Hypericin Production Research Articles

Establishing a simple selection method of high hypericin-producing adventitious root lines from leaf explants of Hypericum perforatum var. Topaz

ABSTRACT Hundreds of adventitious root lines from Hypericum perforatum L. were examined for their hypericin content in order to establish a system for studying the production of hypericin in adventitious root lines. The effects of the medium consistency (solid and liquid) and the effects of three different auxins (Indole-3-butyric acid (IBA), Indole-3-acetic acid (IAA), and 1-Naphthaleneacetic acid (NAA)) on the induction of adventitious roots in leaf explants were studied. In a half-strength MS liquid medium with 2 mg/L IBA concentration, adventitious roots developed from H. perforatum leaves, whereas NAA and IAA caused callus-like and low branch roots. HPLC-UV was used to profile hypericin content variations in 100 H. perforatum adventitious root lines. Hierarchical component analysis (HCA) was used for selecting elite lines. After cluster analysis and selecting high-performance lines in terms of hypericin, five elite lines were chosen and the amount of hypericin production, as well as the phenol and flavonoid content, was analysed.

Non-Enzymatic and Enzymatic Antioxidant Responses of Hypericum perforatum L. Hairy Roots upon Photooxidative Stress

The aim of this study was to evaluate the non-enzymatic and enzymatic antioxidant response of fifteen Hypericum perforatum L. dark-grown (HR1 A-HR1 O) and photoperiod-exposed (HR2 A-HR2 O) hairy root clones. Dark-grown HR1 clones were characterized with high biomass accumulation and up-regulated phenylpropanoid metabolism through phenylalanine ammonia lyase (PAL)-mediated production of flavonoids, flavan-3-ols and anthocyanins. These groups of phenolics showed superior hydrogen-donating capability and significantly contributed to the antioxidant capacity of dark-grown HR1 clones. Photoperiod-exposed HR2 clones showed green coloration with shoot regenerative potential and reduced biomass accumulation. Photoperiod exposition improved the production of hypericins, as well as ferrous chelating properties and lipid peroxidation inhibition activity in HR2 cultures. Furthermore, HR2 clones were represented with enhanced activity of antioxidant enzymes (guaiacol peroxidase, catalase and superoxide dismutase) that resulted in suppression of oxidative stress markers (hydrogen peroxide, superoxide anion and malondialdehyde). These observations revealed the involvement of an efficient antioxidant defense system in the adaptive response of HR to photooxidative stress. Altogether, photoperiod-exposed H. perforatum HR2 clones were considered as a promising alternative for further scale-up production of naphthodianthrones that could be used in the pharmaceutical industry.

Evolutionary Aspects of Hypericin Productivity and Endogenous Phytohormone Pools Evidenced in Hypericum Species In Vitro Culture Model.

Shoot cultures of hypericin non-producing H. calycinum L. (primitive Ascyreia section), hypericin-producing H. perforatum L., H. tetrapterum Fries (section Hypericum) and H. richeri Vill. (the evolutionarily most advanced section Drosocarpium in our study) were developed and investigated for their growth, development, hypericin content and endogenous phytohormone levels. Hypericins in wild-growing H. richeri significantly exceeded those in H. perforatum and H. tetrapterum. H. richeri also had the highest hypericin productivity in vitro in medium supplemented with 0.2 mg/L N6-benzyladenine and 0.1 mg/L indole-3-butyric acid and H. tetrapterum—the lowest one in all media modifications. In shoot culture conditions, the evolutionarily oldest H. calycinum had the highest content of salicylic acid and total jasmonates in some of its treatments, as well as dominance of the storage form of abscisic acid (ABA-glucose ester) and lowest cytokinin ribosides and cytokinin O-glucosides as compared with the other three species. In addition, the evolutionarily youngest H. richeri was characterized by the highest total amount of cytokinin ribosides. Thus, both evolutionary development and the hypericin production capacity seemed to interact closely with the physiological parameters of the plant organism, such as endogenous phytohormones, leading to the possible hypothesis that hypericin productivity may have arisen in the evolution of Hypericum as a means to adapt to environmental changes.

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

ABSTRACT 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.

