Identification and Quantification of Rosmarinus officinalis L. Leaf Extract Phytochemical Profiles Using Gas Chromatography-Mass Spectrometry

Received 29 Aug, 2023

Accepted 04 May, 2024

Published 30 Sep, 2024

Background and objectives: In Palestine, herbal medicine is widespread, with rosemary being one of the most commonly utilized herbs. However, its usage is more closely associated with traditional customs passed down through generations than scientific research. Rosemary contains secondary metabolites that have broad applications in folk medicine and the culinary sector. The research aim was the extraction of Rosmarinus officinalis L. leaves collected from different locations, followed by GC-MS analysis and phytochemical testing. Materials and Methods: Samples of rosemary leaves were gathered from various locations in the Southern Region of the West Bank, including Raqah, Khilt Al-Adrah, Umm Lasfah village, Hebron City and Bani Naim. The air-dried leaves were powdered and subjected to extraction using two different concentrations of methanol (80 and 90%). The evaluation of the methanol extracts of rosemary leaves was done using two extraction methods (room temperature and reflux) using GC-MS while the photochemistry of these extracts was studied using different tests. Results: The GC-MS revealed the presence of numerous volatile compounds, each showing varying levels across the samples. Common volatile compounds detected in all samples included 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate, bornyl acetate and caryophyllene. Phytochemical analysis of the rosemary leaves revealed the presence of compounds such as cardiac glycosides, phenolic groups, coumarin, saponins, steroids, tannins and terpenoids in all samples. Interestingly, alkaloids were found only in the leaves of the Raqaa and Bani Naim samples. Conclusion: The Palestinian rosemary is rich in phytochemicals; thus, rosemary leaves are recommended for medicinal purposes.

INTRODUCTION

Medicinal plants contain phytochemicals like flavonoids, alkaloids, tannins and terpenoids, known for their antibacterial and antioxidant qualities^1,2. Rosemary extracts derived from dried leaves have garnered significant attention in the food and pharmaceutical sectors due to their wide-ranging health benefits, encompassing antioxidant, antibacterial, anti-inflammatory and anticancer properties^3-5.

Rosmarinus officinalis L. consists of bioactive compounds known as phytocompounds, which play a role in carrying out diverse pharmacological functions, including anti-inflammatory, antioxidant, antimicrobial, antiproliferative, antitumor, protective, inhibitory and attenuating activities^6,7.

Limited research has been conducted on Palestinian herbs, leaving their composition, effectiveness and safety largely unexplored. With concerns surrounding the adverse effects of conventional synthetic drugs, including toxicity and carcinogenicity and the growing issue of microbial resistance to existing antimicrobial agents, there has been a notable surge in interest in identifying naturally occurring antioxidant and antimicrobial compounds suitable for food and medicinal applications. Although a few studies in Palestine have demonstrated the positive effects of Rosmarinus officinalis L. on overall health and therapeutic enhancement^8,9, an exploration of the presence and extent of antioxidants, minerals, antibacterial properties and other polyphenols still needs to be light of this, we propose to analyze Rosmarinus officinalis L. leaves using GC-MS to determine the active principles present and to assess and compare the phytochemicals, antioxidants, antimicrobial activity, nutritional composition and biological potential of rosemary leaves. The selection of Rosmarinus officinalis L. for this investigation is rooted in its established medicinal reputation among Palestinians and widespread use. The lack of comprehensive analysis of its volatile and semi-volatile phytochemical composition and mineral content, coupled with the scarcity of pharmacological studies like antioxidant and antimicrobial assessments, has driven the motivation behind this research. The concentration of phytochemicals in rosemary extract varies based on the extraction method employed¹⁰. Rosemary extract contains various chemical groups, including flavonoids, polyphenols, terpenoids and volatile oils¹¹. The phytochemical composition of R. officinalis extracts encompasses compounds such as rosmarinic acid, caffeic acid, ursolic acid, betulinic acid, carnosic acid and carnosol¹². The essential oils of rosemary consist of 1,8-cineole, α-pinene, verbenone, camphor and borneol, although their proportions can significantly differ¹³. This study was carried out to examine the phytochemical composition of Rosmarinus officinalis L. obtained from different regions of Palestine.

MATERIALS AND METHODS

Study area: This research was conducted at the Department of Pharmacy Laboratory, Faculty of Pharmacy and Medical Sciences, University of Hebron, Palestine, between January, 2021 and January, 2022.

Data and sample collection: During the 2021 season (from February to July, 2021), 5 samples of rosemary leaves were collected from different sites in the Southern Region of the West Bank, Hebron-Palestine, including Raqah, Khilt Al-Adrah, Umm Lasfah Village, Hebron City and Bani Naim. The leaves of R. officinalis were dried in the shade at room temperature. The dried samples were stored in airtight paper bags, protected from light and powdered before extraction. From each, 5 g of rosemary powder were extracted with 50 mL of methanol at two concentrations (80 and 90%) and two extraction methods (room temperature and reflux) and analyzed using GC-MS.

Gas chromatography-mass spectrometry (GC-MS) analysis: The GC-MS analysis was carried out using the GC-MS Perkin Elmer Auto System. Used as a DB-5ms capillary column (30 m, 0.25 μm film thickness, 0.25 μm capillary diameter), the injection volume was 1 μL as identified^7,14,15. After being held at 80°C for 2 min, the oven’s temperature was raised to 280°C at a rate of 6°C per min. The injector’s temperature was set to 280°C. Helium is the chosen carrier gas and the gas flow and velocity were kept at 134.3 mL per min and 43.1 cm per min, respectively. The compounds’ molecular masses (m/z), which were obtained at a rate of 70 mv, ranged from 50 to 500 m/z and the MS scan speed was 1000 amu s1.

