Pressurized low polarity water extraction of saponins from cow cockle seed

Journal of Food Engineering 80 (2007) 619–630 www.elsevier.com/locate/jfoodeng

Pressurized low polarity water extraction of saponins from cow cockle seed ¨ zlem Gu¨c¸lu¨-U ¨ stu¨ndag˘ a, John Balsevich b, G. Mazza O a

a,*

National Bioproducts and Bioprocesses Research Program, Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, 4200 Hwy 97 Summerland, BC, Canada V0H 1Z0 b National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9 Received 1 March 2006; received in revised form 20 June 2006; accepted 21 June 2006 Available online 6 September 2006

Abstract Pressurized low polarity water (PLPW) extraction of cow cockle seed was carried out to determine the effect of extraction conditions (temperature (125–175 C), time (15–180 min)), and sample pre-treatment on saponin yield and composition. Accelerated solvent extractions (ASE) and ultrasonic extractions (USE) using water, methanol and ethanol (50%, 80%, 100%) were also carried out to determine the effect of extraction solvent and method on saponin recovery. A higher saponin yield was obtained by ASE compared to USE using pure and aqueous ethanol and methanol. The highest saponin yield, which was obtained by ASE using 80% ethanol, was used to calculate saponin recoveries. The saponin yield of PLPW extracts increased with extraction temperature and time. While only 33.2 wt% of total saponins was extracted from ground seeds at 125 C in 3 h, 60.2 wt% was recovered in the first 15 min at 175 C. Total extraction (1 h) of whole seeds yielded more saponins than ground seeds at 125–160 C. Saponin concentration of the extracts was affected by the extraction solvent and method, sample pre-treatment and to a lesser extent by the time and temperature of PLPW extraction. The highest saponin concentration of PLPW extract was obtained at 125 C using whole seeds (12%). The saponin composition of water extracts differed from that of aqueous ethanol and methanol extracts. Crown Copyright  2006 Published by Elsevier Ltd. All rights reserved. Keywords: Vaccaria segetalis; Saponaria vaccaria; Cow cockle seed; Saponins; Cyclopeptides; Subcritical water extraction; Ultrasonic solvent extraction; Accelerated solvent extraction; Bioactive components; Nutraceuticals

1. Introduction Saponins are glycosides widespread in the plant kingdom and present in a few marine organisms such as starfish and sea cucumber (Hostettmann & Marston, 1995). They are categorized based on their structure containing a steroid or terpenoid aglycone attached to one or more sugar chains. Their structural diversity results in a number of physicochemical and biological properties with various ¨ stu¨ndag˘ & Mazza, in industrial applications (Gu¨c¸lu¨-U press). Mounting evidence on the biological activity of saponins such as cholesterol-lowering and anticancer proper-

*

Corresponding author. Tel.: +1 250 494 6376; fax: +1 250 494 0755. E-mail address: [email protected] (G. Mazza).

ties (Kerwin, 2004; Oakenfull & Sidhu, 1990) has prompted research into investigation of new sources and processing methods for their commercial production (Dobbins, 2002; Muir, Paton, Ballantyne, & Aubin, 2002). Cow cockle seed (Vaccaria segetalis Garcke, Saponaria Vaccaria L., Vaccaria pyramidata) is an annual herb widespread in grain fields of the North Western United States and in the prairie provinces of Canada, Asia and Europe (Bailey, 1976; Mazza, Biliaderis, Przybylski, & Oomah, 1992). Although considered a weed in North America, cow cockle seed, known as Wang-Bu-Liu-Xing, has a prominent role in the traditional Chinese medicine (Sang, Lao, Chen, Uzawa, & Fujimoto, 2003). Its main uses include the promotion of diuresis and milk secretion, activation of blood circulation and the relief of carbuncle (Sang et al., 2003). Cow cockle seeds contain over 55%

0260-8774/$ - see front matter Crown Copyright  2006 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2006.06.024

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starch, 14% protein, 2–3% oil and around 2% saponins (Mazza et al., 1992). The bioactive components of cow cockle seeds include alkaloids, cyclopeptides, phenolic acid, flavonoids and steroids, in addition to saponins (Sang et al., 2003). Processing and properties of cow cockle seed starch, which has a small granular size, have been investigated (Brelsford & Goering, 1971), however, research on the bioactive constituents of cow cockle seed has largely been limited to isolation and identification of individual components (Jia, Koike, Kudo, Li, & Nikaido, 1998; Sang et al., 2003). Saponins isolated from cow cockle seed are listed in Fig. 1 and Table 1. Processing of cow cockle seed for the extraction/concentration of saponins and other bioactive components however has not been investigated. Pressurized low polarity water (PLPW, also known as subcritical, hot, and superheated water) extraction involves the use of pressure to maintain water in the liquid state at temperatures above its normal boiling point. The higher temperatures thus achieved improves the mass transfer properties (faster diffusion rates) and decreases the polarity

of water modifying its solvent power. For example, the dielectric constant of liquid water decreases with increasing temperature from 80 at 25 C to 27 at 250 C, which falls between those of methanol (e = 33) and ethanol (e = 24) (Hawthorne, Yang, & Miller, 1994). Increasing the temperature can also disrupt the solute–matrix interactions, and reduce the viscosity and surface tension of water improving the contact between the solvent and the solute (Richter et al., 1996). The high pressures used can further enhance the extraction of analytes trapped in matrix pores (Richter et al., 1996). Pressurized liquid extraction (PLE, Dionex trade name accelerated solvent extraction (ASE)), which involves the use of pressurized organic solvents, offers similar advantages. The use of pressurized solvents at elevated temperatures can thus improve the efficiency of traditional processes resulting in shorter extraction time and lower solvent consumption. The dependence of solvent power/selectivity on temperature can be exploited to modify extract composition and for fractionation purposes.

