# Evaluation of terpenes rich Hura crepitans extract on glucose regulation and diabetic complications in STZ-induced diabetic rats

Halimat Yusuf Lukman, Yucheng Kuo, Mayowa Solomon Owolabi, Bashir Lawal, Lung-Ching Chen, Olabode T Ajenifujah, Adewale O Fadaka, Femi Olawale, Sunday A Onikanni, Saidu Sani, et al.

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## To cite this version:

Halimat Yusuf Lukman, Yucheng Kuo, Mayowa Solomon Owolabi, Bashir Lawal, Lung-Ching Chen, et al. Evaluation of terpenes rich Hura crepitans extract on glucose regulation and diabetic complications in STZ-induced diabetic rats. Biomedicine and Pharmacotherapy, 2024, 179, 10.1016/j.biopha.2024.117308. hal-05084276

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# Evaluation of terpenes rich *Hura crepitans* extract on glucose regulation and diabetic complications in STZ-induced diabetic rats

Halimat Yusuf Lukman<sup>a,1</sup>, Yucheng Kuo<sup>b,c,1</sup>, Mayowa Solomon Owolabi<sup>d</sup>, Bashir Lawal<sup>e,f</sup>, Lung-Ching Chen<sup>g,h</sup>, Olabode T. Ajenifujah<sup>i</sup>, Adewale O. Fadaka<sup>j</sup>, Femi Olawale<sup>k</sup>, Sunday A. Onikanni<sup>l,m</sup>, Saidu Sani<sup>n</sup>, Michel De Waard<sup>o,p,q</sup>, Dalia Fouad<sup>r</sup>, Gaber El-Saber Batiha<sup>s</sup>, Saheed Sabiu<sup>a</sup>, Alexander T.H. Wu<sup>t,u,w</sup>, Hsu-Shan Huang<sup>v,w,x,y,w,w</sup>

## ARTICLE INFO

**Keywords:**

*Hura crepitans*

Streptozotocin

Oxidative stress

Inflammation

Diabetes complication

Bioactive compounds

## ABSTRACT

The continual increase in global diabetic statistics portends decreased productivity and life spans, thus making it a disease of concern requiring more effective and safe therapeutic options. While several reports on antidiabetic plants, including *Hura crepitans*, are available, there is still a dearth of information on the holistic antidiabetic properties of *H. crepitans* and its associated complications. This study evaluated the antidiabetic potential of methanolic extract of *Hura crepitans* using *in vitro*, *in vivo*, and *in silico* approaches. The extract revealed a dose-dependent *in vitro* effect, with a 47.97 % and 65.34 % decrease in the fasting blood sugar levels of streptozotocin (STZ) induced diabetic rats at 150 and 300 mg/kg BW, respectively. Likewise, the extract increased serum and

<footnote><sup>a</sup> Department of Biotechnology and Food Science, Faculty of Applied Sciences, Durban University of Technology, P.O. Box 1334, Durban 4000, South Africa</footnote>

<footnote><sup>b</sup> Department of Pharmacology, College of Medicine, Taipei Medical University, Taiwan</footnote>

<footnote><sup>c</sup> School of Post-Baccalaureate Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 40604, Taiwan</footnote>

<footnote><sup>d</sup> Department of Breast Cancer, Baylor College of Medicine, USA</footnote>

<footnote><sup>e</sup> UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, USA</footnote>

<footnote><sup>f</sup> Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA</footnote>

<footnote><sup>g</sup> Division of Cardiology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taiwan</footnote>

<footnote><sup>h</sup> School of Medicine, Fu Jen Catholic University, New Taipei 24205, Taiwan</footnote>

<footnote><sup>i</sup> Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA</footnote>

<footnote><sup>j</sup> Department of Biotechnology, University of The Western Cape, Belleville, South Africa</footnote>

<footnote><sup>k</sup> Nano Gene and Drug Delivery Group, University of KwaZulu-Natal, South Africa</footnote>

<footnote><sup>l</sup> Centro de Ciências da Saúde, Instituto de Ciências Biomédicas, Laboratório de Endocrinologia Experimental-LEEx, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-617, Brazil</footnote>

<footnote><sup>m</sup> Department of Chemical Sciences, Biochemistry Unit, Afe-Babalola University, Ado-Ekiti, Ekiti State, Nigeria</footnote>

<footnote><sup>n</sup> Alex Ekwueme-Federal University Ndufu-Alike, Ikwo, Nigeria</footnote>

<footnote><sup>o</sup> Smartox Biotechnology, 6 rue des Platanes, F-38120 Saint-Egrève, France</footnote>

<footnote><sup>p</sup> L'institut du thorax, INSERM, CNRS, UNIV NANTES, F-44007 Nantes, France</footnote>

<footnote><sup>q</sup> LabEx Ion Channels, Science & Therapeutics, Université de Nice Sophia-Antipolis, F-06560 Valbonne, France</footnote>

<footnote><sup>r</sup> Department of Zoology, College of Science, King Saud University, P.O. Box 22452, Riyadh 11495, Saudi Arabia</footnote>

<footnote><sup>s</sup> Department of Pharmacology and Therapeutics, Faculty of Veterinary Medicine, Damanhour University, Damanhour, AlBeheira 22511, Egypt</footnote>

<footnote><sup>t</sup> TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei 11031, Taiwan</footnote>

<footnote><sup>u</sup> The Ph.D. Program of Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan</footnote>

<footnote><sup>v</sup> PhD Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei 11031, Taiwan</footnote>

<footnote><sup>w</sup> Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei 11490, Taiwan</footnote>

<footnote><sup>x</sup> School of Pharmacy, National Defense Medical Center, Taipei 11490, Taiwan</footnote>

<footnote><sup>y</sup> PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan</footnote>

<footnote>* Corresponding author at: The Ph.D. Program of Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan.</footnote>

<footnote>** Corresponding author at: PhD Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei 11031, Taiwan.</footnote>

<footnote>E-mail addresses: chaw1211@tmu.edu.tw (A.T.H. Wu), huanghs99@tmu.edu.tw (H.-S. Huang).</footnote>

<footnote n="1">¹ These authors contributed equally</footnote>
pancreatic insulin levels, and significantly ameliorated neuronal oxidative stress and inflammation by reducing the expression levels of cholinesterase, NF-κB, and COX-2 in the brain of hyperglycemic rats. Serum dyslipidemia, liver, and kidney biomarker indices, and hematological alterations in diabetic rats were also significantly attenuated by the extract. Several constituents, mainly terpenes, were identified in the extract. To further predict the drug-likeness, pharmacokinetics, and binding properties of the compounds, in silico analysis was conducted. Ergosta-2,24-dien-26-oicacid, 18-(acetyloxy)-5,6-epoxy-4, 22-dihydroxy-1-oxo-, delta-1-lactone-4. beta., displayed the highest docking scores for acetylcholinesterase, butyrylcholinesterases, alpha-amylase, and nuclear factor-kB with values of -12.4, -10.9, -10.3, and -9.4 kcal/mol, while ergost-25-ene-6,12-dione, 3,5-dihydroxy-, (3. beta.,5 alpha.) topped for cyclooxygenase-2 (-9.0 kcal/mol). The top-ranked compounds also presented significant oral drug-likeness, pharmacokinetics, and safety properties. Altogether, our data provide preclinical evidence of the potential of *Hura crepitans* in ameliorating diabetes and its associated complications.

# 1. Introduction

The escalating global prevalence of diabetes mellitus (DM) presents a significant public health challenge, with numbers soaring from 151 million cases in 2000–415 million in 2015, further rising to 540 million in 2023. Projections indicate an increase to 643 million by 2030 and 783 million by 2045, underscoring the urgent need for proactive intervention [1–3]. According to the US National Diabetes Statistics Report for 2024, among U.S. adults aged 18 years or older, the prevalence of diagnosed diabetes ranges from 12.1% to 13.6% depending on demographic factors [2].

Type 2 diabetes mellitus (T2DM) constitutes approximately 90–95% of all diabetes cases and is primarily characterized by insulin resistance alongside pancreatic β-cell dysfunction, impairing the secretion of insulin. This peptide hormone is essential for regulating blood glucose levels by facilitating its uptake across cell membranes [4,5]. T2DM typically manifests in the later stages of life, with initial symptoms such as growth impairment, blurred vision, excessive urination, and weight loss often unnoticed until the condition advances, potentially leading to both microvascular (e.g., nephropathies, neuropathies, and retinopathies) and macrovascular (e.g., cardiovascular diseases and ischemic stroke) complications [6].

The pathophysiology of T2DM exacerbates the risk of cardiovascular diseases and disrupts lipoprotein metabolism, characterized by increased triacylglycerides, and low-density lipoproteins (LDLs) levels, alongside reduced high-density lipoproteins (HDLs) levels [7]. Additionally, hyperglycemic conditions foster the production of advanced glycation end-products (AGEs), activation of the nuclear factor (NF)-κB pathway, insulin action inhibition by neurotransmitters, and induction of inflammation and endothelial dysfunction, contributing to the overall diabetic pathology [8–10]. These effects may lead to tissue damage or organ failure, severely impacting life quality.