Agrobacterium rhizogenes-mediated transformation of Hypericum sinaicum L. for the development of hairy roots containing hypericin

Hypericum sinaicum L. is an endangered Egyptian medicinal plant of high importance due to the presence of naphthodianthrones (hypericins), which have photodynamic properties and pharmaceutical potential. We sought to assess H. sinaicum ability to develop hairy roots that could be cultured in contained conditions in vitro and used as a source for hypericin production. We used four A. rhizogenes strains differing in their plasmids and chromosomal backgrounds to inoculate excised H. sinaicum root, stem and leaf explants to induce hairy root development. Additionally, inoculum was applied to shoots held in Rockwool cubes supporting their stand after removal of the root system. All explant types were susceptible to A. rhizogenes although stem explants responded more frequently (over 90%) than other explant types. The A4 and A4T A. rhizogenes strains were highly, and equally effective in hairy root induction on 66-72% of explants while the LBA1334 strain was the most effective in transformation of shoots. Sonication applied to explants during inoculation enhanced the frequency of hairy root development, the most effective was 60 s treatment doubling the percentage of explants with hairy roots. However, shoot transformation was the most effective approach as shoots developed hairy roots within 10 days after inoculation. Molecular analyses confirmed that the established hairy root cultures in vitro were indeed obtained due to a horizontal gene transfer from bacteria. These cultures grew fast and the hypericin content in hairy roots was about two fold higher than in H. sinaicum plants as determined by HPLC.

Rapid Method for In vitro Multiplication of Hypericin Rich Shoots of Hypericum perforatum

Hypericum perforatum is a high value medicinal herb possessing a broad range of pharmacological activities. These medicinal properties are attributed to the presence of major compound hypericin. In current study, the effect of different growth hormones and media combinations along with various growth parameters including light and temperature were analyzed for hypericin production in in vitro grown shoot cultures of H. perforatum. Rapid method for micropropagation with an average of 36.3 shoots per explant in 5-6 days was developed. Highest hypericin content of 0.119 µg/mg was detected in shoots grown on MS medium containing sucrose (3%), IBA (3mg/l), KN (1mg/l) and agar (0.9%) at 25±2 °C under light conditions after two months of incubation. In addition, sustention of in vitro grown shoots after hardening under greenhouse conditions was also observed showing comparable hypericin content in hardened and field grown plants after 2 years. Therefore, current study reports the rapid method for in vitro multiplication for hypericin rich shoots of H. perforatum under optimized light and temperature conditions. To the best of our knowledge, this is the first report on comparative analysis of hypericin production in hardened and field grown shoots of H. perforatum. Hence, the outcome is an endeavor to meet the increasing industrial demands by providing quality rich raw material using tissue culture techniques.

Effect of BAP on total hypericin production in shoot cultures of Hypericum scabroides: An endemic species in the Eastern Anatolia Region of Turkey

Background: Due to the therapeutic importance of hypericin, a number of Hypericum species are being investigated. The aim of this study was to determine the effect of various BAP (6- benzylaminopurine) concentrations on seed germination, shoot proliferation, and total hypericin in a tissue culture of Hypericum scabroides which is endemic to the Eastern Anatolia region, Turkey. Material and Methods: Hypericum scabroides specimens were collected from Eastern Anatolia (Elazig, Turkey). The effects of various BAP concentrations (0.0 (control), 0.5, 1.0, and 2.0 mg/L) on seed germination, shoot multiplication, and the accumulation of total hypericin were determined using tissue cultures of H. scabroides. Murashige and Skoog (MS) medium was used for germination and shoot cultures. Measurements of total hypericin were taken using a UV spectrophotometer. Results: The best germination rate (59.2%) was obtained using hormone-free MS medium (control group). Apical tips of freshly germinated seedlings were proliferated on the MS medium supplemented with various BAP concentrations (0.5, 1.0, and 2.0 mg/L) and the control group (without BAP). The highest number of shoots (42.7 shoot/explant) and longest shoot length (2.50 cm) were obtained on MS medium supplemented with 2.0 mg/L BAP. Total hypericin was found in trace amounts and it was found that the total hypericin was not affected by the concentration of BAP. Conclusions: Our results showed that increasing concentrations of BAP stimulated shoot multiplication but did not affect seed germination rates or total hypericin in in vitro cultures of H. scabroides.