Fig. 1. Structure of cow cockle seed saponins (a) 1. Vaccaroside A, 2. Vaccaroside B, 3. Vaccaroside C, 4. Vaccaroside D; (b) 1. Vaccaroside E, 2. Vaccaroside F, 3. Vaccaroside G, 4. Vaccaroside H (Jia et al., 1998; Koike et al., 1998).

¨ . Gu¨c¸lu¨-U ¨ stu¨ndag˘ et al. / Journal of Food Engineering 80 (2007) 619–630 O Table 1 Saponins isolated from cow cockle seed Saponins

Formula

Aglycone

Ref.

Segetosides Segetoside B Segetoside C Segetoside D Segetoside E Segetoside F Segetoside G Segetoside H Segetoside I Segetoside K

C69H106O33 C56H88O26 C69H106O34 C72H112O34 C67H104O32 C70H110O32 C68H104O33 C68H104O34 C54H86O26

Sang Sang Sang Sang Sang Sang Sang Sang Sang

Segetoside L

C60H98O28

Gypsogenin Gypsogenic acid Quillaic acid Quillaic acid Gypsogenin Gypsogenin Gypsogenin Quillaic acid Olean-12-ene-23a, 28b-dioic acid 3b, 16a-dihydroxy Oleanolic acid

Vaccarosides Vaccaroside A Vaccaroside B Vaccaroside C Vaccaroside D

C54H86O25 C60H94O29 C54H86O25 C54H86O25

Koike Koike Koike Koike

Vaccaroside E Vaccaroside F Vaccaroside G Vaccaroside H Vaccaroid A Vaccaroid B

C66H102O33 C65H102O33 C66H102O32 C65H102O32 C54H86O25 C60H94O29

Gypsogenic acid Gypsogenic acid Gypsogenic acid 3,4-Seco derivative of gypsogenic acid Quillaic acid Segetalic acid Gypsogenin Vaccaric acid Gypsogenic acid Gypsogenic acid

et et et et et et et et et

al. al. al. al. al. al. al. al. al.

(2002) (1999) (1998) (1998) (2000b) (2000a) (2000a) (2000a) (2000c)

Xia et al. (2004) et et et et

al. al. al. al.

(1998) (1998) (1998) (1998)

Jia et al. (1998) Jia et al. (1998) Jia et al. (1998) Jia et al. (1998) Morita et al. (1997) Yun et al. (1997)

Pressurized low polarity water extraction and PLE have been widely investigated for the analysis of environmental samples (Hawthorne et al., 1994), and plant chemicals such as saponins (Benthin, Danz, & Hamburger, 1999; Lee, Koh, Ong, & Woo, 2002; Ong, 2002; Ong & Len, 2003), paclitaxel (taxol) (Kawamura, Kikuchi, Ohira, & Yatagai, 1999), anthocyanins (Ju & Howard, 2003), polyphenols (Alonso-Salces et al., 2001), berberine (Ong, Woo, & Yong, 2000), aristolochic acid (Ong et al., 2000), dianthrons (Benthin et al., 1999), silybin (Benthin et al., 1999), curcumin (Benthin et al., 1999), and thymol (Benthin et al., 1999). The recognition of the efficiency of PLPW extraction coupled with the additional advantages offered by the use of water as an alternative to organic solvents has prompted researchers to investigate the potential of PLPW for industrial-scale extraction of natural products. PLPW extraction of essential oils (Ayala & Luque de Castro, 2001; Basile, Jime´nez-Carmona, & Clifford, 1998) and other phytochemicals such as antioxidants from rosemary (Ibanˇez et al., 2003), boldo leaves (del Valle, Rogalinski, Zetzl, & Brunner, 2005), and yam (Chen, Tu, Wu, Jong, & Chang, 2004), anthocyanins from berries (King, Grabiel, & Wightman, 2003), and dried grape skin (Ju & Howard, 2005), terpene trilactones from Gingko leaves (Wai & Lang, 2003), isoflavones from defatted soybean flakes (Li-Hsun, YaChuan, & Chieh-Ming, 2004), and lignans from flaxseed (Cacace & Mazza, 2006) has been reported. The use of pressurized solvents in saponin processing however has been limited to analytical applications (Benthin et al., 1999; Choi, Chan, Leung, & Huie, 2003; Lee et al., 2002; Ong, 2002; Ong & Len, 2003).

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Therefore, the objectives of this study were: (1) to investigate the potential of PLPW for the extraction of saponins from cow cockle seed, and (2) to determine the effects of PLPW extraction parameters (extraction time and temperature) and sample pre-treatment on saponin recovery, concentration, and composition. 2. Materials and methods 2.1. Materials Milli-Q grade water, HPLC grade methanol (Caledon Laboratories Ltd., Georgetown, Ont., Canada) and anhydrous ethanol (Commercial Alcohols Inc., Brampton, Ont.) were used as extraction solvents. Milli-Q grade water was purged with nitrogen prior to PLPW extraction to remove any dissolved oxygen. HPLC grade methanol and acetonitrile (EM Science, Gibbstown, NJ), Milli-Q water, formic acid (88%, Certified ACS, Fisher Scientific, Springfield, MA) and C18t Sep-Pak cartridges (Waters Limited, Mississauga, Ont., Canada) were used for HPLC analysis. Glycyrrhizic acid ammonium salt (75% purity) was obtained from Sigma–Aldrich Canada Ltd. (Oakville, Ont., Canada). Cow cockle seeds, grown near Saskatoon, SK, Canada, were used as whole or ground (1 mm) using an IKA MF 10 grinder (Rose Scientific Ltd., Edmonton, AB, Canada). 2.2. Extraction procedures 2.2.1. Pressurized low polarity water extraction A schematic diagram of the PLPW extractor, which was built in-house, is given in Fig. 2. Extraction takes place in a stainless steel column (1.27 cm OD, 0.9525 cm ID, 20 cm) placed inside an Isotemp Programmable Oven (Fisher Scientific Co., Nepean, Ont., Canada) for temperature control. The column is connected to processing lines

BPR

C

PG

P

O WP H

EC

T Fig. 2. Diagram of pressurized low polarity water extractor, WP, water pump; PG, pressure gauge; H, heating coil; EC, extraction chamber; O, oven, C, cooling coil; BPR, back pressure regulator; P, product collector; T, thermometer.

