Cytotoxic potential and metabolomic profiling of alkaloid rich fraction of Tylophora indica leaves.

Tylophora indica (Burm f.) Merrill, belong to family Asclepiadaceae, is considered to be a natural remedy with high medicinal benefits. The objective of this work is to assess the metabolomic profile of T. indica leaves enriched in alkaloids, as well as to evaluate the in vitro cytotoxicity of these leaves using the MTT assay on human breast MCF-7 and liver HepG2 cancer cell lines. Dried leaves of T. indica were extracted by sonication, using methanol containing 2 % (v/v) of acetic acid and obtained fraction was characterized by HPTLC and UPLC-MS. The UPLC-MS study yielded a preliminary identification of 32 metabolites, with tylophorine, tylophorine B, tylophorinine, and tylophorinidine being the predominant metabolites. The cytotoxicity of the extract of T. indica was evaluated on HepG2 and MCF-7 cell lines, yielding inhibitory concentration (IC50) values of 75.71 µg/mL and 69.60 µg/mL, respectively. Data suggested that the phytochemical screening clearly showed presence of numerous secondary metabolites with moderate cytotoxic efficacy. In conclusion, the future prospects of T. indica appear promising for the advancement of phytopharmaceutical-based anticancer medications, as well as for the design of contemporary pharmaceuticals in the field of cancer chemotherapy.

Design, Synthesis and in Vitro Evaluation of Tylophorine Derivatives as Possible Antitumor Agents.

Structural simplification and modification of natural products are always very important resources to antitumor drugs. By introducing various aminomethyl groups and amide groups into the phenanthrene ring of tylophorine, a novel series of tylophorine derivatives have been designed and synthesized, and their antiproliferative activities against MCF-7, A549 and HepG-2 cells have been evaluated, too. The results indicated that most of the prepared compounds exhibited good antitumor activities. Especially, one compound with an {ethyl[2-(morpholin-4-yl)ethyl]amino}methyl group at the side chain exhibited the most significant cytotoxic effects.

5-Oxo-1-[(2,3,6,7-tetramethoxy-9-phenanthrenyl)methyl]-L-proline Inhibits Hepatitis C Virus Entry.

Hepatitis C virus (HCV) is the major causative agent of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. The recent development of highly effective direct-acting antivirals (DAAs) has revolutionized the treatment of HCV patients. However, these DAAs are exorbitantly expensive for the majority of HCV patients worldwide. Moreover, these drugs still show genotypic difference in cure rate and have some resistant-associated variants. Tylophorine, a natural compound derived from Tylophora indica plants, is known to have anti-inflammatory and anti-cancerous growth activities. In the present study, we showed that two tylophorine intermediates, 5-Oxo-1-[(2,3,6,7-tetramethoxy-9-phenanthrenyl) methyl]-L-proline (O859585) and 2,3,6,7-tetramethoxy-9-phenanthrenecarboxylic acid (T298875), displayed anti-HCV activity with an EC50 of 38.25 µM for T298875 and 29.11~35.3 µM for O859585 in various HCV genotypes. We demonstrated that O859585 efficiently blocked HCV attachment by neutralizing free viral particles without affecting other stages of the HCV life cycle and interferon stimulation. O859585 interrupted binding between HCV E2 and CD81. Of note, co-treatment of O859585 with either interferon alpha (IFNα) or sofosbuvir exerted either an additive or synergistic antiviral activity in HCV-infected cells with no measurable effect on cell viability. Most importantly, O859585 in combination with IFNα and sofosbuvir exhibited synergistic effects on anti-HCV activity in primary human hepatocytes. Collectively, these data suggest that O859585 may be a novel antiviral agent for HCV therapy.

The Potential Impacts of Tylophora Alkaloids and their Derivatives in Modulating Inflammation, Viral Infections, and Cancer.

