2019 Volume 25 Issue 6 Pages 765-773
Stems of brown seaweed Undaria pinnatifida are not often used because they are too difficult to masticate. This study used conventional liquid solvent extraction methods and supercritical carbon dioxide (SC-CO2) fluid extraction to investigate the yields of various bioactive compounds in Undaria pinnatifida stems. The enzymatic (α-amylase and glucoamylase) inhibitor activities of the extracts were also examined. The SC-CO2 with ethanol extraction produced high amounts of phenolic and flavonoid compounds, fucoxanthin, epicatechin, and gallic acid. Also, the extracts obtained by SC-CO2 with ethanol exhibited the most potent inhibitor of α-amylase and glucoamylase among all the extracts studied, perhaps because of the high content of fucoxanthin. These results suggest that stems of Undaria pinnatifida could have value as a raw material to extract these bioactive substances.
Undaria pinnatifida (Wakame in Japanese), a type of brown seaweed broadly farmed in the Sea of Japan, China, and Korea, is a traditional and healthy food in many countries. Its structure is divisible into the blade (lamina), midrib, sporophyll, and stem (Fig. 1), which collectively contain various major bioactive compounds such as carotenoids, fucoxanthin, fatty acids, and phytosterols (Fung et al., 2013; Prabhasankar et al., 2009; Tiwari and Troy, 2015). Recently, the functionalities of Undaria pinnatifida have attracted much interest globally. In fact, several beneficial effects have been reported, including antioxidant, anti-cancerous, anti-hypertension and anti-hypercholesterolemia properties (Hayato-Maeda, 2008; Heo et al., 2008; Ikeda et al., 2003; Murata et al., 1999; Shiratori et al., 2005; Tiwari and Troy, 2015; Val et al., 2001).
Illustration of Undaria pinnatifida.
The main edible part of Undaria pinnatifida is the blade. Other parts are less used and are often discarded because of their harsh taste. The stem of Undaria pinnatifida is too tough to chew. Moreover, it cannot be swallowed. The amount of Undaria pinnatifida waste is greater than 200,000 tons/year in Japan, leading to mammoth environmental difficulties (Fujii and Korenaga, 2000; Obara et al., 2015). Furthermore, Undaria pinnatifida has been regarded as a dangerous invasive seaweed species worldwide (Casas et al., 2004; Curiel et al., 2002; James, 2016), posing an important threat to marine ecosystems because it can grow rapidly and multiply in various environments (James, 2016). Given this background, making full use of Undaria pinnatifida is an urgent issue to be resolved.
Because Undaria pinnatifida is rich in various bioactive compounds as described above, unused Undaria pinnatifida parts might have some value as raw materials for extraction of these substances. Several works to date have reported fucoxanthin and epicatechin extraction from Undaria pinnatifida, although most such studies did not specify which parts of Undaria pinnatifida were used. Zaharudin et al. (2018) extracted epicatechin and gallic acid from Undaria pinnatifida using water, methanol, and acetone. Roh et al. (2009) reported that usage of supercritical carbon dioxide (SC-CO2) with ethanol as an extraction solvent improved the fucoxanthin yield from Undaria pinnatifida. Nevertheless, little information has been forthcoming about the contents of these substances in Undaria pinnatifida stems. This study was conducted to investigate the yields of various bioactive compounds in Undaria pinnatifida stems: total phenol, total flavonoid, fucoxanthin, epicatechin, and gallic acid. Several extraction methods were used, such as conventional liquid solvent extraction and SC-CO2 fluid extraction. Furthermore, enzymatic (α-amylase and glucoamylase) inhibitor activities of the stem extracts were examined, some of which have been little investigated in earlier studies.
Materials Fresh Undaria pinnatifida (Wakame) stems were collected from Kesennuma, Miyagi Prefecture, Japan. High-purity carbon dioxide gas (99%) was obtained from Showa Denko Gas Products Co., Ltd. (Kawasaki, Japan). α-Amylase from Bacillus sp., glucoamylase from Rhozopus sp., and amylose (average molecular weight: ∼16,000) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Gallic acid, fucoxanthin, and epicatechin standards (≥98% purity) were purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan). Acarbose was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All the water used for experiments was Milli-Q water. All other reagents used for this study were analytical or high-performance liquid chromatography (HPLC) grade.
