Optimisation of asarone removal from Piper sarmentosum Roxburgh leaves using supercritical carbon dioxide extraction: the Box-Behnken design

Abstract

Piper sarmentosum is a herbaceous shrub with numerous pharmacological benefits. However, the presence of two toxic phenylpropanoids (α- and β-asarone) limits the medicinal usage of the plant. In this study, the extraction of three asarone isomers, namely α-, β-, and -asarone was optimised using supercritical carbon dioxide extraction (SC-CO₂) combined with Box-Behnken experimental design. Comparison of asarone contents in different conventional solvent extracts of P. sarmentosum leaves prior to and after SC-CO₂ extraction was performed. The SC-CO₂ method successfully maximised the extraction of α-, β-, and ɣ-asarone at P = 81.16 bar, T = 50.11°C, and t = 80.90 min, yielding 13.91% α-asarone, 3.43% β-asarone, and 14.95% ɣ-asarone. The SC-CO₂ residue of the leaves re-extracted with conventional solvents showed a significant decrease of asarone ranging from 45% to 100% (p<0.001) compared to their counterparts without SC-CO₂ treatment. α-, β-, and ɣ-asarone were completely removed in the ethanol extract of the residue. These findings suggested that the optimised SC-CO₂ extraction parameters may serve as a quick treatment step for the selective removal of asarone from P. sarmentosum to develop safer extracts for the food and nutraceutical industries applications.

Keywords:
Piper sarmentosum; Supercritical fluid extraction; Asarone; Optimisation; Box- Behnken; HPLC

INTRODUCTION

Piper sarmentosum Roxburgh, which belongs to the Piperaceae family, is a herbaceous shrub widely distributed in Southeast Asia with several pharmacological activities such as antioxidant (Sumazian et al., 2010), anti-amoebic (Sawangjaroen, Sawangjaroen, Poonpanang, 2004), antibacterial (Masuda et al., 1991), neuromuscular blocking (Ridtitid et al., 1998), anti-malaria (Rahman et al., 1999), hypoglycaemic (Steinrut, Itharat, 2014), anti- tuberculosis (Hussain et al., 2009), anticancer (Ariffin et al., 2009), and anti-angiogenic activity (Hussain et al., 2008). Several amide alkaloids, phenylpropanoids (α-, β, and ɣ-asarone, Figure 1), lignans, sterols, and flavonoids were identified from the plant (Parmar et al., 1997; Subramaniam et al., 2003). Among the chemical constituents reported, two phenylpropanoids, i.e., α- and β-asarone, were reported to exhibit insecticidal, fungicidal, and neuroprotective activities (Cho et al., 2002; Park, Kim, Ahn, 2003; Shenvi et al., 2011). Nonetheless, they were also reported having carcinogenic, cytotoxic, and genotoxic compounds (Unger, Melzig, 2012; Cartus, Schrenk, 2016). Both asarones are regulated in food and herbal products to ascertain product safety (European Medicine and Health Agency, 2005). Therefore, an extraction technique to reduce the asarone isomers in herbal material is important for developing safer extracts in herbal remedy preparation.

FIGURE 1
Chemical structure of (1) α-asarone, (2) β-asarone, (3) ɣ-asarone.

Supercritical fluid extraction using carbon dioxide (SC-CO₂) as extraction solvent has become a popular alternative extraction technique over traditional liquid- solvent-based extraction, as it offers a short extraction time, leaving no solvent residue in the extract, and could extract thermally labile compounds under mild conditions (Lang, Wai, 2001). SC-CO₂ has been applied to extract medicinal constituents such as carotenes and alkaloids from natural products besides extracting essential oils, flavour, and fragrance compounds (Capuzzo, Maffei, Occhipinti, 2013). In addition, SC-CO₂ could selectively extract compound of interest from the sample matrix by adjusting the pressure and temperature, which changes the CO₂ solvating power and density (Hallgren et al., 2006; Kim et al., 2008). SC-CO₂ also offers an advantage by preserving the chemical components from oxidation, degradation, hydrolysis, and rearrangement, usually in the traditional hydrodistillation method (Bartley, Foley, 1994).

