Comparative volatiles profiling in Marjoram products essential oil as extracted using classical and headspace techniques and in relation to antioxidant and antibacterial effects

doi:10.21203/rs.3.rs-4824314/v1

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Comparative volatiles profiling in Marjoram products essential oil as extracted using classical and headspace techniques and in relation to antioxidant and antibacterial effects

https://doi.org/10.21203/rs.3.rs-4824314/v1

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Recently, the growth of consumer demand for natural herbal products with both safety and efficacy has led to great advances in analytical tools to assess and assure their quality. Marjoram (Origanum majorana L.), also known as "sweet marjoram" or “sweet oregano” is a Mediterranean herbaceous perennial herb cultivated in Egypt. The main goal of this study was to assess volatiles’ variation in marjoram samples collected from two different commercial products using two different extraction techniques viz. HS-SPME and petroleum ether coupled with GC-MS analysis. A total of 20 major aroma compounds were identified in samples extracted with HS-SPME with abundance of monoterpene hydrocarbons and oxygenated compounds. The major volatiles included β-phellandrene (20.1 and 14.2%), γ-terpinene (13.4 and 11.7%), 2-bornene (12.3 and 11.5%), p-cymene (9.8% and 4.6%) terpenen-4-ol (16.4% and 7.5%), sabinene hydrate (16.02% and 8.8%) and terpineol (4.2 and 3.2%) in MR and MI, respectively. Compared with HS-SPME, 51 aroma compounds were identified in marjoram samples extracted with pet. ether, more enriched in aliphatic hydrocarbons (42.8 and 73.8%) in MR and MI, respectively. While higher identification score was observed in case of solvent extraction, SPME appeared to be more selective in recovery of oxygenated terpenes to more account for marjoram aroma. The total phenolic and flavonoid contents in marjoram samples were at (111.9, 109.1 µg GA/mg) and (18.3, 19.5 µg rutin eq/mg) in MR and MI, respectively. Stronger antioxidant effects were observed in MR and MI samples with IC₅₀ at 45.5 and 56.8 µg/mL respectively compared to IC₅₀ 6.57 µg/mL for trolox as assayed using DPPH assay. Moderate anti-bacterial effect was observed in MR and MI samples and expressed as zone of inhibition mostly against Bacillus subtilis (16.03 & 15.9 mm), B. cereus (12.9 & 13.7mm), Enterococcus faecalis (14.03 & 13.97 mm), and Enterobacter cloacae (11.60 & 11.56 mm) respectively.

Biological sciences/Biochemistry/Metabolomics

Physical sciences/Chemistry/Analytical chemistry/Mass spectrometry

Origanum majorana

sweet oregano

HS-SPME

GC-MS

Total phenolic

total flavonoid

DPPH

antibacterial

Natural products are considered as important spirit for the discovery of novel pharmaceutical active metabolites ¹. Labiatae is one of the most significant herbal families that comprises ca. 224 genera and 5600 species of well-known aromatic plants with potential biological and health benefits ². The genus Origanum is one of the most important genera belonging to family Labiatae that includes 42 aromatic species and 18 hybrids widely distributed throughout Europe, Asia, and North Africa ³. Marjoram (Origanum majorana L.), also known as "sweet marjoram" or “sweet oregano” is a Mediterranean herbaceous perennial herb widely cultivated in Egypt ⁴, Europe, northern America, Greece, France, and Asia ⁵. Marjoram is well known for its economic importance being included in pharmaceutical, cosmetic, food production ⁶, and food preservation ⁷. Moreover, marjoram is used as a condiment and spice for special flavoring in foods such as meat, fish, soups, sauces, and canned foods ⁸.

Marjoram is traditionally used to treat several ailments. Marjoram leaves are the widely used part as anti-anxiety, anticonvulsant, and anti-gout ⁹. Leaf decoction was used in the treatment of respiratory infections and as an anti-diabetic ¹⁰, whereas leaf infusion has been used for management of hypertension ¹¹. Mixed leaf and flower infusion exhibited a calming, antispasmodic effect, and relief colds, fever, and headaches ¹², whereas, blended leaf and stem are known to be effective against rheumatism, stomach pain, headache, cough, insomnia, and as antipyretic ¹³, meanwhile, the whole plant has a sedative effect ¹⁴. Marjoram leaf infusion and decoction were used as sedative and to relief nerve pain in Italian traditional medicine, ¹⁵. Moreover, marjoram was used for the treatment of digestive disorders, bug bites, and as a disinfectant ¹⁶.

Considering its favored and rich aroma, marjoram has been previously investigated for its essential oil and phenolics composition ¹⁰. There are various essential oils in two different commercial products using two different extracts such as linalyl acetate and santalene in HS-SPME and γ sitosterol and longipinane in pet. ether extract. In addition to essential oils, several phenolics were identified from marjoram including luteolin-7-O-β-glucuronide and methyl rosmarinate which isolated from the aqueous acetone extracts of the marjoram dried herb cultivated in Poland ¹⁷.

