In Vitro Propagation, Phytochemical Analysis, and Evaluation of Free Radical Scavenging Property of Scrophularia kakudensis Franch Tissue Extracts

Abstract

The current study deals with in vitro propagation, antioxidant property estimation, and assessment of acacetin content in Scrophularia kakudensis Franch. Adventitious shoot induction was achieved from the nodal explant with the highest number of adventitious shoots per explant (17.4) on Murashige and Skoog's (MS) medium fortified with 2.0 mg·L(-1) 6-benzyladenine (BA) and 0.5 mg L(-1) indole-3-acetic acid (IAA). Maximum number of roots per plant (16.5) was noted in half strength MS medium supplemented with 0.5 mg·L(-1) IAA. The regenerated plants displayed successful survival ratio (95%) in the greenhouse. The highest content of acacetin, a pharmaceutically important flavonoid, was observed in the shoot extracts (in vitro: 32.83 µg·g(-1) FW; in vivo: 30.05 µg·g(-1) FW) followed by root extracts. Total phenol and flavonoid contents along with free radical scavenging assays revealed the occurrence of larger amount of antioxidants in shoot extract in comparison with callus and root extracts of S. kakudensis. Thus, the outcome of the present study can be highly beneficial for the germplasm conservation and commercial cultivation of S. kakudensis for therapeutic purposes.

Figure 1

Adventitious shoot organogenesis in S. kakudensis Franch. (a) Clump of green colored protuberances appeared from the nodal explant. (b) Early stage of adventitious shoot induction with small leaves indicated by an arrow. (c) Adventitious shoots induced after three weeks. (d) Shoot elongation. (e) In vitro root induction from the shoot base. (f) Acclimatized plants in a greenhouse.

Figure 2

Phytochemical contents of in vitro and in vivo extracts. (a-b) Total phenol and total flavonoid contents present in vitro shoot extract (ISE), in vitro root extract (IRE), callus extracts (CEs), in vivo shoot extract (SE), and in vivo root extract (RE) of S. kakudensis. Different letters in one measurement indicate statistically significant difference at P ≤ 0.05 by Duncan multiple range test.

Figure 3

HPLC results of acacetin content estimation in in vitro and in vivo plant extracts. The representative peaks obtained for acacetin in (a) in vitro shoot extract, (b) in vivo shoot extract, (c) callus extracts, (d) in vitro root extract, and (e) in vivo root extract of S. kakudensis and (f) reference. (g) The quantification of acacetin in tissue extracts of S. kakudensis. Different letters in one measurement indicate statistically significant difference at P ≤ 0.05 by Duncan multiple range test.

Figure 4

Free radical scavenging potentials of in vitro and in vivo extracts. (a–d) Nitric oxide scavenging, superoxide scavenging, hydrogen peroxide scavenging, and DPPH radical scavenging activities of in vitro shoot extract (ISE), in vitro root extract (IRE), callus extracts (CEs), in vivo shoot extract (SE), and in vivo root extract (RE) of S. kakudensis and ascorbic acid (AA). Different letters in one measurement indicate statistically significant difference at P ≤ 0.05 by Duncan multiple range test.

Figure 5

DNA protection potential of in vitro tissues and commercial extracts. Lane 1: positive control (2 μg ascorbic acid + 50 ng DNA). Lane 2: 20 mM H₂O₂ + 50 ng DNA + 2 μg in vivo shoot extract (SE). Lane 3: 20 mM H₂O₂ + 50 ng DNA + 2 μg in vivo root extract (RE). Lane 4: 20 mM H₂O₂ + 50 ng DNA + 2 μg in vitro shoot extract (ISE). Lane 5: 20 mM H₂O₂ + 50 ng DNA + 2 μg in vitro root extract (IRE). Lane 6: 20 mM H₂O₂ + 50 ng DNA + 2 μg callus extracts (CEs). Lane 7: negative control (20 mM H₂O₂ + 50 ng DNA) of S. kakudensis. Oc: open circular form, Li: linear form, and Sc: supercoiled form of plasmid DNA.