3.1 GC-MS profile
The chromatogramatical study of MeOH extracts of flowers, leave, and roots of P.decaisnei showed different alkaloid contents (Table 1). The tested plant organs namely flowers, leaves, and roots contained 17, 19, and 22 chemical compounds, respectively, accounting for 100% of their volatile compounds (fig. 2, 3, 4).
Table 1. Shows phytochemical contents of roots, leaves, and flowers of P.decaisnei.
NO.
|
RT
|
Components Name
|
Similarity
|
Peak area percentage %
|
PF
|
PL
|
PR
|
1
|
7.55
|
Glycerin
|
9
|
6.41
|
-
|
-
|
2
|
8.666
|
Uracil, 1-N-Methyl-
|
59
|
-
|
1.76
|
-
|
3
|
8.686
|
Thymine
|
72
|
-
|
-
|
0.96
|
4
|
9.003
|
Guaiacol
|
94
|
1.85
|
-
|
0.99
|
5
|
10.259
|
4H-Pyran-4-One, 2,3-Dihydro-3,5-Dihydroxy-6-Methyl-
|
72
|
-
|
-
|
1.19
|
6
|
10.58
|
Silane, Triethylmethoxy-
|
72
|
1.99
|
-
|
-
|
7
|
11.021
|
2-Methyl[1,3,4]Oxadiazole
|
59
|
-
|
-
|
0.72
|
8
|
11.659
|
1,2-Benzenediol
|
81
|
-
|
-
|
0.54
|
9
|
12.038
|
2,3-Dihydro-Benzofuran
|
90
|
-
|
2.68
|
0.77
|
10
|
13.029
|
2H-Pyran-2-One, Tetrahydro-4-Hydroxy-4-Methyl-
|
91
|
4.85
|
-
|
-
|
11
|
13.642
|
Trans-Anethole
|
98
|
-
|
-
|
2.55
|
12
|
14.269
|
2-Methoxy-4-Vinylphenol
|
96
|
2.18
|
4.14
|
1.63
|
13
|
15.079
|
2,6-Dimethoxyphenol
|
95
|
-
|
-
|
0.61
|
14
|
15.592
|
DL-Proline, 5-Oxo-, Methyl Ester
|
78
|
2.80
|
-
|
0.44
|
15
|
16.127
|
Vanillin
|
96
|
-
|
-
|
0.52
|
16
|
17.201
|
6,8-Dioxa-3-Thiabicyclo(3,2,1)Octane 3,3-Dioxide
|
47
|
-
|
8.07
|
2.49
|
17
|
17.958
|
1,6-Anhydro-.Beta.-D-Glucopyranose (Levoglucosan)
|
47
|
2.67
|
-
|
-
|
18
|
18.457
|
2,4-Di-Tert-Butylphenol
|
96
|
-
|
2.80
|
-
|
19
|
18.462
|
Phenol, 2,4-Bis(1,1-Dimethylethyl)-
|
96
|
0.60
|
|
0.84
|
20
|
20.376
|
.Alpha.-Cedrol
|
99
|
-
|
-
|
1.40
|
21
|
21.907
|
3a-Hydroxy-1,2,3,3a,8,8a-Hexahydropyrrole(2,3b)Indole
|
90
|
-
|
-
|
0.45
|
22
|
22.763
|
4-Methoxy-6-Methyl-2-Propylpyridine
|
50
|
-
|
-
|
1.27
|
23
|
23.297
|
Benzyl Benzoate
|
98
|
3.59
|
2.52
|
-
|
24
|
24.496
|
Neophytadiene
|
96
|
-
|
4.79
|
-
|
25
|
25.944
|
Hexadecanoic Acid, Methyl Ester
|
99
|
-
|
1.70
|
-
|
26
|
26.52
|
Hexadecanoic Acid
|
99
|
16.40
|
14.66
|
2.66
|
27
|
28.725
|
Methyl 9,12,15-Octadecatrienoate
|
99
|
-
|
2.19
|
-
|
28
|
28.901
|
Phytol
|
91
|
-
|
3.10
|
-
|
29
|
29.187
|
9,12-Octadecadienoic Acid (Z,Z)-
|
96
|
3.47
|
-
|
1.45
|
30
|
29.192
|
Linoleic Acid
|
97
|
-
|
2.18
|
-
|
31
|
29.29
|
9,12,15-Octadecatrien-1-Ol, (Z,Z,Z)-
|
94
|
12.00
|
25.45
|
-
|
32
|
29.607
|
Octadecanoic Acid
