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Comprehensive Analysis of the Herbal Mixture Made of Juniperus oxycedrus L. Berries, Inner Bark of Betula pendula Roth., and Grains of Avena sativa L.

This is the first report of the high-performance liquid chromatography and gas chromatography–mass spectrometry profile of a herbal mixture (HM) made of Juniperus oxycedrus L. (redberry juniper) berries, inner bark of Betula pendula Roth. (silver birch), and grains of Avena sativa L. (oat), and its effect on the No. of micronuclei (MN) in human lymphocytes and toxicity toward Artemia salina. Constituents represented by over 1000 µg per g of methanol dry extract were gallic acid, protocatechuic acid, and amentoflavone. The methanol extract of the HM at a concentration of 2.0 μg/mL decreased MN frequency by 38.3%, which was more than 3 times greater than that of the radioprotectant amifostine. The essential oil isolated from the HM was composed mainly of β-myrcene (32%) and showed weaker toxicity toward Artemia salina than the positive control after both incubation periods (24 h and 48 h). These findings suggest that the examined HM, beside its ethnopharmacological relevance on the elimination of renal calculi, also significantly reduces the No. of MN in human lymphocytes.


Introduction
Nephrolithiasis is a condition caused by the formation and displacement of crystal agglomerates, also known as stones or calculi, in the urinary tract. 1 The incidence of kidney stone formation is increasing across the world. 2,3 In order to eliminate renal colic and calculus many supplements have emerged. Development of new drug formulations from well-known folk remedies may decrease the side effects of drugs, while maintaining the therapeutic effect. 4 A decoction made of the berries of Juniperus oxycedrus L., inner bark of Betula pendula Roth., and grains of Avena sativa L. is used in the Balkan peninsula to eliminate kidney stones.
Although there are numerous studies that validate the medicinal relevance of the individual mixture components [5][6][7] there are no data for the evaluation of this herbal mixture (HM). Therefore, in the present study, high-performance liquid chromatography (HPLC) analysis of the methanol extracts of the HM and individual mixture components was performed, as well as determination of the protective effect of the methanol extracts on human peripheral blood lymphocytes by cytokinesis-block micronucleus assay (CBMN). Furthermore, gas chromatography-mass spectrometry (GC-MS) analysis and acute toxicity to Artemia salina (brine shrimps) were evaluated for the essential oils of J. oxycedrus berries and HM. The headspace (HS) volatiles of the individual herbal materials and the mixture were also determined. The ratio of HM constituents was the same as in the HM traditionally used to treat kidney stones.
HM were determined using HPLC-UV analysis. Phenolic compounds were identified according to their retention times and UV spectra, which were compared with the available commercial standards. Quantification was performed using calibration curves obtained for phenolic acids (gallic acid), flavonoids (rutin and quercetin), and procyanidin B2. Results are given in Table 1.
The most significant difference between the methanol extracts was the presence of bioflavonoids, detected only in the methanol extract of J. oxycedrus berries and HM.
Kaempferol-3-O-glycoside and gallic acid were the most abundant compounds of the A. sativa methanol extract (1080 and 2060 μg/g of dry extracts, respectively), while several derivatives of kaempferol, apigenin, and luteolin were detected in lower amounts. It was found that oat products possess an impact on uric acid excretion. In the conducted experiments, patients with elevated uric acid levels were treated by giving them an herbal tea containing 75% of A. sativa, which reduced uric acid levels. 8 Birch bark consists of brown inner bark or cambium (approximately 75%) and white outer bark (approximately 25%). In the sample of inner bark examined, cyanidin and a procyanidin derivative were detected, as well as catechin, epicatechin, and syringic acid.
