Establishment of hydroponic root culture systems of Rhinacanthus nasutus and increased rhinacanthin production using elicitation technique

Abstract

The purpose of the study was to determine how Rhinacanthus nasutus natural growth parts and harvesting times affected the production of rhinacanthin-C, -D, and -N in plant raw materials. In addition, a study of the quality of commercially available plant raw materials and tea infusion health products from R. nasutus in terms of rhinacanthin content was carried out prior to establish hydroponic root culture systems of R. nasutus that produce high root biomass and a high content of rhinacanthin-C, -D, and -N. Elicitation techniques were used to improve rhinacanthin production in the hydroponic system of R. nasutus. The rhinacanthin content in different parts of the plant varied significantly. Roots consistently had the highest total rhinacanthin levels, with September harvests yielding the highest concentrations (4.91% w/w). In contrast, leaves contained the second-highest levels of rhinacanthins (4.42% w/w) during the same season. In March, both roots and leaves exhibited lower rhinacanthin levels (3.73% and 3.18% w/w, respectively). On the contrary, R. nasutus powders sourced from ten distinct suppliers and two tea products demonstrated relatively low total rhinacanthin concentrations, spanning from 0.14% to 0.55% w/w. These concentrations were similar to those observed in powders derived from aerial components, falling within the range of 0.27% to 0.53% w/w. According to the optimization of R. nasutus hydroponics, aeration (4.5 L/min) through 10% MS in light-protected growing containers significantly increased dried root biomass (143.31 mg/plant) and total rhinacanthin content (2.21% w/w) compared to those without aeration and in translucent growing containers. An appropriate MS concentration of 20% was found to considerably enhance root biomass (247.03 mg/plant) and rhinacanthin content (2.65% w/w). Based on the harvesting time, the plant grown for three months produced sufficient root biomass (142.91 mg/plant) and rhinacanthin content (2.70% w/w). Notably, increasing the harvesting duration resulted in an increase in root biomass and rhinacanthin content, which reached 232.07 mg/plant and 3.04% w/w at five months, respectively. Additionally, blue-red lighting increased root biomass and rhinacanthin content up to 250.47 mg/plant and 3.31% w/w, respectively. The addition of 0.15 mg/mL chitosan boosted rhinacanthin production to 6.10% dry weight (DW). The productivity obtained was 2.24 times more than that produced with untreated root (2.72% w/w DW). Furthermore, a 1 mg/mL T. harzianum treatment dosage increased rhinacanthin synthesis up to 2.22-fold (6.04% w/w DW) compared to untreated root. While lawsone (6 M) treatment modestly increased rhinacanthin production to (4.19% w/w DW) compared to untreated root. Chitosan, Trichoderma harzianum, and lawsone were the most effective elicitors for increasing rhinacanthin production in a hydroponic system of R. nasutus. In sequential dual elicitation, the root and leaf dry weights were unaffected by any of the treatments. Chitosan at 0.15 mg/mL for 48 h increased rhinacanthin production by 2.24-fold to 6.10% w/w dry weight (DW). The treated leaves generated a substantial quantity of rhinacanthin (5.44% w/w DW) after 24 h of chitosan treatment. Furthermore, a 24 h treatment with 1 mg/mL T. harzianum boosted rhinacanthin production up to 2.22-fold (6.04% w/w DW) in roots and 1.5-fold (6.66% w/w DW) in leaves. While 72 h of lawsone (6 M) treatment boosted rhinacanthin production in roots by (4.55% w/w DW) and leaves by 1.13-fold (5.17% w/w DW). During simultaneous dual elicitation, rhinacanthin production was maximum in roots at 72 h, reaching up to 4.51% w/w DW and 5.76% w/w DW in leaves. When chitosan was applied to T. harzianum-treated roots for 72 hours, the highest rhinacanthin production (9.65%w/w DW) was observed. The current study indicated the best approach for obtaining R. nasutus herbal raw material during the rainy season by employing roots or leaves with the maximum rhinacanthin concentration. R. nasutus hydroponics were developed and improved to overcome raw material scarcity and poor quality. Furthermore, the elicitation strategy utilizing T. harzianum and chitosan yielded a significant rhinacanthin concentration.