Two new methoxylated flavones isolated from Casimiroa edulis La Llave and their MMP-9 inhibitory activity.

Casimiroa edulis La Llave is known to contain unusual 5,6-dimethoxyflavones bearing a variously oxygenated B-ring. Phytochemical investigation of the leaves and the roots of C. edulis achieved the isolation of two new methoxylated flavones, named casedulones A (1) and B (2), together with 12 known analogues. Their unique structures were established with the aid of spectral analyses and total syntheses. Pre-treatment with 20 µM of 1 and 2 suppressed MMP-9 expression in LPS-mediated THP-1 cells, indicating that the characteristic flavonoids in C. edulis could be potential anti-angiogenics for cancer prevention.

Antidepressant-like effect of Casimiroa pubescens root extracts.

In this study, we investigated the antidepressant-like effects of the hexane (HCP), ethyl acetate (ECP) and methanol (MCP) extracts of the roots of Casimiroa pubescens Ramírez (Rutaceae) using the forced swim test (FST). In an initial experiment, each extract was orally administered to mice only once 60 min before to the FST. In a second experiment, doses were administered 24, 7 and 1 h before testing. Our results showed that the triple administration of the extracts provided a stronger effect than single administration. However, the combination of HCP at 7.5 mg/kg and imipramine (IMI) at 12.5 mg/kg showed the greatest effect. The coumarins 3-(1',1', dimethyl allyl)-herniarin, auraptene, 8-geranyl-oxy psoralen, isopimpinellin and the flavonoid zapotin were isolated from the extracts. The hexane extract of C. pubescens showed an antidepressant-like activity, which may inspire further studies on developing new antidepressant agents.

New Methoxyflavone from Casimiroa sapota and the Biological Activities of Its Leaves Extract against Lead Acetate Induced Hepatotoxicity in Rats.

Flavonoids are agents with strong antioxidant properties and ameliorate many diseases associated with oxidative stress. Leaves of Casimiroa sapota were investigated for components and antioxidant/anti-inflammatory activities against lead acetate ((AcO)2 Pb) induced hepatotoxicity in rats. Three groups of male albino rats were administrated orally with vehicle or C. sapota (100 and 200 mg/kg b.w/day) for 28 days; other group was injected with sub-acute dose (100 mg/kg b.w/day) of (AcO)2 Pb. Three protective groups were injected with (AcO)2 Pb (100 mg/kg b.w/day) for 7 days at day 22 after treatment with either C. sapota (100 or 200 mg/kg b.w/day) or silymarin (SILY) for 28 days. We isolated and identified, from C. sapota, a new compound for the 1st time in nature; 5,6,2',3'-tetramethoxyflavone in addition to the rare compound 5,6,3'-trimethoxyflavone (second report of isolation from nature) and the known compound 5,6,2',3',4'-pentamethoxyflavone. There is an improvement in all hemato-biochemical parameters, antioxidant defense system and anti-inflammatory cytokines of protective groups, which received C. sapota in dose dependent manner. The percentage of changes in all parameters measured in (AcO)2 Pb groups that received vehicle, CS100, CS200 or SILY were 109.2, 37.3, 12.5%, and 1.2% compared with the healthy control group. The C. sapota groups confer a better antioxidant activity by preventing oxidative stress and inflammation in (AcO)2 Pb treated rats. The compounds isolated are responsible at least in part for the observed protective effects.

Phenolic compounds from leaves of Casimiroa edulis showed adipogenesis activity.

Casimiroa edulis is known as cochitzapotl, and it belongs to a species of tropical fruiting tree in the family Rutaceae, native to eastern Mexico and Central America south to Costa Rica. In this study, we isolated two furocoumarins and two polymethoxyflavones from leaves of C. edulis and evaluated the functions of glucose and lipid metabolism activity with 3T3-L1 adipocytes. We discovered that the addition of furocoumarins increased glucose uptake and lipid accumulation in 3T3-L1 adipocyte. These results suggest that furocoumarin compounds can be used as functional food-derived compounds, to regulate adipocyte functioning for the management of metabolic syndrome, which is associated with dysfunctions of glucose and lipid metabolism.

