Isodon rugosus (Wall. ex Benth.) Codd In Vitro Cultures: Establishment, Phytochemical Characterization and In Vitro Antioxidant and Anti-Aging Activities

Abstract

Isodon rugosus (Wall. ex Benth.) Codd accumulates large amounts of phenolics and pentacyclic triterpenes. The present study deals with the in vitro callus induction from stem and leaf explants of I. rugosus under various plant growth regulators (PGRs) for the production of antioxidant and anti-ageing compounds. Among all the tested PGRs, thidiazuron (TDZ) used alone or in conjunction with α-napthalene acetic acid (NAA) induced highest callogenesis in stem-derived explants, as compared to leaf-derived explants. Stem-derived callus culture displayed maximum total phenolic content and antioxidant activity under optimum hormonal combination (3.0 mg/L TDZ + 1.0 mg/L NAA). HPLC analysis revealed the presence of plectranthoic acid (373.92 µg/g DW), oleanolic acid (287.58 µg/g DW), betulinic acid (90.51 µg/g DW), caffeic acid (91.71 µg/g DW), and rosmarinic acid (1732.61 µg/g DW). Complete antioxidant and anti-aging potential of extracts with very contrasting phytochemical profiles were investigated. Correlation analyses revealed rosmarinic acid as the main contributor for antioxidant activity and anti-aging hyaluronidase, advance glycation end-products inhibitions and SIRT1 activation, whereas, pentacyclic triterpenoids were correlated with elastase, collagenase, and tyrosinase inhibitions. Altogether, these results clearly evidenced the great valorization potential of I. rugosus calli for the production of antioxidant and anti-aging bioactive extracts for cosmetic applications.

Keywords: Isodon rugosus; anti-aging; antioxidant; pentacyclic triterpenoid; phenolic acid; plant growth regulators.

Figure 1

(A) Callus induction frequency (%) for stem and leaf explants under different PGRs concentrations. Values are means ± SE from three replicates; (B) Effect of 5 weeks old culture media containing various PGRs on callus induction and growth of stem explants; (B1) TDZ 1.0 mg/L; (B2) NAA 4.0 mg/L; (B3) BAP 2.0 mg/L; (B4) 1:1 TDZ 1.0 + NAA 1.0 mg/L; (B5) TDZ 1.0 + BAP 4.0 mg/L; (C) Effect of 5 weeks old culture media containing various PGRs on callus induction and growth of leaf explants; (C1) TDZ 3.0 mg/L; (C2) NAA 3.0 mg/L; (C3) BAP 4.0 mg/L; (C4) TDZ 1.0 + NAA 1.0 mg/L; and (C5) TDZ 1.0 + BAP 5.0 mg/L.

Figure 2

Time-course fresh and dry weight of callus cultures at different PGRs (in mg/L). (A) Fresh weight for stem-derived callus culture (in g/L); (B) dry weight for stem-derived callus culture (in g/L); (C) fresh weight for leaf-derived callus culture (in g/L); and (D) dry weight for leaf-derived callus (in g/L) cultured on MS medium fortified with TDZ, NAA, BAP (1.0–5.0 mg/L), TDZ (1.0 mg/L) + NAA (1.0–5.0 mg/L), TDZ (1.0 mg/L) + BAP (1.0–5.0 mg/L). Values are means of three replicates with standard deviation.

Figure 3

Comparison of total phenolic content (TPC) and total phenolic productivity (TPP) of extracts at different PGRs. (A) TPC (in mg/g DW) for stem-derived callus culture; (B) TPC (in mg/g DW) for leaf-derived callus culture; (C) TPP (in mg/L) for stem-derived callus culture; and (D) TPP (in mg/L) for leaf-derived callus cultured on MS medium fortified with PRG (TDZ, NAA, BAP (1.0–5.0 mg/L), TDZ (1.0 mg/L) + NAA (1.0–5.0 mg/L), TDZ (1.0 mg/L) + BAP (1.0–5.0 mg/L)). Values are means of three replicates with standard deviation.

Figure 4

(A) Chemical structures of main phytochemicals accumulated in callus cultures of I. rugosus caffeic acid (CA, 1) rosmarinic acid (RA, 2) betulinic acid (BA, 3) oleanolic acid and (OA, 4) plectranthoic acid (PA, 5); (B) Magnification of typical HPLC chromatograms showing the correct separation of the main phytochemicals accumulated in callus cultures of I. rugosus.

Figure 5

Hierarchical clustering analysis (HCA) of twelve I. rugosus callus extracts related to their phytochemical profile.

Figure 6

Principal component analysis (PCA) of the different phytochemicals and biological activities of I. rugosus callus extracts. Variance of factor 1 (F1) = 70.13% and of factor 2 (F2) = 18.14%.