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through column end fittings which contain a 100 lm frit to prevent clogging of the lines (Chromatographic Specialties, Inc, Brockville, Ont., Canada). Extraction solvent (Milli-Q water) is introduced into the system using a Waters 515 HPLC pump (Waters Limited, Mississauga, Ont., Canada) and passes through a 4 m heating coil before entering the column from the bottom. Wash solvent can be introduced into the system via tubings connected to tees at the outlet and inlet of the column. The pressure of the system is kept constant at 5.17 MPa (750 psi) using a back pressure regulator (Scientific Products and Equipment, Concord, Ont., Canada) placed at the end of the system, and a pressure gauge is used to monitor the pressure. Two thermocouples (Omega Canada, Laval, QC, Canada) placed in the tees before and after the column and connected to a datalogger (Omega Canada, Laval, QC, Canada) are used to monitor the temperature. Stainless steel tubing (0.125 cm OD, Sigma–Aldrich Canada Ltd./Supelco, Oakville, Ont., Canada) is used to introduce the extraction and wash solvents into the system and for the outlet line. One of the main processing challenges associated with PLPW extraction of cow cockle seeds arises from their composition, particularly their high starch content. The swelling of the seeds in the presence of water at high temperature may lead to overpressure and eventual cessation of flow. The degree of swelling is determined by the amount of seeds and extraction temperature. Hence, preliminary trials were carried out to determine the maximum amount of seeds that could be introduced into the system without blockage at the investigated temperature range. Ground cow cockle seeds (2 g) mixed with glass beads (15 g, 3 mm); and whole seeds (2 g) without any beads were thus used in the experiments to ensure efficient extractions. After cow cockle seeds were loaded into the column, glass wool was placed at the column outlet and inlet to prevent carry-over of solid particles into processing lines. The loaded column was then connected to the system and the system was pressurized by pumping water at 2 mL/min. The temperature of the oven was then set to the desired value. When the oven temperature reached the set temperature, solvent flow, thus the extraction was started. The first 10 mL (equivalent to the dead volume after the column) collected was discarded. Fractions were collected in graduated cylinders throughout the extraction as a function of time. The extraction was stopped by stopping the solvent flow and the system was washed using methanol (30 mL) and milli-Q water. Fraction yields were determined gravimetrically after the extraction solvent (water) was removed by freeze-drying. A subsample of each extract (10 mg) was redissolved in milli-Q H2O (2 mL) and prepared for HPLC analysis as described in the next section. Pressurized low polarity water extractions of cow cockle seed were carried out at least in duplicates to determine the effect of temperature, sample pre-treatment and extraction time on the total extraction yield, saponin yield/recovery

and the composition of the extracted saponin mixture (Table 2). Fractional extraction of ground cow cockle seeds (3 h, 1 fraction/15 min) was carried out to determine the extraction curve for saponins as affected by extraction temperature (125–175 C). Total extraction parameters (1 h, 125–160 C) were chosen based on the results of the fractional extractions. 2.2.2. Pressurized liquid extraction (accelerated solvent extraction, ASE) Accelerated solvent extraction of cow cockle seeds was carried out in duplicates using a Dionex ASE 100 extraction system (Dionex Co., Sunnyvale, CA) according to Benthin et al. (1999) with modifications. The extraction cell (14 mL), which contained a stainless steel frit and a cellulose filter at the outlet, was loaded with ground cow cockle seeds (2 g) and sand (2 g). The parameters of ASE method, which were set and maintained electronically, were: 150 C; 10.34 MPa (1500 psi); two 6-min static cycles with 60% flush volume and 100 s purge time (extraction time: 22 min, solvent volume: 89–193 mL). Investigated solvents included methanol, ethanol and their aqueous solutions (80% methanol, and 50%, 80% ethanol). Extractions using water and 50% methanol could not be completed due to the blockage of the extraction cell, which resulted in the cessation of flow. After the extractions, the solvents were removed from the extracts using a rotary evaporator at 40 C. The extracts were left under house-vacuum overnight to remove any solvent residues. Extract yields were then determined gravimetrically. The extracts were redissolved in milli-Q H2O (to yield a concentration of 5 mg/ mL) and prepared for HPLC analysis as described in the next section. 2.2.3. Ultrasonic solvent extraction Ultrasonic solvent extraction (USE) of cow cockle seeds was carried out in duplicates using water, methanol (50%, 80%, 100%) and ethanol (50%, 80%, and 100%) according to Li, Mazza, Cottrell, and Gao (1996) with modifications.

