E. asperula Zollinger et moritzi cell suspension cultures
Cell suspension cultures were started using the friable callus of
E. asperula Zollinger et Moritzi (Fig 3A). Single cells and small clumps were present in the dark yellow cell suspensions after four weeks of culture (Fig 3B).
RA in E. asperula Zollinger et moritzi cell suspension cultures
This study used spectrophotometry and HPLC methods to determine the RA content in extract samples from
E. asperula Zollinger et Moritzi.
In Table 2, RA is a phenolic compound with significant content in the tested extracts.
In vitro leaves had the highest concentration, followed by callus and field-grown leaves and the lowest concentration was in cell suspension cultures. RA content in the extracts measured by the HPLC method was higher than that measured by the spectrophotometry method.
DPPH test for scavenging free radicals in the extracts
The results showed that the extracts created yellowish solutions on a purple background, demonstrating antioxidant activity in the DPPH experiment (Fig 4).
A comparative analysis of the antioxidant activity of the extracts from leaves of field-grown and
in vitro E. asperula Zollinger et Moritzi plants, callus and biomass of cell suspension cultures was presented in Fig 5.
The inhibition percentage of DPPH free radical scavenging by leaves
in vitro and callus extracts was provided as 90.3% and 90.99% at the concentration of 2000 μg/mL, which have more potent antioxidant activity than the extracts of field-grown leaves (82.24% at the concentration of 2000 μg/mL) or biomass of cell suspension cultures (65.53% at the concentration of 2000 μg/mL) (Fig 5).
From the standard curve showing the linear correlation between the concentration of the sample test and the value of % inhibition of DPPH free radicals (Fig 5), the DPPH inhibition IC
50 value of the experimental extracts was interpolated (Table 3). The leaves
in vitro extracts had the strongest anti-radical activity (IC
50 = 871.54 μg/mL) in the extracts. However, ascorbic acid’s IC
50 value (27.62 μg/mL) was higher than the
E. asperula Zollinger et Moritzi extracts.
In Table 2, RA contents in
E. asperula Zollinger et Moritzi extracts were high. The lowest RA concentration in the biomass of cell cultures was due to a portion of RA dissolved in the liquid medium
(Tram et al., 2022). In particular, the RA contents recorded by the HPLC method were higher than those measured by spectrophotometry. The HPLC method is susceptible, has good quantitative ability and a higher level of accuracy than the spectrophotometry method. The RA content of the extract from field-grown leaves reached 8.94%, equivalent to the RA content from
E. asperula Zollinger et Moritz leaves extracted with 50% ethanol (9.95%), as reported by
Ly (2016). The RA content in
E. asperula Zollinger et Moritzi cell suspension cultures (74.64 mg/g DW) was equivalent to that in
Thymus lotocephalus shoot cultures (78.57 mg/g DW) (
Gonçalves et al., 2019), higher in
Dracocephalum moldavica L. cell suspension cultures (27.2 mg/g DW) (
Weremczuk-Jeżyna et al., 2017).
About the assessment of antioxidant activity, the tested extract samples had antioxidant capacity equivalent to the extracts from
in vitro E. asperula Zollinger et Moritzi plants grown under a fluorescent lamp (IC
50 = 1101.10 µg/mL) in the experiment of
Tram et al., (2018). In particular, the extract from the biomass of cell suspension cultures had a lower antioxidant capacity than others, possibly due to some biologically active substances (especially RA) being secreted from the cells during growth and dissolved in the culture medium
(Tram et al., 2022).
The experimental extract samples of
E. asperula Zollinger et Moritzi also had an antioxidant capacity equivalent to or higher than the antioxidant capacity of some previously studied herbs, such as
Allamanda neriifolia root, stem and leaf extracts with IC
50 values of 713.44, 1397.24 and 936.86 μg/mL, respectively
(Hung et al., 2014), extracts from
Sauropus androgynus,
Polyscias fruticosa,
Portulaca oleracea L. with IC
50 values of 993.85, respectively; 2110.08 and 2835.33 µg/mL
(Mai et al., 2017).
A strong negative correlation was found between the RA contents in the experimental extracts and the IC
50 values (r = -0.934 for the spectrophotometric method, r = -0.756 for the HPLC method). RA plays an essential role in the bioactivity of
E. asperula Zollinger et Moritzi extracts. The antioxidant activity of RA is well known due to its four phenolic hydrogens and two catechol (1,2-dihydroxy benzene) rings
(Bhatt et al., 2013). A significant correlation between total phenolic contents (especially RA contents) from the cultures
in vitro and antioxidant activity has been reported in many previous studies
(Shiga et al., 2009; Samarakoon et al., 2016; Weremczuk-Jeżyna et al., 2017;
Gonçalves et al., 2019).