The presence of specific alkaloids in Ruta species was underlined more than 60 years ago with the isolation of the first furanocoumarin and furoquinoline alkaloids, such as dictamnine and evolitrine ^[1][29]. But these products were isolated mainly from R. graveolens L. and R. montana Mill. ^[2][30]. The presence and bioactivities of alkaloids in R. angustifolia Pers. was unambiguously established much later, in 2014 with the isolation of the quinolinone alkaloid pseudane IX and the characterization of its activity against the HCV virus ^[3][21]. Pseudane IX was found to inhibit HCV replication, predominantly at the post-entry step (IC₅₀ = 3.0 µg/mL post-entry versus 11.5 µg/mL at the entry step, in Huh7.5 cells infected with HCV). The compound functions as an inhibitor of HCV RNA replication and viral protein synthesis. In the viral system used (HCV strain J6/JFH1-P47), pseudane IX turned out to be more potent that the reference product ribavirin (IC₅₀ = 1.4 and 2.8 µg/mL, respectively) and the alkaloid only exerted a modest cytotoxic action (CC₅₀ = 26 µg/mL).

The case of the furoquinoline alkaloids, kokusaginine merits a mention because the compound, found in diverse plants, has been shown to inhibit tubulin formation and assembly. It binds to the colchicine site on tubulin, thereby inhibiting the growth of breast cancer cells, including multidrug resistant sublines such as MCF-7/ADR and MDA-MB-231/ADR ^[4][37]. Kokusaginine also displays modest cholinesterase inhibitory activities (IC₅₀ = 41.46 and 70.24 µg/mL against butyrylcholinesterase and acetylcholinesterase, respectively) ^[5][6][38,39]. The compound has been used as a starting point to the design of antiparasitic molecules active against Trypanosoma cruzi responsible for the Chagas disease ^[7][40]. Kokusaginine is an isomer of skimmianine and a close analogue of dictamnine, both furoquinolines commonly found in Ruta species and endowed with anticancer properties ^[8][9][10][41,42,43]. There is a sound basis to investigate further the anticancer properties of kokusaginine and related furoquinoline alkaloids.

Many quinoline alkaloids have been isolated from Ruta species, such as dictamnine, pteleine, skimmianine, rutacridone, maculosidine, graveoline, graveolinine ^[11][44]. The quinolone alkaloid graveoline, initially isolated from the common rue (Ruta graveolens) ^[12][11], is a major phytotoxin from Ruta plants ^[13][45]. It is found in many Ruta, including the leaves of R. angustifolia ^[14][15][14,19]. In this specie, graveoline has been characterized recently as an antifungal agent inhibiting the expression of the enzyme isocitrate lyase 1 (ICL1) in the fungus Candida albicans, as observed with the reference antifungal product fluconazole ^[15][19]. However, the mechanism of action of this compound is multifactorial because it has been characterized as an inhibitor of photosynthesis ^[16][46] and as an antitumor agent capable of inducing production of reactive oxygen species (ROS) in cancer cells and triggering both apoptotic and autophagic cell death ^[17][47]. The compound is easily metabolized in the liver, with up to 12 metabolites identified ^[18][48]. The derivative graveolinine displays weaker anti-angiogenic activity than the parent compound graveoline ^[19][49] but it can serve as a starting point for the design of anti-Alzheimer agents ^[20][50]. The compound has been shown to bind to the serotonin 5-HT2B receptor and to inhibit weakly the cyclooxygenase 2 enzyme (COX2, 79% inhibition at 150 µM) ^[21][51].

A quinolin-4-one moiety is also present in arborinine, another alkaloid found in R. angustifolia. As mentioned above, this tricyclic compound can inhibit HCV replication, but it is less potent than pseudane IX ^[3][21], and it contributes to the antifungal activity of the plant extract ^[15][19]. Arborinine can be isolated from diverse plants, including Rutaceae such as Vepris trichocarpa and Vepris teva ^[22][23][52,53], Araliopsis soyauxii ^[24][54] and different Ruta species ^[25][55]. Interestingly, the acridone alkaloid has been shown to exert marked antitumor activities. It inhibits dose-dependently the proliferation of several cancer cell lines ^[26][27][56,57], triggers cell cycle arrests, blocks cancer cell migration, and induces apoptosis ^[28][29][58,59]. It displays a sub-micromolar activity against drug-resistant SGC-7901 gastric cancer cells resistant to adriamycin (SGC-7901/ADR: IC₅₀ = 0.24 μM) or to vincristine (SGC-7901/VCR: IC₅₀ = 1.09 μM) ^[30][60]. Arborinine has been characterized as a selective and reversible inhibitor of histone lysine-specific demethylase 1 (LSD1), an enzyme frequently overexpressed in cancer cells. LSD1 is a key enzyme, implicated in the epithelial-mesenchymal transition (EMT) and in tumor progression. Arborinine has the capacity to repress EMT in cancer cells, modulating the expression of specific markers (upregulation of E-cadherin, downregulation of N-cadherin and vimentin). The blockade of LSD1 with arborinine induces a dose-dependent accumulation of methylated histone H3 in cells (H3K4me1, H3K9me1, H3K9me2), thereby inducing inhibition of cancer cell migration, invasion and proliferation.

