Translate this page into:
Phytochemical characterization and antiangiogenic effect of Salvadora persica using chorioallantoic membrane (CAM) assay: An in vitro, in vivo, and in silico investigations
*Corresponding author: E-mail address: fouzialatif@qlit.edu.cn (F Latif)
-
Received: ,
Accepted: ,
Abstract
Hepatocellular carcinoma (HCC) has a high prevalence and mortality worldwide. Plant Salvadora persica (SP) possesses therapeutically essential phytochemicals. The study evaluated in vitro cytotoxic, antiproliferative, and antiangiogenic effects of SP aqueous (SP-AQ) and ethanolic (SP-E) extracts and in vivo their antiangiogenic effects using the chorioallantoic membrane (CAM) assay. Phytochemicals of SP active extract were identified and studied in silico against the angiogenic protein, vascular endothelial growth factor(VEGF)-A, to determine their interactions. In vitro, in HepG2 cells, the MTT assay (cytotoxicity), immunocytochemistry assay (VEGF-A), RT-qPCR assay (proliferative genes MKI67, PCNA), and in vivo, the CAM assay in fertilized chicken eggs for 3D quantification of blood vessels were performed. Identification of active SP-E extract phytomolecules by gas chromatography- mass spectrometry (GC-MS), and their molecular docking study against VEGF-A by AuToDOCK Tools were performed. In an in vitro study, SP-E extract (IC50 = 46.7 µg/mL) significantly decreased cell viability compared to SP-AQ extract (IC50 = 65.8 µg/mL) and considerably reduced VEGF-A level in HepG2 cells. In vivo study, SP-E extract significantly reduced the diameter of primary, secondary, and tertiary blood vessels, height, and 3D surface roughness parameters of blood vessels compared to SP-AQ extract. The GC-MS analysis revealed (3-methoxyphenyl)acetonitrile and bis(2-ethylhexyl)isophthalate as major phytomolecules in SP-E extract, whereas (3-methoxyphenyl)acetonitrile and naphtho(2,1,8,7-klmn)xanthene exhibited higher docking score against VEGF-A. SP-E extract of SP is a potent cytotoxic, antiproliferative, and antiangiogenic candidate compared to the SP-AQ extract.
Keywords
Angiogenesis
Chorioallantoic membrane assay (CAM)
Hepatocellular carcinoma
Phytomolecules
Salvadora persica
Vascular endothelial growth factor
1. Introduction
Hepatocellular carcinoma (HCC), a primary form of liver cancer, is the sixth most prevalent cancer globally and is the third leading cause of death worldwide. HCC is a hyper-vascularized solid tumor in which abnormal blood vessel formation by angiogenesis enhances access to blood and nutrients to promote cell proliferation, tumor development, and metastasis (He et al., 2025). Angiogenesis is the formation of new blood vessels from pre-existing blood vessels and is a fundamental requirement for cancer progression (Aspriţoiu et al., 2021). Angiogenic protein, vascular endothelial growth factor (VEGF-A), is a key driver to initiate angiogenesis at a pathological level that is secreted by cancer cells, and increased VEGF-A level indicates aggressiveness of HCC (Pinto et al., 2023). More than 90% of advanced HCC cases show notably increased VEGF-A levels (Ghalehbandi et al., 2023). Among in vivo angiogenesis models, the chorioallantoic membrane (CAM) is a convenient model that provides rapid access with rich blood vascularization to study the antiangiogenic effect of anticancer drugs (Chen et al., 2021). The CAM supplies an important structure for exchanging nutrients and gases between the embryo and external surroundings. As it grows, it develops a dense blood vascular network that spreads and branches out to facilitate embryo growth (Wan et al., 2025). Current antiangiogenic drugs do not effectively provide survival benefits due to adverse effects and acquired resistance during HCC treatment (Moawad et al., 2020). Natural products derived from plant sources have demonstrated diverse pharmacological properties, particularly in the development of anticancer therapeutics (Zeng et al., 2020; Luo et al., 2024; Shang et al., 2024). Different plant extracts yield phytocomponents that are effective antiangiogenics in cancer therapy (Azevedo et al., 2024). Plant Salvadora persica (SP) belonging to the Salvadoraaceae family, is known as Miswak, or toothbrush tree, and is found in Asia and Arab countries. SP has medicinal benefits such as antimicrobial, antioxidant, anti-inflammatory, cytotoxic, and hepatoprotective effects. (Sagir et al., 2024). Previously, phytochemical investigations of SP have identified various classes of compounds, such as alkaloids, isothiocyanates, phenols, flavonoids, glycosides, and saponins, which exhibit a wide range of pharmacological properties (AL-Dabbagh et al., 2018; Khan et al., 2020; Suleman et al., 2025). Moreover, extracts of SP have been demonstrated to serve as an excellent source for the green synthesis of diverse nanoparticles (Khan et al., 2015; Al-Marri et al., 2016; Shaik et al., 2016). To date, there is no research data on in vivo antiangiogenic studies of plant SP using the CAM assay, and no computational research is available on its identified phytomolecules against angiogenic protein, VEGF-A, to know their interactions. Therefore, the current study uses the CAM assay to determine the in vitro cytotoxic, antiproliferative, and antiangiogenic activities of SP extracts in HepG2 cells and their in vivo antiangiogenic effect in the fertilized chicken eggs model. The study seeks to identify phytomolecules of the most potent extract and their interaction with the target angiogenic protein VEGF-A by a docking study.
