Introduction
The dramatic demand to locate potential new molecules in anticancer and antimicrobials is associated with increased resistant microbial strains and cancer cells against the existing medications. Screening nature was and still is one of the valuable resources in this field because of its ability to accommodate various external changes. Among the natural sources, herbs with previous records of medicinal benefits are considered an example.
Traditional medicine practitioners widely use plant extracts as remedies for many ailments, including fever, inflammation, and infectious diseases. For instance, ayurvedic medicine provides countless examples of medicinally beneficial herbs containing phytochemicals, such as tannins, flavonoids, and alkaloids [1]. Previous reports have shown the benefits of the tannins and flavonoids against gram-positive cocci and Candida albicans infections [2, 3], and many alkaloids have been used clinically as anticancer agents, as highlighted by the use of vinca alkaloids for treating various lymphomas [1]. In the drug discovery process, screening for bioactive metabolites from known traditional herbs provides great medicinal interest, and different medicinal plant metabolites demonstrated a wide range of potential bioactivities, many of which found their way into clinical practice. A vast number of isolated plant metabolites in the form of crude mixtures exerted antimicrobial and cytotoxic potentials. However, due to the resistance of cancerous cells and microbes to the available medications, there is significant interest in identifying these metabolites [4, 5]. Among the plant secondary metabolites, alkaloids comprise a large group. Most of these agents contain heterocyclic nitrogen skeletons that, in some cases, such as with the pyridine alkaloids, resonate with an aromatic nucleus [6]. The importance of alkaloids in medicine is due to their potent pharmacologic effects at even low concentrations relative to other natural products. Their effects apply to various disorders, including bacterial and fungal infections and some cancers [7]. Pyridine alkaloids are essential members and have recorded medical benefits through demonstrating significant cytotoxic [8], antibacterial [9], and antifungal [10] effects. Cyperus rotundus L. (CR) is a herbaceous medicinal plant of the family Cyperaceae that grows in the Iraqi-Kurdistan Region of the Middle East. The CR crude extract (CE) has been used to treat many ailments, including fever, malaria, gastrointestinal disturbances, hormonal imbalances, and infections [11]. Sesquiterpenes [12], pyridine alkaloids [13], sterol glycosides [14], furochromones, and flavonoids [15] have been isolated from the CR rhizomes. Previous works investigating the cytotoxic [16], antibacterial [17], and antimycotic [18] activities of CR used the rhizomes’ CEs rather than using single isolated compounds, as it is easier, more rapid, and less expensive. Despite the CR beneficial effects, the total extracts were used as remedies for the mentioned ailments; isolated purified entities as active constituents would potentially be more productive therapeutically due to their efficacy at very low concentrations and reduced inter-molecular and intra-molecular interactions that may be associated with total extracts [19]. Rotundine alkaloids have been previously isolated from CR rhizomes, but no record of bioactivity has been reported [13]. The current study screened and evaluated a methanol extract of CR rhizomes to isolate and identify a potential bioactive pyridine alkaloid as a lead compound for anticancer and antimicrobials. The cytotoxic and antimicrobial effects of the alkaloid were then investigated.

Materials and Methods 
Chemicals and reagents
High-performance liquid chromatography (HPLC) graded all chemicals and reagents to ensure precision and accuracy. Mueller Hinton Agar (MHA, LAB039) from Seoul, Korea, and Mueller Hinton Broth (MHB) from MD, USA, were prepared according to the supplier’s recommendations. Sephadex LH-20 was purchased from GE Healthcare (USA), while gentamicin and fluconazole were obtained from TransGen Biotech (Beijing, China). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin, ampicillin, and kanamycin were purchased from TransGen Biotech (Beijing, China). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Karl Roth (Darmstadt, Germany). All the procedures were performed with consideration of laboratory safety operating procedures, chemicals, and biological samples were handled with extra care. The biological wastes were disinfected, autoclaved, and properly discarded. All chemicals were collected, detoxified, and properly discarded.

