Subcritical water extraction for selective recovery of phenolic bioactives from kānuka leaves

J. of Supercritical Fluids 158 (2020) 104721

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Subcritical water extraction for selective recovery of phenolic ¯ leaves bioactives from kanuka Sinemobong Essien, Brent Young, Saeid Baroutian ∗ Department of Chemical and Materials Engineering, The University of Auckland, Auckland 1010, New Zealand

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• SWE effectively extracted pheno• • • •

lic and flavonoid compounds from ¯ kanuka leaves. Gallic acid, quercetin and catechin were identified as the major bioactives. The highest yield was attained at 170 ◦ C for 20 min with 15 g/L sample. Significant correlation existed between antioxidant content and activity. SWE showed selectivity due to changes in polarity with temperature.

a r t i c l e

i n f o

Article history: Received 28 August 2019 Received in revised form 12 December 2019 Accepted 13 December 2019 Available online 24 December 2019 Keywords: Antioxidants Flavonoids ¯ Kanuka Polyphenols Subcritical water extraction Bioactive compounds

a b s t r a c t ¯ Kanuka is a good source of functional ingredients like phenolic compounds. This study investigated ¯ the selective recovery of antioxidant compounds from kanuka leaves using subcritical water extraction (SWE) at different extraction temperatures (150–210) ◦ C, time (0–40) min, and solid-to-solvent ratio, SSR ¯ (15–35) g/L. The maximum total phenolic and flavonoid content in the kanuka leaf extracts were (167.93 ± 2) mg GAE/g dw and (405.4 ± 8.47) mg QE/g dw, respectively. These amounts were obtained using SWE at 170 ◦ C for 20 min with SSR of 15 g/L; the extracts also showed the highest antioxidant activity. Eight compounds were identified, including gallic acid, quercetin, and catechin as major compounds. In comparison with conventional ethanol extraction, SWE proved to be a more effective alternative for extraction of bioactive compounds. The results indicate the potential of SWE for the production of high-quality plant ¯ extracts from kanuka leaves. © 2019 Elsevier B.V. All rights reserved.

1. Introduction ¯ Kanuka, Kunzea ericoides (A. Rich) J. Thompson is a medicinal tree of the Myrtaceae family. Traditional applications and ¯ modern studies on kanuka have shown its potential in the food

∗ Corresponding author. E-mail address: [email protected] (S. Baroutian). https://doi.org/10.1016/j.supflu.2019.104721 0896-8446/© 2019 Elsevier B.V. All rights reserved.

and health sector as well as to the environment [1–3]. A recent review by Essien et al. [4] provides a comprehensive update on the ¯ value-added properties of kanuka, highlighting its underutilised ¯ potential. For instance, kanuka products have been found to possess potent antimicrobial, antioxidants, and immunological effects, which is conferred by various bioactive compounds contained in the plant [1,5–7]. These compounds are responsible for characteristic plant aromas, colours, flavours, and protection against biotic and abiotic stresses [8]. They can also interact with living tissues

2

S. Essien, B. Young and S. Baroutian / J. of Supercritical Fluids 158 (2020) 104721

to produce desired outcomes that promote human health. Several groups of these compounds, mainly terpenoids and phenolic compounds, are exploited in the production of natural medicines and nutraceuticals, fragrances, and as food additives for prevention of fat oxidation and protection of vitamins and enzymes [1–3]. The focus of this research is on phenolic compounds and their role as antioxidants. Extraction of bioactive compounds has gained considerable momentum in recent times. Predominant interest is on environment-friendly extraction techniques that can separate compounds with health-promoting ability in its purest form. Yet, very few research is available on the targeted extraction of bioactive ¯ compounds from kanuka. The focus is mostly on essential oil extraction (non-polar terpene compounds). The one study that aimed at ¯ recovering biologically active compounds from kanuka was conducted by Wyatt et al. [1]. They used ethyl acetate, ammonia, methanol, and dichloromethane organic solvents. The traditional extraction methods, though simple and affordable, require long extraction times and utilise solvents that are often considered toxic and may cause environmental issues [9]. The quality of the extract can also be affected because of the inherent difficulty in screening [10]. Due to the campaign for eco-friendly and sustainable processes, extraction techniques like subcritical water extraction are recommended as alternatives to organic solvent extraction. Subcritical water extraction is an operation that explores the solvating power of water at high temperatures (100–374) ◦ C at pressures high enough to maintain the water in a liquid state. The dielectric constant of water, which is responsible for solubility, reduces under SWE conditions to values similar to organic solvents like ethanol [11,12]. Subcritical water extraction has been used to recover phenolic and antioxidant compounds from plants such as olive leaves [13], Phlomis umbroza Turcz [14], ginger [15], carrot leaves [16], ginseng [17], onion skin [18] and, kiwifruit pomace [19]. However, SWE has not been applied in the recovery of bioactive compounds ¯ from kanuka. As part of the on-going investigation to further the understand¯ ing of the biological activities of kanuka and to promote diversified usage of this plant, studies to recover bioactive compounds from ¯ leaves were conducted. This present work was undertaken kanuka ¯ to investigate the efficiency of SWE of bioactives from kanuka leaves compared to conventional extraction. The impact of SWE operating parameters on the physicochemical and antioxidative properties of the extracts was also studied to determine the optimum conditions for maximum extraction of biologically important ¯ substances. The phenolic profile of the kanuka leaf extract was analysed, and the selectivity of SWE was assessed. The method and results of SWE for quantitative and qualitative extraction of ¯ bioactive from kanuka leaves are presented.

2. Materials and methods 2.1. Materials ¯ Kanuka foliage was obtained from the east coast of the North ¯ Maori landownIsland of New Zealand (provided by Te Tai Rawhiti ers). The leaves, moisture content of 6.8 % on dry basis, were plucked, ground and sieved to a particle size below 0.85 mm. All sieved samples were stored in a sealed bag and kept in a dry place at room temperature. Chemical standards and reagents used for chemical analysis were gallic acid, catechin hydrate, Folin-Ciocalteu’s phenol reagent, DPPH, Trolox (6-hydroxy-2,5,-7,-8-tetramethylchroman-2-carboxylic acid), ABTS, aluminium chloride, sodium carbonate, sodium nitrite, sodium hydroxide, and HPLC-grade solvents from Sigma-Aldrich (New Zealand).

