological changes. Moreover, a computational network pharmacology study was performed using different databases.

Results

Metabolomic profiling of Lobophytum sp.

Metabolomic profiling of Lobophytum sp. crude extract was done by using LC-ESI-HR-MS, which revealed the presence of a panel of several metabolites, including diterpenoids, sesquiterpenoids, steroids, and steroid glycosides. LC-HR-ESI–MS dereplication led to the identification of 20 compounds (Table 1). The exact masses of these metabolites were compared to those found in databases, such as the Dictionary of Natural Products (DNP) and Metabolite and Chemical Entity (METLIN), in order to identify them. Table 1 Tentatively identified secondary metabolites from Lobophytum sp. using LC-HR-ESI–MS.

No.	Compound name	Molecular formula	RT (min)	Ionization (ESI+/ESI−)	Molecular weight	Observed (m/z)	Ref.	
1	Nardol	C15H26O	3.5970	[M+H]+	222.1983	223.1981	10	
2	Lobophytolide F	C24H32O6	6.1859	[M+H]+	416.2198	417.2196	11	
3	Pregnedioside A	C26H42O6	6.2038	[M−H]−	450.2963	449.2961	12	
4	Lobophytol	C20H28O4	6.2166	[M−H]−	332.1987	331.1981	13	
5	Alismoxide	C15H26O2	6.7678	[M−H]−	238.1932	237.1934	10	
6	Denticulatolide	C22H30O6	7.3301	[M−H]−	390.2042	389.2044	14	
7	Durumhemiketalolide B	C22H30O6	7.4550	[M−H]−	390.2042	389.2040	15	
8	Chalinasterol	C28H46O	7.5001	[M+H]+	398.3540	399.3504	10	
9	Durumolide P	C21H32O5	8.1299	[M+H]+	364.2241	365.2243	16	
10	Lobophytol A	C29H50O	9.6505	[M+H]+	414.3861	415.3853	17	
11	Lobocrassin B	C20H30O3	10.074	[M+H]+	318.2194	319.2198	18	
12	Cembrene A	C20H32	11.2029	[M−H]−	272.2504	271.2507	10	
13	Dendrinolide	C21H32O3	11.6346	[M+H]+	332.2351	333.2352	19	
14	Lobophytrol B	C20H36O4	11.7204	[M−H]−	340.2613	339.2614	6	
15	Nephalsterol C	C30H48O4	11.8611	[M−H]−	472.3552	471.3552	10	
16	Lobocrassin D	C22H34O3	12.1638	[M+H]+	346.2507	347.2507	18	
17	Lobophytolide A	C20H28O2	12.2356	[M−H]−	300.2089	299.2088	20	
18	Lobophytosterol	C28H46O4	12.3222	[M−H]−	446.3396	445.3398	21	
19	Lobophysterol C	C29H50O5	13.7442	[M−H]−	478.3674	477.3601	22	
20	Depresosterol	C28H48O4	15.2692	[M+H]+	448.3552	449.3553	21	
RT: retention time; min: minute.

Acute toxicity study

Several concentrations of the extract were used for the acute toxicity study up to 5000 mg/kg b. wt. No mortality and no toxicity signs were observed up to 5000 mg/kg over 24 hr so the LD50 was shown to be higher than 5000 mg/kg. The selected dose used was 40 mg/kg b. wt.

Histological examination

Group I

Corpus striatum (CS)

Using hematoxylin and eosin stain (H&E), the brain tissue "corpus striatum" had a striped appearance which mainly formed of a cluster of interconnected neurons, and a consequence of diverging fibers. The cellular contents were predominantly medium sized spiny projection neurons, with the remaining several types of interneurons. Their cytoplasm was moderate to intense basophilic (Fig. 1A) with large pale nearly rounded nuclei. Neurons appeared multipolar or bipolar, with infrequently branching dendrites. Intercellular neuropil showed neuroglia cells that appeared smaller and darker than other neurons and had dense nuclei (Fig. 1A).Figure 1 A photo-micrograph showing the brain tissue (corpus striatum, CS) of all studied groups: Group I, (A); Control Group; showing cluster of branched multipolar neurons (thick arrows) with moderate-dense basophilic cytoplasm and large pale nuclei. Normal blood vessels (arrows) and slight neuroglia cells infiltration (g). (B1,2,3); Parkinsonism Group; showing many neurons appear shrunken surrounded by perineuronal hallows (tailed arrows). Notice the increased interstitial spaces, dilated blood vessels (arrows) and infiltration by excess neuroglia cells (g). The neuropil has many vacuoles (V) and acidophilic aggregation of Lewy bodies (striped arrow) and gliosis (star) and a group of macrophages with pigmented material (macrophages). Group III, (C); (Lobophytum-treated Group); showing neuronal normal picture with intense basophilic cytoplasm and vesicular nuclei (arrows). Group IV, (D); (Rotenone + Lobophytum-treated Group); showing less apoptotic neuronal cells but excess infiltration of neuroglia cells (g). Some cells still shrunken and surrounded with empty hallows (arrows) (H&E staining ×200 and 400).

Substantia nigra pars compacta (SNC)

The substantia nigra pars compacta (SNC) appeared as a tiny cluster of closely packed neurons that had variable sizes and shapes. Their cytoplasm showed moderate to intense basophilia besides black cells, which were the neuro-melanin-containing dopaminergic neurons (Fig. 2A).Figure 2 A photo-micrograph of the brain tissue (substantia nigra pars compacta, SNC) of all studied groups, to show a closer view of the darkly pigmented dopaminergic neurons. GI, (A) Control Group; showing cluster of closely packed neurons (red arrows) and black cells are the neuromelanin-containing dopaminergic neurons (yellow arrows). Group II, (B) Parkinsonism Group; showing severe loss of dopaminergic cells. Cells showing prominent aggregation of acidophilic plaques “Lewy bodies” (black arrows). GIII, (C); (Lobophytum-treated Group); have apparently the same picture of the control group. GIV, (D) (rotenone + Lobophytum-treated Group); show mild cell dropout accompanied by moderate aggregation of Lewy bodies (black arrows). (H&E staining ×200 and 400).

Group II

Corpus striatum (“CS”)

The brain tissue CS showed apparently increased interstitial spaces between neurons that appeared with varying sizes and shapes and most of the neurons had darkly stained nuclei. The cytoplasm was paler and was characterized by chromatolysis, moreover neuronal processes appeared lost or retracted (Fig. 1B1). Many neurons were shrunken and surrounded by perineuronal spaces. However, some neurons with rounded lightly-stained nuclei and basophilic cytoplasm were also observed. The main neurodegenerative features of the neuropil were aggregates of eosinophilic, round, and elongated (Lewy bodies) accompanied with scars of dense fibrous network of neuroglia cells (gliosis). The presence of macrophages with pigmented material was obviously seen (Figs. 1B2,B3, and 3).Figure 3 Histological findings in the brain corpus striatum “CS” and subtantia nigra pars compacta “SNC” immune-stained with tyrosine hydroxylase “TH” of all studied groups: “CS” (A), Control Group; showing densely packed strong positive immune-reactive neurons (thick arrows) and dense positive TH immunoreactive neuropil (stars) “CS” (B1 and 2), Parkinsonism Group; showing less numerous TH immune-positive neurons, peri-nuclear aggregates of dense inclusions (tailed arrows) and beaded or swollen processes (linear arrow). B2; showing immune-negative areas of neuropil (black star) and other areas are immune-positive (yellow star). “CS” (C), (lobophytum-treated Group); have apparently the same picture of the Control Group; strong cytoplasmic TH immune-reaction (thick arrows) and strong immune-reactive neuropil (star). “CS” (D), (Rotenone + Lobophytum-treated Group); neurons showing strong cytoplasmic immune-reaction (arrows) and mild “TH” immune-reactive neuropil (star). “SNC” (E); Control Group; showing densely packed strong positive immune-reactive neurons (thick arrows) and dense positive TH immunoreaction “SNC” (F); Parkinsonism Group; showing few “TH” immune-positive cells (thick arrows) and the neuropil shows beaded or swollen processes (linear arrows) “SNC” (G); (Lobophytum-treated Group); showing have apparently the same picture of the control group. Strong immune-positive neuronal cytoplasmic expression (thick arrows) and strong immune-reactive neuropil. “SNC” (H); (Rotenone + Lobophytum-treated Group); shows strong immune-reactive cytoplasmic expression (arrows) but mild immune-reactive neuropil (tyrosine hydroxylase “TH” immune-staining × 200 and 400).

Substantia nigra pars compacta (SNC):

Marked and selective loss of dopamine-producing neurons in the substantia nigra pars compacta and the presence of intracytoplasmic aggregation of Lewy bodies (LBs) in surviving neurons (Fig. 2B).

Group III

Corpus striatum "CS"

In the group treated with Lobophytum sp. extract, many neurons appeared with their basophilic cytoplasm, rounded pale nuclei, and prominent nucleoli while other cells were still shrunken with small dense nuclei and surrounded with wide hallows (Fig. 1C).

Substantia nigra pars compacta (SNC)

Brain tissue (SNC) of this group had apparently the same picture of the control group. Cells showing aggregation of Lewy bodies were rarely seen (Fig. 2C).

Group IV

Corpus striatum (CS)

The group treated with Lobophytum sp. extract showed a pronounced improvement in the histological picture; there was restoration of nearly the normal picture the neurons, e.g. intense granular basophilic cytoplasm and vesicular nuclei. Moreover, some neurons appeared with darkly stained nuclei and surrounded by empty hallow but excess infiltration of neuroglia cells (Fig. 1D).

Substantia nigra pars compacta (SNC)

Minimal loss of dopamine-producing neurons in the substantia nigra pars compacta (SNC) and cells showing aggregation of Lewy bodies (LBs) were rarely seen (Fig. 2D).

