In vitro and in silico antiproliferative potential of isolated ﬂ avonoids constitutes from Pistacia

: Cancer is one of the most demanding domains for innovative, e ﬀ ective, safe, and a ﬀ ordable therapeutically active chemicals. The main aim of this study is to research new phytochemicals with anticancer activity. The current experiment identi ﬁ ed and analyzed six compounds for anti-cancer potential supported by molecular simulation studies. The defatted methanolic extract underwent column chromatography, resulting in the isolation of six ﬂ avonoids. These include 3,5,7,4 ′ -tetrahydroxy- ﬂ avanone ( 1 ), naringenin ( 2 ), 3,5,4 ′ -trihydroxy-7-methoxy- ﬂ avanone ( 3 ), sakuranetin ( 4 ), spinacetin ( 5 ), and patuletin ( 6 ). The isolated compounds ( 1 – 6 ) were assessed for in vitro anti-cancer activity against various cell lines such as HepG2 (hepatoma G2), A498 (kidney), NCI-H226 (lungs), and MDR2780AD (human ovarian). The maximum anti-proliferative e ﬀ ect was against HepG2 and MDR2780AD. When compounds 6 , 5 , and 1 were compared to a standard anti-cancer medicine (paclitaxel) with an IC 50 of 7.32, it was shown that compounds 6 , 5 , and 1 exhibited signi ﬁ cant activity against HepG2 with IC 50 values of 14.65, 20.87, and 27.09 µM, respectively. All tested compounds showed an IC 50 of less than 1 µM and had notable e ﬀ ects against MDR2780 AD cell lines. Compound 6 exhibited notable potency against the HepG2, A498, and MDR2780AD cell lines, among the six compounds that were evaluated. In contrast, compound 3 demonstrated the most pronounced impact on the NCI-H226 cell line. Docking investigations were performed using tubulin as the speci ﬁ c target concerning PDB ID 4O2B. The six compounds under investigation interact hydrophobically and hydrophilically with tubulin-binding site amino acid residues.