2.2. Subcritical water extraction (SWE) Subcritical water extraction was performed in a 1.0 L batch highpressure autoclave reactor (Amar Equipment, India) equipped with a PID controller and liquid sampling system. The vessel was filled ¯ with 600 mL of the aqueous mixture of kanuka leaf powder (15–35) g/L and purged with nitrogen (N2 ) to remove air, thus avoid any thermo-oxidation reaction. Extraction was conducted at temperature conditions between 150 ◦ C to 210 ◦ C, agitation rate of 500 min−1 , and pressure of 40 bar. Samples were taken in predetermined intervals beginning when the interior temperature of the reactor reached the setpoint up to 40 min. An average of 20 min was taken to reach each set temperature. The SSR, temperature, and time conditions yielding the maximum extraction efficiency was chosen as the optimum conditions. The control samples were prepared by mixing ground leaves in distilled water at similar SSR for 30 min at room temperature. All samples were centrifuged then vacuum filtered using 0.45 × 10-3 mm qualitative filter papers (Whatman No. 1). The extracts were stored at 4 ◦ C for further analysis. 2.3. Ethanol extraction Ethanol extraction was performed as a benchmark to check the efficiency of subcritical water extraction. Ethanol was used because it is one of the common solvents for solid-liquid extraction. It also has a similar dielectric constant to water at subcritical conditions. Powdered samples (300–700) mg were mixed with 20 mL of ethanol (50 % to 80 %) in glass bottles and placed on the shaker bath extractor (New BrunswickTM Excella® E24 Incubator Shaker) at a constant speed of 200 min−1 . The selection of ethanol concentration was based on our preliminary experiments, which showed that 50 % to 80 % ethanol is the optimal range. The solvent extraction was performed at a fixed temperature of 30 ◦ C and different retention times of 1 h–24 h. Here, the conditions yielding the maximum extraction efficiency was taken as the optimum conditions. The extracts were centrifuged, filtered under vacuum, and stored at 4 ◦ C for further analysis. 2.4. Total phenolic content (TPC) The TPC was determined following the procedures described in Tang et al. [20] and Munir et al. [18]. Briefly, 25 × 10−3 mL of diluted ¯ kanuka leaf extracts and gallic acid standards were mixed with 125 × 10−3 mL of 10-fold freshly diluted Folin-Coilcalteu’s reagent in 96 well plates. After 8 min, 125 × 10−3 mL of 7.5 % sodium carbonate was added, and the plates were incubated in the dark for 60 min at room temperature. Well-plate vortex equipment was used to carry out the necessary mixing. The absorbance was measured at 765 nm using an ultraviolet-visible (UV–vis) microplate reader (PerkinElmer 2300 EnSpire Multimode Reader, USA). The total phenolic content in obtained extracts was calculated using the standard curve of gallic acid (50 mg/L to 500 mg/L) and expressed as mil¯ ligram gallic acid equivalent per gram dry kanuka leaves (mg GAE/g dw). 2.5. Total flavonoid content (TFC) ¯ The TFC of kanuka leaf extract was determined based on the pro¯ cedure described in Tang et al. [20]. The kanuka extracts (25 × 10−3 mL) were mixed with 100 × 10−3 mL of distilled water and 10 × 10−3 mL of 5 % sodium nitrite. After 5 min, 15 × 10−3 mL of 10 % AlCl3 was added and allowed to stand for 6 min before adding 50 × 10−3 mL of 1 mol/L NaOH. Finally, distilled water (50 × 10−3 mL) was added, and the absorbance was measured against a reagent blank at 510 nm after 60 min incubation in the dark at room tem-

S. Essien, B. Young and S. Baroutian / J. of Supercritical Fluids 158 (2020) 104721

perature. Well-plate vortex equipment was used to carry out the necessary mixing. Results were expressed as milligram quercetin ¯ equivalents per gram dry kanuka leaves (mg QE/g dw).

for green and +a* for red) while b* denotes the yellow/blue value (–b* for blue and +b* for yellow). The browning index (BI) and the total colour change (E) were calculated using Eqs. (2) and (4), respectively [21,22].

2.6. DPPH scavenging activity The method of determining the DPPH free radical scavenging activity of the extract was adapted from Munir et al. [18]. Briefly, 200 × 10−3 mL of DPPH solution (dissolved in ethanol to a concentration of 40 mg/L) was added to a 96 well plate containing 10 × 10−3 mL of extract samples, standard solutions, or blank. The mixture was kept in the dark for 60 min, and the absorbance was measured at 517 nm using the same microplate reader. The scavenging capacity was calculated according to Eq. 1 %DPPH =

Ablank − Asample Ablank

× 100

(1)

where, Asample is the absorbance of the sample and DPPH at 517 nm; Ablank is the absorbance of DPPH without the sample at 517 nm. The assay was performed in triplicate. The results were expressed as ¯ leaves (mg TE/g dw). milligrams Trolox per gram dry kanuka 2.7. Ferric reducing antioxidant power (FRAP) The FRAP analysis was performed according to the method in ¯ Kheirkhah et al. [19]. The diluted kanuka leaf extract or trolox standards (10 × 10−3 mL) and FRAP reagent (200 × 10−3 mL) were mixed and incubated at room temperature for 60 min. The FRAP reagent was prepared by mixing TPTZ solution (10 mmol/L TPTZ in 40 mmol/L HCl), 20 mmol/L FeCl3 , and 300 mmol/L sodium acetate buffer (pH 3.6) at a ratio of 1:1:10. The absorbance was read at 593 nm against a reagent blank. The assay was performed in triplicate. ¯ The reducing power of the kanuka leaf extract was expressed as ¯ milligrams Trolox per gram dry kanuka leaves (mg TE/g dw). 2.8. Determination of individual bioactive compounds ¯ The identification of phenolic profile in kanuka leaf extracts was conducted using a Shimadzu Prominence HPLC apparatus with a diode array detector between 210 nm to 325 nm according to the ¯ compound’s maximum wavelength. Kanuka leaf extracts obtained at the optimum SSR condition (15 g/L) were used for this analysis. A Zorbax C18 column (4.6 mm × 150 mm, 5 × 10−3 mm pore size) at 30 ◦ C was used for phenolic separation. The binary mobile phase consisted of 0.1 % acetic acid in water (solvent A) and 0.1 % acetic acid in acetonitrile (solvent B) with gradient flow as follows: 5 % B at 0 min, 25 % B at 15 min, 40 % B at 20 min, 15 % B at 25 min and to 5 % at 26 min to equilibrate before next injection. The flow rate and injection volume were 1 mL/min and 10 × 10−3 mL, respectively. Peaks were monitored at 220 nm, 280 nm, 325 nm for different phenolic compounds. Phenolic compounds and flavonoids commonly identified in vegetal matrices like tea trees were used as standards. They include gallic acid, chlorogenic acid, catechin, quercetin, trans-ferulic acid, syringic acid, caffeic acid, 4hydroxybenzoic acid (4-HB), and 2-hydroxycinnamic acid (2-HC). These compounds were identified and quantified by comparing retention times of unknown peaks to those of external standard phenolic compounds. 2.9. Colourimetric analysis Colour measurements were conducted using a Kinoca Minolta CR-400 colourimeter calibrated on a standard white calibration plate with the D65 light standard. The CIE L*a*b* coordinates were measured. In the CIE L*a*b* coordinates, L* defines the lightness from 0 (black) to 100 (white), a* represents red/green value (–a*

3

BI =

100(x − 0.31) ×100 0.17

(2)

x=

at +1.75Lt 5.645Lt + aw − 3.012bt

(3)

where, aw is the initial colour measurement of calibration white plate and Lt , at , and bt are the colour measurements at the specified extraction conditions.



