Steroidal Alkaloids from the Roots of Veratrum mengtzeanum Loes. with Their Anti-Inflammatory Activities

The phytochemical investigation of Veratrum mengtzeanum Loes. roots resulted in the isolation and characterization of two novel, namely Mengtzeanines A (1), Mengtzeanines B (2), and eight known steroidal alkaloids (3–10). Their structural properties were assessed though extensive spectroscopic techniques. All constituents 1–10 were analyzed for suppression of NO formation in LPS-induced RAW264.7 macrophages. Among them, constituent 6 (Verazine) showed inhibition against LPS-induced NO production (IC50 = 20.41 μM). Additionally, compound 6 could inhibit the secretion of IL1β, IL6, and TNFα, and downregulate the productions of iNOS and COX2 in LPS-induced RAW264.7 macrophages. Further experiments revealed that 6 exhibited a potent anti-inflammatory level in LPS-stimulated RAW264.7 macrophages via inhibiting NF-κB, and triggering of Keap1/Nrf2/HO-1 axis, implying that compound 6 may be a promising candidate for treating inflammatory disorders.

Inflammation, mainly refers to a reaction of the body to defense stress caused by the stimulation of various factors, such as tissue damage and infection, which can lead to severe ailments, including cancer, diabetes, and atherosclerosis [17][18][19].
To identify more herbs with therapeutic potential against various ailments, in ing those linked to inflammation and oxidative stress, we conducted a comprehe chemical analysis of the rhizomes and roots of V. mengtzeanum Loes.This effort expe the extraction of two novel Veratramine-group alkaloids, Mengtzeanines A (1) and together with eight known analogues (3)(4)(5)(6)(7)(8)(9)(10). Furthermore, we assessed the ti-inflammatory properties of 1-10 through examining their suppressive consequenc LPS-induced NO formation.Additionally, we investigated the most promising pound (6) to unravel its anti-inflammatory mechanisms in LPS-stimulated RAW macrophages.In the current report, we present the process of extraction, struc characterization, and biological assessment of these constituents.

Isolation and Structural Assessment
Chromatographic isolation of the acid-soluble and alkali-precipitation fractio MeOH extract of roots and rhizomes of V. mengtzeanum Loes. on CC and HPLC affo two novel alkloids-Mengtzeanines A (1), and Mengtzeanines B (2)-and eight kn constituents 3-10 (Figure 1).
Mengtzeanines A (1) was obtained as a white and shapeless powder, which played a quasi-molecular ion [M + H] + at m/z 428.3165 (calcd for C27H41NO3, 428.315 its HRESIMS spectrum, with eight magnitudes of unsaturation.The IR spectra reve the existence of a hydroxyl group (3437 cm −1 ), and a carbonyl group (1702 cm −1 ). 1 H-N On the other hand, overproduction of ROS can cause oxidative stress, which leads to an inflammatory response.Heme oxygenase (HO)-1, which prevents oxidative stress, includes a protective mechanism against ROS-induced oxidative damage.The Keap1/Nrf2 axis is a crucial molecular target for inducers of antioxidant enzymes, leading to upregulation of HO-1 and decreased susceptibility to oxidative stress-associated damage, including inflammation [24][25][26][27][28].
To identify more herbs with therapeutic potential against various ailments, including those linked to inflammation and oxidative stress, we conducted a comprehensive chemical analysis of the rhizomes and roots of V. mengtzeanum Loes.This effort expedited the extraction of two novel Veratramine-group alkaloids, Mengtzeanines A (1) and B (2), together with eight known analogues (3)(4)(5)(6)(7)(8)(9)(10). Furthermore, we assessed the anti-inflammatory properties of 1-10 through examining their suppressive consequences on LPS-induced NO formation.Additionally, we investigated the most promising compound (6) to unravel its anti-inflammatory mechanisms in LPS-stimulated RAW264.7 macrophages.In the current report, we present the process of extraction, structural characterization, and biological assessment of these constituents.

Isolation and Structural Assessment
Chromatographic isolation of the acid-soluble and alkali-precipitation fractions of MeOH extract of roots and rhizomes of V. mengtzeanum Loes. on CC and HPLC afforded two novel alkloids-Mengtzeanines A (1), and Mengtzeanines B (2)-and eight known constituents 3-10 (Figure 1).