Recently, the growth of consumer demand for herbal products with exact composition revealed warrants for the development of analytical tools to assess and assure their quality ¹⁸. Metabolomics tools are widely used for quality assessment of herbal products owing to the large metabolite’s matrix ¹⁹. Among metabolomics techniques, GC-MS analysis is well adopted for profiling of volatiles ²⁰. Several extraction techniques are widely used for essential oil recovery form herbal materials among which head space solid phase microextraction (HS-SPME) and solvent extraction are widely used ²¹. To aid for comparison among different samples of origin or derived using various extraction methods in an untargeted manner,, multivariate data analysis including principal component analysis (PCA) is widely used to better assess such data matrix ¹⁸.

The main goal of this study was to assess volatiles’ differences from marjoram samples collected from two different commercial products in the Egyptian market (MR and MI) using two different extraction techniques viz. HS-SPME and solvent extraction. Moreover, assessment of total phenolics and flavonoids in marjoram methanol extract was performed to more account on non-volatile phenolic portion in marjoram. In vitro assays of antioxidant and antibacterial potential in marjoram samples was performed.

Chemical composition of marjoram samples extracted using HS-SPME

The volatile profiles in two commercial marjoram were analyzed using HS-SPME coupled with GC-MS analysis. A total of 20 peaks (Fig. 1) were identified belonging to alcohol, ester, ketone, monoterpene hydrocarbon, oxygenated monoterpene, and sesquiterpene hydrocarbon (Table 1, Fig. 2).