TH immunohistochemical study

Group I

Corpus striatum shows a group of tyrosine hydroxylase (TH) immune-reactive neurons that occur among the predominant interneurons (Fig. 3A).

Immunohistochemistry of SNC showed densely packed strong positive immune-reactive neurons. The brown material is neuromelanin, which occurs naturally in neurons of the substantia nigra. A dense positive TH immunoreaction neuropil was also observed (Fig. 3E).

Group II

CS showed a number of degenerative changes, which include no/faint immune-reactivity of most neurons and the TH immune-positive neurons appeared less numerous than those of the control group. Perinuclear aggregates of dense inclusions within the immune-positive neurons and beaded or swollen processes were also observed (Fig. 3B1). The neuropil showed some areas with a positive immune-reactivity with relative reduction in intensity and other areas with a negative immune-reactivity (Fig. 3B2). Dilated blood vessels and many vacuolations within the neuropil were also noticed (Fig. 3B1,2). SNC showed few immune-positive cells (arrows) and the neuropil showed beaded or swollen processes (Fig. 3F).

Group III

TH immune-histochemical study: neurons of the “CS” showed strong immune-reactive cytoplasmic expression and strong immune-reactive neuropil (Fig. 3C).

Enlarged nerve cell processes appeared diffusely distributed along the neuropil (Fig. 3C). Neurons of the “SNC” exhibited strong immune‐reaction nearly identical to that of the control group. Few cells demonstrated reduced expression of tyrosine hydroxylase (Fig. 3G).

Group IV

Neurons of the “CS” showed strong immune-reactive cytoplasmic expression (Fig. 3D). Neurons of the SNC showed strong immune-reactive cytoplasmic expression, but mild immune-reactive neuropil in comparison with that of the control group. Diffusely distributed enlarged nerve cell processes appeared along the neuropil (Fig. 3H).

Assessment of superoxide dismutase (SOD) and malondialdehyde (MDA) levels in the brain

The assessment of the antioxidant effect of the extract involved the evaluation of the activity of antioxidant enzymes specifically within the midbrain region. The levels of SOD exhibited a significant decrease (1.7 units per gram) in rats injected with rotenone (ROT) in comparison to rats in the normal control group (p < 0.05). However, the administration of the extract treatment resulted in a significant improvement (p < 0.05) in the activities of SOD levels, measuring at 3.6 units per gram as compared to the rats that only received the ROT injection (Fig. 4A). Additionally, the administration of ROT resulted in a significant elevation of the MDA level, measuring 5.5 nmol/g, in comparison to the control rats (p < 0.05). It is worthy to mention that the extract derived from Lobophytum sp. demonstrated a protective effect against oxidative stress, as evidenced by the significantly decreased levels of MDA at 3.3 nmol/g, in comparison to rats treated with ROT (p < 0.05) (Fig. 4B).Figure 4 The impact of the Lobophytum sp. extract on oxidative stress parameters was assessed in a rat model of Parkinson disease. (A): SOD activity, (B): MDA levels. The values are expressed as the mean ± SD. A one-way ANOVA was utilized, followed by a Tukey’s test for conducting multiple comparisons. *p < 0.05 as compared to the control group; #p < 0.05 as compared to the ROT-alone group.

Estimation of cerebral acetylcholinesterase activity

Cholinesterase activity was assessed in brain homogenate in both the control and treated rats. The cholinesterase activity exhibited a statistically significant decrease (p < 0.05) in rats induced with rotenone, measuring at 9.2 µmol/g. Upon administration of the extract, a notable augmentation in cholinesterase activity (13.1 µmol/g) was observed in comparison to the rats treated with rotenone (p < 0.05) (Fig. 5).Figure 5 Effect of extract on cholinesterase activity. Values were represented as mean ± SD. A one-way ANOVA test was employed followed by Tukey’s test for multiple comparisons. ****p < 0.0001 as compared to the control group; ###p < 0.001 as compared to the ROT-alone group.

Evaluation of apoptosis markers

As depicted in Fig. 6, the administration of rotenone led to a notable upregulation of the Bcl-2-associated X protein (BAX) gene expression by a factor of 3.3 in the brain tissues, in comparison to the control group (p < 0.05). Fascinatingly, the administration of the extract exhibited a notable ability to effectively hinder the process of rotenone-induced apoptosis. This was achieved by reducing the expression of the BAX gene to twofold when compared to the group treated solely with rotenone (p < 0.05) (Fig. 6A). On the contrary, the administration of rotenone led to a notable reduction in the expression of the Bcl-2 gene to 0.35-fold in the brain tissues, when compared to the control group (p < 0.05). It is intriguing to note that the administration of Lobophytum sp. Extract resulted in a notable increase in the expression of the Bcl-2 gene, reaching a level of 0.84-fold compared to the group treated with rotenone (p < 0.05) (Fig. 6B).Figure 6 Gene expression of (A): Bcl-2-associated X protein (BAX), (B): Bcl-2 in all study groups. To analyze gene expression in injured tissues, qRT-PCR was used. Following normalization to GAPDH, the data were displayed as fold change compared to the control group. The bars on the graph depict the mean ± SD. One-way ANOVA test was done to calculate statistical difference followed by Tukey’s test for multiple comparisons. *p < 0.05 as compared to the control group; #p < 0.05 as compared to the rotenone-alone group.

Evaluation of TNF-α, IL-1β, IL-6, and NF-ƘB

In order to examine the regulatory effect of the Lobophytum sp. extract on inflammation, we conducted a study where we analyzed the gene expression of various inflammatory mediators (TNF-α, IL-6, IL-1β, and NF-ƘB) in brain tissues using real-time PCR. Our findings showed that the application of rotenone significantly increased the gene expression of TNF-α, IL-6, IL-1β, and NF-ƘB in the brain by 5.44-fold, 4.3-fold, 4.13-fold, and 6.39-fold, respectively, compared to the control group (p < 0.05). Conversely, when the Lobophytum sp. extract was administered, it significantly reduced the neuroinflammation induced by rotenone. This was evident by the decreased expression of pro-inflammatory mediators (TNF-α, IL-6, IL-1β, and NF-ƘB) to 2.36-fold, 1.89-fold, 1.57-fold, and 2.58-fold, respectively (p < 0.05), compared to the group treated with rotenone (Fig. 7).Figure 7 Gene expression analysis of pro-inflammatory cytokines in all study groups. (A): TNF-α, (B): IL-6, (C): IL-1β and (D): NF-ƘB. To analyze gene expression in injured tissues, qRT-PCR was used. Following normalization to GAPDH, the data were  displayed as fold change compared to the control group. The bars on the graph depict the mean ± SD. One-way ANOVA test was done to calculate statistical difference followed by Tukey’s test for multiple comparisons. *p < 0.05 as compared to the control group; #p < 0.05 as compared to the ROT-alone group.

Effect of Lobophytum sp. extract on α-synuclein

Immunoblotting was conducted to assess the expression of α-synuclein, serving as an indicator of protein-mediated toxicity within the experimental cohorts. Rats subjected to rotenone treatment exhibited heightened α-synuclein expression. In terms of quantity, there was a notable augmentation in the levels of brain α-synuclein to 27.3 ng/mg protein when compared to the control group (p < 0.05). The administration of an extract derived from Lobophytum sp. exhibited a preventive effect on the observed increase. There was a notable reduction in brain α-synuclein levels to 20.45 ng/mg protein in comparison to the group treated with rotenone (p < 0.05) (Fig. 8).Figure 8 Effect of the Lobophytum sp. extract on protein expression of α-synuclein. The bars indicate the mean ± SD. Statistical significance between groups was calculated using one-way ANOVA test and Tukey’s test, where *p < 0.05 indicated significance when compared to the control group, and #p < 0.05 when compared to the ROT group.

Network pharmacology study

To determine the genes related to the Lobophytum sp. soft corals that affect Parkinson's disease, a network pharmacology that determined the  CYP19a1, AR, CYP17A7, and PDE10A genes were the top-represented genes related to Parkinson's disease. The mechanism of anti-parkinsonism activity was suggested as related to different pathways. The VGEF signaling pathway was the top signaling pathway.

Soft coral-metabolite network

A network was constructed to visualize the identified metabolites from the Lobophytum sp. soft corals (Fig. S1).

Metabolite-genes network

The genes identified by metabolites of Lobophytum sp. were obtained from the Swiss Target Prediction and Binding DB databases. A network was constructed to find the relations (edges) between the identified metabolites and the corresponding genes. The results confirmed the identification of 446 genes as targets for the Lobophytum sp. soft corals metabolites. The formed network consisted of 466 nodes (representing metabolites and identified genes) and 1021 edges, with a network centralization of 0.198 and a characteristic path length of 3.408 (Fig. S2).

Gene-Parkinsonism network

The associations between the studied set of genes (446 genes) and parkinsonism disease were obtained through the DisGeNET database, which resulted in 66,574 gene-disease associations. The number of the actual gene-parkinsonism associations was 172 associations. There were only 109 genes among the gene set associated with the parkinsonism disorder. This network consisted of 115 nodes (the identified genes, and parkinsonism disorders) and 172 edges with a characteristic path length of 2.157 and a network centralization of 0.902 (Fig. 9).Figure 9 Gene-Parkinsonism’s disease network; a network describing the identified genes related to Parkinsonism’s disease, the blue rectangles represent genes, and the yellow rectangles represent Parkinsonism’s disease.

Metabolites-Parkinsonism genes

After identifying the genes related to parkinsonism among the studied set of genes, a network (Metabolites-Parkinsonism Genes) was constructed. From this network, all the metabolites were associated with genes related to parkinsonism except for the compound cembrene A. The CYP19A1 gene was the most represented gene with 17 edges followed by AR, CYP17A1, and PDE10A genes, which have 11, 9, and 9 edges respectively. Among the identified 20 metabolites, dendrinolide and denticulatolide, were the top compounds targeting high numbers of parkinsonism disorder genes with 27 and 37 edges, respectively. The formed network consisted of 130 nodes and 344 edges with a characteristic path length of 3.003 and a network centralization of 0.250 (Fig. 10).Figure 10 Metabolites-Parkinsonism Genes; a network describing the metabolites links to genes related to parkinsonism; yellow circles are the identified metabolites from Lobophytum sp. The bigger the circle, the more edges, green circles represent the genes related to parkinsonism, and the bigger the circle, the more edges.

