Decoupling of Plant Growth and Accumulation of Biologically Active Compounds in Leaves, Roots, and Root Exudates of Hypericum perforatum L. by the Combination of Jasmonate and Far-Red Lighting.

The plant hormone jasmonic acid (JA) fine tunes the growth-defense dilemma by inhibiting plant growth and stimulating the accumulation of secondary compounds. We investigated the interactions between JA and phytochrome B signaling on growth and the accumulation of selected secondary metabolites in Hypericum perforatum L., a medically important plant, by spraying plants with methyl jasmonate (MeJA) and by adding far-red (FR) lighting. MeJA inhibited plant growth, decreased fructose concentration, and enhanced the accumulation of most secondary metabolites. FR enhanced plant growth and starch accumulation and did not decrease the accumulation of most secondary metabolites. MeJA and FR acted mostly independently with no observable interactions on plant growth or secondary metabolite levels. The accumulation of different compounds (e.g., hypericin, flavonols, flavan-3-ols, and phenolic acid) in shoots, roots, and root exudates showed different responses to the two treatments. These findings indicate that the relationship between growth and secondary compound accumulation is specific and depends on the classes of compounds and/or their organ location. The combined application of MeJA and FR enhanced the accumulation of most secondary compounds without compromising plant growth. Thus, the negative correlations between biomass and the content of secondary compounds predicted by the growth-defense dilemma were overcome.

Subject: UV-B radiation and low temperature promoted hypericin biosynthesis in adventitious root culture of Hypericum perforatum.

The hypericin is assumed as a highly demanded and key bioactive compound, which has antiviral, antimicrobial, antioxidant, and antitumor properties isolated from Hypericum perforatum. Nowadays, increasing bioactive molecules' contents through generating novel compounds is one of the major research objectives of H. perforatum biotechnology; however, this plant remains recalcitrant and unmanageable to Agrobacterium mediated transformation and genetic improvement programs. In order to overcome these challenges, many researchers have focused on this unruly herb using biotic and abiotic eliciting strategies. Therefore, two experiments were separately designed for the evaluation of two types of abiotic elicitors, aiming at increasing the productivity of hypericin in the adventitious root suspension culture of H. perforatum. The first one was accomplished to evaluate the effect of UV-B light elicitors (the exposure time of 30, 60, and 90 min) and the recovery treatment (with or without) on hypericin content while the second one was assessed the effect of various temperatures (4°C, 8°C, 16°C, and 25°C) in three different exposure times (24 h, 72 h, and 7 d). Based on the results, UV-B (60 min) treatment followed by the recovery produced 0.430 µg/g DW hypericin and was distinguished as the most effective UV-B elicitation treatment. In addition, a temperature of 4°C for a period of 72 hours is required to get the highest amount of hypericin content. These findings indicate the fact that hypericin biosynthesis is notably affected by UV-B exposure time and Low-temperature. The data also clearly elucidate further mechanisms of hypericin production in H. perforatum adventitious root culture.

Effect of different quality of light on growth and production of secondary metabolites in adventitious root cultivation of Hypericum perforatum.

Naphthodianthrone derivatives that produced in Hypericum perforatum (St. John's Wort) are valuable secondary metabolites for depression treatment and photodynamic therapy. However, the traditional cultivation of this plant does not meet both quantitatively and qualitatively the high demand of the pharmaceutical industry. So, the adventitious root culture along with elicitation has been introduced as an alternative for production of such valuable bioactive compounds. The aim of this study was to evaluate the effect of darkness and red, blue and fluorescent light on growth and production of secondary metabolites in the adventitious root cultivation of H. perforatum. Our results showed that biomass production was significantly higher in the cultures grown under dark and red light, but in terms of hypericins production, red light was the best. Despite the inhibitory effect of five weeks blue light treatment on both biomass and secondary metabolite production of adventitious roots, one-week blue light treatment of four-weeks grown roots is an effective stimulator for increasing total phenolic compounds and hypericins. Interestingly, the roots were regenerated under red light and stems and leaves were formed.

Local climate explains degree of seed dormancy in Hypericum elodes L. (Hypericaceae).