Table 1

Relative area percentage (%) of volatile metabolites in two commercial Marjoram leaf products analyzed via HS-SPME GC–MS (n = 3).
No.		Rt	RI	Compound name	Class		R (%)	Sd	I (%)	sd
14		13.003	1041	4-Thujanol	Alcohol		1.66	0.04	2.93	0.01
15		14.678	1137	Terpinen-4-ol	Alcohol		7.58	0.03	16.47	0.03
Total alcohol							9.24	0.07	19.40	0.04
13		12.403	1272	Linalyl acetate	Ester		0.41	0.01	1.95	0.01
16		15.01	1330	Terpinyl formate	Ester		2.33	0.02	3.36	0.01
19		18.316	1327	4-Terpinenyl acetate	Ester		1.43	0.01	1.25	0.02
Total ester							4.17	0.04	6.56	0.03
17		16.586	1158	Carvenone	Ketone		0.00	0.00	2.86	0.14
Total ketone							0.00	0.00	2.86	0.14
1		7.442	902	α-Thujene	Monoterpene Hydrocarbon		6.91	0.02	6.17	0.02
2		7.618	948	α-Pinene	Monoterpene Hydrocarbon		3.34	0.02	1.31	0.02
3		8.684	964	β-Phellandrene	Monoterpene Hydrocarbon		20.10	0.05	14.26	0.03
4		9.273	943	β-Pinene	Monoterpene Hydrocarbon		2.96	0.02	1.59	0.02
5		9.613	873	β-Thujene	Monoterpene Hydrocarbon		0.92	0.02	1.19	0.02
6		10	932	2-Bornene	Monoterpene Hydrocarbon		12.34	0.05	11.59	0.03
7		10.085	1042	p-Cymene	Monoterpene Hydrocarbon		4.63	0.02	9.86	0.03
8		10.374	943	Camphene	Monoterpene Hydrocarbon		2.32	0.01	1.62	0.03
9		11.266	998	γ-Terpinene	Monoterpene Hydrocarbon		13.40	0.30	11.74	0.03
11		12.18	919	(+)-4-Carene	Monoterpene Hydrocarbon		2.06	0.01	1.78	0.01
18		17.139	948	(+)-3-Carene	Monoterpene Hydrocarbon		0.60	0.01	1.28	0.02
Total Monoterpene hydrocarbon							69.59	0.51	62.39	0.26
10		11.356	1191	γ-Terpineol	Oxygenated Monoterpene		4.24	0.02	3.28	0.02
12		12.262	1041	cis-Sabinene hydrate	Oxygenated Monoterpene		11.78	0.02	5.59	0.02
Total oxygenated monoterpene							16.02	0.03	8.87	0.04
20		21.919	1211	Santalen	Sesquiterpene hydrocarbon		0.88	0.01	0.00	0.00
Total sesquiterpene hydrocarbon							0.88	0.01	0.00	0.00
Table 2. Relative area percentage (%) of volatile metabolites in two commercial Marjoram leaf products pet. ether extract analyzed via head space GC–MS (n = 3).
Peak No.	Rt		RI	Compound name	Class	MR (%)	sd	MI (%)	sd
1	3.137		794	3-Hexen-1-ol	Alcohol	3.25	0.02	5.14	0.00
9	11.25		1191	γ-Terpineol	Alcohol	2.69	0.03	0.28	0.01
12	12.937		1041	4-Thujanol	Alcohol	0.89	0.02	0.00	0.00
13	14.649		1137	Terpinen-4-ol	Alcohol	4.37	0.02	2.08	0.01
28	36.732		2052	Linoleyl alcohol	Alcohol	0.00	0.00	0.58	0.01
50	55.632		3942	1-Heptatriacotanol	Alcohol	0.00	0.00	4.12	0.04
Total alcohol						11.20	0.09	12.20	0.07
3	3.469		752	3-Methylheptane	Aliphatic Hydrocarbon	0.00	0.00	0.15	0.01
4	3.512		842	1,3-Dimethylcyclohexane	Aliphatic Hydrocarbon	0.00	0.00	0.12	0.01
5	3.985		816	Octane	Aliphatic Hydrocarbon	0.00	0.00	0.35	0.02
10	12.838		1185	2,4-Dimethylundecane	Aliphatic Hydrocarbon	0.00	0.00	0.17	0.02
23	31.856		2027	3-Eicosyne	Aliphatic Hydrocarbon	0.00	0.00	0.33	0.02
31	37.276		3508	17-Pentatriacontene	Aliphatic Hydrocarbon	0.27	0.01	0.00	0.00
39	47.34		2009	Eicosane	Aliphatic Hydrocarbon	0.87	0.02	1.65	0.01
40	48.407		3401	Tetratriacontane	Aliphatic Hydrocarbon	6.07	0.14	10.91	0.05
42	49.684		1448	5-Methyltetradecane	Aliphatic Hydrocarbon	0.00	0.00	0.22	0.02
43	52.4		2080	Octacosane	Aliphatic Hydrocarbon	0.58	0.02	2.33	0.03
45	52.883		3500	Pentatriacontane	Aliphatic Hydrocarbon	8.78	0.01	11.11	0.02
46	53.889		3600	Hexatriacontane	Aliphatic Hydrocarbon	9.62	0.05	10.01	0.03
49	55.417		1746	3-Methylheptadecane	Aliphatic Hydrocarbon	0.91	0.01	0.05	0.03
52	55.821		3997	Tetracontane	Aliphatic Hydrocarbon	12.83	0.02	35.02	0.09
53	58.849		4395	Tetratetracontane	Aliphatic Hydrocarbon	2.85	0.02	1.36	0.03
Total Aliphatic hydrocarbon						42.79	0.31	73.77	0.38
25	32.037		1774	Neophytadiene	Diterpene	0.72	0.01	0.00	0.00
35	41.067		2247	Dehydroabietinol	Diterpene	1.78	0.01	2.15	0.02
Total diterpene						2.50	0.02	2.15	0.02
14	14.986		1333	alpha.-Terpinyl acetate	Ester	1.44	0.03	0.76	0.01
16	16.975		1381	γ-Terpinyl acetate	Ester	0.82	0.02	0.47	0.02
17	17.114			Linalyl acetate	Ester	0.32	0.02	0.00	0.00
18	18.3		1327	4-Terpinenyl acetate	Ester	0.26	0.01	0.17	0.02
33	39.425		2370	4-epi-Dehydroabietinol acetate	Ester	0.55	0.02	0.00	0.00
34	39.525		2404	Butyl citrate	Ester	0.21	0.01	0.00	0.00
36	41.45		2193	Linoleyl acetate	Ester	0.00	0.00	0.18	0.02
41	48.937		2192	trans-Geranylgeraniol	Ester	2.15	0.02	0.61	0.05
24	32.023		4085	1,1-Bis(dodecyloxy)hexadecane	Ether	0.00	0.00	0.53	0.02
Total ester						5.74	0.12	2.72	0.13
20	25.634		2609	10,12-Tricosadiynoic acid, methyl ester	Fatty acid/ester	0.00	0.00	0.73	0.02
27	33.528		1878	Palmitic acid, methyl ester	Fatty acid/ester	0.00	0.00	0.95	0.02
29	36.815		2101	Methyl linolenate	Fatty acid/ester	0.34	0.01	1.27	0.02
30	36.91		1886	7-Hexadecenoic acid, methyl ester	Fatty acid/ester	0.00	0.00	0.38	0.01
32	38.585		2177	Palmitic acid, butyl ester	Fatty acid/ester	0.00	0.00	0.57	0.02
37	41.527		2300	n-Propyl linolenate	Fatty acid/ester	0.00	0.00	0.56	0.02
48	55.371		2980	Eicosyl nonyl ether	Fatty acid/ester	0.00	0.00	1.48	0.03
Total fatty acid/ester						0.34	0.01	5.95	0.14
15	16.553		1158	Carvenone	Ketone	0.00	0.00	0.54	0.02
Total ketone						0.00	0.00	0.54	0.02
6	8.485		964	β-Phellandrene	Monoterpene hydrocarbon	0.38	0.01	0.00	0.00
7	9.931		1042	p-Cymene	Monoterpene hydrocarbon	0.00	0.00	0.45	0.03
8	11.145		998	γ-Terpinene	Monoterpene hydrocarbon	0.36	0.03	0.22	0.01
Total monoterpene hydrocarbon						0.74	0.04	0.67	0.04
21	25.655		1569	Isospathulenol	Oxygenated Sesquiterpene	1.33	0.02	0.00	0.00
22	25.783		1507	Caryophyllene oxide	Oxygenated Sesquiterpene	0.56	0.02	0.00	0.00
47	54.731		1484	Cubebol	Oxygenated Sesquiterpene	0.34	0.02	0.00	0.00
Total oxygenated sesquiterpene						2.23	0.06	0.00	0.00
19	21.916		1494	Caryophyllene	Sesquiterpene hydrocarbon	1.03	0.02	0.00	0.00
26	32.539		1393	Longipinane	Sesquiterpene hydrocarbon	0.28	0.01	0.00	0.00
Total sesquiterpene hydrocarbon						1.31	0.03	0.00	0.00
51	55.701		2731	γ-Sitosterol	Sterols	4.55	0.00	24.63	0.25
Total sterols						4.55	0.00	24.63	0.25

