Elicitation Induced α-Amyrin Synthesis in Tylophora indica In Vitro Cultures and Comparative Phytochemical Analyses of In Vivo and Micropropagated Plants

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.

Keywords: GC–MS analysis; HPTLC; Tylophora indica; biochemical analyses; elicitors; in vitro culture; α-amyrin.

Figure 1

Callus-mediated indirect organogenesis from leaf-derived callus. (a) Callus induction from leaf explant, (b) callus proliferation (after 4 weeks of culture), (c) multiple shoot buds; regeneration medium contained 2.0 mg L⁻¹ BAP + 0.5 mg L⁻¹ NAA, (d) rooting of shootlets in 1.0 mg L⁻¹ IBA (Bar (a): 1.5 cm, (b): 1.0 cm, (c): 2.0 cm, (d): 1.5 cm).

Figure 2

GC–MS chromatogram of methanolic leaf extract of in vivo plants of T. indica.

Figure 3

GC–MS chromatogram of methanolic leaf extract of in vitro-developed plants of T. indica.

Figure 4

Sugar, protein and proline contents in mg g⁻¹ FW in the root, callus and leaf parts of T. indica treated with different CdCl₂ treatments (a–c) (T0: Control; T1: 0.1; T2: 0.2; T3: 0.3; T4: 0.4 mg L⁻¹) and different chitosan treatments (d–f) (T0: Control; T1: 1; T2: 5; T3: 25; T4: 50 mg L⁻¹). Values are expressed as means ± standard errors of three replicates. Means followed by same letters are significantly different at p ≤ 0.05 according to Duncan’s multiple range test (DMRT). T0: Control; T1: 0.1; T2: 0.2; T3: 0.3; T4: 0.4 mg L⁻¹.

Figure 5

SOD, APX and CAT activities in EU mg⁻¹ protein min⁻¹ in the root, callus and leaf parts of T. indica treated with different CdCl₂ treatments (a–c) (T0: Control; T1: 0.1; T2: 0.2; T3: 0.3; T4: 0.4 mg L⁻¹) and different chitosan treatments (d–f) (T0: Control; T1: 1; T2: 5; T3: 25; T4: 50 mg L⁻¹). Values are expressed as means ± standard errors of three replicates. Means followed by same letters are significantly different at p ≤ 0.05 according to Duncan’s multiple range test (DMRT).

Figure 6

The steps followed for the preparation of the solvent extract in the Tylophora plant. (a) Small pieces of dried leaf, callus and roots; (b) coarse powder of dried tissues; (c) sample extract in methanol (at 25 °C for 48 h); (d) concentrate of methanolic sample extract.

Figure 7

(a) The six-point calibration curve of α-amyrin with linear regression correlation coefficient r = 0.997 and regression equation y = 0.078 × x + 11.693, where y is the spot area and x is the concentration in ng/spot. (b) HPTLC separation of standard (α-amyrin) and leaf, callus and root samples of regenerated T. indica.

Figure 8

(a) HPTLC densitograms displaying single, sharp and flat peaks of the α-amyrin standard at Rf = 0.62, measured at wavelength = 580 nm; (b–d) show HPTLC densitograms of α-amyrin in leaf, callus and root extracts of field-grown plants displaying similar peaks at Rf = 0.62.

Figure 9

HPTLC densitograms of α-amyrin content of leaf (top), callus (middle) and root (bottom) tissues on control and on elicitation of T3 treatment of CdCl₂ in T. indica.

Figure 10

HPTLC densitograms of α-amyrin content of leaf (top), callus (middle) and root (bottom) tissues on control and on elicitation of T3 treatment of chitosan in T. indica.