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 6-benzyl adenine (BA) and 2 mg l α-naphtaleneacetic acid (NAA) and 1 mg l (BA) + 0.4 mg l 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 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, while in cell suspension cultures these rates were 0.0018 and 0.0016 mg g, suggesting that the accumulation of this compound in cell suspension needs further modifications.


Introduction
The genus Hypericum belongs to the Clusiaceae (Guttiferae) family which encompasses approximately 460 species accommodate in 36 sections throughout the world [1]. The genus Hypericum includes both annual and perennial herbaceous plants, as well as woody, shrubs and small trees. H. triquetrifolium Turra., which is important plant of Hypericum genus, widespread in warm-temperate areas throughout the world and well represented in the Mediterranean and in the Near East area. H. triquetrifolium is a herbaceous perennial plant, and it usually grows on dry stony or sandy places [2]. The genus are of great economical and medicinal importance for containing of phytochemical constituents [3].
Properties of callus tissue are an important factor in the establishment of cell cultures. These features allow to form single or clusters of cells in liquid culture and are further increased quality of cell suspension culture. Such cells dispersed in the liquid medium have a potential to synthesize any of the compounds normally associated with the intact plant [14]. Division of cells in suspension cultures was much higher and more affected by changing media conditions in comparison with cells in callus culture [15].
Production of secondary metabolites via plant cell and tissue cultures has various advantages such as obtaining desirable secondary metabolites and large scale production [16]. In recent years, plant cell culture techniques has been commonly applied to the production of many useful secondary metabolites, including pharmaceuticals, pigments, and other bioactive compounds [17,18]. Several valuable studies have been performed on the production of useful secondary metabolites in plant cell cultures and organ cultures of some Hypericum species. The relationship beetwen biosynthesis of hypericin compounds and in vitro development of differentiated structures in Hypericum plants has also been investigated [19][20][21][22].
The aim of this work was to develop an efficient procedure for the establishment of callus and cell suspension cultures from H. triquetrifolium and determine hypericin production of these cultures. The role of plant growth regulator treatments has been particularly studied.

Plant Material and Culture Conditions
The seeds of H. triquetrifolium were collected in September 2008 from Diyarbakir, Turkey. The seeds were surface sterilized by immersion in a 70% (v/v) ethanol solution for 30 s, followed by immersion in a 53% aqueous solution of 5% sodium hypochlorite in water for 10 min and five times rinses with sterile distilled water. After sterilization process, the seeds were germinated on Murashige and Skoog basal MS medium [23] devoid plant growth regulator in Magenta vessels (GA-7). The pH of the media was adjusted to 5.8 and it was supplemented 0.7 % agar for media solidification prior to autoclaving at 1 atm 121 º C for 25 min. The cultures were incubated under a photoperiod of 16 h light/8 h darkness in a growth chamber at 25 ± 2 º C. After 4 weeks initiation of in vitro cultures, plantlets obtained from the germinated seeds were transferred to an MS basal medium containing 1 mg l -1 BA, agar 0.7% (w/v) and 3% (w/v) sucrose and they were grown in this medium for proliferation.

Callus Induction
To induce callus formation, leaves of in vitro grown plantlets were used (Fig. 3a). Leaf explants excised from seedlings approximately 4 cm high were divided into four parts and aseptically transferred with the abaxial side-down to the culture mediumn. In order to test the effect of different growth regulators on calli induction, the different combinations of auxins (NAA and 2,4-D) and cytokinins (BA and Kin) were tested and the frequency of callus induction and growth rates was evaluated 4 weeks after culture initiation ( Table 1). The cultures of calli were regularly subcultured every 28 days.

