Comparative Volatile Composition, Phenolic Contents, and Antimicrobial Activities of the Essential Oils and Solvent Extracts of Orthomnion Rostratum and Orthomnion Medium

Abstract

The present study aimed to investigate the chemical composition of essential oils (EOs) and methanol extracts of Orthomnion rostratum and Orthomnion medium species. The EOs were obtained using hydrodistillation (HD) and microwave distillation (MWD) and analyzed by GC-FID-MS. Octanal (24.12%, HD; 20.22%, MWD), pentanal (31.45%, HD), and n-pentanol (25.50%, MWD) were found to be significant compounds, respectively. Analysis of the phenolic constituents of the methanol extracts of O. rostratum and O. medium yielded apigenin glucoside derivatives as the major compounds, with concentrations of 1.355 and 0.382 mg/g, respectively. The antimicrobial activities of the EOs and solvent extracts (n-hexane, chloroform, methanol, and water) of both species were evaluated against nine microorganisms. EOs obtained using MWD from both species exhibited the strongest antimicrobial activity against Staphylococcus aureus (15.9 µg/mL and 15.0 µg/mL), Enterococcus faecalis (31.9 µg/mL and 30.0 µg/mL), Bacillus cereus (15.9 µg/mL and 15.0 µg/mL), respectively.

Keywords:
Orthomnion rostratum; Plagiomnium medium; Essential oils; Phenolic compounds; Antimicrobial activity.

INTRODUCTION

Bryophytes (mosses) are considered the second largest group of terrestrial plants, and are classified into three groups (Bryophyta, Marchantiophyta, and Anthocerophyta) (Goffinet, Buck, Shaw, 2009; Kürschner, Frey, 2011).Approximately 24.000 bryophyte species exist worldwide (Asakawa, Ludwiczuk, Nagashima, 2013). Bryophytes have been traditionally used in folk medicine in various countries, including China, India, and among Native American communities (Klavina et al., 2015; Glime, 2017). Several wild taxa of Bryophyta have been used for ethnomedical purposes (Motti, Palma, de Falco, 2023), with herbal treatment for skin and hair care being the most commonly reported applications (Pejin et al., 2013; Aslanbaba et al., 2017). according to the literature, many bryophyte extracts and isolated compounds exhibit antimicrobial, antiviral, and cytotoxic activities (Motti, Palma, de Falco, 2023).

The genus Orthomnion (Syn: Plagiomnium) belongs to the family Mniaceae (Koponen, Sun, 2016). Eight species of the Orthomnion genus are found in Türkiye (Erdağ, Kürschner, 2021), including Orthomnion affine (Blandow ex Funck) T. J. Kop. & Yu Sun, O. confertidens (Lindb. & Arnell), T.J. Kop. & Yu Sun, O. cuspidatum (Hedw.) T.J. Kop. & Yu Sun, O. elatum (Bruch & Schimp.) T.J. Kop. and Yu Sun, O. ellipticum (Brid.) T.J. Kop. & Yu Sun, O. medium (Bruch & Schimp.) T.J. Kop. & Yu Sun, O. rostratum (Schrad.) T.J. Kop. & Yu Sun and O. undulatum (Hedw.) T.J. Kop. & Yu Sun. Orthomnion species inhabit shaded streambanks on rock and forest floors, thriving in humus content (Goffinet, Buck, Shaw, 2009; Kürschner et al., 2012; Erdağ, Kürschner, 2021). O. medium is specifically known as alpine thyme moss. (Edwards, Sean, 2012).

Essential oils derived from various plants contain valuable secondary metabolites, predominantly terpenes. These oils are widely used in cosmetic, pharmaceutical, and food industries. Recent studies have highlighted the biological importance of mosses, prompting increased investigations into their volatile components (Li et al., 2022; Valarezo et al., 2018; Yayıntaş et al., 2019; Sarıtaş et al., 2001; Anhut et al., 1992; Mitra, Burger, Poddar-Sarkar, 2013) and their antibacterial properties of various moss species (Semerjyan, Semerjyan, 2022; Marques et al., 2022; Önder et al., 2022; Vollar et al., 2018; Nikolajeva et al., 2012).

Currently, no studies have focused on the of essential oils (EOs), phenolic content, and antimicrobial activities of extracts obtained from O. rostratum and O. medium. Therefore, this study aimed to (i) determine the chemical composition of the EOs from O. rostratum and O. medium species, (ii) analyze the correlation between the chemical composition of EOs obtained by hydrodistillation (HD) and microwave distillation (MWD) methods, (iii) assess the antimicrobial activities of all extracts, and (iv) evaluate the phenolic content of methanolic extracts of O. rostratum and O. medium.

MATERIAL AND METHODS

Plant

The green, young leafy parts of O. rostratum (350 g) and O. medium (445 g) were collected from Trabzon, Düzköy, Kayabaşı Plateau on September 22^nd, 2022. The collection sites were located at 40°51'00.59"N, 39°28'08.37"E (1715 m) for O. rostratum and 40°50'42.56"N, 39°28'09.57"E, (1756 m) for O. medium. The plants were gathered from forested areas near stream edges or in wet soil. Voucher specimens were deposited in the Herbarium of Karadeniz Technical University, Department of Biology (KTUB), Trabzon, Türkiye (O. rostratum KTUB 1622 and O. medium KTUB 1623) (Kürschner et al., 2012; Erdağ et al., 2021).

Chemicals and reagents

The following chemicals and reagents were used in this study: n-Hexane, chloroform, methanol, acetonitrile, acetic acid, dimethyl sulfoxide solvents from Fluka Chemie GmbH (Buchs, Switzerland). Additional reagents included Na₂SO₄, gallic acid, protocatechuic acid, p-hydroxy benzoic acid (p-OH benzoic acid), chlorogenic acid, vanillic acid, caffeic acid, syringic acid, vanillin, epicatechin, p-coumaric acid, ferulic acid, rutin, luteolin-7-glycoside, naringin, hesperidin, apigenin-7-glycoside, rosmarinic acid, fisetin, eriodictyol, luteolin, quercetin, naringenin, hesperetin, apigenin, kaempferol, ampicillin, streptomycin, and fluconazolepurcahsed from Merck(Darmstadt, Germany) and Sigma-Aldrich (Missouri, ABD). all chemicals were of analytical grade.

