GC/MS Analysis, Cytotoxicity, and Antiviral Activities of Annona glabra Hexane Extract Supported by In Silico Study

Annona glabra Linn is employed in conventional medicine to treat a number of human disorders, including cancer and viruses. In the present investigation, the significant phytochemical components of Annona glabra hexane extract were identified using gas chromatography–mass spectrometry (GC-MS) analysis. Three major compounds were identified in the hexane extract: tritriacontane (30.23%), 13, 17-dimethyl-tritriacontane (22.44%), and limonene (18.97%). MTT assay was used to assess the cytotoxicity of the extract on six human cancer cell lines including liver (HepG-2), pancreas (PANC-1), lung (A-549), breast (MCF-7, HTB-22), prostate (PC-3), and colon (CACO-2, ATB-37). The extract exhibited significant cytotoxic activity against both CACO-2 and A-549 cancer cell lines (IC50 = 47 ± 0.74 μg/mL and 56.82 ± 0.92 μg/mL) in comparison with doxorubicin (IC50 = 31.91 ± 0.81 μg/mL and 23.39 ± 0.43 μg/mL) and of SI of 3.8 and 3.1, respectively. It also induced moderate-to-weak activities against the other cancerous cell lines: PC-3, PANC-1, MCF-7, and HepG-2 (IC50 = 81.86 ± 3.26, 57.34 ± 0.77, 80.31 ± 4.13, and 57.01 ± 0.85 μg/mL) in comparison to doxorubicin (IC50 = 32.9 ± 1.74, 19.07 ± 0.2, 15.48 ± 0.84 and 5.4 ± 0.22 μg/mL, respectively) and SI of 2.2, 3.1, 2.2, and 3.1, respectively. In vitro anti-HSV1 (Herpes simplex 1 virus) and HAV (Hepatitis A virus) activity was evaluated using MTT colorimetric assay with three different protocols to test protective, anti-replicative, and anti-infective antiviral activities, and three separate replications of each experiment were conducted. The plant extract showed promising protective and virucidal activity against HSV1 with no significant difference with acyclovir (79.55 ± 1.67 vs. 68.44 ± 7.62 and 70.91 ± 7.02 vs. 83.76 ± 5.67), while it showed mild protective antiviral activity against HAV (48.08 ±3.46) with no significant difference vs. acyclovir (36.89 ± 6.61). The selected main compounds were examined for their bioactivity through in silico molecular docking, which exhibited that limonene could possess the strongest antiviral properties. These findings support Annona glabra’s conventional use, which is an effective source of antiviral and anticancer substances that could be used in pharmaceuticals.


