Fig 1.
Peanut hairy root cultures of the K2-K599 line treated with a combination of chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD), shown in three biological replicates (R1, R2, R3).
(a) Hairy root cultures prior to elicitation (0 hour), (b) hairy root cultures after 72 hours of elicitation, (c) culture medium collected after 72 hours of elicitation. Chemical structures of the major stilbenoids: (d) trans-arachidin-1 (Ara-1) and (e) trans-arachidin-3 (Ara-3).
Table 1.
Stability assessment of antioxidant activity, total phenolic content, and stilbene compounds (trans-arachidin-1 and trans-arachidin-3) in peanut hairy root crude extracts (PCE) stored at room temperature (RT), 4 °C, and –20 °C over a three-month period. Different letters within the same column indicate statistically significant differences (p < 0.05) among storage times under the same temperature condition.
Fig 2.
Stability of (a) ABTS antioxidant activity and (b) total phenolic content in peanut hairy root culture crude extracts (PCE) stored at room temperature (RT), 4 °C, and –20 °C over a three-month period.
Samples were prepared from a PCE batch, aliquoted, and stored at the 3 months period prior to analysis. Bars represent mean ± SD (n = 3). Different letters within the same temperature condition indicate statistically significant differences (p < 0.05) among storage periods.
Fig 3.
HPLC chromatograms of (a) standard trans-arachidin-1 (Ara-1), (b) standard trans-arachidin-3 (Ara-3), and the peanut hairy root culture crude extracts (PCE) under different storage conditions: (c) freshly prepared extract at 0 month (initial time point), (d) extract stored for 3 months at room temperature (RT); (e) extract stored for 3 months at 4 °C; and (f) extract stored for 3 months at –20 °C.
Chromatographic signals were recorded as photodiode array detector voltage output (V), which is proportional to UV absorbance.
Fig 4.
Quantification and stability of (a) trans-arachidin-1 (Ara-1) and (b) trans-arachidin-3 (Ara-3) in peanut hairy root culture crude extracts (PCE) stored at room temperature (RT), 4 °C, and −20 °C over a three-month period. Stilbenoid contents were quantified by HPLC-UV analysis based on integrated peak areas and calculated using authentic Ara-1 and Ara-3 reference standards.
Data are presented as mean ± SD (n = 3). Different letters within the same storage temperature indicate statistically significant differences among storage time points (p < 0.05).
Fig 5.
Concentration–response effects of peanut hairy root culture crude extracts (PCE) on SW480 colon cancer cell viability following storage under different temperature conditions.
(a–c) show concentration–response curves expressed as percentage cell viability compared with the vehicle control after storage at room temperature (RT), 4 °C, and −20 °C, respectively, at 0, 1, 2, and 3 months. (d) summarizes the corresponding IC₅₀ values derived from these curves. Data represent mean ± SD from three independent experiments. In panels (a–c), *p< 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences compared with the vehicle control. In panel (d), *p < 0.05, **p < 0.01, and ***p < 0.001 indicates a statistically significant difference compared with the corresponding initial time point (0 month).
Fig 6.
Quantification and stability of predominant stilbenoids in (a) peanut hairy root culture crude extracts (PCE), (b) partially purified fraction enriched in trans-arachidin-1 (Ara-1), and (c) partially purified fraction enriched in trans-arachidin-3 (Ara-3) stored at −20 °C over a six-month period.
Stilbenoid contents were quantified by HPLC-UV analysis based on integrated peak areas and calculated using Ara-1 and Ara-3 reference standards. Samples were prepared from a purification batch, aliquoted, and stored at the indicated time points prior to analysis. Bars represent mean ± SD (n = 3). Different lowercase letters indicate statistically significant differences (p < 0.05) among storage periods for Ara-1, while different uppercase letters indicate statistically significant differences (p < 0.05) among storage periods for Ara-3.
Fig 7.
Stability of ABTS antioxidant activity in peanut hairy root culture crude extract (PCE) and partially purified fractions of trans-arachidin-1 (Ara-1) and trans-arachidin-3 (Ara-3) after storage at –20 °C for six months.
Samples were prepared from a purification batch, aliquoted, and stored at the 6 months period prior to analysis. An asterisk (*) denotes a statistically significant difference (p < 0.05) relative to the initial measurement (0 month).
Fig 8.
Cytotoxic activity of peanut hairy root culture crude extract (PCE) and partially purified fractions of trans-arachidin-1 (Ara-1) and trans-arachidin-3 (Ara-3) against KKU-100 cholangiocarcinoma cells following storage at −20 °C.
(a) and (b) show concentration–response effects expressed as percentage cell viability at the initial time point (0 month) and after 6 months of storage, respectively. (c) presents a comparison of IC₅₀ values between 0 and 6 months for each sample. Data represent mean ± SD from three independent experiments. In panels (a) and (b), *p < 0.05 and ***p < 0.001 indicate statistically significant differences compared with the vehicle control. In panel (c), *p < 0.05 indicates a statistically significant difference compared with the corresponding initial time point (0 month), while ns indicates no significant difference.
Fig 9.
Microscopic images of KKU-100 cholangiocarcinoma cells (10x magnification) after 48 hours of treatment with peanut hairy root culture crude extracts (PCE) and partially purified fractions of trans-arachidin-1 (Ara-1) and trans-arachidin-3 (Ara-3) at concentrations ranging from 100 to 500 µg/mL, after six months of storage.