E =

2

2

(L∗ ) + (a∗ ) + (b∗ )

2

(4)

2.10. Analysis of Maillard reaction products The formation of Maillard reaction products was studied by measuring the absorbance changes at 420 nm and the concen¯ tration of 5-hydroxymethylfurfural (5-HMF). Kanuka leaf extracts obtained at the optimum time and SSR conditions (20 min and 15 g/L) for all levels of temperature were analysed. The absorbance changes at 420 nm were measured following the procedure explained in Lee et al. [23]. The Shimadzu Prominence HPLC system and a Zorbax C18 column (4.6 mm × 150 mm, 5 × 10−3 mm pore size) at 30 ◦ C was used to quantify 5-HMF. The mobile phase was a mixture of solvent A (0.1 % acetic acid in water) and solvent B (0.1 % acetic acid in acetonitrile). The flow rate of the mobile phase in the gradient program was 1 mL/min, and the injection volume was 10 ␮L. The results were expressed as mg/100 ¯ leaves. g dried kanuka 2.11. Statistical analysis All experiments were conducted in three replications, and the results were presented as mean values ± standard deviation. Analysis of variance (ANOVA) was performed, and the difference between means was analysed using Duncan’s method of multiple comparisons. Statistical significance was considered at P <0.05. Correlation between bioactive content and antioxidant activity was analysed using Pearson’s correlation test. All statistical analysis was performed using SigmaPlot (version 12, Systat Software, Inc). 3. Results and discussion 3.1. Subcritical water extraction The effects of extraction temperature, solid-to-liquid ratio, and extraction time on the quality of the extract were studied. The indicators of extract quality used were the antioxidant content (TPC and TFC), antioxidant capacity (DPPH and FRAP), and MRP formation. A ¯ pictorial view of the control and SWE kanuka leaf extracts are displayed as supplementary data (Figure A.1). The results of TPC, TFC, and antioxidant activity of the control samples are presented in Table 1, while Figs. 1 and 2 show the variation in polyphenol con¯ tent and antioxidant activity of kanuka leaf extracts during SWE. ¯ extract increased From these results, the TPC and TFC of the kanuka from 39.39 mg ± 0.77 mg GAE and 91.52 mg ± 5.28 mg QE, respectively for the control samples, to 172.81 mg ± 1.9 mg GAE, and 405.4 ¯ mg ± 8.47 mg QE per g dry kanuka leaves, respectively when SWE was applied. Likewise, the antioxidant activities of the extracts also increased by more than 4-fold for both FRAP and DPPH when SWE was used. These results indicate that temperature less than 170 ◦ C greatly influenced the solvation power of water. Moreover, the highest antioxidant activity was obtained at similar SWE conditions

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Table 1 ¯ leaf control sample. TPC, TFC, DPPH, and FRAP of kanuka Solid-Solvent ratio (g/L)

TPC(mg GAE/g dw)

TFC(mg QE/g dw)

DPPH(mg TE/g dw)

FRAP(mg TE/g dw)

15 25 35

44.12 ± 0.70a 41.62 ± 0.89ab 39.39 ± 0.77b

91.52 ± 5.28 a 54.53 ± 2.42 b 38.22 ± 2.8 c

109.78 ± 3.54a 69.41 ± 1.53b 50.38 ± 0.72c

246.37 ± 2.70a 142.02 ± 2.72b 110.33 ± 5.54c

Results are expressed as means ± standard deviation. Means with different letters in a column are significantly different at P = 0.05.

Fig. 1. The effect of extraction temperature [150 ◦ C(䊏), 170 ◦ C (䊉), 190 ◦ C (), 210 ◦ C ()], extraction time and SSR [15 g/L (A, D), 25 g/L (B, E), 35 g/L (C,F)] on total phenolic ¯ content (TPC) and total flavonoid content (TFC) in kanuka leaf extracts during subcritical water extraction. Values are mean ± standard deviation (n = 6).

Fig. 2. The effect of extraction temperature [150 ◦ C(䊏), 170 ◦ C (䊉), 190 ◦ C (), 210 ◦ C ()], extraction time and SSR [15 g/L (A, D), 25 g/L (B, E), 35 g/L (C,F)] on DPPH and FRAP ¯ activity of kanuka leaf extracts during subcritical water extraction.Values are mean ± standard deviation (n = 6).

S. Essien, B. Young and S. Baroutian / J. of Supercritical Fluids 158 (2020) 104721

5

Table 2 Pearson correlation analysis results. Assay

SSR

Subcritical water extraction −0.593** TPC TFC −0.172** FRAP −0.740** −0.910** DPPH Ethanol extraction −0.331** TPC 0.083 TFC FRAP −0.786** DPPH 0.974**-

EtOH

Temperature

Time

TPC

TFC

FRAP

DPPH

– – – –

0.456** 0.515** 0.231** 0.179**

−0.260** −0.323** −0.214** −0.104

1 0.831** 0.914** 0.808**

– 1 0.709** 0.492**

– – 1 0.898**

– – – 1

−0.531** −0.118 −0.383** −0.002

– – – –

0.119 0.311** 0.013 −0.018

1 0.279** 0.775** 0.317**

– 1 0.104 0.144

– – 1 0.768**

– – – 1

*Correlation is significant at the 0.05 level (2-tailed). ** Correlation is significant at the 0.01 level (2-tailed).

where the highest TPC and TFC were obtained. Similar results were reported in Munir et al. [18], Kheirkhah et al. [19], and Yan et al. [24], which suggests the existence of a correlation between the TPC, TFC, DPPH, and FRAP. Table 2 shows data obtained from the linear correlation analysis using the two-tailed Pearson correlation. A significant positive correlation was observed for TPC and TFC in SWE samples with their DPPH and FRAP activity, respectively, indicating the involve¯ ment of these compounds in the antioxidant activity of kanuka leaves extracts. Phenolic and flavonoid compounds are well-known for their antioxidant potential; hence are often used to determine the antioxidant activity of plant extracts. The antioxidant activity was mainly contributed by phenolic acids, as seen in the correlation analysis. A strong positive correlation (r = 0.831) existed between TPC and TFC as expected since TPC represents the total polyphenols content, including flavonoids. Negative yet significant correlations were obtained between the extraction variables (time and SSR) and all extract quality indicators in support of experimental observations. There was a significantly positive correlation between extraction temperature and TPC, FRAP, TFC and DPPH ¯ extracts. activity in the SWE kanuka Temperature and SSR were the most significant operating parameters, and a statistically significant interaction between both parameters (P ≤ 0.001) was obtained. Discussion on their respective effects are detailed in Sections 3.1.1 through 3.1.3. For ethanol extracts, the correlation between TPC and TFC with antioxidant activity was significant for the former but not for the latter. This correlation was weak for DPPH activity but moderate to strong for FRAP values. It may be that the assay was not sensitive enough, especially since ethanol was used in dissolving the radical during analysis. Regarding extraction variables, these results support the idea that variation in extraction time did not have much significant effect on the extract quality.

3.1.1. Effect of solid-to-solvent ratio (SSR) The solid-to-solvent ratio had a significant impact (P < 0.05) on the extract properties. It provides the driving force for mass transfer, which is the concentration gradient between the solid sample ¯ leaves) and the bulk solvent (water) [25]. Increasing the (kanuka SSR decreased the concentration gradient resulting in a decrease in the diffusion rate. As observed in the control sample (Table 1), the TPC and TFC decreased when SSR increased from 15 g/L to 35 g/L at room temperature. Same trend was observed for subcritical water extracts (Fig. 1). The margin of difference between the ratio effects reduced as the SSR increased for SWE. These results are in good agreement with results reported in Wu et al. [26], Gong et al., (2015), and Yan et al., (2019). Similar to the TPC and TFC, a significant (P < 0.05) increase in the antioxidant capacity for DPPH scavenging and FRAP, with a decrease in SSR, were observed (Fig. 2). The DPPH inhibition concentration for all SSR

studied was higher than the minimum inhibition concentration of 50 % (IC50 ), which was 0.104 mg TE/mL. According to mass transfer principles, SSR also influences the equilibrium condition of the SWE process thus, it affects the solubility of solutes during SWE. The equilibrium concentration for TPC and TFC was dependent on the duration of extraction and the process temperature. Furthermore, Table 3 demonstrates significant changes in measured and calculated colour properties with respect to variation in SSR. An understanding of the effect of SSR in phytochemicals extraction, and proper selection thereof will aid in the efficient usage of solvent, boost cost-effectiveness through solvent waste reduction, help prevent diluted solutions that may require extra concentration steps. Based on these observations, the ideal SSR was selected as 15 g/L.

