Antimicrobial and Cytotoxic Isohexenylnaphthazarins from Arnebia euchroma (Royle) Jonst. (Boraginaceae) Callus and Cell Suspension Culture

The phytochemical investigation of the n-hexane extract from callus and cell suspension culture of Arnebia euchroma (Royle) Jonst. resulted in the isolation of nine isohexenylnaphthazarins: deoxyalkannin (1), alkannin (2), acetylalkannin (3), isobutyrylalkannin (4), β-hydroxyisovalerylalkannin (5), 2''-(S)-α-methylbutyrylalkannin (6), propionylalkannin (7), teracrylalkannin (8) and acetylshikonin (9). Their structures were determined by MS and NMR spectroscopy. Pigments 2–8 are isolated for the first time from Arnebia in vitro cultures, 4 and 7 are reported in the present work as novel metabolites within the Arnebia genus, while 9 is a known constituent of both natural roots and in vitro cultures of A. euchroma. Moreover, methyl jasmonate and 1-monoglyceryl olate, palmitate and stearate are reported for the first time within the Boraginaceae family. The antimicrobial and cytotoxic activities of all isolated pigment compounds were tested, revealing a very interesting profile.


Introduction
Alkannin, shikonin and related isohexenylnaphthazarin compounds are natural lipophilic red pigments that occur in many species of the Boraginaceae family. The chiral pairs of alkannins/shikonins are potent pharmaceutical substances that have shown significant biological activities including wound healing, antimicrobial, antiinflammatory, antioxidant, anticancer and antithrombotic properties [1][2][3][4]. Because of the importance of the alkannin/shikonin-related compounds in the pharmaceutical and cosmetic industry, cultivation of Boraginaceous species, including the genus Arnebia, have been studied using biotechnological approaches. Arnebia euchroma (Royle) Jonst. tissue culture was pioneered in Russia due to the plant's high content of shikonin/alkannin derivatives [5]. In this study we were focused on the isolation of isohexenylnaphthazarin compounds from callus culture and cell suspension culture of A. euchroma, elucidation of their structures by modern spectral techniques and the evaluation of their antimicrobial and cytotoxic activities.
Regarding their natural abundance within the Arnebia genus, a review of the literature lead to the following findings: (a) compound 1 is abundant in the roots of various Arnebia species, including A. euchroma, and has also been isolated from cell cultures of the latter species [1]; (b) compound 2 is found in the roots of A. hispidissima, A. nobilis, A. tinctoria [1,15] and A. euchroma [13]; (c) compound 3 is found in the roots of A. euchroma, A. hispidissima and A. nobilis [1]; (d) compound 4 is not contained either in the natural roots or in any cell culture so far; (e) compound 5 occurs in the roots of A. euchroma and A. hispidissima [1]; (f) compound 6 occurs in the roots of A. densiflora [13,14,16] and A. euchroma [13]; (g) compound 7 is not produced in either natural roots or in in vitro cultures; (h) compound 8 is found in the roots of A. densiflora [15]; (i) compound 9 is a known constituent of both natural roots and in vitro cultures of A. euchroma [1].
Apart from naphthoquinone pigments, linoleic acid [17] and β-sitosterol [18] have been identified in both in vitro cultures. Additionally, methyl linolate [17] has been isolated from the cell suspension culture (also detected by GC-MS in the roots of other Boraginaceous species [19,20]) as well as methyl jasmonate (10) [21,22] (for the first time within the Boraginaceae family), a triglyceride mixture of palmitic, stearic and 8-Ζ-or 9-Ζ-or 11-Ζ or 9-Ε-octadecenoic acid (as determined after transesterification/methylation and GC-MS analysis of the resulting fatty acid methyl esters [23]) and also a mixture of 1-monoglycerides of palmitic, stearic and oleic acid (as determined after transesterification/methylation and GC-MS analysis of the resulting fatty acid methyl esters [23], as well as after GC-MS analysis of the silylated derivatives which were identified as 2,3-O-bis(trimethylsilyl)-1-glyceryl palmitate, stearate and oleate respectively) probably for the first time within Boraginaceae family.