Cancer chemotherapies or antitumor agents mainly remain the backbone of current treatment based on killing the rapidly dividing cancer cell such as tylophora alkaloids and their analogues which have also demonstrated anticancer potential through diverse biological pathways including regulation of the immune system. The introduction of durable clinically effective monoclonal antibodies, however, unmasked a new era of cancer immunotherapies. Therefore, the understanding of cancer pathogenesis will provide new possible treatment options, including cancer immunotherapy and targeted agents. Combining cytotoxic agents and immunotherapies may offer several unique advantages that are complementary to and potentially synergistic with biologic modalities. Herein, we highlight the dynamic mechanism of action of immune modulation in cancer and the immunological aspects of the orally active antitumor agents tylophora alkaloids and their analogues. We also suggest that future cancer treatments will rely on the development of combining tumor-targeted agents and biologic immunotherapies.

A General Cp*CoIII -Catalyzed Intramolecular C-H Activation Approach for the Efficient Total Syntheses of Aromathecin, Protoberberine, and Tylophora Alkaloids.

Herein, we report a Cp*CoIII -catalyzed C-H activation approach as the key step to create highly valuable isoquinolones and pyridones as building blocks that can readily be applied in the total syntheses of a variety of aromathecin, protoberberine, and tylophora alkaloids. This particular C-H activation/annulation reaction was achieved with several terminal as well as internal alkyne coupling partners delivering a broad scope with excellent functional group tolerance. The synthetic applicability of this protocol reported herein was demonstrated in the total syntheses of two Topo-I-Inhibitors and two 8-oxyprotoberberine cores that can be further elaborated into the tetrahydroprotoberberine and the protoberberine alkaloid core. Moreover these building blocks were also transformed to six different tylophora alkaloids in expedient fashion.

Screening of promising chemotherapeutic candidates from plants against human adult T-cell leukemia/lymphoma (IV): phenanthroindolizidine alkaloids from Tylophora tanakae leaves.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature peripheral T lymphocytes caused by human T-cell lymphotropic virus type 1 (HTLV-1). There are an estimated 5 million to 20 million HTLV-1-infected individuals worldwide; their lifetime risk of developing ATL is 3-5 %, and high HTLV-1 proviral loads have been shown to be an independent risk factor. Although conventional chemotherapeutic regimens used against other malignant lymphomas have been administered to ATL patients, the prognosis is often poor. In previous studies, we screened 459 extracts from 344 plants to isolate components exhibiting antiproliferative activity against HTLV-1-infected T-cell lines (MT-1 and MT-2). In our continuing search for potential anti-HTLV-1 natural products, 15 extracts of Asclepiadaceae plants were further tested against MT-1 and MT-2 cells. The MeOH extract of aerial parts of Tylophora tanakae showed antiproliferative activity. Activity-guided fractionation resulted in the isolation of 6 phenanthroindolizidine alkaloids (including a new compound), and we examined their antiproliferative activity against MT-1 and MT-2 cells. The EC50 value of some of the alkaloids was in the low nanomolar range, comparable to that of the clinically used antineoplastic drug doxorubicin. Structure-activity relationship analyses suggested that a 14ß-hydroxy moiety is essential for activity against HTLV-1-infected T cells. In contrast, the presence of a 2-methoxy moiety, a 7-methoxy moiety, or an N-oxide moiety appears to reduce the potency of the antiproliferative activity against HTLV-1-infected T cells.

Tylophorine, a phenanthraindolizidine alkaloid isolated from Tylophora indica exerts antiangiogenic and antitumor activity by targeting vascular endothelial growth factor receptor 2-mediated angiogenesis.

BACKGROUND: Anti-angiogenesis targeting VEGFR2 has been considered as an important strategy for cancer therapy. Tylophorine is known to possess anti-inflammatory and antitumor activity, but its roles in tumor angiogenesis, the key step involved in tumor growth and metastasis, and the involved molecular mechanism is still unknown. Therefore, we examined its anti-angiogenic effects and mechanisms in vitro and in vivo. METHODS: We used tylophorine and analyzed its inhibitory effects on human umbilical vein endothelial cells (HUVEC) in vitro and Ehrlich ascites carcinoma (EAC) tumor in vivo. RESULTS: Tylophorine significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, migration, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR2 tyrosine kinase activity and its downstream signaling pathways including Akt, Erk and ROS in endothelial cells. Using HUVECs we demonstrated that tylophorine inhibited VEGF-stimulated inflammatory responses including IL-6, IL-8, TNF-α, IFN-γ, MMP-2 and NO secretion. Tylophorine significantly inhibited neovascularization in sponge implant angiogenesis assay and also inhibited tumor angiogenesis and tumor growth in vivo. Molecular docking simulation indicated that tylophorine could form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR2 kinase unit. CONCLUSION: Tylophorine exerts anti-angiogenesis effects via VEGFR2 signaling pathway thus, may be a viable drug candidate in anti-angiogenesis and anti-cancer therapies.

