Leishmania

Isopimpinellin (oral gavage, 35-150 mg/kg) inhibits the formation of DMBA-DNA adducts and B[a]P-DNA adducts in SENCAR mice with skin tumor[1].

Female SENCAR mice (7-9 weeks of age) were fed AIN-76A semi-purified diet (Dyets, Bethlehem, PA) for 2 weeks prior to and during the study[1]. 35–150 mg/kg. Oral gavage, suspended in 0.1 mL corn oil at 24 h and 2 h prior to topical treatment with [3H]B[a]P (200 nmol, 1 Ci/mmol) or [3H]DMBA (10 nmol, 10 Ci/mmol) (each in 0.2 mL acetone).

Toxicity Summary
Identification and Uses: Isopsoralen is a coumarin derivative isolated from the fruit extract of Cnidium monnieri. It has been used as an experimental chemopreventive therapy. Human Studies: Isopsoralen inhibits coumarin 7-hydroxylase (COH) activity in mice, but has no effect on humans. Animal Studies: Isopsoralen has been reported to induce a sustained photostimulation response in mice. However, synthetic isopsoralen containing only trace amounts of bergamot lactone (5-methoxypsoralen) and xanthotoxin (8-methoxypsoralen) did not show phototoxicity in chicken skin bioassay systems. These findings contradict earlier studies showing phototoxicity of isopsoralen in chicken skin and support the conclusion that isopsoralen lacks photobiological activity. Other reports of photoactivity in isopsoralen are likely due to contamination with small amounts of highly active psoralens (such as bergamot lactone or xanthotoxin). Isopsoralen inhibits the activity of mouse coumarin 7-hydroxylase (COH) both in vitro and in vivo. Isopsoralen strongly inhibits insulin-stimulated lipogenesis. It induces the expression of hepatic glutathione S-transferases (GSTs) and is a potent inhibitor of cytochrome P450 1A1/1B1. Oral administration of isopsoralen blocks 7,12-dimethylbenzo[a]anthracene-induced DNA adduct formation and skin tumor development in SENCAR mice and mouse mammary glands (L579). Many furanocoumarins act based on their ability to form photoadducts with DNA and other cellular components such as RNA, proteins, and various membrane proteins, including phospholipases A2 and C, calcium-dependent and cAMP-dependent protein kinases, and epidermal growth factor. Furanocoumarins can intercalate between DNA base pairs and form cycloadducts upon UVA irradiation. (L579)