Preparation of lyophilized Undaria pinnatifida stems Fresh Undaria pinnatifida stems were washed with water until any visible attached salt and sea sand were removed. After smashing the stems into small pieces using a meat chopper (MS-12B; Nantsune Co., Ltd., Osaka, Japan), they were frozen at −80 °C, and were then lyophilized for 72 h in a freeze dryer composed of a vacuum oven (Eyela VOC-300D; Tokyo Rikakikai Co., Ltd., Tokyo, Japan), a cold trap (Eyela Cool Ace CA-1200; Tokyo Rikakikai Co., Ltd.), and a vacuum pump (GCD-051X; Ulvac, Inc., Chigasaki, Japan). The lyophilized sample was ground in an electrical grinder (ECG 62; Melitta Japan Ltd., Tokyo, Japan) and then sieved carefully on 180-µm stainless steel sieving mesh. We stored samples at -80 °C in a deep freezer before use.
Conventional solvent extraction Water, ethanol, methanol, acetone, and acetone–methanol (acetone: methanol = 7:3, v/v) were used as extraction solvents. The ratio of the solvent to the freeze-dried stem was set to 20:1 (v/w). They were mixed, then stirred in the dark for 24 h at 22 ± 1 °C on a magnetic stirrer at 800 rpm. Then the crude extract was filtered using filter paper (JIS P 3801, No. 2, Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The filtrate was kept in a 4 °C refrigerator. The mixing and filtration were conducted twice. Finally, the extraction filtrates were mixed. The solvent of the crude extract was evaporated at 40 °C in a rotary vacuum evaporator (N-1110; Tokyo Rikakikai Co., Ltd.) connected with a cooling water circulator (CCA-1111; Tokyo Rikakikai Co., Ltd.). The dried extracts were stored at -80 °C in a deep freezer and were used within 48 h for subsequent analyses.
Supercritical carbon dioxide extraction A supercritical fluid extraction system (Teledyne ISCO Inc., Lincoln, NE, USA) was used (Fig. 2). The freeze-dried stem sample (5 g) and ethanol (5 mL) were put into a 10-mL sample cartridge. Two cartridge filters (2 µm; Teledyne ISCO Inc.) were placed at the two sides of the extraction cartridge. Extraction was conducted using static and dynamic extraction. First, carbon dioxide was pumped using a syringe pump (260D; Teledyne ISCO Inc.) to maintain the pressure (27.58 MPa) and temperature (40 °C) in the extraction chamber for 60 min. During this static extraction, the chamber outlet valve was closed. No CO2 leaked out. After the valve was opened, CO2 was pumped into the extraction chamber at 1 mL/min with constant pressure (27.58 MPa) and temperature (40 °C) (Roh et al., 2009) and the eluted CO2 was passed through ethanol (about 4.5 mL) in a glass tube to trap crude extract. These steps are referred to as dynamic extraction. The dynamic extraction time was set as 10 min. After the extraction process, the dried crude extract was obtained by evaporation of ethanol at 40 °C using a rotary vacuum evaporator connected to a cooling water circulator. The extracts were stored at −80 °C in the freezer and were used within 48 h for subsequent analyses.
Schematic diagram of supercritical carbon dioxide extraction apparatus
Total phenolic content and total flavonoid content assay Total phenolic contents (TPC) in the dried crude extracts were estimated using the Folin–Ciocalteu colorimetric method, as described by Li et al. (2008) and by Wong et al. (2006) with slight modification. An aliquot (1 g) of the dried crude extract of stem was dissolved in 10 mL ethanol. Then 1 mL of the solution was mixed with 1 mL of Folin–Ciocalteu reagent (1:1 v/v, in water). After standing for 4 min, 800 µL of 7.5% (w/v) sodium carbonate solution was added to the mixture. Subsequently, the mixture was vortexed for 5 s and then stored at 22 ± 1 °C in a dark environment for 2 h. A blank was also prepared using 1 mL of water. Absorbance of the mixture was measured at 765 nm against the blank using a microplate reader (Versa Max; Molecular Devices Corp., San Jose, CA, USA). As a calibration standard, gallic acid solutions (0.1–2.0 mg/mL) were also prepared and processed in the same way to obtain the calibration standard curve. Results are expressed as mg of gallic acid equivalent in 1 g total dried crude extract.