Protein–protein interaction (PPI) network

The interactions between the proteins of the identified genes associated with parkinsonism were determined using the STRING database. The formed network consisted of 109 nodes and 811 edges. The average node score was 15 and the average local clustering coefficient was 0.498 (Fig. 11A). The top 10 genes arranged according to node degree are summarized in Fig. 11B. The Bcl-2 gene was the top gene, with 50 node degrees.Figure 11 (A) PPI Network of the identified genes associated with parkinsonism; (B) the top 10 proteins arranged according to the node degree.

Complete pharmacology network

As a tool to summarize the above networks a complete pharmacology network was constructed. This network was formed by merging three networks: (Soft Coral—Metabolite Network), (Gene—Parkinsonism Network) and (Metabolites—Parkinsonism Genes Network). This network consisted of 136 nodes and 532 edges, the characteristic path length was 2.256 and the network centralization was 0.716 (Fig. 12).Figure 12 Complete pharmacology network: a network describing the interaction between the Lobophytum sp. extract, the identified metabolites, and the genes related to parkinsonism and parkinsonism conditions. The orange rectangle is Lobophytum sp., the yellow oval shapes represent the metabolites, green diamonds represent genes related to parkinsonism, and blue rectangles represent parkinsonism conditions.

Gene ontology (GO) and enrichment analysis

The gene ontology analysis for the identified genes that correlated to parkinsonism among the studied set of genes were 109. These genes were used as the input data to figure out the GO in terms of biological processes, cellular components, and molecular functions. Each term component was arranged in descending order according to fold enrichment. These terms were identified by the ShinyGO 0.77 database. The database defined 1000 biological processes. The top 5 biological processes are trans-synaptic signaling by lipid, modulating synaptic transmission, trans-synaptic signaling by endocannabinoid, modulating synaptic transmission, regulation of serotonin secretion, negative regulation of serotonin secretion, and phospholipase C-activating serotonin receptor signaling pathway (Fig. S3). The database identified 213 cellular components. The top 5 cellular components were the serotonin receptor complex, the G protein-coupled serotonin receptor complex, the phosphatidylinositol -3-kinase complex, class IA, the endolysosome lumen, and the phosphatidylinositol -3-kinase complex, class IB (Fig. S4). There were 437 identified molecular functions. The top 5 molecular functions were nitric-oxide synthase activity, tetrahydrobiopterin binding, phosphatidylinositol-3,4-bisphosphate -5-kinase activity, Gq/11-coupled serotonin receptor activity, and cholinesterase activity (Fig. S5).

A total of 203 KEGG (Kyoto Encyclopedia of Genes and Genomes) biological pathways were identified. The top identified pathways according to fold enrichment were: type II diabetes mellitus, VEGF signaling pathway, prolactin signaling pathway, central carbon metabolism in cancer, and EGFR (Epidermal Growth Factor Receptors) tyrosine kinase inhibitor resistance (Fig. 13 and Table S1). The top signaling pathway was the VEGF signaling pathway (Fig. 14).Figure 13 The top 20 KEGG biological pathways.

Figure 14 VEGF signaling pathway (the top identified signaling pathway).

Discussion

Metabolomic profiling of Lobophytum sp

LC-HR-ESI–MS dereplication led to the identification of 20 compounds (Table 1, Fig. 15). The exact masses of these metabolites were compared to those found in databases such as the Dictionary of Natural Products (DNP) and Metabolite and Chemical Entity (METLIN), to identify them.Figure 15 Chemical structures of dereplicated compounds from Lobophytum sp.

Diterpenes were found to represent the majority of the dereplicated compounds from Lobophytum sp. crude extract, which include six cembranolide diterpenes at mass ion peaks 299.2088 [M−H] −; 331.1981 [M−H]−; 365.2243 [M+H]+; 389.2040 [M−H]−; 389.2044 [M−H]–, and 417.2196 [M+H]+, in accordance with the molecular formulas C20H28O2, C20H28O4, C21H32O5, C22H30O6, C22H30O6, and C24H32O6, were identified as lobophytolide A20; lobophytol13; durumolide P16; durumhemiketalolide B15; denticulatolide14, and lobophytolide F11, respectively. Metabolite 4 (lobophytol) was the signature metabolite identified in the negative mode of Lobophytum sp. LC-HR-ESI–MS. Moreover, three cembranoid diterpene (cembrene A, lobocrassin B, and lobocrassin D) were identified at molecular ion peaks at 271.2507 [M-H]−, 319.2198 [M+H]+, 347.2507 [M+H]+ for the predicted molecular formulas C20H32, C20H30O3, and C22H34O3, respectively10,18. Furthermore, one capnosane and one dendrinolide diterpene type were dereplicated at mass ion peaks of 339.2614 [M−H]–, and 333.2352 [M+H]+ for the predicted molecular formula C20H36O4, C21H32O3, respectively, and they were distinguished as lobophytrol B, which had formerly been reported in Lobophytum sp.6, and dendrinolide, which had been previously identified in the antarctic sponge Dendrilla membranosa19. Additionally, two sesquiterpenoid compounds, identified at mass ion peaks m/z 223.1981 [M+H]+ and 237.1934 [M−H]−, having the molecular formulas C15H26O and C15H26O2 respectively, were identified as nardol and alismoxide from the dereplicated data. Both sesquiterpene compounds had previously been isolated from Lobophytum sp.10. On the other hand, steroids were found to be the second-major class dereplicated from Lobophytum sp. extract. From the dereplicated data, three steroid metabolites had previously been reported in Lobophytum sp., with mass ion peaks at m/z 399.3504 [M+H]+, 471.3552 [M−H]−, and 477.3601[M−H]− for the suggested molecular formulas C28H46O, C30H48O4, and C29H50O5, respectively, were identified as chalinasterol10, nephalsterol C10, and lobophysterol C22. The metabolite with the molecular formula C29H50O, known as lobophytol A, was dereplicated from the mass ion peak at m/z 415.3853 [M+H]+, that previously isolated as a new sterol from the Chinese soft coral Lobophyton sp.17 and it was the signature metabolite identified in the positive mode of Lobophytum sp. by LC-HR-ESI–MS. Besides, two metabolites with molecular formulas C28H46O4 and C28H48O4 and with mass ion peaks at m/z 445.3398 [M−H]– and 449.3553 [M+H]+, were detected as lobophytosterol and depresosterol, respectively, both compounds had formerly been isolated from the Red Sea soft coral Lobophytum depressum21. Additionally, the steroid glycoside metabolite pregnedioside A was dereplicated from its mass ion peak at m/z 449.2961 [M−H]–, in accordance with the molecular formula C26H42O6; it had previously been isolated from an Okinawan soft coral of Alcyonium sp.12. As terpenes and saponins are the major secondary metabolites reported in the Lobophytum sp. extract, terpenes have demonstrated encouraging outcomes in reducing a number of degenerative PD-related processes, including dopaminergic neuronal loss, oxidative stress, and neuroinflammation23.

Additionally, the multifaceted neuroprotective mechanisms of saponins encompass a variety of functions, such as neurotransmitter activity modulation, anti-oxidative qualities, anti-inflammatory, and anti-apoptotic actions. Moreover, they can decrease tau protein phosphorylation, regulate neurotrophic factor levels, modulate calcium influx (attenuation of Ca2+ influx), and support the regeneration of neuronal networks. Each of these mechanisms adds to capacity of saponins for neuroprotection23.

Anti-parkinsonism activity

In this study, the tested rats were subjected to rotenone administration at a dosage of 2 mg/kg/d, subcutaneously, for a duration of 4 weeks. This approach was employed to induce characteristic features of Parkinson's disease in the experimental subjects. Prior research had demonstrated that the administration of this specific dosage regimen yielded phenotypic expressions of Parkinson's disease24. The brain tissue observed in the substantia nigra pars compacta (SNC) of the control group exhibited a conglomeration of densely clustered neurons, displaying a range of sizes and shapes. The cytoplasm exhibited varying degrees of basophilia, ranging from moderate to intense. In the rats having parkinsonism, the neuropil displayed prominent neurodegenerative characteristics, primarily characterized by the presence of Lewy bodies. These aggregates were observed to be eosinophilic, round, and elongated in shape. Additionally, the neuropil exhibited gliosis, which refers to the formation of dense fibrous networks composed of neuroglia cells. The observed results were consistent with the research conducted by Langston et al.25, which demonstrated the existence of acidophilic cytoplasmic structures known as Lewy bodies, which effectively supplant the majority of cellular constituents. The presence of Lewy bodies can be attributed to the accumulation of α-synuclein, an atypical protein. This outcome was consistent with the discovery observed in our investigation. The phenomenon had been the subject of recent investigations by other researchers26. The immunohistochemical analysis of SNC in the group of individuals with parkinsonism had revealed a notable diminishment in the expression of TH immunoreactivity within the neurons. This phenomenon can be elucidated through the investigation conducted by Rausch et al., wherein they posited the initial hypothesis of a deficiency in the TH enzyme as a potential factor contributing to the manifestation of parkinsonism27. The process of neurodegeneration and the subsequent demise of dopaminergic neurons is the underlying pathological mechanism. This leads to a reduction in the levels of TH. The introduction of Lobophytum sp. extract to rats intoxicated with rotenone resulted in the mitigation of the detrimental impact caused by the neurotoxin. Following this intervention, only a subset of neurons exhibiting a dark cytoplasm were observed in the substantia nigra region, while an increased survival rate of TH + neurons in this specific area was observed.

