Papaver Decaisnei: GC-MS Alkaloids Proling, in Vitro Antioxidant, and Anticancer Activity

The Papaver L. plant have been well known as a source of pharmaceutically valuable alkaloids (noscapine, thebaine, codeine, roemerine, papaverine and morphine). The current study investigates the phytochemical, in-vitro antioxidant, and anticancer activities of papaver decaisnei , an endemic plant species to the ora of Kurdistan-Iraq. The chemical analysis of the methanolic (MeOH) extracts of owers, leaves, and roots of papaver decaisnei were made by using gas chromatography-mass spectrophotometry (GC-MS), and the antioxidant activity evaluation done by radical scavenging [on 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2j-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid) (ABTS)], and reducing power [cupric reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP)] assays. The anticancer actions were presented as IC 50 (inhibitory concentration at 50%) on human colorectal adenocarcinoma (Caco-2), mammary cancer cells (MCF-7), and human cervical carcinoma (HeLa) cells. The results of the phytochemical analysis showed 17, 19, and 22 chemical compounds for owers, leaves, and roots of P . decaisnei, respectively. The prevalent organic compounds of P . decaisnei were alkaloids, phenolics, fatty acids, esters, and phytosterols, namely Roemerine (70.44%), Decarbomethoxytabersonine, 9,12,15-Octadecatrien-1-ol, Hexadecanoic acid, 6,8-Dioxa-3-thiabicyclo(3,2,1)octane 3,3-dioxide, and γ -Sitosterol . The antioxidant activity of plant organ extracts was within 39.1-143.5 μ g/ml for DPPH and 123.12-276.4 μ g/ml for ABTS assays, while, the FRAP and CUPRAC values ranged within 12.4- 34.3 and 42.6-75.8 μ g/ml, respectively. The anticancer action of P.decaisnei organ extracts was found against all tested human cell lines (Caco-2, MCF-7, HeLa) with inhibitory concentrations (IC 50 ) values between 125.3-388.4 μ g/ml. The presented data on alkaloid contents and biological activity of P . decaisnei can serve a ground knowledge for the future biomedical synthesis and cancer research projects.


Introduction
The poppy (Papaver L.) belongs to the family Papaveraceae with about 820 species belonging to 43 genera [1]. In the ora of Iraq, the genus Papaver comprises of 15 annual and perennial species and most of them occurred in Kurdistan (80% in Rwanduz district), one of which species are endemic namely: papaver decaisnei Hochst. [2]. The Papaver species share different characteristics with releasing approximately 170 different alkaloids [3]. The alkaloid chemicals (thebaine, morphine, and codeine) of Papaver plant have been used as pharmaceutical and curative agents [4]. Thebaine is commonly used for the production of biologically active chemicals in the Papaver species and Papaver organs aids breeders to select desired poppy genotypes for the purpose of harvesting and breeding process, which could help in developing functional foods with better health bene ts for the consumers. Therefore, the current study aimed to determine the GC-MS phytochemical pro le, the antioxidant and antiproliferative activity of roots, owers, and leaves of P. decaisnei.

Plant collection
The roots, leaves parts, and owers of Papaver decaisnei ( gure 1)were collected at different growing stages during spring of 2021 from Erbil, Iraq (Altitude: 36.609153, Latitude: 44.526220). The plant was identi ed and the voucher specimen was deposited from the Salahaddin University Herbarium-Education College (ESUH). (voucher no. 6548).

Plant extract preparation
The dried roots, owers, and leaves (100 mg each) of P. decaisnei were macerated with 1 L of Methanol (99.9 % absolute methanol) extracting solvent by aluminum foil and ultrasonic incubation at room temperature for 2 hours. The solvent drainage done by rotary evaporator in water bath 40 • C to synthesis the crude extract, then the extracts freeze-dried to remove the solvent completely. The obtained MeOH extract was 21.4, 25.2, 19.6 % (w/w) for owers, leaves, and roots, respectively. The extracts were stored at +4•C for further investigation [14].

Phytochemical Pro ling
The Papaver organ extracts were screened qualitatively for their alkaloid contents using the GC/GC-MS technique. The methanol extract examined by Shimadzu Model QP-2010 GC coupled with MS. GC equipped with HP-5 MS (5% phenylmethyl siloxane), capillary column (30 m × 0.25 mm i.d., lm thickness 0.25μm) with temperature 60°C (2') to 250 °C for 10 minutes at a rate of 20 °C /min, helium ow rate 1.61ml/minute. The sustenance ion source was at 250 °C and 70 eV electron energy. The extracts were mixed with methanol before injecting 1μl into the column. The Wiley GC/MS Library and Adams Library, and Mass Finder Library were used to determine the exact name and molecular weight of the unknown component by comparing their mass spectrum with the reference spectrum [15], [16].