Table 2 Parameters of pressurized low polarity water extraction of cow cockle seed Temperature (C)

Extraction time (min)

Number of fractions

Sample pretreatment

Fractional extractions 125 180 150 180 175 180

1 fraction/15 min 1 fraction/15 min 1 fraction/15 min

Ground Ground Ground

Total extractions 125 125 140 140 150 150 160 160

1 1 1 1 1 1 1 1

Ground Whole Ground Whole Ground Whole Ground Whole

60 60 60 60 60 60 60 60

fraction/60 min fraction/60 min fraction/60 min fraction/60 min fraction/60 min fraction/60 min fraction/60 min fraction/60 min

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Ground cow cockle seeds (200 mg) were extracted using 10 mL solvent for 1 h in an ultrasonic bath (Branson 3200 ultrasonic cleaner, Branson Ultrasonics Corporation, Danbury, CT). After centrifugation, the supernate was dried under nitrogen, weighed to determine the extraction yield and redissolved in milli-Q H2O (to yield a concentration of 5 mg/mL). The extracts were then prepared for HPLC analysis as described in the next section. 2.3. Saponin analysis by high performance liquid chromatography (HPLC) 2.3.1. Sample preparation A solid phase extraction (SPE) procedure was employed for sample purification prior to HPLC analysis. Samples (10 mg in 2 mL water) were filtered using a 0.45 mm PVDF filter (Chromatographic Specialties, Brockville, Ont., Canada) onto SPE cartridges conditioned with 5 mL methanol followed by 5 mL water. Carbohydrates were removed by washing the cartridges with 5 mL water. Saponins were then eluted using 80% methanol. Solvent was removed under nitrogen and the samples were redissolved in 1 mL 20% acetonitrile and filtered using a 0.45 lm hydrophilic Durapore membrane filter (Millipore Corp., Bedford MA) prior to HPLC analysis.

mAU 300

623

2.3.2. Qualitative and quantitative determination An HPLC method developed by Li et al. (1996) for the analysis of ginsenosides has been modified for the analysis of cow cockle seed saponins. Saponin analyses of extracts were carried out using an HPLC (Agilent Technologies, Inc., Mississauga, Ont., Canada) with a Zorbax SB-C18 column (2.1 mm, 150 mm, 5 lm) and a UV–Vis diodearray detector. The solvent systems used were acetonitrile:water:formic acid (88%) (100:900:1) (solvent A) and acetonitrile: formic acid (88%) (1000:1) (solvent B). The parameters of the HPLC method used for saponin analysis were: solvent gradient (%B): 20 at 0 min, 20 at 5 min, 40 at 25 min, 100 at 30 min, 100 at 35 min, 20 at 37 min; run time = 45 min, solvent flow rate = 0.25 mL/min, column temperature = 30 C, injection volume = 20 lL. Peaks were detected at 203 nm (bandwidth = 8). Spectral analysis of HPLC peaks together with information provided by LC–MS analysis of an aqueous methanol cow cockle seed extract by Balsevich, Bishop, Hickie, Dunlop, and Ramirez-Erosa (2005) was used for the qualitative determination of saponins in cow cockle seed extracts. While the spectra of earlier peaks in the HPLC chromatograms were consistent with cyclopeptides (peaks 1 and 2 in Fig. 3), the majority (P95%) of the peaks that appear after peak 2 had spectra consistent with saponins.

DAD1A, Sig=203, 8 Ref=595, 100 (P:\HPLC\MAR15010.D)

Group 2

Group 4

250

Peak 1

Peak 2

200

Group 5

Group 1 150

Group 3

100

50

0 0

b

mAU 80

5

10

15

20

25

min

DAD1A, Sig=203, 8 Ref=595,100 (P:\HPLC\APR14035.D)

Group 2 Peak 1

70

Group 4

Peak 2 Group 1

60

Group 3

50

Group 5

40 30 20 10 0 0

5

10

15

20

25

min

Fig. 3. HPLC chromatograms of ground cow cockle seed extracts obtained using (a) accelerated solvent extraction with 50% ethanol, and (b) pressurized low polarity water extraction at 125 C for 15 min.

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These peaks were grouped into five according to their retention time (Fig. 3) and were used to monitor the effect of extraction procedure (extraction solvent and operating conditions) on the yield and composition of the fractions. Glycyrrhizic acid ammonium salt (75% purity) was used as an external standard for the quantitative determination of saponins. 2.4. Distribution of saponins in the cow cockle seed Cow cockle seeds are made up of three structural components: seed coat, endosperm, and embryo. The embryo occurs around the ‘equator’ of the seed, overlaying the starchy endosperm (Fig. 4). Cow cockle seeds were cracked in a mortar by gently pressing with the pestle using a slow rotational motion to release the embryo. Fifty randomly chosen intact embryos were weighed (=57.2 mg) and subsequently ground with a glass stirring rod and extracted (3 · 3 ml) with 70% methanol over a 24 h period. The extract was concentrated to dryness in vacuo to afford a white solid residue (13.6 mg) which contained no significant lipophilic (ethyl acetate soluble) material. The residue was dissolved in water (1 ml) and applied to a C18 Sep-Pak column (100 mg). The column was eluted with water (3 ml), 20% methanol (3 ml), 40% methanol (3 ml), and methanol (5 ml). Evaporation of the pure methanol fraction afforded saponins largely free of other products as a white solid (7.3 mg, 12.8% of embryo by wt). The saponin content of 50 randomly chosen seeds (373 mg) was also determined using the same procedure to be 10.1 mg (2.7% of seed by wt). These results indicate that 72.3% of the total saponins of cow cockle seed are located in the embryo, whereas the seed coat and endosperm saponins account for 27.7% of total saponins.