Isolation of essential oil by HD and MW methods

The green, young leafy parts of O. rostratum (116 g, fresh) and O. medium (120 g, fresh) were ground into small pieces using a mill. The grounded parts of the plant material underwent hydrodistillation (HD) using a modified Clevenger-type apparatus (7 ^oC, seven h), yielding (v/w): 0.027% and 0.011% essentil oil, respectively. The ground parts of O. rostratum (120 g, fresh) and O. medium (122 g, fresh) were extracted by the MW hydrodistillation method using a modified Clevenger-type apparatus (7 ^oC, two h), yielding (v/w): 0.032% and 0.015% essential oil, respectively. The EOs were extracted with n-hexane (HPLC grade, 0.5 mL), dried over Na₂SO₄ in a brown glass bottle, and stored in a refrigerator at -10 ⁰C until use (Özdemir et al., 2010; Cansu et al., 2013).

Solvent extractions (n-hexane, chloroform, methanol, and water)

The green, young leafy parts of O. rostratum and O. medium (80 g each, dry) were ground and then extracted (~5 g, 10 mL × 2; 24 h each) by maceration with n-hexane, chloroform, methanol, and water in a brown bottle (50 mL) separately. After the solvent filtration, the same solvent extracts were combined and evaporated or lyophilized to yield crude n-hexane (0.1402 g and 0.1632 g), chloroform (0.2140 g, 0.2472 g), methanol (0.2942 g, 0.3125 g), and water extracts (0.1530 g, 0.1626 g) of O. rostratum and O. medium, respectively.

Gas chromatography-mass spectrometry (GC-FID-MS)

GC-FID-MS analysis was performed using a Shimadzu ultra QP2010 (Shimadzu Class-5000 Chromatography Workstation software). A Restek Rxi-5MS column (30 mm x 0.25 mm × 0.25 μm) (USA) was used for the analysis. The sample (1 μL, in HPLC-grade n-hexane) was injected in split mode (1:30) at 230 ^oC. The initial column temperature was 60 °C for 2 min and then increased to 240 °C with a 3ºC/min heating ramp. The final temperature was maintained at 250 ^oC for 4 min. The carrier gas was Helium (99.999%) with a 1 mL/min flow rate. MS detection was performed in the electronic impact mode (EI, 70 eV; scan mode 40-450 m/z) (Adams, 2007; Üçüncü et al., 2010a; Yaylı et al. 2022). The samples were analyzed, and the mean values are reported (Table I).

TABLE I
Essential oil composition of O. rostratum and O. medium species

No	RT	RILit*	RIExp.a	Compounds	Area %b
					O. rosratum		O. medium
					HD	MWD	HD	MWD
1	8.20	715	710	Pentanal	-	-	31.45	-
2	8.25	706	697	n-Pentanol	-	-	-	25.50
3	11.72	802	801	Octane	-	-	3.18	-
4	20.65	1090	1085	2-Nonanone	0.55	-	-	-
5	20.68	1089	1087	α-Terpinolene	-	-	-	0.85
6	21.25	1002	1009	Octanal	24.12	20.22	5.44	-
7	23.68	1099	1101	α-Pineneoxide	-	-	-	4.44
8	24.72	1146	1147	iso-Borneol	1.91	5.91	-	-
9	25.21	1177	1169	Terpinen-4-ol	0.29	-	-	0.93
10	25.86	1189	1193	α-Terpineol	-	-	0.81	0.81
11	26.23	1197	1201	Myrtenol	-	-	-	0.18
12	26.36	1202	1202	Decanal	0.90	1.15	1.19	1.65
13	26.82	1212	1216	2.4-Nonadienal	-	0.46	0.46	0.61
14	27.44	1226	1229	β-Cyclocitral	0.47	0.54	-	-
15	27.45	1229	1236	cis-Carveol	-	-	-	1.10
16	29.15	1269	1263	2-Decenal	0.94	1.87	0.92	1.15
17	29.58	1286	1279	Decyl alcohol	0.64	0.99	-	-
18	29.90	1281	1280	2-Phenylcrotonaldehyde	0.57	-	-	-
19	30.61	1286	1292	iso-Bornyl acetate	-	11.44	8.94	-
20	30.65	1289	1294	Bornyl acatate	20.98	-	-	24.42
21	30.75	1293	1296	2E,4Z-Decadienal	-	-	1.01	-
22	31.34	1307	1308	Undecanal	0.30	2.78	1.17	1.00
23	31.86	1317	1319	2E,4E-Decadienal	0.72	2.04	2.82	3.16
24	34.06	1364	1366	2E-Undecenal	0.35	1.24	1.54	1.19
25	34.40	1370	1374	Undecanol	0.30	-	0.32	0.28
26	35.10	1394	1387	α-Copaene	0.37	-	-	-
27	35.11	1388	1389	β-Cubene	-	-	0.46	0.42
28	35.61	1391	1396	β-Bourbanene	0.29	0.71	-	-
29	35.64	1400	1400	Tetradecane	-	-	0.87	0.60
30	35.76	1391	1401	β-Elemene	-	0.43	0.29	-
31	36.12	1409	1410	Dodecanal	-	-	-	0.18
32	36.55	1423	1420	Geosmin	-	-	0.33	-
33	37.11	1421	1423	β-Cedrene	-	0.31	-	-
34	37.26	1432	1434	β-Copaene	-	-	-	5.70
35	37.27	1437	1437	γ-Elemene	3.67	11.34	4.89	2.48
36	37.50	1440	1442	α-Guaine	0.51	-	2.45	-
37	37.93	1447	1451	2E,6Z-Dodecadienal	-	-	-	0.07
38	38.14	1455	1456	Geranyl acetone	-	0.54	0.21	0.25
39	38.80	1477	1471	α-Humulene	1.70	8.83	2.79	3.53
40	38.97	1471	1475	Dodecanal	2.35	3.04	0.51	1.00
41	39.66	1496	1492	γ-Amorphene	-	-	0.30	-
42	39.91	1489	1494	(E)-β-Ionone	19.90	2.01	1.03	-
43	40.25	1500	1503	Eremophilene	-	-	0.14	-
44	40.64	1510	1513	Tridecanal	-	-	1.92	1.16
45	41.35	1539	1535	α-Cadinene	-	0.95	0.24	0.80
46	41.65	1530	1536	Zonarene	-	-	0.57	0.63
47	43.02	1563	1569	(E)-Nerolidol	-	0.66	0.40	0.75
48	43.15	1583	1574	Caryophyllene	-	-	-	0.16
49	43.41	1569	1576	2E-Tridecanal	-	-	-	1.66
50	44.05	1585	1593	Gleenol	-	1.57	-	-
51	44.06	1605	1597	Spathulenol	0.39	-	0.37	-
52	44.51	1593	1598	Viridiflorol	2.91	0.58	-	-
53	44.52	1608	1604	Humulene epoxide-"II”	-	5.84	5.29	3.54
54	44.94	1613	1615	Tetradecanal	0.76	1.90	9.46	5.00
55	46.67	1646	1652	α-Muurolol	0.26	1.71	0.19	-
56	46.73	1667	1663	Intermedeol	-	-	0.09	1.04
57	47.26	1663	1670	α-Cadinol	2.13	0.51	0.93	-
58	47.49	1673	1678	Tetradecanol	0.43	0.57	1.28	0.53
59	51.41	1774	1780	Pentadecanol	-	0.20	2.69	0.29
60	52.13	1797	1801	Phenanthrene	-	-	-	0.19
61	52.88	1816	1819	Hexadecanol	-	-	1.43	-
62	53.69	1849	1842	Neophytadiene	0.22	-	-	-
63	53.93	1838	1843	Phytone	7.62	8.50	1.34	0.71
64	55.17	1876	1882	Hexadecanol	-	1.08	0.13	-
65	57.59	1948	1952	iso-Phytol	1.00	-	-	-
66	59.78	2000	2001	Eicosane	0.22	-	-	-
67	60.96	2047	2044	Thunbergol	0.53	-	-	-
				Chemical classes	Area %b
				Monoterpenes	-	-	-	0.85
				Monoterpenoids	23.65	17.89	9.75	31.88
				Sesquiterpenes	6.54	22.57	12.13	13.72
				Sesquiterpenoids	25.59	12.88	8.3	5.33
				Diterpenes	0.22	-	-	-
				Diterpenoids	1.53	-	-	-
				Aldehydes	28.66	31.66	60.56	16.83
				Alkanes	-	-	0.87	0.60
				Alcohols	3.72	5.88	6.36	27.6
				Hydrocarbons	0.22	-	-	-
				Aromatic hydrocarbons	-	-	-	0.19
				Ketones	0.55	-	-	-
				Others	7.62	9.04	1.88	0.96
				Total	98.30	99.92	99.85	97.96

