Anti-inflammatory activity and chemical compositions of essential oil of Achillea fragmmentissma.
Natural products once served humankind as the source of all drugs, and higher plants provided most of these therapeutic agents.
Plants provided most of these therapeutic agents. Now, natural product derivatives still represent over 50% of drugs in clinical use, with higher-plant-derived natural products representing 25% of the total.  The World Health Organization estimates that 80% of the people in developing countries of the world depend on traditional medicine for their primary health care, and about 85% of traditional medicines involve the use of plant extracts. This means that about 3.5 to 4 billion people in the world depend on plants as herbal drugs. 
The genus Achillea, consisting of 140 perennial herbs, which is used traditionally in Middle Eastern countries, for treating digestive problems, diseases of liver and gallbladder, menstrual irregularities, cramps, fever, and for healing wounds. 
Achillea fragrantissima, family Asteraceae, is a common plant in the Mediterranean region and easily found growing in fields and on roadsides. It contains high percentage of flavonoids, tannins, volatile oils, sterols, triterpenes, unsaturated amides, and sesquiterpene lactones. Achillea was highly valued as a medicinal plant for its antiseptic properties. It was used to cover cuts and sores and hasten scar tissue formation; the clinical use of Achillea fragrantissima is not described. 
Achillea fragrantissima grows in dry areas, steppe, and desert. People collect it in the Badia region, where it is common. It is the last species of Achillea to flourish, it has a strong smell and an astringent taste, for this reason it is usually mixed with sugar, but the flowers can be eaten alone, for diabetes, stomach (alone, or mixed with Teucrium polium), muscular rheumatism (fumigation), cough (drinking in the morning with sugar). 
Chemical analysis revealed that Achillea genus contains volatile oils, mainly azulene, it also contains several known anti-inflammatory compounds such as flavonoids. 
The anti-inflammatory effect of volatile oil extracted by hydrodistillation of Achillea fragrantissima was studied using carrageenan-induced paw edema. Essential oil (100 mg/kg and 200 mg/kg) was tested; the plant shows high inhibition at 200 mg/kg than 100 mg/kg after 4 h. 
Pervious review shows the importance of known medicinal plant used by many people in the northern region, the used to drink it with a tea (Alqausom in Arabic). Achillea fragrantissima is used by Bedouins in the treatment of fever and for wound healing, so the study of anti-inflammatory effect of the plant extract is important for discovering new drug. The aim of present study, are extraction of volatile oil by two different methods and, differentiate quantitative and quantitative between these methods we use the oil extract (OE) in manufacture of (soap) to used it as anti-inflammatory drug.
MATERIALS AND METHODS
The sample (Achillea fragrantissima) plant was collected from north region, Arar-turaif road, in spring season where the plant was flowering; plant sample was identified in the Botany Department, Faculty of Science, Northern Border University.
Preparation of Extracts
011 extract: The plant sample was collected from north region, 0.6 kg of arial part of the plant was cleaned and grounded to a fine powder, percolated in 1000 mL of olive oil for 15 days. Alter 15 days, the mixture was filtered, then the filtrate was kept till used.
Hexane extract: The plant powder was filtered and solvent was evaporated under reduced pressure.
The constituents of the volatile oils obtained from OE and HE were analyzed by gas chromatography-mass spectrometry (GC-MS) as reported by El-Shazly et al.  Compounds were identified by comparison of their retention indices (RI), (C9-C24 n-alkane mixture) and mass spectra as mentioned in literature. [9-12]
Anti-inflammatory: The method developed by Agbaje,  was employed. Albino Wistar rats of both sex (120-130 g) were randomized to four groups. Animals were deprived of food for 12 h before the experiment and only water was given ad-libitum.
* First group (control group) received 1 mL of distilled water (10 mL/kg)
* Second group received indomethacin (10 mg/kg) suspended in distilled water
* Third group received HE at a dose of 100 mg/kg orally dissolved in distilled water
* Fourth group received OE at a dose of 100 mg/kg orally dissolved in distilled water
After 1 h of administration of the treated extracts, carrageenan suspension (0.1 mL of 1% w/v suspended in 0.9% saline solution) was injected into the subplantar region of the left hind paw of the animals. Directly, the paw volume was measured; initial paw volume was measured using plethysmometer (UGO Basile, 21025 Comerio, Italy) before carrageenan injection. Thereafter, the paw volume was measured after 1, 2, 3, and 4 h after carrageenan administration. The difference between initial and subsequent readings gave the change in edema volume for the corresponding time. Edema volume of control ([V.sub.c]) and volume of treated ([V.sub.t]) were used to calculate percentage (%) inhibition and percentage edema volume using Eq. (1).