Since standards of all identified compounds were not available, their identification was achieved by comparison with available standards as follows: compounds 14 and 15 were identified as kaempferol derivatives, compounds 17 and 18 as apigenin and luteolin derivatives, while compounds 19, 20, and 21 were identified according to literature data. 9 Also, compounds 2, 8, and 10 exhibited characteristic UV absorption bands indicating the presence of procyanidin B2 and cyanidin-3-O-glucoside derivatives, according to available standards.
Our results are in agreement with those previously published. Emmons and Peterson 10 have reported the following phenolic compounds in fractions of oat (groats and hulls): gallic acid, protocatechuic acid, p-hydroxybenzaldehyde, p-coumaric acid, vanillic acid, caffeic acid, vanillin, ferulic acid, sinapic acid, and avenanthramide. Apigenin, luteolin, and tricin are 3 major flavones found in oat flour and were identified in the vegetative plant only as glycosides. 11 Kaempferol glycosides were isolated from the bran of A. sativa. 12 Biflavonoids were detected as the main compounds of J. oxycedrus berries methanol extract, which is in agreement with previously published results. 9 Phenolic acids (syringic acid 4-β-glucopyranoside), flavanols {( + )-catechin 7-O-β-D-xylopyranoside and ( + )-catechin} and procyanidin dimers and trimers were determined in the inner bark using LC/MS analysis. 13,14 It was demonstrated that the inner bark contains high amounts of flavonoids, arylbutanoids, diarylheptanoids, simple phenolic compounds, phenolic acids, lignans, and procyanidins. 15,16 Chemical Composition of HS Volatiles and Essential Oils of J. oxycedrus Berries and HM The qualitative composition and relative abundance of the HS volatile compounds of the berries of J. oxycedrus (HS1), inner bark of B. pendula (HS2), grains of A. sativa (HS3), HM (HS4), essential oils of J. oxycedrus berries (EO 1 ), and HM (EO 2 ) are given in the Table 2.
Biological Activities of J. oxycedrus Berries, Inner Bark of B. pendula, Grains of A. sativa, and HM Cytokinesis-Block Micronucleus Assay. The analysis of micronuclei (MN) in cultured lymphocytes is applied as a method for genotoxic studies . 19 In this study, the methanol extracts of J.  Table 3 and Figure 2.
All tested compounds (in minimal doses of 1 μg/mL) exerted a beneficial effect by decreasing DNA damage of human lymphocytes in the range of 18.2% to 31.1%. Among the tested extracts, the most prominent effect was exhibited by the methanol extract of the HM at a concentration of 2.0 μg/mL, which gave a significant decrease (P <0 .01) in the MN frequency of 38.3%, which was threefold higher than the effect of amifostine (MN frequency of 11.4%). Also, treatment 1054 of the cell cultures with the methanol extract of the HM at concentrations of 1 μg/mL and 4.0 μg/mL affected a significant decrease (P <0 .01) in the frequency of MN (31.1% and 32.8%, respectively) compared with the control cell cultures ( Figure 2). The methanol extracts of individual samples (M1, M2, and M3) at a concentration of 2.0 μg/mL exhibited a twofold higher activity than that of amifostine, while concentrations of 1 μg/mL and 4.0 μg/mL showed slightly lower activity, but were still more effective than amifostine. The tested pure substances were selected due to their widespread representation in plant extracts. Among them, rutin exhibited the highest activity at a concentration of 2.0 μg/mL and gave a decrease in the MN frequency of 41.7%, which was slightly higher than that of the methanol extract of the HM (38.3%), even though it was detected as one of the compounds of the mixture. Hesperidin and naringin caused a slightly lower effect than rutin. It was noticed that the pure substances were slightly more efficient than that of the examined methanol extracts in decreasing the MN frequency.
The comparable cytokinesis-block proliferation index (CBPI) for extracts, positive control, and untreated cells confirmed the absence of the impact on cell proliferation.
The methanol extracts of the HM and mixture constituents showed the most significant reduction in the MN frequency at a concentration of 2.0 μg/mL, whereas concentrations of 1 μg/ mL and 4 μg/mL were less effective, but still more effective than amifostine.