Determination of Hypericin Content in Callus and Cell Suspension Cultures of Hypericum triquetrifolium Turra

Hypericum triquetrifolium Turra. is a valuable medicinal plant owing to source of many bioactive compounds. Hypericin is one of the significant components among these compounds. The aim of this study was to develope an efficient method allowing to improve hypericin production from calli and cell suspension cultures of Hypericum triquetrifolium Turra. Callus formation was obtained from axenic leaf explants grown in Murashige and Skoog (MS) salts supplemented with 1 mg l -1 6-benzyl adenine (BA) and 2 mg l -1 α-naphtaleneacetic acid (NAA) and 1 mg l -1 (BA) + 0.4 mg l -1 2,4-dichlorophenoxyacetic acid (2,4-D). Growth medium of cell suspension culture was the same with callus medium but devoid of agar. The increase of biomass in cell suspension culture was obtained between 6.31-6.28 fold compared to the first day of culture, on the 20 th day. We assayed hypericin content in methanolic extracts of calli and cell suspension cultures of H. triquetrifolium Turra. Hypericin contents of the samples were measured at 589 nm by a UV-VIS spectrophotometer. Analysis of hypericin contents showed that levels found in callus were 0.0527 and 0.0485 mg g -1 , while in cell suspension cultures these rates were 0. 0018 and 0.0016 mg g -1 , suggesting that the accumulation of this compound in cell suspension needs further modifications.

Hypericins: biotechnological production from cell and organ cultures.

Hypericum perforatum L. (St. John's wort), a perennial flowering plant native to Europe, is widely used as a medicinal plant and has a long history of its use in the treatment of various ailments. Currently, H. perforatum is widely used as an herbal remedy for the treatment of mild to moderate depression. Hypericins are natural napthodianthrone compounds produced from H. perforatum (St. John's wort) which are having antitumor, antiviral (i.e., against human immunodeficiency and hepatitis C virus), antineoplastic, and antidepressant properties. Currently, field-grown plant materials are generally used for the commercial production of hypericins. It has been reported that hypericin accumulation in natural plants is influenced by different ecological and environmental conditions including light intensity, nitrogen availability, temperature, seasons, and growing regions. Therefore, up to 17-fold and 13-fold differences in hypericin and pseudohypericin amounts, respectively, are reported in different phytopharmaceutical preparations. Plant cell and organ cultures are effective systems for producing natural products, and attempts were made for the production of biomass and stable concentrations of hypericins through in vitro cultures of H. perforatum. Cell, callus, shoot, plantlet, and adventitious root cultures have been established and various chemical and physical factors which influence the biomass and secondary metabolite accumulation have been investigated. Large-scale plantlet and adventitious root cultures have also been attempted in H. perforatum in bioreactors, and various strategies have been applied for the production of higher biomass and secondary products. This review describes the biotechnological approaches employed for the production of hypericins and focuses upon the challenges and future prospects.

Several factors affecting hypericin production of Hypericum perforatum during adventitious root culture in airlift bioreactors

Hypericum perforatum L. is a traditional medicinal plant for the treatment of depression and wound healing, and hypericin is one of the main effective active substances. To optimize the culture system for producing hypericin in adventitious root, this study used balloon-type airlift bioreactors to investigate the effect of air volume, inoculation density, indole-3-butyric acid (IBA) concentration and methyl jasmonate (MeJA) concentration on hypericin content and productivity during adventitious root culture. Hypericin content and productivity were improved with increasing air volume, and 0.1 vvm (air volume/culture volume/min) was optimal for hypericin production. Inoculation density also had a great effect on hypericin accumulation. Hypericin content and productivity were favorable in an inoculation density of 5.0 g l−1 and decreased when inoculation densities were lower or higher than 5.0 g l−1. Furthermore, 1.25 mg l−1 IBA enhanced hypericin content and productivity, but too low (≤0.50 mg l−1) or too high (≥1.50 mg l−1) IBA concentrations decreased hypericin accumulation. MeJA concentration significantly affected biomass accumulation and hypericin production. The biomass decreased and hypericin production increased with increasing MeJA concentration. Optimum hypericin content (1.61 mg g−1 DW) and productivity (15.57 mg l−1) were obtained at 350 μM MeJA. The hypericin content in bioreactor-grown adventitious roots was lower than in 3-year field-grown plants, but significantly higher than that in in vitro-grown plantlets and 1-year field-grown plants. Thus, the bioreactor culture of adventitious roots can realize rapid and mass production of hypericin in H. perforatum.

Toxic Effects of Pb on Anatomy and Hypericin Content in Hypericum perforatum L.

