GC-MS Analysis of Bioactive Compounds in Methanolic Extracts of Papaver decaisnei and Determination of Its Antioxidants and Anticancer Activities

The Papaver L. plant ( Papaver decaisnei ) has ethnobotanical records in many countries including Iraqi Kurdistan. The current study investigates the methanol (99.9%) extracts (10 μ g/mL) of roots, leaves, and ﬂowers of Papaver decaisnei in terms of phytochemistry by gas chromatography-mass spectrophotometry GC-MS, in vitro antioxidant activity by radical scavenging and reducing power assays, and ﬁnally, the anticancer actions as IC 50 (inhibitory concentration at 50%) against human colorectal adenocarcinoma (Caco-2), mammary cancer cells (MCF-7), and human cervical carcinoma (HeLa) cells. The results showed 22, 19, and 17 chemicals for roots, leaves, and ﬂowers of P . decaisnei , respectively. The prevalent organic compounds of P . decaisnei were alkaloids (62.03%), phenolics (55.43%), fatty acids (42.51%), esters (32.08%), terpenoids (25.59%), and phytosterols (15.68%), namely, roemerine ( 70.44 %), 9,12,15-octadecatrien-1-ol (37.45%), hexadecanoic acid (33.72%), decarbomethoxytabersonine (24.49%), and c -sitosterol (11.22%). The antioxidant activity of plant organs was within 39.1–143.5 μ g/mL for DPPH, 135.4–276.4 μ g/mL for ABTS, 12.4–34.3 μ g/mL for FRAP, and 42.6–75.8 μ g/mL for CUPRAC assays. The anticancer of P. decaisnei was found as 125.3–388.4 μ g/mL against all tested cell lines (Caco-2, MCF-7, and HeLa). The detected alkaloids and bioactivity of P . decaisnei encourage future isolation of those remarkable alkaloids (reomerine) for potential usage in the pharmaceutical industry.


Introduction
e poppy Papaver L., Papaveraceae, has more than 820 species distributed into the 43 genera [1]. e Iraqi flora shows 15 Papaver geniuses with annual and perennial species, mostly growing in Iraqi Kurdistan (an autonomous region in Iraq) particularly about 80% of them were found in Rawanduz district. ey include P. acrochaetum Bornm., P. argemone L., P. armeniaca L., P. bornmuelleri Fedde., morphine) of Papaver species are industrially used for pharmaceutical products as a treatment for different health problems [4]. ebaine is a known chemical for the production of pentacyclic morphinan-based drugs. Morphine, noscapine, and codeine were previously presented as analgesia as well as an antiproliferative drug for their significant remedial potency [5]. In more recent decades, the world's tendency to depend on the Papaver alkaloids and their derivatives as a remedy has remodified the drug production basics of major pharmaceutical companies [6]. Gas chromatography coupled with mass spectrometry is one of the fast and sensitive techniques for the identification of volatile compounds from plant extract. On this occasion in the present work, the GC-MS method was chosen to determination qualitative and quantitative for the complex product [7,8].
Natural antioxidants and herbal medicine usage as antifree radicals become popular among health care due to the downsides of chemically synthetic drugs. e antioxidant potentials of Papaver species have been reported by numerous research studies [7][8][9][10]. is bioactivity of Papaver species has been correlated with their moderate hydrophilic secondary metabolites contents, namely, flavonoids, phenolic acids, and glycosides, as well as lipid-soluble compounds, namely, sterols, zeaxanthins, tocopherols, tocotrienols, and carotenoids, which were stated as antioxidants in several diseases involved with the oxidative stress [11,12]. Alkaloids, namely, berberine, roemerine, amaranthine, thebaine, morphine, and noscapine, of Papaver species attracted scientists for further exploration due to their significant antitumor action and antioxidant activity [12][13][14][15][16]. e chemicals of Papaver species and their enriched alkaloid contents were always the central attention of plant breeders and investigation on different Papaver species and Papaver organs, and enlighten the plant breeders to grow the desired poppy genotypes with the most active functional compounds for the food and pharmaceutical industry and with less side effect for the sake of human health. Enlightened by that the present research explores the phytochemistry and bioactivity of MeOH extracts of roots, leaves, and flowers of P. decaisnei for the first time.

Plant Collection.
e flowers, roots, and leaves of P. decaisnei were collected from Iraqi Kurdistan, Safeen Mountain (Altitude: 36.609153, Latitude: 44.526220). e identification of the plant was done by botanist Dr. Abdullah Sh. Sarder, and the voucher number (6548) was deposited from the Herbarium of the Salahaddin University Education College (HSUEC) as shown in Figure 1.

Plant Extract Preparation.
e plant organs (roots, flowers, and leaves) were dried and an amount of 25 g each separately of P. decaisnei were taken for the extraction process by using 0.5 L of methanol (99.9%) and microwave (Samsung microwave 20 A3010, 10 power levels, frequency 50 MHz, Australia). e separation of solvents by rotary evaporator was performed in a water bath at 40°C for the crude preparation, further drying of the crude to exclude the solvent completely. e final crude extract for flowers, leaves, and roots was 9.72, 12.4, and 12.92% (w/w), respectively. e plant extracts were kept at +4°C for later analysis [17].

