Microwave assisted process intensification and kinetic modelling: Extraction of camptothecin from Nothapodytes nimmoniana plant

Industrial Crops and Products 98 (2017) 60–67

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Microwave assisted process intensification and kinetic modelling: Extraction of camptothecin from Nothapodytes nimmoniana plant Dhiraj M. Patil, Krishnacharya G. Akamanchi ∗ Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga, Mumbai, 400019, India

a r t i c l e

i n f o

Article history: Received 10 November 2016 Received in revised form 23 December 2016 Accepted 16 January 2017 Keywords: Nothapodytes nimmoniana Process intensification Camptothecin Microwave SEM Kinetic model

a b s t r a c t Process intensification of natural products extraction is possible through assistance of microwaves, ultrasound, high pressure, enzyme pretreatments and supercritical fluids. In the present paper microwave-assisted process intensification is investigated for the extraction of camptothecin (CPT) from Nothapodytes nimmoniana plant. CPT is an industrial raw material for the production of anticancer agents topotecan and irinotecan and semisynthetic analogs of CPT. Nothapodytes nimmoniana is found wild in India and is a prime source for CPT. Effects of various process factors such as stirring, solid to liquid ratio, microwave power, irradiation time and particle size on extraction kinetics have been studied. The results showed that stirring, microwave power and the temperature had a significant effect on extraction rate and yields of camptothecin. Overall process intensification has been achieved with extraction yield of 0.41% w/w of CPT, an efficiency of about 97% in very short time of just 2 min as against 6 h by classical string extraction method. Scanning electron microscopy (SEM) analysis of the spent plant material was performed to assess the effect of microwaves and found complete cell disruption has happened. This cell disruption and associated enhancement in mass transfer could be the prime reason for the process intensification. An attempt has been made to fit the extraction kinetics data with different kinetic models such as second order rate model, power law model and two site kinetic models. The second order rate model appears to be the best fit to explain the microwave assisted kinetics. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Process intensification has become imperative due to energy and environmental considerations and to maximize output with minimum inputs. Process intensification of extraction processes of natural products from plant origin has been achieved though the assistance of ultrasound, microwave, high pressure, enzyme pretreatment and supercritical fluids. In particular, microwave assisted intensification is attractive because penetration of microwaves in plant material generates heat due to its interaction with the polar components of plant matrix. The heating of microwave energy acts directly on the molecules by ionic conduction and dipole rotation (Sparr Eskilsson and Björklund, 2000). High pressure and energy created by microwaves in a closed system leads to accelerated extraction, as disruption of the plant matrix and elution of the active components to solvent take place within shortest time (Chen et al., 2010).The accelerated extraction may be attributed to the synergistic action of temperature and concentration gradients tak-

∗ Corresponding author. E-mail address: [email protected] (K.G. Akamanchi). http://dx.doi.org/10.1016/j.indcrop.2017.01.023 0926-6690/© 2017 Elsevier B.V. All rights reserved.

ing place in the same direction from inside to outside as compared with a conventional method where heat transfer takes the place from external surface to inside (Veggi et al., 2013; Yedhu Krishnan and Rajan, 2016). In microwave assisted extraction (MAE) process, heating and microwave power combinations can be tuned for efficient extraction with constant or varying temperature. MAE technique has been widely used to extract different bioactive components such as thymol (Gujar et al., 2010), piperine (Raman and Gaikar, 2002), artemisinin (Hao et al., 2002), flavonoids (Yedhu Krishnan and Rajan, 2016), phenolic compounds (Bhuyan et al., 2015; Dahmoune et al., 2014; Milutinovic´ et al., 2015) Camptothecin (CPT) was first isolated from Camptotheca acuminata by Wall and Wani in 1966 during a screening of plant extracts for antitumor activity (Wall et al., 1966). Later on, it was isolated from different plant sources (Govindachari and Viswanathan, 1972; Gunasekera et al., 1979). CPT exhibits an antitumor activity due to inhibition of topoisomerase I-DNA complex formation (Staker et al., 2002). Even though CPT has shown remarkable activity, it has drawbacks such as intolerable toxicity, and poor pharmacokinetic properties due to insolubility in water. To overcome these drawbacks and to increase the antitumor activity, various CPT analogs are semi-synthesized starting from CPT (Chavan