Peak identification: The identification of compounds was based mainly on matching their MS spectra with the National Institute of Standards and Technology (NIST) mass spectral library. Moreover, the Kovats Retention (KI) calculation was used to support the identification. The KI values were compared with NIST

values from the literature. Excellent agreement was obtained even using different chromatographic conditions. Quantitative analysis of the essential oils was performed once the MS knew the identities of the compounds.

Phytochemicals: Phytochemical tests of the samples were carried out according to the procedure described by Harborne¹⁶ and Mujeeb et al.¹⁷:

	•	Test for anthocyanin: In a test tube, 2 mL of extract was added to 1 mL of (2N) NaOH and heated for 5 min. The formation of a bluish-green color indicates a positive for anthocyanin
	•	Test for coumarin: In a test tube, 1 mL of extract was added to 1 mL of NaOH and kept in a boiling water bath for a few min. The presence of yellow color indicates positive coumarins
	•	Test for saponins: In a test tube, 5 mL of distilled water was shaken with 2 mL of extract and foam formation indicates positive saponins
	•	Test for quinone: As 1 mL of concentrated H2SO4 was added to 1 mL of extract in a test tube. The red coloration is a sign that quinines are present
	•	Test for glycosides: As 2 mL of the extract was mixed with 2 mL of 50% H2SO4 in a test tube. Fehling’s solution is added and boiled in 10 mL after 5 min of water bath heating. A red prick precipitate suggests the presence of glycosides
	•	Test for anthraquinones: In a test tube, 2 mL of the extract and benzene were combined with 1 mL of a 10% NH3 solution. A positive result for anthraquinones is indicated by a red, pink, or violet hue
	•	Test for cardiac glycosides: In a test tube, 2 mL of glacial acetic acid and 1 mL of conc. H2SO4 and a few drops of FeCl3 were added to the 2 mL extract. The formation of the brown ring indicates a positive for glycosides
	•	Test for steroids: As 1 mL of extract was mixed with 2 mL of CHCl3 and 1 mL of H2SO4 in a test tube, the presence of a reddish-brown ring confirms the presence of steroids
	•	Test for flavonoids: In a test tube, 2 mL of extract were combined with a few drops of 1% NH3 solution. Yellow coloration is a sign that flavonoids are present
	•	Test for phenolic groups: As 1 mL of extract was placed in a test tube along with 2 mL of distilled water and a few drops of 10% FeCl3. A positive for phenolic groups is the creation of a blue or black color
	•	Test for tannins: In a test tube, 1 mL of distilled water and 1-2 drops of FeCl3 were added to a 2 mL extract, a green or blow black color indicates a positive for tannins
	•	Test for terpenoids: As 2 mL of CHCL3 and 3 mL conc in a test tube. The H2SO4 was mixed with 2 mL of extract. The formation of a reddish-brown layer indicates a positive for terpenoids
	•	Test for phlobatannins: In a test tube, 1 mL of 10% NaOH was added to the 2 mL extract. The formation of a yellow color indicates a positive for phlobatannins
	•	Test for alkaloids: In a test tube, 1 mL of 1% HCl was added to a 2 mL extract and then a few drops of Meyer’s reagent were added to the mixture. The presence of a white precipitate indicates a positive for alkaloids

RESULTS

Gas chromatography-mass spectrometry (GC-MS) analysis: Different concentrations of methanolic extracts from rosemary leaves were tested by GC-MS and identified by comparing them with the NIST library. The GC-MS analysis revealed the presence of many volatile compounds in each rosemary sample with different values. Despite their geographical location, all rosemary leaves were found to contain volatile compounds. Significant compounds were identified and their molecular formula, weight and retention time were summarized in Table 1-10. The GC-MS analysis of rosemary in the 80% methanol extract at room temperature (Table 1) showed the identification of many compounds. Major volatile compounds detected in all samples were 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate, bornyl acetate and caryophyllene. The Raqaa region sample showed the presence of more components compared with others (Table 1). In addition to the mentioned compounds, major volatile compounds were detected: Vitamin A, aldehyde, naphthalene, beta-humulen, epicedrol, isoparvifuran, kokusaginine, (2 (1H)-Phenanthrenone, 3, 4, 4A, 9, 10, 10A-Hexahydro-6-Methoxy-1,1,4A-) and (Indolo[2,3-A] Quinalizine, 1, 2, 3, 4, 5, 6, 7, 12B-Octahydro-12, 12B-Dimethyl. The other minor volatile compounds identified are shown in Table 1. Nine compounds were found in rosemary leaves with 90% methanol at room temperature in the Raqaa region (Table 1). The significant compounds identified were 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate and caryophyllene. The other minor volatile compounds identified are shown in Table 1. Table 2 shows 14 compounds found in rosemary leaves with 80% methanol in the Khilt Al-Adrah region sample and 15 compounds extracted from rosemary leaves with 90% methanol at room temperature. The additional significant compounds identified were isoparvifuran and 2 (1 hr)-pyridinone, 3, 4, 4A and 10A-hexahydro-6-methoxy-1,1,4A. The major compounds identified were 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate and caryophyllene. The other minor volatile compounds identified are shown in Table 2.