Phytochemical Profiling.
e Papaver crude extracts were qualitatively investigated by using the GC/GC-MS technique to determine their phytochemical and alkaloid constituents. e methanol extract examined by Shimadzu Model QP-2010 GC coupled with MS. GC equipped with a capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm) and HP-5 MS (5% phenylmethyl siloxane) at a helium flow rate of 1.61 mL/minute with the temperature 60°C (2′) to 250°C for 10 minutes at a rate of 20°C/min. e ion source was maintained at 250°C and 70 eV electron energy.
e methanol was added to the extracts before injecting 1 μl into the column. e exact name and molecular weight of unknown compounds were found by comparing their mass spectrum with the reference spectrum available in the Wiley GC/MS Library, Mass Finder Library, and Adams Library [16][17][18][19].
2.6. Statistical Analysis. Statistical analysis of multivariate GC-MS experimental analysis by GC Shimadzu software was applied. e IC 50 numbers were found as drug concentrations causing a 50% decrease in the viability or slowing the bioactivity. e IC 50 numbers were calculated by a logistic curve of the four-parametric form (Sigma Plot 11.0) [26,27]. e biological activity data were calculated by Student's ttest (α � 0.05) and one-way analysis (ANOVA) from the SPSS v. 14.0 program version 24.0 for Windows. e values considered significant if p < 0.05 [28].

GC-MS Profile.
e phytochemical qualitative study of extracts of Papaver organs (flowers, leaves, and roots) resulted in finding various alkaloids and chemicals as shown in Table 1, belonging to different organic classes as shown in Table 2. e chemical profiling of P. decaisnei plant organs, namely, roots, leaves, and flowers, showed 22, 19, and 17 chemicals, respectively, constituting 100% of their volatile contents as shown in  e chemical profiling of MeOH root extracts found Roemerine (70.44%) as the main compound in this organ, while some compounds were detected in a minor amount, including 3′-methyl-1′-phenylspiro (indoline-2,4'-(2) pyrazoline)-5′-one (5.16%), and hexadecanoic acid (2.66%) as shown in Table 1 and Figure 2. e biological and medicinal efficiency (alleviation symptoms of neurodegenerative diseases) of roemerine as a natural alkaloid has been reported by previous research studies [29]. A previous study by Hijazi et al. (2017) had reported roemerine as the main component of P. libanoticum with significant anticancer activity of its extract and alkaloid contents against human breast cancer and human colon cancer cells [30].

Antioxidant Activity.
e investigation of antioxidant activity of P. decaisnei organs revealed the significant potentiality of this species as a strong antioxidant agent. is bioactivity of P. decaisnei could be linked with its phytochemicals, mainly alkaloid and polyphenolic compounds. e antioxidant activity was evaluated by two different methods, radical scavenging activity by DPPH and ABTS assays, and reducing power activity by FRAP and CUPRAC assays, as presented in Table 3. e antioxidant activity presented as IC 50 e antiradical actions of different organs of P. decaisnei can be correlated with their different chemical constituents, namely, alkaloids, polyphenols, and phytosterols. e antioxidant activity of this compound has been also clarified by several research studies [33][34][35][36][37]. e present research found flower extracts as the highest bioactive part in the antioxidant tests by DPPH and ABTS assays, which may be due to its phytochemical constituents, mainly terpenoids, fatty acids, and ester, namely, hexadecanoic acid, decarbomethoxytabersonine, and anthocyanins. e given data results are following the previous findings regarding the antioxidant activity of the mentioned phytochemicals [34,[38][39][40][41][42][43][44][45][46]. e reducing power estimation by FRAP and CUPRAC assays revealed the part of leaves as the most active part in the antioxidant activity.
is bioactivity of leaves can be linked with its high alkaloid, fatty acids, and polyphenolic constituent, mainly 9,12,15-octadecatrien-1-ol, 8.β,13 : 8.α,14-diepoxy-14,15-bisnorlabdane and hexadecanoic acid. A similar study showed significant antioxidant activity of Papaver rhoeas L. leave extracts and linked this action with its increased phenolic contents [47,48]. Furthermore, the another study by Trichopoulou et al. revealed significant antioxidant action of Papaver leaves and linked this action with their efficient flavonoid contents, namely, quercetin, isorhamnetin, kaempferol, and myricetin [49]. e present investigation of P. decaisnei root extract revealed a significant amount of roemerine, a known aporphine alkaloid, which is regarded as an efficient antioxidant and anticancer agent [50]. Furthermore, the antioxidant and anticancer potentials of aporphine compounds like roemerine have also been confirmed by D. Muthna and his colleagues [51]. Similarly, the antioxidant and antitumor capacity of alkaloids from plant sources have been confirmed [9,37,[46][47][48]. e abovepresented record could be enough evidence regarding the antioxidant activity of P. decaisnei.

