Green extraction of Quercus infectoria gall with supercritical CO2 and methanol co-solvent

Green extraction of supercritical liquid CO2 (SCFE-CO2) using co-solvent methanol can produce a more complete phenolic acid composition and a higher quantity when compared to those without using co-solvent. The extract was devoid of toxicity. SCFE-CO2 is carried out by putting 100 g of Quercus infectoria gall of size 0.3 mm into extraction tube at temperature of 60 °C and the pressure of 20 MPa with a CO2 flow rate of 25 ml/min using co-solvent methanol with variation of flow speed 0.05, 0.5, 1.5, 3, and 6 for 60 min. The extract is analyzed using LC–MS/MS, the total phenolic content is determined using the Folin-Ceocateu method, and the toxicity value is determined using the Vero cell. According to the results, the green method of extracting SCFE-CO2 with methanol co-solvent can produce a peak and identify about 27 phenolic compounds, and increasing the rate of flow of methane co-solvent will greatly affect the outcome of the extraction to a flow rate of 0.5 ml/min, while adding a co-solvent with a flow speed above 0.5 does not affect the result. Repeated extraction of some of the largest phenolic peaks provide phenol content with minimal extract variability (div. sd. 0.1%), and the addition of soluble methanol will also increase the TPC concentration but does not increase the IC50 toxicity value above 1000.


Introduction
Quercus infectoria is a plant known to produce gall, where the gall is a special product produced from the interaction between plants and insects called gallflies or Cynips gallae-tinctoriae, so the gall of Q. infectoria is called Quercus infectoria gall (Q.infectoria gall).(Nurul-Ajilah et al. 2019).Q. infectoria gall is popular in Southeast Asia, East Asia, and South Asia as a medicinal plant, for example, to treat inflammatory pain, stomach pain, diarrhea, and postpartum care (Jamal et al. 2011;Parwata 2016).In Southeast Asia, East Asia, and South Asia, the plant is also found in Turkey, Syria, Iran, Cyprus, and Greece (Baharuddin et al. 2015;Ab-Rahman et al. 2015).Then, further using of Q. infectoria gall revealed that this plant has strong antibacterial, antiinflammatory, and antioxidant properties (Do et al. 2014;Dash et al. 2017).
Originally, Q. infectoria gall was used by the community directly by being stung, dried in hot water, and then they drunk it (Khoddami et al. 2013).However, in its development, the extraction process is carried out first before consumption, to increase the concentration of the active substance and eliminate unwanted components (Hadzri et al. 2014;Iylia Arina & Harisun 2019).The advantage of the extraction method makes the bioactive compound of phenolic, for example, tannic acid (TA), gallic acid (GA), and ellagic acid (EA), another advantages can remove other useless compound (Askari et al. 2020;Radzali et al. 2014).The disadvantage of this extraction process leads to decreased effectiveness of the extraction (Khemakhem et al. 2017;Nadia et al. 2018), as a result of some bioactive components being lost during the extraction process (Hamad 1 3 et al. 2017;Khemakhem et al. 2017).The heating process while extraction can decrease the occurrence; therefore, it will reduce the effectiveness of the extract.It can make the compound less effective for drug agent (Manna et al. 2015).The using of methanol solvent is avoided when the extract will be used as a medicinal ingredient.The extract usually still contains the solvent and it can increase the toxicity (Hassim et al. 2015).Meanwhile, removing the solvent from the extract must be done with special handling and can increase the operating costs.But if it is done without special handling, it will also cause environmental problems, although evaporation can reduce the cost (Hartanto et al. 2016a, b;Hassim et al. 2015), while other organic solvents can increase the toxicity of the extract and even make it carcinogenic (Markom et al. 2013;Wang and Weller 2006).
The green extraction method is one of the methods of supercritical fluid extraction CO 2 (SCFE-CO 2 ) and a prospect to overcome the weaknesses of the solvent extraction process, while this extraction has long been developed, and this method can increase the efficiency of extraction (Singh et al. 2016;Sukor et al. 2020;Zi et al. 2016).However, because the extraction of Q infectoria gall by the SCFE-CO 2 method where CO 2 is a nonpolar solvent and is it extracted phenolic compounds, the extracts are less good (Wyrepkowski et al. 2014).To increase the effectiveness and efficiency of phenolic compounds from Q. infectoria gall, the polar co-solvents should be added during extraction.(Haşimi et al. 2017).Methanol is one of co-solvent which improve the effectiveness and efficiency of Q. infectoria gall extraction (Bouhafsoun et al. 2018;Hartanto et al. 2016b;Mota et al. 2008).The disadvantages of adding co-solvent methanol is the extract that will increase the toxicity (Kamarudin et al. 2021;Yulianti et al. 2014); therefore, the extract cannot be used as a herbal ingredient (Shi et al. 2022).The optimization of the methanol during the SCFE-CO 2 extraction will affect the result of the compounds.This research studied about increasing the efficiency of extract using co-solvent methanol and the toxicity.

