Modeling and optimization of microwave-assisted extraction of pentacyclic triterpenes from Centella asiatica leaves using response surface methodology

Industrial Crops & Products 147 (2020) 112231

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Modeling and optimization of microwave-assisted extraction of pentacyclic triterpenes from Centella asiatica leaves using response surface methodology

T

Bancha Yingngama,*, Abhiruj Chiangsomb, Adelheid Brantnerc a

Department of Pharmaceutical Chemistry and Technology, Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand Department of Pharmacology, College of Pharmacy, Rangsit University, Pathum Thani 12000, Thailand c Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Universitaetsplatz 4/1, A-8010 Graz, Austria b

A R T I C LE I N FO

A B S T R A C T

Keywords: Centella asiatica Microwave-assisted extraction Pentacyclic triterpene Response surface methodology Ultraviolet B protection

Efficient plant extraction techniques that provide high yields of the desired molecules are important. This study focused on the optimization of the processing conditions for a microwave-assisted extraction (MAE) to obtain a maximal yield of prominent pentacyclic triterpenes from Centella asiatica. Response surface methodology (RSM) was tested in the experiments to improve the extraction conditions. The proportions of liquid-to-solid ( X1), ethanol concentration ( X2 ), microwave power ( X3 ), and irradiation time ( X 4 ) were defined as the independent variables. The responses were the recovery of madecassoside, asiaticoside, and its sapogenin triterpene acids. The target compound-rich extract was intensified using Diaion HP-20 resin. The results showed that all the independent MAE variables are important for the isolation of specific compounds (p < 0.05). The optimal conditions for X1, X2 , X3 and X 4 were 10 mL/g, 58 % (v/v), 300 W and 3.4 min, respectively. The efficient macroporous resin method was successful at enriching the triterpenes in concentrated extracts. Both MAE crude extract and enriched samples exerted inhibitory effects on matrix metalloproteinases (MMPs)-3 and 9 secretions against ultraviolet B (UVB)-irradiated human dermal fibroblasts (HDFs). The MAE results, when enriched, are beneficial for increasing the amounts of the targeted active ingredients. These findings demonstrate the feasibility of extracting and enriching the target triterpenes.

1. Introduction Centella asiatica (L.) Urb. (Apiaceae family), one of the most important herbaceous plants, has been well documented worldwide as having health benefits. In addition to being used for herbal tea, as a food additive and as a cosmetic excipient, this species has a broad variety of therapeutic applications in the pharmaceutical sector. A number of studies and review articles have described its pharmacological properties, including wound healing (Azis et al., 2017), memory and mood boosting (Orhan, 2012; Wattanathorn et al., 2008), neuroprotective (Sabaragamuwa et al., 2018), anti-inflammatory, antioxidant (Viswanathan et al., 2019), and antiulcer (Abdulla et al., 2010) capabilities. Scar management and wound healing activities are attributed to triterpenes in C. asiatica extract and have been described in the Chinese, European, German, and Indian Pharmacopoeias (Viswanathan et al., 2019) as well as in the Thai Herbal Pharmacopoeia (Thai Herbal Pharmacopoeia, 2016). Thus, the available evidence for the benefits of pentacyclic triterpenes (Fig. S1) is strong. Due to its outstanding characteristics, C. asiatica is currently cultivated in pantropical countries for

⁎

food, cosmetic and medicinal purposes (Azis et al., 2017). Over the years, knowledge of plant extraction techniques has matured. Moreover, research regarding the intensification of these extraction processes is of great interest and has an active research community. Much of the existing literature on C. asiatica applied conventional extraction methods, including dynamic maceration (Monton et al., 2019), Soxhlet (Pravallika et al., 2019), and subcritical water techniques (Kim et al., 2009). However, these methods have limitations: they are labor intensive and time consuming, result in poor triterpene yields, and involve costly procedures (Cassol et al., 2019). In the framework of green chemistry, efficient plant extraction techniques that provide elevated yields of the desired molecules are essential. Similarly, the solvents employed in the extraction process should be safe for humans and should minimize environmental pollution. Researchers have recently focused on microwave-assisted extraction (MAE), which is a key development in consumer healthcare products (Nabet et al., 2019). Microwaves can generate heat in a polar liquid medium, leading to elevated temperatures and high pressures. As a result, processing effectiveness is improved by both heat and mass

Corresponding author. E-mail address: [email protected] (B. Yingngam).

https://doi.org/10.1016/j.indcrop.2020.112231 Received 20 October 2019; Received in revised form 8 February 2020; Accepted 11 February 2020 0926-6690/ © 2020 Elsevier B.V. All rights reserved.

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the applied reagents were of analytical grade.

transfers and can be assumed to enhance the extraction feasibility (Cassol et al., 2019). MAE has been utilized to extract a broad range of phytochemicals, such as phenolic compounds from Hibiscus sabdariffa calyces (Alara and Abdurahman, 2019; Cassol et al., 2019), oil from Cyperus esculentus (Hu et al., 2018), phenolic compounds from Origanum glandulosum and Thymus fontanesii (Nabet et al., 2019), and mangiferin, amarogentin and swertiamarin from Swertia species (Kaur et al., 2019). Conceptually similar works by Puttarak and Panichayupakaranant (2013) extracted triterpenes derived from C. asiatica in absolute ethanol using MAE via a one-factor-at-a-time strategy. Despite the success of this work in certain aspects, the technique still suffers from a lack of data explaining the relationships between the MAE variables. Moreover, the resulting product had a deep green color derived from chlorophyll, which required subsequent removal by activated charcoal adsorption. Due to the complexity of the active molecules in plant matrices, the efficiency of extraction techniques can be improved by using statistical tools. Currently, response surface methodology (RSM) is used to model and optimize the intended procedure, and such techniques are useful in a wide range of applications (Yingngam and Brantner, 2018; Yingngam et al., 2019). This tool helps to solve engineering problems and enhance sample extraction effectiveness by combining experiments with computer simulations. The variables and their possible interactions are considered during the optimization process. Applying this novel approach makes it possible to meet the requirements for a specific purpose, to provide maximum data, and to reduce the number of required experiments (Nabet et al., 2019). Box-Behnken designs (BBDs), a type of RSM conducted using an asymmetric domain, offer some of the benefits of minimal collinearity, practicality, and rotatability. The RSM modeling can provide useful information even from small-scale experimental data (Yingngam et al., 2019). However, to date, no studies have focused on combining MAE and RSM to extract triterpenes from C. asiatica leaves. The authors hypothesized that the use of the RSM model would help maximize the triterpene yields. Therefore, the goals of this research were (i) to improve the MAE method by integrating mathematical modeling and optimization of the extraction procedures using RSM, (ii) to enrich the triterpenes in the extract using macroporous resin, and (iii) to evaluate the possible inhibitory properties of the resulting extract on matrix metalloproteinase (MMP) production in ultraviolet B (UVB)-irradiated normal human dermal fibroblasts (HDFs).