Introduction
For centuries, cultures around the world have learned how to use herbal medicine to improve healthcare regimens. The importance of plant-based products for disease treatment is growing exponentially due to the increased incidence of adverse drug reactions and the development of microbial resistance to available antimicrobial drugs [1]. Natural products possess tremendous potential for medical development. Compounds originated from plants and their semisynthetic, synthetic, and analogous components have helped mankind as a primary source of medications for treating human illnesses. Therefore, for treating disease states where drug therapy is a reasonable course of action, plant materials are acceptable beginning points for developing novel agents [2]. Herbal medicines have a wide range of activities on the human body, including hepatoprotective [3,4], antidiabetic, anti-inflammatory [5], antioxidant, chemoprevention, immunoregulatory, and anti-toxic activities [6]. However, users should consider efficacy, quality, and safety when using natural remedies. A paradigm based on ethnobotanical and ethnopharmacological data would be more advantageous and cost-effective in the viral and cancer drug discovery program for discovering potential antiviral and anticancer molecules than the mass screening of plant species [2].
The family Annonaceae includes the genus Annona, which is a genus of tropical fruit trees. Annonaceae is a family of flowering plants that includes trees and shrubs. Several genera yield fruit that can be consumed, most notably Annona [7]. The family's most significant genus is this one, Annona, which includes approximately 119 species [8]. After Guatteria, it is regarded as the second-largest genus of flowering plants in the family Annonaceae [9]. The Latin word "anon," which means "yearly produce" and refers to the production of fruits by many species in this genus, is the source of the name "Annona" [10]. Annona glabra Linn, commonly named as pond apple [2], is a Florida-native tropical fruit tree (United States of America). It is well recognized that the Annona plant has medicinal properties, including antidiarrheal [11], antiparasitic [12], anticonvulsant [13], antiulcer [14], antioxidant [15], antidiabetic [16], antidepressant [17], antiviral [18], Dengue vector control [19], anticancer [20], and anti-inflammatory [21] activities. In addition, pond apple leaves and bark have historically been employed as insect and parasiticides [12]. The complete plant of the pond apple tree is used as an anticancer medication in China, and the leaves are used to cure chronic bronchitis [2]. An alcoholic seed extract from Annona glabra has demonstrated anticancer action, making it a viable source of chemicals for cancer treatment [22]. From the phytochemical standpoint, the Annonaceous acetogenins, a novel class of chemicals discovered from Annona plants and showing potent anticancer activity, were found to be abundant in the plant. Three different Annona plant extracts-bullatacin, asinicin, and bullatacinone-showed 300-, 80-, and 40-times more anticancer effects than taxol in vivo tests on mice with leukemia. Liu et al. proved the anticancer effect of two extracts from Annona glabra, which is 100,000 times higher than adriamycin. Annona plants have emerged as a new, promising source for cutting-edge cancer treatments [20]. A. glabra's phytochemical study isolated acetogenins, ent-kauranes, and alkaloids [23]. Cunabic acid and ent-Kauran-19-al-17-oic acid, two diterpenoid substances derived from Annona glabra Linn, can clearly stop the growth of HLC cell line SMMC-7721 [20].
Glacin A and glacin B, two novel bioactive mono-THF acetogenins, have shown strong and targeted in vitro cytotoxicities against a variety of human solid cancer cell lines [24]. The hexane extract of A. glabra stem bark also revealed significant antibacterial activity in a preliminary screening [22]. The fruits of Annona glabra showed some action against HIV replication in H9 lymphocyte cells and considerable suppression of HIV-reverse transcriptase through phytochemical analyses [25].
The increase in virus infections and cancer diseases has necessitated the ever-increasing demand for therapeutic natural product drugs due to the lack of vaccines, the emergence of viral strains resistant to antiviral medications, and the side effects of cancer chemotherapy. Antiviral activity is not widely reported from Annona species. In light of the fact that acyclovir, a nucleoside analog, is one of the medications that is endorsed for the treatment of HSV infections, our curiosity to assess the antiviral activity of Annona glabra against some notable and easily cultivable viral pathogens, such as Hepatitis A virus (HAV), Herpes simplex type 1 (HSV1), and New Castle with three different mechanisms, has been peaked.
Herein, using gas chromatography-mass spectrometry (GC-MS), we extracted and specified some of the components of the hexane extract of Annona glabra leaves and assessed the cytotoxic activity of A. glabra against hepatocellular carcinoma (HepG-2), colorectal adenocarcinoma (CACO-2), breast cancer (MCF-7), lung (A-549), prostate (PC-3), and pancreas cancer (PANC-1) cell lines. One chemotherapy drug used to treat cancer is called doxorubicin was used as a positive control. Through intercalation and suppression of macromolecular production, it interacts with DNA. This prevents topoisomerase II, an enzyme that loosens DNA supercoils for transcription, from progressing. Additionally, it keeps the DNA double helix from being released and stops the replication process by stabilizing the topoisomerase II complex after it has broken the DNA chain for replication [26].
The increase in virus infections and cancer diseases has necessitated the ever-increasing demand for therapeutic natural product drugs due to the lack of vaccines, the emergence of viral strains resistant to antiviral medications, and the side effects of cancer chemotherapy. Antiviral activity is not widely reported from Annona species. In light of the fact that acyclovir, a nucleoside analog, is one of the medications that is endorsed for the treatment of HSV infections, our curiosity to assess the antiviral activity of Annona glabra against some notable and easily cultivable viral pathogens, such as Hepatitis A virus (HAV), Herpes simplex type 1 (HSV1), and New Castle with three different mechanisms, has been peaked.
Herein, using gas chromatography-mass spectrometry (GC-MS), we extracted and specified some of the components of the hexane extract of Annona glabra leaves and assessed the cytotoxic activity of A. glabra against hepatocellular carcinoma (HepG-2), colorectal adenocarcinoma (CACO-2), breast cancer (MCF-7), lung (A-549), prostate (PC-3), and pancreas cancer (PANC-1) cell lines. One chemotherapy drug used to treat cancer is called doxorubicin was used as a positive control. Through intercalation and suppression of macromolecular production, it interacts with DNA. This prevents topoisomerase II, an enzyme that loosens DNA supercoils for transcription, from progressing. Additionally, it keeps the DNA double helix from being released and stops the replication process by stabilizing the topoisomerase II complex after it has broken the DNA chain for replication [26].

