Identification of Active Compounds, Phenols and Antioxidants in Rosemary

Abstract

Rosemary (Rosmarinus officinalis L.) is an evergreen shrub with a height around 1 m with erect stems that have fragrant, dark green leaves and blue and white flowers. Rosemary leaves have long been used in traditional medicine for a variety of conditions such as colds and coughs and also as an antispasmodic, hypotensive, antiepileptic and for the treatment of diabetes and parasites. Furthermore, Rosemary has been used in alternative medicine and complementary industries for its digestive properties and as a food preservative, stimulant, astringent, and diuretic. It has also been linked to a wide range of beneficial health effects. Furthermore, Rosemary has been used in alternative medicine and complementary industries for its digestive properties and as a food preservative, stimulant, astringent, and diuretic. It has also been linked to a wide range of beneficial health effects. In this study, we evaluated the total contents of active compounds, antioxidants and phenols of rosemary plant and determined the importance of rosemary plant in the folk medicine. Rosemary extracts obtained by extraction in many ways have shown promise with regard to their incorporation into many foods, cosmetics and pharmaceuticals and chemical compounds have been identified by GC-MS. The extracts were analyzed for compounds showing biological and antioxidant activity. Rosemary extracts have been tested with respect to antioxidants, determination of phenols by the method of HPLC and determination of total phenolic content by Folin-Denis reagent.

Keywords: Rosemary; antioxidants; phenols; mobile phase;

1. Introduction:

Rosemary (Rosmarinus officinalis L.) belongs to the family of L Lamiaceae .It is an evergreen shrub with a height around 1 m with erect stems that have fragrant, dark green leaves and blue and white flowers . This plant is native to the Mediterranean region and Asia, but now cultivated in temperate places around the world as an ornamental plant and a culinary herb. Rosemary leaves have been used to flavor foods such as lamb, pork, chicken, fish, and fillings and to prepare essential oils.

Rosemary leaves have long been used in traditional medicine for a variety of conditions [1]. In the Mediterranean region, aerial parts are used to treat colds and coughs [5] and as an antispasmodic, hypotensive, antiepileptic [4-10], and for the treatment of diabetes [6,9], and parasites [11]. An infusion of alcohol or olive oil is used externally to treat bruises, rheumatism, muscle and joint pain [3-12]

Rosemary extracts, derived mainly from the leaves, are herbal products commonly used as flavorings, antioxidants and in food and cosmetic processing. And as a natural antioxidant, it is preferred over synthetic antioxidants such as butylated hydroxytoluene (BHT). Furthermore, rosemary has been used in alternative medicine and complementary industries for its digestive properties and as a food preservative, stimulant, astringent, and diuretic. It has also been linked to a wide range of beneficial health effects

The aim of this study was to demonstrate that the extracts of rosemary leaves could be equivalent to certain medications and can be an effective adjuvant in the pharmaceutical industry and to evaluate the total contents of rosemary plant of active compounds , antioxidants and phenols and determine the importance of rosemary plant in folk medicine, through this determination, especially that finding natural sources of biologically active substances and methodology of extraction of active ingredients is of great interest in the field of food and cosmetics also active compounds play a role in protecting cells from free radicals and thus play an important role in heart disease, cancer and others.

2. Materials and methods:

2-1 Materials and fittings:

All of the chemicals used in the extraction and analysis processes are of the class of HPLC and GR of Merck Company.

Methanol, acetone, hexane, petroleum ether and dichloromethane with GC grade were obtained from Merck company.

Standard materials to confirm the presence of any rosemary leaf extract Carnosol (>95%), camphor (>97%), α- terpineol (>97%) and linalool (>97%) were purchased from Sigma-Aldrich. Carnosic acid (≥97%), 1,8 cineole _(98%) , borneol (>99%), ethanol and phosphoric acid (85%) were HPLC grade from Merck.

Chemical kits used to determine the capacity of antioxidant substances dissolved in water ACW and materials dissolved in lipids ACL using photochemical illuminating technique were obtained from Analytik Jena AG (Jena, Germany)

2-2 Instrumentations:

An Agilent gas chromatography (model ) with mass spectrometry (model )

Rotary evaporator with vacuum

HPLC Device from Agilent with the pump bilateral and injector automatic and detector PDA with analytic software Chemstation

Photochemical scintigraphy device for determining antioxidants, which is from Ana

Ultrasonic device, model 405 from Power Sonic Company

Centrifuge model 2.0R- A 550Heraeus from Megafuge

1. determination of phenols using high-performance liquid chromatography technology with optical and ultraviolet Matrix detector pda. High-Performance Liquid Chromatography and Detector HPLC-PAD
2. Specifications of the PAD HPLC(Agilent 1260)

