Journal of Medicinal Plants Application of Conventional and New Biotechnological Approaches for Improving of Morphinane Alkaloids Production 1 2 3 Dehghan E (Ph.D. student) *, Hosseini B (Ph.D.) , Naghdi1 Badi H (Ph.D.) , Shahriari Ahmadi F (Ph.D.) 1- Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran 2- Department of Horticulture, Faculty of Agriculture, Urmia University, Urmia, Iran 3- Iranian Academic Center for Education, Culture & Research (ACECR), Institute of Medicinal Plants Research, Karaj, Iran *Corresponding author: Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran Tel: +98-511- 8796818-20, Fax: +98-511- 8787430 Email: [email protected] Receive: 4 Apr. 2010 Acceptance: 30 Aug. 2010 Abstract More than 12,000 alkaloids are known in plants, mostly used as medicine with a world market value of about 4 billion US$. Opium poppy, Papaver somniferum, is the most important economic source of morphinane alkaloids such as morphine, codeine, thebaine, narcotine and papaverine that are exploited by the pharmaceutical industry as analgesics, antitussives and anti-spasmodics. With regard to increasing demand for these compounds, the aim of this review is presenting an outlook of classical breeding programs that successfully applied for enhancing the alkaloid content of opium poppy. The latest biotechnological approaches also are discussed to give an outlook for future trends and possibilities. Keywords: Papaver species, Biotechnological methods, Conventional breeding, Morphinane Alkaloids, Tissue culture, Metabolic engineering Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 Volume 9, No. 35, Summer 2010 33 Application of … Introduction and derivatives are the most important Poppy seems to be one of the few species alkaloids produced by the opium poppy. which was utilized, even as cultivated plant, in Morphine has long occupied an eminent prehistoric times. There is no doubt about the position on the list of useful drugs. Morphine evidence that the poppy was also cultivated by and codeine are prescribed analgesics and Sumerians, Babylonians and Assyrians about cough-suppressing drugs [5]. Morphine exerts 3–6 thousand years BC [1]. The Opium poppy its effects either by hyperpolarizing or Papaver somniferum (Fig. 1) is a multipurpose inhibiting postsynaptic neurons, probably by + 2+ crop which is used as a medicinal or increasing K efflux, or by reducing Ca ornamental plant, as well as a source for seeds influx into presynaptic nerve endings and and seed oil. Opium, as dried latex of unripe thereby reducing transmitter release, including capsules of P. somniferum, contains more than acetylcholine, norepinephrine, dopamine, 80 isoquinoline alkaloids. The main alkaloids serotonin and substance P [6]. Morphine is derived in opium are morphine (4-21%), metabolized into morphine-3-glucuronide and followed by codeine, thebaine, papaverine, morphine 6-glucuronide (M6G) in the human noscapine, and narceine [2]. The Persian body; which the later one possesses analgesic genius Ebne Sina, known in the West as activity. Avicenna (980-1037), who was revered as the Other alkaloids from poppy species have "prince and chief of physicians" and "the various uses: noscapine has antitussive and second teacher after Aristotle," recommended antitumorogenic properties; papaverine is a opium and plants of the nightshade family as vasodilator and smooth muscle relaxant; analgesics and anaesthetics [3, 4]. The sanguinarine is antimicrobial and anti- morphinanes (5-ring benzylisoquinoline inflammatory [5, 7]. alkaloids) include morphine, codeine, thebaine Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 Fig. 1- Opium poppy, P. somniferum and different parts of its vegetative and generative organs [8] Journal of Medicinal Plants, Volume 9, 34 No. 35, Summer 2010 Dehghan & Authors The opiates are industrial commodities of protopines, pavines/isopavines, phthalideisoq- plant origin for which there is still uinolines, protoberberines, the true considerable demand. Globally, opium poppies benzylisoquinolines, the aporphines, the are legally cultivated on around 40,000 ha benzophenanthridines and the morphinanes annually. The major supplier of opium poppies [14]. Common to all the members of these is Tasmania, supplying around 40% of the classes are the first steps in the biosynthetic market. GlaxoSmithKline, Tasmanian pathway that lead to the central intermediate Alkaloids and Johnson-Matthey operate fully (S)-reticuline (Fig. 2). integrated supply chains for opiate production. The presence of morphinane alkaloids Levels of production are controlled by the UN (thebaine, codeine and morphine), together Single Convention on Narcotic