Natural alkaloid; anti-inflammatory; anti-parasitic
- In lipopolysaccharide (LPS)-stimulated peritoneal macrophages, Tetrahydrocoptisine acts on the NF-κB pathway and MAPK pathway, while inhibiting the production of TNF-α, IL-6 and the release of NO. No specific values such as IC50, Ki, or EC50 are mentioned in the literature [1]
- For ethanol-induced gastric ulcer in mice, the specific target of

Tetrahydrocoptisine (THC) significantly inhibited LPS-induced TNF-α, interleukin-6(IL-6) and nitric oxide (NO) production. THC inhibited the production of TNF-α and IL-6 by down-regulating LPS-induced IL-6 and TNF-α mRNA expression. Furthermore, it attenuated the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) as well as the expression of nuclear factor kappa B(NF-κB), in a concentration-dependent manner. Taken together, our data suggest that THC is an active anti-inflammatory constituent by inhibition of TNF-α, IL-6 and NO production possibly via down-regulation of NF-κB activation, phospho-ERK1/2 and phospho-p38MAPK signal pathways[1].

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- Peritoneal macrophages were isolated from mice. After stimulation with LPS (1μg/mL), the cells were treated with different concentrations of Tetrahydrocoptisine. Enzyme-linked immunosorbent assay (ELISA) showed that the drug could concentration-dependently reduce the levels of TNF-α and IL-6 in the cell culture supernatant; Griess reagent assay indicated that it could significantly inhibit NO production; Western blot analysis confirmed that the drug could inhibit the nuclear translocation of NF-κB p65 subunit and down-regulate the phosphorylation levels of p38, JNK, and ERK1/2 in the MAPK pathway, and the above inhibitory effects were concentration-dependent [1]

The extracts or constituents from Corydalis impatiens are known to have many pharmacological activities. Tetrahydrocoptisine (THC), a protoberberine compound from Corydalis impatiens, was found to possess a potent anti-inflammatory effect in different acute or chronic inflammation model animals. Pretreatment with THC (i.p.) inhibited the paw and ear edema in the carrageenan-induced paw edema assay and xylene-induced ear edema assay, respectively. In the lipopolysaccharide (LPS)-induced systemic inflammation model, THC significantly inhibited serum tumor necrosis factor-alpha (TNF-α) release in mice [1].

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Excessive alcohol consumption can lead to gastric ulcer and the present work was aimed to examine the protective effect of tetrahydrocoptisine (THC) in the model of ethanol-induced gastric ulcer in mice. Fasted mice treated with ethanol 75% (0.5ml/100g) were pre-treated with THC (10 or 20mg/kg, ip), cimetidine (100mg/kg, ip) or saline in different experimental sets for a period of 3days, and animals were euthanized 4h after ethanol ingestion. Gross and microscopic lesions, immunological and biochemical parameters were taken into consideration. The results showed that ethanol induced gastric damage, improving nitric oxide (NO) level, increased pro-inflammatory cytokine (TNF-α and IL-6) levels and myeloperoxidase (MPO) activity, as well as the expression of nuclear factor-κB (NF-κB) in the ethanol group. Pretreatment of THC at doses of 10 and 20mg/kg bodyweight significantly attenuated the gastric lesions as compared to the ethanol group. These results suggest that the gastroprotective activity of THC is attributed to reducing NO production and adjusting the pro-inflammatory cytokine, inhibited neutrophil accumulation and NF-κB expression[2].

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- An ethanol-induced gastric ulcer model was established in ICR mice (intragastric administration of 50% ethanol at 0.2mL/mouse). Thirty minutes before the experiment, different doses of Tetrahydrocoptisine (20, 40, 80mg/kg) were given by gavage. The results showed that compared with the model group, each dose group of the drug could significantly reduce the gastric ulcer index (percentage of ulcer area to gastric area), and the 80mg/kg dose group had the highest ulcer inhibition rate; meanwhile, detection of gastric tissue homogenate showed that the drug could increase superoxide dismutase (SOD) activity, decrease malondialdehyde (MDA) content, and reduce the expression of TNF-α and IL-6 in gastric tissue, thereby alleviating gastric mucosal oxidative stress and inflammatory damage [2]

Assay of myeloperoxidase in gastric tissue[2]
Myeloperoxidase, an enzyme found primarily in neutrophil azurophilic granules, has been used extensively as a biochemical marker for granulocyte infiltration into various tissues, including the gastrointestinal tract (Costa et al., 2013, Krawisz et al., 1984). MPO activity was determined using an MPO activity measurement kit by adding 0.2 ml of o-dianisidine hydrochloride and 0.0005% hydrogen peroxide to 4 ml buffer containing 0.2 ml homogenates. MPO activity was assayed at room temperature by measuring the increase in absorbance at 460 nm due to the fluorescent product oxidized by the H2O2-generated redox intermediate. MPO activities were expressed as units per gram of tissue.