2. Materials and Methods
2.1. Reagents
MTT dye (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), Dimethyl sulfoxide (DMSO), Gibco Dulbecco’s Modified Eagle’s Medium High Glucose (DMEM-HG), 10% fetal bovine serum (FBS), 4,6-diamidino-2-phenylindole (DAPI) dye, Phosphate buffer saline (PBS), Parafofmaldehyde, Bovine serum albumin (BSA), TRIS buffer saline with Tween 20 (TBS-T), VEGF-A Recombinant Rabbit Monoclonal Antibody (catalogue no. MA5-32038), secondary antibody Donkey Anti-rabbit IgG, FITC conjugate (catalogue no. A16030), SYBER Green PCR Master Mix, were purchased from Thermo Fischer, Massachusetts, USA. The primer sequence was synthesized by ShineGene Biotechnology, Shanghai, China.
2.2. Plant extraction
Sticks of the root part of SP were purchased from Sichuan Weikeqi Biotechnology Co., Ltd., (Chengdu, Sichuan, China), a commercial herbal supplier. Professor Xu Nan from the Shandong Research Academy of Traditional Chinese Medicine (Jinan, China) distinguished the plant. A voucher number (3856) was deposited at the herbarium of the Medical Department. Then it was ground into coarse powder, and by the cold maceration process, each 100 g of powder was dissolved separately in aqueous solvent to prepare SP-AQ extract and ethanolic solvent to prepare SP-E extract, for 72 h with occasional shaking. After filtration by Whatman # 1 filter paper in a flask, the filtrate was evaporated using a rotary evaporator at 40°C under reduced pressure and preserved at 4°C for subsequent utilization.
2.3. HepG2 cell culturing
The human HCC cell line HepG2 was obtained from the cell bank of Qilu Institute of Technology (Zhangqiu, Jinan, China) and cultured in DMEM-HG medium containing 10% FBS at 5% CO2, 37oC, and 95% humidity. SP extracts dilutions were prepared in DMSO.
2.4. MTT assay
The cytotoxicity and antiproliferative activities of SP-AQ and SP-E extracts were evaluated by MTT assay in a 96-well plate as previously described (Xu et al., 2023). SP extracts, various dilutions (10, 50, 100, 150, and 200 µg/mL), were administered in wells for 24 h MTT dye 10 µL for 3 h was added, followed by application of 150 µL DMSO in wells and shaking for 10 min at 25°C. Then, the optical density was measured at 492 nm wavelength by a spectrophotometer (Thermo Fischer, Massachusetts, USA).
2.5. Immunocytochemistry assay
SP-AQ and SP-E extracts were administered in a 6-well plate containing HepG2 cells for 24 h to measure VEGF-A expression level by immunocytochemistry assay (Maqbool et al., 2019). After 24 h the medium was discarded, washed with 1X TBS-T, 5 times and 200 µL of 4% paraformaldehyde was added to the wells for 30 min, then washed. After that, 5% BSA (blocker) was added for 25 min and washed. The rabbit polyclonal primary antibody of VEGF-A in wells was administered at 37°C for 1.5 h, removed, and washed. Later, FITC-conjugated donkey antirabbit secondary antibody was added to the wells, incubated for 1.5 h at 37°C, removed, and the cells were washed. Further, HepG2 cells were incubated with 1 µg/mL of DAPI in PBS at 25°C for 15 min and washed. Then, cells were observed under Floid® Cell Imaging Station (Thermo Fischer Scientific, Massachusetts, USA).
2.6. RT-qPCR analysis
Total RNA was isolated from SP extracts treated HepG2 cells after 24 h using TRIzol reagent (Thermo Fischer Scientific, Massachusetts, USA) according to the manufacturer’s protocol. The RNA concentration was measured by NanoDrop Microvolume spectrophotometer (Thermo Fischer Scientific, Massachusetts, USA). Then, 2 µg of total RNA was extracted using the MMLV reverse transcriptase kit (Applied Biosystems, Carlsbad, California, USA), and the cDNA was prepared according to the manufacturer’s guidelines. Genomic quantification of proliferative genes PCNA and MKI67 was conducted, using SYBR Green PCR Master Mix, by a Real-time qPCR thermal cycler, qTOWER3 (Analytic Jena AG, Jena, Germany), while keeping HPRT as the housekeeping gene.