Table 1:

Compounds detected in methanol extracts 80 and 90% at RT. of rosemary leaves in the Raqaa region sample with their retention time (RT), molecular weight (MW) and molecular formula (MF)

Methanol extracts 80% at RT				Methanol extracts 90% at RT
RT	Compound	MW	MF	RT	Compound	MW	MF
3.489	3-Carene	136	C10H16	3.489	3-Carene	136	C10H16
3.539	α-Pinene	136	C10H16	3.729	Camphene	136	C10H16
3.729	Camphene	136	C10H16	4.509	Alph-caryophyllene	136	C10H16
4.254	1.3 cyclopentadene	122	C9H14	5.02	Eucalyptol	154	C10H18O
4.825	Benzene, 1 methyl-3-(methylethyl)	134	C10H14	7	Camphor	152	C10H16O
4.905	D-Limonene	136	C10H16	7.436	Borneol	154	C10H18O
4.99	Eucalyptol	154	C10H18O	9.296	Isobornyl acetate	196	C12H20O2
6.99	Camphor	152	C10H16O	11.592	Caryophyllene	204	C15H24
7.441	Borneol	154	C10H18O	12.152	Alpha-Caryophyllene	204	C15H24
7.556	Borneol chloride	172	C10H18OCl
8.076	Bicycle (3,1,1 hepta-3-(-N-2-one)	150	C10H14O
9.031	Cyclopropane carboxylic acid, 2,2-Dimethyl-3-(2-methyl-1-propen)	168	C10H16O2
9.301	Isobornyl acetate	196	C12H20O2
10.352	Cyclohexanmethanol	140	C9H16O
11.337	Vitamin A aldehyde	284	C20H28O
11.612	Humulen-(V1)	204	C15H24
14.989	Naphthalene	204	C15H24
15.324	Beta-Humulen	204	C15H24
16.279	Thunbergol	290	C20H34O
16.924	Aromadendrene oxide-(2)	220	C15H24O
17.29	Caryophyllene	204	C15H24
17.72	Thujopsene	204	C15H24
18.735	Epicedrol	222	C15H26O
20.086	3,4-Diethylphenol	150	C10H14O
20.311	Lumisantonin	246	C15H18O3
20.501	Methoxsalen	216	C12H8O4
20.791	Menthol	156	C10H20O
21.296	Ambrosin	246	C15H18O3
21.771	Isoparvifuran	254	C16H14O3
24.723	Furo[2,3B]Quinoline, 4, 6, 7-trimethoxy (Kokusaginine)	259	C14H13O4N
26.678	2(1H)-phenanthrenone, 3, 4, 4A, 9, 10, 10A-hexahydro-6-methoxy-1,1,4A-.	314	C21H30O2
27.674	Indolo[2,3-A] Quinalizine, 1, 2, 3, 4, 5, 6, 7, 12B-octahydro-12, 12B-Dimethyl	296	C20H28ON2

Table 2:

Compounds detected in methanol extracts 80 and 90% at RT of rosemary leaves in the Khilt Al-Adrah Region sample with their retention time (RT), molecular weight (MW) and molecular formula (MF)

Methanol extracts 80% at RT				Methanol extracts 90% at RT
RT	Compound	MW	MF	Rt	Compound	MW	MF
3.524	3-Carene	136	C10H16	3.534	3-Carene	136	C10H16
3.764	Camphene	136	C10H16	3.729	Camphene	136	C10H16
4.995	Eucalyptol	154	C10H18O	4.509	Alpha-phellandrene	136	C10H16
7.005	Camphor	152	C10H16O	5.015	Eucalyptol	154	C10H18O
7.451	Borneol	154	C10H18O	7.02	Camphor	152	C10H16O
9.296	Isobornyl acetate	196	C12H20O2	7.446	Borneol	154	C10H18O
11.587	Caryophyllene	204	C15H24	7.811	Bornyl chloride	172	C10H17Cl
15.349	Beta-humulene	204	C15H24	8.076	D-Verbenone	150	C10H14O
16.914	Geraniol	430	C10H18O	9.306	Isobornyl acetate	196	C12H20O2
18.76	Longifolenealdehyde	220	C15H24O	11.602	Caryophyllene	204	C15H24
21.761	Isoparvifuran	254	C16H14O3	12.157	Alpha-caryophyllene	204	C15H24
24.752	Furo[2,3B]Quinoline, 4, 6, 7-trimethoxy (Kokusaginine)	259	C14H13NO4	14.143	Vitamin A aldehyde	284	C20H28O
26.658	2(1H)-pyridinone, 3, 4, 4A, 10, 10A-hexahydro-6-methoxy-1, 1, 4A-	314	C21H30O2	15.204	Methyl steviol	204	C21H32O3
27.659	indolo[2,3-A]Quinalizine, 1, 2, 3, 4, 5, 6, 7, 12B-Octahydro-12, 12B-Dimethyl	296	C20H28ON2	23.367	Ferruginol	286	C20H30O
				26.833	Alpha-amyrin	426	C30H50O

Table 3:

Compounds detected in methanol extracts 80 and 90% at RT of rosemary leaves in the Umm Lasfah Village sample with their retention time (RT) (MW) and molecular formula (MF)