In a previous study (Hamil et al., 2016), α- and β-asarone were reported to be present in various amounts in alcohol and hydroalcoholic extracts of P. sarmentosum, thus, limiting the utilisation of the extracts for food and nutraceutical products. The present work aims to optimise the extraction of α-, β-, and ɣ-asarone from P. sarmentosum leaves using SC-CO₂ technology and compare the asarone level of the leaves extracted with conventional solvents prior to and after SC-CO₂ treatment. A Box-Behnken experimental design was used to investigate the effect of pressure, temperature, and extraction time on asarone extraction from P. sarmentosum leaves. This is the first study reported on optimising asarone removal from the P. sarmentosum plant to the best of our knowledge.

MATERIAL AND METHODS

Carbon dioxide with 99.9% purity (Linde, Malaysia) was used as a supercritical fluid. Analytical grade n-hexane, chloroform, acetone, ethyl acetate, methanol, and ethanol were purchased from QRec, New Zealand. HPLC grade methanol and acetonitrile were purchased from Merck, USA. α-asarone (1) and β-asarone (2) were purchased from Sigma-Aldrich, USA, whereas, ɣ-asarone (3) was isolated from the plant. The fresh leaves of P. sarmentosum were collected from Perak, Malaysia. The plant was authenticated by Dr. Rahmad Zakaria from the School of Biological Sciences, Universiti Sains Malaysia, with voucher specimen number USM/Herbarium/11481. The leaves were washed thoroughly with tap water and dried in the oven at 40°C. The moisture content for the leaves was 3.68% ± 0.03. The leaves were ground into powder (0.5 mm diameter) using an electric grinder SM-100 (Retsch, Germany).

Supercritical fluid extraction

SC-CO₂ extraction was employed using a lab-scale SC-CO₂ with 1 L capacity (Separex, France). For the preliminary screening of asarone, three extracts were obtained using different pressure values. Experimental parameters were set as follows: (1) 50°C/100 bar/120 min dynamic extraction time; (2) 50°C/300 bar/120 min dynamic extraction time; and (3) 50°C/700 bar/120 min dynamic extraction time. Optimisation conditions were subsequently designed as follows: pressure (80, 115, and 150 bar), temperature (40°C, 50°C, and 60°C), and dynamic extraction time (30, 75, and 120 min). Static extraction time was fixed at 30 min, with a constant flow rate at 30 g/min and plant mass of 50 g for all conditions. The collection vessel and chiller temperature were set at 50°C and 0°C, respectively. SC-CO₂ crude extracts were collected in a vial (10 mL) and kept at 4°C prior to analysis. The experiments were performed in triplicate.

Conventional extraction

Approximately 10 g of ground leaves were mixed with 200 mL (1:20) n-hexane, chloroform, ethyl acetate, acetone, methanol and ethanol and macerated at 50°C for 24 hours. The extracts were filtered and concentrated using a rotary evaporator and kept at 4°C before analysis. The same method was applied for the extraction of SC-CO₂ residue. The experiments were performed in triplicate.

Isolation of ɣ-asarone

Approximately 18 g of SC-CO₂ extract was subjected to flash column chromatography using the increasing ratio of ethyl acetate in n-hexane (0:100 to 100:0) to obtain 30 fractions. The asarone-rich fraction (F12) was further purified using Shimadzu LC-20AP preparative HPLC (Shimadzu Corporation, Japan) on PrepHT Phenyl- Hexyl Preparative Cartridge (21.2 × 250 mm, 5 µm) column (Agilent Technologies, USA) with 0.1% ortho- phosphoric acid, acetonitrile and methanol as mobile phase. Meanwhile, ɣ-asarone was characterised using UV-Vis, FT-IR, GC-MS, and NMR.

HPLC analysis

Quantification of asarone in the P. sarmentosum extracts was performed using HPLC described by Hamil et al. (2016). Briefly, the analysis was performed on Agilent Technologies 1260 Infinity HPLC system (USA). Elution was achieved using an isocratic mobile phase consisting of 0.1% ortho-phosphoric acid:acetonitrile:methanol (50:40:10 v/v/v), with a flow rate of 1 mL/min on Zorbax Eclipse Plus C-18 column (250 × 4.6 mm, 5 μm; Agilent Technologies, USA). The injection volume was 10 μL, the column temperature was maintained at 30°C, and detection was fixed at 210 nm.