Another characteristic feature of Parkinson's disease is the existence of intracellular structures known as Lewy bodies. These Lewy bodies primarily consist of α-synuclein protein that has undergone misfolding. In its typical state, α-synuclein exists as a soluble protein that can be found within the presynaptic terminals28. The precise functionality of this entity remains incompletely comprehended, although it exhibits potential as a chaperone participating in intracellular transportation and the dynamics of synaptic vesicles29. Mutations in the α-synuclein gene, along with oxidative stress, excessive production, and compromised breakdown mechanisms, can result in the misconfiguration and clumping together of the soluble α-synuclein molecules28,30. This process ultimately culminates in the generation of α-synuclein fibrils, which are insoluble, thereby resulting in the emergence of Lewy bodies in Parkinson's disease31. These aggregates perturb cellular functionality, impede inter-organelle interactions, and hinder organelle axonal transport, ultimately resulting in the demise of neurons32. Administration of rotenone for a duration of 4 weeks has demonstrated the ability to induce the formation of α-synuclein aggregates, as evidenced by prior research33. Fascinatingly, our investigation revealed that the administration of Lobophytum sp. extract alongside rotenone could effectively impede the accumulation of α-synuclein in brain tissues.

Dopaminergic neurons in Parkinson's disease are found to be in a perpetual state of oxidative stress, primarily attributed to the production of free radicals34. The pathogenesis of Parkinson's disease (PD) is significantly influenced by the occurrence of lipid peroxidation and oxidative stress, which are initiated by the presence of free radicals35. The depletion of antioxidant defenses results in an accumulation of reactive oxygen species (ROS)36, which is linked to detrimental impacts on dopaminergic neurons in Parkinson's disease37. Hence, we evaluated the impact of Lobophytum sp. extract administration on the antioxidant defense mechanism. Rats that were subjected to ROT administration exhibited a reduction in their antioxidant defense mechanisms, as evidenced by a decrease in the activities of SOD and an increase in the levels of MDA in the midbrain. The observed reduction in the activities of SOD and the concurrent elevation in the levels of MDA in rats injected with ROT can be attributed to the deactivation of these enzymatic antioxidants by H2O2. Consequently, this deactivation leads to an upsurge in lipid peroxidation, as evidenced by the heightened MDA levels. Nevertheless, the administration of Lobophytum sp. extract to rats injected with ROT resulted in the restoration of SOD and MDA levels, indicating the strong antioxidant properties of the extract. In accordance with prior findings, our investigation has revealed that the neuroprotective efficacy of the Lobophytum sp. extract can be ascribed to its capacity for scavenging free radicals and maintaining the integrity of both enzymatic and non-enzymatic antioxidants.

A multitude of investigations have been conducted to elucidate the precise mechanisms that govern the demise of dopaminergic cells in Parkinson's disease. Mounting evidence indicates a complex interaction between neuronal apoptosis, neuroinflammation, and oxidative stress, as supported by various studies24,37,38. An increased abundance of BAX, a protein known for its proapoptotic properties, has been observed in numerous models of Parkinson's disease24,39. Elevated levels of BAX expression have been observed to stimulate the intrinsic apoptotic pathway40–42. A recent study conducted in vitro demonstrated that the prevention of BAX oligomerization and activation resulted in the inhibition of mitochondrial cytochrome c release into the cytosol, and in the prevention of caspases activation. These effects ultimately led to a reduction in the degeneration of dopaminergic neurons in a cell model of Parkinson disease41. Furthermore, the elevated concentration of the proinflammatory cytokine TNF-α was regarded as a signal that promotes programmed cell death (apoptosis) via the p55 receptor or tumor necrosis factor receptor 1 (TNFR1) mediated extrinsic apoptotic pathway24. Furthermore, the process of dopaminergic neuronal demise is impeded through the upregulation of Bcl-2, a protein known for its anti-apoptotic properties, in animal and cellular models of Parkinson's disease43. Prior research had demonstrated that an elevated BAX/Bcl-2 ratio can induce a proapoptotic milieu, resulting in the demise of dopaminergic cells within the substantia nigra of rats afflicted with Parkinson's disease44. In the current investigation, it was observed that the administration of rotenone over a period of 4 weeks led to a notable augmentation in the expression of BAX, accompanied by a decrease in the expression of Bcl-2 within the midbrains. Consequently, this resulted in a significant elevation of the BAX/Bcl-2 ratio. In the study, it was observed that the administration of Lobophytum sp. extract had a mitigating effect on the elevated BAX/Bcl-2 ratio induced by rotenone in the midbrain of rats. This reduction in ratio contributed to a decrease in cellular apoptosis and ultimately led to improved survival outcomes.

Numerous post-mortem investigations have consistently demonstrated that neuroinflammation plays a significant role in the manifestation of Parkinson's disease45,46. Multiple investigations have unveiled the induction of nuclear factor kappa B (NF-кB) activation in individuals affected by Parkinson's disease (PD) and rats subjected to rotenone treatment24,47. Upon activation, NF-кB undergoes translocation to the nucleus, where it initiates the transcription of multiple proinflammatory factors, including interleukin (IL)-6, IL-1β, and TNF-α, within the microglia48. The presence of these pro-inflammatory cytokines has the potential to elicit detrimental consequences for the dopaminergic neurons49. Furthermore, it has been observed through epidemiological studies that there exists a correlation between an elevated susceptibility to Parkinson's disease and variations in the genetic makeup of the inflammatory cytokines, specifically the genes responsible for encoding IL-6 and TNF-α50. A recent study exhibited that the suppression of TNF-α by lactic acid bacteria in the PD mice model led to noteworthy enhancement of motor skills51. Fascinatingly, the present investigation revealed the inhibitory impact of Lobophytum sp. extract on the augmentation of NF-кB, TNF-α, IL-6, and IL-1B expression in midbrains of rats treated with rotenone.

Based on our empirical findings, we present our observations that the extract derived from Lobophytum sp. exhibited neuroprotective properties in the context of rotenone-induced Parkinson's disease. This neuroprotection was achieved through the inhibition of ROS generation, apoptosis, and the suppression of inflammatory mediators such as IL-6, IL-1β, TNF-α, and NF-кB. Also, the accumulation of α-synuclein proteins was attenuated. Consequently, these actions resulted in the prevention of subsequent neurodegenerative processes. Hence, it is postulated that the extract derived from Lobophytum sp., due to its multifaceted mechanisms, has the potential to be advanced as a prospective pharmacological intervention for the management of Parkinson's disease.

Material and methods

Metabolomic analysis of Lobophytum sp.

Lobophytum sp. collection and identification

Samples of the soft coral Lobophytum sp. samples were gathered from the Egyptian Red Sea off the coast of Hurghada in January 2023. The collected samples were cleaned with seawater, tap water, and distilled water to get rid of any impurities, sand, or salts. The samples were brought to the lab in an ice box, stored in sterile plastic bottles, and kept cold. A voucher specimen (2023-DUPD-12) was kept at the Department of Pharmacognosy, Faculty of Pharmacy, Deraya University, Egypt, where the specimen was identified using common taxonomic keys.

Extraction of Lobophytum sp.

About 350 g of a Lobophytum sp. sample were air dried for a month in the shade. The dried sample was subsequently processed with an OC-60B/60B grinder (60–120 mesh, Henan, Mainland China) to give 285 g fine powder. The powder was extracted using 6 L of 70% ethanol, which were macerated for 5 d at room temperature. A rotary evaporator (Buchi Rotavapor R-300, Cole-Parmer, Vernon Hills, IL, USA) was used to concentrate the mixture under vacuum at 45 °C to provide 50 g of crude extract.

Metabolomic analysis

An Acquity Ultra Performance Liquid Chromatography system connected to a Synapt G2 HDMS quadrupole time-of-flight hybrid mass spectrometer (Waters, Milford, CT, USA) was used to perform high-resolution liquid chromatography-mass (HR-LC–MS) metabolic tentative identification for 1 mg of the Lobophytum sp. extract, which had been weighted using a delicate electric balance (Sartorius, type 1712, Germany) and dissolved in 1 mL of HPLC-grade methanol.

An ACE C18 column, measuring 75 mm by 3.0 mm and 5 µm, was used as the HPLC column (ACE, Mainz, Germany). The mobile phase consisted of HPLC grade water (A), which was obtained in-house, from a direct Q-3 water purification system (Millipore, Watford, UK), and acetonitrile (B) with 0.1% formic acid in each solvent. All reagents were purchased from Fisher Scientific, Hemel Hempstead, UK, and were of analytical grade. The gradient program was 10% B at first, and after 30 min of linear growth to 100% B at a flow rate of 300 µL/min, it was isocratic for 5 min before linearly falling back to 10% B in 1 min. Before the injection, the column was re-equilibrated with 10% B for 9 min. Every sample was analyzed for a total of 45 min. The tray temperature was kept at 12 °C, and the injection volume was 10 µL. Both positive and negative ESI ionization modes of high-resolution mass spectrometry were used to include the highest number of metabolites from the investigated methanol extract, with a capillary temperature of 320 °C and a spray voltage of 4.5 kV. The mass range of 150–1500 m/z was chosen. The dereplication was achieved for each m/z ion peak with metabolites recorded in the customized databases such as METLIN and the Dictionary of Natural Products (DNP) database52 based on established parameters (m/z threshold of ± 3 ppm and retention time), which provided a high level of confidence in metabolites identity. As a result, 20 compounds were dereplicated from Lobophytum sp. methanol extract, and the number of the remaining unknown metabolites were refined. The raw data were processed, aligned, and merged into one dataset according to the method previously developed in our lab53,54.

