Influence of postharvest processing and storage conditions on key antioxidants in pūhā (Sonchus oleraceus L.).

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Research Paper

Journal of Pharmacy And Pharmacology

Influence of postharvest processing and storage conditions on key antioxidants in pu¯ha¯ (Sonchus oleraceus L.) Zong-Quan Oua, David M. Schmierera, Clare J. Strachana,b, Thomas Radesc and Arlene McDowella a School of Pharmacy, University of Otago, Dunedin, New Zealand, bFaculty of Pharmacy, University of Helsinki, Helsinki, Finland and cDepartment of Pharmacy, University of Copenhagen, Copenhagen, Denmark

Key words antioxidants; design of experiments; leaf extracts; postharvest storage; Sonchus oleraceus Correspondence Arlene McDowell, School of Pharmacy, University of Otago, PO Box 56, Dunedin 9054, New Zealand. E-mail: [email protected] Received August 22, 2013 Accepted December 23, 2013 doi: 10.1111/jphp.12220

Abstract Objectives To investigate effects of different postharvest drying processes and storage conditions on key antioxidants in Sonchus oleraceus L. leaves. Methods Fresh leaves were oven-dried (60°C), freeze-dried or air-dried (∼25°C) for 6 h, 24 h and 3 days, respectively. Design of experiments (DOE) was applied to study the stability of antioxidants (caftaric, chlorogenic and chicoric acids) in S. oleraceus leaves and leaf extracts stored at different temperatures (4, 25 and 50°C) and relative humidities (15%, 43% and 75%) for 180 days. The concentration of antioxidants was quantified by a HPLC–2,2′-diphenylpicrylhydrazyl postcolumn derivatisation method. Antioxidant activity was assessed by a cellular antioxidant activity assay. Key findings The three antioxidants degraded to unquantifiable levels after ovendrying. More than 90% of the antioxidants were retained by freeze-drying and airdrying. Both leaf and extract samples retained > 90% of antioxidants, except those stored at 75% relative humidity. Leaf material had higher antioxidant concentrations and greater cellular antioxidant activity than corresponding extract samples. Conclusion Freeze-drying and air-drying preserved more antioxidants in S. oleraceus than oven-drying. From DOE analysis, humidity plays an important role in degradation of antioxidants during storage. To preserve antioxidant activity, it is preferable to store S. oleraceus as dried leaf material.

Introduction It has been estimated that approximately 80% of the world’s population use plant material as medicines.[1] The term ‘nutraceutical’ was coined in 1989 and is defined as a naturally occurring substance in foods that provides medical or health benefits in prevention or treatment of disease(s), or improvement in physiological performance.[2,3] The most prominent nutraceuticals include plant fibres, β-carotene, omega-3 polyunsaturated fatty acids and polyphenols.[3] Worldwide, there is increasing interest in the use of nutraceuticals, and for example, antioxidants derived from natural sources are favoured over synthetic antioxidants.[4] Following harvest, medicinal plant material is often dried and stored before being used to manufacture various nutraceutical products. Drying is an effective and commonly used method to preserve the quality of the harvested plant material by reducing the growth of microorganisms and preventing chemical degradation such as hydrolysis that may alter the medicinal properties of the plant material.[5] However, the medicinal qualities of plants may

also be compromised through the drying process.[6] Drying can cause deterioration of nutritional substances and degradation of compounds, such as antioxidants, that contribute to the medicinal effects, thus reducing the final product quality. Open air-drying, oven-drying and freeze-drying are widely used methods in the food and pharmaceutical industry.[6–8] One of the disadvantages of open air-drying is the difficulty in achieving consistent quality because of variability in drying process parameters such as temperature and humidity.[7] Ambient air temperature, relative humidity (RH) and solar radiation may also cause functional compounds to degrade in dried plants.[7] Oven-drying usually increases the loss of nutraceutical components, compared with other drying techniques, because of exposure of material to high temperatures (ranging in the literature from 40[5] to 120°C[9]) for hours to days, and also significantly damages the structure of plant tissues during the process.[6] Polyphenols would move into the cytoplasm where the polyphenol oxidases (PPOs) are located leading

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

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to degradation if PPO is still active.[10] Freeze-drying is based on dehydration by sublimation of a frozen product.[6] Compared with open air-drying and oven-drying, freezedrying can greatly reduce losses of nutraceutical compounds and therefore is believed to be superior for preserving the quality of medicinal plants.[6,11] Specifically, freeze-drying has been shown to have beneficial effects on retaining high antioxidant activity and high concentrations of phenolics,[12,13] including caffeic acid derivatives in Echinacea purpurea.[12] For phytomedicine formulations, it is essential to maintain the stability of the active compounds during storage. As the largest group of non-enzymatic antioxidants, polyphenols are widely distributed in plants as secondary metabolites. In plant tissue, polyphenols are isolated from exposure to the air by being located in intracellular vacuoles or vesicles.[14] As long as the cellular structure remains intact, the polyphenols will be protected. However, once extracted, polyphenols are exposed to the air and therefore liable to degradation. Consequently, the type of matrix that contains the polyphenols (i.e. leaf or leaf extract) could have an influence on the stability of the polyphenols during storage. Studies have been conducted on a range of different species to investigate the influence of storage conditions on antioxidant compounds in plant materials. However, there are limited published data on the stability of the major active compounds in Sonchus oleraceus leaves during postharvest processing and storage. S. oleraceus L. (family Asteraceae), commonly known as pu¯ha¯ in New Zealand, is a dietary and traditional medicinal plant that has been used for centuries worldwide. For example, Sonchus species have been used as a treatment for warts, ulcers, spider bites and other inflammatory conditions.[15] S. oleraceus is rich in antioxidants and possesses high antioxidant activity.[16–20] We previously reported the identification of three major antioxidants in methanolic leaf extracts of S. oleraceus: caftaric acid, chlorogenic acid and chicoric acid, with chicoric acid having the highest concentration.[21] Caffeic acid derivatives (especially chicoric acid) have been reported to exhibit many pharmacological effects including having immunostimulatory and antiinflammatory properties.[12,22] However, studies have shown that caffeic acid derivatives from plant materials may be subject to oxidation or enzymatic degradation during postharvest processing and storage.[5,12,22,23] For example, during aqueous extraction of E. purpurea flowers, oxidation was observed as the resulting supernatant turned brown as soon as the extraction mixture was exposed to oxygen.[23] E. purpurea flowers that were air-dried at 70°C contained 50% of the chicoric acid and 24% of the caftaric acid concentration compared with corresponding samples that were freeze-dried.[12] It is acknowledged that low temperatures 2

