Hypecoum spp.—Chemistry and Biological Activity of Alkaloids

: Genus Hypecoum Tourn. ex L. belongs to the poppy family Papaveraceae and comprises about 19 species occurring in Europe, Northern Africa and Asia. Hypecoum species have been widely used in traditional medicine as antipyretic, analgesic and anti-inﬂammatory remedies. Their effects are associated with the biologically and pharmacologically active isoquinoline alkaloids in them, such as protopines, protoberberines, benzophenanthridines, aporphines, simple isoquino-lines, secoberbines, spirobenzylisoquinolines and others. In this study, we aimed to review and organize information on ethnomedicinal, phytochemical, chemotaxonomical and pharmacological studies of alkaloids and extracts obtained from Hypecoum plants, and to suggest opportunities for further research.

In recent years, more than 40 alkaloids have been identified in Hypecoum species, and new alkaloid structures, along with their pharmacological activities, are reported every year, enriching the diversity of natural compounds.Some of them have promising properties for the treatment of important diseases such as inflammation, cancer and microbial infections [18,19,22,23,[26][27][28][29].
In this review, the chemical structures and biological activities of the isoquinoline alkaloids that have been reported for the genus Hypecoum over the past fifty years are systematically summarized.For this purpose, the relevant data were collected through the scientific databases "SCOPUS", "Google Scholar", "ISI Web of Knowledge", "ScienceDirect", "PubMed", "SciFinder Scholar" and "Wiley Online Library".
H. erectum is mainly used in China and Mongolia for traditional treatments for inflammation, fever and pain relief [2,4,5,36,37].The whole plant and its root are used for acute pharyngitis, relief from cough and inflamed red eyes [6].In Mongolian medicine, H. erectum is included in various herbal formulations such as Deva-5, which is used to treat acute infectious diseases [36].The plant H. erectum, along with 20 other herbs, is used as a Tibetan medicine-Liu Gan Pills (LGP)-to treat flu, headache, cough, body aches and fever [38].In the past few years, LGP has been widely used in the prevention and treatment of COVID-19 in the Tibetan areas of China [39,40].

Chemistry of Hypecoum Alkaloids
Alkaloids are a large group of N-containing natural compounds with diverse structures and a wide spectrum of pharmacological effects on human health.The Hypecoum species are recognized as important medicinal plants, biosynthesizing isoquinoline alkaloids as major active ingredients.

Protopine Alkaloids
The protopine alkaloids (1-11) represent the major class of alkaloids in the Hypecoum species (Table 2).Their structure includes a tricyclic frame with a carbonyl group at C-14 and methoxy-or methylendioxy-groups at C-2, C-3, C-9 and C-10.Alkaloids

Biosynthesis and Chemotaxonomical Relevance of Isoquinoline Alkaloids
The isoquinoline alkaloids are characteristic secondary metabolites in the Papaveraceae family and their structural types have been used as chemotaxonomical markers to determine relationships between genera [25].However, for a correct chemotaxonomic classification, it is important to understand the biosynthetic pathways of isoquinoline alkaloids in the plant species.
The biosynthetic pathways of the different types of isoquinoline alkaloids identified in Hypecoum species are presented in Figure 1.
The first step in the process is the condensation and cyclization of two tyrosine derivatives, dopamine and 4-hydroxyphenylacetaldehyde, to yield the benzylisoquinoline alkaloid (S)-norcoclaurine, which is the central precursor of all isoquinoline alkaloids.(Figure 1) [55].(S)-Norcoclaurine is sequentially hydroxylated, O-methylated and Nmethylated to give (S)-reticuline.It can subsequently undergo stereochemical inversion to (R)-reticuline, a precursor of the morphinane alkaloids.This inversion is not characteristic of Hypecoum plants, as this type of alkaloid has not been found in the genus [56].
As shown in Figure 1, the transformation of (S)-reticuline via C-C or C-O coupling enzymatic reactions is the branch point that drives the formation of protoberberine and aporphine subclasses [56].
Afterward, the main scaffold of each type undergoes different enzyme reactions such as oxidations, N-and O-methylations or methylenedioxy bridge formation, yielding the structural diversity of alkaloids identified in Hypecoum species (Table 2).Yuan et al. presented a possible biosynthetic pathway of spirobenzylisoquinolines (69-71) C-8 linked via a C-C bond to a 3-aminomaleimide group, in which sibiricine (72) is further attacked by asparagine to hyperectine (70) (Figure 1) [19].