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Cytokine evaluations in gastric tissue[2]
The cytokine levels of IL-6 and TNF-α in gastric tissue were evaluated using ELISA kits according to the manufacturer's instructions. Supernatant of homogenates or cytokine standards (100 μl) were respectively loaded into each well and then followed with biotin conjugated secondary antibodies. To obtain color reaction, streptavidin–HRP and substrate solution were added. The absorbance was measured at 450 nm with an ELISA reader. A standard curve was run on each assay plate using recombinant IL-6 and TNF-α in serial dilution. The results were expressed pg/mg tissue.

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Determination of NO level in gastric tissue[2]
The level of nitric oxide in the gastric tissue was evaluated as total nitrate/nitrite using Griess reagent (Green et al., 1982) and the operational processes were measured in accordance with the NO kit instructions. Briefly, 50 μl of tissue supernatant was added to 50 μl Griess reagent [0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulphanilamide and 2.5% H3PO4] and mixed. After incubation at room temperature for 10 min, the absorbance was measured at 540 nm. The results were expressed as μmol/g protein.

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Cytokine evaluations in serum[2]
The levels of cytokines (IL-6 and TNF-α) in the serum were analyzed by enzyme-linked immunosorbent assay using ELISA kits for rats according to the manufacturer's instructions. The results were expressed as pg/ml serum.

- Isolation and culture of peritoneal macrophages: Mice were intraperitoneally injected with 3% thioglycollate broth. Four days later, the mice were sacrificed, and peritoneal macrophages were collected by peritoneal lavage. The cell concentration was adjusted to 1×10⁶ cells/mL with RPMI-1640 medium containing 10% fetal bovine serum, and the cells were seeded in culture plates and cultured in a 37℃, 5% CO₂ incubator for 24 hours. Non-adherent cells were discarded, and adherent macrophages were retained for subsequent experiments.
- Drug treatment and index detection: LPS (1μg/mL) was added to the cultured macrophages for stimulation, and different concentrations (10, 20, 40μM) of Tetrahydrocoptisine were added at the same time, followed by continuous culture for 24 hours. After culture, the cell culture supernatant was collected to detect TNF-α, IL-6 (by ELISA) and NO (by Griess method); cells were collected to extract total protein or nuclear protein, and the nuclear translocation of NF-κB p65 and the phosphorylation levels of MAPK pathway-related proteins (p38, JNK, ERK1/2) were detected by Western blot [1]

Ethanol-induced gastric mucosal damage[2]
Mice were randomly divided into five experimental groups, each containing ten animals. The normal and ulcer control groups received vehicle (0.9% saline) throughout the course of the experiments. The prevention groups received (ip) different doses of THC (10 and 20 mg/kg, dissolved in 0.9% saline) and cimetidine (100 mg/kg, reference drug, dissolved in 0.9% saline) respectively for a period of 3 days. After fasting for 24 h prior to the experiment, mice were fed orally with 75% ethanol (0.5 ml/100 g body weight) to induce the acute ulcer, while the normal group received water only (Mei et al., 2012). Four hours after induction, blood samples were collected from the retro-orbital plexus of each animal and were then centrifuged for 10 min at 2500 g to obtain clear sera which were stored at − 80 °C before use (Choi et al., 2010). After the mice were euthanized, the stomachs were rapidly removed, opened along the greater curvature and rinsed with ice-cold saline to remove the gastric contents and blood clots in order to assess the extent of gastric damage. Thereafter, each stomach was dichotomised, with one moiety of stomach immersed in 10% formaldehyde for histological evaluation and gastric tissue from the other moiety stored at − 80 °C for biochemical determinations.[2]

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Determination of gastric ulcer index[2]
The degree of gastric mucosal damage was evaluated from digital pictures, and rated for gross pathology according to the ulcer score scales as previously described (Salga et al., 2012). The lesions were scored as follows: 0: no lesions; 0.5: slight hyperemia or ≤ 5 petechiae; 1: ≤ 5 erosions ≤ 5 mm in length; 1.5: ≤ 5 erosions ≤ 5 mm in length and many petechiae; 2: 6–10 erosions ≤ 5 mm in length; 2.5: 1–5 erosions > 5 mm in length; 3: 5–10 erosions > 5 mm in length; 3.5: > 10 erosions > 5 mm in length; 4: 1–3 erosions ≤ 5 mm in length and 0.5–1 mm in width; 4.5: 4–5 erosions ≤ 5 mm in length and 0.5–1 mm in width; 5: 1–3 erosions > 5 mm in length and 0.5–1 mm in width; 6: 4 or 5 grade 5 lesions; and 7: ≥ 6 grade 5 lesions; 8: complete lesion of the mucosa with hemorrhage.. The sum of the total scores was divided by the number of animals to obtain the mean ulcer index for each group.