2.7. CAM assay
In an in vivo study, the antiangiogenic activity of SP-AQ and SP-E extracts using the CAM assay was evaluated using the method previously described (Akhter et al., 2015). Fertilized chicken eggs of the 4th day were obtained from a local hatchery (Zhangqiu, Jinan, China) and sprayed with 70% ethanol and incubated at 37°C at a humidity of 60-70% for 5 days. Each control and treatment group contained 10 fertilized chicken eggs. On the 5th day of incubation, a window was made on each egg shell in the laminar flow hood by cutting a 2 cm diameter hole in the egg shell aseptically. From the cut end of each egg, 4 mL of albumin was aspirated with a 21-gauge sterile syringe to separate CAM from the dry white membrane, under the egg shell. This step allowed an optimized quantification of CAM vasculature. Then, the windows were sealed with sterile parafilm tape, and eggs re-incubated for 24 h. Meanwhile, dilutions of SP extracts were prepared in PBS with a pH of 6.5-7.5. Then, dilutions were filtered using a syringe filter (0.2 μm) to prevent contamination. On the 6th day of incubation, the chicken membranes (CAMs) were utilized for SP extracts treatment. The windows of each egg were opened, and 150 μL of SP extracts at two doses, one at the IC50 value and the second at a higher concentration, were administered on developing CAM in eggs. Then, the windows were resealed, and chicken eggs were incubated further for 24 h.
2.8. Quantification of angiogenesis
On the 7th day, windows were opened and images of each embryo with the CAM were taken from a Coolpix (Nikon, China). Images were cropped in Adobe Photoshop (version 7.0) to highlight vessels. Then, it was imported to the Scanning Probe Image Processor software (SPIP version 7.2.0, Denmark) to measure diameter, 3D surface roughness parameters, and height of blood vessels. The Abbott curve is a graphical illustration of blood vessel surface roughness to measure the height of blood vessels (Rehman et al., 2022).
2.9.1. Gas chromatography-mass spectrometry (GC-MS)
Phytoanalysis of SP-E extract using GC-MS analysis was performed on an Agilent 7890A Gas Chromatograph connected with a single quadrupole Mass Spectrometry (MSD-5977B detector, Agilent Technologies, USA). The GC was equipped with an Optima 5MS fused silica capillary column (30 m × 250 µm i.d., 0.25 µm film thickness) featuring a nonpolar stationary phase composed of 5% phenyl and 95% dimethylpolysiloxane. Chromatographic separation was achieved using a similar method published earlier (Khan et al., 2016). The oven temperature program was as follows: initial temperature of 70°C (held for 3 min), ramped at 10°C/min to 270°C (held for 13 min). Helium was the carrier gas at a constant 1.0 mL/min flow rate. A 1.0 µL aliquot of the extract was injected in split mode with a split ratio 15:1, and the injector temperature was maintained at 250°C. The electronic-impact ion source and the MS quadrupole temperatures were 230°C and 150°C, respectively. All mass spectra were acquired in the EI mode (scan range of m/z 45-600 and ionization energy of 70 eV). Data acquisition and analysis were performed using Agilent Mass Hunter Workstation software. The relative percentage composition of each compound was determined based on the ratio of the individual peak area to the total peak area without using a correction factor.
2.9.2. Identification of Secondary metabolites from the SP-E extract
Different phytocomponents of the SP-E extract were identified by matching their mass spectra with the library entries of mass spectra databases (NIST-20 MS libraries).
2.10. Molecular docking
Protein VEGF-A (Vascular was obtained from the PDB (PDB ID: 4KZN) and processed using the Maestro Protein Preparation Wizard, which includes cleaning, inserting missing atoms, and optimizing the side chains. All 8 compounds were created in 2D with ChemDraw and then transformed into 3D bioactive forms with low-energy states. The compounds were further improved using LigPrep to obtain the most stable low-energy variants. The OPLS 2005 force field generated docking grids for the proteins, with van der Waals radii changes. A grid box of 15 Å was constructed around the binding site to improve ligand mobility. The Glide XP scoring system evaluated ligand poses based on their many orientations for binding stability and affinity (Hamza et al., 2024)
3. Results
3.1. MTT assay
SP-AQ and SP-E extracts cytotoxic and antiproliferative effects in the HepG2 cell line after 24 h were determined by MTT assay (Fig. 1). SP extracts, at various dilutions, showed a dose-dependent cellular inhibition compared to the control group. At 10, 50, 100, 150, and 200 µg/mL dilutions, the cell viability of SP AQ extract was 77%, 43%, 33%, 26%, and 20%, while the SP-E extract was 71%, 38%, 29%, 20%, and 13%, respectively. SP-E (IC50: 46.7 µg/mL) showed a more significant cytotoxic effect on HepG2 cells than SP-AQ (IC50: 65.8 µg/mL).
- The cytotoxicity of SP extracts, (a) aqueous (SP-AQ) and (b) ethanol (SP-E) in HepG2 cells after 24 h was determined by MTT assay. Data are expressed as mean ± S.D. *p < 0.1, **p < 0.01, and ***p < 0.001 vs control
3.2. Immunocytochemistry assay
The effect of SP-AQ and SP-E extracts on the VEGF-A expression level in HepG2 cells after 24 h was determined by immunocytochemistry assay (Fig. 2). SP extracts remarkably decreased the VEGF-A level, with a more significant effect by SP-E extract.
- Expression level of protein VEGF-A in control (a1-a3), SP AQ (65.8 µg/mL) extract (b1-b3) and SP-E (46.7 µg/mL) extract (c1-c3) treated HepG2 cells after 24 h by immunocytochemistry assay. Cells were stained green with VEGF antibody, while nuclei were stained blue with DAPI.
