The Anti-oxidative and Anti-inflammatory Effects of Caffeoyl Derivatives from the Roots of Aconitum koreanum R. RAYMOND (original) (raw)

2009, Biological & Pharmaceutical Bulletin

Free radicals can cause oxidative damage by oxidizing biomolecules and this results in cell death and tissue damage as is observed in atherosclerosis, cancer, emphysema, cirrhosis, and arthritis. 1) Oxidative damage plays a critical pathological role in human diseases, and thus, anti-oxidative supplements are important because they reduce oxidative damage in the human body. 2) Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are known to contribute to inflammatory response via the production of nitric oxide (NO) and prostaglandins. 3,4) These two enzymes, which mediated inflammatory events, are highly expressed by proinflammatory cytokines, such as, interferon-g (IFN-g) and tumor necrosis factor a (TNF-a), and by lipopolysaccharide (LPS), a component of the outer membranes of Gram-negative bacteria. 5) Thus, the screening of components that suppress these proteins and their mRNAs has proven to be a useful strategy for identifying therapeutics capable of combating inflammatory diseases. 6,7) As part of our ongoing screening program to search antioxidative and anti-inflammatory natural products, 8) we examined the roots of Aconitum koreanum (RAK), which was traditionally used to treat pain, rheumatic arthralgia, epilepsia, furunculus, acne, vertigo, tetanus, and cardialgia. 9,10) In addition, diterpene alkaloids, which have been shown to have anti-arrhythmic 11) and myorelaxant 12) effects, also have been isolated from this plant. Furthermore, RAK has been reported to possess analgesic and anti-inflammatory effects. 13) Thus, we undertook to search the components in RAK that scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and inhibit NO production and the expression of iNOS and COX-2. MATERIALS AND METHODS General Experiment TLC was carried out using precoated silica gel 60 F 254 plates (Merck, Darmstadt, Germany) and ethylacetate-water-acetic acid (10 : 9 : 1, volume ratio) or chloroform-methanol-water (6 : 4 : 1, volume ratio). The spots were detected using UV (254 nm) and by spraying with FeCl 3 and 10% H 2 SO 4 , followed by heating. Compounds were identified by 1 Hand 13 C-NMR (Varian Gemini 2000, U.S.A. or Brucker Amx-500), and by LC/MS (API 3000 triple quadrupole mass spectrometry, Canada) and were characterized by performing comparisons with literature values. Plant Material The roots of Aconitum koreanum were collected from Pyung-chang, Kangwondo, South Korea in June of 2007. A voucher specimen (AK2007-6) has been deposited at the herbarium, College of Pharmacy, Chung-Ang University. Extraction, Isolation, and Identification The roots of Aconitum koreanum (3 kg) were extracted three times with 80% aqueous acetone at room temperature. After removing the acetone under vacuum, the residual aqueous solution was filtered. The filtrate was then concentrated (328 g) and applied to an Amberlite XAD-2 column (20-50 mesh, 2000 g, 10ϫ100 cm) (Fluka AG, Buchs, Switzerland) eluted with water containing increasing proportions of MeOH, to afford 6 sub-fractions. Repeated column chromatography of fraction 5, which showed anti-oxidative activity with an IC 50 value of 22.66Ϯ2.23 mg/ml compared with a positive control, L-ascorbic acid (IC 50 ϭ13.92Ϯ3.87 mg/ml) (data not shown) on a Sephadex LH-20 column (25-100 mm, 800 g, 6ϫ75 cm) (Pharmacia, Uppsala, Sweden) eluted with 60% MeOH resulted in two sub-fractions (5-1, 5-2). Sub-fraction 5-1 was further separated on a Sephadex LH-20 column (25-100 mm, 800 g, 6ϫ75 cm) eluted with 40% MeOH to yield caffeic acid (1, 130 mg). Sub-fraction 5-2 on MCI-gel CHP 20P (75-150 mm, 600 g, 5ϫ60 cm) (Mitsubishi, Tokyo, Japan) eluted using a H 2 O-MeOH gradient yielded 4,5-dicaffeoylquinic acid (2, 180 mg), 3,5-dicaffeoylquinic acid (3, 150 mg) and 3,5-dicaffeoylquinic acid methyl ester (4, 100 mg).