3.1.2. Effect of time A progressive darkening of the extracts with an increase in extraction time from 0 min to 40 min, perceptible through close observation and at a glance, can be seen in Figure A.1; and is also indicated by the increase in the total colour change (E) with time ¯ leaf extracts increased (Table 3). The TPC and TFC of the kanuka when extraction time increased from 0 min to 20 min beyond which these concentrations decreased (Fig. 1). Within this 20 min period, an increase in TPC and TFC occurred for 150 ◦ C and 170 ◦ C but a gradual decrease was recorded for 190 ◦ C and 210 ◦ C. Prolonged exposure time to each heating temperature, with an exception for 150 ◦ , resulted in a decrease in TPC, TFC, and antioxidant activity of the extracts. This behaviour is often associated with degradation of already extracted compounds as a result of side reactions since bioactive compounds are thermo-liable [27]. Moreover, the maximum concentration of solutes the solvent can contain in the batch-type extraction had been reached, so the rate of mass transfer declined accordingly. A similar phenomenon was observed for DPPH and FRAP capacity of the extract (Fig. 2). The exception in trend for the time at 150 ◦ signifies that the equilibrium concentration was not yet achieved. Extraction time is one of the key parameters in the recovery of bioactive compounds as it influences the yield of valuable compounds, the volume of solvent, energy and operating cost of the process [22,28]. Statistically, the overall effect of time was significant (P < 0.05) for all qualities. But based on the results of multiple comparison tests, change in extraction time between 0 min and 20 min was not significant (P > 0.05) for TPC, TFC, DPPH, and FRAP. Increase in extraction time beyond 20 min was found to be statistically significantly on these extract properties. According to these results, a moderate extraction time of 20 min was selected, taking energy savings as well as phenolic degradation into consideration. In general, increasing extraction time decreased the TPC, TFC and ¯ the DPPH and FRAP activity of kanuka leaf extract. The time which this decline began and its magnitude thereof, depended mainly on

6

Temp. (◦ C)

Time (min)

0 5 10 20 30 40 0 5 170

10 20 30 40 0 5

190

10 20

25 g/L a*

b*

−0.94 ± 0.55 3.3 ± 0.79 24.24 ± 0.19 a.A −0.51 ± 0.09a.A 2.38 ± 0.11a.A ac.A ab.A 23.96 ± 0.1 −0.14 ± 0.15 2.85 ± 0.13ba.A 23.93 ± 0.51 ac.A −0.16 ± 0.17ab.A 2.66 ± 0.26ba.A

E

BI

–

– 1.05 ± 0.12a.A 8.08 ± 0.13a.A a.A 1.04 ± 0.1 12.03 ± 1.3b.A 1.21 ± 0.23a.A 11.14 ± 2.19b.A

23.41 ± 0.17 bc.A 0.28 ± 0.18bd.A 22.78 ± 0.24 b.A 0.94 ± 0.09c.A 22.8 ± 0.2 b.A 0.55 ± 0.27cd.A

3.23 ± 0.13b.A 1.6 ± 0.21ac.A 16.23 ± 1.41c.A 2.93 ± 0.23ba.A 2.53 ± 0.21b.A 18.41 ± 1.12c.A 3.01 ± 0.08b.A 2.23 ± 0.24bc.A 16.89 ± 1.74c.A 23.69 ± 0.44 a.A −0.05 ± 0.12a.B 2.68 ± 0.44a.A 1.41 ± 0.18a.A 11.89 ± 2.85a.B 22.89 ± 0.27 b.B 0.44 ± 0.45b.B 22.88 ± 0.42 b.B 0.6 ± 0.39b.B 22.28 ± 0.48 bc.B 1.27 ± 0.48c.B 21.76 ± 0.41 c.B 1.13 ± 0.34c.A 22.54 ± 0.8 b.AB 1.07 ± 0.34c.B 22.81 ± 0.59 a.B 0.68 ± 0.5a.C 22.07 ± 0.45 b.C 1.1 ± 0.19a.C 21.39 ± 1.23 b.C 0.94 ± 0.43a.BC 21.69 ± 0.49b.BC 1.1 ± 0.27a.B

2.84 ± 0.23a.A 2.13 ± 0.51b.B 15.46 ± 1.89ac.B 2.98 ± 0.06a.A 2.21 ± 0.56b.B 16.94 ± 2.55c.B 2.39 ± 0.51a.B 3.22 ± 0.79c.B 17.75 ± 0.25b.A 2.59 ± 0.4a.A 3.49 ± 0.28c.B 18.5 ± 0.66bc.A 2.7 ± 0.09a.A 2.86 ± 0.66c.B 2.93 ± 0.02a.A 2.36 ± 0.56a.B 2.59 ± 0.33a.A 3.2 ± 0.53b.C

18.13 ± 2.06c.A 17.13 ± 2.65a.C

0 5

20.7 ± 0.19a.D 1.12 ± 0.15ac.C 21.04 ± 0.44a. C 1.11 ± 0.16ac.C 21.41 ± 0.01ab.C 1.07 ± 0.47abc.B 3.23 ± 0.36bc.A 3.65 ± 0.28ac.B 22.08 ± 4.28b.B 21.85 ± 0.23b.B 0.69 ± 0.1cb.A 3.69 ± 0.46c.B 3.1 ± 0.16bc.BA 22.09 ± 2.04bc.B 22.12 ± 0.18b.AB 0.6 ± 0.3b.BA 3.79 ± 0.29c.B 2.83 ± 0.26b.BA 21.86 ± 2.95c.B

30

E

BI

L*

a*

24.41 ± 0.45 23.3 ± 1.78a.A

−1.12 ± 0.24 4.02 ± 0.73 – 0.02 ± 0.18a.A 3.61 ± 0.98a.A 2.16 ± 1.09a.A a.AC a.A 0.37 ± 0.34 3.52 ± 0.75 2.34 ± 0.93a.A 0.43 ± 0.34a.B 3.13 ± 0.74a.A 2.61 ± 0.97a.A

17.35 ± 6.87a.A 18.75 ± 6.14a.A

0.7 ± 0.69ba.B 3.01 ± 0.84a.A 2.96 ± 0.9a.A 1.11 ± 0.36b.A 2.92 ± 0.3a.A 3.32 ± 0.71a.A 1.08 ± 0.45b.A 2.59 ± 0.42a.A 3.14 ± 0.79a.A 1.36 ± 0.15a.B 2.43 ± 0.57a.B 3.9 ± 0.49a.B

19.75 ± 3.88a.A 17.5 ± 0.74a.A

−1.26 ± 0.36 23.24 ± 0.25a.A −0.12 ± 0.27a.AB a.A 22.94 ± 0.46 0.38 ± 0.6b.A 22.86 ± 0.41a.A 1.04 ± 0.61c.AC

3.91 ± 0.78 – – 3.47 ± 0.07a.A 1.51 ± 0.29a.A 15.55 ± 1.32a.A a.A ab.A 3.43 ± 0.2 2.08 ± 0.73 18.14 ± 3.37a.A 3.01 ± 0.12ab.A 2.79 ± 0.57bc.A 19.22 ± 3.17b.A

