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Pentacyclic triterpenes in birch bark extract inhibit early step of herpes simplex virus type 1 replication.

ABSTRACT

Antiviral agents frequently applied for treatment of herpesvirus infections include acyclovir and its derivatives. The antiviral effect of a triterpene extract of birch bark and its major pentacyclic triterpenes, i.e. betulin, lupeol and betulinic acid against acyclovir-sensitive and acyclovir-resistant HSV type 1 strains was examined. The cytotoxic effect of a phytochemically defined birch bark triterpene extract (TE) as well as different pentacyclic triterpenes was analyzed in cell culture, and revealed a moderate cytotoxicity on RC-37 cells. TE, betulin, lupeol and betulinic acid exhibited high levels of antiviral activity against HSV-1 in viral suspension tests with [IC.sub.50] values ranging between 0.2 and 0.5 [micro]g/ml. Infectivity of acyclovir-sensitive and clinical isolates of acyclovir-resistant HSV-1 strains was significantly reduced by all tested compounds and a direct concentration- and time-dependent antiherpetic activity could be demonstrated. In order to determine the mode of antiviral action, TE and the compounds were added at different times during the viral infection cycle. Addition of these drugs to uninfected cells prior to infection or to herpesvirus-infected cells during intracellular replication had low effect on virus multiplication. Minor virucidal activity of triterpenes was observed, however both TE and tested compounds exhibited high anti-herpetic activity when viruses were pretreated with these drugs prior to infection. Pentacyclic triterpenes inhibit acyclovir-sensitive and acyclovir-resistant clinical isolates of HSV-1 in the early phase of infection.

Keywords:

Betulin

Birch bark

Herpes simplex virus

Acyclovir resistance

Introduction

Plants produce a variety of biochemical constituents with the potential to inhibit viral replication, compounds from natural sources are of interest as possible sources to control viral infection (Khan et al. 2005). Plant extracts have been widely used in traditional medicine to treat a variety of infectious diseases and represent an abundant source of new bioactive secondary metabolites. Triterpenes are biologically active secondary plant substances that display antimicrobial (Pavlova et al. 2003; Woldemichael et al. 2003; Aiken and Chen 2005), hepatoprotective (Shikov et al. 2011), anticancer (Cichewicz and Kouzi 2004) and anti-inflammatory (Fernandez et al. 2011) effects. Betulinic acid mediates a specific cytotoxicity and induces apoptosis in skin cancer cell lines by direct effects on mitochondria (Fulda et al. 1998). Cytotoxic effects for triterpenes were observed in cancer cell lines originating from breast, colon, lung and neuroblastoma as well as antitumor effects in mice (Striih et al. 2013). In particular, the pentacyclic triterpenes betulin, lupeol and betulinic acid display anti-inflammatory activities which accompany immune modulation. Pentacyclic triterpenes are secondary plant metabolites widespread in fruit peel, leaves and stem bark. The outer bark of birches like Betula pendula, B. pubescetis and B. papyrifera consists of cork layers that are rich in pentacyclic triterpenes of the oleanane and lupane types. Among these triterpenes, betulin, a lupane triterpene, predominates with up to 34% of dry weight (Laszczyk et al. 2006). Birch bark also contains lupeol, betulinic acid, erythrodiol and oleanolic acid and is a low-cost waste product in the veneer and paper industry that is usually burned.

Herpes simplex virus (HSV) is differentiated into two antigenic types of type 1 (HSV-1) and type 2 (HSV-2) and infects mucocutaneous membranes. HSV-1 is a wide spread human pathogen, which causes epidermal lesions in and around the mouth, whereas HSV-2 causes genital herpes (Wald et al. 1995). The symptoms caused by herpes infections are usually self-limiting in immunocompetent individuals, but can be extensive and prolonged in immunocompromised patients. Antiviral agents licensed currently for the treatment of herpesvirus infections include acyclovir and its derivatives, nucleoside analogues which function as DNA chain terminators, ultimately preventing elongation of viral DNA (De Clercq 2004). Some of these antiviral agents might produce toxic side-effects. In addition, the emergence of virus strains resistant to commonly used anti-herpesvirus drugs is a growing problem, particularly in immunocompromised patients (Chakrabarti et al. 2000; Chen et al. 2000).