Biological Activities
Based on the long-standing use of Hypecoum species in traditional medicine, different solvent extracts and isolated alkaloids have been evaluated for their effects in the treatment of inflammation, microbial and viral infections, as well as for their analgesic, antitumor, anti-lipase and antioxidant properties.

Anti-Inflammatory Activity
H. erectum is widely used in Tibetan medicine to treat various inflammations, relieve pain and reduce fever [4,5].
In 2021, Yuan et al. [19] verified the traditional use of H. erectum by assessing its anti-inflammatory effect.The authors studied the in vivo anti-inflammatory activity of the methanolic (MeOH) extract, total alkaloid and non-alkaloid fractions of H. erectum at doses of 200, 100 and 50 mg/kg using a carrageenan-induced paw edema model.It was found that the alkaloid fraction showed significant anti-inflammatory activity at all tested doses with inhibition percentages of 60.5%, 54.3% and 47.4%, while the MeOH extract and non-alkaloid fractions showed good effects only at the dose of 200 mg/kg, with inhibition of 53.0% and 43.2%, respectively.
To determine the compounds responsible for the bioactivity of H. erectum, four individual isoquinoline alkaloids-protopine (1), cryptopine (4), allocryptopine (5) and oxohydrastinine (34)-at doses of 100 and 50 mg/kg were assessed for their in vivo antiinflammatory activity using the same carrageenan-induced paw edema animal model [19].The results indicated that compounds 4, 5 and 34 in both doses used possessed significant anti-inflammatory activity equivalent to that of the positive control (aspirin).Therefore, the authors suggest that these isoquinoline alkaloids are the main anti-inflammatory substances in the species.
Other authors also found that the ethanolic extract of H. erectum may reduce LPSinduced inflammation [58].Protopine (1) reduces nitric oxide (NO) production and inhibits the levels of prostaglandin E2 (PGE2) and COX-2 in LPS-stimulated RAW 264.7 murine cell lines in a dose-dependent manner [59].The mechanism of action of 1 involved the suppression of phosphorylation of mitogen-activated protein kinases (MAPKs) and blocking of the activation of nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) [59].

Anticancer Activitiy
The anticancer properties of H. leptocarpum are well known in traditional Tibetan medicine.Therefore, Luo et al. [28], in 2022, evaluated the anticancer activity of the isoquinoline alkaloid corydamine (60).It was found that the alkaloid significantly reduced hepatocellular carcinoma (HCC) growth both in vitro and in vivo.Corydamine (60) dosedependently suppressed HuH7 and LM9 cell proliferation and metastasis in vitro via arresting the cell cycle in the G1/G0 phase and inducing cell apoptosis of the mitochondrial pathway.In vivo studies in LM9 xenografted nude mice showed that a 21 day therapy with 60 at a dose 50 mg/kg/day significantly inhibited tumor growth.These findings suggest that 60 may be used as a natural therapeutic agent for the treatment of HCC.
In 2015, Zang et al. [27] evaluated the cytotoxicity of hypecocarpine (43) and leptopidine (46) isolated from H. leptocarpum in human breast cancer cells (MDA-MB-231) with different concentrations (0, 2, 4, 6, 8, 10 g/mL) of the alkaloids for 24 and 48 h, followed by a cell viability assay.The results showed that only 46 exerted a cytotoxic effect on the growth of MDA-MB-231 cells in a dose-and time-dependent manner.In addition, this alkaloid affected intracellular fatty acid biosynthesis in breast cancer cells via downregulating fatty acid synthase expression.
Other authors in the same year evaluated the cytotoxic activity of the 15 isolated alkaloids from H. leptocarpum against three tumor cell lines-human ovarian cancer cell lines (A2780), human cervical cancer cell lines (HeLa) and human hepatocellular carcinoma cell lines (HepG2) [23].This study established that coptisine ( 14) exhibited significant cytotoxic activity against the A2780 and HeLa tumor cell lines with IC 50 values of 31.49and 30.72 µM, respectively, while leptocaramine (62) exhibited moderate cytotoxicity against HepG2 cell lines with an IC 50 value of 22.16 µM.The cytotoxicity of 14 against A2780 and HeLa tumor cell lines is due to the presence of a positive N-ion in the alkaloid molecule.The moderate cytotoxicity of 62 against the HepG2 cancer cell line is related to the number of electron-donating groups in its structure.The presence of the carboxyl group in the alkaloids coryximine (41), leptopidine (46), hendersine B (49), corydamic acid (61) and leptocarpinine B (79) is the probable reason for their low cytotoxicity against the three tumor cell lines [23].

