Cytotoxic Activities and Fingerprint Analysis of Triterpenes by HPTLC Technique for Distinguishing Ganoderma Species from Vietnam and other Asian Countries

Ganoderma lucidum (Fr.) P. Karst. (Ganodermataceae), commonly called Linhzhi, is traditionally employed in the treatment of human diseases, including hepatitis, liver disorders, hypercholesterolemia, arthritis, bronchitis, and tumorigenic diseases. In this study, the fingerprint profiles of five different strains of G. lucidum originated from Japan, Korea, China, and Vietnam, five samples of G. lucidum growing on Erythrophloeum fordii Oliv. in Vietnam, and five related Linhzhi species (Ganoderma applanatum, Ganoderma australe, Ganoderma clossum, Ganoderma subresinosu, and Ganoderma sp.) were investigated for triterpene derivatives using high-pressure, thin-layer chromatography (HPTLC). The HPTLC fingerprint profiles demonstrated significant differences between G. lucidum and other related Linhzhi species in the presence of triterpene derivatives. Evaluation for the cytotoxicity of these samples against four cancer cell lines, including A549, MCF7, PC3, and HepG2, displayed various levels of cytotoxic effects, with IC50 values of: 15.6–46.3 µg/mL on the A549 cancer cell line, of 18.4–43.6 µg/mL on the MCF7 cancer cell line, of 10.0–32.1 µg/mL on the PC3 cancer cell line, and of 10.6–27.6 µg/mL on the HepG2 cancer cell line. Conclusively, these data contributed to the literature on the cytotoxic activities and fingerprint analysis of triterpenes by the HPTLC technique for distinguishing Ganoderma species from Vietnam and other Asian countries.


HPTLC Analysis
Triterpenes and ergosterol (1-7, Figure 1) are nonpolar compounds; therefore, the solvent system that obtained the optimized resolution of the HPTLC images is dichloromethane:methanol (9:1). A high resolution with fluorescence bands in the chromatogram of 15 tested samples (G1-G15) was observed (Figure 2A). The chromatogram of samples G1-G10 showed 12-13 fluorescence bands with R f values higher than 0.4, which were regarded as triterpenes and ergosterol derivatives. In comparison, chromatograms of samples G11-G15 were significantly different from each other and from the chromatograms of samples G1-G10. solvent system that obtained the optimized resolution of the HPTLC images is dichloromethane:methanol (9:1). A high resolution with fluorescence bands in the chromatogram of 15 tested samples (G1-G15) was observed (Figure 2A). The chromatogram of samples G1-G10 showed 12-13 fluorescence bands with Rf values higher than 0.4, which were regarded as triterpenes and ergosterol derivatives. In comparison, chromatograms of samples G11-G15 were significantly different from each other and from the chromatograms of samples G1-G10.  solvent system that obtained the optimized resolution of the HPTLC images is dichloromethane:methanol (9:1). A high resolution with fluorescence bands in the chromatogram of 15 tested samples (G1-G15) was observed ( Figure 2A). The chromatogram of samples G1-G10 showed 12-13 fluorescence bands with Rf values higher than 0.4, which were regarded as triterpenes and ergosterol derivatives. In comparison, chromatograms of samples G11-G15 were significantly different from each other and from the chromatograms of samples G1-G10.   (G3 and G5), and wildly collected samples in Quang Nam, Vietnam (G6-G10). However, G5-G6 possessed a higher level of lucidenic acid N (1) and lucidadiol (5) compared to those in G1-G5. It is important to note that fluorescence bands with R f values of 0.26-0.3 of G. applanatum (G11), G. clossum (G12), G. subresinosum (G13), G. sp (G14), and G. australe (G15) were different to those of G. lucidum (G1-G10) ( Figure 2B-D). As demonstrated in Figure 2D, only ganodermadiol (6) and ergosterol (7) appeared in five Ganoderma species (G11-G15).

