In Vitro and In Silico Cytotoxic and Antibacterial Activities of a Diterpene from Cousinia alata Schrenk

L.N. Gumilyov Eurasian National University, Nur-Sultan, Kazakhstan S. Seifullin Kazakh Agro Technical University, Nur-Sultan, Kazakhstan Sh. Ualikhanov Kokshetau University, Kokshetau, Kazakhstan Pharmacognosy and Medicinal Plants Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt Pharmaceutical Medicinal Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt Department of Pharmaceutical Sciences, College of Pharmacy, AlMaarefa University, Ad Diriyah, Riyadh 13713, Saudi Arabia E.A. Buketov Karaganda University, Karaganda, Kazakhstan S. Amanzholov East Kazakhstan State University, Oskemen, Kazakhstan Department of Biology, Katholieke Universiteit Leuven, Leuven 3000, Belgium


Introduction
Cancer is a disease that takes place in an organ or tissue of the body because of the uncontrolled growth of abnormal cells invading other parts inside the body and spreads to other tissues [1,2]. According to the WHO, in 2018, with a ratio of one in six deaths, cancer was the second cause of death all over the world [3]. e global increase in antibiotic resistance causes an increase in medical costs and mortality rates, especially with some severe bacterial infections such as pneumonia, tuberculosis, gonorrhea, and salmonellosis [4]. Natural products can be considered as the main source of antimicrobial and anticancer compounds [5]. e activity of natural products either originated from plants [5][6][7], marines [8,9] or fungi [10][11][12][13][14] is owned to a diversity of secondary metabolites that belong to various chemical classes such as flavonoids [15][16][17], saponins [6,18], pyrones [19], isochromenes [20], and alkaloids [21].
Cousinia Cass. is one of the largest genera of the Asteraceae family with approximately 600-700 species and the most diverse species in Central and Southwest Asia [22][23][24]. About 56 species are distributed throughout Kazakhstan, including Cousinia alata Schrenk., which is a perennial plant, up to 50-90 cm high; its stems are straight, freely branched, naked, and winged; wings are with spinydentate margin; leaves are thin and simple; corolla is yellow or pink color. During the flowering period from flower baskets, there is a significant release of rubber. is plant grows on the slopes of low hills, in sandy places, and in plains throughout Kazakhstan's territory. General distribution: China, Mongolia, and Central Asia [25][26][27].
Histone deacetylases (HDAC) are a group of enzymes that are responsible for the deacetylation from the N-acetyl lysine amino acid on the histone protein; this step (deacetylation) is very essential in the process of DNA expression because it allows the histones to wrap the DNA more effectively [38]. HDAC inhibitor is any molecule that increases the acetylation of lysine residues on histone and nonhistone proteins through the inhibition of HDAC enzyme activity. Consequently, HDAC inhibitors could be an excellent choice to discover new anticancer agents [39]. Moreover, the presence of HDAC homologs in bacteria in the form of acetoin utilization proteins (AcuC) has been reported [40]. Also, the use of a histone deacetylase inhibitor in infected mice with M. tuberculosis inhibited bacterial growth, accelerated immune cell recruitment, induced proinflammatory cytokine expression, and suppressed IL-10 expression [41]. Another model of infected mice with E. coli treated with valproic acid (HDAC inhibitor) caused a noticeable decrease in bacterial load exaggeration and cytokine expression [42]. Recently, Grabiec et al reported the antibacterial effects of several HDAC inhibitors through different mechanisms [43].
is study aims to isolate and identify the secondary metabolites which are responsible for the cytotoxic and antibacterial effects of Cousinia alata Schrenk extracts. Additionally, molecular docking studies have been preceded to have a better idea about the mechanism of action of the active compound on a molecular level. e air-dried flowers and leaves of C. alata (500 g) were extracted with chloroform in the water bath at 65°C for one hour. After filtration, the filtrate was evaporated under reduced pressure at 40°C. is procedure was repeated three times and the concentrated extracts were treated with waterethanol (1 : 2) solution to remove ballast substances. After water-ethanol treatment, the obtained extract was subjected to silica-gel column chromatography. Column chromatography runs were performed using silica gel (63-200 µm, 735 g) and a mobile phase of petroleum ether/ethyl acetate with a manner of increasing polarity, which afforded 120 fractions (each 250 ml).
Regarding isolation of grindelic acid (1), 200 g of the aerial parts of C. alata were powdered and extracted with methanol, followed by evaporation under reduced pressure on a rotary evaporator. e crude methanol extract was further fractionated by liquid-liquid extraction with hexane and then dichloromethane. e fractions were then concentrated again using a rotary evaporator to give hexane (12,3 g), dichloromethane (25,8 g), and methanol extracts. A sample was taken from each extract for preliminary screening of antibacterial activity. us, the hexane extract showed antimicrobial activity, which was further subjected to bioassay-guided isolation of antimicrobial compounds. e dried active extract (hexane) was loaded on a silica gel column (60 cm height and 5.5 cm diameter), which was eluted with a step gradient increasing polarity, hexane : ethyl acetate and ethyl acetate : methanol. Aliquots of all fractions were dried and dissolved in DMSO for bioassay. e fractions from 39 to 48 demonstrated activity, so they were combined (4 g) and then the extract was loaded on silica gel column again with the same solvents for elution, each step ten tubes of 15 ml. ree groups of fractions showed activity: 50-57, 59-62, and 64-80. Every group of fractions was subjected to reversed-phase high-performance liquid chromatography equipped with a UV detector (Shimadzu, Japan). e gradient solution system was started from 50% acetonitrile (0,1% TFA) and 50% deionized water (0,1% TFA) over 10 min and then up to 100% acetonitrile (0,1% TFA) in 30 min and maintained 20 min at 100%. Flow rate: 4 ml min −1 ; injection volume: 2 ml; a SunFire prep. C18 column (5 μm, 10 × 250 mm); and detector wavelengths: 214 and 254 nm. ere were collected 60 fractions of 4 ml per minute and then they were tested. All groups of fractions contained the same active peak at 32 minutes (grindelic acid (1)).