Anticancer activity
The Papaver extracts of owers, leaves, and roots were tested for anti-proliferative effectivity by evaluating their minimal inhibitory concentration (IC50) on Caco-2 (human colorectal adenocarcinoma), MCF-7 (human breast adenocarcinoma), and HeLa (human cervical cancer) cell lines using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay as previously explained [18]. The counting of viable cells made at 580nm by ELISA plate reader as equal value to the intensity in light absorbance.
Statistical Analysis.
Multivariate statistical analysis of GC-MS experimental data was performed by GC Shimadzu software. All experiments were carried out in triplicate. The IC 50 values were determined as drug concentrations leading to a 50% reduction in the viability or inhibition of the biological activity. The IC 50 values were calculated using a four-parametric logistic curve (Sigma Plot 11.0). The biological activity data interpretation made by student t-test (α = 0.05) using the SPSS v. 14.0 program. Finally, the data measurement was done by oneway analysis (ANOVA) of SPSS statistical software package, version 24.0 for Windows. The signi cance value is considered as p<0.05.
The main detected chemicals of MeOH root extracts were Roemerine (70.44%), 3'-methyl-1'-phenylspiro (indoline-2,4'-(2)pyrazoline)-5'-onE (5.16%), and Hexadecanoic acid (2.66%) ( Table 1 and Figure 4). Roemerine is a naturally occurring alkaloid that was reported to facilitate in reducing symptoms of Neurodegenerative diseases [22]. Previous work also detected signi cant amount of Roemerine in P. lacerum and P. syriacum and labeled it as possible strong antidepressant drug [23]. The current study also detected a variety of alkaloids in the plant organs as mentioned in the table 1, which were not discussed in this report.
The organic class of major detected chemicals were found as alkaloids, phenolics, and fatty acids, esters, and terpenoids. While, the organic classes of minor detected phytochemicals in the three plant organs were organosulfur, coumaranes, fatty alcohols, and phytosterols (Table 1). Our data results are in agreement with a recent study on the chemical pro ling of P.decaisnei by thin layer chromatography (TLC), which reported alkaloids as the main organic class content in P.decaisnei, namely aporphine-type roemerine and proaporphine-type mecambrine (PD2) [24]. Similar alkaloid contents (roemerine, dehydroromerine, roemerine N-oxide, rhoeagenine) have been reported from P. glaucum [24]. Furthermore, same alkaloid constituents were reported from P. somniferum [25], P. bracteatum [26], and P. rhoeas [27].
To the researcher's best knowledge, there is no previous research on the GC-MS pro ling of P. decaisnei, Therefore, the current study considered as the rst record on the identi cation of speci c phytochemicals in owers, leaves, and roots of P.decaisnei.

Antioxidant activity
The results of the antioxidant activity evaluation of P.decaisnei organs demonstrated that the species possessed profound antioxidant capacity. This may be due to the presence of alkaloid and polyphenolic chemicals. The antioxidant activity measured by radical scavenging (DPPH and ABTS) and reducing activity (FRAP and CUPRAC) assays, as presented in Table 2. The plant extraction needed to inhibit 50% of essay reagents is presented as IC 50 . The lower the IC 50 [29], [30], [31]. The current study shows owers as the most active antioxidant part in DPPH and ABTS assays, these could be correlated with its chemical contents, mainly terpenoids, esters, and fatty acids, namely decarbomethoxy tabersonine, hexadecanoic acid, and anthocyanins. The present outcome agrees with previous claims [29], [32], [33], [34]. Previous studies have also reported alkaloids namely, thebaine, noscapine, morphine, and codeine as antioxidant agents [10], [35].
The antioxidant estimation by FRAP and CUPRAC assays shows leaves as a superior part in antioxidant activity. These increased reducing power activity of leaves could be associated with its alkaloid, fatty acids and polyphenolic contents, mainly 9,12,15-Octadecatrien-1-ol, Hexadecanoic acid, and 8.beta.,13:8.alpha.,14. The previous investigation also showed Papaver leave extracts as a stronger radical scavenging agent [36], [37]. Previous phytochemical screening of Papaver leaves concluded avonols, like quercetin, kaempferol, myricetin, and isorhamnetin, as effective antioxidants [38]. Furthermore, scientists have correlated increased total phenolic content in Papaver rhoeas L. leave extracts with its high antioxidant activity [36].
The current research study detected signi cant amount of Roemerine in MeOH root extracts of P.decaisnei, a known aporphine alkaloid, which was reportedly stated as antioxidant and anticancer drugs [39].
Furthermore, a study by D. muthna and his colleagues con rmed the antioxidant and anticancer e cacy of aporphine members like roemerine [40]. Accordingly, numerous studies reported the antioxidant capacity of alkaloids isolated from different plant species [8], [41], [42]. The data stated above could be a reliable evidence for the antioxidant activity of P.decaisnei.