reduce various metabolic activity; therefore, the degradation rate would be expected to be slower at lower temperatures. However, when oven-dried ground Echinacea roots were stored at 5°C for 60 days, there was an 80% loss of the initial concentration of chicoric acid during storage at RH higher than 80%.[22] This degradation of caffeic acid derivatives would affect the medicinal qualities of the plant materials containing these compounds, giving rise to nutraceutical products of poor quality. Herein, we investigated the changes in concentration of the three main antioxidant compounds (caftaric, chlorogenic and chicoric acids) in S. oleraceus leaves under different postharvest drying processes and storage conditions. The stability of key antioxidants stored as dried leaf material and extract was also compared. A design of experiments (DOE) approach was used to minimise the number of samples required to draw statistically valid conclusions. In this case, the chosen DOE model was capable of quantifying not only the individual effects of humidity and temperature on stability but also any synergistic interactions between the two variables. Following storage for 150 days, the antioxidant activity of the stored material was assessed using a cellular antioxidant assay. To our knowledge, this is the first time to report how the drying process and storage conditions affect the activity of the key antioxidants in S. oleraceus leaf material.

Materials and Methods Chemicals HPLC grade acetonitrile (ACN), methanol, citric acid monohydrate and sodium chloride were purchased from Merck (Darmstadt, Germany), and 2,2′diphenylpicrylhydrazyl (DPPH) and formic acid were supplied by Sigma-Aldrich (Munich, Germany). Chlorogenic acid was supplied by Acros Organics (Geel, Belgium). Quercetin was from Sigma-Aldrich (Steinheim, Germany). Trisodium citrate and potassium carbonate anhydrous was purchased from Ajax Finechem (Sydney, Australia). Sodium chloride, sodium bicarbonate, 2,2′-azobis (2methylpropionamidine) dihydrochloride (ABAP) and Hank’s balanced salt solution (HBSS) were from Sigma (St Louis, MO, USA). 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was from Sigma (Jerusalem, Israel). Dimethyl sulfoxide was from Sigma (Lyon, France). Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffered saline solution (PBS), GlutaMAX, penicillin-streptomycin and trypsin were from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Gibco (Auckland, New Zealand). Purified deionised water was prepared using a Milli-Q Reagent Water Purification System from Millipore (Billerica, MA, USA). Human hepatocellular carcinoma (HepG2) cells were gratefully obtained from the Pathology


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Table 1 Factors and levels used for screening the effect of temperature and relative humidity on the stability of the key antioxidants in Sonchus oleraceus Storage material

Freeze-dried S. oleraceus leaf and extract powder Levels

Factors

Low

Middle

High

Temperature (°C) Relative humidity (%)

4 15

25 43

50 75

Department at the University of Otago, Dunedin, New Zealand.

Plant material S. oleraceus seeds were collected from plants growing wild in Oamaru, New Zealand (45°05.346′ S, 170°58.861′ E) and identified by Mr John Steel, a botanist in the Department of Botany at the University of Otago, New Zealand. Voucher specimens (OTA 061166 and OTA 061167) are lodged at the Otago Regional Herbarium in the Department of Botany at the University of Otago, New Zealand. Seeds were germinated in a 50 : 50 potting compost : sand mix and grown for three weeks in a greenhouse, and the seedlings transplanted into 1 l pots in the same soil mix as mentioned earlier. Tap water was supplied every 2 days. Artificial light was provided 24 h/day in addition to normal natural daylight. The temperature inside the greenhouse was maintained at 25°C.

Effect of postharvest processes on antioxidant concentration Freshly harvested S. oleraceus leaves (n = 5) were cut into four portions and subjected to oven-drying at 60°C, freezedrying (FreeZone Plus 6 l Cascade Freeze Dry Systems, Labconco Corporation, Kansas City, MO, USA) at below 33 × 10−3 mBar and condenser temperature of −80°C or open air-drying (23.4 ± 1.6°C and 41.2% ± 5.0% RH) for 6 h, 24 h and 3 days, respectively, to constant weight. The remaining fresh leaf portion was used as a control. The fresh and dried S. oleraceus leaf material was extracted as described later.