Bacterial and fungal strains and human cell line
Escherichia coli (ATCC No. 8739), Staphylococcus aureus (ATCC No. 6538), and C. albicans (ATCC No. 10231) were purchased from Medya-Med Labs (Erbil, Iraq). The bacterial strains were inoculated into fresh media, 10% glycerin was added, and aliquots were stored at −80°C until use. The human endothelial HeLa cell line was obtained from the fine test (Wuhan, China). The HeLa cell line was thawed and inoculated into fresh DMEM containing 10% FBS in culture flasks and incubated at 37°C and 5% CO2. Cell morphology and growth were checked every 24 h, and subsequently, sub-cultures were prepared.

Plant collection
CR plant was harvested from the Shahrazoor District, Sulaimaniyah-Iraq. Plant identification was carried out at the Kurdistan Botanical Foundation, and a voucher specimen was deposited in the Medicinal Plants Herbarium of the College of Pharmacy, University of Sulaimani, for future reference (voucher No.: Cr 17.01).

Extraction of plant material
The dried CR rhizomes were pulverized, and 250 g was treated with 10% alcoholic potassium hydroxide solution, which was then macerated in 350 mL methanol with shaking at 50°C for 72 h. The solvent was replaced with fresh solvent daily. The extract was filtered and vacuum-concentrated at 40°C in a rotary evaporator. The yield ([dried extract weight/crude plant weight]×100) was calculated, and the CE was re-suspended in 50 mL methanol. The solution was acidified using 1% hydrochloric acid, then vacuum filtered three times using the Whatman No.1 filter paper. The filtrate was extracted using methanol: chloroform (5:95 V:V). The pH adjustment to 9.0 was achieved by adding drops of ammonium hydroxide solution (35.046 g/mol). 

Fractionation
The mixture was partitioned three times using 100 mL 5% methanol: chloroform. Sufficient volumes of Mayer’s and Dragendorff’s reagents were added to aliquots of the aqueous and organic layers on microscope slides, and the mixtures were observed under a microscope for the presence of precipitates. The organic phase was separated and vacuum condensed at 40°C. The final product was weighed and considered the crude alkaloid extract (CAE).

Column chromatography
The CAE was eluted over Sephadex LH-20 using 80 mL of methanol as an isocratic mobile phase. A total of 16×5 mL sub-fractions were collected. Each sub-fraction was tested for alkaloids using Mayer’s and Dragendorff’s reagents. Alkaloid-positive sub-fractions were subjected to thin-layer chromatography (TLC) analysis over silica gel using chloroform: methanol: Diethylamine at a volume ratio of 15:1:0.1. The chromatogram was sprayed with Dragendorff’s reagents. Spots showing positive reactions (orange-brown color) were recovered using preparative-TLC, vacuum-dried, and again column-chromatographed under the same conditions as before, except smaller fraction volumes were collected (25×1 mL). A single spot that reacted positively with the alkaloid-detecting reagents was scraped and solubilized in 5 mL of 5% methanol: chloroform and then filtered. The solvent was evaporated at 40°C. An oily pale-yellow substance was recovered, weighted, and labeled as Alk-1. The Alk-1 sample was stored at 4°C until further analysis [13].

Total oligomer flavonoids analysis
An amount of one-hundred grams of the pulverized CR rhizomes were macerated using acetone: distilled water (2:1 V/V) at room temperature with shaking for 72 h. The solvent was refreshed daily. The extracts were pooled, filtered, and vacuum-condensed at 25°C. The aqueous phase was weighed and then treated with excess sodium chloride solution for 24 h at 5°C to remove the tannins. The obtained supernatant was extracted with ethyl acetate (EtOAc), and the extract was precipitated by adding an excess of chloroform. The precipitate was partitioned and dried, and the total oligomer flavonoids were calculated [20].

Determination of total flavonoid content
One-hundred grams of pulverized CR rhizomes were macerated in 70% ethanol at room temperature for 72 h. The CE was partitioned thrice with equal volumes of n-hexane, EtOAc, and n-butanol. One gram from the EtOAc portion was assayed for total flavonoid content using the aluminum chloride (AlCl3) colorimetric method using quercetin as a standard. Aliquots from the CE and EtOAc fractions were prepared at 1 mg/mL concentrations. Serial dilutions of quercetin (10, 20, 40, 80, 120, and 160 µg/mL) were prepared in 95% ethanol as standard solutions adjusted with sterilized micropipettes. Then, 200 µL of each sample or standard was placed in a test tube and mixed with 0.1 mL of freshly prepared 10% AlCl3, 0.1 mL K+(CH3COO)− solution, and 4.6 mL of distilled water. The mixture was vortex-mixed and left in the dark for 30 min. A blank was prepared for each sample using the same procedure, but distilled water was used instead of AlCl3. The absorbance of the tubes at 415 nm was recorded using a UV-Vis spectrometer.

