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Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions

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  • Macleaya cordata, a medicinal plant in the Papaveraceae family, is rich in bioactive benzylisoquinoline alkaloids. Recent research has elucidated the mechanisms by which these active components promote livestock and poultry growth and exhibit anti-inflammatory effects on the intestines. These findings have led to the development of two raw medicinal materials and two veterinary drug formulations, widely used in China's livestock industry. Advances in multi-omics technologies, such as whole-genome sequencing and transcriptomics, have clarified the chemical composition of alkaloids and the biosynthesis of sanguinarine, enabling its de novo synthesis in yeast. Efforts in plant breeding have focused on cultivar selection and germplasm innovation, establishing DUS (Distinctness, Uniformity, and Stability) testing guidelines. Genetic engineering techniques have re-edited the sanguinarine pathway and induced hairy roots and cell suspension cultures in M. cordata. These advancements reduce production costs, ensure product stability, and promote sustainable production. This paper reviews the species origin, current research status, and prospects of M. cordata, offering guidance for further research on this valuable resource.
  • Traditionally, fish is considered an affordable source of protein of high biological value, including high levels of essential amino acids (lysine, methionine, etc.), lipid-soluble vitamins, minerals (Se, P, Fe, Mg, and K), and rich in highly unsaturated fatty acids (ω3, 6, and 9), such as docosahexaenoic and eicosapentaenoic acid[1, 2]. The lipid fraction and other bioactive components present in fish, have attracted a great deal of attention because of their favorable effects on human health[3], including reducing the risk related to human cardiovascular and chronic neurodegenerative diseases. Accordingly, the World Health Organization and American Heart Association have also recommended consuming 1−2 servings of fish weekly regularly. Nevertheless, quality preservation of fish and fish products is challenging due to their high perishability. Further, due to high water and non-protein nitrogen content, the freshness of post-mortem fish muscle rapidly declines. The deterioration in quality of fish due to spoilage by microorganisms[4]. Further microbial spoilage leads to lipid oxidation resulting deterioration of different quality aspects of fish. In general, fish spoilage is governed by three basic mechanisms namely enzymatic autolysis, microbial growth and lipid peroxidation. All three, in a favourable condition continue simultaneously in a food substrate and after a certain interval, the evidence of spoilage could be noticed.

    Spoilage is considered as one of the major concerns of fish food safety which may cause several negative effects on the health of the consumers. For this reason, commonly, one or more preservation technique(s) is employed to extend their shelf life, with the purpose to prevent and or delay quality changes[5]. Traditional preservation methods, including drying by different means such as salting, smoking, fermentation, etc., low-temperature storage (chilling, freezing), or with chemical preservatives, have been commonly used in the industry. Nowadays, non-thermal physical technologies (pulsed electric fields-PEF, high hydrostatic pressure-HPP, ionizing radiation ultrasonication, cold plasma, and innovative packaging systems) are also being employed for the same purposes[6]. Likewise, PEF and HHP treatments may not ensure food safety, as these can only cause sublethal damage to the bacterial cell wall[7]. Beside processing strategies, various organic acids and their different salts (such as sorbic acid, benzoic acid, propionic acid, sodium benzoates, propionates, potassium sorbates, nitrites, ascorbic acid, citric acid, etc.), and synthetic phenolic compounds such as butylated hydroxyl toluene (BHT), butylated hydroxyl anisole (BHA), tert-butylated hydroquinone (TBHQ), dodecyl gallate are used as food preservatives[8]. Incorporating synthetic antioxidants viz., BHT, BHA, TBHQ at high doses, either to the raw materials or end-products can negatively affect human health[9]. Prolonged use of these synthetic preservatives and compounds are reported to induce cancer, liver and kidney damage, gastrointestinal disorders, asthma, and many allergies[10].

    With increased concerns and negative perceptions among consumers regarding the safety aspects of chemical preservatives and synthetic compounds, the demand for minimally processed ready-to-eat fish products has increased many-fold. To cater to the demand of consumers and to ensure the availability of safe, nutritious, tasty, and convenient food, natural alternatives, such as those involving phytochemicals/phytoextracts, including essential oils, phenolics etc. derived from plants have gained lots of interest. Plants and their parts harbor a complex mixture of bioactive compounds that are a good source of phytochemicals such as polyphenols, phytosterols, alkaloids, nitrogen-containing compounds, terpenoids, organosulfur compounds etc. These compounds possess inherent biological effects such as antimicrobial, antioxidant, antidiabetic, anti-inflammatory, immune-enhancing functions etc. that are reported to offer health benefits[1113] and form the major basis of Ayurveda, Unani, Siddha and the Chinese system of medicine since antiquity.

    As the focus of the processing industry is to maintain the organoleptic and nutritional characteristics and ensure the quality and safety of food products, the phytoextracts/phytochemicals derived from plants are gaining increasing importance nowadays. This paper aims to provide an overview of the recent applications of phytoextracts/phytochemicals for shelf-life extension and nutritional and organoleptic quality and sensory improvement of the fish, and its products.

    Phytochemicals, also called green chemicals, produced by plants through primary or secondary metabolism, are gaining attraction as healthier alternatives to synthetic antioxidants and antimicrobials[14]. The phytochemicals are extracted from various plant parts (leaves, shrubs, seeds, flowers, fruits, bark etc.). They also can be recovered from the residues and leftovers of fruits and vegetables (peels, pulp) generated during harvesting and processing[15] and thus contribute to waste valorization and circular economy[11]. Generally, based on their chemical structure and characteristics, the secondary metabolic products of plants are classified into phenolics, terpenoids, carbohydrates, phytosterols, alkaloids and other nitrogen-containing compounds[1113]. Phenolics are the largest category of phytochemicals being structurally diverse and abundantly distributed in the plant kingdom. They can be divided into phenolic acids (hydroxybenzoic acids − e.g. gallic, ellagic, vanillic acids etc.; hydroxycinnamic acids − e.g. caffeic, chlorogenic, cinnamic, ferulic etc.), flavonoids (flavones, flavonols, flavan-3-ols, isoflavones, anthocyanidins, anthocyanins etc.) and other phenolics (tannins, stilbenes, lignans, xanthones, lignins, chromones etc.)[16]. Among flavonoids, flavonols like quercetic, rutin, myricetin, kaempferol etc. and flavon-3-ols like catechin, epicatechin etc. are common. Apart from flavonols and flavones, anthocyanins and anthocyanidins are major flavonoids widely available in various fruits and vegetables such as grapes, apples, plums, cabbage, purple corn, and different varieties of berries like elderberry, blueberry, blackberry, elderberry, etc. Popular bioactive compounds among anthocyanins and anthocyanidins are cyanidin, malvidin, delphinidin, peonidin, petunidin, pelargonidin, etc.[11].

    Terpenes, also known as terpenoids, exhibit diverse biological and pharmacological properties which are beneficial to humans. Based on the number of C5 isoprene unit, terpenes are grouped as hemiterpenes (prenol and isovaleric acid), monoterpenes (geraniol and limonene), sesquiterpenes (farnesol), diterpenes (quinogolides and taxadiene), sesterterpenes, triterpenes (squalene), tetraterpenes and ployterpenes[17]. Carotenoids such as lycopene, phytoene, phytofluene, lutein, zeaxanthin, β-cryptoxanthin, astaxanthin etc. are tetraterpenes reported to have many biological functions. These terpenoids form the major constituents of various essential oils.

    Essential oils (EOs) obtained from various parts of plants (leaves, barks, stems, roots, flowers, and fruits) are complex mixtures of numerous individual aromatic volatile compounds that can act as defense mechanisms against microorganisms[18]. These volatile compounds belong to various chemical classes: alcohols, ethers or oxides, aldehydes, ketones, esters, amines, amides, phenols, heterocycles, and mainly the terpenes. Thousands of compounds belonging to the family of terpenes have so far been characterized and identified in essential oils[19], such as functionalized derivatives of alcohols (α-bisabolol), ketones (menthone, p-vetivone) aldehydes (citronellal, sinensal), esters (γ-tepinyl acetate, cedryl acetate), and phenols (thymol). Interestingly, EOs also contain non-terpenic compounds which are bio-generated by the phenylpropanoids pathway, like eugenol, cinnamaldehyde, and safrole[20]. Figure 1 illustrates the different kinds of natural bioactive compounds extracted from various plant components.