Brine shrimp cytotoxicity of crude methanol extract and antispasmodic activity of α-amyrin acetate from Tylophora hirsuta wall.

BACKGROUND: We have previously reported that aerial parts of Tylophora hirsuta have antispasmodic profile. The current work is an attempt for isolation of pharmacologically active compound(s) that contribute for its antispasmodic activity. METHODS: Preliminary phytochemical screening for crude methanol extract of Tylophora hirsuta (Th.Cr) is performed. Brine shrimp cytotoxicity of crude methanol extract is performed. Column chromatography was used for isolation of compounds. Mass spectroscopy, H(1) NMR and C(13) NMR were used for structural determination of compounds. α-amyrin acetate was tried for possible spasmolytic activity in rabbit's jejunal preparations and KCl-induced contractions. RESULTS: Th.Cr tested positive for saponins, alkaloids, flavonoids and terpenoids. Compound 1 was isolated as α-amyrin acetate. Compound 2 was heptaeicosanol. Crude methanol extract tested positive for brine shrimp cytotoxicity with LC(50) 492.33± 8.08 mg/ml. Compound 1 tested positive for antispasmodic activity on spontaneous rabbits' jejunum preparations with EC(50) (60 ± 2) × 10(-5)M. The compound also tested positive on KCl induced contractions with EC(50) (72 ± 3) × 10(-5)M. CONCLUSIONS: The present work confirms that α-amyrin acetate is has antispasmodic profile and the relaxant effect may be attributed to α-amyrin acetate which is a major compound.

Left, right, or both? On the configuration of the phenanthroindolizidine alkaloid tylophorine from Tylophora indica.

The alkaloid (-)-tylophorine was isolated from a sample of Tylophora indica, and the crude extract was analyzed by HPLC/MS(n) and chiral HPLC/MS. While the literature states that the naturally occurring form of this alkaloid is the R-enantiomer and that its S-antipode is usually not found in nature, we confirmed the hypothesis of Govindachari and Nagarajan that natural levorotatory tylophorine is indeed a nearly racemic mixture with a slight excess of the R-enantiomer.

Antifungal activity of hydrochloride salts of tylophorinidine and tylophorinine.

The antimicrobial efficacy of two phenanthroindolizidine alkaloids, tylophorinidine hydrochloride (TdnH) and tylophorinine hydrochloride (TnnH), isolated from the plant Tylophora indica (local name, Antamul) was evaluated. These were screened for in vitro antifungal and antibacterial activities. Both compounds exhibited potent antifungal activity displaying minimum inhibitory concentrations (MIC) in the range of 2-4 microg/mL for TdnH and 0.6-2.5 microg/mL for TnnH against Candida species.

Interaction studies of an anticancer alkaloid, (+)-(13aS)-deoxytylophorinine, with calf thymus DNA and four repeated double-helical DNAs.

BACKGROUND: Phenanthroindolizidine alkaloids are a family of plant-derived compounds with significant antineoplastic activity. The specific biomolecular targets of these alkaloids have not yet been clearly identified. (+)-(13aS)-deoxytylophorinine is a new phenanthroindolizidine alkaloid originally extracted from the roots of Tylophora atrofolliculata and Tylophora ovata in our institute. (+)-(13aS)-deoxytylophorinine exerts both in vitro and in vivoanticancer activities. METHODS: The in vivo anticancer effects and toxicity of this compound were investigated in mice, and interactions between this compound and double-helical DNA sequences were studied in detail with circular dichroic spectroscopy and fluorescence spectroscopy. Viscosity measurements were applied to check the interactive mode between this compound and DNA. RESULTS: Potent anticancer effects were observed in vivo. Also, concentration-dependent interactions were observed and this compound seemed to interact in a sequence-specific manner with AT-repeated sequences of double-helical DNA. Such interactions were proved to be intercalating by viscosity measurements. CONCLUSIONS: Anticancer alkaloid (+)-(13aS)-deoxytylophorinine can have sequence-specific interactions with DNA in an intercalating manner.