Total flavonoid content (TFC) in the extract was measured using the method described by Karadeniz et al. (2005) and by Ozsoy et al. (2008) with some modification. Briefly, 1 g of dried crude extract of stem was dissolved in 10 mL of water. Next, 0.5 mL of the solution was mixed with 2.5 mL of water in a test tube, followed by addition of 150 µL of a 5% (w/v) sodium nitrite solution. After standing for 6 min, 300 µL of a 10% (w/v) aluminum chloride solution was added. After the mixture was allowed to stand for an additional 5 min, 1 mL of 1 M NaOH was added. Then, the mixture was filled up to 5 mL by adding 550 µL water. Immediately after mixing well, absorbance was measured at 510 nm. As a calibration standard, (+)-catechin solutions (0.01–1 mg/mL) were also prepared and processed in the same way to obtain the calibration standard curve. Results are expressed as mg of (+)-catechin equivalents in 1 g total dried crude extract.
Analysis of fucoxanthin, epicatechin and gallic acid The fucoxanthin, epicatechin, and gallic acid contents in the extracts were evaluated using HPLC. The HPLC conditions were similar to those used for earlier studies (Roh et al., 2009; Sivagnanam et al., 2015; Zaharudin et al., 2018). All HPLC analyses were conducted using an HPLC system (SCL-10A; Shimadzu Corp., Kyoto, Japan) equipped with a UV/VIS detector (SPD-20A; Shimadzu Corp.) and a hybrid silica-based ODS column (YMC-Triart C18, 5 × 4.6 mm co1umn size, S-5 µm particle size, 12 nm pore size; YMC Co., Ltd., Kyoto, Japan). As a mobile phase, methanol–acetonitrile (6:4, v/v) was used for analysis of fucoxanthin. Acetonitrile-water (6:4, v/v) was used for analyses of epicatechin and gallic acid. All HPLC analyses were conducted at ambient temperature as follows. 1 mg/mL solutions of dried crude extracts (solvent: mobile phase of HPLC) were filtered using a 0.45-µm membrane filter (Minisart CE 0120; Sartorius Stedim Biotech GmbH, Göttingen, Germany). Then 10 µL of the solutions were injected to the device and eluted at 1 mL/min by the mobile phase as described above. The detection wavelength was set as 450 nm for fucoxanthin and epicatechin, and as 270 nm for gallic acid. The standard curve prepared using an authentic standard was used for quantification of fucoxanthin, epicatechin, and gallic acid content in the injected solution of dried crude extract. The obtained value was converted to units of weight (mg) in 1 g dried crude extract.
α-Amylase and glucoamylase inhibition assay The α-amylase and glucoamylase inhibitory activities of the extracts were assayed based on methods described by Fuwa (1954) and Xiao et al. (2006) with modifications. First, four solutions were prepared: A, B, C, and iodine reagent. Solution A was prepared by dissolving the dried crude extract in DMSO. The concentrations were set to be 10 µg/mL, 50 µg/mL, and 100 µg/mL, respectively. Solution B was 0.2% amylose solution. It was prepared by dissolving amylose powder in water at 80 °C for 72 h with stirring. Solution C was an enzyme (α-amylase and glucoamylase) solution (1 mg/mL enzyme concentration, 0.1 M phosphate buffer solvent (pH 7.0)). The iodine reagent was prepared by mixing 5 mM I2 with 5 mM KI (1:1). Then the same aliquot (20 µL) of solution A, solution B, and solution C were added together into PCR tubes (0.2 mL volume) and incubated at 50 °C in a PCR thermal cycler (Dice mini; Takara Bio Inc., Kusatsu, Japan) to activate the enzymatic reaction. Since the extract concentrations in solution A were 10 µg/mL, 50 µg/mL, and 100 µg/mL, the final concentration of the extract during this enzymatic reaction process corresponded to 3.33, 16.67, and 33.33 µg/mL, respectively. After 45 min of incubation, 20 µL of 1 M HCl was added to stop the enzymatic reaction. Subsequently 100 µL of iodine reagent was added for color development. Finally, absorbance at 580 nm was measured using a microplate reader (Versa Max; Molecular Devices Corp.). The enzyme activity was obtained by the difference in absorbance compared with that of the sample without enzyme. The inhibitory activity was expressed as a percentage of the enzyme activity to that of the sample without the extract. To examine the practicality of the extract as an enzymatic inhibitor, the inhibitory activity of acarbose was also evaluated as the positive control. Acarbose inhibits α-amylase and glucoamylase reaction (Kim et al., 1999; Lordan et al., 2013; Yilmazer-Musa et al., 2012), and has been used in practice as a medicine to treat diabetes mellitus (Chiasson et al., 2002; Mertes, 2001). The final concentration of acarbose during the enzymatic reaction process was set to be the same as the extracts (3.33, 16.67, and 33.33 µg/mL).