% Inhibition = [1-([V.sub.t]/[V.sub.c])] x 100 (1)
Data were subjected to SPSS (ver. 8.0). P < 0.05 was regarded as significant.
Soap Manufacturing Process
The soap was produced from OE according to the method by Selinger. [14,15]
Soaps and detergents are widely used in our society, So, can use this soap for the treatment of skin inflammation. Soap is the product of reaction between a fat and sodium hydroxide:
Fat + 3NaOH [right arrow] glycerine + 3 soap
Soap is produced industrially in four basic steps.
OE of Achellia fragrantissima is treated with sodium hydroxide (160 g) and the soap produced is the salt of a long-chain carboxylic acid.
Step 2--Glycerine removal
Glycerine is more valuable than soap, so most of it is removed. Some is left in the soap to help make it soft and smooth.
Step 3--Soap purification
Any remaining sodium hydroxide is neutralized with a weak acid such as citric acid and two-thirds of the remaining water removed.
Additives such as preservatives, color can added and mixed in with the soap then is shaped into cups.
Anti-inflammatory study of two extracts of Achillea plant collected from north region and extracted by two methods, HE and OE, revealed that OE showed good result in anti-inflammatory assay by carrageenan method.
The extracts have a yellow color and a pleasant odor. Most of their components could be identified by GC-MS., by comparison (mass fragmentation and retention index) with literatures. The identified compounds are a mixture of oxygenated monoterpenes and sesquiterpenes, A. fragrantissima oil is rich with sesquisabinene hydrate, bisabolene epoxide, camphor, and caryophyllene oxide; also limonene, menthol, azulene, and thujone are found in moderate amount, while santolina alcohol, lanceol, cedrene, and granny geraniol are found in a minor amounts (Table 1). The essential oil in the OE differs from that in the HE; qualitatively some compounds such as azulene, alpha-bisabolol, and bisbolene epoxide hydrate are found in OE and not in HE, while oxazolidine, viridiflorol, camphor, and thujol are found only in HE; quantitatively some little difference found as in [alpha]-humulene and limonene increased in Hexane Extract (HE) than in Oil Extract (OE).
[FIGURE 1 OMITTED]
Anti-inflammatory activity of extracts (OE and HE) was screened in vivo by inhibition of carrageenan-induced rat paw edema method at aa oral dose of 100 mg/kg. Results are presented in Table 2 as percent of inhibition and percent edema, increase at the right hind paw.
Carrageenan is a useful method for studying new drugs uesd in reducing inflammation. Carrageenan-induced rat paw edema is a nonspecific inflammatory agent resulting from a complex of diverse mediators. Edema of this type is more sensitive to nonsteroidal anti-inflammatory drugs. 
The results showed significant anti-inflammatory activity, with inhibition in edema in the range of 71%-79.9% after 4 h. The standard drug indomethacin has shown 89.5% inhibition after 4 h., the plant shows high inhibition at a concentration 100 mg/kg, OE shows high inhibition than HE, this may be due to the higher amount of some volatile oil compound present in OE than in HE, such as bisbolene epoxide, alpha-bisabolol, and azulene, these compounds are used as anti-inflammatory agents.
Essential oils isolated from aromatic plants have wide applications in perfumery, flavor, cosmetic, and pharmaceutical industries. They have been used since ancient times, and despite many of them being substituted by synthetic ones, the demand for essential oils obtained from natural sources is increasing,  so if the plant contains many important compounds as in Achillea plant, it is very important to study the pharmacological activity of the plant. Menthole is a volatile aromatic compound, it is the major constituent of many plants such as eucalyptus, lemongrass, palmarosa and peppermint. The reports proved it have antibacterial and antifungal activity, the study confirmed that, (-)menthol can inhibit the growth of rat liver epithelial tumor cells.  Laboratory studies have verified that, menthol can inhibit N-acetyltransferase activity in human hepatoma cells. 
The LD50 of a-bisabolol was studied in previous review and it was found it (13.14 g/kg) of body weight orally in adult mice and rats,  also other studies show it is known to possess anti-inflammatory and antibiotic [20,21] gastroprotective,  and antioxidant effects.  In addition, recent studies showed that a-bisabolol inhibits the growth and survival of glioblastoma, prostate cancer, breast cancer, and liver cancer in vitro,  wherever [alpha]-bisabolol showed no toxicity against normal cells.  So the presence of [alpha]-bisabolol in high percent in plant confirmed its important anti-inflammatory activity.
GC-MS analysis revealed the presence of terpenoid compounds (Phorbol, Isochiapin B, stigmasterol acetate, and [beta]-sitosterol). These compounds are well known for their biological activities as antiinsect and antitumor agents.