3. Results and discussion Fractional PLPW extraction of cow cockle seeds at 125– 175 C was carried out for 3 h to determine the effect of extraction time and temperature on the extraction behaviour of saponins. Based on the findings of the fractional extractions, total extractions were then carried out (at 125–160 C for 1 h) to determine the effect of sample pretreatment and temperature on saponin recovery and composition. Pressurized liquid extractions (accelerated solvent extractions, ASE) using pure and aqueous ethanol and methanol, and ultrasonic extractions (USE) using water, aqueous ethanol and methanol solvents were also carried out to determine the effect of extraction solvent and method on saponin yield/recovery and composition. 3.1. Saponin yield The highest saponin yield was obtained by 80% ethanol extraction (ASE) corresponding to 3.3% of feed material (Table 3). This value was used in the calculations of the recovery of the saponins (Figs. 5–7). The highest USE yield was obtained using 50% ethanol. The lowest saponin yield was obtained using pure ethanol (0.1 and 0.9 g/100 g seed for USE and ASE, respectively). The findings on the effect of extraction solvent on saponin yield are in agreement with the solubility/extraction trends reported for other saponins in the literature. In the aqueous ethanol concentration range of 30–100% the highest solubility of soyasaponin Bb was obtained in 60% ethanol (Shimoyamada, Osugi, Shiraiwa, Okubo, & Watanabe, 1993). Similarly, recovery of quinoa saponins obtained using aqueous ethanol and methanol was higher than that of water and pure alcohol solvents (Muir et al., 2002). Maximum ginsenoside yield from ginseng was obtained by 60–75% ethanol in the range of 30–90% ethanol (Kwon, Lee, Be´langer, & Pare´, 2003), while the highest saponin recovery from Glinus lotoides seeds was achieved using 60% methanol (in 0–100% methanol range) (Endale, Schmidt, & Gebre-Mariam, 2004). A higher amount of saponins was recovered by ASE in 22 min (solvent to feed ratio: 0.04–0.1) compared to 1 h Table 3 Saponin yields obtained by ultrasonic solvent extraction (USE: solvent/ feed ratio: 0.05, 1 h) and accelerated solvent extraction (ASE: 150 C, solvent to feed ratio: 0.04–0.1, 22 min) of ground cow cockle seed Solvent

Saponin yield (wt%) g/100 g dried extract

Fig. 4. Structural components of cow cockle seed: embryo, starchy endosperm and seed coat.

Methanol-100% Methanol-80% Methanol-50% Ethanol-100% Ethanol-80% Ethanol-50% Water

g/100 g seed

USE

ASE

USE

ASE

18.8 ± 4.6 28.4 ± 8.0 33.8 ± 0.1 2.1 ± 0.3 37.4 ± 1.6 35.4 ± 3.0 11.2 ± 0.3

28.1 ± 3.8 40.2 ± 0.3 – 15.6 ± 2.8 35.2 ± 0.0 28.8 ± 0.3 –

1.2 ± 0.2 1.9 ± 0.5 2.8 ± 0.1 0.1 ± 0.0 2.5 ± 0.2 2.9 ± 0.3 2.1 ± 0.1

2.3 ± 0.3 3.0 ± 0.5 – 0.9 ± 0.1 3.3 ± 0.0 3.2 ± 0.0 –

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125 °C 140 °C 150 °C 160 °C USE ASE

100

Saponin recovery (wt %)

90 80 70 60 50 40 30 20 10 0 MeOH 100%

MeOH 80%

MeOH 50%

EtOH 100%

EtOH 80%

EtOH 50%

Water

Extraction solvent

Fig. 5. Saponin recovery obtained from ground cow cockle seeds by ultrasonic solvent extraction (USE, 1 h), accelerated solvent extraction (ASE, at 150 C, 22 min), and pressurized low polarity water extraction (PLPWE, at 125–160 C, 1 h).

100 125 °C 150 °C 175 °C

90

Saponin recovery (wt %)

80 70 60 50 40 30 20 10 0 0

30

60

90

120

150

180

Extraction time (min)

Fig. 6. Saponin recovery obtained by pressurized low polarity water extraction of ground cow cockle seeds at 125, 150 and 175 C.

90 80

ground whole

Saponin recovery (%)

70 60 50 40 30 20 10 0 120

130

140

150

160

170

Temperature (°C)

Fig. 7. Saponin recovery obtained by 1 h pressurized low polarity water extraction of ground and whole cow cockle seeds at 125–160 C.

625

USE using pure and aqueous alcohol solvents (50–100% ethanol; 80%, 100% methanol; solvent/feed ratio: 0.05) (Table 3 and Fig. 5). Glycyrrhizic acid (main saponin of licorice) recoveries of pressurized methanol (Ong, 2002) (at 100 C, 20 min, 20–25 mL solvent) and pressurized water (Ong & Len, 2003) (at 95 C) extractions were comparable to or higher than those obtained with a multiple step ultrasonic extraction using 70% methanol (0.6 g sample, 20 mL, at room temperature, 10 min · 3). Saponin recovery obtained by fractional PLPW extraction at 125, 150, and 175 C in 3 h increased with extraction temperature and time (Fig. 6). While only 33.2 wt% of total saponins were extracted from ground seeds at 125 C in 3 h, 62.8 wt% of the total saponins were recovered in the first hour at 150 C. A similar yield (60.2 wt%) was obtained in the first 15 min at 175 C. An increase in extraction yield with temperature was also observed during PLPW extraction of phenolics from flaxseed (Cacace & Mazza, 2006) and hydroxycinnamates from red grape skin (Ju & Howard, 2005). While the yield of PLPW extraction (1 h/60 mL) of flaxseed increased from 21 to 86 wt%, 26 to 87 wt%, and 30 to 82 wt% for secoisolariciresinol diglucoside (SDG), p-coumaric acid glucoside, and ferulic acid glucoside, respectively (Cacace & Mazza, 2006), the yield of total hydroxycinnamates from red grape skin increased from 1.08 to 1.30 as temperature increased from 120 to 160 C (Ju & Howard, 2005). HPLC analysis of the 150 and 175 C fractions obtained after 60 and 30 min, respectively, showed considerable background noise which appeared to be due to a significant increase in the amount of compounds eluting earlier than saponins. The saponin contents of these fractions thus are not included in the total recovery values. This considerable increase in the content of more polar compounds in the extracts with temperature and time can be attributed to the coextraction of breakdown compounds of seed components (starch, proteins and saponins), which in addition to complicating the analysis of the extracts, can have important implications for purification and properties of the extract. Thus, while the mass transfer and solubility enhancement effects associated with PLPW extraction will increase with temperature resulting in higher extraction yields, the extraction efficiency might be adversely affected if thermal degradation occurs at high temperatures. The extraction yield of total anthocyanins from red grape skin decreased by 64% as temperature increased from 110 to 160 C (Ju & Howard, 2005). In the temperature range 100–160 C, the highest glycyrrhizic yield from licorice was obtained at 100 C (Ong, 2002). The efficient extraction of anthocyanins at temperatures higher than predicted by their thermal stability (120 C) was attributed to the low residence time of anthocyanins in the extraction cell (King et al., 2003). The degradation temperature of a soybean extract containing 37% isoflavones was noted to be 140 C as determined by thermogravimetric analysis (TGA) (Li-Hsun et al., 2004).

