Immunomodulatory Effects of Diterpene Quinone Derivatives from the Roots of Horminum pyrenaicum in Human PBMC

Several phytochemicals were shown to interfere with redox biology in the human system. Moreover, redox biochemistry is crucially involved in the orchestration of immunological cascades. When screening for immunomodulatory compounds, the two interferon gamma- (IFN-γ-) dependent immunometabolic pathways of tryptophan breakdown via indoleamine 2,3-dioxygenase-1 (IDO-1) and neopterin formation by GTP-cyclohydrolase 1 (GTP-CH-I) represent prominent targets, as IFN-γ-related signaling is strongly sensitive to oxidative triggers. Herein, the analysis of these pathway activities in human peripheral mononuclear cells was successfully applied in a bioactivity-guided fractionation strategy to screen for anti-inflammatory substances contained in the root of Horminum (H.) pyrenaicum L. (syn. Dragon's mouth), the only representative of the monophyletic genus Horminum. Four abietane diterpene quinone derivatives (horminone, 7-O-acetylhorminone, inuroyleanol and its 15,16-dehydro-derivative, a novel natural product), two nor-abietane diterpene quinones (agastaquinone and 3-deoxyagastaquinone) and two abeo 18 (4 → 3) abietane diterpene quinones (agastol and its 15,16-dehydro-derivative) could be identified. These compounds were able to dose-dependently suppress the above mentioned pathways with different potency. Beside the description of new active compounds, this study demonstrates the feasibility of integrating IDO-1 and GTP-CH-I activity in the search for novel anti-inflammatory compounds, which can then be directed towards a more detailed mode of action analysis.


