An in vitro and Hydroponic Growing System for Hypericin, Pseudohypericin, and Hyperforin Production of St. John’s Wort (Hypericum perforatum CV New Stem)

• Abstract
• Introduction
• Materials and Methods
• Seed germination
• Germplasm lines
• In vivo acclimatization
• Hypericin, pseudohypericin and hyperforin analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

While the interest in medicinal plants continues to grow, there is a lack of basic information with respect to efficient protocols for plant production. Recently, in vitro regeneration protocols have been developed to provide masses of sterile, consistent St. John’s wort. The current study assessed the potential for acclimatization of in vitro grown St. John’s wort plantlets to a nutrient film technique (NFT) hydroponic system in a controlled environment greenhouse. Quantitative analyses of hypericin, hyperforin and pseudohypericin in flower tissues were used as the parameters to assess the quality of the greenhouse-grown plants. The three bioactive compounds were found to be present in similar or higher amounts than previously reported values for field-grown plants. These data provide evidence that greenhouse hydroponic systems can be effectively used for the efficient production of St. John’s wort and other medicinal plants.

Introduction

Whole plant preparations for the treatment or prevention of human ailments are increasingly popular and efficient technologies for medicinal plant propagation, production and biochemical characterization are required. Preparations of St. John’s wort (Hypericum perforatum L.), used for the treatment of depression, had an estimated value of $170 million in the USA in 2000 and St. John’s wort has demonstrated pharmaceutical efficacy attributed to inhibition of monoamine re-uptake [1], mediation of the dopaminergic system [2] or the modulation of interleukin-6 activity [3]. In general, standardization of natural health products of St. John’s wort has been based mainly on the quantification of three secondary metabolites, hypericin, pseudohypericin and hyperforin [4], [5]. However, inconsistency of the concentration of these biomarkers in commercially prepared St. John’s wort capsules has been reported (Consumer Safety Symposium on Dietary Supplements and Herbs, 1998). This example is evidence of several problems that have emerged with St. John’s wort and other plant-based medicines including (i) variation in the concentration of actives, and (ii) contamination with metals, toxic chemicals and microorganisms [6]. Botanical misidentification may account for some of the variability in preparations with reported instances of H. maculatum Crantz, H. barbatum Jacq., H. hirsutum L., H. montanum L., H. tetrapterum Fries and H. calycinum L. [7] in preparations of St. John’s wort. The proportion of the different plant tissues can also affect the efficacy St. John’s wort preparations as the percentage of flowers and fruits in a commercial product gradually increased from 0 % to more than 50 % over a growing season [8]. Environmental factors such as light and available nutrition have also been found to alter the concentration of hypericins in St. John’s wort [9] as have harvest date and season [10].

In Canada, recently announced legislation to address these concerns will require products to be produced in a facility that complies with Good Manufacturing Practices (GMP) regulations, to provide evidence of plant identity, medicinal efficacy and the assurance of a minimal level of medicinal constituents as well as an absence of abiotic or biotic contamination. These measures necessitate the development of new approaches for production of medicinal plants and in vitro propagation schemes provide significant advantages for compliance and to assure the production of pathogen-free, consistent, biochemically characterized and optimized plant tissues [11], [12]. Recently, we reported the establishment of protocols for in vitro regeneration of St. John’s wort from etiolated hypocotyl explants [12] and sterile stem sections [13]. The objectives of the present study were to develop an efficient hydroponic greenhouse system for in vitro grown St. John’s wort plantlets and to assess the quality of plants by the quantification of three major active constituents (hypericin, hyperforin, pseudohypericin) in flower tissues. The flower tissues are a rich source of hypericin, pseudohypericin and hyperforin and a recent report provided data for field grown St. John’s wort flowers at distinct stages [8]. The current experiments were designed to measure the concentration of the same metabolites in the same flower stages grown in an in vitro - in vivo system.

Materials and Methods

Seed germination

An optimized clonally propagated line of St. John’s wort (Hypericum perforatum L. CV New Stem, formerly Anthos; Richter’s Herbs, Goodwood ON) was generated from a wild-harvested seedling regenerated in vitro as previously reported [12], [13]. Seeds were inspected and surface sterilized by sequential immersion in a) 70 % ethanol solution for 5 s, b) 30 % aqueous solution of 5.4 % sodium hypochlorite (Lilo Products, Hamilton, ON) in water with one drop of Tween 20 per 500 ml for 20 min, and c) three washes with sterile distilled water. Sterile seeds were cultured onto water agar (8 g·l^-1; Laboratory Grade Agar, Fisher Scientific, Mississauga, ON) and incubated for 16 days in darkness in a growth cabinet at 24 °C.

