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Abstract

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.

Keywords

herbal mixture HPLC GC-MS methanol extract essential oil micronucleus test

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^5–7 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.

Results and Discussion

HPLC Analysis

The chemical composition of the methanol extracts of J. oxycedrus berries, inner bark of B. pendula, grains of A. sativa, and the 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.

Table 1.

Chemical Composition of Methanol Extracts of Juniperus oxycedrus Berries (M1), Inner Bark of Betula pendula (M2), Grains of Avena sativa (M3), and Herbal Mixture (M4) (μg/g of dry Extract).

S. No.	Compound	M1	M2	M3	M4
1	Tartaric acid	-	-	232	210
2	Procyanidin derivative	-	159	-	136
3	Gallic acid	-	-	2060	1555
4	Catechin derivative	-	32.9	-	15
5	Protocatechuic acid	5360	-	-	3407
6	Catechin	-	52.3	-	38
7	Epicatechin	-	6.9	-	3
8	Cyanidin-derivative	-	84.7	-	78
9	Syringic acid	-	2038	-	956
10	Cyanidin derivative	-	227	-	110
11	Rutin	730	-	-	580
12	Kaempferol-3-O-glycoside	-	-	1080	880
13	Quercetin	-	-	100	72
14	Kaempferol derivative	-	-	100	72
15	Kaempferol derivative	-	-	670	480
16	Apigenin	310	-	-	210
17	Apigenin derivative	-	-	460	251
18	Luteolin derivative	-	-	221	150
19	Cupressoflavone	3250	-	-	670
20	Amentoflavone	3700	-	-	1030
21	Biflavone derivative	1400	-	-	590

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 Peterson¹⁰ 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 (EO₁), and HM (EO₂) are given in the Table 2.

Table 2.

Composition (%) of Headspace (HS) Volatile Compounds of the Berries of Juniperus oxycedrus (HS1), Inner Bark of Betula pendula (HS2), Grains of Avena sativa (HS3), Herbal Mixture (HM) (HS4), and Essential Oils of J. oxycedrus Berries (EO₁) and HM (EO₂).

^aRI	^bAI	Constituents	HS1	HS2	HS3	HS4	EO₁	EO₂	Class
774	762	n-Pentanol	-	6.7	-	-	-	-	O
801	806	n-Hexanal	-	51.0	-	-	-	-	O
866	863	n-Hexanol	-	17.9	-	-	-	-	O
924.8	921	Tricyclene	t	-	-	-	-	-	M
929	924	α-Thujene	t	-	-	-	-	-	M
936.5	932	α-Pinene	23.9	-	9.4	17.2	13	4.1	M
950.6	946	Camphene	0.1	-	-	-	-	-	M
975.6	969	Sabinene	0.2	-	-	0.1	0.2	0.1	M
979	974	β-Pinene	1.4	6.6	3.2	1.2	1	0.5	M
996	988	β-Myrcene	65.4	-	5.6	71.8	37	32	M
1031.8	1029	Limonene	6.8	-	-	7.4	6.0	5.4	M
1061	1054	γ-Terpinene	-	-	-	-	0.1	-	M
1091	1086	Terpinolene	0.3	-	-	0.1	T	0.2	M
1129	1122	α-Campholenal	-	-	-	-	0.1	0.1	MO
1142	1135	trans-Pinocarveol	-	-	-	-	-	0.1	MO
1169	1165	Borneol	-	-	-	-	0.4	-	M
1181	1174	Terpinen-4-ol	t	-	-	-	0.4	0.5	MO
1193.7	1186	α-Terpineol	-	-			0.3	0.1	MO
1200	1195	Myrtenal	-	-	-	-	0.1	-	MO
1214	1204	Verbenone	-	-	-	-	0.1	-	MO
1221	1217^c	trans-Carveol	-	-	-	-	0.1	0.1	MO
1248	1239	Carvone	-	-	-	-	0.1	-	MO
1290	1287	Bornyl acetate	t	-	-	-	0.2	-	O
1355	1345	α-Cubebene	t	-	-	-	0.6	0.8	S
1382	1374	α-Copaene	-	-	-	-	0.3	0.3	S
1397.5	1389	β-Elemene	-	-	-	-	0.2	0.1	S
1429	1417	β-Caryophyllene	0.3	-	-	0.5	8.7	12.7	S
1463	1452	α-Humulene	0.1	-	5.1	0.1	6	/	S
1484	1478	γ- Muurolene	1.2	-	-	-	0.6	0.7	S
1490	1484	Germacrene D	-	-	46.5	1.1	7.7	12.1	S
1507	1500	α-Murolene	-	-	-	-	1.3	1.5	S
1521.9	1513	γ-Cadinene	-	-	-	-	0.4	0.5	S
1530	1522	δ-Cadinene	t	-	-	-	2	2.6	S
1545	1537	α-Cadinene	-	-	-	-	0.1	-	S
1593	1582	Caryophyllene oxide	-	-	-	-	3.5	4.4	SO
1682	1685^c	Eudesma-4 (15),7-dien-1β-ol	-	-	-	-	1.1	1.3	SO
Total amounts (%)			99.7	82.2	69.8	98.5	91.6	80.2