Statistical Analysis The TPC, TFC, HPLC, and enzyme inhibition assays were applied in triplicate. The data are expressed as mean ± standard deviation. All mean values were analyzed using Tukey's multiple comparison. Significant difference was inferred for p < 0.05. Statistical analyses were applied using GraphPad Prism software (ver. 7.0) (GraphPad Software Inc., San Diego, CA, USA), SigmaPlot software (ver. 12.5) (Systat Software Inc., San Jose, CA, USA), or SPSS software (SPSS for Windows ver. 17.0; SPSS Inc., Chicago, IL, USA).
Extraction yield The highest and lowest yield were 1.27 ± 0.13% for acetone extraction and 1.06 ± 0.11% for water extraction, respectively (Fig. 3A). The yields of extraction by various solvents decreased in the following order: acetone > SC-CO2 with ethanol > methanol > ethanol > acetone-methanol > water; however, no statistically significant difference was observed among them.
(A) Extraction yield (%), (B) total phenolic content (TPC) (as gallic acid equivalents) (mg/g total dried extract) and (C) total flavonoid content (TFC) (as catechin equivalents) (mg/g total dried extract) of Undaria pinnatifida stem from various extracts. Error bars represent standard deviation with three replicates.
WE: water extract; EE: ethanol extract; ME: methanol extract; AE: acetone extract; AME: acetone mix methanol extract; SFE: SC-CO2 with ethanol extract. Significant differences are denoted by unshared letters between columns.
Total phenolic contents and total flavonoid contents The amounts of total phenolic contents and total flavonoid contents are presented in Figs. 3B and 3C. The highest phenolic content was found for methanol extraction (62.23 ± 0.59 mg/g dried crude extract). The highest flavonoid content was found in SC-CO2 with ethanol extraction (32.48 ± 0.31 mg/g dried crude extract).
Zaharudin et al. (2018) obtained extracts from Undaria pinnatifida using methanol as an extraction solvent. They reported that the total phenolic content in the extract was 30.8 mg/g dried extract. Schultz et al. (2014) reported that the total phenolic content in the ethanol extract from the same seaweed type was 25.12 mg/g dried extract. Strictly speaking, direct comparison of our results to these values is inadequate because the concentrations of polyphenols and flavonoids in seaweed reportedly depend on many variables such as habitat, season of harvesting, and environmental conditions including light, temperature, and salinity (Quirós et al., 2010). Moreover, the works described above include no description of which Undaria pinnatifida parts were used, which makes detailed comparison difficult. In spite of these uncertainties, our values resembled those obtained from earlier studies, suggesting that Undaria pinnatifida stems are useful as a polyphenol and flavonoid source.
The extraction method using SC-CO2 with ethanol achieved the highest TFC and similar levels of TPC among all of the extraction methods. Additionally, it only used small amounts of organic solvent (5 mL ethanol in the sample cartridge and around 4.5 mL ethanol for trapping the extracts in the eluted CO2). Results suggest that SC-CO2 with ethanol extraction is an efficient, excellent method.
The TPC and TFC by water solvent extraction were lower than those obtained using other organic solvents. For other seaweeds such as Fucus vesiculosus, Limnophila aromatica, and Gracilaria changii, some researchers have reported similar trends (Chan et al., 2015; Do et al., 2014; Wang et al., 2009), perhaps because greater amounts of water-insoluble phenolic compounds exist in these seaweeds, including Undaria pinnatifida.