Isolation and biological activities of phenanthroindolizidine and septicine alkaloids from the Formosan Tylophora ovata.

An investigation of alkaloids present in the leaves and stems of Tylophora ovata led to the isolation of two new septicine alkaloids and one new phenanthroindolizidine alkaloid, tylophovatines A, B, C (1, 2, and 5), respectively, together with two known septicine and six known phenanthroindolizidine alkaloids. The structures of the new alkaloids 1, 2, and 5 were established by means of spectroscopic analyses. These eleven alkaloids show in vitro anti-inflammatory activities with IC50 values ranging from 84 nM to 20.6 µM through their suppression of nitric oxide production in RAW264.7 cells stimulated by lipopolysaccharide and interferon-γ. Moreover, these substances display growth inhibition in HONE-1, NUGC-3, HepG2, SF-268, MCF-7, and NCI-H460 cancer cell lines, with GI50 values ranging from 4 nM to 24.2 µM. In addition, tylophovatine C (5) and 13a(S)-(+)-tylophorine (7) were found to exhibit potent in vivo anti-inflammation activities in a rat paw edema model. Finally, structure­activity relationships were probed by using the isolated phenanthroindolizidines and septicines. Phenanthroindolizidines are suggested to be divided into cytotoxic agents (e.g., 10 and 11) and anti-inflammation based anticancer agents (e.g., 5­9).

Anticancer effect and neurotoxicity of S-(+)-deoxytylophorinidine, a new phenanthroindolizidine alkaloid that interacts with nucleic acids.

Phenanthroindolizidine alkaloids are a family of plant-derived compounds with significant antineoplastic activity as well as other effects like antiamebicidal, antiviral, and anti-inflammatory activities. The specific biomolecular targets of these compounds have not yet been clearly identified. S-(+)-Deoxytylophorinidine (CAT) is a new phenanthroindolizidine alkaloid, originally extracted from the roots of Tylophora atrofolliculata and Tylophora ovata. Potent anticancer activity was observed in vitro and in vivo. Neurotoxicity of CAT was also studied and it was far less serious than that of vinblastine. Interactions between this compound and DNA had been studied in detail in our laboratory previously, and we further studied its interactions with RNA.

Secoiridoids and xanthones from Tylophora secamonoides Tsiang.

A new secoiridoid, secamonoide A (1), and a new xanthone glycoside, secamonoide B (2), together with nine known compounds, were isolated from the aerial parts of Tylophora secamonoides Tsiang. Their structures were elucidated on the basis of spectroscopic methods. An antimicrobial bioassay showed that secamonoides A and B exhibited weak activities (MIC values greater than 100 mg/ml) against some hospital bacteria in vitro.

Structural analogs of tylophora alkaloids may not be functional analogs.

Phenanthroindolizidine-based tylophora alkaloids have been reported to have potential antitumor, anti-immuno and, anti-inflammatory activity. The structure-activity relationships of a series of tylophora alkaloids were studied to guide future drug design. Our results indicate that although these compounds are structural analogs, their potency of cytotoxicity, selectivity against NF-kappaB signaling pathway, and their inhibitory effects against protein and nucleic acid synthesis are different. Because they do not have an identical spectrum of targets, the studied compounds are structural, but may not be functional analogs.

On-line identification of phenanthroindolizidine alkaloids in a crude extract from Tylophora atrofolliculata by liquid chromatography combined with tandem mass spectrometry.

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with sequential mass spectrometry (LC/MS(n)) were applied to identify trace-level phenanthroindolizidine alkaloids in crude extracts from Tylophora atrofolliculata. Based on the relationship between the characteristic fragmentation reactions and the structural features of related compounds of known structure from this plant, the bioactive crude extract was analyzed in detail by positive and negative ion ESI-MS(n), LC/UV-MS and LC/MS(n) techniques. A total of nine constituents in the crude extract were identified rapidly, including several isomers; seven of these constituents are new and two are known compounds. The structures of four of these constituents were subsequently confirmed by nuclear magnetic resonance (NMR) and accurate mass measurements using high-resolution fast-atom bombardment mass spectrometry (FAB-HRMS).