In Vitro Cytotoxicity Assay
The MTT test was used to assess the cytotoxicity of Annona glabra hexane extract against normal Vero and six human cancer cell lines of the liver (HepG-2), breast (MCF-7), colon (CACO-2), pancreas (PANC-1), lung (A-549) and prostate (PC-3). The cytotoxicity experiment was performed 48 h after the extract was inoculated with various doses ranging from 31.25 to 1000 µg/mL. The outcomes revealed that the sample had noncytotoxic action to the normal cells (IC 50 value > 100 µg/mL). Moreover, the investigated sample, in a way depending on concentration, prevented the proliferation of the tumor cells. Due to this, SI of the extract to Vero and different cancer cells was more than that of doxorubicin >1, indicating that the extract was safer. The safer the medicine, the higher the SI value.
The IC 50 , µg/mL (the concentration that results in a 50% reduction in cell viability), and selectivity index (SI) values were used to express the sensitivity of the cell lines to the sample. In general, the drug potency is inversely proportional to the IC 50 values.
The easiest evaluation of IC 50 is to plot x-y and fit the data with a straight line (linear regression). IC 50 value is then evaluated using the fitted line, i.e., Y = a * X + b,

Annona glabra Sample MNTC Determination
The MNTC is known as the maximum nontoxic concentration required to produce optimum biological activity and less of a cytotoxic effect. As shown in Table 3, MNTC was determined for Annona hexane extract as 7.81 µg/mL vs. acyclovir (62.5 µg/mL) as a reference drug.

Antiviral Efficacy of the Annona glabra Extract versus HAV and HSV1
The Minimum nontoxic concentration of Annona glabra sample was tested against each virus to assess the mechanism behind its antiviral action on the basis of three protocols (Tables 4-6).   The antiviral action of acyclovir was demonstrated in the following order: protocol B (92.69 ± 1.32) > protocol A (83.76 ± 5.67) > protocol C (68.44 ± 7.62), as shown in Table 5. On investigating the antiviral performance of Annona glabra extract versus HSV1, it was discovered that the extract showed pronounced antiviral activity with HSV1 using protocol C and protocol A, with no significant difference with acyclovir (79.55 ± 1.67 vs. 68.44 ± 7.62) and (70.91 ± 7.02 vs. 83.76 ± 5.67), and moderate activity with protocol B (61.91 ± 3.51), with no significant difference. Acyclovir showed the highest activity with protocol B (92.69 ± 1.32). Thus, the Annona extract showed pronounced protective and virucidal antiviral activity against HSV1, and moderate anti-replicative activity with no significant difference.

Comparison of the Effect of the Annona Extract versus HAV vs. Acyclovir with Different Protocols
The antiviral action of acyclovir was demonstrated in the following order: protocol B (54.8± 11.7) > protocol A (46.17 ± 1.67) > protocol C (36.89 ± 6.61), as shown in Table 6. On investigating the antiviral performance of Annona extract versus HAV (Table 6), it was found that, for dealing with the hexane extract, HAV's antiviral activity was: protocol C (48.08± 3.46) > protocol A (36.81 ± 2.67), with no significant difference. Furthermore, the least among the protocols was protocol B (20.13 ± 3.1), which showed no antiviral activity against HAV. Thus, Annona extract showed mild protective antiviral activity against HAV.