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et al., 2004; Du, 2003). CPT, 9-Me-CPT and other analogs have demonstrated effectiveness in killing various cancer cells such as small-cell lung cancer, ovarian cancer, and colorectal cancer. Topotecan and irinotecan are the most promising anticancer agents approved by United States Food and Drug Administration (USFDA). Other analogs are currently in different stages of preclinical and clinical trials (Yu et al., 2012). Demand for CPT and its analogs have gone up substantially and in value terms crossed US $1 billion (Ramesha et al., 2008). Currently, demand of CPT is only met by extraction and purification from natural source. N. nimmoniana, plant native to India, was found to contain the highest concentration of CPT among available natural sources (Namdeo and Sharma, 2012), representing potential as raw material for industrial scale extraction and purification of CPT. Thus it is imperative to develop efficient extraction process. Conventionally CPT extraction was carried out by either maceration or stirring methods and which require long extraction time of 5 days (Wu et al., 2008), high solvent consumption up to 20fold (Fulzele and Satdive, 2005) to 200-fold (Ramesha et al., 2008) and three extraction cycles (Fulzele and Satdive, 2005) with poor extraction yields. To overcome these drawbacks, various novel extraction techniques such as microwave, ultrasound, and pressure assisted extraction have been introduced and investigated. These techniques have been claimed to be better in terms of efficiency, extraction time and solvent consumption (Wang and Weller, 2006). Most of the MAE investigations reported thus far have focused on standardisation of the process and in very few cases an attempt made to establish quantitative relationship between the different influencing process factors and extraction kinetics. Kinetic modelling provides insightful information on the extraction behaviors. Several models have been proposed including chemical kinetic based equations (Spigno and De Faveri, 2009), modified flicks law (Gujar et al., 2010) and empirical equations such as power law (Dong et al., 2014), two site kinetic equation (So and MacDonald, 1986) and second order rate law (Ho et al., 2005; RakotondramasyRabesiaka et al., 2007). However models based on second order rate best explain the kinetics of conventional and non-conventional extractions. Present investigations are focused on microwave assisted intensification of extraction process by way of understanding effect of influencing factors such as solvents, stirring, solid to liquid ratio, microwave power, temperature, and particle size on the extraction of CPT (Fig. 1) form N. Nimmoniana. Kinetic data obtained has been studied by different models. SEM analysis on spent material has been performed to assess the changes contributing towards process intensification.

2. Materials and methods 2.1. Materials Nothapodytes nimmoniana (J. Graham) Mabb. stem material was collected from coordinates 17◦ 32 56 N 73◦ 45 11 E in month of January from nearby Mahabaleshwar, Maharashtra state, India. The material was authenticated from botanist. Prior to use it was dried at 60 ◦ C in vaccum oven dryer for 72 h, powdered, defatted and separated into three different classes: 0.84–0.42 mm, 0.42–0.25 mm, P.S < 0.25 mm respectively. The total content of CPT by exhaustive soxhlet extraction, carried out for 72 h till constant extraction yield by HPLC analysis using standard curve method, was found to be 0.42 ± 0.23% w/w. Analytical grade solvents were purchased from S.D. Fine chem. Limited, Mumbai, India. High performance liquid chromatography (HPLC) grade solvents were purchased from Rankem. All solvents were passed through 0.22 ␮m filter prior to use in HPLC analysis. CPT reference standard was prepared by

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Fig. 1. Chemical structure of camptothecin.