A pivotal strategy in managing T2DM involves regulating carbohydrate metabolism by inhibiting α-amylase and α-glucosidase activities in the gastrointestinal tract, thus moderating postprandial blood glucose spikes [11]. Although, synthetic drugs, including biguanides and sulfonylureas, among others, are commonly used, they often fall short in offering a comprehensive antidiabetic therapy, primarily due to safety concerns [12]. In contrast, natural products have been historically utilized for their therapeutic benefits across various ailments, attributed to their efficacy and minimal side effects. Research into medicinal plants has highlighted their potential antidiabetic properties [13]. However, the antidiabetic effects of numerous plants, including *Hura crepitans*, remain either unexplored or inadequately documented.

*Hura crepitans*, known colloquially as the “sandbox tree” among other names, is a member of the family Euphorbiaceae prevalent in tropical regions, growing up to 60 m tall and often planted for its shade. It is recognized for its protein, mineral and oil content. It is traditionally used for antimicrobial, anti-leprosy, anti-inflammatory, hepatoprotective, and emetic purposes [14–16]. Previous research by Igwenyi et al. [17] hinted at the antidiabetic potential of *H. crepitans*, but comprehensive documentation of its effects on diabetic complications and implicated metabolic pathways was lacking. The significance of

using *Hura crepitans* lies in its rich terpene content, which includes compounds known for their diverse biological activities [14]. Terpenes such as phytol and pentadecanoic acid have been reported for their antioxidant, anti-inflammatory, and lipid-modulating properties, which are beneficial in managing diabetes and its complications [15]. This study aims to investigate the comprehensive antidiabetic effects of *Hura crepitans*, focusing on its ability to regulate blood glucose levels, ameliorate oxidative stress, and address lipid and inflammatory dysregulations associated with diabetes.

# 2. Materials and methods

## 2.1. Materials

Experimental rats (*Rattus norvegicus*) weighing 118.78 ± 2.89 g were obtained from the animal holding unit of the Federal University of Technology, Minna (FUT Minna), Nigeria. The liver and kidney function assay kits used in this study were products of Randox Laboratories (Atrium, UK). Enzyme-linked immunosorbent assay (ELISA) kits for cholinesterase, nuclear factor (NF)-κB, serotonin, dopamine, and other biomarkers were products of Elabscience. The ELISA kit for insulin (catalog no: IN374S) was from Calbiotech (El Cajon, CA, USA). The ELISA reader was a product of Sunrise (Tecan, Austria). All other chemicals and reagents used were of analytical grade and were products of Sigma Aldrich (St. Louis, MO, USA).

## 2.2. Methods

### 2.2.1. Collection and preparation of extract

The leaves of *Hura crepitans* was collected within Minna, Nigeria. The plant was identified and authenticated at the herbarium unit of the FUT Minna, Nigeria where a voucher number was deposited. The plant extract was prepared by first rinsing with water, followed by air drying in the laboratory until a constant dry weight was obtained. The dried plant was ground into a fine powder, and 200 g of the powder was soaked in 1000 mL of methanol for three days with constant shaking for complete extraction. The mixture was then filtered through Whatman filter paper (no. 1), and the filtrate was concentrated under reduced pressure to obtain the crude *H. crepitans* methanolic extract (CHCME) which was preserved for future use.

### 2.2.2. In vitro hypoglycemic assays

The percentage glucose uptake by yeast cells was analyzed by incubating mixtures of various glucose concentrations with different concentrations of the CHCME and standard at 37°C for 10 minutes. A yeast suspension was prepared by centrifuging commercial baker's yeast repeatedly at 3000 rpm for 5 minutes with distilled water until a clear supernatant was obtained. This suspension was subsequently used to make a 10% v/v solution in distilled water. A 100 µL aliquot of the yeast suspension was then added to initiate the reaction, which was incubated for another 60 minutes at 37°C before the mixture was centrifuged, and the percentage glucose uptake was measured in the supernatant.

The α-amylase inhibitory effect of the CHCME was evaluated by
<table><caption>**Table 1**
Grouping and treatment administration of experimental rats.</caption><thead><tr><td>Group</td><td>Treatment</td></tr></thead><tbody><tr><td>1: Non-diabetic (normal control)</td><td>2 mL/kg BW normal saline</td></tr><tr><td>2: Diabetic untreated (negative control)</td><td>2 mL/kg BW normal saline</td></tr><tr><td>3: Diabetic + standard drug (positive control)</td><td>100 mg/kg BW metformin</td></tr><tr><td>4: Diabetic + extract</td><td>150 mg/kg BW CHCME</td></tr><tr><td>5: Diabetic + extract</td><td>300 mg/kg BW CHCME</td></tr></tbody></table>

BW: body weight; CHCME: crude *Hura crepitans* methanolic extract.

adding a porcine pancreatic enzyme solution in 0.02 M sodium phosphate buffer (pH 6.9) to the CHCME (50–400 µg/mL) and acarbose (standard) (12.5–100 µg/mL). The mixtures were incubated for 10 minutes at 37°C, and then 500 µL of 1 % starch solution (prepared in 0.02 M sodium phosphate buffer, pH 6.9 with 0.006 M NaCl) was added to initiate the reaction. This was incubated at 37°C for 30 minutes. The reaction was terminated by adding 10 µL of 1 M HCl, while iodine was used for color development. The absorbance was read at 580 nm, and the percentage inhibition was calculated as described by Worthington [18].

### 2.2.3. *In vivo* antidiabetic studies

In accordance with the ethical standards and laboratory conditions set by the FUT Minna and AE-FUNAI, Nigeria ethical committee (Ethical reference Number AE-FUNAI-2020/0099), meticulous care was taken in the handling of experimental animals. This included providing clean water, standard pelletized feed, and maintaining a 12-hour light/dark cycle. Initially, animals were categorized based on their body weight (BW) at Day 0. To induce diabetes, each animal received an intraperitoneal injection of 40 mg of streptozotocin (STZ) per kg of BW, followed by the administration of a 5 % glucose solution 24 hours later. Fasting

blood sugar (FBS) levels were measured on the 3rd day post-STZ administration to confirm the induction of diabetes. Rats exhibiting FBS levels of 250 mg/dL or above were classified as diabetic [19]. Subsequently, these diabetic animals were organized into four distinct groups, each comprising five animals, and were treated as outlined in Table 1. The FBS levels were closely monitored every three days throughout the two-week treatment period to assess the therapeutic effects of the extract. A preliminary acute toxicity test was conducted to determine the safe dose of the extract. The criteria for selecting the doses of CHCME used in the study were based on the safe doses identified from the toxicity test and supported by literature reporting the therapeutic doses of the extract. Specifically, the doses were chosen to ensure they were within the safety margin established during the acute toxicity assessment while being therapeutically relevant as indicated by previous research.

#### 2.2.3.1. Preparation of blood, serum, and tissue homogenates.
On the 14th day of treatment, animals were starved overnight, weighed, and anesthetized with diethyl ether. Blood was collected from the jugular vein into heparinized (for hematological analysis) and non-heparinized bottles. Blood in the non-heparinized bottles was allowed to clot and then centrifuged at 3000 rpm for 15 min to collect the serum for biochemical analysis [20,21]. Organs (liver, kidney, pancreas, and brain) were harvested, cleaned of blood stains, weighed, then homogenized in a 0.25 M sucrose solution [22,23], and centrifuged at 4000 rpm for 10 min to obtain supernatants for biochemical analyses. All samples were properly stored in a refrigerator for further use.

#### 2.2.3.2. Determination of insulin levels.
The serum and pancreatic insulin levels (µIU/mL) were determined with an enzyme-linked

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** *In vitro* hypoglycemic activities of the crude *Hura crepitans* methanolic extract. Values are presented as the mean ± standard error of mean of replicate determinations. Values in each line with different superscripts significantly (p<0.05) differ.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Effect of crude *Hura crepitans* methanolic extract on FBS, body weights of STZ-induced diabetic rats. Values are presented as the mean ± standard error of mean of replicate determinations. Values in each line represent Mean ± SEM of replicate determinations. BW, body weight. \*\*p < 0.01, \*\*\*p < 0.001 significantly different compared with the diabetic untreated control.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Effect of crude *Hura crepitans* methanolic extract on serum and pancreatic insulin levels of STZ-induced diabetic rats. Values are presented as the mean ± standard error of mean of replicate determinations. Each bar represents mean ± SEM of replicate values. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001, significantly difference compared with the diabetic untreated control.</figcaption></figure>

immunosorbent assay (ELISA) kit following the manual's instructions. The insulin ELISA kits (catalog no. IN374S; Calbiotech, El Cajon, CA, USA) was used for the assay and read with an ELISA reader (Sunrise, Tecan, Austria).

