Papaver Plants: Current Insights on Phytochemical and Nutritional Composition Along with Biotechnological Applications

The genus Papaver is highly esteemed in the pharmacy industry, in the culinary field, and as ornamental plants. These plants are also valued in traditional medicine. Among all Papaver species, Papaver somniferum L. (opium poppy) is the most important species in supplying phytochemicals for the formulation of drugs, mainly alkaloids like morphine, codeine, rhoeadine, thebaine, and papaverine. In addition, Papaver plants present other types of phytochemicals, which altogether are responsible for its biological activities. Therefore, this review covers the phytochemical composition of Papaver plants, including alkaloids, phenolic compounds, and essential oils. The traditional uses are reviewed along with their pharmacological activities. Moreover, safety aspects are reported to provide a deep overview of the pharmacology potential of this genus. An updated search was carried out in databases such as Google Scholar, ScienceDirect, and PubMed to retrieve the information. Overall, this genus is a rich source of alkaloids of different types and also contains interesting phenolic compounds, such as anthocyanins, flavonols, and the characteristic indole derivatives nudicaulins. Among other pharmacological properties, numerous preclinical studies have been published about the analgesic, anticancer, antimicrobial, antioxidant, and antidiabetic activities of Papaver plants. Although it highlights the significant impact of this genus for the treatment of a variety of diseases and conditions, as a future prospect, characterization works accompanying preclinical studies are required along with clinical and toxicology studies to establish a correlation between the scientific and traditional knowledge.


Introduction
Many plants are naturally rich sources of phytochemicals with valuable biological properties, which could have significant impact for the treatment of a variety of diseases and conditions and as potential alternative options for synthetic drugs. This is also the case of the genus Papaver (family Papaveraceae), which is known for its medicinal properties attributed to their phytochemical composition ( [1]; [180]. This genus belongs to the family Papaveraceae, which is a cosmopolitan family growing from tropical to alpine ecosystems [1], mainly in the northern hemisphere [2]. The flowers have no style, but on the top of the ovary, a stigmatic tissue is arranged radially on a sessile stigmatic disc. Their similar characteristics in their flower shapes, colors, and fruits complicate the identification based only on morphological characteristics [2], and different number of species is given in literature. For example, it consists of~80-100 species, including annual, biennial, and perennial herbs [1,3]. As the family, the genus Papaver is widely natural distributed, especially in regions with Mediterranean climate [1,4]. In the case of Papaver somniferum L. (opium poppy), the most important species and due to its narcotic properties, it is highly produced in countries such as Afghanistan, Myanmar, Mexico, and Lao PDR (or Laos) [5], but illegally [6]. Alternatively, Turkey is one of the main legal manufacturers of the poppy plant [7], along with Czechia, Spain, etc. Figure 1 shows the world production of poppy seeds in the last twenty years according to the data available from the Food and Agriculture Organization of the United Nations [8].
Other commonly cultivated species of the genus Papaver are Papaver bracteatum Lindl. (Iranian poppy), Papaver rhoeas L. (common poppy or corn poppy), Papaver dubium L., Papaver pseudo-orientale Medw., and Papaver orientale L. P. bracteatum that grow wild in high altitudes in north and northwest of Iran, in Russia and Caucasia regions [9]. P. rhoeas is an important competitive plant in winter cereals in southern Europe under Mediterranean climate [10] and thus called corn poppy. P. dubium is also called long-head poppy. P. dubium is widespread throughout Europe and America and is an important weed in western Iran [11]. P. orientale and P. pseudo-orientale are distributed into the Caucasus area [12].
Concerning the natural product field, P. somniferum was the first source of natural drugs with the obtainment of analgesic morphine drugs [13], including codeine, morphine, and a variety of semisynthetic derivatives, mainly derived from thebaine, such as oxycodone and buprenorphine [14]. These compounds belong to the opiate family that has analgesic properties mainly by binding to the mu-opioid receptor within the central nervous system (CNS) and the peripheral nervous system. It leads to an overall reduction of the nociceptive transmission [15]. However, the latex of the opium poppy is not only used for the treatment of severe pain, but it is also subjected to opioid abuse and drug trafficking due to the narcotic properties of these compounds. Therefore, their production is regulated internationally.
P. bracteatum has high content of thebaine as the main alkaloid, which has been utilized in the production of codeine [16]. Besides these compounds, the nonnarcotic papaverine is another economically important alkaloid with vasodilator properties [1,17].
Apart from the alkaloids, poppy plant is a rich source of phenolic compounds, such as anthocyanins, flavonols, and the characteristic indole derivatives nudicaulins, and essential oil volatiles, which altogether are responsible for its pharmacological activities.
Moreover, Papaver seeds are esteemed in the food sector, e.g., to be used in bakery and desserts and to produce oil. For example, P. somniferum seeds are used in some Central Eastern European countries (European Food Safety Authority, [18]). In this context, poppy can be classified into three main categories depending on the use: industrial poppy intended for alkaloid extraction from the capsule of the plant; culinary poppy when it is grown to obtain seeds and oil; both industrial and culinary poppy [19]. P. rhoeas is also used as garniture in salad in some regions [20], and the seeds of P. bracteatum are used in foods in Central Anatolia [21].

Ethnobotanical Uses of the Papaver Genus in Different Human Cultures
The traditional and medicinal properties of this genus have been documented since 3000 BC. The main analgesic compound "morphine" was isolated from P. somniferum L. by a German pharmacist "Sertüner" in 1905 [22]. The genus Papaver is not popular for traditional medicine due to narcotic and other side effects. Nevertheless, there are several uses such as anti-inflammatory, antidiabetic, analgesic, and remedy for cough and lung infection as described in traditional medicine and detailed in Table 1. This includes the use of the flowers, buds, seeds, fruits, and leaves or aerial parts of the most popular species of Papaver in different countries and cultures.