Establishment of Cell Suspension Cultures
For initiation of cell suspension cultures, calli obtained from in vitro grown leaves were used (Fig. 3b). After 3-5 subcultures, calli (1g F.W.) were inoculated in 100 ml flasks containing 20 ml MS medium and maintained on a rotary shaker at 100-105 rpm. Medium used for suspension culture was the same of callus medium but without agar. Initial subcultures of plant cell cultures performed in 100 ml erlen flasks containing 20 ml of fresh liquid media but the next subcultures were maintained in 500 ml erlen flasks containing 50 ml of fresh media and for 500 ml erlen flasks were used 100 ml fresh media (Fig. 3d). After 15 days in liquid culture, the cells released from calli were transferred into fresh liquid media (Fig. 3c). During the each subculture, microscopic observations were carried out to observe presence of single cells (Fig. 3e) and small cell clusters (Fig. 3f) and morphological appearance of the cells in suspension cultures after filtering through sieves of mesh sizes 500, 250 and 100 µm. Each subcultures were performed every 2 weeks.

Growth Parameters of Cell Suspension Cultures
Cell growth of two media containing MS medium supplemented with 1 mg l -1 BA + 2 mg l -1 NAA and 1mg l -1 BA + 0.4 mg l -1 2,4-D was measured by determining the packed cell volume (PCV/100), fresh weight (FW/100) and dry weight (DW/100) at 4, 8, 12, 16, 20 days. The values of PCV, FW and DW were obtained by compressing the cell mass in a given volume of cell suspension culture (10 ml). Each point on the growth curve represents the mean of three independent measurements ± S.E.

Extraction and Hypericin Analysis Of Samples
The samples of 100 mg (DW) were placed in vials containing chloroform (10 ml) and extracted in a sonicator to remove their chlorophyll contents. Following that, the chloroform was removed in a vacuo. After removing the chloroform, the samples were re-extracted with methanol (10 ml) in the sonicator for 5 min with methanol, the extraction process was repeated three times and then, the methanol was removed from the vacuo. The final samples (supernatants) were dissolved in methanol and 1 ml of each methanolic extract was placed in a test tube for the determination of hypericin content and spectrometric analysis were performed. Absorbance was measured at 589 nm by a spectrometer (Shimadzu UV/Vis-T80+).

Statistical Analysis
The spectrometer analysis was performed by using an eight point calibration curve generated with pure hypericin (Fig. 1). Experiments were repeated 3 times using a complete randomized block design. Analysis of samples was performed and differences between the means of the treatments were determined by Duncan's Multiple Range test at p 0.05 and 0.01. All calibration curves showed good Determination of Hypericin Content in Callus and Cell Suspension Cultures of Hypericum triquetrifolium Turra linear regression (R 2 =0.992) within the test range.

Initation of callus and cell suspension cultures
All media tested formed callus but calli obtained showed different features in terms of both appearance and growth rate. Although the calli obtained kinetin in combination with 2,4-D showed undesired properties due to weak growth and the calli formation rate, BA in combination with 2,4-D and NAA had desired properties (Table 1). In MS media containing 1 mg l -1 BA in combination with 2 mg l -1 NAA and 1 mgl -1 BA + 0.4 mg l -1 2,4-D was the most convenient properties (Table 1). Calli grown in medium containing 1 mg l -1 BA and 2 mg l -1 NAA was green, friable and more rapid in terms of growth (Fig. 3b). The calli grown in medium containing 1 mg l -1 BA and 0.4 mg l -1 2,4-D was yellowish, rapid and friable. These calli grown in different media were used for establishment of cell suspension cultures. Growth medium of cell suspension cultures was the same with callus growth medium but without agar.  Figure 2, biomass values of the suspension cell cultures cultured with MS medium containing 1 mg l -1 BA and 2 mg l -1 NAA enhanced with the elongation of culture time from day 0 to 20. Total biomass showed a sharpe increase between 8-16 th days of cultures. The rate of increase in the culture biomass reached the maximum level on the 20 th day. Compared to the first day, on the 20 th day, a 6.28-fold increase in the biomass of culture was reported. The medium containing 2,4-D realized sharpe increases between 12-16 days and compared to the first day growth rate was 6.31 -fold.

