Biological Activities of Flavonoids Isolated from Chinese Herb Huang

Chapter 7

Biological Activities of Flavonoids Isolated from Chinese Herb Huang Qui: Inhibition of NO and PGE Production by Flavonoids 2

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Yen-Chou Chen , Shing-Chuan Shen , and Foun-Lin Hsu 1

Graduate Institute of Pharmacognosy Science, School of Pharmacy, and Department of Dermatology, School of Medicine, Taipei Medical University, Taipei, Taiwan, Republic of China

Downloaded by COLUMBIA UNIV on July 19, 2012 | http://pubs.acs.org Publication Date: August 5, 2003 | doi: 10.1021/bk-2003-0859.ch007

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Huang Qui is one of the popular Chinese herbs, and has been used in treatment of several human diseases such as inflammation, allergy and artherosclosis for thousands of years. However the active components of Huang Qui are still undefined. Our recent studies demonstrated that flavonoids in Huang Qui including wogonin, quercetin, and oroxylin A showed the significant inhibition on lipopolysaccharide (LPS)induced nitric oxide (NO) and prostaglandin E (PGE ) production, accompanied by inhibiting inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) gene expression. The inhibitory mechanism of these compounds on LPS-induced responses was through inhibiting NF-κB activation. In vivo study showed that wogonin and quercetin were able to suppress LPS-induced NO production in the serum of Balb/c mice. In addition to NO inhibition, wogonin showed the apoptotic effect on human promyeloleukemia cells HL-60 and hepatocellular carcinoma cells SK-HEP-1 cells through activation of caspase 3-dependent cascade, and oroxylin A exhibited the significant relaxative effect in porcine cerebral arteries pre-constricted by U-46619 through activation of potassium channels. Results of our studies demonstrate that wogonin, quercetin, and oroxylin A are active components of Huang Qui and deserve several beneficial biological activities to be explored further. 2

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© 2003 American Chemical Society In Oriental Foods and Herbs; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Medicinal plants have been used as traditional remedies for hundreds of years. Scutellaria baicalensis Georgi (Huang Qui) is one of the important medicinal herbs widely used for the treatment of various inflammatory diseases, hepatitis, tumors and diarrhea in East Asian countries such as China, Korea, Taiwan and Japan (/). The plant has been reported to contain a large number of flavonoids,frequentlyfound as the glucosides and other constituents including phenethyl alcohols, sterols and essential oils and amino acids. Flavonoids have been identified as either simple or complex glycosides in many plants, and humans have been estimated to consume approximately 1 g flavonoids/day (2). Several previous studies (3,4) have demonstrated thatflavonoidsexhibit a wide variety of biological activities including antioxidant, free radical scavenging, anti-cancer and anti-inflammatory activities. Although Huang Qui has been used extensively for a long time in the Chinese society, the active compounds are still undefined. Wogonin, oroxylin A and quercetin are components of Huang Qui, and seldom biological functions are identified. Quercetin is a prototypical polyphenolic pimtflavonoid,and can be derived from rutin though hydrolization by glucosidase. Quercetin was a potent antioxidant and anti-inflammatory agent, prevented cisplatin-induced cytotoxicity in LLC-PK1 cells in vitro, and prevented tubular injury induced by acute renal ischemia in vivo (5). Rangan et al. reported that quercetin inhibited lipopolysaccharide-induced cytokines such as interleukin-ΐβ, tumor necrosis factor-α productions though blocking nuclear factor-kappa Β activation (6). In addition to beneficial effects, quercetin also has been implicated as a strong mutagen without microsomal activation, and the mutagenic activity of quercetin was increased significantly after microsomal activation (7). In contrast to quercetin, biological activities of wogonin and oroxylin A remained unclear.

Inhibition of Nitric Oxide and PGE Production by Wogonin, Oroxylin A and Quercetin 2

The chemical structures of wogonin, quercetin, and oroxylin A are shown in Figure 1. In our study, effects of wogonin, oroxylin A, and quercetin on LPSinduced NO and PGE production in RAW 264.7 macrophages were investigated. Nitrite accumulated in the culture medium was estimated by the Griess reaction as an index for NO synthesis from the cells. When the cells incubated with wogonin, quercetin, and oroxylin A alone, the amount of nitrite in medium was maintained at a background level similar to that in the unstimulated samples (data not shown). After treatment with LPS (100 ng/mL) for 12 hours, nitrite concentration in medium increases remarkable about 10 fold (-30 μΜ). When RAW 264.7 macrophages were treated with different concentration of wogonin, quercetin, and oroxylin A together with LPS (100 ng/mL) for 12 hours, significant concentration dependent inhibition of nitrite production was detected in the presence of wogonin, quercetin, and oroxylin A (Figure 2). Similarly, 2

In Oriental Foods and Herbs; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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Figure 1. Chemical structure of quercetin, wogonin and oroxylin A.

wogonin, quercetin, and oroxylin A showed the inhibition on LPS-induced PGE production (Figure 2). Examination of cytotoxicity of wogonin, quercetin, and oroxylin A in RAW 264.7 macrophages by MTT assay indicated that three compounds, even at the concentration of 40 μΜ, did not decrease cell viability in RAW 264.7 cells (data not shown). Therefore, inhibition of LPS-induced NO and PGE production by wogonin, quercetin, and oroxylin A was not result of their cytotoxicity on cells. 2

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Inhibition of iNOS and COX-2 Gene Expression, but not iNOS and COX-2 Activities, by Wogonin, Oroxylin A and Quercetin Two possibilities of NO and PGE inhibition by wogonin, quercetin and oroxylin A, one is inhibiting iNOS and COX-2 gene expression, the other is blocking iNOS and COX-2 activity. By Western blotting analysis, wogonin, quercetin and oroxylin A inhibited LPS-induced iNOS and COX-2 protein expression in cells (Figure 3). However, iNOS and COX-2 activities induced by LPS did not alter by these compounds by direct and indirect activity assays (8). It is suggested that inhibition of NO and PGE production by wogonin, quercetin, 2

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116 and oroxylin A was mediated by inhibiting iNOS and COX-2 gene expression, but not their activities.