2.3. MAE The MAE was implemented using a modified microwave oven with a variable magnetron input voltage attached to a water-circulating cooling unit (ME711 K, Samsung Thailand Electronics, Chonburi, Thailand). The inner dimensions of the microwave oven were 21.5 × 35 × 33 cm. The microwave power ranged from 0 W to 800 W at a frequency of 2.45 GHz. 2.3.1. Study of the appropriate range of the relevant extraction parameters A single-factor design was used to screen the significant variables and their respective ranges influencing process effectiveness. A plant sample (5 g) was introduced into a glass tube attached to a column condenser. After the extraction, the triterpene content of each sample was analyzed using the high-performance liquid chromatography (HPLC) method described in Section 2.6. The input variables were the ethanol solution-to-solid ratio (10:1, 20:1, 30:1, 50:1, and 70:1 mL/g), ethanol concentration (0, 25, 50, 75 and 100 %), microwave power (300, 450, 600, 700, and 800 W) and irradiation time (0.5, 1, 2.5, 5, 7.5 and 10 min). The output factors were the recovery rate of each triterpene. Subsequently, a suitable range of individual factors was chosen for the experimental design. 2.3.2. Experimental design by RSM A type of RSM, a 4-factor 3-level BBD with a total of 5 replications for the center point, was performed to investigate simultaneously the influence of independent factors on triterpene yields and to identify the optimal conditions. The selected input factors were the liquid-to-solid ratio ( X1, 10−50 mL/g), ethanol concentration [ X2 , 5–95 % (v/v)], microwave power ( X3 , 300−800 W) and irradiation time ( X 4 , 0.5−5 min). The levels of the independent variables used in the experimental design and the optimization constraints are shown in Table 1. The BBD-based RSM was used to perform 29 treatment studies. Five replicates at the center point were applied to check the experimental error (Table 2). The responses included yields of madecassoside (Y1), asiaticoside (Y2 ), madecassic acid (Y3), asiatic acid (Y4 ), and of total triterpenes (Y5). A random order was assigned to the experimental run to minimize any systematic experimental errors and to improve the predictability of the suggested models. The relationship between the target responses and the independent variables was established using the quadratic model in Eq. (1):

2. Experimental 2.1. Plant material

k

Y = β0 +

C. asiatica was grown in Ubon Ratchathani Province, Thailand. The plant material was collected and identified by Asst. Prof. Bancha Yingngam in March 2019. A voucher specimen was deposited at the Herbarium of Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Thailand (No. BCY-UBU API01032019). The plant material was washed with tap water and then dried in a hot-air oven at 50 ± 1 °C for 18 h. The plant leaves were ground and sifted through a 40-mesh size sieve prior to use.

k

∑ βi Xi +∑ ∑ i=1

βij Xi Xj +∑ βii Xi2 , (k = 4)

i=1 j=i+1

(1)

i=1

Table 1 Experimental parameters for the BBD of triterpene extraction from C. asiatica leaves. Factor

2.2. Chemicals Asiaticoside, asiatic acid and madecassoside were purchased from Sigma-Aldrich (St. Louise, MO, USA), while madecassic acid was obtained from ChromaDex, Inc. (CA, USA). Absolute ethanol, acetonitrile, and phosphoric acid were purchased from Carlo Erba Reagents (Val de Reuil, France). Diaion HP-20 resin was purchased from Supelco (Supelco Park, Bellefonte, PA, USA). Dulbecco’s modified Eagle’s medium, fetal bovine serum, penicillin and streptomycin solution, and L-glutamine were obtained from Gibco Invitrogen Corporation (Grand Island, NY, USA). Deionized water was used throughout the study. All 2

Symbol

Unit

Independent variables Liquid-to-solid ratio Ethanol concentration Microwave power Irradiation time

X1 X2 X3 X4

mL/g % v/v W min

Dependent variables Madecassoside Asiaticoside Madecassic acid Asiatic acid Total triterpenes

Y1 Y2 Y3 Y4 Y5

mg/g mg/g mg/g mg/g mg/g

dry dry dry dry dry

basis basis basis basis basis

Level Low −1

Medium 0

High +1

10.00 10.00 300.00 0.50

20.00 50.00 450.00 2.75

30.00 90.00 600.00 5.00

Desirability constraints Maximize Maximize Maximize Maximize Maximize

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Table 2 The layout of the BBD matrix used to extract the bioactive components from C. asiatica leaves and their responses. Run

Independent variable

X1 (mL/g) 1 2cp 3cp 4 5cp 6 7 8 9 10 11 12 13cp 14 15 16 17 18 19 20 21cp 22 23 24 25 26 27 28 29

10.00 20.00 20.00 20.00 20.00 30.00 30.00 10.00 20.00 10.00 10.00 30.00 20.00 20.00 30.00 20.00 20.00 20.00 30.00 30.00 20.00 20.00 20.00 10.00 20.00 20.00 20.00 20.00 10.00

(−1) (0) (0) (0) (0) (+1) (+1) (−1) (0) (−1) (−1) (+1) (0) (0) (+1) (0) (0) (0) (+1) (+1) (0) (0) (0) (−1) (0) (0) (0) (0) (−1)

Yield of pentacyclic triterpenes

X2 (%, v/v) 10.00 50.00 50.00 90.00 50.00 50.00 50.00 50.00 10.00 50.00 50.00 50.00 50.00 50.00 90.00 90.00 90.00 50.00 50.00 10.00 50.00 10.00 50.00 90.00 90.00 10.00 50.00 10.00 50.00

(−1) (0) (0) (+1) (0) (0) (0) (0) (−1) (0) (0) (0) (0) (0) (+1) (+1) (+1) (0) (0) (−1) (0) (−1) (0) (+1) (+1) (−1) (0) (−1) (0)

X3 (W) 450.00 450.00 450.00 450.00 450.00 450.00 300.00 300.00 300.00 450.00 450.00 450.00 450.00 300.00 450.00 600.00 450.00 300.00 600.00 450.00 450.00 450.00 600.00 450.00 300.00 450.00 600.00 600.00 600.00

X 4 , (min) (0) (0) (0) (0) (0) (0) (−1) (−1) (−1) (0) (0) (0) (0) (−1) (0) (+1) (0) (−1) (+1) (0) (0) (0) (+1) (0) (−1) (0) (+1) (+1) (+1)

2.75 2.75 2.75 5.00 2.75 0.50 2.75 2.75 2.75 5.00 0.50 5.00 2.75 5.00 2.75 2.75 0.50 0.50 2.75 2.75 2.75 0.50 0.50 2.75 2.75 5.00 5.00 2.75 2.75

(0) (0) (0) (+1) (0) (−1) (0) (0) (0) (+1) (−1) (+1) (0) (+1) (0) (0) (−1) (−1) (0) (0) (0) (−1) (−1) (0) (0) (+1) (+1) (0) (0)

Y1 (mg/g)

Y3 (mg/g)

Y2 (mg/g) a,d

18.42 ± 0.03 22.17 ± 0.06b 22.24 ± 1.27b 10.34 ± 0.08c 19.49 ± 0.16a,d 18.14 ± 0.10a,d 21.51 ± 0.05b 23.64 ± 0.10b 18.18 ± 0.32a,d 22.39 ± 0.27b 19.45 ± 0.32a,d 19.59 ± 0.11a,d 20.14 ± 0.05a,b 18.92 ± 0.17a,d 9.75 ± 0.03c 8.47 ± 0.93c 6.09 ± 0.34e 17.15 ± 0.15a 19.14 ± 0.11a,d 19.67 ± 0.12a 20.75 ± 0.16b 17.29 ± 0.83a 16.26 ± 0.19a 9.18 ± 0.47c 8.49 ± 0.58c 16.72 ± 0.14a 17.67 ± 1.78a,d 16.07 ± 0.98d 18.04 ± 2.41a,d

a

3.38 ± 0.07 9.89 ± 0.23b 9.64 ± 0.12b 9.44 ± 0.06b 9.58 ± 0.21b 8.99 ± 0.04b 9.49 ± 0.26b 10.39 ± 0.20b 3.31 ± 0.05a 10.14 ± 0.02b 9.37 ± 0.31b 9.44 ± 0.01b 9.69 ± 0.01b 9.29 ± 0.01b 9.10 ± 0.75b 9.28 ± 0.28b 7.80 ± 0.03b 8.93 ± 0.03b 9.13 ± 0.06b 3.51 ± 0.01a 9.37 ± 0.04b 2.26 ± 0.29a 8.61 ± 0.08b 9.41 ± 0.15b 9.20 ± 0.15b 2.55 ± 0.26a 9.10 ± 3.69a,c 2.45 ± 0.45a 9.15 ± 0.01b