Fucoxanthin, epicatechin, and gallic acid contents Fucoxanthin, epicatechin, and gallic acid contents are presented in Fig. 4. The fucoxanthin contents using SC-CO2 with ethanol demonstrated the highest value (178.33 ± 2.98 µg/g freeze dried stem). In the extract obtained using water as an extraction solvent, fucoxanthin was not detected. Sang et al. (2012) and Hwang et al. (2015) also reported the same result. Several studies have examined fucoxanthin extraction from Undaria pinnatifida. Similar to the discussion related to TPC and TFC above, direct comparison of these values with our results is inadequate because of different environmental conditions such as habit and salinity. In spite of these uncertainties, comparison between our values and theirs provides some insights into the improvement of extraction yield and into the usefulness of Undaria pinnatifida stems as a fucoxanthin source. Xiao et al. (2012) reported 726.67 µg per 1 gram dried Undaria pinnatifida using microwave-assisted ethanol extraction. Quitain et al. (2013) reported 145.28 µg per unit of freeze dried Undaria pinnatifida using SC-CO2 with ethanol extraction at 25 °C. Kanda et al. (2014) conducted extraction of fucoxanthin from Undaria pinnatifida using four methods: batch-type extraction with ethanol, semi-continuous flow-type extraction with liquefied dimethyl ether, SC-CO2 extraction, and SC-CO2 with continuous ethanol elution. Each of the best recoveries for these four methods was, respectively, 50, 390, 60.12, and 994.53 µg/g dried Undaria pinnatifida. Those studies did not clarify which part of Undaria pinnatifida was used. However, Kanda et al. (2014) presented a photograph of an Undaria pinnatifida sample in their paper. The photograph strongly suggested that the main part they used were leaves, the main edible part. Assuming that they used leaves, the results suggest that fucoxanthin contents in Undaria pinnatifida stems were of a similar level to those of leaves because the fucoxanthin yield by batch-type extraction with ethanol found in this study (57.21 ± 1.21 µg/g freeze dried stem) was comparable to that found in their study (60.12 µg/g dried Undaria pinnatifida). Our fucoxanthin contents extracted using SC-CO2 with ethanol (178.33 ± 2.98 µg/g freeze dried Undaria pinnatifida) were lower than their reported values (994.53 µg/g dried Undaria pinnatifida), obtained by SC-CO2 with continuous ethanol elution. This lower value might derive from different extraction conditions: CO2 flow rate, extraction time, extraction temperature, and ethanol supply method. They used a higher CO2 flow rate (3 mL/min), a longer extraction time (5 h), a higher extraction temperature (50 °C), and a continuous ethanol supply. Generally, these can be expected to act to increase the fucoxanthin yield. In other words, one expects that improving the fucoxanthin yield from Undaria pinnatifida stems can be achieved by changing extraction conditions as described by Kanda et al. (2014).
(A) Fucoxanthin, (B) epicatechin and (C) gallic acid content (µg/g freeze dried Undaria pinnatifida stem) of Undaria pinnatifida stem from various extracts. Error bars represent standard deviation with three replicates.
WE: water extract; EE: ethanol extract; ME: methanol extract; AE: acetone extract; AME: acetone mix methanol extract; SFE: SC-CO2 with ethanol extract. Significant differences are denoted by unshared letters between columns.
As for epicatechin contents, the highest value was obtained when using methanol extract (352.35 ± 3.17 µg/g freeze dried Undaria pinnatifida), followed by SC-CO2 with ethanol extract (319.36 ± 2.14 µg/g freeze dried Undaria pinnatifida) and acetone–methanol extract (282.51 ± 2.31 µg/g freeze dried Undaria pinnatifida). The water extract contained the lowest epicatechin content (102.50 ± 1.78 µg/g freeze dried Undaria pinnatifida) because epicatechin is more soluble in organic solvents than in water (Cayman Chemical, 2019). Results showing that SC-CO2 with ethanol gave high yields of fucoxanthin (highest) and epicatechin (2nd) demonstrate SC-CO2 with ethanol to be an excellent solvent for extracting these bioactive compounds.
All the solvent extracts contained lower amounts of gallic acid than of fucoxanthin or epicatechin (Fig. 4). These results suggested that fucoxanthin and epicatechin were the two main bioactive compounds in Undaria pinnatifida stems.
Fucoxanthin, the main carotenoid in edible brown seaweed, has strong antioxidant properties that show significant anticancer, antihypertensive, anti-obesity, and anti-inflammatory effects (Das et al., 2005; Hosokawa et al., 2004; Koyanagi et al., 2003; Maeda et al., 2007; Peng et al., 2011; Sugawara et al., 2002). Because Undaria pinnatifida has a higher amount of fucoxanthin than other seaweeds (Gupta and Abu-Ghannam, 2011; Kanda et al., 2014; Kazuki et al., 2008), it has recently attracted much attention by researchers (Kumar et al., 2008; Miyashita and Hosokawa, 2007; Prabhasankar et al., 2009). Few studies have investigated the fucoxanthin contents of Undaria pinnatifida stems. Our results suggest using Undaria pinnatifida stems as a source of fucoxanthin. Furthermore, the combination of SC-CO2 with ethanol as an extraction solvent is expected to contribute to greater amounts of fucoxanthin production from stems of Undaria pinnatifida with less use of organic solvents.
