INTRODUCTION:

Rosemary is a highly variable species that shows differences in morphology, chemical composition, and genetic diversity. Thus, it can be classified into different types based on various criteria, such as origin, biological properties, and chemical composition. One of the most widely recognized types of rosemary is the constitutional rosemary type Rosmarinus officinalis L.^1,2. Moreover, Rosemary has two synonyms, Rosmarinus lavandulaceus De Noé and Rosmarinus laxiflorus De Noé, which were described by De Noé (1854) as distinct species but later considered as synonyms of R. officinalis^3,4.

Additionally, a type of rosemary known as Rosmarinus officinalis subsp. palaui (O. Bolós & Molinier) Malagarriga, was reported by Sabbagh et al⁵^,⁶. This subspecies is endemic to Spain and is distinguished from R. officinalis by its smaller leaves, flowers, and fruits, as well as its lower levels of camphor and α-pinene⁶.

Rosemary is a rich source of bioactive compounds with potential medicinal properties. Studies have reported that rosemary may have antioxidant, anti-inflammatory, antimicrobial, and neuroprotective effects^7,8. To maintain the quality of the plant and the extracted oil, they should be stored away from sun and moisture⁸. Rosemary's diterpenes have been shown to possess antimicrobial properties, while its rosmarinic acid, carnosol, and carnosic acid are potent antioxidants that can protect against oxidative stress. Rosemary's hepatoprotective properties are believed to be due to the presence of various bioactive compounds, including rosmarinic acid and carnosic acid, which have been shown to protect the liver from various toxic substances. Rosemary's anti-inflammatory properties may be due to its ability to inhibit the activity of certain inflammatory enzymes. Rosemary's chemoprotective and anti-mutagenic effects may be due to the presence of various bioactive compounds, including rosmarinic acid, carnosol, and carnosic acid, which have been shown to protect against the development of cancer and mutations in DNA⁹.

Unfortunately, the medicinal use of Rosemary could cause some side effects including allergic skin reactions, uterine contractions and miscarriage in pregnant women, coma, vomiting, gastrointestinal and renal irritation, and death in large doses, Stomach irritation and seizures in susceptible individuals. It is important to note that these side effects are primarily associated with medicinal use of Rosemary. Normal dietary consumption is generally safe^10-11. Rosemary is available in a variety of pharmaceutical forms for internal and external use, including whole plant, powder, tea, wine, tincture, liquid extract, and bath additive^12-14. It can be taken internally or applied externally. For internal use: 4-6 grams per day are taken orally or as a cup of tea several times a day. Alternatively, 20-40 drops of the 1:5 tincture or 2-4 ml of the liquid extract can be taken as a single dose. External use: Applied topically in liquid or semi-solid pharmaceutical forms containing 6-10% of its essential oil, 2-3 times daily.

The drug under study, which is derived from the Rosemary plant, contains a complex mixture of chemical compounds. The volatile oil is one of the main constituents of the drug, which varies in its proportions according to different researchers. The percentages of the volatile oil range from 0.1-2%, 0.5-2%, and 1.0-2.5%. The volatile oil contains various compounds, including camphor (10-25%), alpha-pinen (15-25%), and cineole (20-50%). In addition, it contains bornyl acetate, camphene, beta-caryophyllene, limonene, linalool, borneol miocene, alph-terpineol, and verbenone¹⁵. Furthermore, the drug contains flavonoids, which are a class of compounds known for their antioxidant properties. Flavonoids found in the drug include cirsmarin, diosmin, hesperidin, homoplantiginin, and hegopolin. Another class of compounds found in the drug is diterpenes, which include carnosolic acid picroslavin, isorosmanol, rosmadial, rosmaridiphenol, and rosmariquinone. Additionally, the drug contains triterpenes, mainly oleanolic acid, ursolic acid, and 3-acetyl esters. Moreover, the drug contains caffeine acid derivatives, with rosmarinic acidbeing the main compound. Rosmarinic acid is a depside of caffeic acid and alpha-hydroxy dehydro-caffeic acid. The drug also contains petulin and β-sitosterol with epi-α-amyrin¹⁵. For instance, a comparison of the oil content of Rosemary plants in Syria with a number of Mediterranean countries showed that the juicy white Syrian plant had the highest oil content (0.770%), followed by the juicy violet Syrian plant (0.366%), the French plant (0.500%), the Tunisian plant (0.500%), the Hispanic plant (0.725%), and the Marrakech plant (0.400%) (Table 1)¹⁶.