Y5 (mg/g)

Y4 (mg/g) a

0.03 ± 0.00 2.19 ± 0.07b 1.81 ± 0.07c 2.10 ± 0.11b,g 1.74 ± 0.01c 1.36 ± 0.01d 1.69 ± 0.01c,f,h 2.15 ± 0.02b,g 0.08 ± 0.01a 1.17 ± 0.02e 1.56 ± 0.02f,h 1.80 ± 0.01c 1.80 ± 0.01c 1.70 ± 0.01c,f 1.99 ± 0.01g 1.98 ± 0.03g 1.33 ± 0.01d 1.09 ± 0.01e 1.63 ± 0.01f,h 0.18 ± 0.01a 1.79 ± 0.01c 0.04 ± 0.00a 1.39 ± 0.01d,h 1.80 ± 0.01c 1.73 ± 0.02c 0.08 ± 0.00a 1.70 ± 0.01c,f 0.07 ± 0.02a 1.53 ± 0.22h

a

0.40 ± 0.10 2.57 ± 0.13b 2.23 ± 0.21b,c 2.45 ± 0.12b 2.15 ± 0.14b,c 1.72 ± 0.04c 2.17 ± 0.10b,c 3.19 ± 0.66b 0.00 ± 0.00a 1.35 ± 0.19c 2.26 ± 0.21b,c 2.41 ± 0.10b,c 2.43 ± 0.10b 2.50 ± 0.02b 2.63 ± 0.05b 2.87 ± 0.09b 2.03 ± 0.28b,c 1.62 ± 0.09c 2.44 ± 0.22b 0.10 ± 0.09a 2.65 ± 0.23b 0.19 ± 0.04a 2.10 ± 0.31b,c 2.97 ± 0.15b 2.63 ± 0.09b 0.00 ± 0.00a 2.19 ± 0.01b,c 0.00 ± 0.00a 1.73 ± 0.21c

21.82 ± 0.10a 34.25 ± 0.24b 31.28 ± 2.72b,c 21.88 ± 0.10a 30.81 ± 2.26b 28.49 ± 0.17b 32.69 ± 0.28b 36.19 ± 0.26b 21.58 ± 0.29a 33.70 ± 0.28b 30.78 ± 0.78b 33.41 ± 0.19b 33.87 ± 0.18b 32.35 ± 0.29b 22.99 ± 0.79a 21.92 ± 1.08a 18.42 ± 1.00a 27.17 ± 0.18a,c 31.24 ± 0.34b,c 25.63 ± 0.20a,c 34.31 ± 0.09b,c 22.02 ± 1.21a 28.76 ± 0.24b,c 23.01 ± 0.60a 22.05 ± 0.79a 22.22 ± 0.21a 25.10 ± 6.74a 20.21 ± 1.37a 24.04 ± 5.80a

The data are presented as the mean ± SD (n = 3). Values followed by the same letter in the same column are not significantly different (p > 0.05). cp Center point.

The resulting ethanol aqueous extract was filtered, concentrated under reduced pressure, and freeze-dried. Diaion HP-20 resin was selected to enrich the triterpenes. The resin was allowed to swell in ethanol for 24 h and washed with deionized water before use.

where Y is the expected value of the response; β0 , βi , βii and βij are the regression coefficients of the intercept, linearity, quadratic and interaction terms, respectively; Xi and Xj demonstrate the coded independent variables; and k is the number of independent factors (Yingngam et al., 2019). The significance of each factor was then determined using analysis of variance (ANOVA). Significant levels of linear and quadratic interactions of independent variables were evaluated on the basis of the Fisher (F)-value at a probability (p) of 0.05. In addition, the lack-of-fit 2 test, the determination coefficient (R2 ) and the adjusted R2 (Radj ) were evaluated to determine the adequacy of the proposed models. Threedimensional (3D) surface plots were constructed from mathematical models to assess the details in response to test variables in depth. The desirability function, a popular technique in RSM, was used to optimize simultaneously a series of responses (Yingngam et al., 2019). Normalization was performed by converting the response values’ factors to a free scale between 0 and 1. The larger the value was, the more desirable the corresponding response value was. Attaining values close to 1 was the goal of this study.

2.5.1. Static adsorption and desorption studies The static adsorption and desorption proportions of the four triterpenes on the resin were investigated. The aqueous solution (100 mL) from C. asiatica crude extract (1 g) was added to the hydrated resin (equivalent to 3 g of dry resin) in Erlenmeyer flasks, which were then continuously shaken at 150 rpm for 12 h at 25 °C to achieve adsorption equilibrium. The resin and residual sample solution were separated. HPLC was used to analyze the aqueous solution acquired after the adsorption procedure. Subsequently, the remaining resin was washed with deionized water, followed by desorption with 40 mL of ethanolic solutions of different dilutions (0, 25, 50, 75 and 100 % v/v) at 150 rpm and 25 °C for 6 h. The resulting supernatant was filtered through a 0.22 μm membrane filter prior to the analysis of each triterpene content. The adsorption capacity (Qe ) (mg/g dry resin) and desorption ratio (Qd ) (%) of triterpenes on the resin were calculated as shown in Eqs. (2) and (3):

2.4. Comparison with reference extraction methods The MAE extraction performance obtained from the optimized system was compared with two reference extraction methods: dynamic maceration and MAE with a single-factor approach [referring to Monton et al. (2019) and Puttarak and Panichayupakaranant (2013)]. After extraction, the solution was filtered for further analysis by HPLC. 2.5. Enrichment of triterpenes with the resin

Qe =

(C0 − Ce ) × V0 m

(2)

Qd =

Cd × Vd × 100 (C0 − Ce ) V0

(3)

where C0 , Ce and Cd denote the initial, absorption equilibrium and desorption concentrations of analytes, respectively (mg/mL); V0 and Vd are the original volume of crude extract and the volume of the desorption solution (mL), respectively; and m is the dry weight of the resin (g).

Several batches of C. asiatica crude extract produced using the optimal MAE technique were pooled and partitioned with hexane to remove the chlorophylls until a colorless hexane fraction was obtained. 3

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cells were washed twice with PBS, and MTT (0.5 mg/mL in serum-free culture media) was added to each well. The plates were incubated at 37 °C for 3 h. Formazan crystals that formed were dissolved by the addition of dimethyl sulfoxide (DMSO) (100 μL/well), and the absorbance was measured at 570 nm using a PowerWave XS2 microplate reader (BioTek, Winooski, VT, USA).

