Separation Science and Technology ISSN: 0149-6395 (Print) 1520-5754 (Online) Journal homepage: http://www.tandfonline.com/loi/lsst20 Enrichment, in vitro, and quantification study of antidiabetic compounds from neglected weed Mimosa pudica using supercritical CO2 and CO2Soxhlet Tasnuva Sarwar Tunna, Md. Zaidul Islam Sarker, Kashif Ghafoor, Sahena Ferdosh, Juliana Md Jaffri, Fahad Y Al-Juhaimi, Md. Eaqub Ali, Md. Jahurul Haque Akanda, Md Shihabul Awal, Qamar Uddin Ahmed & Jinap Selamat To cite this article: Tasnuva Sarwar Tunna, Md. Zaidul Islam Sarker, Kashif Ghafoor, Sahena Ferdosh, Juliana Md Jaffri, Fahad Y Al-Juhaimi, Md. Eaqub Ali, Md. Jahurul Haque Akanda, Md Shihabul Awal, Qamar Uddin Ahmed & Jinap Selamat (2018) Enrichment, in vitro, and quantification study of antidiabetic compounds from neglected weed Mimosa pudica using supercritical CO2 and CO2-Soxhlet, Separation Science and Technology, 53:2, 243-260, DOI: 10.1080/01496395.2017.1384015 To link to this article: https://doi.org/10.1080/01496395.2017.1384015 View supplementary material Accepted author version posted online: 25 Sep 2017. Published online: 25 Sep 2017. Submit your article to this journal Article views: 38 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=lsst20 Download by: [Tohoku University] Date: 30 November 2017, At: 21:27 SEPARATION SCIENCE AND TECHNOLOGY 2018, VOL. 53, NO. 2, 243–260 https://doi.org/10.1080/01496395.2017.1384015 Enrichment, in vitro, and quantification study of antidiabetic compounds from neglected weed Mimosa pudica using supercritical CO2 and CO2-Soxhlet Tasnuva Sarwar Tunnaa, Md. Zaidul Islam Sarkera, Kashif Ghafoora, Sahena Ferdosha, Juliana Md Jaffrib, Fahad Y Al-Juhaimic, Md. Eaqub Alid, Md. Jahurul Haque Akandae, Md Shihabul Awalf, Qamar Uddin Ahmeda, and Jinap Selamatg,h Downloaded by [Tohoku University] at 21:27 30 November 2017 a Faculty of Pharmacy, International Islamic University Malaysia, Pahang D/M., Malaysia; bDepartment of Food Science and Nutrition, King Saud University, Riyadh Saudi Arabia; cFaculty of Science, International Islamic University Malaysia, Pahang, Malaysia; dNanotechnology and Catalysis Research Centre (NanoCat), University of Malaya, Kuala Lumpur, Malaysia; eFaculty of Food Science and Nutrition, University Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia; fDepartment of Food Science & Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh; gDepartment of Food Science, Faculty of Food Science and Technology, University Putra Malaysia, Serdang, Selangor, Malaysia; hFood Safety and Food Integrity (FOSFI), Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, Serdang, Selangor, Malaysia ABSTRACT ARTICLE HISTORY Supercritical fluid extraction (SFE) using carbon dioxide (CO2) and liquid CO2 using Soxhlet (CO2-Soxhlet) extraction were employed to extract three (3) antidiabetic compounds viz. stigmasterol, quercetin, and avicularin from Mimosa pudica. Various extraction parameters were studied. Extracts were analyzed pharmacologically, qualitatively and quantitatively to ascertain enrichment levels. All three antidiabetic compounds were effectively enriched under optimized conditions of temperature 60°C, pressure 40 MPa, co-solvent ratio 30%, and CO2 flow rate of 5 ml min−1. SFE was found to be the better method for enrichment of the antidiabetic compounds than the CO2-Soxhlet method. Extraction conditions were seen to affect the enrichment of desired compounds. Received 4 March 2017 Accepted 20 September 2017 Introduction The global threat of diabetes mellitus is increasing day by day. The demand for the improved medications and along with the therapeutics, medicinal plants, and herbal medicines are also availed by the health issues worldwide.[1] The significance of producing such magnitude of medicinal plants and herbal medicine-based products leads to stress on the flora of medicinal nature. Antidiabetic preparations with the traditional usages are in demand worldwide.[2,3] Alternative sources include biomass of lignocellulosic materials such as agricultural and forest waste, weed, seaweed, etc. is renewable, cheap, and abundant.[4] In order to extract medicinally active ingredients from such waste and the demand for clean extractions are of choice to reduce the toxin level and organic traces.[5] M. Pudica has been chosen for this study as the neglected, agricultural weed which has been underutilized and could be used an alternative source for antioxidants and bioactives for food and health industries as well as for cosmetics and toiletries and many other purposes. M. KEYWORDS Mimosa pudica; supercritical fluid extraction (SFE); CO2-Soxhlet; antidiabetic compounds; α-glucosidase assay pudica has been reported to contain antioxidants, flavonoids, phenolic acids, and many anti diabetic compounds.[6,7] Conventional extraction methods such as maceration, reflux under heat, Soxhlet are employing huge organic solvents which are polar and non-polar, nonselective, time consuming and uses of hazardous solvents at higher temperatures which leads to degradation of the bioactive compounds.[8] An ideal extraction process should provide a good yield with non-degraded and healthy bioactive compounds which are easily separable from the plant matrices. Therefore, the importance of employing alternative and green methods, which are environment friendly and safe bio solvents to achieve all the above mentioned objectives, is great.[9,10] Various green technologies are being developed and employed throughout the world to address these issues constructively with regard to recover toxin free and medicinally active agent extracts. Many researchers have reported the modified green technologies such as supercritical fluid extraction (SFE), CONTACT Zaidul Islam Sarker zaidul@iium.edu.my Faculty of Pharmacy, International Islamic University Malaysia, Kuantan Campus, 25200, Pahang D/M., Malaysia. Supplemental data for this article can be accessed here. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lsst. © 2017 Taylor & Francis Downloaded by [Tohoku University] at 21:27 30 November 2017 244 T. S. TUNNA ET AL. soaking and pressure swing method of SFE, sub critical extraction (SCE), pressurized liquid extraction (PLE) etc..[11,12] In this study SFE and CO2-Soxhlet have been conducted first time to extract and enrich the bioactive compounds in a single extract from the neglected weed of M. pudica. To achieve the optimum and efficient extracts, the extraction parameters such as pressure, temperature, and flow rate of the applied solvents need to be carefully considered and optimized in each major stage. Extraction rate may be altered due to factors influencing parameters like real temperature of the extraction fluid, real pressure inside the extraction vessel especially for pressurized fluids, matrixes of the plant materials for which the rate of extraction is usually varied on the solubilization and the diffusion of the solute such as bioactive of the plant matrixes.[13,14] Other factors affecting the yield could be the harvest season, geographical location, plant parts selection, climate, sample collection, and the nature of the extraction solvent as well as the method used. Moreover, green technology is employed for numerous health and nutritional facets, advancement in medicinal therapeutics extraction can be an answer for the shortage and the stress on the medicinal flora, unhealthy extraction methods often carcinogenic in nature, side effects of current medications and a deeper need for newer and more easily available green and healthy products.[9] Soxhlet is a conventional extraction method employing high temperatures usually more than the boiling point of the solvents. Soxhlet recycles the solvent and therefore the solvent is reused in cycles ensuring the completion of the extraction. Whereas, carbon dioxide under subcritical conditions used as a solvent in Soxhlet extraction is regarded as CO2-Soxhlet which is defined as a non-conventional and green method. In the CO2Soxhlet method CO2 is applied in its subcritical conditions. In this study CO2-Soxhlet pressure of 7 MPa and the temperature at 28°C were employed.