Germplasm lines

Hypocotyl sections were excised from sterile etiolated seedlings and cultured on a medium containing MS salts [14], B5 vitamins [15], 30 g·l^-1 sucrose and 5 μmol·l^-1 thidiazuron [TDZ; N-phenyl-N’-(1,2,3-thidiazol-5-yl)-urea]. The pH was adjusted to 5.7 and 3 g·l^-1 gellan gum was added before the medium was autoclaved. After 6 days, explants were subcultured onto the same medium devoid of any plant growth regulators. Cultures were incubated in a growth cabinet with a 16 h photoperiod under cool white light at 40 - 60 μmol·m^-2·s^-1. Germplasm lines were generated from regenerated shoots that were separated from a single explant and subcultured into Magenta boxes containing 50 ml of basal medium for root development. Division of the growing plantlets and subculture onto basal medium was used to perpetuate St. John’s wort germplasm.

In vivo acclimatization

After 2 months, a selected germplasm line (#17) was acclimatized to hydroponic greenhouse conditions. Roots of plantlet clusters with a small amount of residual medium were carefully wrapped in a single layer of cheesecloth for initial protection and slid onto the center groove of a 2-inch plastic track. Tracks were placed into 114 cm × 56 cm plastic trays placed at a slight incline on the greenhouse benches. To maintain simplicity for commercial production, a continuous flow of 25 or 50 % greenhouse fertilizer hydroponic solution [20 - 8-20 commercial fertilizer (Plant Products, Brampton, ON); pH 6.0 in untreated well water] was provided as a nutrient film for the plantlet growth. Humidity of the plantlets was maintained with an intermediate misting system with an initial greater frequency (15 min intervals during daylight, 60 min intervals overnight; 34 times in 24 hours) and reduced after 6 weeks (30 min intervals in daylight, 60 min intervals at night; 20 times in 24 hours). After approximately 14 - 16 weeks, St. John’s wort plants in the greenhouse hydroponic system produced flowers and the analytical experiments were initiated. Flowers were harvested at the same time each morning three times per week at 48-hour intervals between July 18^th and August 3^rd, 2001 and classified into six stages of flower bud development. The mean daytime and nighttime temperatures in the greenhouse during this period were 31.4 °C and 18.3 °C, respectively. There was no supplemental lighting in the greenhouse during this period and the average light level on the benches at the time of collection was 244 μmol·m^-2·s^-1.

Hypericin, pseudohypericin and hyperforin analysis

Chemicals: Reference standards of hypericin, pseudohypericin, and hyperforin were purchased from ChromaDex, Inc. (Laguna Hills, CA). The high performance liquid chromatography (HPLC)-grade acetonitrile, acetone and methanol were purchased from Caledon (Mississauga, ON). Triethylammonium acetate is a product of Sigma-Aldrich Canada (Oakville, ON).

Extraction and HPLC analysis of hypericin, pseudohypericin, and hyperforin: The isolation and analysis method for hypericin, pseudohypericin, and hyperforin was developed by modification of previously described methods [16], [17], under low light intensity and at room temperature. Samples of the six distinct stages of flower development (0.25 g fresh weight) were collected in 1.5 ml Eppendorf tubes, immediately frozen in liquid nitrogen and stored at -80 °C. The frozen flowers were freeze-dried overnight (18 h) using a Labconco Freeze Dry System (10 × 10 ^{- 3} mbar, -40 °C) (Caltec Scientific Ltd., Toronto, CA), ground into fine powder with a polyvinyl mortar, and transferred into an amber-colored 20 ml vial. Extracts were prepared in 5 ml of acetone:methanol (50 : 50, v:v) with 30 min sonification (Ultrasonic FS-14 Sonicator; Fisher Scientific, Nepean, ON). Samples were then centrifuged at 3000 rpm for 10 min (GS-6 series centrifuge, Beckman Instruments Inc, Palo Alto, CA) and particulate matter in 3 ml of the supernatant was removed (0.2 μm nylon syringe filter; Waters Chromatography Inc., Mississauga, ON). Aliquots of each sample (500 μl) were transferred into a clear glass auto-sampler HPLC vial and an amber glass auto-sampler vial and the vials were sealed with Teflon coated aluminum lids to minimize contamination with air. Amber glass vials were immediately analyzed for hyperforin (within 7 h of extraction) while clear glass vials were exposed to a light source (15 cm distant from a 100W tungsten light) for 30 min to complete the conversion of the proto forms of hypericin and pseudohypericin, before analysis.

