WO2007107803A2

WO2007107803A2 - Process and modified media for preparing callus- and cell suspension cultures of hypericum perforatum l.

Info

Publication number: WO2007107803A2
Application number: PCT/HU2007/000026
Authority: WO
Inventors: Adrienne Seres
Original assignee: Adrienne Seres
Priority date: 2006-03-23
Filing date: 2007-03-21
Publication date: 2007-09-27
Also published as: WO2007107803B1; HU227572B1; HUP0600245A2; HU0600245D0; WO2007107803A3

Abstract

The invention relates to a process for preparing callus- and suspension cultures of Hypericum perforatum L. in modified media in order to produce increased amount of biomass and active ingredients. According to the invention the ratio of the macro- and microelements is altered in the known MS, N6 and B5 media, further the media are completed with EDTA-Na-Fe selecton iron and optionally with phytohormones. In such manner, continuously producing in vitro systems are obtained which produce the synergetic, biologically active components of H. perforatum L. simultaneously with the plant material. The invention also relates to the modified media.

Description

Process and modified media for preparing callus- and cell suspension cultures of Hypericum perforatum L.

The invention relates to a process for the preparation of callus- and cell suspension cultures of Hypericum perforatum L. (St. John's wort) in modified media in order to produce biomass and active ingredients in an increased amount. The invention also relates to the modified media. Hypericum perforatum L is a herbaceous perennial species which is found in the temperate zones of Europe, Asia and North-Afrika in the nature and which has been used as a medicinal plant since the Greek-Roman ages. It is well-known for its diuretic, sedative and wound-healing effect [Fitoterapia 66, 43-68 (1995)]. Nowadays, H. perforatum L. is mainly used for the treatment of moderate depression [Eur. Neuropsychopharmacol. 9(6), 501-505 (1999)]. Initially, the hypericin and its relative compounds were considered as the most active substances in the inhibition of the monoamine oxidase and catechol-O-methyltransferase enzymes. The antiviral- and antiretroviral activity of these metabolites were also demonstrated [Antiviral. Res. 15, 101 (1991)], which can be increased by light irradiation. Recently, the attention has been focused on hyperforin as the most active component, that is responsible for the antidepressant activity [Life Sci. 63, 499-510 (1998)]. Nowadays, it seems, that several chemical substances are present in the whole drug extract, such as naphtodiantrones (hypericin and its derivatives), hyperforins, xanthones, flavonoids and procyanidins and their relative bioactivity are responsible for the antidepressant activity. Among the flavonoids rutin, hyperoside, quercitrin and kampferol can be also detected, which compounds have smooth muscle-relaxing, antiphlogistic, diuretic, antimicrobial and antioxidant effect. The quercetin aglikon inhibits the growing of the tumor cells.

Therefore, there is a high demand for therapeutic components derived from H. perforatum and this fact makes necessary to search after alternative biotechnological processes for their production.

Nowadays, plant cells and organ cultures have important role as effective in vitro systems for synthetizing natural products on a large scale. Different factors that influence the plant cultivation, such as climatic conditions, the generation's and taxon's effect for the arrival of the phenological phases, the shooting period depending on the season, the vegetative growing stage, the protracting phenophase of the flowering and the generative phenophases can be avoided by in vitro tissue cultures and cell suspension cultures as alternative technologies for the production of active ingredients. Furthermore, the drug from cultivated St. John's wort is heterogenous morphologically and according to inner content. At the requirements of the purity of drug, it needs to take into consideration the pesticide residues, the microbiological contamination and the heavy metal content as well. Several examinations were carried out in St. John's wort cell- and tissue cultures for studying the production of the secondary metabolites [see e.g. Planta Med. 58, 621-625 (1992); Phytochemistry 48, 1165-1168 (1998)]. In these experiments usually the MS medium and its completed variants were used.

The cell cultures prepared in liquid MS medium containing BAP (10^'6 M) and NES (10^"7 M) according to Kartnig et al. [Planta Med. 62, 51-53 (1996)] at 24°C, under continuous 2000 lux irradiation, by passaging every 3-4 week and with growth rate of 30-500%, differ from strain to strain, did not show relationship between the growth rate and the production of the active ingredient.

Flavonoids and hypericin synthesized in vegetative buds were measured by Pasqua at al. [Plant Science 165, 977-982 (2003)]

Kirakosyan et al. [Phytochemistry 53, 345-348 (2000)] investigated the production of hypericin in case of mannan eliciting as well as the relationship between the biosynthesis of hypericin and the in vitro maturity of the differentiated structures. BAP and NAA as well as 2,4-D and KIN growth regulators were used for the preparation of shoot organ cultures.

Bais et al [In Vitro Cell. Dev. Biol. - Plant 38, 58-65 (2002)] obtained hypericin from the cell aggregates (1000 μm size) of Hypericum perforatum L. cultivating cultures under dark and light conditions.

The total flavonoid content of the callus produced by Dias et al. [Journal of Liquid Chromatography and Related Technologies 22(2), 215-227 (1999)] was 0.5- 0.7 mg/g dry substance; it is much less than the value found in the plants living in the nature, which is 14-70 mg/g dry substance. The calli obtained in the experiments of the above mentioned articles can not be maintained continuously. Furthermore, their active ingredient content relatively low and the cultures become old after a few passages. That is why the demand still exists for preparing cell- and organ cultures of Hypericum perforatum L which are effective in the production of the natural active components.

The aim of the invention is to prepare such in vitro cultivating systems that produce the biologically active components of H. perforatum L together with an increased biomass production.