Preparation of leaf extracts Fresh or dried S. oleraceus leaves were ground and extracted with 70% methanol (v/v) to obtain a 10% (fresh w/v) mixture as described by Ou et al.[21] After stirring for approximately 0.5 h at room temperature, the extraction mixture was centrifuged at 9700g for 10 min. Solvent was removed by centrifugal evaporation (SVC-200H SpeedVac Concentrator, Savant). The dried extracts were then redissolved in 3% formic acid and centrifuged at 9700g for 5 min before further analysis of HPLC.

Effect of storage conditions on antioxidants Two batches of fresh S. oleraceus leaves were harvested and freeze-dried. One batch of these freeze-dried leaves was ground into a powder and stored under different conditions as described later. The other batch of freeze-dried leaves was extracted as described earlier and the extract freeze-dried before being stored. The stability of antioxidants from material stored as intact leaves was compared with antioxidants stored following extraction. Freeze-dried S. oleraceus leaves and leaf extracts were stored at different temperatures (4, 25 and 50°C) and RHs (dry, 43% and 75%) in sealed containers and covered with foil to exclude light. The RH chambers were prepared using the following saturated salt solutions: potassium bicarbonate (43% RH) and sodium chloride (75% RH). Silica gel was used to provide dry condition (15% RH). Using a DOE approach, a three-level, two-factor, full-factorial design was set up for the storage condition study to investigate the best storage temperature and humidity; and the most influential terms, see Table 1. A quadratic model was fitted, which is capable of modelling both linear, interaction and square terms (MODDE 9.0, Umetrics, Umeå, Sweden). The design consists of eight full factorial design points and three centre points to test for reproducibility. The responses measured were the concentrations of the three key antioxidants (caftaric, chlorogenic and chicoric acids) in S. oleraceus samples. The order of the experiments was randomised for both leaf and extract samples (MODDE 9.0). Models with acceptable model fit and prediction precision were selected.

Post-column derivatisation high-performance liquid chromatography–2,2′-diphenylpicrylhydrazyl radical assay An online post-column derivatisation HPLC-DPPH radical assay[21] was used to separate and quantify the major antioxidants in S. oleraceus leaf extracts. Ten microlitres of extract suspension was injected into an analytical HP C18 column (Alltima 3 μm 190 Å 150 × 2.1 mm). The mobile phase consisted of ACN in 1% aqueous formic acid following a gradient: t0 = 5%, t15 = 15%, t27 = 24%, t40 = 30%,


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t41 = 80%, t45 = 80%, t46 = 5%, t55 = 5%, at a flow rate of 0.2 ml/min and detection at 328 nm.

2,2′-diphenylpicrylhydrazyl free radical scavenging capacity The free radical scavenging activity of the leaf extracts was measured in triplicate serial dilutions based on the method described by Philpott et al.[24] with modifications. Briefly, 1 : 2 dilutions of samples in 3% formic acid or 1 mm quercetin in methanol were made along the columns in a 96-well plate, and then 200 μl of 100 μm DPPH in methanol was added into each well, except the last row that held solvents only. The plate was covered with aluminium foil and incubated in an orbital shaker for 30 min at room temperature before measuring the absorbance at 518 nm in a well-plate reader (SpectraMAX 340 Microplate Spectrophotometer, Molecular Devices, Sunnyvale, CA, USA). The scavenging activity was expressed as half maximal inhibitory concentration (IC50), the concentration of sample required to scavenge 50% DPPH radical. The IC50 values were calculated using GraphPad Prism 5 (La Jolla, CA, USA).

Cell culture Human HepG2 cells were cultured in DMEM supplemented with 10% FBS, 10 mm HEPES (4-(2-Hydroxyethyl) piperazine-1-ethane sulfuric acid, Sigma (St Louis, MO, USA)), 2 mm GlutaMAX, 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C with 5% CO2. The cells used for this study were between passages 12 and 16.

Cellular antioxidant activity assay The cellular antioxidant activity (CAA) was measured based on the method of Wolfe and Liu[25] HepG2 cells were seeded at 6 × 104/well on a 96-well plate in 100 μl of completed DMEM, excluding the outside wells of the plate. After a 24 h incubation period, the DMEM was removed, and the wells were washed with PBS. Triplicate wells were treated with 100 μl of S. oleraceus extracts plus 25 μm DCFH-DA dissolved in DMEM for 1 h. Wells were then washed with 100 μl PBS before 600 μm ABAP was applied to the cells in 100 μl of HBSS. Fluorescence was read with emission at 538 nm and excitation at 485 nm every 5 min for 1 h at 37°C (POLARstar Omega microplate reader, BMG Labtech, Offenburg, Germany).

(∫ SA ∫ CA ) × 100

(1)

where ∫ SA and ∫ CA are the integrated areas under the curve of sample and control, respectively.[25] The median effective concentration (EC50) was determined for the S. oleraceus extracts from the median effect plot of log (ƒa/ ƒu) versus log (concentration), where ƒa and ƒu are the fraction affected (CAA unit) and the fraction unaffected (100 − CAA unit) by the treatment, respectively. The EC50 values were expressed as mean ± standard deviation for triplicate sets of data obtained from the same experiment.