Seed dormancy and germination characteristics may vary within species in response to several factors. Knowledge of such variation is crucial to understand plant evolution and adaptation to environmental changes. We examined the correlation of climate and population genetic differentiation (ISSR) with primary seed dormancy and germination behaviour in populations of the Atlantic-European soft-water pool specialist Hypericum elodes. Primary dormancy was measured by analysing seed germination response of fresh seeds and after various periods of cold stratification. Laboratory germination experiments revealed that the single most important factor for promoting germination was cold stratification prior to placing at the germination temperature. However, in agreement with their weaker primary dormancy, the seeds germinated well when fresh, and the benefit of cold stratification was more relaxed for the southern populations. Seeds of all populations demonstrated a near absolute requirement for a light and alternating temperature regime in order to germinate. The promoting effect of alternating temperatures was particularly effective at warm temperatures (mean 20 °C) but not at cool temperatures. Whilst seed germination requirements were similar among populations, the degree of primary dormancy varied considerably and was not associated with population genetic differentiation. Primary dormancy degree was instead associated with local climate: higher temperature in summer and rainfall in winter predicted weak and rapid loss of dormancy. These results suggest that seed maturation environment may play a substantial role in explaining the degree of dormancy in H. elodes, highlighting that physiological dormancy can be modulated by local climate.

Effects of UV-B radiation on total phenolic, flavonoid and hypericin contents in Hypericum retusum Aucher grown under in vitro conditions.

This study was conducted to increase total phenolics, flavonoids and hypericin accumulation in in vitro cultures of Hypericum retusum Aucher to determine the appropriate time of UV radiations. Proliferation of plantlets on Murashige-Skoog medium containing 0.5 mg L(- 1)N-6-benzylaminopurine was achieved under in vitro conditions. Then, the plantlets were exposed to UV-B radiation for different periods (15, 30, 45 and 60 min). The highest total phenolics, flavonoids and hypericin accumulation (43.17 ± 0.8; 35.09 ± 0.8; 2.7 ± 0.05 mg g(- 1), respectively) was achieved at 45 minutes of exposure to UV-B radiation when compared with the contents of naturally growing plants (23.33 ± 0.9, 18.62 ± 0.3 and 1.6 ± 0.01 mg g(- 1), respectively) and control groups (control group was not subjected to UV-B radiation).

Phenolic profile of dark-grown and photoperiod-exposed Hypericum perforatum L. Hairy root cultures.

Hypericum perforatum L. is a medicinal plant considered as an important natural source of secondary metabolites with a wide range of pharmacological attributes. Hairy roots (HR) were induced from root segments of in vitro grown seedlings from H. perforatum after cocultivation with Agrobacterium rhizogenes A4. Investigations have been made to study the production of phenolic compounds in dark-grown (HR1) and photoperiod-exposed (HR2) cultures. The chromatographic analysis of phenolic acids, flavonols, flavan-3-ols, and xanthones revealed marked differences between HR1 and HR2 cultures. The production of quinic acid, kaempferol, and seven identified xanthones was increased in HR2. Moreover, HR2 showed a capability for de novo biosynthesis of two phenolic acids (3-p-coumaroylquinic acid and 3-feruloylquinic acid), three flavonol glycosides (kaempferol hexoside, hyperoside, and quercetin acetylglycoside), and five xanthones (tetrahydroxy-one-methoxyxanthone, 1,3,5-trihydroxy-6-methoxyxanthone, 1,3,5,6-tetrahydroxy-2-prenylxanthone, paxanthone, and banaxanthone E). On the other side, HR1 cultures were better producers of flavan-3-ols (catechin, epicatechin, and proanthocyanidin dimers) than HR2. This is the first comparative study on phenolic profile of H. perforatum HR cultures grown under dark and photoperiod conditions.

Effects of UV-B on secondary metabolites of St. John's Wort (Hypericum perforatum L.) grown in controlled environments.

The medicinal plant industry is under increasing scrutiny due to wide variance in active ingredient (AI) concentration from values claimed on labels. Reasons for this disparity include environmental and genotypic variation which influence AI concentration. St. John's wort (Hypericum perforatum) is a popular herbal remedy which also exhibits marked variance in AI concentration among products. This study evaluated concentration changes of three biologically active metabolites of H. perforatum after exposure to UV light while plants were still vegetative. Treatments were performed with 55-day-old plants grown under 400 µmol m(-2) s(-1) PAR for 16 h a day. Three UV light treatments were evaluated: a single dose, a daily dose and an increasing daily dose. Concentrations of hyperforin, pseudohypericin and hypericin were monitored for 7 days after each treatment. A daily dose and an increasing daily dose did not produce significantly greater increases in secondary metabolites compared to single dose treatments. These results suggest the small but significant transient metabolite concentration increases in H. perforatum can be induced by UV light exposure. Information from this study can be useful in optimizing total biomass and metabolite production in controlled environments.