---

Interactions
We investigated several natural coumarins previously found to effectively inhibit ethoxyhalogen-O-deethylase (EROD) and/or pentohalogen-O-dealkylase (PROD) in mouse liver, examining their effects on the formation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzo[a]anthracene (DMBA) DNA adducts in mouse epidermis, and their influence on the development of skin tumors induced by these polycyclic aromatic hydrocarbons (PAHs). Topical application of the potent hepatic EROD inhibitor bergamot lactone 5 minutes before the initial dose of B[a]P significantly reduced the total covalent binding of B[a]P to DNA in a dose-dependent manner within 24 hours post-treatment. 400 nmol of bergamot lactone reduced the covalent binding of benzo[a]pyrene (B[a]P) by 72%. 400 nmol of coumarin also significantly reduced the total valent binding of B[a]P by 59%. Furthermore, both coumarins selectively reduced the formation of the major (+) anti-B[a]P-diol epoxide-N2-dGuo adduct. Conversely, 400 nmol or 800 nmol of bergamot lactone and coumarin did not significantly reduce the covalent binding of DMBA to epidermal DNA. Isoimperatorin and isoimperatorin were more potent inhibitors of hepatic PROD activity, significantly reducing the total binding of DMBA to epidermal DNA by 67% and 52%, respectively, at a dose of 400 nmol. Both coumarins also inhibited the formation of B[a]P-DNA adducts at similar doses, but to a weaker degree. 400 nmol of isoprenone significantly reduced the formation of covalent DNA adducts derived from the trans and cis-diol epoxides of DMBA. Bergamot lactone is a potent inhibitor of B[a]P-induced tumorigenesis, while citronellol is less effective. Isoprenone is an effective inhibitor of DMBA-induced skin tumorigenesis and can completely inhibit the carcinogenic effects of this polycyclic aromatic hydrocarbon. At doses higher than those inhibiting DMBA, isoprenone also inhibits B[a]P-induced tumorigenesis. These results indicate that several naturally occurring coumarins possess the ability to block the formation of DNA adducts and tumorigenesis induced by polycyclic aromatic hydrocarbons such as B[a]P and DMBA. The mechanism of reduced DNA adduct formation and tumorigenesis appears to be related to the inhibition of P450 enzymes involved in the metabolic activation of these hydrocarbons. Finally, the different effects of certain coumarins on the formation of benzo[a]pyrene (B[a]P) and dimethylbenzo[a]anthracene (DMBA) DNA adducts and tumor initiation may help elucidate the role of specific cytochrome P450s in their metabolic activation. Natural coumarins (NOCs) inhibited polycyclic aromatic hydrocarbon-induced skin tumor initiation in mice by blocking the bioactivation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzo[a]anthracene (DMBA) mediated by cytochrome P450 (P450). Bergamot lactone selectively inhibited B[a]P-induced tumor initiation, while isoimperatorin and isoimperatorin inhibited tumor initiation induced by both carcinogens. This study aimed to investigate the ability of NOCs to inhibit human P450 in vitro and to determine whether NOCs with anticancer activity in mice could block the bioactivation of carcinogens in cultured human cells. In the initial experiments, we used an incubation system containing 5 μM P450, P450 substrate, NADPH generation system, and NOCs to determine the concentration (IC50) at which each inhibitor inhibited 50% of P450 activity. The results confirmed that NOCs could inhibit multiple human P450s and were selective for certain human P450 subtypes. In subsequent experiments, we investigated the effects of bergamot lactone, isoimperatorin, and isoimperatorin on the formation of DMBA and B[a]P DNA adducts in the human breast cancer MCF-7 cell line. Co-incubation of cells with these three different NOCs significantly inhibited the formation of DMBA DNA adducts by 29% to 82% at doses ranging from 2 to 10 μM; and significantly inhibited the formation of B[a]P DNA adducts by 37% to 80% at doses ranging from 20 to 80 μM. High-performance liquid chromatography analysis of DNA hydrolysates showed that the inhibition of DNA adducts corresponded to the inhibition of major B[a]P and DMBA diol epoxide-derived adducts. Although bergamot lactone was not effective in blocking the bioactivation of DMBA in a mouse skin model, its effect was similar to that of isoimperatorin and isoimperatorin in MCF-7 cells. These results indicate that natural coumarins (NOCs) found in citrus fruits and other human dietary components can inhibit carcinogen-metabolizing enzymes and block the bioactivation of B[a]P and DMBA in MCF-7 cells. Natural coumarins (NOCs) also exhibit anticancer activity in a mouse skin model. To characterize the chemopreventive