Anticancer Activity.
e anticancer action of P. decaisnei extracts was expressed as IC 50 , a value that ranged between 125.3 and 388.4 μg/mL based on the plant's ability to inhibit the growth of MCF-7 (human breast adenocarcinoma), HeLa (human cervical cancer), and Caco-2 (human colorectal adenocarcinoma) as shown in Table 4. Moreover, doxorubicin as a standard reference was used on the mentioned cancer cell lines.
In recent years, herbal medicine and plant-derived compounds become a strong alternative source of synthetic drugs for the treatment of several tumor diseases such as hepatocellular carcinoma, human colorectal adenocarcinoma, and breast cancer. e main reason is because of fewer side effects of these natural compounds than chemical drugs. For that, multiple flowering herbs including Papaver species were used for medicinal and pharmaceutical purposes due to its increased alkaloids and aromatic hydrocarbon contents [52][53][54][55].
e Papaver alkaloids, namely, narcotine, morphine, codeine, narceine, and thebaine, from P. somniferum, and P. libanoticum are considered nitrogenous waste-producing chemicals with several pharmaceutical and biological activities including antidepressant, analgesia, and anticancer activity [15,30,39]. e antitumor action of herbal alkaloids brings more hope to the scientists in handling severe diseases due to the lower side effects of natural-based cures than chemically synthetic therapy. us, in the past few years, scientists have raced against the time to find an alkaloid with the highest indole ring activity and hydrocarbon chains against different cancer cell lines, because those properties have significant roles in drug delivery process [54,[56][57][58].
e anticancer action of MeOH root extracts of P. decaisnei against HeLa cell lines was higher (125.3 μg/mL) than that (165.3 μg/mL and 228.4 μg/mL) for leaves and flowers, respectively.
Our data regarding highlighting the two mentioned compounds as anticancer agents are following the previous herbal bioactive studies [30,[61][62][63]. Furthermore, the cytotoxic mechanism of roemerine as the main alkaloid component of P. libanoticum and Nelumbo nucifera against several cancer cells has been linked to its ability to stimulate apoptosis, increase membrane permeability, and facilitate in cleavage of caspase-3, caspase-7, and caspase-9 in human cancer cells as clarified previously [30,50,64]. Similarly, to our results, researchers have also shown the superiority of MeOH root extracts of P. somniferum in reducing the growth of HeLa cell lines [15,58,65]. Moreover, based on several previous studies, the Papaveraceae members can be emphasized as a strong candidate for anticancer pharmaceutical production because of their high alkaloid contents [3,9,16,40,44,50,52]. e systematic Google search and

Journal of Food Quality
SciFinder did not find any previous anticancer record for P. decaisnei; therefore, the present work is considered as the first project studying the anticancer of P. decaisnei.

Conclusion
e present article showed GC-MS analysis, antiradical, and antitumor of roots, flowers, and leaves of P. decaisnei as the first report. e prevalent detected alkaloids of flowers were decarbomethoxytabersonine, hexadecanoic acid, and anthocyanin. Moreover, the analysis of MeOH leaves extract showed 9,12,15-octadecatrien-1-ol, hexadecanoic acid, and c-sitosterol as their prevalent alkaloids. e prevalent alkaloid content of methanolic root extract was roemerine (70.44%), an alkaloid commonly used due to its antidepressant and antianxiety action. e antioxidant assays revealed the Papaver flower as the most active organ in DPPH and ABTS assays, while root extracts had the highest activity in reducing power activity by FRAP and CAPRAC assays. e antiproliferative analysis of roots, flowers, and leaves of P. decaisnei revealed significant potentials of Papaver organs against the proliferation of HeLa, Caco-2, and MCF-7 cell lines. e current research presents a ground knowledge on the phytochemical profile and bioactivity of P. decaisnei that could be used for future biomedical analysis and cancer study programs; however, a more detailed study   e values indicated by different superscripts within the same column are different according to Tukey's honestly significant difference test at 5% significance level (value as mean ± standard deviation). 2 IC50 (μg/mL), inhibition concentration at which 50% of the DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radicals were scavenged and the ferrous ion-ferrozine complex was inhibited. 3 EC50 (μg/mL) is effective concentration at which the absorbance was 0.5 for CUPRAC (cupric ion reducing antioxidant capacity) and FRAP (ferric reducing antioxidant power) assays. 4 EDTA: ethylenediaminetetraacetic acid (disodium salt). 5 ND: not detected. is required to investigate the toxicity and drawbacks of P. decaisnei as a potential remedy for different human diseases.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Ahmed Aj. Jabbar and Fuad O. Abdullah have structured, designed, and done the methodology. Kamaran K. Abdulrahman analyzed the results, and A. A Jabbar wrote the article with the guidance of all authors. All authors participated equally in reviewing and the finalizing manuscript.