High-performance liquid chromatography analysis
The purity of the isolated alkaloid was determined using integrated HPLC analysis (Waters e 2695 Alliance HPLC systems, USA) with an Intersil C-18 column (150 mm×4.6 mm, 5 µm; GL Science, Japan). The chromatography was performed at 30ºC using a mobile phase of acetonitrile: Distilled water at a 60:40 volume ratio and a 1 mL/min flow rate. The injection volume was 50 µL. The detection was performed at 254, 302, and 365 nm wavelengths. System precision, detector linearity, injector linearity and accuracy, injector carryover test, flow-rate linearity, compositional precision, noise, and drift test were previously tested for the instrument, and the method selection was based on previous literature [21]. The water used was purified using a Millipore Mill-Q system. The isolated alkaloid was stored at −86°C until structural elucidation and performance of bioactivity assays. Similar conditions and standard concentrations of authentic samples were applied in determining the amounts of nootkatone, quercetin, chlorogenic acid, catechin, and β-sitosterol in the CE.

Detection by chemical tests
One mg aliquot of the oily extract was tested with Mayer’s and Dragendorff’s reagents (dropwise addition), and the precipitate was examined under a microscope for confirmation.

Structure elucidation
Fourier-transform infrared (FTIR) spectrum analysis was performed using the KBr pellet method (Vmax in cm-1) and a Perkin-Elmer FTIR spectrometer. Data interpretation was done using Spectrum-A software (ChemDraw Professional/ PerkinElmer, Ver. 19.1). The proton nuclear magnetic resonance (H-NMR) spectrum was recorded using an Avance-III 400 MHz NanoBay spectrophotometer (Bruker, Switzerland) and 2 mg of the alkaloid. The samples for chemical and biological analyses were dissolved in methanol. The following conditions were used: Spectrometer frequency=400.1500 MHz, acquisition time=4.0894465 s, relaxation delay (D1)=1.0 s, right pulse width=45°, spectral width=8012.820 Hz, digital resolution=0.3 Hz, and Fourier transform size=298.1 K. Chemical shifts (δ) were calculated as parts per million (ppm) relative to the D-glucose molecule (CDCl3, δH 7.24, δC 77.0). The data were recorded using the Topspin software, version 3.2.
The mass spectrum analysis was applied using liquid chromatography with tandem mass spectrometry on an API-3200 system. The electrospray ionization technique was used, with the sample being directly injected with a continuous flow rate of 10 µL/mL for a total run time of 1 min. Ions were detected at a positive mode (m/z +1) using a carrying diluent of methanol: Distilled water (50:50 V/V), and the sample solution concentration was adjusted to 5 µg/mL. Detection parameters were DP=40, EP=10, IS=5500 v, and ion source spray gas 30. Data interpretation was done using the Analyst software, version 1.6.3.

Minimum inhibitory concentration and minimum bactericidal concentration assays
MIC values of the isolated compound were tested against non-fastidious microorganisms S. aureus, E. coli, and C. albicans according to the method previously established by the Clinical and Laboratory Standard Institute at a microbial cell density of 0.5×108 cells/mL. Briefly, the microorganisms were inoculated into sterile MHB and incubated for 24 h at 37°C. Microbial cultures were suspended in sterile normal saline (0.9%), and the concentrations were adjusted to an optical density of 0.5 McFarland turbidity standard [22]. Serial dilutions of the CE and the isolated compound were also prepared. The antibiotic gentamicin and antifungal fluconazole were used as standards, accordingly. The MIC assay was performed using sterile MHB with the microorganism cultures adjusted to 200 µL/well in 0.1% DMSO.