Vasodilation and radical-scavenging activity of imperatorin and selected coumarinic and flavonoid compounds from genus Casimiroa.

Hypertension is a very widespread condition which is not strictly considered as an illness but if not countered, progressively causes damage to all tissues and loss in their functionality. For this reason the find of new antihypertensive agents is prominent and medicinal plants and their derivatives are valuable for the purpose. The genus Casimiroa (Rutaceae) includes plants from Central America and Mexico; among these, Casimiroa edulis Llave et Lex. and Casimiroa pubescens Ramirez are the most relevant species, even for their medicinal uses. The decoction of leaves and seeds is traditionally taken as a tea mainly to lower blood pressure. The object of this research was the study of vascular activity of coumarinic and flavonoid compounds isolated from seeds of Casimiroa spp. in comparison with Casimiroa edulis and Casimiroa pubescens extracts. The phenolic compounds isolated from Casimiroa were herniarin (Her), imperatorin (Imp), 8-geranyloxypsoralen (GOP) and 5,6,2',3',4'-pentamethoxyflavone (PMF). All these compounds induced vasorelaxation on rat arterial tissues although with different effectiveness. To study the cellular mechanisms of the vasorelaxation exhibited by imperatorin, we used selective inhibitors of different receptors and enzymes, such as atropine, pyrilamine, nifedipine, L-NAME and DETC. In a further step of this research, we evaluated the radical-scavenging activity of Casimiroa extracts and isolated compounds by means of DPPH assay. In general, we observed that the scavenging activities increased in a concentration-dependent manner for all substances. The phenolic compounds highlight a synergism of vasodilation and antioxidant activity which may be very useful in the management of cardiovascular diseases. Among the evaluated compounds, imperatorin shows a significant vasorelaxant activity even higher than acetylcholine and similar to nitrite, and also useful antiradical capabilities. All these properties suggest its possible role against hypertension and vasculopathies, even if in vivo studies are needed to determine the actual applications.

New biological activities of Casimiroa edulis leaf extract and isolated compounds.

The phytochemical investigation of Casimiroa edulis Llave et Lex (Rotaceae) afforded four coumarins: umbelliferone (1), esculetin (2), imperatorin (3) and xanthotoxol (4). The identification of these compounds was achieved by using a combination of m.p., UV, EI-mass, ¹H NMR and ¹³C NMR spectroscopy. Essential oil extracts were analysed by GC/MS leading to the identification of 60 components. Sesquiterpene hydrocarbons accounted for the major make up of the oil. Microbiological screenings of the oil and successive plant fractions were performed, showing promising activity against a number of microorganisms with Minimum inhibitory concentrations (MIC) comparable to the standard antibiotics such as chloramphenicol and kanamycin. The plant ethanol extract (400 mg/kg) and the isolated coumarins (60 mg/kg) showed anticoagulant activity. Analyses to determine the activity of the extracts on liver and kidney function were performed, revealing no negative or detrimental effects.

Age-dependent vasorelaxation of Casimiroa edulis and Casimiroa pubescens extracts in rat caudal artery in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaf and seed decoctions of Casimiroa spp. are used in Mexican traditional medicine to treat high blood pressure. Previous researches showed as Casimiroa extracts are able to induce relaxation of rat aortic and caudal arteries. To study the influence of the aging, we determined the vascular effect induced by extracts of Casimiroa edulis and Casimiroa pubescens in arterial tissues from young and old rats. MATERIALS AND METHODS: The activity of Casimiroa edulis extracts: hexanic-leaf (Ce5), methanolic-leaf (Ce6), hexanic-seed (Ce7) and methanolic-seed (Ce8), and Casimiroa pubescens: hexanic-leaf (Cp9), methanolic-leaf (Cp10), hexanic-seed (Cp11) and methanolic-seed (Cp12) were investigated in precontracted caudal arteries of young (4 months) and old (20 months) rats. RESULTS: The Casimiroa extracts tested at 20 µg/ml induced vasorelaxation in phenylephrine-precontracted arterial tissues both in young and old arterial tissues. Methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12) caused a higher relaxation in young than in old arterial tissues. Nifedipine (0.01 µM) did not change the vasorelaxation induced by Casimiroa edulis extract either in young and old rat arterial tissues. CONCLUSIONS: The vasorelaxation induced by Casimiroa edulis and Casimiroa pubescens extracts is decreased from aging since the effects were higher in young than in old rat arterial tissues. However, the methanolic-seed extracts of both plant species induced a relevant vasorelaxation also in old arterial tissues. Thus the results support the traditional use of Casimiroa decoctions as antihypertensive, also in elderly.