In the course of our experiments we have found that if the ratio of macro- and microelements is altered in the known MS, N6 and B5 media and the modified media are completed with EDTA-Na-Fe selecton iron and optionally phytohormones, continuously producing in vitro systems are obtained which produce also the synergetic, biologically active components of H. perforatum L., such as rutin, hyperoside, quercitrin, quercetin, kampferol among the flavonoids and hypericin among the naphtodiantrons, simultaneously with the plant material.

One aspect of the present invention relates to the modified MS-62 medium suitable for preparing callus- and cell suspension cultures of Hypericum perforatum

L which can be characterized in that it contains the macro- and microelements in an altered ratio compared to the basic medium and it is completed with 5-45 mg/l EDTA- Na-Fe(III) and 0.1-10 mg/l one or more phytohormones.

Preferably, the modified MS-62 medium according to the invention compared to the basic medium

(a) contains half amount of boric acid and it is completed with 40 mg/l EDTA-Na-Fe as well as with 1 mg/l IAA, 0.2 mg/l NAA and 0.1 mg/l KIN phytohormones (Medium 1); or

(b) contains half amount of boric acid and it is completed with 40 mg/l EDTA-Na-Fe as well as with 0.1-10 mg/l IAA, 0.1-0.4 mg/l NAA, 0.1-2.0 mg/l 2,4-D, 0.5 mg/l BAP or 0.2-0.5 KIN phytohormones (Medium 2); or

(c) contains half amount of boric acid and it is completed with 40 mg/l EDTA-Na-Fe as well as with 0.8 mg/l NAA and/or 1 mg/l 2,4-D and 0.5 mg/l KIN phytohormones (Medium 3); or (d) contains half amount of boric acid and it is completed with 40 mg/l EDTA-Na-Fe as well as with 1 mg/l NAA, 1 mg/l 2,4-D and 0.1 mg/l KIN phytohormones (Medium 4); or

(e) contains quarter amount of macroelement, half amount of boric acid as well as it is completed with 10 mg/l EDTA-Na-Fe and with 0.8 mg/l IAA and 1.6 mg/l

BAP phytohormones (Medium 5); or

(f) contains quarter amount macroelement, half amount of boric acid and it is completed with 10 mg/l EDTA-Na-Fe as well as with 0.8 mg/l 2,4-D and 1.6 mg/l BAP phytohormones (Medium 6); or (g) contains quarter amount of macroelement, double amount of microelement, identical amount of boric acid and it is completed with 10 mg/l EDTA- Na-Fe as well as with 0.5-0.5 mg/l IAA and KIN phytohormones (Medium 7).

More preferably, the modified MS-62 medium according to the invention is one of the Media 1-7 with the composition indicated in Table 1. Another aspect of the present invention relates to a modified MS-62 medium suitable for preparing callus- and cell suspension cultures of Hypericum perforatum L. which can be characterized in that it does not contain Mo, Cu and Co compared to the basic medium but contains 0-1650 mg/l NH₄NO₃, quarter amount of boric acid, fifth amount of MnSO₄, seventh amount of ZnSO₄, 1 -5-fold amount of inositol and vitamins, and it is completed with 250-500 mg/l asparagine or proline, 35-45 mg/l EDTA-Na-Fe and 0-1.0 mg/l 2,4-D phytohormone.

According to a preferred embodiment, the modified MS-62 medium of the present invention is Mo-, Cu-, Co- and NH₄NO₃-deficient, it contains 1.6 mg/l H3BO3, 4.4 mg/l MnSO₄XH₂O, 1.5 mg/l ZnSO₄x7H₂O, 40 mg/l EDTA-Na-Fe, 100 mg/l inositol, 1 mg/l nicotinic acid, 1 mg/l pyridoxine.HCI, 10 mg/l thiamine. HCI, 0.0/0.5/1.0 mg/l 2,4-D, 500 mg/l asparagine, as well as 30 g/l sucrose and 0-1.0 g/l caseine (Medium 8).

According to another preferred embodiment, the modified MS-62 medium of the present invention is Mo-, Cu- and Co-deficient, but contains 1650 mg/l NH₄NO₃, 1.6 mg/l H₃BO₃, 4.4 mg/l MnSO₄XH₂O, 1.5 mg/l ZnSO₄x7H₂O, 40 mg/l EDTA-Na-Fe, 500 mg/l inositol, 1 mg/l nicotinic acid, 1 mg/l pyridoxine.HCI, 10 mg/l thiamine. HCI, 1.0 mg/l 2,4-D, 250 mg/l proline and 30 g/l sucrose (Medium 9). Another aspect of the present invention relates to a modified N6 medium suitable for preparing callus- and cell suspension cultures of Hypericum perforatum L. which can be characterized in that it is completed with molybdenum, copper and cobalt in the form at B5-microelements as well as with 35-45 mg/l EDTA-Na-Fe, 0.5- 1 ,0 mg/l nicotinic acid and 20-30 g/l sucrose and contains optionally a half amount of macroelements compared to the basic medium.

Preferably, the modified N6 medium according to the present invention is a N6-78 hormone-free medium which contains 0.25 mg/l Na₂MoO₂XH₂O, 0.025 mg/l CuSO₄x5H₂O and 0,025 mg/l CoCI₂x6H₂O as well as 40 mg/l EDTA-Na-Fe, 0.5 mg/l nicotinic acid and 30 g/l sucrose (Medium 10) as supplements.

According to another preferred embodiment, the modified N6 medium contains solely a half amount of macroelements.

A further aspect of the present invention relates to a modified B5 medium suitable for preparing callus- and ceil suspension cultures of Hypericum perfortum L. which can be characterized in that it contains increased amount of CaCI₂ and 35-45 g/l EDTA-Na-Fe, 0.5-1.0 mg/l nicotinic acid, 0-1.0 mg/l 2,4-D hormone and 15-25 g/l sucrose as supplements compared to the basic medium.

