Plant-derived isoquinoline alkaloids that target ergosterol biosynthesis discovered by using a novel antifungal screening tool.

The ergosterol pathway is a prime antifungal target as it is required for fungal survival, yet is not involved in human homeostasis. Methods to study the ergosterol pathway, however, are often time-consuming. The minimum inhibitory concentration (MIC) assay is a simple research tool that determines the lowest concentration at which a novel antimicrobial is active in vitro with limited scope to determine the mechanism of action for a drug. In this study, we show that by adding hydrogen peroxide, an oxidative stressor, or glutathione (GSH), an antioxidant, to modify a commonly performed MIC assay allowed us to screen selectively for new antifungal drugs that target ergosterol biosynthesis in fungi. A human pathogen and dermatophyte, Microsporum gypseum, was used as a test organism. When exposed to ergosterol targeting drugs, the hydrogen peroxide treatment significantly decreased fungal survival by reducing ergosterol in the cell wall, whereas GSH increased survival of M. gypseum. Further, by performing a series of experiments with M. gypseum and Trichophyton rubrum, it was determined that the oxidative stress from hydrogen peroxide causes cell death at different developmental stages based on fungal species. These findings allow us to describe a simple, high-throughput method for simultaneously screening new antifungal drugs for activity and effects on the ergosterol pathway. By using this tool, two isoquinoline alkaloids were discovered to be potent inhibitors of ergosterol biosynthesis in vitro by reducing the amount of ergosterol without affecting the expression of 1,3-ß-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo.

Systematic identification of compounds in Macleaya microcarpa by high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry combined with mass spectral fragmentation behavior of Macleaya alkaloids.

RATIONALE: Macleaya microcarpa (Maxim.) Fedde belongs to the genus Macleaya of the Papaveraceae family. Benzylisoquinoline alkaloids (BIAs) are considered the main bioactive constituents of M. microcarpa. METHODS: Using high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (HPLC/QTOFMS/MS) we identified BIAs in the aerial parts of M. microcarpa in the early flowering stage. Target profiling and identification of BIAs in the extracted samples from the fresh aerial parts of M. microcarpa were exclusively based on a personal, accurate, mass database of known compounds and the mass spectral fragmentation behavior of Macleaya alkaloids. RESULTS: A total of 97 alkaloids, comprising 7 benzyltetrahydroisoquinolines, 1aporphine, 9 tetraprotoberberines, 3 protoberberines, 2 N-methyltetrahydroprotoberberines, 4 protopines, 47 dihydrobenzophenanthridines, and 24 benzophenanthridines, were identified from the fresh aerial parts of M. microcarpa, and 77 of these were detected for the first time in M. microcarpa. In addition, some of the screened alkaloids were related to the biosynthetic pathways of sanguinarine and chelerythrine. CONCLUSIONS: The integrated method is sensitive and reliable for screening and identifying trace or ultra-trace isoquinoline alkaloids and has contributed to a better understanding of BIAs in the fresh aerial parts of M. microcarpa.

Anticancer and Reversing Multidrug Resistance Activities of Natural Isoquinoline Alkaloids and their Structure-activity Relationship.

The severe anticancer situation as well as the emergence of multidrug-resistant (MDR) cancer cells has created an urgent need for the development of novel anticancer drugs with different mechanisms of action. A large number of natural alkaloids, such as paclitaxel, vinblastine and camptothecin have already been successfully developed into chemotherapy agents. Following the success of these natural products, in this review, twenty-six types of isoquinoline alkaloids (a total of 379 alkaloids), including benzyltetrahydroisoquinoline, aporphine, oxoaporphine, isooxoaporphine, dimeric aporphine, bisbenzylisoquinoline, tetrahydroprotoberberine, protoberberine, protopine, dihydrobenzophenanthridine, benzophenanthridine, benzophenanthridine dimer, ipecac, simple isoquinoline, pavine, montanine, erythrina, chelidonine, tropoloisoquinoline, azafluoranthene, phthalideisoquinoline, naphthylisoquinoline, lycorine, crinane, narciclasine, and phenanthridone, were summarized based on their cytotoxic and MDR reversing activities against various cancer cells. Additionally, the structure-activity relationships of different types of isoquinoline alkaloid were also discussed. Interestingly, some aporphine, oxoaporphine, isooxoaporphine, bisbenzylisoquinoline, and protoberberine alkaloids display more potent anticancer activities or anti-MDR effects than positive control against the tested cancer cells and are regarded as attractive targets for discovery new anticancer drugs or lead compounds.