purification through silica gel column chromatography (#60-120, eluent chloroform: methanol 24:1). 2.2. Extraction methods 2.2.1. Stirring extraction (SE) Stirring extraction was carried out by modification of reported method (Fulzele and Satdive, 2005). Mechanical overhead stirrer with paddle blade of standard laboratory size was used for stirring. For stirring extraction, 1 g of sample was extracted at solid to liquid ratio of 1:50 g ml−1 , stirring speed 1000 rpm, particle size 0.84-0.42 mm and at temperature 40 ◦ C for 24 h. 2.2.2. Microwave assisted extraction (MAE) A microwave oven (Multiwave PRO sample preparation system, Anton Paar, Graz, Austria) equipped with temperature probe with infrared sensors, immersed pressure sensors and 16 high pressure ceramic vessels was used. The internal volume of vessels was 100 ml and maximum operational temperature and pressure at 310 ◦ C and 115 bar respectively. Multiwave PRO is closed vessel with provision of maximum power output of 1500 W and precise temperature and pressure control. To study influence of different process factors on MAE process, solvents (Methanol, ethanol, chloroform and acetone), stirring (No and low stirring), solid to liquid ratio (1:30,1:40,1:50,1:60 and 1:70 g ml−1 ), irradiation power (60,90,120 and 150 W), temperature (40,50 and 60 ◦ C) and particle size (0.84–0.42, 0.42–0.25, P.S < 0.25 mm) were taken into consideration. To investigate effect of different parameters on extraction kinetics, 1 g of the sample was extracted by changing one factor at a time and keeping other parameters constant. One factor at a time approach was considered because it provides detail investigation and understanding of effect of individual factors. 2.3. Analytical method Characterization of CPT standard was done by IR, 1 HNMR and Mass spectras (Supplementary information) IR (PerkinElmer, L1280032, KBr): 3434.3, 2927.29, 2851.54, 1743.6, 1652.91, 1604.77, 1580.41, 1439.07, 1406.99, 1345.25, 1230.76, 1200,

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1156.42, 1042.01, 917.48, 833.56, 772.80. 1 HNMR (Agilent Technologies, 400-MR, 400 MHz,CDCl3 ), ␦1.1(3H,t,J = 7.5 Hz), 1.9(2H, m), 5.32(1H, d, J = 16 Hz), 5.35 (1H, s), 5.75(1H, d, J = 16 Hz), 7.65 (1H,t,J = 7.5 Hz), 7.72 (1H,s), 7.82 (1H,t,J = 7.5 Hz), 7.91 (1H,d,J = 7.5 Hz), 8.23 (1H,d,J = 7.5 Hz), 8.4 (1H,s) and MS (Agilent Technologies, 6400 series, m/z): 349.6 (M + H)+ . For CPT quantification, Jasco HPLC system equipped with UV/Vis detector, 20 ␮l loop injector (PU 1575) were used in isocratic mode. The chro® matographic conditions: column, LiChrospher 100 RP-18, 5 ␮m (250 mm × 4.6 mm i.d, Merck, Darmstadt, Germany); mobile phase, ACN-Water (35:65) at a flow rate of 0.75 ml min−1 ; the column temperature, 30 ◦ C; sample volume injected, 20 ␮l; detector wavelength, 254 nm for detection of CPT.

Standard curve (regression equation for the area under the curve, y = 243310+ 51212x; r2 = 0.999) was prepared in the concentration range of 20 ␮g ml−1 to 200 ␮g ml−1 by HPLC analysis and employed for estimation of extraction yield. The extraction yield (mg g−1 ) of CPT was calculated by following formula: Wt (by HPLC) of the extracted CPT (mg) (1) Wt of the dried plant powdered material (g)

2.5. Scanning electron microscopy (SEM) The spent material recovered after SE and MAE were assessed for morphological changes of surface using field emission scanning electron microscope (SEM) (SU30 Camera microscope, JEOL, Japan). 2.6. Kinetic model fitting In present study second-order rate model, power law model and two site kinetic model were employed to find best fit. 2.6.1. The second-order rate model The second order rate model is prominently used in modelling extraction due to its suitable representation of process (Ho et al., 2005; Rakotondramasy-Rabesiaka et al., 2007; Yedhu Krishnan and Rajan, 2016). Therefore, this model is studied to fit the experimental data. The dissolution rate of CPT can be expressed as dCt /dt = k(Cs -Ct )2

(2)

Where, Ct is concentration of CPT in extraction liquid (mg L−1 ) at a specific time t (s), Cs is concentration of CPT at saturation in extraction liquid (mg L−1 ), t is the extraction time (s) and k is the extraction rate constant (L g−1 s−1 ). The initial extraction rate defined as h (mg L−1 s−1 ), when t approaches to 0 it can be expressed as, h = k Cs 2