Introduction
Medicinal herbs are utilized by an estimated 80 % of the global population to address their fundamental health needs.The relationships between humans and plants and medical procedures from plants may be used to recap human history.Herbal treatments are primarily derived from plants.It is believed that plants contain substances that might be employed in contemporary medicine to heal illnesses that now have no known cure.Pakistan has a wide variety of flora, particularly in Khyber Pakhtunkhwa.Nationwide, there is an extensive array of over 6000 distinct species of wild plants, with an estimated 400 to 600 species believed to possess therapeutic properties.In the last decade, there has been a significant global expansion in recognizing and utilizing conventional medical systems.Despite the availability of modern medicine in numerous developing nations, prevailing estimates indicate that a considerable proportion of the population in these countries continues to rely on traditional healers for their healthcare needs predominantly.The continued need for herbal therapies can be attributed to historical and cultural influences [1].
Numerous secondary metabolites or mixtures, such as flavonoids, terpenoids, tannins, and alkaloids, are present in medicinal plants and primarily impact their antibacterial effects.Similar phytochemicals, such as flavonoids and tannins, have also demonstrated antimicrobial activity against pathogenic bacteria [2].The Anacardiaceae family includes Pistacia integerrima, also known as Kakar Singh.It is offered for sale in several countries, including Pakistan, Afghanistan, and India [3].The single-stem, deciduous P. integerrima tree still only reaches a height of around 25 m.Pemphigus pests form robust, multi-branched structures [4].The pharmacological properties of the P. integerrima tree are widely recognized and generally acknowledged.Due to its pharmacological properties as a hematological purifier, expectorant, anti-inflammatory agent, anti-diabetic medication, and digestive enhancer, it can be employed in a diverse range of therapeutic applications.
P. integerrima, a botanical of considerable importance in traditional medicine, exhibits potential therapeutic properties in mitigating oxidative stress and addressing hyperuricemia [5].The aforementioned botanical remedy exhibits potential medicinal properties in managing pyrexia, bronchial asthma, respiratory distress, pertussis, gastrointestinal disturbances, and emetic episodes within the geographical region of India [6].The constituent parts of P. integerrima were analyzed for their phytochemical composition, revealing the presence of diverse bioactive secondary metabolites, including tannins, terpenoids, alkaloids, flavonoids, and other compounds.The roots, leaves, and bark of P. integerrima contain tannins and terpenoids, while the bark also contains flavonoids and terpenoids [7].The pistagremic acid found in the P. integerrima plant has been demonstrated to possess potent antibacterial, enzyme-inhibitory, and antipyretic effects [8].The isolated P. integerrima and its crude extracts have promising properties concerning its potential anti-cancer, anti-oxidant, and xanthine oxidase inhibitory capabilities [9,10].The galls of the plant were found to contain three newly discovered phytoconstituents, along with the previously identified chemical compound b-sitosterol.The compounds were n-decan-30-ol-yl-neicosanoate, n-octadecan-9,11-diol-7-one, and 3-oxo-9b-lanost-1,20 (22)-dien-26-oic acid [11].Hemeg et al. discovered the in vitro α-glycosidase inhibition of all six isolated derivatives of P. integerrima.Among them, patuletin was found to be the most potent, followed by spinacetin and 3,5,7,4′-tetrahydroxy-flavanone [12].Ruh revealed that Naringenin is a bioflavonoid with a slight antiestrogenic effect [13].Naringenin and its derivatives also exhibited modest antimicrobial effects [14].In 2016, Rani et al. revealed the anticancer properties of naringenin [15].Sakuranetin and naringenin possess strong anti-inflammatory, anti-microbial, and anti-allergic properties [16].In their study, Vicente Silva and colleagues found that sakuranetin has a strong anticonvulsant impact at very low doses [17].According to Ji et al., spinacetin has strong antiinflammatory effects [18].
This study aimed to assess the antiproliferative potential of isolated flavonoids from P. integerrima through in vitro and in silico methods.
2 Materials and methods 2.1 Plant collection P. integerrima specimens were acquired from the garden at the University of Peshawar, Pakistan.Dr. Muhammad Ilyas, a faculty member from the Department of Botany, University of Swabi, Khyber Pakhtunkhwa, Pakistan, identified the plant specimen.The voucher specimen with the code UOS/Bot-102 is stored in the herbarium at the Department of Botany, University of Swabi, Khyber Pakhtunkhwa, Pakistan.

Extractions and isolation
The botanical specimens were subjected to a thorough aqueous rinsing procedure to eliminate any particulate matter that adhered to their surfaces effectively.Subsequently, the specimens were subjected to a controlled desiccation process under shaded conditions for 21 solar cycles.The dried plant (6.98 kg) was treated for 14 days of cold methanol extraction.This methanol extraction was carried out three times.The methanolic extract was obtained by concentrating it at low temperature and pressure, followed by filtration through the filter paper.This process resulted in a methanolic extract of the plant, yielding 134.76 g from 6.98 kg of the plant material.The liquid partitions of the methanolic extract (6.98 kg) with chloroform, n-hexane, and ethyl acetate produced, in that order, the soluble fractions of chloroform (79.43 g), n-hexane (40.8 g), ethyl acetate (65.65 g), and residue fraction (residue fraction = 6.98 kg).The ethyl acetate fraction, weighing 26 g, was subjected to column chromatography using silica gel as the stationary phase.The column was subsequently subjected to elution utilizing a mixture of chloroform and methanol in a ratio of 100:00:90.The obtained subfractions were subjected to iterative chromatographic analysis using a mixture of methanol and chloroform in a ratio of 100:00:88.Consequently, six distinct compounds were obtained.The physical and spectral data were meticulously scrutinized and cross-referenced with existing published data to ascertain the precise chemical structures of each isolated substance [19].

