κ Opioid Receptor/KOR; μ Opioid Receptor/MOR

In the present study, researchers have confirmed that matrine significantly suppresses the growth of human lung cancer A549 and hepatoma SMMC-7721 cells in vitro and ex vivo. Furthermore, they have also demonstrated that the induction of apoptosis by reducing ratios of the Bcl-2/Bax protein levels in A549 and SMMC-7721 cells is one of the important mechanisms of action of matrine against cancer cell growth. In addition, our result indicates that matrine reduces the rate of A549 cell migration more than 30–48% at concentrations without affecting cell viability. Moreover, our present result has also suggested that the reduction of VEGF-A secretion in A549 cells by matrine may be one of important mechanisms of action of matrine against the migration and growth in A549 cells. More importantly, researchers show that matrine enhances the anticancer activity of the anticancer agent TSA by reducing the viability and/or the Bcl-2/Bax protein ratio in A549 cells. All these findings suggest that matrine may have the wide therapeutic and/or adjuvant therapeutic application in the treatment of human NSCLC and hepatoma.[1]

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In vitro activity: Matrine ((+)-Matrine) is an alkaloid that is present in plants belonging to the Sophora family. It possesses multiple pharmacological effects, such as acting as a kappa opioid receptor agonist and having anti-cancer properties. Human non-small cell lung cancer A549 and hepatoma SMMC-7721 cells are highly inhibited in their growth by marrine, which also induces apoptosis by dramatically lowering the viability and Bcl-2/Bax protein ratio in A549 cells.[1] matrine may activate the descending dynorphinergic neuron, which in turn activates the spinal cord's kappa-opioid receptors (KORs), which in turn causes mice to exhibit antinociception.[2]

Matrine (200, 100, and 50 mg/kg) administered orally greatly reduced the left ventricular dysfunction and cardiac necrosis caused by isoproterenol. When mice were administered LPS, a high dose of matrine significantly decreased their mortality rate. Matrine treatment improved the histopathologic changes in the lung caused by LPS, reduced the production of inflammatory mediators such as TNF-α, IL-6, and HMGB1, alleviated pulmonary edema, and stopped lung vascular leak.

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In the present study, we found that i.c.v. administration of either (+)-matrine- or (+)-allomatrine induced antinociceptive effects in the mouse tail-flick and warm-plate test, whereas these alkaloids when given spinally failed to induce antinociception. In the guanosine-5'-O-(3-[(35)S]thio)trisphosphate ([(35)S]GTPgammaS) binding assay, we demonstrated that neither (+)-matrine nor (+)-allomatrine produced the stimulation of [(35)S]GTPgammaS binding in the membranes of the spinal cord, indicating that (+)-matrine- and (+)-allomatrine-induced supraspinal antinociceptive actions was not due to a direct stimulation of KORs by these alkaloids. Therefore, we next investigated the involvement of dynorphin A (1-17) release at the spinal or supraspinal site in (+)-matrine- or (+)-allomatrine-induced antinociception. The i.c.v. pretreatment with an antiserum against dynorphin A (1-17) could not affect the antinociceptive effect induced by s.c. treatment of (+)-matrine. In contrast, the s.c.-administered (+)-matrine- and (+)-allomatrine-induced antinociceptive effect was significantly attenuated by i.t. pretreatment of an antiserum against dynorphin A (1-17). The present data suggest that either (+)-matrine or (+)-allomatrine when given i.c.v. may stimulate the descending dynorphinergic neuron, resulting in the stimulation of KORs in the spinal cord, and this phenomenon in turn produces the antinociception in mice [2].

Cell culture and in vitro and ex vivo cell growth assays [1]
The A549 lung adenocarcinoma and hepatoma SMMC-7721 cell lines were used. The human cancer cell lines A549 and SMMC-7721 were cultured in RPMI 1640 and DMEM medium, respectively, containing 10% heat-inactivated fetal bovine serum (FBS), glutamine (2 mM), penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in a humidified incubator with 95% air/5% CO2 atmosphere. The in vitro and ex vivo assays were done according to our published methods (Zhang et al. 1999, 2001). The cells in control group were treated with DMSO (0.1%, final concentration). The cells were respectively incubated in RPMI 1640 and DMEM medium supplemented with 10% FBS (in the case of in vitro assay) containing different concentrations of matrine, or in the absence or presence of the existing anticancer agents (TSA and Bay), or 10% rabbit sera (in the case of ex vivo assay) obtained at different time points after matrine was orally intubated to rabbits. Cell viability was measured 24, 48, and 72 h after the treatments using MTT assay kit. The MTT method is based on the method of Zhang et al. (1999). Each experiment was repeated three times.