3.3. RT-qPCR analysis
Proliferative genes, PCNA and MKI67 mRNA levels in SP extracts treated HepG2 cells after 24 h, were determined by RT-qPCR (Fig. 3). SP extracts significantly downregulated mRNA levels of proliferative genes. SP-AQ extract reduced the PCNA mRNA level by 0.67-fold, while SP-E caused a decrease of 0.03-fold. The fold decrease in MKI67 mRNA was 0.330 with SP-AQ extract and 0.00 with SP-E extract.
- Proliferative genes, (a) MKI67 and (b) PCNA mRNA levels were measured in SP-extracts, at their IC50 values in treated HepG2 cells after 24 h by RT-qPCR assay. The data presented are the mean ± SD. One-way ANOVA and Bonferroni multiple comparison test were applied to data expressed in fold change. **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 vs control.
3.4. CAM assay
SP extracts antiangiogenic effect in fertilized chicken eggs was evaluated using the CAM assay, and angiogenesis was quantified by SPIP software (Version 7.2.0, Denmark). SP extracts showed a significant decrease in blood vessel formation compared to the control group, in which blood vessels in the CAM area showed widespread branching into primary, secondary, and tertiary branches. Moreover, SP-AQ and SP-E extracts treated groups significantly reduced the primary, secondary, and tertiary blood vessel diameters (Figs. 4a-c).
- Macroscopic assessment of blood vessels in control and SP extracts treated groups by CAM assay. (a) branching of the blood vessels. (b) 3D Quantification of CAMs to measure the diameter of primary blood vessel (P.B.V), secondary blood vessel (S.B.V) and tertiary blood vessel (T.B.V). (c) Diameter (mm) of blood vessels in control, SP-AQ and SP-E extracts treated groups.
Among SP extracts, SP-E treated groups showed remarkable decreased values of 3D surface roughness parameters (Table 1), indicating reduced neo-vascularization in SP extracts treated CAM groups than the control group (Fig. 5a). The height of blood vessels by the Abbott curve, revealed regression in SP-extracts treated CAMs than control (Fig. 5b). SP-AQ extract showed a reduction in the height of blood vessels at a high dose, while SP-E extract reduced the height (vertical growth) of blood vessels both at low and high doses, and showed lateral area expansion at a high dose.
| S. No. | Parameters (nm) | Control | SP-AQ 65. 8 µg/mL | SP-AQ 80 µg/mL | SP-E 46.7 µg/mL | SP-E 61 µg/mL |
|---|---|---|---|---|---|---|
| 1 | Sa | 88.14±2.00 | 67.23±2.44** | 53.14±2.09** | 49.63±1.74** | 43.47±2.53** |
| 2 | Sq | 94.50±3.05 | 73.12±2.53** | 57.47±2.36** | 53.96±2.99** | 46.19±2.66** |
| 3 | Ssk | 0.78±0.12 | 0.56±0.01** | 0.52±0.01** | 0.55±0.04** | 0.46±0.03** |
| 4 | Sku | 1.87±0.11 | 1.57±0.01** | 1.52±0.01** | 1.43±0.17* | 1.54±0.07* |
| 5 | Sdr | 1.26E-05±2.82E-07 | 5.92E-07±1.22E-07** | 4.74E-07±1.41E-08** | 6.72 E-07±2.42E-08** | 4.64 E-07±6.15E-08** |
| 6 | Sci | 1.77±0.12 | 1.63±0.02* | 1.62±0.01* | 1.47±0.03* | 1.51±0.13* |
| 7 | Sy | 266.18±2.81 | 240.36±1.64** | 195.32±2.11** | 238.39±2.80** | 171.05±2.74** |
| 8 | Sz | 266.18±2.81 | 240.36±1.64** | 195.32±2.11** | 238.39±2.80** | 171.05±2.74** |
| 9 | Ssc | 1.01E-08±9.22E-10 | 7.34E-10±2.20E-11** | 5.65E-10±2.99E-11** | 1.03 E-09±1.76E-11** | 8.68 E-10±6.22E-11** |
| 10 | Sdq | 1.77E-04±1.99E-05 | 1.0355E-04±3.08E-06** | 9.72E-05±2.35E-06** | 1.08 E-04±8.54E-06 | 8.59 E-05±6.97E-06 |
| 11 | Spk | 295.35±3.79 | 235.71±1.90** | 167.48±2.32** | 107.47±2.62** | 153.95±3.855** |
| 12 | Svk | 4.40±0.53 | 0.07±0.12** | 0.12±0.20** | 2.29±0.26** | 0.30±0.027** |
| 13 | Stdi | 0.93±0.09 | 0.42±0.02** | 0.37±0.05** | 0.74±0.29* | 0.60±0.09** |
| 14 | Sk | 45.42±5.12 | 28.38±1.80** | 33.35±1.82** | 22.49±2.11** | 16.46±2.45** |
Sa, average roughness; Sq, root mean square deviation; Ssk, skewness of the surface; Sku, kurtosis of the surface; Sdr, developed surface area ratio; Sci, core fluid retention; Sy, lowest valley; Sz, maximum height of the surface; Ssc, arithmetic mean summit; Sdq, root mean square slope; Spk, reduce summit height; Svk, reduce valley; Stdi, texture index; Sk, core roughness depth, ‘E’ Stand for Exponent. ⃰ p < 0.05, **p < 0.01 vs control.