Preferably, the modified B5 medium is a B5-68 hormone-free medium which contains 440 mg/l CaCI₂x2H₂O, 40 mg/l EDTA-Na-Fe, 1 mg/l nicotinic acid and 20 g/l sucrose (Medium 11).

According to another preferred embodiment, the modified B5 medium of the present invention contains 1 mg/l 2,4-D phytohormone and hereby it is suitable for the generation of secunder calli.

The media used in this invention are reviewed in the following articles: MS-62 medium [T. Murashige, F. Skoog: Plant Physiol. 15, 473-397 (1962)], N6 medium [C. Chu et al.: Scient. Sin. I., 18, 659-668 (1975)] and B5 medium [O. L. Gamborg: Exp. Cell. Res. 50, 151-158 (1968)]. The macro- and microelement composition of these media can be also found in the following handbook: Dudits, D. and Heszky, L.: Novenybiotechnolόgia, Mezόgazdasagi Kiadό, Budapest, 1990. Examples for modified media according to the invention are the following media with the compositions indicated in Table 1 : MS-62 medium 1-9; N6-78 medium 10; and B5-68 medium 11. In this table the conditions of the cultivation are also indicated. In contradiction to the plant cultivation, by the in vitro H. perforatum L. cell- and tissue cultures according to the present invention, it is possible to produce plant substances under standardized, controlled, optimalized conditions, independently from the weather and pathogens, among regulated circumstances. Additionally, it is possible to prepare continuously growing cultures by proper grafting and continuous sustenance, while it can be to examine the factors influencing the biosynthesis of the active ingredients and to increase the output of the active ingredients.

The meaning of the abbreviations used in the description is the following: BAP = benzylaminopurine 2,4-D = 2,4-dichlorophenoxy acetic acid IAA = indol-3-acetic acid KIN = kinetin

NAA = naphthalene acetic acid Rt = retention time

Another aspect of the present invention relates to a process for preparing callus cultures of H. perforatum L. which process comprises the following steps: the seeds of H. perforatum L are germinated on a wet filter paper or hormone-free modified B5 medium at 20-23⁰C, then are inoculated onto sterile modified MS-62 medium (pH 5.8) and calli are induced in darkness at 24.5-25.5⁰C, then secondary calli are generated from the primary calli and the callus cultures are cultivated on a modified MS-62 medium at pH 5.0-5.8, at 23-27⁰C, under alternation of dark-light cycles, then they are inoculated in a fresh medium or are harvested and the dry substance content and active ingredient content are determined. In another preferred embodiment in vitro callus cultures are prepared from the explants of cotyledon, young foliage-leaf and roots and leafy shoots of an in vitro regenerated plant of H. perforatum L (inoculation onto a solid medium), as well as from the leaves developed in the upper third of the flowery shoots from the intact plant by initiation on the modified media according to the invention: for example, in Medium 2 primary callus is induced at pH 5.8 and 24.5-25.5⁰C in darkness, then secondary calli are prepared from the primary calli by incubating at 24.5-25.5°C in the media according to the invention, such as in modified MS-62 medium 2 with 2,4- D, IAA, NAA, BAP or KIN hormones at pH 5.0-5.8, cultures are grown in dark and light; in the modified MS-62 medium 6 with BAP and 2,4-D hormones at pH 5.0, cultures are grown in light; in the modified MS-62 medium 3 with 2,4-D and KIN hormones at pH 5.8 cultivating in dark and light; in the modified MS-62 medium 7 with IAA and KIN hormones at pH 5.8, cultivating the cultures in light; or in the modified B5 medium 11 with 1 mg/l 2,4-D hormone cultivating the cultures in light; or in the modified N6 medium 10 comprising half amount of macroelements, cultivating the cultures in dark and light. In darkness white-coloured embryogenic calli are obtained, but getting to the light the calli turn green or red.

The stimulating materials, plant hormones used in the above processes influence the respiration, the enzyme activity and the nitrogen metabolism. During our experiments we have discovered that IAA, NAA and KIN block the wood formation of St. John's wort on Medium 2. The high IAA-concentration increase the juvenile character in the callus in Medium 2 with increased hormone content. The darkness promotes the enzymatic conversion. By decreasing the amount of the macroelements to its quarter and at the same time double the microelement content and decreasing the amount of sucrose to 5 g/l and supplementing with 0.5-0.5 mg/l IAA and KIN hormones, the active ingredient production can be increased in Medium 7. The reduction of pH value from 5.8 to 5.0 in Medium 2 and the use of pH 5.0 in the Media 6 and 7 induces growth, eliminates the suboptimal physical conditions, as well as guarantees genetic stability and long-distance regenerating ability causing rapid biomass production.

According to the invention the suspension cultures are prepared in such manner that the callus lines showing the best growing character are inoculated, then a suspension is prepared from the callus in a media according to the invention of different composition and hormone content, such as in modified MS-62 medium 1 with IAA, NAA, KIN hormones at pH 5.8; in modified MS-62 medium 2 with 2,4-D₁ IAA, NAA, BAP or KIN hormone composition at pH 5.0-5.8; in modified MS-62 medium 3 with NAA, KIN hormones at pH 5.8; in modified MS-62 medium 4 by adding 2,4-D, NAA, KIN hormones at pH 5.8; in modified MS-62 medium 5 with BAP, IAA hormones at pH 5.0; in modified MS-62 medium 8 by the use of 2,4-D hormone at pH 4.8-5.2; in modified MS-62 medium 9 with 2,4-D hormone at pH 5.8. The suspensions are cultivated in Erlenmeyer flask at 23.5-25⁰C in dark and light at 100 rpm on a rotary shaker equipment. After further callus incubation for 21-28 days and suspension shaking for 14-21 days, the calli, cell suspensions are inoculated on a fresh medium or harvested in order to determine the dry substance content and to examine the active ingredient content. The determination of the dry substance content is carried out by lyophilisation and the investigation of the active material content is carried out by HPLC. in the course of our examination we have found that Medium 1 according to the invention at pH 5.8, in light, at 23.5-25⁰C results in abundant, uniforming, reddening yellowish-green embryogenic cell aggregates with a few lysis.