    Figure 1.  Schematic diagram of different components in phytochemicals.

    After harvest, different activities namely oxidative and enzymatic autolysis in fish cause deteriorative changes in sensory and nutritional value. These changes not only cause the loss of freshness as perceived by consumers but also limit the shelf-life during storage. Again above deteriorative changes with loss of freshness are mainly due to lipid oxidative products and protein degradation, which accelerate the undesirable changes in color, flavor and texture in fish. A wide range of plant extracts rich in polyphenols have been used for the preservation of fish and fish products, because of their antioxidant and antimicrobial activities. These extracts are applied either as dip treatment or as a coating in raw, chilled, and frozen stored products[21]. Various packaging methods viz. vacuum packaging and modified atmospheric packaging (MAP) are employed for extending the shelf life during the storage and retailing of fish and fish products. Different plant extracts are also being incorporated into ice to enhance the quality attributes of fish during storage[22]. In a recent study, the application of oregano essential oil (OEO) vapors under vacuum conditions, immediately before packaging, has been reported to be more effective than conventional dipping and topical application in maintaining the freshness and quality of fish products[23]. The effect of various phytochemicals on the quality aspects of fish and fish products is depicted in a schematic diagram shown in Fig. 2.

    Figure 2.  Schematic diagram showing the effect of various phytochemicals on quality aspects of fish and fish products.

    Physicochemical properties like pH, water holding capacity, emulsion stability, cooking yield, etc., play important roles in case of emulsion-based muscle foods, including fish products. Amongst these, pH and water holding capacity are regarded as critical quality parameters in muscle food products due to their closest adherence to texture, cooking loss, juiciness, tenderness, and microbial quality of the products. Several reports on phytoextracts and their effects on the pH, water holding capacity, yield, total volatile basic nitrogen (TVB-N), trimethylamine (TMA-N) etc. of fish and fish-based products are available in the literature (Table 1).