2.5.2. Investigation of adsorption kinetics The adsorption kinetics of each triterpene was determined at 25 ± 1 °C by maintaining the proportion of crude extract and resin at 1:5 (w/w) and adding 50 mL deionized water. The Erlenmeyer flasks were shaken at 150 rpm, and the triterpene content in the solution was determined by HPLC at specific time intervals up to equilibrium. The adsorption kinetic curves of each triterpene were plotted, and the pseudo-first-order and pseudo-second-order kinetic models were then calculated by Eqs. (4) and (5), respectively:

Qt = Qe − Qe e−k1 t Qt =

2.7.2. Determination of MMP-3 and MMP-9 secretions HDFs were seeded in 6-well plates at a density of 2 × 105 cells/well and allowed to attach overnight. After incubation, the cells were pretreated with different concentrations (0–0.1 mg/mL) of each extract for 12 h prior to irradiation with 30 mJ/cm2 of UVB (Honle UV America, MA, USA) for 2 h. The supernatants were then collected, centrifuged at 10,000 g for 5 min to remove the particulate matter and stored at −80 °C. The concentration of proteins in the supernatants was determined using the Bradford method. The active MMP-3 and MMP-9 samples were quantified using Sigma MMP-3 and MMP-9 ELISA kits (St. Louis, MO, USA) according to the manufacturer’s protocols.

(4)

k2 Qe2 t 1 + k2 Qe t

(5)

where Qe is the adsorption/desorption capacity of the resin at equilibrium; Qt denotes the concentration of triterpene absorbed at time t ; and k1 and k2 are the rate constants of the pseudo-first-order and pseudo-second-order, respectively. 2.5.3. Dynamic adsorption and desorption study Before studying the effect of ethanol concentration on the desorption efficacy, the dynamic leakage profiles of triterpenes on the resin were evaluated. The triterpenes were fully adsorbed by the resin before the volume of the sample solution reached 3 BV. The sample was then carefully introduced to a glass column (22 mm × 300 mm) packed with pretreated resin (equivalent to 3 g of dry resin) at 4 °C overnight. The column was thoroughly washed with deionized water (5 BV) to remove water-soluble impurities. The gradient elution was implemented on the adsorbate-loaded column with 5 BV of successive series of aqueous ethanol solutions (25 %, 50 %, 75 %, and 100 %) at a 2 BV/h flow rate. The resulting eluates were collected manually (5 BV) at each concentration, and the content of the triterpenes in the desorption solution was determined by HPLC.

2.8. Statistical analysis All the experiments were performed at least in triplicate, and the data are reported as the mean and standard deviation of the mean. The BBD and ANOVAs were conducted using Statistica software version 10.0 (StatSoft Inc., Tulsa, OK, USA). Tukey’s test was used to compare the mean values. A p-value below 0.05 was considered to indicate a statistically significant difference. 3. Results and discussion 3.1. Screening of the relevant factors by single-factor test Many factors can affect the extraction efficiency during the MAE process, such as the solvent-to-material ratio, solvent composition, microwave power and irradiation time. These factors not only directly influence the effectiveness of extraction but also engage with each other. In this work, the extraction temperature was not considered as a test factor affecting the extraction rate in MAE. This is because an open microwave system was applied for the extraction; the temperature in the crude extract has always been at the boiling point of the implemented aqueous ethanol mixture (Teslić et al., 2019). A single-factor technique was used to select the suitable range of each independent variable; the results are graphically depicted in Fig. 1.

2.6. Quantification of triterpenes In this study, the triterpene content was determined using the HPLC method proposed by Monton et al. (2019) with some modifications. The HPLC device consisted of a binary pump, an autosampler, a column compartment, a photodiode array detector and a Chromeleon™ Chromatographic Data System software (version 7.2 RS4) (Dionex Ultimate 3000 UHPLC, Thermo Fisher Scientific Company, USA). Properly diluted samples were injected into the guard column (4.0 mm × 2.0 mm) attached to a C18 Luna analytical column (250 mm × 4.6 mm, 5 μm particle size) (Phenomenex, Torrance, USA). The mobile phase consisted of 0.01 % aqueous phosphoric acid (solvent A) and acetonitrile (solvent B). The linear gradient elution was performed as follows: maintain 20 % B for 5 min, from 20 % B to 37 % B over 10 min, from 37 % B to 45 % B over 5 min, maintain 45 % B for 15 min, and return to 20 % B over 2 min. Other conditions were held constant as follows: injected sample volume, 20 μL; flow rate, 1 mL/min; column temperature, 35 °C; and UV–vis spectrum wavelength, 200–500 nm. The content of each triterpene was quantified with the corresponding standards. The calibration curves of the reference standards were carried out at 8 points within a range of 1.56 and 200 μg/mL for each compound and were detected at 210 nm.

3.1.1. Effect of liquid-to-solid ratio The effect of the liquid-to-solid ratio on the yield of triterpenes was studied at a fixed X2 of 75 % (v/v), X3 of 700 W and X 4 of 3 min, whereas the liquid-to-solid ratio ranged from 10 to 70 mL/g. As shown in Fig. 1a, an increase in the triterpene yield was observed when the liquid-to-solid proportion increased from 10 to 20 mL/g (p < 0.05). No significant increase occurred in the recovery of the target compounds in excess of 20 mL/g (p > 0.05). This result could be related to the sufficient solvency of the target compounds in a larger volume of extraction solvent, as was reported by previous results (Alara and Abdurahman, 2019). In the subsequent experiments, the liquid-to-solid ratio was restricted to 20 mL/g. 3.1.2. Effect of ethanol concentration The impact of ethanol concentration on the content of triterpenes was investigated by setting X3 and X 4 to 700 W and 3 min, respectively. Fig. 1b reveals that the triterpene content increased gradually as the ethanol concentration increased from 0 to 50 % (v/v), after which the triterpene contents decreased. The combination of water and ethanol tends to boost the solubility of triterpene acids with less polarity. This observation agrees with the extraction of several phytochemicals in ethanol and water mixtures, similar to the results reported in (Nabet

2.7. Inhibition of MMP-3 and MMP-9 secretions 2.7.1. Cytotoxicity study The protective effect of C. asiatica extracts against UV-induced cytotoxicity to HDFs was determined using a 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, HDFs were seeded into 96-well culture plates at a density of 5 × 103 cells/well and incubated overnight. The cells were then treated with different concentrations of each extract (0−1 mg/mL). After incubation for 24 h, the 4

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Fig. 1. Effects of (a) the liquid-to-solid ratio, (b) ethanol concentration, (c) microwave power, and (d) irradiation time on the extraction of triterpenes from C. asiatica leaves.

through regression analysis. Table 3 summarizes the ANOVA results of the quadratic model. The statistical significance of the suggested model was assessed by F-test and p-value. The F-value of each term was calculated by dividing the mean square by the residual mean square: larger values of F with lower values of p indicate a greater term significance and are sufficient to illustrate the relationship between the actual and expected values (Nabet et al., 2019). These results clearly showed that the F-values of all the models were highly important, with very small pvalues (< 0.0001) and F-values above 15, indicating that there is only a 0.01 % probability that the developed models are affected by noise. Lack-of-fit variance is another statistic that demonstrates the accuracy of the suggested regression model. The lack-of-fit F-values for madecassoside, asiaticoside, madecassic acid, asiatic acid, and total triterpenes were calculated to be 0.8700 (p = 0.6139), 2.3100 (p = 0.2175), 1.2900 (p = 0.4346), 2.1500 (p = 0.2399), and 1.2700 (p = 0.4413), respectively, without any significant differences in variance. This result indicates the excellent proximity of the proposed models for predicting the variations (Yingngam et al., 2019). The adequacy precision ratio of all the proposed models was above 4, indicating their acceptability, and the proposed model can be used to navigate the design region. The adequacy of each of the above models was validated using four diagnostic plots: the normal probability against internally studentized residuals, internally studentized residuals against predicted, internally studentized residuals against the run number, and predicted against actual values. A normal distribution of the residuals of total triterpenes was verified, and the findings show that all the points were spread along a straight line. This observation indicates that the error terms are normally distributed and independent of each other. The points of the internally studentized residual plots against the predicted values and the deleted studentized residuals against the run numbers were scattered between −3.0 and +3.0 except for outliers, suggesting that the quadratic model effectively formed a link between the causal variables

