Natural antitussive alkaloids

The experiment in vitro showed that NTS significantly reduced the arginase-1 (marker for M2) expression in a dose-dependent manner but down-regulated the iNOS (marker for M1) expression only at 100μM. In conclusion, our study demonstrated for the first time that NTS has a significant protective effect on BLM-induced pulmonary fibrosis through suppressing the recruitment and M2 polarization of macrophages.[1]

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Neotuberostemonine (NTS) greatly attenuated pulmonary fibrosis induced by bleomycin.[1]

NTS significantly relieved macrophage infiltration in bleomycin-induced lung tissue.[1]

NTS mainly inhibited the M2, rather than M1, polarization both in vivo and in vitro.[1]

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In the mouse macrophage RAW 264.7 cell line, Neotuberostemonine showed no cytotoxicity up to 100 μM as determined by MTT assay.[1]
Neotuberostemonine (1, 10, 100 μM) reduced the production of nitric oxide (NO) induced by LPS stimulation, with inhibition rates of 8.6%, 10.4%, and 14.6%, respectively.[1]
Western blot analysis showed that Neotuberostemonine at 100 μM significantly down-regulated the expression of iNOS (a marker for M1 macrophages) in LPS-stimulated RAW 264.7 cells.[1]
Neotuberostemonine (1, 10, 100 μM) significantly suppressed the IL-4-induced expression of arginase-1 (Arg-1, a marker for M2 macrophages) in RAW 264.7 cells in a dose-dependent manner.[1]
ELISA results indicated that Neotuberostemonine (1, 10, 100 μM) significantly inhibited the elevated levels of TGF-β1 induced by IL-4 in RAW 264.7 cells.[1]

NTS decreases BLM-induced weight loss, mortality and lung index in mice[1]
Administration of BLM in mice is the most commonly model for experimental lung fibrosis. As shown in Fig. 1B, the BLM-injured mice showed significant decrease in the body weight compared with the sham mice. Treatment of these mice with NDN at 40 mg/kg moderately increased the body weight, which was similar with the literature result. NTS treatment at 40 mg/kg (NTS-40) could reduce the body loss induced by BLM during the whole treatment period, and the terminal body weight was...
Neotuberostemonine (NTS) is one of the main antitussive alkaloids in the root of Stemona tuberosa Lour. This study aimed to investigate the effects of NTS on bleomycin (BLM)-induced pulmonary fibrosis in mice and the underlying mechanism. After BLM administration, NTS were orally administered to mice at 20 and 40mg/kg per day from days 8 to 21, with nintedanib as a positive control. The effect of NTS on BLM-induced mice was assessed via histopathological examination by HE and Masson's trichrome staining, TGF-β1 level and macrophage recruitment by immunohistochemical staining, expression of profibrotic media and M1/M2 polarization by western blot. RAW 264.7 cells were used to evaluate whether NTS (1, 10, 100μM) directly affected macrophages. The results revealed that NTS treatment significantly ameliorated lung histopathological changes and decreased inflammatory cell counts in the bronchoalveolar lavage fluid. The over-expression of collagen, α-SMA and TGF-β1 was reduced by NTS. Furthermore, NTS markedly lowered the expression of MMP-2 and TIMP-1 while raised the expression of MMP-9. A further analysis showed that NTS was able to decrease the recruitment of macrophages and to inhibit the M2 polarization in mice lung tissues.

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Pulmonary fibrosis may be partially the result of deregulated tissue repair in response to chronic hypoxia. In this study we explored the effects of hypoxia on lung fibroblasts and the effects of neotuberostemonine (NTS), a natural alkaloid isolated from Stemona tuberosa, on activation of fibroblasts in vitro and in vivo. PLFs (primary mouse lung fibroblasts) were activated and differentiated after exposure to 1% O2 or treatment with CoCl2 (100 μmol/L), evidenced by markedly increased protein or mRNA expression of HIF-1α, TGF-β, FGF2, α-SMA and Col-1α/3α, which was blocked after silencing HIF-1α, suggesting that the activation of fibroblasts was HIF-1α-dependent. NTS (0.1-10 μmol/L) dose-dependently suppressed hypoxia-induced activation and differentiation of PLFs, whereas the inhibitory effect of NTS was abolished by co-treatment with MG132, a proteasome inhibitor. Since prolyl hydroxylation is a critical step in initiation of HIF-1α degradation, we further showed that NTS treatment reversed hypoxia- or CoCl2-induced reduction in expression of prolyl hydroxylated-HIF-1α. With hypoxyprobe immunofiuorescence staining, we showed that NTS treatment directly reversed the lower oxygen tension in hypoxia-exposed PLFs. In a mouse model of lung fibrosis, oral administration of NTS (30 mg·kg-1·d-1, for 1 or 2 weeks) effectively attenuated bleomycin-induced pulmonary fibrosis by inhibiting the levels of HIF-1α and its downstream profibrotic factors (TGF-β, FGF2 and α-SMA). Taken together, these results demonstrate that NTS inhibits the protein expression of HIF-1α and its downstream factors TGF-β, FGF2 and α-SMA both in hypoxia-exposed fibroblasts and in lung tissues of BLM-treated mice. NTS with anti-HIF-1α activity may be a promising pharmacological agent for the treatment of pulmonary fibrosis.Reference: Acta Pharmacol Sin. 2018 Sep;39(9):1501-1512. https://pubmed.ncbi.nlm.nih.gov/29645000/