• RT: Retention time;
• ^*
  KI_Lit.: literature retention index values;
• ^a
  KI_Exp.: Experimental Retention Index calculated from retention times relative to n-alkane series (C₆ -C₃₀);
• ^b
  %: percentages obtained by FID peak-area normalization.

HPLC-DAD analyses

The extracts were dissolved in methanol and diluted to 50% aqueous concentration suitable for HPLC-DAD analysis (O. rosratum: 16 mg/mL and O. medium: 11 mg/mL). HPLC analysis was performed using a Dionex (Thermo Scientific, Germering, Germany) Ultimate 3000 high-performance liquid chromatography. A Thermo Acclaim C30 column (diameter: 150 mm. 3mm id. 3µm pd) was used with a Macherey Nagel (3 mm id) guard column. Gradient elution was used for the mobile phases: A, 2% acetic acid in water; B, 70% acetonitrile-30% water. The flow rate was 0.37 mL/min, and the injection volume was 10 μL. The column temperature was maintained at 25 °C. The following 25 phenolic standards were used to calibrate and validatethe HPLC-DAD method: gallic acid, protocatechuic acid, p-OH benzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, vanillin, epicatechin, p-coumaric acid, ferulic acid, rutin, luteolin-7-glycoside, naringin, hesperidin, apigenin-7-glycoside, rosmarinic acid, fisetin, eriodictyol, luteolin, quercetin, naringenin, hesperetin, apigenin, and kaempferol. (Turumtay et al., 2022). They were diluted from their stock solution to five different concentrations of 0.625, 1.25, 5.0, 10.0, and 20.0 µg/mL in a 1:1 methanol-water solution. An external calibration method was used, and the regression coefficient was found to be at least 0.99. The repeatability of the retention time and peak areas were measured as the coefficient of variation (CV), which was under 0.61 for retention times and 4.00 for areas of the peaks. Limit of detection and quantification values of the peaks were under 0.18 and 0.52 µg/mL for all standards. Chromatograms were processed at 254, 280, 315, and 370 nm with a DAD which operated 200-400 nm. Since the UV-Vis spectra were available with DAD, the peaks were identified by comparing the retention times and UV-Vis spectra with those of standard phenolic compounds, which were used for the calibration and validation of the method (Table II). Some peaks had the same or very similar UV spectra as some standards but with different retention times. They were defined as derivatives of standards with similar UV spectra and were quantified as equivalent to those standards (Table II).