[15,16] Thus, the solvent is in liquid state and since the targeted bioactive are both polar and non-polar in nature could be trapped to separate from the sample matrixes. For CO2-Soxhlet ethanol was introduced as modifier and direct spiking method was used. Various ratios at 2:1, 2:1.5, and 1:1 of sample to modifier was used and the sample was directly spiked with designated amount of ethanol for overnight to let the solvent soak in and later to make it easier for the CO2 to separate the bioactive compounds. The aim of this study was to perform comparisonbased enrichment of antidiabetic compounds along with others secondary metabolites in a single extract to overcome the isolation method for the individual compound from M. pudica. Moreover, enzyme inhibitory activity was checked for this enriched extract of the bioactive compounds of obtaining by supercritical CO2 extraction (SFE) and subcritical CO2-soxhlet extraction which was found more active than the extracted and isolated compounds from M. pudica using conventional extraction method using organic solvent at high temperature. In this study four parameters such as temperature, pressure, co-solvent, and CO2 flow rate of solvent and co-solvent were tested using SFE and modifier ratio by subcritical CO2 Soxhlet methods. For the SFE run Box Behnken Design (BBD) was employed in the experimental design to optimize the yield. Liquid chromatography mass spectra (LCMS) and High performance liquid chromatography (HPLC) were used for qualitative and quantitative analyzes to find the extracts with the highest concentration of the bioactive compounds such as stigmasterol, quercetin, and avicularin (Fig. 1). Methodology Chemicals DPPH (2,2-diphenyl-1-picrylhydrazyl), Folin Ciocalteu reagent and solvents were bought from Fisher and Merck (Australia). The enzyme α-glucosidase type 1 (from baker’s yeast), ρ-nitrophenyl-α-D-glucopyranoside (ρ-NDG), potassium phosphate monobasic, dipotassium phosphate, Na2CO3 etc. were bought from SigmaAldrich (Germany). Halogen moisture analyzer from USA was used (HB43, Mettler Toledo, Columbus). Ultra pure water was used wherever water was needed as the solvent. An ultraviolet visible microplate reader Spectrophotometer from Tecan Nano Quant, Infinite M200, Austria was used. Pure carbon dioxide (99.99%) was purchased from Malaysian oxygen (MOX), Malaysia. For HPLC-UV analysis the solvents (Methanol, Acetic acid) of HPLC-UV grade was used. Sample preparation Plant material collection Fresh aerial parts of M. pudica were collected during flowering season (March till July every year) from the vicinity of the International Islamic University Malaysia campus (IIUM), Kuantan, Pahang DM, Malaysia. The plant was identified by taxonomist at Kulliyyah of Pharmacy, IIUM, Malaysia. The voucher specimen (NMPC-QU037) has been deposited in the Herbarium, Faculty of Pharmacy, IIUM, Kuantan, Pahang DM, Malaysia. Grinding The fresh aerial parts of M. pudica (3.5 kg) were dried in a PROTECH laboratory air dryer (FDD-720-Malaysia) at SEPARATION SCIENCE AND TECHNOLOGY Downloaded by [Tohoku University] at 21:27 30 November 2017 A C 245 B D Figure 1. The chemical structures of Stigmasterol (A), Quercetin (B), Avicularin (C), and acarbose (D). 40°C for 7 days and pulverized using Fritsch Universal Cutting Mill-PULVERISETTE 19-Germany. It was then stored in a dessicator at 2°C until further use. Approximately 1.1 kg powdered material was obtained from the fresh sample followed by which particle size was determined using a sieve. The particle size was kept at ≤ 1mm. Moisture content determination For moisture content determination an automatic Halogen moisture analyzer (HB43, Mettler Toledo, Columbus, USA) was used. 1g of dried powder was placed in a tarred aluminum plate. The sample was heated to 106°C for 10 min. The machine determines the moisture loss after heating and states the moisture content after heating period is over. The process was repeated thrice and the mean value was calculated and expressed as percentage of 100 g sample as dry weight basis. Extraction Supercritical fluid extraction. Supercritical fluid extraction (SFE) system comprised of CO2 cylinder, cool water circulator (VTR-620, Jeio Tech., Seoul, Korea), column thermostat (CO-1560, JASCO Corporation, Tokyo, Japan), both the CO2 and modifier pumps (PU-1580, JASCO), UV/VIS detector (UV-1575, JASCO) and back pressure regulator (880–81, JASCO), and water bath (Heidolph WB-2000). Three levels (low, medium and high) of each of the four parameters were tested thrice individually. The parameters were temperatures (40°, 50°, and 60°C), pressure (20, 30, and 40 MPa), co-solvent ethanol (10, 20, and 30% of main solvent CO2) and CO2 flow rate (1, 3, and 5 ml min−1). Ten grams of dried, powdered sample of M. pudica was kept in the extraction vessel and placed in the column thermostat set for each predetermined temperature. Predetermined pressure was adjusted at the back pressure regulator and solvent pumps. CO2 gas from the cylinder is pumped by the CO2 pump with a cooling jacket for cooling and condensing, through the chiller which is a low temperature bath circulator (631D, Tech-Lab Manufacturing Sdn. Bhd., Selangor, Malaysia) containing a mixture of ethylene glycol: water at 50:50 (v/v) to −6.5°C. The flow rates for CO2 and co-solvent were fixed, respectively, based on experimental design following BBD. Once the desired temperature and pressure (at solvent pumps and back pressure regulator) were achieved after turning on the injection valve and the system was in equilibrium, the extraction was carried out for 2 hours. The extract was separated from the supercritical CO2 via reduction of pressure by an expansion valve. The extract was collected in an amber vial placed at the collector and later stored at 4°C before further analysis for the extract yield and bioactive compounds. The percent of extract yield was measured by drying the liquid extract at 50°C until constant weight of the extract was obtained. Downloaded by [Tohoku University] at 21:27 30 November 2017 246 T. S. TUNNA ET AL. Carbon dioxide Soxhlet extraction (CO2-Soxhlet). CO2-Soxhlet extraction is an innovative extraction technique used to test the extractability of the predetermined bioactive compounds. This method combines the benefits of conventional Soxhlet and the CO2-extraction. The process includes the use of liquid CO2 at its subcritical state at a temperature of 28 oC, pressure of 7 MPa. CO2 is circulated in and out to the extraction vessel for 300 cycles for each run. During the process of extraction CO2 is continuously regenerated via boiling and condensation and does not use a CO2-pump instead naturally circulates the system. The modifier (co-solvent) was introduced via direct spiking method. For each run 250 gm of dried powdered sample was used and before each run the ethanol (at predetermined ratio) was added and allowed to soak for 12 h. The next day the wet sample was put into the extraction vessel (1L) and CO2 was supplied to the system and collected at the condenser. The extraction started when the liquid CO2 flowed into the extraction chamber. The estimated mass of the liquid CO2 used for the system was 6 kg and the solvent to feed ratio about 24:1. Next the sensor 1 (S1) (Fig. 2) transmitted a signal to the pneumatic valve 1 (V1) (Swagelok, USA) which in turn opens the gateway for CO2 to flow back to the reboiler which closes after 2 min to ensure the passage of all the CO2 back to the reboiler. The CO2 vapor is formed in the reboiler and the gas send to the condenser for cooling and conversion back to liquid state to complete one[1] extraction cycle. During Figure 2. The schematic diagram of CO2 Soxhlet method. the process the extract remained back at the bottom of the extraction vessel. The extraction continued until the extract percentage dropped below 1% this was done to ensure complete extraction. The extract was collected at the cyclone separator via the valve 2 (V2) (Parker A-Lok, USA). Temperature was controlled by the temperature sensors TS1, TS2 and TS3. The study was performed at three[3] ratios of sample: solvent at 2:1, 2:1.5, and 1:1 w/ v. After the duplicate cycles were completed the extract was collected in pre-weighed vials and stored at 4 oC chiller to be stored until further use.