Pseudohypericin is necessary for the light-activated inhibition of prostaglandin E2 pathways by a 4 component system mimicking an Hypericum perforatum fraction.

Hypericum perforatum (Hp) has been used medicinally to treat a variety of conditions including mild-to-moderate depression. Recently, several anti-inflammatory activities of Hp have been reported. An ethanol extract of Hp was fractionated with the guidance of an anti-inflammatory bioassay (lipopolysaccharide (LPS)-induced prostaglandin E2 production (PGE2)), and four constituents were identified. When combined together at concentrations detected in the Hp fraction to make a 4 component system, these constituents (0.1microM chlorogenic acid (compound 1), 0.08microM amentoflavone (compound 2), 0.07microM quercetin (compound 3), and 0.03microM pseudohypericin (compound 4)) explained the majority of the activity of the fraction when activated by light, but only partially explained the activity of this Hp fraction in dark conditions. One of the constituents, light-activated pseudohypericin, was necessary, but not sufficient to explain the reduction in LPS-induced PGE2 of the 4 component system. The Hp fraction and the 4 component system inhibited lipoxygenase and cytosolic phospholipase A2, two enzymes in the PGE2-mediated inflammatory response. The 4 component system inhibited the production of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), and the Hp fraction inhibited the anti-inflammatory cytokine interleukin-10 (IL-10). Thus, the Hp fraction and selected constituents from this fraction showed evidence of blocking pro-inflammatory mediators but not enhancing inflammation-suppressing mediators.

Influence of the habitat altitude on the (proto)hypericin and (proto)pseudohypericin levels of hypericum plants from Crete.

Environmental factors are known to influence strongly the accumulation of secondary metabolites in plant tissues. In a previous paper, we studied the contents of (pseudo)hypericin and its immediate precursors in wild populations of various HYPERICUM species on the island of Crete, Greece, in dependence on their developmental stage. In this study, we investigated the effect of the habitat altitude on the total hypericins content of the plants, which is defined as the sum of protohypericin, hypericin, protopseudohypericin and pseudohypericin. Taking into account our previous finding that the highest accumulation is found during the flowering period in June, we collected the aerial parts of spontaneously growing H. PERFORATUM L. , H. TRIQUENTRIFOLIUM Turra , H. EMPETRIFOLIUM Willd. and H. PERFOLIATUM L. during that time frame at elevations between 100 and 600 m above sea level, however, bearing in mind the time lag in development with increasing altitude. HPLC analysis of the plant material, separated again into a flowers and a leaves/petioles fraction, revealed great differences in the total hypericin content in dependence on the altitude of the habitat. Specifically, a clear trend was revealed, showing an increase of the total hypericin content with increasing altitude. However, no changes could be observed in the ratio of hypericin to protohypericin and in that of pseudohypericin to protopseudohypericin. The habitats of the employed plants were again randomly distributed all over Crete. It is proposed that higher light intensities accompanied by enhanced UV-B radiation and lower air temperature might be responsible for the increasing levels of total hypericins with increasing altitude

Effect of light on hypericins contents in fresh flowering top parts and in an extract of St. John's Wort (Hypericum perforatum).

St. John's Wort is a medicinal plant increasingly used for its antidepressive activity. Hypericins are considered as one of the compounds contributing to the activity of the extract. These naphthodianthrones exist in various forms in Hyperici herba. Protopseudohypericin and protohypericin (protopigments) are converted into pseudohypericin and hypericin (pigments) under the action of light. The aim of this work is to study the influence of light on the phototransformation of protopigments into pigments. Two experiments were carried out. The studies were performed on one hand, on plant material in order to know the proportion of these substances in various plant parts and the possibility of transforming the protopigments into pigments under the action of sunlight; on the other hand, in the extract to determine the optimal wavelength allowing this transformation. Three parts of the fresh plant (buds, flowers, leaves) were treated with sunlight on three levels of exposure. Liquid extracts were exposed to various types of light with wavelengths ranging between 480 and 660 nm by means of diodes. The flowering tops of St. John's Wort contain a share of approximately 30% hypericins in the form of protopseudohypericin and protohypericin: buds (48%), flowers (30%), leaves (17%). After an exposure of fresh buds to sunlight for 16 hours the share of protopigments was then 32%. In the extract, the transformation of the protopigments is total and requires less energy than in the plant material. The optimal wavelength for the transformation of the protopigments in the extract is around 515 nm (green), close to the optimum absorption level of protopigments.