Biological Studies 2.3.1. Anti-Inflammatory Property through the Suppression of LPS-Induced NO Formation
To evaluate the anti-inflammatory activities of the constituents identified from the rhizomes and roots of V. mengtzeanum Loes., we suppressed NO generation in LPS-induced RAW264.7 cell lines.The inhibitory effects of 1-10 on NO production by macrophages are presented in Figure 4 and LMMA was employed as a positive control.Among the isolated constituents, 6 and 10 had a NO inhibitory effect on LPS-stimulated RAW264.7 murine macrophages (Figure 4A).At a concentration of 50 µM, compound 6 showed a similar effect to the positive control.Importantly, at a concentration of 50 µM, compound 6 did not exert any significant impact on RAW264.7 macrophage viability (Figure 4B).Compound 6, identified as Verazine, a veratraminetype isosteroidal alkaloid category, demonstrated the most potent inhibitory activity, as evidenced by an IC 50 value of 20.41 µM (Figure 4C), which was comparable to the positive control L-NMMA (IC 50 = 21.86 µM).Therefore, compound 6 (Verazine) was selected for further analysis.
ules 2023, 28 To evaluate the anti-inflammatory activities of the constituents identified from rhizomes and roots of V. mengtzeanum Loes., we suppressed NO generatio LPS-induced RAW264.7 cell lines.The inhibitory effects of 1-10 on NO productio macrophages are presented in Figure 4 and LMMA was employed as a positive con Among the isolated constituents, 6 and 10 had a NO inhibitory effect on LPS-stimu RAW264.7 murine macrophages (Figure 4A).At a concentration of 50 µM, compou showed a similar effect to the positive control.Importantly, at a concentration of 50 compound 6 did not exert any significant impact on RAW264.7 macrophage viab (Figure 4B).Compound 6, identified as Verazine, a veratramine-type isosteroidal alk category, demonstrated the most potent inhibitory activity, as evidenced by an IC50 v of 20.41 µM (Figure 4C), which was comparable to the positive control L-NMMA (I 21.86 µM).Therefore, compound 6 (Verazine) was selected for further analysis.To assess whether the suppressive effect of Verazine on NO formation attributable to the downregulated translation of iNOS and COX-2, we conducte examination of the productions of these enzymes.iNOS and COX-2 are pivotal in pathogenesis of inflammatory diseases as they enhance vascular permeability and in tissue damage.In our study, it was observed that Verazine significantly downregu the productions of both iNOS and COX-2 in LPS-induced RAW264.7 macrophages ure 5A-C).Apart from NO, several pivotal pro-inflammatory cytokines, including TN IL1β, and IL6, are involved in orchestrating inflammatory responses (including tissue degeneration, exudation, and proliferation), causing damage to cells and tis The inhibition of these mediators is a promising strategy for treating inflammatory orders.In our work, we found a dose-dependent decrease in the release of TNFα, I and IL6 in LPS-stimulated RAW264.7 macrophages following treatment with Vera (Figure 5D-F), and BAY was employed as a positive control.These findings strongl dicate that Verazine s anti-inflammatory effects are closely linked to its ability to press these pro-inflammatory cytokines.

Verazine Inhibits the Release of TNFα, IL1β, and IL6, and Suppresses the Production of iNOS and COX2
To assess whether the suppressive effect of Verazine on NO formation was attributable to the downregulated translation of iNOS and COX-2, we conducted an examination of the productions of these enzymes.iNOS and COX-2 are pivotal in the pathogenesis of inflammatory diseases as they enhance vascular permeability and induce tissue damage.In our study, it was observed that Verazine significantly downregulated the productions of both iNOS and COX-2 in LPS-induced RAW264.7 macrophages (Figure 5A-C).Apart from NO, several pivotal pro-inflammatory cytokines, including TNFα, IL1β, and IL6, are involved in orchestrating inflammatory responses (including local tissue degeneration, exudation, and proliferation), causing damage to cells and tissues.The inhibition of these mediators is a promising strategy for treating inflammatory disorders.In our work, we found a dose-dependent decrease in the release of TNFα, IL1β, and IL6 in LPS-stimulated RAW264.7 macrophages following treatment with Verazine (Figure 5D-F), and BAY was employed as a positive control.These findings strongly indicate that Verazine's anti-inflammatory effects are closely linked to its ability to suppress these pro-inflammatory cytokines.