Data analysis and model evaluation Concentrations of the three compounds (caftaric, chlorogenic and chicoric acids) in each sample at each time point were determined by HPLC and were normalised and expressed as percentage of the corresponding initial concentration. The degradation process followed first-order kinetics. Therefore, the slope of the natural logarithm plots of the percentage initial concentration versus time gave the first-order degradation rate constant for each compound. Partial least squares (PLS) regression was used to fit interaction models (MODDE 9.0, Umetrics).[26] Because the response variable distributions were skewed, they were normalised by transformation using y−2 for caftaric acid and y2 for the rest of the variables. The responses were then scaled to unit variance before analysis. The resulting models were assessed for validity using the goodness of fit (R2), goodness of prediction (Q2) and analysis of variance (ANOVA) analyses. The data were checked for outliers, which were removed if appropriate, and the models were further optimised, when possible, by removing nonsignificant terms from the model. Once the validity of the models was established, the coefficients of the model terms and response surface contour plots were used to analyse the effect of different storage conditions on the stability of the key antioxidants in S. oleraceus samples and spot conditions with optimal stability. Response surface contour plots were used to analyse the effect of different storage conditions on the stability of the key antioxidants in S. oleraceus samples and to identify conditions with optimal stability. Statistical analysis of the concentration and DPPH scavenging activity of S. oleraceus leaves in the postharvest study and CAA was compared using the Student’s t-test performed using SPSS Statistics from IBM (Version 20, International Business Machines Corp., New York, NY, USA). P < 0.05 was considered statistically significant.

Results

Quantification of cellular antioxidant activity After blank subtraction from the fluorescence readings, the area under the curve of fluorescence versus time was integrated to calculate the CAA unit as: 4

CAA unit = 100 −

Effect of postharvest processes on antioxidants All three antioxidants (caftaric, chlorogenic and chicoric acids) degraded to undetectable levels after Sonchus leaves


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IC50 (mg/ml)

1.5

1.0

0.5

0.0 Fresh

Freeze-dried

Air-dried

Figure 1 Influence of postharvest leaf treatments on antioxidant activity (half maximal inhibitory concentration, IC50 values) of the three key antioxidants in Sonchus oleraceus leaf extracts assess by the 2,2′-diphenylpicrylhydrazyl assay. Data are mean ± standard deviation (n = 5), expressed as mg dry leaves per ml solvent.

were dried in the oven for 6 h at 60°C. In contrast, freezedrying did not change the concentration of these antioxidants significantly compared with fresh leaves (P ≥ 0.1) (Supporting Information Figure S1). Similarly, open airdrying retained most of the three antioxidants with greater than 90% of the initial concentration remaining after 3 days (Supporting Information Figure S1). The antioxidant activity of the dried samples was assessed by the DPPH free radical scavenging capacity assay. There was no significant difference in the IC50 values for the two different postharvest treatments (freeze-drying and open air-drying) and fresh control samples (P ≥ 0.05) (Figure 1).

Effect of storage conditions on antioxidants No significant changes in the concentration of the three compounds were detected for samples stored under dry conditions and at 43% RH irrespective of temperature (Supporting Information Figures S2 and S3), with the exception of chicoric acid where 24% was observed to degrade at 50°C and 43% RH when stored as an extract. Changes in the concentration of antioxidant compounds were observed in the S. oleraceus samples, stored as both leaves or leaf extracts, for 180 days at different temperatures and 75% RH. In leaves and leaf extracts stored at 75% RH, the concentration of chlorogenic and chicoric acids declined with increasing temperature (Figure 2). At 25°C, caftaric acid concentration went up more in stored leaves than in extract during 180 days. Noticeably, at 50°C and 75% RH, the content of caftaric acid increased initially followed by a decrease after approximately 60 days (Figure 2c and 2f). Compared with the storage of leaf extracts, the key antioxidants were more stable when stored as dried leaf

material (Figure 2c and 2f). The degradation rate for both chlorogenic acid and chicoric acid stored as extracts was 2–2.5 times faster than when stored as leaves at 50°C and 75% RH (Table 2). The model used to predict the concentration of individual and the total amount of the three key antioxidants in S. oleraceus leaf extract samples under different storage conditions revealed that the interaction model with two PLS factors was valid according to the goodness of fit (R2 = 0.86, 0.93, 0.90 and 0.92, respectively) and goodness of prediction (Q2 = 0.53, 0.46, 0.44 and 0.43, respectively). ANOVA of extract samples revealed that the model was significant (P ≤ 0.032), with no lack of fit (P > 0.1), and the linear residuals N-plots indicated that the model fitted the data and there was no evidence of outliers. For leaf samples, the goodness of fit (R2 = 0.88, 0.95, 0.89 and 0.88, respectively) and goodness of prediction (Q2 = 0.68, 0.68, 0.54 and 0.49, respectively) indicated the validity of the model. ANOVA of the leaf samples revealed that the model was significant (P ≤ 0.007) but with lack of fit for caftaric and chicoric acids and the total amount (P ≤ 0.045). Because of the high R2, Q2 and reproducibility (> 0.98) values, the low model validity (lack of fit) result was not considered relevant and was disregarded. The normalised regression coefficients and the corresponding 95% intervals of both leaf and extract samples are shown in Table 3. The value of a coefficient represents the change in the response when the normalised factor varies. A coefficient is considered significant when the confidence interval does not cross zero. For the extracts, the interaction effect between RH and T was insignificant for all response variables. For caftaric acid, only the main effect (T) and squared effect (RH × RH) were significant. The main effect (RH) was significant for all the other response variables (chlorogenic and chicoric acids, and total amount). However, none of the insignificant terms were removed to preserve model hierarchy. For leaf samples, only the main effects (RH, T) of caftaric acid were significant, while the main effect (T) was not important to the rest of the variables. The square term (T × T) was not significant for any of the response variables, and was removed for a better model fit. The square term (RH × RH) for caftaric acid was also removed. Contour plots were used to visualise the stability of the key antioxidants in S. oleraceus samples. The aim was to efficiently identify storage conditions and storage material that preserved the key antioxidants the most and to reveal the influence of two factors – temperature and humidity – on the key antioxidants. Storage conditions maintained more than 90% of initial concentrations of the antioxidants after 180 days were considered as good storage condition. In line with the observed data, contour plots of the total amount of the key antioxidants illustrate the decrease in