Anti-cancer activity
The evaluation of cytotoxicity for the isolated compounds was conducted using the MTT procedure.The RPMI 1640 medium, composed of Gibco BRL, was fortified with streptomycin sulfate at a concentration of 100 μg/ml, penicillin sodium salt at a concentration of 100 μg/ml, Na 2 CO 3 at a concentration of 2 mg/ml, and 10 % fetal bovine serum (FBS) procured from Gibco, Institute of Bioinformatics, National Chiao-Tung University, Hsinchu, Taiwan.The formulated medium was employed for the cultivation and sustenance of three discrete human cancer cell lines, specifically NCI-H226 (non-small cell lung), human A498 (renal), human hepatoma (HepG2), and MDR human ovarian cancer 2780AD cell lines.Mice hepatocytes were co-cultured with HepG-R (2 × 10 4 cells) and HepG2 (9 × 10 3 cells) in 96-well plates.The cellular specimens were conserved utilizing the specified concentrations of compounds ranging from 1.5 to 100 μM, or the control group was treated with a vehicle solution containing 0.2 % DMSO.Following preservation, the cells were incubated for 48 h.The examination employed an MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) tracer procured from Sigma in St. Louis, MO, USA.A similar assay was performed on the remaining cell lines.The IC 50 values of the isolated compound were acquired from the concentration-effect curves observed on different cell lines.Paclitaxel, a pharmacological agent obtained from Sigma, was employed as the positive control.
The in vitro cytotoxicity effect was assessed by employing LCMK-2 monkey kidney epithelial cells and mice hepatocytes.The compounds underwent a 24 h incubation period, during which the cell viability was evaluated using MTT protocols.The cellular specimens were preserved in a culture medium known as RPMI 1640, which was supplemented with 10 % fetal bovine serum (FBS) sourced from Gibco BRL.The medium was fortified with 110 μg/ml of penicillin sodium salt, a sodium bicarbonate solution at a concentration of 2 mg/ml, and 100 μg/ml of streptomycin sulfate.The primary inoculation encompassed the introduction of 7.1 × 103 LCMK-2 cells and 8.6 × 103 mice hepatocytes into 96-well plates.
The cellular specimens were preserved using various test sample concentrations, along with a vehicle solution containing 0.2 % DMSO.Subsequently, these specimens were incubated for 48 h, followed by the implementation of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, as per the established protocol provided by Sigma [20].

Docking studies
To explore possible targets for cancer therapy, we used the Molecular Operating Environment (MOE) program to conduct docking experiments on the tubulin protein of interest, identified explicitly by its PDB ID 4O2Bthe research aimed to evaluate the docking of six compounds isolated from P. integerrima.
The process of docking using MOE included utilizing the coordinates of the crystallized ligands to position and align the binding sites accurately.The Amber10EHT forcefield was implemented using software to analyze the protein structure obtained from the downloaded dataset.Subsequently, a 3D protonation technique was used to process the protein, followed by energy minimization up to a gradient of 0.1.The Amber10EHT force field was used to perform energy minimization on the structure, achieving a gradient convergence threshold of 0.00001.
Before docking the isolated compounds, the docking procedure was validated using the re-dock method.Co-crystallized ligand colchicine was re-docked into the tubulin binding site, and root-mean-square deviation was computed.The docking method with an RMSD value less than 1.0 Å was used for further studies.
The triangular matcher docking approach was used to dock the compound to the catalytic site of the protein and produce diverse conformations of the protein-ligand complex.The process of flexible docking, also known as induced fit, was conducted to achieve the structure with the lowest energy for the ligand.The GBVI/WSA computation was performed to get the binding energy scores for ligand ranking.The GBVI/WSA technique is used to calculate the free binding energy of a ligand based on its specific location.For all scoring functions, lower scores are better.Functions are scored in kilocalories per mole.Following the docking process, the molecular interactions and generation of three-dimensional structures for the protein-ligand complex were assessed utilizing the ligand interaction module in MOE and Discovery Studio software [21][22][23].