(3)

Kinetic parameters (k and Cs ) were obtained by integrating Eq. (2) under the initial and boundary conditions t = 0 to t and Ct = 0 to Ct Ct = (Cs 2 kt)/(1 + k Cs t)

(4)

The linear form derived from Eq. (4) is, t/Ct = (1/k Cs 2 ) + (t/Cs )

(5)

Cs and k can be calculated by plotting graph of t/Ct vs.t Influence of temperature on rate of extraction was assessed by Arrhenius law. It can described by the following eq. k = k0 exp(-Ea /RT)

lnk = lnk0 + (-Ea /R)(1/T)

(7)

2.6.2. The power law model To model the solid-liquid extraction process, the power law has been successfully used (Dong et al., 2014). The power law model can be expressed as follows: Ct = Btn

2.4. Extraction yield determination

Extraction yield =

Where, k is the extraction rate constant (L g−1 s−1 ), k0 is the temperature-independent factor (L g−1 s−1 ), Ea is the activation energy for the extraction (J mol−1 ), R is gas constant (8.314 J mol−1 K). Linear form of Eq. (6), as ln k against 1/T plot, will provide the value of activation energy and temperature independent factor (k0 ) by eq

(6)

(8)

Where, Ct is a concentration of CPT in extraction liquid (mg L−1 ) at a specific time t (s), n is power law exponent (<1) and B is constant related to extraction rate (L g−1 s−1 ). On solving the equation (8), lnCt = lnB + n lnt

(9)

Values of B and n calculated by plotting graph of lnCt vs. lnt 2.6.3. Two site kinetic model So and Macdonald proposed two site kinetic equation is widely used to model the extraction process (So and MacDonald, 1986). Model assumes that the extraction process is taking place in two stages, first is washing stage and second is diffusion stage. The two site equation is given as follows: Ct = Cw [1-exp (-kw t)] +Cd [1-exp (-kd t)]

(10)

Where, Ct is a concentration of CPT in extraction (mg L−1 ) at a specific time t (s), Cw and Cd are a concentration of CPT in extraction liquid (mg L−1 ) in washing stage and diffusion stage respectively. Kw and Kd are coefficient of extraction (L g−1 s−1 ) during washing stage and diffusion stage respectively 2.7. Statistical analysis and model evaluation All experiments were performed in triplicate and statistical analysis were carried out using “Originpro 8”. (Originlab Corporation, USA). The values of kinetic model parameters were calculated by “Originpro 8”. (Originlab Corporation, USA). The experimental values and model values were compared by means of adjusted correlation coefficient (Radj 2 ) and root mean square deviation (RMSD).Higher values of Radj 2 and lower values of RMSD denotes that the model fits better to the experimental values. 3. Results and discussion 3.1. Microwave assisted process intensification 3.1.1. Effect of solvents The selection of suitable solvent in MAE process mainly depends upon the solvent properties such as dielectric constant, viscosity, polarity, solvent penetration and interaction with plant matrix. The solvent with high dielectric constant can absorb more microwave energy and can transfer certain amount of it for disruption of plant matrix. More polar solvents can effectively dissolve polar components. Action of microwaves generates temperature rise, decreases the solvent viscosity and surface tension and hence leads to better interaction with components which are present deep inside of plant matrix due to enhanced solvent penetration. CPT-solvent interaction mainly governed by location of CPT within plant matrix. CPT concentration is high in exterior parts of plant mainly epidermal and cortical tissues while interior parts such as xylem,

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Table 1 Physical properties of different solventsa and extraction yieldb . Solvents

Polarity index

Dielectric constant

Viscosity (cp)

Extraction yield (mg g−1 )

Ethanol Methanol Acetone Chloroform

5.2 5.1 5.1 4.1

24.60 32.60 21.01 4.81

1.20 0.60 0.32 0.57

2.987 ± 0.010c 3.349 ± 0.007d 2.991 ± 0.018c 2.576 ± 0.002e

Extraction conditions: 1 g of sample, particle size 0.84-0.42 mm, temperature 40 ◦ C, solid to liquid ratio 1:50 g ml−1 ,power, time–2 min. Different alphabet c, d, e are statistically significant (P < 0.05) a Physical property values are taken from Literature (Reichardt, 2003). b Extraction yield determined by HPLC, experiments performed in triplicate and yield ± SD is given.