ABSTRACT Lead (Pb) is one of the most common heavy metal contaminants in the environment. The present study was therefore undertaken to determine the effects of Pb on structural characteristics and hypericin production in Hypericum perforatum. Mature plants were treated with contaminated soil in seven treatments (75, 150, 300, 600, 800, 1000, and 1500 mg/kg Pb in soil) with three repeats per treatment every 14 days. Maximum observed Pb content in shoot parts was observed in the treatments with 600 and 1500 mg/kg Pb. The Pb concentration in roots was higher than in shoot parts, enhanced with increasing Pb concentration in the soil. In this study, Pb treatment significantly influenced the morphology, anatomy, and hypericin content in the plant. Anatomical characteristics of leaf, stem, and root affected by Pb contamination, as well as scanning electron microscopy (SEM) studies, revealed structural changes in stomata and epicuticular waxes. Under Pb toxicity, anatomical symptoms occurred in leaves, including increase in sizes of epidermal cells, mesophyll tissue, and diameter of stems and roots, as well as amplified vascular bundles and pith area. This, therefore, indicated that metal contamination can change the chemical composition of this plant. Maximum hypericin content was observed in the treatment containing 600 mg/kg Pb in soil, which then decreased.

Stimulation of the production of hypericins in in vitro seedlings of Hypericum adenotrichum by some biotic elicitors

The goal of this work was to study the stimulating effects of mannan and pectin on the biosynthesis of hypericins in in vitro seedlings of Hypericum adenotrichum Spach. Seedlings of H. adenotrichum were grown on a modified MS medium containing mannan (10, 50, and 100 mg/L) or pectin (10, 50, and 100 mg/L) for 15 and 30 days. Seedlings of H. adenotrichum were extracted with methanol. The methanol extracts of the seedlings were analysed by HPLC to investigate the changes in hypericin and pseudohypericin levels. The best results were obtained by exposure to 50 mg/L mannan or pectin for 15 days. Mannan stimulated pseudohypericin production up to 2.8-fold and hypericin production up to 1.7-fold. Pectin stimulated pseudohypericin production up to 4.8-fold and hypericin production up to 2.7-fold. The increases in hypericins in response to mannan and pectin demonstrate that these elicitors can be evaluated for production of secondary metabolites in Hypericum species.

English

Background: Hypericum adenotrichum contain many biologically active compounds, some of which, especially hypericins (hypericin and pseudohypericin), have antidepressant, antimicrobial, antiviral, and antitumor properties. In this paper, we report the effects of osmotic stress on the production of hypericins in H. adenotrichum under in vitro conditions. Osmotic stress is an abiotic elicitor that can alter the physiological and biochemical properties of plants, as well as decrease or increase the concentrations of secondary metabolites in plant tissues. Material and Methods: Sucrose and polyethylene glycol (PEG) were used to cause osmotic stress. Seedlings of H. adenotrichum were grown on a modified MS medium containing sucrose (15, 30, 45, and 60 g/L) or PEG (2.5, 10, and 15 g/L) for 15 and 30 days. Then, H. adenotrichum seedlings were extracted with methanol. These extracts were analysed by HPLC to investigate the changes in hypericins levels. Results: Under osmotic stress conditions, the concentrations of hypericins changed in seedlings of H. adenotrichum. Treatment with 10 g/L PEG for 15 days increased production of hypericin (2.1-fold) and pseudohypericin (2.3-fold), but PEG treatment for 30 days affected less hypericins levels when compared to PEG treatment for 15 days. The amount that the hypericins increased was minimal and proportional with the amount of sucrose up to treatment with 45 g/L sucrose, and then the hypericins decreased at 60 g/L of sucrose treatment for 15 days. In sucrose treatment, the highest hypericins levels were observed in the control seedlings at 30 days treatment period of sucrose. Conclusions: These results can be evaluated in experimental botany and in the technology of Hypericum species cultivation for pharmaceutical applications.

Influence of vitamins on polyphenolic content, morphological development, and stress response in shoot cultures of Hypericum spp

Hypericin is a widely studied phytopharmaceutical produced by the more developed representatives of the Hypericum genus. We established that Gamborg’s (G5) vitamins significantly raised hypericins levels in H. rumeliacum and H. tetrapterum shoot cultures in comparison with the Murashige and Skoog’s (MS) vitamin supplementation. To elucidate this effect, we further examined the impact of the two vitamin formulations on the polyphenolic compounds, morphological development, hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels in the two species, as well as in hypericin non-producing H. calycinum. It was found that in all three species Gamborg vitamins decreased the polyphenolic compounds content, stimulated multiplication, decreased leaf compactness and increased dark glands density per square millimeter (for the two hypericin producing species) as compared to MS vitamins. In addition, the raised levels of hypericins in H. rumeliacum and H. tetrapterum, were also connected to increased content of H2O2 and MDA in vitro. Moreover, the values of these two parameters were considerably lower in hypericin non-producing H. calycinum, implying that oxidative stress either by increased metabolic activity, connected with higher hypericin production, or by a possible auto-toxic effect of the high amounts of pro-oxidant hypericin itself could be induced in Hypericum in vitro.