Data analysis for HR-ESI–MS

The MassConvert tool from ProteoWizard was first used to split the raw data into two data sets based on the ionization mode (positive and negative modes). In MZmine 2.1055, the sliced data sets were imported and processed utilizing predefined settings to extract features from raw data. With MZmine, the following data processing steps were carried out: peak detection (using chromatographic builder and mass detection), deconvolution, deisotoping, filtering, alignment, and gap filling. Adducts and complexes were identified, and formula prediction procedures were performed to minimize feature misassignment by removing adducts and complexes and to forecast potential chemical formulae for each feature (see Supplementary Information for full details of all settings and procedures utilized to process data in MZmine). After that, the data was exported as a CSV file for additional cleanup. Antibase® (February 2013) and Marinlit® (September 2013) molecular formula data sets were used, with an algorithm being used. The provided molecular weights do not distinguish between monoisotopic, average, and most abundant masses in these versions of the manually curated databases. Next, exact monoisotopic masses of each metabolite were computed and used to create the customised library. Peak identification and dereplication were performed using the integrated Excel macro in the customized library, which integrated the processed data from MZmine. Unidentified peaks and ―Hits‖ were cross-checked with the MS raw data in Xcalibur 2.2. Excel macros were created to enable the combination of positive and negative ionisation mode data files produced by MZmine, as well as the elimination of background peaks. Ion peaks from the medium were removed whereas features with peak intensities 20 times higher in the samples than in the medium were kept. This was done by using an algorithm to determine the intensity of each m/z in both the bacterial extracts and the media extracts. Through the use of RT and an m/z threshold of ± 3 ppm, the Excel macro was able to dereplicate each m/z ion peak with compounds in the customized database, providing information on the putative identities of all the metabolites in the Lobophytum sp. extract and sequentially sorting the number of unknowns that remained for the extract. Hits from the database were accessed using ChemBioFinder version 13 (PerkinElmer Informatics, Cambridge, UK). After that, the data was converted into a CSV file and exported to SIMCA-P V 13.0 Umetrics, Umeå, Sweden. As a result, each feature in the extract was given a feature ID number, ionization mode, m/z, retention time, potential molecular formulas, and peak intensity54.

Biological study

Animals

A total of 32 male Sprague Dawley rats, at the age of 8 weeks, with a weight range of 200–250 g, were selected as subjects for this research investigation. The subjects were confined within polystyrene enclosures maintained at a consistent temperature of 25 ± 2 °C, while being subjected to a regular 12-h cycle of alternating light and darkness. The animals were provided with a diet consisting of standard chow pellets and unrestricted access to water. One week prior to the experiment, the animals were acclimated to the laboratory environment.

Extract preparation

The Lobophytum sp. extract was dried and dissolved in 0.5% carboxymethylcellulose (CMC). It was administered orally by gavage at a dose of 40 mg/kg daily throughout the entire duration of the experiment. A solution containing rotenone was prepared, with a concentration of 2 mg/mL in sunflower oil. The rats in the group receiving rotenone were given subcutaneous injections of 2 mg/kg/d.

Ethics approval

All animal treatments adhered rigorously to the institutional and international ethical guidelines for the utilization and welfare of laboratory animals, and all experiments were conducted in accordance with the ARRIVE guidelines. The experimental protocols underwent approval by the Experimental Animal Center and Research Ethical Committee, Deraya University, Minia, Egypt.

Acute toxicity study

An acute toxicity study was performed according to the method outlined by Lorke17. During the initial phase, a group of rats was divided into three smaller groups, each consisting of three rats. The groups were administered the extract at a dosage of 10, 100, or 1000 mg/kg body weight. The animals were closely monitored for a period of 24 h to detect any indications of toxicity or mortality.

In the second phase, the rats were divided into three groups. The extract was then administered to all three groups at a dose of 1600, 2900, and 5000 mg/kg. The LD50 was determined based on the findings of the final phase, using the following formula:LD50=(D0×D100)

D0 is the highest dose that resulted in no mortality, D100 is the lowest dose that resulted in mortality.

Experimental design

A population of rats was subjected to random allocation into four distinct groups, each containing eight individuals. Group 1 was administered a solution containing 0.5% CMC orally and sunflower oil subcutaneously for a duration of 4 weeks. Group 2 was administered the oral extract dissolved in 0.5% CMC via gavage on a daily basis, along with subcutaneous administration of sunflower oil, over a period of 4 weeks. Group 3 was administered 0.5% CMC via the oral route, while also receiving rotenone (Sigma Aldrich) (2 mg/kg) per day, administered subcutaneously. This treatment regimen continued for a duration of 4 weeks. Group 4 was administered the oral extract via gavage on a daily basis, along with rotenone (2 mg/kg/d, s.c.), for a duration of 4 weeks24. After a period of 24 h following the final injection, the rats were anesthetized using thiopental sodium at a dose of 50 mg/kg56 and scarified by decapitation. The entire brains were then carefully dissected, and the midbrains and striata from one hemisphere were removed. These brain regions were subsequently stored at a temperature of − 80 °C for preservation purposes. The stored samples were later utilized for neurochemical analysis, while the second brain was immersed in a 10% formalin solution (pH 4.0) for the purpose of histological assessment.

Histological examination

Hematoxylin and eosin (H&E) staining

The paraffin beeswax tissue blocks underwent sectioning at a thickness of 4 μm using a sliding microtome. The specimens were obtained and mounted onto glass slides, followed by the removal of paraffin. Subsequently, a staining technique involving hematoxylin and eosin was employed. Finally, an impartial researcher scrutinized the samples using a light microscope, which was equipped with a camera57.

Immunohistochemistry

Paraffin tissue sections with a thickness of 5 μm were meticulously prepared following the established protocol outlined by Johansson et al. in their seminal work published in 200258. Immunohistochemical staining was performed in accordance with the manufacturer's protocols utilizing a TH rabbit monoclonal antibody (1:200 dilution, Abcam, Cambridge, UK; catalog number ab75875).

Assessment of SOD and MDA levels in the brain

The estimation of the activities of the endogenous antioxidant enzyme superoxide dismutase (SOD) was conducted in accordance with the protocols provided by the manufacturer's kits (Bio-Diagnostic, Giza, Egypt). The expression of SOD activities is quantified in units per gram tissue59,60. Cerebral MDA content assessment was conducted utilizing commercially available kits in accordance with the protocols specified by the manufacturer (Bio-Diagnostic, Giza, Egypt).

Estimation of cerebral acetylcholinesterase activity

The colorimetric assay involved the evaluation of the hydrolysis process of butyrylthiocholine into butyrate and thiocholine, which occurred in the presence of cholinesterase. The formation of thiocholine was subsequently subjected to reaction with DTNB, resulting in the acquisition of a pigmented product. This product was subsequently quantified by measuring its absorbance at a wavelength of 405 nm. The magnitude of absorbance augmentation is contingent upon the level of cholinesterase activity present within the homogenate61.

Gene expression analysis by real-time PCR

In order to ascertain the mRNA expression levels of the target genes, the total RNA was extracted utilizing a TRIzol reagent (Invitrogen, Waltham, MA, USA) in accordance with the guidelines provided by the manufacturer. The quantification of the isolated RNA was performed utilizing the Nanodrop 1000 instrument (Thermo Fisher Scientific Inc., Waltham, MA, USA) to determine its concentration. The process of cDNA synthesis was carried out utilizing a High-Capacity cDNA Reverse Transcription Kit. To conduct a quantitative analysis, we employed real-time PCR to identify the overall mRNA transcripts of the target genes. This was done using the step-one real-time PCR platform and the maxima SYBR green master mix from (Thermo Fisher Scientific Inc., Waltham, MA, USA). The primer sequences of the target genes were acquired from the National Center for Biotechnology Information (NCBI) and are presented in Table 2. The assessment of target gene expression was conducted utilizing the 2-ΔΔCt method, which involves normalizing the results to the housekeeping gene GAPDH. Table 2 Primers list for real-time PCR.

IL-1β	Forward	5ʹ-GTG ATG AAA GAC GGC ACA CC-3ʹ	
Reverse	5ʹ-TCC TGG GGA AGG CAT TAG GA-3ʹ	
GAPDH	Forward	5ʹ-CTC TCT GCT CCT CCC TGT TC-3ʹ	
Reverse	5ʹ-CGA CAT ACT CAG CAC CAG CA-3ʹ	
TNF-α	Forward	5ʹ-CCT CTC TGC CAT CAA GAG CC-3ʹ	
Reverse	5ʹ-GGC TGG GTA GAG AAC GGA TG-3ʹ	
IL-6	Forward	5ʹ-TCT GGT CTT CTG GAG TTC CGT-3′	
Reverse	5ʹ-GGA TGG TCT TGG TCC TTA GCC-3′	
Bcl-2	Forward	5ʹ-GGG CTA CGA GTG GGA TAC TG-3ʹ	
Reverse	5ʹ-GAC CCC ACC GAA CTC AAA GA-3ʹ	
BAX	Forward	5ʹ-CAC GTC TGC GGG GAG TC-3ʹ	
Reverse	5ʹ-TGT TGT CCA GTT CAT CGC CA-3ʹ	
NF-ƘB	Forward	5ʹ-CAG CAG ATG GCC CAT ACC TT-3ʹ	
Reverse	5ʹ-CTG TCA TCC GTG CTT CCA GT-3ʹ	

Enzyme-linked immunosorbent assay (ELISA)

Using an ELISA plate reader and a commercially available ELISA kit (Cusabio), the quantity of α-synuclein in rat brain tissues was  assessed. The results are represented as ng/mg protein.

Statistical analysis

The data were reported in the form of the mean ± standard deviation (SD) and subjected to statistical analysis using a one-way ANOVA followed by the Tukey’s test as a post-hoc analysis. The statistical analyses were performed using GraphPad Prism software 9 (GraphPad Software, Inc. La Jolla, CA, USA). The statistical significance of the results was determined based on probability values below 0.05.

Network pharmacology

Soft coral-metabolite network

The metabolites identification, carried out by the LC–HR–ESI–MS technique, tentatively identified 20 metabolites, and a basic network, linking the soft coral (Lobophytum sp.) to the identified metabolites was constructed.