Molecular Docking 2.4.1. Docking into Cyclin-Dependent Kinase 2 Active Site
Cyclin-dependent kinases (CDKs) are a class of serine/threonine kinases that work with cyclins to ensure the development of the cell cycle [29]. CDKs are the key managers of cell division and are capable of creating a heterodimer with cyclins to catalyze the phosphorylation of diverse substrates, resulting in the change of the cell cycle at different phases [30]. Intriguingly, CDKs have significant roles in antiviral responses; the present investigation targeted CDK2 as a potential therapeutic target in an effort to develop drugs with antiviral efficacy against HSV-1. Tritriacontane, limonene, and 13,17-dimethyltritriacontane, the major compounds of hexane extract, were docked and linked to the ATP binding site of the CDK2 enzyme as roscovitine, which is a cocrystal ligand (Figures 2-8; Table 7). Tritriacontane forms two alkyl bonds with the essential binding residues Lys89 and Lys88 [31] ( Figure 2; docking scores −6.6 kcal/mol). 13,17-dimethyltritritritriacontane exhibited a favorable interaction by establishing six hydrophobic bonds in the CDK2 active site (Figure 4). The binding mode of limonene in the active site of CDK2 kinase is depicted in Figure 8, with the highest docking score of −7.5 kcal/mol coming from roscovitine. Limonene docked similarly to roscovitine ( Figure 5) at the hinge region of CDK2 and interacts hydrophobically with key residues, including Val18, Lys33, Ala31, Val64, Leu134, and Ala144 [31]. Additionally, it interacts with the gatekeeper residue Phe80 through a Pi-sigma interaction. Figure 6 displayed a significant RMSD of 0.48 Å for the cocrystal ligand (roscovitine) in its docked position. through a Pi-sigma interaction. Figure 6 displayed a significant RMSD of 0.48 Å for the cocrystal ligand (roscovitine) in its docked position.   Molecules 2023, 28, x FOR PEER REVIEW 10 of 22 through a Pi-sigma interaction. Figure 6 displayed a significant RMSD of 0.48 Å for the cocrystal ligand (roscovitine) in its docked position.             The HAV 3C protease (3Cpro) is in control of the majority of cleavages within the viral polyprotein, allowing for viral replication and propagation. HAV 3Cpro is one of the primary targets for the development of anti-HAV medications due to its critical role in viral replication and proliferation [32]. The three docked compounds and the inhibitor drug (Z10325150) were anchored in the catalytic site, with docking scores ranging from −6.3 to −6.9 kcal/mol (Figures 7-10; Table 7). All of them interact with His44, except for 13,17-dimethyltritriacontane (Figures 7-10), which displayed three hydrophobic interactions with Lys146, Val144, and Val28 ( Figure 9). Limonene formed seven alkyl bonds with His44, Cys172, His145, and Met29 (Figure 8), which showed the highest docking score of −6.9 kcal/mol coming from the Z10325150 compound.

Docking into Hepatitis A Virus 3C (HAV 3C) Protease Enzyme Active Site
The HAV 3C protease (3Cpro) is in control of the majority of cleavages within the viral polyprotein, allowing for viral replication and propagation. HAV 3Cpro is one of the primary targets for the development of anti-HAV medications due to its critical role in viral replication and proliferation [32]. The three docked compounds and the inhibitor drug (Z10325150) were anchored in the catalytic site, with docking scores ranging from −6.3 to −6.9 kcal/mol (Figures 7-10; Table 7). All of them interact with His44, except for 13,17dimethyltritriacontane (Figures 7-10), which displayed three hydrophobic interactions with Lys146, Val144, and Val28 (Figure 9). Limonene formed seven alkyl bonds with His44, Cys172, His145, and Met29 (Figure 8), which showed the highest docking score of −6.9 kcal/mol coming from the Z10325150 compound.

Discussion
Viral infections continue to be a major cause of morbidity and mortality around the world. With more organ transplants, blood transfusions, and the use of hypodermic syringes, the number of patients diagnosed with viral infections rises alarmingly each year. Traditional antiviral drugs, such as ribavirin and interferon, are effective in vitro against

Discussion
Viral infections continue to be a major cause of morbidity and mortality around world. With more organ transplants, blood transfusions, and the use of hypodermic ringes, the number of patients diagnosed with viral infections rises alarmingly each y Traditional antiviral drugs, such as ribavirin and interferon, are effective in vitro aga