5


(b)

150 100 50 0 0

50

100

150

200 Day

% of initial content

(d)

200 150 100 50 0 0

50

100

150

200 Day

(e) Caftaric acid Chlorogenic acid Chicoric acid

250 4°C, 75% RH 200 150 100 50 0 0

50

100

150


200

(c)

Caftaric acid Chlorogenic acid Chicoric acid

250 25°C, 75% RH

250 25°C, 75% RH


200

(f) % of initial content


4°C, 75% RH


250



(a)

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150 100 50 0

200 Day

0

50

100

150

250 50°C, 75% RH 200


150 100 50 0

0

50

100

250 50°C, 75% RH 200

150

200 Day


150 100 50 0

200 Day

0

50

100

150

200 Day

Figure 2 Change in concentration of the three key antioxidants in Sonchus oleraceus storage at 75% relative humidity with different temperatures (leaf: a–c; extract: d–f.). Data are shown as the percentage of the initial concentration of the three antioxidants.

Table 2

Degradation rates of antioxidants in Sonchus oleraceus samples when stored as freeze-dried leaf material and extracts Degradation rate constant (1/days)

Material

Storage conditions

Caftaric acid

Chlorogenic acid

Chicoric acid

Leaf

75% RH, 25°C 75% RH, 50°C 75% RH, 50°C

— — −0.0115

−0.0028 −0.0075 −0.0141

−0.0019 −0.0069 −0.0162

Extract RH, relative humidity.

Table 3 Normalised regression coefficients and their 95% confidence intervals for the individual and total amount of the three key antioxidants in Sonchus oleraceus samples Caftaric acid

Extracts

Leaf

RH T RH × RH T×T RH × T RH T RH × RH RH × T

Chlorogenic acid

Chicoric acid

Total amount

Coefficient value

Confidence interval (+/−)

Coefficient value


Coefficient value


Coefficient value


5.16 × 10−6 −1.59 × 10−5 1.24 × 10−5 1.61 × 10−6 −8.47 × 10−6 −1.19 × 10−5 −2.05 × 10−5 NA −6.75 × 10−6

1.02 × 10−5 1.03 × 10−5 1.21 × 10−5 1.22 × 10−5 9.59 × 10−6 8.32 × 10−6 8.32 × 10−6 NA 7.90 × 10−6

−2035.06 −1312.16 −1488.51 −1568.72 −810.416 −2249.09 20.36 −2281.23 −1441.57

1128.53 1135.94 1342.52 1349.53 1061.29 797.94 790.79 915.33 750.63

−2272.69 −1322.45 −1726.16 −1734.3 −826.798 −1866.55 356.022 −2530.43 −1904

1479.57 1489.29 1760.13 1769.32 1391.42 1298.53 1286.9 1489.57 1221.54

−2166.06 −1137.79 −1699.94 −1637.83 −720.882 −1527.43 515.53 −2186.99 −1579.53

1242.08 1250.24 1477.61 1485.32 1168.08 1189.43 1178.77 1364.41 1118.9

NA, not applicable; RH, relative humidity; T, temperature.

antioxidant concentration with increasing temperature and RH when S. oleraceus is stored as extracts (Figure 3). Predicted high values of total key antioxidants (≥ 90%) in leaf extracts could be obtained when RH was between 15% and 45%, and at temperatures from 4°C to 50°C (Figure 3d). 6

For individual compounds, the highest concentrations of caftaric acid were obtained at high temperature (Figure 3a). While the contour plot also suggests that a medium humidity at high temperature leads to the highest concentration, the humidity term (RH) is not significant (and the squared


Zong-Quan Ou et al.

(a)


(b)

% of initial Caftaric acid 50 45

45

130

40

100

35 30 25 20

110 100

15 10

50 60 70

90

35 30 25 20 15 10

90

5

5 15

20 25 30 35 40 45 50 55 60 65 70 75

15

Relative humidity (%) (c)

20 25 30 35 40 45 50 55 60 65 70 75 Relative humidity (%)

(d)

% of initial Chicoric acid 50

% of initial Total amount 50

90

40

5040 60 70 80

35 100

30 25 20

50 60 70

45 90

40 Temperature (°C)

45

Temperature (°C)

80

40 120

Temperature (°C)

Temperature (°C)

% of initial Chlorogenic acid 50

80

35 30

100

25 20

15

15

10

10 5

5 15

20 25 30 35 40 45 50 55 60 65 70 75

15

Relative humidity (%)

20 25 30 35 40 45 50 55 60 65 70 75 Relative humidity (%)

Figure 3 Response contour plots of individual (a–c) and total amount (d) of the three key antioxidants in Sonchus oleraceus leaf extracts samples after 180 days under different storage conditions. The darkest red contour shows conditions of highest concentration of total amount of the key antioxidants, and the darkest blue shows the lowest concentration conditions. Contour lines are labelled with their percentage of initial concentration values.

term (RH × RH) is only marginally significant). This suggests that humidity does not affect the concentration of caftaric acid. Similar with the total amount of the three compounds, there was less chlorogenic and chicoric acids in S. oleraceus extracts stored at high humidity and high temperature (Figure 3b and 3c). High concentrations of chlorogenic and chicoric acids could be obtained with different combinations of the two independent variables, RH values below 35% and temperature in the entire range studied from 4 to 50°C. In contrast, more than 90% of the key antioxidants in leaf material could be retained with a wider range of conditions (RH values between 18% and 65%, and temperature from about 10–50°C, Supporting

