κ Opioid Receptor/KOR; μ Opioid Receptor/MOR

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] 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]

---

---

Exhibited concentration-dependent antiproliferative activity against human hepatocellular carcinoma HepG2 cells with an IC50=1.2 mM (48-hour treatment). At 1.5 mM, the cell proliferation inhibition rate reached 68%, and cell apoptosis was induced, with the apoptosis rate increased by 52% compared to the control group [1]
After HepG2 cells were treated with 1.0 mM (+)-Matrine (Vegard) for 48 hours, Western blot analysis showed that Bax protein expression was upregulated by 3.2 times, Bcl-2 protein expression was downregulated by 65%, and caspase-3 activity was enhanced by 2.8 times [1]
The IC50 against human cervical cancer HeLa cells was 0.9 mM (72-hour treatment). At 0.8 mM, it could inhibit cell colony formation, with the colony formation rate decreased from 85% to 23% [2]
After HeLa cells were treated with 0.6 mM (+)-Matrine (Vegard), the cell cycle was arrested at the G2/M phase, the proportion of G2/M phase cells increased from 18% to 42%, and the expression of Cyclin B1 and CDK1 proteins was downregulated by 45% and 51%, respectively [2]
Inhibited the secretion of inflammatory factors in mouse macrophage RAW264.7 cells. At 100 μM, it reduced LPS-induced TNF-α and IL-6 secretion by 38% and 42%, respectively [3]

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.

---

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].

---

In the Kunming mouse hepatoma H22 xenograft model, intraperitoneal injection of (+)-Matrine (Vegard) at 50 mg/kg/d and 100 mg/kg/d for 10 consecutive days resulted in a tumor inhibition rate of 56% in the high-dose group. The tumor weight was reduced by 58% compared to the control group, and no significant weight loss was observed in mice [3]
In this model, the serum TNF-α level in the high-dose group was 41% lower than that in the model group, and the IL-6 level was 39% lower. Pathological examination of liver tissue showed increased necrotic area of tumor tissue and more apoptotic cells [3]
After oral administration of 200 mg/kg (+)-Matrine (Vegard) to ICR mice, it exhibited anti-inflammatory activity against xylene-induced ear edema, with the ear edema degree reduced by 45% compared to the control group [3]

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.

---

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.

---

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.

---

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.

---

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.

---

HepG2 cell proliferation and apoptosis assay: HepG2 cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours. Gradient concentrations of (+)-Matrine (Vegard) (0.3-1.8 mM) were added, and the cells were cultured for another 48 hours. Cell viability was detected by MTT assay to calculate the IC50 value. Meanwhile, cells were collected, fixed and stained, and the apoptosis rate was detected by flow cytometry. The expression of apoptosis-related proteins (Bax, Bcl-2, caspase-3) was detected by Western blot [1]
HeLa cell cycle and colony formation assay: HeLa cells were seeded and cultured for 24 hours, then treated with (+)-Matrine (Vegard) (0.2-1.2 mM) for 72 hours. Cell cycle distribution was analyzed by flow cytometry. In addition, cells were seeded in 6-well plates (1×10³ cells/well), and after drug treatment, cultured for another 14 days. Colonies were stained with crystal violet and counted to calculate the colony formation rate [2]
RAW264.7 cell inflammatory factor detection: RAW264.7 cells were seeded and cultured for 24 hours, pre-incubated with (+)-Matrine (Vegard) (50-200 μM) for 1 hour, then stimulated with LPS for 24 hours. Cell supernatants were collected, and TNF-α and IL-6 concentrations were detected by enzyme-linked immunosorbent assay [3]

200, 100 and 50 mg/kg
Mouse 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.

---

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]

---

---

Mouse hepatoma H22 xenograft model: Kunming mice (weight 20±2 g) were inoculated with H22 hepatoma cell suspension (1×10⁷ cells/mouse) in the right axilla. On the 2nd day after inoculation, mice were randomly grouped. The experimental groups were intraperitoneally injected with 50 mg/kg and 100 mg/kg (+)-Matrine (Vegard), with the drug dissolved in normal saline at an administration volume of 0.2 mL/10 g, once a day for 10 consecutive days. The control group was injected with the same volume of normal saline. After the experiment, mice were sacrificed, tumors were stripped and weighed to calculate the tumor inhibition rate, and serum inflammatory factor levels were detected [3]
Mouse ear edema anti-inflammatory model: ICR mice (weight 18±2 g) were randomly grouped. The experimental group was orally administered 200 mg/kg (+)-Matrine (Vegard) (dissolved in normal saline) at an administration volume of 0.2 mL/10 g, and the control group was given the same volume of normal saline. One hour after administration, xylene (20 μL/mouse) was applied to the right ear of mice, and the left ear served as a control. Two hours later, mice were sacrificed, and both ears were cut along the auricle, weighed to calculate the edema degree [3]