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Thermal stability of C-27 steroidal (furastanol, spirostanol, and spirosolane) saponins and sapogenins upto 150– 170 C has been demonstrated by thermal analysis (Bobeyko & Kintia, 1996). Gradual degradation was observed with further increase in temperature resulting in complete destruction of saponins at temperatures >270 C (Bobeyko & Kintia, 1996). Hydrothermolysis of triterpenoid and steroid saponins occurred upon heating in water at 100–140 C for extended periods of time (17–90 h) resulting in the production of aglycones, prosapogenins, and sugars (Kim, Higuchi, & Komori, 1992). Thermal degradation of other seed components such as starch and proteins might also take place at elevated temperatures. The production of organic acids and amino acids from fish protein at 200–400 C (Yoshida, Terashima, & Takahashi, 1999); and the production of glucose, maltose, fructose, 5hydroxymethylfurfural, furfural, and oligomers with various degrees of polymerization from starch at 180 and 240 C (Nagamori & Funazukuri, 2004) in the presence of water have been documented. These results are consistent with the findings of our study which suggest thermal degradation of seed components at temperatures P150 C. The high total extraction yields obtained at higher temperatures (73 wt% at 175 C in 1 h) can in part be attributed to the higher solubility of breakdown compounds in PLPW. Total extraction of whole and ground seeds was carried out at 125–160 C for 1 h. Saponin yield of 1 h PLPW extraction increased by a factor of 4.0 (from 13% to 53%) and 2.3 (from 30% to 69%) for ground and whole seeds, respectively, as temperature increased from 125 to 160 C (Fig. 7). Higher amounts of saponins were extracted from whole seeds at all the temperatures investigated (Fig. 7). Sample pre-treatment to reduce particle size of plant materials has been commonly used to improve the rate/yield of the extraction process (del Valle, Germain, Uquiche, Zetzl, & Brunner, 2006). Packed beds with small particles however can decrease extraction efficiency due to low density of packing and agglomerating tendency of the sample, and poor distribution of solvent flow (e.g., channelling) (del Valle et al., 2006). The distribution of saponins in the cow cockle seed should also be considered while interpreting these results. Our analysis revealed that the majority of saponins (72.3%) were present in the embryo, which is located between the starchy endosperm interior and the seed coat exterior of the seed (Fig. 4). Grinding of quinoa seeds, saponins of which are concentrated in the bran portion, also decreased the saponin yield of 50% ethanol extraction (at 50 C) by 28% (Muir et al., 2002). 3.2. Saponin concentration Saponin concentration of the cow cockle seed extracts was affected by the extraction solvent, method, sample pre-treatment, and time and temperature of PLPW extraction (Fig. 8, Tables 3 and 4). The most concentrated extract

16

Saponin concentration of dried extracts (wt %)

626

whole ground

14 12 10 8 6 4 2 0 120

125

130

135

140

145

150

155

160

165

Temperature (°C)

Fig. 8. Saponin concentration (wt%) of whole and ground seed extracts obtained by 1 h pressurized low polarity water extraction at 125–160 C.

Table 4 Saponin concentration of fractions obtained by pressurized low polarity water (PLPW) extraction of ground cow cockle seeds (2 g) at 125–175 C (flow rate: 2 mL/min) Extraction time (min)

Saponin concentration

125 C

150 C

175 C

15 30 45 60 120 180

4.5 ± 1.3 5.4 ± 0.2 5.3 ± 1.8 4.9 ± 2.2 6.0 ± 1.5 6.7 ± 0.8

5.2 ± 0.4 3.6 ± 0.0 5.1 ± 1.3 4.6 ± 1.0

4.9 ± 2.6 5.7 ± 2.3

g/100 g dried extract

was obtained using ASE with 80% methanol (40.2%) and USE with 80% ethanol (37.4%) (Table 3). Ethanol (100%) extract had the lowest saponin content for both methods (2.1, 15.6%), followed by the water extract obtained by USE (11.2%) (Table 3). The lower saponin concentration of water extract is indicative of the higher selectivity of water to non-saponin components (such as carbohydrates) than the other solvents investigated. Saponin concentrations of the PLPW fractions obtained from ground seeds at 125–175 C were in the range of 4.5–6.7 at 125 C, 3.6–5.2 at 150 C, and 4.9– 5.7 g/100 g dry extract at 175 C (Table 4). Saponin concentration of whole seed extracts, which was higher than that of ground seed extracts at 125 C, decreased with temperature to levels in ground seed extracts at higher temperatures (>140 C) (Fig. 8). The most concentrated extract was thus obtained by PLPW extraction of whole seeds at 125 C. This can be attributed to the higher concentration of saponins in the outer embryo fraction. The decrease in saponin concentration of whole seed extracts with temperature can be attributed to the change in the seed structure with temperature, which overrides the extractive advantage of saponin concentration in the outer layers.