Medium 2 according to the invention increases the amount of biomass, influences the development of the colour as well as the enhancement of the active ingredient level at pH 5.8. This medium at pH 5.0 increases the embryogenic character and the active ingredient production, as well. In the same medium (pH 5.0) when 2,4-D, IAA, NAA, KIN hormone variation is used, a deep-green callus is generated on a natural light at 24.5-26.5⁰C, while in darkness at 24.5-25°C a well- proliferating, yellowish-white callus is developed. The 2,4-D, IAA, NAA, BAP hormone composition produces a white-coloured embryogenic callus and abundant cell mass in darkness at 24.5-25⁰C; however, in the light it produces a green-coloured callus with cells that later slightly become red at 24.5-25⁰C and it induces the regeneration of the shoots. The dry substance content of the callus increases significantly compared to the initial inoculation level.

Furthermore, we have found that Medium 2 according to the invention with decreased 2,4-D, IAA, NAA and KIN hormon content in suspension results in organogenic aggregates and suspension containing small cells with light brown lysis on filter light with shaking for a short time. Shoots can be regenerated from the aggregates of the suspension in modified B5 solid medium 11 in light.

Medium 3 according to the invention with 1 mg/l 2,4-D and 0.5 mg/l KIN hormone content promotes the production of a well-developing, whitish-yellow callus at pH 5.8 in darkness at 24.5-25⁰C, but a green-coloured callus is induced in light at 24.5-26.5⁰C. In the suspension the substitution of 1 mg/l 2,4-D with 0.8 mg/l NAA results in an abundant biomass- and active ingredient production in dark at 23.5- 25⁰C.

In Medium 4 according to the invention supplemented with 2,4-D, NAA and KIN hormones and 250 g/l ascorbic acid an abundant, slightly reddening, embryogenic cell mass is generated in light, at 23.5-25°C and pH 5.8, in suspension. In Medium 5 according to the invention containing 1.6 mg/l BAP, 0.8 mg/l IAA and 0.25 MS macroelement content (a macroelement content decreased to a quarter), an abundant biomass is grown which contains yellow, almost uniform, smaller-sized aggregates with embryogenic character at pH 5.0 and 23.5-25⁰C, in suspension. In Medium 6 according to the invention with 1.6 mg/l BAP and 0.8 mg/l 2,4-D complementation and at the same time 0.25 MS macroelement content a yellowish- white biomass with a great efficiency is produced at pH 5.0 and 23.5-25⁰C, and the production of the active ingredient begins in shaked culture in light and dark conditions. When using callus culture in light a deep-green cell mass with reddening surface is generated at 24.5-26⁰C and the active ingredient production also increases.

In Medium 7 according to the invention the IAA and KIN hormone's combination, the 0.25 MS macroelement's, the double MS microelement's and the decreased sucrose's content increases the active ingredient production and the formation of the colour material at pH 5.8 but inhibits the growing of the callus in light at 24.5-26⁰C.

In Medium 8 according to the invention with 0.5 - 1 mg/l 2,4-D concentration or without it, with asparagine, caseine or without them, respectively, at pH 4.8-5.2 and

23.5-25⁰C lysis as well as formation of an intensive purplish-brown colour can be observed simultaneously with the biomass production, which fact refers to a high active ingredient (hypericin) content.

Medium 9 according to the invention with 1 mg/l 2,4-D and proline instead of asparagine and 500 mg/l mesoinositol results in an abundant, yellowish cell mass at pH 5.8 and 23.5-25⁰C in light in suspension.

Medium 10 according to the invention is a hormone-free medium containing 30 g/l sucrose and 0.5 mg/l nicotinic acid; it results in a rapid shoot-elongation during 3-7 days at pH 5.8 and 24.5-25⁰C in light on intensive green coloured embryogenic callus pieces placed on a lean medium which calli were previously grown in light at pH 5.0 in a medium containing BAP.

If desired, medium 10 can contain macroelements in a half amount, 350 mg/l inositol, 900 mg/l glutamine, 250 mg/l ascorbic acid and vitamins, preferably vitamin H. Medium 11 according to the invention is a hormone-free medium containing

440 mg/l CaCI₂ and 20 g/l sucrose which is effective to regenerate embryogenic callus derived from a medium containing hormones, at pH 5.8 and 14-26.5⁰C in light in order to obtain shoot/rooty shoot.

Maintenance experiments are performed on 11 different media under different environmental conditions at pH 4.6-5.8. The incubation parameters are the following: 24 hours dark (control) at 24.5-25⁰C; a photoperiod of 16 hours light/8 hours darkness at 23.5-28⁰C, by the use of fluorescent lamp F1 Cool Day Light 36W.

The biomass increment of the callus- and suspension culture of H. perforatum L. can be monitored well by drawing a diagram about growth plotted against time on the examined Line II. In the experiments performed at 25⁰C the secondary callus tissues with 0.2 g inoculation mass are placed into Petri-dishes containing 40 ml Medium 2 according to the invention. The measurement is made every 3-5 days from dry substance mass of the randomly selected fresh tissue that was lyophylised for 72 hours. The measurements are performed in case of calli for 28 days and for 21 days in case of suspension. The results are showed on the Figures 1 a)-c).

