Isolation and structure elucidation of constituents of Citrus limon, Isodon japonicus, and Lansium domesticum as the cancer prevention agents

In the course of our research to investigate the cancer prevention potency of natural products derived from plant materials, we isolated fifty-five compounds, including twenty-one new compounds from the peels of Citrus limon, aerial parts of Isodon japonicus, and leaves of Lansium domesticum. The chemical structures of the isolated compounds were elucidated by chemical/physicochemical evidence, and nuclear magnetic resonance spectroscopy and mass spectrometry results. Moreover, the absolute stereochemistry of the new compounds were elucidated by various techniques such as chemical synthesis, modified Mosher’s method, Cu-Kα X-ray crystallographic analysis, and comparison of experimental and predicted electronic circular dichroism data. The antimutagenic effects of the isolated and structure-elucidated compounds against heterocyclic amines, 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp-P-1) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), were evaluated by the Ames test and in vivo micronucleus test. In this review, we present the comprehensive results of the antimutagenic effects of the isolated natural products.


Background
Cancer is the leading cause of death worldwide and one of the major risk factors is exposure to the agents that damage the genetic material. These agents are known as genotoxins and, according to their mode of action, are classified into mutagens, carcinogens, or teratogens [1,2]. Trp-P-1 and PhIP are well known mutagenic and carcinogenic heterocyclic amines that are found in cooked meat. Therefore, it is difficult to completely avoid these risk factors in daily life. On the other hand, previous case-control studies have suggested that the consumption of some plant derived foods such as citrus fruits is associated with a reduced all-cancer incidence [3].
Based on these studies, we searched for antimutagenic materials derived from foods. In the course of this study, we found using the Ames test that the methanolic (MeOH) extracts of C. limon [4,5], aerial parts of I. japonicus [6], and leaves of L. domesticum [7,8] showed antimutagenic effects against Trp-P-1 and PhIP. Therefore, we directed our efforts toward the isolation of their constituents and evaluation of the antimutagenic effects of the isolated constituents using the Ames test and in vivo micronucleus test.

Review
Compounds obtained from the peels of C. limon The fruits of Citrus limon (L.) Burm.f. contain important natural chemical components, such as flavonoids, furanocoumarins, and limonoids [9]. Previous studies have described the biological activities of these compounds, such as the antioxidative, anti-inflammatory, antiallergic, antiviral, antiproliferative, anticarcinogenic, and antimutagenic activities of flavonoids [10], the suppressive effect of limonin on intestinal polyp development in Apc-mutant Min mice [11], and the inhibitory effects of furanocoumarins on human CYP 3A4 [12]. In addition, the essential oil of C. limon leaf has been shown to act as a central nervous system depressant and anticonvulsant in animal models [11].

Evaluation of the antimutagenic effects of isolated compounds using the Ames test
The antimutagenic effects of the isolated compounds were evaluated against Trp-P-1 and PhIP by the Ames test using the S. typhimurium TA98 strain (Tables 1, 2, 3 and 4). Trp-P-1 and PhIP are well known mutagenic and carcinogenic heterocyclic amines found in cooked meat. We used nobiletin as the positive control that have been reported to have antimutagenic effects using the Ames test [44]. As shown in Tables 2 and 4, among the compounds isolated from the peels of C. limon, furanocoumarins, (+)-tert-O-methyloxypeucedanin hydrate   . Each point represents the mean and standard deviation of five mice. a Normal or sample feeds that included limonin (19) at low or high dose (0.02% or 0.04%, w/w) were given ad libitum. b Normal or sample feeds that included lansionic acid (53) at low or high dose (0.03% or 0.06%, w/w) were given ad libitum [4,8] activity of tested compounds against the S. typhimurium TA98 strain were tested using nutrient agar containing NaCl and all compounds showed weak [44: 23.2% inhibition at 400 nmol/plate, 54: 23.0% inhibition at 400 nmol/plate] or no (other compounds: < 5.0% inhibition at highest concentration in Table 1) antibacterial activities [4][5][6][7][8].
Evaluation of antimutagenic effects of limonin (19) and lansionic acid (53) using in vivo micronucleus test To examine the antimutagenic effects of limonin (19) and lansionic acid (53) in vivo, we conducted a micronucleus test using the peripheral blood of male ICR mice. These two compounds were isolated enough amount and are the major constituents of C. limon and L. domesticum. The micronucleus test detects chromosomal damage induced by genotoxic/carcinogenic compounds, and it has been used to evaluate antimutagenic agents in vivo. We gave either normal feed or sample feed that included the limonin (19) or lansionic acid (53) at low or high dose. The mouse tail vein blood (5 μL) was taken prior to the administration of PhIP, and at 24, 48, and 72 h after administration. As a result, limonin (19) and lansionic acid (53) significantly decreased the frequency of micronucleated reticulocytes (MNRETs) treated with PhIP at 24 h and 48 h after the administration (Fig. 4) [4,7].

Conclusions
In conclusion, twenty-one new compounds and thirtyfour known compounds including coumarins, furanocoumarins, oximes, ent-kaurane diterpenoids, and onoceranoid-type triterpenoids were isolated from the peels of C. limon, aerial parts of I. japonicus, and leaves of L. domesticum. Among them, furanocoumarins showed the strongest antimutagenic effects in the Ames test. It is important to note that furanocoumarins were well known Cytochrome P450 (CYP) 1A2 inhibitors [45], and the mutagenicity of Trp-P-1 and PhIP depends on their bioactivation by CYP 1A2 [46]. These facts suggest that one of the mechanisms of the antimutagenic effects of furanocoumarins against Trp-P-1 and PhIP may be the inhibition of the CYP 1A2 enzyme. In addition, the oral intake of limonin (19) and lansionic acid (53), which are the major constituents of C. limon and L. domesticum, respectively, showed significant antimutagenic effects against PhIP in the in vivo micronucleus test. These results suggest that these compounds and plant materials are likely useful agents for cancer prevention.