Phytoconstituents
The identification of the phytochemical composition of medicinal plants is highly important to provide a best known of the active compounds. It involves complex mixtures of natural compounds with different organic structures and varies depending on the plant source [48]. Papaver species contain alkaloids, phenolic compounds, and essential oil volatiles, among other components [49]. These classes found in different parts of the Papaver plants are discussed in the following subsections.
4.1. Alkaloids. As other phytochemicals, the production of alkaloids in poppy plants is induced by environmental stress conditions, but the details about regulatory processes are not well known and subjected of ongoing studies [50]. Moreover, the alkaloid composition varies even within the same species [51]. This makes that the Papaver genus yields more than 170 alkaloids [52,53]. As an example, Figure 2 summarizes the type of alkaloids found in the genus Papaver with their chemical structures.
In particular, P. somniferum presents interesting benzylisoquinoline alkaloids, such as papaverine, and the morphinanes morphine, codeine, and thebaine (Table 2, Figure 2), as mentioned in Section 1. Since P. somniferum has been extensively utilized illegally, its cultivation is strictly regulated by the International Narcotics Control Board [54,55]. The latex of the opium poppy, which surrounds the seed capsule [56], may contain up to 80 alkaloids, but the latter compounds, morphine, codeine, and thebaine, along with narcotine and narceine are generally the main alkaloids [17,18].
P. bracteatum is also a source of the alkaloid thebaine, the precursor of the opiate analgesics codeine, buprenorphine, oxymorphone, and oxycodone [14,57,58]. The plant capsule of this species shows high concentrations of morphine and oripavine (another morphinan) as compared to the stem tissues. It seems that the origin and even the latitude affect the thebaine, morphine, and oripavine [59]. This makes that it has intraspecies variation. For example, another study found that the major alkaloids in this species were salutaridine (promorphinan) and thebaine [53]. Poppy seed production (tonnes) G e r m a n y A u s t r i a B u l g a r i a C z e c h i a C r o a t i a S l o v a k i a S p a i n F r a n c e H u n g a r y 3 Oxidative Medicine and Cellular Longevity Concerning P. rhoeas, phytochemical composition has showed rhoeadine alkaloids as major compounds, including rhoeadine and rhoeagenine [60,61]. Recent trends based on mass spectrometry (MS) analysis enabled the identification of a high number of phytochemicals from Papaver samples. For alkaloid profiling, electrospray ionization in the positive ionization modes generally leads to richer and complex chromatographic profiles with more intense signals for elucidation purposes [62]. Using this technique, for example, 55 alkaloids were characterized in the aerial parts of P. rhoeas and P. somniferum. This included benzophenanthridine, protoberberine, benzylisoquinoline, aporphine, and rhoeadine-type alkaloids (see examples, in Figure 2). The most characteristic feature was that rhoeadine alkaloids were observed only in P. rhoeas samples, and codeine and morphine were tentatively identified in P. somniferum [180] ( Table 2).
In the latter work, different solvents were tested for extraction including ethyl ether with 10% ammonia, pure ethanol, and methanol, as well as aqueous-methanol 50% and 80%. Among them, ethanol can be applied to extract the aerial parts of P. rhoeas and P. somniferum, with advantages due to its high extraction efficiency [181] and as its low toxicity. Similarly, four Papaver species (Papaver Lacerum Popov, Papaver syriacum Boiss. & Blanche, Papaver glaucum Boiss. & Hausskn., and P. rhoeas) were collected from different sites in Turkey and the aerial parts were extracted using methanol. By using LC-tandem MS, two alkaloids, pronuciferine (proaporphine type) and roemerine (aporphine type), were determined in the selected species [63]. The latter compound was the major one in some P. rhoeas samples [64]. Recent studies showed that both alkaloids increase brainderived neurotrophic factor (BDNF) protein expression in hippocampal SH-SY5Y cells demonstrating that besides the Pakistan Herb (narcotic, stimulant, to increase performance, cough suppressant); fruit, leaf (analgesic, narcotics); seed (narcotic, analgesic, sedative, increases excitement and physical vigor) common poppy alkaloids, the former alkaloids could also be interesting [65]. Other compounds identified were salutaridine (promorphinan type), coulteropine (protopine type), and rhoeadine derivatives (epiglaucamine, glaudine, and rhoeagenine) [64]. Furthermore, using a combination of LC-MS and molecular networking, isoquinoline alkaloids in Papaver nudicaule L. and P. rhoeas aerial parts were clustered. 42 and 16 compounds were characterized, respectively, and a variation was observed depending on the color of the flowers [66].

Oxidative Medicine and Cellular Longevity
Rhoeadine is another group of alkaloids which is very common and widespread in the genus Papaver and contains at least 25 types. Particularly, alpinigenine, alpinine, and epialpinine were isolated from the Papaver alpinum L., whereas epiglaudine was isolated from the P. glaucum. Other rhoeadine-type alkaloids include glaucamine, glaudine, isorhoeadine, isorhoeagenine, isorhoeagenine-D-glucoside, Nmethylporphyroxigenine, oreodine, oreogenine, papaverrubines A, B, C, D, E, F, G, H, rhoeadine, and rhoeagenine which are extracted from different species of Papaver. In general, all rhoeadines are characterized by a benzazepine system fused with six-membered acetal or hemiacetal moieties [69].