22.31 ± 0.4ab.A 1.64 ± 0.15d.A 22.31 ± 0.24ab.A 1.3 ± 0.46d.CA 21.73 ± 0.06b.AB 1.59 ± 0.04d.A

2.73 ± 0.28b.A 3.62 ± 0.23c.A 3.07 ± 0.3ab.A 3.24 ± 0.57c.A 2.07 ± 0.67c.A 4.17 ± 0.3ab.A

22.67 ± 0.27a.AB 0.4 ± 0.48a.A 22 ± 0.26ab.B 0.78 ± 0.45a.B 21.47 ± 0.13bc.B 1.43 ± 0.29b.A

2.95 ± 0.12a.AB 2.4 ± 0.49a.B 2.76 ± 0.19a.B 3.16 ± 0.45ab.B 17.41 ± 1.63ab.A 20.71 ± 0.33b.BC 2.46 ± 0.2ab.AB 4.04 ± 0.33b.B 19.65 ± 0.98ab.A 20.21 ± 0.17b.B 2.03 ± 0.56b.B 4.41 ± 0.69ab.AB 18.47 ± 1.99b.B 20.04 ± 0.05b.B 2.08 ± 0.48b.B 4.83 ± 0.26b.B 19.63 ± 2.42ab.AB 19.92 ± 0.07b.B

21.38 ± 0.68bc.B 1.63 ± 0.13b.A 1.67 ± 0.14b.A

20.74 ± 0.21c.B 20.96 ± 0.09c.B

1.54 ± 0.12b.A 1.62 ± 0.22a.AB

2.07 ± 0.31b.A 4.6 ± 0.16ab.A 2.32 ± 0.39a.B 3.91 ± 0.36a.C 2.18 ± 0.43a.B 4.18 ± 0.38a.C

21.34 ± 0.84b.A 21.4 ± 1.03b.A 18.24 ± 3.35b.A 15.95 ± 2.6a.A

20.89 ± 0.29b.B 1.27 ± 0.2a.A 21.24 ± 0.2ab.C 1.14 ± 0.15ab.B 21.26 ± 0.37ab.B 1.26 ± 0.19b.D 20.57 ± 0.58a.B 20.64 ± 1.32a.B 22.1 ± 1.59bc.A 22.4 ± 1.2c.A

1.32 ± 0.07b.B 1.17 ± 0.22ab.A 0.88 ± 0.27ab.A 0.69 ± 0.63a.B

22.96 ± 1.24a.A 22.85 ± 1.43a.A 22.62 ± 1.2a.A 22.31 ± 0.93a.A 22.7 ± 0.54a.A 21.88 ± 0.34a.B

b*

1.26 ± 0.12a.BC 1.94 ± 0.56a.B 1.16 ± 0.09a.A 1.74 ± 0.54a.B 1.34 ± 0.16a.A 1.75 ± 0.41a.B

18.57 ± 1.45b.A 18.8 ± 2.41b.A

1.19 ± 0.12a.A 1.66 ± 0.22a.B 19.9 ± 0.18b.B 1.39 ± 0.15a.A 1.88 ± 0.05a.A 22.24 ± 0.53a.BA 1.33 ± 0.65ab.B 2.72 ± 0.39a.AB 21.74 ± 0.19a.B 1.72 ± 0.21b.B 2.15 ± 0.16a.B 21.68 ± 0.47a.C 1.43 ± 0.17ab.A 2.38 ± 0.55a.AB

17.47 ± 0.6c.B 17.35 ± 2.84c.B

21.43 ± 0.14a.C 21.6 ± 0.29a.A

18.61 ± 1.25a.A 19.38 ± 0.94a.B

2.07 ± 0.26a.A 4.48 ± 0.42a.A 17.27 ± 0.33c.A 2.62 ± 0.12a.B 3.95 ± 0.21a.C 19.19 ± 1.01a.B 2.37 ± 0.47a.B 4.11 ± 0.46ab.C 18.51 ± 1.53.A 2.18 ± 0.29a.B 4.71 ± 0.5ab.B 18.38 ± 1.59a.A

1.26 ± 0.1a.D

40

24.1 ± 0.69

b*

2.71 ± 0.27a.A 3.22 ± 0.34b.B 17.99 ± 0.91ac.A 2.73 ± 0.57ab.A 4.24 ± 0.12a.C 20.85 ± 3.14a.D 2.49 ± 0.37a.A 4.35 ± 0.28a.D 18.94 ± 1.29a.C 3.03 ± 0.32ab.A 3.97 ± 0.44a.C 21.54 ± 2.43a.C

1.1 ± 0.24a.A 0.93 ± 0.39a.B

20.88 ± 0.06a.C

20

35 g/L a*

2.22 ± 0.55a.B 4.22 ± 0.24a.B 1.91 ± 0.34a.B 4.67 ± 0.32a.B 2.06 ± 0.75a.B 4.54 ± 0.62a.B

21.61 ± 0.37b.B 21.93 ± 0.51b.B

10

L*

22 ± 0.27a.BC 18.09 ± 0.53ac.BC 21.52 ± 0.06ab.B 1.52 ± 0.28a.C 2.94 ± 0.53a.A 3.69 ± 0.94b.C 19.88 ± 2.37bc.BC 21.43 ± 0.34ab.B 1.5 ± 0.18a.B 2.69 ± 0.27a.AB 3.48 ± 0.56b.B 18.97 ± 1.61b.A 20.9 ± 0.1b.B 1.48 ± 0.23a.A 2.37 ± 0.48a.A 3.61 ± 0.53b.B 17.35 ± 1.03c.A 20.86 ± 0.32b.B 1.29 ± 0.18a.A

40

30

210

L* 24.43 ± 0.86

Control

150

15 g/L

2.16 ± 0.13a.AB 4.61 ± 1.16ab.B 17.51 ± 1.81b.B 2.69 ± 0.58a.BA 3.33 ± 1.2ab.A 17.49 ± 0.71c.B 2.56 ± 0.37a.A 2.99 ± 1.17a.B 15.72 ± 3.03c.A

E

–

17.23 ± 6.42a.A 18.1 ± 6.33a.A

18.73 ± 2.13a.A

4.89 ± 0.11ab.BC 16.75 ± 3.79a.A 5.31 ± 0.23b.B 15.47 ± 2.58a.A 5.51 ± 0.22b.B 16.65 ± 2.46a.A 5.58 ± 0.11b.B 5.59 ± 0.09b.B

15.41 ± 1.33a.A 17.72 ± 0.79a.A

3.52 ± 0.92a.B 4.33 ± 0.25a.C

19.82 ± 1.8a.A 19.22 ± 0.52a.A

4.1 ± 0.59a.C 1.58 ± 0.18ab.A 2.02 ± 0.36a.B 4.5 ± 0.34a.B 1.4 ± 0.29ab.B 1.94 ± 0.84a.BC 4.34 ± 0.73a.A 21.78 ± 0.12a.AC 1.07 ± 0.23a.A 2.47 ± 0.28a.A 3.77 ± 0.23a.AC a.C 20.09 ± 0.2 0.84 ± 0.14a.C 1.1 ± 0.21a.C 5.58 ± 0.2a.C 19.85 ± 0.4a.C 0.83 ± 0.36ac.A 1.33 ± 0.63a.B 5.67 ± 0.47a.B 20.59 ± 0.51ba.BC 1.11 ± 0.25a.A 2.1 ± 0.87b.B 4.87 ± 0.6ab.BC 20.97 ± 0.2ba.BC 1.19 ± 0.28a.A 2.37 ± 0.44b.AB 4.48 ± 0.25b.B 21.56 ± 1.62b.A 0.78 ± 0.64a.B 2.63 ± 1.13b.AC 3.75 ± 1.93b.A 21.05 ± 0.21ba.C 1.14 ± 0.1a.A