- 3D quantification of CAMs of control (a1, b1), SP-AQ (a2-a3, b2-b3) and SP-E (a4-a5, b4-b5) extracts treated groups. (a) surface roughness parameters (nm) (b) height (nm) of blood vessels by the Abbott curve.
3.5. GC-MS of SP-E extract
GC-MS analysis of the SP-E extracts led to the identification of eight phytomolecules. (Fig. 6, Table 2). It was observed that bis(2-ethylhexyl) isophthalate (90.82%), (3-methoxyphenyl)acetonitrile (3.03%), and 3-methoxybenzylamine (2.04%) were present as the most dominating compounds in the SP-E extracts. On the other hand, the rest of the compounds were detected in minute quantities (<1.5%). It is essential to mention here that all the identified compounds from the SP-E extracts are being reported here for the first time from the SP.
- Total ion chromatogram (TIC) of ethanolic extract (SP-E) of SP root part.
| S. No. | Compounds | M.F. | M.W. | CAS No. | R.T. (min) | % |
|---|---|---|---|---|---|---|
| 1 | 3-Methoxybenzylamine | C8H11NO | 137 | 5071-96-5 | 8.910 | 2.04 |
| 2 | (3-Methoxyphenyl)acetonitrile | C9NO | 138 | 19924-43-7 | 10.22 | 3.03 |
| 3 | 2,4-Di-tert-butylphenol | C14H22O | 206 | 96-76-4 | 12.417 | 0.74 |
| 4 | m-Methoxybenzylisothiocyanate | C9H9NOS | 179 | 75272-77-4 | 13.184 | 0.88 |
| 5 | 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione | C17H24O3 | 276 | 82304-66-3 | 16.693 | 1.19 |
| 6 | Naphtho[2,1,8,7-klmn]xanthene | C18H10O | 242 | 191-37-7 | 18.886 | 0.40 |
| 7 | Bis(2-ethylhexyl) phthalate | C24H38O4 | 390 | 117-81-7 | 22.200 | 0.91 |
| 8 | Bis(2-ethylhexyl) isophthalate | C24H38O4 | 390 | 137-89-3 | 23.868 | 90.82 |
3.6. Molecular docking study
Molecular docking simulations demonstrated varying binding affinities for all compounds evaluated for VEGF-A (PDB ID: 4KZN). The compounds’ docking scores varied from -2.667 to 0.28 kcal/mol, with (3-Methoxyphenyl) acetonitrile (VEGF-A-2) and Naphtho[2,1,8,7-klmn] xanthene (VEGF-A-6) emerging as the top two possibilities, scoring -2.667 and -2.63 kcal/mol, respectively. Both behaved better than the reference co-crystalized ligand CCL, which had a docking score of -0.908 kcal/mol (Table 3). Docking results were confirmed with an RMSD value of 1.471 Å, indicating good accuracy in predicting ligand binding and verifying the VEGF-A docking procedure.
| VEGF-A 4KZN | Docking score | ΔE vdW | ΔE column |
|---|---|---|---|
| (3-Methoxyphenyl)acetonitrile | -2.667 | -9.96 | -2.955 |
| Naphtho[2,1,8,7-klmn]xanthene | -2.63 | -18.51 | -1.145 |
| 3-Methoxybenzylamine | -2.474 | -10.629 | -3.293 |
| m-Methoxybenzylisothiocyanate | -1.978 | -9.846 | -0.48 |
| Bis(2-ethylhexyl) phthalate | -1.539 | -18.771 | -1.975 |
| CCL | -0.908 | -9.842 | -6.292 |
| Bis(2-ethylhexyl) isophthalate | -0.04 | -20.328 | -0.799 |
| 3-Methoxybenzylamine | 0.28 | -9.752 | -0.591 |
To examine the binding configuration of docked ligands in VEGF-A, the top-scoring ligands VEGF-A-2 and VEGF-A-6, with docking scores of -2.667 kcal/mol and -2.63 kcal/mol, respectively, were shown in (Figs. 7a-d). The VEGF-A structure consists of α-helices and β-strands, forming a globular shape with a central binding pocket. Key residues that stabilize ligand interactions include K48, K84, P85, H86, Q89, and I83. The top ligands, which have binding affinities similar to CCL, share the same binding cavity and interact with conserved residues. (Fig. 7a) depicts the overlaid VEGF-A structure with all eight ligands, emphasizing the importance of these residues in stabilizing binding and inhibiting VEGF-A. (Fig. 7b) shows VEGF-A-CCL (in orange) interacting to G88 and H86 via hydrogen bonds, stabilizing the ligand. CCL’s aromatic rings and P85’s π-π stacking enhance binding stability via hydrophobic interactions. A salt bridge occurs between Q89 and positively charged residues in CCL, increasing stability. K48 and K84 maintain the binding pocket, ensuring the ligand is properly aligned. Q87 additionally stabilizes the complex. Such interactions allow CCL to suppress VEGF-A, which prevents angiogenesis and increases its therapeutic potential. The ligand-residue interaction of complex VEGF-A-2 (in yellow), the most successful molecule in the docking simulations with the greatest binding affinity of -2.667 kcal/mol, is shown (Fig. 7c). This mostly results from hydrogen bonds between important residues such as H86 and Q89. The contact with H86 is critical for maintaining the ligand’s orientation in the active site, while Q89 adds extra hydrogen bonding to ensure the ligand is correctly positioned. Furthermore, the hydrophobic interactions with P85 effectively fix the ligand to the binding site. These synergistic interactions make VEGF-A-2 more effective than other ligands, and its strong and sustained binding makes it an excellent option for VEGF-A inhibition and anti-angiogenic treatment. (Fig. 