[15,16] The subcritical Soxhlet instrument used for this study had fixed subcritical conditions of temperature 28°C and pressure 7 MPa. Due to the equipment restraint of fixed temperature and pressure as well as flow rate and volume of solvent (CO2 usage) the only other parameter left to alter was the co-solvent ratio. Three ratio of cosolvent was tested and based on the trial and error it was found that a ratio of CO2: ethanol below 2:1 gave negligible yield and a ratio more than 1:1 did not improve yield much so the higher ratios were not studied extensively. Concurrently sample preparation can be studied for future improvement of this technique. Experimental design for supercritical fluid extraction and CO2-Soxhlet. For the supercritical Carbon Dioxide (Sc-CO2) method, SFE was used for the enrichment of the pre-isolated and identified antidiabetic compounds from M. pudica, as green extraction method. Downloaded by [Tohoku University] at 21:27 30 November 2017 SEPARATION SCIENCE AND TECHNOLOGY Temperature (°C), pressure (MPa), CO2 flow rate (ml min−1), and percentage of co-solvent (ethanol %) were the four parameters employed for the process design.[17] For SFE each run were duplicated and a total of 27 separate runs based on the parameters were tested at three levels (low, medium, and high). For experimental design and optimization purpose Box–Behnken Design (BBD) was used employing response surface methodology (RSM). The co-solvent parameter tested was in percentage (10–30%) employed in the ratio with the flow rate of CO2. The dependent variable was extract yield (Y) and independent variables were temperature (X1), pressure (X2), co-solvent % (X3), and CO2 flow rate (X4). Based on various trial and error process and literature review the parameters were set. Within the input ranges the change of extract yield (Y) was found to be significant from the trial error process and also based on the literature review (Table 1). The experimental design of BBD has four parameter and 27 experimental trials (run). A randomized experimental order was followed to reduce the overall impact of change or variation of the extraneous variable in the responses observed. A second order quadratic polynomial regression model was used for predicting the extract yield (Y). The equation is as follows: 247 Y ¼ βo þ Σβi Xi þ Σβii Xi 2 þ ΣΣβij Xi Xj (1) where Y is the response variable, βo is a constant, βi βii and βij represent the linear, quadratic and interactive coefficients, respectively. Xi and Xj represents independent variables. The Minitab software (version 16) was used for multiple regression analysis, analysis of variance (ANOVA) and the coefficient of determination (R2) was measured to find the fit of the regression model. The t-value for the estimated coefficients as well as the related probabilities was included. Statistical significance with a confidence level of 95% was accepted as the criteria for the total error. Threedimensional response surface graph and two-dimension contour plots were used for the study to analyze the effect of the parameters on the yield of the extract and their individual interactions as well. Response surface graphs allows the user to find the maximum, minimum, and the saddle points but it is not suitable to determine the level of variables necessary for desired response. On the other hand contour plots help us to obtain the levels and interpret the variables for optimization.[18] For the 2nd method to be tested, CO2-Soxhlet was studied using three ratio of modifier to sample as 2:1, 2:1.5, and 1:1 of sample:modifier. Table 1. Extract yield from SFE runs at various factors and levels. Conditions applied Run order of Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Temperature, X1 (°C) 60 50 50 50 60 40 50 60 60 40 50 50 50 50 40 50 50 50 40 50 50 50 60 50 60 40 40 Pressure, X2 (MPa) 20 20 30 20 40 40 20 30 30 20 40 40 40 20 30 30 40 30 30 30 30 30 30 30 30 30 30 Co-solvent, X3(%) 20 30 20 20 20 20 10 10 30 20 20 30 20 20 20 10 10 30 10 20 10 30 20 20 20 30 20 CO2 flow rate X4 (ml min−1) 3 5 3 3 3 3 1 1 5 3 3 5 3 3 3 1 1 5 1 3 1 5 3 3 3 5 3 Experimental yield, Y (%) 1.950 0.876 2.346 1.122 5.070 2.245 0.987 2.390 3.738 2.750 2.567 5.113 4.620 1.022 1.766 1.124 1.731 1.475 1.100 2.641 2.667 5.490 4.678 2.334 2.478 3.790 3.009 Estimated yield (%) 1.325 1.201 2.44. 1.482 5.244 2.491 1.200 2.800 3.876 2.197 2.512 5.054 4.486 1.303 2.177 0.685 1.560 1.535 1.188 2.440 2.567 5.211 4.839 2.440 2.221 3.606 3.001 Identity of Chosen sample (SFE) 6 12 5 10 2 8 3 7 1 11 9 4 Downloaded by [Tohoku University] at 21:27 30 November 2017 248 T. S. TUNNA ET AL. In- vitro analysis DPPH free radical scavenging activity. DPPH (2,2diphenyl-1-picrylhydrazyl) assay is performed to determine the radical scavenging potential of a sample with respect to find out its inhibitory effect on the free radical in the form of DPPH. The ethanol extract (EtOH) was evaluated for the free radical scavenging activity following the method described by Nickavar et al., 2006 with some modifications.[19] In short, 1 mL of various concentrations of MeOH and extracts in methanol (3 μg mL−1–100 μg mL−1) were prepared and treated with 2 mL of 0.1 mM of DPPH (prepared fresh with methanol) and diluted using 1mL of ultrapure water. The mixture was kept in an incubator at 30°C (found to be optimum) for 30 min after which absorbance was taken using a UV spectroscope at 517 nm. Methanol was employed as blank and DPPH, methanol and water (2:1:1) were employed as controls. Quercetin was used as standard and IC50 values in μg mL−1 were determined for all the samples and standard deviation was calculated. DPPH as percentage scavenging activity was calculated using the following equation: Scavenging activity ¼ Control Absorbance x100 (2) Control Total phenolic content assay. This assay was performed to determine the total amount of phenolic compounds present in the sample with respect to a standard phenolic compound, in this case, gallic acid. TPC was determined using Folin Ciocalteu (FC) method by following the procedure described by Singleton et al. (1965) with minor modifications.[20] To evaluate the TPC, the sample (0.5 mL) was mixed with 2.5 mL of FC reagent (10 times dilution with deionized (DI) water) in amber glass vials and kept aside for 6 min. Subsequently, 2 mL of 7.5% Na2CO3 was added and the media was vortexed and then kept for incubation at 30° C for 30 min. After incubation, the supernatants were collected and the absorbance was taken using UV-Vis spectrometer at 760 nm. Experiments were performed in triplicate. Gallic acid was used as the standard and the TPC was calculated using the following equation: TPC ðmg=g Þ ¼ GAE x V x ðDx10 6 x 100Þ Sw (3) GAE – gallic acid equivalent (mg); V – Vol. of sample (mL); D = dilution factor; Sw – sample weight in grams. Total flavonoid content assay. This assay was performed to determine the total amount of flavonoids present in a particular sample with respect to a standard flavonoid (Quercetin). TFC assay was performed using AlCl3 colorimetric method by following the method described by Zishen et al. (1999) with some modifications.[21] In amber glass tubes, 500 μL of EtOH extract was mixed with 2 mL DI water and 15 μL of 5% NaNO3 and incubated at room temperature for 6 min. Subsequently, 150 μL of 10% AlCl3, 2 mL of 2 M NaOH and 200 μL of water were added. The reaction media was vortexed and incubated at 30°C for 30 min. After incubation, absorbance was measured at 415 nm. Quercetin was used as standard and appropriate blanks were used. Experiments were done in triplicate. TFC was calculated using the following equation: TFC ðmg=g Þ ¼ QE x V x ðDx10 6 x 100Þ Sw (4) GAE – gallic acid equivalent (mg); V – Vol. of sample; D = dilution factor; Sw – sample weight in grams. Α-glucosidase inhibitory assay. α-glucosidase enzyme inhibitory assay was performed by following the standard protocols from Apostolidis et al. (2007) with slight modifications.