2.2.3.3. *In vivo* antioxidant analyses. Superoxide dismutase (SOD) activity was assessed using 0.05 mol/L carbonate buffer (pH 10.2), 0.2 mL of the tissue homogenate, and 0.3 mmol/L epinephrine, monitoring

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** *In vivo* anti-inflammatory effects of crude *Hura crepitans* methanolic extract (CHCME) on (a) acetyl cholinesterase (ACHE), (b) butyryl cholines-terase (BCHE) activities in the brain of STZ-induced diabetic rats. Values are presented as mean ± standard error of mean of replicate determinations. \* *p*<0.05, \*\* *p*<0.01, \*\*\* *p*<0.001.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** *In vivo* antioxidant effects of crude *Hura crepitans* methanolic extract on superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and malondialdehyde (MDA) concentrations in the brain and pancreas of STZ-induced diabetic rats. Values are presented as mean ± standard error of mean of replicate determinations. \* *p*<0.05, \*\* *p*<0.01, \*\*\* *p*<0.0001. BW, body weight.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** *In vivo* anti-inflammatory effects of the crude *Hura crepitans* methanolic extract (CHCME) on (a) cyclooxygenase (COX)-2 activity, (b) nuclear factor (NF)-κB level, and (c) nitrogen oxide (NO) level in the brains of rats with STZ-induced diabetes. Values are presented as mean ± standard error of mean of replicate determinations. \* *p*<0.05, \*\* \*p*<0.001, \*\*\* \*p*<0.0001.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Serum lipid profile of rats with streptozotocin-induced diabetes treated with the crude *Hura crepitans* methanolic extract (CHCME). Values are presented as the mean ± standard error of mean of replicate determinations. Values are presented as mean ± standard error of mean of replicate determinations. \*\*\* \*p*<0.0001. BW, body weight.</figcaption></figure>

absorbance changes at 480 nm over 90 seconds, following Misra [24]. Catalase (CAT) activity measurement involved mixing 1 mL of 0.01 M phosphate buffer (pH 7.0) with 0.4 mL of 0.2 M H2O2 and 0.1 mL of the tissue homogenate, terminating the reaction with dichromate acetic acid reagent, and reading absorbance at 620 nm as per Sinha [25]. Reduced glutathione (GSH) levels were determined by precipitating the tissue homogenate with trichloroacetic acid (TCA), the resulting supernatant was mixed with phosphate buffer and DNTB, incubated at 37 °C, and the absorbance was measured at 412 nm to calculate GSH concentration using Kum-Tatt's colorimetric method [26]. Lipid peroxidation was quantified by reacting the sample with thiobarbituric acid, HCl, and TCA, incubating, cooling, centrifuging, and measuring the supernatant's absorbance at 535 nm against a blank, according to Shagirtha et al. [27].

### 2.2.3.4. Cholinesterase and inflammatory biomarker analyses.
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured by adding phosphate buffer (0.1 M, pH 8.0), DTNB (10 mM), 50 µL of cytosol, and 150 mM of acetylthiocholine iodide (AChE assay) or 150 mM of butyryl thiocholine iodide (BChE assay) to the tissue

homogenate and incubated. The changes in the absorbance were monitored at 412 nm for 3 min as described Ellman *et al.* [28]. Cyclo-oxygenase (COX)-2 activity was measured using a rat prostaglandin endoperoxide synthase 2 (PTGS2)/COX-2 ELISA Kit (catalog no. E-EL-R0792, Elabscience, USA), and NF-κB was measured using a rat nuclear factor NF-kappa-B p105 subunit ELISA kit (catalog no. E-EL-R0673, Elabscience). This was based on color development when rat NF-κB-p105 was conjugated with a rat NF-κB-p105-specific biotinylated detection antibody and avidin-horseradish peroxidase (HRP) conjugate. Nitrate was reduced to nitrite to measure levels of nitric oxide (NOx) as described by Miranda *et al.* [29].

### 2.2.3.5. Hematological analyses.
Serum analyses were carried out on an Olympus AU800 automated analyzer (Olympus, Japan) using standard protocols; these include alanine aminotransferase (ALT) [30], aspartate aminotransferase (AST) [30], alkaline phosphatase (ALP) [31], total protein (TP) [32], total and direct bilirubin (TB and DB) [33], albumin (ALB) [34], creatinine [35], and urea [36]. The lipid profiling of the serum was conducted using colorimetric analyses and included the
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** : Effects of crude *Hura crepitans* methanolic extract (CHCME) treatment on liver function biomarkers in the serum of rats with streptozotocin-induced diabetes. Values are presented as the mean ± standard error of mean of replicate determinations. Values in each bar with different superscripts significantly (p<0.05) differ. BW, body weight; ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase; TP, total protein; ALB, albumin; TB, total bilirubin; DB, direct bilirubin. Values are presented as mean ± standard error of mean of replicate determinations. *** p<0.001, **** p<0.0001.</figcaption></figure>

high-density lipoprotein (HDL)-cholesterol [37], triglycerides (TGs) [38], and total cholesterol (TC). Low-density lipoprotein (LDL)-cholesterol (mg/dl) was calculated as TC – (HDL + very LDL (VLDL)) [39], and the serum electrolyte concentration was measured as described by Tietz [40]. A full blood count analysis was carried out with an automated hematologic analyzer (Sysmex, KX-21, Japan) which included hemoglobin (HGB), packed cell volume (PCV), RBCs, mean corpuscular hemoglobin (MCH), mean cell volume (MCV), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBCs), and platelet (PLT) counts.

### 2.2.4. Characterization of the CHCME with a gas chromatographic-mass spectrometry (GC-MS) analysis

The CHCME was prepared by diluting with chloroform: methanol (2:1), followed by vortexing, sonication at room temperature for 30 minutes, and centrifugation at 3000 rpm for 1 minute. The extract was then dried under nitrogen, reconstituted in pyridine and Bis (trimethylsilyl) trifluoroacetamide (BSTFA), and analyzed using GC-MS (6890 N, Agilent Technologies with MSD 5975, Agilent Technologies, and ZB-5MS capillary column). Helium served as the carrier gas at 1 mL/min. The injector and oven temperatures were set to 250 °C and programmed from 100 °C (4 min) to 300 °C at 7 °C/min, holding for 6 min.

The MSD operated in EI mode at 70 eV, scanning from 40 to 650 m/z.

## 2.3. *In silico* analysis

### 2.3.1. Molecular docking analysis

The crystal structures of cholinesterase AChE (PDB ID: 1H23), BChE (PDB ID: 5LKR), inflammatory COX-2 (PDB ID: 5F19), NF-kB (PDB ID: 1NFK), NOx (PDB ID: 1B1C), and antidiabetes alpha-amylase (PDB ID: 4GQR)] targets were obtained from the RSCB Protein Data Bank (PDB) (https://www.rcsb.org/) (accessed on 05/04/2024) while the compound structures were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) (accessed on 04/04/2024). The targets and compound structures were prepared and saved for docking using UCSF Chimera software V1.14 [41] and Open babel plug-in of PyRx, respectively [42]. The docking was done using the Autodock Vina plug-in of the PyRx software [42] with consistent grid box space and coordinates (x, y, z) for each target. The docking process was validated by ensuring that the compounds bind at target active sites as the native ligands. Root mean square deviation (RMSD) of 0.5 Å was obtained after optimal superimposition of the native ligand and the top-ranked compounds with each target (Figure S1 and Table S1) [43]. Validation of each resulting complex was done using Discovery Studio v21.1.0 [44].
<figure><img src="image_12.png" /><figcaption>**Fig. 9.** Effects of crude *Hura crepitans* methanolic extract (CHCME) treatment on kidney function biomarkers and electrolyte concentrations in the serum of rats with streptozotocin-induced diabetes. Values are presented as mean ± standard error of mean of replicate determinations. \*\* *p*<0.01, \*\*\* \*p*<0.0001.</figcaption></figure>

#### 2.3.1.1. Pharmacokinetics, drug-likeness, and toxicity studies.
The absorption, distribution, metabolism, elimination and toxicity (ADMET) and drug-likeness properties of the top-ranked compounds of CHEME were predicted using their SMILES (retrieved from PubChem) on the SWISS ADME server (http://www.swissadme.ch/) and the ProTox server (https://tox-new.charite.de/protox_II/) (accessed on 07/04/2024) [45,46].

### 2.3.2. Data analyses

For the *in vitro* and *in vivo* analyses, the result presented are expressed as the mean ± standard error of the mean (SEM) of three determinations. One-way analysis of variance (ANOVA) using Duncan's multiple-range test was considered statistically different at a significance level of *p*<0.05. Graph pad prism (v 8.0) was used for all the statistical analyses.

## 3. Results

### 3.1. Hypoglycemic effects of crude *Hura crepitans*: *in vitro* analysis of glucose uptake and alpha-amylase inhibition

The *in vitro* hypoglycemic activities of *Hura crepitans* methanolic extract are illustrated in Fig. 1. There was a notable decrease in glucose uptake by cells with an increasing concentration of the extract, in the

presence of varying yeast cell concentrations. Specifically, at a yeast concentration of 5 mM, glucose uptake increased by 31.50%±0.48% at an extract concentration of 50 mg/dL and by 95.13%±0.08% at 400 mg/dL. Conversely, at a yeast concentration of 25 mM, the glucose uptake enhancement was 17.26%±0.14% at 50 mg/dL and 48.39% ±0.02% at 400 mg/dL (Fig. 1a). Moreover, a dose-dependent increase in the inhibition of alpha-amylase activity was observed with escalating concentrations of the extract, indicating the potential mechanism of its hypoglycemic effect (Fig. 1b).