RI—experimental linear retention indices relative to C₈–C₄₀ alkanes on an HP-5MS column calculated in accordance with experimental conditions for the GC-MS analysis. ^bAI—Adam's retention indices.¹⁷ ^cSalido et al¹⁸ t: traces (<0.1%); (-): not detected.

Abbreviations: EO₁, essential oil of J. oxycedrus berries; EO₂, essential oil of the herbal mixture; HS1, dried berries of J. oxycedrus; HS2, inner bark of B. pendula; HS3, grains of A. sativa; HS4, herbal mixture;MH, monoterpene hydrocarbons; MO, oxygenated monoterepenes; SH, sesquiterpene hydrocarbons; SO, oxygenated sesquiterpenes; O, Others.

The most dominant component of sample HS1 was β-myrcene (65.4%), accompanied by α-pinene (23.9%) and limonene (6.8%). The dominant component of the essential oil of the berries was β-myrcene (37%), accompanied by α-pinene (13%), β-caryophyllene (8.7%), α-humulene (6%), and germacrene D (7.7%). The amount of β-myrcene was twofold smaller than that of the HS sample. Similarly, the essential oil isolated from the HM was composed mainly of β-myrcene (32%), but the amount of monoterpenes (43.2%) was twofold smaller than that of the HS volatiles of the HM (96.8%). The main monoterpene hydrocarbons of the HM (HS4) were β-myrcene (71.8%), α-pinene (17.2%), and β-pinene (1.2%), followed by a small amount of the sesquiterpene hydrocarbon, germacrene D (1.1%). Dominant HS volatiles of the inner bark of B. pendula (HS2) were primary aldehyde and alcohols n-hexanal (51.0%), n-hexanol (17.9%), and n-pentanol (6.7%), while monoterpenes were detected in significantly lower amounts compared to the other 2 samples (6.6%). The only detected monoterpene was β-pinene (6.6%).

Sesquiterpenes composed the major part of the HS volatiles of A. sativa grains, amounting to 51.6% of the total HS volatiles. The most abundant compounds (HS3) were the sesquiterpenes, germacrene D (46.5%), and α-humulene (5.1%), followed by the monoterpenes α-pinene (9.4%) and β-myrcene (5.6%).

The essential oil of the HM contained similar amounts of monoterpenoids and sesquiterpenoids (43.2% and 37.0%, respectively). Higher amounts of β-caryophyllene (12.7%) and germacrene D (12.1%) were detected in the essential oil of the HM than in the essential oil of the berries (8.7% and 7.7%, respectively), as shown in Figure 1.

Figure 1.

Main volatile compounds of essential oils of Juniperus oxycedrus berries (EO₁) and herbal mixture (HM) (EO₂).

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. oxycedrus berries (MJO), inner bark of B. pendula (MBP), grains of A. sativa (MAS), HM, and pure compounds at concentrations of 1.0, 2.0, and 4.0 µg/mL were subjected to CBMN assay. The frequencies and distribution of MN in human lymphocytes were scored. The results are presented in Table 3 and Figure 2.

Figure 2.

Frequency of micronuclei (MN) in cell cultures of human lymphocytes treated with different concentrations of methanol extracts of Juniperus oxycedrus berries (M1), inner bark of Betula pendula (M2), grains of Avena sativa (M3), herbal mixture (HM) (M4), and pure compounds.

Table 3.