[22] In 96-well plate 50 μL of sample (1mg mL−1) was added to 100 μL of (l U mL−1) α-glucosidase enzyme (Sigma-Aldrich) in 0.1 M potassium phosphate buffer (pH 6.9). The mixture was incubated at 25°C for 10 min after which 50 μL of ρNDG was added at 5 s intervals and further incubated at 25°C for 5 min. Readings were taken using Micro plate reader at 405 nm. Blanks were initial extracts, sub-extracts with solvents instead of enzyme and control is enzyme and solvent in place of initial mother extract and sub extracts. Acarbose at 1 mg mL−1 (in sodium phosphate buffer) was used as standard. Enzyme inhibition was calculated using the following equation:   Abs S Inhibition ð%Þ ¼ 1 x100 (5) Abs E Abs S – Absorbance of Sample; Abs E – Absorbance of Enzyme Liquid chromatography mass spectra analysis. Liquid chromatography was done using a Dionex Ultimate 3000 model attached to a Bruker Micro Q tof (United States of America). The column was a Hypersil Gold Thermo Scientific (250mm x 2.1mm, 5 micron), column temperature was set at 27°C. For analysis of MS data Hystar Version 3.2 software was used. At precise time 0, 3, 10, 10, 25, 26, and 30 the ratio of solvent used was 90:10, 90:10, 10:10, 10: 90, 10:90, 90:10, and 90:10, respectively. HPLC-UV analysis. The extracts from all the 15 runs of SFE and CO2-Soxhlet were analyzed using HPLC-UV Downloaded by [Tohoku University] at 21:27 30 November 2017 SEPARATION SCIENCE AND TECHNOLOGY which was composed of water 600 pump controller, 9486 tuneable absorbance UV detector and equipped with an Eclipses XDR-C18 reversed phase column (250 mm × 4.6 mm × 5 μm, Supelco, USA). The volume for the injection loop was 20μl and Classis Millennium software 2010 was used for data processing. The mobile phases were solvent A: acetic acid buffer pH 2.5 in deionized RO purified water and solvent B: 100% methanol (HPLC-UV grade). For washing deionized water was used. The compounds were detected at 280 nm. The temperature was set at 27°C and flow rate 1.0 ml min−1. The gradient method was used at the following ratios solvent A: solvent B as follows 95:5; 75:25; 50:50; 25:75; 5:95. The isolated pure compounds were used as standards and their profiling were conducted before the runs were tested. Later on quantitative calculations for the presence of these compounds were made according to the linear calibration curves made with the pure compounds. The R2 values >0.97 were obtained. Statistical analysis. The TFC, TPC, and DPPH assays were performed in triplicate and the results were expressed as means ±SD using Microsoft Excel. The enzyme analysis was performed in six times replication and evaluated by analysis of variance by one-way ANOVA followed by post hoc analysis via Tukey’s post t-test and Dunnett using IBM SPSS. For the ANOVA analysis as well as quadratic equation-based experimental yield calculation Response Surface Methodology (RSM) was employed. p < 0.01 was regarded to be very significant whereas a significance of p < 0.05 was significant. Minitab version 16 was used for RSM. Results and discussion 249 The chosen extracts (column 8) are the ones which underwent the in vitro and qualitative as well as quantitative analysis for enrichment. From Table 1, it is evident that the effect of the parameters at the various levels studied gave significantly different yields. The yields were seen to be essentially three types as low yield, medium and then high yields based on the low, medium and high levels of the parameters. Evidently high levels of the parameters were giving yields in the approximate range of 4–5.5%, whereas medium levels giving yields in the approximate range of 2–3% and lower levels of the parameters giving yields in the approximate range around 1% were obtained. Despite the results being commendable, yield (Table 1) for run number 5 was very low due to spilling problem during decompression stage of extraction. The reason for this could be spilling observed during the decompression stage. Spilling of extract during decompression stage could be caused due to the probable fact that at lower pressure the plant sugar (carbohydrates) and waxy components get clogged in the collection nozzle. Since the flow rate of CO2 and co-solvent was in medium it probably couldn’t create enough force for the extract to be pushed out from the nozzle and separate out from the solvent during the decompression.[4] Table 2 summarizes the multiple regression coefficients obtained by the regression analysis to predict the second-order polynomial model used for extract yield. The coefficient of determination (R2) was 0.96 meaning that the regression model for the extract yield was satisfactory and fits the experimental results adequately. The predicted yields were obtained by the plotting of the experimental yields in the polynomial Eq. (6) to give Eq. (7). As depicted in Table 2 the experimental yields were seen to be close to the Supercritical fluid extraction Supercritical and subcritical CO2 extractions were employed for the enrichment of the desired antidiabetic compounds of M. pudica in this study. The study was conducted to check and ascertain the efficacy and extractability of the desired antidiabetic compounds. The aimed compounds were stigmasterol, quercetin, and avicularin that were previously isolated and their antidiabetic activity checked through inhibitory assay against digestive enzyme α-glucosidase responsible for the rise of post-prandial blood glucose. Factors like temperature, pressure, percentage of ethanol as co-solvent, and CO2 flow rate were studied. Table 1 shows the parameters tested and the respective yields obtained against each run, which are a combination of the parameters. All the runs were performed for 120 min each keeping the time constant. Table 2. Regression analysis of BBD for SFE extraction. Term β0 X1 (β1) X2 (β2) X3 (β3) X4 (β4) X12 (β11) X22 (β22) X32 (β33) X42 (β44) X1 X2 (β12) X1 X3 (β13) X1 X4 (β14) X2 X3 (β23) X2 X4 (β24) X3 X4 (β34) R2 Adj. R2 a Coefficients Standard error T p- valuea 2.44033 0.47033 1.05325 0.44850 0.2378 0.1189 0.1189 0.1189 10.263 3.956 8.859 3.772 0.000 0.002 0.000 0.003 0.87358 0.49375 −0.12013 0.12550 −0.06662 0.90625 0.86076 −0.33550 0.53825 0.87325 1.38950 0.9594 0.9121 0.1189 0.1783 0.1783 0.1783 0.1783 0.2059 0.2059 0.2059 0.2059 0.2059 0.2059 7.348 2.769 −0.674 0.704 −0.374 4.401 4.180 −1.629 2.614 4.241 6.748 0.000 0.017 0.513 0.495 0.715 0.001 0.001 0.129 0.023 0.001 0.000 p<0.001: highly significant; 0.001 <p<0.005 significant; p>0.005 not significant. 250 T. S. TUNNA ET AL. Downloaded by [Tohoku University] at 21:27 30 November 2017 predicted yields. From the regression analysis the linear effect of the parameter of pressure and CO2 were seen to be very significantly effecting the rate of extraction (p < 0.01) while the effect of the parameters of temperature and co-solvent were found to be significant (p < 0.05). The quadratic effects were found to be non-significant. The interaction between temperature and pressure, co-solvent %, and CO2 flow rate, respectively, was found to be highly significant (p < 0.01) while the interaction between pressure and co-solvent was found to be non-significant. The interactions between pressure and CO2 flow rate and cosolvent and CO2 flow rate were found to be highly significant (p < 0.01). The second order polynomial equation for the extraction can be written as follows with coefficient: Y ¼ 2:44033 þ 0:47033X1 þ 1:05325X2 þ 0:44850X3 þ 0:87358X4 þ 0:49375X1 2 0:12013X2 2 þ 0:12550X3 2 0:06662X4 2 (7) þ 0:90625X1 X2 þ 0:86075X1 X3 0:33550X1 X4 þ 0:53825X2 X3 þ 0:87325X2 X4 þ 138950X3 X4 Effect of temperature Temperature is the most important feature and parameter of all the factors affecting the rate of extraction. For both conventional and non-conventional methods temperature plays a crucial and vital role in extracting bioactive compounds from the sample matrices. Temperature is believed to increase the extractability of bioactive and secondary metabolites. The higher the temperature the better the extraction yield both in terms of amount and types of compounds extracted. The intermolecular and inter-matrix bonds between the bioactives and the sample matrix depend on the polarity and non-polarity of the substance. Temperature helps in breaking the bonds between the compounds and sample matrices and frees the substances while the solvent carries them outside of the matrices. Temperature for supercritical CO2 (ScCO2) is also crucial because with temperature the density of the fluid decreases and the forces of breaking the said bonds are decreased hence lower the extraction yield. Thus, it was important to optimize the temperature, so that the extraction rate remains high by keeping the density of ScCO2 optimum by applying pressure.