### 3.2. CHCME reduced blood glucose levels, improved body weights and insulin levels of STZ-induced diabetic rats

Streptozotocin (STZ) induction led to elevated fasting blood sugar (FBS) levels in rats, signifying the onset of diabetes. Notably, subsequent treatment with CHCME resulted in a significant (*p*<0.001) reduction in FBS levels, showcasing a significant improvement over the standard treatment (Fig. 2a). Furthermore, CHCME treatment notably enhanced body weights (BWs) of diabetic rats (*p*<0.01), in stark contrast to the untreated diabetic group, which experienced a weight decline of 31.76% from day 1 to day 18 of the study (Fig. 2b). While the liver to body weight ratio exhibited noticeable changes, no significant differences (*p*>0.05) were observed in other organ weights when compared to the untreated diabetic rats (Figure S2). Serum insulin levels were lower
<figure><img src="image_13.png" /><figcaption>**Fig. 10.** Hematological parameters in the plasma of rats with streptozotocin-induced diabetes treated with the crude *Hura crepitans* methanolic extract (CHCME). Values are presented as mean ± standard error of mean of replicate determinations. \* *p*<0.05, \*\* *p*<0.01. Hb, hemoglobin; PCV, packed cell volume; MCV, mean cell volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RBCs, red blood cells; PLC, platelet count; TWBCs, total white blood cells; N, neutrophils; L, lymphocytes; M, monocytes; E, eosinophils.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>Fig. 11. GC-MS chromatogram of the crude *Hura crepitans* methanolic extract.</figcaption></figure>

in the untreated group but significantly increased upon treatment with CHCME (Fig. 3a). Analysis of pancreatic insulin levels revealed that rats treated with metformin and 150 mg/kg BW of CHCME displayed higher insulin levels, unlike the 300 mg CHCME/kg BW group, which did not significantly (p>0.05) differ from the untreated diabetic rats (Fig. 3b).

### 3.3. *In vivo antioxidant activity-stimulatory effects of the CHCME in rats with STZ-induced diabetes*

The antioxidative properties and lipid peroxidation (LPO) biomarker effects of CHCME are detailed in Fig. 4 and S3. Impressively, the extract exhibited beneficial effects against oxidative stress in both neuronal (Fig. 4a) and pancreatic tissues (Fig. 4b) of rats with STZ-induced diabetes. CHCME significantly (p<0.01) enhanced the free radical-scavenging activities, as evidenced by dose-dependent increases in superoxide dismutase (SOD) and catalase (CAT) activities across all examined tissues, compared with the diabetic untreated group. Similarly, an upsurge in glutathione (GSH) production was recorded in the extract-treated groups across all tissues (Fig. 4 and Figure S3). Furthermore, an elevation in malondialdehyde (MDA) levels, indicative of lipid peroxidation, was noted in STZ-induced diabetic rats. However, CHCME treatment led to significant reductions in MDA levels (p<0.001), aligning them closely with those of the normal control group (Fig. 4 and Figure S3).

### 3.4. Modulation of neuronal cholinesterase activities and neuroinflammatory markers by CHCME in STZ-induced diabetic rats

Figs. 5 and 6 illustrate the effects of CHCME on neuronal cholinesterase activities and neuroinflammatory markers in STZ-induced

diabetic rats. Compared to normal controls, diabetic rats exhibited increased activities of both AChE (Fig. 5a) and BChE (Fig. 5b). Treatment with CHCME at both 150 mg/kg and 300 mg/kg body weight effectively mitigated these heightened cholinesterase activities, aligning them closely with those observed in the metformin-treated group and the normal control. The attenuation of AChE activity by CHCME treatment was noted to be dose-independent, showing effectiveness similar to that of the normal control group. Moreover, neuroinflammatory markers such as COX-2, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and NOx levels, which were significantly higher in untreated diabetic rats, were markedly reduced following CHCME treatment, reaching levels akin to those in the normal control group (Fig. 6a-c). Despite these changes in inflammatory markers and cholinesterase activities, the levels of neurotransmitters serotonin and dopamine remained consistent across all experimental groups, indicating no significant impact of the treatment on these neurotransmitters (Figure S4).

### 3.5. Lipid profiles of rats with STZ-induced diabetes treated with the CHCME

Fig. 7 showcases the lipid profiles of rats with STZ-induced diabetes following treatment with CHCME. In diabetic untreated rats, decrease level of high-density lipoprotein (HDL), and elevated triglycerides (TAG), low-density lipoprotein (LDL), and total cholesterol levels were observed. The administration of the standard treatment or CHCME notably improved these lipid parameters, restoring them to levels akin to those observed in healthy, normal rats. Notably, the most pronounced effects were seen in the group treated with 300 mg/kg body weight (bw) of CHCME, indicating its potent lipid-modulating capacity.
<table><caption>**Table 2**
Gas chromatographic-mass spectrometric analysis of the crude *Hura crepitans* methanolic extract.</caption><thead><tr><th>Ret. time (min)</th><th>Area (%)</th><th>Height (%)</th><th>A/H</th><th>Name</th><th>Molecular weight</th><th>Similarity index</th></tr></thead><tbody><tr><td>11.37</td><td>0.25</td><td>0.31</td><td>2.84</td><td>Farnesene epoxide</td><td>220</td><td>72</td></tr><tr><td>11.421</td><td>0.33</td><td>0.31</td><td>3.6</td><td>Eicosane</td><td>282</td><td>86</td></tr><tr><td>11.819</td><td>0.13</td><td>0.24</td><td>1.81</td><td>Phenol,2,6-bis(1,1-dimethylethyl)-</td><td>206</td><td>80</td></tr><tr><td>11.949</td><td>0.68</td><td>1</td><td>2.34</td><td>6,10-Dodecadien-1-yn-3-ol,3,7,11-trimethyl-</td><td>220</td><td>84</td></tr><tr><td>12.556</td><td>0.29</td><td>0.28</td><td>3.52</td><td>2-Carene</td><td>136</td><td>82</td></tr><tr><td>14.75</td><td>0.44</td><td>0.48</td><td>3.18</td><td>Tetradecanoic acid</td><td>228</td><td>91</td></tr><tr><td>15.046</td><td>0.22</td><td>0.3</td><td>2.48</td><td>2,6,10-Dodecatrien-1-al,12-(acetoxy)-2,6,10-trimethyl-, (E,E,E)-</td><td>278</td><td>76</td></tr><tr><td>15.383</td><td>0.41</td><td>0.57</td><td>2.48</td><td>Methyl4,7,10,13-hexadecatetraenoate</td><td>262</td><td>78</td></tr><tr><td>15.474</td><td>4.41</td><td>8.28</td><td>1.84</td><td>Phytol, acetate</td><td>338</td><td>93</td></tr><tr><td>15.6</td><td>0.24</td><td>0.38</td><td>2.15</td><td>Benzene (1-propyldecyl)-</td><td>260</td><td>77</td></tr><tr><td>15.795</td><td>0.42</td><td>0.36</td><td>3.99</td><td>1,2-Benzenedicarboxylicacid,bis(2-ethylpropyl)ester</td><td>278</td><td>77</td></tr><tr><td>15.925</td><td>2.55</td><td>3.47</td><td>2.54</td><td>3,7,11,15-Tetramethyl-2-hexadecen-1-ol</td><td>296</td><td>92</td></tr><tr><td>16.292</td><td>1.24</td><td>2.03</td><td>2.11</td><td>5,9,13-Pentadecatrien-2-one,6,10,14-trimethyl-, (E, E)-</td><td>262</td><td>88</td></tr><tr><td>16.396</td><td>0.49</td><td>0.96</td><td>1.76</td><td>Hexadecanoic acid, methylester</td><td>270</td><td>91</td></tr><tr><td>16.901</td><td>6.51</td><td>6.11</td><td>3.68</td><td>Pentadecanoic acid</td><td>242</td><td>92</td></tr><tr><td>17.043</td><td>0.64</td><td>0.61</td><td>3.58</td><td>1,5,9-Decatriene,2,3,5,8-tetramethyl-</td><td>192</td><td>82</td></tr><tr><td>17.183</td><td>0.47</td><td>0.57</td><td>2.83</td><td>Androst-1-en-3-one,17-hydroxy-, (5. alpha.,17.beta.)-</td><td>288</td><td>73</td></tr><tr><td>17.225</td><td>0.41</td><td>0.42</td><td>3.38</td><td>9-Methyl-Z-10-pentadecen-1-ol</td><td>240</td><td>60</td></tr><tr><td>17.366</td><td>0.53</td><td>0.45</td><td>4.03</td><td>2,6,9,11-Dodecatetraenal,2,6,10-trimethyl-, (E, E, E)-</td><td>218</td><td>72</td></tr><tr><td>17.433</td><td>0.35</td><td>0.41</td><td>2.95</td><td>Nerolidylacetate</td><td>264</td><td>82</td></tr><tr><td>17.695</td><td>0.2</td><td>0.37</td><td>1.91</td><td>5-Methyl-Z-5-docosene</td><td>322</td><td>63</td></tr><tr><td>17.833</td><td>1.01</td><td>0.82</td><td>4.27</td><td>Eicosanoic acid</td><td>312</td><td>87</td></tr><tr><td>17.961</td><td>0.68</td><td>0.59</td><td>3.94</td><td>10-Methyldodecan-4-olide</td><td>212</td><td>74</td></tr><tr><td>18.136</td><td>1.01</td><td>1.08</td><td>3.24</td><td>8,11,14-Docosatrienoic acid, methylester</td><td>348</td><td>86</td></tr><tr><td>18.258</td><td>4.52</td><td>5.91</td><td>2.64</td><td>Phytol</td><td>296</td><td>97</td></tr><tr><td>18.375</td><td>0.72</td><td>1.01</td><td>2.46</td><td>Methylstearate</td><td>298</td><td>93</td></tr><tr><td>18.619</td><td>4.68</td><td>4.54</td><td>3.56</td><td>9,12,15-Octadecatrienoic acid, (Z,Z,Z)-</td><td>278</td><td>90</td></tr><tr><td>18.829</td><td>4.06</td><td>4.23</td><td>3.31</td><td>Octadecanoic acid</td><td>284</td><td>96</td></tr><tr><td>19.014</td><td>1.3</td><td>0.95</td><td>4.73</td><td>E-2-Octenyltiglate</td><td>210</td><td>69</td></tr><tr><td>19.319</td><td>0.65</td><td>0.58</td><td>3.87</td><td>6-epi-shyobunol</td><td>222</td><td>73</td></tr><tr><td>19.397</td><td>0.44</td><td>0.56</td><td>2.7</td><td>Geranylisovalerate</td><td>238</td><td>70</td></tr><tr><td>19.479</td><td>1.02</td><td>0.88</td><td>3.97</td><td>Eicosanoicacid,2-hydroxyethylester</td><td>356</td><td>83</td></tr><tr><td>19.809</td><td>0.69</td><td>0.56</td><td>4.23</td><td>1-Decanol,5,9-dimethyl-</td><td>186</td><td>64</td></tr><tr><td>20.146</td><td>0.58</td><td>0.55</td><td>3.65</td><td>Alpha-farnesene</td><td>204</td><td>60</td></tr><tr><td>20.41</td><td>0.78</td><td>0.56</td><td>4.75</td><td>1,6,10,14,18,22-Tetracosahexaen-3-ol,2,6,10,15,19,23-hexamethyl-, (all-E)-</td><td>426</td><td>73</td></tr><tr><td>20.492</td><td>1.7</td><td>1.33</td><td>4.39</td><td>4,8,12,16-Tetramethylheptadecan-4-olide</td><td>324</td><td>84</td></tr><tr><td>20.682</td><td>2.13</td><td>1.89</td><td>3.88</td><td>trans-Geranylgeraniol</td><td>290</td><td>92</td></tr><tr><td>21.156</td><td>0.9</td><td>0.51</td><td>6.04</td><td>2-(2,7-Dimethylocta-2,7-dienyl) malonic acid, diethyl ester</td><td>296</td><td>72</td></tr><tr><td>21.355</td><td>0.51</td><td>0.46</td><td>3.82</td><td>Octadecanoicacid,2-hydroxyethylester</td><td>328</td><td>68</td></tr><tr><td>21.725</td><td>0.38</td><td>0.4</td><td>3.26</td><td>Ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3.beta.,5.alpha.)-</td><td>444</td><td>58</td></tr><tr><td>21.971</td><td>0.49</td><td>0.56</td><td>3.06</td><td>Ergosta-2,24-dien-26-oicacid,18-(acetyloxy)-5,6-epoxy-4, 22-dihydroxy-1-oxo-, delta-lactone,(4.beta.,</td><td>512</td><td>47</td></tr><tr><td>24.051</td><td>3.21</td><td>1.2</td><td>9.25</td><td>2-methylhexacosane</td><td>380</td><td>86</td></tr><tr><td>24.49</td><td>1.53</td><td>0.45</td><td>11.85</td><td>Fumaric acid, decylpropargylester</td><td>294</td><td>64</td></tr></tbody></table>