Incidence of Micronuclei (MN), Cytokinesis-Block Proliferation Index (CBPI), Distribution of MN per Cells, and Frequency of MN in Cell Cultures of Human Lymphocytes Treated With Different Concentrations of Methanol Extracts of Juniperus oxycedrus Berries (M1), Inner Bark of Betula pendula (M2), Grains of Avena sativa (M3) , Herbal Mixture (M4), and Pure Compounds.

Sample	Conc. µg/mL	MN/1000 Bn cell	% Bn cell with MN	MN/Bn cell	CBPI	Frequency of MN
Control		26.26 ± 0.34	2.12 ± 0.11	1.17 ± 0.06	1.66 ± 0.02	100%
AM	1.0	21.93 ± 0.62 ^a,c	1.80 ± 0.12	1.22 ± 0.07	1.61 ± 0.04	83.51%
MMC	0.2	34.59 ± 0.95 ^a,b	3.05 ± 0.17	1.13 ± 0.03	1.66 ± 0.05	131.72%
M1	1.00	19.13 ± 0.41 ^a,b,c	1.58 ± 0.10	1.17 ± 0.07	1.64 ± 0.02	72.85%
M1	2.00	18.15 ± 0.30 ^a,b,c	1.58 ± 0.05	1.15 ± 0.04	1.66 ± 0.03	69.12%
M1	4.00	18.59 ± 0.98 ^{a,b ̽,c}	1.64 ± 0.11	1.08 ± 0.04	1.64 ± 0.01	70.79%
M2	1.00	20.17 ± 0.49 ^a,c	1.64 ± 0.04	1.16 ± 0.05	1.66 ± 0.01	76.80%
M2	2.00	18.87 ± 0.85 ^{a,b ̽,c}	1.64 ± 0.10	1.15 ± 0.04	1.64 ± 0.02	71.86%
M2	4.00	19.73 ± 0.80 ^a,c	1.61 ± 0.03	1.22 ± 0.06	1.63 ± 0.02	75.10%
M3	1.00	21.48 ± 0.71 ^a,c	1.85 ± 0.05	1.15 ± 0.02	1.71 ± 0.05	81.80%
M3	2.00	20.42 ± 0.70 ^a,c	1.70 ± 0.08	1.20 ± 0.06	1.61 ± 0.01	77.76%
M3	4.00	21.00 ± 0.48 ^a,c	1.76 ± 0.06	1.20 ± 0.06	1.65 ± 0.01	80.00%
M4	1.00	18.09 ± 0.67 ^a,b,c	1.54 ± 0.06	1.11 ± 0.04	1.66 ± 0.03	68.90%
M4	2.00	16.21 ± 0.42 ^a,b,c	1.50 ± 0.03	1.10 ± 0.03	1.61 ± 0.02	61.73%
M4	4.00	17.65 ± 0.93 ^a,b,c	1.65 ± 0.11	1.07 ± 0.03	1.58 ± 0.03	67.20%
Hesperidin	1.00	17.98 ± 0.27 ^a,b,c	1.41 ± 0.05	1.27 ± 0.05	1.61 ± 0.01	63.18%
Hesperidin	2.00	17.44 ± 0.36 ^a,b,c	1.45 ± 0.07	1.20 ± 0.04	1.65 ± 0.03	61.28%
Hesperidin	4.00	17.66 ± 0.53 ^a,b,c	1.60 ± 0.07	1.10 ± 0.02	1.68 ± 0.04	62.05%
Naringin	1.00	19.86 ± 0.24^a,b,c	1.60 ± 0.10	1.26 ± 0.09	1.66 ± 0.00	69.78%
Naringin	2.00	19.44 ± 0.76 ^a,b,c	1.67 ± 0.14	1.18 ± 0.11	1.64 ± 0.01	68.31%
Naringin	4.00	19.68 ± 0.57 ^a,b,c	1.59 ± 0.06	1.29 ± 0.07	1.63 ± 0.02	69.15%
Rutin	1.00	17.41 ± 0.37 ^a,b,c	1.54 ± 0.10	1.14 ± 0.08	1.61 ± 0.07	61.17%
Rutin	2.00	16.58 ± 0.43 ^a,b,c	1.54 ± 0.08	1.14 ± 0.05	1.70 ± 0.05	58.26%
Rutin	4.00	16.98 ± 0.58 ^a,b,c	1.54 ± 0.10	1.17 ± 0.08	1.62 ± 0.03	59.66%

Note: MN/1000 Bn cells: incidence of MN in 1000 binucleated cells (examined for each concentration); % Bn cells with MNi; MN/Bn cells: incidence of MN in binucleated cells; Frequency of MN: incidence of MN present like % from control groups in cell cultures of human lymphocytes treated with different concentrations of extracts.

a—compared with control groups, statistically significant difference P <0 .01; a*—compared with control groups, statistically significant difference P <0 .05; b—compared 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.