Phenolic Compounds.
Phenolic compounds are natural antioxidants and other interesting phytochemicals found in Papaver plants. For example, petals of P. rhoeas flowers present flavonoids, which are responsible for their color, including white, yellow orange, white, and red colors. Particularly, the red flowers of this species contain anthocyanins [70]. This agreed with the results obtained by Soulimani et al. [71], who showed that a lyophilized ethanolic aqueous extract of P. rhoeas petals has anthocyanins, whereas no alkaloids were detected. Anthocyanins such as pelargonidin glycosides have been detected in red and orange petals of the plant [72].
Among the solvents, water, ethanol, and aqueous ethanol can be applied to extract high amounts of phenolics, but among them, the water extract showed the highest phenolic content. It was found that the aqueous extract of P. somniferum stalk contains high amount of phenolics, including flavonoids. The methanol and aqueous extracts presented considerable amounts of the flavanol (−)-epicatechin and the benzoic acid syringic acid [76]. Moreover, the aerial parts of P. macrostomum had the flavone luteolin ( Figure 3) [67].
The essential oil of the aerial parts of P. rhoeas that was gathered from the Elazig region in Turkey was obtained by hydrodistillation and analyzed using gas chromatography. Twenty-one constituents comprised the 98.6% of the total essential oil volatiles extracted from the plant. The major ones were phytol (52.8%), tricosane (7.8%), 2-pentadecanone (6%), and heneicosane (5.3%) ( Figure 3); some of them are in common with P. somniferum [79]. Among them, the diterpene phytol is another interesting bioactive compound [80].
Moreover, the triglyceride composition of P. somniferum seed oil has been analyzed by matrix-assisted laser desorption/ionization-time-of-flight-MS and electrospray ionization ion trap-MS/MS. It enables the determination of the major triglyceride components, which were composed of linoleic, oleic, and palmitic acid, comprising approximately 70% of the oil [78]. The presence of high amount of unsaturated fatty acids makes the poppy seed oil suitable for its application in foods for maintaining the cardiovascular health.

Phytochemical
Variation. The type of phytochemical and its content mainly depend on the part used and solvent applied for the extraction, as it was discussed in the previous sections. Also, intraspecific variation occurs [51,59], for example, due to different locations [59], growth stage, and conditions [181]. This is extremely important for standardization or to choose those plants with strong enough potency to be applied to obtain functional ingredients.
For example, in a relevant study, empty poppy capsules (poppy straw) of 15 cultivars of P. somniferum were studied for the phytochemical profile. The seeds were raised in randomised block design with 3 replications during three consecutive years in 2007, 2008, and 2009. The extracts from the poppy straw were prepared using 5% acetic acid under sonication and then analyzed using liquid chromatography-MS. The overall results showed that the ratio of the alkaloids, morphine, codeine, narcotine, papaverine, and thebaine was highly variable in the selected 15 poppy cultivars, more than the difference found between the years [81].

Biological Activities of the Papaver Genus
Papaver forms part of the traditional system of medicines that plays an important role in providing health care to large section of the world population. Therefore, in this section, we discuss the updated snapshot of the bioactivities and therapeutic applications of Papaver genus, some of them related to its traditional use (Table 1).

Analgesic Activities.
Few studies have already recognized that the treatment addressed to the immune system modulates the analgesic effect of the opiates isolated from poppy plant. It seems that during illness, the inhibition of morphine analgesia is due not only to the offsetting of analgesia by enhanced pain sensitivity but the action of endogenous antianalgesic mechanisms can be implied. The role of N-methyl-D-aspartate and central opioid receptors was established by Johnston 7 Oxidative Medicine and Cellular Longevity and Westbrook [82], as well as the glial activation in the spinal cord. Other Papaver species have revealed some analgesia properties. Ibrar and group [83] reported the analgesic activity of the Papaver pavoninum C.A. Mey. extract. The study was completed on a mouse model, and the results demonstrated that plant extract significantly reduced pain in mice at all the three doses (50, 100, and 150 mg/kg body weight), as indicated by reduction in number of writhes as 36.91,  Oxidative Medicine and Cellular Longevity 57.01, and 68.39%, respectively. The reduction in pain was dose dependent; hence, the 150 mg/kg dose proved to be most effective than the standard analgesic drug. Similarly, the ethanolic extract from the aerial parts of Papaver libanoticum Boiss., an endemic plant to Lebanon, exhibited a potent dose-dependent analgesic activity, which involved activation of opioid receptors in the central nervous system. This activity could be attributed due to the presence of alkaloids, different to morphine or its derivatives, and phenolic compounds [178].
Alternatively, besides to have mild opioid activity [178], Shams et al. [84] tested the effect of the administration of a hydroalcoholic extract from P. rhoeas to mice to evaluate the analgesic tolerance induced by morphine (1-10 mg/kg) using the tail-flick method. The results indicated that the extract of P. rhoeas showed no effects on analgesia at 25-100 mg/kg. However, treated animals with different doses of the extract (25-100 mg/kg) before the administration of morphine were effective to decrease the analgesic tolerance promoted by morphine.