Ret., retention; A/H,

<table><caption>**Table 3**
Docking scores of the top-five ranked compounds of *Hura crepitans* with cholinesterase, inflammatory and diabetes targets.</caption><thead><tr><th rowspan="2">Compounds</th><th colspan="6">Docking score (kcal/mol)</th></tr><tr><th>AChE</th><th>BChE</th><th>COX-2</th><th>NFKb</th><th>NOx</th><th>A-amylase</th></tr></thead><tbody><tr><td>Ergosta-2,24-dien-26-oicacid,18-(acetyloxy)-5,6-epoxy-4, 22-dihydroxy-1-oxo-, delta-lactone,(4.beta., Ergosta-25-ene-6,12-dione,3,5-dihydroxy-, (3.beta.,5.alpha.)</td><td>-12.4</td><td>-10.9</td><td>-8.8</td><td>-9.4</td><td>NA</td><td>-10.3</td></tr><tr><td>Trans-geranylgeraniol</td><td>-10.7</td><td>-9.4</td><td>-9.0</td><td>-9.0</td><td>NA</td><td>-8.4</td></tr><tr><td>Androst-1-en-3-one,17-hydroxy-, (5. alpha.,17.beta.)</td><td>-9.2</td><td>NA</td><td>-7.9</td><td>-7.1</td><td>NA</td><td></td></tr><tr><td>5,9,13-Pentadecatrien-2-one,6,10,14-trimethyl-, (E, E)-</td><td>-9.8</td><td>-9.5</td><td>NA</td><td>-7.2</td><td>-6.6</td><td>-9.1</td></tr><tr><td>4,8,12,16-Tetramethylheptadecan-4-olide</td><td>-8.9</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>2,6,10-Dodecatrien-1-al,12-(acetoxy)-2,6,10-trimethyl-, (E,E,E)-</td><td>NA</td><td>NA</td><td>-7.9</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Alpha-farnesene</td><td>NA</td><td>NA</td><td>NA</td><td>-6.8</td><td>NA</td><td>NA</td></tr><tr><td>2,6,9,11-Dodecatetraenal,2,6,10-trimethyl-, (E, E, E)-</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>-6.5</td><td>NA</td></tr><tr><td>6-epi-shyobunol</td><td>NA</td><td>-7.4</td><td>NA</td><td>NA</td><td>-6.5</td><td>-6.4</td></tr><tr><td>Tetradecanoic acid</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>-6.5</td><td>-</td></tr><tr><td>Phenol,2,6-bis(1,1-dimethylethyl)-</td><td>NA</td><td>-7.6</td><td>-7.8</td><td>NA</td><td>NA</td><td>-6.8</td></tr></tbody></table>

NA: Not applicable
<figure><img src="image_15.png" /><figcaption>**Fig. 12a.** 2D interaction plots of acetylcholine with (a) trans-geranylgeraniol (b) 5,9,13-Pentadecatrien-2-one, 6,10,14-trimethyl-, (E, E)- (c) ergost-25-ene-6,12-dione, 3,5-dihydroxy-, (3.beta., 5.alpha.), (d) ergosta-2,24-dien-26-oicacid, 18-(acetyloxy)-5,6-epoxy-4,22-dihydroxy-1-oxo-, delta-lactone, (4.beta., and (e) androst-1-en-3-one, 17-hydroxy-, (5. alpha., 17.beta.).</figcaption></figure>

**3.6. The CHCME improved associated dysfunction in liver and kidney biomarkers in rats with STZ-induced diabetes**

The STZ-induced diabetic rats that were left untreated exhibited significantly elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin. However, these elevated biomarkers were significantly reduced (p<0.05) following the administration of CHCME, demonstrating the extract's potential in mitigating liver damage. Additionally, CHCME treatment led to improvements in total protein and albumin (ALB) levels among the diabetic rats (Fig. 8). The kidney biomarkers and serum electrolyte concentrations, which were significantly elevations in the diabetic rats, were notably reduced (p<0.05) following treatment with CHCME (Fig. 9). This underscores CHCME's beneficial effects not only on liver function but also in addressing renal stress markers and maintaining electrolyte balance in STZ-induced diabetic rats.

**3.7. Hematological parameters in the plasma of rats with STZ-induced diabetes**

Fig. 10 shows the effect of crude *Hura crepitans* methanolic extract (CHCME) on the hematological parameters in STZ-induced diabetic rats. In untreated diabetic rats, notable decreases (p<0.05) were observed in hemoglobin (HGB), packed cell volume (PCV), mean corpuscular hemoglobin (MCH), and red blood cell (RBC) counts, alongside lymphocyte, and monocyte counts. However, these parameters exhibited significant improvement following CHCME treatment. Conversely, the

total white blood cell, neutrophils, mean corpuscular volume (MCV), and plateletcrit (PLC), counts, which were elevated in diabetic rats, were significantly (p<0.05) reduced upon administering CHCME, aligning closer to levels seen in healthy controls. Notably, the treatment with CHCME resulted in PCV, MCV, RBC, and monocyte counts that were comparable to those observed in the normal control group.

**3.8. GC-MS chromatogram of CHCME**

The gas chromatography-mass spectrometry (GC-MS) analysis of the crude *Hura crepitans* methanolic extract identified a diverse array of bioactive compounds (Fig. 11, Table 2), shedding light on its potential therapeutic applications. Among the compounds, phytol (4.52 %), pentadecanoic acid (6.51 %), phytol acetate (4.41 %), 9,12,15-octadecatrienoic acid (ZZZ)- (4.68 %), and octadecanoic acid (4.06 %) stood out based on their area (%), indicating their significant presence in the extract. However, phytoconstituents have various biological importance, hence all the compounds profiled were docked against the biomarkers studied in the *in vitro* and *in vivo* analysis.