Anti-cancer effect
The isolated compounds were tested against various cell lines such as HepG2 (hepatoma G2), A498 (kidney cell lines), NCI-H226 (lung carcinoma), and MDR2780 AD (human ovarian carcinoma cell), as shown in Table 1.

Effect on HepG2
Compared with the standard anti-cancer drug (paclitaxel), a significant anti-cancer effect against HepG2 was demonstrated by compounds 6, 5, and 1 with IC 50 values of 14.65, 20.87, and 27.09 µM, respectively.The tested compound 1 exhibited the maximum anti-cancer effect and might be a significant drug candidate for liver carcinoma.

Effect on A498
Different isolated compounds demonstrated a mild anticancer effect, as shown in Table 1.The impact of standard chemotherapeutic drugs was maximum, as compared to tested compounds.The IC 50 value (108.33 µM) of compound 6 was comparatively near to the standard drug (92.89µM).

Effect on NCL-H226
The lung carcinoma cell lines were also significantly attenuated by standard drugs and compound 3 (57.09µM).The IC 50 value of the standard was 58.09 µM.

Effect on MDR2780 AD
The isolated compounds (Figure 1) showed a significant anticancer effect against human ovarian carcinoma cell lines (MDR2780 AD), as shown in Table 1.The standard anti-cancer drug and the other tested compounds showed a significant effect with the best IC 50 values (less than 1 µM).The isolated compounds exhibited maximum anti-cancer effect against MDR2780 AD cell lines among the tested cell lines.Compound 6 (0.56 µM) was the most antiproliferative comparatively.

Docking studies
Molecular docking analysis was used to determine the binding affinity of each isolated drug to tubulin's colchicine binding site.The docking studies for all six compounds were conducted on our target PDB ID 4O2B.The results of re-dock experiments are shown in Figure 2a.In comparison, the interaction of colchicine with amino acid residues is shown in Figure 2b.
Compound 1 shows conventional hydrogen bonding with two residues that include CYS241 and ASP251.These interactions are at a distance of 2.06 Å and 2.49 Å.This also shows π-sulfur interaction with residue MET259 at a distance of 4.92 Å (Figure 3a).Compound 2 shows conventional hydrogen bonding with three residues that are VAL238, ASP251, and ASN258 at a distance of 2.59 Å, 2.76 Å, and 2.86 Å, respectively.Compound 2 also shows π-σ interaction with LEU255 residue at a distance of 2.81 Å (Figure 3b).Compound 3 shows conventional hydrogen bonding with three residues that are VAL238, CYS241, and THR353 at a distance of 1.98 Å, 2.24 Å, and 2.25 Å respectively.This compound also shows π-σ bond with the residue LEU 255 at a distance of 2.56 Å (Figure 3c).
Compound 4 exhibits three different interactions with different residues.First, it shows conventional hydrogen bonding with two residues THR353 and VAL238.These interactions are at a distance of 2.26 Å and 1.96 Å.Second, it shows π-sulfur interaction with residue CYS241 at a distance of 4.44 Å.The compound also shows π-σ interactions with residue LEU255 at a distance of 2.62 Å (Figure 3d).Compound 5 shows conventional hydrogen bonding with three different residues that are CYS241, MET259, and VAL315 at a distance of 2.54 Å, 2.81 Å, and 2.71 Å, respectively (Figure 3e).Compound 6 shows conventional hydrogen bonding with three residues that are MET259, VAL315, and ASP251 at a distance of 2.66 Å, 2.96 Å, and 3.09 Å, respectively.It also shows π-sulfur interaction with residue MET259 at a distance of 5.16 Å (Figure 3f).