Antimicrobial Activity
According to our results ( Table 1) the n-hexane extract of both callus and cell suspension culture of A. euchroma, showed a very interesting broad antimicrobial profile against all the assayed microorganisms, while among the pure tested compounds the most active compounds were alkannin, acetylshikonin, β-hydroxyisovalerylalkannin and isobutyrylalkannin. It is noteworthy that acetylshikonin was much more active in comparison with its enantiomer acetylalkannin.

Cytotoxic Activities
To study the cytotoxicity of the isolated naphthoquinone derivatives, they were subjected to a viability assay using different cancer cell lines. IC 50 values of active compounds were determined ( Figure 2 and Table 2). Among 1-9, 6 showed the highest activity against all assayed cancer cell lines and was even more active than 2 which is believed to be the most active isohexenylnaphthazarine. Compounds 3 and 4 also exhibited higher cytotoxicity than 2, while 1, 5, 7 and 8 exhibited similar IC 50 values, with the exception of 7, which was not active against U251 cells up to 100 µM. Our results indicate that the naphthoquinone moiety is more important for activity than the side chain and that the side chain is a kind of modulator of the activity, an assumption that was also made by Cui et al. [24]. Similar observations were also made by Papageorgiou [25] concerning antimicrobial activity. Lu et al. [26] reported that also the chiral center does not affect activity. Our findings support this because 1, which lacks the chiral center, showed comparable activity with the other derivatives and the activities of enantiomers 3 and 9 did not differ significantly.

Chemicals and Reagents
Silica gel 60 and TLC plates Kieselgel 60, 0.2 mm layer thickness, were purchased from Merck Chemical Co. (Darmstadt, Germany). Bands on TLC plates were detected under UV light (254 and 366 nm) and/or after spraying with a 2.5% H 2 SO 4 and 2.5% vanillin methanolic solutions and heating at 105 °C for 5 min. Dichloromethane was used as an extraction solvent for the compound bands obtained from preparative TLC, using 20 cm × 20 cm plates each one loaded with ≤15 mg of sample. Solvents including n-hexane, cyclohexane, ethyl acetate, dichloromethane, diethyl ether and toluene were purchased from Lab Scan Ltd (Dublin, Ireland). BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) was purchased from Merck (Darmstadt, Germany).

Instruments and Methods
The GC-MS analysis was performed with a Hewlett Packard Gas Chromatograph 5890 Series II Plus linked to a Hewlett Packard 5972 mass spectrometer system equipped with a 30 m long  0.25 mm i.d. and 0.5 µm film thickness HP5-MS capillary column. The temperature was programmed from 100 to 300 °C at a rate of 4 °C/min. Helium was used as a carrier gas, flow rate 0.7 mL/min. Split ratio 1:20, injector temperature 220 °C, ionization voltage 70 eV. Silylated samples for GC were obtained after addition of excess of BSTFA in pyridine followed by heating at 80 °C for 20 min. ESI-MS spectra were obtained using a 3200 QTRAP LC/MS/MS Applied Biosystems mass spectrometer using an ion spray voltage of +4,500 V, declustering potential of 50 V and entrance potential of 10V. 1 H-and 13 C-1D, HSQC, and COSY 2D-NMR spectra were recorded on Bruker Avance 400 and 200 MHz FT-NMR spectrometers.
[α] D values were measured in dichloromethane using a Perkin Elmer 341 polarimeter.

Plant Callus and Cell Suspension Culture
Callus tissue of A. euchroma was maintained on MSA solid medium [5]

Compound Characterization
All structures were identified by MS (samples dissolved in methanol) and NMR spectroscopy.  effect on their growth and viability. In case of CCRF-CEM cells, aliquots (100 µL) of 1 × 10 5 cells/mL were seeded in 96-well plates (flat bottom) and substances were added immediately. All cells were incubated with the substance of interest for 72 h before XTT solution was added. Vinblastine served as a positive control. XTT is a yellow tetrazolium salt (sodium 3'-[1-(phenylaminocarbonyl)-3,4tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) and cleaved by metabolic active cells into an orange formazan dye. This colour change occurs only in viable cells and can be directly quantified using a scanning multiwell spectrophotometer [28]. Numbers of viable cells were determined with the following formula expressed as percentage of control: (absorbance of treated cells/absorbance of untreated cells) × 100 [29]. IC 50 values were calculated using SigmaPlot 11.0 (Systat Software Inc., San Jose, CA, USA) and the four parameter logistic curve.