Methods of Producing Ganoderma Lucidum Mycelia Having Enhanced Ganodermanondiol Content

Abstract

Provided is a method of culturing ganoderma lucidum mycelia in which the content of ganodermanondiol serving as an inhibitor to melanin biosynthesis is enhanced to improve skin whitening function.

Claims

1. A culture medium of ganoderma lucidum mycelia including oriental raisin tree extract to enhance ganodermanondiol content contained in ganoderma lucidum mycelia.

2. A method of producing ganoderma lucidum mycelia in which ganodermanondiol content is enhanced by adding oriental raisin tree extract.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings are included to provide a further understanding of the present invention, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present invention and, together description, serve to explain principles of the present invention. In the drawings:

[0013] FIG. 1 is a graph illustrating an increase of ganodermanondiol content contained in ganoderma lucidum mycelia according to a concentration of oriental raisin tree extract added therein;

[0014] FIG. 2 is a graph illustrating cytotoxicity of ganoderma lucidum mycelial extract according to a concentration of oriental raisin tree extract added therein; and

[0015] FIG. 3 is a graph illustrating inhibitory activity of ganoderma lucidum mycelial extract to melanin biosynthesis according to a concentration of oriental raisin tree extract added therein.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0016] Hereinafter, the present invention will be described in more details with reference to accompanying embodiments. It is noted, however, that the scope of the present invention should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be illustrative and exemplary.

EXAMPLE 1.

Producing ganoderma lucidum mycelia

[0017] Ganoderma lucidum No. 2 bought from Mushroom Department of National Institute of Horticultural and Herbal Science, Rural Development Administration, was used as the ganoderma lucidum mycelia. The Ganoderma lucidum No. 2 was aseptically separated and cultured in a PDA culture medium (containing 15 g of agar, 4 g of potato extract, and 20 g of glucose per 1 L culture solution).

EXAMPLE 2.

Producing Oriental Raisin Tree Extract

[0018] A 35 g of oriental raisin tree was ground and added with 350 g of purified water to conduct hot water extraction, which was repeated twice. The filtered extracts were obtained and used as an oriental raisin tree extract (100%).

EXAMPLE 3.

Culture Condition and Producing a Culture Medium for Liquid Culture

[0019] In order to produce a culture medium for liquid culture to enhance ganodermanondiol content of ganoderma lucidum mycelia, 100 ml of PDB culture medium (containing 4 g of potato extract and 20 g of glucose per 1 L of culture solution) and the oriental raisin tree extract (100%) prepared in Example 2 are mixed in a 250-ml triangular flask to make concentrations of 1, 5, 10 and 15 respectively. Then, the ganoderma lucidum mycelia obtained in Example 1 are ascetically sliced in a short size and placed in the 250-ml triangular flask to perform shaking culture at 100 revolution per minute (rpm) for 5 days.

EXAMPLE 4.

Producing ganoderma lucidum mycelia Extracts

[0020] The cultured ganoderma lucidum mycelia are separated from the culture medium and dried with hot air. Next, 0.2 g of the dried specimen is added with 50 ml of 70% ethanol to conduct reflux extraction, which is repeated twice, and filtered extraction liquid can be obtained. The extraction liquid may be concentrated in a reduced pressure to obtain 50 mg of ganoderma lucidum mycelia.

EXAMPLE 5.

Content Analysis of ganodermanondiol in ganoderma lucidum mycelia

[0021] Quantitative analysis of ganodermanondiol was performed using a HPLC (SPD-20A UV/VIS Deteor, LC-20AT Pumping System, Manual Injection Value 20 μl Loop, Shimadzu Corp., Japan) equipped with Phenomenex Luna C18UV(2) column (4.6×250 mm, 5 μm, USA). A moving phase thereof was measured with acetonitrile and 0.2% acetic acid, while setting flow rate at 0.8 ml/min, using an ultraviolet detector at 245 nm.

[0022] In addition, the quantitative analysis of ganodermanondiol contained in the ganoderma lucidum mycelia was also performed using ganodermanondiol (99% purity) purchased from ChemFace® (China) as a standard material.

[0023] The content of ganodermanondiol in the ganoderma lucidum mycelia according to a concentration of oriental raisin tree extract added in the ganoderma lucidum culture medium was analyzed and the results thereof are illustrated in FIG. 1 and. TABLE 1 below.

TABLE-US-00001 TABLE 1 Concentration of oriental Ganodermanondiol Specimen raisin tree extract (%) contents (mg/g) Ganoderma 0 1.02 ± 0.05 lucidum 1 1.14 ± 0.09 mycelia 5 1.51 ± 0.04 10 2.19 ± 0.11 15 2.81 ± 0.15

[0024] The ganodermanondiol content of the ganoderma lucidum mycelia according to a concentration of oriental raisin tree extract is described in TABLE 1. As shown in TABLE 1 and FIG. 1, the ganodermanondiol content of the ganoderma lucidum mycelia increases as the concentrations of the oriental raisin tree extract increases.