Monoterpene and sesquiterpene hydrocarbons

Monoterpene hydrocarbons represented by 11 peaks were detected as the most abundant in marjoram leaf from the two commercial sources at level of 69.5 and 62.4% in MR and MI samples. β-Phellandrene (peak 3) was identified as the major form in both samples at levels 20.1 and 14.2%, respectively. In contrast, p-cymene (peak 7) was detected at higher level in marjoram sample (MI) at 9.8% versus 4.6% in marjoram sample (MR). Comparable levels were detected in case of (peak 9) and 2-bornene (peak 6) in both samples MR and MI 12–13% and 11–12% and suggestive of no pattern in monoterpene profile for each sample %Unlike monoterpene hydrocarbons, sesquiterpenes were detected at trace level only in MR samples, and absent in MI samples.

Previous investigation of marjoram samples revealed detection of α-thujene, α-terpineol, borneol, carvacrol, β-caryophyllene, eucalyptol, linalool, myrcene, p-cymene, phellandrene, sabinene, terpinene, and terpinolene ¹⁰.

Alcohol, ketone, ester, and oxygenated monoterpene

Alcohols represented by two peaks were detected at higher levels in MI samples at 19.4% compared to 9.2% in MR samples. Terpenen-4-ol (peak 15) was detected as the major alcohol in both marjoram samples with two-folds higher level in MI (16.4%) compared to MR sample (7.5%). Ketones represented by carvenone (peak 17) was detected only in MI samples at 2.8%. Likewise, ester represented by 3 peaks was detected in MI samples at 6.5% compared to 4.2% in MR sample. Terpinyl formate (peak 16) was the abundant ester compound in both marjoram samples. In Contrast, oxygenated monoterpenes were detected at higher level in MR samples at 16.02% compared to 8.8% in MI samples. Sabinene hydrate (peak 12), previously detected at high levels in O. majorana ⁴. was found abundant in MR at 11.8% compared to 5.6% in MI sample The oil composition of marjoram cultivated in India was reported by ²² revealing the presence of terpinen-4-ol, sabinene hydrate, p-cymene, sabinene, and α-terpineol as the most abundant volatiles. γ-Terpineol (peak 10) was detected in both marjoram samples at 3–4%. The essential oil composition of three marjoram accessions cultivated in Egypt was previously investigated using hydro-distillation technique ⁴, with major volatiles to include sabinene hydrate (15.4–34.3%), α-terpinen (8.9–18%), 4-terpineol (15.2–35%), terpinolene (10.3–11.8%), and sabinene (7.4–8.4%) ⁴.

The oil composition of marjoram cultivated in India was studied by ²² revealing the detection of terpinen-4-ol, sabinene hydrate, p-cymene, sabinene, and α-terpineol as the most abundant volatiles. Marjoram leaf collected from Egypt extracted by hydro-distillation and supercritical CO₂ using GC-MS analysis revealed the abundance of terpinen-4-ol and sabinene ²³. Terpinen-4-ol was detected as the most abundant volatile detected in marjoram essential oil ²⁴. The abundance of cis-sabinene is a chief determinant of high quality of essential oils in O. majorana (sweet oregano) ⁴. In another study, analysis of essential oil composition in sweat marjoram revealed the detection of terpinene-4-ol (20.9%), linalool (15.7%), linalyl-acetate (13.9%), limonene (13.4%), and α-terpineol (8.57%) ²⁵. Previous reports indicated that carvacrol was abundant in Turkish marjoram, whereas the Iranian variety was rich in linalyl acetate. Marjoram varieties grown in Reunion Island, Greece, and Egypt are rich in terpinen-4-ol and sabinenes ¹⁶. In another study, marjoram essential oil was reported to be rich in carvacrol, thymol, terpinen-4-ol, trans-caryophyllene, γ-terpinene, and p-cymene ²⁶. Study done by ²⁷ on the essential oil composition of Iranian variety revealed the detection of linalool, thymol, p-cymene, terpinen-4-ol, sabinene, β-myrcene, β-caryophyllene, and γ-terpinene.