Table 1: Comparison of rosemary oil content in Syria with that of several Mediterranean countries.

Oil Source		Oil Percentage %
Spanish		0.725
Frenche		0.5
Tunisian		0.5
Marrakchi		0.4
Syrian White	Luffa Plant	0.77
	Dried	0.42
Syrian Violet	Luffa Plant	0.366
	Dried	1.2

The chemical composition of rosemary^17-19, which contributes to its medicinal and culinary properties, can be influenced by environmental factors, including soil salinity. Soil salinity is a significant abiotic stress in agricultural soils, particularly in arid and semi-arid regions. It can alter plant growth, yield, and quality by affecting the availability of water and nutrients to the plant and the production of secondary metabolites such as essential oils^20-21. Recent studies have demonstrated that soil salinity can affect the chemical ingredients and volatile oil of rosemary. Salinity stress can reduce the growth and yield of rosemary and induce changes in the levels and composition of its essential oils. These changes may be due to altered gene expression and enzyme activity, as well as oxidative damage caused by reactive oxygen species (ROS)^21-22. Understanding the effects of soil salinity on the chemical composition of rosemary is crucial for maintaining the quality and medicinal properties of this herb. This study investigates the impact of different levels of soil salinity on the chemical ingredients and volatile oil of rosemary. The findings could provide valuable insights into the optimal growing conditions for rosemary and help develop strategies to mitigate the adverse effects of soil salinity on plant growth and quality²³.

MATERIAL AND METHODS:

Wild rosemary plant samples:

The study was conducted at the laboratory of postgraduate studies in the Department of Pharmacognosy at the Faculty of Pharmacy, University of Damascus. Several wild rosemary plant samples were collected from different geographical areas in Syria, including the mountainous region of As-Suwayda - Salkhad (900m above sea level), the coastal area of Tartous - Sheikh Badr (350m above sea level), the inland region of Qara - Qalamoun (1250m above sea level).

Cultivation of Rosemary (Rosmarinus officinalis): A Study of SalinityTolerance:

To investigate the salinity tolerance of Rosemary, four groups of constitutional Rosemary were cultivated over a four-month period from July to October. Each group consisted of four samples, with 3 kilos of soil placed in each pot. The plants were watered with graduated saline solutions, starting from a concentration of 25mmol/l and increasing to 50mmol/l and 100mmol/l for each group separately. The fourth group, which was used as a control, was watered with plain water. The watering rate was half a liter once every three days.

After cultivation, plant samples were weighed and their weights were recorded. One sample of plant leaves was frozen in the refrigerator (-20°C) (College of Agriculture sample). For comparison, two samples of the plant were dried in one of the following two ways:

1. Drying in the oven: The leaves and flowers were separated from the stems, placed on filter paper, and then placed in the oven at a temperature of 35 °C for four hours. One of the samples grown was dried in this way.

2. Drying in the shade: After the plant was collected, it was spread on filter papers in the shade in a well-ventilated room to ensure good drying and not to lose the essential oil for a period of 7 days. After the leaves became dry, they were separated from the stem by passing the fingers of the hand over the entire stem, and the dried leaves were kept in paper bags at room temperature, away from light and moisture until the time they were used for extraction.

The study aimed to investigate the salinity tolerance of Rosemary and to determine the best method for plant drying. The results of this study can provide valuable information for the cultivation and production of Rosemary in different regions with varying levels of salinity.