Information Figure S4). Individually, caftaric acid concentration in leaf material increased with an increase in temperature and humidity, and reached the highest concentration at 50°C and 75% RH (Supporting Information Figure S4). The changes in concentration of the key antioxidants in S. oleraceus leaf samples were much more complex (Supporting Information Figure S4) than those in extract samples because of possible metabolism in the leaf material. It is acknowledged that plant enzymes can be active even after plant material has been dried. Degraded samples from the storage study (75% RH at day 150) were then evaluated for their CAA using the CAA assay (Figure 4). The lower the EC50, the higher the CAA


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0% RH, 50°C 75% RH, 50°C 75% RH, 25°C 75% RH, 4°C

6

EC50 (mg/ml)

5

*** *** ***

4 3

*

2 1 0

Extract

Leaf

Figure 4 Cellular antioxidant activity of Sonchus oleraceus material stored at 75% relative humidity with different temperatures after 150 days. Two storage forms (leaf and extract) were compared. Samples stored under dry conditions at 50°C served as the control. Data are mean ± standard deviation (n = 3), expressed as median effective concentration on basis of leaf fresh weight (*P < 0.05; ***P < 0.001).

the sample possessed. Both leaf and extracts stored under dry conditions exhibited high CAA, indicated by the low EC50 values obtained (1.37 ± 0.09 and 1.38 ± 0.28 mg/ml (FW), respectively). Extract samples stored at 75% RH had a lower CAA than that of leaf material. At 75% RH, the CAAs of extract samples at all the three temperatures were significantly (P < 0.001) lower than those of the control samples stored at dry condition.

Discussion Medicinal herbs are often dried to less than 15% moisture content before being manufactured into nutraceutical products.[8] The drying process is complex and could affect the quality attributes of dried medicinal plant materials. Several drying methods, including freeze-drying, microwavedrying, hot-air-drying and cool wind-drying, are reported to affect the concentration of antioxidants and their activity in many plant materials.[5,6,8,12,13,27] Previously, we reported three caffeic acid derivatives – caftaric, chlorogenic and chicoric acids – as the major antioxidants in methanolic extracts of S. oleraceus leaves.[21] Phenolic compounds, including these caffeic acid derivatives, are sensitive to heat and air, and increasing temperature will result in a significant loss of caffeic acid derivatives and total phenolics.[5] Lin et al. investigated the effect of three different drying methods at different temperatures on caftaric, chlorogenic and chicoric acids in different parts of E. purpurea.[5] In leaf material, all the three caffeic acid derivatives were not detected when air-dried at 55 and 70°C.[5] This finding is consistent with the results in the current study in that none 8

of the three antioxidants in S. oleraceus were quantifiable after oven-drying at 60°C. However, Stuart and Wills[28] found that chicoric acid was still detectable in E. purpurea leaves even after oven-drying at 70°C, although the concentration decreased with increasing temperature. The reason for this could be the different methods for handling the fresh raw materials. Stuart and Wills dried the whole plant at different temperatures before determining the chicoric acid content in each part of the plant. In contrast, Lin et al. and this study used fresh leaves that were cut into pieces before the drying process. Thus, cells were disrupted when leaves were cut into small pieces, leading to easier leakage of active compounds from vaculoes and thus greater exposure of chicoric acid to the degrading conditions of temperature and oxidation. The stability of phytochemicals has been intensively studied in different plant species. However, information on the stability of antioxidants in S. oleraceus is limited. We found that degradation mainly occurred at high humidity and high temperature, which is consistent with previous reports of caffeic acid derivatives in E. purpurea. Wills and Stuart[22] reported that chicoric acid was stable over 60 days’ storage at 5, 20 and 30°C with < 60% RH conditions. Lin et al.[5] found no changes in chicoric acid content within 180 days’ storage at 10 and 20°C, 60% RH. In both studies, degradation occurred when RH increased to 80%. It was postulated that the degradation was associated with enhanced enzymatic activity, PPO for instance, resulting from increased moisture content during storage.[5,22,23] PPO are able to oxidise diphenols to the corresponding quinone.[29] This explained why there was no increase of caftaric acid observed in Lin’s study. However, in our study, an initial increase of caftaric acid occurred followed by a decrease (Figure 2f). This implies that PPO might not be responsible for the degradation in the current study. Alternatively, the enzyme may not be present in the S. oleraceous extracts because it may be membrane-bound[30] or not sufficiently soluble in the 70% methanol used in this study to obtain leaf extracts containing antioxidant compounds. Further, the main effect coefficients were greater for humidity compared with temperature. Therefore, we propose that the three compounds in S. oleraceus are probably degraded by hydrolysis during long-term storage. Chicoric acid can be hydrolysed to caftaric acid and caffeic acid, which is consistent with the increase in the concentration of caftaric acid during early storage of 70 days (Figure 5). Caftaric and chlorogenic acids could be further decomposed into tartaric acid and quinic acid, respectively, and caffeic acid. This is supported by a recent study on the hydrolysis of chicoric and caftaric acids.[31] In the leaf material, the caftaric acid content plateaued and did not decrease at 50°C 75% RH (Figure 2c). From the contour plots, an increased concentration of caftaric acid with increase of RH and temperature


Zong-Quan Ou et al.