Metabolite-genes network

A network (metabolite-genes) was constructed, based on chemical data extracted for each compound from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/)62 (last accessed on 10-08-2023) and SwissTargetPrediction database http://www.swisstargetprediction.ch/result.php?job=215444691&organism=Homo_sapiens63 (last accessed on 11–8-2023) was used to find out the targets of each identified compound related to the human species (Homosapien). The top genes were selected in the SwissTargetPrediction database with a probability score > 0.

Gene-Parkinsonism network

DisGenet (https://www.disgenet.org/)64 (last accessed on 13-8-2023) online database was used to find out the target genes related to parkinsonism. A filter option was chosen in the database, and a word filter (Parkinson) was applied.

Metabolites–Parkinsonism genes

A network was formed based on extracted from the DisGenet. The determined genes related to parkinsonism were selected and in a backward step, each identified gene related to parkinsonism was referred to its corresponding metabolite.

Protein–protein interaction (PPI) network

Interactions between proteins of the identified genes related to parkinsonism were obtained from the STRING database62. The graphical diagram was obtained from the same database.

Complete pharmacology network

A complete pharmacology network was formed by combining the Soft Coral-Metabolite Network (Gene-Parkinsonism Network) and (Metabolites–Parkinsonism Genes Network).

Networks construction visualization and analysis

The networks were constructed, visualized, and analyzed using the software Cytoscape 3.9.0. (https://cytoscape.org/download.html)65.

Gene ontology (GO) and enrichment analysis

The gene ontology and enrichment analysis were performed on the genes related to parkinsonism among the gene set identified by metabolites. The determination of the gene ontology in terms of the cellular components, biological processes, molecular function, and biological pathways that are related to this set of genes was performed using ShinyGO 0.7566.

Conclusion

In conclusion, this work offers strong evidence for the neuroprotective potential of Lobophytum sp. extract in a rotenone-induced rat model of neurodegeneration resembling Parkinson's disease (PD). The results demonstrate that the extract targets several pathways involved in the pathophysiology of Parkinson's disease, successfully mitigating neuron loss and exerting a neuroprotective impact. The results of the experiment indicate that the extract from Lobophytum sp. inhibits the production of reactive oxygen species (ROS), lowers apoptosis, and suppresses NF-кB activation as well as inflammatory mediators like IL-6, IL-1β, and TNF-α. These benefits aid in preventing the neurodegeneration linked to Parkinson's disease. Tyrosine hydroxylase immunochemistry and histopathological examination provide more evidence for the neuroprotective properties of the extract. Furthermore, metabolomic profiling using HR-LC–ESI–MS was carried out to characterize the potential bioactive compounds responsible for the neuroprotective properties of the extract through the computational network pharmacology study. Although the study investigated various pathways and mechanisms involved in the neuroprotective effects of the extract, it does not provide a comprehensive understanding of the underlying molecular mechanisms. Further studies are required to elucidate the specific bioactive compounds responsible for the observed effects and their precise mechanisms of action. Moreover, the study suggests the potential therapeutic benefits of Lobophytum sp. extract for Parkinson's disease, it acknowledges the need for additional investigation and clinical studies. The translation of findings from preclinical animal models to human patients is a complex process, and further research is necessary to evaluate the safety, efficacy, and optimal dosage of the extract in human subjects.

To conclude, although the study shows encouraging results regarding the neuroprotective potential of Lobophytum sp. extract in a rat model of neurodegeneration similar to Parkinson's disease, more human studies are required to establish the safety of the extract and its efficacy as a therapeutic agent for treating Parkinson's disease and to determine its clinical applicability.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-66781-9.

Acknowledgements

We thank Deraya University for providing laboratory space.

Author contributions

Conceptualization: U.R.A., G.B., H.T.B., F.H.A., N.A.A., M.A.A.; methodology: F.M. Abd., D.H.A., R.H.A., FA. M., E.A.S.; software: D.H.A., R.H.A, FA. M.; formal analysis: U.R.A., G.B., F.H.A., N.A.A.; investigation: U.R.A., F.M. Abd., D.H.A., R.H.A., E.A.S.; resources: U.R.A., D.H.A.; data curation: U.R.A., G.B., H.T.B., F.H.A., N.A.A., M.A.A.; writing—original draft: all authors; writing—review and editing: U.R.A., G.B, F.M. Abd.; all authors have read and agreed with the final version of the manuscript.