The description of Figures: Figure 1a): this figure shows the growing diagram of H. perforatum callus in Medium 2. It can be seen that the fresh mass of the callus with 28 days old cultivation is 5.28 g and its lyophylised dry substance content is 0.48 g. It means that the fresh mass of the callus culture increases 26.4-fold compared to the 0.2 g inoculation mass, its dry substance, however, increases 48-fold compared to .. the 0.01 g dry substance content of the originally inoculated mass.

Figure 1b): this figure shows the growing diagram of the H. perforatum suspension in

Medium 2. It can be seen that yellowish-brown, small-cellular suspensions containing smaller aggregates and shaken in darkness for 21 days in Medium

2 produce 10.56 g fresh mass (in 80-100 milliliters) and 0.74 g dry substance after initial inoculation by 2 g. It corresponds 5.3-fold increase and 3.9-fold increase, respectively (compared to the 0.19 dry substance of initial material).

Figure 1c): this figure shows the growing of the H. perforatum suspension in Medium

8 which culture is multicellular and reddish-brown, and it contains aggregates, as well. From the curve it can be seen that after the initial 2 g inoculation 21.95 g fresh mass as well as 1.33 g dry substance are originated by the 21^st day. Thus, the fresh mass increases to 11 -fold and the dry substance content increases to 7.8-fold (compared to the 0.17 g dry substance of the initial material).

The biomass produced by Line Il was monitored during a 2-year period. The fresh mass of the total callus during the 2 years is 1068 g, in the 1^st year is relatively small but in the 2^nd year increased 25-fold. The fresh mass of the total suspension during the 2 years is about 2200 g from which the production of 1^st year increased to about 550-fold. The average of dry mass is 8.52% by weight of callus and of suspension 7.79% by weight in the 2 years period (see Table 4). From this Table it can be seen that the callus- and suspension cultures prepared in modified media according to the invention are able to produce biomass in a very considerable quantity during a long-lasting period. The active ingredient content of the biomass (callus, suspension and folium) produced in media according to the invention was determined by HPLC and it was compared to a Hyperici herba commercial product [producer: Gyόgynoveny-kutatό Intezet (Institute for Medicinal Plant Research), Budakalasz, Hungary], The results obtained with Lines I₁ Il and III are shown in Tables 2 and 3. The total measured flavonoid content is 13.35%, the hypericin content, however, is 0.062% on the examined media according to the invention (Table 1) based on the average values of HPLC measurements (Tables 2 and 3) during the above mentioned two-years period (the average of 3 samples, two replicates). The total flavonoid content detected in cultures is 8.49-8.86% at callus and 0.33-2.12% in case of suspension in an one-year period. The percentage means percent (%) by weight.

TABLE 2. Active ingredient spectrum of H. perforatum callus, suspension and folium according to the invention on the basis of HPLC determination

(H herba, commercial product, F folium of a leafy shoot initiated from the seed of raised plant Il and III Com ared to Folium/Herba + increase in value, ~ almost consistent value

TABLE 3. Comparision of flavonoid and hypericin content between the cultures of callus lines I, Il and III and suspension line Il cultivated on different media and the sam le of Herba in the eriod 2003-2005. + Growth compared to herba

TABLE 4 Biomass production of H. perforatum Line Il in the period 2003-2005

The data of the tables show that NAA and KIN increased the synthesis of rutin on Medium 3 compared to the 2,4-D, IAA, NAA and BAP hormones used in Medium 2 (control). Similarly, the MS-macroelements content of Medium 6 in amount of 0,25% and the ratio of BAP and 2,4-D increased the accumulation of rutin in shaked culture in dark and light. The rutin content also increased in Medium 8 using of 2,4-D in suspension, in light. The BAP, IAA, NAA and 2,4-D content of Medium 2 similarly induced the increase of rutin in callus grown in dark and light conditions.

The amount of quercitrin also increased compared to the control (Medium 3) in suspension on Medium 2 with altered pH containing 2,4-D, IAA, NAA and BAP hormones in dark and light as well as in Medium 6 containing 2,4-D in light. In Media

2 with altered pH (pH 5.0-5.8) in callus the quercitrin level is also increased under dark an light conditions.

The amount of quercetin also increased in suspension in Medium 2 in light, in Medium 6 in dark and light, in Medium 8 in light compared to the control (Medium 2) shaked in dark. In callus quantitative increase of quercetin appeared under dark and light conditions.

The synthesis of kampferol is shown to be optimal in suspensions: such as in

Medium 3 in dark, in Medium 6 in dark and light and in Medium 8 in dark. In callus the culture cultivated in Medium 2 showed the increase of the kampferol level in light and dark compared to cultures cultivated in Medium 2 but started in another point of time.

The hypericin production in Media 6 and 8 in suspension shaked in dark and light was higher compared to the control (Medium 2).

The rutin production which depends on the Hypericum species, in the folium started from seed increased at Line Il with 0.2% and at Line III with 1.26%.

The amount of commonly detected rutin and hyperoside increased to 1.82% in callus in Medium 6 in a culture cultivated in light. In the Hyperici herba GYNKI 315

Ph. Eur. 4 commercial sample the total amount of rutin and hyperoside together was

1.035%. It means that a very considerable enhancement of rutin plus hyperoside can be achived in the modified media according to the invention.

The hyperoside content in folium started from seed at the examined Line Il is 1.11 % and at Line III is 0.76%. The hyperoside level in callus runs between 0.1 % and 0.61 % in Medium 2 in dark, 0.4% in Medium 6 in light at Line Il and considerably higher, 2.02% in Medium 6 in light at Line III.