**3.9. Docking scores of the top-five ranked compounds of *Hura crepitans* with cholinesterase, inflammatory and antidiabetic targets**

The docking scores of AChE, BChE, COX-2, NFKb, NOx, and alpha-amylase with the top-ranked compounds of *H. crepitans* are presented in Table 3. Noteworthy, all the top-ranked compounds had higher docking scores than the standard reference. For AChE, BChE, NFKb, and
<figure><img src="image_16.png" /><figcaption>**Fig. 12b.** 2D interaction plots of butyrylcholinesterase with (a) ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3.beta.,5 alpha.), (b) ergosta-2,24-dien-26-oicacid,18-(acetyloxy)-5,6-epoxy-4,22-dihydroxy-1-oxo-, delta-lactone,(4.beta., (E, E)- (c) 6-epi-shyobunol (d) androst-1-en-3-one,17-hydroxy-, (5. alpha.,17.beta.) and (e) phenol,2,6-bis(1,1-dimethylethyl)-.</figcaption></figure>

alpha-amylase, all the top-five compounds displayed significant high docking scores with ergosta-2,24-dien-26-oicacid,18-(acetyloxy)-5,6-epoxy-4, 22-dihydroxy-1-oxo-, delta-lactone,(4.beta., (Erg-2,24) having the highest score of -12.4, -10.9, -9.4, and -10.3 kca/mol for the targets, respectively (Table 3 and Tables S1). Notably, metformin did not bind with AChE. For COX-2, ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3.beta.,5 alpha.) ranked top among the five compounds and the standard with a score of -9.0 kcal/mol while androst-1-en-3-one,17-hydroxy-, (5. alpha.,17.beta.) had the highest docking score for NOx (Table 3).

The 2D interaction plots of the targets with their top-ranked compounds of *H. crepitans* is displayed in Fig. 12a-f. Several numbers of interactions and bonds were observed in each docked complex. With AChE, trans-geranylgeraniol had 19 interactions including 1-hydrogen (H), 13 van der Waals (VDW), 3 pi-alkyl and 2 pi-sigma bonds. 5,9,13-Pentadecatrien-2-one,6,10,14-trimethyl-, (E, E)- had 16 interactions comprising 2-H bonds, 10 VDW, 3 pi-alkyl and 1 pi-sigma bonds, while ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3.beta.,5.alpha.) presented 13 interactions with 1 H, 10 VDW, 1 pi-alkyl and pi-sigma bonds each. The Erg-2,24 compounds interacted with AChE with 20 interactions of 3-H, 13 VDW, 1 unfavourable donor-donor (UDD), and 3 pi-sigma bonds while androst-1-en-3-one,17-hydroxy-, (5. alpha.,17.beta.) had 13 interactions of 11 VDW and 2 pi-alkyl bonds (Fig. 12a). The 2D docking interaction of the top-five compounds of *H. crepitans* with BChE is presented in Fig. 12b. Ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3.beta.,5. alpha.) interacted with 17 bonds including 1-H, pi-alkyl, pi-sigma and 14 VDW bonds, Erg-2,24 interacted with 19 bonds comprising 3-H, 1

unfavorable acceptor-acceptor (UAA) and 15 VDW bonds, 6-epi-shyobunol interacted with 12 bonds of 3 alkyl, 2 sigma and 7 VDW. Androst-1-en-3-one,17-hydroxy-, (5. alpha.,17.beta.) formed 15 interactions with 3-H, 1 pi-sigma, 2-alkyl and 9 VDW bonds while phenol,2,6-bis(1,1-dimethylethyl)- had 12 interactions comprising 1 pi-sigma, 2 pi-alkyl and 9 VDW bonds (Fig. 12b).

The 2D docking interaction of the top-five compounds of *H. crepitans* with COX-2 is presented in Fig. 12c. Ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3.beta.,5.alpha.) interacted with 16 bonds including 1-H, pi-alkyl, and 14 VDW bonds, Erg-2,24 interacted with 13 VDW bonds, trans-geranylgeraniol interacted with 21 bonds of 1-H, 9 alkyl, and 11 VDW. phenol,2,6-bis(1,1-dimethylethyl)-formed 15 interactions with 6 pi-alkyl and 9 VDW bonds while 4,8,12,16-Tetramethylheptadecan-4-olide had 19 interactions comprising 1-H, 9 pi-alkyl and 9 VDW bonds (Fig. 12c). For NFKb ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3. beta.,5.alpha.) interacted with 23 bonds including 1-H, 1 UAA, and 21 VDW bonds, Erg-2,24 interacted with 22 bonds including 1-H, 1 pi-UAA/UDD, and 20 VDW bonds, trans-geranylgeraniol interacted with 20 bonds including 1-H, 2 pi-alkyl, and 17 VDW bonds, androst-1-en-3-one,17-hydroxy-, (5. alpha.,17.beta.) interacted with 12 bonds including 1UAA and 11 VDW bonds while 2,6,10-Dodecatrien-1-al,12-(acetoxy)-2,6,10-trimethyl-, (E,E,E)- interacted with 21 bonds including 4-H, 1 pi-alkyl, and 16 VDW bonds (Fig. 12d).

With NOx, androst-1-en-3-one,17-hydroxy-, (5. alpha.,17.beta had 8 interactions comprising 1-H, 2 pi-alkyl and 5 VDW bonds, tetradecanoic acid had 20 interactions comprising 4-H, 3 pi-alkyl and 13 VDW bonds, alpha-farnesene had 17 interactions comprising 4 pi-alkyl and 13 VDW
<figure><img src="image_17.png" /><figcaption>**Fig. 12c.** 2D interaction plots of cyclooxygenase-2 with (a) ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3.beta.,5 alpha.), (b) ergosta-2,24-dien-26-oicacid,18-(acetyloxy)-5,6-epoxy-4,22-dihydroxy-1-oxo-, delta-lactone, (4.beta., (c) trans-geranylgeraniol (d) phenol,2,6-bis(1,1-dimethylethyl)- and (e) 4,8,12,16-Tetramethylheptadecan-4-olide.</figcaption></figure>

bonds, 6-epi-shyobunol had 12 interactions comprising 1-H, 2 pi-sigma, and 9 VDW bonds and 2,6,9,11-Dodecatetraenal, 2,6,10-trimethyl-, (E, E, E)- had 15 interactions comprising 2 H, 3 pi-alkyl and 10 VDW bonds (Fig. 12e). Regarding alpha-amylase, ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3.bet.,5 alpha.) 13 interactions comprising 1-H, 2 pi-alkyl, 1 pi-sigma, 1 UAA and 8 VDW bonds, Erg-2,24 18 interactions comprising 5-H, 1 pi-alkyl, and 12 VDW bonds, androst-1-en-3-one, 17-hydroxy-, (5 alpha., 17. beta.) had 14 interactions comprising 2 pi-alkyl, 1 pi-sigma and 11 VDW bonds, phenol, 2,6-bis(1,1-dimethylethyl)- 8 interactions comprising 1-H, 2 pi-alkyl, 2 pi-sigma and 4 VDW bonds, and 6-epi-shyobunol had 13 interactions comprising 1-H, 2 pi-alkyl, 1 pi-sigma and 9 VDW bonds (Figure 12 f).

### 3.10. Pharmacokinetics, drug-likeness, and toxicity profiles of the top-ranked compounds of *H. crepitans*

The ADMET properties of the top-ranked compounds of H. crepitans obtained from the docking analysis are presented in Tables 4 and 5. All the compounds showed less than two violations of Lipinski rule for oral drug-likeness. Similarly, the compounds had high gastrointestinal absorption except 4,8,12,16-tetramethylheptadecan-4-olide and alpha-farnesene. Overall, the blood-brain barrier permeability, phosphoglycoprotein substrate, synthetic accessibility, and cytochrome P450 inhibitory profiles of the compounds were relatively good (Table 4). Interestingly, the compounds were mostly inactive against the organ and end-point toxicities properties analyzed (Table 5).

## 4. Discussion

The comprehensive study utilizing crude *Hura crepitans* methanolic extract (CHCME) reveals significant insights into its hypoglycemic and antioxidant capabilities, its effects on lipid metabolism, liver and kidney function biomarkers, hematological parameters, and its modulation of neuronal cholinesterase activities and neuroinflammatory markers in STZ-induced diabetic rats. These findings not only underscore the multifaceted therapeutic potential of CHCME but also align with and extend the current understanding of natural remedies in the treatment of diabetes and its complications.

The observed dose-dependent increase in glucose uptake and alpha-amylase inhibition suggests CHCME's potential in enhancing cellular glucose utilization and slowing carbohydrate digestion, respectively, which are critical mechanisms in managing hyperglycemia [47,48]. These effects are consistent with other studies showing similar hypoglycemic actions of plant extracts, underscoring the vital role of natural products in diabetes management [49]. Furthermore, the significant improvement in body weight and reduction in fasting blood sugar levels in diabetic rats treated with CHCME parallels findings from Kimani, et al. [50], which demonstrated the antidiabetic properties of another medicinal plant through mechanisms possibly involving pancreatic beta-cell regeneration or insulinotropic effects.