Methanol extracts 80% at RT				Methanol extracts 90% at RT
RT	Compound	MW	MF	RT	Compound	MW	MF
3.479	3-Carene	136	C10H16	3.434	3-Carene	136	C10H16
3.754	Camphene	136	C10H16	3.729	Camphene	136	C10H16
4.995	Eucalyptol	154	C10H18O	4.509	Alpha-phellandrene	136	C10H16
7.005	Camphor	152	C10H16O	5.005	Eucalyptol	154	C10H18O
7.436	Borneol	154	C10H18O	7.01	Camphor	152	C10H16O
8.076	D-Verbenone	150	C10H14O	7.441	Borneol	154	C10H18O
9.301	Isobornyl acetate	196	C12H20O2	7.801	Bornyl chloride	172	C10H17Cl
11.582	Caryophyllene	204	C15H24	8.066	D-Verbenone	150	C10H14O
15.199	Humulen-(V1)	204	C15H24	9.306	Isobornyl acetate	196	C12H20O2
20.791	Menthol	156	C10H20O	11.597	Caryophyllene	204	C15H24
21.751	Isoparvifuran	254	C16H14O3	12.152	Alpha-caryophyllene	204	C15H24
26.653	2 (1H)-phenanthrenone, 3, 4, 4A, 10, 10A-hexahydro-6-	314	C21H30O2	15.194	Methyl steviol	204	C21H32O3
	methoxy-1, 1, 4A-			23.362	Ferruginol	286	C20H30O

Table 3 presents the 12 compounds extracted from rosemary leaves in 80% methanol. The 13 compounds found in rosemary leaves in 90% methanol at room temperature in the Umm Lasfah Village Region. The significant compounds identified were 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate, bornyl acetate and caryophyllene and 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate and caryophyllene in 80 and 90% methanol, respectively. The other minor volatile compounds identified are shown in Table 3. Table 4 shows the 13 compounds in rosemary leaves in 80% methanol. The nine compounds found in rosemary leaves in 90% methanol at room temperature in the Hebron region. The significant compounds identified were 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate, bornyl acetate and caryophyllene for 80% methanol and 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate and caryophyllene for 90% methanol. The other minor volatile compounds identified are shown in Table 4.

Table 4:

Compounds detected in methanol extracts 80 and 90% at RT of rosemary leaves in the Hebron sample with their retention time (Rt) (MW) and molecular formula (MF)

Methanol extracts 80% at RT				Methanol extracts 90% at RT
RT	Compound	MW	MF	RT	Compound	MW	MF
3.474	3-Carene	136	C10H16	3.489	3-Carene	136	C10H16
3.719	Camphene	136	C10H16	3.734	Camphene	136	C10H16
5.005	Eucalyptol	154	C10H18O	4.509	Alpha-phellandrene	136	C10H16
7.005	Camphor	152	C10H16O	5.025	Eucalyptol	154	C10H18O
7.446	Borneol	154	C10H18O	7.015	Camphor	152	C10H16O
7.806	Bornyl chloride	172	C10H17Cl	9.301	Isobornyl acetate	196	C12H20O2
8.076	D-Verbenone	150	C10H14O	11.597	Caryophyllene	204	C15H24
9.301	Bornyl acetate	196	C10H20O2	12.157	Alpha-caryophyllene	204	C15H24
11.587	Caryophyllene	204	C15H24	23.362	Ferruginol	286	C20H30O
15.209	Beta-humulen	204	C15H24
20.801	Menthol	156	C10H20O
21.766	Isoparvifuran	254	C16H14O3
24.552	2-Bromo-5,8-dimethoxy-3- methyl-1-naphthol	296	C13H13O3Br

Table 5:

Compounds detected in methanol extracts 80 and 90% at RT. of rosemary leaves in the Bani Naim sample with their retention time (RT) (MW) and molecular formula (MF)

Methanol extracts 80% at RT				Methanol extracts 90% at RT
RT	Compound	MW	MF	RT	Compound	MW	MF
3.474	3-Carene	136	C10H16	3.494	3-Carene	136	C10H16
3.759	Camphene	136	C10H16	3.734	Camphene	136	C10H16
4.99	Eucalyptol	154	C10H18O	4.179	Alpha-pinene	136	C10H16
7.005	Camphor	152	C10H16O	4.514	Alpha-phellandrene	136	C10H165
7.446	Borneol	154	C10H18O	5.015	Eucalyptol	154	C10H18O
7.806	Bornyl chloride	172	C10H17Cl	7.015	Camphor	152	C10H16O
8.081	D-Verbenone	150	C10H14O	7.446	Borneol	154	C10H18O
9.301	Isobornyl acetate	196	C12H20O2	8.116	D-Verbenone	150	C10H14O
11.582	Caryophyllene	204	C15H24	9.306	Isobornyl acetate	196	C12H20O2
15.204	Beta-humulen	204	C15H24	11.597	Caryophyllene	204	C15H24
20.796	Menthol	156	C10H20O	15.204	Vitamin A aldehyde	284	C20H28O
21.766	Isoparvifuran	254	C16H14O3	23.367	Ferruginol	286	C20H30O
24.552	2-Bromo-5, 8-dimethoxy-3- methyl-1-naphthol	296	C13H13O3Br

Table 5 shows 13 compounds found in rosemary leaves in 80% methanol and 12 compounds extracted from rosemary leaves in 90% methanol at room temperature in the Bani Naim region sample. The significant compounds identified were 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate, bornyl acetate and caryophyllene. The 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate and caryophyllene for 80 and 90% methanol, respectively. The other minor volatile compounds identified are shown in Table 5. Table 6 indicates that 11 components were found in the methanol extracts at 80 and 13 were found in the methanol extract at 90% reflux of rosemary leaves in the Raqaa region sample. The major volatile compounds identified were 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate, borneol acetate and caryophyllene for 80% methanol and 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate and caryophyllene for 90% methanol reflux method. The other minor volatile compounds identified are shown in Table 6.