The quercitrin content is 2.73% at the examined Line Il and 0.8% at Line III. Hyperici herba commercial product contains 0.156% quercitrin. The amount of quercitrin is 0.41 % - 1.01% in the media according to the invention in calli cultivated in light which value considerably exceeds the one of the known product.

The quercetin content in folium is 0.21 % at the examined Line Il and 0.20% at Line III. The quercetin content of the Hyperici herba commercial product detected similarly by HPLC is 0.105%. The quercetin content in the media according to the invention in callus cultivated in dark is 0.3%, while in callus cultivated in light is 1.01 %. This fact indicates that the quercetin content of the callus is multiplied in the media according to the invention.

The amount of kampferol is 0,071% in Hyperici herba. The kampferol content of the callus developed in light on Medium 2 according to the invention is about the half of the value of the known product (0.036%). However, in suspension both in dark and light a very considerable kampferol content (0.19%) can be detected in Media 6 and 8 which value is more than 2.5 times more than the value of the commercial product.

The amount of hypericin is 0.219% in the commercial sample of Hyperici herba. In contrary, the hypericin content in the folium according to the invention is

0.61% at the examined Line Il and 0.51 % at Line III. The hypericin content of the suspension cultures according to the invention is 0.06-0.20% and the hypericin content of the calli is 0.01-0.04%. According to the article of Bais et al. mentioned in the background of the invention the hypericin content of cell suspension cultures of H. perforatum cultivated in light and dark is 0.012%. Therefore, the hypericin content of the cultures cultivated in the media according to the invention exceeds the hypericin content of the suspension cultures cultivated in Bais's medium.

Summarizing the above mentioned it can be stated that in the folia, calli as well as cell suspension cultures according to the invention the amount of individual active ingredients significantly increased, thus the total active ingredient content increased.

A further aspect of the present invention relates to a process for in vitro regeneration of Hypericum perforatum L. from a culture of embryogenic character which process can be characterized in that shoots or rooty-shoots are regenerated from a callus prepared in modified MS-62 medium, in modified N6-78 or in modified, hormon-free B5-68 medium in light.

The adventages of the modified media according to the invention prepared for the cultivation of the cultures of Hypericum perforatum L, can be summarized as follows: they are continuously producing in vitro systems by which de-differentiated or differentiated structures can be prepared and maintained continuously; the active ingredients of H. perforatum can be produced with callus- and suspension cultures prepared in the systems according to the invention and with in vitro regenerated plants; the cultivation in them results in an increased biomass production and simultaneously increased active ingredient (flavonoid and hypericin) production; they are well-controlled systems under standardized, controled and optimalized conditions; they are suitable for cultivation in a large scale.

The invention is illustrated in detail in the following examples, without limiting the scope of the invention. Example 1

Preparation of callus- and cell suspension cultures of H. perforatum L.

From cotyledon, young foliage-leaf, root and leafy shooting of in vitro regenerated plant or leaf developed in the upper third (30 cm) of the flowery shooting of intact plant of H. perforatum L 0.5 cm segments are prepared, and they are sterilized for 5 minutes with a solution with 80% ethyl alcohol content, for 10 minutes with sodium hypochlorite solution, then rinsed five times with sterile distilled water, dried on sterile filter paper, and inoculated onto 30-40 ml modified MS-62 medium (e.g. Medium 2 with a composition indicated in Table 1 , pH 5.8) according to the invention solidified with agar-agar on a Petri-dish with 10 cm diameter in order to initiate primary callus. The Petri-dishes are kept at 24.5-25⁰C on dark, and the pH of the medium is regulated with 0.2 N NaOH or 0.1 N HCI. After 3-6 weeks of the incubation primary callus is induced, then after 3-4 weeks the calli are inoculated further into a fresh medium and the culture is kept in dark, as a result of this secondary callus is proliferated.

After further 3.5-4 weeks the cultures kept continuously in dark are inoculated onto fresh medium in such manner that only the best growing calli are selected. After further 3.5-4 weeks the one half of the calli are cultivated forth in dark at

24.5-25⁰C, for example, in Medium 2 (pH 5.8, control) or Medium 3 (pH 5.8), while another half of the calii are incubated in light at 24.5-25°C, for example, in Medium 2 (pH 5.0 and pH 5.8), in Medium 6 (pH 5.0), in Medium 3 (pH 5.8) or in Medium 7 (pH 5.8). After further 3-4 weeks the calli cultivated in dark are selected further, and the selected calli are cultivated in dark again at 24.5-25⁰C, for example, in Medium 2 (pH 5.8) or Medium 3 (pH 5.8). Meanwhile from a part of them suspension is prepared in dark in an Erlenmayer flask coated by foil in 60 ml medium at 23.5-25°C under laboratory conditions, with shaking at 100 rpm on a laboratory shaking machine, for example, in Medium 2 (pH 5.0 or 5.8), Medium 3 (pH 5.8), Medium 6 (pH 5.0) or Medium 8 (pH 4.8-5.2). One part of the suspensions shaken in dark is cultivated also with 16/8 hours photoperiodicity in the same media.

Among the calli cultivated in light the ones with the best growing capacity are inoculated further after 3-4 weeks; the parameters of the incubation are as follows: 25-26.5°C, relative humidity about 70%, photoperiodicity 16/8 h, photon lux density 50-60 μmol/m²/s between November and February (for somatic embryogenesis), 14- 21⁰C; 80-100 μmol/m²/s between March and May (for green, reddening calli), 21.5- 24.5⁰C; 24 hours illumination on a lightself in a plant room, the illumination 30 μmol/m²/s (Lightmeter, Li-189, USA), under Fl Cool Day Light 36 W fluorescent lamp, 40 cm shelf distance, relative humidity 20% (for light-green and purplish-red calli), 24- 25⁰C in Medium 8; and 16/8 h photoperiodicity, for example, in Medium 2 (pH 5.0), in Medium 2 (pH 5.8), in Medium 6 (pH 5.0), in Medium 3 (pH 5.8), in Medium 5 (pH 5.0) or in Medium 7 (pH 5.8).