    Table 1.  Effect of phytochemicals as bioactive compounds on physicochemical, microbiological and sensory quality of fish and fish products.
    Fish and fish productsPhytochemical usedParameters studied Key findingsReferences
    Salted sardines
    (Sardina pilchardus)
    Lemon essential oil (EO) micro-emulsion at 3 and 10 g/kgChemical, microbiological and sensory parameters of salted sardines during the entire period of ripening (150 d)• Retarded the growth of Enterobacteriaceae by 0.95 CFU/g, Staphylococci by 0.59 CFU/g, and rod lactic acid bacteria by 1.5 log cycles
    • Lowered the accumulation of histamine
    • Registered highest scores for flavor and overall acceptability
    [46]
    Common carp (Cyprinus carpio) filletsCinnamon essential oil (1 g/kg)Physicochemical, spoilage microbes and sensory attributes of fillets stored at 4 ± 1 °C for 14 d• Decreased the relative abundance of Macrococcus (51.8% vs 33.4%)
    • Effective in inhibiting the increase of TVB-N and the accumulation of biogenic amines.
    • Extended the shelf life of vacuum-packed fillets
    [40]
    Fillets of Sardinella longiceps and Rastrelliger kanagurtaColeus aromaticus and Sargassum wightii leaf pasteProximate, microbiological, and sensory characters of fillets under chilled storage (4 ± 1ºC) conditions for 7 d• Significantly (P ˂ 0.05) improved proximate parameters with reduced moisture and TVC content as compared to control
    • Treated fillets had the best appearance, smell, color, texture, and taste compared to control
    S. wightii proved to be a better preservative than C. aromaticus
    [27]
    Smoked tilapia
    (Oreochromis niloticus) fish
    Ginger, garlic and clove powder
    (5 g/kg)
    Microbial activity, shelf life and safety of fish during 8 weeks of storage• Preservatives treated samples had reduced microbial load, and longer shelf-life (8 weeks)
    • Treated samples recorded zero/no total coliform count (TCC) growth and were accepted by the consumers
    [47]
    Frozen rainbow trout filletsClove oil (5 and 10 g/kg) used as natural preservative
    Microbiological and sensory quality of frozen and vacuum-packed rainbow trout fillets stored at -18 °C for six months
    • Microbial growth was high for frozen storage for control samples
    • Samples with clove oil had longer shelf life than normal
    • Clove oil can be used as a natural protective and influential antibacterial in conjunction with a vacuum pack to augment the quality
    [64]
    Founder (Paralichthys orbignyanus) filletsFillets packed in agar film with fish protein hydrolysate
    (FPH) (500g FPH/ kg agar) or with film containing clove EO (500 g EO/kg agar)
    Microbiological quality and shelf life of fillets stored at 5 °C for 15 d• Fillets packaged with film containing clove EO had better microbiological quality than packaged in agar film with FPH
    • Both the films effective in extending the shelf-life of fillets
    [65]
    Fish surimi from
    O. niloticus
    Treating of surimi gel by immersion with colored plant extracts- CPEs
    (2.5 g/L)- Hibiscus sabdariffa calyces, Curcuma longa rhizomes, and Rhus coriaria fruits
    Microbiological and sensory attributes of samples aseptically packaged into polyethylene bags and stored at 4 °C for 7 d
    H. sabdariffa extract was the most effective antimicrobial
    • CPEs enhanced sensorial attributes of surimi during storage study
    • CPEs application as colorants and antibacterial and quality enhancing agents recommended for biopreservation of seafood
    [54]
    Fish patties from Hake fillets (Merluccius capensis, Merluccius paradoxus)Extract from pomegranate peel, rosemary, citric, and hydroxytyrosol (obtained from vegetable waters of olive tree)
    (@ 0.2 g/kg)
    Physicochemical, microbiological properties, sensory analysis, and shelf life of patties under chilled storage for 14 d• Patties with rosemary extract had a high level of protein (140 g/kg),
    α-linolenic acid (up to 400 g/kg), selenium minerals, and low fat
    (< 20 g/kg) compared to the control
    • Extracts effective against protein oxidation of patties than commercial preservatives added to the control
    • Patties with pomegranate extract had a longer life (7 to 11 d) than others (4–6 d)
    [28]
    Grass carp (Ctenopharyngodon idellus) filletsTreatment with essential oils (EOs)- oregano (Origanum vulgare), thyme (Thymus mongolicus Ronn.), and star anise (Illicium verum) @ 1 g/L for 30 min at room temperatureMicrobial composition and quality of fillets stored at 4 ± 1 °C• EOs effective in inhibiting microbial growth (TVC) by 0.59 log CFU/g, delaying lipid oxidation, and retarding the increase of TVB-N, putrescine, hypoxanthine, and K-value
    • Samples with EOs had a less fishy smell and firmer texture compared to the control
    • EOs extended the shelf-life of fillets by 2 more days compared to the control
    • Treatment with EOs can effectively inhibit the degradation of ATP and maintain a high quality of fish products
    [4]
    Sea bream (Sparus aurata) fresh filletsApplication of oregano essential oil (OEO) in the vapor phase (67 µL/L) under vacuum (5–10 hPa) immediately before MAP fillet packagingMicrobial and sensory product quality of fish fillets stored at 4 ºC for 28 d• OEO vapor treated samples had better physicochemical parameters (pH, TMA-N and WHC) as well as freshness compared to dipping
    • Shelf life of vapor OEO treated MAP fish fillets was extended up to 28 d compared to control (7 d)
    • Microbial quality of fish fillets is well preserved with the innovative OEO vapor injection under vacuum
    [23]
    Sardine (Sardinella albella) muscleBetel leaf (Piper betle) extracts (BLE)
    @ 0.5 and 1 g/kg in the ice medium
    Microbial, biochemical, and sensory score of fish during 14 d of chilled storage• BLE at both concentrations inhibited the microbial proliferation and fish deterioration and extended the shelf life of fish for at least 3 d compared to the control sample
    • BLE incorporated into ice improved the sensory score and chemical (pH, TVB-N, and TMA-N) quality
    [30]
    Indian mackerel (R. kanagurta)Methanolic extract of the red alga Gracilaria verrucose-GC @ (0.67 and 2.5 g lyophilized alga/L aqueous solution) in the icing mediumMicrobial, chemical, and sensory study of fish chill stored for 15 days• GC significantly (P < 0.05) inhibited the mesophilic and psychrophilic bacteria and chemical markers (pH, TVB-N, TMA-N, and biogenic amines) of fish deterioration relative to the control
    • Icing medium containing GC extract improved the sensory acceptability, quality, and safety of fish compared to control
    • The seafood industry can explore icing medium containing GC as a biopreservative
    [66]
    Hairtail fish ballAqueous solution containing 1 g/kg sage extract, 1 g/kg oregano extract and
    0.1 g/L grape seed extract (GSE)
    Quality and volatile flavor component of
    fish balls stored at 4 °C up to 15 d
    • GSE stabilized meatball pH of hairtail fish
    • The extract also reduced fishy odor, TBARS values, and TVB-N
    • Inhibited bacterial growth compared to control
    [67]
    Wallago attu fish nuggetsTreated with guava (Psidium guajava L.), bael (Aegle marmelos L.) pulp and dragon fruit (Hylocereus undatus L.) peel powder @ 15 g/kgVarious physicochemical,
    textural and sensory attributes of fish nuggets refrigerated stored up to 10 d
    • Fruits powder @ 15 g/kg significantly reduced the pH of the nuggets compared to control
    • Increased emulsion stability, cooking yield, moisture, fat, and protein percentage
    • Slowed down the lipid peroxidation of fish nuggets
    • Textural attributes were improved in treated nuggets
    [25]
    Minced meat of Indian mackerel (R. kanagurta)Pomegranate peel extract (PPE)
    @ 1, 1.5, and 2 g/kg
    Oxidative stability of samples packed in polythene bags and stored at 4 °C• PEE @ 2 g/kg ppm increased oxidative stability of minced meat
    • Improved shelf life of fish meat up to 8 days compared to 4 d in control
    [41]
    Canned common barbel (Barbus barbus) fish burgersCystoseira compressa and Jania adhaerens powder @ 5, 10, and
    15 g/kg
    Texture and sensory characteristics of fish burgers stored at 4 ºC for further analyses (8 months)• Treated formulations had improved nutritional content WHC, and enhanced texture stability
    • Burgers containing 10 g/kg algae had better texture and sensory properties (P < 0.05)
    • Algae could be considered as nutritious additives and natural flavoring and coloring agents to produce fish-based products
    [68]
    Cobia (Rachycentron canadum) filletsPsidium guajava extract (PGE)
    @ 0.3 g/kg (w/v) for 30 min
    Physicochemical and microbiological changes in fillets packed and stored in ice for 15 d• PGE @ 0.3 g/kg showed a significantly lower increment of pH values during storage
    • Treated fillets showed significantly higher sensory properties, lower PV and TBARs compared to the control
    [24]
    Bighead carp (Aristichthys nobilis) filletsAqueous pomegranate peel extract (APPE) @ 0.5 g GAE/L and ethanolic pomegranate peel extract (EPPE)
    @ 0.5 g GAE/L
    Microbiological and quality changes in fillets stored at 4 °C for 8 d• PPE decreased the TVC of fish spoilage bacteria such as Pseudomonas, Aeromonas, and Shewanella
    • APPE is more effective in retarding the
    increase of TVB-N and K-value
    • EPPE was relatively better in inhibiting biogenic amines
    [52]
    Atlantic mackerel (Scomber scombrus) filletsFillets immersed in 10 g/L of rosemary or basil essential oils (EOs) for 30 min at 2 °CPhysicochemical quality of fillets stored at 2 °C up to 15 d• Rosemary and basil treatments effectively inhibit the formation of TVB-N and lipid oxidation products during storage.
    • Significantly lower pH values were observed for the basil group than others, indicating antimicrobial effects
    • Compared to the control group, fillets treated with rosemary and basil EOs had extended shelf life by 2 and 5 d
    [69]
    Common Carp (C. carpio) filletsEdible coating (C + EC), edible coating +, 5 g/kg chitosan (C + ECCh) and edible coating + 15 g/kg chitosan + 100 g/kg peppermint (C + ECChP)Quality and shelf life of common carp (C. carpio) during refrigerated storage (4 ± 1 °C) for 9 d• ECChP coating treatment extended the shelf life of carp by about 4 days compared with the control
    • (C + ECCh) and (C + ECChP) significantly effective (P < 0.05) in delaying hydroperoxide production of fillets during refrigerated storage, reducing lipid oxidation
    [36]
    Fish (S. scombrus) minceGreen tea
    extract (GTE), grape seed extract (GSE), and pomegranate rind extract (PRE) at a level of 0.1 g/kg equivalent phenolics
    Changes in quality of fish mince during frozen storage at −18 ± 1 °C for 6 months
    • PRE effectively inhibited lipid oxidation with lower peroxide and TBARS values
    • Minced fish containing PRE had lower carbonyl and higher sulfhydryl contents
    • GTE was not effective against lipid and protein oxidation
    • PRE could be utilized as an antioxidant to extend the storage period in raw minced fish tissue
    [34]
    Fried fillets of Nile tilapia (O. niloticus)Fillets treated with rosemary extract-RE (1, 2, 3 g/kg) and Vitamin E 1 g/kgPhysicochemical and sensory quality of fried fillets stored for 15 d at
    4 ± 1 °C and 3 months at −18 ± 2 °C
    • TMA-N and TVB-N, values of RE and vitamin E treated samples were significantly lower than control samples (P < 0.05)
    • R.E. @ 3 g/kg retarded oxidative changes in chilling and frozen fried fillets
    • Significant (P < 0.05) enhancement in sensory quality attributes in samples treated with RE and vitamin E
    [59]
    Whole rainbow trout (Oncorhynchus mykiss)Effect of ice coverage comprised of Reshgak (Ducrosia anethifolia) extract (RE) @ 3 mg/L and Reshgak essential oil (REO) @ 15 g/LChemical, microbiological and shelf life study during a 20-day storage period.• Treated samples had lower bacterial counts and chemical indices than ice coverage without extract
    • Fish stored in ice containing REO had a longer shelf-life (> 16 d) than RE (16 d) and lot stored in traditional ice (12 d)
    [70]
    TMA-N = Trimethylamine; TVB-N = Total volatile basic nitrogen; TVC = Total viable count; TBARS = Thiobarbituric acid reactive substances; WHC = Water holding capacity; MAP = Modified atmosphere packaging.
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    The cobia (Rachycentron canadum) fish fillets treated with guava leaf extract had significantly lower pH values than the control samples during storage for up to 15 d[24]. Likewise, the incorporation of different fruit powders viz. guava and bael pulp at 15 g/kg were found to significantly reduce the pH of fish nuggets[25]. The application of oregano EO in the vapor phase (67 μL/L) under vacuum (5−10 hPa) immediately before MAP of sea bream (Sparus aurata) fresh fillets has also been reported to maintain the physicochemical parameters like pH, TMA-N, water holding capacity and freshness[23]. The lower pH, TMA-N and freshness in treated fillets might be due to inhibition of spoilage microorganisms by EO vapors and reduced accumulation of alkaline compounds from protein degradation and decarboxylation of amino acids. Furthermore, the acidic nature and ascorbic acid content of plant extracts applied in powder form contributed to lowering the pH of fish products.

    Sardine (Sardinella aurita) fillets marinated with pomegranate (Punica granatum L.) peel and artichoke (Cynara cardunculus L.) leaves extracts had the lowest pH value, TVB-N, and histamine content at the end of the storage time, resulting in less microbial spoilage compared to the control group[26]. In another study, the Coleus aromaticus leaf and microalga (Sargassum wightii) paste significantly improved protein, lipid, and carbohydrate contents while reducing moisture content of fish fillets of Sardinella longiceps and Rastrelliger kanagurta stored under chilled conditions for 7 d compared to control[27]. Apart from exhibiting antioxidative and antimicrobial activities, phytoextracts also preserved/improved the nutritional value of products. For example, fish patties incorporated with rosemary extract at 0.2 g/kg were found to have higher protein (140 g/kg), phosphorus and selenium minerals, alpha-linoleic acids up to 400 g/kg and low-fat contents (< 20 g/kg), compared to the control sample[28].

    Fish nuggets with dragon fruit peel powder (10, 15 and 20 g/kg) have been reported with lower pH values, and significantly improved emulsion stability and cooking yield compared to the control[29]. This might be due to the water and fat-binding properties of dietary fibre present in dragon fruit peel powder. Betel leaf (Piper betle) extracts (0.5 and 1 g/kg) in ice medium have also been reported to improve the chemical quality such as pH, TVB-N, TMA-N and extend the shelf life of sardine muscle[30]. Essential oils like oregano, thyme, and star anise (1 g/L) were reported to be effective in delaying lipid oxidation, biogenic amines formation and TVB-N putrescine, and hypoxanthine of grass carp fillets. Besides, these EOs also delay the degradation of ATP and IMP which in turn helps in maintaining the quality of fish products[4].

