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Three new steroidal alkaloids, veratrasines A - C (1-3), along with ten known analogues (4-13) were isolated from the roots of Veratrum stenophyllum. Their structures were elucidated by NMR and HRESIMS data and comparison with the reported data in the literatures. A plausible biosynthetic pathway for 1 and 2 were proposed. Compounds 1, 3, and 8 showed moderate cytotoxic activity against MHCC97H and H1299 cell lines., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Veratrazine A (1), a steroidal alkaloid with a unique 6/5/5 triheterocyclic scaffold as the side chain, was isolated from Veratrum stenophyllum , and its structure was established via spectroscopic analyses and X-ray diffraction. A plausible biosynthetic pathway for 1 is proposed. Bioassy exhibits moderate anti-inflammatory activities in vitro and in vivo .

Ethnopharmacological Relevance: "Li-Lu", the roots and rhizomes of Veratrum grandiflorum (Melianthiaceae), has been historically used as a traditional folk medicine for the treatment of wrist pain, fractures, sores, and inflammation in Yunnan Province, China. However, the anti-inflammatory and analgesic studies of this plant have seldom reported., Aim of the Study: To evaluate the anti-inflammatory and analgesic properties related to the traditional usage of V. grandiflorum both in vitro and in vivo, and further explore the accurate bioactive compounds from the medicinal plant., Materials and Methods: Phytochemical investigation was carried out by chromatographic methods and their structures were established based on extensive spectra and comparison with corresponding data in the reported literatures. Anti-inflammatory activities were assessed by the suppression of lipopolysaccharide-activated inflammatory mediators in RAW 264.7 macrophage cells in vitro. Furthermore, anti-inflammatory and analgesic effects were evaluated based on carrageenan-induced paw edema and acetic acid-stimulated writhing in mice., Results: The methanol extract (ME) of V. grandiflorum significantly alleviated the paw edema caused by carrageenan and the writhing numbers induced by acetic acid. Subsequent phytochemical investigation led to isolated of 21 steroidal alkaloids, including seven new compounds, veragranines C-I (1-7). Anti-inflammatory test indicated that steroidal alkaloids could decrease the expression of cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells at a concentration of 5.0 μg/ml in vitro, comparable to DXM. Moreover, five new steroidal alkaloids (2, 4, 5, 6, and 7) and two major steroidal alkaloids (9 and 13) significantly decreased the numbers of writhing in mice at the doses of 0.5 and/or 1.0 mg/kg (p < 0.01/0.05), roughly comparable to Dolantin™ at 10.0 mg/kg., Conclusions: The investigation supported the traditional use of V. grandiflorum and provided new steroidal alkaloids as potent analgesic agents., (Copyright © 2022. Published by Elsevier B.V.)

Tuberindines A and B ( 1 and 2 ), truffle alkaloids with intriguing structures, were isolated from Tuber indicum and detected in other truffle species. They appear to arise biosynthetically from amino acid and isosaccharinic units. Compound 1 upregulated the expression of organic anion transporters OAT1 and ABCG2 and significantly exhibited antihyperuricemic bioactivity in vitro and in vivo , which might support the value of truffles as a dietary supplement.

Hyperectumine ( 1 ), the first C 19 benzylisoquinoline alkaloid with a complicated ring system, was isolated from Hypecoum erectum and structurally characterized. Its biosynthetic origin should involve a hybrid pattern of C 8 + C 8 + C 1 + C 2 , from which a C 17 benzylisoquinoline alkaloid might be further attacked by a malonamic acid and undergo decarboxylation and cyclization to produce 1 . Compound (-)- 1 exhibited moderate anti-inflammatory activity via suppression of LPS-activated inflammatory mediators in RAW 264.7 macrophage cells.