Chemical composition of pet. ether extract of marjoram samples

Analysis of volatile profile of marjoram samples from two commercial sources extracted with pet. ether using GC-MS analysis revealed the identification of 51 metabolites (Fig. 1, Table 2). The identified volatiles belonged to aliphatic hydrocarbons, alcohols, esters, monoterpene and sesquiterpene hydrocarbons, oxygenated sesquiterpenes, fatty acid/esters, ketones, and sterols (Fig. 2).

Aliphatic hydrocarbons

Aliphatic hydrocarbons represented by 15 peaks were detected as the major metabolite class detected in marjoram samples extracted with pet. ether to account for ca. 73.8 and 42.8% in MI and MR samples, respectively. Tetracontane (peak 52) was the major aliphatic hydrocarbon detected in marjoram samples at higher level in MI sample at 35.02% compared to 12.8% in MR samples. Moreover, other contane-derivatives including tetratriacontane (peak 40), pentatriacontane (peak 45), and hexatriacontane (peak 46) were detected at level range 6.1–9.6% in MR compared to higher level range at 10.01–11.1% in MI samples.

Alcohol, ketone, and esters

Alcohols were detected at comparable levels 11–12% in marjoram samples MR and MI. Compared to SPME extraction, several alcohols were detected with this solvent extraction including 3 -Hexen-1-ol, 1-heptatriacontanol, linoleyl alcohol, terpene-4-ol, 4-thujanol, and γ terpineol. 3-Hexen-1-ol (peak 1) was detected at a high level 5.1% in MI samples compared to 3.3% in MR sample, whereas terpene-4-ol (peak 13) was detected in MR at a higher level (4.4%) than MI samples (2.1%). Fatty alcohols including 1-heptatriacontanol (peak 50) and linoleyl alcohol (peak 28) were detected only in MI samples extracted with pet. ether. Similar to SPME results, carvenone was detected as major ketone in marjoram samples MI. Esters represented by 9 peaks were detected at 5.7% in MR compared to 2.7% in MI. Geranylgeraniol (peak 41) and terpinyl acetate (peak 14) were the abundant esters.

Monoterpene, diterpenes, and sesquiterpene hydrocarbons

Monoterpene hydrocarbons represented by β-phellandrene, p-cymene, and γ-terpinene were detected at trace levels in both marjoram samples extracted with pet. Ether. Likewise, diterpenes including neophytadiene and dehydroabietinol were detected at 2.1–2.5% in marjoram samples. Sesquiterpene hydrocarbons were detected only in MR samples (1.3%) and in accordance with SPME extraction method. Caryophyllene (peak 19) and longipinane (peak 26) represented the sesquiterpenes detected in MR extracted with pet. Ether, while santalene was the one which detected with SPME extraction.

Fatty acids/esters and sterols

Fatty acids were detected at high levels in marjoram samples MI 5.6% compared to traces in MR samples indicating the effect of solvent in the extraction of nonpolar compounds in marjoram. Sterols were detected at higher levels in MI samples at 24.6% represented by γ-Sitosterol compared to much lower level of 4.5% in MR samples.

PCA analysis of marjoram volatile metabolites analyzed with GC-MS

Multivariate data analysis using principal component analysis (PCA) was further used for better assessment of volatiles distribution among marjoram samples from two commercial sources (Fig. 3). A PCA model (Fig. 3A) of marjoram volatiles extracted by HS-SPME showed discrimination of MR clusters at the left side of PC1 versus MI samples clustering towards the right side of PC1. The corresponding loading plot (Fig. 3B) revealed that γ-terpinene, β-phellandrene, and 2-bornene were enriched in MR samples, versus enrichment of oxygenated terpenes i.e, terpinen-4-ol, carvenone, and linalyl acetate in MI samples, in addition to β-thujene and cymene and accounting for its segregation. The abundance of monoterpene hydrocarbon and oxygenated compound in samples extracted using HS-SPME revealed the efficiency of extraction technique for evaluation of the quality of sweet oregano as it in agreement with the hydro-distillation method ⁴.

Another PCA model (Fig. 3C) of marjoram volatiles extracted by pet. Ether showed discrimination of MR clusters at the left side of PC1 while MI samples were clustered towards the right side of PC1. The corresponding loading plot Fig. 3D revealed that γ-terpinene, 4-terpinenyl acetate, and γ-terpineol were enriched in MR samples. Aliphatic hydrocarbons including tetracontane, hexatriacontane, tetratriacontane along with 1-heptatriacotanol and γ-sitosterol were more enriched in MI samples and accounting for their segregation toward the right side. However, identification of large number of metabolites in marjoram extracted with pet. Ether method, large number of non-polar compounds was extracted which may interfere with the quality assessment of marjoram samples. Hence, HS-SPME is the most suitable for assessment of the quality of marjoram samples.