Antibacterial and Antifungal Activity
An in vitro study of the antimicrobial activity of 306 Iranian plants against Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Pseudomonas aeruginosa, Morganella morganii (Gram-negative bacteria), Bacillus subtilis and Staphylococcus aureus (Gram-positive bacteria) and Candida albicans, established that the methanolic extract of H. pendulum L. exhibited a significant effect against E. coli, M. morganii and S. aureus [60].
The antimicrobial activity of tertiary and quaternary alkaloid mixtures from H. ponticum was screened against Gram-positive bacteria (S. aureus 209), Gram-negative bacteria (E. coli and P. aeruginosa) and C. albicans [18].The quaternary alkaloid mixture containing Nmethylstylopine (15), N-methylcanadine (16) and hypepontine (47) showed potent in vitro antibacterial activity against Gram-negative and Gram-positive bacteria, with MIC values of 0.036 mg/mL and 0.072 mg/mL, respectively.Furthermore, the mixture of quaternary alkaloids had remarkable antifungal activity (MIC 0.018 mg/mL), higher than the reference antibiotic, and may be a good candidate for future pharmacological tests.It was noticed that the activity of the isolated alkaloids against all tested microorganisms was lower than the activity of the quaternary alkaloid mixture.This effect is probably induced by a synergistic interaction among the quaternary alkaloids.Only alkaloid 47 demonstrated a good inhibitory effect against Gram-negative bacteria P. aeruginosa, with an MIC value of 0.064 mg/mL.

Antiviral Activity
The plant H. erectum, along with four other Asian plants (Terminalia chebula, Polygonum bistorta, Momordica cochinchinensis and Gentiana decumbens), is included in the herbal formulation Deva-5, used in traditional Mongolian medicine to treat acute infectious diseases [61].The aqueous extracts of these plants were investigated for inhibitory activity against influenza A virus subtype H3N8 [3].The in vitro study revealed that H. erectum extract had strong antiviral activity at concentrations of 0.5-1%, but further research is needed to identify the biologically active components with direct activity.

Analgesic Effect
The analgesic effect of three different fractions (methanolic, alkaloidal and nonalkaloidal) and four major isoquinoline alkaloids (protopine (1), cryptopine (4), allcryptopine (5) and oxyhydrastinin (34)) from H. erectum was evaluated using the acetic acidinduced writhing test [19].The alkaloidal fraction exhibited significant analgesic activity at a dose of 100 mg/kg.At the same dose, only compounds 1 and 34 significantly decreased the amount of stretching or twisting in the acetic acid-induced writhing animal model.These findings support the traditional application of H. erectum for the treatment of inflammatory and pain disorders.

Anti-Lipase Activity
Bustanji et al. [62] studied 29 medicinal plants, including H. dimidiatum Delile, to evaluate their inhibitory potential against hormone sensitive lipase (HSL), which is involved in the regulation of lipid metabolism in the treatment of insulin resistance.In this study, the methanolic extract of H. dimidiatum showed weak antilipase activity, with an inhibitory effect of only 25.0%, compared to the plants Malva nicaeensis All., Haplophyllum buxbaumii (Poir.)G. Donand and Anchusa italica Retz., whose inhibitory potential was over 55% [62].