Table 7 presents the 12 compounds extracted from rosemary leaves in the methanol (80% reflux method) and the 12 compounds extracted from the methanol extract (90% reflux method) in the Khilt Al-Adrah region sample. Reflux method in the Khilt Al-Adrah region sample. The major volatile compounds identified were 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate, bornyl acetate and caryophyllene for 80% methanol and 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate and caryophyllene for 90% methanol reflux. The other minor volatile compounds identified are shown in Table 7. Table 8 shows the 11 compounds found in the methanol extracts at 80%. The 13 compounds found in the methanol ( 90% reflux) of rosemary leaves in the Umm Lasfah village region sample. The major volatile compounds identified were 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate, bornyl acetate and caryophyllene for 80% methanol and 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate and caryophyllene for 90% methanol reflux method. The other minor volatile compounds identified are shown in Table 8.

Table 6:

Compounds detected in methanol extracts 80 and 90% by reflux method of rosemary leaves in the Raqaa region sample with their retention time (RT) (MW) and molecular formula (MF)

	Methanol extracts 80% at RT			Methanol extracts 90% at RT
RT	Compound	MW	MF	RT	Compound	MW	MF
3.479	3-Carene	136	C10H16	3.489	3-Carene	136	C10H16
3.754	Camphene	136	C10H16	3.734	Camphene	136	C10H16
4.99	Eucalyptol	154	C10H18O	4.179	Alpha-pinene	136	C10H16
7.000	Camphor	152	C10H16O	4.509	Alpha-phellandrene	136	C10H16
7.441	Borneol	154	C10H18	5.02	Eucalyptol	154	C10H18O
7.806	Bornyl chloride	172	C10H17Cl	7.015	Camphor	152	C10H16O
8.076	D-Verbenone	150	C10H14O	7.451	Borneol	154	C10H18O
9.296	Borneol acetate	196	C12H20O2	8.076	D-Verbenone	150	C10H14O
11.582	Caryophyllene	204	C15H24	9.296	Isobornyl acetate	196	C12H20O2
12.152	Alpha-caryophyllene	204	C15H24	11.597	Caryophyllene	204	C15H24
15.209	Humulen-(V1)	204	C15H24	12.152	Alpha-caryophyllene	204	C15H24
15.204	Vitamin A aldehyde	284	C20H28O
				23.362	Ferruginol	286	C20H30O

Table 7:

Compounds detected in methanol extracts 80 and 90% by reflux method of rosemary leaves in the Khilt Al-Adrah sample with their retention time (RT) (MW) and molecular formula (MF)

Methanol extracts 80% at RT				Methanol extracts 90% at RT
RT	Compound	MW	MF	RT	Compound	MW	MF
3.529	3-Carene	136	C10H16	3.479	3-Carene	136	C10H16
3.764	Camphene	136	C10H16	3.764	Camphene	136	C10H16
4.995	Eucalyptol	154	C10H18O	4.179	Alpha-pinene	136	C10H16
7	Camphor	152	C10H16O	4.499	Alpha-phellandrene	136	C10H16
7.441	Borneol	154	C10H18O	5.005	Eucalyptol	154	C10H18O
7.806	Bornyl chloride	172	C10H17O2	7.015	Camphor	152	C10H16O
8.076	D-Verbenone	150	C10H14O	8.076	D-Verbenone	150	C10H14O
9.301	Borneol acetate	196	C12H20O2	9.296	Isobornyl acetate	196	C12H20O2
11.587	Caryophyllene	204	C15H24	11.597	Caryophyllene	204	C15H24
12.152	Alph-caryophyllene	204	C15H24	12.152	Alpha-caryophyllene	204	C15H24
15.209	Humulen-(V1)	204	C15H24	15.199	Vitamin A aldehyde	284	C20H28O
23.362	Ferruginol	286	C20H30O	23.362	Ferruginol	286	C20H30O

Table 8:

Compounds detected in methanol extracts 80 and 90% by reflux method of rosemary leaves in the Umm Lasfah village sample with their retention time (RT) (MW) and molecular formula (MF)

Methanol extracts 80% at RT				Methanol extracts 90% at RT
RT	Compound	MW	MF	RT	Compound	MW	MF
3.524	3-Carene	136	C10H16	3.479	3-Carene	136	C10H16
3.764	Camphene	136	C10H16	3.769	Camphene	136	C10H16
4.99	Eucalyptol	154	C10H18O	4.544	Alpha-phellandrene	136	C10H16
5.365	Alpha-pinene	136	C10H16	5	Eucalyptol	154	C10H18O
7.015	Camphor	152	C10H16O	7.01	Camphor	152	C10H16O
7.441	Borneol	154	C10H18O	7.446	Borneol	154	C10H18O
7.801	Bornyl chloride	172	C10H17O2	7.806	Bornyl chloride	172	C10H17Cl
8.071	D-Verbenone	150	C10H14O	8.076	D-Verbenone	150	C10H14O
9.301	Isobornyl acetate	196	C12H20O2	9.311	Isobornyl acetate	196	C12H20O2
11.587	Caryophyllene	204	C15H24	11.592	Caryophyllene	204	C15H24
12.147	Alph-caryophyllene	204	C15H24	12.152	Alpha-caryophyllene	204	C15H24
			15.209	Humulen-(V1)	204	C15H24
				23.367	Ferruginol	286	C20H30O