Cytotoxicity Studies and Anticancer Activity.
Several studies have shown that Papaver genus, including P. somniferum, P. rhoeas, Papaver lacerum Popov, and P. nudicaule, can provide anticancer compounds, but most studies were performed in vitro or in silico ( [85,86]; [179]; [87][88][89]), as shown in Table 3. Their efficacy depends again on the part and solvent used [88]. Moreover, among the studied compounds, alkaloids have shown anticancer properties [86,87]. Nonetheless, the most active alkaloids were berberine and macranthine; importantly, they demonstrated low toxicity against the Vero cell line, a noncancerous model. P. somniferum-based nanoparticles (PbO and Fe 2 O 3 ) have shown cytotoxicity in HepG2 cell lines in order to treat hepatic carcinoma [87]. PbO-based nanoparticles demonstrated higher cytotoxicity (~79% inhibition) owing to more penetration due to its smaller size as compared to Fe 2 O 3 nanoparticles (61% inhibition).
In another work, the chemical extracts from the petals of P. rhoeas have recently been tested for potential in the prevention of skin cancer. Sublethal UVB-mediated lesions at both DNA and RNA levels in human keratinocytes were observed, and thus, derived sunscreen based on the extracts of Papaver petals could be promising [90]. As commented before, petals can have phenolic compounds, other potential active compounds.
The lethality to brine shrimp can be applied as prescreen to existing cytotoxicity and antitumor assays [91]. In this context, other studies have tested Papaver extracts in brine shrimp eggs [91,92]. It was established that the most active extract was obtained from P. pavoninum whole plant extracted with ethanol (lethal concentration 50% or LC 50 = 2:54 μg/mL) compared to P. rhoeas seed extracts obtained with dichloromethane (LC 50 = 24 μg/mL) and methanol (LC 50 = 26 μg/mL) [83,92]. Since the latter LC 50 values were lower than 30 μg/mL, these extracts displayed significant cytotoxicity, according to Khalighi-Sigaroodi et al. [91], who tested extracts from other 23 plant species of the Leguminosae family.
Concerning in vivo studies, cytotoxicity has been mainly focused on concrete alkaloids and also the mechanisms of action studied in cancerous cell lines. Besides the aforementioned studies, the nonnarcotic alkaloids noscapine and papaverine have been found as potent anticancer agents against different human cancers such as breast, liver, bone, prostate, colorectal, and fibrosarcoma by inhibiting the cell proliferation, inducing apoptotic cell death, and causing cell cycle arrest [93].
Noscapine has been found to suppress the cell proliferation, migration, and invasion as well as also induce apoptosis. The supplementation of noscapine at the rate of 320 μM concentration to human skin cancer cell line (A-431) induced 80% cell death and induced the structural change in human serum albumin protein [94,95]. Noscapine also presents strong anticancer potential against human epithelial ovarian and prostate cancers via inducing apoptosis in a receptor-dependent but radical oxygen species-(ROS-) independent manner [96]. Noscapine has anticancer activity against two LNCaP and PC-3 human prostate cancer cell lines, but it was combined with paclitaxel. This combination produced significantly lowering the mRNA expression of Bcell CLL/lymphoma (Bcl-2) and increasing the mRNA expression of Bcl-2-associated X protein (Bax), and Bax/ Bcl-2 ratio, among other effects [97]. In this regard, the apoptosis of cancerous cells is regulated by the members of the Bcl-2 family (Bax, Bcl-2). Bcl-2 factors inhibit the apoptosis whereas Bax factors promote it; hence, the ratio of both the factors decides the fate of cancerous cells. Noscapine also improved its therapeutic anticancer potential in colon cancer SW480 cells through inducing apoptotic cell death by blocking the liver-intestine cadherin (CDH17) gene. It also shows a significant effect on the levels of proteins related to apoptosis (Cyt-c, Bax, Bcl-2, and Bcl-xL) [98]. In human SW480 colon cancer cells, noscapine markedly decreased the colony-forming ratio and cell viability, up-regulated the expression levels of cleaved-poly (ADP-ribose) polymerase and cleaved-caspase-3, inhibited cell proliferation, and promoted cell apoptosis [99]. Alternatively, another study proved that noscapine has been found effectively to inhibit proliferation and invasion of MG63 cell line by suppressing the phosphorylation of epidermal growth factor receptor (EGFR) gene and its downstream pathway [100].
There are also numerous studies on the anticancer effects of papaverine in cells. For example, papaverine exhibited anticancer activity on human glioblastoma (GBM) temozolomide (TMZ; as a first-line anticancer medicine)-sensitive U87MG and TMZ-resistant T98G cells via preventing tumor cell growth, suppressed cell migration, and significantly inhibited the cell proliferation. It was also reported that papaverine has a dose-dependent cytotoxic effect on human prostate cancer cells (PC-3) through inducing early and late apoptosis along with inducing sub-G1 cell cycle arrest, lowering the expression levels of Bcl-2 proteins, increasing the Bax protein levels, reducing the NF-κB levels, and downregulating the PI3K and phospho-Akt expression [101,102]. This observation is in line with Antonarakis et al. [103] who also reported other mechanisms such as an enhancement in the expression levels of Bax protein, the release of 9 Oxidative Medicine and Cellular Longevity Papaver alkaloids (amurine, armepavine, berberine, isocorydine, isothebaine, macranthine, mecambrine, mecambridine, narkotine, orientalidine, oripavine, salutaridine, and thebaine) In vitro study HeLa, and Vero cell lines (i) Berberine and macranthine were the most active alkaloids in all 13 compounds (ii) Dose-dependent studies were applied and revealed IC 50 values of 12.08 μg/mL (HeLa) and 71.14 μg/mL (Vero) for berberine, and 24.16 μg/mL (HeLa) and IC 50  10 Oxidative Medicine and Cellular Longevity cytochrome C into the cytoplasm, reduction in the expression levels of X-linked inhibitor of apoptosis protein, and induction of apoptosis. Papaverine was also found effective against hepatic carcinoma by inhibiting the telomerase through downregulation of telomerase reverse transcriptase in humans in HepG-2 cells [104]. Likewise, noscapine and papaverine have an anticancer effect on human MCF-7 and MDA-MB-231 cell lines via enhancing apoptosis, causing cell cycle at G2/M phase, and arresting cell cycle at G 0 / G 1 phase [105]. Moreover, papaverine in combination with lowfrequency ultrasound improved the blood-brain barrier, which is involved in the maintenance of brain homeostasis and compromised in brain tumors [106,107]. This combination was able to reduce the expression levels of zonula occluden-1, occludin, and claudin-5, enhancing the permeability of blood-tumor barrier. This can be a strategy for selective crossing this barrier by chemotherapeutic drugs [107]. Another in vivo study showed that papaverine also markedly delayed the tumor growth in a U87MG xenograft mouse model [108,109].
Besides the latter compounds, sanguinarine is another promising anticancer compound effective against a variety of multidrug-resistant cancers and combined with chemotherapeutic agents to synergistically enhance their sensitivity [110]. Also, berberine has shown anticancer potential in cells ( [87]; [179], among others, as Table 3 shows.