Quantification of Hypericin Compounds
Calli and cell suspensions were analysed for quantification of hypericin compounds and the data was compared in terms of hypericin contents. Hypericin contents of the samples are shown in Table 2. The level of hypericin in cell suspension cultures was very low compared to callus cultures.

Discussion
The calli grown in medium containing 1 mg l -1 BA and 2 mg l -1 NAA (% 92.3±4.63)1 and 1 mg l -1 BA and 0.4 mg l -1 2,4-D (% 56.2±3.42) were used for the initiation of cell suspension due to friable and granuler structure calli.. Biomass in cell suspension culture was higher in medium containing 2,4-D (6.31 fold) compared to that of NAA (6.28 fold). [24] reported that MS basal media containing BA in combination with 2,4-D showed the highest calli formation, while Walker et al. (2002) [25] reported that BA in combination with 2,4-D was efficient for calli formation. Ayan et al. (2005) [26] obtained that the highest frequency of callus induction obtained from the leaf discs cultivated on the medium supplemented with of 2,4-D and kinetin. According to previous reports, in combinations with 2,4-D and kinetin or BA proved to be effective plant growth regulators for callus induction and cell suspension cultures from H. perforatum. But our findings showed that the combinations of NAA and BA was most efficient for calli induction and establish cell suspension cultures for H. triquetrifolium.
Hypericin content of calli in MS medium containing 2,4-D were 0.0527 ± 0.0043 mg g -1 ,while this rate was 0.0018 ± 0.00026 mg g -1 in suspension cells. Hypericin content of calli in the medium containing NAA was 0.0485 ± 0.0077 and in suspension cells, this rate was 0.0016 ± 0.00021. Several studies have been reported on callus and shoot cultures of H. triquetrifolium. A similar study on hypericin content in callus cultures of H. triquetrifolium Turra. conducted by Oluk et al.(2010) [27] and they detected 0.048 mg g -1 amount of hypericin in calli of H.triquetrifolium. In another study, Santarem and Astarita (2003) [28] reported that the level of hypericin in callus of H. perforatum was very low, representing only 0.11% of the amount found in the field-grown plants. Kartnig and Göbel (1992) [29] reported that cell cultures of H. perforatum, H. Maculatum and H. tomentosum produced hpericin and pseudohypericin.
Moreover, it has also been reported that in cell cultures of some Hypericum species were detected presence of hypericin, but this rate was very low in comparison with ex vivo plants [30,31]. It has been reported that callus initiated from stamens of H. perforatum showed only traces of hypericin or pseudohypericin [32]. Another result we obtained Alhough biomass in the MS medium containing NAA was higher than 2,4-D (Fig. 2), in cell suspension culture, hypericin production of medium containing 2,4-D and NAA was close to each other (Table 1). Pasqua et al, (2003) [33] reported that although undifferantiated calli and suspension cultures in MS medium containing 1.105 mgI -1 2,4-D, 0.215 mgI -1 Kinetin and 0.186 mgI -1 NAA was not produce but callus with vegetative buds produced hypericin (0.015 %) and pseudohypericin (0.050 %).
In our study, spectrophotometric analysis of in vitro cultures of H. triquetrifolium showed that hypericin contents was different from each other, suggesting that this in vitro culture system can be improvement for selection of plants with high levels of hypericin. Some researchers reported the utilization of elicitors influenced the hypericin production in calli and cell suspension cultures. Xu et al. (2011) [34] reported that ozone treatment to cell susspension cultures enhanced hypericin production about 4-fold. presence of salycilic acid in calli and suspension cells, hypericin production enhanced 2-fold [35]. Xu et al (2005) [36] reported that nitric oxide elicitation enhanced hypericin production.

Conclusions
The present study has also shown that cell cultures of H. triquetrifolium provide an alternative for hypericin production due to presence of hypericin. However, the commercial application of cell culture for hypericin is limited due to the low production. It needs optimization of the used culture media. To perform this, it needs elisitation of the media.