phloem and pith have shown good concentration (Patil and Akamanchi unpublished data). Thus solvent which can diffuse through all this tissue and dissolve CPT can increase the efficiency. Various solvents (methanol, ethanol, chloroform and acetone) were studied for maximum extraction at conditions of solid to liquid ratio 1:50 g ml−1 , power 150 W, temperature 40 ◦ C and particle size0.84–0.42 mm. Maximum extraction yield (3.349 ± 0.007 mg g−1 ) obtained for methanol, could be correlated to its higher dielectric constant; lower viscosity and high polarity index (Table 1). So methanol was preferred as solvent for further studies. 3.1.2. Effect of extraction time Once the solvent is selected, time optimization represents other most important criteria, as over exposure of microwave radiation leads to decrease the extraction yield by means of loss of chemical structure of the active components (Chen et al., 2010; Hao et al., 2002). MAE is taking place in two stages first stage is washing and second stage is diffusion. Washing of CPT is happening initially till 60 s as observed with rapid rise. Diffusion stage is observed inbetween 60 and 300 s indicated by slow rise of CPT .After 300 s it reached to plateau indicating maximum CPT extracted (Fig. 2). The reason for shortest time of extraction would be microwave power resulting into consistent cell disruption leading to process intensification. For all process factor optimization 300 s was taken as optimum

3.1.4. Effect of solid to liquid ratio An optimum solid to liquid ratio ensures homogenous mixing and effective microwave heating. Whereas excessive solvent causes poor microwave heating as the microwave radiation would be absorbed by the solvent (Mandal and Mandal, 2010). At lower ratios (1:30, 1:40) initial rise of CPT was almost similar to higher ratios (1:60 and 1:70). Increasing solvent helped to rise the CPT concentration in diffusion stage (Fig. 4). The reason could be low ratios of solvent hindered the mass transfer of CPT from interior of plant matrix. But in higher solvent ratios such as 1:60 and 1:70, rise in CPT

Fig. 2. Comparison of SE and MAE, (A) SE conditions: 1 g of sample, particle size 0.84–0.42 mm, solid to solvent ratio 1:50 g ml−1 , stirring speed 1000 rpm and temperature 40 ◦ C (B) MAE conditions: 1 g of sample, particle size 0.84-0.42 mm, solid to solvent ratio 1:50 g ml−1 , power 150 W and temperature 40 ◦ C.

4.5 4 Extraction yield of CPT (mg/g)

3.1.3. Effect of stirring Stirring speed is another factor which plays major role. The significance of this parameter is rarely explored and very few findings have been reported (Deng et al., 2006; Liazid et al., 2011). The stirring effect influences the mass transfer by means of accelerating desorption and dissolution of active components bound within plant matrix into solvent leading to enhanced extraction (Veggi et al., 2013; Ruan and Li, 2007). When stirring was not applied the washing of CPT happened to certain extent but maximum dissolution of CPT in diffusion stage is promoted by introducing stirring (Fig. 3). The probable reason would be stirring influenced the movement of the solvent into deep interior parts such as xylem, phloem, dissolving CPT into solvent. The penetration of solvent would have hindered in absence of stirring. Thus the mass transfer barrier for CPT from concentrated CPT regions (epidermal, xylem and phloem) into solvent was minimized resulted into increase extraction yields of CPT in washing as well as diffusion stage

3.5 3 2.5 No stirring 2

Low stirring

1.5 1 0.5 0 0

50

100

150 200 Time (s)

250

300

350

Fig. 3. Effect of stirring on the kinetics of microwave-assisted extraction of CPT. Experimental conditions: Methanol, solid to liquid ratio 1:50 g ml−1 , power 150 W, temperature 40 ◦ C and particle size-0.84–0.42 mm.