Discussion
The increasing prevalence of natural products can be attributed to their favorable safety profile and costeffectiveness.One of the main reasons behind this popularity is that most of the world population considers these plant-based medicines safe and devoid of any adverse effects.Compared to natural products, the general population considers synthetic drugs as toxic, leading to poor compliance, which compels them to switch from synthetic drugs to alternative medicines.The plants accumulate various chemical constituents with different pharmacological effects.Modern pharmaceuticals have been isolated from plants, such as vincristine [24] and paclitaxel [25] isolated from multiple plants.Most anti-cancer drugs are notorious for the side effects, especially nausea, vomiting, and diarrhea caused by cisplatin [26].In addition to these GIT-related side effects, anti-cancer drugs are mostly considered toxic, leading to poor patient compliance.Screening natural products is essential to improve patient compliance and discover safe, effective, and economical anti-cancer.In the current research work, the isolated compounds of P. integerrima were subjected to anti-cancer effects using various cancer cell lines.This plant's crude extract and multiple fractions have been reported to have significant anti-cancer effects [9].Based on this anti-cancer study, the current study was designed to test the isolated constituents for different carcinomas.
Interestingly, this plant is locally used to treat emesis [27].It means that using these isolated constituents and extracts will help treat cancer without emesis as a side effect.Cisplatin causes a significant loose stools in the patient under treatment, while this plant is an excellent antidiarrheal [28].P. integerrima is locally used to treat various types of cancers [29].The above discussion means that using the P. integerrima extract and its isolated compounds might help find a safe and effective anticancer.However, further mechanistic study is recommended for these isolated compounds.The structure-activity relationship (SAR) study is also recommended for these isolated compounds with the best hope of finding safe, effective, and economical anti-cancer drug candidates.
In this study, we employed docking simulations on the tubulin protein, with paclitaxel serving as the standard drug.Paclitaxel, a known tubulin-targeting agent, was utilized as a reference for comparison.Our docking analysis revealed that all six compounds investigated interacted with crucial amino acid residues within the tubulin binding site through a combination of hydrophobic and hydrophilic interactions.The tubulin protein is an important target in cancer therapeutic research.The microtubule is essential for mitosis and cell proliferation.Medications that attach to tubulin molecules interfere with the dynamic behavior of microtubules, restricting the process of mitosis during cellular division and ultimately resulting in the eradication of cancerous cells.The investigation of various tubulin inhibitors, with a particular focus on αβ-tubulin, has shown the presence of four main classifications of compounds that bind to separate and unique locations: laulimalide, taxane, vinca alkaloid, and colchicine.The colchicine binding site (CBS) inside the tubulin has notable characteristics in the form of a cavity, facilitating its interaction with small molecules.A noteworthy limitation associated with microtubule-targeting drugs in developing drug resistance may manifest either as an inherent trait or as a result of acquired mechanisms.The x-ray studies of the colchicine site of the tubulin protein have some important residues for ligand interaction that include Tyr202, Val238, Leu248, and Asn249 [30][31][32].Paclitaxel is the standard drug used in this study.Paclitaxel is a drug that targets tubulin.Therefore, we performed docking simulations on tubulin.All six compounds under investigation interact with the key amino acid residues in the tubulin binding site via hydrophobic and hydrophilic interactions.These findings underscore the potential of the investigated compounds as tubulin-targeting agents, with their ability to interact with key residues in the colchicine binding site.
The results of the anticancer studies present significant implications for potential applications in therapeutics.Understanding the molecular mechanisms of action also facilitates the development of more precise and effective anticancer drugs with potentially fewer side effects compared to traditional chemotherapy.Additionally, the use of natural products like P. integerrima and its isolated compounds offers advantages in terms of safety, tolerability, and patient acceptance, aligning with the growing interest in complementary and alternative medicine approaches and potentially reducing the risk of adverse effects and improving patient compliance.While the in vitro results are promising, further translational research is necessary to validate the therapeutic potential of these compounds in clinical settings, including preclinical studies using animal models to assess efficacy and safety profiles, as well as clinical trials to evaluate their effectiveness in human patients.Exploring synergistic interactions between these natural compounds and existing anticancer drugs could lead to combination therapies with enhanced efficacy and reduced toxicity, advancing the quest for more effective and tolerable cancer treatments.