Enhancing hypericin production of Hypericum perforatum cell suspension culture by ozone exposure

Accumulation of secondary metabolites is one of the common reactions of plants to ozone exposure in nature. To investigate the effect of ozone on the production of desired compounds of plant cell cultures, we assayed hypericin production of Hypericum perforatum suspension cell cultures treated with different doses of ozone at different culture phases. The results show that hypericin contents of the cells treated with 60 to 180 nL L(-1) ozone are significantly higher than those of the control, showing that ozone exposure may stimulate hypericin synthesis. Hypericin production of the cells treated with ozone at exponential phase is higher than that of lag and stationary phase, which suggests that exponential phase cell cultures are more responsive to ozone exposure than lag and stationary phase cells. The highest hypericin production is obtained by the cells exposed to 90 nL L(-1) ozone at late exponential phase for 3 h, being about fourfold of the control.

Effects of plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of Hypericum hirsutum and Hypericum maculatum

We investigated the effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 6-γ,γ-dimethylallylaminopurine (2iP), thidiazuron (TDZ) and α-naphthaleneacetic acid (NAA)], modified Murashige and Skoog (MS) medium containing 10 mM NH4 + and 5 mM NO3 − and supplemented with 2iP, BA, Kin and NAA (MSM medium), and two elicitors [jasmonic acid (JA), and salicylic acid (SA)], on plant growth and accumulation of hypericins (hypericin and pseudohypericin) and hyperforin in shoot cultures of Hypericum hirsutum and H. maculatum. Our data suggested that culture of shoots on MS medium supplemented with BA (0.4 mg l−1) or Kin (0.4 mg l−1) enhanced production of hypericins in H. maculatum and hyperforin in H. hirsutum. Hypericins and hyperforin concentrations decreased in both species when TDZ (0.4 mg l−1) was added to the MS medium. Also, TDZ induced hyperhydric malformations and necrosis of regenerated shoots. Cultivation of H. maculatum on MSM medium resulted in approximately twofold increased production of hypericins compared to controls, and the growth of H. hirsutum shoots on the same medium led to a 6.16-fold increase in hyperforin production. Of the two elicitors, SA was more effective in stimulating the accumulation of hypericins. At 50 μM, SA enhanced the production of hypericin (7.98-fold) and pseudohypericin (13.58-fold) in H. hirsutum, and, at 200 μM, enhanced the production of hypericin (2.2-fold) and pseudohypericin (3.94-fold) in H. maculatum.

The hyp-1 gene is not a limiting factor for hypericin biosynthesis in the genus Hypericum.

Biosynthesis of the hypericins that accumulate in the dark glands of some members of the genus Hypericum is poorly understood. The gene named hyp-1, isolated from Hypericum perforatum L. has been proposed as playing an important role in the final steps of hypericin biosynthesis. To study the role of this candidate gene in relation to the production of hypericins, the expression of this gene was studied in 15 Hypericum species with varying ability to synthesise hypericin. While the accumulation of hypericins and emodin, an intermediate in the respective pathway, was associated with the dark glands in the hypericin-producing species, the hyp-1 gene was expressed in all studied species regardless of whether hypericins and emodin were detected in the plants. The coding sequences of hyp-1 cDNA were isolated from all species and showed more than 86% similarity to each other. Although, in general, an increased level of the hyp-1 gene transcript was detected in hypericin-producing species, several of the hypericin-lacking species expressed comparable levels as well. Our results question the role of the hyp-1 gene product as a key enzyme responsible for biosynthesis of hypericins in the genus Hypericum. The function of the hyp-1 gene may not be restricted to hypericin biosynthesis only, or some additional factors are necessary for completion of hypericin biosynthesis.