– Column ODS -1 18, inner diameter mm 1.3

– Particle dimensions 3.5 x u, column length 2 250

– Double automatic pump – ultraviolet detector

– Flow rate 0.9l K / min

– Injected sample size 50 microliters

3- Extraction of phenols from the leaves of the rosemary plant

100g of dry and ground rosemary leaves were soaked in about 200 ml solution 50/50 ( ETH /water) and put in a dark flask with a water bath at 40 degrees Celsius for half an hour with stirring . After filtration, the filtrate was acidified in 2N and the phenols were extracted by 50 ml ETH acetate with an ultrasound bath for half an hour. After that the ethyl acetate layer was separated and dried by evaporation with water acidified with 0.5% acetic acid by 80% acetonitrile and 20% acidified water and the phenols extracted by the HPLC technology and were determined according to the following analytical conditions:

3. Results:

Mobile phase:

– Phase A: Acetic acid (0.5% v/v) water

– Phase B: Acetonitrile

Table (1) showing the ratios of the mobile phase distribution over time

B	A	Time
0%	100%	0 min
20%	80%	9 min
30%	70%	15 min
45%	55%	23 min
25%	75%	29 min
0%	100%	37 min

Table (2): Polyphenolic compounds values in rosemary extract identified by HPLC

Phenols	Retention time	Concentration μg/ml
Caffeic acid	4.3	33.1
Medioresinol	5.6	27.5
p-Coumaric acid	9.2	46.8
Luteolin-rutinoside	14.5	37.1
Luteolin-hexoside	17.3	42.6
Isorhamnetin-3-O-hexoside	27.9	34.9
Apigenin-7-O-glucoside	33.9	49.4

These substances are antibacterial, antioxidant, have a cancer cell inhibitory effect, protective effect of transplanted liver and anti-inflammatory

2. Determination of the total phenolic content in the ethanolic extract of rosemary leaf powder:

Rosemary leaf powder was extracted by Soxhlet device and in the presence of ethanol and continued extraction for four hours after which the sample was evaporated with a rotary evaporator and the sample size became 5 ml

For the assay of phenolic dog, was used reagent Folin-Denis according to the method cited by Jiao and Liu and Wang (2005). Quantification was carried out based on the standard tannic acid curve where a linear calibration curve of tannic acid with a value of 0.9995 was created. A volume of 5 ml of the reagent was then added to 5 ml of the sample solution. After 3 minutes, 10 ml of saturated sodium carbonate and 80 ml of distilled water were added and mixed thoroughly. After exactly 30 minutes, the absorbency was measured at a wavelength of 760 Nm. The result was calculated according to the standard. The total phenolic content was expressed as tannic acid equivalents (TAE) in milligrams per gram of extract. The total phenolic content ranged from 79 to 94.36 g of TAE / 100 g of extract, with a total average of 85.71 g of Tae / 100 g of extract

3 determination of Carnosic acid and methylcarnosic acid -methylcarnosic acid12 – O وginquanin in the leaves of a plant rosemary by HPLC

4. Methanol extraction:

A soxhlet device was used as follows: 100 g of dried powder of rosemary leaves was weighed and placed in the extraction tube, then 150 g of methanol was placed in the extraction tank and the extraction process continued until the organic solvent in the extraction tube became colorless .After that, the extract obtained by the rotary evaporator was evaporated to dry and then dissolved by 2 ml acetonitrile in preparation for analysis with the technology OFL G according to the following analytical conditions :

Analytical column (r xr xr-1001 18 25 cm 4 4.6 mm and 5 micrometers(

Mobile phase A: acetonitrile

B: 0.1% phosphoric acid in water

Table (3): the ratios of the mobile phase distribution over time

B	A	Time
0%	100%	0 min
77%	23%	8 min
25%	75%	25 min
0%	100%	35 min

The flow rate was constant at 0.9 mL / min. The volume of the syringe is 20 microliters

The signal was stored at wavelengths of 230, 280 and 350 nm.

The results of HPLC analysis of rosemary are shown in Table 4.