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4.5

4.5

4

4

3.5 3 S/L-1:30 g/ml 2.5

S/L-1:40 g/ml

2

S/L-1:50 g/ml

1.5

S/L-1:60 g/ml S/L-1:70 g/ml

1

Extraction yield of CPT (mg/g)

Extraction yield of CPT (mg/g)

64

0.5

3 2.5

T-40°C

2

T-50°C T-60°C

1.5 1 0.5

0 0

50

100

150 Time (s)

200

250

0

300

Fig. 4. Effect of solid to liquid (S/L) ratio on the kinetics of microwave-assisted extraction of CPT. Experimental conditions: Methanol, low stirring, power 150 W, temperature 40 ◦ C and particle size-0.84–0.42 mm.

concentration in washing as well as diffusion stage was achieved much faster. In case lower ratios (1:30, 1:40) equilibrium yields 3.759 ± 0.005 and 3.909 ± 0.021 mg g−1 was obtained. This might be due to stirring effect which would have influenced the diffusion of CPT leading to increased extraction efficiency. With increasing ratio till 1:60, equilibrium yield increased up to 4.023 ± 0.025. So solid to liquid ratio 1:60 was taken as optimum. 3.1.5. Effect of microwave power Microwave power creates localized heating, absorption and transfer of microwave energy from solvent to the plant matrix leads to cell disruption (Wang and Weller, 2006). Temperature and microwave power are interrelated as high microwave power increases the extraction temperature of the system (Veggi et al., 2013). Most of the papers reported that the increase in microwave power increases the extraction yield up to certain limit after that it becomes insignificant (Mandal and Mandal, 2010). The CPT concentration increased rapidly with increasing power during the washing stage (till 60 s), followed by more consistent increase during diffusion stage (60–300 s) of extraction. Increase in microwave power (from 60W to 120W) acted as a driving force for MAE for rise of CPT concentration in washing stage itself from 2.660 ± 0.016 mg g−1 to 3.458 ± 0.017 mg g−1 (Fig. 5). The extraction yields obtained at microwave power 120 W and 150 W were almost similar revealed that power increase above 120W is insignificant.

0

50

100

150 200 Time (s)

250

300

350

Fig. 6. Effect of temperature on the kinetics of microwave-assisted extraction of CPT. Experimental conditions: Methanol, low stirring, solid to liquid ratio 1:60 g ml−1 , power 120 W and particle size-0.84–0.42 mm.

3.1.6. Effect of temperature With increase in temperature, CPT concentration in washing stage was slightly increased while in diffusion stage very slow and steady rise was observed (Fig. 6). The increase in temperature would have facilitated the motion of solvent into interior parts as well as increased CPT solubility in solvent. Interactive effect of power and temperature would have caused consistent disruption of plant matrix which in turn facilitated slow but steady rise in CPT concentration. Equilibrium yields were almost similar at 50 and 60 ◦ C. So 50 ◦ C was considered as optimum temperature. 3.1.7. Effect of particle size Particle size reduction increases the surface area for extraction. As more surface area is available it facilitates the solvent diffusion. Also more no. of cells directly exposed to microwaves results into spontaneous cell disruption. Particle size reduction would have contributed to rise of concentration of CPT in the solvent (Fig. 7). In first 60 s, rapid rise was observed. Whereas very slow diffusion was observed in between 60 and 120 s and no significant yield increase were observed after 120 s. 3.1.8. Activation energy To achieve solute transfer to solvent, it has to overcome the energy barrier; indicated as activation energy. Solute-solute

4.5

4.5

4

4

3.5 3 P-60W

2.5

P-90W

2

P-120W 1.5

P-150W

1 0.5

Extraction yield of CPT (mg/g)

Extraction yield of CPT (mg/g)

3.5

3.5 3 2.5

Particle size -0.84-0.42 mm

2

Particle size-0.42-0.25 mm

1.5

Particle size<0.25 mm

1 0.5

0

0

0

50

100

150 200 Time (s)

250

300

350

Fig. 5. Effect of microwave power on the kinetics of microwave-assisted extraction of CPT. Experimental conditions: Methanol, low stirring, solid to liquid ratio 1:60 g ml−1 , temperature 40 ◦ C and particle size-0.84–0.42 mm.