Introduction
Throughout the history of mankind, natural product extracts were used for the treatment of a broad range of inflammation-associated symptoms such as infections, fever, or pain [1].Nowadays, natural products are innovative sources for the development of modern therapeutic drugs [2].Approximately 25% of the modern medications originate from plants and/or phytochemicals [3].Still, there is an enormous interest in the identification and analysis of phytochemicals to discover more effective agents for the treatment of diseases or the alleviation of symptoms that are associated with immune activation and inflammation.Specific therapeutic targets can be addressed with bioactivity-guided fractionation strategies, which have emerged as useful tools in the identification of active metabolites from plant extracts and complex mixtures.
Immune activation and oxidative stress is linked with a number of chronic diseases such as infections, autoimmune syndromes, malignancies, neurodegenerative and cardiovascular disorders.The modulation of processes by which oxidative stress induces inflammation and vice versa is suggested to be a general property of substances, which have anti-inflammatory, anti-aging, and health promoting effects [4,5].
Several plants of the Lamiaceae family, which is rich in diterpene compounds, are known for their antioxidant and/ or anti-inflammatory activities, including sage, thyme, rosemary, and lavender [6].After an initial screening, a dichloromethane extract of the roots of Horminum pyrenaicum L. (syn.: Pyrenean Dead-nettle or Dragon's mouth), a European representative of the Lamiaceae family, was chosen for bioguided isolation.
Genetic studies positioned the genus Horminum within the tribe Mentheae into a close relation to the genera Prunella and Cleonia [7].H. pyrenaicum is the only representative of this monophyletic genus.It grows on chalky and stony soils as well as on rough pastures in a height around 1400 till 2500 m and is domestic in the south of the Alps and in the Pyrenees [8].It is a perennial herbaceous plant, which is able to reach 10 to 30 cm height with a strong ligneous tap-root and a plain or weakly branched rhizome [9].Known constituents of H. pyrenaicum are diterpenes like horminone (=7-hydroxyroyleanone) and 7-O-acetylhorminone in the aerial plant parts as well as agastaquinone, coleon U 12methylether and 3-deoxyagastaquinone.So far, the latter compound is only described from this species, and it is contained in the sub-aerial plant parts [10].
Quinones are ubiquitous in nature and derivatives of quinones are common components of biologically relevant molecules such as K vitamins or compounds that are directly involved in oxidative metabolism like coenzyme Q10.
The diterpene derivatives (phytocannabinoids) present in Cannabis plants (Cannabis sativa L., Cannabis indica Lam.) were previously reported to exert potent anti-inflammatory and immunomodulatory effects, which could also be demonstrated in an in vitro setting of mitogen-induced peripheral blood mononuclear cells (PBMC), by applying neopterin formation and tryptophan breakdown as sensitive and convenient readouts [11].This in vitro model has been used to monitor immunomodulatory activities for a broad variety of phytochemicals, drugs, or plant extracts [12].
Briefly, PBMC are isolated from whole blood, treated with test substances and stimulated with mitogens such as phytohaemagglutinin (PHA), which efficiently induce the release of interferon-γ (IFN-γ) and initiate T helper (Th) type 1 responses.Consequently, tryptophan breakdown and neopterin formation via the enzymes indoleamine 2,3-dioxygenase-1 (IDO-1) and GTP-cyclohydrolase 1 (GTP-CH-I) is initiated [13,14].The measurement of these biomarkers allows evaluation and quantification of the immunomodulatory properties of compounds in an in vitro setting, where the interplay between human T-cells and monocytes/macrophages is mimicked.
Activation of the biochemical pathways of tryptophan catabolism and neopterin formation are central events during the cellular (Th1-type) immune reaction [15].Dysregulation and/or overactivation of these pathways is associated with infections, cardiovascular diseases, neurodegenerative disorders, with many different types of cancers, and with the process of aging [16][17][18][19].The Th1-type cytokine IFN-γ is the major inducer of these and many other proinflammatory signaling cascades [20].IDO-1 catalyzes the rate-limiting step in the conversion of the essential amino acid tryptophan into kynurenine [14,21].The kynurenine to tryptophan ratio (Kyn/Trp) is indicative for the activity of IDO-1, when additional inflammation markers such as neopterin are present [22].Neopterin is formed by the enzyme GTP-CH-I in response to IFN-γ, and has been applied as biomarker of Th1-type immune response in several in vitro and clinical settings [23].High Kyn/Trp and high neopterin levels usually coincide in patients suffering from various Th1-related immunopathologies [19].
Within this study, bioactive diterpene quinones derived from H. pyrenaicum were identified through a bioactivityguided separation and isolation process, by focusing on immunomodulatory, preferentially anti-inflammatory properties.The bioactivity screen was based on the test system described above, using mitogen-stimulated human PBMC and tryptophan breakdown as well as neopterin formation, two important immunometabolic pathways of the cellular immune response, as readout.