A 20 μl aliquot of sample was injected onto a Shimadzu 10AD HPLC system consisting of an SCL-10A system controller, SIL-10A auto-injector, SPD-M10AV photodiode array detector at 270 nm (hyperforin) and 588 nm (hypericin and pseudohypericin and a CTO-10A column oven (Shimadzu, Canada) with separation on a Phenomenex Hypersil C₁₈ column (3.0 μm; 4.6 × 100 mm) with a C₁₈ guard column (4 × 3 mm) (Phenomenex, Torrance, CA). The analytes were separated from the extracts with isocratic flow of 0.1M triethylammonium acetate and acetonitrile (33 : 67, v:v) at 1 ml·min^-1 rate.

Significant linear calibration curves (r2 > 0.989) were used for quantification for the each compound. The limits of detection of hypericin, pseudohypericin, and hyperforin were 0.1, 0.1, and 1.0 μg/ml, respectively. A reference sample (ground flowers) was spiked with authentic hypericin (5 μg), pseudohypericin (10 μg), and hyperforin (50 μg) for the calculation of the percent recovery. Recovery of hypericin and hyperforin was above 91 % and pseudohypericin was consistently recovered at greater than 65 %. All results are presented on a % wet weight basis for accuracy and there was no significant difference in the %dry matter of the flowers at different stages.

Results

In vitro-grown St John’s wort plantlets (Fig. [1] A) were hardened to greenhouse conditions over a period of 2 - 4 weeks with high humidity and constant nutrient recirculation. There was no difference between the two fertilizer concentrations (25 or 50 % of 20 : 8:20 NPK) for plantlet survival. Initially, all plantlets suffered a stress manifested as wilting and some leaf senescence but quickly recovered and grew to approximately 20 cm in 6 weeks (Fig. [1] B). Flowers were first observed after 12 weeks of greenhouse growth and flowering was continued for up to 2 months (Fig. [1] C, D). Similar to the field-grown plants, six sequential stages of flower development were observed (Fig. [1] D) beginning with a green bud approximately 3 mm in length and culminating with a fully open yellow flower with a radius of approximately 32 mm.

The need for sequential application of extraction protocols for the quantification of hypericin and hyperforin has previously been described as light is required for photoconversion of the proto-forms of hypericins [18]. Accumulation of hypericin was clearly visible as coloured glands on the anthers and petal margins of gently opened St. John’s wort buds at all stages of flower development (Fig. [1] E). The accumulation of all three secondary metabolites during the flower development showed a similar pattern (Fig. [2]). In general, the compounds were quantified at the highest concentrations in the second stage of flower development with a subsequent decline. The concentration of hypericin was significantly higher in buds at stages 2,3,4, and 5 than in either the youngest or oldest buds (Fig. [2] a). Similarly, pseudohypericin was also present at significantly lower concentrations in the early and late stages of development (Fig. [2] b). The absolute concentration of hyperforin was significantly higher during stages 2 - 5 than in the smallest buds or mature flowers (Fig. [2] c) but hyperforin was present at concentrations upto 100 fold greater than hypericin at the earliest stages of flower growth (Fig. [2] a and c). The relative ratio of hyperforin:hypericin declined in a predictable manner throughout the developmental period (y = 33.113Ln(x) + 90.574; R² = 0.9655).

Discussion

In vitro regeneration systems provide novel, optimized germplasm free from abiotic or biotic contamination [11]. The application of in vitro regeneration technologies for the nature health product industry requires the development of additional protocols for large-scale production. Potential approaches to large-scale production include development of aseptic bioreactors [19], controlled environment production [20], [21] or acclimatization to greenhouse hydroponic systems. In the current research, in vitro grown St. John’s wort plantlets were acclimatized to greenhouse conditions with intermittent misting protocol and continuous nutrient recycling. A modified nutrient film technique was used in which bare-root St. John’s wort was taken directly from culture and grown in a long, narrow channel with a shallow stream of recirculating nutrient solution [22]. It was found that the simple measure of bundling the roots in a single layer of cheesecloth provided effective protection for the in vitro regenerated plantlets. St. John’s wort plantlets suffered some initial shock after transfer from the culture room but quickly recovered in the high-humidity environment. This approach has great potential for acclimatization of in vitro-grown plantlets and medicinal plants, especially those where the active constituents are produced in roots [22]. To our knowledge, this is the first study to report hypericin, psuedohypericin and hyperforin production in cloned St. John’s wort plantlets grown in a greenhouse hydroponic system.