Drugs, which with the secophthalidisoquinoline alkaloids limits production to reasonable saleable (narceine, nornarceine, narceinimide), quantities with allowance made for phthalidisoquinoline alkaloids (narcotine, contingency stocks [9]. narcotoline), benzyltetrahydroisoquinoline In the last few decades, the demand for alkaloids (reticuline, laudanosine, codamine, poppy-derived alkaloids has raised tetrahydropapaverine) and aromatic continuously, in particular the need for benzylisoquinoline alkaloids (papaverine, thebaine, which has increased by ~ 67% in the pacodine) is characteristic of Papaver last 6 years [10]. somniferum L. The carbon skeleton of benzy- With five centers of chirality, the structures lisoquinoline alkaloids is derived from two of morphinane alkaloids present a complexity molecules of tyrosine [15, 16]. The aromatic that renders commercial synthesis uneconomic amino acids, phenylalanine, tyrosine and [11] and cultivation of poppy, in spite of some tryptophan are formed via the shikimate pathway. limitation, continues to be the most effective The availability of tyrosine for alkaloid means to produce opiate analgesics [12]. biosynthetic pathways is an important Higher alkaloid content in the poppy crop determinant of the endogenous level of alkaloids. would enhance the financial return to growers The biosynthesis of benzylisoquinolines and make the industry more competitive [13]. (BIAs) starts with the condensation of two For these purposes, different efforts by tyrosine derivatives leading to the first conventional and new biotechnological tetrahydrobenzylisoquinoline norcoclaurine Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 methods have been applied to enhance alkaloid [17, 18]. Subsequent reactions include the content of P. somniferum. Thus in this methylation at position 6 of norcoclaurine by communication we will try to present an norcoclaurine 6-Omethyltransferase (6-OMT), overview of morphinanes biosynthesis in the methylation of the nitrogen by coclaurine opium poppy and different aspects of genetics, N-methyltransferase (CNMT), and the breeding, tissue culture and metabolic hydroxylation at the 3′-position by the P450 engineering of these valuable compounds. monooxygenase (S)-N-methylcoclaurine 3′-hydroxylase (Cyp80B1). One additional Benzylisoquinoline alkaloids and biosyn- methylation at the 4′position by 3′-hydroxy-N- thesis of morphinanes methylcoclaurine 4′-O-methyltransferase The benzylisoquinoline alkaloids comprise (4′-OMT) finally leads to (S)-reticuline. From a group of about 2,500 compounds, which can this central intermediate the pathway be divided into nine classes: the rhoeadines, 35 Application of … Fig. 2- Benzylisoquinoline biosynthesis pathway. NCS (norcoclaurine synthase); 6-OMT (O-methyl transferase); NMT (N-methyltransferase); CPY80B3 (cytochrome P450); 4OMT (O-methyl transferase) BBE Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 (berberine bridge enzyme); STS (salutaridinol synthase); SAT (salutidarinol-7-O-acetyltransferase), COR (codeine reductase) [19] bifurcates into the different benzylisoquinoline A series of at least 17 enzymatic steps classes and a multitude of rearrangements and contributed in the biosynthesis of morphine. modifications of the benzylisoquinoline The latter steps in the pathway that lead backbone takes place leading to the high specifically from (S)-reticuline, a central structural diversity of these alkaloids. intermediate of isoquinoline alkaloid Interestingly, whereas the biosynthesis of all biosynthesis, to morphine (Fig. 2) involve classes of benzylisoquinolines starts directly three NADPH-dependent oxidoreductases from (S)-reticuline, the conversion of [11], most probably three cytochromes P-450, stereochemistry to (R)-reticuline is a and an acetyl-CoA-dependent prerequisite for the production of morphinans. acetyltransferase [11, 19]. Journal of Medicinal Plants, Volume 9, 36 No. 35, Summer 2010 Dehghan & Authors Although more than 60 species or lower 100 species and is affiliated to the section taxonomic units of the genus Papaver have Mecones comprising five species, among been studied, the presence of morphine has which Papaver setigerum (2n=44) is a close been only detected in two species from the relative and probably the ancestor of the section of Mecones , i.e. P. somniferum L. and opium poppy [28]. Papaver somniferum is P. setigerum DC. The presence of morphine considered to be a predominantly self- has been also demonstrated in other plants, pollinating species with various rates of out- such as hay and lettuce and recently detected crossing depending upon variety and in mammalian tissues. Since the isolation of