0

50

100

150 Time (s)

200

250

300

350

Fig. 7. Effect of particle size on the kinetics of microwave-assisted extraction of CPT. Experimental conditions: Methanol, low stirring, solid to liquid ratio 1:60 g ml−1 , power 120 W and temperature 50 ◦ C.

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Table 2 Radj 2 and RMSD values of the models at different extraction conditions. Second order rate model

Power law model

Radj 2

RMSD

Radj 2

RMSD

Radj 2

RMSD

0.999 0.998

0.057 0.121

0.939 0.977

0.054 0.033

0.989 0.995

0.183 0.153

Solid to liquid ratio (g ml−1 ) 1:30 0.998 1:40 0.998 1:50 0.998 1:60 0.997 1:70 0.998

0.113 0.097 0.114 0.136 0.098

0.953 0.955 0.919 0.950 0.934

0.051 0.046 0.062 0.063 0.060

0.995 0.996 0.996 0.997 0.997

0.120 0.130 0.145 0.186 0.178

Microwave power (W) 60 90 120 150

0.995 0.993 0.996 0.997

0.110 0.163 0.139 0.107

0.906 0.921 0.950 0.966

0.063 0.091 0.064 0.049

0.986 0.958 0.968 0.939

0.141 0.184 0.173 0.176

Temperature (◦ C) 40 50 60

0.997 0.998 0.999

0.099 0.070 0.057

0.958 0.968 0.980

0.053 0.040 0.045

0.988 0.998 0.999

0.122 0.105 0.100

Particle size (mm) 0.84−0.42 0.42−0.25 P.S < 0.25

0.999 1 1

0.067 0.011 0.021

0.972 0.910 0.946

0.037 0.056 0.035

0.989 0.999 0.999

0.180 0.102 0.098

MAE conditions

Effect of stirring No stirring Low stirring

Two site kinetic model

Table 3 Experimental and predicted CPT saturation concentration of the second order rate model at different extraction conditions. MAE conditions

Extraction constant (k) (L g−1 s−1 )

Intial extraction rate (h) (mg L−1 s−1)

Experimental conc.of CPT at saturation Cs (mg L−1 )

Predicted conc. of CPT at saturation Cs (mg L−1 )

Effect of stirring No stirring Low stirring

0.038 0.027

0.209 0.174

74.326 80.300

72.525 77.301

Solid to liquid ratio (g ml−1 ) 1:30 1:40 1:50 1:60 1:70

0.028 0.027 0.026 0.026 0.025

0.439 0.257 0.168 0.120 0.088

125.311 97.723 80.464 67.953 59.531

119.808 94.027 77.149 66.030 57.107

Microwave power (W) 60 90 120 150

0.029 0.024 0.023 0.024

0.082 0.101 0.112 0.118

53.282 65.081 69.575 70.357

50.584 62.433 67.032 67.408

Temperature (◦ C) 40 50 60

0.026 0.033 0.035

0.125 0.584 0.635

69.581 70.039 70.707

66.818 68.414 69.182

Particle size (mm) 0.84−0.42 0.42−0.25 P.S < 0.25

0.033 0.058 0.066

0.579 1.051 1.181

70.113 70.616 70.618

68.336 70.401 70.405

cohesive and solute-solid adhesive interactions are the major contributors to activation energy (Alupului et al., 2012). At higher temperatures, these interactions become quite weak, which results in increased extraction rate constant (Yedhu Krishnan and Rajan, 2016). If the Ea values are lower than 20 kJ mol−1 , the extraction process is governed by the diffusion (Wang et al., 2002), and if the value is higher than 40 kJ mol−1 , the process is controlled by the solubilization reaction (González-Centeno et al., 2015). Whereas activation energy situated between 20 and 40 kJ mol−1 is governed by a mixed reaction and diffusion regime (Lazar et al., 2016). In case of CPT extraction activation energy required was 12.7 kJ mol−1 ; positive value indicates endothermic nature of process. This value signifies that CPT extraction process is diffusion controlled process.