Discussion
Viral infections continue to be a major cause of morbidity and mortality around the world. With more organ transplants, blood transfusions, and the use of hypodermic syringes, the number of patients diagnosed with viral infections rises alarmingly each year. Traditional antiviral drugs, such as ribavirin and interferon, are effective in vitro against most viruses but are frequently ineffective in patients. There is currently no approved treatment for many types of viruses, and vaccination is limited to mumps, hepatitis A, and varicella [33]. Furthermore, in most cases, their use is accompanied by the appearance of side effects or the formation of resistant viral mutants, making therapy ineffective. Therefore, turning to nature to find effective therapies is a good solution to this problem. Thus, the need to discover novel antiviral herbal remedies has been mandated [1]. Numerous studies have been conducted on the cytotoxic and antiviral activities of natural products. In this study, the GC-MS investigation of Annona extract showed the presence of 14 phytochemical compounds, in which tritriacontane, limonene, and 13,17-dimethyltritriacontane are the major peaks.
The investigated extract exhibited a broad cytotoxic activity range against the cancer cell lines under examination. Such differences in cytotoxicity between different types of cells can be explained owing to the variations in their morphology, genomes, and origins, which lead to variations in how susceptible they are to chemotherapy. Thus, the pronounced cytotoxicity of the examined extract is a direct result of the high concentration of compounds in the plant extract. The extract exhibited significant cytotoxic activity against both CACO-2 and A-549 cancer cell lines in comparison with doxorubicin and SI of 3.8 and 3.1, respectively. It also induced moderate-to-weak activities against the other cancerous cell lines, i.e., PC-3, PANC-1, MCF-7, and HepG-2, when compared to doxorubicin and SI of 2.2, 3.1, 2.2, and 3.1, respectively. High selectivity was achieved when the SI was ≥ 3. As the selective index (SI) demonstrates the differential activity of the sample, the greater the SI value is, the more selective it is. An SI value less than two indicates general toxicity of the sample [22]. So, the extract exhibited no cytotoxic activity against normal cells and is considered selective to CACO-2, A-549, PANC-1, and HepG-2.
Moreover, we assessed the plant extract's antiviral capacity and mechanism of action, which was designed to measure the protective, anti-replicative, and anti-infective activity. The MNTC of plant extract vs. acyclovir, a reference medicine utilized as a positive control, was employed in all antiviral assays.
This study proved that the plant extract showed promising protective and virucidal activity against HSV1 (79.55 ± 1.67 and 70.91 ± 7.02) with no significant differences with acyclovir, while the plant extract showed moderate anti-replicative activity against HSV1 (61.91 ± 3.51) with no significant difference. Moreover, the plant extract showed mild protective antiviral activity against HAV (48.08 ± 3.46) with no significant difference vs. acyclovir (36.89 ± 6.61) and no anti-replicative antiviral activity versus HAV (20.13 ± 3.1).
The results offer experimental proof that Annona glabra extract exhibited higher antiviral efficacy versus HSV1 than HAV for protocol C and A. Acyclovir, as a positive control, is one of the medications that has been approved for use as a first-line treatment for HSV infections. It works by blocking DNA synthesis and viral DNA polymerase by targeting viral thymidine kinase, which prevents viral replication [37]. These activities are correlated to the compounds with high concentrations inside the plant extract. From previous studies, it was reported that Annona glabra displayed mild activity versus HIV replication in H9 lymphocyte cells and considerable inhibition of HIV-reverse transcriptase [25]. Moreover, Maqian essential oils showed promising antiviral activities owing to the presence of Llimonene and D-limonene [38]. Essential Oils from Citrus Aurantium L. have virucidal activity against Influenza A Virus H1N1 due to the presence of D-and L-limonene, which may be employed as an efficient disinfectant against viruses [39]. Monoterpenes betapinene and limonene have been reported to show antiherpetic activity and might be used as powerful antiviral agents in recurrent herpes infections [40].
In silico molecular docking is one of the best techniques to determine new ligands for proteins with known structure and thus plays a significant role in the development of drugs based on structure [41]. Intriguingly, CDKs have significant roles in antiviral responses; a recent publication review summarizes the actions of CDKs in antiviral innate immunity. CDKs are potential cellular targets, as they are implicated in the transcription and replication of viral genomes [42,43]. Since CDK inhibitors have manageable side effects in clinical studies for the treatment of cancer and other disorders, they have been investigated as antivirals. Antigen expression study indicates that latent HSV1 reactivation is associated with CDK2 expression in neurons. It has shown that roscovitine (CDK2 inhibitor) inhibits the reactivation of HSV-1 in ex vivo neurons. On the basis of this information, the present investigation targeted CDK2 as a potential therapeutic target in an effort to develop drugs with antiviral efficacy against HSV-1 [44]. Tritriacontane, limonene, and 13,17-dimethyltritriacontane were docked and linked to the ATP binding site of the CDK2 enzyme as roscovitine, which is a cocrystal ligand. Tritriacontane forms two alkyl bonds with docking scores −6.6 kcal/mol [31]. The binding mode of limonene in the active site of CDK2 kinase had the highest docking score −7.5 kcal/mol, which came from roscovitine. Limonene was docked similarly to roscovitine at the hinge region of CDK2 and interacts hydrophobically with key residues. 13,17-dimethyltritriacontane and tritriacontane exhibited less binding activity than limonene (−7.3, −6.6 vs. −7.5). HAV 3Cpro is one of the main targets for the development of anti-HAV medications [32]. The discovery of natural molecules with biological activity is crucial for the identification of new drug candidates and the study of biological systems. Furthermore, limonene formed seven alkyl bonds with His44, Cys172, His145, and Met29 and showed the highest docking score of −6.9 kcal/mol with the Z10325150 compound for HAV 3C protease. All these results revealed that, notably, the hexane extract showed interactions with CDKS and HAV3C protease. These interactions may provide an important foundation for its development as a natural viral defense candidate. To build in silico results with in vivo activities, more research is required.