et al., 2019). As a result, a 50 % (v/v) ethanol solution was adopted for the subsequent experiments. 3.1.3. Effect of microwave power The influence of varying the microwave power on the triterpene content is shown in Fig. 1c. The X1, X2 and X 4 parameters were set to 20 mL/g, 50 % (v/v) and 3 min, respectively. The triterpene yields improved as the microwave power increased from 300 to 600 W. This result can potentially be explained as the power increase improving the solubility and diffusion of the principle targets out of the plant matrix. In addition, the elevated pressure inside the cell wall promotes explosions that release the target compounds (Yingngam and Brantner, 2018). Beyond 700 W of microwave power, a reduced triterpene content was found, which can be ascribed to degradation. In subsequent experiments, the microwave power was set to 600 W. 3.1.4. Effect of irradiation time The effect of the irradiation time on the yield of triterpenes was achieved under the conditions where X1, X2 and X3 were set to 20 mL/g, 50 % (v/v) and 600 W, respectively. As shown in Fig. 1d, the best triterpene yield was reached after 3 min of exposure to microwave irradiation. This could be because this exposure time disrupts cell walls, releasing the soluble molecules from inside the cell to the surrounding medium (Alara and Abdurahman, 2019). No increase in the recovery of triterpenes was observed using irradiation times longer than 3 min. The outcome of the preceding experiments led to the conclusion that the suitable range for each parameter, X1, X2 , X3 and X 4 , was 10–30 mL/g, 10–90% (v/v), 300–600 W and 0.5–5 min, respectively. These values were adopted for subsequent experiments using the BBD. 3.2. RSM modeling The experimental data acquired from the BBD were evaluated 5

0.6800 180.1100 9.2200 7.8200

0.0870 1.9400 0.4100 0.8100 4.2900 0.0250

1.7900

229.7300

8.2400

0.1200 2.6200 0.5600 1.1000 5.8000 0.0340

2.4200

310.4800

11.1300

X12

X22

6

X32 1.5100

19.8900 14.7200 0.0018 X42 Residual 18.9200 Lack of fit 12.9400 0.8700 0.6139 Pure error 5.9800 Cor total 635.93 Parameters used for the adequacy check of the model C.V. (%) 6.8100 PRESS 83.8800 0.9702 R2 0.9903 0.9751 41.4800

0.9405

0.8681

20.0000

2 RAdj

2 RPred Adeq Precision

3.3200 5.0600 0.9951

0.9800 0.8400 0.1500 202.9800

0.2100

79.9300

0.3000

0.0470 0.2000 0.0250 0.2300 0.4600 0.0044

0.3900 112.6600 0.7000 1.3200

0.0124

< 0.0001

0.2023

0.7724 0.1854 0.5318 0.3831 0.0572 0.8771

0.4243 < 0.0001 0.0089 0.0143

202.0000

0.9200 243.4200 12.4600 10.5700

< 0.0001

32.6100

617.0100

β0 Linear X1 X2 X3 X4 Interaction X1 X2 X1 X3 X1 X 4 X2 X3 X2 X 4 X3 X 4 Quadratic

2.3100

21.4300

3.0200

1136.1400

4.3200

0.6700 2.7700 0.3500 3.2200 6.5000 0.0620

5.6000 1601.3300 9.9000 18.8300

205.0800

F-value

Sum of squares

F-value

Sum of squares

P-value probability

Asiaticoside (Y2 )

Madecassoside (Y1)

Source of variation

0.2175

0.0004

0.1042

< 0.0001

0.0564

0.4263 0.1181 0.5609 0.0943 0.0232 0.8065

0.0329 < 0.0001 0.0071 0.0007

< 0.0001

P-value probability

Table 3 ANOVA data showing the effects of the MAE conditions on the responses of triterpene yields.

14.7150

0.8159

0.9227

14.6000 2.6400 0.9614

0.5500 0.4200 0.1300 14.3400

0.4900

0.0620

3.7700

0.0400

0.0006 0.0790 0.1700 0.0190 0.1300 0.0210

0.0140 9.1300 0.0015 0.2600

13.7900

Sum of squares

1.2900

12.4700

1.5600

95.2800

1.0000

0.0140 1.9900 4.3800 0.4700 3.2900 0.5200

0.3500 230.6100 0.0370 6.6100

24.8800

F-value

Madecassic acid (Y3 )

0.4346

0.0033

0.2322

< 0.0001

0.3342

0.9070 0.1804 0.0551 0.5044 0.0910 0.4815

0.5621 < 0.0001 0.8498 0.0222

< 0.0001

P-value probability

14.6950

0.7822

0.9146

15.5500 5.9900 0.9573

1.1700 0.9900 0.1800 27.5100

0.8000

0.0027

5.4600

0.0056

0.0005 0.7400 0.6400 0.0140 0.0920 0.1600

0.0150 18.5000 0.0500 0.0790

26.3400

Sum of squares

2.1500

9.5100

0.0320

65.1000

0.0660

0.0055 8.8400 7.6400 0.1600 1.0900 1.8800

0.1800 220.4700 0.6000 0.9400

22.4300

F-value

Asiatic acid (Y4 )

0.2399

0.0081

0.8602

< 0.0001

0.8007

0.9421 0.0101 0.0152 0.6909 0.3132 0.1923

0.6777 < 0.0001 0.4527 0.3477

< 0.0001

P-value probability

11.9460

0.7084

0.8773

6.8500 232.8600 0.9387

48.9800 37.2400 11.7400 798.4100

27.72

31.2300

602.1200

1.0200

3.6400 28.6700 1.0000 0.3900 2.6600 19.5600

2.0000 0.8600 35.9000 14.1300

749.4300

Sum of squares

1.2700

7.9200

8.9300

172.11

0.2900

1.0400 8.2000 0.2900 0.1100 0.7600 5.5900

0.5700 0.2500 10.2600 4.0400

15.3000

F-value

Total triterpenes (Y5 )

0.4413

0.0138

0.0098

< 0.0001

0.5979

0.3249 0.0125 0.6013 0.7446 0.3982 0.0330

0.4618 0.6282 0.0064 0.0641

< 0.0001

P-value probability

B. Yingngam, et al.

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Fig. 2. Contour plots showing the combined effects of the MAE conditions on the yield of total triterpenes from C. asiatica leaves: (a) X1 and X2 , (b) X1 and X3 , (c) X1 and X 4 , (d) X2 and X3 , (e) X2 and X 4 and (f) X3 and X 4 . The third and fourth independent variables are held constant at the middle level.