Extraction of Essential Oil from Whole Rosemary Plant (Rosmarinus officinalis):

The essential oil extraction process for all samples was carried out using the Salvin Meek low-density volatile oil extraction apparatus, a widely used method for extracting essential oils from plant materials²⁴. The weighted sample was placed in a flask, and distilled water was added to it at the rate of 1 liter per 100g of wet leaves or leaves kept in ice, or 50g of dried leaves²⁵. The leaves were first washed, dried in the shade for two days, and then crushed using a mortar and pestle before being used for extraction²⁶. Shortly after boiling the mixture, distillation began, and the distillation continued until the amount of extracted oil became stable. Then, the oil was separated from the water and collected in sterile, opaque, and airtight glass tubes. The collected oil was stored in a cool, dry place, away from light and heat until further analysis. This method of essential oil extraction from Rosemary has been widely used in previous studies^24-26. and is recognized for its simplicity, efficiency, and ability to produce high-quality essential oils. To determine the volume of essential oil extracted from Rosemary samples: Calibrate a volumetric flask with xylol, Distill 5ml of xylol in 3 liters of distilled water, Place 100g of Rosemary plant material in the flask with the water and xylol, Extract the essential oil using a suitable method, Calculate the volume of essential oil extracted from 100g of the plant material.

Analyze the essential oil using gas chromatography (GC):

GC-MS Scan model, type Agilent 5973, Mass range: 500-100m/z, Column: HP5, Carrier gas: Helium, Flow rate: 0.9ml/min, Sample volume: 1 microliter, Detector: Mass spectrophotometer, Spallation energy: 70 eV, Electronic source temperature: 230 degrees Celsius. GC analysis is a widely recognized method for providing a detailed and accurate analysis of the chemical composition of essential oils. This method has been used in previous studies to analyze the essential oil of Rosemary and to identify its major chemical components^27-29.

RESULTS:

A study was conducted to investigate the concentration of essential oil in rosemary plants grown between November 2022 and February 2023 in a fresh state, based on the different concentrations of salt added to the soil during planting. The results are presented in Table 2, where the concentration of essential oil in the plants was measured at different time points (11, 12, 1, and 2 months) after planting, and at varying concentrations of salt (25, 50, and 100mmol/L). The weight of the sample from which the oil was extracted was 100 g of fresh weight. The arithmetic mean of the concentration of essential oil in the plants was calculated for each salt concentration.

Table 2: Comparison of essential oil concentration in fresh rosemary plants cultivated between November 2022 and February 2023, with different concentrations of salt added to the soil during planting.

Harvest time (month)	Control	25 *	50 *	100 *
11	1.5	1.6	1.7	1.8
12	1.8	1.9	2.1	2.3
1	1.9	2.4	2.14	2.5
2	2.6	2.5	2.9	3
Average	1.95	2.1	2.21	2.4

The results show that the concentration of essential oil in the rosemary plants increased with time and with increasing concentrations of salt added to the soil during planting. For example, at a salt concentration of 100 mmol/L, the concentration of essential oil increased from 1.7% at 11 months to 2.9% at 2 months after planting. The highest concentration of essential oil was observed at a salt concentration of 100 mmol/L, where the arithmetic mean was 2.4%.

Figure 1: Chart showing the high concentration of essential oil in a cultivated plant.

In addition, another study was carried out to investigate the concentration of essential oil in rosemary plants in a dried state, between September and October 2022 (Figure 1). The weight of the sample from which the oil was extracted was 50g of dry weight. The results are presented in Table 3, where the concentration of essential oil in the plants was measured at different time points (9, 10, and 11 months) after planting in three different locations (Qalamoun, As-Suwayda, and Sheikh Badr). The arithmetic mean of the concentration of essential oil in the plants was calculated for each location.

Table 3: High concentration of total rosemary essential oil between September 2022 and October 2022 - in dried state (based on 50g dry weight of the sample).

Harvest time (month)	Sheikh Badr	Sweida	Qalamoun
9	0.7	1.4	0.9
10	0.9	1.9	1.4
11	1.1	2.1	1.6
Average	0.9	1.8	1.3

The results show that the concentration of essential oil in the rosemary plants varied depending on the location, with the highest concentration observed in As-Suwayda. For example, at As-Suwayda, the concentration of essential oil increased from 1.4% at 10 months to 2.1% at 11 months after planting. The arithmetic mean of the concentration of essential oil in the plants was highest in As-Suwayda (1.8%), followed by Qalamoun (1.3%), and Sheikh Badr (0.9%) (Figure 2).