Antimicrobial Activity and Antiviral
Activities. The antimicrobial activity of several extracts from Papaver plants is shown in Table 4. Among these studies, P. somniferum seed extracts, containing alkaloids and phenolic compounds, among other components, have shown the highest antimicrobial activity for the methanol extract against Staphylococcus aureus and Aspergillus species [111], whereas the aqueous and ethanolic extracts against root rot fungi at 5% [112]. In another work, AMA of P. somniferum in nanosystem was evaluated when it was used for the green synthesis of nanoparticles based on lead oxide (PbO) and iron oxide (Fe 2 O 3 ). Both the nanoparticles resulted in effective antimicrobial activity against all the pathogenic microbial strains (Bacillus subtilis, Staphylococcus epidermidis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Fusarium solani, Aspergillus flavus, Aspergillus fumigates, and Aspergillus niger) in a dose-dependent manner (4 to 10 mg/mL concentration) [102]. However, Papaver-based fabrication of PbO nanoparticles resulted in higher antibacterial property due to its small size than Fe 2 O 3 -based nanoparticles.
In a comparison study performed by Ünsal and coworkers [113], the antimicrobial extracts obtained with various solvents from the aerial parts of P. argemone, P. dubium, P. rhoeas, and Papaver clavatum Boiss. & Hausskn. ex Boiss. were recently investigated. Among the solvent tested, P. dubium extracted by petroleum ether and diethyl ether showed a higher effectiveness against S. aureus, with a minimum inhibitory concentration (MIC) of 9.76 and 19.52 μg/mL, respectively, compared to chloroform, ethanol, and acetone. Even, lower values have been reported for the tertiary alkaloids obtained from the aerial parts of P. rhoeas when it was tested against six bacterial species (S. aureus, S. epidermidis, Escherichia coli, K. pneumoniae, P. aeruginosa, and Proteus mirabilis), and three Candida strains (C. albicans, C. parapsilosis, and C. tropicalis) were studied using a microbroth dilution method. In this study, the plant samples were collected from 11 different sites, obtaining the best antimicrobial activity against S. aureus and C. albicans with an MIC value of 1.22 and 2.42 μg/mL in the site with the higher content of roemerine alkaloid [64]. Additionally, Table 4 displays the antimicrobial activity of other Papaver species. Among them, the results of Papaver pseudocanescens M. Pop extracts as an antiviral agent seem promising [114].
In a similar way, the antiviral activities of active compounds of P. rhoeas pollen against influenza H1N1, H3N2, and H5N1 viruses have been evidenced. Total, six flavonoids, including kaempferol derivatives and luteolin, and one alkaloid, chelianthifoline, were isolated and revealed neuraminidase inhibitory activities, reducing the ability of the virus to spread. The concentration required for 50% inhibition (IC 50 ) ranged from 10.7 to 100.5 μM for H1N1, 25.6 to 143.2 μM for H3N2, and 12.6 to 151.1 μM for H5N1. Among all tested compounds, luteolin was found to be the most active [115]. The antimicrobial activity of nudicaulin derivatives (synthesized in vitro and in vivo in P. nudicaule) has also been evaluated, but only one derivative (17-methyl-5,7,11,3′,4′-penta-O-methylnudicaulin) was slightly active [86].