Inhibition of HIV-1 reverse transcriptase associated activities by the hydroalcoholic extract of Casimiroa edulis seeds.

The hydroalcoholic extract obtained from the seeds of Casimiroa edulis cultivated in Sardinia (Italy) have been assayed on the two enzymatic-associated activities of the HIV-1 reverse transcriptase (RT), the RNA-dependent DNA polymerase (RDDP) and the ribonuclease H. In biochemical assays, the extract inhibited both activities in a dose-dependent manner, showing a 10-fold more potent inhibition of the HIV-1 RT RDDP activity. Furthermore, the extract was cytotoxic on K562 cell replication.

Vasorelaxation by extracts of Casimiroa spp. in rat resistance vessels and pharmacological study of cellular mechanisms.

AIM OF THE STUDY: Casimiroa spp. are Mexican plants traditionally used for treatment of hypertension. To study their antihypertensive action, we determined the arterial dilatation induced by extracts from leaves and seeds of Casimiroa calderoniae F. Chiang & Medrano, Casimiroa edulis Llave et Lex, and Casimiroa pubescens Ramirez. MATERIALS AND METHODS: The vascular effects of Casimiroa spp. extracts were investigated on rat caudal and aortic arteries. In addition, the extracts were characterized by HPLC using heraclenol, isopimpinellin, heraclenin and phellopterin as external standards. The methanolic extract of Casimiroa pubescens seeds (Cp12) was also studied by H-NMR and LC-MS (ESI-TOF) for the determination of casimiroin and zapotin. RESULTS: The hexanic and methanolic extracts of Casimiroa spp. (20 µg/ml) showed vasorelaxation in arterial tissues precontracted by phenylephrine (0.5 µM); the extracts from seeds always caused a greater relaxation in comparison to those from leaves. The most active were the methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12). To study the pharmacological mechanisms of vasodilatation we used various inhibitors selective to different receptor subtypes or intracellular enzymes. The vasorelaxant effect of Ce8 (20 µg/ml) remained unaffected by the pretreatment with pyrilamine (10 µM), an antagonist of histamine H(1) receptors, but was inhibited by atropine (0.1 µM), a muscarinic receptor antagonist. Therefore, to determine muscarinic receptor subtypes, we used pirenzepine (1 µM), a selective inhibitor of M(1) receptor, and 4-diphenylacetoxyl-N-methylpiperidine methiodide (DAMP, 0.01 µM), a selective inhibitor of M(3) receptor. Only the latter reduced the vasodilatation by Ce8 and Cp12. To investigate the role of the nitric oxide synthase (NOS), we used N(G)-nitro-l-arginine methyl ester (l-NAME, 10 µM), a selective NOS inhibitor, which decreased the dilatation induced by Ce8 and Cp12. Finally, we studied the action of (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) (ODQ, 3 µM), a selective guanylyl cyclase inhibitor, which inhibited the dilatation by Casimiroa extracts. CONCLUSION: The data show that methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12) induce vasorelaxation by M(3) receptor through the activation of cGMP-dependent NO signaling. These results support the traditional use of Casimiroa decoctions for antihypertensive treatments in the Mexican ethnomedicine.

Chemical composition of the leaf essential oil of Casimiroa edulis La Llave & Lex. (Rutaceae) from Monteverde, Costa Rica.

The leaf essential oil of Casimiroa edulis (Rutaceae), collected from Monteverde, Costa Rica, has been obtained by hydrodistillation and analyzed by GC-MS. The leaf oil was dominated by sesquiterpene hydrocarbons, predominately germacrene D (16-22%) and (E)-caryophyllene (16-17%), consistent with the traditional use of this plant as a sedative, sleep inducer and hypotensive.