potential of NOCs against breast cancer, we first examined their effects on the formation of 7,12-dimethylbenzo[a]anthracene (DMBA)-DNA adducts in mouse mammary glands. We hypothesized that NOCs that both inhibit cytochrome P450 1A1/1B1 and induce hepatic glutathione S-transferases (GSTs) are most effective in blocking the formation of DMBA-DNA adducts in mouse mammary glands. To test this hypothesis, we compared simple coumarin compounds (e.g., coumarin and limonene, which induce liver GSTs in mice but have little effect on P450 1A1/1B1) with linear furanocoumarins (e.g., isopentenone and isoimperatorin, which induce liver GSTs and are potent inhibitors of P450 1A1/1B1). Mice were pretreated with NOCs (150 mg/kg body weight, by gavage) before receiving a single dose of DMBA (50 μg) or multiple doses of DMBA (20 μg daily for 3 and 6 weeks, respectively). The formation of DMBA-DNA adducts in mammary tissue was quantified using a nuclease P1-enhanced 32P post-labeling method. After a single DMBA administration, coumarin, limonene, isoimperatorin, and isoimperatorin inhibited the formation of DMBA-DNA adducts by 50%, 41%, 79%, and 88%, respectively. After 3 weeks of DMBA administration, coumarin, limonene, and isoimperatorin inhibited the formation of DMBA-DNA adducts by 36%, 60%, and 66%, respectively; after 6 weeks of DMBA administration, the inhibition rates were 0%, 49%, and 55%, respectively. In a 6-week dose-response study, researchers investigated specific NOCs and 7,8-benzoflavonoids (a potent P4501 inhibitor with minimal effect on GST), showing that the formation of DMBA-DNA adducts was inhibited by 0%, 43%, and 24% in the limonene group, respectively. In the isopyridone group, the inhibition rates were 26%, 26%, and 69%, respectively; and in the 7,8-benzoflavonoid group, the inhibition rates were 80%, 96%, and 97% (at doses of 35, 70, and 150 mg/kg, respectively). In summary, these results indicate that linear furanocoumarins have a stronger inhibitory effect on the formation of DMBA-DNA adducts in mouse mammary glands compared to simple coumarins, and their main role is likely the inhibition of the P4501 enzyme. This study aimed to determine the mechanistic basis of the differential effects of natural furanocoumarins on 7,12-dimethylbenzo[a]anthracene (DMBA)-induced skin tumorigenesis. Female SENCAR mice were pre-treated with bergamot lactone, isoimperatorin or isoimperatorin (100-3200 nmol), 7,8-benzoflavonoids (7,8-BF, 5-40 nmol, a known DMBA mouse skin carcinogen inhibitor), or acetone (solvent control) five minutes before topical application of DMBA (10 nmol). The results showed that isoimperatorin, isoimperatorin, and 7,8-BF significantly inhibited the formation of DMBA-DNA adducts, while bergamot lactone had no such effect. High-performance liquid chromatography (HPLC) analysis showed that bergamot lactone preferentially inhibited the formation of trans-DMBADE-derived DNA adducts, while isoimperatorin and isoimperatorin inhibited both trans- and cis-derived adducts. 7,8-BF showed some selectivity in reducing cis-DMBADE-DNA adducts. Mouse embryonic fibroblasts C3H/10T1/2 (10T1/2) and mouse hepatocellular carcinoma cell line 1c1c7 (Hepa-1), which preferentially expressed P450 1b1 and P450 1a1, respectively, were co-incubated with 2 μM bergamot lactone, isoimperatorin, isoimperatorin, 7,8-BF, and 2 μM DMBA. Hepa-1 cells (P450 1a1) primarily formed trans-DMBADE-DNA adducts. Conversely, 10T1/2 cells (P450 1b1) primarily formed cis-DMBADE-DNA adducts. Bergamot lactone inhibited the metabolism of DMBA to DMBA-3,4-diol and blocked the formation of DNA adducts in Hepa-1 cells, but had little effect on 10T1/2 cells. Conversely, 7,8-BF completely blocked DMBA metabolism and DNA adduct formation in 10T1/2 cells, but had almost no effect on Hepa-1 cells. Both isoimperatorin and isoimperatorin inhibited the bioactivation of DMBA in both cell lines. These results indicate that bergamot lactone is a selective inhibitor of P450 1a1, but overall, its inhibitory effect on DMBA metabolic activation in mouse epidermis is weak. In contrast, isoimperatorin, isoimperatorin, and especially 7,8-BF, blocked DMBA metabolic activation in mouse epidermis and appear to be more selective for P450 1b1. Based on our studies using 10T1/2 and Hepa-1 cells, P450 1a1 is primarily responsible for converting DMBA-3,4-diol to trans-DMBADE, while P450 1b1 is primarily responsible for converting DMBA-3,4-diol to cis-DMBADE. These data demonstrate the roles of P450 1a1 and 1b1 in the metabolic activation of DMBA in mouse epidermis and provide a mechanistic explanation for the different effects of natural furanocoumarins (and 7,8-BF) on the skin carcinogenicity of polycyclic aromatic hydrocarbons.

