*4.2. Quantification of Secondary Metabolites and Antioxidant Activity of In Vitro-Induced Calli and Conventionally Propagated Nodal Segment of B. glabra*

The results of phytochemicals and antioxidant activities analysis revealed that total phenolic acid, total flavonoid content, DPPH free radical scavenging activity, and ABTS scavenging activity of aqueous extract of the conventionally propagated nodal segment were significantly different from other extraction solvents of in vitro-induced calli of *B. glabra*. The interrelating effect of the extraction solvent and source of the sample has been studied by Mahendra et al. [57] in conventionally propagated plant parts and calli induced under the light regime of *Decalepis arayalpathra*. They revealed maximum TPC and TFC from aqueous extract of the conventionally propagated nodal segment and calli

induced under photoperiod, respectively. Similarly, the current study results confirm the findings of Esmaeili et al. [58] that high polar solvents such as water of conventionally propagated plant parts produced higher TPC, and TFC compared with in vitro induced calli. In addition, node cultures of *Eucalyptus camaldulensis* produced under a 16-h light photoperiod revealed an increase in the level of phenolic compounds [59]. On the other hand, a study conducted by Zahid et al. [60] indicated the hexane extract of micropropagation and conventional propagated 'Bentong' ginger *Zingiber officinale* Roscoe produced the lowest amount of TPC and TFC compared to other extraction solvents, which are in agreement with our findings.

In the antioxidant activities, the aqueous extract of conventionally propagated nodal segment produced higher DPPH free radical scavenging activity and ABTS scavenging activity compared to in vitro-induced calli. This is in agreement with the findings of Esmaeili et al. [58] that the highest antioxidant properties was obtained from the aqueous extract of field-grown plant parts compared to the in vitro induced calli. Moreover, previous studies by Islam et al. [61] on *B. glabra*, Murali and Prabakaran [62] on *Ociumum basilicum* L., and Mahendra et al. [57] on *Salacia macrosperma* showed that the aqueous and ethanol extracts of the conventionally propagated plant parts exhibited the higher DPPH free radical scavenging and ABTS scavenging activity compared to the in vitro induced calli of less polar solvents such as acetone and hexane. Furthermore, studies conducted by López-Laredo et al. [63] on *Tecoma stans*, Shah et al. [64] on *Silybum marianum*, Mohammad et al. [65] on *Olea europaea* L., and Rameshkumar et al. [51] on *Nilgirianthus ciliate* indicated that the calli induced under the photoperiod produced a higher antioxidant activity than the calli induced under dark incubation conditions which are in line with our findings. On the other hand, a study conducted by Zahid et al. [60] indicated that the hexane extract of micropropagated and conventional propagated 'Bentong' ginger *Zingiber officinale* Roscoe produced the lowest inhibition of DPPH than other extraction solvents, which are also in agreement with our findings.

In contrast to other antioxidant activities conducted in which aqueous and other node extracts produced higher antioxidant activities, the iron (II) chelating activity was produced from the aqueous extract of the calli induced under dark conditions. It is clear that the chelating powers of water extracts of the calli induced under dark conditions were higher than the other three extracts and sources of samples. Similarly, a study conducted by Hakkim et al. [66] reported that the in vitro induced callus had a higher iron (II) chelation than that of the field-grown stem, leaves, and inflorescence of *Ocimum sanctum* L. Furthermore, according to Costa et al. [67], the synergistic effect of solvents and the source of the sample exhibited that the aqueous extract of the in vitro-induced callus showed higher iron (II) chelation than conventionally propagated plant parts and other extraction solvents of *Thymus lotocephalus*. In addition, study conducted by Song et al. [68] confirmed that shoot of the field-grown plant of *Mertensia maritima* L. produced higher TPC, TFC, DPPH free radical scavenging activity, and ABTS scavenging activity. However, the in vitro-induced calli chelate had more iron (II) than field-grown shoots, supporting our findings. So, the in vitro-induced callus can be a good source of iron (II) chelation, and this indicates that polyphenols are not primary chelating particles [69].