Box-Behnken Design

The experiment was performed based on the developed design using Design Expert® (Version 7.1.5, Stat-Ease Inc, Minneapolis.). In this study, the Box- Behnken design consisting of 17 runs, 3 factors, and 3 levels were employed for constructing a polynomial model for optimisation of the maximum extraction of α-, β-, and ɣ-asarone.

RESULTS AND DISCUSSION

Identification of ɣ-asarone

Compound 3 (21.4 mg) was obtained as a viscous liquid. UV-Vis showed maximum absorption at λmax 205, 234, and 291 nm. The FT-IR spectra showed vibrational signals at 2926, 1710, 1641, and 912 cm^-1 associated with the methylene group of CH₂ and CH₃; 1610, 1512, 860, and 754 cm^-1 attributed to 2,4,5-tetra substituted moiety; and 1205, 1178, and 1037 cm^-1 due to the C-O-C stretching of phenolic ether. ¹H NMR (CDCl₃ ) showed signals at δ 6.71 (1H, s, H-6), 6.55 (1H, s, H-3), 6.01 (1H, m, H-2’), 5.08 (2H, m, H-3’), 3.90 (3H, s, 2-OCH₃) 3.85 (3H, s, 4-OCH₃), 3.82 (3H, s, 5-OCH₃), and 3.35 (2H, d, J = 6.5 Hz, H-1’); the ¹³C NMR (CDCl₃ ) spectra showed signals at δ 151.34 (C-2), 147.92 (C-4), 143.04 (C-5), 137.32 (C-2’), 120.07 (C-1), 115.18 (C-3’), 114.02 (C-6), 98.08 (C-3), 56.62 (4-OCH₃ & 5-OCH₃), 56.25 (2-OCH₃), and 33.65 (C-1’); EIMS analysis indicated a molecular formula C₁₂ H₁₆O₃ (m/z 208.1 [M]⁺), and fragmentation ions at 193, 181, 165, 124, 91, and 69. Based on the spectral data and comparing with reported literature (Sinha, Acharya, Joshi, 2002; Varma et al.,2002), 3 was identified as ɣ-asarone (Data presented in supplementary section).

Extraction yield of asarone using SC-CO₂ and conventional solvent extraction

The extraction yield of α-, β-, and ɣ-asarone using SC-CO₂ was compared with those using n-hexane, chloroform, acetone, ethyl acetate, methanol, and ethanol as extraction solvents. A total of nine extracts were obtained. Asarone was quantified using HPLC, and the results are listed in Table I. Our findings showed that α-asarone was the dominant isomer in P. sarmentosum, followed by ɣ-asarone. In contrast, β-asarone was found in a smaller amount in all extracts analysed. For the conventional solvent extraction, α-, β-, and ɣ-asarone were extracted at the highest amount using n-hexane (7.48%, 1.22%, and 3.81%), followed by acetone, chloroform, ethyl acetate, methanol, and ethanol. Total asarone content extracted from the conventional solvents ranged from 2.03% to 12.51%. For the SC- CO₂ extraction, a set of different pressure values were studied to determine the ideal condition for the highest asarone extraction. A negative correlation between pressure and asarone was observed, whereby asarone recovery was increased as the pressure decreased. This effect was observed by manipulating the pressure as a variable, and other parameters were fixed. SFE100 yielded the highest asarone content with 13.52%, 3.03%, and 13.84% for α-, β-, and ɣ-asarone. Total asarone yield for SC-CO₂ ranged from 26.11% to 30.39%, more than two-fold compared to other solvent extraction methods. Asarone is a volatile compound that belongs to the phenylpropanoid group. The compounds showed favourable solubility in non-polar solvent compared to solvent at a higher polarity (Hamil et al., 2016). From the preliminary screening, we found that SC-CO₂ extraction at a lower pressure was the most effective method to maximise recovery of asarone; thus, further optimised using the response surface model.