Total phenolics and flavonoids in marjoram from two commercial sources

The total phenolic assay of the two marjoram samples revealed comparable levels of 109–112 µg GA/mg in MR samples (Table 3). Likewise, total flavonoid content was detected at 18.3 µg rutin eq/mg in MR samples compared to 19.5 µg rutin eq/mg in MI samples. Compared to a previous report on Spanish marjoram total phenolics revealed that marjoram contained 163 mg GAE/L ²⁸. Profiling of Egyptian marjoram showed the presence of apigenin, methyl rosmarenate, rosmarenic acid, luteolin-7-O-rutinose, as a major compound, meanwhile p-coumaric acid, gallic acid, chlorogenic acid, caffeic acid, and ferulic acid were detected using HPLC analysis²⁹. Additionally Tunisian marjoram aerial parts revealed the presence of phenolic acids viz. (E)-2-hydroxycinnamic, rosmarinic, vanillic, chlorogenic, gallic, and cinnamic, whereas flavonoids including amentoflavone, apigenin, quercetin, luteolin, coumarin, and rutin ³⁰.

Table 3

Results of total phenolic contents, total flavonoid content, and DPPH radical scavenging capacity of two commercial Marjoram products (R and I) methanolic extract.
Sample	Total phenolic content (µg GA/mg sample)	Total flavonoid content (µg rutin eq/mg sample)	DPPH IC₅₀ (µg/mL)
MR	111.88 ± 3.11	18.32 ± 0.98	45.53 ± 0.66
MI	109.11 ± 8.99	19.54 ± 1.49	56.78 ± 0.86
Trolox (µg/mL)			6.57 ± 0.45

DPPH radical scavenging capacity of marjoram methanolic extracts

MR samples showed radical scavenging capacity with IC₅₀ value of 45.5 µg/mL compared to 56.8 µg/mL for MI samples indicating slightly stronger antioxidant effect of MR (Table 3, Fig. 4), likely attributed for its richness in phenolics and terpenes ²⁸. Next to phenolics, predominant monoterpene hydrocarbons viz. β-phellandrene, p-cymene, and γ-terpinene detected in marjoram essential oil may also contribute to antioxidant activity ²⁸. In agreement with other previous studies, Tunisian O. majorana revealed high antioxidant activity ³¹. Moreover, Iranian O. majorana revealed similar composition of cis-sabinene hydrate and terpeniol, with potential antioxidant capacity ³².

Anti-bacterial activities of petroleum ether extract of marjoram samples from two commercial sources

Essential oil containing herbal extracts are well recognized for their potential antibacterial activity against both Gram-positive and Gram-negative bacteria and the results compared to gentamycin as standard antibacterial drug ³³. Consequently, antibacterial activity of marjoram samples was tested against five Gram-positive bacterial strains and three Gram-negative strains (Table 4). Both marjoram samples revealed moderate antibacterial activities as concluded from the zone of inhibition compared to standard antibacterial agent. MR and MI extracts showed activity against Bacillus subtilis with inhibition zone of 16.03 and 15.9 mm, respectively. Against Bacillus cereus, MR extract showed inhibition zone of 12.9 mm compared to 13.7 mm for MI extract. Moreover, both extracts showed inhibition effect against Enterococcus faecalis with comparable inhibition zone of 14.03 mm. Both Staphylococcus aureus and Streptococcus mutants were found resistant against both marjoram extracts. Regarding Gram-negative bacteria, both marjoram extracts showed inhibition only against Enterobacter cloacae with inhibition zone of 11.6 mm. The antibacterial activity of marjoram oil was previously investigated against S. aureus, E. faecalis, E. coli, and K. pneumoniae ³⁴. Results revealed that among volatiles in O. majorana essential oil, cis-sabinene hydrate contributed towards inhibition of bacterial growth ³⁴. In another study, Pakistani sweet marjoram exhibited antibacterial activity against S. aureus, B. cereus, B. subtilis, P. aeruginosa, and E. coli ²⁵.

Table 4

Comparative antibacterial activity of two marjoram commercial products (MR and MI) pet. ether extract expressed as zone of inhibition (mm).
Evaluated microorganism/sample	MR (Av ± sd)	MI (Av ± sd)	Gentamycin
Gram-positive bacteria
Staphylococcus aureus	N/A	N/A	24.01 ± 0.01
Bacillus subtilis	16.03 ± 0.35	15.91 ± 0.40	26.02 ± 0.03
Streptococcus mutants	N/A	N/A	20.01 ± 0.02
Bacillus cereus	12.97 ± 0.25	13.71 ± 0.40	24.97 ± 0.06
Enterococcus faecalis	14.03 ± 0.45	13.97 ± 0.35	26.09 ± 0.01
Gram-negative bacteria
Enterobacter cloacae	11.60 ± 0.53	11.56 ± 0.50	27.03 ± 0.06
Escherichia coli	N/A	N/A	29.90 ± 0.01
Salmonella typhimurium	N/A	N/A	17.02 ± 0.03
N/A; no activity. The test was done using agar technique, well diameter: 6.0 mm (100 µl was tested), samples was tested at 100 mg/ml and positive control gentamycin 4 µg/ml

Sample collection

Two marjoram commercial products labeled as sweet marjoram (Origanum majorana L.) were provided from wo different companies Royal (MR (and Isis (MI) was collected from a local market in Cairo, Egypt.