of the MAE method and the triterpene yield. The points in the plot of the predicted and actual values are distributed in a straight line, demonstrating that the quadratic model can be used to accurately predict the actual values. An acceptable R2 value should be higher than 0.75, and the most appropriate regression is the method proposed in (Mohammadpour et al., 2019). High (R2 ) values for Y1, Y2 , Y3, Y4 and Y5 were found in this study with values of 0.9702, 0.9951, 0.9614, 0.9573 2 and 0.9387, respectively. The predicted R2 (RPred ) combined with the 2 adjusted R2 (RAdj ), which does not rely on the R2 value, was used to 2 illustrate the predictability of the regression. The values of RPred were 2 high and comparable to those of RAdj . Thus, the proposed models are both reliable and precise, as confirmed by the variation coefficient. The quadratic model was deemed sufficient to represent the experimental data and can be reported in terms of the coded values shown in Eqs. (6)–(10):

asiaticoside (Y2, mg /g ) = 9.63 − 0.18X1 + 3.06X2 − 0.24X3 + 0.33X 4 + 0.34X2 X 4 − 3.51X22 − 0.48X42

madecassic acid (Y3, mg / g ) = 1.86 + 0.87X2 + 0.15X 4 −

−

−

1.75X42

(7)

− 0.28X42 (8)

asiatic acid (Y4, mg / g ) = 2.41 + 1.24X2 + 0.43X1 X3 + 0.40X1 X 4 − 0.92X22 − 0.35X42

(9)

and

total triterpenes (Y5, mg /g ) = 32.90 − 1.73X3 + 1.09X 4 + 2.68X1 X3 − 2.21X3 X 4 − 9.63X22 − 2.19X32 − 2.07X42 (10)

madecassoside (Y1, mg /g ) = 20.96 − 4.50X2 − 1.02X3 + 0.94X 4 − 6.92X22 1.31X32

0.76X22

3.3. Response surface analysis

(6)

3.3.1. Overview analysis The experimental data shown in Table 3 were used to explore the effects of the variables. A negative coefficient reflects an antagonistic 7

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impact, while a positive coefficient demonstrates a synergistic effect of the test parameters on the responses (Yingngam and Brantner, 2018). Regarding the madecassoside yield, the linear terms [ X2 , X3 (negative effect), X 4 (positive effect)], the interaction terms [ X1 X2 (positive effect , X3 X 4 (negative effect)], and the negative effect of the quadratic terms ( X22 , X32 , X42 ) of the independent parameters considerably affected the yield of this compound (p < 0.05). Asiaticoside was impacted by some of the studied MAE conditions [linear term coefficients ( X2 , X2 , X3 , X 4 ), interaction term ( X2 X 4 ), and quadratic terms ( X22 , X42 )] (p < 0.05). The linear coefficients of ethanol concentration ( X2 , p < 0.0001) and irradiation time ( X 4 , p = 0.0222) and the quadratic terms of X22 and X42 influenced the yield of madecassic acid. No interactions occurred between the parameters studied in this molecule (p > 0.05). These coefficients, including the linear term coefficient ( X3 ) and interaction terms ( X1 X3 and X3 X 4 ), influenced the yield of asiatic acid. The linear term coefficients ( X3 , X 4 ), interaction terms ( X1 X3 , X3 X 4 ) and quadratic terms ( X22 , X32 , X42 ) substantially impacted the yield of total triterpenes (p < 0.05). The remaining terms were found to be nonsignificant (p > 0.05). Overall, all the independent parameters tested trigger clear changes in triterpene yields in different ways.

Table 4 The optimum conditions of the MAE and the results of the predicted and validated values of each bioactive component using the RSM approach. Optimized conditions

Predicted values

Validated values

X1 = 10 mL/g X2 = 58 % (v/v) X3 = 300 W X 4 = 3.4 min

Y1 = 21.61 ± 1.49 Y2 = 10.80 ± 0.34 Y3 = 1.91 ± 0.25 Y4 = 3.02 ± 0.37 Y5 = 35.52 ± 2.39

Y1 = 23.09 ± 0.12nd Y2 = 10.76 ± 0.11nd Y3 = 2.01 ± 0.16nd Y4 = 2.94 ± 0.07nd Y5 = 36.01 ± 0.09nd

The data are presented as the mean ± SD (n = 3). nd denotes a nonsignificant difference of the validated values compared to the predicted values (p > 0.05).

3.5. Comparison of MAE with other methods In line with the results of this research, we report that the optimal MAE conditions minimize the extraction time and provide satisfactory triterpene recovery. The methods described by Monton et al. (2019) and Puttarak and Panichayupakaranant (2013) were used for comparison. The total triterpenes in our results (36.01 ± 0.09 mg/g) appeared to be advantageous compared to maceration (5.81 ± 0.24 mg/g) and conventional MAE (26.71 ± 0.31 mg/g). Such procedures resulted in lower triterpene yields, greater time-consumption, and high solvent consumption. Most previous studies have shown MAE benefits for extracting phytochemicals that are consistent with our findings (Cassol et al., 2019; Hu et al., 2018; Kaur et al., 2019; Nabet et al., 2019). The MAE has a greater recovery capability for triterpene extraction due to its ability to destroy cell walls. The heat and internal pressure occurring inside the plant cells caused by microwave energy can contribute to the breakdown of plant cell walls. The rapid mass transfer rate of the target compounds inside the matrices increase when microwaves interact with polar solvents both outside and inside the cells, releasing triterpenes to the surrounding system (Yingngam and Brantner, 2018). The combination of ethanol and water as an extraction solvent is also considered to be a low cost and has a low toxicity. However, there is an important caveat to consider when drawing conclusions regarding the optimized MAE conditions. The result shows that a successful limit is achieved using the specific processing conditions and equipment used in this study. In addition, it would be worth testing how well the method performs with different microwave apparatuses.

3.3.2. 3D and contour plots The influence of the operating conditions on the yield of total triterpenes in depth is depicted by 3D and contour plots in which the third and fourth independent variables are held constant at the middle level. The concentration of ethanol had a major impact on the recovery of target compounds compared to other compounds. The pattern of changes in madecassoside content was the same as that of asiaticoside, and similar behaviors were observed for madecassic acid and asiatic acid. As shown in Figs. S2 and S3, the recovery of triterpene glycosides (madecassoside and asiaticoside) at a low-to-medium proportion of ethanol from 15 to 50 % (v/v) was comparatively high, but the content of these compounds diminished after 50 % (v/v). In contrast, an increase in the ethanol concentration resulted in a greater recovery of triterpene aglycone (> 60 %, v/v) (Figs. S4 and S5). With respect to total triterpenes, the addition of a moderate concentration of ethanol in water enhanced the extraction efficiency. The influence of the liquid-to-solid ratio on the recovery of the total triterpenes was negligible. The X1 of 10–30 mL/g, X2 of 45–60 % (v/v), X3 of 300–500 W and X 4 of 3–4 min seemed to be appropriate to obtain the best yields (Fig. 2a–f). Madecassoside and asiaticoside are composed of pentacyclic triterpenes with conjugated sugar moieties, and compared to their respective aglycones, exhibit a good water solvency. This behavior may be the reason why the four substances had different concentrations when extracted using solvents with distinct polarities. However, the triterpene recovery gradually decreased after reaching peak values as both variables continued to increase. This may be triggered by the degradation of heat-labile compounds due to longer contact times.

3.6. Macroporous resin enrichment The plant material was extracted by the optimum conditions and subjected to drying using the freeze-drying method. The four triterpenes were simultaneously determined by HPLC. The contents of madecassoside, asiaticoside, madecassic acid, asiatic acid, and total triterpenes were 102.78 ± 2.33, 28.35 ± 0.72, 5.70 ± 0.14, 6.65 ± 0.19, and 143.47 ± 2.29 mg/g of extract, respectively. The presence of the target compounds in the freeze-dried extract was compared to that of a commercial crude extract. The total triterpene content was approximately 38-fold higher than the total triterpene content quantified in the commercial sample (purchased from a supplier in Thailand) extracted in ethanol (Table 5).