Antioxidant Activity.
The in vitro antioxidant activity of P. somniferum has been reported by using different methods, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2 ′ -azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and chelating assays [119] (Table 5). Zhang and coworkers from China described the antioxidant activity of the powdered poppy capsule extractive by using DPPH assay and its relationship with quantitative fingerprinting. Morphine and codeine were among the components that have a positive influence in this bioactivity [120]. This agreed with the results obtained by other authors [121]. Moreover, a recent study evaluating different parts of the plant suggests that   [122]. In another context, the antioxidant activity of PbO and Fe 2 O 3 nanoparticles synthesized using P. somniferum 13 Oxidative Medicine and Cellular Longevity was evaluated. Using free radical scavenging assay (FRS), total reducing power assay (TRP), and total antioxidant capacity assay (TAC), it was observed that both the nanoparticles of P. somniferum exhibited concentration-dependent activity. PbO nanoparticles revealed the significant antioxidant activity in terms of FRS (54%), TRP (16.8 mg ascorbic acid equivalents/mg), and TAC (106.1 mg ascorbic acid equivalents/mg) while Fe 2 O 3 nanoparticles showed 52% FRS activity, 16.8 mg ascorbic acid equivalents/mg TRP, and 131.1 ascorbic acid equivalents/mg TAC, respectively [102].
The antioxidant activity in vivo of P. somniferum has been also evaluated through the seed oil administered to rats, observing limited oxidative damage [123]. Concerning other species, the antioxidant activity of P. rhoeas has also been reported by three methods: DPPH, ABTS, and ferric reducing antioxidant power (FRAP) assays. The results clearly indicated that leaf extract demonstrated significant antioxidant activity with a half-maximal effective concentration (EC 50 ) 28.72 mg/100 g dry weight in DPPH, 185.29 mM Fe 2+ /100 g DW in FRAP, and 12.07 mM Trolox equivalents (TE)/100 g dry weight in ABTS [124]. The water extract of this plant has also been evaluated for antioxidant potential using DPPH and superoxide anions assays. The IC 50 value of P. rhoeas was 4.81 mg/mL for DPPH assay, and it was the highest antioxidant activity in the anti-ROS assay [125]. Moreover, the hydrophilic and lipophilic antioxidant activity of P. rhoeas was studied by using the TEAC assay. The leaves of wild P. rhoeas displayed the highest total antioxidant activity (1326 μmol TE/100 g fresh weight) among other assessed plant species, which correlated with their total phenolic and flavonoid content [126].
The antioxidant action of Papaver plants depends on the genotype as shown by Krošlák and coworkers. Their results suggested that there were differences in the antioxidant activity of P. somniferum seeds by using DPPH, ABTS, FRAP, and reducing power (RP) assays. The genotype major displayed the best antioxidant activity in all the assays, namely, DPPH (126.29), ABTS (31.05), FRAP (31.61), and RP (146.56) mg of TE. Alternatively, the genotype MS-423 showed the high inhibition against trypsin, thrombin, and collagenase enzymes [127]. Other important factor is the solvent used for extraction. The results by Selen Isbilir & Sagiroglu [20] indicated that water extract (WE) of P. rhoeas was the most effective compared to ethanol (EE), and acetone extracts (AE); total antioxidant activity of all the extracts was recorded to be 96.01% (WE), 94.98% (EE), and 89.07% (AE), respectively. The TRP of extracts was as follows: WE > EE > AE [20]. Other studies on antioxidant potential of Papaver genus are presented in Table 5, showing the selected solvent for extraction. Although it is difficult to compare all the solvents due to the use of different assays and units, it seems that the methanolic extract was higher in antioxidant activity compared to other solvents for the aerial parts of P. bracteatum.
From the aforementioned studies and Tables 4 and 5, it can be summarized that the bioactivity of the alkaloids derived from the Papaver genus depends on the extraction conditions, as the phytochemical composition. Extraction condition may include the type of the solvent, extraction time, temperature, and other input factors. In addition, Papaver alkaloids or phytochemical extracts demonstrated more effective bioactivities in the nanoforms. Smaller nanoparticles penetrate more easily into the bacterial membrane and dissociate into respective ions causing oxidative stress, membrane leakage, and killing bacterial cells with more perfection. The use of well-established alkaloids for the treatment of various ailments in the human body may be utilized as nanoformulation to enhance the efficacy of the drug. There is further need to develop the field of nanotechnology with respect to Papaver-based drug formulations. In addition, testing other isolated compounds is required to assess the antimicrobial activity and antioxidant potential and their contribution in order to select most active plant extracts.

Antidiabetic Activity.
It is well known that α-amylase and α-glucosidase are key enzymes for the catabolism of complex carbohydrates into glucose and thus target to explore antidiabetic drugs. In this sense, the α-glucosidase inhibitory activity of P. somniferum seeds (aqueous and ethanol extracts) was also demonstrated. It was found that both the extracts showed less than 5% inhibitory activity [132]. Also, the α-amylase enzyme inhibition activity using P. somniferum pod-based PbO and Fe 2 O 3 nanoparticles showed insignificant inhibition as 3% and 25%, respectively [102].
Apart from this, large number of researchers documented the antidiabetic potential of this genus in literature through traditional medicine knowledge [133][134][135][136]. Most of these studies referred to P. rhoeas and particularly to the seeds. For example, boiled seeds capsule of P. rhoeas were used by the communities with 22.85% frequency based on the information collected by 35 healers in Iran [37]. Alternatively, the antidiabetic effects of opium were low in experimental diabetic animals at an oral dose of 10 mg/kg body weight for 90 days, as reported by Ahmed and group. Although opium increased serum insulin and decreased serum glucose, the effect was not significant; this was due to metabolic disorders in diabetic animals. In addition, it is suggested that opium consumption in diabetic patient is not useful [137]. Similarly, Sadeghian et al. [138] reported the effects of available opium substance on glucose and lipid metabolism in streptozotocin-induced-diabetic rats by testing opium contained in the juice of the seed capsule of the P. somnniferum. The test rats were treated with normal opium (20 mg), starting on the fifth day after induction of diabetes for 30 days. The results demonstrated that glycaemia levels in the rats treated with opium (544.8 mg/dl) were similar to the levels determined in the control rats (524.6 mg/dL). In addition, the level of other parameters was similar: serum, total cholesterol, high-density lipoprotein, and triglyceride. Indeed, more studies are needed to clarify the role of Papaver, specifically, P. rhoeas, in the antidiabetic action and the active chemical components. 5.6. Properties in Fertility. The role of P. rhoeas extract (dried petals macerated with 50% ethanol) on fertility has also been investigated in mouse oocytes [139]. The cumulus-oocyte complexes were cultured in a maturation 14 Oxidative Medicine and Cellular Longevity medium supplemented with different concentrations (low: 10-25 μg/mL; high: 50-200 μg/mL) of P. rhoeas extract. Low concentrations of extract showed moderate effects; however, higher concentration (100 μg/mL) significantly improved the rate of oocyte maturation and embryo development in mouse oocyte maturation medium [139]. In another study, similar findings were obtained while working on sheep oocytes [140]. The results demonstrated that plant extract displayed dose-dependent activity in a maturation medium. The concentration of 50 μg/mL was effective and improved the sheep oocyte maturation rate when the extract was supplemented in a maturation medium [140]. Flavonoids, including anthocyanins, have been associated with these effects, which can protect intracellular glutathione levels in oocytes [141].