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Morphological evaluation of apoptotic cells [1]
This was done according to our published methods (Zhang et al. 2000). In brief, A549 and SMMC-7721 cells at 70% confluence were respectively treated for 48 h with matrine at concentrations of 0 (0.1% DMSO, vehicle as control), 100 and 500 μg/mL (in the case of A549 cells) and 0.5 and 1 mg/mL (in the case of SMMC-7721 cells). The treated cells were fixed with 1% glutaraldehyde in PBS for 30 min at room temperature, washed in PBS, and stained with 1 mM Hoechst 33258 for 30 min at room temperature. The morphological changes in the nuclear chromatin were observed under a fluorescent microscope (Nikon, TE2000-U, Japan), using 40× lens.

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Western blot analysis [1]
This was performed according to the method of (Chen et al. 2001). In brief, A549 and SMMC-7721 cells were treated with matrine at different concentrations in the absence or presence of trichostatin A (TSA, 5 μg/L). The cells in control group were treated with DMSO (0.1%, final concentration). Bay (2.5 μM) and celecoxib (S, 10 μM) were used as positive control. The treated cells were collected at 48 h. Equal amounts of cell extracts were resolved by SDS–PAGE, transferred to nitrocellulose membranes, and probed with primary antibodies to human Bcl-2, Bax, and β-Actin and then horseradish peroxidase-conjugated secondary antibodies, respectively. Anti-β-Actin antibody was used as a loading control. Detection was done using an enhanced chemiluminescence system.

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In vitro migration assay [1]
Cancer cell migration was measured by examining cell migration through fibronectin-coated polycarbonate filters, using modified transwell chambers. In brief, A549 cells (5 × 104) were seeded into the upper chamber in 200 μL of serum-free medium containing matrine at concentrations of 0–100 μg/mL, respectively; the cells in control group were treated with DMSO (0.1%, final concentration); the lower compartment was filled with 0.66 mL of RPMI 1640 medium supplemented with 10% of FBS (as a chemoattractant). After incubation for 6 h at 37°C, the cells that migrated to the lower surface of the filter were fixed and stained using propidium iodide. The cells on the upper side of the filter were removed using a rubber scraper. The migrated cells on the underside of the filter were counted and recorded for images under a fluorescent microscope. Experiments were performed in triplicate.

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ELISA for detection of human VEGF-A secretion in A549 cells For detection of the effects of matrine on the secretion of vascular endothelial growth factor A (VEGF-A) in A549 cells, the cells were treated for 24 h with matrine at the concentrations of 50–500 μg/mL. Then each supernatant of the cell culture was respectively collected and analyzed by ELISA using kit (VEGF-A) from R & D Systems. ELISA was done according to the instructions of the manufacturer. Each experiment was repeated three times.

Animal experimentation and preparation of sera from matrine-treated rabbits [1]
These were done according to our published methods with slight modifications (Zhang et al. 2000, 2001). In brief, New Zealand White rabbits (3.5–4 kg) were used. Matrine was orally intubated to the rabbits once daily at a dose of 100 mg/mL/kg body weight for 3 days. On the third day, the blood was then collected at 0, 0.5, 1, and 2 h from the rabbits (fasted for 16 h) after oral intubation of matrine. The collected blood was left to clot for 2 h at room temperature and centrifuged twice at 3000×g at 4 °C for 20 min. The sera were sterilized by filtration and then heated at 56 °C for 30 min. The prepared sera were aliquoted, and stored at −80 °C until ex vivo cell growth assay.

