Bioactive principles from Carduus crispus and Matricaria recutita L. with anti-adipogenic and anti-inflammatory activities

Abstract

Thistles, belonging to the Asteraceae, are too varied a group for generalization

many are troublesome weeds, including some invasive species of Cirsium, Carduus, Silybum, and Onopordum. Carduus crispus, native to Europe and Asia, is a traditional herbal medicine used for treating inflammatory disorders in Korea. Obesity is characterized by a state of chronic inflammation with increased inflammatory markers along with the expression and release of inflammation-related adipokines. Most anti-obesity drugs have been developed based on this concept. This research for anti-obesity agents derived from Carduus crispus utilized the 3T3-L1 cell line. The methanol extract was initially screened and exhibited significant inhibition of adipogenesis in 3T3-L1 adipocytes. Among five liquid-liquid partition fractions, the ethyl acetate (EA) fraction showed the most potent suppressive effect compared with hexane, chloroform, n-butanol, and water. The EA fraction was considered for further study because of the presence of abundant polyphenols, including flavonoids. To isolate the active components from the EA fraction, elution-extrusion counter-current chromatography (EECCC) was used. Among the seven fractions from the EA layer, fraction 6 inhibited lipid accumulation as well as CCAAT/enhancer-binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ) protein expression levels. The active component apigenin (fraction 6) was confirmed using high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), and one-dimensional nuclear magnetic resonance (1D-NMR) spectroscopy. The present data suggest that apigenin is one of the main bioactive compounds from Carduus crispus for inhibiting adipogenesis in 3T3-L1 adipocytes via the activation of AMP-activated protein kinase. Using the marker substance pectolinarin, isolated from Cirsium japonicum, one of the representative type of thistles, a dose-dependent decrease on lipid accumulation was identified in 3T3-L1 adipocytes. This inhibitory effect on adipogenesis was shown to be induced by the suppression of a sterol regulatory element binding protein-1 (SREBP-1) and the activation of adenosine monophosphate-activated protein kinase (AMPK). The findings that the generation of intracellular reactive oxygen species (ROS) and the expression of heme oxygenase-1 (HO-1) proteins were inhibited suggest that this anti-adipogenic effect resulted from lipid oxidation. The present study demonstrates that apigenin from Carduus crispus and pectolinarin from Cirsium japonicum have reduced the lipid accumulation with their significantly inhibition of PPARγ and C/EBPα protein expressions and the activation of AMPK. However, apigenin exhibited cytotoxicity at 50 μM not in pre-adipocytes but in mature adipocytes and pectolinarin has no cytotoxic effect on both types of cells. In summary, this study shows that apigenin has shown the apoptotic 3T3-L1 cell death via the activation of AMPK and caspase-3. Whereas pectolinarin, against apigenin, has shown the lipid oxidation via the inhibition of intracellular ROS and HO-1 generation. Another member of Asteraceae, Matricaria recutita L. (chamomile) was also studied for the anti-inflammatory activity and bioactivity-guided isolation to search the active component. The inhibition effects of nitric oxide (NO) production and NF-κB secretory alkaline phosphatase (SEAP) activity of chamomile methanol extract exhibited better than those of water and ethanol extracts. Because methanol is less polar than water, the methanol extract contains more essential oils than the water extract. To identify the main volatile constituents of the essential oil in chamomile, dried chamomile powders were extracted by head space-solid-phase micro extraction (HS-SPME) and were analyzed by gas chromatography-mass spectrometry (GC-MS). Parameters affecting the solid-phase micro extraction (SPME) procedure including extraction temperature and time, amount of the sample, and desorption time have been evaluated and optimized. In addition, methanol and water extracts of chamomile, and hexane fractionation from the methanol extract of chamomile were analyzed to compare. A total of 41 components were identified. It is well known the principal biologically active compounds in chamomile oils were bisabolol oxide A, bisabolol oxide B, α-bisabolol, chamazulene, spathulenol, and β-farnesene. The powderized sample of dried chamomile was rich in bisabolol oxide B (21.0 %), β-farnesene (17.9 %), and α-bisabolol (9.0 %). Herniarin (10.1 %) was predominant in the water extract. In case of the methanol extract and hexane layer, bisabolol oxide B (each of 21.7 %) was the most abundant component. α-bisabolol was detected only in dried chamomile powder. The compounds identified in the hexane layer fractionated from the methanol extract were found to be very similar to the volatile essential oils identified in the methanol extract. Based on the identification of essential oil components, the organic solvent extracts containing large amounts of essential oil components are thought to have a higher anti-inflammatory effect. Therefore, the non-volatile compound of methanol extract from chamomile was also analyzed to identify anti-inflammatory compounds. Fractionation using liquid-liquid separation was performed to identify non-volatile active compounds as well. The methanol extract of chamomile was fractionated into hexane, EA, BuOH, and water layers. The less polar hexane and EA-soluble layers were shown, as expected, to have higher inhibitory effects on NO generation and NF-κB expression, but the hexane-soluble layer was also found to have cytotoxicity. Therefore, the EA-soluble layer containing large amount of flavonoids was selected for further separation. The EA-soluble layer was separated into seven fractions by silica gel column chromatography. Of those seven fractions, 5th fraction with no toxicity and high yield was selected to be further separated. In the next step, 5th fraction was separated into a total of 5 fractions using Diaion HP-20 column chromatography. From those fractions, 3 main components - dicaffeoyl quinic acids enriched (50% MeOH, DCQ), apigenin 7-O-glucoside derivatives enriched (70% MeOH, A7G) and tetracoumaroyl thermospermine enriched (90% MeOH, TCTS) – were identified, among which TCTS was shown to have the highest NO inhibition effect. Within the range of concentrations at which no cytotoxicity was observed, TCTS was assayed for the expression of proteins associated with inflammation using Western blot analysis. TCTS was shown to have higher inhibitory effects on the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) compared to those of DCQ and A7G and, in particular, was found to inhibit COX-2 selectively without affecting cyclooxygenase-1 (COX-1) expression. In this study, it was also found that the inhibition of COX-2 of TCTS was exerted via extracellular signal-regulated kinases (ERK)/Akt and signal transducer and activator of transcription 3 (STAT3) pathway. The purpose of this study included the identification of essential oil components and the bioactivity-guided isolation for non-volatile active compounds responsible for chamomiles anti-inflammatory effect. The result showed that the dried German chamomile contained large amounts of anti-inflammatory volatile compounds such as α-bisabolol oxide B, β-farnesene and α-bisabolol. The higher anti-inflammatory effect shown in the methanol extract than in the water extract was deemed to be due to the greater amount of essential oil components, in particularly α-bisabolol oxide B, contained in the methanol extract. The bioactivity-guided isolation performed to explore non-volatile active components showed that TCTS is mainly responsible for the anti-inflammatory effect of chamomile with COX-2 inhibition via ERK/Akt and STAT3 pathway.