|
98
|
-
|
2.98
|
-
|
33
|
35.32
|
8.Beta.,13:8.Alpha.,14
|
86
|
-
|
8.81
|
-
|
34
|
35.325
|
Decarbomethoxytabersonine
|
56
|
24.49
|
-
|
-
|
35
|
35.382
|
Roemerine
|
58
|
-
|
-
|
70.44
|
36
|
36.145
|
3-(3-Methoxyphenyl)Propenenitrile, 2-(Diethoxyphosphinyl)-
|
95
|
-
|
-
|
1.23
|
37
|
36.98
|
Butriptyline
|
72
|
-
|
2.25
|
-
|
38
|
36.98
|
Amitriptylinoxide
|
59
|
3.60
|
-
|
-
|
39
|
39.279
|
4-Methoxybenzene, 1-(2-Hydroxynaphthylmethylenamino)-
|
95
|
-
|
2.08
|
-
|
40
|
39.284
|
3'-Methyl-1'-Phenylspiro(Indoline-2,4'-(2)Pyrazoline)-5'-One
|
72
|
3.18
|
-
|
-
|
41
|
39.289
|
3'-Methyl-1'-Phenylspiro(Indoline-2,4'-(2)Pyrazoline)-5'-One
|
72
|
-
|
-
|
5.16
|
42
|
43.139
|
3,4-Dihydro-6,7-Dimethoxyisoquinoline 2-Oxide
|
43
|
5.62
|
-
|
-
|
43
|
43.238
|
Gibberellin A3
|
58
|
-
|
2.55
|
-
|
44
|
44.695
|
γ -Sitosterol
|
95
|
4.31
|
5.31
|
1.69
|
Table 1. (continue)
|
|
|
Compound type (Total number in 3 plant organs)
|
% and (number) in PF
|
% and (number)in PL
|
% and (number) in PR
|
|
|
Alkaloids (12)
|
15.78 (3)
|
23.52 (4)
|
22.72 (5)
|
|
|
Phenolics (11)
|
10.52 (2)
|
17.64(3)
|
27.27 (6)
|
|
|
Fatty acids (8)
|
15.78 (3)
|
17.64(3)
|
9.09 (2)
|
|
|
Esters (6)
|
15.78 (3)
|
11.76 (2)
|
4.54 (1)
|
|
|
Terpenoids (5)
|
21.05 (4)
|
0 (0)
|
4.54 (1)
|
|
|
Phytosterol (3)
|
5.26 (1)
|
5.88 (1)
|
4.54 (1)
|
|
|
Coumaranes (2)
|
5.26 (1)
|
0 (0)
|
4.54 (1)
|
|
|
Organosulfur (2)
|
5.26 (1)
|
0 (0)
|
4.54 (1)
|
|
|
Alcohols (2)
|
5.26 (1)
|
5.88 (1)
|
0 (0)
|
|
|
Oxapanes (1)
|
0 (0)
|
5.88(1)
|
0 (0)
|
|
|
Aromatics (1)
|
0 (0)
|
0 (0)
|
4.54 (1)
|
|
|
Others (5)
|
0 (0)
|
11.76 (2)
|
13.63 (3)
|
|
|
Total
|
100%
|
100%
|
100%
|
a: Retention time (tR [min]) on a Restek Rtx-5 column. Peak area percentage calculated from the GC-FID chromatogram. PF: Papaver flower, PL: Papaver leaves, PR: Papaver roots.
|
The chemical profiling of flower extracts showed 17 phytochemicals (shown in Table 1 and Figure 2), includes majorly Decarbomethoxytabersonine (24.49%), Hexadecanoic acid (16.40%), 9,12,15-Octadecatrien-1-ol, (Z,Z,Z)- (12%), Glycerin(6.41%), 3,4-dihydro-6,7 dimethoxyisoquinoline 2-oxide(5.62%), 2H-Pyran-2-one, tetrahydro-4-hydroxy-4-methyl-(4.85%), γ -Sitosterol(4.31%), Benzyl benzoate (3.60%), and Amitriptylinoxide (3.59%). Researchers have declared that Decarbomethoxytabersonine acts as a strong alkaloid that could play important role as anticancer and reduce free radicals during oxidative stress conditions [19].