Birch bark has been traditionally used as a diction, wash, or bath additive to treat small wounds and various skin diseases (Kim et al. 2008). Triterpenes are promising leading compounds for the development of new multi-targeting pharmaceutical agents and show anti-inflammatory, anti-microbial and wound-healing properties (Laszczyk 2009; Harish et al. 2008). A synergistic antiviral effect for betulin combined with acyclovir had been observed during intracellular replication (Gong et al. 2004). Oleanolic acid showed an antiviral effect against HSV during intracellular replication similar to acyclovir, as determined by PCR resulting in reduced viral load (Mukherjee et al. 2013). Glycyrrhizin, an oleanane-type triterpenoid, showed moderate in vitro anti-HSV-1 activity (Ikeda et al. 2005) and may attenuate inflammatory responses in HSV infection (Huang et al. 2012). Yu et al. (2013) demonstrated the inhibition of hepatitis C virus entry into cells by oleanolic acid. A previous report described disruption of HIV-1 fusion to cells in a post-binding step through interaction of betulinic acid with the viral glycoprotein gp41 as well as disruption of assembly and budding of HIV-1 particles (Cichewicz and Kouzi 2004). Betulinic acid was targeting the V3 loop of HIV gpl20 thereby inhibiting viral entry (Lai et al. 2008).

Monoterpenes and sesquiterpenes demonstrated anti-herpetic activities as recently described (Astani et al. 2010, 2011), an antiviral activity of triterpenes betulin, betulinic and betulonic acids has been demonstrated previously (Pavlova et al. 2003). However, the antiviral mechanism and the step, at which viral replication was interfered by triterpenes, has not been elucidated. In the present study we have analyzed the antiviral activity of a birch bark extract as well as pentacyclic triterpenes against herpes simplex virus type 1. The infectivity of HSV was significantly reduced in vitro, and the mode of antiviral action was analyzed at different steps in the viral infection cycle.

Materials and methods

Triterpene extract and pentacyclic triterpenes

The outer bark of birch contains pentacyclic triterpenes of the oleanane and lupane types. The raw material for the extract originated from Esthonian veneer industry, the birches belong to Betula pendula Roth, Betula pubescens Ehrh., hybrids of both species and other Betula species with white bark, all belonging to the family Betulaceae. A voucher specimen has been deposited. All Eurasian white-barked birches have the same triterpene content according to Krasutsky et al. (2006). The TE was obtained by a standardized, continuous extraction procedure with n-heptane including a clarification crystallization, the yield of extraction was 75%. No further purification was performed. The extract contained 75.4% betulin, 4.1% lupeol, 3.7% betulinic acid, 0.8% erythrodiol and 0.7% oleanolic acid. Other components in birch cork are betulinic aldehyde, betulonic aldehyde, betulonic acid, acetyloleanolic acid, ursolic acid, and sitosterol (Fig. 1). Quantification of silylated triterpenes within the extract (TE) was performed by GC-FID with external standard calibration in dependence on the method published by Laszczyk (2009). Reference standard material, i.e. betulin, lupeol and betulinic acid were provided by Birken company, structural formulas of these compounds are shown in Fig. 2. The purity of the compounds betulin, lupeol and betulinic acid was determined with HPLC and GC at >98%, >95% and >97%, respectively. TE and the pentacyclic triterpenes were dissolved in 99% ethanol for preparation of 10% stock solutions. For cell culture experiments, TE and triterpenes were further diluted resulting in a final ethanol concentration below 1% which is not toxic for cells and has no antiviral effect.

Acyclovir

Acyclovir was purchased from GlaxoSmithKline (Bad Oldesloe, Germany) and dissolved in sterile water.

Cell culture and viruses

RC-37 cells (African green monkey kidney cells) were grown in monolayer culture with Dulbecco' modified Eagle' medium (DMEM) supplemented with 5% foetal calf serum (FCS), 100 [micro]g/ml penicillin and 100 [micro]g/ml streptomycin. The monolayers were serially passaged whenever they became confluent, cells were plated into 96-well and 6-well culture plates for cytotoxicity and antiviral assays, respectively, and incubated at 37[degrees]C in an atmosphere of 5% C[O.sub.2] (Schnitzler et al. 2008). Acyclovir-sensitive herpes simplex virus type 1 strain KOS and two clinical, acyclovir-resistant isolates were used for experiments (Schnitzler et al. 2007). Viruses were routinely grown on RC-37 cells as described previously (Heidary Navid et al. 2013).