The use of salt in the soil during planting has been shown to have a positive effect on the concentration of essential oil in rosemary plants^30,31. Moreover, the effect of location on the concentration of essential oil in rosemary plants has been previously reported^27,29.

Figure 2: Chart showing the high concentration of essential oil in the Total plant.

Essential Oil Extraction Results and Analysis:

Samples obtained from different regions and harvested at different dates. Weight of each sample and average amount of extracted oil (in ml) recorded. Percentage of oil calculated based on the weight of the sample. Percentage of oil in the samples varied, with the highest percentage observed in As-Suwayda (1.2%) and the lowest in Sheikh Badr (0.66%). Average percentage of oil across all samples was 0.9%. To determine the amount of oil extracted from 100g of the drug, volumetric calibration of oil was performed using xylol as the solvent. Amount of oil extracted with xylol in the device was 6.1 ml. Subtracting the amount of xylol (5 ml) used in the extraction, the amount of oil obtained was calculated to be 1.1%. The percentage of essential oil in Rosemary samples varied depending on the region and harvest date. The highest percentage of oil was observed in As-Suwayda (1.2%), and the lowest percentage was observed in Sheikh Badr (0.66%). The average percentage of oil across all samples was 0.9%.

Table 4: Results of oil extraction from different samples and the percentage of oil in each sample.

Region	Date of harvesting	Weight	Average amount of oil extracted	Percentage
Region	Date of harvesting	Weight	(ml)	Oil (%)
Qalamoun	02-09-2011	200	1.3	0.65
Sheikh Badr	20-10-2011	135	0.9	0.66
Sweida	04-11-2011	150	1.8	1.2
control	03-11-2011	200	1.95	0.97
Group 1	06-01-2012	225	2.1	0.93
Group"2"	08-01-2012	225	2.21	0.98
Group 3	02-02-2012	250	2.4	0.96
			Average Percentage	0.9

Figure 3: shows a diagram of the most important ingredients found in the studied essential oils

Table 5: shows the most important chemical components in the studied essential oil samples.

Chemical compounds	group 1%	Group 3%	group 2%	control%	Qalamoon%	Sheikh Badr %	Sweida %
Pinene	6.95	8.2	7.32	6.96	8.55	6.94	3.25
Camphore	10.8	16.44	12.59	10.97	3.95	13.98	4.25
Boreneol	13.92	13.5	13.96	13.91	14.09	14.7	13.91
Pinocmphone	3.45	5.24	3.44	1.3	_	3.11	2.11
Verbenone	24.68	24.38	24.7	24.66	24.84	_	24.65
Eucalyptol	_	_	_	_	_	10.8	_
Camphene	10.03	22.36	13.36	10.01	_	_	5.24
D-limonene	_	9.18	9.83	_	_	_	6.92
O-Cyemene	6.75	6.81	6.63	6.96	_	_	_
Linalool	7.35	10.64	7.99	7.02	_	6	6.25
Caryophyllene	1.55	1.75	1.55	1.52	_	0.69	_
Caryophyllene oxide	1.7	1.81	1.73	1.7	_	0.91	_
Bornyl acetate	0.72	8.12	0.75	0.71	_	_	_
B-Phellandrene	_	_	_	_	_	0.95	_
Terpinene-4-ol	1.53	1.72	1.55	1.23	_	_	_
P-menth-1-en-8-ol	_	_	_	_	_	4.53	_
P-menth-1-en-4-ol	_	_	_	_	_	3.15	_
Octacosane	_	_	_	_	18.92	_	_
Borneol acetate	_	_	_	_	_	_	1.51
Benzaldehyde	_	_	_	_	_	_	3.08
2-Thujene	2.52	2.61	2.55	2.3	_	_	_
1-Octadecyne	_	_	_	_	5.3	_	_
1-Therpinen-4-ol	_	_	_	_	1.17	_	_
3-Thujen-2-ol	0.85	1.01	0.98	0.95	_	_	_

The analysis of essential oil components was carried out using gas chromatography (GC). The chromatograms and their tables show the retention time (Ret. Time) of the components in the essential oil samples and the areas of peaks (Area) of those components. The identification and quantification of the components were based on their retention times and comparison with standard reference materials^24,25. The use of GC analysis in the identification and quantification of essential oil components is a widely recognized and commonly used method^24,25. GC analysis provides detailed information about the chemical composition of essential oils and can be used to identify and quantify the individual components in the oil²⁴ (Table 5 and Figure 7).