BI

2.65 ± 0.73b.A 4.31 ± 0.35b.C

19.01 ± 2.06a.A 18.11 ± 1.23a.A 16.64 ± 2.83a.A 17.57 ± 1.54a.A 10.38 ± 1.73a.B 11.67 ± 5.12ac.BC 16.82 ± 5.56bc.A 18.38 ± 1.36b.A 17.09 ± 1.39b.A 19.6 ± 4.35b.A

Results are expressed as mean ± standard deviation. Means followed by lower case letter within a column per temperature and means followed by same upper case letter within a row per time are not significantly different at (p < 0.05).

S. Essien, B. Young and S. Baroutian / J. of Supercritical Fluids 158 (2020) 104721

Table 3 ¯ leaf extracts on temperature, time and solid-solvent ratio. Dependence on colour properties of kanuka

S. Essien, B. Young and S. Baroutian / J. of Supercritical Fluids 158 (2020) 104721

7

the temperature and the solid-to-solvent ratio. Therefore, careful consideration of these parameters should be taken into account when deciding the optimal extraction time. 3.1.3. Effect of temperature Temperature is a crucial factor in the selective extraction of bioactive compounds under subcritical water conditions. Increase in temperature disrupts and hydrolyses the plant matrix, decreases the viscosity, surface tension, dielectric constant, and consequently, the polarity of water [29,30]. As described in Fig. 1, TPC and TFC increased as the extraction temperature increased up to 170 ◦ C. When the temperature increased from 23 to 170 ◦ C, the dielectric constant (␧) of subcritical water significantly decreases from 78 to 39 nearing that of methanol (␧ = 33) or ethanol (␧ = 25) [31]. These changes facilitate solubility of phenolic and flavonoid compounds in subcritical water, thereby enhancing extraction rate and efficiency [22,28]. Further increase in temperature, in this case from 190 ◦ C, caused a considerable reduction in TPC and TFC of all the ¯ kanuka leaf extracts possibly due to degradation of some phenolic compounds, reaction with other components, or molecular transformation at such high temperature. An exception in this trend is shown in Fig. 1 (C) where TPC at 35 g/L decreases for all temperature as the extraction progresses with time. This is also based on the same reason as previously discussed. The antioxidant activity of ¯ extracts increased with increase in temsubcritical water kanuka perature up to 170 ◦ C and a substantial decrease was recorded with further increase in extraction temperature. The steepness depended on the SSR with 15 g/L showing the most significant changes with temperature. This trend has also been reported in related studies on SWE using different plant materials [16,27,32]. The difference in DPPH between 150 ◦ C and 170 ◦ C was slightly above the significance level. Similar observations were noted for FRAP but, the differences between each temperature at an SSR of 35 g/L was insignificant. The extraction temperature of 170 ◦ C gave the maximum TPC (172.8 mg ± 2 mg GAE/g dw), TFC (405.4 mg ± 8.5 mg QE/g dw), and antioxidant activity (DPPH: 223.8 mg ± 2.7 mg TE/g dw; FRAP: 478.8 mg ± 2.1 mg TE/g dw). This indicates the ¯ kanuka leaf phenolics are better recovered in subcritical water at relatively moderate temperature. Regarding colour intensity, the control sample had a light greenish colour as indicated by their negative a* values. (Table 3). The a* value increased with temperature, denoting an increase in redness, up to 190◦ where it began to decrease. The effect of temperature on the b* value, although significant, varied with time and SSR. ¯ On average, the b*value of the kanuka leaf extracts decreased with temperature but remained in the positive range, meaning the yellowness was maintained. There was also an overall decrease in L* value with increase in temperature and SSR. These changes meant ¯ that the kanuka leaf extracts followed a colour pattern from a yellowish-green colour to a more reddish -ellow colour. According to Tomˇsik et al. [22] and Lee et al. [33], this colour change signifies a non-enzymatic Maillard browning and the formation of brown pigments. Tomˇsik et al. [22] had suggested that L* value be used as an indicator of browning. At these high temperatures, a characteristic burning smell was perceived as representative of possible degradation of phenolic compounds due to higher heating intensity [34]. From the obtained results, extraction temperature had ¯ marked effects on the antioxidant content and activity of kanuka leaf extracts obtained from SWE. 3.2. Comparison between subcritical water extraction and ethanol extraction Phenolic compounds are polar compounds that can be extracted by solid-liquid extraction using organic solvents like ethanol. The comparison of subcritical water extraction with ethanol extraction

Fig. 3. Subcritical water extraction ( ) versus ethanol extraction ( ) samples at optimum conditions versus control sample ( ). The units are mg GAE/g for TPC, mg QE/g for TFC, and mg TE/g for DPPH and FRAP.

¯ for recovery of bioactive kanuka leaf extract at optimum conditions are presented in Fig. 3. An increasing trend was observed in TPC, TFC and antioxidant activity when ethanol concentration increased up to 60 %. Above 60 %, these concentrations remained reasonably constant for TFC but decreased for TPC and antioxidant activity. Also, prolonging the extraction time beyond 8 h did not cause any significant changes in TPC, TFC, and antioxidant activities. Therefore, the highest yield of bioactive compounds, TPC (83.0 ± 1.56) mg GAE/g dw and TFC (140.3 ± 9.42) mg QE/g dw, with high antioxidant activities were obtained for ethanol extraction at 8 h using 60 % ethanol at 15 g/L. Fig. 3 showed a remarkable improvement in yield when subcritical water was utilised. With the same SSR, SWE at 170 ◦ recovered twice as much phenolic compounds and nearly thrice as much flavonoid compounds than ethanol extraction. A substantial increase in antioxidant DPPH and FRAP activity was also observed when SWE was applied. Moreover, the extraction time for SWE (20 min) was over twentyfold shorter than the time needed for ethanol extraction (8 h) to obtain its maximum composition. A similar conclusion was reached for other plant products like liquorice [28]. As previously stated, the high temperature of subcritical water enables thermodynamic changes that enhances the recovery of these bioactive compounds. Together, these outcomes make SWE a more effective technique for the recovery of bioactive compounds ¯ from kanuka leaves. Besides, it has excellent advantages of utilising eco-friendly and non-toxic solvent. 3.3. Maillard reaction product level The complexity of non-enzymatic browning reaction makes it difficult to describe its kinetic characteristics clearly. However, absorbance at 420 nm and the concentration of 5-HMF are often used to study this reaction. Measurement of absorbance at 420 nm is done to monitor the browning intensity caused by brown polymeric substances, such as melanoidins, which are formed at the final phase of Maillard reaction [35]. This subset of Maillard reaction products contributes to the colouration of many processed food. Fig. 4 (A) shows the absorbance measurement data obtained for ¯ SWE kanuka leaves extracts. The brown colour intensity of these extracts increased with elevation in temperature supporting the occurrence of Maillard reaction during SWE processing. A similar trend is recorded in most of the literature [19,23]. Fig. 4 (B) shows chromatogram of 5-HMF as detected by HPLC¯ DAD while Fig. 4 (C–D) shows 5-MHF levels in the kanuka leaves extracts. 5-HMF is a popular marker for Maillard reaction and an