Objective: To investigate the effects of matrine on the anti-tumor efficiency of H22 murine hepatocarcinoma cell-based vaccine modified by TIM2 gene in vivo., Method: The combinant eukaryotic expression vector pIRES2-EGFP-TIM2 was constructed and transfected into H22 cells by lipofectamin. The monoclone of the positive H22-TIM2 cells and negative control H22-EGFP cells were selected by G418 pressure and limited dilution method in turn. The H22 whole-cell-based vaccine were inoculated to establish the tumor-bearing mouse model, and its oncogenicity and immunogenicity were observed in vivo. Then the matrine was administered to the tumor-bearing mice inoculated by H22-TIM2 cells, H22-EGFP cells and H22 cells, and the inhibitory effect of matrine on tumor was studied., Result: The co-expression of EGFP protein and TIM2 mRNA were detected in H22-TIM2 cells. The rate of tumor formation in mice injected of H22-TIM2 cells was 41%, lower than that of H22 cells and H22-EGFP cells injection (92%) in mice. The growth of tumor were significantly inhibited vaccinated with H22-TIM2 cells in mice. The inhibitory rate of tumor (IR) was 69.2% in mice of H22-TIM2 group, higher than that of mice treated with matrine and H22 cells injection, the later was 67.5%. Matrine could dramatically strengthen the anti-tumor efficiency of H22 cells modified by TIM2 gene, with the highest tumor inhibitory rate (IR) (90.6%) in all the experimental mice. The spleen index, populations of CD4-positive lymphocytes and the ratio of CD4-positive to CD8-positive lymphocytes of spleen in mice vaccinated of H22-TIM2 cells were obviously higher than those in the other groups., Conclusion: The oncogenicity of H22 cells is markedly impaired after modified by TIM2 gene. Matrine can strengthen the inhibitory effect of H22-TIM2 cells on tumor in mice. These data give us important clues to further study the biological role of TIM2 gene in tumor immunity and explore the molecular mechanism of matrine in suppressing tumor.

Objective: To investigate the effects of matrine on the anti-tumor efficiency of TIM2 gene-modified murine hepatocarcinoma H22 cells., Methods: A combined eukaryotic expression vector pIRES2-EGFP-TIM2 was constructed and transfected into H22 cells by lipofectamin. The monoclone of positive H22-TIM2 cells and negative control H22-EGFP cells transfected with pIRES2-EGFP vector were selected by G418 pressure and limited dilution method in turn and were inoculated to establish the tumor-bearing mouse model. Next, matrine was administered to the tumor-bearing mice and the inhibitory effect of matrine was determined., Results: The co-expression of EGFP protein and TIM2 gene was detected in H22 cells selected after TIM2 gene transfecion. After subcutaneous injection of H22-TIM2 cells, the rate of tumor formation (41%) was lower than that of H22 cells and H22-EGFP cells injection (92%) in mice. The tumor growth was significantly inhibited in mice vaccinated with H22-TIM2 cells. After the experiment was completed, the volume of tumors in mice of H22-TIM2 group was 31.34 +/- 9.21 mm3, smaller than those in H22-EGFP group (98.25 +/- 25.23)mm3 and H22 cells group (114.08 +/- 36.45)mm3 (P < 0.01). Matrine dramatically enhanced the anti-tumor efficiency of TIM2 gene-modified H22 cells, with the highest tumor inhibitory rate (IR) 90.6% among the H22-TIM2 group, matrine treatment group and H22-EGFP cells combined with matrine treatment group (69.2%, 67.5% and 70.8%, respectively) in the experimental mice., Conclusion: The tumorigenesity of H22 cells has been markedly impaired after modification by TIM2 gene. Matrine can enhance its inhibitory effect on tumors of H22-TIM2 cells in vivo. These data indicate importance to further study on the biological role of TIM2 gene in tumor immunity and explore the molecular mechanism of matrine in suppressing of tumor growth.

Objective: To investigate the inhibitory effect of matrine on tumor growth in tumor-bearing mice and explore its possible mechanisms of anti-tumor action in vivo., Methods: Hepatocellular carcinoma cells H(22) were subcutaneously injected into BALB/c mice and matrine was administered to the tumor-bearing mice. The kinetics of tumor formation and tumor growth were measured, tumor growth inhibition rate (IR) was calculated, and tumor tissue samples were taken and examined by light and electron microscopy to assess the inhibitory effects of matrine on tumor growth in the mice., Results: Marked inhibitory effect of matrine on the transplanted hepatocellular carcinoma H(22) was observed in the tumor-bearing mice. The inhibitory rates were 62.5% and 60.7% in the groups treated with high and low dosage of matrine, respectively (P < 0.01 vs. control group). The tumor formation was significantly retarded and tumor growth was inhibited in matrine-treated groups compared with those in control mice. Histopathological examination revealed widespread necrosis with massive accumulation of infiltrating lymphocytes and plasmacytes in the tumors. Numerous apoptotic cells and apoptotic bodies were observed in the tumors under the electron microscope., Conclusion: Matrine has marked inhibitory effects on tumor growth in vivo, which is probably related to inhibition of cell division and tumor cell proliferation, directly killing of tumor cells and/or induction of apoptosis and modulation of anti-tumor immune responses.

