Inhibition of nitric oxide production on LPS-activated macrophages by kazinol B from Broussonetia kazinoki

doi:10.1016/S0367-326X(03)00062-5

Fitoterapia

Volume 74, Issue 4, June 2003, Pages 350-354

https://doi.org/10.1016/S0367-326X(03)00062-5 Get rights and content

Abstract

The ethyl acetate soluble fraction of Broussonetia kazinoki at 50 μg/ml showed a significant inhibition (78.2%) of nitric oxide (NO) in lipopolysaccharide activated macrophages. Kazinol B, an isoprenylated flavan, was identified as the active principle. It inhibits the NO synthesis with an IC₅₀ of 21.6 μM.

Introduction

The branches, leaves and fruits of Broussonetia kazinoki Sieb have been used as a diuretic, a tonic and a suppressant for edema in Chinese folk medicine. The cytotoxic activity of prenylated flavonols and flavanes from leaves [1] and root bark [2] was evaluated against several different cell lines. Antioxidant and inhibitory activity against tyrosinase [3], [4] was also reported from the root bark of B. kazinoki. Kazinol B, an isoprenylated flavan was found to be able to stimulate the respiratory burst by the synergism of PKC activation and Ca²⁺ elevation in rat neutrophils [5] and inhibit the cyclooxygenase [6], [7]. Alkaloids [8], [9], [10], [11] and flavonoids [12], [13], [14], [15] were previously isolated from B. kazinoki.

The aim of this work is to evaluate the effect of the ethyl acetate soluble fraction of B. kazinoki on the NO production in lipopolysaccharide activated fraction. A further aim is to isolate the relevant active principle by activity-guided chromatographic procedures.

Section snippets

Plant material

B. kazinoki (Moraceae) was collected in the suburbs of Suwon city, the southern part of Kyung-Gi Province, Korea in November 1997 and authenticated by Prof. T.H. Kim at the College of Pharmacy, Sookmyung Women's University. A voucher specimen was deposited in the herbarium of the Sookmyung Women's University, Seoul, Korea (voucher specimen No. SPH 97009).

Extraction and isolation

The ethanolic extract (374 g) of dried and powdered root bark (4.4 kg) was fractionated to yield an ethylacetate soluble fraction (250 g)

Results and discussion

The ethylacetate soluble fraction of B. kazinoki exhibited 78.2% inhibition of LPS-induced NO production at the concentration of 50 μg/ml in cell culture media. Stimulation of macrophages by LPS may increase the inducible nitric oxide synthase enzyme (iNOS). The NO and its derivatives such as peroxynitrite and nitrogen dioxide, produced in large amounts by the iNOS play a role in inflammation and also possibly in the multistage process of carcinogenesis [19]. When kazinol B from ethylacetate

Acknowledgments

This research was supported by Sookmyung Women's University Research Grants 2001.

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Flavans and diphenylpropanes with PTP1B inhibition from Broussonetia kazinoki
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Its 1H NMR spectrum showed typical signals of a 7-hydroxyflavan skeleton with a prenyl group [δH 3.33 (2H, overlapped), 5.06 (1H, t, J = 7.0 Hz), 1.62 (3H, s), 1.66 (3H, s)] and a 2, 2-dimethylpyran ring [δH 5.69 (1H, d, J = 9.8 Hz), 6.35 (1H, d, J = 9.8 Hz), 1.39 (3H, s), 1.39 (3H, s)] (Table 1). Following combination of the NMR, MS and related references, 4 was defined as a prenylated flavan, kazinol B [21]. It also showed two peaks in the chiral HPLC chromatogram with a peak-area ratio of approximately 3:1 (Fig. 3).
Enantiomers account for quite a large percentage of compounds in natural products. Our team is interested in the separation and biological activity of racemic compounds. In this report, four pairs of prenylated flavan enantiomers [(±)-1–(±)-4], including five new compounds, were isolated from the stem and root bark of Daphne giraldii, and separated successfully by using chiral chromatographic column. Their planar structures and absolute configurations were established by comprehensive spectroscopic analyses as well as circular dichroism (CD) spectroscopy. The isolates had a selective cytotoxicity towards hepatic carcinoma cell lines. Among them, new compound (+)-4 showed a more potent inhibitory effect on Hep3B cells with an IC₅₀ value of 30.3 μM, compared with its racemic mixture 4. Therefore, the action mechanism of (+)-4 in vitro was subsequently investigated. The morphological observation and Western blot analysis demonstrated that (+)-4 could markedly induce apoptosis through intrinsic and extrinsic pathways, and also cause autophagy by increasing the phosphorylation of AMP-activated protein kinase (AMPK) in Hep3B cells. After treatment with the autophagic inhibitor bafilomycin A1 (Baf A1), (+)-4-induced apoptosis increased significantly, suggesting that the autophagy induced by (+)-4 performed a protective effect on apoptotic cell death.
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Inhibition of nitric oxide production on LPS-activated macrophages by kazinol B from Broussonetia kazinoki

Abstract

Introduction

Section snippets

Plant material

Extraction and isolation

Results and discussion

Acknowledgments

Br J Pharmacol

Biochem Pharmacol

Biochemistry

J Nat Prod

J Nat Prod

Yakhak Hoechi

Cosmet Toiletries

J Nat Prod

Chem Pharm Bull