Data availability

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Marras C Dihydropyridine calcium channel blockers and the progression of parkinsonism Ann. Neurol. 2012 71 3 362 369 10.1002/ana.22616 22451203
Marras, C. et al. Dihydropyridine calcium channel blockers and the progression of parkinsonism. Ann. Neurol. 71(3), 362–369 (2012).22451203
2. Levin J The differential diagnosis and treatment of atypical parkinsonism Dtsch. Arztebl. Int. 2016 113 5 61 26900156
Levin, J. et al. The differential diagnosis and treatment of atypical parkinsonism. Dtsch. Arztebl. Int. 113(5), 61 (2016).26900156
3. Roy, S. P. et al. Antiparkinsonian activity of Tabebuia impetiginosa bark and biochemical analysis of dopamine in rat brain homogenates. In Annales Pharmaceutiques Françaises. (Elsevier, 2022).
4. Yahr MD Treatment of parkinsonism with levodopa Arch. Neurol. 1969 21 4 343 354 10.1001/archneur.1969.00480160015001 5820999
Yahr, M. D. et al. Treatment of parkinsonism with levodopa. Arch. Neurol. 21(4), 343–354 (1969).5820999
5. Roy PK New Casbane and cembrane diterpenoids from an Okinawan soft coral, Lobophytum sp Molecules 2016 21 5 679 10.3390/molecules21050679 27223275
Roy, P. K. et al. New Casbane and cembrane diterpenoids from an Okinawan soft coral, Lobophytum sp. Molecules 21(5), 679 (2016).27223275
6. Zhang Q Three new capnosane-type diterpenoids from the South China Sea soft coral Lobophytum sp Fitoterapia 2019 133 70 74 10.1016/j.fitote.2018.12.003 30553829
Zhang, Q. et al. Three new capnosane-type diterpenoids from the South China Sea soft coral Lobophytum sp. Fitoterapia 133, 70–74 (2019).30553829
7. Fattorusso E Lobozoanthamine, a new zoanthamine-type alkaloid from the Indonesian soft coral Lobophytum sp Tetrahedron Lett. 2008 49 14 2189 2192 10.1016/j.tetlet.2008.02.028
Fattorusso, E. et al. Lobozoanthamine, a new zoanthamine-type alkaloid from the Indonesian soft coral Lobophytum sp. Tetrahedron Lett. 49(14), 2189–2192 (2008).
8. Ye F New steroids from the south China sea soft coral Lobophytum sp Chem. Biodivers. 2020 17 6 e2000214 10.1002/cbdv.202000214 32314539
Ye, F. et al. New steroids from the south China sea soft coral Lobophytum sp. Chem. Biodivers. 17(6), e2000214 (2020).32314539
9. Chen SH Huang H Guo YW Four new cembrane diterpenes from the hainan soft coral Lobophytum sp Chin. J. Chem. 2008 26 12 2223 2227 10.1002/cjoc.200890395
Chen, S. H., Huang, H. & Guo, Y. W. Four new cembrane diterpenes from the hainan soft coral Lobophytum sp. Chin. J. Chem. 26(12), 2223–2227 (2008).
10. Al-Footy KO Antibacterial and cytotoxic properties of isoprenoids from the red sea soft coral, Lobophytum sp Trop. J. Pharm. Res. 2016 15 7 1431 1438 10.4314/tjpr.v15i7.11
Al-Footy, K. O. et al. Antibacterial and cytotoxic properties of isoprenoids from the red sea soft coral, Lobophytum sp. Trop. J. Pharm. Res. 15(7), 1431–1438 (2016).
11. Chen SH Six new cembranolides from the Hainan soft coral Lobophytum sp Helvetica Chimica Acta 2008 91 5 873 880 10.1002/hlca.200890091
Chen, S. H. et al. Six new cembranolides from the Hainan soft coral Lobophytum sp. Helvetica Chimica Acta 91(5), 873–880 (2008).
12. Kobayashi M Five new steroidal glycosides, pregnedioside-A,-B, and their three monoacetates, from an Okinawan soft coral of Alcyonium sp Tetrahedron Lett. 1984 25 34 3731 3734 10.1016/0040-4039(84)80117-3
Kobayashi, M. et al. Five new steroidal glycosides, pregnedioside-A,-B, and their three monoacetates, from an Okinawan soft coral of Alcyonium sp. Tetrahedron Lett. 25(34), 3731–3734 (1984).
13. Iguchi K Stereostructures of unique 13-membered carbocyclic cembranolides from the soft coral Lobophytum pauciflorum Chem. Lett. 1991 20 2 319 322 10.1246/cl.1991.319
Iguchi, K. et al. Stereostructures of unique 13-membered carbocyclic cembranolides from the soft coral Lobophytum pauciflorum. Chem. Lett. 20(2), 319–322 (1991).
14. Uchio Y Denticulatolide, an ichthyotoxic peroxide-containing cembranolide from the soft coral Lobophytum denticulatum Tetrahedron Lett. 1985 26 37 4487 4490 10.1016/S0040-4039(00)88937-6
Uchio, Y. et al. Denticulatolide, an ichthyotoxic peroxide-containing cembranolide from the soft coral Lobophytum denticulatum. Tetrahedron Lett. 26(37), 4487–4490 (1985).
15. Cheng S-Y Unprecedented hemiketal cembranolides with anti-inflammatory activity from the soft coral Lobophytum durum J. Nat. Prod. 2009 72 1 152 155 10.1021/np800686k 19177635
Cheng, S.-Y. et al. Unprecedented hemiketal cembranolides with anti-inflammatory activity from the soft coral Lobophytum durum. J. Nat. Prod. 72(1), 152–155 (2009).19177635
16. Cheng S-Y New cembranolides from the Dongsha atoll soft coral Lobophytum durum Mar. Drugs 2011 9 8 1307 1318 10.3390/md9081307 21892346
Cheng, S.-Y. et al. New cembranolides from the Dongsha atoll soft coral Lobophytum durum. Mar. Drugs 9(8), 1307–1318 (2011).21892346
17. Chengsheng C Structural determination of lobophytol A isolated from the soft coral Acta Scientiarum Naturalium Universitatis Sunyatseni 1999 38 5 118 120
Chengsheng, C. et al. Structural determination of lobophytol A isolated from the soft coral. Acta Scientiarum Naturalium Universitatis Sunyatseni 38(5), 118–120 (1999).
18. Kao C-Y Lobocrassins A-E: New cembrane-type diterpenoids from the soft coral Lobophytum crassum Mar. Drugs 2011 9 8 1319 1331 10.3390/md9081319 21892347
Kao, C.-Y. et al. Lobocrassins A-E: New cembrane-type diterpenoids from the soft coral Lobophytum crassum. Mar. Drugs 9(8), 1319–1331 (2011).21892347
19. Fontana A Scognamiglio G Cimino G Dendrinolide, a new degraded diterpenoid from the Antarctic sponge Dendrilla membranosa J. Nat. Prod. 1997 60 5 475 477 10.1021/np960712w
Fontana, A., Scognamiglio, G. & Cimino, G. Dendrinolide, a new degraded diterpenoid from the Antarctic sponge Dendrilla membranosa. J. Nat. Prod. 60(5), 475–477 (1997).
20. Karlsson R Lobophytolide, a cembranolide diterpene Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 1977 33 7 2032 2034 10.1107/S0567740877007687
Karlsson, R. Lobophytolide, a cembranolide diterpene. Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 33(7), 2032–2034 (1977).
21. Carmely S Kashman YJT Isolation and structure elucidation of lobophytosterol, depresosterol and three other closely related sterols: Five new c28 polyoxygenated sterols from the red sea soft coral Lobophytum depressum Tetrahedron 1981 37 13 2397 2403 10.1016/S0040-4020(01)88896-7
Carmely, S. & Kashman, Y. J. T. Isolation and structure elucidation of lobophytosterol, depresosterol and three other closely related sterols: Five new c28 polyoxygenated sterols from the red sea soft coral Lobophytum depressum. Tetrahedron 37(13), 2397–2403 (1981).
22. Zhang Q Cytotoxic polyhydroxylated steroids from the South China Sea soft coral Lobophytum sp Steroids 2019 141 76 80 10.1016/j.steroids.2018.11.015 30513321
Zhang, Q. et al. Cytotoxic polyhydroxylated steroids from the South China Sea soft coral Lobophytum sp. Steroids 141, 76–80 (2019).30513321
23. Bhusal, C. K. et al. Unveiling Nature's potential: Promising natural compounds in Parkinson's disease management. Parkinsonism Relat. Disord. 105799 (2023).
24. Habib CN The potential neuroprotective effect of diosmin in rotenone-induced model of Parkinson's disease in rats Eur. J. Pharmacol. 2022 914 174573 10.1016/j.ejphar.2021.174573 34656609
Habib, C. N. et al. The potential neuroprotective effect of diosmin in rotenone-induced model of Parkinson’s disease in rats. Eur. J. Pharmacol. 914, 174573 (2022).34656609
25. Langston JW Multisystem Lewy body disease and the other parkinsonian disorders Nat. Genet. 2015 47 12 1378 1384 10.1038/ng.3454 26620112
Langston, J. W. et al. Multisystem Lewy body disease and the other parkinsonian disorders. Nat. Genet. 47(12), 1378–1384 (2015).26620112
26. Alam G Richardson JR Regulation of tyrosine hydroxylase: Relevance to Parkinson's disease Genetics, Neurology, Behavior, and Diet in Parkinson's Disease 2020 Elsevier 51 66
Alam, G. & Richardson, J. R. Regulation of tyrosine hydroxylase: Relevance to Parkinson’s disease. In Genetics, Neurology, Behavior, and Diet in Parkinson’s Disease 51–66 (Elsevier, 2020).
27. Rausch W-D Wang F Radad K From the tyrosine hydroxylase hypothesis of Parkinson’s disease to modern strategies: A short historical overview J. Neural Transm. 2022 129 5–6 487 495 10.1007/s00702-022-02488-3 35460433
Rausch, W.-D., Wang, F. & Radad, K. From the tyrosine hydroxylase hypothesis of Parkinson’s disease to modern strategies: A short historical overview. J. Neural Transm. 129(5–6), 487–495 (2022).35460433
28. Dar KB Elucidating critical proteinopathic mechanisms and potential drug targets in neurodegeneration Cell. Mol. Neurobiol. 2020 40 3 313 345 10.1007/s10571-019-00741-0 31584139
Dar, K. B. et al. Elucidating critical proteinopathic mechanisms and potential drug targets in neurodegeneration. Cell. Mol. Neurobiol. 40(3), 313–345 (2020).31584139
29. Melki R Role of different alpha-synuclein strains in synucleinopathies, similarities with other neurodegenerative diseases J. Parkinson's Dis. 2015 5 2 217 227 10.3233/JPD-150543 25757830
Melki, R. Role of different alpha-synuclein strains in synucleinopathies, similarities with other neurodegenerative diseases. J. Parkinson’s Dis. 5(2), 217–227 (2015).25757830
30. Stefanis L α-Synuclein in Parkinson's disease Cold Spring Harb. Perspect. Med. 2012 2 2 a009399 10.1101/cshperspect.a009399 22355802
Stefanis, L. α-Synuclein in Parkinson’s disease. Cold Spring Harb. Perspect. Med. 2(2), a009399 (2012).22355802
31. Emamzadeh FN Role of apolipoproteins and α-synuclein in Parkinson’s disease J. Mol. Neurosci. 2017 62 3–4 344 355 10.1007/s12031-017-0942-9 28695482
Emamzadeh, F. N. Role of apolipoproteins and α-synuclein in Parkinson’s disease. J. Mol. Neurosci. 62(3–4), 344–355 (2017).28695482
32. Teil M Targeting α-synuclein for PD therapeutics: A pursuit on all fronts Biomolecules 2020 10 3 391 10.3390/biom10030391 32138193
Teil, M. et al. Targeting α-synuclein for PD therapeutics: A pursuit on all fronts. Biomolecules 10(3), 391 (2020).32138193
33. Javed, H. & Ojha, S. Therapeutic potential of baicalein in Parkinson’s disease: Focus on inhibition of α-synuclein oligomerization and aggregation. In Synucleins-Biochemistry and Role in Diseases (IntechOpen, 2019).
34. Olufunmilayo EO Gerke-Duncan MB Holsinger RD Oxidative stress and antioxidants in neurodegenerative disorders Antioxidants 2023 12 2 517 10.3390/antiox12020517 36830075
Olufunmilayo, E. O., Gerke-Duncan, M. B. & Holsinger, R. D. Oxidative stress and antioxidants in neurodegenerative disorders. Antioxidants 12(2), 517 (2023).36830075
35. Leathem A Evidence for oxidative pathways in the pathogenesis of PD: Are antioxidants candidate drugs to ameliorate disease progression? Int. J. Mol. Sci. 2022 23 13 6923 10.3390/ijms23136923 35805928