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 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—EO₁ and HM—EO₂) are summarized in Table 4. The final concentrations of the tested EO₁ and EO₂ 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).

Table 4.

Acute Toxicity of Examined Samples and SDS (Positive Control) on Artemia salina, Expressed as LC₅₀ in µg/mL After 24 h and After 48 h.

Sample	LC₅₀ 24 h (95% confidence interval)	LC₅₀ 48 h (95% confidence interval)
EO₁	27.63 ± 0.0244 (±0.09%)	25.78 ± 0.0838 (±0.3%)
EO₂	47.45 ± 0.0733 (±0.2%)	37.31 ± 0.00924 (±0.02%)
SDS	33.82 ± 0.0141 (±0.04%)	8.24 ± 0.00924 (±0.1%)

Abbreviations: EO₁, essential oil of the berries; EO₂, essential oil of the herbal mixture; LC₅₀, lethal concentration to 50% of nauplii; SDS, sodium dodecyl sulfate.

The statistical significance of difference between the data pairs was evaluated by analysis of variance (ANOVA), followed by the Tukey test. Statistical difference was considered significant at P < 0.0001. There is a statistically significant difference (P < 0.0001) in activity between each of the oils and the SDS, as well as between the oils, SDS, and control sample (dimethyl sulfoxide [DMSO]).

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, EO₁ showed higher toxicity than both the remaining samples, EO₁/EO₂ (almost twofold stronger) and EO₁/SDS (1.2 times higher). Higher amounts of α-pinene and β-myrcene were detected in the essential oil of the berries, EO₁ (13% and 37%, respectively) than in the essential oil of the HM, EO₂ (4.1% and 32%), while the amounts of β-caryophyllene and germacrene D (8.7% and 7.7%, respectively for EO₁) were smaller (12.7% and 12.1%, respectively for EO₂). 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 (LC₅₀) value of 489.47 ± 27.8 µg/mL.²¹ For the incubation period of 48 h, both oils exhibited lower toxicity than SDS, EO₁ being about 3 times less and EO₂ about 5 times lower.

Experimental

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 21.8989° E, Serbia). The voucher specimens have been deposited in the Herbarium collection at the Department of Biology and Ecology, Faculty of Science and Mathematics, University of Niš under the acquisition numbers: 14611-F, 14612-F, 14613-F, respectively.

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 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 40:1. Post-run: back flash for 1.89 min, at 280 °C, with helium pressure of 50 psi. MS conditions were as follows: ionization voltage of 70 eV, acquisition mass range of 40 to 440, and scan time 0.32 s. The percentage composition of the samples was computed from the TIC peak areas without any corrections. Constituents were identified by comparison of their linear retention indices (relative to C8–C40 n-alkanes on the HP-5MS column) with literature values and their MS with those from Wiley 6, NIST11, and Agilent Mass Hunter Workstation B.06.00 software by application of the AMDIS software (Automated Mass Spectral Deconvolution and Identification System, Ver. 2.1, DTRA/NIST, 2011).

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).

Cytokinesis-Block Micronucleus Assay

CBMN was performed as previously described.^19,23 The cell culture lymphocytes were treated with 1.0, 2.0, and 4.0 μg/mL of the examined methanol extracts and available pure compounds (hesperidin, naringin, and rutin; Sigma-Aldrich).

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 artificial seawater. One hundred microliters of tested oil of each concentration (14.62; 7.31; 3.65; 1.83; 0.91 mg/mL for EO₁ and 25.5; 13; 6.5; 3.25; mg/mL for and EO₂) was added into Petri dishes as well. So, the final concentrations of the tested EO₁ and EO₂ in the Petri dishes were in the range of 4.57 of 73.125 μg/mL and 16.25 of 127.5 μg/mL, respectively. Brine shrimps were not fed during the test. The tested samples were not aerated, and the test dishes were left at room temperature, under constant illumination. DMSO was inactive against A. salina nauplii and it was used as a negative control, whereas varying concentrations of sodium dodecyl sulfate (SDS) were used as the positive control. Further, 3 Petri dishes were filled only with the equal volume (as for all other tests) of seawater and all nauplii survived after 24 h and after 48 h. Brine shrimps were not fed during the test. The tested samples were not aerated, and the test dishes were left at room temperature, under constant illumination. All the tests were performed in triplicate. Dead nauplii were counted after 24 h and 48 h. LC₅₀ was determined after statistical analysis according to Hamilton et al²⁵

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.