TABLE I
The comparison of the extraction yield of α-, β-, and ɣ-asarone in P. sarmentosum extracted using SC-CO₂ and conventional solvents

Solvent	Extraction yield (%)	α-asarone (%)	β-asarone (%)	ɣ-asarone (%)	Total asarone content (%)a
Hexane	1.70 ± 0.03	7.48 ± 0.06	1.22 ± 0.001	3.81 ± 0.36	12.51 ± 0.42
Chloroform	3.30 ± 0.02	6.69 ± 0.01	0.42 ± 0.02	1.53 ± 0.002	8.64 ± 0.03
Acetone	2.68 ± 0.03	5.26 ± 0.01	1.03 ± 0.01	2.23 ± 0.002	8.52 ± 0.001
Ethyl acetate	2.80 ± 0.12	3.14 ± 0.001	0.15 ± 0.003	1.05 ± 0.003	4.34 ± 0.004
Methanol	9.58 ± 0.02	2.71 ± 0.01	0.15 ± 0.001	0.68 ± 0.001	3.54 ± 0.01
Ethanol	7.11 ± 0.05	1.66 ± 0.002	0.08 ± 0.003	0.29 ± 0.002	2.03 ± 0.003
SFE100	1.15 ± 0.02	13.52 ± 0.03	3.03 ± 0.01	13.84 ± 0.05	30.39 ± 0.10
SFE300	2.22 ± 0.01	13.27 ± 0.01	2.79 ± 0.06	12.85 ± 0.004	28.91 ± 0.01
SFE700	2.89 ± 0.01	11.43 ± 0.02	2.00 ± 0.01	12.68 ± 0.07	26.11 ± 0.10

• SFE100: SC-CO₂ at 100 bar; SFE300: SC-CO₂ at 300 bar; SFE700: SC-CO₂ at 700 bar; ^a: sum of α-, β- and ɣ-asarone.

Optimisation of SC-CO₂ using Box-Behnken experimental design

The Box-Behnken experimental design was developed to optimise the SC-CO₂ extraction for maximum recovery of α-, β-, and ɣ-asarone from P. sarmentosum leaves. The various parameters of design and asarone yield are summarised in Table II. Several variables that could potentially influence the extraction efficiency were chosen, such as pressure (P), temperature (T), and dynamic extraction time (t). The results obtained from the Box-Behnken experimental design provided a statistical model used to identify asarone solubility patterns from the extraction process. The equation below illustrated the relationship between the three variables: P, T, and t with asarone content.

Equation for α-asarone:

$Y=15.98-0.85\left(P\right)-0.086\left(T\right)-0.019\left(t\right)+0.58\left(P\right)\left(T\right)-0.54\left(P\right)\left(t\right)+0.075\left(T\right)\left(t\right)-3.11\left(P^2\right)-2.83\left(T^2\right)-3.03\left(t^2\right)$ (1)

Equation for β-asarone:

$Y=3.07-0.57\left(P\right)+0.20\left(T\right)-0.026\left(t\right)+0.12\left(P\right)\left(T\right)-0.097\left(P\right)\left(t\right)-0.055\left(T\right)\left(t\right)-0.21\left(P^2\right)-0.52\left(T^2\right)-0.32\left(t^2\right)$ (2)

Equation for ɣ-asarone

$Y=10.75-2.69\left(P\right)+0.63\left(T\right)-0.24\left(t\right)-0.22\left(P\right)\left(T\right)-0.87\left(P\right)\left(t\right)-0.22\left(T\right)\left(t\right)+1.41\left(P^2\right)-0.22\left(T^2\right)+0.28\left(t^2\right)$ (3)

TABLE II
Box-Behnken experimental design order for optimisation of α-, β-, and ɣ-asarone in P. sarmentosum

Run Order	Pressure	Temperature	Time	Yield	α-asarone	β-asarone	ɣ-asarone
	(bar)	(°C)	(min)	(%)	(%)	(%)	(%)
1	115	50	75	1.13	16.14	3.05	11.01
2	115	60	30	0.93	10.24	2.51	12.10
3	115	40	30	0.93	10.07	1.97	10.07
4	115	50	75	1.12	15.72	3.09	10.62
5	80	50	30	0.29	10.20	3.06	14.54
6	80	40	75	0.48	11.77	2.85	13.86
7	80	50	120	0.64	11.28	3.17	15.87
8	150	50	120	2.09	8.40	1.81	8.60
9	115	60	120	1.33	10.32	2.38	11.10
10	115	50	75	1.12	15.93	3.07	10.53
11	150	60	75	2.01	9.49	2.08	9.58
12	115	40	120	1.60	9.85	2.06	9.96
13	150	50	30	1.11	9.49	2.09	10.75
14	115	50	75	1.10	16.04	3.06	10.81
15	115	50	75	1.08	16.09	3.08	10.77
16	80	60	75	0.45	9.94	2.97	15.23
17	150	40	75	1.83	8.99	1.47	9.08