TABLE II
Phenolic components in methanol extracts of O. rostratum (Or, 16 mg/mL), and O. medium, (Om, 11 mg/mL) by HPLC analysis

No	Compounds		RT	nm (max.- -min.)		Area %		nm	mg/L	mg STD/g extract
1.	Or p-OH Benzoic acid Om		18.19 18.46	255-218 256-217		11.730 22.30		254 254	2.14 4.10	0.134 0.373
2.	Vanilic acid Or		22.55	293-261-224		6.667		254	2.98	0.186
	Om		22.57	292-261-224		4.70		254	2.21	0.201
3.	Apigenin glucoside D. Or		25.26	339-271-227		16.980		315	21.69	1.355
4.	Apigenin glucoside D. Or		25.62	339-272-339		3.697		315	4.80	0.300
5.	Apigenin glucoside D. Or		25.90	341-270-226		2.911		315	3.80	0.237
6.	p-Coumaric acid D. Or		28.15	315-228		11.630		315	2.45	0.153
	Om		28.17	315-228		1.90		315	0.39	0.035
7.	Apigenin glucoside D. Om		28.69	338-272-226		2.714		315	3.55	0.382
8.	Apigenin glucoside D. Or		29.64	335-272		3.145		315	4.09	0.256
9.	Apigenin glucoside D. Or		30.14	341-271		5.055		315	6.52	0.408
10.	Naringin D. Om		31.28	284-228		2.358		280	2.30	0.209
11.	Apigenin glucoside D. Or		31.89	338-289		14.144		315	18.08	1.130
12.	Apigenin glucoside D. Or		32.29	338-269		2.113		315	2.78	0.174
13.	p-OH Benzoic acid D. Om		32.22	295-225		3.959		254	0.70	0.064
14.	p-OH Benzoic acid D. Om		32.59	256		10.70		254	1.95	0.177
15.	p-OH Benzoic acid D. Om		33.53	254		0.497		254	0.06	0.005
16.	Luteolin glucoside D. Or		32.89	348-268		1.116		254	1.08	0.068
17.	Luteolin-7-O-glucoside Or		33.62	347-267		2.507		254	1.82	0.114
18.	p-OH Benzoic acid D. Or		34.86	264		0.941		254	0.14	0.009
	Or		35.23	336-267		0.544		315	0.79	0.049
19.	Apigenin glucoside D. Om		34.87	336-267-224		0.610		315	0.87	0.079
20.	Luteolin glucoside D. Or		35.80	338-266		0.804		254	0.92	0.057
	Om		35.80	348-265-216		0.576		254	0.80	0.072
21.	Luteolin glucoside D. Om		36.05	346-266-253		2.246		254	1.68	0.153
	Or		39.88	315-226		0.858		315	0.17	0.011
22.	p-Coumaric acid D. Om		39.88	315-226		1.982		315	0.41	0.037
23.	p-Coumaric acid D. Or		40.60	312-227		3.166		315	0.66	0.041
	Om		40.59	312-227		8.477		315	1.78	0.162
	Or		41.91	315-212-204		1.597		315	0.33	0.020
24.	p-Coumaric acid D. Om		41.89	315-266-221		1.197		315	0.24	0.022
25.	p-Coumaric acid D. Om		42.51	315-226		0.362		315	0.06	0.006
26.	p-Coumaric acid D. Or		48.07	316-288-220		1.598		315	0.33	0.020

Antimicrobial activities

All test microorganisms: Escherichia coli ATCC35218, Yersinia pseudotuberculosis ATCC911, Pseudomonas aeruginosa ATCC43288, Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC29212, Bacillus cereus 709 Roma, Mycobacterium smegmatis ATCC607, Candida albicans ATCC60193, and Saccharomyces cerevisiae RSKK 251 were obtained from the Hıfzısihha Institute of Refik Saydam (Ankara, Türkiye). The antimicrobial screening test (agar-well diffusion method) was performed as described (Üçüncü et al., 2010b; Barry, 1999; Woods et al., 2011). The tested microorganisms were suspended in a Brain Heart Infusion (BHI). They diluted approximately 10⁶ colony-forming units (per mL), which were “flood-inoculated” on the surface of BHI agar and Sabouraud Dextrose Agar (SDA) and then dried. SDA was used to treat C. albicans. The EOs and solvent extracts (n-hexane, chloroform, methanol, and water) were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions (12.300-108.900 μg/mL). n-Hexane and dimethyl sulfoxide were used as the solvent controls at a dilution of 1:2. Ampicillin, streptomycin, and fluconazole were used as positive controls at ten µg/mL, ten µg/mL, and five µg/ mL concentrations, respectively (Table III).

TABLE III
Antimicrobial activity (MIC) of the EOs and solvent extracts of O. rostratum and O. medium by agar well diffusion method (µg/ml)

Sample Ex-tracts	Const. (µg/mL)	Gram (-)			Gram (+)			No Gr.		Fungi
O. rostratum		Ec	Yp	Pa	Sa	Ef	Bc	Ms	Ca	Sc
n-Hexane	63500	-	-	-	-	-	-	-	-	-
Chloroform	8000	400	400	-	100	-	-	-	-	-
Methanol	67000	875	1750	875	218.7	218.7	218.7	1750	-	1750
Water	35000	-	-	-	-	-	-	-	875	437.5
HD	22300	1115	278.8	-	17.4	34.8	17.4	-	139.4	-
MWD	20400	1020	255	-	15.9	31.9	15.9	-	127.5	-
O. medium
n-Hexane	75200	-	-	-	3760	-	-	-	-	-
Chloroform	6900	345	345	-	172.3	-	-	-	-	-
Methanol	65000	3250	1625	10	3250	-	1625	-	1625	-
Water	42000	-	-	-	-	-	-	-	-	-
HD	21500	1075	263	-	16.8	33.6	16.8	-	134.4	-
MWD	19200	960	240	-	15	30	15	-	120	-
Ampicillin	10	10	18	>128	10	35	15
Streptomycin	10							4
Fluconazole	5								8	8