Compounds
Identification. Proton nuclear magnetic resonance ( 1 H NMR), carbon-13 nuclear magnetic resonance ( 13 C NMR), 1 H-1 H correlation spectroscopy (COSY), heteronuclear multiple-bond correlation (HMBC), and heteronuclear single quantum correlation (HSQC) spectra were measured on a Bruker 500 SB UltraShield TN plus NMR spectrometer, operating at 400 MHz for 1 H and 100 MHz for 13 C. e determination of the melting point of the isolated compounds was carried out with OptiMelt Stanford Device.

Determination of the Cytotoxic Activity.
e 55 ml separatory funnel was filled with artificial seawater and added 200 mg eggs of Artemia salina. en, it was kept with a soft supply of air for three days until the crustaceans hatch from nauplii. One side of the funnel was covered with aluminum foil, and after 5 minutes, the larvae, which moved on the bright side of the separatory funnel, were removed with a Pasteur pipette. 20-40 nauplii were placed into each of the 24 microtiter plates with 990 μl of seawater. Dead larvae were counted under a microscope. 10 μl of dimethylsulfoxide solution per 10 mg ml −1 sample was added. Actinomycin D or staurosporine was used as a standard comparison reagent, and DMSO was a negative control. After 24 h of incubation and further maintaining microtiter plates for 24 hours (to ensure immobility) the dead nauplii were counted under the microscope.
Mortality P was determined by the following formula: where A is the number of dead nauplii after 24 h; N is the number of nauplii that died before the test; and B is the average amount of nauplii died in a negative control.

Antimicrobial Activity.
e antimicrobial assay performed a broth microdilution method against 4 bacterial strains, i.e., Gram-positive Staphylococcus aureus 6532, Bacillus cereus, Gram-negative Salmonella enteritidis, and yeasted strain Candida albicans sc5314. In this assay, standard antibiotics (ampicillin 5 mg ml −1 for bacteria and fluconazole for Candida albicans) and DMSO/water were served as positive and negative controls for the sensitivity of the tested bacteria, respectively.
A single colony of bacteria was inoculated from an agar plate in 3 ml MH medium (0.2% beef extract, 1.75% casamino acids, and 0.015% soluble starch) and in YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) for C. albicans. e tubes were incubated overnight in a shaking incubator (200 rpm) at 37°C.
Ten μL of the test sample was transferred into the wells of a 96-well plate, as well as the positive control (ampicillin, stock 5 mg/mL) and blank (solvent) controls (DMSO and water). Each well of a microdilution plate was then inoculated with 190 μL of the diluted standardized inoculum (OD � 0.003 at 620 nm). Control wells were prepared with 190 μL MH broth and 10 μL extract to correct any absorption due to extracting components. e microdilution plates were placed in a shaker-incubator at 37°C for 24 h and then read on microplate reader at 620 nm. e OD was measured at a wavelength of 620 nm. e relative inhibition (%) of the test sample was calculated by the following formula: where A is the OD value of the sample; B is the OD value of the noninoculated sample control; and C is the average OD value of the solvent (DMSO) [44].

