Gas Chromatography-Mass Spectrometry study of the pulp of Garcinia tinctoria fruit

The aim of this work was to identify the compounds in the hexane extract of the mature fruits of Garcinia tinctoria by means of capillary gas chromatography-mass spectrometry (GC-MS). The study allowed identifying 51 chromatographic peaks. The mass spectra allowed the identification of 20 paraffins, 13 carboxylic acids and 15 phenolic and/or alcoholic compounds in the volatile fraction of hexane extract of pulp. The major compounds were: the stearic, palmitic and oleic acids.


Introduction
The large tropical genus Garcinia (Clusiaceae) contains about 400 species of polygamous trees or shrubs, distributed in the tropical Asia, Africa and Polynesia (Chattopadhyay and Kumar, 2006).Garcinia species are characterized by the production of a yellow latex in the endocarp of the fruit, in the bark and perhaps also in the wood (Negi et al., 2008).Fruits of Garcinia can be widely used for many culinary purposes and as folk medicine to treat skin infections, wounds, and diarrhea (Mahabusarakam, et al., 1987;Joseph et al., 2005).
G. tinctoria (Wight) is an important underutilized crop distributed in the lower hills of Eastern Himalayas, Western Ghats and Andaman Islands (Rema and Krishnamoorthy, 2000).G. tinctoria is well known as yellow mangosteen and its fruits are highly acidic and are used to flavour curries.The dried fruits and leaves have been used widely as a traditional folk medicine for bilious condition, diarrhea, and dysentery (Pedraza-Chaverri et al., 2008).
There is little information about the chemical composition of G. tinctoria (yellow mangosteen) fruits.In this work the compounds from the hexane extract of the mature fruits of Garcinia tinctoria were identified using GC-MS.

Plant material
Fresh mature fruits of Garcinia tinctoria were collected in the Jardí n Botánico Nacional (Habana, Cuba) in February-April 2012.A voucher specimen has been deposited at HAJB Herbarium (Havana, Cuba) under number 700.

Sample preparation and extraction
Fruits were washed and peeled in order to separate the peels from pulps and seeds.Pulp was ground into a paste using a mortar and pestle and it was preserved in refrigeration (4°C) until the moment of utilization.In order to evaluate chemical composition of the apolar extract of Garcinia tinctoria fruits pulp, it was prepared maceration (1g of pulp) with enough hexane during 7 days at room temperature.Sample was filtered using Whatmann No. 4 filter paper and concentrated under reduced pressure at 45°C using a rotary vacuum evaporator.The dried extract was weighed and stored until the analysis moment.

Determination of the components from the hexane extract
It was weighed 10 mg of the extract and was dissolved in 0.5 mL of internal standard solution (tridecanoic acid at 0.05 mg/mL in chloroform, Sigma, USA).The solution was derivatized with 100 μL of N metil N trimetilsililtrifluoroacetamide (MSTFA, Sigma, USA ) to 70°C during 30 min in a dry thermostat Multiblock and flow control (LabLine Instruments Inc., EE UU ).
Sample was separated on a HP-5 Ms 30 m x 0.25 mm installed on a gas chromatograph 6890N with a mass detector 5975 (Agilent, EE UU).
The temperature was programmed as follows: isothermic initial 60°C for 2 min, then temperature was increased up to 200°C at 20°C/min, of 200°C up to 320 at 8°C/min, and isothermic final at 320 for 30 min.The carrier gas was helium at a flow rate of 1.0 mL/min.Injector temperature was 320°C.The energy of ionization was of 70 eV.Mass spectra were obtained in mode scan since 40 to 800 m/z.The volume of injection was of 0.5 µL.
Identification was made by comparison of retention times (tr) and spectra from available commercial standards and similar compound spectra at the libraries Wiley MS, 6ª ed. and NIST 11.The quantification was made using internal standard.

Statistical analysis
Three independent analyses were done on five samples.Statistical analysis was performed by Mann -Whitney U Test.

Identification of the constituents from the apolar extract of pulp
The analysis of the apolar extract of G. tinctoria allowed identifying fifty one compounds.The compounds, their percentages and retention times are listed in Table 1.Three compounds were identified as the major constituents: stearic acid (21.72%), palmitic acid (20.31%) and oleic acid (19.32%) and these represented approximately 61% of all the compounds of the fraction.Ajayi and Adesanwo (2009) reported that the principal fatty acids of pulp and seed of Dacryodes edulis were oleic and palmitic acids.Oleic acid is the most widely distributed fatty acid on nature and it's the principal responsible of health benefits of the Mediterranean diet.Some investigations have demonstrated that oleic acid can reduce the risk to suffer breast cancer and other diseases (Win, 2005).
The abundance of fatty acids could contribute to acidity that characterizes the fruits of Garcinia species, particularly Garcinia tinctoria (Cavalcante et al., 2006;Rittirut and Siripatana, 2006).In a chemical study of volatile constituents of Garcinia dulcis fruits using gas chromatography (Pino et al., 2003), it was reported a higher amounts of fatty acids that could be responsible for the acidic and pungent notes observed in fruit.
In general, it's was observed that most of compounds are derived of paraffins with different degrees of oxidation.The composition of the classes of compounds was as follows: organic acids (22%); phenols (15%); alcohols (11%) and others (13%).The presence of phenols corresponds with the antioxidant activity detected in G. tinctoria using techniques like DPPH y FRAP (Arazo et al., 2012).Numerous investigations have determined the ability of the extracts from fruits of Garcinia species to capture free radicals (Yu et al., 2007;Okonogi et al., 2007;Sulaiman and Udaya, 2009).
Very few studies on the composition of volatile compounds in Garcinia genus have been accomplished (Pino et al., 2003).However, there are no data in the literature concerning the possible pharmacological effects and the chemical constituents of this plant.