The major phytochemicals of methanolic leave extracts were found as 9,12,15-octadecatrien-1-ol (25.45%), hexadecanoic acid (14.66%), 8.beta.,13:8.alpha.,14(8.81%), 6,8-dioxa-3-thiabicyclo(3,2,1)octane 3,3-dioxide (8.07%), γ -sitosterol (5.31%), neophytadiene (4.79), 2-methoxy-4-vinylphenol (4.14%), and phytol (3.10%) (table 1 and figure 3). previous researches have showed different biological activities such as antioxidant and anticancer of plant extracts enriched in 9,12,15-Octadecatrien-1-ol and Hexadecanoic acids [20]. The antioxidant and anti-proliferative effect of γ -Sitosterol has been also reported previously by the researchers. [21]. The analysis of methanolic extract of P.decaisnei leaves also showed some chemicals in low amount including octadecanoic acid (2.98%), 2,4-di-tert-butylphenol (2.80%), 2,3-dihydro-benzofuran (2.68%), gibberellin a3 (2.55%), and few others, which were not discussed in this article.
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 significant 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 profiling 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 profiling of P. decaisnei, Therefore, the current study considered as the first record on the identification of specific phytochemicals in flowers, leaves, and roots of P.decaisnei.
3.2 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 IC50. The lower the IC50 value of an extract indicates its efficiency as an antioxidant agent. In antioxidant activity measurement by DPPH and ABTS assay, flowers (39.1 and 135.4 μg /mL trolox) were superior to leaves (81.35 and 245.6 μg /mL trolox) and roots (143.5 and 276.4 μg /mL trolox), respectively. In the antioxidant evaluation by FRAP and CUPRAC assays, leaves recorded the highest values (12.4 and 42.6 μg /mL trolox) followed by roots (18.3 and 68.1 μg /mL trolox) and flowers (34.3 and 75.8 μg /mL trolox), respectively.
Table 2. Antioxidant activity of MeOH extracts of roots, leaves, and flowers of P.decaisnei 1.
Plant extracts
|
DPPH scavenging2
|
ABTS scavenging2
|
FRAP reducing3
|
CUPRAC reducing3
|
Flowers
|
39.1 ± 0.53b
|
135.4 ± 0.78b
|
34.3 ± 0.05b
|
75.8 ± 0.9b
|
Leaves
|
81.35 ± 0.111a
|
245.61 ± 0.23a
|
12.4±0.08a
|
42.6±0.3a
|
Roots
|
143.5 ± 3.06a
|
276.4±0.045a
|
18.3±1.023c
|
68.1±0.9c
|
Trolox
|
1.4±0.02c
|
2.29±0.02c
|
2435.1±0.01a
|
2255.2±0.02a
|
EDTA4
|
ND5
|
ND
|
ND
|
ND
|
1The values indicated by different superscripts within the same column are different according to the Tukey’s honestly significant difference post hoc test at 5% significance level. (value as mean ±standard deviation)
2IC50 (μg /mL), inhibition concentration at which 50 % of the DPPH (2,2-Diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) radicals were scavenged and the ferrous ion-ferrozine complex were inhibited.
3 EC50 (μg /mL): 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 nf: Not detected.
The antioxidant activity shown by different organs of P.decaisnei could be linked to their phytochemical contents. The alkaloid and polyphenolic of different plant species have been reported to possess antioxidant activity [28],[29],[30],[31]. The current study shows flowers 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 flavonols, 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 significant 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 confirmed the antioxidant and anticancer efficacy 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.
3.3 Anticancer activity
The anti-proliferative activity of P.decaisnei organs presented as IC50 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.
Table 4. The Anti-proliferative activity IC50 (μg/mL) of extract on human cell lines after 24 hr of treatment.
Cell line
|
IC50 values (μg/mL)1
|
Flowers MeOH extract
|
Leaves MeOH extract
|
Roots MeOH extract
|
DOX 2
|
Caco-2
|
223.4±2.1
|
176.2±3.8
|
194.7±6.5
|
6.7±0.4
|
MCF-7
|
306.5±9.8
|
268.2±12.3
|
388.4±11.2
|
18.8±0.3
|
HeLa
|
228.4±3.1
|
165.3±2.3
|
125.3±4.2
|
14.0±0.1
|
Key: Caco-2 (human colorectal adenocarcinoma), MCF-7 (human breast adenocarcinoma), and HeLa (human cervical cancer). (* value as mean±standard deviation (n = 3).)
1Mean value ±S D of IC50 (μg/mL), inhibition concentration at which 50 %
2 DOX: Doxorubicin.
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 flowering 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 find specific alkaloids with the most active indole ring and hydrocarbon chains against cell lines, as these properties significantly affect the delivery of drug [45],[46]. Therefore, we investigated the potency of MeOH extracts of flowers, 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 flower 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 flowers, 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 significant 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 findings, 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 efficacy 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 first record regarding the anticancer of P.decaisnei plant organs.