Synthesis of casimiroin and optimization of its quinone reductase 2 and aromatase inhibitory activities.

An efficient method has been developed to synthesize casimiroin (1), a component of the edible fruit of Casimiroa edulis, on a multigram scale in good overall yield. The route was versatile enough to provide an array of compound 1 analogues that were evaluated as QR2 and aromatase inhibitors. In addition, X-ray crystallography studies of QR2 in complex with compound 1 and one of its more potent analogues has provided insight into the mechanism of action of this new series of QR2 inhibitors. The initial biological investigations suggest that compound 1 and its analogues merit further investigation as potential chemopreventive or chemotherapeutic agents.

Chemical composition and antispasmodic effect of Casimiroa pringlei essential oil on rat uterus.

The Casimiroa pringlei essential oil was analyzed to determine its chemical composition. Its effect on rat uterine smooth muscle was studied and compared with verapamil. Pure commercial piperitone, eucalyptol, and alpha-terpineol, the major constituents of C. pringlei essential oil, were tested on the uterine tonic contraction induced by high-potassium depolarizing solution (KCl 60 mM).

Synthesis and cancer chemopreventive activity of zapotin, a natural product from Casimiroa edulis.

An efficient method has been developed to synthesize zapotin (5,6,2',6'-tetramethoxyflavone), a component of the edible fruit Casimiroa edulis, on a multigram scale. The synthesis utilizes a regioselective C-acylation of a dilithium dianion derived from a substituted o-hydroxyactophenone to afford a beta-diketone intermediate that can be cyclized to zapotin in good overall yield, thus avoiding the inefficient Baker-Venkataraman rearrangement pathway. Zapotin was found to induce both cell differentiation and apoptosis with cultured human promyelocytic leukemia cells (HL-60 cells). In addition, the compound inhibits 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity with human bladder carcinoma cells (T24 cells), and TPA-induced nuclear factor-kappa B (NF-kappaB) activity with human hepatocellular liver carcinoma cells (HepG2 cells). These data suggest that zapotin merits further investigation as a potential cancer chemopreventive agent.

A new blend of white sapote fruit volatiles as potential attractant to Anastrepha ludens (Diptera: Tephritidae).

The behavioral and electrophysiological responses of nonirradiated male and female Anastrepha ludens (Loew) (Diptera: Tephritidae), to white sapote, Casimiroa edulis Oerst. (Rutaceae), volatiles were investigated. Females flew upwind and landed more often on fruit than on artificial fruit in wind tunnel bioassays. Males flew upwind (but not landed) more frequently on fruit than on artificial fruit. Porapak Q volatile extracts of white sapote also elicited upwind flight and landing on artificial fruit for both sexes. Gas chromatography-electroantennographic detection analysis of white sapote extracts revealed that antennae of both sexes responded to eight compounds. Two peaks were unidentified because they did not separate from the solvent. Subsequent peaks were identified by gas chromatography-mass spectrometry as styrene, myrcene, 1,2,4-trimethylbenzene, 1,8-cineole, and linalool in a proportion of 50: 21: 0.5: 27: 1.5, respectively. Eight peaks were tentatively identified as beta-trans-ocimene. The number of A. ludens captured in multilure traps baited with the synthetic white sapote blend was higher than the flies captured by the multilure unbaited traps (control) in field cages. However, the number of flies captured by traps baited with the white sapote blend was not different from that of flies captured by traps baited with hydrolyzed protein. Using standard chemical ecology techniques, we found potential attractants from wild sapote fruit for monitoring and management of A. ludens population.

A furanocoumarin and polymethoxylated flavonoids from the Yucatec Mayan plant Casimiroa tetrameria.

As part of an ongoing study of the medicinal plants of the Yucatec Maya, Casimiroa tetrameria was investigated for its phytochemistry. From an ethyl acetate partition of an ethanol extract of the leaves, eight flavonoids and a furanocoumarin were isolated and characterised as 5,6,2',3',5',6'-hexamethoxyflavone, 5,6,2',3',6'-pentamethoxyflavone and 5-methoxy-8-(3''-hydroxymethyl-but-2''-enyloxy)-psoralen using a combination of 1H, 13C NMR and NOESY spectroscopy.