Sample Preparation

HS-SPME

Fibers used in SPME volatile extraction are stable flex coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 µm) or PDMS (polydimethylsiloxane) were purchased from Supelco (Oakville, ON, Canada). Volatile standards were provided from Sigma Aldrich (St. Louis, MO, USA). Marjoram leaf sample (100 mg) were placed in an SPME screw-cap vial (1.5 mL) spiked with 10 µg (Z)-3-hexenyl acetate with fibers inserted manually above and placed in an oven kept at 50 ºC for 30 min. HS-SPME analysis of the volatile compounds was performed as reported in ³⁵ with slight modifications. The fiber was subsequently withdrawn into the needle and then injected manually into the injection port of a gas chromatography–mass spectrometer (GC–MS).

Solvent extraction using pet. ether

The powdered marjoram leaf sample (20 g each) was extracted with pet. ether (150 mL x 3) with cold sonication at 35 ºC (using Biomall sonicator, India) for volatile extraction. Pet. ether was selected considering its non-polar nature more suited to recover volatile terpenes with less interference from other less volatiles phytochemicals ³³. The pet. ether extract was filtered and concentrated under reduced pressure using Rotary evaporator (Hahin-shin, Japan) at 40 ºC till complete evaporation of pet. ether to yield concentrated extract used for GC-MS analysis.

GC-MS analysis of marjoram aroma profile

GC/MS analysis was performed using Shimadzu GCMS-QP2020 (Tokyo, Japan). The GC was equipped with Rtx-1MS fused bonded column (30 m x 0.25 mm i.d. x 0.25 µm film thickness) (Restek, USA) and a split–splitless injector. The initial column temperature was kept at 45°C for 2 min (isothermal) and programmed to 300°C at a rate of 5°C/min and kept constant at300°C for 5 min (isothermal). The injector temperature was 250°C. Helium carrier gas flow rate was 1.41 ml/min. All the mass spectra were recorded applying the following condition: (equipment current) filament emission current, 60 mA; ionization voltage, 70 eV; ion source, 200°C. Diluted samples (1% v/v) were injected with split mode (split ratio 1: 15).

Identification of essential oil chemical composition

Identification was based on calculation of retention indices of each component, mass spectra matching with NIST-11 and Wiley library database as well as published data in literature ^25,36. Retention indices (RI) were calculated relative to a homologous series of n-alkanes (C8-C28) injected under the same conditions.

Total phenolics assay

The total phenolic content was determined using Folin–Ciocalteu method as described by ³⁷. Samples were prepared at the concentration of 4 mg/mL in methanol. Briefly, the procedure consisted of mixing 10 µL of sample/standard with 100 µL of Folin-Ciocalteu reagent (Diluted 1: 10) in a 96-well microplate. Then, 80 µL of 1M Na₂CO₃ was added and incubated at room temperature (25°C) for 20 min in the dark. At the end of incubation time the resulting blue complex color was measured at 630 nm. Data are represented as means ± SD. Gallic acid standard curve was used for calculation of total phenolic content. The results were recorded using microplate reader FluoStar Omega.

Total flavonoids assay

The total flavonoids content was determined using the aluminum chloride method as described by ³⁸, with minor modifications. Samples were prepared at 4 mg/mL in methanol. Briefly, 15 µL of sample/standard was placed in a 96-well microplate, then, 175 µL of methanol was added followed by 30 µL of 1.25% AlCl₃. Finally, 30 µL of 0.125 M C₂H₃NaO₂ was added and incubated for 5 min. At the end of incubation time the resulting yellow color was measured at 420 nm. Data are represented as means ± SD. Rutin standard curve was used for calculation of total flavonoid content. The results were recorded using microplate reader FluoStar Omega.

DPPH free radical assay

DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical assay was carried out according to the method of ³⁹. Briefly, 100µL of freshly prepared DPPH reagent (0.1% in methanol) were added to 100 µL of the sample in 96 wells plate (n = 6), the reaction was incubated at room temp for 30 min in dark. At the end of incubation time the resulting reduction in DPPH color intensity was measured at 540 nm. Data are represented as means ± SD according to the following equation:

percentage inhibition= ((Average absorbance of blank-average absorbance of the test)/(Average absorbance of blank))*100.

Trolox standard curve was used as positive control for antioxidant assay. The results were recorded using microplate reader FluoStar Omega. Samples MI, and MR were prepared at final the concentration of 15.625, 31.25, 62.5, 125 and 250 µg/ml in Methanol. Trolox stock solution of 20 µg/mL was prepared in 100% methanol from which 5 concentrations were prepared including 10, 7.5, 5, 2.5 and 1.25 µg/mL.