3.4. Optimizing the MAE conditions The optimal MAE constraints should yield the maximum content for all the target triterpenes. In this investigation, the optimal solutions determined by the developed RSM model were 10 mL/g X1 , 58 % (v/v) X2 , 300 W X3 and 3.4 min X 4 , with a desirability function value of 0.9320 (Table 4). Three additional sets of experiments were conducted to validate the conditions. As expected, under the optimum conditions there was no significant difference in the extracted triterpene contents compared to the theoretical values (p > 0.05) (Table 4). The RSM approach is therefore reliable and can be considered acceptable for predicting future results. These findings verify that the methods generate values in suitable tolerance ranges.

3.6.1. Static adsorption and desorption experiments A good adsorbent should have a high adsorption capacity with a good desorption efficiency towards the adsorbent molecules (Tang et al., 2018). Diaion HP-20 resin was selected for this study because—compared to other resins—it has a polarity close to that of the target molecules and a large specific surface with a high adsorption ability for triterpenes (Puttarak and Panichayupakaranant, 2013). The static adsorption capability and the desorption ratio of the target compounds were then assessed in detail. Fig. 3a displays the static adsorption capacity of the analysts on the resin, which reached nearly 8

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Table 5 The extracted quantities of individual triterpenes determined in different samples and their physical appearances. Sample

MAE crude extract Triterpene-enriched extract (BV11-15) Triterpene-enriched extract (BV16-20) Commercial plant extract

Triterpene contents (mg/g plant extract) Madecassoside

Asiaticoside

Madecassic acid

Asiatic acid

Total triterpenes

Appearance

102.78 ± 2.33 706.67 ± 15.36 0.00 ± 0.00 1.58 ± 0.06

28.35 ± 0.72 234.59 ± 5.32 0.00 ± 0.00 1.77 ± 0.03

5.70 ± 0.14 13.42 ± 1.10 97.11 ± 5.54 0.08 ± 0.00

6.65 ± 0.19 5.77 ± 0.33 178.47 ± 22.38 0.31 ± 0.01

143.47 ± 2.29 960.45 ± 21.60 275.58 ± 27.38 3.74 ± 0.08

Yellowish powder Colorless to pale yellow powder Colorless to pale yellow powder Dark green gummy

The data are expressed as the mean ± SD (n = 3).

Fig. 3. Static adsorption and desorption experiments of target triterpenes for Diaion-HP20 macroporous resin: (a) adsorption capacity and (b) desorption ratio of target compounds using different concentrations of ethanol solution [0, 25, 50, 75, 95 and 100 % (v/v)].

100 % for all target compounds. These values were calculated to be 20.56 ± 0.47, 5.67 ± 0.14, 1.14 ± 0.03, and 1.33 ± 0.04 mg/g of dry resin for madecassoside, asiaticoside, madecassic acid, and asiatic acid, respectively. Thus, the static saturated adsorption capacity of the resin used was 28.69 ± 0.46 mg total triterpenes per g of dry resin. These results are satisfactory because the adsorption capacity of resin with a large specific surface area should be over 10 mg/g of dry resin for the separation method (Wang et al., 2018). The reason is likely explainable by the medium with respect to the lower polarity of triterpenes, allowing the compounds to be adsorbed through polar and/or nonpolar interactions with the resin. Thus, the effect of ethanol concentration on the desorption efficiency of the target compounds from Diaion-HP20 was tested. Clearly, the desorption ratio of triterpenes is highly dependent on the ethanol concentration (Fig. 3b). No analytes were found when using 0% and 25 % aqueous ethanol solutions. The difference between madecassoside and asiaticoside was minor in that these substances were completely eluted at 104.47 ± 8.09 % and 103.29 ± 1.62 %, respectively, in a 50 % ethanol aqueous solution. The desorption ratio of the sapogenin triterpene acids gradually increased as the ethanol aqueous solution increased from 50 % to 95 % (p < 0.001), and it decreased significantly when using pure ethanol as an eluent (p < 0.01). A satisfactory result was achieved because the desorption ratios of total triterpenes were 83.90 ± 2.84 % and 83.56 ± 1.33 % when using 75 %

Fig. 4. Kinetic curves of individual triterpenes adsorbed on microporous resin: (a) madecassoside and madecassic acid and (b) asiaticoside and asiatic acid.

and 95 % ethanolic solutions, respectively, as eluents. The resin used not only offered a high adsorption capability but also showed a satisfactory desorption of the target substances. 3.6.2. Dynamic adsorption investigation The adsorption kinetics of each triterpene was studied at 25 °C by maintaining a constant ratio of C. asiatica crude extract and resin at 1:5 (w/w), filled with 50 mL of deionized water. As shown in Fig. 4a and b, the adsorption of the four triterpenes increased rapidly with the adsorption time, followed by a slight increase in the adsorption capacity, and eventually, adsorption equilibrium. The adsorption behavior of the triterpenes in the glycoside and aglycone forms towards the resin used was different. Similar results were found for madecassoside and asiaticoside. These active principle targets rapidly reached saturated adsorption within 1 h of incubation. On the other hand, madecassic acid and asiatic acid shared a more similar behavior over a longer period of time than did their glycoside forms (requiring 10 h to achieve saturated adsorption). This observation could be associated with the polarity properties of the analytes. Some of the results in previous studies are not consistent with the results reported here (Jia and Lu, 2008). The difference is probably due to the use of different types of resin, leading 9

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Table 6 Kinetic parameters for the adsorption of pentacyclic triterpenes on Diaion HP-20 resin. Triterpene

Madecassoside Asiaticoside Madecassic acid Asiatic acid

Qe Experimental value (mg/g)

20.56 6.67 1.14 1.33

Pseudo-first-order kinetic model

Pseudo-second-order kinetic model

Qe Calculated value (mg/g)

k1 (min−1)

R2

Qe Calculated value (mg/g)

k2 ( g / mg × min−1)

R2

20.97 6.68 1.08 1.28

0.1430 0.1520 0.0640 0.0560

1.0000 1.0000 0.9489 0.9450

20.97 6.68 20.91 1.36

0.1420 0.1440 0.0040 0.0050

1.0000 1.0000 0.9986 0.9985

madecassoside and asiaticoside were detected. The content of these molecules improved by 22.31-fold (27.56 % purity) compared to the amount in the crude extract (p < 0.001). The total triterpene recovery was calculated to be 84 %. Thus, the enrichment method of the triterpenes from crude extract should be washed to remove sugars and other water-soluble substances with deionized water, followed by the 25 % (v/v) ethanol solution, for which each 5-bed volume should have a flow rate of 2 BV/h. Resin desorption with the 50 % (v/v) ethanol solution provided madecassoside and asiaticoside-rich fractions with almost complete separation. The 5-bed volume of the 75 % (v/v) ethanol aqueous solution was suitable for acquiring the target madecassic acidand asiatic acid-rich fraction. Finally, reusability was investigated by washing away the impurities remaining on the resin after chromatography to verify reuse in possible practical applications. The results showed that 6 BV of 95 % (v/v) ethanol or absolute ethanol followed by deionized water to remove alcohol residue were sufficient for adsorbent regeneration.

to distinct adsorption profiles of the compounds eluted. The adsorption behavior of the target chemicals can be explained by the pseudo-first-order or pseudo-second-order model (Tang et al., 2018). In this study, the pseudo-second-order equation was better correlated and had a high R2 value (> 0.99) to explain the adsorption of the target compounds (Table 6). The findings of this experiment are in accordance with earlier studies (Tang et al., 2018; Wang et al., 2018). As a result, 10 h was deemed sufficient for the entire system to achieve adsorption equilibrium; thus, 10 h was adopted for subsequent studies. 3.6.3. Dynamic desorption of target triterpenes The dynamic desorption behavior of the target molecules was studied by setting a flow rate of 2 BV/h while the ethanol concentration was varied from 0% to 100 % (v/v). Fig. 5 clearly demonstrates that the desorption capability of the eluant used varied because the four target compounds had different polarity properties. All the target compounds barely dissolved in deionized water. A small quantity of madecassoside and asiaticoside was detected in the 25 % (v/v) ethanol aqueous solution. However, both madecassoside and asiaticoside were eluted rapidly in the presence of the 50 % (v/v) ethanol solution. Thus, this concentration was proposed as the optimal concentration for desorbing the triterpene glycosides. In contrast, elution with the 75 % (v/v) ethanol solution was preferable for madecassic acid and asiatic acid.

