Chemical composition analysis and antibacterial-antiinflammatoryactivity tests of tamanu seed oil extracted by supercritcial fluid technology

The natural product of tamanu oil is valuable in medicine and cosmetic. In this study, the tamanu oil was extracted by supercritical CO2 (sCO2) and Soxhlet (using hexane solvent) methods. The fatty acids and coumarins compound of the obtained tamanu oil were analyzed by gas chromatography–mass spectrometry (GC-MS). The antibacterial and anti-inflammatory activities were also investigated by the diffusion method and performed on the white mice ears (Swiss albino). Content of fatty acid in tamanu oil extracted using sCO2 (72.62 %) was higher than that using Soxhlet method. The compound of coumaron (2H-1-bezopyran-2-one, C9H6O2) was identified belonging the simple coumarin group and its content in the tamanu oil extracted using sCO2 was the highest (169.69 μg/g), 1.79 and 2.04 times as much as that using Soxhlet method and the commercial oil, respectively. Besides, the results of investigation of the antibacterial activity suggested that the tamanu oil extracted using sCO2 was able to against some of strains namely: Staphylococcus aureus (S. aureus) ATCC 25923, S. aureus ATCC 29213, methycilin-resistant S. aureus (MRSA) ATCC 43300, and Pseudomonas aeruginosa (P. aeruginosa) ATCC 277853. In term of the anti-inflammatory activity, the tamanu oil extracted using sCO2 was antiinflammatory effectively with reducing flammatory: 21.41 – 25.39 % as for the thickness of mice ears and 33.88 – 39.40 % as for the weight of mice ears.

compound of the obtained tamanu oil were analyzed by gas chromatography-mass spectrometry (GC-MS).The antibacterial and anti-inflammatory activities were also investigated by the diffusion method and performed on the white mice ears (Swiss albino).Content of fatty acid in tamanu oil extracted using sCO 2 (72.62 %) was higher than that using Soxhlet method.The compound of coumaron (2H-1-bezopyran-2-one, C 9 H 6 O 2 ) was identified belonging the simple coumarin group and its content in the tamanu oil extracted using sCO 2 was the highest (169.69µg/g), 1.79 and 2.04 times as much as that using Soxhlet method and the commercial oil, respectively.Besides, the results of investigation of the antibacterial activity suggested that the tamanu oil extracted using sCO 2 was able to against some of strains namely: Staphylococcus aureus (S. aureus) ATCC 25923, S. aureus ATCC 29213, methycilin-resistant S. aureus (MRSA) ATCC 43300, and Pseudomonas aeruginosa (P.aeruginosa) ATCC 277853.In term of the anti-inflammatory activity, the tamanu oil extracted using sCO 2 was antiinflammatory effectively with reducing flammatory: 21.41 -25.39 % as for the thickness of mice ears and 33.88 -39.40 % as for the weight of mice ears.

INTRODUCTION
Tamanu with scientific name of Calophyllum inophyllum L is widely dispersed throughout the tropics including: Thailand, Myanmar, Malaysia, South India, Sri Lanka, Melanesia, and Polynesia….In Vietnam, tamanu is mainly planted in coastal areas of Hai Phong, Ba Ria-Vung Tau, and Mekong Delta Omega is a mixture of essential fatty acids, it is one of the most important compositions in tamanu oil helping to treat various skin injuries, cell regeneration.Therefore, it plays an important role in cosmetic, omega usually includes 3 main categories [7,8]: + Omega 3: -linolenic, eicosapentaenoic acid and docosahexaenoic acid; are necessary for skin texture, reduce the dehydration of skin so that the skin could become softer.+ Omega 6: linoleic acid,  -linolenic acid and arachidonic acid; have anti-inflammatory and analgesic properties, heal the injuries and treat the skin diseases.
+ Omega 9: oleic acid, eicosenoic acid, and erucic; help to reduce the cholesterol content, sugar steady and prevent the cardiovascular deseases.
Coumarins are classified as a member of the benzopyrone family.All of which consist of a benzene ring joined to a pyrone ring.The benzopyrones can be subdivided into the 5,6benzo-alpha-pyrone to which the coumarins belong and the benzo-gamma-pyrone.
Courmarins were isolated in 1820 and identified the structure.There are four main coumarin subtypes: the simple coumarins, furanocoumarins, pyranocoumarins, and the pyrone-substituted coumarin.The coumarins are great important due to their biological properties such as: the antibacterial, anti-inflammatory, anti-fungal, antioxidant properties that make these compounds more attractive [9].
In addition, the tamanu oil was used as massage oil and a skin moisturizer, tamanu oil has been traditionally used for treating various skin injuries such as scrapes, burns, insect bites, sunburn, and diseases such as dry skin, psoriasis, eczema, ringworm, and even diaper rash.Besides, the results of some studies have even shown that Calophyllum inophyllums extracted from tamanu oil, could inhibit HIV reverse transcriptase in a novel way, which indicates that some day they may be used as part of a combination therapy for AIDS [10].