EXAMPLE 6.

Effect on Cell Survival of Melanoma B16F10 Cell Line

[0025] A melanoma B16F10 cell line purchased from American Type Culture Collection (ATCC, 6475) was used. The cell line was cultured in 5% of CO.sub.2 culture medium at a temperature of 37□ using Dulbecco's Modified Eagle Medium (DMEM) containing 10% of Fetal Bovine Serum (FBS). All cells during experiments were experimented at 80 to 90% of confluency.

[0026] MT assay was performed to confirm whether ganoderma lucidum mycelial extracts and the oriental raisin tree extract have cytotoxicity on melanoma B16F10 cells or not. A 100 μl of DMEM containing 10% of FBS is dispensed in a 96-well plate such that approximately 3×10.sup.3 of cells can be placed in each well, which is then cultured under the condition of 5% of CO.sub.2 for 24 hours. Then, the culture medium was removed and the extracts having different concentrations were processed such that each group was triplicated and cultured under the condition of 5% of CO.sub.2 at 37° C. for 72 hours.

[0027] After culturing, 10 μl of 5-mg/ml MTT reagent was dispensed in each well of the culture medium including the experiment solution and the 96-well plate was cultured for 3 hours without light. The culture medium including the MTT reagent was then removed and 100 μl of DMSO was added to dissolve formazan crystal, which is followed by measuring 570 nm absorbance to determine the cell survival rate.

Cell Survival Rate (%)=(Absorbance of Control Group/Absorbance of Experimental Group)×100

[0028] As shown in FIG. 2, the ganoderma lucidum mycelial extract and mycelial extract added and cultured with oriental raisin tree extracts according to an embodiment of the present invention did not show cytotoxicity to melanoma B161-10 cells in a concentration range of 50 to 200 μg/ml.

EXAMPLE 7.

Inhibitory Effect of Melanoma B16F 10 Cell Line on Melanin Synthesis

[0029] Melanoma B16F10 cells are dispensed in a 6-well plate to become a concentration of 1×10.sup.5 cells/well using DMEM containing 10% of FBS and cultured under the condition of 5% of CO.sub.2 at 37□ for 24 hours. Next, the culture medium is removed, and ganoderma lucidum extracts and the compound having different concentrations are placed in DMEM culture solution containing 10% of FBS, which is followed by adding α-MSH (100 nM) as an induction agent thereto and cultured under the condition of 5% of CO.sub.2 at 37□ for 72 hours.

[0030] The culture medium is removed therefrom after culturing and it is rinsed with PBS twice, which is followed by dispensing 1 ml of PBS in each well to recover a cell pellet. The recovered pellet is centrifuged at 14,000 rpm for 20 minutes and its supernatant faction is removed. Next, the cell pellet is dried at 60□ for 1 hour and processed in a constant temperature bath of 60□ to dissolve melanin within cells, while adding 150 μl of 1 M NaOH containing 10% of DMSO. Then, 100 μl of the solution is dispensed in a 96-well plate and absorbance at 405 nm is measured using an ELISA reader. The result is compared with a standard curve obtained by using melanin standard solution to quantify the melanin content (FIG. 3).

Inhibition Rate of Melanin Content (%)=1−(Melanin Content of Control Group/Melanin Content of Experimental Group)×100

TABLE-US-00002 TABLE 2 Concentration Inhibition Rate Specimen (μg/ml) (%) No treatment 0 0 Ganoderma 50 21.77 lucidum 100 30.13 mycelium 150 39.37 200 45.09 Mycelium added 50 27.93 with oriental 100 25.95 raisin tree 150 34.53 extract 5% 200 33.21 Mycelium added 50 47.51 oriental 100 62.25 raisin tree 150 67.09 extract 15% 200 71.27 Albutin 500 47.07 Mycelium added 50 19.78 oriental 100 20.23 raisin tree 150 35.85 extract 1% 200 44.65 Mycelium added 50 51.91 oriental 100 52.35 raisin tree 150 58.07 extract 10% 200 64.01

[0031] Inhibitory effect of melanin content in the melanoma B16F10 cells is described in TABLE 2.

[0032] As shown in FIG. 3, ganoderma lucidum mycelial extract plays a significant role in inhibiting the melanin content produced by the α-MSH induction, depending on concentration, as the concentration of the oriental raisin tree extract added therein increases (TABLE 2).