Acute Toxicity-Artemia Salina Test
Acute toxicity against Artemia salina is used for the preliminary assessment of the safety and potential pharmacological application of natural products. The results of the bioassay could provide information about the cytotoxic properties of the tested extract considering good correlation between the assay and cytotoxic activity against some human solid tumors. 20,21 The results of the acute toxicity assay of the tested essential oils (berries-EO 1 and HM-EO 2 ) are summarized in Table 4. The final concentrations of the tested EO 1 and EO 2 in the Petri dishes were in the range of 4.57 to 73.125 μg/mL and 16.25 to 127.5 μg/mL (respectively), while the final concentration of Dimethyl sulfoxide (DMSO) was less than 1% (v/v).
To the best of our knowledge, there is no study regarding the acute toxicity of the essential oils of J. oxycedrus berries and the HM in the Artemia salina test. Based on the survival of nauplii after 24 h of incubation, EO 1 showed higher toxicity than both the remaining samples, EO 1 /EO 2 (almost twofold stronger) and EO 1 /SDS (1.2 times higher). Higher amounts of α-pinene and β-myrcene were detected in the essential oil of the berries, EO 1 (13% and 37%, respectively) than in the essential oil of the HM, EO 2 (4.1% and 32%), while the amounts of β-caryophyllene and germacrene D (8.7% and 7.7%, respectively for EO 1 ) were smaller (12.7% and 12.1%, respectively for EO 2 ). Previously, the methanol extract of the berries of Juniperus drupacea from Turkey was found to be moderately toxic against brine shrimps, with a Lethal concentration to 50% of nauplii (LC 50 ) value of 489.47 ± 27.8 µg/mL. 21 For the incubation period of 48 h, both oils exhibited lower toxicity than SDS, EO 1 being about 3 times less and EO 2 about 5 times lower.

Plant Materials
Berries of J. oxycedrus were collected from Klobuk, province of Trebinje (coordinates 42°42 ′ 28.19 ′′ N and 18°31 ′ 33.59 ′′ E, R. Srpska). Red-brown mature fruits were air-dried, lyophilized, and stored in a dark place. Grains of A. sativa were purchased at a health food store. Inner bark of B. pendula was collected in city park Vranje (coordinates 42.5521°N and 21.

Preparation of Methanol Extracts of Berries, Inner Bark, Grains, and HM
The extractions of individual species were performed in triplicate. Two g of each sample was chopped and subjected to ultrasound-assisted extraction with 20 mL of methanol (Sigma-Aldrich; purity ≥ 99.9%) using an ultrasound bath (UZK 8; Maget,) for 30 min; after that, the extracts were left in the dark (room temperature) overnight. Dry residues of the extracts were obtained using a rotary evaporator (KNF Laboxact) with the water bath set at 40°C. The mixture of chopped dried berries (1.1 g), inner bark of B. pendula (0.5 g) and grains of A. sativa (1.6 g) was extracted with 30 mL of methanol in the same way as the individual components of the mixture. The extract yields were 17.4 ± 0.1% for J. oxycedrus berries, 1.33 ± 0.05% for B. pendula inner bark, 1.78 ± 0.04% for grains of A. sativa, and 18.01 ± 0.1% for mixture. The dry methanol extracts were prepared prior to HPLC analysis and MN assay.

Isolation of Essential Oils
The air-dried samples (11 g J. oxycedrus berries, 5 g B. pendula inner bark, and 16 g A. sativa grains) were cut into small pieces and subjected to hydrodistillation using a Clevenger-type apparatus for 2 h. The obtained essential oil was extracted with diethyl ether and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and stored at +4°C prior to analysis. The essential oil of J. oxycedrus berries was obtained in the same way as the HM. The yield of essential oils was 0.66% and 0.61%, respectively, based on the dried weight of the samples.

Sample Preparation for HS Analysis
For HS analysis of the HM, chopped berries of J. oxycedrus (0.5 g), inner bark of B. pendula (0.25 g), and grains of A. sativa (0.8 g) were placed in a vial for HS analysis and moistened with 1 mL a-compared with control groups, statistically significant difference P <0 .01; a*-compared with control groups, statistically significant difference P <0 .05; bcompared with amifostine-WR 2721, statistically significant difference P <0 .01; b* -compared with amifostine-WR 2721, statistically significant difference P <0 .05; c-compared with mitomycine C, statistically significant difference P <0 .01; c*-compared with mitomycine C, statistically significant difference P <0 .05. The statistical significance of difference between the data pairs was evaluated by analysis of variance (one-way ANOVA) followed by the Tukey test. Statistical difference was considered significant at P < 0.01; < 0.05. Abbreviations: AM, amifostine; MMC, mitomycine C.
of distilled water. Samples of the individual ingredients of the mixture were prepared in the same way using 1 g of each ingredient.