Remarkably, the compound was shown to exert a significant antitumor effect in vivo, in two xenografted murine model of gastric cancer (with SGC-7901 cells sensitive or resistant to adriamycin). At the oral dose of 40–80 mg/kg, arborinine reduced tumor growth in mice, without causing any apparent toxicity ^[30][60]. By the same token, very recently arborinine has been shown to suppress ovarian cancer development through inhibition of LSD1. The blockade of LSD1 leads to an increased expression of methylated histone H3K4m1 in SKOV3 ovarian cancer cells, thereby reducing the migration and invasion capacities of the cells due to a blocked EMT ^[31][61]. The effect is not specific to ovarian cancer cells because the LSD1 enzyme (also known as lysine (K)-specific demethylase 1A, or KDM1A) is present and frequently overexpressed in many cancer cell types. Recently, it was demonstrated that arborinine can block LSD1/KDM1A activity in clear-cell renal cell carcinoma ccRCC cell lines, sensitive or resistant to the kinase inhibitor sorafenib. In this case again, the compound blocked cell migration and invasion, cell cycle progression, and induced apoptosis ^[32][62]. A molecular modeling analysis suggested that arborinine could bind directly to the active site of LSD1, and blocks downstream signaling activities, notably the expression of the ubiquitin-conjugating enzyme E2O (UBE2O), an important protein for cancer cell survival and proliferation ^[32][62]. The observation that arborinine represses LSD1/UBE2O signaling in ccRCC opens novel perspectives for research. Firstly, in terms of drug design, it opens a field of research to discover other lysine demethylase inhibitors with an acridone or quinolin-4-one moiety. There exist many naturally-occurring analogues of arborinine, such as evoxanthine, rutacridone, quinolactacins A-C, (iso)acronycine, melicopicine and others tri/tetracyclic products. Bicyclic products derived from echinopsine shall be considered as well, notably the panel of 1-methylquinolin-4-one alkaloids such as eduline, japonine and others. Secondly, in terms of clinical applications, LSD1/KDM1A is aberrantly overexpressed in several cancer types, with different LSD1 inhibitors currently developed to treat solid tumors and hematological malignancies ^[33][34][35][63,64,65]. The antitumor activity of arborinine shall be investigated using a variety of experimental models, to define the optimal conditions and the most suitable applications in humans. Thirdly, outside oncology, LSD1/KDM1A is an enzyme of interest to treat pathologies such as myelofibrosis ^[36][66], autism ^[37][67] and other diseases ^[38][68]. The targeting of LSD1 with arborinine may have broad therapeutic implications; recently, Merck paid US$1.35 billion for LSD1 inhibitors ^[39][69]. Parenthetically, another mechanism of action has been evoked with arborinine: the binding to heme leading to a reduction of oxidative stress and inflammation ^[40][70]. However, the heme binding affinity is weak (K_a = 3.9 × 10⁴ M⁻¹) compared to the level of LSD1 inhibition. Nevertheless, arborinine exhibits anti-inflammatory activities. It is able to reduce the production of nitric oxide (NO) in activated macrophages ^[41][71].

Rutamarin is an anticancer furocoumarin derivative isolated from R. angustifolia Pers. ^[42][72] and other Ruta species ^[43][73]. The compound is moderately cytotoxic to colorectal adenocarcinoma HT29 cells (IC₅₀ = 5.6 μM), and induces cell cycle perturbations and caspase-dependent apoptosis ^[43][73]. At the molecular level, rutamarin is believed to function as a catalytic inhibitor of human topoisomerase II (not a classical Topo II poison), binding to the ATPase domain of the enzyme, thereby blocking DNA replication ^[44][74]. This mechanism accounts for both the anticancer and antiviral activities of rutamarin, and probably for its antiprotozoal activity as well ^[45][75]. Rutamarin inhibits DNA replication of Epstein-Barr virus (EBV) (IC₅₀ = 7.0 μM) ^[46][47][76,77] and Kaposi’s sarcoma-associated herpesvirus (KSHV) (IC₅₀ = 1.12 μM), at least the (+)-enantiomer ^[44][74]. Beyond TopoII, two additional targets for rutamarin have been proposed on the basis of a molecular modeling analysis: the protein tyrosine phosphatase 1B (PTP1B) and the retinoid X receptor α (RXRα). Rutamarin would be an inhibitor of PTP1B and a RXRα agonist. The molecular models have been validated experimentally. Rutamarin effectively inhibits the PTP1B enzyme (IC₅₀ = 6.4 µM), acting as a competitive inhibitor capable of enhancing the insulin-induced translocation of the glucose transporter 4 (GLUT4) in CHO/GLUT4 cells ^[48][78].