Medicinal plants of the genus Macleaya (Macleaya cordata, Macleaya microcarpa): A review of their phytochemistry, pharmacology, and toxicology.

In the genus Macleaya, Macleaya cordata and Macleaya microcarpa have been recognized as traditional herbs that are primarily distributed in China, North America, and Europe and have a long history of medicinal usage. These herbs have been long valued and studied for detumescence, detoxification, and insecticidal effect. This review aims to provide comprehensive information on botanical, phytochemical, pharmacological, and toxicological studies on plants in the genus Macleaya. Plants from the genus of Macleaya provide a source of bioactive compounds, primarily alkaloids, with remarkable diversity and complex architectures, thereby having attracted attention from researchers. To date, 291 constituents have been identified and/or isolated from this group. These purified compounds and/or crude extract possess antitumor, anti-inflammatory, insecticidal, and antibacterial activities in addition to certain potential toxicities. Macleaya species hold potential for medicinal applications. However, despite the pharmacological studies on these plants, the mechanisms underlying the biological activities of active ingredients derived from Macleaya have not been thoroughly elucidated to date. Additionally, there is a need for research focusing on in vivo medical effects of Macleaya compounds and, eventually, for clinical trials.

Mass spectrometry-guided isolation of two new dihydrobenzophenanthridine alkaloids from Macleaya cordata.

Two new alkaloids 6-hydroxyethyldihydrochelerythrine (1) and 6-hydroxymethyl-7,8-demethylenedihydrochelerythrine (2) together with two analogues named maclekarpine E (3) and 6-hydroxymethyldihydrosanguinarine (4) were detected primarily from the leaves of Macleaya cordata by their characteristic mass spectrometry (MS) and tandem mass spectrometry (MS/MS). And then isolation of four targeted-compounds was performed by column chromatography and preparation HPLC under the guiding of MS. Finally, their structures were determined by spectrum analysis. Alkaloids 3 and 4 were isolated from this plant medicine for the first time.

Systematic identification of flavonols, flavonol glycosides, triterpene and siraitic acid glycosides from Siraitia grosvenorii using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry combined with a screening strategy.

The fruits of Siraitia grosvenorii are considered to be health-promoting because of the diversity of their bioactive ingredients. In the present study, a screening method, using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC-Q-TOF-MS) combined with a screening strategy, has been established. The technology was used to systematically screening the targeted metabolites, primarily from the complex matrix of S. grosvenorii. The compounds were then identified by their exact masses and characteristic fragment ions, in comparison with the fragmentation behaviors of 19 references. Finally, 122 compounds, including 53 flavonols and flavonol glycosides, 59 triterpene glycosides and 10 siraitic acid glycosides, were screened and identified in 10-, 50- and 80-day fruits, roots, stems and leaves of S. grosvenorii. 98 of them were reported for the first time. Additionally, the distribution of all identified components in different parts of the plant was determined and metabolic networks for flavonol and triterpene glycosides were proposed.

Mass spectrometry-guided isolation of two new benzoquinoline alkaloids from Macleaya cordata.

Two new alkaloids, named 2,3-methylenedioxy-7,10-dimethyl-7,8,9,10-tetrahydro-benzoquinoline (1) and 2,3-methylenedioxy-7,10-dimethyl-8-carboxyl-benzoquinoline (2), were detected primarily from the fruits of Macleaya cordata by their different fragmentation pathways. And then isolation of the two compounds was performed by column chromatography and preparative HPLC under the guiding of mass spectrometry. Finally, their structures were determined by spectroscopic analysis.

[Study on Alkaloids of Fruit from Macleaya cordata].

Objective: To study the alkaloids from the fruit of Macleaya cordata. Methods: The alkaloids were isolated and purified by silica gel column chromatography and preparative high performance liquid chromatography,and the structures were elucidated based on the spectral data. Results: Eight alkaloids were obtained from the fraction of ethanol extract and identified as 6-cyanodihydrosanguinarine( 1),6-acetonyldihydrosanguinarine( 2),6-acetonyldihydrochelerythrine( 3),chelerythrine( 4),dihydrochelerythrine( 5),13,14-dehydrogen-N-methyl-coptisine( 6),N-methyl-canadine( 7) and berberine( 8). Conclusion: Compounds 1 and 6 are new nature products,and compound 7 is isolated from this plant for the first time.

[Chemical Constituents from the Roots of Macleaya cordata].

