A New Stilbene Glucoside from Biotransformation-Guided Purification of Chinese Herb Ha-Soo-Oh

Abstract

Ha-Soo-Oh is a traditional Chinese medicine prepared from the roots of Polygonum multiflorum Thunb. The herb extract has been widely used in Asian countries as a tonic agent and nutritional supplement for centuries. To identify new bioactive compounds in Chinese herbs, the biotransformation-guided purification (BGP) process was applied to Ha-Soo-Oh with Bacillus megaterium tyrosinase (BmTYR) as a biocatalyst. The result showed that a major biotransformed compound could be purified using the BGP process with preparative high-performance liquid chromatography (HPLC), and it was confirmed as a new compound, 2,3,5,3',4'-pentahydroxystilbene-2-O-β-glucoside (PSG) following mass and nucleic magnetic resonance (NMR) spectral analyses. PSG was further confirmed as a biotransformation product from 2,3,5,4'-tetrahydroxystilbene-2-O-β-glucoside (TSG) by BmTYR. The new PSG exhibited 4.7-fold higher 1,1-diphenyl-2-picrylhydrazine (DPPH) free radical scavenging activity than that of TSG. The present study highlights the potential usage of BGP in herbs to discover new bioactive compounds in the future.

Keywords: Ha-Soo-Oh; Polygonum multiflorum; antioxidant; biotransformation; hydroxylation; melanogenesis; tyrosinase.

Figure 1

High-performance liquid chromatography (HPLC) analysis of the methanol extracts (5 mg/mL) from four different brands, Sun Ten (a), Ko Da (b), Chuang Song Zong (c), and Min Tong (d), of Ha-Soo-Oh herb medicine. The HPLC operation procedure is described in Section 2.4.

Figure 2

HPLC analysis of the methanol extract of Sun Ten Ha-Soo-Oh herb medicine (a) and the biotransformed products by using BmTYR (b). The biotransformation mixture—containing 120 μg/mL of the purified recombinant BmTYR enzymes, 2 mg/mL of the extract, 10 mM of ascorbic acid, and 500 mM of borate buffer at pH 9—was incubated at 50 °C and shaken at 200 rpm for 1.5 h. At the end of the reaction, one-fifth of the volume of 1 M HCl and an equal volume of MeOH were added to stop the reaction, and it was analyzed using HPLC. The HPLC operation procedure is described in Section 2.4. The retention times of the major peak in (a) and compound (1) in (b) are 23.2 and 20.5 min, respectively.

Figure 3

Chemical structure of the biotransformation product (1), 2,3,5,3′,4′-pentahydroxystilbene-2-O-β-glucoside (PSG).

Figure 4

HPLC analysis of 3,5,4′-trihydroxystilbene-2-O-β-glucoside (TSG) (a) and the biotransformed product by using BmTYR (b). The biotransformation and HPLC conditions were the same as those in the legend of Figure 1, with the replacement of the enzyme substrate (2 mg/mL of Ha-Soo-Oh crude extract) with a TSG standard (2 mg/mL). The retention times of TSG in and the biotransformed product are 22.3 and 20.5 min, respectively.

Figure 5

The biotransformation process of TSG by BmTYR.

Figure 6

1,1-Diphenyl-2-picrylhydrazine (DPPH) free radical-scavenging activity of TSG, PSG, and ascorbic acid. The DPPH scavenging activity was determined as described in Materials and Methods. The mean (n = 3) is shown, and the standard deviation (S.D.) is represented by error bars. The IC₅₀ values represent the concentrations required for 50% DPPH free radical-scavenging activity.