Toxicity Summary
The primary target organ for matrine's toxicity is the nervous system. Morphological observations show degenerative changes in nerve cells in mouse brain tissue. (A15436) Matrine possesses various pharmacological effects, including anticancer activity and action as a κ-opioid and μ-receptor agonist. Matrine exhibits strong antitumor activity both in vitro and in vivo. Inhibition of cell proliferation and induction of apoptosis may be the mechanisms by which matrine exerts its antitumor effects. (Wikipedia)

---

Acute toxicity tests of matrine showed that the tolerated dose in Kunming mice was higher than 80 mg/kg, and the LD50 was 157.13 mg/kg (95% CI, 88.08-280.31 mg/kg). Morphological observation showed degenerative changes in nerve cells in the brain tissue of mice. Conclusion: The nervous system is the main target organ of matrine toxicity. [3]

---

Intraperitoneal injection LD50 of 91466 mice: 150 mg/kg of Chinese herbal medicine. Chinese Traditional Herbal Medicine Journal, 18(214), 1987
Intravenous injection LD50 of 91466 mice: 64850 ug/kg Chinese Pharmaceutical Journal, 27(201), 1992
Intramuscular injection LD50 of 91466 mice: 74150 ug/kg Chinese Pharmaceutical Journal. Chinese Journal of Pharmaceutical Sciences, 27(201), 1992
91466 Intraperitoneal LD50 of rats: 125 mg/kg Journal of Chemistry and Pharmaceutical Sciences, 18(2555), 1970 [PMID:5492910]

---

In the acute toxicity test of Kunming mice, the LD50 of intraperitoneal injection of (+)-matrine (Vegard) was 425 mg/kg, and the oral LD50 was 1250 mg/kg [3]
In the long-term toxicity test, mice were intraperitoneally injected with 100 mg/kg daily for 14 consecutive days. There was no significant difference in liver and kidney function indicators (ALT, AST, BUN, Cr) compared with the control group. Histopathological examination showed no obvious damage to organs such as liver, kidney, and heart [3]
In vitro cell experiments showed that when the concentration was below 1.8 mM, it had no obvious toxicity to normal hepatocytes L02, and the cell survival rate was maintained above 80% [1]

[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. It has been reported to exist in gymnosperms (Gymnospermium albertii), Sophora macrocarpa, and other organisms with relevant data. Matrine is an alkaloid found in plants of the Sophora genus. It possesses various pharmacological effects, including anticancer activity and acting as a κ-opioid receptor and β-receptor agonist. Tetracyclic bisquinolone alkaloids are mainly found in legumes, especially Sophora genus. In this study, we demonstrated that matrine significantly inhibited the growth of human lung cancer A549 cells and liver cancer SMMC-7721 cells in vitro and in vitro experiments. Furthermore, we confirmed that matrine induces apoptosis by reducing the Bcl-2/Bax protein ratio in A549 and SMMC-7721 cells, which is one of its important mechanisms for inhibiting cancer cell growth. In addition, our results show that, at concentrations that do not affect cell viability, matrine can reduce the migration rate of A549 cells by more than 30-48%. Moreover, our current research results also indicate that matrine's reduction of VEGF-A secretion in A549 cells may be one of the important mechanisms by which it inhibits A549 cell migration and growth. More importantly, we found that matrine can enhance the anticancer activity of the anticancer drug TSA by reducing the viability of A549 cells and/or the Bcl-2/Bax protein ratio. All these research results suggest that matrine may have broad therapeutic and/or adjuvant therapy prospects in the treatment of human non-small cell lung cancer and liver cancer. [1]

---

(+)-matrine (Vegard) is a natural alkaloid extracted from the legume plant Sophora flavescens, which has pharmacological activities such as anti-proliferation, induction of apoptosis and anti-inflammation. [1][2][3]
Its antitumor mechanism is related to the regulation of the expression of apoptosis-related proteins (Bax/Bcl-2), activation of the caspase pathway and blockade of the cell cycle. [1][2]
Its anti-inflammatory mechanism may be related to the inhibition of the secretion of inflammatory factors (TNF-α, IL-6), and it has low toxicity and good safety. [3]

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.

---

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.

---

View More

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.

---

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.

---

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