Quantification of hypericin, hyperforin and pseudohypericin is the accepted standard for natural health products utilizing St. John’s wort. However, the concentration of hypericins in St. John’s wort tissues varied with the date of harvest with a maximal concentration in the summer months [10]. In both the current experiments and previous work [8], St. John’s wort flowers were collected within the same approximate timeframe of late July. In the previous studies, the hypericin content of field-grown flowers varied between 0.1 - 0.45 % of the dry weight with the highest concentration quantified in mature buds and just opened flowers [8]. By comparison, the values reported for the field-grown flowers were between 25 - 90 % of the hypericin contents determined in the greenhouse-grown plants with a similar pattern to the concentrations except for the fully opened flowers where hypericin declined in the greenhouse. This decline may reflect several factors including (i) the increase in tissue weight as the flowers matured without a corresponding increase in hypericin, (ii) the overhead misting in the NFT system that would wash opened flowers and may remove hypericin glands or (iii) the degradation of hypericin as the flowers mature. For pseudohypericin, the reported content of the field-grown flowers was approximately 2 - 8 % of the greenhouse values, and hyperforin values in field grown flowers were 9 - 50 % of the corresponding hydroponic tissues. While the interpretation of these data may be complicated by many factors including (i) differences in the source material of St. John’s wort, and (ii) differences in the environmental and geographic parameters of the two studies, these data provide the first evidence that greenhouse production of St. John’s wort can result in a product that is of equal or better quality than field-grown tissues.

The future production of plant-based medicines will require new technologies for optimized production of high-quality plant materials. The results of this study provide new protocols to address the ongoing concerns of governments and the public with respect to the safety and efficacy of medicinal plant preparations as well as the practical concerns of efficient production. Greenhouse production facilities could be maintained throughout the year, leading to increased production capability and shorter storage times between harvest and manufacture. As well, it is likely that further optimization of other hydroponic parameters such as nutrient composition, CO₂ enrichment, light and temperature will improve the concentration of biologically active phytochemicals and the medicinal capacity of the plant tissues.

Acknowledgements

The financial support of the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs are gratefully acknowledged.

References

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  CrossrefPubMedSearch in Google Scholar
• 19 Son S H, Paek K Y. Large-scale production of medicinal plant species: The application of bioreactors for production of ginseng roots. In: Development of Plant Based Medicines: Conservation, Efficacy and Safety. P.K. Saxena (Ed.) Kluwer Academic Press 2001: pp 139-50
  Search in Google Scholar
• 20 Murch S J, KrishnaRaj S, Saxena P K. Production of medicinal plant species in sterile controlled environments In: Transplant Production in the 21st Century. Kluwer Academic Pres 2000: pp 160-5
• 21 Hahn E -J, Kin S J, Paek K Y, Lee Y B. Growth and acclimatization of chrysanthemum plantlets using bioreactor and hydroponic culture techniques. Transplant Production in the 21st Century. Kluwer Academic Press 2000: pp 274-8
  Search in Google Scholar
• 22 Cooper A. The ABC of NFT: Nutrient Film Technique. Caspar Publications Pty Ltd 1996: pp. xv, 134
  Search in Google Scholar