Footnotes

Acknowledgments

The authors acknowledge the Ministry of Education, Science and Technological Development of Serbia for financial support (Agreement No. 451-03-9/2022-14-200124).

Author’s Contribution

Dr Ivana Zlatanović: Literature research; Designing a study; Experimental work (plant collecting, HPLC analysis); Analysis of the results; and Writing the paper. Dr Miroslava Stanković: cytokinesis block micronucleus test. Jovana Ickovski: acute toxicity in Artemia salina (brine shrimps) test. Ivana Zrnzević: GC/MS analysis. Dr Gordana Stojanović: Analysis of the results; Writing the paper; And Supervision of all of the work.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Not applicable, because this article does not contain any studies with human or animal subjects.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Ministry of Education, Science and Technological Development of Serbia (grant number No. 451-03-68/2022-14/200124).

Informed Consent

Not applicable, because this article does not contain any studies with human or animal subjects.

Trial Registration

Not applicable, because this article does not contain any clinical trials.

ORCID iD

Gordana Stojanović

References

Ramello

Vitale

Marangella

. Epidemiology of nephrolithiasis. J Nephrol. 2000;13(3):S45-S50. https://www.researchgate.net/publication/12196979_Epidemiology_of_nephrolithiasis

Stamatelou

Francis

Jones

Nyberg

Curhan

. Time trends in reported prevalence of kidney stones in the United States: 1976–1994. Kidney Int. 2003;63(5):1817-1823. doi.org/10.1046/j.1523-1755.2003.00917.x

Worcester

Coe

. Nephrolithiasis. Prim Care. 2008;35(2):369-391. doi.org/10.1016/j.pop.2008.01.005

Rizwan

Yesmine

Banu

Chowdhury

Hasan

Chatterjee

. Renoprotective effects of stevia (Stevia rebaudiana Bertoni), amlodipine, valsartan, and losartan in gentamycin-induced nephrotoxicity in the rat model: biochemical, hematological and histological approaches. Toxicol Rep. 2019;6:683-691. doi.org/10.1016/j.toxrep.2019.07.003

Moreno

Bello

Primo-Yúfera

Espluges

. In vitro studies of methanol and dichloromethanol extracts of Juniperus oxycedrus L. Phytother Res. 1997;11(4):309-311. doi.org/10.1002/(SICI)1099-1573(199706)11:4%3C309::AID-PTR87%3E3.0.CO;2-6

Šarić-Kundalić

Dobeš

Klatte-Asselmeyer

Saukel

. Ethnobotanical study on medicinal use of wild and cultivated plants in middle, south and west Bosnia and Herzegovina. J Ethnopharmacol. 2010;131(1):33-55. doi.org/10.1016/j.jep.2010.05.061

Orhan

Aslan

Pekcan

Orhan

Bedir

Ergun

. Identification of hypoglycaemic compounds from berries of Juniperus oxycedrus subsp. oxycedrus through bioactivity guided isolation technique. J Ethnopharmacol. 2012;139(1):110-118. doi.org/10.1016/j.jep.2011.10.027

Singh

Belkheir

. Avena sativa (oat), a potential neutraceutical and therapeutic agent: an overview. Crit Rev Food Sci Nutr. 2013;53(2):126-144. doi.org/10.1080/10408398.2010.526725

Taviano

Marino

Trovato

, et al. Juniperus oxycedrus L. Subsp. oxycedrus and Juniperus oxycedrus L. Subsp. macrocarpa (Sibth. & Sm.) Ball. “berries” from Turkey: comparative evaluation of phenolic profile, antioxidant, cytotoxic and antimicrobial activities. Food Chem Toxicol. 2013;58:22-29. doi.org/10.3109/13880209.2011.560161

10.

Emmons

Peterson

. Antioxidant activity and phenolic contents of oat groats and hulls. Cereal Chem. 1999;76(6):902-906. doi.org/10.2135/cropsci2001.1676.

11.