    Fish oils contain unsaturated fatty acids, especially polyunsaturated fatty acids (PUFA) which are easily susceptible to oxidative changes. Hence fish and fish products are preserved for longer periods of time with additives having antioxidant properties. During the storage of food products, peroxide value (PV) and thiobarbituric acid (TBA) are considered useful indicators to determine the degree of lipid oxidation. In a study conducted by Mazandrani et al.[31], the peroxide and TBA values of silver carp fillets treated with liposomal encapsulated fennel extracts were significantly lower than the control during storage, suggesting that the fennel extract after encapsulation in liposome may be more effective in lowering lipid oxidation. The preservative effect of dried red beetroot peel (DRBP) extract was studied to monitor the quality changes, such as TBA and sensory values in Nile tilapia fish fillet. The fillets treated with DRBP extract at 1 g/L had reduced TBA content and acceptable sensory scores compared to non-treated samples[32]. The antioxidant potential of the peel extract primarily could be due to the presence of betaines, phenolic and flavonoid compounds containing amino and hydroxyl groups, and other active components such as carotenoids and glycine. In another study, aqueous extracts of seaweed (Padina tetrastromatica) applied at 2% as an additive has been reported to reduce the meat discoloration and fat oxidation in Pangasius fish fillets, and thus extending their storage life[33]. This could be due to tannic acids in seaweed extracts which reduced the myoglobin oxidation resulting in higher redness values. Pomegranate rind extract (PRE) at 0.1 g/kg equivalent phenolics was also reported to significantly reduce lipid oxidation (with lower peroxide and thiobarbituric acid reactive substances, TABRS) and protein oxidation (with lower carbonyl and higher sulfhydryl contents of fish mince during frozen storage)[34]. Protein oxidation can be assessed continuously by analyzing the carbonyl and sulfhydryl contents to understand the extent of protein damage during storage[35].

    Edible coating of chitosan and peppermint has been reported to extend the shelf life of common carp (Cyprinus carpio) fillets during refrigerated storage, by delaying the hydroperoxide production, and also by reducing lipid oxidation[36]. The reduction of lipid oxidative products or delaying hydroperoxide production could be due to antioxidants present in chitosan and peppermint coating by quenching fatty acids or hydroxy radicals. In another study, basil leaf extract (10 g/L) was effective in inhibiting the formation of TVB-N and other lipid oxidative products in fillets of Atlantic mackerel (Scomber scombrus) during storage. Again, basil leaf essential oil combined with ZnO nanoparticles significantly lowered the production of TVB-N, biogenic amines, peroxide, and TBARS values during storage of sea bass (Lates calcarifer) slices[37]. The lower production of TVB-N in treated sample could either be due to the rapid inhibitory effect of bacterial growth or decreased bacterial capacity for oxidative de-amination of non-protein nitrogenous compounds, or both especially by basil leaf EO-ZnO nanoparticles film. Recently, hemp essential oil reinforced in nanoparticles with whey and mung bean proteins complex has been reported to inhibit the microbial activity, lipid oxidation and TVB-N in rainbow trout fillets during refrigerated storage[38]. Nisin and EO from Mentha pulegium. L. when used in free and nanoliposome forms minimized the growth of spoilage microorganisms, TVB-N production and improved sensory properties of minced fish[39]. In the aforesaid studies, amino acid decarboxylase inhibiting properties of EOs might be the reason for the low levels of TVB-N and biogenic amines in treated fish products during storage[40]. Pal et al.[41] reported the presence of high content of phenolic compounds like punicalagin, punicalin, gallic acid, and ellagic acid in pomegranate peel extract (PPE) which extended the shelf life of Indian mackerel mince (by up to 8 d). This could be due to higher antioxidant activity of ethanolic extract of pomegranate peel, when used at a concentration of 2 g/kg as compared to control.

    Betel leaf extract in ice medium has been reported to significantly extend the shelf life of sardine muscle by reducing the production of TVB-N and TMA-N during storage[30]. Recently, extracts from various fruits such as blueberry, acerola, and grape have significantly inhibited the formation of heterocyclic aromatic amines (HAAs) in roasted yellow croaker. Particularly, blueberry extract was more effective in reducing the Norharman (94.85%) and heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine (PhIP) (71.15%) content compared to fruit extracts[42]. Different fruits extracts possibly hindered the pyridines and pyrazines via Strecker degradation, derived from various precursors, including amino acids and glucose, responsible for formation of HAAs[43]. Interestingly, the use of herbs such as parsley (40 g/kg), chives (40 g/kg), and their mixture (Brazilian cheiro-verde) effectively inhibited the formation of cholesterol oxidation products (COPs) in grilled sardine fish[44]. As the formation of higher PhIP content in cooked muscle foods is considered mutagenic and carcinogenic, natural extracts from plant components could be an effective approach to minimize the formation of HAAs in muscle food products.

    Available reports suggest that essential oils from plants, when applied as natural preservatives, show good antimicrobial activity, and maintain the quality of fish and fish products during storage. Antimicrobial effect of EOs is mainly due to interaction of hydrophobic part oil with lipid components of cell membrane of the bacteria resulting in change in sequences of metabolic function and cell death[45]. The addition of lemon EO micro-emulsions retarded the growth of Enterobacteriaceae, Staphylococci, and rod-shaped lactic acid bacteria in salted sardines[46]. Likewise, hot smoked tilapia (Oreochromis niloticus) treated using EOs (extracts of ginger, garlic, clove) had significantly lower total viable, total psychotropic, lactic acid bacteria count for 7 weeks storage period[47]. The inhibitory effects of olive by-products (such as olive leaf extract-OL, olive cake-OC, and black water-BW) were studied on fish spoilage bacteria from anchovy, mackerel, and sardine. Kuley et al.[48] reported that OL extract was more sensitive to fish spoilage bacteria and reference strains such as Enterobacter cloacae, Serratia liquefaciens, Proteus mirabilis, Photobacterium damseale, Pseudomonas luteola, Pantoea spp., Vibrio vulnificus, Stenotrophomonas maltophila, Acinetobacter lwoffii, Pasteurella spp., and Citrobacter spp. The effect of salep gum containing orange peel essential oil (2.5 and 5 g/kg) coating on the microbial growth and shelf life of rainbow trout (O. mykiss) fish fillets stored for 16 d under refrigerated conditions was investigated[49]. Samples treated with 5g/kg orange essential oil had improved shelf life and low numbers of total aerobic mesophilic, psychrophilic, coliforms and lactic acid bacteria, which can be ascribed to the presence of antimicrobial compounds (limonene and other minor compounds) in orange essential oil, exhibiting antimicrobial effects.

    The EO of Zataria multiflora Boiss (ZMB) was reported to be more sensitive, particularly to Gram-negative more than Gram-positive bacteria that cause seafood spoilage, hence can be used as a natural additive for food preservation[50]. However, Hosseini et al.[51] indicated that the highest concentration with sensory acceptability of ZMB EOs when used in rainbow trout cannot inhibit the growth of L. monocytogenes at room, and optimum growth temperature. The variation in antibacterial effect of EOs may be due to factors such as the type, concentration, and form of EOs (liquid or vapor), number of microorganisms and influence of food matrix such as low pH value, level of sodium chloride etc. The vapor phase of various EOs has also been reported to have antimicrobial activity in various food systems. In a recent study, the vapor phase of oregano EO under vacuum immediately before MAP packaging has been reported to limit the microbial growth and maintain the quality of sea bream fresh fillets during refrigerated storage for 28 d. Chemically, the vapor phase of EOs being hydrophobic in nature are accumulated in the lipid component of microbial cell membrane hampering its functional properties, thus leading to structural damage[23].