Material and Methods
2.1.General Experimental Procedures.All reagents used were of analytical grade and were purchased from Sigma Aldrich (Vienna, Austria).HPLC solvents were of gradient grade.Technical grade solvents were distilled before use.Water was produced by reverse osmosis followed by distillation.Optical rotations were measured using a Perkin-Elmer (Wellesley, MA) 341 polarimeter.1D-and 2D-NMR spectra were acquired with a Bruker (BrukerBiospin, Rheinstetten, Germany) Ultrashield plus 600 spectrometer or a Bruker DRX 300 spectrometer using CDCl 3 or deuterated acetonitrile (containing 0.03% TMS) (Euriso-Top, Saint-Aubin, France).Chemical shift values were referenced to the residual solvent signals.HPLC analyses were carried out using an HP 1050 system (Agilent, Waldbronn, Germany) equipped with autosampler, DAD, and column thermostat.Separations were performed on a Phenomenex (Torrance, CA) Luna 3 μm Phenyl-Hexyl column (150 × 3.00 mm).A mobile phase consisting of 0.02% TFA and 1% THF in H 2 O (v/v) (solvent A) and 25% THF in MeOH (v/v) (solvent B) was employed with gradient elution (0 min, 40% B; 5 min, 40% B; 15 min, 65% B; 40 min, 80% B; 50 min, 99% B; 70 min, 99% B).The detection wavelength was 280 nm, and the thermostat was set at 45 °C.The injection volume was 10 μL; the flow rate was 0.3 mL/min.For LC-ESI-MS experiments, the HPLC system was coupled to a Bruker (Bruker Daltonics, Bremen, Germany) Esquire 3000plus iontrap, ) was deposited at the Herbarium of the Institute of Pharmacy/Pharmacognosy, University of Innsbruck, Austria.The air-dried root material (842 g) was grounded and defatted by exhaustive maceration with petroleum ether at room temperature yielding 4.62 g of red-brown petroleum ether extract (0.55%).For a prescreen for potential biological activity in the cell-based assay system described below, 25.0 g of the defatted plant material was re-extracted by sonication for 15 minutes with 200 mL dichloromethane (DCM).This procedure was repeated three-times, yielding 341 mg after evaporation of the solvent (1.36%).For compound isolation, 750 g of the dried plant material were re-extracted by Soxhlet extraction with dichloromethane for 24 hours at room temperature, yielding 14.1 g of extract.
A part of the fraction (195.2 mg) eluted at 580 mL to 650 mL with hexane/DCM (95 + 5), was used for further separation.The obtained fraction was purified by HSCCC using a system of heptane/MeCN/DCM (10 + 6 + 3, v/v/v) using the upper phase as mobile phase (head to tail) with a flow-rate of 1.00 mL/min, 800 rpm, in a 230 mL coil.The eluate was combined to 14 fractions.Fraction 5 (eluted at 270-315 mL) contained 7.8 mg of pure compound 8. Fraction 10 (eluted at 595-680 mL) was purified by preparative TLC using chloroform as mobile phase, yielding 3.6 mg of compound 3.The same procedure was applied to fraction 9 (eluted at 535-590 mL) revealing 4.1 mg of compound 2. Fraction (eluted at 340-415 mL) was further purified by semipreparative HPLC; 2.0 mg of compound 6 and 3.5 mg of compound 7 could be purified in this way.Fraction 8 (eluted at 420-530 mL) was further purified by Sephadex-LH20 CC using DCM/acetone (85 + 15, v/v) as mobile phase yielding 8.9 mg of compound 1 as well as a fraction (13 mg) containing compounds 4 and 5. Due to the small amount of the obtained mixture, the structure elucidation of compounds and 5 was performed by means of LC-SPE-NMR [24].Therefore, the compound mixture (13.2 mg) was dissolved in 1.00 mL methanol.2.5.Cell Culture and PBMC Isolation.The study was performed in accordance with the Declaration of Helsinki.PBMC were isolated from healthy donors who gave written consent that their donated blood may be used for scientific purposes, in case when it was not selected for transfusion.The PBMC were isolated by density gradient centrifugation as described earlier [12].After isolation, the cells were Oxidative Medicine and Cellular Longevity washed three times with phosphate buffered saline containing 1 μM ethylenediaminetetraacetic acid and subsequently they were maintained in RPMI 1640 (Sigma Aldrich, Vienna, Austria) supplemented with 2 mM L-glutamine, 10% heatinactivated fetal calf serum (Biochrom, Germany) and 50 μg/mL gentamicin (Lonza-BioWhittaker, USA), at 37 °C in a humidified atmosphere containing 5% CO 2 .
The cells were seeded in 48-well plates (1.5 × 10 6 cells/mL/ well) and treated either with increasing concentrations of the extract, the individual fractions or the pure compounds, dissolved in DMSO.The PBMC were either stimulated with 10 μg/mL of the mitogen lectin phytohaemagglutin (PHA) after 30 minutes of treatment, or were left unstimulated.In addition to PHA, the stimulatability of the PBMC was controlled for each experiment by treatment with the lectin concanavalin A (ConA) at a concentration of 10 μg/mL.After 48 h of incubation, cell culture supernatants were collected and stored at −20 °C until analysis.In addition, 1-methyl-Dtryptophan (Sigma-Aldrich, Vienna, Austria) was used as a postive control for the suppression of IDO-1 acitivity [25].