Neurological/Mental
Effects. Supplementation of P. rhoeas hydroalcoholic extract reduced depression and increased the neurotransmitters involved in depression, including dopamine, serotonin, and norepinephrine [142]. Depression is also linked with stress and increases glucocorticoid secretion into the blood. Nonetheless, the administration of a hydroalcoholic extract of P. rhoeas (15-60 mg/kg in male mice) enhanced the secretion of glucocorticoids, but it could reduce the side effects of stress [143]. In other work, P. rhoeas distillate decreased anorexia and improved learning ability, but it again increased the corticosterone levels [144]. These positive neurological/mental effects agreed with recent results that suggest that P. rhoeas hydroalcoholic extract has a reducing effect on depression in mice after short-term administration. In this sense, the antidepressant effect of P. rhoeas may not be due to the inhibition of the hypothalamic-pituitary-adrenal stress system, while it could be caused, at least in part, by the inhibition of glutamate or through antiopioid and anticholinergic effects [145].
Furthermore, sedative effects of P. rhoeas aqueous and alcoholic extracts have also been observed, being more marked when 10% ethanol was used as solvent for extraction [71].

Other
Bioactivities. The antiulcerogenic activity of P. rhoeas root extract was assessed by using the ethanolinduced ulcerogenesis model in rats. The plant extract (670 mg/kg) exhibited statistically significant (95.6%) gastroprotective effects. In addition, histopathological studies confirmed the positive results of the extract in vivo [146]. The anti-inflammatory activity of extracts from P. nudicaule aerial parts and its mode of action in RAW264.7 macrophage cells have been also tested. Interestingly, in this work, the aerial parts were selected according to different colors (white, orange, yellow, scarlet, and pink) and under two different growth stages (after 60 and 90 days). All of the extracts of P. nudicaule displayed significant effects in reducing lipopolysaccharide-(LPS-) induced nitric oxide; the white flower extract-90 showed the best results. This extract also decreased the LPS-induced nitric oxide synthase 2 and cyclooxygenase 2. It inhibited the LPS-induced activation of nuclear factor-κB and signal transducer and the activator of transcription 3 signalling pathway [147]. As commented before, the phenolic composition depends on the color and the white ones have petals rich in kaempferol glycosides, but with a lack of pelargonidin glycosides and nudicaulins [73]. Moreover, P. rhoeas extracts prevented pain and inflammation due to their potential activity on opioid, glutamate, and nitric oxide systems, as well as elevated the plasma corticosterone concentration [148]. Furthermore, P. somniferum seeds have inhibitory activities against trypsin, thrombin, and collagenase, suggesting more vast pharmacological possibilities [127].

Safety and Adverse Effects
An undesired harmful effect resulting from a medication or other intervention such as surgery is known as an adverse effect. It may be termed a "side effect," when considered to be secondary to the therapeutic effect [149]. In contrast, dangerous, unintended reactions of medicines that occur at doses normally used for treatment are called adverse drug reactions (ADRs), even can lead to death in many countries [149]. In the case of morphine alkaloids, the pharmacologic properties of these compounds differ widely and their medicinal applications have some safety and adverse effects [150].
Various wanted and unwanted effects of opium consumption are discussed in the encyclopedia Canon of Medicine by Avicenna (980-1037 AD). Avicenna has mentioned on the mechanism of opioid-related respiratory depression, due to respiratory muscle spasm for respiratory failure. Similarly, it is mentioned in Canon of Medicine that opium can cause abnormal and difficult breathing, which can lead to death. A respiratory suppression side effect was observed with patients suffering from fever associated with tuberculosis due to the use of the topical opioid application on the chest. Constipation and painful bowel obstruction were other adverse effects of opium-based [177]. Avicenna has also mentioned poisoning, sluggishness, sedation, and abdominal contractions. Opium has highly addictive qualities and is reported to cause memory and reasoning dysfunction [177].
Remarkably, modern studies have confirmed the adverse effect of morphine alkaloids described by Avicenna [151][152][153]. Some of them are related to the binding to μand κ-opioid receptors, the accumulation of neuroexcitatory opioid metabolites, etc. One of the side effects of opioidbased pain relievers, including morphine and its derivatives, is severe constipation [154]. Kohberg et al. [155] and Rocker et al. [156] also reported constipation as the most frequent adverse effect. Moreover, other effects of morphine are on the CNS mediated by its high affinity to the μ-opioid receptor, such as nausea, vomiting, sedation, euphoria, miosis, respiratory depression, drowsiness, and obstipation [157]. Additional adverse effects are endocrinopathies and sleep disorders. Furthermore, long-term use of opioid can lead opioid tolerance (increased dose needed for analgesia) and hyperalgesia (paradoxical increase in pain with opioid administration) that involves μ-opioid receptor signalling pathways [158][159][160]. Wound healing can also be delayed by chronic morphine intake by inhibiting immune cell recruitment followed by wounding [161].
As codeine is a precursor of morphine, they share some pharmacological features with also direct activity at the opioid receptors, but the former has much lower potency. The most frequent side effects of codeine are constipation and nausea, and addition potential. Nevertheless, in some paediatric patients, the genotype predisposing to ultrarapid metabolism of codeine into morphine by the isoenzyme CYP2D6 can occur [157]. Codeine and morphine can be distributed into breast milk with complications for breastfed infants of mothers receiving codeine [162], even a case of severe neonatal toxicity in a breastfed infant has been reported [163]. In the case of noscapine, it is used as a centrally acting antitussive compound and no toxicological properties have been characterized, but it can present headache and dizziness [157].
Poppy seeds from P. somniferum are commercially available in some countries and widely used as ingredients for various kinds of food, especially in Eastern Europe [164]. Poppy seeds for food uses are generally obtained from cultivars bred to accumulate lower amounts of opium alkaloids [56] and normally contain low levels (2-251 μg/g of morphine and 0.4-57 μg/g of codeine) [165]. The opioid concentrations come primarily from the alkaloid residue retained on the seeds [166]. Therefore, although the consumption of poppy seeds in foods is really in small amounts, EFSA set a general safe level of 10 μg per kilogram of body weight based on the morphine content of poppy seeds [157]. In this sense, only a rare case of death has been published consuming between 64 and 587 times the volume of poppy seeds (around 900 g). This extremely high ingest led to death due to complications of a bowel obstruction, but it did not cause lethal opiate toxicity [165].
Alternatively, extracts or infusions concentrated in opium alkaloids from poppy seeds can have adverse effects [56,165], but there are few reports on this topic [166]. In any case, some authors have attempted the reduction of the content of opium alkaloids in the seeds using different treatments. For example, while the levels of opium alkaloids were not affected by baking or steam application, a high reduction of these compounds can be obtained by water washing or extended thermal treatment [56].
In another context, immunoglobulin E-mediated sensitization to poppy seeds is rare, but if it occurs, the clinical symptoms can be severe, e.g., due to cross-sensitizations events [167,168].
Concerning other Papaver species, some case studies in humans suggest that unconscious ingestion of P. rhoeas can cause acute liver toxicity [169] and intoxication with different effects (nausea, restlessness, dyspnoea, contractions unconsciousness, numbness, etc.) [170]. Alternatively, an in vivo study performed by Soulimani and coworkers [71] suggests that extracts from P. rhoeas petals (without the presence of alkaloids) showed a lethal dose (LD 50 ) of 4000 mg/kg and thus very low toxicity. However, sedative effects were observed. A study in vitro showed that P. rhoeas leaf extract also showed promising antimutagen/anticlastogen activity [171] and thus suggesting low toxicity. Therefore, although Papaver extracts can have some beneficial effects, toxicity studies are further required to establish dosage and side effects. Nonetheless, the culinary use of some parts and Papaver plants indicates that the safety issues are controversial or the dosage is a prerequisite. This includes P. somniferum seeds, with the aforementioned exceptions [56,165]; the shoots of P. rhoeas, the aerial parts of this species, and Papaver strictum Boiss. & Balansa are added to salads, minestra, etc. [32,35,172]. Additionally, in Turkey, poppy flowers are used as food colorant and for enhancing the flavour of herbal teas [64].