    Various researchers have reported the antimicrobial effect of pomegranate peel extract (PPE) on the quality and shelf life of fish and fish products, mainly due to the presence of higher phenolic components such as punicalagin, gallic acid, ellagic acid, chlorogenic acid, caffeic acid, catechin, epicatechin, rutin, quercetin, and galangal[26]. Zhuang et al.[52] reported that bighead carp (Aristichthys nobilis) fillets treated with PPE at 0.5 g GAE/L had significantly decreased total volatile compounds (TVC) of fish spoilage bacteria such as Pseudomonas, Aeromonas, and Shewanella, during chilled storage. The efficacy of pomegranate peel powder breaded on ready-to-cook cod sticks was studied against total mesophilic, psychrotrophic and other spoiling bacteria, Pseudomonas spp., Shewanella putrefaciens and Photobacterium phosphoreum[53]. The study reported a delayed microbial growth in treated samples stored for 17 d under refrigerated conditions, which could be due to higher bioactive compounds such as polyphenols, tannins, flavonoids and anthocyanins in peel powder, exhibiting antibacterial activity. Even colorants obtained from plant extracts such as Hibiscus sabdariffa calyces, Curcuma longa rhizomes, and Rhus coriaria fruits are reported to exert an antimicrobial effect on standard microbial stains like Escherichia coli (ATCC 25922), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 25923), and Pseudomonas aeruginosa (ATCC 27853) in surimi from O. niloticus[54]. Dip treatment of Deccan mahseer (Tor khudree) steaks with beetroot (Beta vulgaris) peel extract (200 g/L) has been reported to exhibit a positive effect by retarding spoilage, thereby extending shelf life up to six months during frozen storage study[55]. The addition of Simira ecuadorensis plant extract (80 g/kg) significantly reduced the aerobic mesophilic bacteria count in a fish burger[56]. The extract reduced the pH of fish burger thereby increased the microbiological safety during further storage periods. Betel leaf extract has also been reported to significantly lower microbial proliferation and prolongs the shelf life of sardine muscle for at least 3 more days compared to the control[30]. In a similar study, the fillets of O. niloticus treated with ethanolic extracts of betel leaf at 0.4 and 0.6 g/kg had reduced microbial growth and quality deterioration during 12 d of storage at refrigerated temperature, compared to untreated samples[57]. The minimum inhibitory concentration (MIC) of various phytochemicals against different fish spoilage microorganisms is presented in Table 2.

    Table 2.  Minimum inhibitory concentration (MIC) of phytochemicals against fish spoilage microorganisms.
    Component of plantsFish productsMIC values Target microorganismsReferences
    Curcuma longa rhizome powder (200 g/L of 70 % aqueous ethanolic solution)
    Surimi gel of tilapia2.2 g/L
    1.8 g/L
    1.8 g/L
    1.2 g/L
    Salmonella Typhimurium
    Staphylococcus aureus
    Escherichia coli
    Pseudomonas aeruginosa
    [54]
    Gabsi pomegranate peel powder (5 g powder in
    150 mL methanol for methanol pomegranate peel extracts)
    fresh fish152 g/LE. coli
    Saccharomyces cerevisiae
    [71]
    Olive leaf extract (OL), olive cake (OC), black
    water (BW)
    Fresh anchovy, mackerel, sardine3.0 g/L
    6.0 g/L
    12.5 g/L
    E. coli
    Salmonella Paratyphi A
    S. aureus
    [48]
    Betel leaf (Piper betle) powder (water extract)Sardine fish meat0.5 g/LPsychrophilic bacterial count[30]
    Hibiscus sabdariffa
    Calyces powder (200 g/L of 70% aqueous ethanolic solution)
    Surimi gel of tilapia1.6 g/L
    1.0 g/L
    1.2 g/L
    1.6 g/L
    S. Typhimurium
    S. aureus
    E. coli
    P. aeruginosa
    [54]
    Thyme essential oilMinced fish meat8 g/kgListeria monocytogenes[72]
    Lavender essential oilCatfish2 g/L,
    1–1.2 g/L
    E. coli,
    S. aureus
    [73]
    Kakadu plum bark powder (methanol extract)
    Chilled fish1 g/LS. aureus[74]
    Fruits and culinary herbs of Australian plant powder (methanolic extract)Fresh fish5 g/LShewanella putrefaciens[75]
    Dried, fragmented leaves of rosemary, thyme and
    dried fruits of anise (Pimpinella anisum)
    Canned fish10 g/L (rosemary)
    1.25 g/L (thyme)
    10 g/L (anise)
    Clostridium perfringens[76]
    Simira ecuadorensis leaf powder (ethanol extract)Fish hamburger80 g/LCampylobacter jejuni
    and S. putrefaciens
    [56]
     | Show Table
    DownLoad: CSV

    The use of phytochemicals in extract, powder, or oil forms influences the sensory attributes of fish and fish products at different levels of efficiency. Fish paste (pollack meat, cuttlefish meat, shrimp meat) received the best score in terms of taste and overall preference, when treated with different levels (10, 30, 50, and 70 g/kg) of C. longa powder[58]. Fish nuggets treated with dragon peel powder at 15g/kg had improved shelf life and sensory attributes compared to others during 15 d of storage[29]. Fillets of S. longiceps and R. kanagurta treated with leaf paste of C. aromaticus and S. wightii had the best appearance, smell, color, texture, and taste compared to control[27]. Even ethanolic extracts of betel leaf at 0.4 or 0.6 g/kg has been found to extend shelf life without any change in taste or discoloration of Nile tilapia (O. niloticus) fillets up to 9 d[57]. In a recent study, fried fillets of Nile tilapia (O. niloticus) treated with rosemary extract (1, 2, 3 g/kg) and vitamin E (1 g/kg) has shown significant enhancement in sensory characteristics[59]. Likewise, salted sardines (Sardina pilchardus), treated with lemon essential oil microemulsion received the highest scores for flavor and overall acceptability[46]. Berizi et al.[60] reported that a 10 g/kg concentration of methanolic pomegranate peel extract (MPPE) had the highest sensory rating and chewiness of chilled gutted rainbow trout. MPPE is, therefore, recommended as a natural agent to improve the textural properties of frozen fish during the first six months of storage. Panza et al.[61] adopted a zero- waste approach by utilizing the whole pomegranate (juice, peel, and seed) in varied proportions to find the effect on spoilage microorganisms and sensory quality of fish burgers. The researchers corroborated that pomegranate treated fish burgers had delayed microbial proliferation and maintained the sensory attributes with prolonged shelf life, due to the antibacterial action of tannins and phenolic acids present in the formulation.

    As far as the impact on sensory acceptability, the effect of oregano EO at 12.5 g/L on texture, color, and sensory acceptability of balls prepared from Tambaqui (Colossoma macropomum) fish was evaluated. It was found that OEO improved the color and aroma as sensory attributes[62]. This could be due to the antimicrobial and antioxidative properties of EOs. The EOs inhibit the H2S-producing bacteria, and chemical reactions which are responsible for the development of off-odors. Besides, when treated with thyme and star anise essential oil (1 g/L) at room temperature for 30 min, grass carp fillets had a less fishy smell and firmer texture than the control[4]. Although the above concentrations (v/v) were acceptable by the panelists.

    To overcome this, combination of various plant derived EOs is suggested, that possess high phenolic content but effective at low concentrations, so that their synergistic effect may offer better antimicrobial and antioxidant activities, and consequently maintaining the balance between sensory properties of fish products and odor factor of EOs. Encapsulation technique is also reported to be effective in masking the strong odor and flavor of EOs, as it (i) maintains the inherent flavor characteristics of food, (ii) prevents the evaporation of volatile compounds, (iii) enhances solubility for effective release and better distribution[11, 63]. It is recommended that materials used for encapsulation should have low reactivity with EOs, to ensure limited impact on the sensory attributes of foods.

    Natural preservatives are safer and more effective agents for retarding the deterioration process of fish products. Because of this, plant-derived bioactive compounds and secondary metabolites, also called phytochemicals/green chemicals, with antioxidant and antimicrobial characteristics are now preferred over their synthetic counterparts. The application of phytochemicals as food additives extends the shelf life by delaying lipid oxidation and inhibiting microbial growth, thereby ensuring better nutritional value, and improved textural properties of fish and fish products. The increased demand for high quality fish products and the concept of 'Green consumerism' gaining momentum are boosting the application of bioactive phytochemicals, obtained from available, cheap, and underutilized resources. Modern and green extraction methods, including ultrasound-assisted extraction, microwave-assisted extraction, supercritical fluid extraction, and pressurized liquid extraction may be employed to obtain enhanced yields and stability of the phytochemicals. Further, the suitability of the phytochemicals, and their synergistic effect in combination with other natural preservatives or non-thermal technologies (irradiation, high pressure, retort pouch processing) and innovative packaging technologies may be explored to increase the degree of quality, functionality, sensory acceptability as well as shelf life of the fish and fish products, and thus meet consumer expectations.