Induction of Apoptosis by Wogonin in Human Promyeloleukemia Ceils and Hepatocellular Carcinoma Cells Apoptosis is a programmed cell death, and cells from a variety of human malignancies have a decreased ability to undergo apoptosis in response to apoptotic stimuli (9-11). Therefore, developing various kinds of effective agents that can enhance the extent of apoptosis might be a promising strategy in the treatment of cancer. Apoptosis is characterized by cellular morphological change,

Figure 2. Inhibition of LPS-induced NO and PGE2 production by quercetin, wogonin and oroxylin A in RA W264.7 macrophages.



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Figure 3. Inhibition of LPS-induced iNOS and COX-2 protein by quercetin, wogonin and oroxylin A in RA W264,7 macrophages.



118 chromatin condensation, and apoptotic bodies which are associated with DNA cleavage into ladders (12,13). Our recent study demonstrated that wogonin showed the effective apoptosis induction activity in human promyeloleukemia cells HL-60, and wogonin-induced apoptosis is caspase 3-dependent (14). P53 is a tumor suppressor gene and play an important role in the progression of apoptosis (75). In order to identify if p53 involved in wogonin-induced apoptosis, human hepatocellular carcinoma cells, which with wild-type p53 gene, were used in our study. Interestingly, wogonin induced apoptosis in SK-HEP-1 cells, accompanied by an increase in p53 protein and p53-controlled gene p21. Activation of caspase 3, but not caspase 1, was involved in wogonin-induced apoptosis in SK-HEP-1 cells (16). These data suggested that wogonin is an effective apoptosis inducer and deserves to be developed as an anti-cancer agent.

Induction of Vasorelaxation by Oroxylin A in Porcine Cerebral Arteries In additional to NO and PGE inhibition by oroxylin A, oroxylin A also showed the vasorelaxative effect in porcine cerebral arteries. As shown in Figure 4, addition of oroxylin A (0.1-30 μΜ) to the incubation medium resulted in concentration-dependent relaxation in intact arteries of circle of willis precontracted by U46619 (1 μΜ). Oroxylin A was able to induce maximal relaxation, and the medium effective concentration (EC50) was 7.1 ±2.4 μΜ. DMSO, a dissolution medium of oroxylin A, at the concentrations used (as much as 0.1 % vol/vol) had no effets on vasoreactivity precontracted by U46619 (data not shown). In endothelium-denuded arteries, oroxylin A show the same relaxation effects as that in intact arteries and EC50 was 5.87±3.1 μΜ. In order to study the cytotoxic effect of oroxylin A on arteries, two consecutive experiments were performed in same arteries with 60 min interval and 3 washes between two applications. The reproducible relaxations were obtained and they were not significant different. These results show that the relaxation elicited by oroxylin A are concentration-dependent and endothelium-independent, and oroxylin A did not have any cytotoxic or desensitive effect on arteries. Furthermore, vasorelaxative effect of oroxylin A was not appeared in KC1preconstricted arteries, and blocked by potassium channel blockers tetraethylammonium (TEA), 4-aminopyridine (4-AP) and iberiotoxin (IBT). It is suggested that oroxylin Α-induced vasorelaxation might be mediated by activation of potassium channels. (Figure 4) 2



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Figure 4. Attenuation of oroxylin Α-induced Vasorelaxation by potassium channel blockers 4-AP andlBT.Eeicosatriyonic acid (ETI, JO μΜ), 4aminopyridine (4-AP, 10 mM), glipizide (GLP, 30 μΜ), miconazole (MCN, 5 μΜ), indomethacine (INDO, 30 μΜ), N-nitro-L-arginine (L-NNA, 60 μΜ), iberiotoxin (IBT, 100 ng/mL).

Conclusion Results of our studies demonstrated that wogonin, quercetin and oroxylin A showed the potent inhibitory activities on LPS-induced responses including NO and PGE production in RAW264.7, primary macrophages, and Balb/c mice. In addition to anti-inflammatory effects, wogonin showed the effective apoptosis inducing activity in tumor cells including human promyeloleukemia cells HL-60 and hepatocellular carcinoma cells SK-HEP-1 through a caspase 3-dependent pathway. And, oroxylin A exhibited vasorelaxative activity in cerebral arteries. These data provide scientific evidences to demonstrate that wogonin, quercetin and oroxylin A are functional compounds of Chinese herbs Huang Qui with a potential to be developed further. 2


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Acknowledgement This study was supported by the National Science Council of Taiwan (NSC 89-2314-B-038-035, NSC89-2320-B-038-054 and NSC89-2320-B-038-041).

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Biological Activities of Flavonoids Isolated from Chinese Herb Huang

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