[1]. Oral administration of the citrus coumarin, isopimpinellin, blocks DNA adduct formation and skin tumor initiation by 7,12-dimethylbenz[a]anthracene in SENCAR mice. Carcinogenesis. 2002 Oct;23(10):1667-75.

Isopsoralen is one of the psoralen compounds. It has been reported that isopsoralen is present in rue, Japanese angelica, and other organisms with relevant data. Isopsoralen is found in angelica. It is also found in the seeds of parsnip. Isopsoralen belongs to the furanocoumarin class of compounds. These compounds are polycyclic aromatic compounds containing a furan ring partially fused with coumarin. See also: Top (part) of Ming Dynasty angelica (Angelica keiskei). Mechanism of Action: Cytochrome P450 (P450) and glutathione S-transferase (GST) are two major families of enzymes involved in the metabolism of carcinogens. Typically, P450 plays an activating or detoxifying role, while GST primarily functions as a detoxifying enzyme. We have previously demonstrated that oral administration of the linear furanocoumarins isoimperatorin and imperatorin modulates P450 and GST activity in various tissues of mice. This study aimed to compare the ability of a wider range of naturally occurring coumarin compounds (including simple coumarins as well as linear and angular furanocoumarins) to modulate the activity of drug-metabolizing enzymes in the mouse liver after oral administration. We found that all the coumarin compounds tested (coumarin, limonene, hesperidin, angelicin, bergamotin, imperatorin, and isoimperatorin) induced hepatic GST activity, while the linear furanocoumarins showed the strongest ability to induce hepatic P450 activity, particularly P450 2B and 3A. Both of these effects corresponded to increased expression of the corresponding enzyme proteins. The xenobiotic-induced expression of P450 2B10, 3A11, and 2C9 is typically due to activation of the pregnane X receptor (PXR) and/or constitutive androstenedione receptor (CAR). Using the pregnane X receptor reporter system, our results indicate that isopidinine activates PXR and its human homologue SXR by recruiting the coactivator SRC-1 in transfected cells. In CAR transfection experiments, isopidinine antagonized the inhibitory effect of androstanol on the full-length mCAR (Gal4-mCAR ligand-binding domain fusion protein) and restored coactivator binding. Oral administration of isopidinine induced the expression of Cyp2b10, Cyp3a11, and GSTain mRNA in the liver of wild-type CAR(+/+) mice. Conversely, in CAR(-/-) mice, isopidinine-induced attenuated Cyp2b10 mRNA expression, suggesting that isopidinine induces Cyp2b10 via the CAR receptor. Overall, the current data suggest that naturally occurring coumarin compounds, based on their chemical structures, exhibit different activities in inducing a variety of exogenous metabolic enzymes.