    Thanks to the Director, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, India and the Station In-charge, Eastern Regional Station, ICAR-IVRI, Kolkata, India for their encouragement in writing this manuscript.

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Huang P, Cheng P, Sun M, Liu X, Qing Z, et al. 2024. Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions. Medicinal Plant Biology 3: e020 doi: 10.48130/mpb-0024-0019
    Huang P, Cheng P, Sun M, Liu X, Qing Z, et al. 2024. Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions. Medicinal Plant Biology 3: e020 doi: 10.48130/mpb-0024-0019

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Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions

Medicinal Plant Biology  3 Article number: e020  (2024)  |  Cite this article

Abstract: Macleaya cordata, a medicinal plant in the Papaveraceae family, is rich in bioactive benzylisoquinoline alkaloids. Recent research has elucidated the mechanisms by which these active components promote livestock and poultry growth and exhibit anti-inflammatory effects on the intestines. These findings have led to the development of two raw medicinal materials and two veterinary drug formulations, widely used in China's livestock industry. Advances in multi-omics technologies, such as whole-genome sequencing and transcriptomics, have clarified the chemical composition of alkaloids and the biosynthesis of sanguinarine, enabling its de novo synthesis in yeast. Efforts in plant breeding have focused on cultivar selection and germplasm innovation, establishing DUS (Distinctness, Uniformity, and Stability) testing guidelines. Genetic engineering techniques have re-edited the sanguinarine pathway and induced hairy roots and cell suspension cultures in M. cordata. These advancements reduce production costs, ensure product stability, and promote sustainable production. This paper reviews the species origin, current research status, and prospects of M. cordata, offering guidance for further research on this valuable resource.

    • Macleaya cordata (Willd.) R. Br., commonly known as boluohui in China, is a perennial, upright herbaceous plant belonging to the Papaveraceae family (Fig. 1)[1]. Modern pharmacological research has identified that plants of the Macleaya genus are rich in benzylisoquinoline alkaloids (BIAs), which exhibit significant anti-inflammatory and antibacterial properties, regulate intestinal microflora, and promote animal growth[2]. The European Food Safety Authority (EFSA) has approved M. cordata as a safe plant for the manufacture of feed additives. In China, four alkaloids found in M. cordata, including sanguinarine (SAN), chelerythrine (CHE), allocryptopine (ALL), and protopine (PRO) have been successfully developed into veterinary medicines (Fig. 1).

      Figure 1. 

      Morphological characteristics of M. cordata. The figure shows various parts of M. cordata, commonly known as boluohui. The main image displays a field of mature plants with characteristic upright growth. The top left inset highlights a single leaf with a broad, lobed structure. The top middle inset shows the flower of M. cordata, featuring clustered blooms with delicate petals. The top right inset illustrates the capsule, the fruiting body containing seeds. These images collectively depict the key morphological features of M. cordata, which is valued for its rich content of benzylisoquinoline alkaloids.

      This review aims to provide a comprehensive overview of the latest advancements in the study of M. cordata. It will cover the plant's resources, chemical composition, pharmacological activities, biosynthesis mechanisms, and breeding strategies. By synthesizing current knowledge, this review seeks to offer a broad perspective on future research and potential applications of M. cordata in various fields.

    • The Macleaya genus comprises two species: M. cordata, and M. microcarpa. M. cordata is primarily distributed in China and Japan, originating in the regions south of the Yangtze River and north of the Nanling Mountains in China. It extends south to Guangdong, west to Guizhou, and northwest to southern Gansu. M. cordata predominantly grows in hills, low mountain forests, shrubs, or grasses at altitudes between 150 and 830 m. It is notably found in grasslands or shrubs on slopes at elevations ranging from 450 to 1,600 m in provinces such as Jiangsu, Jiangxi, Shanxi, Gansu, and Shaanxi.

      Both M. cordata, and M. microcarpa are upright herbaceous plants with a woody base, producing a milky yellow sap. Their stems are hollow, with the upper parts branching out, and the leaves are broad ovate or nearly circular. They possess large conical inflorescences that can be both terminal and axillary, with a flowering and fruiting period from June to November. Plants of the Macleaya genus are rich in various chemical constituents, including alkaloids, flavonoids, terpenes, and phenylpropanoids, with alkaloids being the predominant components[3]. Benzylisoquinoline alkaloids, such as sanguinarine, are among the most active components in this genus. Sanguinarine is also abundant in other plants like poppy (Papaver somniferum) and bloodroot (Sanguinaria canadensis); however, due to regulatory restrictions and over-exploitation, these plants are no longer viable sources. Consequently, M. cordata has emerged as the primary commercial source of sanguinarine.

    • Japanese researchers such as Tani & Takao[4] and Takao[5] isolated alkaloids like sanguinarine, chelerythrine, berberine, and coptisine from M. cordata, initiating the study of its chemical constituents. Subsequently, numerous researchers contributed to the isolation and structural identification of secondary metabolites in the Macleaya genus[69]. From 2009 to 2024, Qing et al.[10] developed a new method for the discovery and structural identification of alkaloids in the Macleaya genus using mass spectrometry, identifying over 200 alkaloids. These alkaloids belong to ten major categories: benzylisoquinoline alkaloids, tetrahydroprotoberberine alkaloids, N-methyl-tetrahydroprotoberberine alkaloids, protopine alkaloids, protoberberine alkaloids, 7,8-dihydroprotoberberine alkaloids, aporphine alkaloids, benzophenanthridine alkaloids, dihydrobenzophenanthridine alkaloids, and benzophenanthridine dimers (Fig. 2). Using mass spectrometry-guided techniques, over 20 new compounds were isolated from Macleaya species, established a mass spectrometry database and metabolic profile for alkaloids in Macleaya, and proposed new biosynthetic pathways for berberine and sanguinarine[1013]. In addition to a large number of alkaloids, Macleaya species also contain small amounts of flavonoids and polyphenols[3]. The rich chemical composition of Macleaya endows it with various biological activities. Modern pharmacological studies have shown that Macleaya possesses antibacterial, anti-inflammatory, insecticidal, antitumor, antifibrotic, hepatoprotective, antiviral, antioxidant, immune-enhancing, gut microbiota-regulating, animal growth-promoting, wastewater-purifying, and soil erosion-preventing properties[1316].

      Figure 2. 

      Types of compounds identified in Macleaya species.

    • The primary active secondary metabolites in Macleaya cordata are protopine alkaloids and quaternary benzo[c]phenanthridine alkaloids. To date, there have been no reports in the literature regarding the total chemical synthesis of protopine alkaloids. The total chemical synthesis of quaternary benzo[c]phenanthridine alkaloids is generally achieved through multi-step reactions using conventional chemical reagents[17]. In most reported synthetic routes, the cyclization reactions to form rings B and C constitute the final steps in constructing the tetracyclic system. Key chemical reactions for constructing ring B include Heck coupling to form the C10a-C11 bond[18], Pictet-Spengler reaction (PS reaction) or Bischler-Napieralski reaction (BN reaction) to form the C6-C6a bond[19,20], amidation to form the N5-C6 bond[21], and electrocyclization to form the N5-C5a bond[22]. For ring C, key reactions include enamide-aldehyde cyclization to form the C11-C11a bond[23] and Friedel-Crafts reaction to form the C12-C12a bond[24]. These reactions have been widely applied in the synthesis of benzo[c]phenanthridine derivatives with anticancer activity (Fig. 3).

      Figure 3. 

      Total synthesis of quaternary benzo[c]phenanthridine alkaloids, the major active components of M. cordata. This figure illustrates the chemical structure of quaternary benzo[c]phenanthridine alkaloids, highlighting the tetracyclic framework composed of rings A, B, C, and D. These alkaloids are notable for their complex ring system and significant biological activities. The positions of the rings are labeled for clarity, facilitating understanding of the synthetic pathways and structural modifications discussed in the study.