Peterson

. Oat antioxidants. J Cereal Sci. 2001;33(2):115-129. doi.org/10.1006/jcrs.2000.0349.

12.

Zhang

Jie-Kun

Zhang

Guan-Hua

. Flavonoids from the bran of Avena sativa. Chin J Nat Med. 2012;10(2):110-114. doi.org/10.3724/SP.J.1009.2012.00110.

13.

Liimatainen

Karonen

Sinkkonen

Helander

Salminen

. Characterization of phenolic compounds from inner bark of Betula pendula. Holzforschung. 2012;66(2):171-181. doi.org/10.1515/HF.2011.146

14.

Liimatainen

Karonen

Sinkkonen

. Procyanidin xylosides from the bark of Betula pendula. Phytochemistry. 2012;76:178-183. doi.org/10.1016/j.phytochem.2012.01.008

15.

Liimatainen

Karonen

Sinkkonen

, et al. Phenolic compounds of the inner bark of Betula pendula: seasonal and genetic variation and induction by wounding. J Chem Ecol. 2012;38(11):1410-1418. doi.org/10.1007/s10886-012-0199-2

16.

Liimatainen

Sinkkonen

Karonen

Pihlaja

. Two new phenylbutanoids from inner bark of Betula pendula. Magn Reson Chem. 2008;46(2):195-198. doi.org/10.1002/mrc.2163

17.

Adams

. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. 4th ed. Allured Publishing Corporation; 2007.

18.

Salido

Altarejos

Nogueras

, et al. Chemical studies of essential oils of Juniperus oxycedrus ssp. badia. J Ethnopharmacol. 2002;81(1):129-134. doi.org/10.1016/s0378-8741(02)00045-4

19.

Fenech

Morley

. The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat Res. 1993;285(1):35-44. doi.org/10.1016/0027-5107(93)90049-L

20.

dos Santos

JHM

Oliveira

de Carvalho

, et al. Evaluation of native and exotic Brazilian plants for anticancer activity. J Nat Med. 2010;64(2):231-238. doi.org/10.1007/s11418-010-0390-0

21.

Miceli

Trovato

Marino

, et al. Phenolic composition and biological activities of Juniperus drupacea Labill. Berries from Turkey. Food Chem Toxicol. 2011;49(10):2600-2608. doi.org/10.1016/j.fct.2011.07.004

22.

Petrović

Stamenković

Kostevski

Stojanović

Mitić

Zlatković

. Chemical composition of volatiles; antimicrobial, antioxidant and cholinesterase inhibitory activity of Chaerophyllum aromaticum L. (Apiaceae) essential oils and extracts. Chem Biodivers. 2017;14(5):e1600367. doi.org/10.1002/cbdv.201600367

23.

Stojanović

IŽ

Stanković

Jovanović

Petrović

Šmelcerovic

Stojanović

. Effect of Hypogymnia physodes extracts and their depsidones on micronucleus distribution in human lymphocytes. Nat Prod Commun. 2013;8(1):109-112. doi.org/10.1177/1934578X1300800125.

24.

Radulović

Mladenović

Blagojević

, et al. Toxic essential oils. Part III: identification and biological activity of new allylmethoxyphenyl esters from a Chamomile species (Anthemis segetalis Ten.). Food Chem Toxicol. 2013;62:554-565. doi.org/10.1016/j.fct.2013.09.017

25.

Hamilton

Russo

Thurston

. Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environ Sci Technol. 1977;11(7):714-719. doi.org/10.1021/es60130a004

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

Abstract

Keywords

Introduction

Results and Discussion

HPLC Analysis

Chemical Composition of HS Volatiles and Essential Oils of J. oxycedrus Berries and HM

Biological Activities of J. oxycedrus Berries, Inner Bark of B. pendula, Grains of A. sativa, and HM

Cytokinesis-Block Micronucleus Assay

Acute Toxicity—Artemia Salina Test

Experimental

Plant Materials

Preparation of Methanol Extracts of Berries, Inner Bark, Grains, and HM

Isolation of Essential Oils

Sample Preparation for HS Analysis

GC-MS Analysis

HPLC Analysis

Cytokinesis-Block Micronucleus Assay

Acute Toxicity—Artemia Salina Test

Conclusion

Footnotes

Acknowledgments

Author’s Contribution

Declaration of Conflicting Interests

Ethical Approval

Funding

Informed Consent

Trial Registration

ORCID iD

References