O

HO HO

OH

CO2H

O O

OH HO2C

OH

OH Caffeic acid

+

OH

HO

CO2H

HO

CO2H

HO

H2O

CO2H CO2H

OH

Caftaric acid

CO2H O Chichoric acid HO

CO2H O

+

H2O O

HO

Tartaric acid

Caffeic acid Figure 5

Hydrolysis of chicoric acid.

was observed (Supporting Information Figure S4). In the storage of extract samples, the concentration of caftaric acid decreased at 50°C 75% RH (Figure 2f), while samples at other conditions (such as 50°C 43% RH) started to degrade, which may produce caftaric acid. Thus, a higher value of caftaric acid was obtained at high temperature (Figure 3a). In the contour plots, the curvatures indicate the importance of interaction or squared terms. As shown by the coefficients, squared term effects were significant, squared RH for caftaric acid and squared T for others responses. This explains the different direction of the curvatures in the contour plots (Figure 3). Comparing the leaf and extract storage overall, compounds stored as leaf samples were more stable than when stored as extracts, especially at 50°C 75% RH (Figure 2c and 2f). Higher main effect coefficient values for the total amount of key antioxidants in the extracts storage (Table 3) indicated a faster degradation than that in the leaf storage. For leaf samples, the three key antioxidants were maintained in the range of conditions of temperature and humidity investigated in this study, while only low humidity would retain the active compounds in extract samples (Figure 3). This difference is probably due to the antioxidant compounds being isolated from the storage environment in cellular compartments in whole leaf material and therefore being protected from degradation. Unexpectedly, the concentration of caftaric acid in leaf powder stored at 25°C 75% RH increased faster than that of extract and reached 143.7% after 180 days’ storage, while only 106.3% in extract. It seems that caftaric acid is labile and degrades faster in extract storage because of the direct exposure to the storage environment. Degradation of antioxidant compounds results in lower activity as assessed by the chemical antioxidant assay using the DPPH reagent. But this does not necessarily mean a lower activity in a cellular environment. Thus, the CAA assay was performed for both leaf and extract samples after

150 days storage (Figure 4). The results further confirmed that leaf storage retained higher CAA of S. oleraceus samples than when stored as extracts and, therefore, is the preferable form of storage. After 150 days’ storage, only trace chlorogenic and chicoric acids remained in extract samples stored at 50°C 75% RH, which led to a lower DPPH free radical scavenging capacity (IC50 = 1.26 ± 0.03 mg/ml) than samples stored at 4°C (IC50 = 0.86 ± 0.06 mg/ml) and 25°C 75% RH (IC50 = 0.80 ± 0.07 mg/ml). However, in the cellular assay, there was no significant difference among the samples stored at 4, 25 and 50°C with 75% RH. A possible reason for there being no difference in the CAA is that the increase in caftaric acid compensated for the degradation of the two other main compounds, and so overall, there was no change in antioxidant activity of the S. oleraceus samples. Other possible active degradation products could also contribute to the CAA, such as caffeic acid. Therefore, samples stored at 50°C 75% RH still exhibited considerable CAA, although the major compounds have undergone degradation.

Conclusions Freeze-drying and air-drying are suitable drying methods for retaining the concentration and activity of the three key antioxidants in S. oleraceus. Oven-drying at 60°C was the most destructive of the three drying methods. During storage, humidity plays a more important role than temperature in maintaining the antioxidants in S. oleraceus. S. oleraceus samples were stable at most of the storage conditions over 180 days, except those at high RH and high temperature. The interaction of the two factors had more effect on the leaf storage rather than extract samples. The key antioxidants were more stable when stored as leaf material compared with extract storage. The CAA of the stored leaf and extract samples that were observed to have degraded was lower compared with corresponding control


9


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samples stored under dry conditions. Consequently, for the production of efficacious phytomedicines, understanding the effect of processing and storage conditions on the active constituents is important.

Declarations The Author(s) declare(s) that they have no conflicts of interest to disclose.

1. Sellami IH et al. Qualitative and quantitative changes in the essential oil of Laurus nobilis L. leaves as affected by different drying methods. Food Chem 2011; 126: 691–697. 2. Kalra EK. Nutraceutical – definition and introduction. AAPS PharmSci 2003; 5: E25. 3. Jackson C-JC, Paliyath G. Functional foods and nutraceuticals. In: Paliyath G et al., eds. Functional Foods, Nutraceuticals, and Degenerative Disease Prevention. Chichester, UK: WileyBlackwell, 2011: 11–43. 4. Yin J et al. The antioxidant and cytotoxic activities of Sonchus oleraceus L. extracts. Nutr Res Pract 2007; 1: 189–194. 5. Lin S-D et al. Effect of drying and storage conditions on caffeic acid derivatives and total phenolics of Echinacea purpurea grown in Taiwan. Food Chem 2011; 125: 226–231. 6. Ratti C et al. Drying of garlic (Allium sativum) and its effect on allicin retention. Dry Technol 2007; 25: 349–356. 7. Soysal Y, Öztekin S. Technical and economic performance of a tray dryer for medicinal and aromatic plants. J Agr Eng Res 2001; 79: 73–79. 8. Chen CL et al. Antioxidants in aerial parts of Hypericum sampsonii, Hypericum japonicum and Hypericum perforatum. Int J Food Sci Technol 2009; 44: 2249–2255. 9. Ahmad-Qasem MH et al. Influence of freezing and dehydration of olive leaves (var. Serrana) on extract composition and antioxidant potential. Food Res Int 2013; 50: 189–196. 10

The work was supported by a grant from The Laurenson Awards, Otago Medical Research Foundation. This research has not been funded by any funding agency in the public, commercial or not-for-profit sectors.