By computation, the optimal points to maximise the extraction of three asarone isomers were predicted as follows: P = 81.16 bar, T = 50.11°C, and t = 80.90 min, which yielded 13.91% α-asarone, 3.43% β-asarone, and 14.95% ɣ-asarone. The percentage yield of SC-CO₂ extract at the optimised parameter was 0.54%. The predicted optimal points were validated by running the extraction using these conditions in triplicate. The percentage yield of the extract was 0.55%. A mean value of 13.99%, 3.44%, and 14.93% (α-, β-, and ɣ-asarone) were obtained, which were in agreement with the predicted values of the asarone isomers at p>0.05. The experimental results confirmed that the response model was adequate for reflecting the expected optimisation with satisfactory accuracy. Figure 2 shows the contour plot of the percentage yields of α-, β-, and ɣ-asarone against the independent variables analysed. At the constant temperature of 50°C, α-asarone in the extracts increased from 14.03% to 15.99%, as the pressure increased to 115 bar. After this point, α-asarone was observed to decrease gradually as the pressure was increased. However, the opposite trend was observed for β- and ɣ-asarone, which showed decreasing recovery from 3.43% to 3.08% and 14.79% to 10.75% for the same operating parameters.

FIGURE 2
Contour plot of (A) pressure against temperature for α-asarone, (B) pressure against extraction time for α-asarone, (C) pressure against temperature for β-asarone, (D) pressure against extraction time for β-asarone, (E) temperature against time for β-asarone, (F) pressure against extraction time for ɣ-asarone.

The increase in temperature showed that the improved extraction was successful for all three asarone isomers until the optimal temperature at 50.11°C was reached. There was a decrease in the percentage at a constant optimal pressure of 81.16 bar after this point. Temperature plays an important role in SC-CO₂ extraction. Generally, an increase in temperature tends to reduce the CO₂ density; thus, decreasing the efficiency of SC-CO₂ extraction. However, as the temperature rises, the vapour pressure of highly volatile asarone increased, resulting in increased solubility until an optimal temperature was achieved (Dai, Ha, Shen, 2008). After the optimal temperature, a further increase in temperature did not improve asarone extraction. This could be due to the reduced solubility as the density decreased. On the other hand, a higher temperature can also be attributed to the degradation of thermally labile compounds (Ahmadian-Kouchaksaraie, Niazmand, 2017).

At the constant pressure of 81.16 bar, the increase of dynamic extraction time until 80.90 min enhanced α-, β-, and ɣ-asarone percentage from 10.43% to 13.92%, 3.05% to 3.43%, and 14.35% to 14.95%. As the extraction time was prolonged, the percentage of α-asarone was decreased gradually while β-asarone was slightly decreased. In contrast, the percentage of ɣ-asarone continue to increase to 120 min (15.51%). An excessive time is not efficient in maximising the extraction of all the asarone isomers. According to Chen, Zhao, and Yu (2003), a longer extraction time was not favourable for asarone, as other compounds will be co-extracted with it. Therefore, an optimised extraction time is crucial to maximising the extraction of asarone with minimal effect on other chemical compounds. We managed to obtain 32.29% of total asarone from the predicted optimised conditions, with an extraction yield of less than 0.6%.

Statistical analysis

The analysis of variance (ANOVA) was performed to fit the model for each variable. The statistical analysis for the regression of coefficients of different factors in all models is shown in Table III. According to the ANOVA, the F-values indicated that the regression equation might explain most response variations. This indicated that the model term is significant at a 95% confidence interval. It was observed that P significantly influenced the recovery of α-, β-, and ɣ-asarone (p<0.0001) in their linear models with negative coefficient values. On the other hand, T and t significantly influenced the extraction of β- and ɣ-asarone with p<0.0001 and p<0.05 in their linear forms. The model adequacy was calculated using the coefficient of determination and lack-of-fit test. The model was statistically significant, with a satisfactory coefficient of determination (R ² = 0.9945-0.9989). In addition, the values of adjusted determination of coefficients (_adj R ² = 0.9874-0.9974) also indicate that the model adequacy is highly significant. The lack-of-fit test did not show significant differences for all three asarones. The interaction effects of P and T significantly influenced the extraction of α- and β-asarone in the quadratic forms. Significant interactions were observed between P and t for all asarone isomers ( p<0.01 to p<0.001). The interaction between T and t was significant (p<0.01) for the extraction of β-asarone only. Furthermore, the coefficient of variation (CV) was within the acceptable range (1.11-2.52), indicating that the model exhibited better reproducibility.