Plant Material
The whole plant of Annona glabra (Annonaceae) was collected from Mazhar Botanic Garden, Egypt, in January 2020. The plant was identified by Mrs. Trease Labib, a senior botanist specializing in plant taxonomy, Orman Garden, Giza, Egypt, as well as by comparison with reference herbarium specimens. At the Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt, a voucher specimen (PHG-P-AG-381) was deposited.

Preparation of the Crude Extract
One kilogram of the air-dried plant was ground into fine particles and thoroughly extracted at room temperature using hexane (5 L × 3 times) for 3 days each. The resultant hexane extract was then dried completely using a rotatory evaporator at a low temperature (55 • C) and reduced pressure. This produced a sticky dark greenish extract (280 g).

GC-FID and GC-MS Analysis of A. glabra Hexane Extract
A Shimadzu GC with an FID detector and an HP-5 fused silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm) were used to perform the GC-FID analysis. The initial oven temperature was 50 • C (with a hold for 2 min and then programmed to 300 • C at 5 • C/min). Both the injector and the detector were at a temperature of 280 • C. Helium (1.0 mL/min) was employed as the carrier gas. A split ratio of 1:15 and an automatic sample injection (1 µL) was employed. Peak area normalization was used to determine the percentages that represented the relative proportions of the hexane extract's constituents. GC-MS analysis was implemented on a Shimadzu GCMS-QP 2010 (Koyoto, Japan) equipped with an Rtx-5MS capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) (Restek, Bellefonte, Pennsylvania, USA). A quadrupole mass spectrometer was directly coupled to the capillary column (SSQ 7000; Thermo-Finnigan, Bremen, Germany). The injector temperature was 250 • C, and the oven temperature was kept at 50 • C for 2 min (isothermal), then programmed to 300 • C at 5 • C/min and kept constant at 300 • C for 5 min (isothermal). As a carrier gas, helium was used with a constant flow rate of 1.41 mL/min. Diluted samples (1% v/v) were injected with a split ratio of 1:15, and the injected volume was 1 µL. The MS operating parameters were as follows: ion source temperature: 200 • C, interface temperature: 280 • C, EI mode: 70 eV scan range: 35-500 amu. Components were identified based on their mass spectral data and Kovats retention indexes (KI). According to a homologous series of n-alkanes (C8-C28) injected under the same circumstances, retention indexes were calculated. The peak area percent of each compound relative to the area percent of the entire FID chromatogram (100%) was calculated.

Preparation of Stock Solution of the Plant Hexane Extract
We dissolved 1 g of the extract in 10 mL of Eagle's minimal essential medium (MEM), sterilized the mixture, and stored it at stock concentrations of 100 mg/mL. For use in the in vitro investigations, further dilutions were prepared in a cell culture medium.

In Vitro Cytotoxicity Assay (Viability Assay)
Six human cancer cell lines were employed to test the cytotoxicity of the hexane leaf extract using the MTT assay [45].