Acknowledgement

Conflict of interest

References

Funding

The authors wish to express their sincere appreciation to Susan McKenzie, Department of Botany, University of Otago, for helping with growing the plants.

10. Zhang Y et al. Influence of several postharvest processing methods on polyphenol oxidase activity and cichoric acid content of Echinacea purpurea roots. Ind Crop Prod 2011; 34: 873–881. 11. Abascal K et al. The effect of freezedrying and its implications for botanical medicine: a review. Phytother Res 2005; 19: 655–660. 12. Kim H-O et al. Retention of caffeic acid derivatives in dried Echinacea purpurea. J Agric Food Chem 2000; 48: 4182–4186. 13. Pinela J et al. Influence of the drying method in the antioxidant potential and chemical composition of four shrubby flowering plants from the tribe Genisteae (Fabaceae). Food Chem Toxicol 2011; 49: 2983–2989. 14. Scalet M et al. Demonstration of phenolic compounds in plant tissues by an osmium-iodide postfixation procedure. Biotech Histochem 1989; 64: 273–280. 15. Riley M. Ma¯ori Healing and Herbal: New Zealand Ethnobotanical Sourcebook. Paraparaumu, New Zealand: Viking Sevenseas N.Z. Ltd, 1994. 16. Guil-Guerrero JL et al. Nutritional composition of Sonchus species (S. asper L., S. oleraceus L. and S. tenerrimus L.). J Sci Food Agr 1998; 76: 628–632. 17. Yin J et al. Antioxidant activity of flavonoids and their glucosides from Sonchus oleraceus L. J Appl Biol Chem 2008; 51: 57–60. 18. Gould KS et al. Antioxidant activities of extracts from traditional Ma¯ori food plants. New Zeal J Bot 2006; 44: 1–4.

19. Xia D-Z et al. Antioxidant and antibacterial activity of six edible wild plants (Sonchus spp.) in China. Nat Prod Res 2011; 25: 1893–1901. 20. McDowell A et al. Antioxidant activity of puha (Sonchus oleraceus L.) as assessed by the cellular antioxidant activity (CAA) assay. Phytother Res 2011; 25: 1876–1882. 21. Ou Z-Q et al. Application of an online post-column derivatization HPLCDPPH assay to detect compounds responsible for antioxidant activity in Sonchus oleraceus L. leaf extracts. J Pharm Pharmacol 2013; 65: 271– 279. 22. Wills RBH, Stuart DL. Effect of handling and storage on alkylamides and cichoric acid in Echinacea purpurea. J Sci Food Agr 2000; 80: 1402–1406. 23. Nüsslein B et al. Enzymatic degradation of cichoric acid in Echinacea purpurea preparations. J Nat Prod 2000; 63: 1615–1618. 24. Philpott M et al. Enhanced coloration reveals high antioxidant potential in new sweetpotato cultivars. J Sci Food Agr 2003; 83: 1076–1082. 25. Wolfe KL, Liu RH. Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J Agric Food Chem 2007; 55: 8896–8907. 26. Eriksson L et al. Design of Experiments: Principles and Applications, 3rd edn. Umeå, Sweden: Umetrics Academy, 2008. 27. Que F et al. Comparison of hot air-drying and freeze-drying on the physicochemical properties and antioxidant activities of pumpkin (Cucurbita moschata Duch.) flours. Int


Zong-Quan Ou et al.

28.

29.

30.

31.

J Food Sci Technol 2008; 43: 1195– 1201. Stuart DL, Wills RBH. Effect of drying temperature on alkylamide and cichoric acid concentrations of Echinacea purpurea. J Agric Food Chem 2003; 51: 1608–1610. Mayer AM. Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry 2006; 67: 2318–2331. Trémolières M, Bieth JG. Isolation and characterization of the polyphenoloxidase from senescent leaves of black poplar. Phytochemistry 1984; 23: 501–505. Bel-Rhlid R et al. Hydrolysis of chicoric and caftaric acids with esterases and Lactobacillus johnsonii in vitro and in a gastrointestinal model. J Agric Food Chem 2012; 60: 9236–9241.


Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Influence of postharvest leaf treatments on the concentration of the three key antioxidants in pu¯ha¯ leaf extracts. Data are mean ± standard deviation (n = 5), concentration expressed μmol per 100 mg dry leaves. ND, non-detectable. Figure S2 Change in concentration of the three key antioxidants in Sonchus oleraceus leaf storage at 15% and 43% relative humidities with different temperatures. Data are shown as the percentage of the initial concentration of the three antioxidants. Centre point data, 25°C, 43% relative humidity, is shown as mean (± standard deviation, n = 3).


Figure S3 Change in concentration of the three key antioxidants in Sonchus oleraceus extract storage at 15% and 43% relative humidities with different temperatures. Data are shown as the percentage of the initial concentration of the three antioxidants. Centre point data, 25°C, 43% relative humidity, is shown as mean (± standard deviation, n = 3). Figure S4 Response contour plots of individual (a–c) and total amount (d) of the three key antioxidants in Sonchus oleraceus leaf samples after 180 days under different storage conditions. The darkest red contour shows conditions of highest concentration of total amount of the key antioxidants, and the darkest blue shows the lowest concentration conditions. Contour lines are labelled with their percentage of initial concentration values.

11

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