Table (4): HPLC analysis of different rosemary compounds and their retention times

Compounds	Retention time	Concentration μg/ml
Carnosic acid	7.3	48.9
12O-methylcarnosic acid	13.2	33.7
Rosmarinic acid	23.4	56.2
Ginquanin	29.7	27.4

From studies it was found that Carnosic acid Compound has properties of anti-proliferation of cells and has a protective effect of photoreceptor cells and is anti-inflammatory and has the inhibitory effect of digestive enzymes lipase, alpha-amylase, and alpha-glucosidase and the suppressive effect of lipogenesis and the rosmarinic acid compound is antidepressant, anxiolytic, anticancer, antifungal

5. identification of volatile compounds in rosemary extract :

The rosemary leaves were extracted hydrolyzed by means of a clevenger Micro-Device as follows:

100 grams of rosemary leaf powder were placed in the extraction clevenger device that containes 250 ml of distilled water with boiling regulators. The extraction was done by steam extraction in presence of heating for 3 hours. After that the polyphenolic extract was collected by density difference with water and then place in dark flask and a pinch of sodium sulfate anhydrous was added to withdraw the entire moisture and stored at a temperature of 4 degrees Celsius in preparation for analysis with the GC-MS technique. The extraction yield was 2.7%

Analysis using (GC-MS) Gas chromatography _ mass spectrometry

Gas chromatography technology connected to mass spectrometry is an effective method for the separation and detection of volatile organic compounds.

– Device type Agilent 5973

Analysis was performed GC-MS on a gas chromatography with a detector, mass spectrometry, and were resolved using capillary column (non-polar column) HP-5 MS with the conductors of the following : length ,30 m, diameter 0.25 mm, the thickness of Phase fixed 0.25 nm according to the following conditions:

Program oven heat from 55 degrees Celsius waiting (2 minutes) then to 280 degrees Celsius at a rate of 3 degrees per minute . Helium carrier gas (99.9996%) with a flow rate of 1 mL / min.

– Injector temperature 275 degrees Celsius

Detector temperature 250 degrees Celsius

Syringe size one microliter

Injection model segmentation at a rate of 1:20

Ionization energy 70 electron volts

The compounds were identified by matching their mass spectra with those in the Wiley and NIST

Table (5): the results of the analysis in the technique of GC-MS for the extract of the rosemary plant

RT	compound name	peak area%	RI	Identification
6.23	α–Thujene	0.16	933	GC/MS, RI
6.52	α-Pinene	4.92	935	GC/MS, RI
6.71	β-Pinene	2.43	938	GC/MS, RI
7.85	Camphene	2.21	950	GC/MS, RI
8.17	1-Octen-3-ol	0.47	967	GC/MS
8.68	3 Octanone	0.73	978
8.93	β- Myrcene	2.48	987	GC/MS
9.32	α-Phellandrene	0.46	1 004	GC/MS, RI
10.21	Δ-3-Carene	0.22	1 007	GC/MS, RI
11.54	Limonene	1.98	1010	GC/MS, RI
12.39	1.8 Cineole	49.57	1019	GC/MS, RI
14.98	Linalool	1.21	1020	GC/MS, RI
15.84	Camphor	11.65	1 123	GC/MS, RI
18.98	α-Terpinene	1.57	1 026	GC/MS, RI
20.14	p-Cymene	2.39	1 035	GC/MS
21.84	1-Terpinen-4-ol	1.76	1053	GC/MS, RI
22.38	α-Terpinolene	4.26	1 082	GC/MS, RI
23.14	Borneol	5.28	1 150	GC/MS, RI
24.97	Verbenone	3.11	1195	GC/MS, RI
25.17	Bornyl Acetate	0.87	1 270	GC/MS, RI
28.91	α-Copaene	0.27	1 379	GC/MS, RI
31.01	β-Caryophyllene	5.31	1 421	GC/MS, RI
35.34	α- Humulene	0.35	1 455	GC/MS, RI
36.11	γ-Murolene GC/MS	0.19	1 479	GC/MS, RI
37.45	Δ-Cadinene	0.21	1 520	GC/MS, RI
38.62	Linalool	0.71	1 553	GC/MS, RI

RI = retention index on HP-5 MS

GC/MS = gas chromatography coupled to mass spectrometry;

The proportion of these vehicles Monoterpene hydrocarbonsOxygenated contains monoterpenes and Sesquiterpene hydrocarbons and Oxygenated sesquiterpenes 99.22%

Column analytical (non-polar column) HP-5 MS

These compounds were also identified by gas chromatography with the flame ionization detector GC-FID using the same analytical column and applying the same analytical conditions to Calculate RIs

Using pure calibrated materials, the percentage composition of active compounds separated from peak regions was calculated; the compounds were identified by matching with the search Nist and Willy present in the analytical program.

By the results it turns out there is 1 1.8 EL by 57.49.And Camphor increased by 11.65 and α-Terpinolene by 4.26 and β- Caryophyllene increased by 5.31 and Borneol increased by 5.28 and α-Pinene increased by 4.92

They are important medicinal compounds with anti-cancer, antibacterial, anti-fungal, anti-inflammatory and antioxidant applications.