Anticancer activity
The anti-proliferative activity of P.decaisnei organs presented as IC 50 value, which ranged from 165.2-388.4 μg/mL on cancer cells derived from Caco-2 (human colorectal adenocarcinoma), MCF-7 (human breast adenocarcinoma), and HeLa (human cervical cancer) (Table 4). Moreover, Doxorubicin was used as a reference against same cancer cell lines. Over the last decades, many cancer diseases like human colorectal adenocarcinoma, hepatocellular carcinoma were tried to be treated by plant-derived compounds as a useful alternative source with less downside effects than synthetic drugs. Several owering plants including Papaver species produces enormous alkaloids and aromatic hydrocarbon compounds [43]. Furthermore, alkaloids (narcotine, morphine, codeine, narceine and thebaine) are nitrogenous waste producing compounds which have medicinal importance including antidepressant and pain relieving effects for humans and animals [44]. The anticancer activity of plant alkaloids raises scientist's hope in dealing with this deadly disease because of its less side effects than synthetic chemo-therapy. Furthermore, recent decodes witness an intense race between the scientists to nd speci c alkaloids with the most active indole ring and hydrocarbon chains against cell lines, as these properties signi cantly affect the delivery of drug [45], [46]. Therefore, we investigated the potency of MeOH extracts of owers, leaves, and roots of P.decaisnei against the growth of Caco-2, MCF-7, and HeLa cell lines. The results show higher anticancer activity of leaves extracts (176.2, 268.2 μg/mL) against Caco-2 and MCF-7 cell lines as compared to that activity of roots (194.7, 388.4 μg/mL), and ower extract (223.4, 306.5 μg/mL) against the same cell lines, respectively. This leave superiority as anticancer agent could be related to its higher content of alkaloids, fatty acids, and phytosterols namely 9,12,15-Octadecatrien-1-ol (25.45%), Hexadecanoic acid (14.66%), 8.beta.,13:8.alpha.,14(8.81%), and γ -Sitosterol (5.31%), which were reportedly considered as the strong anticancer agents [47], [48], [49], [50].
The methanolic extract of P.decaisnei roots was superior in anticancer activity against HeLa cell lines (125.3 μg/mL), which were higher than that (165.3 μg/mL) and (228.4 μg/mL) for leaves and owers, respectively. This root extract superiority against certain cell lines could be ascribed to its increased content of alkaloids and phenols namely roemerine (70.44%) and 3'-methyl-1'-phenylspiro(indoline-2,4'-(2) pyrazoline)-5'-one (5.16%), which were already highlighted as a signi cant anti-proliferative agent by previous natural product studies [51], [39], [52]. The effectivity of roemerine to reduce the proliferation and migration of different human cell lines and stimulated their apoptosis in different degrees have also reported previously [11]. Similar to our ndings, researches have also reported the superiority of P.somniferum roots against HeLa cell lines [12]. Moreover, it could be emphasized that Papaveraceae members are rich with alkaloid contents and many studies have shown their anticancer e cacy against several human cancer cell lines [52], [53]. The systematic search showed no previous anticancer study of P.decaisnei, therefore, the current work considered as the rst record regarding the anticancer of P.decaisnei plant organs.

Conclusion
The current study reports GC-MS pro ling, antioxidant, and anticancer of owers, leaves, and root extracts of P.decainsei for the rst time. The major alkaloids of owers were found as Decarbomethoxytabersonine, Hexadecanoic acid, and anthocyanin. Furthermore, the MeOH leave extract analysis showed 9,12,15-Octadecatrien-1-ol, Hexadecanoic acid, and γ -Sitosterol as their main alkaloid constituents. The main alkaloid compound of root extract was roemerine (70.44%), a known alkaloid as antidepressant and antianxiety-like remedy. The antioxidant analysis showed ower extracts as superior part in DPPH and ABTS assays and root extracts as the most effect plant organ in reducing power activities by FRAP and CAPRAC assays. The anticancer investigation of owers, leaves, and roots of P.decaisnei showed signi cant potency of plant organs against the growth of Caco-2, MCF-7, and HeLa cell lines. Our study provides a detailed phytochemistry and biological activities of P.decaisnei that can be used for numerous biomedical production and cancer research however, further research is needed to explore the toxicity and down side effects of P.decaisnei as a possible curative remedy for various human diseases.