Suspension is also prepared from the calli cultivated in light under laboratory conditions, on shaking machine at 100 rpm shaking, in filter-light, at 23.5-25⁰C, in a 250 ml flask in 60 ml medium, for example, in Medium 1 (pH 5.8), in Medium 2 (pH 5.0), in Medium 4 (pH 5.8), in Medium 5 (pH 5.0), in Medium 6 (pH 5.0), in Medium 8 (pH 4.8-5.2) or in Medium 9 (pH 5.8). After further 3.5-4 weeks a growing diagram is taken from the callus cultures cultivated in dark, which diagram means the increase of the crude biomass and dry substance plotted against time. The callus cultivated in dark is started with 0.2 g starting crude mass and it is incubated for 28-29 days, meanwhile the content of 3 Petri-dishes is eliminated on every 3 day in order to determine the fresh material and dry substance. After 14-21 day cultivation the growing rate and the passage period of the cell suspension cultivated in dark are determined. In this case the starting mass is 2 g in 60 ml medium. The fresh biomass and dry substance (by lyophilisation) produced in dark are determined, for example in Medium 2 (pH 5.8) and in Medium 8 (pH 5.2).

Example 2

The preparation of the leafy shoot (folium) of an intact plant

Seeds from H. perforatum L are germinated on wet filter paper at 23-25⁰C. From one part of the seedlings callus is prepared after surface sterilization on an agar medium, from another part of them leafy seedling is prepared by sticking it in a common blackmould. The folium (leaf) is harvested from this and from one part of them callus is induced, from another part dry substance determination is performed by lyophilisation and active ingredient determination is performed for the comparative examinations.

Example 3

In vitro regeneration of H. perforatum L

By the end of the following 3.5-4 weeks from embryogenic calli obtained in Example 1 and cultivated in light shoots and roofy shoots are regenerated during the following 4-6 weeks in Medium 11 according to the invention, which is a modified B5- 68 hormone-free medium, at pH 5,8.

From embryogenic callus pieces placed into suspension it is possible to regenerate leafy shooting during 3-7 days, in natural light, at pH 5.8 in Medium 10, which is a modified N6-78 hormone-free medium or in Medium 11 , which is a modified B5-68 medium. Example 4

Determination of the biomass

The calli obtained according to Example 1 , cultivated in dark and light, are harvested and their fresh mass and their dry substance after 72 hours lyophilisation are determined.

The shaked suspensions obtained according to Example 1 , cultivated in dark and light, are harvested after 14-21 days and the fresh mass of the biomass is determined by weighing, then the dry substance content is also determined after 72 hour's lyophilisation. For the production of the biomass of H. perforatum Line Il the following average values were measured in a two-years period: in callus the fresh mass is 1068.38 g, the dry substance is 91.02 g (8.52%); in case of suspension the fresh mass is 2198.19 g, the dry substance is 171.34 g (7.79%).

Example 5

Determination of active ingredient content

The calli or suspensions shaken in light according to Example 1 are harvested after 14-21 days (depending on medium), then lyophilised and the biologically active components of H. perforatum are determined. The HPLC measurements are performed as follows.

Extraction: 0.2 g lyophilised dry substance is sonicated in 3x5 ml methanol for 3x30 minutes, then centrifuged in a centrifuge precooled to -5⁰C for 10 minutes, at 5000 rpm. Hereupon the sample is concentrated in a vacuum concentrator and taken up in methanol in an 1 ml Eppendorf tube, centrifuged and the supernatant is transferred onto a reverse phase C18 column.

HPLC system: programable Pharmacia LKB liquid chromatograph equipped with solvent pumps and UV-detector. The active ingredients are eluated in room temperature across a reverse phase C18 column containing silica gel. The isocratic mobile phase is an eluent containing 85% (v/v) acetonitrile and 15% (v/v) sodium acetate buffer (pH 4) and 0.1 M ammonium acetate buffer. The flow rate is 1 ml/minute, the detection takes place at 254 nm, 270 nm and 590 nm, respectively.

Claims

1. Modified MS-62 medium suitable for preparing callus- and suspension cultures of Hypericum perforatum L, characterized in that it contains the macro- and microelements in an altered ratio compared to the basic medium and it is completed with 5-45 mg/l EDTA-Na-Fe(III) and 0.1-10 mg/l one or more phytohormones.

2. Modified MS-62 medium according to claim 1 characterized in that compared to the basic medium it

(a) contains half amount of boric acid and it is completed with 40 mg/l EDTA-Na-Fe(III) as well as with 1 mg/l IAA, 0.2 mg/l NAA and 0.1 mg/l KIN phytohormones; or

(b) contains half amount of boric acid and it is completed with 40 mg/l EDTA-Na-Fe(III) as well as with 0.1-10 mg/l IAA, 0.1-0.4 mg/l NAA, 0.1-2.0 mg/l 2,4- D, 0.5 mg/l BAP or 0.2-0.5 mg/l KIN phytohormones; or (c) contains half amount of boric acid and it is completed with 40 mg/l

EDTA-Na-Fe(III) as well as with 0.8 mg/l NAA and/or 1 mg/l 2,4-D and 0.5 mg/l KIN phytohormones; or

(d) contains half amount of boric acid and it is completed with 40 mg/l EDTA-Na-Fe(III) as well as with 1 mg/l NAA, 1 mg/l 2,4-D and 0.1 mg/l KIN phytohormones ; or

(e) contains quarter amount of macroelement, half amount of boric acid and it is completed with 10 mg/l EDTA-Na-FE(lll)-mal as well as 0.8 mg/l IAA and 1.6 mg/l BAP phytohormones; or

(T) contains quarter amount macroelement, half amount of boric acid and it is completed with 10 mg/l EDTA-Na-Fe(III) as well as with 0.8 mg/l 2,4-D and 1.6 mg/l BAP phytohormones; or

(g) contains quarter amount of macroelement, double amount of microelement, identical amount of boric acid and it is completed with 10 mg/l EDTA- Na-Fe(III) as well as with 0.5-0.5 mg/l IAA and KIN phytohormones.