Cytotoxicity and plaque inhibition assay

For cytotoxicity assays, 5 x [10.sup.4] cells were seeded into 96-well plates per well and incubated for 24 h at 37[degrees]C. The medium was removed and fresh DMEM (Dulbecco's modified minimal essential medium) containing the appropriate dilution of TE or triterpenes was added onto subconfluent cells in eight replicates for each concentration of the drugs. Wells containing medium with 1% ethanol but no drug were also included on each plate as controls. After 3 days of incubation, the growth medium was removed and viability of the drug treated cells RC-37 was determined in a standard neutral red assay (Soderberg et al. 1996). The mean OD of the cell-control wells was arbitrarily assigned to 100%. The cytotoxic concentration of the drug which reduced viable cell number by 50% ([CC.sub.50]) and the maximum noncytotoxic concentration of each drug were determined from dose-response curves. Inhibition of HSV replication was evaluated with plaque reduction assays. Usually 100 plaque forming units (pfu) were incubated with different concentrations of TE or selected compounds for 1 h at room temperature, afterwards treated viruses were allowed to adsorb to RC-37 cells for 1 h at 37[degrees]C. The residual inoculum was then discarded and infected cells were overlaid with medium containing 0.5% methylcellulose. Each assay was performed in six replicates. After incubation for 3 days at 37[degrees]C, monolayers were fixed with 10% formalin, stained with 1 % crystal violet and subsequently clearly visible plaques were counted visually. By reference to the number of plaques observed in virus control monolayers without addition of drugs, the concentration of test compound which inhibited plaque numbers by 50% ([IC.sub.50]) was determined from dose-response curves (Koch et al. 2008).

Mode of antiviral activity

Cells and viruses were incubated with drugs at different stages during viral infection cycle in order to trace the mode of antiviral action. Cells were pretreated with drugs prior to infection with HSV, or viruses were incubated with TE or triterpenes for 1 h at room temperature prior to infection, or the infected cells were incubated for 1 h after infection of HSV with drugs for 72 h (Koch et al. 2008). In all experiments, drugs were used at the maximum non-cytotoxic concentration, untreated virus infected cells were used as control. The number of plaques of treated cells and viruses were compared to untreated controls to calculate the extent of plaque reduction and acyclovir was used as positive control in all assays. Untreated controls always contained 1 % ethanol in order to exclude any effect of ethanol on cells or viruses.

[FIGURE 1 OMITTED]

Detection of protein expression by immunoblot

To evaluate the effect of the compounds on HSV-1 protein expression at different steps of viral replication, western blot analysis was performed. Cells were infected with HSV-1 KOS at a multiplicity of infection (MOI) of 0.1 for 1 h and then treated with maximum noncytotoxic concentration of compounds at 1, 4, 8 h p.i. The plates were incubated for 18 h, then cells were lysed and samples were separated electrophoretically on a 12% SDS-PAGE gel and electroblotted onto polyvinylidene difluoride membranes. After blocking, membranes were incubated overnight with anti-ICP27, anti-UL42, anti-gB or anti-gD antibodies. After incubation with the corresponding secondary antibodies conjugated to horseradish peroxidase, protein bands were revealed by chemiluminescence. Anti-[alpha]-tubulin was used as a control for cellular protein expression (Cardozo et al. 2011).