Involvement of NADPH oxidase-mediated H2O2 signaling in PB90-induced hypericin accumulation in Hypericum perforatum cells

PB90 is a novel protein elicitor isolated from Phytophthora boehmeriae. Here, we report that treatment of PB90 stimulates hypericin production and hydrogen peroxide (H2O2) generation in Hypericum perforatum L. cells and demonstrate that H2O2 is essential for PB90-induced hypericin production. To further study the source of PB90-triggered H2O2, we have investigated activities of plasma membrane NADPH oxidase in Hypericum perforatum L. cells subjected to PB90 treatment. It is revealed that treatment of the cells with PB90 significantly increases NADPH oxidase activity. NADPH oxidase inhibitors suppress not only the PB90-stimulated NADPH oxidase activity but also the PB90-triggered H2O2 generation and PB90-induced hypericin production, showing that NADPH oxidase is involved in PB90-triggered H2O2 generation and hypericin production. Moreover, the suppression of NADPH oxidase inhibitors on PB90-induced hypericin production can be reversed by H2O2, although H2O2 per se has no effects on hypericin production of the cells. Together, the data demonstrate that PB90 may induce hypericin production of H. perforatum cells through the NADPH oxidase-mediated H2O2 signaling pathway.

Signal interaction between nitric oxide and hydrogen peroxide in heat shock-induced hypericin production of Hypericum perforatum suspension cells

Heat shock (HS, 40 degrees C, 10 min) induces hypericin production, nitric oxide (NO) generation, and hydrogen peroxide (H(2)O(2)) accumulation of Hypericum perforatum suspension cells. Catalase (CAT) and NO specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) suppress not only the HS-induced H(2)O(2) generation and NO burst, but also the HS-triggered hypericin production. Hypericin contents of the cells treated with both NO and H(2)O(2) are significantly higher than those of the cells treated with NO alone, although H(2)O(2) per se has no effects on hypericin production of the cells, which suggests the synergistic action between H(2)O(2) and NO on hypericin production. NO treatment enhances H(2)O(2) levels of H. perforatum cells, while external application of H(2)O(2) induces NO generation of cells. Thus, the results reveal a mutually amplifying action between H(2)O(2) and NO in H. perforatum cells. CAT treatment inhibits both HS-induced H(2)O(2) accumulation and NO generation, while cPTIO can also suppress H(2)O(2) levels of the heat shocked cells. The results imply that H(2)O(2) and NO may enhance each other's levels by their mutually amplifying action in the heat shocked cells. Membrane NAD(P)H oxidase inhibitor diphenylene iodonium (DPI) and nitric oxide synthase (NOS) inhibitor S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU) not only inhibit the mutually amplifying action between H(2)O(2) and NO but also abolish the synergistic effects of H(2)O(2) and NO on hypericin production, showing that the synergism of H(2)O(2) and NO on secondary metabolite biosynthesis might be dependent on their mutual amplification. Taken together, data of the present work demonstrate that both H(2)O(2) and NO are essential for HS-induced hypericin production of H. perforatum suspension cells. Furthermore, the results reveal a special interaction between the two signal molecules in mediating HS-triggered secondary metabolite biosynthesis of the cells.

Effects of cytokinins and elicitors on the production of hypericins and hyperforin metabolites in Hypericum sampsonii and Hypericum perforatum

Hypericum perforatum L. (St. John’s wort) and Hypericum sampsonii Hance are medicinal plants used in China in the treatment of viruses and other disorders. In the current study, we investigated the effects of cytokinins 6-benzylaminopurin (BA), zeatin (ZT) and thidiazuron (TDZ) on plant growth and production of hypericins (pseudohypericin and hypericin) and hyperforin. Our data suggested that culture of H. perforatum in modified MS (Murashige and Skoog) medium, with a 50% reduction in ammonium nitrate and potassium nitrate, and supplemented with BA (0.44 μM) and indolebutyric acid (IBA, 0.049 μM), resulted in increased production of hypericins. Similar results were noted with H. sampsonii with minor changes to the medium (0.46 μM ZT and 0.049 μM IBA). There were approximately 2.95-, 2.62-fold increases in H. perforatum pseudohypericin and hypericin production by TDZ (0.45 μM) induction compared to the controls. No enhancement of hypericins and hyperforin production was elicited by TDZ in H. sampsonii. The elicitor methyl jasmonate (MJA, 50 μM) and its analog, 2,3-dihydroxypropyl jasmonate (DHPJA, 50 μM), were also used in H. perforatum and H. sampsonii shoot culture to increase secondary metabolite production, eliciting an increase in the production of hypericins and hyperforin. While leaf senescence and biomass inhibition were observed in cultures induced by MJA, no such effects were observed with DHPJA.