Table 5 Composition of the saponin mixture obtained by accelerated solvent (ASE) and ultrasonic solvent extraction (USE) Saponin group

ASE

USE

MeOH-100%

MeOH-80%

EtOH-100%

EtOH-80%

EtOH-50%

MeOH-100%

MeOH-80%

MeOH-50%

EtOH-100%

EtOH-80%

EtOH-50%

Water

19.9 ± 0.5 21.4 ± 0.2 7.3 ± 0.2 25.0 ± 0.5 26.4 ± 0.0

19.5 ± 0.2 25.4 ± 0.3 7.9 ± 0.9 25.3 ± 0.1 21.9 ± 0.4

22.9 ± 0.4 16.5 ± 0.2 7.5 ± 0.1 22.7 ± 0.0 30.4 ± 0.3

19.0 ± 0.1 24.3 ± 0.1 7.3 ± 0.3 24.5 ± 0.0 24.9 ± 0.2

20.4 ± 0.2 24.5 ± 0.2 7.9 ± 0.5 23.9 ± 0.2 23.3 ± 0.3

16.2 ± 0.5 19.5 ± 0.0 6.9 ± 0.1 27.3 ± 0.1 30.2 ± 0.6

16.0 ± 0.6 23.7 ± 1.4 6.8 ± 0.0 26.7 ± 0.0 26.8 ± 0.8

20.1 ± 0.2 26.2 ± 1.2 8.5 ± 0.2 24.4 ± 0.3 20.9 ± 1.9

23.8 ± 7.2 17.2 ± 2.0 9.0 ± 0.4 20.1 ± 2.3 29.9 ± 6.6

16.3 ± 0.2 22.6 ± 0.9 6.8 ± 0.2 26.6 ± 0.1 27.7 ± 1.2

19.4 ± 0.0 25.2 ± 1.2 7.6 ± 0.1 24.8 ± 0.1 23.1 ± 1.4

41.0 ± 1.8 35.2 ± 0.5 13.7 ± 1.3 6.9 ± 1.0 3.2 ± 0.0

Table 6 Composition of the saponin mixture obtained by fractional pressurized low polarity water (PLPW) extraction of ground cow cockle seeds at 125–175 C Extraction time (min)

15 30 45 60 120 180

Saponin composition (wt%) 125 C

150 C

175 C

Saponin group

Saponin group

Saponin group

1

2

3

4

5

1

2

3

4

40.4 ± 3.1 44.0 ± 0.8 47.3 ± 3.4 47.8 ± 2.1 43.0 ± 2.5 39.7 ± 0.7

35.3 ± 0.6 38.0 ± 0.5 40.5 ± 2.2 44.4 ± 3.1 41.8 ± 2.1 43.7 ± 1.4

7.1 ± 0.4 6.8 ± 1.0 6.2 ± 0.8 5.4 ± 1.6 8.8 ± 0.6 9.7 ± 1.1

13.4 ± 1.8 10.2 ± 0.5 6.0 ± 2.1 2.3 ± 3.1 5.2 ± 0.9 4.9 ± 0.6

3.8 ± 1.2 1.0 ± 0.2 0.09 ± 0.14 0±0 1.2 ± 1.01 2.0 ± 0.3

39.4 ± 2.4 42.6 ± 2.2 44.0 ± 2.8 47.4 ± 3.8

43.6 ± 1.4 42.8 ± 3.2 37.4 ± 4.4 34.7 ± 2.6

8.2 ± 2.5 10.7 ± 1.4 11.3 ± 0.4 12.6 ± 0.1

7.3 ± 2.3 3.5 ± 0.7 4.3 ± 0.4 3.6 ± 0.3

5 1.5 ± 0.7 0.3 ± 0.3 3.0 ± 0.9 1.8 ± 0.9

1

2

3

4

5

46.2 ± 1.7 55.0 ± 3.7

38.0 ± 6.7 30.4 ± 7.1

9.0 ± 2.9 13.0 ± 2.5

5.2 ± 0.2 1.5 ± 1.0

1.7 ± 2.4 0.0 ± 0

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1 2 3 4 5

Saponin composition (wt%)

627

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628

3.3. Saponin composition In this study, selective extraction of different cow cockle seed saponin groups was studied as a function of extraction solvent and PLPW extraction parameters. As the physicochemical and biological properties of saponins are determined by the structure of individual saponins and composition of the saponin extract, processing parameters can be optimized to produce extracts tailored for specific applications. The composition of the saponin mixture was dependent on the extraction solvent, and the conditions of PLPW extraction (Tables 5 and 6, Figs. 9 and 10). While differences between the composition of the alcohol and alcohol:water extracts obtained using ASE and USE were small, water extracts had a different saponin profile (Fig. 9, Tables 5 and 6). Groups 1 and 2, which contained the most polar saponins, made up 76% of the ultrasonic water extract, and 76–92% of PLPW extracts/fractions of ground cow cockle seeds compared to 36–46% of the alcohol extracts (Tables 5 and 6, Fig. 10). Composition of PLPW and pressurized ethanol and aqueous ethanol extracts obtained by ASE at 150 C are shown in Fig. 9. While an increasing and a decreasing trend with water content was observed for group 2 and 5 saponins, respectively; a minimum and a maximum was observed for group 1 and 4 saponins, respectively, at 80% ethanol (Fig. 9). Similar trends were observed for ultrasonic extracts (Table 5). The effect of extraction solvent on saponin composition has also been observed during the extraction of ginsenosides (Du, Wills, & Stuart, 2004), and quinoa saponins

