Flavonoids, Sterols and Lignans from Cochlospermum vitifolium and Their Relationship with Its Liver Activity

The sterols β-sitostenone (1), stigmast-4,6,8(14),22-tetraen-3-one (2), β-sitosterol (3) and stigmasterol (4), the aromatic derivatives antiarol (5) and gentisic acid (6), the phenylpropanes coniferyl alcohol (7), epoxyconiferyl alcohol (8) and ferulic acid (9), the apocarotenoid vomifoliol (10), the flavonoids naringenin (11), 7,4′-dimethoxytaxifolin (7,4′-dimethoxydihydroquercetin, 12), aromadendrin (13), kaempferol (14), taxifolin (dihydroquercetin, 15), prunin (naringenin-7-O-β-d-glucoside, 16), populnin (kaempferol-7-O-β-d-glucoside, 17) and senecin (aromadendrin-7-O-β-d-glucoside, 18) and the lignans kobusin (19) and pinoresinol (20), were isolated from the dried bark of Cochlospermum vitifolium Spreng (Cochlospermaceae), a Mexican medicinal plant used to treat jaundice, liver ailments and hepatitis C. Fourteen of these compounds were isolated for the first time from this plant and from the Cochlospermum genus. Compounds 3–4, 6–7, 9–11, 13–17 and 20 have previously exhibited diverse beneficial liver activities. The presence of these compounds in C. vitifolium correlates with the use of this Mexican medicinal plant.

Previous studies have been conducted to demonstrate the antihypertensive [19][20][21]23], hypoglycemic and antidiabetic [22], immunomodulatory [23] and anti-inflammatory [24] effects of Cochlospermum vitifolium extracts. An exhaustive revision of the existing literature indicated that several of the compounds isolated in this research ( Figure 1) can be associated to its popular use as different liver treatments. Due to the fact that this plant is broadly used as a treatment for jaundice, hepatitis C and other liver ailments in Mexican traditional medicine, its methanol extract was administrated at a dose of 100 mg/kg to bile duct-obstructed rats, to determine its hepatoprotective activity, showing a statistically significant decrease of serum glutamic-pyruvic transaminase (PGT, 45%) and alkaline phosphatase (APh, 15%) [19].
The importance of such activities lie in the fact that hepatic diseases (which comprise several conditions, such as: cirrhosis, hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, cholestatic and drug-induced liver diseases and liver cancer) are extremely high-priced in terms of human suffering, loss of productivity, and medical or hospital consultations. In fact, chronic liver diseases are the major cause of mortality worldwide. In 2013, 29 million people in Europe suffered from a chronic liver condition [25] and more than 30 million Americans had hepatic disease [26]. In China, liver diseases, viral hepatitis (predominantly hepatitis B virus), non-alcoholic fatty liver and alcoholic liver disease affect approximately 300 million people [27]. From a physiopathological perspective, most chronic liver diseases begin as an inflammatory process which evolves into focal fibrosis, and afterwards, to complete fibrosis of the gland (hepatic cirrhosis), which increases the risk Six of these 20 compounds (3, 4, 11, 13, 15 and 20) had been previously isolated from Cochlospermum vitifolium, however all other compounds were isolated here for the first time from this plant and from this genus. Compounds 1-20 belong to either the sterols; C 6 , C 6 -C 1 and C 6 -C 3 aromatic compounds; apocarotenoids; flavonoids and lignans groups, which are the most frequently identified compounds in this genus.
Previous studies have been conducted to demonstrate the antihypertensive [19][20][21]23], hypoglycemic and antidiabetic [22], immunomodulatory [23] and anti-inflammatory [24] effects of Cochlospermum vitifolium extracts. An exhaustive revision of the existing literature indicated that several of the compounds isolated in this research ( Figure 1) can be associated to its popular use as different liver treatments. Due to the fact that this plant is broadly used as a treatment for jaundice, hepatitis C and other liver ailments in Mexican traditional medicine, its methanol extract was administrated at a dose of 100 mg/kg to bile duct-obstructed rats, to determine its hepatoprotective activity, showing a statistically significant decrease of serum glutamic-pyruvic transaminase (PGT, 45%) and alkaline phosphatase (APh, 15%) [19].
The importance of such activities lie in the fact that hepatic diseases (which comprise several conditions, such as: cirrhosis, hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, cholestatic and drug-induced liver diseases and liver cancer) are extremely high-priced in terms of human suffering, loss of productivity, and medical or hospital consultations. In fact, chronic liver diseases are the major cause of mortality worldwide. In 2013, 29 million people in Europe suffered from a chronic liver condition [25] and more than 30 million Americans had hepatic disease [26].
In China, liver diseases, viral hepatitis (predominantly hepatitis B virus), non-alcoholic fatty liver and alcoholic liver disease affect approximately 300 million people [27]. From a physiopathological perspective, most chronic liver diseases begin as an inflammatory process which evolves into focal fibrosis, and afterwards, to complete fibrosis of the gland (hepatic cirrhosis), which increases the risk of liver cancer. This leads to severe hepatic injury and ultimately to liver failure and other complications.
Thirteen of the twenty compounds isolated from Cochlospermum vitifolium have previously exhibited in vitro and in vivo beneficial liver effects. β-Sitosterol (3) decreased hepatofibrosis [28], protecting against CCl 4 -induced hepatotoxicity [29] in animal models. Stigmasterol (4) induced apoptosis in hepatocarcimona (HepG2) cells being a potential antineoplastic therapeutic agent [30]. Gentisic acid (6) showed anti-inflammatory and antimutagenic properties, demonstrating protective effects against induced genotoxicity and hepatotoxicity [31]. On the other hand, coniferyl alcohol (7) had a moderated anti-hepatitis B virus (HBV) activity [32]. Ferulic acid (9) had an in vivo hepato-protective effect against the CCl 4 -and formaldehyde-induced hepatotoxicity [33] and also a capacity to inhibit the development of hepatic fibrosis by activation of Hepatic Stellate Cells (HSCs) in the presence of liver damage [34]. Vomifoliol (10) showed moderate activity against human hepatocarcinoma Hep3B cells [35]. Naringenin (11) had a potent lipid-lowering effect reducing the hepatic lipogenesis in rats and acting as an insulin sensitizer in vivo [36], thus, preventing rat liver damage caused by lead acetate, arsenic and high glucose. Furthermore, this same compound suppresses the metastatic potential of hepatocellular carcinoma [37]. Additionally, aromadendrin (13) possessed radical scavenging and activity against inflammatory, tumor and diabetic processes [38]. Kaempferol (14) had hepatoprotective effects in CCl 4 -, drug-and alcoholic-induced liver injury, constituting a promising therapeutic option for patients with atherosclerotic disease [39]. Taxifolin (15) had antioxidant and cytoprotective effects that prevent and help treat fulminant hepatitis and hepatitis caused CCl 4 [40]. This natural flavonoid is licensed as Silymarin (Legalon ® ), a drug used for the treatment of toxic liver damage, chronic inflammatory liver disease and liver cirrhosis. Prunin (16) showed activity against the hepatitis B (HBV) virus with an IC 50 of 41.59 µM [41]. Administered at a dose of 25 mg/kg, populnin (17) exhibited in vivo hepatoprotective effects against CCl 4 -and D-GalN-induced hepatotoxicity, preventing the development of hepatic lesions [42]. Finally, at 50 and 100 mg/kg, the lignane pinoresinol (20) showed hepatoprotective effects improving CCl 4 -induced liver injury [43]. All these liver beneficial effects from the compounds isolated from Cochlospermum vitifolium directly correlate with its traditional use in Mexican medicine.