The antioxidant effects of CHCME, evidenced by the enhancement of superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) activities alongside the reduction in malondialdehyde (MDA) levels highlight the extract's ability to mitigate oxidative stress. This is pivotal, as oxidative stress plays a crucial role in the pathogenesis and progression of diabetes and its complications [51,52]. The modulation
<figure><img src="image_18.png" /><figcaption>**Fig. 12d.** 2D interaction plots of nuclear factor kappa b with (a) ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3.beta.,5 alpha.), (b) ergosta-2,24-dien-26-oicacid,18-(acetyloxy)-5,6-epoxy-4,22-dihydroxy-1-oxo-, delta-lactone, (4.beta., (c) trans-geranylgeraniol (d) androst-1-en-3-one,17-hydroxy-, (5. alpha.,17.beta.) and (e) 2,6,10-Dodecatrien-1-al,12-(acetoxy)-2,6,10-trimethyl-, (E,E,E)-.</figcaption></figure>

of neuronal cholinesterase and inflammatory markers further indicates CHCME's neuroprotective potential, possibly through anti-inflammatory pathways and cholinesterase inhibition, akin to the mechanisms proposed by previous studies [53–55], and for other medicinal plants with neuroprotective effects. The lipid-modulating capacity of CHCME, leading to improved lipid profiles in the diabetic rats suggests the potential of phytocompounds in correcting dyslipidemia associated with diabetes. Similarly, the normalization of liver and kidney biomarkers underscores CHCME’s protective effect on these organs, which are often compromised in diabetes due to prolonged hyperglycemia and oxidative stress. Moreover, the hematological improvements post-CHCME treatment suggest its beneficial impact on the hematopoietic system, a critical aspect given the susceptibility of diabetic individuals to hematological alterations and infections [56].

The GC-MS analysis identifying bioactive compounds within CHCME, including phytol and pentadecanoic acid among others, provides a chemical basis for the observed pharmacological activities. These compounds have been individually noted for their antidiabetic, antioxidant, and anti-inflammatory properties in various studies [57,58], supporting the integrated effects seen in this study. The compounds of CHCME comprise saturated, mono- and poly- unsaturated fatty acids. These groups of compounds have been associated with various biological effects. For example, phytol, a saturated fatty acid, is reported to reduce the risk of cardiovascular diseases; phytol acetate, an esterified form of phytol, enhances the extract's antioxidant profile; 9,12,15-octadecatrienoic acid (ZZZ)-, known for its anti-inflammatory and heart health benefits while octadecanoic acid, a saturated fatty acid possesses

anti-inflammatory properties. All these biological properties further underscore the extract's potential multifaceted health benefits.

To further substantiate the applicability of compounds as potential drug candidates, computational predictions are important [59]. Molecular docking is used to predict the binding fitness and interactions of target with its ligand [60]. The lower the docking score the higher the interaction of the ligand with the target active site. Notably, majority of *H. crepitans* compounds exhibited lower docking scores than metformin depicting better interaction with the target profiled against and hence better potential to produce therapeutic effect. Surprisingly, the top three compounds with higher percentage areas had lower docking scores against the targets profiled. However, ergosta-2,24-dien-26-oicacid, 18-(acetyloxy)-5,6-epoxy-4, 22-dihydroxy-1-oxo-, delta-lactone, (4. beta., ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3.bet.,5 alpha.), trans-geranylgeraniol, androst-1-en-3-one,17-hydroxy-, (5. alpha.,17. beta.), 5,9,13-Pentadecatrien-2-one,6,10,14-trimethyl-, (E, E)-, 4,8,12, 16-Tetramethylheptadecan-4-olide, 2,6,10-Dodecatrien-1-al,12-(acetoxy)-2,6,10-trimethyl-, (E,E,E)-, alpha-farnesene, 2,6,9,11-Dodecatrienal,2,6,10-trimethyl-, (E, E, E)-, 6-epi-shyobunol, tetradecanoic acid, phenol,2,6-bis(1,1-dimethylethyl) were found most interacting with the targets suggesting their significance in the antidiabetic effect demonstrated by the CHCME. Several intermolecular interactions and interacting bonds necessary for the stability of the complexes were present. Hydrogen bonds contribute to the stability and 3D conformation of a target [61].

Similarly, these compounds produced oral drug-likeness profiles by exhibiting molecular weights < 500 g/mol, hydrogen bond acceptor and
<figure><img src="image_19.png" /><figcaption>**Fig. 12e.** 2D interaction plots of nitric oxide with (a) androst-1-en-3-one, 17-hydroxy-, (5. alpha.,17.beta.) (b) tetradecanoic acid (c) alpha-farnesene (d) 6-epi-shyobunol and (e) 2,6,9,11-Dodecatetraenal, 2,6,10-trimethyl-, (E, E, E)-.</figcaption></figure>

donor of ≤ 10 and 5 respectively, lipophilicity value ≤ 4.15 and bioavailability score of < 0.55 [62]. Additionally, these compounds demonstrated high gastrointestinal absorption collaborating the bioavailability results. The polarity of a compound largely influences its absorption, high absorption rate of compound is proportional to high bioavailability [63]. The controlled transport of drugs that are impermeable to the brain and central nervous system ensure brain functionality and prevention of multidrug-resistance malignancy [64,65]. Their relatively non-inhibitory effects on the xenobiotic metabolizing enzymes (CYP450) implies their potential biotransformation into more polar excretable products [66]. Furthermore, some compounds produce potential pathological conditions at certain concentrations including inflammation, carcinogenic, mutagenic, cytotoxic, and metabolic disorders [67]. However, the profile *H. crepitans* compounds were non-BBB permeant, non-glycoprotein substrate and non-toxicity depicting potential biological and less toxic effects. This finding is in line with the study of [68] for potential drug candidates.

The *in silico* results align well with the *in vivo* findings, suggesting that the identified compounds could be contributing to the observed therapeutic effects through their interactions with key metabolic and inflammatory pathways. For instance, the significant docking scores of compounds like ergosta-2,24-dien-26-oicacid, 18-(acetyloxy)-5,6-epoxy-4, 22-dihydroxy-1-oxo-, δ-lactone (4β) for acetylcholinesterase, butyrylcholinesterase, alpha-amylase, and NF-κB, coupled with their drug-likeness profiles, indicate their potential to modulate enzymatic activities and inflammatory responses observed in the *in vivo* studies. This correlates with the biochemical analyses showing reductions in oxidative stress markers and improvements in lipid and glucose metabolism, highlighting the extract's multifaceted mechanisms in exerting its antidiabetic effects.

The identification of these compounds not only highlights the

chemical richness of the *Hura crepitans* methanolic extract but also provides a foundation for future research into their individual and synergistic effects on health and disease. Moreover, the diverse chemical nature of these compounds—ranging from diterpenes to fatty acids—offers a broad spectrum of mechanisms by which they might exert therapeutic effects, providing a comprehensive approach to tackling diabetes. Overall, the findings from this study contribute to the growing body of evidence supporting the use of plant extracts in the development of natural therapeutic agents and specifically establish the use of *Hura crepitans* as a potential plant in managing diabetes and diabetic-induced inflammation.

## 5. Conclusions

The results of this investigation into the effects of CHCME on STZ-induced diabetic rats offer promising evidence of its potential as a comprehensive therapeutic agent against diabetes and its complications. The extract displayed a significant reduction in fasting blood glucose, improved glucose uptake and insulin levels, and ameliorated diabetes-induced complications, including oxidative stress, dyslipidemia, and inflammation. The *in silico* results showed the compounds of *H. crepitans* as viable potential antidiabetic drug candidates. The findings not only validate traditional uses but also open avenues for future studies aimed at clinical translation. Emphasizing the need for deeper exploration into the mechanisms of action and the therapeutic efficacy of CHCME in human subjects will pave the way for its potential use as a natural antidiabetic therapy.

## Supplementary materials

**FIGURE S1:** Superimposed structure of docked top-ranked
<figure><img src="image_20.png" /><figcaption>**Fig. 12f.** 2D interaction plots of alpha amylase with (a) ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3-beta.,5-alpha.), (b) ergosta-2,24-dien-26-oicacid,18-(acetyloxy)-5,6-epoxy-4,22-dihydroxy-1-oxo-, delta-lactone,(4.beta., (E, E)- (c) androst-1-en-3-one,17-hydroxy-, (5. alpha.,17.beta.) (d) Phenol,2,6-bis(1,1-dimethylethyl)-, and (e) 6-epi-shyobunol.</figcaption></figure>

compounds of crude methanolic extract of *H. crepitans* within the catalytic amino acid residues of (a) AChE (b) BChE (c) COX-2 (d) NFkB (e) NOx (e) A-amylase on their native inhibitor (black). All had the same RMSD value of 0.5 Å. **FIGURE S2:** Effect of CHCME organ (liver, kidney, pancreas, and brain) to body weights of STZ-induced diabetic rats. **FIGURE S3:** *In vivo* antioxidant effects of crude *Hura crepitans* methanolic extract on superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and malondialdehyde (MDA) concentrations in the serum and liver of STZ-induced diabetic rats. **FIGURE S4:** *In vivo* anti-inflammatory effects of crude *Hura crepitans* methanolic extract (CHCME) on serotonin and dopamine levels in the brain of STZ-induced diabetic rats. **Table S1:** Color codes of the compounds used in the docking validation. **Table S2:** Docking scores of CHCME against cholinesterase, inflammatory and T2DM targets.