3. Modified MS-62 medium suitable for preparing callus- and suspension cultures of Hypericum perforatum L, characterized in that compared to the basic medium it does not contain Mo, Cu and Co but contains 0-1650 mg/l NH₄NO₃, boric acid decreased to quarter, MnSO₄ decreased to the fifth, ZnSO₄ decreased to the seventh, 1-5 fold amount of inositol and vitamins and it is completed with 250-500 mg/l asparagine or proline, 30-40 mg/l EDTA-Na-Fe(III) and 0-1.0 mg/l 2,4-D hormone.

4. Modified MS-62 medium according to claim 3 characterized in that it is Mo-, Cu-, Co- and NH₄NO₃-deficient and contains 1.6 mg/l H₃BO₃, 4.4 mg/l MnSO₄XH₂O,

1.5 mg/l ZnSO₄x7H₂O, 40 mg/l EDTA-Na-Fe(III), 100 mg/l inositol, 1 mg/l nicotinic acid, 1 mg/l pyridoxine.HCI, 10 mg/l thiamine. HCI, 0.0-0.5-1.0 mg/l 2,4-D, 500 mg/l asparagine, 30 g/l sucrose and 0-1.0 g/l caseine.

5. Modified MS-62 medium according to claim 3 characterized in that it is Mo-, Cu- and Co-deficient and contains 1650 mg/l NH₄NO₃, 1.6 mg/l H₃BO₃, 4.4 mg/l

MnSO₄XH₂O, 1.5 mg/l ZnSO₄x7H₂O, 40 mg/l EDTA-Na-Fe(III), 500 mg/l inositol, 1 mg/l nicotinic acid, 1 mg/l pyridoxine.HCI, 10 mg/l thyamine.HCI, 1.0 mg/l 2,4-D, 250 mg/l proline>^3G g/l sucrose.

6. Modified N6 medium suitable for establishing callus- and suspension culture of Hypericum perforatum L characterized in that it is completed with Mo, Cu and Co in the form of B5-microelements as well as with 35-45 mg/l EDTA-Na-Fe(III), 0.5-1.0 mg/l nicotinic acid and 20-30 g/l sucrose and contains optionally a half amount of macroelements compared to the basic medium.

7. Modified N6 medium according to claim 6 which is a N6-75 hormone-free medium and contains one by one 0.25 mg/l Na₂MoO₂XH₂O, 0.025 mg/l CuSO₄x5H₂O and 0.025 mg/l CoCI₂x6H₂O, further 40 mg/l EDTA-Na-Fe(III), 0.5 mg/l nicotinic acid and 30 g/l sucrose as supplements.

8. Modified Nβ medium according to claim 7 which contains a half amount of macroelements.

9. Modified B5 medium suitable for establishing callus- and suspension culture of Hypericum perforatum L characterized in that it contains compared to the basic medium increased amount of CaCI₂ as well as 35-45 g/l EDTA-Na-Fe(III), 0.5-1.0 mg/l nicotinic acid, 0-1.0 mg/l 2,4-D hormone and 15-25 g/l sucrose as supplements.

10. Modified B5 medium according to claim 8 which is a B5-68 hormone-free medium containing 440 mg/l CaCI₂x2H₂O, 40 mg/l EDTA-Na-Fe(III), 1 mg/l nicotinic acid and 20 g/l sucrose.

11 Modified B5 medium according to claim 8 which contains 440 mg/l CaCl2x2H₂O, 40 mg/l EDTA-Na-Fe(III), 1 mg/l nicotinic acid, 1 mg/l 2,4-D and 20 g/l sucrose.

12. A method for preparing callus- and cell suspension cultures of Hypericum perforatum L, characterized in that primary callus is induced from the explants of cotyledon, young foliage leaf, root or leafy shoots of the in vitro regenerated plant of H. perforatum L or from leaves developed in the upper third of the flowery shooting of the intact plant by initiation in a medium according to claim 1 at pH 5.8 and 24.5- 25.5°C in dark; then secondary callus is prepared by inoculation into a medium according to claim 1 (pH 5.0-5.8), at 23-27⁰C in light and dark, the secunder callus is selected and inoculated further into a medium according to claim 8 or 11, or after the selection a suspension culture is preprared in a medium according to claims 1 or 2, which is cultivated at pH 5,0-5.8 and 23.5-25⁰C in dark and under a photoperiod of 16 h light/8 h dark.

13. A method for in vitro regeneration of Hypericum perforatum L from a culture with embryogenic character characterized in that shoots or rooty shoots are regenerated from an embryogenic callus prepared according to claim 12 in light in modified N6-78 or B5-68 hormone-free medium.

14. A method according to claim 13 characterized in that the regeneration is carried out in a modified N6-78 medium according to claim 7.

15. A method according to claim 13 characterized in that the regeneration is carried out in modified B5-68 medium according to claim 10.