TABLE III
Statistical variable for optimisation of P. sarmentosum SC-CO₂ extraction

Variables	α-asarone		β-asarone		ɣ-asarone
	F	p	F	p	F	p
Model	170.60	<0.0001	685.45	<0.0001	140.54	<0.0001
Linear
P	66.20	<0.0001	3262.56	<0.0001	989.80	<0.0001
T	0.68	0.4375	389.80	<0.0001	54.44	0.0002
t	0.032	0.8630	6.80	0.0350	7.98	0.0256
Quadratic
P 2	462.83	<0.0001	234.52	<0.0001	143.73	<0.0001
T 2	383.85	<0.0001	1377.47	<0.0001	3.54	0.1019
t 2	441.48	<0.0001	548.57	<0.0001	5.70	0.0483
Interaction
PT	15.45	0.0057	74.04	<0.0001	3.24	0.1147
Pt	13.40	0.0081	46.90	0.0002	51.91	0.0002
Tt	0.26	0.6283	14.93	0.0062	3.40	0.1079
R 2	0.9955	-	0.9989	-	0.9945	-
R 2 adj	0.9896	-	0.9974	-	0.9874	-
Lack of fit	6.03	0.0576	6.23	0.0547	2.64	0.1855
CV%	2.52	-	1.11	-	2.11	-

• P: pressure; T: temperature; t: extraction time; F: Fisher-test value; p: significant level.

HPLC analysis of SC-CO₂ residue

The content of asarone in the extracts varies, as depicted in Figure 3. In this study, P. sarmentosum powder residue from the optimised SC-CO₂ was extracted with conventional solvents to compare the quantity of asarone with their relative extracts without SC-CO₂ extraction. Extraction yields are slightly decreased from the relative solvent extracts; however, there is a significant decline in asarone contents ( p<0.001), with the percentage of removal ranging from 45% to 100% in the extracts (Table IV). β-asarone was removed successfully in the residue re-extracted with methanol and ethanol. Interestingly, the optimised SC-CO₂ extraction could remove all three asarone isomers in R-ethanol. This indicated the potential of SC-CO₂ to remove asarone from P. sarmentosum leaves effectively. In addition, the yield of optimised SC-CO₂ extract was about 0.5%, indicating minimal changes in the constituents and properties of the plant. However, further study is needed to compare the metabolite profile and pharmacological activities between SC-CO₂ residue and the extracts without SC-CO₂ treatment.

FIGURE 3
HPLC chromatogram of (A) mix asarone standard; 1=β-asarone, 2=ɣ-asarone, 3=α-asarone; (B) optimised SC-CO₂ extract; (C) overlay chromatogram of P. sarmentosum extracts with and without SC-CO2 treatment; H=hexane, R-H=R-hexane, C=chloroform, R-C=R-chloroform, A=acetone, R-A=R-acetone, EA=ethyl acetate, R-EA=R-ethyl acetate, M=methanol, R-M=R-methanol, E=ethanol and R-E=R-ethanol.

TABLE IV
Percentage removal of α-, β- and ɣ-asarone from P. sarmentosum SC-CO₂ residue as compared to the respective solvent extracts