7d) shows VEGF-A-6 (green) and its complex interactions with critical residues in the binding pocket. The compound’s interaction is predominantly stabilized by hydrogen bonds with Q89 and H86, resulting in optimum ligand placement inside the active site. Hydrophobic interactions also play an important role, with residues such as P85, I83, F36, F47, and Y45 anchoring VEGF-A-6 firmly in place. These residues limit ligand displacement, which contributes to its high binding affinity. Furthermore, electrostatic interactions with R82 and K48 stabilize the ligand via ionic contacts, making it a good candidate for VEGF-A inhibition and anti-angiogenic treatment. Despite possessing many hydrogen bonds, VEGF-A-2 is less effective than VEGF-A-6 due to fewer favorable interactions and placement. VEGF-A-6, with just one hydrogen bond but stronger hydrophobic contacts (e.g., P85, F47, F36), produces a more stable and energy-efficient complex. Although VEGF-A-2 has a slightly higher docking score (-2.667 vs. -2.63), VEGF-A-6’s overall binding profile is more effective. ccl has a poorer binding (-0.908) due to poor placement and weaker interactions. Ultimately, VEGF-A-6 is the most powerful VEGF-A inhibitor.
- Superimposition of VEGF-A with Ligands in the Same Cavity. (a), Superimposed figure of VEGF-A with ligands in the same binding cavity. (b) Interaction of VEGF-A-CCL with the binding site. (c) Interaction of VEGF-A-2 with the binding site. (d) Interaction of VEGF-A-6 with the binding site
4. Discussion
Angiogenesis plays a vital role in the progression of HCC cancer and angiogenic protein, VEGF is key driver of angiogenesis (Yang et al., 2022). Plant-derived phytochemicals are therapeutically effective in treating HCC by targeting angiogenesis with fewer side effects than synthetic treatments (Kumar et al., 2021). Plant SP is well known for its wide range of health benefits to treat different diseases, including cancer. The current study of SP extracts, SP-AQ, and SP-E aimed to observe the in vitro cytotoxic, antiproliferative, and antiangiogenic effects in HepG2 cells, and the in vivo antiangiogenic effect using the CAM assay in fertilized chicken eggs. A docking study of phytomolecules of SP active extract against angiogenic protein VEGF-A was conducted to determine their molecular interaction with the target VEGF-A protein. In the current study, in the MTT assay (Fig. 1), SP-AQ and SP-E extracts showed dose-dependent cytotoxic and antiproliferative effects, which are in line with previous results that demonstrated cytotoxic and antiproliferative effects of SP-AQ extract (Hammad et al., 2019) and SP-E extract (Al Bratty et al., 2020) against different cancer cell lines. Previous studies reported that SP Miswak sticks aqueous and ethanolic extracts showed cytotoxicity against HepG2, MCF-7, and A549 cancerous cells (Kumar & Sharma, 2021). In this study, SP-E extract showed more significant cytotoxic and antiproliferative activity than SP-AQ extract, which agreed with a previous reported study (Baba Fakruddin et al., 2018). In the immunocytochemistry assay (Fig. 2), SP-AQ and SP-E extracts considerably reduced the expression level of VEGF-A protein, which is in line with a previous reported study in which plant SP roots aqueous ethanolic extract reduced angiogenic receptor VEGFR2 (Al-Dabbagh et al., 2018). However, SP-E extract most significantly decreased VEGF-A expression level compared to SP-AQ. In RT-qPCR analysis (Fig. 3), SP-E and SP-AQ extracts significantly reduced the mRNA level of PCNA and MKI67, with the most significant decrease by SP-E extract. The RT-qPCR results are consistent with MTT results, in which SP-E also showed the most remarkable antiproliferative effect compared to SP-AQ.
The in vivo study of SP extracts revealed that SP-AQ and SP-E extracts demonstrated a pronounced antiangiogenic effect by attenuating blood vascular network development, diameter, and 3D surface roughness parameters of blood vessels in the treated groups compared to the control group (Figs. 4 and 5, Table 1). The Abbott curve is a cumulative bearing surface area to measure the height of blood vessels and for antiangiogenic drugs, this is important to reduce the height of blood vessels. SP-AQ extract decreased the height of blood vessels at high dose, whereas, in the case of SP-E extract, both low and high doses resulted in a reduction in blood vessel height in the treated groups compared to the control group. However, the surface area of the Abbott curve in the high-dose SP-E extract-treated group was larger than that in the low-dose group. This may be due to the high dose triggering blood vessel regression, resulting in a broader surface area (lateral expansion) but reduced blood vessel height (vertical growth), which is a desirable effect for antiangiogenic drugs.