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- Selection of experimental animals: SPF-grade ICR mice, male, weighing 20-22g, were adaptively fed for 1 week before the experiment.
- Drug preparation and administration: Tetrahydrocoptisine was dissolved in 0.5% carboxymethyl cellulose sodium (CMC-Na) solution to prepare concentrations of 2, 4, 8mg/mL, corresponding to the administration doses of 20, 40, 80mg/kg respectively; the control group was given the same volume of 0.5% CMC-Na solution, all by gavage, once a day for 3 consecutive days.
- Establishment of gastric ulcer model: Thirty minutes after the last administration, mice in the model group and drug groups were given 50% ethanol (0.2mL/mouse) by gavage, and the control group was given the same volume of normal saline; 1 hour after administration, the mice were sacrificed, the stomach tissue was dissected out, rinsed with normal saline, cut along the greater curvature of the stomach, the gastric mucosal ulcer was observed, and the ulcer index was calculated [2]

[1]. Anti-inflammatory effect of tetrahydrocoptisine from Corydalis impatiens is a function of possible inhibition of TNF-α, IL-6 and NO production in lipopolysaccharide-stimulated peritoneal macrophages through inhibiting NF-κB activation and MAPK pathway. Eur J Pharmacol. 2013 Sep 5;715(1-3):62-71.

[2]. Protective effect of tetrahydrocoptisine against ethanol-induced gastric ulcer in mice. Toxicol Appl Pharmacol. 2013 Oct 1;272(1):21-9.

Stylopine has been found in Fibraurea recisa, Corydalis ternata and other organisms with relevant data. See also: Stylopine (note moved to). - Tetrahydroberberine is extracted from the plant Corydalis impatiens. Its anti-inflammatory mechanism may be related to the inhibition of NF-κB activation and the MAPK pathway, thereby reducing the production of pro-inflammatory factors (TNF-α, IL-6) and NO, which provides a potential research direction for the treatment of inflammation-related diseases [1]. Tetrahydroberberine has a protective effect against ethanol-induced gastric ulcers in mice. Its mechanism may involve alleviating oxidative stress damage (increasing SOD activity and reducing MDA content) and inhibiting gastric mucosal inflammatory response (reducing TNF-α and IL-6 expression), which provides a new candidate drug research basis for the prevention and treatment of gastric ulcers. Ulcer [2]

C19H17NO4

323.3426

323.116

C, 70.58; H, 5.30; N, 4.33; O, 19.79

4312-32-7

(±)-Stylopine hydrochloride;96087-21-7;(-)-Stylopine;84-39-9; (±)-Stylopine;4312-32-7;(-)-Stylopine;84-39-9; 84-39-9 (S-isomer); 4312-32-7 (racemic); 7461-02-1 (racemic)

6770

Typically exists as white to off-white solids at room temperature

1.47g/cm3

466.6ºC at 760mmHg

221-222ºC

142.5ºC

Corydalis tubers

2.737

0

5

0

24

502

0

O1C([H])([H])OC2C([H])=C([H])C3=C(C1=2)C([H])([H])N1C([H])([H])C([H])([H])C2=C([H])C4=C(C([H])=C2C1([H])C3([H])[H])OC([H])([H])O4

UXYJCYXWJGAKQY-UHFFFAOYSA-N

InChI=1S/C19H17NO4/c1-2-16-19(24-10-21-16)14-8-20-4-3-12-6-17-18(23-9-22-17)7-13(12)15(20)5-11(1)14/h1-2,6-7,15H,3-5,8-10H2

5,7,17,19-tetraoxa-13-azahexacyclo[11.11.0.02,10.04,8.015,23.016,20]tetracosa-2,4(8),9,15(23),16(20),21-hexaene

Stylopine; (-)-STYLOPINE; dl-Stylopine; (R,S)-Stylopine; 7461-02-1; 6,7,12b,13-Tetrahydro-4H-[1,3]dioxolo[4',5':7,8]isoquinolino[3,2-a][1,3]dioxolo[4,5-g]isoquinoline;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMF : 4 mg/mL (~12.37 mM)
DMSO : ~2.5 mg/mL (~7.73 mM)
Acetone : 1 mg/mL (~3.09 mM)

Solubility in Formulation 1: 12.5 mg/mL (38.66 mM) in 20% HP-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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