[23] Also, most of the time much high temperature may negate the effect by degrading compounds like volatile and essential oils, fats and oils as well as low molecular weight phenolic acids. Referring to Table 1 and 2 the fact is enforced that temperature plays a positive role in extraction yield at three temperature points at 40°C, 50°C, and 60°C. Relatively high yields were obtained at 60°C. Temperature beyond 60° C was not employed as higher temperature is connected to degradation of thermolabile components of sample. The result agrees with the report of Brunner, (2005) that extraction yields increases with temperature.[24] Effect of pressure Pressure has proportional relationship with yield for supercritical CO2 and usually yield increases with pressure for the supercritical fluid. Along with the increase in pressure the intermolecular spaces between the fluid solvent molecules decreases therefore increasing the density of the fluid. The density of the fluid increases with pressure therefore increasing the penetrating power of the solvent into the sample matrices and the intermolecular energy is raised hence the bonds are finally broken releasing the solutes from the matrices. The increased fluid density therefore carries higher impact during bond breaking stage of the solutes such as bioactive and other compounds from the sample matrices.[25] High pressure favors extraction of high molecular weight compounds like flavonoids and flavonoidic classes. In Table 1, the three pressure values used were 20, 30, and 40 MPa and it is seen that the yield increases with pressure. Pressure is seen to have a positive linear effect on the extraction yield. Pressure is always an important and somewhat the chief influential parameter for any fluid or compressible liquid solvent.[26] An interesting issue is the combined effect of increased temperature and pressure. The density of CO2 decreases with temperature where the negation of the solvent density is decreasing with temperature can be overcome by increasing pressure, which in turn increases the density hence stabilizing the extraction process. Moreover, high pressure disrupts the plant cellular matrices and help to extract further. Run no 11 has 40 MPa pressure and only 40°C temperature and the extract yield was seen to be only 1.100 (%) contrarily for run no 12 the pressure was 40 MPa where the temperature was applied to 60°C and the yield was seen to be increased to 5.070%. This result agrees with the fact that with higher temperature and lower pressure the yield decreases while increasing the pressure seems to negate the fall in solvating power. Thus, the combination of pressure and temperature need to be optimized in concern to the density of the CO2 and clearly pressure is the most important factor seen here[9] SEPARATION SCIENCE AND TECHNOLOGY Downloaded by [Tohoku University] at 21:27 30 November 2017 Effect of co-solvent percentage Co-solvent percentage influences the extraction yield of bioactives. Co-solvent has a positive linear effect whereby the yield increases with the flow rate of the co-solvent. Co-solvent is an important factor since the CO2 is non polar solvent and it is not suitable to target and extract polar compounds completely. Flavonoidic compounds with polar nature require polar solvent to extract them out from the sample matrix.[27] Addition of ethanol, a polar biosolvents, was seen to drastically increase the extraction yield of classes such as phenolics and flavonoidic extracts. Ethanol works in two ways, first by interacting with the analyte complex to promote rapid desorption in the CO2 and secondly by enhancing the solubility properties of the solutes in CO2[17]. As seen in Table 1, the higher percentage of 30% favors the extraction yields whereas a 10% is inefficient for maximum extraction. A 20% co-solvent level is effective when the other factors like temperature and pressure is higher to support extraction. Therefore, in lieu to the theory of the need of cosolvent to extract polar characters from the sample matrices this study was in alignment that higher the co-solvent percentage employed, higher the yield was obtained. Effect of the CO2 flow rate The carbon dioxide (CO2) flow rate as seen in Tables 1 and 2 had a positive and significant effect on the extraction yield. The results indicate that a lower flow rate of 1 ml min−1 is insufficient to extraction whereas a flow rate of 5 ml min−1 was much effective. Carbon dioxide (CO2) being a non-polar entity was not sufficient for targeted bioactive compounds to be extracted and was very much dependent on its flow rate and percentage of the co-solvent. The co-solvent was introduced in to the reaction vessel using a co-solvent pump and was at its’ subcritical conditions (room temperature and atmospheric pressure). The CO2 was introduced via the CO2 pump and was at its’ supercritical phase where the extraction properties of the gas was enhanced.[28] The fluid in conjunction with the cosolvent as seen in the Table 1 is giving relatively high yields at high ranges. Although it is clear from run numbers 2 until 7 that the flow rate of the CO2 and co-solvent alone or together was not the main factors as for those runs where the temperature and pressure played vital roles for the extraction. These two factors enhanced the extraction by being the carrier to extract out the bioactives. A higher CO2 flow rate enhanced the yields of the sample extracts. 251 Combined effect of the parameters of temperature, pressure, CO2 (solvent) flow rate, and solvent (CO2) – co-solvent ratio studied At this juncture it is important to discuss the combined effects of the four parameters tested in this study. The factors employed were temperature at 40, 50, and 60°C, pressure 20, 30, and 40 MPa, co-solvent at 10, 20, and 30% of the solvent which is CO2 and the flow rate of solvent, CO2 at 1, 3, and 5 ml min−1. For extraction and reactions temperature is essential for the intermolecular and inter-matrix bonds of the solvents and the molecules of the bioactive compounds with that of each other and the matrices to be broken for them to diffuse out of the system for the extraction.[9] Higher temperature aids extraction therefore a rise in temperature usually produces a rise in extraction yield and extraction is completed earlier. However, there is another affecting factor and that is the solvating power of the scCO2 which depends on its density. Higher temperature reduces the density of the supercritical fluid and hence can reduce extraction yield. Also, excess heat can degrade the extract and be not in lieu with the concept of green technology is unable. Moreover, too much diluted solvent at higher temperature is unable to penetrate into the sample matrices and extract bioactive compounds from the sample matrices.[29] On the other hand, pressure has a directly proportional effect on the density. The yield increases due to the increment of density of the CO2. For both these two parameters as seen in Table 1 relatively higher yields are obtained at 60°C and 40 MPa pressure due to their combined effect on the density of the solvent. Figure 3 depicts the contour and surface plots of the interaction between the significant parameters and their effect on the yield of extract. Figure 3a shows that at lower temperature even highest pressure applied did not produce high yield necessarily. Even at lower temperature if high pressure is applied the yield will increase significantly.