    • In recent years, with the deepening research on the chemical constituents of M. cordata, a series of 6-substituted dihydrobenzo[c]phenanthridine alkaloids have been successively isolated and structurally confirmed (Fig. 4)[6,9,25,26]. Currently, studies on the biological activities of these alkaloids are still in their infancy, and no biosynthetic pathways for 6-substituted dihydrobenzo[c]phenanthridine alkaloids have been reported. Based on the structural types and chemical properties of the C-6 substituents, it is speculated that these alkaloids may be biosynthesized in plants through radical reactions or nucleophilic substitution pathways. As shown in Fig. 4, quaternary benzo[c]phenanthridine alkaloids or dihydrobenzo[c]phenanthridine can be converted to α-amino carbon radicals through single-electron reduction[27,28] or oxidation[29,30]. These radicals can then undergo various types of radical reactions to achieve functionalization at C-6 of the benzo[c]phenanthridine ring structure, resulting in the semi-synthesis of the aforementioned alkaloids[31]. Additionally, the N5=C6 double bond in quaternary benzo[c]phenanthridine alkaloids can accept nucleophilic reagents, leading to the formation of 6-substituted dihydrobenzo[c]phenanthridine alkaloids.

      Figure 4. 

      Novel 6-substituted dihydrobenzophenanthridine alkaloids in Macleaya and their semi-synthesis.

    • To date, nearly 300 secondary metabolites, including 204 isoquinoline alkaloids have been identified from M. cordata using untargeted LC-MS metabolomics technology[10]. This breakthrough surpasses the limitations of traditional methods for isolating and identifying secondary metabolites in plants, guiding the targeted analysis of alkaloid metabolism and metabolic profiling in M. cordata. Using untargeted LC-MS metabolomics technology, nearly 2,000 characteristic ions were discovered in M. cordata. Through stable isotope 13C6-labeled tyrosine tracing experiments, 179 13C6-labeled compounds were identified[1]. A database search for the non-labeled counterparts of these 13C6-labeled compounds quickly identified 40 alkaloids and four non-alkaloids, providing evidence for determining the biosynthetic pathways of sanguinarine and chelerythrine in M. cordata.

      Targeted analysis of the metabolic accumulation patterns of intermediates in the biosynthesis of sanguinarine and chelerythrine in different developmental stages and organs of M. cordata, combined with transcriptomics, proteomics, and metabolomics analysis revealed the biosynthetic tissues and gene expression patterns of alkaloid biosynthesis at different growth stages. This study elucidated the biosynthetic pathways of sanguinarine and chelerythrine and discovered the pattern of synthesis by 'root-pod compartmentalized synthesis', where precursor compounds like protopine are primarily synthesized in the roots and then transported to the pods for the synthesis of sanguinarine[1,32]. Laser microdissection with fluorescence detection was used to separate different tissue cells in the roots of M. cordata. Combined with LC-MS targeted analysis of trace alkaloids in each tissue, this approach accurately identified the storage cells of the alkaloids, clarifying the 'synthesis-storage-transport-resynthesis' biosynthetic mechanism of sanguinarine, chelerythrine, and their precursors[33] (Fig. 5).

      Figure 5. 

      Isotope tracing of the biosynthetic pathways of sanguinarine and chelerythrine in Macleaya.

    • In 2013, Zeng completed an integration of transcriptomic, proteomic, and metabolomic data to elucidate the biosynthesis of alkaloids in M. cordata and M. macrocarpa[32]. This study investigated the potential mechanisms of alkaloid biosynthesis in Macleaya species by analyzing transcriptomic, proteomic, and metabolomic data from 10 different samples collected at various times, tissues, and organs. The assembled and clustered transcriptomic data yielded 69,367 unigenes for M. cordata and 78,255 unigenes for M. macrocarpa. Through a multi-level comparative analysis of key gene expressions controlling enzymes in the alkaloid metabolic pathway, the research identified homologous genes for all functional genes in the sanguinarine biosynthesis pathway. This annotation provided foundational data for cloning functional genes in the sanguinarine pathway of M. cordata.

      In 2017, researchers first mapped the complete genome of M. cordata, making it the first Papaveraceae plant to have its whole genome sequenced[1]. The genomic results indicated that the genome size of M. cordata is 540.5 Mb with a heterozygosity rate of 0.92%, predicting 22,328 protein-coding genes, of which 43.5% are transposable elements. Additionally, 1,355 non-coding RNA (ncRNA) genes were identified in the M. cordata genome, including 216 rRNA, 815 tRNA, 75 miRNA, and 249 snRNA genes. Comparative genomics revealed a significant expansion of the flavoprotein oxidase gene family closely associated with sanguinarine synthesis, providing crucial insights into the evolutionary origin of the sanguinarine biosynthetic pathway in Macleaya. Through co-expression analysis of genes and metabolites, researchers successfully identified 14 genes, including flavoprotein oxidases, methyltransferases, and cytochrome P450 enzymes, involved in the biosynthesis of sanguinarine.

    • Recent advancements in genetic engineering have led to a series of modifications on the berberine bridge enzyme (McBBE), a rate-limiting enzyme in the biosynthesis of sanguinarine in M. cordata. These modifications included codon optimization and the design of N-terminal truncated mutants to enhance expression efficiency in heterologous hosts. By employing CRISPR-Cas9 gene editing technology, these optimized genes were successfully integrated into the genome of Saccharomyces cerevisiae. This integration resulted in a significant increase in the production of the key precursor, chelerythrine, achieving a yield 58 times higher than the original level[34]. This accomplishment not only underscores the tremendous potential of genetic engineering in enhancing the synthesis of secondary metabolites but also provides a crucial foundation for subsequent industrial production. Furthermore, the research team developed innovative methods for producing sanguinarine through a 'plant-microbe' co-fermentation system. This system involved the co-cultivation of engineered yeast strains with the non-medicinal parts of M. cordata — specifically, the leaves — in a specially designed co-fermentation reactor[35]. This novel approach effectively converted precursor substances from the leaves into sanguinarine, maximizing the utilization of the non-medicinal parts of M. cordata and reducing production costs. This co-fermentation system not only offers an efficient and sustainable new pathway for the production of valuable bioactive compounds like sanguinarine but also exemplifies the integration of plant biology and microbial biotechnology.

      In 2022, significant progress was made by Jiachang Lian's team, who successfully achieved the de novo biosynthesis of sanguinarine, reaching a yield of 16.5 mg/L[36]. After comprehensive metabolic engineering modifications, the current best-engineered yeast strain achieved a production titer of 448.64 mg/L for sanguinarine[37]. This achievement highlights the immense potential of modern biotechnology in promoting the effective utilization and industrial production of components from traditional medicinal plants. By leveraging advanced genetic engineering techniques, researchers have been able to enhance the efficiency of secondary metabolite production, opening up new possibilities for the pharmaceutical and biotechnology industries.

      The innovative approaches employed in this research, such as codon optimization, N-terminal truncation, and the use of CRISPR-Cas9 for gene editing, represent significant advancements in the field of synthetic biology. These methods not only improve the expression efficiency of key enzymes but also pave the way for the scalable production of complex bioactive compounds. The successful integration of plant and microbial systems in a co-fermentation setup demonstrates a promising strategy for sustainable and cost-effective production processes. This research not only advances our understanding of biosynthetic pathways but also sets the stage for future developments in the industrial application of traditional medicinal plant components

    • In the fields of plant science and biotechnology, artificial genetic transformation technology has become a key tool for molecular breeding. In 2016, a genetic transformation system for Macleaya cordata based on Agrobacterium tumefaciens was successfully established[38]. Utilizing this system, key genes involved in sanguinarine biosynthesis, such as berberine bridge enzyme MCBBE[39] and MCP6H[40], have been overexpressed, demonstrating the significant potential of genetic engineering in enhancing the production of plant secondary metabolites. Additionally, a hairy root culture system for M. cordata and M. microcarpa mediated by A. rhizogenes has also been successfully developed[41,42]. Recently, a CRISPR/Cas9 gene editing system for M. cordata has been established. By re-editing the sanguinarine biosynthesis pathway, researchers have achieved a significant increase in sanguinarine content in transgenic materials[43]. These advancements highlight the potential of molecular breeding techniques to enhance the yield of valuable secondary metabolites in M. cordata, providing a foundation for future research and industrial applications.