Preparation and dilution of stock solutions
Stock solutions were prepared in sterile test tubes with screw caps by dissolving 80 µg of each test sample or standard in 0.1% DMSO/sterile phosphate buffer saline to a final volume of 200 µL. The samples were then homogenized by vortex mixing. Each dilution was adjusted to a total volume of 200 µL/well, which included 50 µL serially diluted samples 1, 2, 4, 8, 16, 32, 64, and 100 µg/mL.

Bacterial sensitivity assay using microplate assembly
Sterile 96-well microplates were prepared with media alone (blank) or microbial suspension (20 µL/well, control) and serial dilutions of antibiotic or antifungal standards at concentrations of 1, 2, 4, 8, 16, 32, 64, and 100 µg/mL. Serial dilutions of aliquots of the isolated compound (1–100 µg/mL) were added to the blank and microbial suspensions. Each well was adjusted to a total volume of 200 µL using micropipettes. The plates were incubated overnight at 37°C with shaking adjusted to 200 rounds per minute. The plates were then examined for bacterial growth, and readings were taken at 600 nm using an enzyme-linked immunosorbent assay microplate reader. The tests were performed in triplicate [23]. To determine the MBCs, inoculated samples from dilutions showing bacterial inhibition onto MHA plates were incubated overnight at 37°C and then examined for bacterial growth [23]. The blank, which included the growth medium, was used only as background, and the control included the growth medium with cultures of the tested microorganisms.

Determination of cytotoxicity using MTT cell proliferation assays
The isolated compound’s cytotoxicity and cell viability were determined using MTT cell proliferation assays. HeLa cells were seeded into culture flasks containing DMEM supplemented with 10% FBS, ampicillin (1 µL/mL), and kanamycin (0.5 µL/mL) and incubated in a humidified atmosphere of 5% CO2 at 37°C. Sub-cultures were prepared on alternate days when confluence reached 70%, and cell morphology was examined. The HeLa cells were inoculated into sterile 96-well microplates at a density of 1×104 cells/well and incubated at 37°C for 24 h in a 5% CO2 incubator. Following incubation, the cells were examined for adherence and morphology and then treated with serial dilutions of the isolated compound at concentrations of 20, 40, 80, 160, and 200 µg/mL. The blank consisted of DMEM supplemented with 10% FBS (cell-free), while the control was media + HeLa cells [16]. The absorbance values of the blank wells were subtracted as background from the absorbance values of the sample wells, and the IC50 values were calculated. The plates were incubated for 24 h under the same conditions. The culture media and the treatment dilutions were then replaced with freshly prepared BSA-free DMEM MTT reagent added to each well (0.5 mg/mL) and incubated for 4 h at 37°C. DMSO (100 µL) was then added to dissolve the developed formazan crystals, and the absorbance of each well was measured at 540 nm using an enzyme-linked immunosorbent assay microplate reader. The absorbance values of the blank wells were subtracted as background from those of the sample wells, and the IC50 was calculated. Each assay was performed in triplicate.

DAPI assay
DAPI dye was prepared by dissolving 20 mg of the pigment in 1 mL of distilled water. The suspension was homogenized in the dark with sonication using an ultrasonic water bath. The solution was stored at 4°C until use. HeLa cells were incubated in a humidified ambient environment with 5% CO2 at 37°C for 24 h. After adherence, the cells were treated with the isolated compounds at 20–200 µg/mL concentrations and then fixed for 15 min in 4% paraformaldehyde, followed by washing with PBS. The DAPI dye was added to the cells (control and treated) and left undisturbed in the dark for 5 min. Chromatin changes (apoptosis) were then recorded using an inverted fluorescence microscope [24].

Statistical analyses
All experiments were independently performed four times, and the data were expressed as Mean±SD. Statistical analysis was carried out using one-way analysis of variance followed by the Tukey post hoc test of the MTT assay results with nonlinear regression analysis of the IC50 results. Differences were considered statistically significant at P<0.05.

Results
Phytochemical characterization of the crude extract 
The CAE yield comprised 0.016% of the total CE, while the isolated oily Alk-1 obtained by column chromatography (RF=0.4) was 20 mg, which was 0.008% of the total weight of the rhizomes CE (Table 1).




