Proteomic approach to identify the differentially abundant proteins during flavour development in tuberous roots of Decalepis hamiltonii Wight & Arn.

2-Hydroxy-4-Methoxy Benzaldehyde (2H4MB) is a structural isomer of vanillin produced in the tuberous roots of D. hamiltonii. Both vanillin and 2H4MB share the common phenylpropanoid pathway for their synthesis. Unlike vanillin, in which the biosynthetic pathway was well elucidated in V. planifolia, the 2H4MB biosynthetic pathway is not known in any of its plant sources. To find the key enzymes/proteins that promote 2H4MB biosynthesis, a comparative proteomic approach was adapted. In this case, two developmental stages of tuberous roots of D. hamiltonii were selected, where the flavour content was highly variable. The flavour content in the two stages was estimated using quantitative HPLC. The flavour content in the first and second stages of tuber development was 160 and 510 µgg-1, respectively. Two-dimensional electrophoresis (2-DE) was performed for these two stages of tubers; this was followed by PDquest analysis. A total of 180 protein spots were differentially abundant of which 57 spots were selected and subjected to MALDI-TOF-TOF analysis. The largest percentage of identified proteins was involved in stress and defence (27.9%), followed by proteins related to bioenergy and metabolism (23.2%), Cellular homeostasis proteins (18.6%), signaling proteins (11.6%), Plant growth and development proteins (9.3%). Holistically, we found the upregulation of methyltransferase, cell division responsive proteins, plant growth and development proteins which directly relate to flavour development and maturation. Similarly, stress-responsive and signaling proteins, vacuole proteins and ATPases were down-regulated with an increase in flavour content. In this study, we could not identify the specific 2H4MB metabolic pathway proteins, however, we could be able to study the changes in physiological and primary metabolic proteins with 2H4MB accumulation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02714-x.

Neuroprotective effect of Decalepis hamiltonii aqueous root extract and purified 2-hydroxy-4-methoxy benzaldehyde on 6-OHDA induced neurotoxicity in Caenorhabditis elegans.

In this study, we investigated the possible neuroprotective efficacy of Decalepis hamiltonii tuber extract against 6-Hydroxy dopamine (6-OHDA) induced neurotoxicity and associated effects in Caenorhabditis elegans. The major component of flavour rich extract from D. hamiltonii is 2-hydroxy-4-methoxy benzaldehyde (2H4MB) which is an isomer of vanillin. We have conducted preliminary experiments with different types of extracts and subsequently DHFE (D. hamiltonii Fresh Tuber Extract) and DHPF (D. hamiltonii purified 2H4MB fraction) were used for further studies. Here we attempted to enumerate the neuroprotective efficacy of the above compounds in worms by evaluating behavioural and mitochondrial function, dopamine content and selective degeneration of dopaminergic neurons in BZ555 strains in comparison with control and 6-OHDA treated organisms. The relative expression levels of selected antioxidant genes involved in defence mechanism like SOD-3, GST-2 and GST-4 were evaluated along with those of CAT-2 and DOP-2 at mRNA level. We observed that both DHPF and DHFE exhibited significant levels of neuroprotective property against 6-OHDA induced neurotoxicity, which was evident in mitochondrial/dopaminergic function and antioxidant defence mechanism.

Biochemical characterization of a key step involved in 2H4MB production in Decalepis hamiltonii.

Decalepis hamiltonii is widely known for its flavour molecule 2-Hydroxy-4-Methoxy Benzaldehyde (2H4MB), a structural isomer of vanillin. As the biosynthetic pathway of 2H4MB is not known, we hypothesised 2H4MB origins could be from phenylpropanoid pathway (PPP). Accordingly, a study was conducted using PPP inhibitors (viz. piperonylic acid, MDCA and propanil) against in vitro root cultures of D. hamiltonii to find the branch of PPP which catalyses the 2H4MB formation. HPLC analysis was carried out to quantify 2H4MB levels in control and respective inhibitor treated root cultures in vitro. The results obtained revealed that piperonylic acid did not inhibit 2H4MB biosynthesis in the given period, whereas MDCA and propanil had the marked inhibitory effect. The inhibitory effect was evident with 13.2, 33.6 and 37.9% decrease in 2H4MB levels at 50, 100 and 150mM concentration of MDCA respectively in comparison with control roots. Similarly, the inhibitory effect of propanil on 2H4MB biosynthesis was obvious with 23.7, 49.5 and 57.9% decrease in 2H4MB levels at 50, 100 and 150µM concentration of inhibitor respectively when compared with control roots. Propanil showed a greater slow down effect on 2H4MB biosynthesis compared to MDCA. Incorporation of 0.1, 0.5 and 1.0mM ferulic acid as a precursor to in vitro root cultures of D. hamiltonii showed an increase in 2H4MB levels at the rate of 3.1, 107 and 94.1% respectively as quantified by HPLC analysis. However, ferulic acid in conjunction with propanil did not show any increase in 2H4MB levels. This clearly explains that ferulic acid is channelled through the 4-CL (4-coumarate CoA ligase) enzyme, where it would be converted to feruloyl-CoA and could be further converted to 2H4MB in D. hamiltonii.