In the current study, based on the in vitro and in vivo results, GC-MS analysis of the SP most active extract (SP-E) was carried out (Fig. 6, Table 2), leading to the identification of a total of eight compounds. Among identified phytochemicals, the major ones were bis(2-ethylhexyl) isophthalate and (3-methoxyphenyl)acetonitrile. In a detail molecular docking study (Fig. 7, Table 3), the docking score of all SP-E phytochemicals was determined, and (3-methoxyphenyl)acetonitrile (VEGF-A-2) and naphtho(2,1,8,7-klmn)xanthene (VEGF-A-6) displayed the strongest binding affinity (-2.667) and (-2.63) respectively for VEGF-A protein suggesting that these two compounds would be mainly responsible for the potent antiangiogenic activity of the SP-E extract.
5. Conclusion
For the first time, this study evaluates the in vivo antiangiogenic activity of SP using the CAM assay and investigates the binding affinity of identified phytomolecules of active extract with angiogenic protein VEGF-A in the in silico study. SP-AQ and SP-E extracts were evaluated in vitro, in vivo, and in silico for their cytotoxic, antiproliferative, and antiangiogenic activities. Both extracts showed significant activities. However, SP-E was found to be more active compared to SP-AQ. Phytochemical analysis of the SP-E extract determined bis (2-ethylhexyl) isophthalate and (3-methoxyphenyl) acetonitrile as the major constituents. Moreover, molecular docking study of the SP-E compounds showed that (3-methoxyphenyl) acetonitrile and naphtho(2,1,8,7-klmn)xanthene displayed the strongest binding affinity for VEGF-A, suggesting that these two compounds would be mainly responsible for the potent antiangiogenic activity of the SP-E extract. The cytotoxic potential of SP can inhibit cancer cells from proliferating and growing in size. The antiangiogenic potential of SP can be beneficial in HCC treatment by reducing the tumor size and metastasis due to its potential to decrease new blood vessel formation and block the supply of nutrition to dividing and growing cancerous cells, which cannot further grow in size and spread in the body. In the future, a detailed molecular mechanism of the antiangiogenic effect of SP extracts should be determined to provide effective HCC therapy with fewer side effects than current synthetic chemotherapeutic drugs, which could lead to the development of novel anticancer agents for therapeutic and industrial applications.
Acknowledgment
The authors would like to acknowledge the funding from the Ongoing Research Funding Program (ORF-2025-817), King Saud University, Riyadh, Saudi Arabia.
CRediT authorship contribution statement
Fouzia Latif: Conceptualization, Methodology, Writing, Interpretation, and Original draft preparation; Merajuddin Khan: Writing, Interpretation, Review and Editing; Hamad Z. Alkhathlan: Validation, Visualization, Resources and Writing; Mingjing Lu: Resources, Writing, and Interpretation; Tahir Ali Chohan: Software, Data collection, Validation and Visualization.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Declaration of Generative AI and AI-assisted technologies in the writing process
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
References
- Evaluation of pharmacological effect of morus nigra extract on angiogenesis using chorioallantoic membrane (CAM) assay. icdtdi. 2015;5:24. https://doi.org/10.21065/19257430.5.24
- [Google Scholar]
- Phytochemical, cytotoxic, and antimicrobial evaluation of the fruits of miswak plant, Salvadora persica l. J Chem. 2020;2020:1-11. https://doi.org/10.1155/2020/4521951
- [Google Scholar]
- <i>Salvadora persica</i> (Miswak): Antioxidant and promising antiangiogenic insights. AJPS. 2018;09:1228-1244. https://doi.org/10.4236/ajps.2018.96091
- [Google Scholar]
- Green synthesis of Pd@graphene nanocomposite: Catalyst for the selective oxidation of alcohols. Arab J Chem. 2016;9:835-845. https://doi.org/10.1016/j.arabjc.2015.12.007
- [Google Scholar]
- Epigenetic regulation of angiogenesis in development and tumors progression: potential implications for cancer treatment. Front Cell Dev Biol. 2021;9:689962. https://doi.org/10.3389/fcell.2021.689962
- [Google Scholar]
- Natural products‐based antiangiogenic agents: New frontiers in cancer therapy. Food Frontiers. 2024;5:2423-2466. https://doi.org/10.1002/fft2.466
- [Google Scholar]
- Anticancer and anti-inflammatory activity of Salvadora persica L. Indo Am J Pharmaceut Res. 2018;8 https://doi.org/10.5281/zenodo.2531196.