Table 9:

Compounds detected in methanol extracts 80 and 90% by reflux method of rosemary leaves in the Hebron sample with their retention time (RT) (MW) and molecular formula (MF)

Methanol extracts 80% at RT				Methanol extracts 90% at RT
RT	Compound	MW	MF	RT	Compound	MW	MF
3.534	3-Carene	136	C10H16	3.494	3-Carene	136	C10H16
3.769	Camphene	136	C10H16	3.734	Camphene	136	C10H16
4.995	Eucalyptol	154	C10H18O	4.509	Alpha-phellandrene	136	C10H16
7.005	Camphor	152	C10H16O	5.01	Eucalyptol	154	C10H18O
7.441	Borneol	154	C10H18O	7.005	Camphor	152	C10H16O
7.556	Bornyl chloride	172	C10H17O2	7.446	Borneol, heptafluorobutyrate (ester)	350	C14H17O2F7
7.806	P-Menth-1-en-8-ol	154	C10H18O	8.081	D-Verbenone	150	C10H14O
8.076	D-Verbenone	150	C10H14O2	9.301	Isobornyl acetate	196	C12H20O2
9.296	Bornyl acetate	196	C12H20O2	11.592	Caryophyllene	204	C15H24
11.587	Caryophyllene	204	C15H24	12.162	Alpha-caryophyllene	204	C15H24
15.214	Humulen-(V1)	204	C15H24	15.204	Vitamin A aldehyde	284	C20H28O
				23.367	Ferruginol	286	C20H30O

Table 10:

Compounds detected in methanol extracts 80 and 90% by reflux method of rosemary leaves in the Bani Naim sample with their retention time (RT) (MW) and molecular formula (MF)

Methanol extracts 80% at RT				Methanol extracts 90% at RT
RT	Compound	MW	MF	RT	Compound	MW	MF
3.524	3-Carene	136	C10H16	3.494	3-Carene	136	C10H16
3.764	Camphene	136	C10H16	3.739	Camphene	136	C10H16
4.529	Alpha-phellandrene	136	C10H16	4.129	Alph-pinene	136	C10H16
5.005	Eucalyptol	154	C10H18O	4.514	Alpha-phellandrene	136	C10H16
7	Camphor	152	C10H16O	5.02	Eucalyptol	154	C10H18O
7.446	Borneol	154	C10H18O	7.005	Camphor	152	C10H16O
7.801	Bornyl chloride	172	C10H17O2	7.476	Borneol, heptafluorobutyrate (ester)	350	C14H17O2F7
8.081	D-Verbenone	150	C10H14O2	8.126	D-Verbenone	150	C10H14O
9.321	Bornyl acetate	196	C12H20O2	9.316	Isobornyl acetate	196	C12H20O2
11.592	Caryophyllene	204	C15H24	11.597	Caryophyllene	204	C15H24
12.177	Alph-caryophyllene	136	C10H16	12.162	Alpha-caryophyllene	204	C15H24
		15.204	Vitamin A aldehyde	284	C20H28O
				23.367	Ferruginol	286	C20H30O

Table 9 indicates that 11 components were found in the methanol extracts at 80% and 12 were found in the methanol at 90% reflux method of rosemary leaves in the Hebron region sample. The major volatile compounds identified were 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate, borneol acetate and caryophyllene for 80% methanol and 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate and caryophyllene for 90% methanol reflux method. The other minor volatile compounds identified are shown in Table 9. Table 10 presented the 11 compounds extracted from rosemary leaves in the methanol extracts with 80 and 12 compounds extracted from rosemary leaves in the methanol with 90% reflux method in the Bani Naim region sample. The major volatile compounds identified were 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate, bornyl acetate and caryophyllene for 80% methanol and 3-carene, camphene, Eucalyptol, camphor, borneol, isobornyl acetate and caryophyllene for 90% methanol reflux method. The other minor volatile compounds identified are shown in Table 10.

Qualitative phytochemical screening: The results of the rosemary leaf qualitative phytochemical screening tests were carried out and showed that the methanol extract of the samples contains a wide range of phytochemical groups such as cardiac glycosides, phenolic groups, alkaloids, coumarin, saponins, steroids, tannins and terpenoids in all regions. Interestingly, alkaloids were found only in the leaves of the Raqaa and Bani Naim samples. However, other groups, such as anthocyanins, anthraquinone, flavonoids, glycosides and phlobatnnins, were absent. The qualitative results of the phytochemical compounds in rosemary samples were expressed in terms of their presence and (-) absence, as presented in Table 11.