Verazine Suppresses the Triggering of NF-κB Signaling
The activation of NF-κB, a transcription factor comprising p65 and p50 subunits, translocates into the nucleus, and induces various pro-inflammatory mediators or cytokines, including IL6, IL1β, and TNFα, triggering a series of inflammatory responses.In order to ascertain whether Verazine s anti-inflammatory efficacy was linked to alterations in NF-κB signaling, we conducted an initial investigation into its impact on key components, including p65, p-p65, and pIκBα (Figure 6).As anticipated, exposure to LPS led to a substantial increase in the degradation and phosphorylation of IκBα, a critical inhibitor of NF-κB activation.In contrast, treatment with Verazine (2.5-30 µM) effectively suppressed the phosphorylation and degradation of IκBα.Additionally, LPS considerably elevated the phosphorylation of NF-κB p65, a modification associated with its transcriptional activity and the initiation of NF-κB-mediated responses in macrophages.Importantly, Verazine treatment conspicuously abrogated the phosphorylation of NF-κB p65.Given NF-κB s pivotal role in the context of inflammation, we thus postulated that Verazine s suppression of NF-κB activity translated into the inhibition of target gene transcription, including COX-2, iNOS, IL6, IL1β, and TNFα, thereby conferring upon it a potent anti-inflammatory effect.

Verazine Suppresses the Triggering of NF-κB Signaling
The activation of NF-κB, a transcription factor comprising p65 and p50 subunits, translocates into the nucleus, and induces various pro-inflammatory mediators or cytokines, including IL6, IL1β, and TNFα, triggering a series of inflammatory responses.In order to ascertain whether Verazine's anti-inflammatory efficacy was linked to alterations in NF-κB signaling, we conducted an initial investigation into its impact on key components, including p65, p-p65, and pIκBα (Figure 6).As anticipated, exposure to LPS led to a substantial increase in the degradation and phosphorylation of IκBα, a critical inhibitor of NF-κB activation.In contrast, treatment with Verazine (2.5-30 µM) effectively suppressed the phosphorylation and degradation of IκBα.Additionally, LPS considerably elevated the phosphorylation of NF-κB p65, a modification associated with its transcriptional activity and the initiation of NF-κB-mediated responses in macrophages.Importantly, Verazine treatment conspicuously abrogated the phosphorylation of NF-κB p65.Given NF-κB's pivotal role in the context of inflammation, we thus postulated that Verazine's suppression of NF-κB activity translated into the inhibition of target gene transcription, including COX-2, iNOS, IL6, IL1β, and TNFα, thereby conferring upon it a potent anti-inflammatory effect.In the aforementioned experiments, we observed that Compound 6, also known as Verazine, exhibited a moderate inhibitory effect on the release of inflammatory factors induced by LPS.This led us to hypothesize that Verazine might play a role in mitigating LPS-mediated oxidative stress.Our observations, as depicted in Figure 7A, revealed a substantial increase in green fluorescence in RAW264.7 macrophages following LPS treatment.However, upon treatment with Verazine, the fluorescence intensity gradually diminished, indicating a significant reduction in the levels of ROS.This reduction was further confirmed through both fluorescence microscopy and flow cytometry analyses.
Nrf2 and NF-κB represent significant transcription factors responsible for governing the expression of antioxidant and pro-inflammatory genes, respectively.Oxidative stress can produce excessive ROS, leading to oxidative damage to cells, which is associated with the occurrence of inflammation disease.These findings collectively demonstrate that Verazine effectively inhibits the production and accumulation of ROS induced by LPS in RAW264.7 macrophages.Oxidative stress can be alleviated through the Nrf2/HO-1 pathway.Antioxidant molecules enter cells, Nrf2 separates from Keap1, translocates to the nucleus, and upregulates HO-1 expression, exerting an anti-inflammatory effect.In this study, Verazine can significantly reduce ROS generation in LPS-induced RAW264.7 macrophages.However, the nuclear level of Nrf2 and HO-1 was significantly increased In the aforementioned experiments, we observed that Compound 6, also known as Verazine, exhibited a moderate inhibitory effect on the release of inflammatory factors induced by LPS.This led us to hypothesize that Verazine might play a role in mitigating LPS-mediated oxidative stress.Our observations, as depicted in Figure 7A, revealed a substantial increase in green fluorescence in RAW264.7 macrophages following LPS treatment.However, upon treatment with Verazine, the fluorescence intensity gradually diminished, indicating a significant reduction in the levels of ROS.This reduction was further confirmed through both fluorescence microscopy and flow cytometry analyses.
Nrf2 and NF-κB represent significant transcription factors responsible for governing the expression of antioxidant and pro-inflammatory genes, respectively.Oxidative stress can produce excessive ROS, leading to oxidative damage to cells, which is associated with the occurrence of inflammation disease.These findings collectively demonstrate that Verazine effectively inhibits the production and accumulation of ROS induced by LPS in RAW264.7 macrophages.Oxidative stress can be alleviated through the Nrf2/HO-1 pathway.Antioxidant molecules enter cells, Nrf2 separates from Keap1, translocates to the nucleus, and upregulates HO-1 expression, exerting an anti-inflammatory effect.In this study, Verazine can significantly reduce ROS generation in LPS-induced RAW264.7 macrophages.However, the nuclear level of Nrf2 and HO-1 was significantly increased after treatment with Verazine, while the expression of Keap1 was reduced.This suggests that Verazine alleviates oxidative stress in LPS-induced RAW264.7 macrophages via activating the Nrf2/HO-1 pathway (Figure 7B).
after treatment with Verazine, while the expression of Keap1 was reduced.This suggests that Verazine alleviates oxidative stress in LPS-induced RAW264.7 macrophages via activating the Nrf2/HO-1 pathway (Figure 7B).