Molecular
Docking. e crystal structure of the target HDAC (PDB ID: 2VQM; resolution: 1.80Å) was downloaded from Protein Data Bank (http://www.pdb.org). Molecular Operating Environment (MOE) was used for the docking analysis [45]. In these studies, the free energies and binding mode of the examined molecule against HDAC were determined. At first, the water molecules were removed from the crystal structure of HDAC, retaining only one Journal of Chemistry chain and zinc atoms which are essential for binding. e cocrystallized ligand (hydroxamic acid derivative, HA3) was used as a reference ligand. en, the protein structure was protonated and the hydrogen atoms were hidden. Next, the energy was minimized and the binding pocket of the protein was defined [46][47][48]. e structures of the examined compound and the cocrystallized ligand were drawn using ChemBioDraw Ultra 14.0 and saved in SDF format. en, the saved file was opened using MOE software and 3D structures were protonated. Next, the energy of the molecules was minimized. e validation process was performed for the target receptor by running the docking process for only the cocrystallized ligand. Low RMSD values between docked and crystal conformations indicate valid performance [49,50]. e docking procedures were carried out utilizing a default protocol. In each case, 10 docked structures were generated using genetic algorithm searches [51][52][53].

Molecular Docking.
ree different reasons encouraged us to run a molecular docking study for the grindelic acid (1) against histone deacetylase (HDAC) enzyme. First is the promising cytotoxic and antibacterial results of grindelic acid which derived us to think deeper to find out the mechanism of action on a molecular level. Second is the presence of the essential pharmacophoric features in   Journal of Chemistry grindelic acid's chemical structure. Finally, the reported cytotoxic and antibacterial effects of HDAC inhibitors [43,61]. e reported histone deacetylase (HDAC) inhibitors must possess three main pharmacophoric features, comprising the zinc-binding group (ZBG) that chelated the zinc atom in the active site, a linker that accommodates the tubular access of the active site, and a capping group for interactions with the surface recognition motif connected by a small connecting unit to the linker (Figure 2(a)) [62,63]. All these features were found in grindelic acid (Figure 2(b)). Docking studies were carried out for compound 1 against HDAC (PDB ID: 2VQM; resolution: 1.80Å) to examine its mode of binding with the proposed target. e cocrystallized ligand (HA3) was used as a reference molecule. e results of docking studies revealed that the docked compound has a good binding affinity against HDAC with a binding free energy of −18.70 kcal/mol. Such compound exhibited a binding mode of interaction similar to that of the cocrystallized ligand (Table 2; Figure 3). e crystallized ligand (HA3) showed a binding energy of −14.02 kcal/mol. e detailed binding mode of the crystallized ligand was as follows: the N-hydroxycarboxamide moiety occupied the zinc-binding region forming two hydrogen bonds with His158 and Gly167. Besides, it formed   Table 2; Figures 4 and 5 ). Compound 1 occupied the active cavity of HDAC showing a binding energy of −18.70 kcal/mol. Acetic acid moiety occupied the zinc binding and the carboxyl group of acetic acid moiety acted as zinc coordinator forming one hydrogen bond with Gly167. Also, it formed an electrostatic interaction with the Zn atom of the cocrystallized ligand. Additionally, (S)-2-methyltetrahydrofuran moiety occupied the linker region of HDAC. Furthermore, the (4aR, 8aS)-1,1,4a-trimethyl-1, 2, 3, 4, 4a, 5, 8, 8a-octahydronaphthalene moiety occupied the cap pocket of the receptor (Figures 4 and 6).

Conclusions
Bioassay-guided isolation of methanolic extract of Cousinia alata led to the identification of grindelic acid (1). Grindelic acid exhibited promising cytotoxic activities against Artemia salina nauplii and antibacterial activities against S. aureus, B. cereus, and S. enteritidis. e pharmacofeatures and molecular docking studies revealed that (1) has a binding mode of interaction similar to that of the cocrystallized ligand and occupied the active cavity of HDAC enzyme with a binding energy of −18.70, which may explain its noticeable biological activities. Additionally, six flavonoids (2-7) have been isolated from the chloroform extract of Cousinia alata. All compounds were isolated for the first time from Cousinia species.
Data Availability e supporting data (NMR of compounds 1-7) are openly available.

Conflicts of Interest
e authors declare no conflicts of interest. Journal of Chemistry 9