[FIGURE 2 OMITTED]

Virucidal activity, attachment and penetration assay

To determine the effect of TE and its components on direct inactivation of virus particles, HSV-1 (2 x [10.sup.5] pfu/100 [micro]l) was treated with an equal volume of drugs at room temperature. After 1 h, 1000-fold dilutions of the mixtures were added to RC-37 cell monolayers for 1 h at 37[degrees]C. The cell monolayers were overlaid with media containing 0.5% methylcellulose and 2% FBS to be plaque-assayed. For the attachment assay, RC-37 cell monolayers were grown in 6-well culture plates and then pre-chilled at 4[degrees]C for 1 h. The medium was aspirated and the cell monolayer was infected with 100 pfu/well in the absence or presence of serially diluted drugs up to the maximum noncytotoxic drug concentrations. After further incubating the infected cell monolayer at 4[degrees]C for another 3h, the medium was aspirated to remove unadsorbed virus. Cell monolayer was then washed with cold PBS and overlaid with medium containing 0.5% methylcellulose and plaque assayed (Cheng et al. 2004). To determine the effect of drugs on viral penetration, an assay was conducted according to Gescher et al. (2011). Cells were prechilled at 4[degrees]C for 1 h followed by infection with 100pfu of HSV per well for 2 h at 4[degrees]C. After washing off the unbound virus, the drugs were added for another 30 min at 4[degrees]C. Then, temperature was shifted to 37[degrees]C to allow penetration. After 30 min at 37[degrees]C, cells were treated with citrate buffer (135 mM NaCl, 10nM KCl, 40 mM Na-citrate, pH = 3) to stop penetration and to inactivate attached, unpenetrated virions.

Statistical analysis

All experiments were performed in triplicate and statistical analysis was performed by Microsoft Excel 2010. The means and standard errors were recorded.

Results

Cytotoxicity

TE and pentacyclic triterpenes were serially diluted and added to cell culture medium to examine the effect on the growth and viability of tissue culture cells. All drugs were further diluted in medium for cell culture experiments, always resulting in an ethanol concentration below 1% which had no effect on cells and viruses. Cell monolayers were grown in medium containing different concentrations of these drugs. After 3 days of incubation, viability of RC-37 cells was determined with the neutral red assay. The maximum noncytotoxic concentrations of the tested drugs were determined at 1 [micro]g/ml for TE, betulin, lupeol, and betulinic acid (Table 1). [CC.sub.50] values were determined using increasing drug concentrations for cell incubation (Fig. 3).

Virus inhibition

The potential antiviral effect of triterpene extract and its major constituents was determined against herpes simplex virus type 1 (HSV-1) in vitro. HSV-1 was pretreated for 1 h with various concentrations of the tested drugs. In all assays untreated virus-infected cells without addition of drugs were used as negative control. HSV-1 was incubated with serially diluted drugs for 1 h at room temperature. Subsequently, aliquots of each dilution were added on the cells and incubated for 1 h at 37[degrees]C. In all experiments untreated virus infected cells incubated with 1% ethanol were used as control, to exclude any influence of ethanol on viral replication. The percent reduction was calculated relative to the amount of virus produced in the absence of the drug. The 50% inhibitory concentrations ([IC.sub.50]) for HSV-1 of TE and its constituents were in the range between 0.2 [micro]g/ml and 0.5 [micro]g/ml. The results are shown as percentage of the control and represent the mean of three independent experiments. In plaque reduction assays, all drugs exhibited a concentration-dependent antiviral effect (Fig. 4). Using maximal noncytotoxic concentrations of the extract or the isolated compounds in viral suspension assays, plaque formation for HSV-1 was reduced by >90%. From the obtained data, the selectivity indices for tested drugs were calculated as the [CC.sub.50]/[IC.sub.50] ratio and are shown in Table 1.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Mechanism of antiviral activity