In Vitro Cytotoxic Activity
Three cultivated Linhzhi (G1-G4), a wild-collected Linhzhi (G6), and five related Linhzhi species (G11-G15) have been evaluated for the inhibition of in vitro cytotoxic effects on four cancer cell lines, including A549, MCF7, PC3, and HepG2. Results are shown in Table 2. According to the US NCI rules on the plant extracts/pure compounds' in vitro cytotoxicity, a plant extract is considered to be toxic to a cell line if its IC 50 value is <20 µg/mL after an incubation time of 48 h, whereas this value should be <10 µg/mL for the pure compounds [33]. As shown in Table 2, among the four cultivated Linhzhi, G1, G2, G3, and G4, which originated from Japan, China, Vietnam, and Korea, G1 showed a potent cytotoxic effect on PC3 and HepG2 cancer cell lines. G2 and G4 showed significant inhibitory activity on a PC3 cancer cell line and a moderate inhibitory activity on HepG2 and A549 cancer cell lines. G1, G2, and G4 displayed non-cytotoxicity against the MCF7 cancer cell line, with an IC 50 value > 50 µg/mL. G3 presented a moderate inhibitory activity on the MCF7 cell line, with an IC 50 value of 33.8 ± 3.4 µg/mL, and was inactive on A549 and PC3, with IC 50 values > 50 µg/mL. For the comparison between cultivated Linhzhi (G1-G4) and a Linhzhi sample collected from nature (G6), a similar effect on the inhibition of three cancer cell lines (MCF7, PC3, and HepG2) was observed. A difference was seen in the highest inhibitory activity of G6 on the A549 cell line, with an IC 50 value of 9.12 ± 1.5 µg/mL, as compared to those of G1-G4.
Five related Linhzhi species, including G. applanatum (G11) and G. clossum (G12), displayed moderate cytotoxic activity against the A549 cancer cell line with IC 50 values of 46.3 and 24.8 µg/mL, respectively. G. subresinosum (G13) and G. sp (G14) showed considerable cytotoxic activities against the A549 cancer cell line with IC 50 values of 15.6 and 17.7 µg/mL. G. australe (G15) exhibited cytotoxic inactivity on this cell line at IC 50 > 50 µg/mL. G13-G15 displayed significant cytotoxic activity against the MCF7 cancer cell line with IC 50 values within 18.4-30.7 µg/mL. G11-G12 did not demonstrate any significant cytotoxic activity against the MCF7 cancer cell line. Four samples (G12, G13, G14, and G15) displayed a moderate inhibitory effect on PC3 with IC 50 values ranging from 23.6 to 32.1 µg/mL, and G11 presented no cytotoxic activity on this cell line. G11, G13, and G15 did not show any significant cytotoxic activity against the HepG2 cell line. On the other hand, G12 and G14 displayed considerable cytotoxic effects against HepG2, with IC 50 values of 20.2 and 23.5 µg/mL, respectively.

Discussion
In the present work, we have reported, for the first time, the fingerprint profiles of four Linhzhi strains originated from Vietnam, Japan, and China that were successively cultivated in Vietnam, one Korean Linhzhi strain that was cultivated in Korea, five wildharvesting Linhzhi in Vietnam, and five related Linhzhi strains that were successively cultivated in Vietnam. It can be implied that different strains possess different chemical constituents due to differences in the geographical distributions, growth conditions, and substrates. In our study, the profiles of lanostan triterpenes differed considerably in the five different strains (G1-G10) and were distinguishable from the five related Linhzhi species (G11-G15). Similar data have been reported in the literature. The growth conditions might be the major factors contributing to the differences between the Iranian and Chinese Linhzhi strains in producing various ganoderic acids [24]. Besides, the quality assessment of Linhzhi and its respective commercial products was performed based on small (triterpenes and nucleic acids) and macro (polysaccharides) molecular bioactive compounds by using HPLC, high-performance size-exclusion chromatography evaporative light scattering detector (HPSEC-ELSD), and HPTLC. The data also displayed the obvious variations among Linhzhi strains or products [34,35]. Notably, the Italian G. lucidum possesses different phytochemical contents, namely protein and polysaccharide, compared to the same Chinese species cultivated in similar medium. Thus, this fact re-confirms that the bioactive components and the therapeutic activities of Linhzhi are heavily dependent on the climatic and geographical conditions. In our research, phytochemical investigation demonstrated significant differences between Linhzhi strains and related Linhzhi species. In addition, the dissimilarity of cytotoxicity against four human cancer cell lines: A549, MCF7, PC3, and HepG2, was observed from ten Linhzhi strains and five related Linhzhi species cultivated in Vietnam. Ganoderma sp. (G14), and Ganoderma australe (G15) were gifts from Linh chi Vina Company, Vietnam. Ganoderma lucidum (G4) was purchased from Longevity Linhzhi Farm, Korea. Ganoderma lucidum (G5) was purchased from the Vietnam Academy of Agricultural Sciences. Ganoderma lucidum (G6-G10) was wildly collected from Tienphuoc district (or Tien Phuoc), Quangnam province (or Quang Nam), Vietnam, known locally as Natural Green Lim mushroom (Nấm Lim Xanh). The samples (G1-G3, G11-G15) were botanically identified (Table 1)