Total oligomer flavonoids and total flavonoid content results and the amounts of nootkatone, quercetin, chlorogenic acid, catechin, and β-sitosterol according to HPLC analysis are shown in Table 1. HPLC analysis of the isolated compound showed a retention time (RT) of 3.0 with a single peak (Figure 1a).
Fourier-transform infrared analysis
FTIR assay results are illustrated in Figure 1b and Table 2.


FTIR spectrum revealed a broadband at 3550 cm-1, which indicates an absorbance due to O-H stretching of multiple alcoholic hydroxyl groups, primarily glucose hydrogens bonded with water. Furthermore, the strong broadband suggested intermolecular bonding. The absorption at 1375 cm-1 is for O-H stretching of the multiple glucose hydroxyls. A band at 1255 cm-1 for C-O stretching of ether supported the presence of alcohol, most likely for the sugar-free hydroxyl alcohol groups [25]. The peak at 2873 cm-1 is for C-H stretching of aliphatic hydrocarbons (-CH2, -CH3). The bands at 3064 cm-1, 3032 cm-1, and 2929 cm-1 were the results of C-C stretching of aromatic compounds, further supported by an overtone at 2000–1700 cm-1 and a C-C stretching at 1602 cm-1. The shift in absorbance can be attributed to ring activation by the nitrogen atom, which is typical for pyridine. This was evidence for the presence of pyridine rather than benzene. The band at 1498.85 cm-1 demonstrates the C-C bending of aromatic compounds, while the absorbance at 728 cm-1 and 693 cm-1 (out-of-plane bending) were due to multiple aromatic substitutions. The band at 462 cm-1 was the result of traces of halogens from the solvent (chloroform). The free alcoholic hydroxyl proton of the Alk-1 at C-14 was overlapped by the glucose hydroxyls [26, 27].

Proton nuclear magnetic resonance analysis
Assignment of the protons was done using H-NMR (400 MHz, methanol, d) with the integration calculated for each proton as (H+=0.5), according to the glucose protons. The results for Alk-1 showed the following δs: 1.00 (d, J=4 Hz, 3 H), 1.31 (s, 6 H), 1.4 (m, J=4 Hz, 2 H), 1.64 (m, J=8.0 Hz, 2 H), 1.7 (m, J=164 Hz, 1 H), 2.28 (d, J=4.0 Hz, 2 H), 3.5 (d, 2 H), 3.55 (d, 2 H), 3.68 (s, 1 H), 3.86 (d, 1 H), 3.92 (d, 1 H), 4.1 (m, 2H), 4.15 (d, 1 H), 4.16 (d, 1 H), 3.5-4.16 (m, J=8 Hz, 11 H), 6.97 (d, 1 H), 8.13 (d, 1 H) (Figure 1c). The δ at 8.13 ppm, s (1 H) and δ at 6.97 ppm, s (1 H) were caused by the protons attached to C-2 and C-5 of the pyridine ring, respectively. The proton appeared downfield (>8.0 ppm) as the ring was activated. The proton attached to C-2 appeared in the lower field as it was also adjacent to the nitrogen atom [25, 27]. The pyridine ring was substituted with a cyclopentane ring at C-3 and C-4. The two C-7 protons appeared in the region 2.28 ppm, d (2 H) and were coupled (J=4.0 Hz). These two protons split the C-8 proton (J=164 Hz), shown in the region 1.77 ppm, m. The protons of the two methyl groups (C-10 and C-11) attached to C-9 of the cyclopentane appeared in the region 1.31 ppm, s (6 H). These were adjacent and symmetric, displaying the same integration (geminal protons). The assigned 2-butanol protons attached to the cyclopentane ring at C-8 showed δ at 1.64 ppm, m (2H) for the C-13 protons. These protons appeared in this region as they were adjacent to the alcoholic C-14, adjacent to each other and coupled (J=8.0 Hz), and split the protons attached to C-12 and C-14 (J=88.0 Hz). The broad multiplet peak below 2.0 ppm was from the C-14 proton being un-shielded by the alcoholic hydroxyl. The alcoholic hydroxyl proton attached to C-14 overlapped with the alcoholic or glucose hydroxyl protons as they appeared in the same region [25]. The C-12 protons appeared in the region 1.42 ppm, m (2 H). These protons were coupled and adjacent to each other (J=4 Hz). The three protons attached to C-15 appeared in the upfield region at 1.0 ppm, d (3 H), and were coupled to each other (J=4 Hz).
The H-NMR analysis showed the presence of glucose protons in the region 4.3–3.5 ppm (11 H). The proton attached to the sugar anomeric carbon sugar was shielded and appeared downfield for the adjacent electronegative oxygens (anomeric and C-5′). The C-(2′, 5′) protons were shifted downfield because of the neighboring oxygen atoms. These protons overlapped with the methanol (solvent) proton and appeared in the region 4.16-3.5 ppm. The two protons attached to C-6′ appeared in the region 3.49-3.51 ppm, d (1 H) and split each other and the adjacent hydroxyl proton (H-C-OH; J=8.0 Hz).