Trang 148
Mechanical pressing is the traditional and popular method for extraction of tamanu oil.
Accordingly, tamanu seeds were dried to a certain humidity before being entered the presses.Although, this method is simple and convenient, the crude tamanu oil is not pure and content of oil is not high.Besides, tamanu oil is also separated by Soxhlet method using organic solvents.The crushed tamanu seeds were extracted with various solvents: ethanol, nhexane, petroleum ether,… However, this method is toxic, consume a large amount of solvents, last for a long time and have the impurities of solvent in product [11].On the other hand, the extraction using supercritical CO 2 (sCO 2 ) has been attractive the attention of many researchers as for the separation of essential oil and flavonoids from natural materials.CO 2 is generally the most desirable solvent for supercritical fluid extraction.The critical temperature of CO 2 is only 304 K, which makes it attractive for the extraction of heat sensitive compounds.In addition, it is an inert, non-flammable, non-explosive, inexpensive, odorless, colorless, and clean solvent those leave no solvent residue in the product, it is also nontoxic and is generally accepted as a harmless [12].
The supercritical CO 2 solvent has been utilized to extract essential oils from different plants as: red pepper, cinnamon, Melaleuca, Momordica cochinchinensis….which were reported in the previous literatures [10][11][12].In this study, the tamanu oil was extracted from seeds using supercritical CO 2 and the obtained oil was analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the content and composition of oil.The properties of tamanu oil extracted using supercritical CO 2 were also compared with the commercial oil and the oil extracted using Soxhlet method.Finally, the antibacterial property was investigated using the diffusion method on agar plate and investigation of anti-inflammatory was performed on ear skin of white mice.

Materials
Tamanu seeds and commercial tamanu oil were provided by production facility at 25/21 Hau Giang, Ward 4, Tan Binh District, HCM city with resources collected from the Mekong Delta.

Using solvent of n-hexane
The tamanu seeds were dried at 40C and crushed to certain particle size about 1-2 mm.
These crushed tamanu seeds were placed in the thimble of Soxhlet extractor and the extraction was carried out for 6 hours at 20C.The obtained product was concentrated using rotary evaporator (Buchi R-215) at 37C.

Composition analysis of tamnanu oil
The fatty acid composition of the tamanu seed oil was analyzed by GC-MS Agilent technology DB5-MS system coupled with a flame ionization detector (FID) with a capillary column (HP-INNOWax, 30m x 0.25µm x 0.25 mm).

Investigation of antibacterial and antiinflammatory activities of tamnanu oil
Investigation of antibacterial activity of tamanu oil using the agar diffusion method [14][15][16]

The composition of fatty acid
From Table II, there are total 18 fatty acids including: 9 saturated fatty acids (caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, and lignoceric) and 9 unsaturated fatty acids (OA, LA, ALA, GLA, eicosenoic, ARA, EPA, erucic DHA) which were identified in tamanu oil.According to these results, the tamanu oil extracted using supercritical CO 2 has the total content of omega (3, 6, and 9) of 72.62%, being higher than that of tamanu oil extracted by Soxhlet (71.18%) and the commercial oil (71.38%).It was explained that the fatty acids were able to dissolve and easily diffused into the supercritical CO 2 fluid.Therefore, using supercritical CO 2 fluid technology is one of the advance method for extraction of natural products.

Identification and quantification of coumarin
The compound of 2H-1-bezopyran-2-one (coumaron, C 9 H 6 O 2 ) belong to the simple coumarin group and its chemical structure was represented at Figure 1.

Antibacterial activity
Table 4 indicated that the extracted oil against S. aureus ATCC 25923, P. aeruginosa ATCC 277853 at the concentration of 10 0 , 10 -1 , and 10 -2 ; when diluting further to concentration of 10 -3 , the oil just has the inhibition for P. aeruginosa ATCC 277853.As for C. albicans The oil that separated by Soxhlet method appeared the inhibition on S. aureus ATCC 25923, P. aeruginosa ATCC 277853 and did not against C. albicans ATCC 10231.The Table 6 shows that the commercial oil just against S. aureus ATCC 25923 at high concentration and was inactive as for the others.

The anti-inflammatory activity on mice ears
Tables 8 and 9 indicated that the tamanu oil was extracted by supercritical CO 2 is antiinflammatory effectively (reducing flammatory: 21.41 % as for the thickness of mice ears and 33.88 % as for the weight of mice ears).
tinted green, thick, and woodsy or nutty smelling and is easily absorbed into the skin [3].Tamanu oil is mainly extracted from seeds containing roughly 50.3-73% of tamanu oil.The extracted tamanu oil has high amount of many actives like: calophyllic acid and lactone with the antibacterial, anti-inflammatory activities, and promote healing the scars.This was discovered by the French researcherprofessor of Lederer [4].All species of tamanu tree have various pharmacological uses.The tamanu oil has been used in medicine, cosmetic, energy, and some other industries[5,6].The tamanu oil provides plenty of valuable compounds namely: fatty acid, coumarin, calophyllolid, and calophyllic acid.
were carried out by Pasteur Institute of HCM City, Vietnam, and Microbiology Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Medicine and Pharmacy.The anti-inflammatory activity was tested on mice ears-Swiss albino by Microbiology Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Medicine and Pharmacy, HCMC.

Table 1 .
Operating parameters of experiment

Table 2 .
The fatty composition in tamanu oil

Table 3 .
The quantification of courmarin in tamanu oil From Table3, the amount of coumarin in tamanu oil extracted by supercritical CO 2 was higest (169.68 µg/g), being 1.79 and 2.04 times as much as that of tamanu oil extracted by Soxhlet and commercial oil, respectively.This suggested that the supercritical CO 2 fluid technology could be utilized to extract the precious compounds more effective in comparison with the other traditional methods.

Table 7 .
Results of investigation of antibacterial activity of oil at low concentration 10-4