Characterization of kaempferia species and activityguided selection of Kaempferia parviflora Wall. Ex baker for agronomic improvement

Kaempferia is a genus of gingers has promising potential to be discovered for their ornamental and medicinal value. Nuclear ribosomal internal transcribed spacer (ITS) sequences and morphological characteristics were used to characterize six Kaempferia species including Kaempferia parviflora Wall. ex Baker, Kaempferia galanga Linn., Kaempferia elegans Wall. ex Baker, Kaempferia pulchra Ridl., Kaempferia angustifolia Roscoe, Kaempferia marginata Carey ex Roscoe. Sequence data showed nucleotide diversity (0.458), estimated values of transition/transversion bias (0.89) and divergence pattern in the Kaempferia genus was inferred using maximum parsimony analysis. The resulting phylogenetic tree from morphological and molecular data both showed congruence with three distinctive groupings within selected Kaempferia species. ITS 4 and 5 sequences are proposed as DNA barcodes for identification of Kaempferia species. In terms of morphological traits, petiole length, plant habit, rhizome colour and leaf variation can be used for preliminary identification of this genus. Preliminary screening for phytochemical and bioactivity comparing Kaempferia extracts have provided baseline information on activities and potentials of each species. Plant extracts from each of the above-mentioned species were screened with cytotoxicity, anti-inflammatory and anti-histamine bioassays. All extracts were evaluated for total phenolic content, total flavonoid content and antioxidant activity. Most of the extracts were found to have phenolic compounds, flavonoids and anti- oxidant potential. Some extracts have shown positive activity in all three bioassays. Among the selected species, K. parviflora showed positive activity in three bioassays and higher phytochemical activity in comparison to other species. K. parviflora was selected for further studies. K. parviflora cultivars, KPM (K. parviflora from Malaysia) and KPT (K. parviflora from Thailand) both gave the most selective cytotoxic activity against HCT116 cells (IC50 9-13 μg mL⁻¹). KPM and KPT extracts also exhibited high β-hexosaminidase inhibitory effects (>50%) at concentrations 7.5 to 10 µg mL⁻¹ without affecting cell viability in RBL-2H3 cells. Both extracts exhibited high inhibition against NO production at the highest concentration (50 µg mL⁻¹) but affected the viability of RAW 264.7 macrophage cells (<80%). Total phenolic content (TPC) and total flavonoid content (TFC) was higher in KPM 3.14 mg gallic acid equivalent (GAE)/g DW, 1.83 mg rutin equivalent / g DW. The results demonstrate that K. parviflora extracts have therapeutic properties, but its beneficial qualities may differ between cultivars. To improve propagation of K. parviflora, multiple shoot regeneration was found most successful on Murashige and Skoog (MS) medium supplemented with 35.52 µM N⁶-benzyladenine (BA) in terms of highest number of shoots (22.4 ± 1.84), leaves (29.27 ± 1.30), and roots (17.8 ±1.72) per explant. A batch of regenerated in vitro plantlets were then successfully induced to form micro rhizomes in MS medium free of plant growth regulators with an optimal concentration of 6% (w/v) sucrose. Increase in micro rhizome biomass (35.7 ± 2.59 g per flask), number of micro rhizomes (5.2 ± 0.78), shoots (8.5 ± 1.58) and roots (8.5 ± 1.58) were observed under this treatment. Light is one of the main limiting factors for phenolic compound biosynthesis in plants. KPM and KPT were investigated for growth and secondary metabolite content under four shade levels (0%, 30%, 50%, and 70%). Photosynthetic capability, plant biomass and total flavonoid content were highest in both accessions under 30% shade after nine months of growth cycle. 5,7-dimethoxyflavone (DMF) a chemical marker for K. parviflora, was greatest in KPM grown under 30% shade. KPT had higher biomass than KPM under 30% but lower DMF content. K. parviflora is a semi shade loving plant suitable to grow under 30% shading for high yield and secondary metabolite production. This research gives an overall overview and comprehensive comparison of Kaempferia species for further research.

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