6. determination of the exchange capacity of antioxidants :

100 g of leaves of plants wild rosemary Syrian and after grinding were soaked in 200 ml solution of 50/50 (ethanol /water) and in need of dark spots in the water bath at the temperature of 40 ° C for half an hour, stirring, then stir constantly offers in the bathroom of the waves of the ultrasonic for 25 minutes then instructs the liquid flow in the amount of one drop per second. get to the bottom of the liquid transferred to a rotary evaporator and warmly without 30ºC until the yield limits of 5 ml then add 30 ml of an aqueous solution metal 50 % Then we put the beaker containing the feed plant in a bath Ultra sonic for two minutes, then vaporize methanol, best of the rotor to a lesser extent than 30ºC review We get the water extract to be placed in the refrigerator in preparation for analysis

Antioxidant measurement:

The method developed by L V [5] applied the total antioxidant capacity protocol for substances dissolved in water .in this work , where the antioxidant effectiveness of all components found in the roots and leaves of the Syrian wild plant Akol was measured, I purchased the necessary reagents kits from the company analytic Jena – Germany. The first three reagents are: reagent 1 (solvent), reagent 2 (aqueous solution=10.5), and reagent 3(photosensitive). The working solution of reagent 3 (3-WS) is prepared by taking the solution of reagent 3 and extending it by 750l L of reagent 2. The working solution of reagent 4 (4-WS) is prepared by taking reagent 4, adding μ 490 of reagent 1 and mixing it with 10 of sulfuric acid at a concentration of 95-97% from Merck. Stir for several seconds, take from the previous mixture 10L D and extend by 990 D from reagent 1 to obtain a working solution 4 (4-WS) . Table 4 shows all procedures and volumes used in the analysis.

Table 6: sizes used in different measurements

Reagent	1	2	3-WS	4-WS	Sample
Control	1500l	1000l	25l	0	0
Calibration	1500l-x	1000l	25l	x	0
Measurements	1500l-y	1000l	25l	0	y

x= 10, 15, 20µl, y= 10µl, WS (working solution)

The measurements were calibrated according to the standard kit protocol as shown in the table, and the measurements were made with a photochem (Nm) device from analytic Jena – Germany. The used sizes were prepared in microliters and the measurements were repeated twice. A light emission curve was recorded in 240 seconds using an inhibitor as a parameter to estimate the antioxidant capacity, and the antioxidant capacity is determined by taking the integration given by the previous curve and expressed in mmol/L ascorbic acid used as the criterion for obtaining the calibration curve.

Table 7 shows the biodegradable antioxidant capacity in water equivalent to the efficiency expressed in nanomol equivalent of ascorbic acid per gram of the studied product.

Table 7: total antioxidant rated with ascorbic acid equivalents using photochemical luminescence technology

Measurements	Antioxidant in leaf extract of wild Syrian rosemary(Ascorbic Acid equivalent(nmol/g*(
1st duplicate	117.9
2nd duplicate	115.4
3rd duplicate	119.2
Average	117.5

* One gram of dry matter for fruits or flowers

** Average score for three measurements

It is clear from Table 7 that the highest antioxidant capacity values of the leaf extract of the Syrian wild rosemary plant expressed in ascorbic acid are 117.5 (nmol/G) .

2. Discussion

The total phenolic content and antioxidant properties of Syrian wild rosemary leaf extract were verified and through the results we obtained good positive linear correlations; between phenols and antioxidants the current study represents the first comprehensive analysis of Syrian wild rosemary leaf extract in Syria. The method described here has proven to be sensitive, selective, and quick to analyze. The results obtained can explain the past and current use of the leaves of the Syrian rosemary plant as found in folk medicine, and may also support its other uses in health and nutrition as a functional food, comprehensive research and health-promoting functional ingredients. Where the content of phenolic high floor was to make sure that the HPLC and through the way that you think the reagent Folin-Denis, and the results were compatible which confirms the presence of high content of antioxidants this is confirmed by the results of the analysis show that the anti-oxidant kidney bottom of the leaves of a plant rosemary Syrian destined been acid for.5 (nmol/g)**, which confirm the validity of the use of a rosemary plant as an antidote to oxidation of the natural in industrial applications, taking into account the importance of the pharmaceutical and medicinal. Many of the components of the pilot essential oil have also been identified by the technology.

Through the results indicate the presence of 1.8 Cineole increased by 57.49 and Camphor increased by 11.65 and α-Terpinolene by 4.26 and β-Caryophyllene increased by 5.31 and Borneol increased by 5.28 and α-Pinene increased by 4.92

Thus, the study confirmed that the extracts of the leaves of the rosemary plant can be equivalent to the available drugs and effective supplement in the pharmaceutical industry, and has a promising importance in the treatment of some diseases, especially since it has antioxidants and multiple phenols and compounds effective for the treatment of many diseases.

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