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The purpose of this study is to evaluate the content of bioactive pigments in coloured callus of Azadirachta indica and to understand the correlation between the callus colours with their bioactive constituents, antioxidant properties and cytotoxicity. These assessments will yield valuable insight into the use of in vitro-derived pigments for possible use as functional natural colourants.

In this study, the authors have successfully developed a protocol to produce leaf-derived callus of various colours with enhanced content of bioactive pigments in A. indica through plant tissue culture. Comparative analysis of the pigments content (chlorophyll, carotenoid, phenolics and anthocyanins) in the coloured callus was conducted, followed by evaluation of its bioactive properties. The antioxidant properties against 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radicals, ferric reducing antioxidant power and cytotox activities of the coloured callus extracts were also reported.

Callus of various colours were successfully produced in A. indica through plant tissue culture, and their valuable pigment content and bioactivity were evaluated. The green callus contained the highest amount of anthocyanin, followed by brown and cream callus. The total anthocyanin contents in both the green and brown callus was more than two-fold higher than that in cream callus. Contrasting observation was obtained for total phenolic content (TPC), where the TPC of cream callus was significantly higher than that in brown callus. Nevertheless, the green callus also exhibited the highest TPC. Green callus also contained the highest amount of total chlorophyll and carotenoid, as well as exhibited the highest antioxidant potential, and was found to be the only extract with active cytotox activity against SKOV-3 cells. Correlation analysis revealed that the excellent bioactivity exhibited by the coloured extracts was strongly correlated with the bioactive pigments present in the callus.

The major bioactive compounds identified in the methanolic extracts of A. indica coloured callus are anthocyanins, phenolics, chlorophylls and carotenoids. Future research work should include improvements in the extraction and identification methods, which may lead to detection and determination of other compounds that could attribute to its bioactivity, to complement the findings of the current study.

This analysis provides valuable information on the application of plant tissue culture as an alternative source for sustainable production of major pigments with medicinal benefits in A. indica for possible use as functional natural colourants.

A comparative study on bioactive pigment production in coloured callus from A. indica leaves and its antioxidant potential and cytotoxicity is original. To the best of the authors’ knowledge, this is the first report detailing a comparative evaluation on the production of coloured callus in A. indica and its relative biochemical composition and bioactive properties.

The purpose of the present study is to understand the role of auxin and cytokinin in stimulating the production of pigmented callus in Orthosiphon stamineus and to gain correlation between the callus colours with their antioxidant capacity and bioactive constituents.

In this study, plant tissue culture was used to induce production of callus of various colours from leaf explants of O. stamineus, via manipulation of plant hormones (0-2.0 mg L⁻¹ indole-3-acetic acid [IAA] and Kinetin [Kin]). The coloured callus was subjected to solvent extraction and used for quantification of its carotenoid, chlorophyll, anthocyanin and phenolic contents. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of the extracts was also evaluated, before and after four weeks of storage at −20°C.

The highest mean (per cent) explants that produced roots (93.33 ± 0.05 per cent) were observed when the cultures were supplemented with 2.0 mg L⁻¹ IAA. The colour of the callus changed with time, from green to cream to brown after two and four months of culture, respectively. Optimum production of green callus was achieved with addition of 2.0 mg L⁻¹ Kin plus 1.0-2.0 mg L⁻¹ IAA to the media, while cream callus in 0.5 mg L⁻¹ Kin plus 2.0 mg L⁻¹ IAA and brown callus in 0.5 mg L⁻¹ Kin plus 1.5 mg L⁻¹ IAA. Green callus was found to contain the highest amount of chlorophylls, carotenoid and anthocyanin, while cream callus contained the highest amount of phenolic compounds. The amount of pigments and secondary metabolites in the callus extracts decreased after four weeks of storage, except anthocyanin. The antioxidant potential of the extracts also increased after storage.

The major compounds identified in the methanolic extracts of O. stamineus-coloured callus are chlorophylls, carotenoids, flavonoids and phenolic acids. Future research work should include improvements in the extraction and identification methods which may lead to detection of other compounds that could attribute to the antioxidant capacity, to complement the findings of the current study.

This analysis provides valuable information on the application of IAA and Kinetin (Kin) to manipulate the content of major pigments with medicinal benefits in O. stamineus by using the plant tissue culture system.

A comparative study on antioxidant capacity and bioactive constituents of pigmented callus from O. stamineus leaves is original. To the best of the authors’ knowledge, this is the first attempt of comparative evaluation on antioxidant potential of O. stamineus-coloured callus produced using IAA and Kin.