[30] For the parameters of the CO2 flow rate and cosolvent percentage (Fig. 3b), better yield was observed at higher levels and the results of run no. 2, 9 and 4 depicts the finding (Table 2). Table 2 shows the relationship between carbon dioxide flow rate with cosolvent percentage to be highly significant (p < 0.001). At higher co-solvent percentage if the CO2 flow rate is not high then the yield will be considerably very low at high temperature (60°C) and pressure (40 MPa). For this interaction to be effective all four parameters are predicted from the plots (see supplementary data of the surface and contour plots for the regression analysis) be needed to be at highest then yield will be obtained Downloaded by [Tohoku University] at 21:27 30 November 2017 252 T. S. TUNNA ET AL. Figure 3. a) Contour plots for yield as a function of temperature and pressure (b) Contour plots for function of co-solvent and CO2 flow rate (c) Surface plots of co-solvent and temperature (d) Surface plot of pressure and CO2 flow rate. higher. The other two significant interaction found were that between temperature and co-solvent (Fig. 3c) and CO2 flow rate and pressure (Fig. 3d). RSM was also used to find optimization parameters and their levels for the extraction. The optimization graph was obtained. The optimized conditions for the process were found to be 60°C, 40 MPa, 30% co-solvent (ethanol), and 5 ml min−1. The desirability of the optimized conditions were found to be 1.0000 and the maximized yield (Y) was found to be 9.9510% on dry basis. The highest yield from the experimental set obtained through SFE was 5.490% at 50°C temperature, 30 MPa pressure, 30% co-solvent, and 5 ml min−1 CO2 flow rate. The optimized conditions obtained agree with the results, so far that extraction is increased and yield improved with high levels of the parameters tested. The finding is in line with the assumptions made that for extractability to increase the parameters such as temperature, pressure, co-solvent percentage and CO2 flow rate need to be at their high levels. Carbon dioxide Soxhlet (CO2-Soxhlet) extraction Soxhlet extraction with CO2 at its subcritical state (CO2-Soxhlet) was also studied to compare the effectiveness and suitability of the extraction of bioactives from M. pudica sample with SFE. Study like this using modifier with medicinal plant for bioactive extraction for CO2-Soxhlet is relatively newer technology and was done for the 1st time for sample. The ratio of the modifier was chosen based on trial and error at various rations. Since without modifier the yield was negligible and a ratio more than 2:1.5 reduced the yield so ratios higher than 1:1 was not chosen. A total of four final runs were conducted in duplicate and their respective yields are shown in Table 3. SEPARATION SCIENCE AND TECHNOLOGY Downloaded by [Tohoku University] at 21:27 30 November 2017 Table 3. Results of extraction for CO2-Soxhlet. Ratio used Extract obtained (mg) % yield No modifier 2:1 2:1.5 1:1 1.56 123 976 673 0.000624 0.0492 0.3904 0.2692 It is clear from Table 3 that the modifier used was very crucial for the CO2-Soxhlet as the carbon dioxide (CO2) was in its subcritical and gaseous state, which was not very suitable for the polar bioactive extraction. CO2-Soxhlet instrument used in this study, has fixed pressure, cycle rates and temperatures so the only other parameter that could be tested was the modifier ratio. For CO2-Soxhlet low level of modifier unlike SFE, where a higher amount of co-solvent employed, was used. The results show that a middle ratio of modifier is giving higher yield than the higher ratio of modifier. This could be due to the fact that the dynamics and equilibrium was somewhat attained at a ratio of 2:1.5 samples: solvent and the CO2 could work well synergistically with the modifier. The reason for this could be because high amount of solvent could have made it harder for the gas to carry out that extract load during the decompression stage and the equilibrium may be attained earlier. Porto et al. (2014) verified that mass transfer decreases with the increase of flow rate and vice versa.[30] Since the CO2 employed is in gaseous state therefore higher mole from the solvent can cause reverse stress and cause product dissociation.[10,31] These may be the reasons for the contrary results of higher modifier ratio decreasing the extract yield. Hence, the modifier ratio of 2:1.5 was seen to be the optimum for the maximum extraction yield of bioactive compounds from M. pudica using CO2- Soxhlet. Various factors affect the extraction like the particle size of the vegetal biomass, shape and porosity of the sample particle all are important factors influencing and affecting the mass-transfer rate.[32] Different time of collection, geographical factor, particle size, biomass maturity affects the amount of extraction of targeted compounds and the difference between the SFE extracts and CO2-Soxhlet could also be due to the stated above factors.[33] Thus far, from the discussion on the parameters and extraction yield it is seen that for supercritical CO2 extraction (SFE) a combined effect of high temperature, pressure, CO2 flow rate, and co-solvent percentage is essential to attain better yields of extracts as compared to single parameters or squared parameters. Among the 4 parameters, temperature and pressure has been seen to be predominantly important for extraction and the co-solvent flow rate closely following. The regression 253 and ANOVA data shows the linear effect of pressure and CO2 flow rate to be highly significant (p < 0.001) and temperature and co-solvent to be significant (p < 0.005). The quadratic interactions were found to be non-significant. The interaction data for temperature combined with pressure and co-solvent were highly significant as well as CO2 flow rate combined with pressure and co-solvent. The interaction between pressure and co-solvent as well as temperature and CO2 flow rate was found to be non-significant. In vitro studies For the in vitro assays (DPPH, TFC, and TPC and the αglucosidase enzyme inhibitory assays) along with the qualitative and quantitative enrichment studies employing HPLC and LCMS, only 12 of the 27 experimental SFE runs were studied. Whereas all three of the CO2Soxhlet experimental runs were studied. The runs were chosen based on the experimental yield values taking four high yields, four medium yields, and four low yields based on the three levels of parameters tested. The reason for taking 12 runs rather than 27 was to reduce the consumption of enzymes, chemicals and solvents. The point here was to try to get study-based results from the three levels reducing the time and materials involved. All the antioxidant assays were done in triplicates and 6 replicates for enzyme inhibition. Free radical scavenging (DPPH) assay 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay is a standard antioxidant assay utilized worldwide to evaluate the antioxidant property of extracts, bioactive compounds, and lead molecules. This study employed DPPH assay as one of the three tests performed to ascertain the antioxidant levels and free radical scavenging properties of the various extracts prepared by the SFE and the CO2-Soxhlet extractions. Table 4 shows the results from the in vitro assays for the SFE and CO2-Soxhlet runs as compared to the run conditions. A lower IC50 value suggests better activity means the extract is more potent in smaller concentrations. Run no 2, 5, 6, and 12 of SFE showed the best activity, while for CO2-Soxhlet the 2nd run showed the best activity (Refer to Table 4). Compounds typically responsible for showing activities are phenolic compounds, flavonoid groups, glycosides, terpenoidal components, and sometime fatty acids as well.[7,34,35] 254 T. S. TUNNA ET AL. Table 4. Depiction of IC50 of DPPH assay for SFE and CO2-Soxhlet extracts. Extract SFE 1 SFE 2 SFE 3 SFE 4 SFE 5 SFE 6 SFE 7 SFE 8 SFE 9 SFE 10 SFE 11 SFE 12 CO2-Soxhlet A CO2-Soxhlet B CO2-Soxhlet C Run conditions 50°C, 30 MPa, 30%, 5ml min−1 60°C, 30 MPa, 20%, 5 ml min−1 50°C, 40 MPa, 20%, 5ml min−1 40°C, 30 MPa, 30%, 5ml min−1 50°C, 30 MPa, 20%, 3ml min−1 60°C, 20 MPa, 20%, 3ml min−1 50°C, 40 MPa, 20%, 3ml min−1 40°C, 20 MPa, 20%, 3ml min−1 50°C, 30MPa, 30%, 5ml min−1 60°C, 30 MPa, 10%, 1ml min−1 40°C, 30 MPa, 10%, 1ml min−1 60°C, 40MPa, 30%, 5ml min−1 2:1 sample: solvent ratio 2:1.5 sample: solvent ratio 1:1 sample: solvent ratio DPPH, IC 50 (mg ml−1) 1.62 (±0.01) 0.52 (±0.03) 1.36 (±0.01) 1.76 (±0.01) 1.29 (±0.03) 1.32 (±0.04) 1.51 (±0.96) 2.53 (±0.02) 1.41 (±0.01) 2.62 (±0.02) 1.14 (±0.35) 0.18 (±0.68) 3.73 (±0.01) 0.98 (±0.01) 1.22 (±0.03) Downloaded by [Tohoku University] at 21:27 30 November 2017 Total phenolic content assay The total phenolic content assay (TPC) was the 2nd antioxidant assay used for this study and it was performed to ascertain the content of phenols and phenolic compounds in extracts and samples. Phenolic compounds are the secondary metabolites produced by the plants in response to stressors like infections, wound and UV radiation.