Materials
Quercus infectoria gall was purchased from the local market in Indonesia, then Q. infectioria galls was washed with boiling water and dried in the oven at a temperature of 40 °C for about 24 h.After it is cooled to room temperature, it is folded to obtain a size of about 0.30 mm.Galls of a size of 0.3 mm are put into plastic bags and sealed and stored in the freezer, and this material is used for extraction.

Extraction of solvents
Ten grams and 0.3 mm of Q. infectoria gall are inserted into a chemical glass; the solvent is added in a volume ratio of 1:10, and the mixture is heated to a temperature of 50 °C on the hot plate while being mixed with a magnetic mixer at 250 rpm for 8 h (Slimen et al. 2017).During the extraction process, the glass is closed to reduce evaporation.The result is filtered, and the filterware evaporates until the solvent is exhausted, allowing measurement of the result, total phenolic content, composition, and toxicity.The data diagram can be seen in Fig. 1.

Extraction of supercritical liquid CO 2 with a co-solvent
One hundred grams of Q. infectoria gall size 0.3 mm are put into the extraction vessel, and the thermal jacket is run with hot water (60 °C).Liquid and co-solvent CO 2 is pumped into the extraction tube with a CO 2 /methanol ratio of 500:1 and a pressure of 20 MPa.The extraction is carried out for 60 min, and the output flow rate is 25 mL/min.The extracts are collected in extract storage tubes that are also cooled with dry ice, and the extraction results are weighed gravimetrically.Extraction was avoided for the ratio of CO 2 and other methanol (Al-Maqtari et al. 2021;Qi et al. 2020).The data diagram can be seen in Fig. 1.

Analysis of extract compounds with LC-MS/MS
Measurement of the composition of the extract Q. infectoria gall is carried out using LC-MS/MS with Phenomenex Prodigy 5. Detection of galactic acid, ellagic acid, quinic acid is set at 270 nm and tannic acid at 280 nm.As the mobile phase is 1 ml/min (phosphate amounts: acetonitrile 9:1) in the isocratic course, the standard curve is arranged at a concentration of 10-60 ppm (Amr et al. 2021;Aras et al. 2016;Kaur et al. 2016;Sun et al. 2014).

Total phenolic content (TPC) extract of Galle Q. infectoria by Folin-Ciocalteu method
This method uses the Folin-Ciocalteu reagent, in which the Folin-Ciocateu reactor is produced by dilution of Folin-Ciocalteu with fermented water at a ratio of 1:10 v/v.0.5 mL of Q. infectoria gall extract is mixed with 5 mL of Folin-Ciocalteu reagents and inserted into a clove.After 5 min, 4 ml of sodium carbonate (Na 2 CO 3 ) is added to the clove and mixed until homogeneous.The solution was then measured with an UV photometer at 765 nm against an empty solution without an extract.The content of TPC is expressed as the mass equivalent of gallic acid acid (equivalent to milligrams of gallic acid per gram of extract) (Slimen et al. 2017).

Measurement of toxicity of Q. infectoria gall extract with MTT assay
The toxicity test of Q. infectoria gall extract was performed using the reagents 3- Based on the test, Vero cell seeding was first done on 96-well plates with a density of 2.4 104 cells per well.The test was carried out by putting Q. infectoria gall extract to the Vero cell and then incubating it in an incubator for 48 h at a temperature of 37 °C and with a 5% CO 2 content.After incubation, the old media is removed and new media containing 10% MTT is added and incubated again for 4 h.Deposits are added with DMSO to dissolve them and formed for the MTT reaction.The quantity of MTT reaction results with Q. infectoria gall extract was determined by measuring absorption at wavelengths of 560 nm and 750 nm using the Glo-Max Microplate Multi-Detection Reader (Dos Santos et al. 2018).

Measurement of methanol residual in Q. infectoria gall extract with GC
Analysis was performed in a GC instrument, equipped with a flame ionization detector.Column: retention gap 1 m × 0.32 mm I.D. connected to 50 m WCOT fused silica CP-WAX 57 CB capillary column.GC conditions are as follows: carrier He 2 mL/min, FID temperature 250 °C (H 2 35 mL/min, air 350 mL/min, makeup gas N 2 30 mL/min), injector temperature 240 °C; oven temperature 50 °C (6 min isotherm), ramp program 10 °C/min rate to 210 °C (15 min isotherm); injection volume 1 μL; split ratio 1:20.Result calculation was carried out as described on GC.