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In a bleomycin (BLM)-induced pulmonary fibrosis mouse model, oral administration of Neotuberostemonine (20 and 40 mg/kg/day, from days 8 to 21 post-BLM) significantly attenuated body weight loss and improved the cumulative survival rate compared to the model group.[1]
Neotuberostemonine treatment (40 mg/kg) significantly decreased the lung index (lung weight/body weight ratio) in BLM-challenged mice.[1]
Histopathological examination (H&E staining) showed that Neotuberostemonine treatment reduced inflammatory cell infiltration, alveolar septal thickening, and structural destruction in lung tissues, leading to lower inflammation scores.[1]
Neotuberostemonine treatment significantly reduced the total white blood cell count, as well as the counts of macrophages/monocytes and lymphocytes in the bronchoalveolar lavage fluid (BALF) of BLM-induced mice.[1]
Masson's trichrome staining revealed that Neotuberostemonine (20 and 40 mg/kg) significantly reduced collagen deposition in the lungs of BLM-induced mice.[1]
Immunohistochemical staining and western blot analysis demonstrated that Neotuberostemonine treatment down-regulated the expression of the pro-fibrotic markers TGF-β1 and α-smooth muscle actin (α-SMA) in lung tissues.[1]
Western blot analysis showed that Neotuberostemonine treatment modulated the expression of extracellular matrix regulators: it decreased the BLM-induced overexpression of MMP-2 and TIMP-1, increased the expression of MMP-9, and consequently elevated the MMP-9/TIMP-1 ratio in lung tissues.[1]
Immunohistochemical staining for F4/80 (a macrophage marker) showed that Neotuberostemonine treatment reduced macrophage infiltration in fibrotic lung tissues.[1]
Western blot analysis of lung tissues indicated that Neotuberostemonine treatment significantly decreased the expression of arginase-1 (M2 marker), with a less pronounced effect on iNOS (M1 marker).[1]

Immunofluorescent assay: Hypoxyprobe™-1 is a substituted 2-nitroimidazole named pimonidazole. It will bind to cells if the p O2 levels are less than 10 mmHg, so it is often used as a probe for detecting cell hypoxia. PLFs were cultured to 80% confluence. After 12 h of starvation, PLFs were incubated with 200 μmol/L hypoxyprobe and 10 μmol/L Neotuberostemonine (NTS) under normoxia, hypoxia (1% O2) or 100 μmol/L CoCl2 treatment at 37 °C for 12 h. PLFs were fixed and blocked with 3% bovine serum albumin (BSA), then treated with anti-hypoxyprobe and DAPI. The blank control was treated in the same manner. All the treated PLFs were examined under confocal scanning microscopy.Reference: Acta Pharmacol Sin. 2018 Sep;39(9):1501-1512. https://pubmed.ncbi.nlm.nih.gov/29645000/

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Cell Viability (MTT assay): RAW 264.7 cells were seeded in 96-well plates at a density of 2 × 10⁵ cells/mL. After incubation for 6 hours, the cells were treated with various concentrations of Neotuberostemonine for 24 or 48 hours. Subsequently, MTT solution was added to each well and incubated for 4 hours. The formazan crystals formed were dissolved with DMSO, and the absorbance was measured at 570 nm using a microplate reader to assess cell viability.[1]
Nitric Oxide (NO) Production: RAW 264.7 cells were pretreated with Neotuberostemonine for 1 hour and then stimulated with LPS (1 μg/mL) for 24 hours. The concentration of NO in the culture supernatant was measured using Griess reagent.[1]
Western Blot Analysis for M1/M2 Markers: RAW 264.7 cells were pretreated with Neotuberostemonine for 1 hour, followed by stimulation with LPS (1 μg/mL for 24 hours) to induce M1 polarization or IL-4 (20 ng/mL for 48 hours) to induce M2 polarization. Cells were then lysed. Protein concentrations were determined, and equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against iNOS (M1 marker) or arginase-1 (Arg-1, M2 marker). Protein bands were visualized using HRP-conjugated secondary antibodies and ECL reagent.[1]
ELISA for TGF-β1: The concentration of TGF-β1 in the supernatant of RAW 264.7 cells treated with Neotuberostemonine and IL-4 was determined using a commercial ELISA kit according to the manufacturer's instructions.[1]