[36] The results for the TPC assay performed for this study is given in Table 4. For this study SFE showed good phenolic content for the run no 1, 2, 4, 6 and 9 (Table 4) and is similar to the DPPH assay the 2nd run of CO2-Soxhlet showed remarkable activity. The CO2-Soxhlet showed the highest amount of phenolic content (39.004 mg/g of sample) as compared to SFE. On an average the subcritical Soxhlet showed higher phenolic content in all the extracts as compared with SFE as some of the SFE runs yielded lower than the CO2-Soxhlet. This high value may be attributed to the lower temperature (29° C) used for the extraction process which helped to keep the integrity and quantity of the phenolic contents.[37,38] Smaller phenolic compounds are heat labile and thermosensitive since lower molecular weight equals lower melting point so extraction techniques that employs less temperature is suitable to preserve the amount and quality of the phenolic compounds present in a sample.[23] Total flavonoid content assay The total flavonoid content assay was the 3rd antioxidant assay performed in this study. The flavonoid content assay determines the amount of flavonoid present in extract of any sample. Flavonoids are the large group of plant secondary metabolites present in various plant tissues, inside the cells or on the surface. Flavonoids are the most common polyphenolic compound class responsible for aging, carry out cellular TPC (mg g−1) 23.9 (±0.01) 24.96 (±4.03) 22.45 (±2.73) 27.39 (±5.58) 19.27 (±0.13) 25.54 (±3.85) 16.45 (±3.69) 21.05 (±3.80) 20.83 (±4.12) 14.08 (±2.46) 15.56 (± 2.32) 20.48 (±3.18) 22.20 (±5.57) 39.00 (±0.08) 24.82 (±2.34) TFC (mg g−1) 0.83 (±0.20) 0.71 (±0.05) 0.49 (±0.05) 0.75 (±0.11) 0.12 (±0.02) 0.38 (±0.09) 0.81 (±0. 25) 0.87 (±0. 23) 0.62 (±0.09) 0.50 (±0.15) 0.85 (±0.20) 1.25 (±0.11) 0.32 (± 0.09) 0.66 (± 0.18) 0.452 (± 0. 2) IC 50 (mg ml−1) 1.27 (±1.14) 1.07 (±2.28) 1.04 (±0.85) 1.23 (±1.37) 0.56 (±2.14) 2.45 (±2.08) 1.91 (±4.99) 1.69 (±1.41) 1.08 (±1.89) 2.59 (±1.78) 1.88 (±1.98) 1.05 (±1.09) 5.32 (±1.36) 2.19 (±1.42) 3.25 (±0.91) metabolic reactions, coloring agents etc.[39] For this study the TFC values as depicted in Table 4 shows SFE to be the better method of extracting flavonoids than the CO2-Soxhlet.[23] For SFE, most of the conditions showed pretty high yields specially for the run no. 1, 7, 8, and 11 and the highest flavonoid content was found for run no 12. Some of the SFE conditions such as run no. 3, 5, 6, 9, and 10 showed lower flavonoid content. As seen in the other assays the 2nd subcritical Soxhlet run showed higher flavonoid content and came out to be the best method of using sample to solvent ratios of 2:1.5.[40] Parab et al. (2013) tested the relationship between pressure and TFC determination and the results showed that flavonoid content was seen to increase with the increase in pressure.[41] Run no 7, 8, 11, and 12 showed very high TFC value and those runs used the pressure of 40 MPa. Flavonoid extraction was seen to be improved at medium to high temperature range but mostly temperature of 40–50°C. It is clear from the results that high pressure improves the total flavonoid content and overall extraction of flavonoids. Enzyme inhibition assay α-Glucosidase enzyme is responsible for digesting the disaccharides, priory broken down from the polysaccharides by α-amylase, to monosaccharides and release them in the blood which in turn increases the “blood glucose level” in other words post prandial diabetes.[42] These digestive enzymes are the key players for carbohydrate digestion leading to diabetic sugar-crash which is harmful as that leads to glycosylated hemoglobin. Currently, acarbose is the only FDA approved enzyme inhibitor prescribed the world over to control post prandial diabetes.[43] Many medicinal plants have been studied to link extraction factors with digestive enzyme inhibition potential.[42,44,45] Downloaded by [Tohoku University] at 21:27 30 November 2017 SEPARATION SCIENCE AND TECHNOLOGY The previously isolated and identified antidiabetic compounds (in our previous study) were aimed to be concentrated using non conventional “green technology” like SFE and CO2-Soxhlet extraction methods to obtain enriched extracts of M. pudica. Enzyme assays were employed to check the degree and efficacy of each enriched extracts for all the 15 runs of SFE and CO2Soxhlet. The result is tabulated in Table 4. Five[5] of the 12 SFE runs were enriched and have shown good activities against α-glucosidase enzyme and the 2nd run of CO2-Soxhlet showed the best activity amongst the rest of the CO2-Soxhlet runs. In terms of enrichment, SFE came out be the better extracting technique as compared to CO2-Soxhlet as it showed better enzyme inhibition. Run no 2, 3, 5, 9, and 12 showed good inhibitions. Along with that the conditions are mostly the high levels of the parameters of temperature (50 and 60°C), pressure (30 and 40 MPa), co-solvent percentage (20 and 30%), and CO2 flow rate (3 and 5 ml min−1). For CO2-Soxhlet the 2nd run showed the best activity and was also from the run giving highest yield at sample: solvent ratio 2:1.5. Temperature and pressure were the most important factors for extraction followed by the solvent and co-solvent percentage and ratios used. From the results it is seen that enzyme inhibition is favored by the samples which were produced by employing high temperature, pressure, solvent, and co-solvent parameters. Parameters at higher levels increase the efficiency of the process of pulling out or extracting the desired compounds (in this case antidiabetic) from the plant matrices and hence give better inhibitory activity.[9] Enzyme inhibitory activity of extracts were shown by mostly phenolics, polyphenolics and the flavonoid classes of compounds including the glycosides which was not limited but divulged to alkaloids, gums, amino acids, peptidoglycans etc.[46] Many studies linked flavonoids, flavonoidic glycosides and polyphenolic compounds to antidiabetic activities.[47] Many well established compounds are now known antidiabetics like Metformin which is galegine derived from Galega officinal.[48] This study showed that the extracts of M. pudica containing the mentioned classes of compounds have shown antidiabetic activity and the strength of the extracts depend on the concentration and the types of the compounds present. The concentration of antidiabetic compounds depends on some factors like extraction time, pressure, temperature, amount of sample used, flow rate of solvent and co-solvent involved, pre-treatments, season and method of sample collection, processing and storage and other factors that may directly or indirectly affect the quantity and the quality of antidiabetic compounds[49] 255 In this study, quantitative analysis using HPLC-UV and LCMS was also performed to ascertain the level of the antidiabetic compounds present in each of the runs. The next stage was to find the amount and the presence of these 3 isolated compounds in the 12 SFE and 3 CO2-Soxhlet runs to find the enriched extracts. Evaluation of the enrichment The aim for this study was comparing and obtaining enriched extracts employing SFE and CO2-Soxhlet that will contain the pre-isolated antidiabetic compounds stigmasterol, quercetin, and avicularin. Liquid chromatography mass spectra qualification For this study each of the samples was prepared and analyzed using the method described in the methodology. The three isolated compounds of stigmasterol (mass: 412), quercetin (mass: 302), and avicularin (mass: 434) were detected by matching the fragmentation pattern of reference with the run data. Fifteen samples were analyzed and the results of the presence of the compounds at the highest concentration for SFE 2, 5, 7, 12, and CO2-Soxhlet B are shown in Fig. 4(A–E). Liquid chromatography coupled with mass spectra (LCMS) was used for qualitative detection of those three compounds using the retention time