RESULTS AND DISCUSSION

The EOs of mosses (O. rostratum and O. medium) were extracted using hydrodistillation and microwave distillation methods and analyzed by GC-FID-MS using a Rxi-5MS capillary column. Chemical constituents were identified by comparing their RI and MS data with NIST, Wiley7NL, FFNSC1.2, and W9N11 libraries (Adams, 2007; Cansu et al., 2013). The structures of the compounds, percentages, and retention indices of all compounds in O. medium and O. rostratum are presented in Table I. GC-FID-MS analysis of the EOs yielded 32, 31, 40, and 37 compounds, accounting for 98.30%, 99.92%, 99.85%, and 97.96% of the total components, respectively. Octanal (24.12%), bornyl acetate (20.98%), β-E-ionone (19.90%), phytone (7.62%), and γ-element (3.67%) were found to be the main components in the EO (HD) of O. rostratum, whereas octanal (20.22%), iso-bornyl acetate (11.44%), γ-elemen (11.34%), and α-humulene (8.83%) were detected as the main compounds in the MWD oil of O. rostratum. Pentanal (31.45%), tetradecanal (9.46%), iso-bornyl acetate (8.94%), octanal (5.44%), and humulene epoxide-II (5.29%) were found as the highest amounts in the HD EO of O. medium, but n-pentanol (25.50%), bornyl acetate (24.42%), β-copaene (5.70%), tetradecanal (5.00%), and α-pinene oxide (4.44%) were detected as major compounds in MWD EO of O. medium.

The identified compounds in O. rostratum and O. medium plants were classified according to their component classes (Table I). In the EOs (HD and MWD) of O. medium, aldehydes and monoterpenoids were seen as the main components, at a rate of 60.56% (11 compounds) and 31.88% (6 compounds), respectively. Aldehydes were detected as the main components in both HD and MWD EOs of O. rostratum at the rates of 28.66% (seven compounds) and 31.66% (eight compounds) (Table I). The amounts of terpenic components in the EOs of O. rostratum was determined as 57.53% (HD), 53.34% (MWD), and O. medium was 30.18% (HD) and 51.78% (MWD), respectively. The MWD method was more effective in obtaining EO from O. medium containing terpenic components. However, the terpenic content was found to be quite similar in both the HD and MWD methods in O. rostratum. Different extration techniques such as HD, MWD, and pressing are used to obtain EOs, and it is known in the literature that there is an awareness of volatile compounds according to the techniques used. GC-MS analysis of EO components obtained according to HD and MWD methods for both species revealed chemical component differences. Compounds found only in HD EOs (12 and 16 compounds) or MWD EOs (11 and 12 compounds) of O. rostratum and O. medium are shown in Table I, respectively. Bornyl acetate (20.98%) and iso-bornyl acetate (11.44%) in the EOs of O. rostratum, and pentanal (31.45%) and pentanol (25.50%) in the EOs of O. medium were the major compounds (Table I).

Among the terpenic components, sesquiterpenoids (25.59%) and sesquiterpenes (22.57%) were seen in the highest amount in HD and MWD EOs of O. rostratum, respectively. In both methods, seven terpenic compounds, namely iso-borneol, β-cyclocitral, δ-elemen, α-humulene, β-E-ionone, viridifluorol, and α-cadinol, were observed at varying concentrations. Sesquiterpenes (12.13%) and monoterpenoids (31.88%) were the predominant terpenic components in HD and MWD EOs of O. medium, respectively. In both methods (HD and MWD), nine terpene/terpenoid components (α-terpineol, β-cubebene, β-elemen, α-humulene, α-cadinene, zonarene, E-nerolidol, humulene epoxide-II, and intermedeol) of O. medium were seen in different ratios. EOs analyses of the O. rostratum and O. medium revealed that terpenic components accounted for 57.53%, 53.34%, 30.18%, and 51.78%, respectively. Although they belong to the same plant, they contain different components, depending on the method used to obtain EOs.

Recent research has highlighted the biological importance of mosses, prompting increased scientific research on the volatile components and antibacterial properties of various moss species. Literature has shown that Phytol was the main component of the GC-FID-MS analysis of Rhodobryum ontariense using an HP-5-MS column (Budke et al., 2018). EOs of moss plants (Brachythecium salesroom, Eurhynchium pulchellum, and Plagiomnium undulatum) have been reported by GC-FID-MS. A total of 39 compounds from the EOs of B. salebrosum, E. pulchellum, and P. undulatum at a rate of 85.2%, 80.9%, and 88.8%, and n-nonanal (66.3% and 36.2%) and γ-elemen (24.10%) were reported as the main components, respectively. Sesquiterpenes (51.7%) in P. undulatum and aliphatic aldehydes (73.3% and 57.9%) in B. salebrosum and E. pulchellum were reported as the main components (Özdemir et al., 2009). The EO components of Breutelia tomentose (Sw. ex-Brid.) A. Jaeger, Leptodontium viticulosoides (P. Beauv.) Wijk and Margad, Macromitrium perreflexum Steere, Campylopus richardii Brid., Rhacocarpus purpurascens (Brid.) Paris, and Thuidium peruvianum Mitt. were reported, and epizonarene (8.7%), β-selinene (13.5%), selina-3,11-dien-6-α-ol (19.7%), epi-α-muurulol (15.1%), and phytol (21.7%) were reported as the main components, respectively (Valarezo et al., 2018). GC-FID-MS analysis of Brachythecium albicans (Hedw.) Schimp., Bryum pallescens Schleich. ex Schwagr, and Syntrichia intermedia Brid. produced nonanal (41.0%, 29.3%) and E-2-tetradecene-1-ol (9.9%) as the main compounds, respectively (Özdemir et al., 2010). EOs analysis of Tortula muralis Hedw. Homalothecium lutescens (Hedw.) H. Rob., Hypnum cupressiforme Hedw., and Pohlia nutans (Hedw.) Lindb. gave the nonanal as a major compound in the ratios of 18.3%, 36.8, 12.5, and 7.8, respectively (Üçüncü et al., 2010a). β-Pinene (11.6%) and α-pinene (8.9%) in Hylocomium splendens (Hedw.) and non-anal (26.8%), and heptanal (13.7%) in Leucodon sciuroides (Hedw.) Schwagr has been reported as the main component. Additionally, antimicrobial activity of EOs has been reported to be 428-857 µg/mL against C. albicans (Cansu et al., 2013). GC-FID-MS analysis of Grimmia trichophylla Grew and G. decipiens (Shultz) Lindb. reported 23 and 32 compounds, and 2E-nonenal (9.8%), nonanal (6.3%), trichosane (5.1%), and cyclo tetradecane (3.7%) were reported to be the main components of G. trichophylla and G. decipiens, respectively (Cansu et al., 2010). A total of 13, 33, and 40 components were identified by GC-FID-MS analysis of Tortella inclinata var. densa, Tortella tortusa, and Pleurochaete squarrosa mosses. It has been reported that aldehydes are the leading group of compounds. As a result of antimicrobial studies have shown that essential oils have moderate activity, and their MIC values are within the range of 405-4650 μg/ μL (Tosun et al., 2014). Fatty acids have been reported in the EO of Cyathophorella adiantum (Griff.) M. Fleisch. (Mitra, Burger, Poddar-Sarkar, 2013).