Parameter	Yield (%)	Asarone content (%)				Percentage removal (%)
Extract		α-asarone	β-asarone	ɣ-asarone	Total asarone	α-asarone	β-asarone	ɣ-asarone	Total asarone
Optimised SC-CO2	0.55±0.02	13.99±0.01	3.44±0.005	14.93±0.01	32.36±0.01	NA	NA	NA	NA
R-Hexane	0.96±0.06	4.49±0.004*	0.61±0.01*	1.78±0.001*	6.88±0.01*	39.97±0.50	50.00±0.87	53.28±4.53	45.00±1.80
R-Chloroform	2.55±0.04	1.42±0.004*	0.16±0.002*	0.52±0.001*	2.10±0.001*	78.77±0.02	61.90±1.56	66.01±0.003	75.69±0.06
R-Acetone	2.43±0.06	1.03±0.001*	0.09±0.005*	0.24±0.001*	1.36±0.001*	80.42±0.003	91.26±0.01	89.24±0.03	84.04±0.001
R-Ethyl Acetate	2.61±0.04	0.60±0.002*	0.04±0.003*	0.39±0.004*	1.03±0.01*	78.57±0.05	73.33±0.28	62.86±0.49	74.06±0.16
R-Methanol	9.16±0.09	0.33±0.01*	NA	0.08±0.01*	0.41±0.01*	87.82±0.26	100±0.00	88.24±0.88	88.42±0.37
R-Ethanol	6.97±0.04	NA	NA	NA	NA	100±0.00	100±0.00	100±0.00	100±0.00

• NA: not available; R: SC-CO₂ residue; * indicated significant different as compared to the solvent extracts without SC-CO₂ treatment at p<0.001. Percentage removal was calculated using the following formula: [1-(organic solvent extract after SC-CO₂/ organic solvent extract before SC-CO₂)] × 100.

Ethanol is a very polar molecule due to its hydroxyl group, with a high electronegativity of oxygen that allows hydrogen bonding to occur with other molecules. Ethanol is the best solvent for extracting phenolics, flavonoids, and alkaloids from a sample matrix (Ivanovska, Philipov, 1996; Do et al., 2014). In the SC-CO₂ system, the addition of ethanol as a co-solvent is commonly used to increase the solubility of polar compounds (Dobbs et al., 1987). In this study, we compared the solubility of asarone in organic solvents with different polarities. As we increased the polarity of solvents from n-hexane to ethanol, the asarone content in the extracts was gradually decreased. After the SC-CO₂ treatment, the amount of asarone and non-polar components in the sample were substantially decreased as CO₂ removed them. According to Vatai, Škerget, and Knez (2009), the high-pressure SC-CO₂ could break the plant cell walls, resulting in an abundant release of phenolic compounds. As more metabolites are available in the samples, they readily available to be extracted by ethanol, which produces an asarone-free extract.

Several studies have reported that α- and β-asarone possess psychoactive, carcinogenic, genotoxic, and cytotoxic properties. Their presence was reported in Acorus calamus, Acorus gramineus, Asarum europaeum, Guatteria gaumeri, and Piper sarmentosum up to 95% in various extracts and essential oils (Authority, 2009; Hamil et al., 2016). The administration of products with a high level of asarone could pose health issues among consumers with observed side effects such as tachycardia, dizziness, tremor, irregular breathing, pallor, anxiety, nausea, and vomiting. (Zuba, Bryska, 2012). Several regulatory bodies developed the guidelines on the maximum intake of asarone to ascertain safe asarone-related products. The cut-off value for β-asarone in alcoholic products that contained calamus was limited to 0.5 mg/kg (Council of Europe, 2005). According to the European Medicine and Health Agency (2005), α- and β-asarone should be reduced as minimum as possible. Permissible daily intake of herbal products containing β-asarone should be less than 115 µg/day or 2 µg/kg bw/day.

CONCLUSION

In this study, SC-CO₂ extraction was optimised using the Box-Behnken experimental design for maximum removal of α-, β-, and ɣ-asarone from P. sarmentosum leaves. Optimised extraction conditions using Box- Behnken experimental design was achieved at P = 81.16 bar, T = 50.11°C, and t = 80.90 min, which yielded 13.91% α-asarone, 3.43% β-asarone, and 14.95% ɣ-asarone. The HPLC data indicated that all three asarone isomers were reduced significantly (p<0.001) in the residue extracted with conventional solvents compared to their respective extracts without SC-CO2 treatment. The asarone-free extract was obtained from SC-CO₂ residue extracted using ethanol. It can be concluded that the optimised SC- CO₂ technique may serve as a quick treatment step for the removal of asarone from P. sarmentosum to develop safer extracts for the food and nutraceutical industry applications.

ACKNOWLEDGMENTS

This research has been funded with Bridging Research Grant from Universiti Sains Malaysia with grant number 304/PFARMASI/6316172.

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Publication Dates

• Publication in this collection
  06 May 2022
• Date of issue
  2022

History

• Received
  28 Feb 2019
• Accepted
  03 Jan 2021