Objective: To compare gene expression profile in K562 cells induced by matrine, so as to screen for differentiation related genes., Methods: K562 cells were exposed to 0.1 mg/ml matrine for 3 hours, and gene expression profiles in matrine-treated and non-treated cells were studied by cDNA microarray., Results: From 8465 screened genes, 30 differentially expressed genes were found. Among them 18 showed decreased expression including oncogene and proto-oncogene, signal transduction gene, DNA binding and transcription gene etc, 12 showed increased expression including cell receptor gene, immune-associated gene, metabolism-associated gene etc in matrine-treated K562 cells as compared with that in non-treated cell., Conclusion: The genes, that are closely associated with differentiation of can-cer cell, could be the potential targets for cancer treatment.

Background & Objective: The differentiation of K562 cells can be induced by a given concentration of matrine. This study was designed to investigate the mechanism of signal transduction in induced differentiation of K562 cells., Methods: ELISA coupled with streptavidin-biotin system was used to dynamically detect the activity of protein tyrosine kinase and phosphatase in the cytoplasm and the membrane of K562 cells treated by matrine., Results: Matrine inhibited the activity of protein tyrosine kinase in K562 cells, and the inhibitive effect depended on matrine of the concentration within 0.1 mg/ml. The effects of different concentration of matrine on the protein tyrosine phosphatase activity had no significant difference. Activity of the protein tyrosine phosphatase decreased transiently accompanied with the activity of the protein tyrosine kinase in K562 cells treated with 0.1 mg/ml matrine., Conclusions: The change of protein tyrosine kinase activity is involved in the differentiation of K562 cells induced by matrine. The tyrosine kinase activity in cell membrane decreased more rapidly than that in cell cytoplasm, suggesting there be a transmembrane signal transduction. The change of tyrosine phosphatase activity following the kinase indicates the real time regulation of phosphorylation and dephosphorylation. Moreover, the inhibitory effect of matrine on the protein tyrosine kinase shows the characters of specificity and saturation, suggesting the exist of matrine-associated receptor in K562 cells.

Background & Objective: It has been reported that matrine had the anti-tumor activity, and our previous study have provided that matrine had the effects of inhibiting proliferation and inducing differentiation in K562 cell line, but its molecular mechanism is unknown yet., Method: The expression of several proto-oncogenes(c-myc, c-jun, H-ras, p21, and HNF-1 alpha) in K562 cell line treated by 0.2 mg/ml matrine were measured by RT-PCR., Result: The c-myc, c-jun, and HNF-1 alpha mRNA of K562 cells treated by 0.2 mg/ml matrine were dramatically decreased at the early stage (3 h), while the H-ras and p21 mRNA were increased obviously at the same time., Conclusion: The changes of several proto-oncogenes in K562 cells treated by 0.2 mg/ml matrine may be related to inhibiting proliferation and inducing differentiation.

We investigated the effects of Matrine on proliferation by trypan blue exclusion and differentiation by benzidine staining positive cells in K-562 cells, assayed the telomerase activity using PCR-ELISA assay, analyzed cell cycle by fluorescence-activated cell sorter analysis of the DNA content, and also determined the gene expression level of c-myc, N-ras and p53 by northern blot and dot blot analysis. The results showed that with the addition of 0.1 mg/ml Matrine, cell growth was inhibited significantly by 4 days, benizidine-positive cells rose from 1% to 2% in control cells to 15% in treated cells on day 5; treatment of K-562 cells with 0.1 mg/ml Matrine for 5 days resulted in a marked inhibition in telomerase activity, in a manner that correlated with the extent of differentiation; after exposure to Matrine for 72 h, 64.6% cells were arrested in the G1-phase of the cell cycle, the fraction of cells in S-phase had decreased from 56.9% in control cells to 24.4% in differentiated cells, and the levels of N-ras and p53 mRNA were remarkably increased for 24 and 48 h, respectively, c-myc mRNA expression level declined for 24 h and was inhibited significantly for 48 h. Our study confirmed that Matrine plays a significant effect on the inhibition of proliferation cells and inducing differentiation in K-562 cells.