In the literature, P. acutum is a moss plant used for cancer treatment in alternative medicine in China, and its essential oils and antitumor effects have been reported. GC-MS analysis of EO obtained from Orthomnium acutum (Lindb.) T.J. Kop. and Yu Sun identified 74 compounds. It was stated that The volatile components consisted of diterpenes (25.6%), sesquiterpenes (23.89%), and alcohols (21.81%). Moreover, EO showed significant cell growth inhibition against HepG2 and A549 cells in cancer testing (Li et al., 2022). Volatile compounds in Oxytegius tenuirostris, Eurhynchium striatum W. P. Schimper, and Rhynchostegium murale (Hedw.) Schimp., and antioxidant effects of solvent extracts and total phenolic substances have been reported (Yayıntaş et al., 2019). The EOs of mosses belonging to the genera Homalia, Plagiothecium, Taxiphyllum, Orthomnium (Plagiomnium), and Mnium were identified using GC-MS. New sesquiterpene compounds were also identified. Although the amounts of volatile substances in mosses are lower than those in liverworts, they are reported to be similar in terms of terpenoid components. However, it has been reported that they are richer in aliphatic compounds (Sarıtaş et al., 2001). Pharmacognosic studies on mosses identified two new luteolin C-glycosides, chrysoeriol 8-C-rhamnosyl, and four known flavone C-glycosides from P. elatum. Apigenin, luteolin, and chrysoeriol glycosides have also been reported in P. cuspidatum (Ahnut et al., 1992). Twenty-two flavonoid compounds have been identified in seven different species of Orthomnium (Plagiomnium), and flavonoids are apigenin C-glycosides (Wyatt, Lane, Stoneburner, 1991). Three new dihydrobiflavones have been reported in P. cuspidatum (Ahnut et al., 1989). Dihydroxyrobustaflavone and luteolin have been identified in Antitrichia curtipendula and Racomitrium lanuginosum, and a new flavone was reported in P. elatum (Geiger, 1988). Isoorientin glycosides have also been reported in P. affine (Freitag et al., 1986). In addition to the two known biflavonoid compounds, two new biflavonoids have been reported from P. undulatum (Rampendahl et al., 1996). Sterol-based compounds in six moss species have been reported, and the main components are sitosterol, stigmasterol, clionasterol, cholesterol, and campesterol (Chiu, Patterson, Fenner, 1985). The literature shows that Seasonal awareness research has been conducted on secondary metabolite amounts in Marchantia polymorpha, Fissidens taxifolin, Grimmia pulvinate, Polytrichum strictum, and P. undulatum. Metabolite profile differences between seasons, life strategies, growth forms, light, and soil are among the most important ecological determinants (Peter et al., 2018).

The phenolic constituents of the methanol extract of O. rostratum and O. medium were analyzed using the HPLC-DAD method developed using 25 phenolic compounds as standards. The results indicated that each moss had similar phenolic compounds (19 and 15 compounds, respectively), except that the methanol extract of O. rostratum did not contain a naringin derivative (Table II). Apigenin glucoside derivatives (RT: 25.26 (1.355 mg/g) and 28.69 (0.382 mg/g), respectively) were the major phenolic compounds in the methanolic extract of both plants.

The antimicrobial activities of the EOs and solvent extracts of O. rostratum and O. medium against three Gram (-) bacteria (E. coli, Y. pseudotuberculosis, and P. aeruginosa), three Gram (+) bacteria (S. aureus, E. faecalis and B. cereus), one no gram (M. smegmatis), and two yeast fungi (C. albicans and Saccharomyces cerevisiae) were investigated (Table III) (Üçüncü et al., 2010b; Barry, 1999; Woods et al., 2011).

The antimicrobial activities of EOs (HD and MWD) of O. rostratum and O. medium resulted in being most effective against S. aureus, E. faecalis, and B. cereus with IC₅₀ values of 17.4 /15.9 µg/mL and 16.8/15.0 µg/mL, 34.8/31.9 µg/mL and 33.6/30.0 µg/mL, and 17.4/15.9 µg/ mL and 16.8/15.0 µg/mL, respectively. Among the solvent extracts, chloroform was the most effective against E. coli (IC₅₀, 400/345 µg/mL), Y. pseudotuberculosis (IC₅₀, 400/345 µg/mL), and S. aureus (IC50, 100/172.3 µg/mL) in O. rostratum and O. medium, respectively. The other solvent extracts and EOs showed low or no activity against other tested microorganisms. The high amount of terpenic components in the EOs of O. rostratum and O. medium influences antimicrobial activity.

The literature reported that the Aqueous and ethanolic extracts of Bryophyta and Marchantiophyta species showed activity against S. aureus, E. coli, and B. cereus (Nikolajeva et al., 2012). The solvent extract of Lophocolea heterophylla has been shown to inhibit the growth of B. cereus. None of the tested extracts grew against E. coli. 70% of bryophyte species have been reported to show no specific activity against S. aureus. 73% of the ethanolic extracts and 39% of the aqueous extracts of Dicranum scoparium, Atrichum undulatum, and Rhytidiadelphus squarrosus mosses exhibited the highest degree of antibacterial activity against S. aureus. It has been reported that the bactericidal effects of aqueous extracts have not been determined. A previous antimicrobial assessment of crude extracts obtained from mosses showed the greatest inhibition zone diameter against S. aureus (Nikolajeva et al., 2012). In our case, the EOs of O. rostratum and O. medium were more active against S. aureus, E. faecalis and B. cereus, which could be due to the species, location, and time of the plant's collection.