Determination of Cytotoxicity by MTT Assay
The MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazollium bromide] technique was used to assess the cytotoxicity [45]. The 96-well tissue culture plate was seeded with 1 × 10 5 cells/mL (100 µL/mL) and incubated at 37 • C for 24 h to form a complete monolayer sheet. The growth medium was then decanted from the 96-well microtiter plates, and 0.1 mL of each dilution (made by two-fold dilutions in RPMI medium with 2% serum maintenance medium) was examined in different wells, leaving 3 wells as control, receiving only a maintenance medium. The plates were incubated at 37 • C in an atmosphere with 95% relative humidity and 5% CO 2 gas. After 2 days, 20 µL of MTT solution (5 mg/mL in PBS, BIO BASIC INC, Markham, Canada) prepared in cell culture medium was added to each well; then, the wells were placed on a shaking table for 5 min to fully include the MTT into the media, and the plates were cultured for 4 hours to enable the metabolization of the MTT. Each well received 200 µL of DMSO to dissolve the formazan crystals after the MTT solution had been removed without disturbing the cells (MTT metabolic product). After giving the plates a light shake (150 rpm/5 min), the crystals were entirely disintegrated. At a wavelength of 560 nm, using a microplate reader, the optical density (OD) of each well was calculated.

Cell Culture and Viruses
For the antiviral experiments, Vero cells isolated from the kidney of an African green monkey (CCL-81; American Type Culture Collection, USA) were cultured in MEM containing fetal calf serum (FCS; 10% v/v), penicillin (100 U/mL), L-glutamine (2 nM), and streptomycin (100 µg/mL). Cells were multiplied for up to 4 weeks before being cultured as a cell line stock at 37 • C in a humidified environment containing 5% CO 2 . The Laboratory of Virology, Microbiology Department, Faculty of Medicine (for Girls), Al-Azhar University, Cairo, Egypt, kindly donated HAV and HSV1.

Virus Stock Preparation
Each virus was used to multiply in Vero cells by infecting a monolayer of confluent Vero cells in 75 cm 2 culture flasks and letting them adsorb for one hour. The infected cells were then covered with 20 mL of 2% MEM (maintenance media) and incubated until a full cytopathogenic effect (CPE) was seen daily for up to 4-6 days. Nonadherent particles were then rinsed off using 2% MEM. This process was performed twice, after which the plaque formation assay [46,47] was used to calculate the challenge dosage of each virus, and each viral harvest was kept at a temperature of −20 • C until use.

Cytotoxicity Assay
The tested material was diluted ten times in MEM with FCS, going from 100 mg/mL to a 10-6 dilution, to determine the extract's maximum nontoxic concentration (MNTC). Confluent Vero cells were then treated with 0.2 mL of the dilution in four of the wells of a 96-well Falcon plate (Falcon; Corning, USA). In addition to evaluating MNTC, acyclovir (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in distilled water. At 37 • C, the plates were incubated. A daily inverted microscope examination of the cells was conducted to ascertain the minimum concentration necessary to cause changes in cell morphology. Even after 7 days, there were no morphological alterations between the treated and control Vero cells at the MNTC. As previously mentioned, a Colorimetric MTT assay (Bio Basic Inc., Markham, Canada) was carried out [45].

Antiviral Protocols
The virus's challenge dose (CDV) was introduced into cultures of Vero cells that received treatment using the extract's MNTC and its serial dilution in accordance with three protocols in order to examine the mechanism of the antiviral activity of the plant extract [48][49][50][51]. Each protocol was independently repeated three times to ensure consistency, and the mean of the three experiments for each protocol was then given.
Protocol A: Virus Pretreatment (Anti-Infective Activity) [51] The virucidal activity of the extract was examined using this procedure. The challenge virus was subjected to an equal volume of a nonlethal dilution (MNTC) of the tested extract (1:1 v/v) for one hour at 37 • C. Then, 0.5 mL of media containing the extract and an equivalent volume of the CDV viral suspension of each virus were placed on top of confluent monolayers of Vero cells in 96-well flat-bottomed microtiter plates. The viralinduced CPE was measured using the MTT colorimetric test for 72 h under an inverted microscope after the plates were incubated at 37 • C for 60 min following virus adsorption.
Protocol B: Postinfection Treatment (Anti-Replicative Activity) [48,50] This technique examined how the extract affected the spread of viruses. The CDV (100 µL) of each virus was incubated with a confluent layer of Vero cells in 96-well plates. A layer of agarose containing the (MNTC) of the tested extract was applied to the cells after the cells had been adsorbed for 60 min. After 72 h, CPEs were found on the monolayer. Using the formazan absorbance value utilized in the MTT inclusion experiment, as was said earlier, the viability of both infected and healthy cells was evaluated [45]. Additionally, the IC 50 , or 50% inhibitory concentration, was computed.
Protocol C: Cell Pretreatment (Protective Activity) [49,50] By preventing adhesion to the cell surface, this approach was utilized to check for viral penetration into the host cells. Confluent Vero cell monolayers were pretreated for about 48 h on a 96-well flat-bottomed microtiter plate using a medium containing the tested extract (MNTC). The cells were then challenged with the CDV of each virus after being rinsed twice with phosphate-buffer saline. The monolayer was covered with agarose solution containing the culture media and allowed to incubate after 60 min of adsorption at 37 • C. The virus-induced CPE was graded, as previously stated, after 72 h.
Three wells in each treatment were left uninoculated to serve as a negative control, while three additional wells were infected with MNTC of the antiviral drug acyclovir (positive control).

