Obtaining of-Sitosterol from Cardamom by Supercritical CO 2 Extraction

Cardamom is plant of the Zingiberaceae family. It has been used for the treatment of many diseases such as migraine, bronchitis, stomach and intestinal disorders. Cardamom contains triterpenes, resins, starch and fatty compounds. Phytosterols (stigmasterol, campesterol and β-sitosterol) are a group steroid alcohol in plants. They are used food, medicine and cosmetic industry. They are protective effects against some types of cancer too. Phytosterols are found in the vegetable oil such as the spindle, corn and soybean oil. This paper deals with the maximum oil and β-sitosterol yield were investigated by means of the supercritical CO2 extraction of cardamom. The effect of operating parameters as temperature, pressure and CO2 flow rate were investigated on oil yield. The amount of β-sitosterol was analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) at optimized conditions. The optimized conditions were recorded as temperature of 40 o C, pressure of 200 bar and CO2 flow rate of 4 L/min. The maximum oil yield and amount of β-sitosterol were found as 74.83 mg oil/g seed and 4.73 mg β-sitosterol/g seed cardamom under these conditions.


Introduction
Cardamom is a general name of genera Elettaria and Amomum in the family Zingiberaceae.Cardamom seeds are dark brown, lentil-sized and pugnent smelling.Its triterpenes contain resin, starch and essential oils [1][2][3].Cardamom oil has antimicrobial effect for this reason it is used for treatment in throat ache, earache and headache; asthma, bronchitis; snakebite, scorpion sting and disases of bladder, intestine, kidney.In addition, cardamom increases the number of healthy cells by fighting cancer cells, so that it shows anti-cancer properties [4][5][6].
Many important compounds which are used in various fields such as medicine and food and which are difficult to produce in laboratory conditions can easily be obtained by classical extraction method from plants [7].However, organic solvents used during classical extraction are not completely removed from these compounds and the purification of the product becomes difficult [8].The supercritical fluid extraction method, which shortens the extraction period, performs the separation at low temperature and has high separation efficiency compared to the classical extraction, has attracted interest in recent years.[9].
In this method known as environmentally friendly, carbon dioxide, which is generally an inert gas, is used as a chemical solvent.This gas is not flammable, explosive and doesn't lead to chemical reaction.Since high yields can be obtained at low temperatures, many compounds such as essential oils, vitamins are extracted without being decomposition [10].
Many studies have been reported in the literature such as obtaining essential oil from yarrow [11] and boldo leaves [12], obtaining β-sitosterol from melon and peach seed [13], obtaining silybin from thistle [14] and obtaining caffeine from tea [15,16] by supercritical CO 2 extraction.
There is much research in the literature about oil and essential oils extraction from cardamom by different techniques.Compounds such as essential oils and / or oleresine have been obtained from the cardamom by methods such as solvent extraction and water vapor distillation [17].In recent years, supercritical fluid extraction has been used to extract oleresine and essential oils [18][19][20].However, in the literature, it has not been found any study about obtaining β-sitosterol from cardamom by supercritical CO 2 extraction.
The purpose of this study was to obtain maximum oil by supercritical CO 2 extraction from the cardamom and to determine the amount of β-sitosterol in this oil.
For obtaining maximum yield of oil, extraction parameters effect such as temperature, pressure and flow rate was investigated and it was determined optimum conditions on the supercritical CO 2 extraction process.The amount of β-sitosterol in the optimum condition was analyzed by Gas Chromatography-Mass Spectroscopy (GC-MS).

Method
Cardamom; were separated from their shells and dried till fixed weighing at 25°C in a laboratory ambient.Separated and dried cardamom was ground with BOSCH MKM6000 just before the experiments it is stated that the particle size should be within a minimum range of 0.25-2 mm at much research which is the supercritical fluid extraction of various natural sources in the literature.When working with a smaller particle size; it has been seen that some of the solvents did not come into contact with the extraction material as it penetrated through the channels in the extraction vessel, causing loss of efficiency in process and causing some obstructions in the channels in the extraction bed [9,10].Previous work by our group has reported that increasing the particle size (d p ) of seeded natural materials has a negative effect oil yield by supercritical CO 2 extraction [13,14].Taking into account the findings of these studies, grinded seeds were sieved from sieves complying with ASTM standards and particle size in the range of 0,185 <d p <0,425 mm was obtained.
The experimental supercritical CO 2 extraction system consists of a supercritical fluid extraction unit, a compressor, a CO 2 tube with siphon, a modified pump, and a cooling water bath (Figure 1).The supercritical extraction device is "Applied Separations' Spe-ed SFE" oven model.The device consists of three parts; pump, extraction unit in the oven, collection and control units.The extraction vessel is a stainless steel cylindrical tube with a volume of 24 mL.Four special gaskets which are resistant to temperature and pressure were used between the connections of the extraction vessel for preventing leakage in the vessel.This vessel is placed vertically inside the oven.There are 3 valves, V 1 (inlet valve), V 2 (outlet valve) and V 3 (solvent discharging valve in the system) on the oven.There is a flow meter designed to measure the velocity of the fluid exiting the outlet valve V 2 .A sample collection vessel is present at the exit of the oven to collect the product obtained.pressure, and 3-5 mL/min CO 2 flow rate ranges.Operating conditions were determined by using the previous work carried out by our group [13,14].On the whole of the experiments; oil and β-sitosterol analyzes were repeated 3 times.Saponification and derivatization pretreatments were performed to analyze the βsitosterol contained in the extract.The amount of β-sitosterol in the extract obtained was analyzed by Gas Chromatography-Mass Spectroscopy (GC-MS).Details of the analysis were given in another study conducted by our group [13].