Preparation of Extracts for Cytotoxic Test
The air-dried fruiting bodies of each sample of Ganoderma species (50 g) (summary in Table 1) were extracted with MeOH (3 × 0.6 L). Each extract was passed through a No. 1 Whatman filter (Whatman Inc., Hillsboro, OR, USA) and the filtrate was evaporated to dryness under a vacuum at 40 • C to obtain the MeOH extracts. The sample extracts were stored at −20 • C until the cytotoxic test.

Preparation of Sample Solution for HPTLC Analysis
For the HPTLC analysis, 0.5 g of powder of each Ganoderma species was accurately weighted into a conical flask, respectively, and refluxed with 60 mL of MeOH for 1 h. The extract solution was filtered, and the residues were washed with 20 mL of MeOH, twice. This extraction process was repeated two times. The extracts were then combined and concentrated to dryness under a vacuum. The dried residue was dissolved in 2 mL of MeOH, filtered through a 0.45 µm membrane filter, and subjected to HPTLC analysis.

MTT Assay
Regarding the cytotoxicity tests on the cancer cell lines, the MTT assay was employed. To this end, the cancer cells were seeded in 96-well plates, incubated for 24 h at 37 • C, and the plant extracts (at concentrations ranging from 1 to 100 µg/mL) were added to each well. The mixtures were then incubated for another 48 h, and the MTT solution was subjected to the wells. The formed formazan crystals in viable cells were dissolved in DMSO, and its UV-Vis absorbance at 550 nm was measured by a microplate reader. The percentages of cell viability were calculated based on the absorbance values, in relation to the negative control (i.e., cells exposed to the control vehicle) [36].

Chromatography
For the HPTLC, the sample and references were applied band-wise (track distance 8 mm, band length 6 mm) on the precoated silica gel plates. Then, the plates were desiccated in a vacuum trunk for 2 h, followed by HPTLC with the low layer of dichloromethane:methanol (9:1), for 85 mm at room temperature, in a Camag twin-trough chamber. After that, the plates were visualized with 10% H 2 SO 4 in ethanol, under heating at 105 • C. Finally, the plate was observed under UV exposure at a wavelength of 366 nm, and the HPTLC chromatograms were recorded. The corresponding digital scanning profile was generated with the selfdeveloped software by our research team. The method was critically validated following the ICH guideline [43].

Statistical Analysis
The data were analyzed using the unpaired Student's t-test between the control and compounds. Data were compiled from three independent experiments and the values were expressed as mean ± standard deviation (SD).

Conclusions
In summary, along with other analysis methods such as HPLC or GC, HPTLC is also feasible for standardization and quality control of various Linhzhi samples or Linhzhi products, based on differences in the fingerprint profiles of triterpenes on HPTLC chromatograms. The unique fingerprint profiles were observed for cultivated Linhzhi strains originated from Korea, Japan, China, and Vietnam, wild-collected Linhzhi in Vietnam, and other related Linhzhi species, including G. applanatum, G. australe, G. clossum, G. subresinosu, and Ganoderma sp. These distinctive triterpene components could be readily used for the rapid differentiation of these Linhzhi and related Linhzhi species. In addition, evaluation of the cytotoxicity of these species against four cancer cell lines, including A549, MCF7, PC3, and HepG2, displayed various levels of cytotoxic effect. This research contributes to the literature on the cytotoxic activities and fingerprint analysis of triterpenes by the HPTLC technique for distinguishing Ganoderma species from Vietnam and other Asian countries.