Plants
Veratrum mengtzeanum predominantly inhabits vegetation types characterized by limestone shrubland.The rhizomes and roots of V. mengtzeanum Loes., harvested from the Yinshan Mountains (24.492582 • N, 103.166481 • E), Gejiu City, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province, China, in October 2020, and authenticated by Professor Guo-dong Li, YNUCM, were processed for extraction and isolation.A voucher specimen (20200012) was meticulously prepared at the Yunnan Agricultural University.

Cell Culture
Cell line was cultivated in DMEM fortified with 10% FBS and penicillin-streptomycin, and maintained at 37 • C and 5% CO 2 .

MTT Assays
MTT assays were assessed using a prior method with slight modifications [39].

NO Inhibition Assays
NO inhibition assays were assessed using a prior method with slight modifications [40].

ELISA
RAW264.7 macrophages (1 × 10 6 cells/well) were raised in a platter, followed by overnight incubation at 37 • C.After pre-treatment with Verazine for 2 h, the cells were triggered with LPS (1 µg/mL) for an additional 18 h.Then, 440 µL of RIPA buffer containing 1 mM PMSF was used to treat the lysed cells, and the cells were ruptured immediately on ice.After 30 min, the protein was scraped with a clean tip.The protein was harvested and centrifuged (15,000× g, 4 • C, 10 min).The levels of IL1β, IL6, and TNFα were determined using ELISA kits (ABclonal, Technology, Beijing, China).

Western Blot
After pre-treatment with Verazine for 2 h, RAW264.7 macrophages were endured to LPS for an additional 18 h.The cells were ruptured with RIPA buffer comprising 1% phosphatase and protease inhibitors.Protein concentrations were detected using a BCA kit.

Figure 4 .
Figure 4. Evaluation of the anti-inflammatory properties of isolated compounds on LPS-stimu NO generation in RAW264.7 macrophages.After pre-treatment with the tested compound µM) for 2 h, RAW264.7 macrophages were exposed to LPS for an additional 18 h.The NO co (A) and cell viability (B) were assessed with Griess reagent and MTS reagent, respectively.(C IC50 value of Verazine against LPS-stimulated NO generation in RAW264.7 macrophages wa termined (inhibition rate reaches 50% for the dashed line).Means ± SD, n = 3.

Figure 4 .
Figure 4. Evaluation of the anti-inflammatory properties of isolated compounds on LPS-stimulated NO generation in RAW264.7 macrophages.After pre-treatment with the tested compounds (50 µM) for 2 h, RAW264.7 macrophages were exposed to LPS for an additional 18 h.The NO content (A) and cell viability (B) were assessed with Griess reagent and MTS reagent, respectively.(C) The IC 50 value of Verazine against LPS-stimulated NO generation in RAW264.7 macrophages was determined (inhibition rate reaches 50% for the dashed line).Means ± SD, n = 3.
a : 125 MHz, δ in ppm, J in Hz; b : 500 MHz, δ in ppm, J in Hz.