45

Ethanol

Composition of the saponin mixt

ure (wt %)

40

80% Ethanol 35

50% Ethanol Water

30

25

20 15 10 5 0 1

2

3

Saponin

group

4

5

Fig. 9. Saponin composition of pressurized ethanol and aqueous ethanol extracts obtained by accelerated solvent extraction and water extract obtained by pressurized low polarity water extraction at 150 C.

Saponin composition (wt %)

60 whole ground

50 40 30 20 10 0 1

2

3

125 °C

4

5

1

2

3

4

140 °C

5

1

2

3

150 °C

4

5

1

2

3

4

5

160 °C

Saponin Group Temperature

Fig. 10. Saponin composition of 1 h total extracts obtained by pressurized low polarity water extraction as a function of temperature (at 125–160 C) and sample pre-treatment.

(Muir et al., 2002). The ratio of neutral to malonyl ginsenosides in aqueous ethanol extract of American ginseng increased with the proportion of ethanol in the solvent (Du et al., 2004). While maximum extraction of neutral ginsenosides was obtained with 70% ethanol, the highest yield of malonyl ginsenosides was achieved using 40% ethanol resulting in the highest total ginsenoside yield with 60% ethanol (Du et al., 2004). Differential extraction of saponins from quinoa bran using pure water and alcohol solvents was reflected in the differences in the saponin composition of the extracts (Muir et al., 2002). The composition of the saponin mixture was also affected by temperature and time of PLPW extraction and sample pre-treatment (Fig. 10 and Table 6). The % of group 1 saponins increased with extraction time at 125, and 150 C (for 1 h) and at 175 C (for 30 min); whereas a decreasing trend with time was observed for group 4 saponins at all temperatures (Table 6). The content of group 3 saponins followed a similar trend to group 1 saponins at 175 and 150 C however a decrease with time was observed at 125 C (Table 6). No clear trends could be determined for group 5 saponins. Temperature effect was also dependent on extraction time and type of saponins (Table 6). While % of group 1 and 3 saponins increased with temperature, an opposite trend was observed for group 4 saponins. The effect of temperature on % of group 2 saponins was affected by extraction time. The effect of sample pre-treatment on saponin composition was most evident at 125 C. At this temperature whole seed extracts were more concentrated in group 3– 5 saponins compared to ground seed extracts (Fig. 10). The effect of sample pre-treatment can be explained by the distribution of individual saponins within the seed. HPLC analysis of whole seed, embryo, seed coat and endosperm fractions revealed that the endosperm contained mainly Group 1 and 2 saponins, whereas the latter eluting saponins were concentrated mainly in the embryo fraction.

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In addition to solubility differences, the generation and degradation of individual saponins induced by heating also contribute to the differences in the composition of PLPW extracts. For example, steaming of raw ginseng at temperatures >100 C resulted in changes in composition of the saponin extracts enhancing their biological properties (Kim et al., 2000). 4. Conclusion The findings of this study provide information on the effect of PLPW extraction conditions (temperature, time, sample pre-treatment) on saponin yield, concentration and composition of PLPW extracts. The increase in saponin yield with extraction temperature and time (up to a value of 63% in 1 h at 150 C and 60% at 175 C in 15 min), and the degradation of seed components, which became apparent at temperatures P150 C, should be considered while choosing an extraction time and temperature. Extraction efficiency was not improved by sample pretreatment as total extraction (1 h) of whole seeds yielded more saponins than ground seeds at 125–160 C. The effects of PLPW extraction conditions and extraction solvent on saponin composition suggest that the processing conditions can be optimized to produce extracts tailored for specific applications. Further research is in progress in our laboratory to assess the influence of processing methods and process variables on the bioactivity and functionality of cow cockle seed saponins and saponin rich extracts. Acknowledgements The authors acknowledge the following scientists and staff for providing technical assistance: Tony Cottrell, Rod Hocking, and Juan Eduardo Cacace at the Pacific Agri-Food Research Center, Summerland, British Columbia, and Greg Bishop and Leah Deibert at National Research Council of Canada, Plant Biotechnology Institute, Saskatoon, Saskatchewan. References Alonso-Salces, R. M., Korta, E., Barranco, A., Berrueta, L. A., Gallo, B., & Vicente, F. (2001). Pressurized liquid extraction for the determination of polyphenols in apple. Journal of Chromatography A, 933, 37–43. Ayala, R. S., & Luque de Castro, M. D. (2001). Continuous subcritical water extraction as a useful tool for isolation of edible essential oils. Food Chemistry, 75, 109–113. Bailey, L. H. (1976). Hortorium. Hortus third: a concise dictionary of plants cultivated in the United States and Canada. New York: MacMillan Publishing Co. Inc, pp. 1142. Balsevich, J. J., Bishop, G. G., Hickie, R. A., Dunlop, D. M., & RamirezErosa, I. J. (2005). Identification of new quillaic acid and gypsogenin bidesmosidic saponins in a Saponaria vaccaria accession via LC–MS– DAD. Eighty-eighth Canadian Chemistry Conference Abstracts. http://www.csc2005.ca/abstracts/00000838.htm Accessed 14.11.2005.

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