Herpesvirus replication is characterized by a complex sequence of different steps at which antiviral agents might interfere. In order to investigate the inhibitory effects on herpes simplex virus in detail, TE and its major compounds were added at different steps during viral infection with acyclovir-sensitive and acyclovir-resistant HSV-1 strains. Tested drugs did not show a significant effect on HSV when host cells were pretreated prior to infection (Fig. 5A). Preincubation of HSV-1 with the tested drugs caused a significant suppression of HSV multiplication. At maximum noncytotoxic concentrations of the tested drugs, infectivity was significantly reduced for the acyclovir-sensitive as well as the acyclovir-resistant strains (Fig. 5B). In contrast, when the extract or single triterpenes were added to the overlay medium after penetration of the viruses into the host cells, plaque formation was not significantly reduced (Fig. 5C). For comparison, all untreated controls contained the same concentration of ethanol as the drug-treated viruses, in order to exclude any influence of ethanol. Acyclovir showed the highest antiviral activity when added during the intracellular replication period with inhibition of the viral replication of >98% of the acyclovir-sensitive strain. However, acyclovir was not active against both acyclovir-resistant clinical isolates. This drug inhibits specifically the viral DNA polymerase during the replication cycle when new viral DNA is synthesized. Viral protein expression was analyzed when drugs were added during the intracellular replication by immunoblot. TE and the pentacyclic triterpenes did not inhibit expression of herpetic proteins of immediate early, early and late ([alpha], [beta] and [gamma]) HSV-1 genes. Only acyclovir inhibited expression of HSV-1 late proteins (Fig. 6A). In order to determine a potential virucidal effect of extract and compounds against virus particles, HSV-1 was treated at room temperature with the maximum noncytotoxic drug concentration. After 1 h, these drug-treated viruses were highly diluted with medium as typically performed in virucidal assays and added to RC-37 cell monolayers. TE and triterpenes revealed low virucidal activity (data not shown). We next evaluated the ability of TE and pentacyclic triterpenes to affect viral attachment and penetration into cells. The attachment assay was carried out at 4[degrees]C, which allows viral binding but not viral entry. Our results showed only marginal interference of all tested drugs during attachment (Fig. 6B) or penetration of virus particles (data not shown). Heparin, an attachment inhibitor for HSV-1, interfered with all tested HSV-1 strains (Fig. 6B).

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Time-dependent antiviral activity

In order to examine the time-dependence of the antiviral effect, acyclovir-sensitive and acyclovir resistant HSV-1 strains were incubated with maximum noncytotoxic concentrations of tested drugs at different periods of time. After 5, 10, 15, 20, 30 and 60 min, an aliquot was applied on confluent monolayers of RC-37 cells. A clearly time-dependent activity could be demonstrated for triterpene extract and pentacyclic triterpenes for all tested HSV-1 strains. The results for acyclovir-resistant strain 1 are shown in Fig. 7. After 60 min of incubation with tested drugs, the infectivity of HSV-1 was nearly completely abolished.

Discussion

We have investigated a birch bark triterpene extract (TE) in comparison to its major pentacyclic triterpenes in vitro for their antiviral activity against acyclovir-sensitive and acyclovir-resistant clinical isolates of HSV-1. Experiments to assess the cytotoxicity of TE and pentacyclic triterpenes indicated a low toxic behaviour in cell cultures (RC 37) according to Halle and Gores (1987). This is in accordance with Gong et al. (2004), who demonstrated also a low cytotoxicity for betulin in cell culture on Vero cells. We have analyzed the in vitro inhibitory effect of TE and related pentacyclic triterpenes on HSV-1 strains using plaque reduction assays. At maximum noncytotoxic concentration of these drugs, plaque formation of herpesvirus was significantly reduced. Similar results have been reported previously for triterpenoids (Li et al. 2007), e.g. saponin, however the antiviral effect was achieved with about 50 fold higher concentrations of these drugs when compared to the pentacyclic triterpenes used in our experiments. In order to determine the mode of antiviral action, time-on addition experiments have been performed at different steps of the herpesvirus replication cycle. Pretreatment of host cells with TE or pentacyclic triterpenes and addition of these drugs during intracellular replication of HSV revealed only minor effects on viral replication. Interactions between phytochemicals and a multi-target therapeutic concept of phytotherapy have been demonstrated recently (Efferth 2009). In our experiments, triterpenes achieved only minor effects during intracellular replication. Virudicity of TE and triterpenes as well as the effect of these drugs on viral attachment and penetration were only marginal. Cells treated with neem bark extract inhibited HSV-1 glycoprotein-mediated cell-cell fusion and polykaryocytes formation suggesting an additional role at the viral fusion step (Tiwari et al. 2010). A phytochemical analysis of the neem bark extract has not been performed, thus the underlying antiviral compounds were not identified. However a drastic decrease in viral infectivity was detected for HSV-1 in our experiments, when viruses were treated with TE and pentacyclic triterpenes prior to infection, thus a high antiviral activity probably due to direct drug-virus interaction was detected. Jaki et al. (2008) explored the variability of biological responses from the perspective of sample purity and introduced the concept of purity-activity relationships in natural product research. The plant triterpene ursolic acid was analyzed for anti-tuberculosis activity in their study, varying inpurities in ursolic acid preparations were the likely cause of antimycobacterial potential. However, the purity of our compounds was determined to be very high, thus the biological activity is reliable. Pentacyclic triterpenes betulin, lupeol and betulinic acid inhibited acyclovir-resistant clinical HSV-1 isolates, indicating a different antiviral mechanism. Some essential oils, e.g. from thyme, were shown to be effective against acyclovir-resistant HSV, too (Schnitzler et al. 2007).