Mass spectrum analysis
The mass of the molecular ion for the isolated Alk-1 was M+=397. The other ion fragments are shown in Figure 1d. Mass spectrum analysis of the isolated Ak-1 (C20H31NO7) using the high-resolution LC-MS/MS ABSCIEX system revealed ion fragments m/z 163 and m/z179 for glucose-OH. The mass spectrum analysis also showed the fragment mz=396.6 (M+). 

Antimicrobial assays (minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations assays (MBC))
MIC analysis of the Alk-1 compound against S. aureus showed that it inhibited bacterial growth at a 64 µg/mL concentration. The results for the MBC assay mean was 64 µg/mL with no growth being detected (bactericidal). The same results were seen at higher concentrations of the Alk-1. The MIC for Alk-1 against E. coli was 100 µg/mL. No inhibition of E. coli was detected in the MBC assay at the same concentration. The antifungal activity of Alk-1 against C. albicans was recorded as 64 µg/mL, and no growth (fungicidal) was detected in the MBC assay (Figure 2 and Table 3).

The MIC assays for Alk-1 against both S. aureus and C. albicans showed increased inhibition with increasing concentrations from 64 µg/mL to 100 µg/mL. The same results were obtained for the MBC assays against the tested microorganisms, with no growth detected on the agar plates; however, the antibacterial activity of Alk-1 against E. coli was seen only at 100 µg/mL, with no apparent inhibition of growth on the agar plates. The blank wells showed no growth, and the control wells showed no inhibition. The MIC and MBC assay results of the tested alkaloid showed selective bactericidal activity against S. aureus and C. albicans, while bacteriostatic action against was observed against E. coli.

Cytotoxicity assays
MTT assays
The MTT assay results showed significant activity at 80 µg/mL and highly significant cytotoxicity at 160–200 µg/mL for the isolated Alk-1 against HeLa cells. The IC50 value was 203.7 for Alk-1 (Figure 3). As shown in Figure 4, the results of the cell viability analysis in response to increasing concentrations of the test compound showed a significant decrease (P=0.0133) in the cell numbers. For both antimicrobial and cytotoxicity assays, low concentrations were considered to show clinical significance and productivity compared to human utilization and eligible pharmaceutical dosage formulations. 