Measurements of Tryptophan and Kynurenine
Concentrations.Briefly, the HPLC analysis of tryptophan and kynurenine was performed on a ProStar Varian system (USA) using rp-18 columns (Merck, Germany) and acetate buffer as eluent (flow-rate: 0.9 mL/min) according to the protocol described earlier [26,27].3-Nitro-L-tyrosine (Sigma Aldrich, Austria) was used as an internal standard.Kynurenine and tryptophan standards were purchased from Sigma-Aldrich (Austria).Kynurenine and 3-nitro-L-tyrosine were detected by UV-absorbance at 360 nm wavelength (Shimadzu SPD-6A UV detector, Austria), tryptophan was detected by its fluorescence with an excitation wavelength of 286 nm and an emission wavelength of 366 nm (ProStar 360 detector, Varian, USA).The Kyn/Trp was calculated, which is an estimate of IDO-1 activity [12] and expressed in μmol Kyn / mmol Trp.The sensitivity of the measurements was 0.5 μmol/L kynurenine and 0.1 μmol/L tryptophan.2.7.Neopterin Measurements.Neopterin concentrations were measured by ELISA (BRAHMS, Germany), according to the manufacturers' instructions with the detection limit of 2 nmol/L.

IC 50
Calculations and Statistical Analysis.The half maximal (50% inhibitory) concentration (IC 50 ) was calculated by using the CalcuSyn software (Biosoft, UK) according the concept of Chou and Talalay [28].The Statistical Package for the Social Sciences (SPSS, version 21, Chicago, USA) was used for the analysis of data with Friedman and Wilcoxon signed-rank tests, as not all data showed normal distribution.P-values below 0.05 were considered to indicate significance.Results are expressed as percent of unstimulated and PHAstimulated control cells and are shown as mean ± standard error of the mean (SEM).

3.2.
Bioactivity-Guided Fractionation.The DCM extract of H. pyrenaicum roots was separated by Sephadex-LH20 column chromatography to obtain 8 fractions (HOR-a1 to HOR-a8).Fractions with similar compound spectra were pooled.Fractions HOR-a3, HOR-a4, HOR-a6, HOR-a7, and HOR-a8 were tested for their immunomodulatory activity in the PBMC cell model to guide the subsequent isolation of bioactive constituents.Several fractions contained bioactive compounds, which dose-dependently suppressed the target pathways (see supplemental figure S1).The half maximal inhibitory concentrations (IC 50 ) for neopterin formation and tryptophan breakdown of the most active fractions HOR-a3, HOR-a4, and HOR-a6 can be found in Table 1.The strongest suppressive effect on both mitogen induced pathway was obtained with fraction HOR-a3, for which neopterin formation was suppressed to 50% at a treatment concentration of 63.5 μg/mL and tryptophan breakdown was reduced to 50% with 7.02 μg/mL HOR-a3 (Figure 2).
In unstimulated cells, there was a minor but significant suppressive effect on both pathways upon exposure to HOR-a3, however at higher treatment concentrations only.Due to the stronger relative effect on Kyn/Trp compared to neopterin formation, the tryptophan breakdown pathway was selected as primary decision making-parameter for further subfractionation of the most active fraction HOR-a3.
In addition to these diterpene quinones, of which only compounds 1 and 2 are known constituents of H. pyrenaicum, the bioguided separation process yielded fraction HP-r4 comprising two major compounds, which were difficult to separate with the used chromatographic techniques.Therefore, in this case, we did not follow the classical approach, that is, separation and isolation, NMR measurement and identification, and biological testing of the pure compounds, but another approach, that is, testing of the compound mixture and subsequent identification of the compounds by LC-SPE-NMR according to a procedure described by Sturm et al. [24].One of the compounds comprised in the mixture was identified as 15,16-dehydroagastol (5) [38].The compound has already been isolated from Agastache rugosa [36].However, it represents a newly identified compound in the species H. pyrenaicum.By means of 1D and 2D-NMR-spectroscopy, the second constituent of fraction HP-r4 was identified as 15,16-dehydroinuroyleanol (4), which is a new natural compound (Figure 4).
Consequently, the isolated diterpenchinones 1-3 and 6-8 as well as fraction HP-r4 comprising a mixture of compounds 4 and 5 were tested on their ability to influence Th1-type immune responses.