Yield and composition
The graphical of supercritical extraction figured out in Fig. 1.The overall result of the supercritical CO 2 (SCFE-CO 2 ) extraction of Q. infectoria gall with variations in cosolvent methanol diluted between 0 and 6 ml/min (0-24%) against the flow of CO 2 (25 ml/min), then the results of qualitative analysis with LC-MS of the extract Q. infectoria gall using the method of supercritical SCFE-CO 2 extraction with co-solvent methanol, resulting more than 20 peaks identified with MS were obtained as phenolic compounds.Then, the SCFE-CO 2 extraction without methanol co-solvent, only 12 peaks were obtained.Both chromatograms of these analysis results are illustrated in Fig. 2, and a qualitative tabulation of compounds contained in both Q. infectoria gall extracts can be seen in Table 1.
The chromatographic results of the Q. infectoria gall extract (Fig. 1) were compared between the SCFE-CO 2 extract without co-solvent of methanol and the extract with a methanol co-solvent.Both graphics showed that the chromatogram of the Q. infectoria gall extract with the addition of co-solvent produced more peaks compared to one without the added methanol.This is reasonable because when methanol is added during extraction, there will be a change in CO 2 polarity, which will cause the polar compounds to be easily attracted to the CO 2 solvent, thereby causing the compound taken to be more volatile.While on SCFE-CO 2 without the addition of solvent, the obtained chromatogram appears to only extract fewer components, and those only contain large amounts.Further analysis of the peaks that appear with MS showed the types of compounds listed in Table 1.Overall, by studying the peak pattern of the existing MS compounds obtained from the constituent of the Q infectoria gall extract.
Variations in the addition of methanol as a co-solvent in supercritical CO 2 extraction (SCFE-CO 2 ), Q. infectoria gall also studied: the composition of the four main  compounds.The total phenolic content (TPC), as well as the toxicity of the extract, can be seen in Table 2.In this table, a comparison is also shown for supercritical extraction without using co-solvent, as well as the results of extraction analysis using the CSE method.
The extraction of CSE technique is used as a control in the extraction of Q. infectoria gall.The extract obtained yield of 4.29% and standard deviation of ± 2.1% for five times of extraction.While the extraction using SCFE-CO 2 technique, the extract obtained yield of 8-10% and standard deviation of about 0.1% for five times of extraction.Based on the yield of extraction, it can be concluded the extraction of SCFE-CO 2 have good reproducibility than the CSE.The further analyzed is LC-MS/MS.The quantitative of the major phenolic compounds in the extract which have the largest peak of LC-MS/MS.It contains tannic acid, gallic acid, ellagic acid, and quinic acid.The CSE extraction has less increasing yield.The using of methanol has similar result with the one without methanol.The phenolic compounds also have optimal without methanol.Compared to the SCFE-CO 2 extraction, the using of methanol was very effective than the one without using methanol.It is caused the undissolved extract using CO 2 can be carried out by adding methanol.Many phenolic compounds with high retention appear in the LC-MS/MS analyzed.

Effect of addition methanol co-solvent in SCFE-CO 2
The using of methanol as co-solvents in supercritical CO 2 extraction are expected to increase CO 2 polarity so can be produced the optimum extract.This extraction used 200 MPa of pressure, 60 °C as the operating temperature, and 3 mm of Q. infectoria gall material.The addition of co-solvent methanol is done at 0-6 ml/min, or 0-24% of the amount of CO 2 discharged.The results of co-solvent on Q. infectoria gall extraction can be seen in Fig. 3.
In the extraction of Q. infectoria gall by the SCFE-CO 2 technique, the addition of co-solvent methanol by only 0.05 ml/min or 0.2% increases yields in the 1% range, and the increase in co-solvent addition of methanol does not increase linearly.So, the addition of the smallest cosolvent methanol may already be able to increase the yield range of 1%.In addition, compounds with a high retention time on probation with LC-MS or greater than 6 min will appear and obtain a phenolic compound.It is interesting because the addition of small methanol created a lot of technical problems during the flowing of co-solvent.But it will disappear when the rate was enhanced.Therefore, even the additional of small co-solvent has good result, the technical problems should be calculated.
In the observation, the addition of methanol around 0.05-3 ml/min has no residues in the extract.This is very important because the extract is used for the raw material of the drug.The residues of methanol can be analyzed using GC-MS.It is used the different analyzed from the extract because while using LC-MS-MS, methanol is used carrier solvent.
Based on the results of the analysis, the contents of the residual methanol is figured out in Table 3.Where the methanol residues are contained in the Q. infectoria gall extract, for a high co-solvent flow rate of above 3 ml/min and after evaporation for 10 min, the residual content of methanol will disappear.Based on the literature, the methanol co-solvent will also disappear while the SCFE-CO 2 is carried out at a pressure above 300 MPa (Al-Maqtari et al. 2021;Nadia et al. 2018;Wang & Weller 2006).