3.7. Inhibitory activity against MMP-3 and MMP-9 production The production of MMPs is well established in photodamaged human skin (Kolakul and Sripanidkulchai, 2018). The sample effects on MMP proteins in UVB-irradiated HDFs using ELISA were examined to promote the potential benefits of C. asiatica extracts. Cell viability was measured to determine the noncytotoxic concentration of samples prior to the study of their inhibitory properties. As shown in Fig. 6a, samples including the MAE crude extract, BV11–15 fraction and BV16–20 fraction did not demonstrate toxic effects in HDFs at concentrations of 0.0001–0.1 mg/mL, with cell viability > 90 %. A significant reduction in cell viability of < 20 % was observed for all samples at 1 mg/mL (p < 0.001). A positive control (200 μM H2O2) with cell viability of 56–59 % was also evaluated in parallel to check the validity of the method. Concentrations of samples up to 0.1 mg/mL were therefore used in further UVB protection tests. Fig. 6b and c respectively demonstrate the MMP-3 and MMP-9 secretion-inhibiting activity of the test samples. These results are consistent with Kurt-Celep et al. (2019), who have reported a rise in MMP

3.6.4. Optimization of the resin chromatography The HPLC chromatograms of the triterpene standards and tested samples before and after separation on resin are shown in Fig. S6a–f. Many unwanted chemicals were removed from the crude MAE extract (Fig. S6b) when compared to those in the enriched fractions [(BV11-15) (Fig. S6e) and (BV16-20) (Fig. S6f)]. The physical appearance of the triterpene-rich extract after a single-run resin treatment ranged from colorless to pale yellow for both fractions (Table 5). The initial concentration of total triterpenes in the sample collected under optimized MAE conditions was 143.47 ± 2.29 mg/g of crude extract. The total triterpenes improved significantly by 6.70-fold—from 143.47 mg/g to 960.45 ± 21.60 mg/g of plant extract (96.05 % purity) (p < 0.001) for triterpene-rich extract (BV10–15). In the BV16–20 samples, no

Fig. 5. Dynamic desorption curves of four triterpenes from column chromatography via a column packed with Diaion HP-20 resin using a stepwise elution process with different concentrations of ethanol. 10

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Fig. 6. Effect of C. asiatica extract obtained from the optimized MAE conditions and its fractions on UVB-irradiated HDFs: (a) cytotoxicity in HDFs, (b) MMP-3 secretion, and (c) MMP-9 secretion. In the same graph, values followed by the same letter do not differ significantly (p > 0.05).

resin. The mathematical modeling obtained through the BBD-based RSM shows the relationships between the test process variables and their responses. The MAE process accelerates the mass transfer rate and improves the extraction yield while consuming less solvent. The proportion of ethanol in water has the greatest effect on the extracted quantity of the target compounds. To enrich the triterpenes, a gradient elution mode consisting of different ethanol concentrations was applied. The optimal conditions for adsorption and desorption were as follows: 20 mg/mL crude extract, column temperature 25 °C; flow rate, 2 BV/h; and ethanol solution, 0, 25, 50, 75 and 95 % (v/v). MAE crude extract and enriched samples display an inhibitory effect on MMP-3 and MMP-9 production in UVB-irradiated HDFs. The application of the experimental design with MAE supports the hypothesis that the method would produce a superior efficiency for triterpene recovery. These green and efficient procedures should be a promising option to guide industrial design for the production of triterpene-rich plant extracts.

levels following exposure to UVB. In contrast to the UVB-treated control group, all three samples suppressed an elevation in UVB-induced MMP secretion. This phenomenon tended to be concentration-dependent: a high concentration resulted in a strong potency. The attenuation of UVB-induced MMP levels by the C. asiatica samples was considerably lower than that of 50 μM ascorbic acid (positive control) (p < 0.05). It is possible to assume that C. asiatica extract and its enriched fractions caused the inhibition of MMP production by UVB radiation through the contained triterpenes and other compounds. These properties make them potential ingredients for helping to prevent skin cell photoaging caused by UVB.

4. Conclusions In this study, triterpenes were successfully extracted from the leaves of C. asiatica utilizing MAE, followed by enrichment via macroporous 11

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CRediT authorship contribution statement

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Bancha Yingngam: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Writing original draft, Writing - review & editing, Visualization, Project administration, Funding acquisition. Abhiruj Chiangsom: Formal analysis, Investigation, Data curation, Writing - original draft, Funding acquisition. Adelheid Brantner: Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment This work was supported by the Research Council of Thailand (grant number 16330/16340). 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.indcrop.2020.112231. References Abdulla, M.A., Al-Bayaty, F.H., Younis, L.T., Hassan, M.A., 2010. Anti-ulcer activity of Centella asiatica leaf extract against ethanol-induced gastric mucosal injury in rats. J. Med. Plants Res. 4 (13), 1253–1259. Alara, O.R., Abdurahman, N.H., 2019. Microwave-assisted extraction of phenolics from Hibiscus sabdariffa calyces: kinetic modelling and process intensification. Ind. Crops Prod. 137, 528–535. Azis, H.A., Taher, M., Ahmed, A.S., Sulaiman, W.M.A.W., Susanti, D., Chowdhury, S.R., Zakaria, Z.A., 2017. In vitro and in vivo wound healing studies of methanolic fraction of Centella asiatica extract. S. Afr. J. Bot. 108, 163–174. Cassol, L., Rodrigues, E., Noreña, C.P.Z., 2019. Extracting phenolic compounds from Hibiscus sabdariffa L. calyx using microwave assisted extraction. Ind. Crops Prod. 133, 168–177. Hu, B., Zhou, K., Liu, Y., Liu, A., Zhang, Q., Han, G., Liu, S., Yang, Y., Zhu, Y., Zhu, D., 2018. Optimization of microwave-assisted extraction of oil from tiger nut (Cyperus esculentus L.) and its quality evaluation. Ind. Crops Prod. 115, 290–297. Jia, G., Lu, X., 2008. Enrichment and purification of madecassoside and asiaticoside from Centella asiatica extracts with macroporous resins. J. Chromatogr. A 1193 (1–2), 136–141. Kaur, P., Pandey, D.K., Gupta, R.C., Dey, A., 2019. Simultaneous microwave assisted extraction and HPTLC quantification of mangiferin, amarogentin, and swertiamarin in Swertia species from Western Himalayas. Ind. Crops Prod. 132, 449–459.

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