Anti-Inflammatory Effects of the Isolated Compounds.
All of the known and identified compounds in this study except inuroyleanol (6) were able to influence tryptophan breakdown in stimulated PBMC at micromolar concentrations and in a dose-dependent manner, however, with different intensities (Table 2, Figure 5).

Conclusion
This study reports the isolation process of H. pyreanicum compounds in a bio-guided way, where fractions with high immunomodulating properties were used to direct further subfractionation processes.This guided separation of bioactive compounds successfully led to the identification of potent diterpene quinone derivatives, which were able to suppress central Th1-type immunometabolic pathways.A variety of phytochemicals were shown to interfere with IDO-1 activity previously, for example, the antioxidants resveratrol [12], the lavender oil constituents (−)-linalool, (+)-α-pinene and (+)-limonene [39], the lignans arctigenin and trachelogenin [40] or the benzylisoquinoline alkaloid berberine [41].In addition, commonly used antiinflammatory medications were shown to interfere with the IDO-1 and GTP-CH-I activity, for example, acetylsalicylic acid (aspirin) [42].These studies further confirm the importance of these pathways to be investigated in mode of action studies of phytochemicals.Furthermore, it should be considered that the suppressive effect can be either indirect by the generation of a more reductive milieu due to the antioxidant nature of several phytochemicals, thereby counteracting T cell activation leading to less IFN-γ production [43], or directly as has been shown for example for arctigenin and trachelogenin that interact with the binding site of the enzyme [44].The investigation of neopterin formation gives a further hint towards the mode of action, as in human macrophages neopterin is produced at the expense of tetrahydrobiopterin (BH4), an essential cofactor for several monooxygenases [45].
Inflammation and immune activation play an important role in the pathogenesis of a variety of diseases and are of importance especially in chronic conditions, when the induction of regulatory T-cells (Treg) counteracts the initial immune activation cascade and contributes to the maintenance of an immunosuppressed state, which is associated with fatal outcome, for example, in patients with cardiovascular disease but also in cancer.This adverse situation is well indicated by increased neopterin and Kyn/Trp levels in patients that are associated with increased mortality [46,47].Hence, the regulatory influence of plant compounds could be of importance to normalize the immunological situation in such patients.Central immunological cascades are redox sensitive, thus targeting these processes could be a successful strategy.However, several recent studies point towards the adverse   [48,49].This might be considered when botanical extracts or phytochemical antioxidants are used without medical consideration, in excess and/or over long periods.Bioactivity guided fractionation of the root material of H. pyrenaicum, enabled the identification of four abietane-diterpene quinone derivatives as horminone (1), 7-O-acetylhorminone (2), inuroyleanol (6) and its 15,16dehydro-derivative (4).Additionally, the nor-abietanes agastaquinone (3) and 3-deoxyagastaquinone (8), and the abeo 18 (4 → 3) abietanes agastol (7) and its 15,16-dehydro-derivative (5) were identified.NMR-data of compounds 1-3 and 5-7 were in agreement with previous reports mentioned above, while compounds (4) and (8) were identified as new natural products.Compounds 1-3 and compounds 6-8 as well as fraction HP-r4 comprising a 1 : 1 mixture of compounds (4) and (5) were tested in the in vitro assay for their immune-modulating properties.Except of inuroyleanol (6), all compounds affected mitogen-induced tryptophan breakdown dose-dependently in the tested concentration range; however, the suppressive effect was of different intensity (Table 2, Figure 5).
Interestingly, fraction HP-r4, which comprises compounds (4) and (5), had the strongest effect on human PBMC with an IC 50 for Kyn/Trp suppression of 25.8 μM.Further investigations on the mode of action of compounds 4 and 5 would be necessary to evaluate their potential as IDO-1 inhibitors.
The here reported properties of the identified compounds clearly support follow-up studies to investigate their effects on further immunological cascades, including neopterin formation.Higher neopterin levels and accelerated tryptophan breakdown is frequently observed in patients with various diseases associated with chronic immune activation [19,23], thus, the here selected target pathways are of high relevance also for the in vivo situation.
The occurrence of so far already known diterpene quinones and the finding of newly identified compounds clearly warrant further investigations of the bioactivities of H. pyrenaicum extracts and contained phytochemicals.
Moreover, the results of this study underline that the model system of mitogen induced PBMC using the  7 Oxidative Medicine and Cellular Longevity immunometabolic pathways of tryptophan breakdown and neopterin formation, is a reliable screening approach for a bio-guided fractionation and isolation of immunomodulatory compounds.By using human PBMC from healthy donors, the aspect of crosstalk between different immune competent cells, most importantly between T-cells and monocytes/macrophages is taken into account [12].Moreover, by using unstimulated and mitogen-stimulated conditions, immune activating and anti-inflammatory properties can be analyzed in parallel.As both neopterin and Kyn/Trp are biomarkers, which have been used for the monitoring of several human diseases, there is a close connection to the in vivo situation.Due to the limitations of every in vitro system, further research will be needed to extrapolate the findings to the human system.In this regard, the combination of the here reported pathwayfocused strategy with an unbiased functional genomics approach could be advantageous in building an interaction model of the compounds of interests with other important immunological pathways, as well as to identify potential unwanted off target effects [50].In conclusion, the here reported approach justifies future studies for an in depth analysis of the immunomodulatory properties of the reported diterpene quinones.) of the isolated compounds on IDO-1 activity, as indicated by the kynurenine to tryptophan ratio in phytohemagglutinin stimulated PBMC, calculated according Chou and Talalay [28] (n.d.: concentration-dependent activity could not be determined in the tested concentration range; cursive: estimated values, only limited repetitions were possible due to sample limitations).