Leathem, A. et al. Evidence for oxidative pathways in the pathogenesis of PD: Are antioxidants candidate drugs to ameliorate disease progression?. Int. J. Mol. Sci. 23(13), 6923 (2022).35805928
36. Abdelaleem ER Apple extract protects against indomethacin-induced gastric ulcers in rats by suppressing oxidative stress—The implication of Nrf-2/HO-1 signaling pathway: In silico and in vivo studies J. Funct. Foods 2024 112 105926 10.1016/j.jff.2023.105926
Abdelaleem, E. R. et al. Apple extract protects against indomethacin-induced gastric ulcers in rats by suppressing oxidative stress—The implication of Nrf-2/HO-1 signaling pathway: In silico and in vivo studies. J. Funct. Foods 112, 105926 (2024).
37. Eddin LB Limonene, a monoterpene, mitigates rotenone-induced dopaminergic neurodegeneration by modulating neuroinflammation, hippo signaling and apoptosis in rats Int. J. Mol. Sci. 2023 24 6 5222 10.3390/ijms24065222 36982297
Eddin, L. B. et al. Limonene, a monoterpene, mitigates rotenone-induced dopaminergic neurodegeneration by modulating neuroinflammation, hippo signaling and apoptosis in rats. Int. J. Mol. Sci. 24(6), 5222 (2023).36982297
38. Cabral-Costa J Kowaltowski A Neurological disorders and mitochondria Mol. Aspects Med. 2020 71 100826 10.1016/j.mam.2019.10.003 31630771
Cabral-Costa, J. & Kowaltowski, A. Neurological disorders and mitochondria. Mol. Aspects Med. 71, 100826 (2020).31630771
39. Bové J BAX channel activity mediates lysosomal disruption linked to Parkinson disease Autophagy 2014 10 5 889 900 10.4161/auto.28286 24686337
Bové, J. et al. BAX channel activity mediates lysosomal disruption linked to Parkinson disease. Autophagy 10(5), 889–900 (2014).24686337
40. Nazmy MH Assessing the antiproliferative potential of a novel combretastatin A4 derivative via modulating apoptosis, MAPK/ERK and PI3K/AKT pathways in human breast cancer cells Front. Biosci. 2023 28 8 185 10.31083/j.fbl2808185
Nazmy, M. H. et al. Assessing the antiproliferative potential of a novel combretastatin A4 derivative via modulating apoptosis, MAPK/ERK and PI3K/AKT pathways in human breast cancer cells. Front. Biosci. 28(8), 185 (2023).
41. Chung Y Dysregulated autophagy is linked to BAX oligomerization and subsequent cytochrome c release in 6-hydroxydopmaine-treated neuronal cells Biochem. Biophys. Res. Commun. 2021 548 20 26 10.1016/j.bbrc.2021.02.045 33631669
Chung, Y. et al. Dysregulated autophagy is linked to BAX oligomerization and subsequent cytochrome c release in 6-hydroxydopmaine-treated neuronal cells. Biochem. Biophys. Res. Commun. 548, 20–26 (2021).33631669
42. Fricker M Neuronal cell death Physiol. Rev. 2018 98 2 813 880 10.1152/physrev.00011.2017 29488822
Fricker, M. et al. Neuronal cell death. Physiol. Rev. 98(2), 813–880 (2018).29488822
43. Liu J Liu W Yang H Balancing apoptosis and autophagy for Parkinson’s disease therapy: targeting BCL-2 ACS Chem. Neurosci. 2018 10 2 792 802 10.1021/acschemneuro.8b00356
Liu, J., Liu, W. & Yang, H. Balancing apoptosis and autophagy for Parkinson’s disease therapy: targeting BCL-2. ACS Chem. Neurosci. 10(2), 792–802 (2018).
44. Li C Chen X Zhang N Changes of expression of apoptosis-related proteins Bcl-2 and Bax in Parkinson's disease rat induced by rotenone Zhongguo Shiyan Dongwu Xuebao 2009 17 1 50 52
Li, C., Chen, X. & Zhang, N. Changes of expression of apoptosis-related proteins Bcl-2 and Bax in Parkinson’s disease rat induced by rotenone. Zhongguo Shiyan Dongwu Xuebao 17(1), 50–52 (2009).
45. Öberg M The role of innate immunity and inflammation in Parkinson s disease Scand. J. Immunol. 2021 93 5 e13022 10.1111/sji.13022 33471378
Öberg, M. et al. The role of innate immunity and inflammation in Parkinson s disease. Scand. J. Immunol. 93(5), e13022 (2021).33471378
46. Rocha, N. P., De Miranda, A. S. & Teixeira, A. L. Insights into neuroinflammation in Parkinson’s disease: from biomarkers to anti-inflammatory based therapies. BioMed Res. Int. 2015 (2015.).
47. Thakur P Nehru B Inhibition of neuroinflammation and mitochondrial dysfunctions by carbenoxolone in the rotenone model of Parkinson’s disease Mol. Neurobiol. 2015 51 209 219 10.1007/s12035-014-8769-7 24946750
Thakur, P. & Nehru, B. Inhibition of neuroinflammation and mitochondrial dysfunctions by carbenoxolone in the rotenone model of Parkinson’s disease. Mol. Neurobiol. 51, 209–219 (2015).24946750
48. Kim ME Anti-neuroinflammatory effects of vanillin through the regulation of inflammatory factors and NF-κB signaling in LPS-stimulated microglia Appl. Biochem. Biotechnol. 2019 187 884 893 10.1007/s12010-018-2857-5 30097802
Kim, M. E. et al. Anti-neuroinflammatory effects of vanillin through the regulation of inflammatory factors and NF-κB signaling in LPS-stimulated microglia. Appl. Biochem. Biotechnol. 187, 884–893 (2019).30097802
49. Zhang Q-S Pathological α-synuclein exacerbates the progression of Parkinson’s disease through microglial activation Toxicol. Lett. 2017 265 30 37 10.1016/j.toxlet.2016.11.002 27865851
Zhang, Q.-S. et al. Pathological α-synuclein exacerbates the progression of Parkinson’s disease through microglial activation. Toxicol. Lett. 265, 30–37 (2017).27865851
50. Krüger R Genetic analysis of immunomodulating factors in sporadic Parkinson's disease J. Neural Transm. 2000 107 553 562 10.1007/s007020070078 11072751
Krüger, R. et al. Genetic analysis of immunomodulating factors in sporadic Parkinson’s disease. J. Neural Transm. 107, 553–562 (2000).11072751
51. Visñuk DP Neuroprotective effects associated with immune modulation by selected lactic acid bacteria in a Parkinson's disease model Nutrition 2020 79 110995 10.1016/j.nut.2020.110995 32977125
Visñuk, D. P. et al. Neuroprotective effects associated with immune modulation by selected lactic acid bacteria in a Parkinson’s disease model. Nutrition 79, 110995 (2020).32977125
52. Zahran EM Wound restorative power of halimeda macroloba/mesenchymal stem cells in immunocompromised rats via downregulating inflammatory/immune cross talk Marine Drugs 2023 21 6 336 10.3390/md21060336 37367661
Zahran, E. M. et al. Wound restorative power of halimeda macroloba/mesenchymal stem cells in immunocompromised rats via downregulating inflammatory/immune cross talk. Marine Drugs 21(6), 336 (2023).37367661
53. Tawfike A New bioactive metabolites from the elicited marine sponge-derived bacterium Actinokineospora spheciospongiae sp. nov AMB Express 2019 9 1 12 10.1186/s13568-018-0730-0 30680548
Tawfike, A. et al. New bioactive metabolites from the elicited marine sponge-derived bacterium Actinokineospora spheciospongiae sp. nov. AMB Express 9(1), 12 (2019).30680548
54. Macintyre L Metabolomic tools for secondary metabolite discovery from marine microbial symbionts Mar. Drugs 2014 12 6 3416 3448 10.3390/md12063416 24905482
Macintyre, L. et al. Metabolomic tools for secondary metabolite discovery from marine microbial symbionts. Mar. Drugs 12(6), 3416–3448 (2014).24905482
55. Pluskal T MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data BMC Bioinform. 2010 11 1 11 10.1186/1471-2105-11-395
Pluskal, T. et al. MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinform. 11, 1–11 (2010).
56. Flecknell P Anaesthesia of animals for biomedical research Br. J. Anaesth. 1993 71 6 885 894 10.1093/bja/71.6.885 8280560
Flecknell, P. Anaesthesia of animals for biomedical research. Br. J. Anaesth. 71(6), 885–894 (1993).8280560
57. Bancroft JD Gamble M Theory and Practice of Histological Techniques 2008 Elsevier Health Sciences
Bancroft, J. D. & Gamble, M. Theory and Practice of Histological Techniques (Elsevier Health Sciences, 2008).
58. Johansson AC Enhanced expression of iNOS intratumorally and at the immunization site after immunization with IFNγ-secreting rat glioma cells J. Neuroimmunol. 2002 123 1–2 135 143 10.1016/S0165-5728(01)00468-4 11880158
Johansson, A. C. et al. Enhanced expression of iNOS intratumorally and at the immunization site after immunization with IFNγ-secreting rat glioma cells. J. Neuroimmunol. 123(1–2), 135–143 (2002).11880158
59. Fossati P Prencipe L Berti G Use of 3, 5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine Clin. Chem. 1980 26 2 227 231 10.1093/clinchem/26.2.227 7353268
Fossati, P., Prencipe, L. & Berti, G. Use of 3, 5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine. Clin. Chem. 26(2), 227–231 (1980).7353268
60. Nishikimi M Rao NA Yagi K The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen Biochem. Biophys. Res. Commun. 1972 46 2 849 854 10.1016/S0006-291X(72)80218-3 4400444
Nishikimi, M., Rao, N. A. & Yagi, K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys. Res. Commun. 46(2), 849–854 (1972).4400444
61. Alharthy KM Barbigerone potentially alleviates rotenone-activated Parkinson’s disease in a rodent model by reducing oxidative stress and neuroinflammatory cytokines ACS Omega 2023 8 5 4608 4615 10.1021/acsomega.2c05837 36777578
Alharthy, K. M. et al. Barbigerone potentially alleviates rotenone-activated Parkinson’s disease in a rodent model by reducing oxidative stress and neuroinflammatory cytokines. ACS Omega 8(5), 4608–4615 (2023).36777578
62. Ahmed SR A Network Pharmacology Analysis of Cytotoxic Triterpenes Isolated from Euphorbia abyssinica Latex Supported by Drug-likeness and ADMET Studies 2022 ACS Omega
Ahmed, S. R. et al. A Network Pharmacology Analysis of Cytotoxic Triterpenes Isolated from Euphorbia abyssinica Latex Supported by Drug-likeness and ADMET Studies (ACS Omega, 2022).
63. Abou-Taleb HA Network pharmacological analysis of the red sea sponge hyrtios erectus extract to reveal anticancer efficacy of corresponding loaded niosomes Mar. Drugs 2022 20 10 628 10.3390/md20100628 36286452
Abou-Taleb, H. A. et al. Network pharmacological analysis of the red sea sponge hyrtios erectus extract to reveal anticancer efficacy of corresponding loaded niosomes. Mar. Drugs 20(10), 628 (2022).36286452
64. Piñero J The DisGeNET knowledge platform for disease genomics: 2019 update Nucleic Acids Res. 2020 48 D1 D845 D855 31680165
Piñero, J. et al. The DisGeNET knowledge platform for disease genomics: 2019 update. Nucleic Acids Res. 48(D1), D845–D855 (2020).31680165
65. Franz M Cytoscape. js: A graph theory library for visualisation and analysis Bioinformatics 2016 32 2 309 311 10.1093/bioinformatics/btv557 26415722
Franz, M. et al. Cytoscape. js: A graph theory library for visualisation and analysis. Bioinformatics 32(2), 309–311 (2016).26415722
66. Ge SX Jung D Yao R ShinyGO: A graphical gene-set enrichment tool for animals and plants Bioinformatics 2020 36 8 2628 2629 10.1093/bioinformatics/btz931 31882993
Ge, S. X., Jung, D. & Yao, R. ShinyGO: A graphical gene-set enrichment tool for animals and plants. Bioinformatics 36(8), 2628–2629 (2020).31882993
