Production of Tetrahydrocurcumin From Curcumin by Cultured Plant Cells and Inhibitory Effects of Curcuminoids on NF-κB Activity

Abstract

Biotransformation of curcumin was investigated using cultured plant cells of Morus sp. Cultured Morus cells converted curcumin into dihydrocurcumin and tetrahydrocurcumin. The activity of nuclear factor-κB was inhibited by curcumin and tetrahydrocurcumin.

Cultured plant cells have been studied with respect to biotransformation reactions because of their biochemical potential to produce specific secondary metabolites such as flavors, pigments, and agrochemicals. The reactions that are associated with the biotransformation of organic compounds by cultured plant cells include oxidation, reduction, hydroxylation, esterification, methylation, isomerization, hydrolysis, and glycosylation. ¹ Reduction by cultured plant cells has been the subject of increasing attention, since regioselective and/or stereoselective reduction of organic compounds by plant cultured cells provides useful synthetic materials. Additionally, the reduction of bioactive compounds can improve their bio- and pharmacological properties.

Turmeric derived from the rhizome of Curcuma longa Linn. has been used for the treatment of inflammatory disorders such as arthritis, colitis, and hepatitis. Curcumin is a representative phenolic constituent found in C. longa and exhibits antioxidant, anti-inflammatory, and anticancer properties. ^{2 -6} Curcumin is synthesized through polyketide biosynthesis pathway. Its starting material is acetyl-CoA, which is also a key substrate of terpene biosynthesis. On the other hand, recent paper reported that tetrahydrocurcumin showed higher anticancer activity than curcumin. ⁷

Nuclear factor-κB (NF-κB) is a dimeric transcription factor that induces the expression of genes involved in the inflammatory process. ⁸ So, compounds that inhibit NF-κB are of great interest as lead structures for the treatment of acute and chronic inflammation.

Herein, we report the production of tetrahydrocurcumin by reduction of curcumin with plant cultured cells of Morus sp. and the effects of curcuminoids on NF-κB activity.

Curcumin (1) was subjected to the biotransformation system using cultured plant cells of Morus sp. as follows: The substrate 1 was administered to ten 300-mL conical flasks (10 mg/flask) containing Morus suspension cells. The cultures were incubated at 25°C for 2 days on a rotary shaker. After 2 days incubation, the Morus cells and medium were divided by filtration. Compounds 2 and 3 were purified from the extracts of the medium with ethyl acetate by HPLC. The chemical structure of the product was determined on the basis of ESIMS and NMR spectroscopic methodology. The product 2 was identified as dihydrocurcumin (2) (Figure 1). On the other hand, the reduction product 3 of 2 was determined as tetrahydrocurcumin (3).

Figure 1

Reduction of curcumin (1) by Morus sp.

Next, the effects of curcuminoids, ie, curcumin and tetrahydrocurcumin, on NF-κB activity were examined. After 24-hour treatment of HeLa cells with curcuminoid sample, the NF-κB activity was measured as described in the Experimental section. The NF-κB activity was inhibited by curcumin with 64% inhibition. On the other hand, tetrahydrocurcumin decreased NF-κB activity with 25% inhibition.

Thus, it was demonstrated that cultured plant cells of Morus sp. can reduce curcumin to dihydrocurcumin and tetrahydrocurcumin. We recently reported that cultured Phytolacca americana cells glycosylated polyphenols to the corresponding mono- and diglycosides. ^9,10 The Morus cultured cells transform polyphenol curcumin to reduction products. The NF-κB inhibitory effect of curcumin was higher than tetrahydrocurcumin. This is the first report that describes the inhibitory effects of tetrahydrocurcumin on NF-κB activity.

Experimental

General

Curcumin was purchased from Wako Pure Chemicals Co. and was used without further purification. The cultured plant cells of Morus sp. were subcultured at 4-week intervals on solid medium containing 2% glucose, 1 ppm 2,4-dichlorophenoxyacetic acid, and 1% agar (adjusted to pH 5.7) in the dark. A suspension culture was started by transferring 20 g of the cultured cells to 300 mL of liquid MS medium in a 500-mL conical flask.

Biotransformation Procedure

The suspension plant cells (20 g) were incubated in conical flasks for 2 days. The cultured cells in the stationary growth phase have been used for experiments. After the cultivation period, 10 mg of the substrate was added. The transformation was performed by incubating the mixture at 25°C on a rotary shaker for 2 days. The culture medium and cells were separated by filtration. The culture medium was extracted with ethyl acetate. The cells were extracted (×3) by homogenization with methanol. The methanol fraction was concentrated and partitioned between H₂O and ethyl acetate. The ethyl acetate fractions were concentrated and analyzed by HPLC.

NF-κB Activity

At the day before transfection, 50 × 10⁴ HeLa cells were seeded on a 60 mm dish. pNF-κB-secreted alkaline phosphatase (pNF-κB-SEAP, 1.0 µg) and pGL3-control vector (1.0 µg) were co-transfected with an Effectene transfection reagent. Cells were harvested and seeded onto 96-well white plate at 16 hours after transfection and then ligands were added. After 24 hours, luciferase and SEAP activities were assayed using a microplate reader. Secreted alkaline phosphatase data were normalized to an internal luciferase control and the reported values are the means of triplicate assays.