Figure 1 :
Figure 1: Concentrations of neopterin (Neo), tryptophan (Trp), kynurenine (Kyn) as well as the Kyn/Trp, a measure of IDO-1 activity, in the supernatant of unstimulated PBMC and cells stimulated with 10 μg/mL concanavalin A (Con A) or phytohaemagglutinin (PHA) for 48 h.Results shown are the mean values ± SEM of seven independent experiments run in duplicates ( * p < 0 05, compared to unstimulated cells; please note log-scale).

Figure 2 :
Figure 2: Bioactivity of the Horminum pyrenaicum root extract: Unstimulated (grey squares) and phytohemagglutinin (PHA)-stimulated peripheral mononuclear cells (PBMC; black squares) were treated with increasing extract concentrations for 48 h.Neopterin formation (a) and tryptophan breakdown to kynurenine, indicated by the Kyn/Trp (b), was measured in the cell supernatants.Results shown are the mean values ± SEM of three independent experiments run in duplicates.* p < 0 05 indicates significant differences compared to the respective unstimulated or stimulated control cells (set as 100%).

Table 1 :
Half maximal inhibitory concentration (IC 50 ) of Horminum pyrenaicum extract sub-fractions (HOR-a3 to HOR-a6) that had the largest effect on neopterin formation and IDO-1 activity, as indicated by the kynurenine to tryptophan ratio in phytohemagglutinin stimulated PBMC.More details are shown in the supplemental figure S1.