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Objective: To investigate the acute toxicity and assess the median lethal dose (LD50) of matrine in Kunming mice. Methods: Matrine at different doses were administered in Kunming mice via intraperitoneal injection, and the toxic reactions and LD50 of matrine was observed and determined.[3]

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200, 100 and 50 mg/kg

Mouse

Toxicity Summary
The nervous system is the main target organ by the toxicity of matrine. Morphological observation revealed degenerative changes of the nerve cells in the brain tissue of the mice. (A15436) Matrine has a variety of pharmacological effects, including anti-cancer effects, and action as a kappa opioid receptor and µ-receptor agonist. Matrine possesses strong antitumor activities in vitro and in vivo. Inhibition of cell proliferation and induction of apoptosis are the likely mechanisms responsible for matrine's antitumor activities. (Wikipedia)

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Toxicity Summary
The nervous system is the main target organ by the toxicity of matrine. Morphological observation revealed degenerative changes of the nerve cells in the brain tissue of the mice. Matrine has a variety of pharmacological effects, including anti-cancer effects, and action as a kappa opioid receptor and µ-receptor agonist. Matrine possesses strong antitumor activities in vitro and in vivo. Inhibition of cell proliferation and induction of apoptosis are the likely mechanisms responsible for matrine's antitumor activities.

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The acute toxicity test of matrine indicated that the tolerable dose of matrine was above 80 mg/kg in Kunming mice, and the LD50 was 157.13 mg/kg (95%CI, 88.08-280.31 mg/kg). Morphological observation revealed degenerative changes of the nerve cells in the brain tissue of the mice. Conclusion: The nervous system is the main target organ by the toxicity of matrine.[3]

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91466 mouse LD50 intraperitoneal 150 mg/kg Zhongcaoyao. Chinese Traditional and Herbal Medicine., 18(214), 1987
91466 mouse LD50 intravenous 64850 ug/kg Zhongguo Yaoxue Zazhi. Chinese Pharmacuetical Journal., 27(201), 1992
91466 mouse LD50 intramuscular 74150 ug/kg Zhongguo Yaoxue Zazhi. Chinese Pharmacuetical Journal., 27(201), 1992
91466 rat LD50 intraperitoneal 125 mg/kg Chemical and Pharmaceutical Bulletin., 18(2555), 1970 [PMID:5492910]

[1]. Cytotechnology . 2009 Apr;59(3):191-200.

[2]. Biol Pharm Bull . 2005 May;28(5):845-8.

[3]. Nan Fang Yi Ke Da Xue Xue Bao . 2010 Sep;30(9):2154-5.

Matrine is an alkaloid.
Matrine has been reported in Gymnospermium albertii, Sophora macrocarpa, and other organisms with data available.
Matrine is an alkaloid found in plants from the Sophora genus. It has a variety of pharmacological effects, including anti-cancer effects, and action as a kappa opioid receptor and μ-receptor agonist.
Tetracyclic bis-quinolizidine alkaloids found in the family LEGUMINOSAE, mainly in the genus SOPHORA.

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In the present study, we have confirmed that matrine significantly suppresses the growth of human lung cancer A549 and hepatoma SMMC-7721 cells in vitro and ex vivo. Furthermore, we have also demonstrated that the induction of apoptosis by reducing ratios of the Bcl-2/Bax protein levels in A549 and SMMC-7721 cells is one of the important mechanisms of action of matrine against cancer cell growth. In addition, our result indicates that matrine reduces the rate of A549 cell migration more than 30–48% at concentrations without affecting cell viability. Moreover, our present result has also suggested that the reduction of VEGF-A secretion in A549 cells by matrine may be one of important mechanisms of action of matrine against the migration and growth in A549 cells. More importantly, we show that matrine enhances the anticancer activity of the anticancer agent TSA by reducing the viability and/or the Bcl-2/Bax protein ratio in A549 cells. All these findings suggest that matrine may have the wide therapeutic and/or adjuvant therapeutic application in the treatment of human NSCLC and hepatoma.[1]

C15H24N2O

248.36

248.188

C, 72.54; H, 9.74; N, 11.28; O, 6.44

519-02-8

91466

White to off-white solid powder

1.2±0.1 g/cm3

396.7±31.0 °C at 760 mmHg

77°C

172.7±17.2 °C

0.0±0.9 mmHg at 25°C

1.581

1.44

0

2

0

18

356

4

O=C1CCC[C@]2([H])[C@@]3([H])CCCN4[C@@]3([H])[C@](CCC4)([H])CN21

ZSBXGIUJOOQZMP-JLNYLFASSA-N

InChI=1S/C15H24N2O/c18-14-7-1-6-13-12-5-3-9-16-8-2-4-11(15(12)16)10-17(13)14/h11-13,15H,1-10H2/t11-,12+,13+,15-/m0/s1

(1R,2R,9S,17S)-7,13-diazatetracyclo[7.7.1.02,7.013,17]heptadecan-6-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.07 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.07 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.07 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 37.5 mg/mL (150.99 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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