### CRediT authorship contribution statement

**Halimat Yusuf Lukman:** Conceptualization, Methodology, Formal analysis, Software, Investigation, Validation, Writing – original draft preparation, Writing – reviewing & editing.
**Yucheng Kuo:** Conceptualization, Methodology, Formal analysis, Investigation, Validation, Software, Writing – original draft.
**Mayowa Solomon Owolabi:** Methodology, Formal analysis, Software, Investigation.
**Bashir Lawal:** Conceptualization, Methodology, Formal analysis, Software, Investigation, Validation, Writing – original draft, Writing – reviewing & editing.
**Lung-Ching Chen:** Data curation, Investigation, Software, Validation, Writing – reviewing & editing.
**Olabode T. Ajenifujah:**

Conceptualization, Formal analysis, Software, Supervision, Writing – reviewing & editing.
**Adewale O. Fadaka:** Methodology, Formal analysis, Data curation, Software, Investigation, Writing – original draft.
**Femi Olawale:** Methodology, Formal analysis, Software, Investigation, Writing – original draft.
**Sunday A. Onikanni:** Conceptualization, Methodology, Formal analysis, Software, Investigation.
**Saidu Sani:** Data curation, Investigation, Software, Validation, Writing – reviewing & editing.
**Michel De Waard:** Conceptualization, Methodology, Funding acquisition, Investigation, Writing – original draft.
**Dalia Fouad:** Conceptualization, Methodology, Funding acquisition, Investigation, Writing – original draft.
**Gaber El-Saber Batiha:** Conceptualization, Supervision, Software, Funding acquisition, Writing – reviewing & editing.
**Saheed Sabiu:** Software, Supervision, Investigation, Writing – reviewing & editing.
**Alexander T.H. Wu:** Data curation, Investigation, Software, Validation, Supervision, Funding acquisition, Writing – reviewing & editing.
**Hsu-Shan Huang:** Conceptualization, Methodology, Formal analysis, Software, Funding acquisition, Writing – original draft.

### Declaration of Competing Interest

The authors declare no competing financial interest or personal relationship that could influence the work reported in this paper.

### Data availability

The data presented in this study are available in the article.
<table><caption>**Table 4**
Pharmacokinetics and drug-likeness properties of the top-ranked compounds of crude methanolic extract of *H. crepitans*.</caption><thead><tr><th>Compounds</th><th>MW (g/mol)</th><th>NHA</th><th>NHD</th><th>LP</th><th>BS</th><th>LVN</th><th>GIA</th><th>BBP</th><th>PgpS</th><th>CYP1A2 Inb.</th><th>CYP1A2 Inb.</th><th>CYP2C9 Inb.</th><th>CYP2C6 Inb.</th><th>CYP3A4 Inb.</th><th>SA</th></tr></thead><tbody><tr><td>Ergosta-2,24-dien-26-oicacid,18-(acetyloxy)-5,6-epoxy-4,22-dihydroxy-1-oxo-,delta-lactone,(4,beta., Ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3. beta.,5.alpha.)</td><td>470.6</td><td>6</td><td>2</td><td>3.39</td><td>0.55</td><td>0</td><td>High</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>6.83</td></tr><tr><td>Trans-geranylgeraniol</td><td>290.48</td><td>1</td><td>1</td><td>4.75</td><td>0.55</td><td>1</td><td>High</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>3.65</td></tr><tr><td>Androst-1-en-3-one,17-hydroxy-, (5. alpha.,17. beta.)</td><td>302.45</td><td>2</td><td>1</td><td>3.03</td><td>0.55</td><td>0</td><td>High</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>4.82</td></tr><tr><td>5,9,13-Pentadecatrien-2-one,6,10,14-trimethyl-, (E, E)-</td><td>262.43</td><td>1</td><td>0</td><td>3.67</td><td>0.55</td><td>1</td><td>High</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>3.47</td></tr><tr><td>4,8,12,16-Tetramethylheptadecan-4-olide</td><td>324.54</td><td>2</td><td>0</td><td>4.15</td><td>0.55</td><td>1</td><td>Low</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>4.12</td></tr><tr><td>2,6,10-Dodecatrien-1-al,12-(acetoxy)-2,6,10-trimethyl-, (E,E,E)-</td><td>278.39</td><td>3</td><td>0</td><td>3.77</td><td>0.55</td><td>0</td><td>High</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td><td>3.26</td></tr><tr><td>Alpha-farnesene</td><td>204.35</td><td>0</td><td>0</td><td>3.89</td><td>0.55</td><td>1</td><td>Low</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>3.72</td></tr><tr><td>2,6,9,11-Dodecatetraenal,2,6,10-trimethyl-, (E, E, E)-</td><td>218.33</td><td>1</td><td>0</td><td>3.33</td><td>0.55</td><td>0</td><td>High</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>3.43</td></tr><tr><td>6-epi-shyobunol</td><td>222.37</td><td>1</td><td>1</td><td>3.17</td><td>0.55</td><td>0</td><td>High</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>3.84</td></tr><tr><td>Tetradecanoic acid</td><td>228.37</td><td>2</td><td>1</td><td>3.32</td><td>0.85</td><td>0</td><td>High</td><td>Yes</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2.09</td></tr><tr><td>Phenol,2,6-bis(1,1-dimethylethyl)-Metformin</td><td>220.35</td><td>1</td><td>1</td><td>4.3</td><td>-</td><td>0</td><td>High</td><td>Yes</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2.09</td></tr><tr><td></td><td>129.16</td><td>2</td><td>3</td><td>0.34</td><td>0.55</td><td>0</td><td>High</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>3.02</td></tr></tbody></table>

MW: Molecular weight; NHA: Number hydrogen bond acceptor; NHD: Number hydrogen bond donor; LP: Lipophilicity (MLogP); BS: Bioavailability score; LVN: Lipinski number of violations; GIA: Gastrointestinal absorption; BBP: Blood barrier permeant; PgpS: Phosphoglycoprotein substrate; CYP: Cytochrome P450; I: inhibitor; SA: Synthetic accessibility

<table><caption>**Table 5**
Toxicity profile of top-ranked compounds of crude methanolic extract of *H. crepitans*.</caption><thead><tr><th>Compounds</th><th></th><th>LD 50 (mg/kg)</th><th>Class</th><th>Hepatotoxicity</th><th>Carcinogenicity</th><th>Immunogenicity</th><th>Mutagenicity</th><th>Cytotoxicity</th></tr></thead><tbody><tr><td>Ergosta-2,24-dien-26-oicacid,18-(acetyloxy)-5,6-epoxy-4,22-dihydroxy-1-oxo-,delta-lactone,(4,beta., Ergost-25-ene-6,12-dione,3,5-dihydroxy-, (3.beta.,5.alpha.)</td><td></td><td>300</td><td>3</td><td>Inactive</td><td>Inactive</td><td>Active</td><td>Inactive</td><td>Active</td></tr><tr><td>Trans-geranylgeraniol</td><td></td><td>2500</td><td>5</td><td>Inactive</td><td>Inactive</td><td>Active</td><td>Inactive</td><td>Inactive</td></tr><tr><td>Androst-1-en-3-one,17-hydroxy-, (5. alpha.,17.beta.)</td><td></td><td>5000</td><td>5</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td></tr><tr><td>5,9,13-Pentadecatrien-2-one,6,10,14-trimethyl-, (E, E)-</td><td></td><td>1860</td><td>4</td><td>Active</td><td>Inactive</td><td>Active</td><td>Inactive</td><td>Inactive</td></tr><tr><td>4,8,12,16-Tetramethylheptadecan-4-olide</td><td></td><td>5000</td><td>5</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td></tr><tr><td>2,6,10-Dodecatrien-1-al,12-(acetoxy)-2,6,10-trimethyl-, (E,E, E)-</td><td></td><td>4400</td><td>5</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td></tr><tr><td>Alpha-farnesene</td><td></td><td>5000</td><td>5</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td></tr><tr><td>2,6,9,11-Dodecatetraenal,2,6,10-trimethyl-, (E, E, E)-</td><td></td><td>3650</td><td>5</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td></tr><tr><td>6-epi-shyobunol</td><td></td><td>4950</td><td>5</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td></tr><tr><td>Tetradecanoic acid</td><td></td><td>5000</td><td>5</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td></tr><tr><td>Phenol,2,6-bis(1,1-dimethylethyl)-Metformin</td><td></td><td>900</td><td>4</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td></tr><tr><td></td><td></td><td>650</td><td>4</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td></tr><tr><td></td><td></td><td>680</td><td>4</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td><td>Inactive</td></tr></tbody></table>

## Acknowledgements

Alexander TH Wu is funded by the National Science and Technology Council (NSTC, 113-2320-B-038-034-, and 113-2314-B-038-010-). Hsuan-Huang is funded by The National Science and Technology Council, Taiwan (NSTC112-2314-B-038-006), SKH-TMU-113-02 and A-112-071. We express our profound gratitude to The National Council for Scientific and Technological Development (CNPq) Brazil, and Universidade Federal do Rio de Janeiro. The authors would like to extend their gratitude for the research funding support by the King Saud University, through Researcher Support Project number RSPD2024R965. M.D.W. thanks the Agence Nationale pour la Recherche for the financial support to the

laboratory of excellence Ion Channels, Science and Therapeutics (Grant Number ANR-11-LABX-0015).

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2024.117308.

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