    • Previously, wild M. cordata capsules were the primary source of raw materials for M. cordata-related products. However, the popularity of these products has led to a significant decline in wild resources year by year. In response, domestic research teams have developed a standardized cultivation technology system for M. cordata. This system encompasses various techniques, including seedling raising, field management, pest and disease control, harvesting, and primary processing, facilitating the shift from wild to cultivated varieties. Additionally, comprehensive research on the current state of resources and breeding has led to the establishment of 30 traits which form the M. cordata DUS testing guidelines. As a result of these efforts, the team has preliminarily identified a superior strain, 'Meibo 1', known for its high fruit yield and elevated haematoxylin content.

    • Sanguinarine from M. cordata as a bioactive compound was initially used in Europe as feed additives to enhance flavor and stimulate appetite in food animals[44]. Studies have shown that M. cordata extracts are safe, with no adverse effects observed in target animals even at ten times the recommended clinical dose. Pharmacodynamic and clinical data indicate that long-term addition of M. cordata extracts at recommended doses has anti-inflammatory and growth-promoting effects, and total protopine alkaloids are effective in treating Escherichia coli-induced diarrhea in poultry.

      Research on the mechanism of antibiotic substitution revealed that M. cordata extract enhances intestinal health by increasing the abundance of lactic acid bacteria, which inhibit pathogenic microorganisms through competitive exclusion[2]. Sanguinarine inhibits the phosphorylation and ubiquitination of I-κB proteins, reducing the dissociation of p50 and p60, thereby inhibiting the activation of the NF-κB signaling pathway. This results in decreased expression of pro-inflammatory cytokines and reduced inflammation levels in animals[45]. Additionally, sanguinarine was found to enhance protein synthesis in animals by inhibiting tryptophan decarboxylase activity, thereby increasing amino acid utilization[46,47]. Based on the growth-promoting effects of M. cordata extract as an antibiotic substitute, Jianguo Zeng's team has developed a comprehensive feed technology focused on 'intestinal health, anti-inflammatory properties, and growth promotion'. This approach avoids the risk of antibiotic resistance associated with traditional growth-promoting antibiotics while still enhancing animal growth performance. This technology supports the development of antibiotic-free feed solutions and the use of non-antibiotic inputs in animal husbandry. Furthermore, chelerythrine in M. cordata, when used as an animal feed additive, interacts with phospholipids on bacterial membranes, increasing membrane fluidity and impairing respiration by disrupting proton motive force and generating reactive oxygen species, leading to reduced intracellular ATP levels. This dual action of downregulating the mobile colistin resistance gene mcr-1 and associated genes offers a new strategy to circumvent colistin resistance. These findings provide strong technical support for the development of antibiotic-free feeding practices and the creation of alternative growth-promoting solutions in animal production[48].

      M. cordata extract is the first traditional Chinese veterinary medicine product approved for feed use in China. It is widely used to improve animal production performance, enhance animal health, and replace growth-promoting antibiotics in feed. Weaning stress is a major factor causing diarrhea and growth inhibition in piglets. Adding M. cordata extract to their diet can improve small intestine morphology, enhance intestinal barrier function, and regulate intestinal microbiota homeostasis, thereby reducing diarrhea and improving growth performance[4952]. During pregnancy, progressive oxidative stress and parturition stress can lead to metabolic disorders and increased inflammation in sows. Supplementing their diet with M. cordata extract can reduce inflammation, enhance antioxidant capacity, shorten farrowing duration, increase feed intake and milk production during lactation, and ultimately increase the weaning weight of piglets[53,54]. Environmental stress and pathogenic infections are significant threats to poultry intestinal health. Adding M. cordata extract to poultry feed can improve intestinal mucosal morphology, enhance intestinal barrier function, regulate microbial populations, and improve broiler growth performance[5558]. High-fat and high-protein feed can cause intestinal damage in fish. Supplementing their diet with M. cordata extract can enhance intestinal antioxidant capacity, alleviate intestinal barrier damage, improve microbiota homeostasis, and thus increase survival and growth rates[5961]. Ruminants have a unique rumen physiology where the microbiota plays a crucial role in nutrient digestion and metabolism. Studies have shown that adding M. cordata extract to dairy cow diets can regulate rumen microbial populations and reduce methane emissions, alleviate intestinal inflammation in weaned lambs[62], promote growth in meat sheep[63], increase milk yield in dairy goats, reduce somatic cell counts in milk, and improves milk quality[64].

      In addition to its applications in animal production, M. cordata extracts have been developed into various products due to their significant insecticidal and antibacterial properties. In 2021, they were registered as a new botanical pesticide in China and the United States for controlling greenhouse plant diseases such as powdery mildew and leaf spot disease. In the bioenergy sector, the combination of M. cordata extracts with trace elements cobalt and nickel can effectively accelerate the fermentation process of agricultural waste, increasing biogas production. Furthermore, Macleaya alkaloids are used in toothpaste and mouthwash for their sustained antibacterial activity, which helps treat periodontal disease and prevent bad breath[65]. These findings highlight the diverse applications and benefits of M. cordata in improving animal health and productivity, controlling plant diseases, and enhancing bioenergy production (Fig. 6).

      Figure 6. 

      This illustration showcases the versatile applications of alkaloids derived from M. cordata. The molecular structures of key alkaloids—chelerythrine, sanguinarine, allocryptopine and protopine—are depicted in the center. These alkaloids are utilized in enhancing digestion and improving feed efficiency in ruminants, boosting immunity and promoting growth in pigs, promoting health and growth in fish, improving health in poultry, serving as natural pesticides for crops, reducing methane emissions from ruminants to lower greenhouse gases, acting as antibacterial ingredients in toothpaste and mouthwash, and exhibiting potential anti-cancer activity in cancer treatment. The alkaloids from M. cordata demonstrate significant potential across various fields, from agriculture to medicine.

    • M. cordata, a plant rich in bioactive compounds, holds vast potential for applications in medical and agricultural fields. However, future research faces multiple challenges and opportunities. Firstly, although substantial research has been conducted on the primary alkaloids in M. cordata, their regulatory mechanisms remain unclear. Future studies need to integrate multi-omics data and systems biology approaches to elucidate the molecular mechanisms regulating alkaloid biosynthesis in M. cordata, providing a theoretical foundation for precise metabolic pathway regulation.

      Secondly, the application of molecular breeding and metabolic engineering in M. cordata is still in its infancy. Effectively increasing the yield of specific bioactive compounds through genetic engineering, particularly achieving efficient biosynthesis and accumulation of key alkaloids like sanguinarine, will be a focus of future research. Despite the achievement of de novo synthesis of sanguinarine in yeast, the scalability of this production method remains challenging. It is necessary to assess the economic feasibility of these methods to ensure they can meet industrial demands without compromising quality or sustainability. Additionally, the long-term stability and consistency of genetically engineered strains and their products need to be ensured to avoid potential issues in production and application.

      Lastly, as the medicinal value of M. cordata becomes better understood, ensuring its sustainable use and the conservation of wild resources, while developing more high-value products, will be crucial for future development. Large-scale cultivation and utilization of M. cordata may pose potential environmental impacts, including changes in soil health, biodiversity, and local ecosystems, which are not sufficiently studied and require further investigation and evaluation.

      In summary, while the research and application prospects of M. cordata are promising, they also present a series of scientific and technical challenges. By fostering interdisciplinary collaboration and enhancing the integration of basic and applied research, these challenges can be overcome, enabling the efficient utilization and industrial development of M. cordata resources. Addressing the limitations in current research, particularly in ecological impact and production methods, will provide a more balanced perspective and guide future research toward sustainable and scalable solutions.

    • The authors confirm contribution to the paper as follows: study conception and design: Huang P, Zeng J; draft manuscript preparation: Huang P, Cheng P, Sun M, Liu X, Qing Z, Liu Y, Yang Z, Liu H, Li C, Zeng J. All the authors reviewed the results and approved the final version of the manuscript.

    • Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

      • This work was supported by Hunan Provincial Natural Science Foundation of China (2023JJ40367 & 2023JJ30341).

      • The authors declare that they have no conflict of interest.

      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
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    Huang P, Cheng P, Sun M, Liu X, Qing Z, et al. 2024. Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions. Medicinal Plant Biology 3: e020 doi: 10.48130/mpb-0024-0019
    Huang P, Cheng P, Sun M, Liu X, Qing Z, et al. 2024. Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions. Medicinal Plant Biology 3: e020 doi: 10.48130/mpb-0024-0019

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