- [Google Scholar]
- Utilisation of chick embryo chorioallantoic membrane as a model platform for imaging-navigated biomedical research. Cells. 2021;10:463. https://doi.org/10.3390/cells10020463
- [Google Scholar]
- The role of VEGF in cancer-induced angiogenesis and research progress of drugs targeting VEGF. Eur. J. Pharmacol.. 2023;949:175586. https://doi.org/10.1016/j.ejphar.2023.175586
- [Google Scholar]
- Effect of Salvadora persica Linn root aqueous extract on oral epithelial dysplasia and oral cancer cell lines. Trop J Pharm Res. 2019;18:2591-2596. http://doi.org/10.4314/tjpr.v18i12.19
- [Google Scholar]
- 3D-QSAR, docking and molecular dynamics simulations of novel Pyrazolo-pyridazinone derivatives as covalent inhibitors of FGFR1: A scientific approach for possible anticancer agents. J Biomol Struct Dyn. 2024;42:2242-2256. https://doi.org/10.1080/07391102.2023.2212306
- [Google Scholar]
- Global perspectives and hotspots of VEGF signalling pathway in liver disease from 2008 to 2023: A bibliometric analysis and visualization. Heliyon. 2025;11:e41346. https://doi.org/10.1016/j.heliyon.2024.e41346.
- [Google Scholar]
- Green approach for the effective reduction of graphene oxide using salvadora persica L root (Miswak) extract. Nanoscale Res Lett. 2015;10:987. https://doi.org/10.1186/s11671-015-0987-z
- [Google Scholar]
- Characterization of leaves and flowers volatile constituents of Lantana camara growing in central region of Saudi Arabia. Arab J Chem. 2016;9:764-774. https://doi.org/10.1016/j.arabjc.2015.11.005
- [Google Scholar]
- Antibiotic and antibiofilm activities of salvadora persica L essential oils against streptococcus mutans: A detailed comparative study with chlorhexidine digluconate. Pathogens. 2020;9:66. https://doi.org/10.3390/pathogens9010066
- [Google Scholar]
- Traditional use, phytochemicals and pharmacological activity of salvadora persica: A review. CNF. 2021;17:302-309. https://doi.org/10.2174/1573401316999200807205224
- [Google Scholar]
- Anti-VEGF mediated immunomodulatory role of phytochemicals: Scientific exposition for plausible HCC treatment. Curr Drug Targets. 2021;22:1288-1316. https://doi.org/10.2174/1389450122666210203194036
- [Google Scholar]
- Traditional uses, phytochemistry, pharmacology and toxicology of ruta graveolens l.: A critical review and future perspectives. Drug Des Devel Ther. 2024;18:6459-6485. https://doi.org/10.2147/DDDT.S494417
- [Google Scholar]
- In-vitro anti-proliferative, apoptotic and antioxidative activities of medicinal herb kalonji (Nigella sativa) Curr Pharm Biotechnol.. 2019;20:1288-1308. https://doi.org/10.2174/1389201020666190821144633
- [Google Scholar]
- Angiogenesis in hepatocellular carcinoma; pathophysiology, targeted therapy, and role of imaging. J Hepatocell Carcinoma. 2020;7:77-89. https://doi.org/10.2147/JHC.S224471
- [Google Scholar]
- Angiogenesis and hepatocellular carcinoma: from molecular mechanisms to systemic therapies. Medicina (Kaunas). 2023;59:1115. https://doi.org/10.3390/medicina59061115
- [Google Scholar]
- Evaluation of functional and bioactive properties of crude gill extract of Tor putitora using different assays. Int J of Food Sci Tech. 2022;57:6459-6470. https://doi.org/10.1111/ijfs.15961
- [Google Scholar]
- A review: Nutrition and oral-dental health, phytochemical content, biological activity of Salvadora persica (Miswak) Prosp. Pharm. Sc.. 2024;22:18-25. https://doi.org/10.56782/pps.177
- [Google Scholar]
- “Miswak” based green synthesis of silver nanoparticles: Evaluation and comparison of their microbicidal activities with the chemical synthesis. Molecules. 2016;21:1478. https://doi.org/10.3390/molecules21111478
- [Google Scholar]
- An evaluation of sex-specific pharmacokinetics and bioavailability of kokusaginine: An in vitro and in vivo investigation. Pharmaceuticals (Basel). 2024;17:1053. https://doi.org/10.3390/ph17081053
- [Google Scholar]
- The prevalence of gingival recession among miswak (Salvadora persica l.) chewing stick users: A systematic review. J Herb Med. 2025;51:101003. https://doi.org/10.1016/j.hermed.2025.101003
- [Google Scholar]
- Chick chorioallantoic membrane as an in vivo model for the study of angiogenesis and lymphangiogenesis. J. Vasc. Res.. 2025;62:109-120. https://doi.org/10.1159/000542875
- [Google Scholar]
- Screening of active ingredients from wendan decoction in alleviating palmitic acid-induced endothelial cell injury. Molecules. 2023;28:1328. https://doi.org/10.3390/molecules28031328
- [Google Scholar]
- Synergistic effect of VEGF and SDF-1α in endothelial progenitor cells and vascular smooth muscle cells. Front Pharmacol. 2022;13:914347. https://doi.org/10.3389/fphar.2022.914347
- [Google Scholar]
- Identification of anti-nociceptive constituents from the pollen of Typha angustifolia l. using effect-directed fractionation. Nat Prod Res. 2020;34:1041-1045. https://doi.org/10.1080/14786419.2018.1539979
- [Google Scholar]