Chemical and pharmacological investigation of micropropagated Hygrophila pogonocalyx produced from leaf explants

Abstract

Background: An optimized method for indirect shoot organogenesis from the leaf explants of Hygrophila pogonocalyx, a rare and endemic species in Taiwan, was developed to supply enough quantity of plant materials for the first chemical and pharmacological investigation.

Results: Incubation of the young leaves on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (0.5 mg/l) and indole-3-acetic acid (0.1 mg/l) resulted in the best multiplication rate for organogenesis. The average number of adventitious buds per leaf was 22.8 ± 1.9 after 8-week culture. The adventitious buds rooted and developed into plantlets when cultured simply on MS medium. Using this protocol, up to 37,600 plants were produced from a single leaf explant in one year. From the ethanol extract of the leaves of this micropropagated plant, 13 compounds were isolated and identified, including two flavones (1, 11), four flavonols (9, 10, 12, and 13), three phenylethanoid glycosides (6-8), two alkylated glycosides (2-3), and two steroids (4-5). Of these, acteoside (7) exhibited anti-tyrosinase activity in human epidermal melanocytes and luteolin 7-O-β-D-glucopyranoside (11) exhibited the greatest neurocytoprotective activity.

Conclusions: The method, indirect shoot organogenesis from leaf explants of H. pogonocalyx, could be developed to supply enough quantity of plant materials for the chemical and pharmacological investigation. In the present study, the isolated active compounds may develop for whitening agents or treating neurodegenerative diseases in the future.

Keywords: Anti-melanogenesis; Constituents; Hygrophila pogonocalyx; Indirect shoot organogenesis from leaf explants; Neurocytoprotection.

Figure 1

Callus and adventitious shoot regeneration of leaf explants of Hygrophila pogonocalyx cultured with different concentrations (mg/l) of plant growth regulators. L1: BA (0.1) + NAA (0.1); L2: BA (0.5) + NAA (0.1); L3: BA (1) + NAA (0.1); L4: BA (0.1) + IAA (0.1); L5: BA (0.5) + IAA (0.1); L6: BA (1) + IAA (0.1); L7: BA (0.1) + IAA (0.1) + 2iP (1) ; L8: BA (0.5) + IAA (0.1) + 2iP(1); L9: BA (1) + IAA (0.1) + 2iP (1) (bar = 1 cm).

Figure 2

The adventitious buds rooted and regenerated into plantlets when cultured on MS medium without plant regulators. Bar = 7.5 cm.

Figure 3

Structures of the isolated compounds from leaf explants of H. pogonocalyx .

Figure 4

Cytotoxicity and cellular anti-tyrosinase activity of the isolated constituents of H. pogonocalyx in human epidermal melanocytes (HEMn cells). HEMn cells (1 × 10⁵) were treated with the positive control arbutin (Ar) and the isolated compounds (100 μM) for 24 h. Afterward, (A) the supernatant was removed and incubated with WST-8 cell counting reagent for 4 h at 37°C. The absorbance was measured using a microplate reader at 450 nm. (B) The lysates (equal amount of proteins) were incubated with l-dopa at a final concentration 2.5 mM for 1 h at 37°C. Each determination was made in triplicate, and the data shown represent means ± SD. *P-value < 0.05 when compared with control.

Figure 5

Cytotoxicity and neurocytoprotective activity of the isolated constituents of H. pogonocalyx in 6-OHDA-induced NGF-differentiated PC12 cells. (A) NGF-differentiated PC12 cells (2 × 10⁵) were treated with 6-OHDA (175 μM) and tested samples (100 μM) for 24 h. (B) The NGF-differentiated PC12 cells (2 × 10⁵) were treated with the tested constituents (100 μM) for 6 h and then were treated with 6-OHDA (175 μM) for 24 h. The cell-protective activity was calculated as follows: (OD₄₅₀ of the sample/OD₄₅₀ of 6-OHDA) × 100. Each determination was made in triplicate, and the data shown represent means ± SD. *P-value < 0.05 when compared with control.