ICR male mice were randomly divided into 5 groups (n=20), ie, the sham, model, Neotuberostemonine (NTS), PND and Dig groups. After 12 h of fasting, mice were intratracheally injected with BLM (3.5 U/kg in 0.9% NaCl) or 0.9% NaCl (sham group) after anesthesia with 4% chloral hydrate (10 mL/kg). After 7 d of model formation, BLM-treated mice were orally administered Neotuberostemonine (NTS) (30 mg/kg), PDN (6.5 mg/kg) and an equivalent volume of the same menstruum (sham and model group) or intraperitoneally injected with Dig (1 mg/kg) once a day for 7 or 14 consecutive days. The lungs were excised on d 15 and 22 after BLM treatment.Reference: Acta Pharmacol Sin. 2018 Sep;39(9):1501-1512. https://pubmed.ncbi.nlm.nih.gov/29645000/

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BLM-induced Pulmonary Fibrosis Model: Male C57BL/6 mice were anesthetized. Pulmonary fibrosis was induced by a single intratracheal instillation of bleomycin (BLM, 5 U/kg, dissolved in 0.9% NaCl). The day of BLM administration was designated as day 0. Mice in the sham control group received an equal volume of 0.9% NaCl.[1]
Drug Treatment: Neotuberostemonine was suspended in 0.5% carboxymethyl cellulose sodium (CMC-Na) solution. Treatment was administered by oral gavage once daily at doses of 20 and 40 mg/kg, starting from day 8 after BLM instillation and continuing until day 21. A positive control group received nintedanib (40 mg/kg, orally) on the same schedule. Mice in the model and sham groups received the vehicle (0.5% CMC-Na) only.[1]
Sample Collection: On day 22, mice were euthanized. Bronchoalveolar lavage fluid (BALF) was collected for inflammatory cell analysis. Lung tissues were harvested, weighed, and divided: one portion was fixed in formalin for histology, and the remainder was frozen for protein analysis.[1]

[1]. Neotuberostemonine attenuates bleomycin-induced pulmonary fibrosis by suppressing the recruitment and activation of macrophages. Int Immunopharmacol. 2016 Jul;36:158-164.

Neotuberostemonine is an alkaloid with the effects of a metabolite. It has been reported to exist in Stemona japonica, Stemona phyllantha, and other organisms with relevant data. Neotuberostemonine is an alkaloid isolated from the root of Stemona tuberosa Lour., a traditional Chinese medicine used to treat respiratory diseases such as cough and asthma. [1] This study is the first to demonstrate that Neotuberostemonine has a protective effect against bleomycin-induced pulmonary fibrosis in mice. The mechanism involves inhibiting the recruitment of macrophages to the lungs and suppressing their polarization towards the pro-fibrotic M2 phenotype, thereby reducing the production of pro-fibrotic mediators such as TGF-β1. [1] In this model, the anti-fibrotic efficacy of Neotuberostemonine (40 mg/kg) was comparable to that of the positive control drug nintedanib (40 mg/kg). [1]

C22H33NO4

375.509

375.24

C, 70.37; H, 8.86; N, 3.73; O, 17.04

143120-46-1

11667940

White to off-white solid powder

1.2±0.1 g/cm3

554.2±50.0 °C at 760 mmHg

289.0±30.1 °C

0.0±1.5 mmHg at 25°C

1.556

Roots of Stemona tuberosa Lour; Stemona japonica; Stemona phyllantha

2.28

0

5

2

27

636

10

O1C([C@@]([H])(C([H])([H])[H])[C@]2([H])[C@@]1([H])[C@]([H])(C([H])([H])C([H])([H])[H])[C@@]1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N3[C@]([H])([C@]4([H])C([H])([H])[C@]([H])(C([H])([H])[H])C(=O)O4)C([H])([H])[C@]2([H])[C@]31[H])=O

GYOGHROCTSEKDY-UEIGSNQUSA-N

InChI=1S/C22H33NO4/c1-4-13-14-7-5-6-8-23-16(17-9-11(2)21(24)26-17)10-15(19(14)23)18-12(3)22(25)27-20(13)18/h11-20H,4-10H2,1-3H3/t11-,12-,13+,14+,15-,16-,17-,18-,19+,20+/m0/s1

(2S,7aR,8R,8aR,11S,11aR,11bS,11cR)-8-ethyldodecahydro-11-methyl-2-[(2S,4S)-tetrahydro-4-methyl-5-oxo-2-furanyl]-furo[2,3-h]pyrrolo[3,2,1-jk][1]benzazepin-10(2H)-one

Neotuberostemonine; Tuberostemonine LG; CHEBI:69386; (1S,3S,9R,10R,11R,14S,15R,16R)-10-ethyl-14-methyl-3-[(2S,4S)-4-methyl-5-oxooxolan-2-yl]-12-oxa-4-azatetracyclo[7.6.1.04,16.011,15]hexadecan-13-one; CHEMBL479493; SCHEMBL19197265; DTXSID501316122; (+)-Neotuberostemonine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~133.16 mM)
H2O : < 0.1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)