ABSTRACT
Fungal elicitation could improve the secondary metabolite contents of in vitro cultures. Herein, we report the effect of Fusarium oxysporum on vinblastine and vincristine alkaloid yields in Catharanthus roseus embryos. The study revealed increased yields of vinblastine and vincristine in Catharanthus tissues. Different concentrations, i.e., 0.05% (T1), 0.15% (T2), 0.25% (T3), and 0.35% (T4), of an F. oxysporum extract were applied to a solid MS medium in addition to a control (T0). Embryogenic calli were formed from the hypocotyl explants of germinating seedlings, and the tissues were exposed to Fusarium extract elicitation. The administration of the F. oxysporum extract improved the growth of the callus biomass, which later differentiated into embryos, and the maximum induction of somatic embryos was noted T2 concentration (102.69/callus mass). A biochemical analysis revealed extra accumulations of sugar, protein, and proline in the fungus-elicitated cultivating tissues. The somatic embryos germinated into plantlets on full-strength MS medium supplemented with 2.24 µM of BA. The germination rate of the embryos and the shoot and root lengths of the embryos were high at low doses of the Fusarium treatment. The yields of vinblastine and vincristine were measured in different treated tissues via high-pressure thin-layer chromatography (HPTLC). The yield of vinblastine was high in mature (45-day old) embryos (1.229 µg g-1 dry weight), which were further enriched (1.267 µg g-1 dry weight) via the F. oxysporum-elicitated treatment, especially at the T2 concentration. Compared to vinblastine, the vincristine content was low, with a maximum of 0.307 µg g-1 dry weight following the addition of the F. oxysporum treatment. The highest and increased yields of vinblastine and vincristine, 7.88 and 15.50%, were noted in F. oxysporum-amended tissues. The maturated and germinating somatic embryos had high levels of SOD activity, and upon the addition of the fungal extracts, the enzyme's activity was further elevated, indicating that the tissues experienced cellular stress which yielded increased levels of vinblastine and vincristine following the T2/T1 treatments. The improvement in the yields of these alkaloids could augment cancer healthcare treatments, making them easy, accessible, and inexpensive.
ABSTRACT
Andrographis paniculata (Burm.f.) Wall. (Acanthaceae) is revered for its medicinal properties. In vitro culture of medicinal plants has assisted in improving both the quantity and quality of their yield. The current study investigated the effects of different surface sterilization treatments, plant growth regulators (PGRs), and elicitors on culture establishment and axillary shoot multiplication of A. paniculata. Subsequently, the production of andrographolide in the in vitro plantlets was evaluated using high-performance liquid chromatography (HPLC) analysis. The shoot-tip explant was successfully sterilized using 60% commercial bleach for 5 min of immersion with a 90% survival rate and 96.67% aseptic culture. The optimal PGR for shoot growth was 6-benzylaminopurine (BAP) at 17.76 µM, supplemented into Murashige and Skoog (MS) media, producing 23.57 ± 0.48 leaves, 7.33 ± 0.10 shoots, and a 3.06 ± 0.02 cm length of shoots. Subsequently, MS medium supplemented with 5 mg/L chitosan produced 26.07 ± 0.14 leaves, 8.33 ± 0.07 shoots, and a 3.63 ± 0.02 cm length of shoots. The highest andrographolide content was obtained using the plantlets harvested from 5 mg/L chitosan with 2463.03 ± 0.398 µg/mL compared to the control (without elicitation) with 256.73 ± 0.341 µg/mL (859.39% increase). The results imply that the protocol for the shoot-tip culture of A. paniculata was developed, and that elicitation enhanced the herbage yield and the production of andrographolide.
ABSTRACT
Tylophora indica (Burm. f.) Merrill is an endangered medicinal plant that possesses various active agents, such as tylophorinine, kaempferol, quercetin, α-amyrin and beta-sitosterol, with multiple medicinal benefits. α-amyrin, a triterpenoid, is widely known for its antimicrobial, anti-inflammatory, gastroprotective and hepatoprotective properties. In this study, we investigated the metabolite profiling of tissues and the effects of cadmium chloride and chitosan on in vitro accumulation of alkaloids in T. indica. First, the callus was induced from the leaf in 2,4-D-, NAA- and/or BAP-fortified MS medium. Subsequent shoot formation through organogenesis and in vitro roots was later induced. Gas chromatography-mass spectrometry (GC-MS)-based phytochemical profiling of methanolic extracts of in vivo and in vitro regenerated plants was conducted, revealing the presence of the important phytocompounds α-amyrin, lupeol, beta-sitosterol, septicine, tocopherol and several others. Different in vitro grown tissues, like callus, leaf and root, were elicited with cadmium chloride (0.1-0.4 mg L-1) and chitosan (1-50 mg L-1) to evaluate the effect of elicitation on α-amyrin accumulation, measured with high-performance thin layer chromatography (HPTLC). CdCl2 and chitosan showed improved sugar (17.24 and 15.04 mg g-1 FW, respectively), protein (10.76 and 9.99 mg g-1 FW, respectively) and proline (7.46 and 7.12 mg g-1 FW), especially at T3 (0.3 and 25 mg L-1), in the leaf as compared to those of the control and other tissues. The antioxidant enzyme activities were also evaluated under an elicitated stress situation, wherein catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) displayed the highest activities in the leaf at T4 of both of the two elicitors. The α-amyrin yield was quantified with HPTLC in all tested tissues (leaf, callus and root) and had an Rf = 0.62 at 510 nm wavelength. Among all the concentrations tested, the T3 treatment (0.3 mg L-1 of cadmium chloride and 25 mg L-1 of chitosan) had the best influence on accumulation, irrespective of the tissues, with the maximum being in the leaf (2.72 and 2.64 µg g-1 DW, respectively), followed by the callus and root. Therefore, these results suggest future opportunities of elicitors in scaling up the production of important secondary metabolites to meet the requirements of the pharmaceutical industry.