CONCLUSIONS

The EOs and phenolic composition of the aerial parts of O. rostratum and O. medium were identified using GC-FID-MS and HPLC. A total of 67 compounds have been identified from the EOs of O. rostratum and O. medium at a rate of 98.30%, 99.92%, 99.85%, and 97.96%, and terpenic components accounted for 57.53%, 53.34%, 30.18%, and 51.78%, respectively. Octanal (24.12%, HD and 20.22%, MWD), pentanal (31.45%, HD), and pentanol (27.50%, MWD) were reported as the main compounds in the EOs (HD and MWD) of O. rostratum and O. medium. Aldehydes (HD) and monoterpenoids (MWD) were the main class of components in the EOs of O. medium. The major class of compounds detected was aldehydes in both the HD and MWD EOs of O. rostratum. Apigenin glucoside derivatives (1.355 mg/g and 0.382 mg/g) were the main phenolic compounds in the methanol extracts of both mosses. The EOs of both plants were the most effective among the extracts against S. aureus, E. faecalis, B. cereus, and C. albicans within the range of 15-25 mm inhibition zones. Thus, the antimicrobial assay results showed that the EOs and methanol extracts of both mosses could be promising for pharmaceutical applications. In future studies, bio-guided isolation studies should be carried out on O. rostratum and O. medium extracts to determine their bioavailability.

ACKNOWLEDGMENTS

We thank KTU-BAP (8881 and 6714) for their financial support.

DATA AVAILABILITY STATEMENT

All data is available within the article or its supplementary material.