Molecular Docking
The Protein Data Bank provided the crystal structures of CDK2 (pdb ID.: 2a4l) [52], and HAV 3C protease (pdb ID.: 2cxv) [53] was accomplished utilizing AutoDock 4.2 [54]. The preparation of ligands and protein files, grid, and docking parameters files was conducted following the reported method [55]. The protein and the ligand were prepared and saved as pdbqt files from AutoDock software. A 3D grid box of 60 × 60 × 60 Å size with the space of 0.37 Å was placed at 100, 101, and 79 Å for docking into CDK2 and at 30.0, 0.24, and 0.27 Å for docking into HAV 3C protease. Biovia Discovery Studio 4.5. was used in the evaluation and visualization of the docking outcomes.

Statistical Analysis
Data of the Annona extract and doxorubicin (positive control) cytotoxicity against a number of cell lines including Vero, HepG-2, Caco-2, Pc-3, Panc-1, Mcf-7, and A-549 have been investigated. The antiviral activity of Annona extract and Acyclovir (positive control) were also investigated against HAV and HSV1 viruses. A trio of protocols (A, B, and C) was used to combat HAV, HSV1, and Newcastle viruses. All analyses were executed using Minitab 20 and SPSS 28. Before performing any statistical analyses, the data were cleaned. The data were also reviewed for missing data and typographical errors. Three replicates for each level were used to obtain descriptive statistics such as mean, standard deviation (SD), standard error (SE), minimum (min), first quartile (Q1), third quartile (Q3), median, and maximum (max). The outcomes of various groups were compared using inferential statistics. The box-cox transformation for non-normal dependent variables was implemented whenever necessary using the optimal λ approach, and all variable parametric assumptions were tested. R 2 results showed a value of over 97%, while the adjusted R 2 showed a value of over 95% for all cytotoxicity results, antiviral activity results showed an R 2 value of over 65% and adjusted R 2 over 60%, and the Newcastle virus results showed low R 2 and adjusted R 2 . Under the fit general linear model, one-way and two-way analyses of variance were used for a number of comparisons (ANOVA). Results indicated a good fit for several models, and after data transformation, normal residual probability plots displayed a linear attitude for all studies. p-values were deemed significant at α < 0.05. Using the Tukey test for pairwise comparisons, a post hoc analysis of all group interactions was conducted. Results of the post hoc analyses are shown as letters, with groups sharing the same letter being nonsignificant different from one another and groups with distinct letters expressing statistically different characteristics.

Conclusions
The goal of the current inquiry was to identify different bioactive chemicals from the hexane leaf extract of Annoa glabra by GC-MS analysis. Tritriacontane, limonene, and 13,17dimethyltritriacontane are the main substances in charge of the various pharmacological and therapeutic activities. We have additionally offered proof of the hexane leaf extract of A. glabra for its antiviral and cytotoxic activities.
This study showed that the plant extract exhibited a high variation of the cytotoxic effects against tested tumor cell lines. The reported activity was detected against the colon, lung cancer cell lines. Antiviral activity is not widely reported from Annona species. In this study, it was proved that an Annona glabra hexane extract had a pronounced protective and virucidal antiviral activity against HSV1 and mild protective antiviral activity against HAV. In addition, moderate anti-replicative activity was exerted by the plant extract against the HSV1 virus. Limonene showed the highest docking score for both HSV1 and HAV. Our research suggests that the plant could help us create trustworthy and potent medications to fight viral infections and cancer. For the development of new medication formulations, additional studies to evaluate their bioactivity and clinical trials are required.