Table 11:

Phytochemical screening for the methanol extracts from rosemary leaf samples

Phytochemical screening tests

Rosemary sample location

Parts

Cardiac glycosides

Phenolic groups

Alkaloids

Anthocyanin

Couarin

Saponins

Anthraquinone

Quinones

Steroids

Tannins

Terpenoids

Flavonoids

Glycosides

Phlobatnnins

Raqaa

Leaves

+

+

+

-

+

+

-

-

+

+

+

-

-

-

Khilt Al-Adrah

+

+

-

-

+

+

-

-

+

+

+

-

-

-

Umm Lasfah Village

+

+

-

-

+

+

-

-

+

+

+

-

-

-

Hebron

+

+

-

-

+

+

-

-

+

+

+

-

-

-

Bani Naim

+

+

+

-

+

+

-

-

+

+

+

-

-

-

-:Absence and +: Presence

Table 12:

Major compounds detected in all samples by GC-MS analysis of rosemary leaves

Name of the compound	Nature of the compound	Compound structure	Activity
3-Carene	Monoterpene		Antimicrobial, antioxidant, anticancer, semiochemical and fumigant properties18
Camphene	Monoterpene (terpenoid)		Antibacterial, antifungal, anticancer, antioxidant, antiparasitic, antidiabetic, antiinflammatory, hypolipidemic, anti-leishmanial, hepatoprotective, antiviral and anti-acetylcholinesterase19
Eucalyptol	Monoterpenoid		Antiinflammatory and antioxidant20
Camphor	Monoterpene (terpenoid)		Antiviral, antimicrobial, antitussive and analgesic agent21
Borneol	Monoterpenoid (terpene)		Antiinflammatory and cell penetration enhancing effect22
Isobornyl acetate	Monoterpene (terpenoid)		Antimicrobial23
Bornyl acetate (ester of borneol)	Monoterpenoid (terpene)		Antimicrobial24
Caryophyllene	Sesquiterpene		Antimicrobial, Anticarcinogenic, Antiinflammatory, Antioxidant and Anxiolytic, Local anesthetic25

Table 12 shows the chemical structures, nature of the compounds and biological activities of the main constituents in all studied samples of rosemary.

DISCUSSION

Rosemary plant samples from all regions tested for phytochemicals were found to have cardiac glycosides, phenolic groups, alkaloids, coumarin, saponins, steroids, tannins and terpenoids. The volatile components of 80 and 90% methanol at room temperature and reflux for rosemary leaf extracts were detected using GC-MS with the Electron Impact (EI) mode and compared to the NIST database. The main components of the rosemary plant samples are Eucalyptol, camphor, 3-carene, camphene, borneol, isobornyl acetate, bornyl acetate and caryophyllene. These results were also compatible with the results obtained by Verma et al.²⁶ and Masumoto et al.²⁷. Borneol, bornyl acetate, camphor, 1,8-cineole (Eucalyptol) and verbenone are the main volatile components that contribute to rosemary extracts’ distinctive flavor and aroma²⁸. Rosemary contains several phytochemicals that could be used to treat diseases or disorders. It was found that the significant chemotypes of Mediterranean-grown rosemary are Eucalyptol and campho ²⁹. Eucalyptol has several drug properties that are gaining medical attention and evidence of its anti-inflammatory and antioxidant modes of action³⁰. Eucalyptol (1,8-cineole) is also a potent cytokine inhibitor with a significant improvement in anti-inflammatory activity, according to research by Juergens et al.³¹. Volatile components like Eucalyptol and α-pinene have an anti-hyperglycemic impact because they lower plasma glucose levels, raise insulin levels and aid in glucose utilization by cells³². Camphor is also widely used on the skin for its antipruritic, analgesic and anti-irritant properties³³. Phytochemicals can vary greatly depending on plant parts (stems, leaves, flowers and roots), extraction circumstances (time, solvents and extraction method), processing procedures and environmental conditions in which plants grow^34-36.

There is variation in the phytochemicals in the different samples tested and analyzed, this might be due to varying extraction concentrations and methods. In addition to the various locations of the samples collected, which seems to be an essential factor for the variation of the components in rosemary leaves used in this study, the concentration of the extraction solvent and the method or conditions of the extraction method play a significant role in the components that are present in the plant, i.e., 80% methanol and RT extraction are optimum for enhancing the quality and the quantity of the phytochemicals presents in the plant. For example, ferruginol was only present in the sample tested when 90% methanol was used as the solvent of extraction and with the reflux method, only the exceptions were in the Khilt Al-Adrah sample, it was present in both solvent concentrations (Table 7) and it is not present in the Raqaa region sample (Table 1). However, if ferruginol is required to be obtained, the hot extraction method with 90% methanol was used. Ferruginol is a diterpene phenol with antibacterial, antitumor, antimalarial and cardioprotective properties³⁷. The differences in the composition between the samples analyzed were given in Table 1-10, the blue-colored text in the tables indicates the standard components in common between each sample at different extraction conditions.

The present study concluded that the plants contained many cardiac glycosides, phenolic groups, alkaloids, coumarin, saponins, steroids, tannins and terpenoids (Table 12). According to a study by Tabassum et al.³⁸, the leaves of rosemary plants present tannins, flavonoids, terpenoids, alkaloids, cardiac glycosides, phenols and saponins. In addition, Johar et al.³⁹ showed that rosemary has terpenoids, flavonoids and saponins but not tannins. Phenolic compounds may help prevent several chronic illnesses, including diabetes, cancer, cardiovascular disease and infections caused by bacteria and parasites. The rosemary leaf extracts tested in this current study show that rosemary also contains tannins, which have antioxidant properties, enhance wound healing and are beneficial against peptic ulcers. The presence of terpenoids in rosemary leaf extracts may also have cardioprotective and antioxidant properties⁴⁰. Another secondary metabolite detected in rosemary leaf extracts was steroids, which aid in reducing cholesterol and improving airway inflammation in asthma⁴¹. Saponins have historically been used as natural detergents, too. Their physicochemical and biological properties are exploited in food, cosmetics and medicine⁴². It was reported that alkaloids have pharmacological activities like antimicrobial, analgesic, antioxidant and anti-inflammatory⁴³. Rosemary has been demonstrated to reduce iron absorption and utilization. Thus, it should be used cautiously in patients at risk of iron deficiency because the extract is rich in phenol. According Samman et al.⁴⁴, the biological activities and the nutritional composition of the methanol-extracted rosemary leaves, which include antibacterials, antioxidants, minerals, etc., should be determined.