Studies on [alpha]-bisabolol and [alpha]-bisabolol oxide found in flower of chamomile plant in 1%-2% of volatile oils showed antiinflammatory and antiphlogistic activity. [26,27]
The bisabolol, bisabolol oxide, mentho, menthol, and b-caryophyllene are the major constituents of the Achillea extract so concern the discusses the role of these compounds as antitumor and anti-inflammatory agent.
The results suggested that the excellent performance of essential oil might be attributable to the effects of major constituents or synergistic effects among the constituents. To decipher the mechanism of oil in anti-inflammatory action need more investigation and research.
The aim of the present study was to use essential oils extracted by different methods from Achillea fragrantissima (HE and OE) in the manufacture of soap, which can used as anti-inflammatory agent. The two extracts were analyzed by GC and GC-MS. There are differences in the chemical component of volatile oil of the two extracts.
The author thank the Authority of Food and Drug, Saudi Arabia, for the financial support.
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Source of Support: Nil, Conflict of Interest: None declared.
Eman Elsharkawy (1,2)
(1) Department of Environment and plant pasture, Desert Research Center, Mathef El-Mataria, Egypt.
(2) Department of Chemistry, Science Faculty, Girls section, Northern Border University-Arar, North Region, Saudi Arabia.
Correspondence to: Eman Elsharkawy, E-mail: firstname.lastname@example.org
Received February 23, 2016. Accepted March 16, 2016
Table 1: Chemical constituents of essential oil extracts of (Achillea fragrantissima) by GC-MS Chemical Structure AF Hexane AF Oil compound extract extract Eucalyptol C10H18O 0.28 - Santolina Alchol C10H18O --- 0. 3 Camphor C10H16O 4.8 -- Viridiflorol C15H26O 2.95 --- Oxazolidine C3H7NO 0.93 --- Benzyl alcohol C7H8O 0.46 1.6 Limonene oxide C10H16O 2.93 --- Limonene C10H16 2.9 1.62 Limonene dioxide C10H16O2 --- 1.52 [beta]-Caryophyllene C15H24 2.0 0.87 Caryophllene oxide C15H22O2 1.54 1.84 Globulol C15H26O 6.51 ---- Thunbergol C20H34O 8.95 ---- [alpha]-Selinene C15H24 1.2 --- [alpha]-Humulene C15H24 0.28 0.67 Flacarinol C17H24O 1.35 -- [alpha]-Phellandrene C10H16 - 0.38 Thujol C10H18O 0.39 --- Thujone C10H16O - 0.30 Lanceol C18H36O 0.86 0.86 Bergamotol C15H24O -- 0.86 Cederne C15H24 - 0.36 [alpha]-Cubebene C15H24 - 0.36 [alpha]-Bisabolol C15H26O - 1.95 Geramcerene B - 0.1 Azulene C10H8 1.36 0.67 Spathulenol C15H24O - 0.08 Farnesol C15H24O - 0.27 Gerinol C10H18O - 0.12 [alpha]-Tocopherol C29H50 O - 0.08 [??]-Tocopherol C29H50 O - 0.08 Bisbolone epoxide C15H24 O - 1.54 Menthol C10H20 O 1.35 1.5 Phorbol C20H28O6 - 1.2 Isochiapin-B C19H22O6 1.0 2.0 [beta]-Sesquiphellandrene C15H24 - 0.4 Coumarin C9H6O2 - 0.9 Table 2: Anti-inflammatory activity of Achillea fragrantissima extracts % Edema Time (H) 1st 2nd Control 54.70 [+ or -] 2.9 67.11 [+ or -] 4.4 Indomethacin 49 [+ or -] 4.2 37 [+ or -] 4.1 * AF1 58 [+ or -] 2.7 49.5 [+ or -] 2.3 * AF2 50 [+ or -] 2.3 42.8 [+ or -] 1.9 * % Edema Time (H) 3rd 4th Control 79.58 [+ or -] 4.1 83.22 [+ or -] 4.7 Indomethacin 23 [+ or -] 3.0 * 8.6 [+ or -] 0.9 * AF1 30 [+ or -] 3.8 * 23. [+ or -] 2.3 *(a) AF2 28 [+ or -] 2.4 * 17 [+ or -] 1.7 * % Inhibition Time (H) 1st 2nd 3rd 4th Control Indomethacin 10.5 44 70 89 AF1 0.78 32 54.6 71.9 AF2 2.3 36 58.6 79.4 AF1: Hexane extract; AF2: oil extract
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|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||May 1, 2016|
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