8

S. Essien, B. Young and S. Baroutian / J. of Supercritical Fluids 158 (2020) 104721

¯ Fig. 4. (A) Absorbance changes at 420 nm for kanuka leaf extract from SWE at different temperature for 20 min extraction time and 15 g/L SSR. (B) HPLC detection of 5-HMF ¯ leaf extract (15 g/L) at different extraction temperature [150 ◦ C(䊏), 170 ◦ C (䊉), 190 ◦ C (), 210 ◦ C ()], and time; (D) at at 285 nm; (C) Concentration of 5-HMF in kanuka optimum extraction time (20 min).

important end product in acid conditions. It should be noted that ¯ the pH for the kanuka leaf extracts was in the range of 3.81 – 4.18. 5-HMF is used as flavouring agents in the food industry; it also plays essential roles in the production of several industrial materials like diesel-like fuel, resins [36,37]. The data in Fig. 4 (C–D) show that 5-HMF was not detected in SWE extracts at 150 ◦ C but significantly ¯ leaves, with (p < 0.05) increased, up to 368 mg/100 g dried kanuka increasing extraction temperature and static extraction time. A significant increase in 5-HMF content was observed at 210 ◦ C after 10 min. These results are in agreement with results from Kheirkhah et al. [19] who also found that the presence of this compound did not contribute to the antioxidant nor antiproliferative activity of their extract contrary to popular belief. Several concerns have been raised regarding the effect of 5-HMF on health. Some 5HMF metabolites have been found to be genotoxic, yet in vitro data found that it poses no adverse effects on human health [38,39]. Maillard reaction provides unique aroma profiles and desired changes in food quality parameters but, too much of it results in some undesirable changes such as protein glycation in health and medical sciences [40,41]. 3.4. Identification of bioactive compounds Table 4 shows the variations in the concentration of the selected ¯ bioactive compounds during SWE from kanuka leaves. Their LC chromatograms are presented as supplementary data. From the results, gallic acid and catechin first increased then decreased as extraction temperature increased, which is similar to observations recorded for TPC and TFC in this study (3.1.2 and 3.1.3). This trend is expected since these compounds are often used as standards for TPC and TFC analysis. They had the highest concentration amongst compounds of interest for this study. The same trend was observed with increase in extraction time for gallic acid while a decrease in concentration was noted for catechin. Catechin and trans-ferulic

acid exhibited the same trend that was observed for TFC and TPC. Others like syringic acid increased with increasing temperature while the remaining compounds decreased with increase in temperature. From the results, syringic and 4-hydroxybenzoic acids were not present in the control sample but were identified in SWE extracts (Table 4). These results illustrate the capacity of subcritical water to extract compounds that were not soluble in water at room temperature. The main reason is due to a reduction in the polarity of water as temperature increased, leading to its ability to solvate nonpolar compounds [42]. On the other hand, it is possible that some of the bioactive compounds may have formed as a result of the breakdown of larger compounds with the increase of subcritical water temper¯ ature [12]. Fig. 5 presents the chromatogram of kanuka extracts at optimum SWE conditions. Most of these compounds play vital roles in the formation or are derivatives of more complex bioactive compounds. For instance, syringic acid is a naturally occurring phenolic acid, and a derivative of gallic acid [43]. From the results, gallic acid was identified in the control extract, and the concentration decreased with increasing temperature while syringic acid increased sharply at higher temperature. These results demonstrate that the SWE enabled the breakdown of gallic acid to form syringic acid. The physiochemical characteristics, like the chemical structure and melting point of the compounds, also play a key role in its extraction, especially under high temperature. According to Ko et al. [44], some of these compounds can convert back to their isomers under high temperature and pressure conditions, and this conversion is reversible. In a more recent study, they noted an increase in 6-shogaol due to conversion of 6-gingerol as results of increasing temperature and time during SWE [34]. This mechanism can also be attributed to 2hydroxycinnamic acid, which recorded a sharp increase after 190 ◦ . The 2-hydroxycinnamic acid is said to be prevalent within plants in four basic molecules and is mostly derived from cinnamic acid.

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9

Table 4 ¯ leaves extract at different extraction temperature and time. Concentrations of individual bioactive compounds in SWE kanuka Time (min) Control sample 0 5 10 20 30 40 Control sample 0 5 10 20 30 40 Control sample 0 5 10 20 30 40 Control sample 0 5 10 20 30 40

150◦

170◦

Quercetin – 614 ± 48a.A 608 ± 21ab.A 602 ± 8a.A 652 ± 78a.A 603 ± 16a.A 650 ± 69ab.A 605 ± 30a.A 635 ± 59abc.A 618 ± 22a.A 684 ± 45bc.A 603 ± 71a.A 634 ± 12c.A Trans-ferulic acid 8.37 ± 1.8C 24 ± 3a.A 26 ± 2ab.A 25 ± 3a.A 28 ± 3ab.A 27 ± 3a.A 29 ± 4ab.A 27 ± 3a.AC 31 ± 4a.A 27 ± 1a.A 26 ± 3ab.A 28 ± 5a.AC 24 ± 2b.AB Catechin – 1585 ± 80a.A 326 ± 39a.B 896 ± 17b.A 235 ± 41b.B 294 ± 19c.A 757 ± 67c.B 211 ± 11d.A 733 ± 41cd.B 141 ± 18d.A 913 ± 0e.B 128 ± 15d.A 664 ± 99d.B Syringic acid – 222 ± 1a.A 64 ± 1a.AB 63 ± 0ab.A 0 ± 0a.A 0 ± 0b.B 0 ± 0a.B 0 ± 0b.A 73 ± 0a.A 0 ± 0b.A 93 ± 27a.AC 0 ± 0b.A 102 ± 25a.A