Results and Discussion
In this work, obtaining the maximum yield of oil was studied by supercritical CO 2 extraction of cardamom.The effects of temperature, pressure and CO 2 flow rate parameters on oil yield were investigated.Optimum conditions were determined and the amount of β-sitosterol in these conditions was analyzed.As can be seen in Figure 2, the rate and yield of extraction is increasing with decreasing temperature.As the temperature is reduced from 80 ⁰C to 40 ⁰C, the density of carbon dioxide decreases from 839.9 kg/m 3 to 594.1 kg/m 3 at 200 bar pressure.
Supercritical carbon dioxide reduces the density of cardamom, so the solubility of the oil decreases in supercritical CO 2 [21].Similarly, in the study by Goash et al., the crossover pressure was determined to be 200 bar at the eugenol extraction with supercritical CO 2 from the Ocimum sanctum Linn and it was stated that the extraction yield decreased with increasing temperature at lower pressures than 200 bar.At low pressures, the increase of vapor pressure with temperature is more effective than decrease of supercritical CO 2 density and solving power [22].

Effect of Pressure
The effect of pressure on the supercritical CO 2 extraction of cardamom plant was examined for values of 180, 200 and 220 bar, and the findings obtained were given in Figure 4. extraction with pressure increased and the highest yield was obtained at 30 MPa [20].
The findings obtained are consistent with results of many studies such as obtaining βsitosterol from melon and peach seed [13], obtaining silybin from thistle [14] and obtained caffeine from tea [15,16] with supercritical CO 2 extraction.increases, so the solubility of the CO 2 molecules increases by more contacting cardamom with CO 2 molecules.As the CO 2 flow rate increases, the intra-particle diffusion resistance dominates.In the increasing flow rate from 4 L/min to 5 L/min, while the external film transfer coefficient is decreased, the interaction time between cardamom and CO 2 is shortened.As a result of this situation, the second effect is predominant, so the efficiency is affected in the negative direction as shown in Figure7.and intra-particulate diffusion resistance dominates [25].In the supercritical carbon dioxide extraction study of jojoba seed oil, it has been noted that the increase of the CO 2 flow rate increases the initial extraction rate and there is no significant change in the extraction efficiency [26].Findings of the effect of flow rate on the yield are supported by similar results in the literature.

Analysis of β-sitosterol
For the determination of the amount of β-sitosterol contained in the oil sample obtained after cardamom with supercritical CO 2 extraction, the oil sample was purified by pre-treatment of saponification and derivatization [13] and analyzed by GC-MS.
The amount of β-sitosterol in the extract obtained under optimal conditions was found to be 4.73 mg β-sitosterol/g seed.

Conclusion
In this study, it was investigated β-sitosterol extraction with supercritical CO 2 from cardamom which is used in the treatment of many diseases such as bronchitis, stomach and intestinal diseases, migraine etc.For this purpose, the effects of temperature, pressure and CO 2 flow rate on oil yield were investigated.Optimum

Figure 1 .
Figure 1.Flow diagram of the supercritical extraction set-up

Figure 3
Figure3shows the amount of oil (mg oil / g seed), obtained at the end of the

Figure 3 .
Figure 3.The temperature effect on oil yield at the end of the extraction process

Figure 5 . 3 . 3 .
Figure 5.The pressure effect on oil yield at the end of the extraction process

Figure 6 .
Figure 6.Effect of carbon dioxide flow rate on supercritical extraction processes

Figure 7 .
Figure 7.The carbon dioxide flow rate effect on oil yield at the end of the extraction conditions for the extraction of cardamom plant with supercritical CO 2 were found as 40°C temperature, 200 bar pressure and 4 mL/min carbon dioxide flow rate.The oil yield and β-sitosterol amount in this condition are 74.85 mg oil/g seed and 4.73 mg βsitosterol/g seed.Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 1 November 2017 doi:10.20944/preprints201711.0005.v1