Effects of Culture Period and Plant Growth Regulators on In Vitro Biomass Production and Phenolic Compounds in Seven Species of Hypericum

Abstract

This study evaluated biomass accumulation and phenolic compound production in seven Hypericum species (H. androsaemum, H. calycinum, H. hirsutum, H. kalmianum, H. olympicum, H. perforatum, and H. triquetrifolium) cultivated in vitro under varying growth regulator treatments and culture periods. Shoots were grown on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) or meta-topoline (mT) and analyzed after 40 and 60 days. MS medium supplemented with 0.2 mg/L BA was the most effective condition for promoting biomass across all species, with shoot fresh weight increasing significantly at 60 days, particularly in H. olympicum, H. perforatum, and H. triquetrifolium. High-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) identified 13 phenolic compounds, including flavonols, hydroxycinnamic acids, anthocyanins, phloroglucinols, and naphthodianthrones. Phenolic profiles were species-specific and influenced by culture period. H. kalmianum accumulated the highest total phenolic content (37.6 mg/g DW), while H. olympicum was the top producer of hypericin and pseudohypericin. These results highlight the crucial role of culture conditions in regulating both biomass and phytochemical production and provide a promising approach for producing bioactive metabolites in Hypericum species through in vitro systems.

Keywords: HPLC; benzyladenine; bioactive compounds; hypericin; naphthodianthrones; shoots culture.

Figure 1

Morphology of cluster shoots from Hypericum species in vitro cultures in Murashige and Skoog medium supplemented with 0.2 mg/L 6-benzyladenine after a 60-day growth cycle: (a)—H. androsaemum; (b)—H. calycinum; (c)—H. hirsutum; (d)—H. kalmianum; (e)—H. olimpicum; (f)—H. perforatum; (g)—H. triquetrifolium.

Figure 2

Representative HPLC-DAD profiles of methanolic extracts from (a) H. olympicum (60 days, highest hypericin content) and (b) H. kalmianum (60 days, highest total phenolic content). Peak numbers correspond to the identified compounds listed in Table 4.

Figure 3

Individual phenolic compound content of seven Hypericum species biomass extracts cultivated in vitro for 40 and 60 days on MS + 0.2 mg/L BA: (a)—3-Caffeoylquinic acid; (b)—cyanidin-glucoside (c)—cyanidin-rhamnoside; (d)—hyperforin; (e)—protopseudohypericin; (f)—pseudohypericin; (g)—quercetin-galactoside; (h)—quercetin-rhamnoside; (i)—hyperfirin; (j)—adhyperforin; (k)—adhyperfirin; (l)—quercetin; (m)—hypericin; (n)—total phenolic compound. HPLC-DAD-MS-ESI was used for the analysis, and the values are expressed as milligrams per gram of dry weight ± standard error (mg/g DW ± SE). The bars marked with different lowercase letters denote significant differences between samples at p < 0.05.

Figure 3

Individual phenolic compound content of seven Hypericum species biomass extracts cultivated in vitro for 40 and 60 days on MS + 0.2 mg/L BA: (a)—3-Caffeoylquinic acid; (b)—cyanidin-glucoside (c)—cyanidin-rhamnoside; (d)—hyperforin; (e)—protopseudohypericin; (f)—pseudohypericin; (g)—quercetin-galactoside; (h)—quercetin-rhamnoside; (i)—hyperfirin; (j)—adhyperforin; (k)—adhyperfirin; (l)—quercetin; (m)—hypericin; (n)—total phenolic compound. HPLC-DAD-MS-ESI was used for the analysis, and the values are expressed as milligrams per gram of dry weight ± standard error (mg/g DW ± SE). The bars marked with different lowercase letters denote significant differences between samples at p < 0.05.

Figure 4

Heat map showing the content of each phenolic compound in the biomass extracts of seven Hypericum species at two different culture periods (40 and 60 days) on MS + 0.2 mg/L BA. The color intensity in each cell corresponds to the concentration of the respective phenolic compound, with a gradient ranging from black (lowest concentration) to red (intermediate concentration) to purple/blue (highest concentration).