DISCUSSION:

In this study, several observations were made regarding the yield of essential oil in the samples of “Rosmarinus officinalis”. The samples were subjected to both oven drying and shade drying, resulting in weight losses of 38% and 36%, respectively. The percentage of essential oil extracted from the samples fell within the range of 0.65% to 1.2%, with an average of approximately 0.9%. The effect of humidity, rainfall, altitude, and soil characteristics on the production of volatile oil was investigated, and it was found that the As-Suwayda and Qalamoun regions yielded the highest quantities of oil. The higher the humidity, the higher the yield of essential oil, which can be explained by the hydration of plants and the protective barrier against direct solar radiation provided by a water vapor layer in humid regions. Altitude also influences the essential oil yield, with higher altitudes resulting in higher oil percentages due to decreased temperatures and increased atmospheric humidity. The addition of "turp" fertilizer to the soil led to an increase in the amount of essential oil extracted from the cultivated samples. Salinity was also investigated, and it was found that the addition of saline irrigation water at low concentrations led to a gradual increase in the amount of essential oil, with the plant acclimatizing to the salt stress. Gas chromatography analysis of the essential oil revealed the presence of various compounds, including Borneol, Camphor, α-pinene, and Pinene, with some samples containing Camphene, Limonene, Verbenone, PinoCamphone, Terpineol, or O-cymene. Eucalyptol, one of the main components of rosemary essential oil, was present in the sample from the Sheikh Badr indoor area^32-36. For the cultivated samples, a higher percentage of oxygen compounds, particularly camphor, borneol, and verbenone, was generally observed. These compounds have significant pharmacological implications, with camphor serving as an antiseptic and α-Pinene used as a urinary tract antiseptic. It is evident that soil improvement and the addition of salinity within permissible limits can lead to an enhanced yield of essential oil in the rosemary plant, providing the pharmaceutical industry with vital active substances. This study highlights the potential for modifying environmental conditions to yield greater amounts of volatile oil^35,36.

CONCLUSION:

This study provides valuable insights into the yield of essential oil in Rosmarinus officinalis and the factors that influence its production. The results indicate that the percentage of essential oil extracted from the samples fell within the range of 0.65% to 1.2%, with an average of approximately 0.9%, which is consistent with previous findings. The investigation of environmental factors such as humidity, rainfall, altitude, and soil characteristics revealed that the As-Suwayda and Qalamoun regions yielded the highest quantities of oil due to the protective barrier against direct solar radiation provided by a water vapor layer in humid regions and decreased temperatures and increased atmospheric humidity at higher altitudes. The use of "turp" fertilizer and the addition of saline irrigation water at low concentrations were found to enhance the yield of essential oil in cultivated samples. Gas chromatography analysis revealed the presence of various compounds, including Borneol, Camphor, α-pinene, and Pinene, with some samples containing other compounds such as Camphene, Limonene, Verbenone, PinoCamphone, Terpineol, or O-cymene. The presence of oxygen compounds such as camphor, borneol, and verbenone in cultivated samples suggests their potential use in pharmacological applications. Overall, the findings of this study demonstrate the potential for modifying environmental conditions to enhance the yield of volatile oil in Rosmarinus officinalis. The results provide valuable information for the pharmaceutical industry, which can utilize these active substances in various applications. Further research is warranted to investigate the potential of other environmental factors and fertilizer types on the yield and quality of essential oil in this plant.

FUNDING:

This research is funded by Damascus University-funder No. 501100020595.

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Received on 21.09.2023 Modified on 14.11.2023

Research J. Pharm. and Tech 2024; 17(5):2282-2288.

DOI: 10.52711/0974-360X.2024.00358