General Procedures
Compounds 1-20 were purified by successive open column chromatography (CC) using silica gel (70-230 and 230-400 mesh, Sigma-Aldrich, Toluca, México). The isolation procedures and purity of compounds were monitored by thin layer chromatography (TLC) using precoated silica gel 60 F 254 aluminium sheets, visualizing with UV-light and subsequently spraying the plates with (NH 4 ) 4 Ce(SO 4 ) 4 in 2 N H 2 SO 4 (Sigma-Aldrich, Toluca, México). All 1 H-, 13 C-and 2-D NMR experiments were performed in CDCl 3 on a Varian Unity 400 spectrometer (Varian, Inc., Palo Alto, CA, USA) equipped with a 5 mm inverse detection pulse field gradient probe at 25 • C, at 400 MHz for 1 H-NMR and 100 MHz for 13 C-NMR. Chemical shifts were referenced to tetramethylsilane as an internal standard.

Plant Material
The wood of Cochlospermum vitifolium was collected from "Sierra de Huautla" (20 •

Conclusions
Cochlospermum vitifolium biosynthesizes among other compounds the sterols 3 and 4, the aromatic compounds 6, 7 and 9, the apocatrotenoid 10, the flavonoids 11 and 13-17, and the lignan 20, which have demonstrated beneficial activity to alleviate different liver diseases. The presence of these compounds in the plant agrees with its traditional use in Mexican medicine and some are even included in commercial pharmaceutical formulations used in the treatment of hepatopathies. Their presence within Cochlospermum vitifolium extracts indicates that this plant could be an active hepatoprotective agent. However, the human consumption of this plant must be subjected to toxicity, pharmacodynamic and pharmacokinetic studies to determine how it can be a health contributor.
The isolated metabolites in this study and the chemical composition previously reported for Cochlospermum vitifolium agree with the metabolic content within other Cochlospermum species. The flavonoids, sterols, carotenoids, apocarotenoids and lignans isolated here have chemotaxonomic significance within this genus. This is the first report of compounds 1-2, 5-10, 12, 14, 16-19 from Cochlospermum vitifolium.