Prof. Praveen K. Saxena

Department of Plant Agriculture

Edmund C. Bovey Complex

University of Guelph

Guelph

Ontario

Canada

N1G 2W1

Phone: +1-519-824-4120 ext. 2495

Fax: +1-519-767-0755

Email: psaxena@uoguelph.ca

References

• 1 Perovic S, Muller W EG. Pharmacological profile of Hypericum extract: Effect of serotonin uptake by postsynaptic receptors.  Arzneimittel-Forsch. 1995;  45 1145-8
  PubMedSearch in Google Scholar
• 2 Butterweck V, Petereit F, Winterhoff H, Nahrstedt A. Solubilized hypericin and pseudohypericin from Hypericum perforatum exert antidepressant activity in the forced swimming test.  Planta Med. 1998;  64 291-4
  Thieme ConnectPubMedSearch in Google Scholar
• 3 Caputi P. Interleukin-6 involvement in antidepressant action of Hypericum perforatum .  Pharmacopsych. 2001;  34 S8-10
  PubMedSearch in Google Scholar
• 4 Chatterjee S S, Noldner M, Koch E, Erdelmeier C. Antidepressant activity of Hypericum perforatum and hyperforin: The neglected possibility.  Pharmacopsychiat. 1998;  31 7-15
  PubMedSearch in Google Scholar
• 5 Orth H C, Rentel C, Schmidt P C. Isolation, purity analysis and stability of hyperforin as a standard material from Hypericum perforatum L.  J Pharm Pharmacol. 1999;  51 193-200
  CrossrefPubMedSearch in Google Scholar
• 6 Murch S J, KrishnaRaj S, Saxena P K. Phytopharmaceuticals: Problems, limitations and solutions.  Scientific Reviews of Alternate Medicine. 2000;  4 33-8
  PubMedSearch in Google Scholar
• 7 St. John’s Wort Monograph. American Herbal Pharmacoepea and Theraputic Compendium HerbalGram. American Botanical Council 1997 40: 37-45
  Search in Google Scholar
• 8 Tekel’ova D, Repcak M, Zemkova E, Toth J. Quantitative changes in dianthrones, hyperforin andflavonoids content in the flower ontogenesis of Hypericum perforatum .  Planta Med. 2000;  66 778-80
  Thieme ConnectPubMedSearch in Google Scholar
• 9 Briskin D P, Gawienowski M G. Differential effects of light and nitrogen on production of hypericins and leaf glands in Hypericum perforatum .  Plant Physiol Biochem. 2001;  39 1075-81
  CrossrefPubMedSearch in Google Scholar
• 10 Southwell I A, Bourke C A. Seasonal variation in hypericin content of Hypericum perforatum L. (St. John’s wort).  Phytochemistry. 2001;  56 437-41
  CrossrefPubMedSearch in Google Scholar
• 11 Murch S J, KrishnaRaj S, Saxena P K. Phytopharmaceuticals: Mass-production, standardization and conservation.  Scientific Reviews of Alternate Medicine. 2000;  4 39-43
  PubMedSearch in Google Scholar
• 12 Murch S J, Choffe K L, Victor J MR, Slimmon T Y, KrishnaRaj S, Saxena PK Thidiazuron-induced regeneration from hypocotyl cultures of S t. John’s wort (Hypericum perforatum cv. Anthos).  Plant Cell Reports. 2000;  19 576-81
  CrossrefPubMedSearch in Google Scholar
• 13 Murch S J, KrishnaRaj S, Saxena P K. Tryptophan is a precursor for melatonin and serotonin biosynthesis in in vitro regenerated St. John’s wort (Hypericum perforatum L. cv. Anthos) plants.  Plant Cell Reports. 2000;  19 698-704
  CrossrefPubMedSearch in Google Scholar
• 14 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures.  Physiol Plant. 1962;  15 473-97
  CrossrefPubMedSearch in Google Scholar
• 15 Gamborg O L, Miller R A, Ojima K. Nutrient requirement of suspension cultures of soybean root cells.  Exp Cell Res. 1968;  50 150-8
  PubMedSearch in Google Scholar
• 16 Liu F F, Ang C YW, Springer D. Optimization of extraction conditions for active components in Hypericum perforatum using response surface methodology.  J Agric Food Chem. 2000;  48 364-71
  PubMedSearch in Google Scholar
• 17 Gray D E, Rottinghaus G E, Garrett H EG, Pallardy G. Simultaneous determination of the predominant hyperforins and hypericins in St. John’s Wort (Hypericum perforatum L.) by liquid chromatography.  J AOAC Inter. 2000;  83 944-9
  PubMedSearch in Google Scholar
• 18 Pourtaraud A, Lobstein A, Girardin P, Weniger B. Improved procedure for the quality control of Hypericum perforatum L.  Phytochem Anal. 2001;  12 355-62
  CrossrefPubMedSearch in Google Scholar
• 19 Son S H, Paek K Y. Large-scale production of medicinal plant species: The application of bioreactors for production of ginseng roots. In: Development of Plant Based Medicines: Conservation, Efficacy and Safety. P.K. Saxena (Ed.) Kluwer Academic Press 2001: pp 139-50
  Search in Google Scholar
• 20 Murch S J, KrishnaRaj S, Saxena P K. Production of medicinal plant species in sterile controlled environments In: Transplant Production in the 21st Century. Kluwer Academic Pres 2000: pp 160-5
• 21 Hahn E -J, Kin S J, Paek K Y, Lee Y B. Growth and acclimatization of chrysanthemum plantlets using bioreactor and hydroponic culture techniques. Transplant Production in the 21st Century. Kluwer Academic Press 2000: pp 274-8
  Search in Google Scholar
• 22 Cooper A. The ABC of NFT: Nutrient Film Technique. Caspar Publications Pty Ltd 1996: pp. xv, 134
  Search in Google Scholar

Prof. Praveen K. Saxena

Department of Plant Agriculture

Edmund C. Bovey Complex

University of Guelph

Guelph

Ontario

Canada

N1G 2W1

Phone: +1-519-824-4120 ext. 2495

Fax: +1-519-767-0755

Email: psaxena@uoguelph.ca