In vitro antibacterial assay

The antibacterial activity of tested samples was investigated in vitro against eight bacterial strains and results were investigated in term of zone of inhibition. The culture medium is Mueller-Hinton agar recommended by National Committee for Clinical Laboratory Standards. The standard antimicrobial agent was gentamicin; as an antibacterial agent obtained from the Regional Center for Mycology and Biotechnology, Al-Azhar University, Egypt. The microorganisms were obtained from the Regional Center for Mycology and Biotechnology, Al-Azhar University, Egypt. Gram-positive bacteria Staphylococcus aureus ATCC 25923, Bacillus subtilis RCMB 015 (1) NRRL B-543, Bacillus cereus RCMB 027 (1), Streptococcus mutants RCMB 017 (1) ATCC 25175, Enterococcus faecalis ATCC 29212. Gram-negative bacteria Enterobacter cloacae RCMB 001(1) ATCC 23355, Escherichia coli ATCC 25922, and Salmonella typhimurium RCMB 006 (1) ATCC 14028.

Statistical analysis

The results were displayed as mean ± standard deviation of the mean (SD). Data was analyzed using Microsoft Excel® and the IC₅₀ value was calculated using Graph pad Prism 6® by converting the concentrations to their logarithmic value and selecting non-linear inhibitor regression equation (log (inhibitor) vs. normalized response – variable slope equation).

Assessment of volatile metabolite variations in marjoram samples collected from two different commercial products (MR and MI) using two different extraction techniques viz. HS-SPME and petroleum ether coupled with GC-MS analysis was studied herein. A total of 20 aroma compounds and 51 volatile metabolites were identified in samples extracted with HS-SPME and pet. Ether, respectively. HS-SPME revealed the abundance of monoterpene hydrocarbons and oxygenated compounds. The major identified aroma compounds were β-Phellandrene (20.1 and 14.2%), γ-terpinene (13.4 and 11.7%), 2-bornene (12.3 and 11.5%), p-cymene (9.8% and 4.6%) terpenen-4-ol (16.4% and 7.5%), Sabinene hydrate (16.02% and 8.8%) and terpineol (4.2 and 3.2%) in MR and MI, respectively. Extraction with pet. Ether revealed the abundance of aliphatic hydrocarbons (42.8 and 73.8%) in MR and MI, respectively. PCA analysis revealed variation in volatiles between both samples with a distinct quality in MR samples with higher cis-sabinene hydrate. The abundance of monoterpenes and oxygenated compounds in HS-SPME analyzed samples revealed the efficiency of the extraction in evaluation of marjoram essential oil composition, while pet. Ether increased the recovery of non-polar compounds. The total phenolic and flavonoid contents in marjoram samples were (111.9, 109.1 µg GA/mg) and (18.3, 19.5 µg rutin eq/mg) in MR and MI, respectively. MR samples showed DPPH inhibition with IC₅₀ at 45.5 µg/mL compared to 56.8 µg/mL for MI samples which indicate the antioxidant effect. The antibacterial activity of marjoram samples was tested against Gram-positive and Gram-negative strains and revealed moderate antibacterial activities. Hence, HS-SPME is recommended for quality assessment of marjoram essential oil. Further LC-MS based analysis is recommended for profiling of the phenolics and flavonoids in marjoram.

Author Contributions

M. B. A.: Methodology, Data Curation. E. M. S.: Writing Original draft. O. E.: Methodology, Data Curation. M.A. F. Writing-Review editing. M. H. B.: Conceptualization, Supervision, Writing Original draft, Writing-Review editing.

Conflict of interest.

No conflict of interest

Data availability

All data generated or analyzed during this study are included in this published article.

Funding

None

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Comparative volatiles profiling in Marjoram products essential oil as extracted using classical and headspace techniques and in relation to antioxidant and antibacterial effects

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Abstract

Figures

Introduction

Results and discussion

Chemical composition of marjoram samples extracted using HS-SPME

Monoterpene and sesquiterpene hydrocarbons

Alcohol, ketone, ester, and oxygenated monoterpene

Chemical composition of pet. ether extract of marjoram samples

Aliphatic hydrocarbons

Alcohol, ketone, and esters

Monoterpene, diterpenes, and sesquiterpene hydrocarbons

Fatty acids/esters and sterols

PCA analysis of marjoram volatile metabolites analyzed with GC-MS

Total phenolics and flavonoids in marjoram from two commercial sources

DPPH radical scavenging capacity of marjoram methanolic extracts

Anti-bacterial activities of petroleum ether extract of marjoram samples from two commercial sources

Material & Methods

Sample collection

Sample Preparation

HS-SPME

Solvent extraction using pet. ether

GC-MS analysis of marjoram aroma profile

Identification of essential oil chemical composition

Total phenolics assay

Total flavonoids assay

DPPH free radical assay

Statistical analysis

Conclusion

Declarations

References

Additional Declarations

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