Viscum album var coloratum Ohwi V album var C

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Various chemical components have been isolated and identified from the extracts of V. album var. C., such as lectins, steroids, triterpenes, sesquiterpene lactones, carbohydrates, flavonoids, organic acids and amines, alkaloids, amino acids and peptides.

Lectins

An agglutinin from Viscum album var. C. harvested in Korea (VCA) was isolated by gel filtration using Sephadex-G 75 (Park et al, 1994a), affinity chromatography using acid-treated Sepharose 4B (Park et al., 1997a). VCA was also isolated using Sepharose 4B modified by lactose-bovine serum albumin (BSA) conjugate synthesised by reductive amination of ligand (lactose) to e-amino groups of lysine residues of spacer (BSA) after reduction by NaCNBH3 (Park et al., 1998-a). Recently the VCA was efficiently isolated from Korean mistletoe by affinity chromatography using asialofetuin immobilised Sepharose 4B. Using this optimised isolation procedure the Korean mistletoe lectin (VCA) binds to galactose and lactose, similar to ML I isolated from V. album L. The molecular weight of the VCA determined by SDS-PAGE was 60 kDa, with a 31.5 kDa A-chain and a 34.5 kDa B-chain, and has an isoelectric point of 8.0-8.7 (Pfuller and Park, in preparation).

The investigation of binding kinetics and sugar inhibition of VCA by BIACore (Pharmacia) showed different association and dissociation steps and sugar specificity as compared to those of European mistletoe lectins (ML I, ML II, ML III). Both, association and dissociation rate of VCA to asialofetuin immobilised on sensorchip CM5 (Pharmacia) was lower than those of European ML, and the recognition of VCA by asialofetuin was inhibited by D-galactose and lactose, which is similar to ML I. Using an ELLA system (asialofetuin-lectin-polyclonal antibody-streptoavidin/ peroxidase-OPD/H2O2), VCA reacts with polyclonal anti-ML antibodies. Further investigations using sandwich ELISA with monoclonal anti-ML antibodies revealed

Table 2 Lymphocyte stimulating activity of VCA.

Mitogen MTT assay

Concanavalin A 0.201

VCA from unfermented sample 0.236

VCA from fermented crude extract (Id) 0.181

VCA from fermented crude extract (2 d) 0.159

VCA isolated from fraction 1* of sample fermented for 3 d 0.157

VCA isolated from fraction 2* of sample fermented for 3d 0.131

Metabolic activity of lymphocytes stimulated with VCA (2 HU) for 24 h was measured by MTT assay as described (Park et al., 1995).

* fraction from ion exchange chromatography.

that VCA was recognised by an antibody detecting ML I (MNA9-TA5b), but not by a monoclonal antibody to ML II/III (C12-H11b) (Pfuller and Park, in preparation), indicating that VCA shares distinct epitopes with ML from European mistletoe.

The VCA was recognised to increase the metabolic activity of lymphocytes, as measured by MTT assay, more strongly than the mitogenic lectin concanavalin A (Park et al., 1994a). In contrast, an additional lectin of 18.5 kDa MW, identified by fermentation with Lactobacillus plantarum, decreased the metabolic activity of lymphocytes (Park et al., 1995) (Table 2). An apoptosis-inducing four chain lectin from Korean mistletoe (termed KML-C) with 27.5, 30, 31 and 32.5 kDa was described by Yoon et al. (1999).

The effects of pH, temperature and guanidine chloride on the activities of VCA were investigated by measuring its intrinsic fluorescence and compared with its hemagglutinating activities. There are significant relationships between activities and conformations of the lectin (Park et al., 1998a): The hemagglutinating activity of the lectin was stable at the pH range of 4.0 to 8.0, decreased up to 50% at pH 9.0, and disappeared completely at pH 10.0. It is noticed that the activity was enhanced to 200% at the pH range of 4.0 to 6.0. The enhancing of lectin activity induced by pH changes in fluorescence position of spectral maximum evidently indicated the significant structural transition in the environment of tryptophan residues. Blue shift was detected on the acidic pH, which suggested unfolding of the protein structure near tryptophan residues. Similar changes also occurred at the alkaline pH range of 8.0 to 10.0. These changes corresponded to the decrease of activity in the alkaline pH. From these results it is assumed that high activity of lectin at pH 4.0 to 6.0 was caused by more suitable folding structure of lectin. The hemagglutinating activity of lectin was stable at a wide range of temperature (0-45°C). Half of the activity was maintained at 55°C, but the activity disappeared over 65°C. The increase in temperature resulted in a typical denaturational shift of the protein spectra towards a position characteristic of free tryptophan (353 nm). From these results, it was assumed that there may be a relationship between activity and conformation of lectin. In denaturing conditions, such as high concentration of guanidine hydrochloride, tryptophan emission profile of lectin showed typical denaturational red shift, which also corresponded to the conformation and activity of lectin.

Organic acids palmitic acid, lignoceric acid, cerotic acid, octacosanoic acid, succinic acid, ferulic acid, caffeic acid and protocatechuic acid (Kong et al., 1989).

Steroids

^-sitosterol and daucosterol (Tseng et al., 1957; Kong et al., 1987a, b). Carbohydrates mesoinositol; the acetate has hypotensive action (Tseng et al., 1957).

Triterpenes oleanolic acid, ¡¡-amylin, ¡¡-amyrin palmitate, ¡¡-acetylamyrin, erythordiol, betulinic acid and lupeol (Tseng et al., 1957); oleanolic acid, olea-12-en-3^-ol and olean-12-en-3^-ol acetate and lup-20(29)-en-3-one (Ahn, 1996).

Flavonoids flavoyandrinin-A, flavoyandrinin-B and homo-flavoyandrinin-B (Ohta et al., 1970); rhamnazin-3-O-^-D-glucoside, homoeriodictyol-7-O-^-D-glucoside; rhamnazin (Kong et al., 1987a); homoeriodictyol, viscumneoside III (homoeriodictyol-7-O-^-apiosyl-1-2-^-D-glucopyranoside) (Kong et al., 1988b); viscumneoside IV (rhamnazin-3-O-¡-D-(60-£-hydroxy-£-methylglutaryl)glucoside (Kong et al., 1988c, 1990a); viscumneoside V (homoeriodictyol-7-O-^-apiosyl-1-5-^-D-apiosyl-1-2^-D-glucopyranoside), viscumneoside VI (homoeriodictyol-7-O-^-D-(60-O-acyl)glucopyranoside (Kong et al., 1988a, b); viscumneoside VII (rhamnazin-3-O-^-D-apiosyl-1-2 (60-O-(3-hydroxy-3-methylglutarate)glucoside) (Kong et al., 1990b); viscoside A (Li et al., 1985); isorhamnazin-3-O-^-D-glucoside; isorhamnazin-7-O-^-D-glucoside; rhamnazin-3,49-di-O-glucoside, homoeriodictyol-7-O-(apiosyl-1-2 glucoside) (Fukunaga et al., 1989a, b).

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