The dual effect of curcumin nanoparticles encapsulated by 1-3/1-6 β-glucan from medicinal mushrooms Hericium erinaceus and Ganoderma lucidum

1-3/1-6 β-glucan (Glu) used in this project is extracted from Lion's mane mushroom (Hericium erinaceus) and Lingzhi mushroom (Ganoderma lucidum) cultured in Vietnam. Other chemicals include curcumin (Sigma-Aldrich), ethanol (Merk) and several standard chemicals for laboratory purposes.

Cell lines were supplied by Institute of Natural Product Chemistry including human hepatocellular carcinoma (Hep-G2) and human lung adenocarcinoma (LU-1).

1-3/1-6 β-glucan encapsulated curcumin nanoparticles (NanoGluCur) were prepared by nano-precipitation method. In the first step of the method, 10 ml of 1 mg ml⁻¹ solution of curcumin in ethanol was dropped into 10 ml solution of 20 mg 1-3/1-6 β-glucan in deionized water. The mixture then had been stirred at room temperature for 48 h. Then, solvent was evaporated by a rotary evaporator. Next, product was centrifuged at 400 rpm to eliminate free curcumin. The mixture that contains 1-3/1-6 β-glucan encapsulated curcumin was then dried by Freeze Dry System (Labconco).

The structure of NanoGluCur was investigated by Fourier transform infrared spectroscopy (FTIR). In addition, UV–vis absorption and emission spectroscopy were used to confirm the formation of nanoparticles. The morphology and size of nanoparticles were characterized by field emission scanning electron microscope (FESEM) and dynamic light scattering (DLS) imaging.

Loading capacity (LC) was calculated using UV–vis absorption spectroscopy. First, curcumin was separated from nanoparticles by adding ethanol. Then the mixture was centrifuged to eliminate polymer and insoluble components. Curcumin concentration in ethanol was calculated based on absorbance standard curve. LC was calculated by the following formula:

$$\begin{eqnarray*}&&LC( \% )=\displaystyle \frac{Encapsulated\,curcu\,{\min }}{Total\,weight\,of\,components}\times 100.\end{eqnarray*}$$

Solubility of NanoGluCur was determined after dissolving to saturation freeze dried NanoGluCur powder and eliminating insoluble component by centrifuging. Solubility of curcumin in the NanoGluCur solution is the concentration (C₀) of the saturated solution quantified by UV–vis spectroscopy. Solubility of NanoGluCur was calculated by the following formula:

$$\begin{eqnarray*}&&{S}_{p}=\displaystyle \frac{{C}_{0}}{LC},\end{eqnarray*}$$

with ${C}_{0}=k{X}_{1},$ k is dilution factor, X₁ is concentration of curcumin after dilution.

Cancer cells were cultured following the protocol described by Skehan et al [16]. Cytotoxicity of test samples was determined by sulforhodamine B (SRB) colorimetric method [17]. The method has been carried out in Department of Experimental Biology from Institute of Natural Product Chemistry (INPC, VAST) since 1996.

Anti-tumor promoting assay is a technique of three dimensional tissue culture models in cancer biology based on the formation of tumors from seed cancer cells in soft agar. The assay has been conducted by Department of Experimental Biology (INPC, VAST) since 2010 following methods that described in earlier studies of Kim [18] and Gao et al [19]. Briefly, test samples were incubated (37 °C, 10–20 days) in micro-titer plate with cancer cells (Hep-G2 cell line) in DMEM-soft agar-medium at appropriated concentrations. Effects of test samples were determined by measuring sizes and densities of forming tumors in each well by light converted microscope (Olympus MX51). Cells and tumors were stained by SRB and crystal violet dyer.

FTIR spectra and characteristic peaks of curcumin (Cur), 1-3/1-6 β-glucan (Glu) and NanoGluCur are shown in figure 3 and table 1.

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Figure 3 and table 1 showed shifts from curcumin characteristic vibrations at 3510 cm⁻¹ (O−H bond), 1628 cm⁻¹ (C=O bond) and 1509 cm⁻¹ (C=C bond) to 3398, 1635, 1500 cm⁻¹ of curcumin contained in nanoparticles. Also, peaks at 3402 và 860 cm⁻¹, corresponding to O-H bond and β-bridge of free glucan, shifted to peaks of 3398 and 849 cm⁻¹ of β-glucan in NanoGluCur. In addition, the spectrum of interval 1026–1150 cm⁻¹ that characterizes the glucose link at 3-O in glucan structure shifted to that of 1014–1142 cm⁻¹ of nanoparticles. These evidences induce the formation of β-glucan encapsulated curcumin.

To further confirm the formation of NanoGluCur, fluorescene and UV–vis spectra were compared between curcumin before and after encapsulated. It was observed that emission peak of curcumin in ethanol at 540 nm is shifted to 529 nm after encapsulated by 1-3/1-6 β-glucan (figure 4(a)). Similarly, the maximum absorption wavelength of curcumin in ethanol at 430 nm was shifted to 417 nm after encapsulated by 1-3/1-6 β-glucan (figure 4(b)). These shifts in fluorescence and UV–vis spectra could be attributed to the formation of intermolecular hydrogen bonding between curcumin and 1-3/1-6 β-glucan.

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