The implications and application of the phytochemical analysis of Rosmarinus officinalis L. (rosemary) leaves using GC-MS can be multifaceted and significant for many aspects such as, the identification of bioactive compounds in rosemary leaves. This knowledge can help researchers and industries understand the plant’s chemical composition, which can have implications for its medicinal, culinary, or cosmetic uses. Besides, by identifying the phytochemicals in rosemary, researchers can explore its potential medicinal properties. Rosemary has been traditionally used for its antioxidant, anti-inflammatory and antimicrobial properties. Understanding the specific compounds responsible for these effects can lead to the development of new herbal medicines or supplements. In addition, phytochemical analysis can provide information about the nutritional value of rosemary leaves. This data can be used to incorporate rosemary into diets for potential health benefits or to enhance the flavor of various food products. The findings can be valuable for the food industry. Rosemary extracts and essential oils are used as natural flavorings and preservatives. A detailed analysis can help improve the quality and consistency of such products. The study can inform agricultural practices by highlighting the compounds responsible for rosemary’s resistance to pests or diseases. This information can be used to develop more robust and sustainable cultivation methods. The identification of bioactive compounds can inspire the development of pharmaceuticals or nutraceuticals. For example, if a specific compound shows potent antioxidant properties, it might be incorporated into pharmaceuticals or dietary supplements. Understanding the phytochemical profile of Rosmarinus officinalis can also have implications for its conservation. If certain compounds are found to be unique or important, it may influence conservation efforts to protect this species and its natural habitat. The analysis contributes to the broader scientific understanding of plant chemistry and biochemistry. It can serve as a reference for future research on similar plant species and their potential applications. In summary, the phytochemical analysis of Rosmarinus officinalis L. leaves using GC-MS can have wide-ranging implications and applications, spanning from medicine and nutrition to agriculture and industry as mentioned. There are general recommendations that might apply to the study such as optimizing extraction methods to obtain a more comprehensive and representative sample of phytochemicals from rosemary leaves. This could involve experimenting with different solvents, temperatures and extraction times. Identify and characterize the most abundant and bioactive compounds found in rosemary leaves. Provide detailed information on their chemical structures, concentrations and potential health benefits. It is also recommended to focus on specific compounds that exhibit noteworthy properties, such as antioxidants or antimicrobial agents. The development of quality control standards to ensure consistent and safe product formulations. It is also recommended further investigations into the development of herbal medicines or dietary supplements containing rosemary extracts. Dosage recommendations and potential therapeutic applications may be explored too. Using phytochemical analysis data to enhance the flavor, shelf life, or nutritional content of food products. This might involve incorporating rosemary extracts into recipes or using them as natural preservatives. Guiding using rosemary leaves in cooking to maximize their flavor and potential health benefits. Safety assessments and regulatory considerations may be necessary. Additionally, collaboration with experts in related fields such as pharmacology, nutrition, or culinary arts may be necessary to translate the findings into practical applications. The GC-MS is a valuable technique, but it has some limitations and challenges such as limited identification as GC-MS can identify compounds based on their mass spectra, but it may not provide unequivocal identification of all compounds. Some compounds may have similar mass spectra, making it challenging to distinguish them accurately. The quantification of the phytochemicals can be challenging. Plant extracts can contain a complex mixture of compounds and GC-MS may not be able to separate and identify all of them. Co-elution of compounds can occur, making it challenging to distinguish individual peaks. The GC-MS is best suited for the analysis of volatile and semi-volatile compounds. It may not effectively analyze non-volatile or high molecular weight compounds, limiting the scope of the analysis. Despite these limitations, GC-MS remains a powerful tool for phytochemical analysis when used appropriately and in conjunction with other analytical techniques.

CONCLUSION

Two extraction methods with different solvent concentrations were used to detect and identify the phytochemicals in rosemary leaves. The GC-MS technique was utilized and found to be accurate and reliable in separating and identifying components of the methanol extracts of Rosmarinus officinalis L. The study established the chemical composition of the rosemary leaves from different locations and compared them. The main phenolic and volatile compounds in a rosemary plant in Palestine have been identified. A comparable study has been conducted between the samples using different extraction methods. The major volatile compounds detected were Eucalyptol and camphor in all samples. Palestinian rosemary is rich in phytochemicals, thus, rosemary leaves are recommended for medicinal purposes.

SIGNIFICANCE STATEMENT

The study established and compared the chemical composition of the rosemary leaves from different locations. The main phenolic and volatile compounds in a rosemary plant in Palestine have been identified. A comparable study has been conducted between the samples using different extraction methods. Palestinian rosemary is rich in phytochemicals, thus, rosemary leaves are recommended for medicinal purposes.

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