and matching the mass: charge ratio profiles. The ESI-MS (Mass Spectra) data of the three compounds (studied by authors previously) were used as the compound profiles to analyze the LCMS data. The height of the peaks determines the concentration of that compound. The peaks for stigmasterol were found to be the highest amongst the other detected compounds specially avicularin and quercetin, respectively. In some extracts the concentration of quercetin was seen to be higher than avicularin like in SFE 5 and CO2-Soxhlet B. The results are somewhat similar to the findings of the HPLC-UV. Liquid chromatography coupled with mass spectra (LCMS) was used for qualitative analysis only that is to detect the presence of the compounds and projection of the probable amount. High performance liquid chromatography (LCMS) quantification High performance liquid chromatography (HPLC-UV) is one of the common, fast, sensitive methods of qualitative and quantitative assay of the compounds specifically phenolic acids and polyphenolic compounds but can work for flavonoids also. HPLC-UV uses reverse 256 T. S. TUNNA ET AL. SFE: 2 / Stigmasterol- 4; Quercetin- 2; Avicularin- 5 A Downloaded by [Tohoku University] at 21:27 30 November 2017 SFE: 5 / Stigmasterol- 4; Quercetin- 2; Avicularin- 5 B SFE: 7 / Stigmasterol- 8; Quercetin- 6; Avicularin- 9 C SFE: 12 / Stigmasterol10; Quercetin-8; Avicularin-11 D CO2-Soxlet B / Stigmasterol- ND; Quercetin- 8; Avicularin-11 E Figure 4. (A–E): The LCMS analysis depiction of chosen extracts (extracts showing best activities) the compounds (Compound profiling for stigmasterol, quercetin and avicularin). Downloaded by [Tohoku University] at 21:27 30 November 2017 SEPARATION SCIENCE AND TECHNOLOGY phase C-18 column, UV or Diode Array Detector (DAD), polar acidified solvents like methanol, acetonitrile, acetic, or trifluoro acetic acids etc. This is pretty sensitive and before running any sample an array of precautions are often taken. This study employed HPLC-UV for quantification purpose to determine the level of enrichment in each runs by comparing the presence and amount of the isolated compounds stigmasterol, quercetin, and avicularin in them. After trial and error and developing the method the compounds were successfully profiled and linear calibration curves obtained. The equations were used for the calculation of the compounds. The compound profiling for stigmasterol, quercetin, and avicularin are given in Fig. 5. The equations derived from the profiling which has been used for quantification of the compounds in each HPLC-UV run are given below: Stigmasterol : x ¼ Quercetin : x ¼ Avicularin : x ¼ ðy þ 76:77Þ 11:44 257 (8) ðy 3:334Þ 11:28 (9) ðy 5:024Þ 0:511 (10) The results (Table 5) showed that stigmasterol was present in highest amount next to avicularin and lastly quercetin. Supercritical fluid extraction run no. 2, 3, 5, 9, 12 and CO2-Soxhlet B showed the best enzymatic activities whereas SFE run no. 2, 4, 5, 7, 11, 12 showed most enrichment in the descending order of run no. 12 > 4 > 2 > 5 > 11 while CO2-Soxhlet B and C showed interchangeable results. CO2-Soxhlet B showed the better activity and the enrichment shows the presence of Stigmasterol A Quercetin B Avicularin C Figure 5. HPLC-UV profiling of stigmasterol (A), quercetin (B), and avicularin (C). 258 T. S. TUNNA ET AL. Table 5. HPLC Quantification of isolated antidiabetic compounds in SFE and CO2-Soxhlet. Sample Downloaded by [Tohoku University] at 21:27 30 November 2017 SFE 1 SFE 2 SFE 3 SFE 4 SFE 5 SFE 6 SFE 7 SFE 8 SFE 9 SFE 10 SFE 11 SFE 12 CO2-Soxhlet A CO2-Soxhlet B CO2-Soxhlet C Stigmasterol mg g−1 Quercetin mg g−1 Avicularin mg g−1 86.73 ± 0.04 310.06 ± 0.01 287.64 ± 0.05 323.33 ± 0.04 409.91 ± 0.01 386.48 ± 0.02 400.09 ± 0.03 464.92 ± 0.06 227.37 ± 0.10 170.74 ± 0.17 289.01 ± 0.11 490.14 ± 0.09 0 0 0 14.45 ± 0. 85 84.21 ± 0. 26 7.44 ± 0. 711 148.07 ± 0. 54 17.93 ± 0. 16 12.72 ± 0.23 17.03 ± 0.10 6.36 ± 0.80 5.74 ± 0.77 0 8.45 ± 0.50 56.05 ± 0.20 0 60.73 ± 0.44 14.46 ± 0.51 77.78 ± 0.82 80.04 ± 0.69 21.28 ± 0.10 83.68 ± 0.80 98.89 ± 0.29 38.89 ± 0.90 87.19 ± 0.27 39.33 ± 0.87 29.85 ± 0.57 17.12 ± 0.72 98.77 ± 0.65 98.54 ± 0.67 0 16.54 ± 0.87 77.78 ± 0.82 quercetin to be high in that extract. Consequently, for SFE run 2, 5 and 12, showed high inhibitory activity. Comparison of Tables 4 and 5 showed that the runs with high amount of quercetin are predominantly giving better inhibition then stigmasterol, avicularin, and acarbose. Avicularin supported quercetin which amounted to the inhibitory potential of run no. 2, 3, 5, 9, 11, and 12 of SFE. Stigmasterol on the other hand was predominant in all the runs and it can be deduced that supercritical fluid extraction was the best method for the extraction of stigmasterol as well as quercetin and avicularin. Comparing the extraction conditions based on the levels of the parameters (Table 2) with the in vitro results (Table 4) and the enrichment analysis (Table 5) it can be deduced clearly that extraction parameters play vital role in the in vitro activity as well as enrichment of antidiabetic compounds. Higher levels of temperature, pressure, cosolvent, and CO2 flow rate positively affects the amount of antioxidants and antidiabetic compounds such as stigmasterol, quercetin, and avicularin extraction. Subsequently the free radical scavenging potency and diabetic enzyme α-glucosidase inhibitory efficacy relies on the amount of the stated compounds present as depicted by the in vitro results. Among these SFE run no 2, 5, 12, and CO2-Soxhlet B have been seen to consistently give high inhibitory effect and high enrichment and the conditions respective to the runs are shown here: For both the methods and among these experimental runs by the results of enriched extracts and antidiabetic assay the run order could be regarded as: SFE 12 > SFE 2 > SFE 5 > CO2-Soxhlet b Hence it can be concluded that supercritical fluid extraction is the better method for the enrichment of M. pudica for the compounds stigmasterol, quercetin, and avicularin as compared to CO2-Soxhlet. SFE and CO2-Soxhlet were both green technology and could be tweaked to obtain the desired result without any harmful or hazardous organic solvent trace or minute presence. Conclusion The study aimed at finding the best extract enriched with the chosen (pre-isolated) antidiabetic compounds stigmasterol, quercetin, and avicularin using supercritical extraction (SFE) and subcritical CO2 Soxhlet (CO2-Soxhlet) at various extraction conditions. Temperature, pressure, co-solvent percentage and CO2 flow rate were tested for SFE and modifier ratio (ethanol: sample) for CO2-Soxhlet have been tested at three levels. High performance liquid chromatography and LCMS has been run to ascertain the degree of enrichment of the pre-isolated antidiabetic compounds of stigmasterol, quercetin, and avicularin. In vitro DPPH, TFC, and TPC along with α-glucosidase enzyme inhibitory assay were performed for all 15 extracts (runs) SFE and CO2-Soxhlet. The results for the enzyme and the antioxidant assays vary a little in the LCMS and HPLC-UV analysis. SFE runs 2, 5, 7, 9, 12, and CO2Soxhlet B showed good antioxidative potential and enzyme inhibition. Overall, it can be concluded that SFE is the better technique, than CO2-Soxhlet, for enriching desired compounds to obtain organic solvent free greener medicinal extracts from an alternative source M. pudica hence reducing the stress on medicinal flora of the world. Declaration of interest SFE 2: 60°C temperature, 30 MPa pressure, 20% co-solvent and 5 ml min−1 CO2 ● SFE 5: 50°C temperature, 30 MPa pressure, 20% co-solvent and 3 ml min−1 CO2 ● SFE 12: 60°C temperature, 40 MPa pressure, 30% co-solvent and 5 ml min−1 CO2 ● CO2-Soxhlet B: sample: solvent 2: 1.5 direct spiking, 300 cycles of subcritical conditions of CO2 (28°C, 7 MPa) ● None to declare. Funding The work was funded by the exploratory research grant scheme, no. ERGS13-028-0061 of Ministry of Higher Education, Malaysia. The authors extend their appreciation to the International Scientific Partnership Program (ISPP) at King Saud University, Riyadh, Saudi Arabia, for funding this research study through ISPP# 0026. 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