REFERENCES

• Adams RP. Identification of essential oil components by gas chromatography/mass spectrometry: Allured Publ., Carol Stream, IL. 2007.
• Anhut S, Biehl J, Seeger T, Mues R, Zinsmeister HD. Flavone-C-glycosides from the mosses Plagiomnium elatum and Plagiomnium cuspidatum Z Naturforsch C. 1992;47(9-10):654-660.
• Asakawa Y, Ludwiczuk A, Nagashima F. Phytochemical and biological studies of bryophytes. Phytochemistry. 2013;91:52-80.
• Aslanbaba B, Yilmaz S, Tonguc YÖ, Özyurt D, Öztürk BD. Total phenol content and antioxidant activity of mosses from the Yenice forest (Ida mountain). J Sci Perspect. 2017 1:1-12.
• Barry AL. Methods for determining bactericidal activity of antimicrobial agents: approved guideline. National Committee for Clinical Laboratory Standards: National Committee for Clinical Laboratory Standards Wayne, PA. 1999.
• Budke JM, Bernard EC, Gray DJ, Huttunen S, Piechulla B, Trigiano RN. Introduction to the special issue on bryophytes. Crit Rev Plant Sci. 2018;37(2-3):102-112.
• Cansu TB, Ucuncu O, Kahriman N, Özdemir T, Yaylı N. The essential oil composition of Grimmia trichophylla Grew and G. decipiens (Shultz) Lindb. Grown in Turkey. Asian J Chem. 2010;22(9):7280-7284.
• Cansu TB, Yaylı B, Özdemir T, Batan N, Karaoğlu ŞA, Yaylı N. Antimicrobial activity and chemical composition of the essential oils of mosses (Hylocomium splendens (Hedw.) Schimp. and Leucodon sciuroides (Hedw.) Schwägr.) growing in Turkey. Turk J Chem. 2013;37(2):213-219.
• Chiu P-L, Patterson GW, Fenner GP. Sterols of Bryophytes. Phytochemistry. 1985;24(2):263-266.
• Edwards, Sean R. English Names for British Bryophytes British Bryological Society Special Volume. Vol. 5 (4 ed.). Wootton, Northampton: British Bryological Society. 2012.
• Erdağ A, Kürschner H. Türkiye bitkileri listesi:(Karayosunları) Ali Nihat Gökyiğit Vakfı. 2021; 8: 32-37.
• Freitag P, Mues R, Brill-Fess C, Stoll M, Zinsmeister HD, Markham KR. Isoorientin 3′-O-sophoroside and 3′-O-neohesperidoside from the moss Plagiomnium affine Phytochemistry. 1986;25(3):669-671.
• Geiger H. Biflavones from some mosses. Z Naturforsch C. 1988;43:1-4.
• Glime JM. Medical uses: biologically active substances. Chapter. 2-2. In: Glime JM, editor. Bryophyte ecology. Vol. 5. Michigan-USA: E-book sponsored by Michigan Technological University and the International Association of Bryologists. 2017.
• Goffinet B, Buck WR, Shaw AJ. Morphology and Classification of the Bryophyta. In Goffinet, B, Shaw, J. (eds.). Bryophyte Biology (2nd ed.). New York: Cambridge University Press. pp. 55-138. 2008.
• Klavina L, Springe G, Nikolajeva V, Martsinkevich I, Nakurte I, Dzabijeva D, Steinberga I. Chemical composition analysis, antimicrobial activity, and cytotoxicity screening of moss extracts (Moss Phytochemistry). Molecules. 2015;20:17221-17243.
• Koponen T, Sun Y. Preliminary study on phylogenetic position and delimitation of the ciliate arthrodontous genera of the moss family Mniaceae. J Bryol. 2016;39(1):23-38.
• Kürschner H, Frey W. Liverworts, mosses, and hornworts of southwest Asia (Marchantiophyta, Bryophyta, Anthocerotophyta). In der Gebr. Borntraeger verlagbuchhandlung, Stuttgart. 2011.
• Li M, Wang L, Li S, Hua C, Gao H, Ning D, et al. Chemical Composition, Antitumor Properties, and Mechanism of the Essential Oil from Plagiomnium acutum T. Kop. Int J Mol Sci. 2022;23(23):14790.
• Marques RV, Sestito SE, Bourgaud F, Miguel S, Cailotto F, Reboul P, et al. Anti-Inflammatory Activity of Bryophytes Extracts in LPS-Stimulated RAW264.7 Murine Macrophages. Molecules 2022;27:1940.
• Mitra S, Burger BV, Poddar-Sarkar M. Headspace volatile oxylipins of Eastern Himalayan Moss Cyathophorella adiantum extracted by sample enrichment probe. Lipids. 2013;48:997-1004.
• Motti R, Palma A, de Falco B. Bryophytes used in folk medicine: An ethnobotanical overview. Horticulturae. 2023;9:137.
• Nikolajeva V, Liepina L, Petrina Z, Krumina G, Grube M, Muiznieks I. Antibacterial activity of extracts from some bryophytes. Adv Microbiol. 2012;2:345-353.
• Önder A, Yildiz A, Cinar AS, Zengin G, Ak G, Ozenoglu H. The comparison of the phytochemical composition, antioxidant and enzyme inhibition activity of two moss species: Plagiomnium ellipticum (Brid.) T. Kop. and Antitrichia californica Sull., from the southwest ecological region in Turkey. Nat Prod Commun. 2022;36:2660-2665.
• Özdemir T, Üçüncü O, Cansu T, Kahriman N, Yaylı N. Volatile Constituents in Mosses (Brachythecium albicans (Hedw.) Schimp., Bryum pallescens Schleich. ex Schwagr and Syntrichia intermedia Brid.) Grown in Turkey. Asian J Chem. 2010;22:7285-7290.
• Özdemir T, Yaylı N, Cansu TB, Volga C. Essential oils in mosses (Brachythecium salebrosum, Eurhynchium pulchellum, and Plagiomnium undulatum) grown in Turkey. Asian J Chem. 2009;21:5505-5509.
• Pejin B, Bogdanovic-Pristov J, Pejin I, Marko SM. Potential antioxidant activity of the moss Bryum moravicum Nat Prod Commun. 2013;27:900-902.
• Peters K, Gorzolka K, Bruelheide H, Neumann S. Seasonal variation of secondary metabolites in nine different bryophytes. Ecol Evol. 2018;8:9105-9117.
• Rampendahl C, Seeger T, Geiger H, Zinsmeister HD. The bioflavonoids of Plagiomnium undulatum Phytochemistry. 1996;41:1621-1624.
• Sarıtaş Y, Sonwa MM, Iznaguen H, König WA, Muhle H, Mues R. Volatile constituents in mosses (Musci). Phytochemistry. 2001;57:443-457.
• Semerjyan GH Semerjyan IH. Antioxidant activity of extract from three species of mosses: Plagiomnium cuspidatum, Mnium spinosum, Anomodon vticulosus. Hayastani Kensabanakan Handes Biol J Armen. 2022;4(74):79-86.
• Tosun G, Yaylı B, Özdemir T, Batan N, Yaylı N, Karaoglu SA. Chemical composition and antimicrobial activity of essential oils from Tortella inclinata var. densa, T. tortusa, and Pleurochaete squarrosa Asian J Chem. 2014;26:2001-2004.
• Turumtay H, Tombuloglu H, Er H, Sandallı C, Akyuz Turumtay E. Elucidation of Bioactive Compounds in Flower Extracts of Camellia sinensis by HPLC-DAD-MS/ MS and Their Inhibitory effects on Replicative Bacterial DNA Polymerases. Ind Crops Prod. 2022;188:115528.
• Üçüncü O, Cansu TB, Özdemir T, Karaoğlu ŞA, Yaylı N. Chemical composition and antimicrobial activity of the essential oils of mosses (Tortula muralis Hedw., Homalothecium lutescens (Hedw.) H. Rob., Hypnum cupressiforme Hedw., and Pohlia nutans (Hedw.) Lindb.) from Turkey. Turk J Chem. 2010a;34:825-834.
• Üçüncü O, Kahriman N, Terzioğrlu S, Karaoğrlu ŞA, Yaylı N. Composition and antimicrobial activity of the essential oils from flowers of Senecio othonnae, S. racemosus, and S. nemorensis Nat Prod Commun. 2010b;5:831-834.
• Valarezo E, Vidal V, Calva J, Jaramillo SP, Febres JD, Benitez A. Essential oil constituents of mosses species from Ecuador. J Essent Oil-Bear Plants. 2018;21:189-197.
• Vollar M, Gyovai A, Szucs P, Zupko I, Marschall M, Csupor-Loffler B, et al. Antiproliferative and antimicrobial activities of selected bryophytes. Molecules. 2018;23:1520-1535.
• Yayintaş Ot, Ozyurt D, Demirata B, Yuceer YK, Yilmaz S. Chemical Composition and Antioxidant Capacities of Mosses Gathered from Mount Ida (Çanakkale, Türkiye). Fresenius Environ Bull. 2019;2413-2420.
• Yaylı N, Öksüz E, Korkmaz B, Erik I, Fandaklı S, Faiz Ö. Volatile and phenolic contents, antimicrobial and tyrosinase activities of two endemic species Scorzonera pisidica and Scorzonera sandrasica L. grown in Turkey. Rec Nat Prod. 2022;1:46-57.
• Wyatt R, Lane DM, Stoneburner A. Chemosystematics of the Mniaceae. II. Flavonoids of Plagiomnium Section Rosulata. Bryologist. 1991;94(4):443-448.
• Woods GL, Brown-Elliott BA, Conville PS, Desmond EP, Hall GS, Lin G, et al. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes. Approved Standard M24-A2. 2011;31:5.

Edited by

• Associate Editor:
  Severino Matias de Alencar

Publication Dates

• Publication in this collection
  17 July 2026
• Date of issue
  2026

History

• Received
  05 Dec 2024
• Accepted
  24 Mar 2025