190◦

210◦

150◦

658 ± 84a.A 617 ± 40ab.A 636 ± 32ab.A 637 ± 60b.A 603 ± 23bc.A 584 ± 14b.A

640 ± 22c. B 616 ± 21d.A 593 ± 17da.A 604 ± 26a.A 575 ± 20ba.A 584 ± 8b.A

28 ± 1a.A 26 ± 4a.A 20 ± 5b.B 23 ± 5ab.BC 20 ± 4b.B 19 ± 9b.B

25 ± 2a.A 21 ± 2a.A 21 ± 2a.B 20 ± 1a.B 24 ± 1a.AB 23 ± 0a.BC

827 ± 0a.C 1086 ± 111b.C 865 ± 35a.C 844 ± 16a.C 575 ± 64c.C 335 ± 74d.C

991 ± 14a.D 914 ± 13a.A 423 ± 0b.D 495 ± 0b.D 466 ± 0b.D 465 ± 0b.D

0 ± 0c.B 82 ± 6cb.A 94 ± 14cb.AB 96 ± 3cb.A 182 ± 7ab.BC 225 ± 20a.B

81 ± 19a.AB 117 ± 15a.A 256 ± 5a.A 332 ± 1c.B 519 ± 41b.B 661 ± 52b.B

170◦

Chlorogenic acid 27.7 ± 0D 44 ± 3a.A 46 ± 4a.AB 47 ± 2ab.A 55 ± 7ac.A 49 ± 2ab.A 65 ± 4acb.A 51 ± 0ab.A 71 ± 6bcd.B 53 ± 1b.A 81 ± 7bd.B 58 ± 3b.A 91 ± 10d.B 2-hydroxycinnamic acid 32.75 ± 5.7B 124 ± 2a.A 128 ± 14a.A 115 ± 22a.A 146 ± 29a.A 134 ± 21a.A 121 ± 2a.A 134 ± 19a.A 117 ± 3a.A 143 ± 29a.A 64 ± 7a.A 190 ± 88a.A 67 ± 2a.B 4- hydroxybenzoic acid – 587 ± 139a.A 204 ± 41b.B 302 ± 19ab.A 303 ± 57ab.A 247 ± 19b.A 291 ± 74a.B 174 ± 72b.A 200 ± 50b.A 167 ± 63b.A 222 ± 18b.A 164 ± 49b.A 214 ± 32b.A Gallic acid 852 ± 0A 1139 ± 537a.A 1615 ± 46a.AC 1419 ± 42a.A 2472 ± 191b.B 2137 ± 107b.A 2872 ± 98b.B 2347 ± 287bc.AB 2749 ± 42b.A 2469 ± 196bc.B 2450 ± 35b.B 2872 ± 101b.B 2386 ± 89b.B

190◦

210◦

61 ± 8a.B 87 ± 19b.B 102 ± 18bcd.B 111 ± 11c.C 114 ± 7c.C 107 ± 18cb.BC

96 ± 10a.C 124 ± 10b.C 120 ± 23b.B 107 ± 14ab.C 110 ± 15ab.C 113 ± 21ab.C

103 ± 59a.A 101 ± 5a.A 135 ± 6a.A 110 ± 12a.A 117 ± 18a.A 104 ± 14a.B

56 ± 6a.A 75 ± 40a.A 96 ± 1a.A 105 ± 28a.A 85 ± 6a.A 83 ± 4a.B

217 ± 16a.AB 261 ± 34a.A 253 ± 61a.A 226 ± 43a.A 244 ± 25a.A 214 ± 35a.A

320 ± 56a.AB 266 ± 33a.A 266 ± 9a.AB 290 ± 54a.A 276 ± 28a.A 249 ± 0a.A

2787 ± 18a.B 2403 ± 78ab.B 2251 ± 110bd.A 1855 ± 201bc.B 1591 ± 4cd.C 1362 ± 84d.C

2711 ± 371a.C 1972 ± 54a.B 1650 ± 56ab.A 1091 ± 71b.C 0 ± 0c.A 0 ± 0c.A

¯ Values are means of triplicate injections ± standard deviation expressed as mg/100 g dry kanuka leaves. Means followed by lower case letter within a column per temperature and means followed by same upper-case letter within a row per time for each compound are not significantly different at (p < 0.05).

¯ Fig. 5. HPLC chromatogram (A - 220 nm, B - 280 nm, C - 325 nm) of the kanuka extracts after 20 min SWE at 170 ◦ C.

3.5. Selectivity of SWE The quantitative composition of the extracts allowed evaluation of the selectivity of SWE at the different extraction temperatures. Polyphenols with different polarities were identified in subcritical ¯ water extracts of kanuka. Fig. 6 shows the selectivity of SWE to ¯ recovering selected polyphenols from kanuka leaves. The dielectric constant of water indeed reduces with increase in temperature as well as the viscosity and surface tension [45]. These changes enhance the diffusion rate, especially of less polar compounds, in water. The sigma (␴) profile, which describes polarity of water and the selected compounds, are shown in Fig. 7. When the screening charge density exceeds ±0.0084 eA−2 in the ␴-profile, the molecule is considered sufficiently polar while compounds with peaks within that range (0.0084 < ␴ < 0.0084) eA−2 are non-polar. The two outer regions in Fig. 7 are the hydrogen

bonding regions. Quercetin had the highest peak in the non-polar region and small peaks in regions the hydrogen bonding regions. This means that it was the weakest for hydrogen bond interactions and also insoluble in water, which makes it the most non-polar of the nominated compounds. By changing the polarity of water, via increase in temperature from 150 ◦ C to 190 ◦ C, non-polar compounds, for instance, quercetin, were solubilised in water, making it one of the major components of the extracts. Moreover, syringic acid showed high peak in the non-polar region and relatively small in the hydrogen bonding areas. From the results, the concentration of syringic acid compound increased as the dielectric constant reduced due to increased temperature. In related studies, Gabaston et al. [46] observed improvements in yield of complex stilbenes dimers and tetramers with temperature increase. Mlyuka et al. [11], Fan et al. [28] and Ko et al. [44] also recorded similar observations.

10

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perature and time. A low SSR was favourable for the extraction of phenolic compounds. The results proved that degradation, and in general, the extraction efficiency was not only dependent upon temperature but also on the residence time and solid-to-solvent ratio. A strong interrelationship was noted between the antioxidant activity of SWE extracts and the polyphenol content. From the findings, SWE certainly affected the optical properties ¯ of kanuka extracts due to the release of phenolic compounds and possible degradation at elevated conditions. The efficiency of SWE was found to be significantly dependent on the extraction variables. The best condition for SWE was extraction temperature 170 ◦ C, SSR 15 g/L and extraction time of 20 min. In addition, the following phenolic compounds were identified in SWE extracts: gallic acid, quercetin, trans-ferulic acid, syringic acid, 4-hydroxybenzoic acid, chlorogenic acid, 2-hydroxycinnamic acid, catechin. These are well-established compounds known to provide various food and health benefits. Overall, SWE was more effective than traditional ethanol extraction for recovery of bioactive compounds from ¯ leaves. The information from this study is needed to prokanuka ¯ mote further research and the value-added potential of kanuka. This research satisfies the desire of using environmentally-friendly solvents to separate compounds with health-promoting ability from plant materials. Future studies will look into identifying more unique compounds present in this extract. Declaration of Competing Interest ¯ Fig. 6. Selectivity of SWE to recovering selected polyphenols from kanuka leaves. (A) selective extraction of quercetin ( ), catechin ( ), and gallic acid ( ); (B) selective extraction of chlorogenic acid ( ), trans-ferulic acid ( ), 2-HC acid ( ), and 4-HB acid ( ) and formation of 5-HMF( ). Dielectric constants (- - -) at 40 bar were obtained from Uematsu and Frank [31].

The authors declare no conflict of interest in the publication of this study. Acknowledgement ¯ communiThe authors acknowledge Dr Kiri Dell and the Maori ¯ for providing the samples utilised ties of East Cape (Te Tai Rawhiti) in this study. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.supflu.2019. 104721. References

Fig. 7. Sigma profile of chlorogenic acid ( ), gallic acid ( ), catechin ( ), 4-HB ( ), 2-HC ( ), caffeic acid ( ), quercetin ( ), syringic acid ( ), trans-ferulic acid ( ), 5-HMF ( ), and water ( ) at 25 ◦ C.

4. Conclusions The application of subcritical water extraction for recovery ¯ bioactive compounds from kanuka leaf extracts was investigated. Different extraction conditions were evaluated and found to have a significant effect on extracts. The results showed that the efficiency of SWE was significantly dependent on the extraction variables. The effect of time was strongly dependent on extraction temperature, the most dominant parameter in this SWE process. The yield of phenolic bioactives decreased with an increase in extraction tem-

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