Toxicity of Q. infectoria gall extract and environment
The addition of co-solvent in the SFCE-CO 2 resulted the increasing of the yield.Based on the experiment, the optimum range is on the 0.5-1.5 ml/min.The result is figured out in Fig. 4. In Fig. 4, it is shown that the quantity increased after addition of methanol, which showed an increase in the percentage of yield.The addition of methanol was not affected for increasing the yield.The addition from 0.05 to 6 ml/min only increased 1% of the yield.It did not indicate an improvement of the yield.Meanwhile, the phenolic composition has the same composition of compounds and the same quantity.
In Fig. 4, the correlation between of the extract, the total phenolic content, and the toxicity properties of the SCFE-CO 2 extract of Q. infectoria gall is illustrated.The result which can be observed from the graphic are (1) the extract of Q. infectoria gall obtained does not increase with the addition of the co-solvent flow during extraction, while the total phenolic content increases significantly with the increase in the flow of co-solvent.( 2) An increased addition of the cosolvent methanol affects the toxicity of the extract.The two results above, based to the result can be seen from Fig. 2, increase in the amount of TPC and yield is less affected by toxicity.Comparing the data extraction using the CSE technique (as a control), TPC is the lowest but has the highest toxicities.It is related with the CSE technique used as a control using 100% methanol; it can be produced the Q. infectoria gall extract from SCFE-CO 2 with large amount of addition methanol, but the extract will be very toxic, whereas a good extraction is when the yield has low toxicity.
The result of the high content of TPC is related by the level of toxicity of the Q. infectoria gall extract.The total phenolic content is determined by the Folin-Ciocalteu method as the standard of gallic acid.Based on the above phenolic determination methods, if the extract has other fractions and is mixed with the extracts, it can contribute to increased toxicity.Thus, the toxicity The extracts with the SCFE-CO 2 technique did not show a small number of IC 50s , all above 1000, although co-solvents used methanol.This is in accordance with the classification toxicity for natural materials is grouped as having IC 50 < 10 μg/mL (cells 106 /mL) is a highly toxic compound, 10 μg/mL < IC 50 < 100 μg/mL is a toxic composition, 100 μg/mL < IC 50 < 1000 μg/mL a moderate compounds, and the latter is IC 50 > 1000 μg/ml discarding non-toxic extracts (Yudhisi et al. 2019;Nguta and Mbaria 2013); Leung and Ballantyne 1999).
Based on the extraction improvement of the SCFE-CO 2 technique with co-solvent methanol, whose addition is very small (0.05 ml/min) and produces very small toxicity, this extraction technique is excellent for the environment.Even without the flow of methanol, the results obtained decreased only by 1% and were of good quality.

Conclusion
The supercritical CO 2 or SCFE-CO 2 extraction method using methanol as a co-solvent yielded Q. infectoria gall extract 7-8% and the quantity of phenolic compounds were be increased.The addition of the smallest of methanol co-solvent, 0.05 ml/min, produced an extract containing 27 phenolic compounds, and increasing the amount of methanol co-solvent did not make the result increase significantly.The addition of co-solvent at 6 ml/ min produce the extract which containing methanol residue.But it will be evaporated away.The total phenolic content will increase, and it is followed by the toxicity which also increased.It showed the phenolic compound's exist.According the toxicity of the plant extract, all of the six extracts are described non-toxic.It caused the IC 50 is more than 1000 mg/L (Yudhisi et al. 2019).

Fig. 2
Fig. 2 Chromatogram of Q. infectoria gall extract extracted from the SCFE-CO 2 technique using co-solvent methanol and without co-solvent

Table 1
Characteristics of the composition of the Q. infectoria gall extract using the (SCFE) technique at CO 2 speed of 25 ml/min and with co-solvent of 0.05 ml/min

Table 2
The characteristic of the extract by conventional solvent extraction (CSE) and supercritical extraction of CO 2 (SCFE) with a velocity of CO 2 25 ml/min

Table 3
Methanol residues in extract Q. infectoria gall after and before evaporating of the Q. infectoria gall extract can be caused to some factors, the involvement of solvents, the participation of the highly toxic compounds extracted, and the total phenolic content.As in the SCFE-CO 2 method with co-solvent methanol extracted by Q. infectoria gall, it does not have toxic properties because when the extract is separated from CO 2 , the methanol is carried by CO 2 , so that the extracts are not contaminated with methanol.Instead, by increasing the rate of flow of co-solvent methanol, Q. infectioria gall extract will increase the content of TPC; this is understandable because co-solvent methanol can bind phenolic compounds and produce more results in the extraction.The highest TPC content of the extract is 1756 mGAE, which is a SCFE-CO 2 engineering extract with methanol as a co-solvent.Toxicity is tested using the MTT test.The most toxic extracts are shown in the CSE technique with methanol as a solvent with IC 50 713.