α-Amylase and glucoamylase inhibition Enzyme activity data obtained in the presence of Undaria pinnatifida stem extracts with different concentrations are presented in Figs. 5A (α-amylase) and 5B (glucoamylase). They are expressed as percentages against uninhibited enzyme activity.
(A) α-Amylase and (B) glucoamylase activities in the presence of Undaria pinnatifida stem extract and acarbose. Error bars represent standard deviation with three replicates (ap < 0.01, bp < 0.05 relative to control, cp < 0.01, dp < 0.05 relative to acarbose).
WE: water extract; EE: ethanol extract; ME: methanol extract; AE: acetone extract; AME: acetone mix methanol extract; SFE: SC-CO2 with ethanol extract.
As for α-amylase activity, the extract obtained using SC-CO2 with ethanol showed the lowest enzymatic activity among all extracts. At the highest extract concentration (33.33 µg/mL), less than 60% activity was obtained, corresponding to greater than 40% inhibition. The extract obtained using SC-CO2 with ethanol also showed the lowest glucoamylase activity. At 33.33 µg/mL, less than 70% activity, which was equivalent to more than 30% inhibition, was observed. The ethanol, methanol, acetone, and acetone–methanol extracts exhibited similar inhibition ratios for both enzymes. The water extract showed no clear inhibition, even at the highest concentration. Enzyme activity data in the presence of acarbose (positive control) are also shown in Fig. 5. Acarbose exhibited more inhibitory activity than the stem extracts, but the inhibition ratio of the extract obtained using SC-CO2 with ethanol was similar at the same concentration. These data suggest that the Undaria pinnatifida stem extract obtained using SC-CO2 with ethanol might be a practically potent and effective inhibitor of α-amylase and glucoamylase as acarbose. Extraction by SC-CO2 with ethanol exhibited the strongest inhibition among all extracts. Furthermore, the fucoxanthin content in the extract obtained by SC-CO2 with ethanol was the highest. Fucoxanthin was not detected in the water extract and no enzymatic inhibition was readily apparent. These results suggest contributions of fucoxanthin to the inhibition of α-amylase and glucoamylase in the presence of stem extracts. Zaharudin et al. (2019) identified the α-glucosidase inhibitory compound found in Undaria pinnatifida as fucoxanthin. That finding also supports our inference, although that earlier report did not describe which parts of Undaria pinnatifida were used. From Fig. 3A and Fig. 4A, the fucoxanthin content in the dried extract obtained by SC-CO2 with ethanol is estimated to be 1.47% (w/w). It seems unreasonable that this small content of fucoxanthin in the extract showed similar enzymatic inhibitory activity to that of acarbose. However, Zaharudin et al. (2019) also reported that fucoxanthin exhibited more than 10 times higher α-glucosidase inhibitory activity (IC50 value: 0.047 ± 0.001 mg/mL) than acarbose (IC50 value: 0.6 ± 0.01 mg/mL). That means that the small amount of fucoxanthin in the extract can show comparable inhibitory activity as acarbose in principle. Actually, the SC-CO2 with ethanol extract is a complex mixture of various substances. Therefore, future work must be done to ascertain which compounds in the extract obtained by SC-CO2 with ethanol contribute to α-amylase and glucoamylase inhibition.
This study showed SC-CO2 with ethanol extraction to be useful for extracting high contents of phenolic and flavonoid compounds, fucoxanthin, epicatechin, and gallic acid from Undaria pinnatifida stems: inedible and usually discarded parts of Undaria pinnatifida. Extraction by SC-CO2 with ethanol, perhaps because it has higher fucoxanthin content, produced a more potent inhibitor of α-amylase and glucoamylase than extraction by any other solvent. Because Undaria pinnatifida stems are inexpensive and ubiquitous waste products, they present the potential for use in food production, particularly to produce functional foods with bioactive compounds that are effective at reducing hyperglycemia.