Clinical Trials
Besides the aforementioned case reports studies, there are a very limited number of clinical studies reporting the health 16 Oxidative Medicine and Cellular Longevity beneficial effects of Papaver plants, as far as we know. One of them tested the iodized poppy-seed oil as vehicle of the drug epirubicin against hepatocellular carcinoma [173,174], but the anticancer effects of poppy have not been evidenced in humans. In the ClinicalTrials.gov database, there are two studies based on the administration of California poppy (Eschscholzia californica Cham.) (NCT03364101) but only one refers to the Papaver genus. In this work, ground poppy seeds were baked into a bran muffin and administered to evaluate the effect on postprandial blood glucose response, vascular, appetite, and sensory parameters (NCT01579656), but the results have still not been posted. Furthermore, a recent study on P. rhoeas combined with other herbs in syrup has improved sexual experience of men following consumption of this mixture with no drug-related serious adverse events. Therefore, the authors suggest that this aphrodisiac syrup can be applied alternatively to other chemical sexual drive enhancers with complicated side effects [175].

Conclusions and Future Perspectives
Besides the pharmacological interest of P. somniferum, the traditional use of different Papaver plants is widely established in different cultures and countries. This fact makes this genus attractive as a source of pharmacoactive extracts and compounds (alkaloids, phenolic compounds, and essential oil). These compounds are responsible for the multifaceted biological activities of the Papaver genus including anticancer, antioxidant, antimicrobial, and analgesic. The finding from different studies also demonstrated that these useful compounds are present throughout the plant including agro-residue generated from the Papaver plants. Nonetheless, pharmacological studies on extracts from these plants should be reinforced with characterization studies to know the active molecules, or if synergism exists that makes more interesting the use of the whole extracts. For that, bioassay-guided fractionation or even chemometrics with MS-based methodologies and HPLC with MS/MS can be applied to identify the overall profile of the Papaver plant extracts. This is especially important since the phytochemical composition and content as well as the bioactivity depend on several factors, including the genotype, the growth stage, and even the color of the flower. The phytochemical profile also depends on the method of extraction, input factors used for the extraction, and also on the style of preparation of the sample for analysis. Moreover, little is known about the bioactivity of the essential oil from these plants, even though some authors suggest the presence of phytol. This compound is valuable as a fragrance and exhibits a broad range of bioactivities [80]. Moreover, applications of this genus in nanotechnology seem promising, for example, to synthesize nanoparticles for different pharmacological purposes but further work is required, including more toxicity studies. In this sense, the use of plant extracts is increasing in green synthesis and the type of compounds present on these extracts can modulate the nanoparticle shape (Vijayaraghavan et al., [176]) and probably its functionality. Finally, although some preclinical results are promising, more clinical studies are needed to provide scientific evidence of the traditional use of Papaver plants before consumption and to avoid intoxication events. Overall, these studies along with a better known of the active molecules through comprehensive characterization and bioguided fractionation studies should be undertaken in future research.

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