[FIGURE 7 OMITTED]

Glycyrrhizin reduced hepatocellular damage in chronic hepatitis B and C infection and virus-induced cirrhosis was reduced in chronic hepatitis C. Mechanism for antiviral activity of glycyrrhizin include reduced transport to the membrane and sialylation of hepatitis B virus surface antigen and reduction of membrane fluidity leading to an inhibition of fusion of the viral membrane of HIV-1 with the cell (Fiore et al. 2008). Similar results have been reported for the monoterpene terpinene (Astani et al. 2010) and sesquiterpene [beta]-caryophyllene (Astani et al. 2011). Our results indicate that pentacyclic triterpenes affected viruses before adsorption and in a different manner than acyclovir. Attachment and penetration of HSV to host cells was only marginally affected. It remains to be determined whether the antiherpetic effect of is due to binding of the drugs to viral proteins involved in early steps of viral infection. However, minor triterpenes present in the TE extract need to be analyzed for their antiherpetic potential in future experiments. Triterpene extract from birch bark has been evaluated for toxicity in rats and dogs and provides a high safety profile and its betulin is bioavailable (Jager et al. 2008). Also a betulin-based emulsion without preservatives and detergent emulsifiers was prepared from birch bark extract and applied successfully for the treatment of a severe necrotizing herpes zoster in an immunosuppressed patient who had not responded to a conventional topical treatment. The emulsion demonstrated impressive skin tolerance and anti-zoster properties (Weckesser et al. 2010).

The birch bark triterpene extract demonstrated antiherpetic activity in vitro and was also highly active against acyclovir-resistant herpesvirus strains. Clinical trials are necessary to evaluate the in vivo antiherpetic efficacy.

ARTICLE INFO

Article history:

Received 4 August 2013

Received in revised form 5 May 2014

Accepted 9 June 2014

http://dx.doi.org/10.1016/j.phymed.2014.06.007

Acknowledgement

We would like to thank Dr. A. Sauerbrei, University of Jena, for providing clinical HSV-1 isolates.

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M. Heidary Navid (a), M.N. Laszczyk-Lauer (b), J. ReichlingS P. Schnitzler (a), *

(a) Department of Infectious Diseases, Virology, University of Heidelberg, im Neuenheimer Feld 324, 69120 Heidelberg, Germany

(b) Birken AG, Streiflingsweg 11, Niefem-Oschelbronn, Germany

(c) Department of Biology, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

* Corresponding author. Tel.: +49 6221 56 50 16; fax: +49 6221 56 50 03.

E-mail addresses: Paul_Schnitzler@med.uni-heidelberg.de,

Paul.Schnitzler@med.uni-heidelberg.de (P. Schnitzler).
Table 1
Cytotoxicity, anti/HSV/1 activity, and selectivity index of
triterpene extract, betulin, lupeol and betulinic acid.
[CC.sub.50]: 50% cell cytotoxicity, [IC.sub.50]: 50%
inhibitory concen/tration, SI: selectivity index
[CC.sub.50]/[IC.sub.50].

Compound             Max. nontoxic    [CC.sub.50]
                     concentration     ([micro]
                     ([micro]g/ml)       g/ml)

Triterpene extract         1               4
Betulin                    1              22
Lupeol                     1               5
Betulinic acid             1               5

Compound              [IC.sub.50]         SI
                        ([micro]
                         g/ml)

Triterpene extract        0.5              8
Betulin                   0.32           68.8
Lupeol                    0.2             25
Betulinic acid            0.32           15.6

Statistical analysis
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Author:Navid, M. Heidary; Laszczyk-Lauer, M.N.; Reichling, J.; Schnitzler, P.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Sep 25, 2014
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