Antioxidant Activity
The antioxidant activity of Hypecoum species is associated with phenolic compounds [63][64][65].However, there is one study in which the free radical scavenging ability of the alkaloidsprotopine (1), cryptopine (4) and coptisine ( 14)-and the new nonalkaloid compoundhypecoumic acid, isolated from H. erectum-was examined [20].The antioxidant activity was assessed using the DPPH-scavenging assay and the determination of IC 50 values related to the amount of sample required to reduce 50% of free radicals.The most effective antioxidant was found to be hypecoumic acid, with an IC 50 of 86.3 µM, followed by the alkaloids 14, 1 and 4 with IC 50 values 252.6, 345.2 and 430.1 µM, respectively.

Hepatoprotective Effect
The H. erectum aqueous extract had a marked hepatoprotective effect characterized by inhibition of the disturbances in the cholate-synthetic functions of the liver, increase of the bile secretion rate and preservation of the cholate concentration in the bile.The significant increase of the total concentration of secreted bile, as well as the amount of the main constituents in the bile-bilirubin, bile acids and cholesterol-indicated that the choleretic properties of the extract are in the dose range of 50-200 mg/kg [66,67].Also, the H. erectum extract, at an experimental dose of 50 mg/kg, limited liver degeneration in tetracycline-induced hepatitis [68].

Anti-Hyperglycemic Activity
The dipeptidyl peptidase-4 (DPP 4) is essential in the regulation of diabetes mellitus (DM), and DPP4 inhibitors can be used as potential drugs for this disease.Gong and collaborators implemented a new artificial intelligence protocol to test and validate potential inhibitors from the Traditional Chinese Medicine Database, among which was an acidified form of corydamine (60) from H. leptocarpum [54].Combining the constructed models and algorithms, the authors reported that 60 was a potent inhibitor and that H. leptocarpum can be used as a potential and effective medicinal herb for the treatment of DM.

Insecticidal Activity
H. leptocarpum extracts obtained via two extraction methods (constant temperature oscillation method and Soxhlet extraction method) with different polarity solvents (ethanol, methanol, hexane and dichloromethane) were tested for insecticidal activity against Mythimna seperata, a pest of a number of agricultural plants [69].The extracts were found to show a strong growth inhibitory effect on the third instar larvae of M. seperata, and, after 24 to 48 h of feeding, the inhibition rate was over 70%, while with the ethanol extract it was the highest-over 90%.All types of extracts had low gastric and contact toxicity.In another study, acetone extract of H. leptocarpum also showed clear insecticidal activity against M. seperata [70].

Conclusions and Future Perspectives
This review is a summary of the traditional Chinese, Tibetan and Mongolian medicines produced by plants of the genus Hypecoum.To date, the isoquinoline alkaloids are the most-studied natural compounds isolated from the Hypecoum spp., and a total of 86 of them have been identified and classified by structural type.The structural type of isoquinolines are used for chemotaxonomic purposes to determine the relationships between Hypecoum species and other genera in the Papaveraceae family.Pharmacological studies of the isolated alkaloids, as well as some Hypecoum extracts, show that they have anti-inflammatory, antitumor, antibacterial, antiviral, hepatoprotective and antioxidant properties, among others.The alkaloids protopine (1), cryptopine (4), allcryptopine (5), oxyhydrastinin (34) and corydamine (60) have shown multiple potent biological activities and could be good candidates for future pharmacological tests.
Although recent years have seen an increase in the interest of researchers in the plants of the genus, they are still relatively poorly studied.Phytochemical research has only focused on ten Hypecoum species, and data on the composition and biological activity of the remaining species and subspecies belonging to the genus are still lacking.Future studies on the alkaloid profile of these plants would contribute to enriching the diversity of biologically active natural compounds with the potential to improve human health.Furthermore, studies of the chemical composition should include not only the determination of the structural type of compounds but also the study of biosynthesis and metabolic processes, which are essential for the discovery of new medicinal resources.In addition, pharmacological studies of the genus are insufficient and most of the active studies remain only at the level of in vitro experiments of extracts or individual compounds, which hinders their future applications to some extent.Further assessments and in vitro and in vivo studies should be performed to confirm the bioactive compounds, as well as their toxicity, mechanisms of action and the safety of effective doses in humans.Therefore, in the next stage of research, the alkaloids of the Hypecoum species should be subjected to wider phytochemical and pharmacological analyses that will improve the knowledge of the genus.