DAPI analysis
The DAPI assay results showed morphological changes, including chromatin changes in treated cells with the isolated compound, condensed chromatin, fragmented nuclei, and apoptotic bodies. These effects are characteristics of apoptosis and were dose-dependent, as shown in Figure 5.
Discussion
The relative amount of CAE (0.016%) obtained from the CE of CR rhizomes followed the results obtained previously (0.017%) [13]; however, the percentage of the isolated alkaloid per extract weight in our study (0.008%) was 2.5-fold higher compared to that of previous reports [13]. This difference may be accredited to a longer extraction period and an elevated extraction temperature with shaking. The amounts of nootkatone were 20-fold less in the current study compared to previous studies [18], which may be attributed to differences in the detection method, plant echo system, extraction, and isolation processes. The amounts of quercetin, chlorogenic acid, catechin, and β-sitosterol in the CEs and EtOAc fraction also showed variations compared to the previous reports. Earlier studies detected these compounds in the CE. Still, the results of the current study differed in the type and quantity of each component, likely due to environmental factors, the solvents used, and the techniques applied in the extraction and isolation processes [20, 28-30]. In this work, the polyphenolic contents (TOF and TFC) were obtained from the EtOAc fraction, while previous studies assayed the total extract [20, 31-33]. This might have caused the three-fold decrease in yields in the amounts of polyphenols in the current study compared to the previous reports.
The δ pattern from H-NMR analysis previously presented for rotundine B [13], an alkaloid isolated from CR rhizomes (not glycosylated), is highly relevant to our current results. Variations in the proton shifts may be attributed to the isolated compound due to the glycosylation effect on the solubility and the overlapping of alcoholic hydroxyl protons with the alcoholic hydroxyl at C-14. Furthermore, glycosylation may affect the structural pattern of the attachment of pyridine and the cyclopentane ring at C-3 and C-4, which varied in our study compared to that reported in the previous study [13]. Structural elucidation of the isolated compound illustrated the chemical formula of the oily yellow alkaloid, which was also confirmed by the positive reaction to alkaloid-detecting reagents. 
The structural variation of Alk-1 (norrotundin-6-O-glucoside) compared with the previously reported rotundine B [34] may be attributed to differences in the growth conditions and environmental changes that may affect protein expression and modify biogenetic assemblies. This may contribute to further post-translation modification involving the addition of a glucose moiety by glucosyltransferase (Figure 6). The final metabolite should serve a biological purpose to the plant [35].
S. aureus, E. coli, and C. albicans are opportunistic pathogens and are the common causes of a wide range of infections in the community in general and hospitalized patients particularly. The increased resistance to antimicrobial agents is a trait related to biofilm formation, and these organisms are all capable of producing biofilm, especially on prosthetic materials and catheters, contributing to great risk in immunocompromised patients.
The antimicrobial activity of the isolated compound Alk-1 showed inhibitory activity against S. aureus at 64 µg/mL with no growth on MHA plates at this concentration, indicating it was bactericidal [17]. The detected MIC against E. coli was 100 µg/mL, and the MBC assay exhibited bacteriostatic activity. The MIC and MBC values were 64 µg/mL against C. albicans. Previous studies demonstrated antimicrobial activity for the CE from CR rhizomes [17]. However, there are no reports about testing the antimicrobial properties of isolated rotundine. Previous studies have shown relevant antibacterial activities of different carbazole alkaloids against S. aureus with an isolated harman dianamine having an MIC value of 64 µg/mL. At the same time, other related isomers express greater antibacterial potency [36]. The noticeable antimicrobial effects of Alk-1 against S. aureus and C. albicans suggest that this compound should be tested against multi-drug-resistant species of the same microorganisms. As the initial study using whole plant extract demonstrated activity only against the gram-positive bacteria S. aureus and the yeast C. albicans, the purified compound was also expected to exhibit activity against only these two microorganisms. The absence of an inhibitory effect of the CE against E. coli growth may be attributed to inter, or intra-molecular deactivation of the mixture components, leading to lower activity than the purified compound.
Earlier studies investigated the CR rhizomes’ CE cytotoxicity and recorded activity against various human cancer cell lines, but they failed to attribute the activity to any specific metabolite [16, 37]. Screening for molecules showing cytotoxic properties is inevitable because of the high global mortality rates caused by different cancer types [34]. In the current study, evaluation of the isolated norrotundine-6-O-glucoside demonstrated dose-dependent cytotoxicity against HeLa cells, a human endothelial cancer cell line at 80 µg/mL, with increased activity at 160–200 µg/mL and an IC50 of 203.7 µg/mL after exposure to increasing concentrations (20–200 µg/mL). Evidence of condensed chromatin, fragmented nuclei, and formation of apoptotic bodies were all observed, in which chromatin condensation can be regarded as the normal transformation stages of genetically active chromatin to an inactive highly condensed form, followed by fragmentation and packaging into apoptotic bodied during apoptotic execution [38]. The mechanisms underlying the desensitization of mutated cells to apoptotic proteins may be attributed to intrinsic and extrinsic factors. Studies on cancer molecular targeting are focused on resetting apoptotic sensitization through the studying various proteins involved in this cascade, such as the anti-apoptotic Bcl-2, FLICE-inhibitory protein, c-FLIP, inhibitor of apoptosis proteins, and caspases [39]. The cytotoxic effect of CR rhizome extract has been evaluated previously, and the results showed promising cytotoxicity against various cancer cell lines [16]. HeLa cells have been used to develop many drugs and treatments and have been instrumental in gene mapping and studying human diseases, especially cancer. Our current study showed concentration-dependent cytotoxicity and enhanced apoptosis by the isolated alkaloidal glycoside. However, previous studies have reported lower IC50 values for different isolated alkaloids against HeLa cells. For instance, the steroidal alkaloids tomatidine and solasodine inhibit HeLa cells with IC50 values at 146.5 µg/mL and 104.3 µg/mL, respectively [22]. In contrast, other alkyl pyridine alkaloids inhibit HeLa cells at even lower concentrations (IC50=9.07 µg/mL) [40]. This study’s lower cytotoxic potency of the isolated norrotundine-6-O-glucoside may be attributed to lower penetration capacity than the previously reported alkaloids. Such variations in the alkaloid’s cytotoxicity could be greatly influenced by their diverse chemical structures and cellular macromolecular targeting sites. The observed differences in the current study compared to previous ones in terms of the amount and types of the screened metabolites providing that various environmental conditions and separation techniques are applied, such findings emphasize the capacity of living systems like plants to adapt to unusual conditions through new proteins transcriptions with modified functions to evolve new compounds with new therapeutic potentials.

