Antioxidant, antityrosinase and antitumor activity comparison: the potential utilization of fibrous root part of Bletilla striata (Thunb.) Reichb.f

Abstract

This study was carried out to evaluate the utilization probability of the fibrous root part (FRP) of Bletilla striata, which was usually discarded and harvesting pseudobulb part (PSP). The chemical composition, total phenolic content, DPPH radical scavenging activity, Ferric-reducing antioxidant power and tyrosinase inhibition activity were compared between FRP and PSP. Antioxidant and pro-oxidant effect as well as antitumor effect of the extract of FRP and PSP were analyzed by in vitro cell system as well. Thin layer chromatography and high performance liquid chromatography analysis indicated that the chemical compositions in the two parts were similar, but the content in FRP was much higher than PSP. Meanwhile, the FRP extracts showed higher phenolic content, stronger DPPH scavenging activity, Ferric-reducing antioxidant capacity and tyrosinase inhibition activity. Sub-fraction analysis revealed that the distribution characteristic of phenolic components and other active constituents in FRP and PSP were consistent, and mainly deposited in chloroform and acetoacetate fractions. Especially, the chloroform sub-fraction (sch) of FRP showed extraordinary DPPH scavenging activity and tyrosinase inhibition activity, with IC50 0.848 mg/L and 4.3 mg/L, respectively. Besides, tyrosinase inhibition activity was even stronger than the positive compound arbutin (31.8 mg/L). Moreover, In vitro cell system analysis confirmed that FRP extract exerts comparable activity with PSP, especially, the sub-fraction sch of FRP showed better antioxidant activity at low dosage and stronger per-oxidant activity at high dosage, and both sch of FRP and PSP can dose-dependent induce HepG2 cells apoptosis, which implied tumor therapeutic effect. Considering that an additional 0.3 kg FRP would be obtained when producing 1.0 kg PSP, our work demonstrated that FRP is very potential to be used together with PSP.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. The underground part of a three-year-old Bletilla plant.

Red, green and blue arrows indicate pseudobulb, fibrous roots and shoots of Bletilla respectively.

Figure 2. Analysis of the constituents of the PSP and FRP by TLC method.

The plate was air dried and recorded under UV light (panel A), then developed with I2/KI under room temperature (panel B), and 5% H2SO4-ethanol in 110°C for five minutes (panel C) sequentially. The lane 1, 3 and 5 were loaded with the sub-fractions of spe, sch and sac from the FRP; lane 2, 4 and 6 were loaded with the sub-fractions of spe, sch and sac from the PSP. Red arrows indicated the components that are absent in the PSP.

Figure 3. The HPLC results of tubers and fibrous roots of B. striata.

The black and red curve represent for PSP (15 µL injection) and FRP (5 µL injection) 95% ethanol extract, respectively. Peak marked was identified as the same compound both existed in PSP and FRP by UV-VIS spectrum.

Figure 4. The phenolic content in sub-fractions of the PSP and FRP (*, P<0.05, n = 3).

Figure 5. DPPH scavenging activity and Ferric-reducing antioxidant power of the extracts.

The symbol □, ○, Δ in panel A and C represent for 95% ethanol extract of the PSP, 95% ethanolic extract of the FRP and the positive control of gallic acid, respectively; Panel A and B, DPPH scavenging activity; Panel C and D, Ferric-reducing antioxidant power; #, P<0.05; *, P<0.01; n = 3.

Figure 6. Correlation analysis between the DPPH scavenging activity (IC50) and the phenolic content for sub-fractions of the PSP and FRP (P<0.05).

Figure 7. Effect of sub-fractions on intracellular ROS levels in HepG2 cell line.

HepG2 cells were stimulated for 1.5 h, intracellular ROS levels were assessed as described in the ‘Materials and Methods’ section. (A) Dose-dependent effect of H2O2 on intracellular ROS levels; (B,C and D) Intracellular ROS levels in cultured HepG2 in the absence (CTRL) or presence of the indicated concentration of (B) spe, (C) sch and (D) sac sub-fractions of both FRP and PSP. (A–D) * Significantly different from the control, P<0.05; n = 3.

Figure 8. Sub-fractions induce HepG2 impairment and apoptosis.

HepG2 cells were exposure in different sub-fractions and concentrations for 24h, and then cell viability and apoptosis were assessed as reported in the ‘Materials and Methods’ section. (A) Cell viability, (B) apoptosis and (C) necrosis in presence of the indicated sub-fraction concentration. Data are expressed as percent of control (CTRL). (A–D) * Significantly different from the control, P<0.05; n = 3.

Figure 9. sch-fractions induce HepG2 apoptosis and necrosis.

HepG2 cells were treated in different sub-fractions and concentrations for 24 h, and then cell were stained with Hoechst/PI as mentioned in the ‘Materials and Methods’ section. (A–C) Cells were absence of any treatment; (D–F) Cells were exposure to 6.25 µg/ml sch of FRP; (G–I) Cells were exposure to 50 µg/ml sch of PSP; (J–L) Cells were exposure to 50 µg/ml sch of FRP; magnification:×200.