---

Therapeutic Uses
/Exploratory Treatment/ Objective: To screen antitumor active compounds, drug-like compounds, or lead compounds from traditional Chinese medicinal materials. Methods: Eleven coumarin compounds were isolated from traditional Chinese medicinal materials. Their in vitro antitumor activity was studied by determining their inhibition rate on the growth of human bladder cancer cell line EJ. Results: The results showed that umbelliferone, scopolamine, desmethylfurantoin, isopyrrolizine, fumarate, coumarin, dicustin, and glycyrrhizin all inhibited the growth of human bladder cancer cell line EJ in vitro, and their activity showed a concentration-effect relationship. Fumarate, coumarin, dicustin, and umbelliferone had the strongest inhibitory effects, with IC50 values of 7.50×10⁻⁷, 2.30×10⁻⁶, 6.00×10⁻⁶, and 1.30×10⁻⁶ mol/L, respectively. However, flavoxel, aescin, and sphenanthrene did not inhibit the growth of the human bladder cancer cell line EJ under these experimental conditions. Conclusion: These results suggest that fobesoxidine, succinoside, aescin, dicustin, and umbelliferone may be effective against human bladder cancer or could be considered potent drug-like compounds or lead compounds. Previous studies have found that several common natural coumarin compounds found in the human diet can inhibit the P450-mediated metabolism of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzo[a]anthracene (DMBA) in vitro, block the formation of DNA adducts in mouse epidermis, and, when applied topically to mice, inhibit B[a]P and/or DMBA-induced skin tumors. This study aimed to investigate the effects of oral administration (70 mg/kg, for four consecutive days) of two linear furanocoumarin compounds (isoimperatorin and isoimperatorin) on the activities of P450 and glutathione S-transferase (GST) in different tissues of mice, as well as on the formation of benzo[a]pyrene (B[a]P) and dimethylbenzo[dMBA] (DMBA) DNA adducts. At 1 hour and 24 hours after oral administration, both isoimperatorin and isoimperatorin significantly inhibited the activities of ethoxyhalogen O-deethylase (EROD) and pentoxyhalogen O-dealkylase (PROD) in the epidermis. At 1 hour after the last oral administration, both isoimperatorin and isoimperatorin had a slight inhibitory effect on EROD activity in the lungs and forestomach, and a significant inhibitory effect on PROD activity in the lungs and forestomach. Twenty-four hours after the last oral administration, the activities of EROD and PROD in the epidermis and lungs remained inhibited. However, the P450 activity in the forestomach had recovered to control levels. Interestingly, isoimperatorin and isoimperatorin treatment inhibited hepatic EROD activity at 1 hour, with no effect on PROD activity at this time, but increased the activities of both enzymes at 24 hours. The increase in EROD and PROD activity was consistent with the increase in hepatic P450 content. Compared with the corn oil control group, isoimperatorin and isoimperatorin treatment increased hepatic cytoplasmic GST activity by 1.6-fold at both 1 hour and 24 hours after the last oral administration. Oral administration of isoimperatorin and isoimperatorin also protected against the formation of DNA adducts induced by benzo[a]pyrene (B[a]P) and dimethylbenzo[a]pyrene (DMBA). Isoimperatorin pretreatment reduced the formation of DMBA-induced DNA adducts in the forestomach. Isoimperatorin pretreatment reduced the levels of DNA adducts in liver (B[a]P), lung (B[a]P), and mammary epithelial cells (DMBA). These results suggest that dietary supplementation with isocoumarins and isoimperatorin may have potential chemopreventive effects. This study aimed to evaluate the effects of oral administration of the citrus coumarin isoimperatorin on the development of skin tumors induced by topical application of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzo[a]anthracene (DMBA). To assess the effects of oral isoimperatorin on B[a]P and DMBA-induced skin tumor development, its effect on DNA adduct formation was first evaluated. Female SENCAR mice were pre-administered corn oil or isoimperatorin (70 mg/kg body weight) 24 hours and 2 hours before topical application of B[a]P or DMBA, respectively. Another citrus coumarin, isocoumarin, was also included as a control. Oral administration of isoimperatorin and isoimperatorin significantly inhibited the formation of benzo[a]pyrene-DNA adducts, with inhibition rates of 37% and 26%, respectively. Isopsalicylate also inhibited the formation of dimethylbenzo[a]pyrene-DNA adducts, with an inhibition rate of 43%. In another dose-response study, oral administration of isopsalicylate (35, 70, and 150 mg/kg) inhibited the formation of dimethylbenzo[a]pyrene-DNA adducts, with inhibition rates of 23%, 56%, and 69%, respectively. In tumor studies, mice were pretreated with corn oil or isopsalicylate orally 24 hours and 2 hours before injection of dimethylbenzo[a]pyrene, respectively, and then injected with 12-O-tetradecanoylphorbol-13-acetate (TPA) two weeks later to promote tumor growth. Compared with the corn oil control group, isopsalicylate significantly reduced the average number of papillomas per mouse at doses of 30, 70, and 150 mg/kg body weight, with reduction rates of 49%, 73%, and 78%, respectively. Oral administration of isopsalicylate significantly reduced the incidence of papillomas in mice, a result also observed in the highest dose group (150 mg/kg). For comparison, this study also evaluated the efficacy of topical application of isoimperatorin against DMBA-induced tumorigenesis over a wide dose range. As part of this study, we evaluated various systemic toxicity parameters in mice following oral administration of isoimperatorin and imperatorin. Mice were orally administered corn oil, isoimperatorin, or imperatorin (35, 70, and 150 mg/kg body weight) once daily for four consecutive days. Mice were sacrificed 24 hours after the last administration, and their liver, lungs, and kidneys were histologically evaluated. In addition, we examined urinary nephrotoxicity parameters, blood liver and kidney function parameters, and prothrombin time. Results showed no significant changes in blood coagulation, kidney, or liver function. However, a significant increase in liver weight was observed, accompanied by hepatocyte cytoplasmic vacuolation. No histopathological changes were observed in the lungs and kidneys. Overall, these data suggest that oral administration of isoimperatorin (and imperatorin) has a chemopreventive effect against DMBA-induced skin tumors.

C13H10O5

246.2155

246.052

482-27-9

68079

Light yellow to yellow solid powder

1.4±0.1 g/cm3

448.7±45.0 °C at 760 mmHg

150-151ºC

225.1±28.7 °C

0.0±1.1 mmHg at 25°C

1.612

2.31

0

5

2

18

366

0

DFMAXQKDIGCMTL-UHFFFAOYSA-N

InChI=1S/C13H10O5/c1-15-10-7-3-4-9(14)18-12(7)13(16-2)11-8(10)5-6-17-11/h3-6H,1-2H3

4,9-dimethoxyfuro[3,2-g]chromen-7-one

Isopimpinellin

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: 49~50 mg/mL (199.0~203.1 mM)

Solubility in Formulation 1: 2.5 mg/mL (10.15 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (10.15 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.

---

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