Conclusion
This study was limited by insufficient data analysis of the isolated alkaloid, which was not previously characterized for chemical structure and biological activities. There is need to extend the antimicrobial activity to include a broad spectrum of resistant strains (Ex: Vancomycin-resistant enterococcus, Acinetobacter baumannii, Pseudomonas aeruginosa, and Campylobacter spp.) and examine the IC50 of the isolated compound against more human cancer cell lines (Ex: breast [MCF-7], colorectal [HT-29], liver [Hep G2], and prostate [PC-3]). There is also a need for further structure elucidation of the isolated compound to identify its stereoisomers. 
The plant used in our current study, C. rotundus, is a naturally growing weed in the Kurdistan Region of Iraq. The use of sequential extraction and chromatographic techniques, and spectroscopic analysis using FTIR, H-NMR, and MS led to the isolation of a novel rotundine glycoside from the methanolic crude extract of the plant rhizomes; such finding demonstrated for the first time the presence of a glycoside and positioning of the attachment to the alkaloid nucleus as a structural variation from the previously reported compound. The antimicrobial and cytotoxic assessments were not tested previously for the isolated alkaloid. Furthermore, previous studies examined galenic crude extract rather than a purified compound’s activity assessment, which is more beneficial for the drug discovery process. The isolated rotundine glycoside norrotundine-6-O-glucosidemaybe an excellent candidate antimicrobial and or anticancer drug. However, extended studies are required to confirm its potential. This compound may also be useful as a biomarker for the chemotaxonomic identification of the plant based on the isolated metabolite expressed by this plant species.
The need for new cytotoxic compounds should further drive investigations in the drug discovery field to unravel the relationship between structure and activity of norrotundine-6-O-glucoside. Furthermore, greater insight is needed regarding the molecular mechanism behind the cytotoxicity of norrotundine-6-O-glucoside, such as transcriptional analysis of the genes involved in its induction of the apoptotic pathway and molecular targeting sites involved in the cell toxicity, which includes the proteins involved in cell proliferation, enzymes modulating the DNA or extranuclear factors.

Ethical Considerations
Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Funding
This research was supported by the University of Sulaimani (Grant No.: 7/2/13319).

Authors' contributions
Conceptualization and Supervision: Kawkab Y. Saour and Shwan Rachid; Methodology: Mohammed N. Sabir; Investigation, Writing – original draft, and Writing – review & editing: Mohammed N. Sabir; Selar Izzat; Bahez O. Ismael; Shwan Rachid; Data collection: Shahlaa M Abdullah; and Bahez O. Ismael; Data analysis: Mohammed N. Sabir; Selar Izzat and Shahlaa M Abdullah. 

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors express their appreciation to the University of Sulaimani for supporting this research. The authors  would also like to extend their gratitude to both Emad M. Al-Khafaji and Sewara Jalal for their cooperation and to all who contributed to this work. Profound appreciation to Pioneer Company for Pharmaceutical Industries-Sulaymaniyah/Iraq for help performing the high-performance liquid chromatography analysis.


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