GC-MS Analysis
The samples were analyzed by a 7890/7000B GC/MS/MS triple quadrupole system in MS1 scan mode (Agilent Technologies) equipped with a Combi PAL sampler and HS for G6501B/G6509B. Details about GC-MS analyses, identification, and calculation of relative amounts of volatile compounds have been reported previously. 22 In short, a fused silica capillary column HP-5MS (5% phenylmethylsiloxane, 30 m × 0.25 mm, film thickness 0.25 µm) was used. The injector and interface operated at 250 and 300°C, respectively. Temp. program: from 50 to 290°C at a heating rate of 4°C/min. The carrier gas was helium with a flow of 1.0 mL/min. The 500 µL HS volatile components and 1 µL essential oil solutions in hexane (1:100), respectively, were injected. Split ratio was 40:

HPLC Analysis
HPLC analysis was performed on an Agilent, Zorbax Eclipse XDB-C18, 5 μm, 4.6 mm × 150 mm column using an Agilent 1200 series liquid chromatography (equipped with a diode array detector [DAD]), Chemstation Software (Agilent Technologies), a quaternary pump, an online vacuum degasser, autosampler, and a thermostated column compartment. The mobile phase consisted of 2 solvents: water-formic acid (1%) (A) and methanol (B), starting with 30% B at 5 min, and using a gradient to obtain 70% B at 20 min and 90% B at 25 min and finished with 90% B at 40 min. The flow rate was 0.5 mL/min, the injection volume 20 μL, and the column was thermostated at 25°C. Spectral data from all peaks were accumulated in the range of 190 to 400 nm, and chromatograms were recorded at 254 and 350 nm. Identification was conducted using retention times and UV spectra, which were compared with the corresponding commercial standards. The following standards were used: tartaric acid, procyanidin B2, catechin, gallic acid, syringic acid, protocatechuic, rutin, kaempferol-3-O-glycoside, quercetin, kaempferol, apigenin, and luteolin (all standards were supplied by Sigma-Aldrich). Quantification was performed using calibration curves obtained of any standard except for derivatives of the corresponding compound for which calibration curve of that compound was used and also for cupressoflavone, amentoflavone, and biflavone derivative for which quercetin calibration curve was used. The amount of identified compounds was expressed as μg per g of dry extract weight (μg/g).
Amifostine WR-2721 (98% S-2[3-aminopropylamino] ethylphosphothioic acid; Marligen-Biosciences) at a concentration of 1 μg/mL was used as a positive control. The alkylating agent mitomycin-C (MMC, Bristol-Myers Squibb), prepared by diluting the drug in phosphate buffer (PBS), was added at a final concentration of 0.2 µg/mL to the lymphocytes cultures and used as a negative control. Three experiments were performed for each sample. The results are expressed as the means ± standard deviation (SD). The statistical analysis was performed using Origin software package version 7.0. The statistical significance of difference between the data pairs was evaluated by analysis of variance (one-way ANOVA), followed by the Tukey test. Statistical difference was considered significant at P < .01 and P < .05.

Acute Toxicity-Artemia Salina Test
Acute toxicity in Artemia salina (brine shrimps) was evaluated using the method previously described by Radulović and colleagues. 24 Two teaspoons of lyophilized cysts of Artemia salina were added to 1 L of the artificial seawater. The suspension was thermostated at 28°C, aerated with a strong airflow, and kept under constant illumination for 48 h, after which most of the cyst hatched. Freshly hatched nauplii (20 individuals) were transferred into Petri dishes containing 20 mL of

Conclusion
Results obtained by HPLC analysis of the methanol extract of the HM and individual mixture components revealed the presence of phenolic acids, flavonols, flavonols glycosides, bioflavonoids, flavones, and flavan-3-ols derivatives. Major volatile components of the HM essential oil were β-myrcene (32%), β-caryophyllene (12.7%), and germacrene D (12.1%). Methanol extracts of the HM and its constituents reduced the frequency of MN in a concentration-dependent manner, with the medium concentration of 2.0 μg/mL being the most effective and even more effective than the positive control. The essential oil of the HM showed weaker toxicity than the positive control after both time periods (24 h and 48 h). These findings suggest that this HM, beside its ethnopharmacological relevance on the elimination of renal calculi, also significantly reduces the No. of MN in human lymphocytes. As the No. of MN serves as an indicator of DNA damage, the results indicate that the tested extracts at a concentration of 2 μg/mL show a protective effect on DNA.