The dihydrofuranocoumarin chalepin can be isolated from the leaves R. angustifolia Pers. together with its furanocoumarin analogue chalepensin ^[49][13]. They are present in different Rutaceae plants ^[50][6]. They bear a 3-prenyl side chain, as in rutamarin. Chalepin displays a moderate antibacterial activity against B. substilis (Gram positive) ^[51][80] and a weak antiparasitic activity against Trypanosoma cruzi, the pathogen responsible for the Chagas disease. Chalepin and related coumarins can bind to the active site of T. cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) ^[52][53][54][81,82,83]. However, the binding affinity is weak and no anti-trypanosomal activity has been reported. Chalepin is better known for its anticancer properties. The compound displays antiproliferative activities, notably toward A549 lung cancer cells (IC₅₀ = 27.6 μM), and triggers apoptosis with specific alterations of mitochondrial activities ^[49][13]. For a long time, the compound is known to reduce mitochondrial respiration, acting as a rotenone-like inhibitor of mitochondrial complex I ^[55][84]. It inhibits markedly the pyruvate/malate-supported proton flux in rat liver mitochondria (but is about 10 times less potent than rotenone) ^[56][85]. In cancer cells, chalepin not only alters mitochondria but affects also cell cycle progression and triggers extrinsic apoptosis ^[57][58][86,87].

A few other coumarins have been isolated from the aerial parts of R. angustifolia, such as 6,7,8-trimethoxycoumarin and scoparone ^[59][88]. The latter is a well-known antioxidant and lipid-lowering agent, considered useful to alleviate alcohol- or high-fat diet-induced liver injuries ^[60][61][89,90]. It has osteogenic effects, potentially useful to avoid bone demineralization ^[62][91], and displays antiproliferative effects against tumor cells ^[63][92]. Perhaps the most emblematic (but also confusing) coumarin is angustifolin, present in a very small amount of the aerial part of R. angustifolia ^[59][88]. The name angustifolin of the product is emblematic because it derives directly from Angustifolia, but it is confusing because the same name has been given to a totally different product, an ent-kaurane diterpenoid isolated from the aerial parts of Isodon species ^[64][65][93,94]. There are also four lignans named angustifolin A-D isolated from Kadsura angustifolia ^[66][95], and an alkaloid designated angustifoline (with a “e” at the end to respect the alkaloid terminology) found in Lupinus species ^[67][68][96,97]. Hence, the possible confusion between names Angustifolin from Ruta has been rarely cited, but it cannot be ignored ^[59][88].

Bergapten (5-methoxypsoralen) is a furocoumarin found in many plant species (notably bergamot), including R. angustifolia ^[49][13]. It is a classical antioxidant and anti-inflammatory compound, well known for its hypo-lipidemic, anti-ulcer and antidiarrheal activities ^[69][70][98,99]. It is also considered for the treatment of ischemic stroke ^[71][72][100,101]. Its known phototoxicity can be an issue, but the photoactivation could be exploited to treat skin cancers ^[73][102]. Bergapten displays anticancer properties, notably as a metabolic modulator for breast and colon cancer cells ^[74][75][103,104].

A relatively rare series is called moskachans A-D, four benzodioxone derivatives, discovered initially from the aerial part of R. angustifolia ^[76][105] and later found in R. chalepensis ^[77][106]. The name moskachan comes from ‘moskatxa’, the Basque name for R. angustifolia ^[76][105] but also used for other Ruta species. Moskachan B is not cytotoxic whereas moskachan D displays a weak antiproliferative activity against A549 lung cancer cells (IC₅₀ = 74.4 μM) ^[49][13]. In addition to these four moskachans, other compounds with a methylenedioxyphenyl moiety have been identified from an essential oil of R. angustifolia (from a specimen collected in Malaysia), including compounds structurally close to safrol which is a well-known phenylpropene derivative ^[78][107]. Moskachans have been occasionally found in other species but rarely studied. They could be further considered as ingredients in the food or perfumery industry, as it is the case for other compounds with a methylenedioxyphenyl moiety.