Objective: To investigate the chemical constituents from the roots of Macleaya cordata. Methods: The compounds were isolated and purified by silica gel,recrystallization and semi-preparative HPLC, their structures were eclucidated by physicochemistry properties, MS and NMR. Results: Seven compounds were isolated and identified as 6-cyanodihydrochelerythrine (1),6-cyanodihydrochelilutine (2),dihydrochelirubine (3),6-methoxynorchelerythrine (4),dihydrosanguinarine (5),6-actonyldihydrosanguinarine (6) and stigmasterol (7). Conclusion: Compounds 1,2 are new natural compounds and compound 4 is obtained from Macleaya cordata for the first time.

Systematic identification of alkaloids in Macleaya microcarpa fruits by liquid chromatography tandem mass spectrometry combined with the isoquinoline alkaloids biosynthetic pathway.

Alkaloids in Macleaya microcarpa were characterized systematically by combining liquid chromatography tandem mass spectrometry (LC-MS/MS) with the biosynthetic pathway of isoquinoline alkaloids. The mass spectral fragmentation behaviors of 16 references belonging to eight types of alkaloids that exist in the biosynthetic pathway of isoquinoline were investigated in detail. The benzyltetrahydroisoquinoline and aporphine alkaloids were distinguished by characteristic losses of the NHR1R2 (R1 and R2 represent the substituent groups of the nitrogen atom) radical and the fragment ions below m/z 200. Tetrahydroprotoberberine, N-methyltetrahydroberberine and protopine alkaloids were differentiated by the retro-Diels-Alder (RDA) reaction, α-cleavage and the [M-H2O](+) and [M-CH4](+) ions. Discrimination of protoberberine, benzophenanthridine and dihydrobenzophenanthridine-type alkaloids can be realized through the characteristic [fragment ion-2H](+), [M-H2O](+), [M-CH4](+), [M+H-CH3CH2CH2OH](+) and [M+H-CH3COCH3](+) ions. Forty-one alkaloids, including one benzyltetrahydroisoquinoline, one aporphine, nine protopines, seven protoberberines, one tetrahydroprotoberberine, three N-methyltetrahydroprotoberberines, five benzophenanthridines and fourteen dihydrobenzophenanthridines, were separated and identified simultaneously. Thirty-three of these were reported for the first time in M. microcarpa. The benzyltetrahydroisoquinoline, aporphine, tetrahydroprotoberberine and N-methyltetrahydroprotoberberine-type alkaloids have not been reported previously in M. microcarpa. This method can be applied to the analysis of herbal medicines that possess the biosynthetic pathway of isoquinoline alkaloids.

23,24-Dihydrocucurbitacin C: a new compound regarded as the next metabolite of cucurbitacin C.

Cucurbitacin C, a bitter substance in Cucumis sativus L., was isolated from green leaves by using phytochemical methods. An analytical method using high-performance liquid chromatography (HPLC) was established for the quantification of cucurbitacin C in different parts of the cucumber plant at different growth periods. Cucurbitacin C was detected in the leaves and stems but not in the female flowers, fruits, roots and leafstalks. The level of cucurbitacin C decreased significantly with the process of young leaves turning old. A new compound named 23,24-dihydrocucurbitacin C, regarded as the next metabolite of cucurbitacin C, was determined unambiguously by HPLC-quadrupole-time-of-flight mass spectrometry and nuclear magnetic resonance.

Structural speculation and identification of alkaloids in Macleaya cordata fruits by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry combined with a screening procedure.

RATIONALE: Alkaloids with significant therapeutic effects are the main active constituents of Macleaya cordata, which is a perennial herb plant in the Papaveraceae family. A systematic and novel method for speculating and identifying the structures of alkaloids in M. cordata fruits by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/Q-TOF-MS) with a screening procedure was reported. METHODS: Investigation of mass spectral fragmentation of alkaloids was carried out based on the tandem mass spectrometry (MS/MS) data analyses of eight reference substances. The skeletons of alkaloids were determined by their ultraviolet spectra (UV) and MS/MS data. The substituent groups of the alkaloids were acquired through a screening procedure developed in our laboratory and MS/MS data. The substituent linkage sites were deduced by MS/MS fragmentation behavior, as well as biosynthetic pathways of related alkaloids. RESULTS: The structures of 21 alkaloids were speculated in this study, 10 of which were reported for the first time in M. cordata. Furthermore, benzyltetrahydroisoquinoline and N-methyltetrahydroprotoberberine-type alkaloids were discovered, which indirectly proved that the biosynthetic pathways of benzophenanthridine alkaloids reported in Eschscholtzia california existed in M. cordata as well. CONCLUSIONS: HPLC/Q-TOF-MS combined with a screening procedure was a systematic and reliable method for speculating and elucidating the structures of alkaloids. This study might be useful for the identification of other compounds in herbal medicines.