3.2. Comparison of kinetic models To understand the complex diffusion, mass transfer and thermodynamic parameters affecting the extraction rate, kinetic modelling is of prime importance. To find the best possible fit with experimental data, different kinetic models such as second order rate model, power law model, and two site kinetic model were employed. To determine the appropriateness of mathematical model and evaluate the goodness of fit, Radj 2 and RMSD are frequently used. The high value of Radj 2 and low value of RMSD denotes good fit. Radj 2 and RMSD values for different models are presented in Table 2. Among these, the second order rate model fits the best with experimental values showing the highest average values of Radj 2 (0.993-1) and

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the lowest average values of RMSD (0.011-0.163). For all studied factors, the plots of t/Ct vs t resulted in a linear function. The linearity of the functions is in agreement with second order rate model and allowed in determination of experimental values of kinetic parameters. 3.3. Effect of kinetic parameters on CPT extraction kinetics The kinetic parameters of the second order rate model for CPT extraction at different extraction conditions are represented in Table 3. The increment in initial rate of extraction (h), second order rate constant (k) and saturation concentration (Cs ) were observed for all parameters except solid to liquid ratio. Even though equilibrium yield were higher at higher solid to liquid ratio. It was observed that initial extraction rate and saturation concentration was highest at low ratios and then declined progressively with increasing solid to liquid ratio (Table 3) this could be attributed to inadequate mixing. Microwave enhanced cell disruption and simultaneous stirring effect would have caused sudden rise of CPT concentration in solvent leading higher saturation even at low ratios. Similar results were reported, where flavonoid saturation

concentration and initial rate extraction decreased with increasing solid to liquid ratio (Yedhu Krishnan and Rajan, 2016). The increase in saturation concentration with increase other parameters may be correlated to enhanced diffusion of CPT into solvent by means of either desorption or dissolution. At all process conditions, predicted values matched well with experimental values Thus second order model is satisfactory fit for CPT extraction. 3.4. Comparison of SE and MAE Physical changes happened on particle surface was analysed before and after extraction. The spent material was analysed by scanning electron microscopy before (Fig. 8A) and after (Fig. 8B and Fig. 8C) extraction. In case of SE the SEM image (Fig. 8(B1)) of spent powder showed only slight damage otherwise image were almost similar to the SEM image of stem material before extraction (Fig. 8(A1)). In comparison SEM images of spent material, MAE process showed complete cell disruption (Fig. 8(C1) and (C2)). Due to rapid disruption by MAE process shortened extraction time to just 2 min with more than 95% extraction efficiency, whereas with SE even after 6 h efficiency was only 46.51%.

Fig. 8. Scanning Electron Micrographs (SEM) of the samples; (A1) Control-Ground sample × 250; (A2) Control −Ground sample × 500; (B1) SE sample × 300; (B2) SE sample × 500; (C1) MAE sample × 250; (C2) MAE sample × 500.

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4. Conclusion Even though CPT extraction from Nothapodytes nimmoniana plant is already reported, the use of microwaves for process intensification and kinetic modelling is first time attempted. Process intensification due to action microwaves lead to time reduction to just 2 min. The kinetic studies confirmed that process factors such as stirring, power and temperature has significant effect on extraction efficiency. To find the best possible fit to explain extraction kinetics three models were evaluated. Second order rate model appears to be the best to explain the extraction kinetics. At all the process conditions, predicted values matched well with experimental values. SEM images further provided proof of complete cell disruption justifying the use of microwaves. MAE showed higher yields of CPT as compared with SE by 2-fold at solid to liquid ratio 1:60 g ml−1 , low stirring, microwave power 120 W, particle size-0.42–0.25 mm and at temperature 50 ◦ C. In some cases maximum 97% extraction efficiency achieved. Finding from these studies provides understanding of mechanisms involved in the kinetics of extraction of CPT from N. nimmoniana which has better potential for commercialization. Acknowledgment The authors are thankful to the University Grant Commission (UGC) of India for the financial support under Special Assistance Programme (UGC-SAP). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop.2017.01. 023. References Alupului, A., Cˇalinescu, I., Lavric, V., 2012. Microwave Extraction of active principles from medicinal plants. UPB Sci. Bull. Ser. B Chem. Mater. Sci. 74, 129–142. Bhuyan, D.J., Van Vuong, Q., Chalmers, A.C., van Altena, I.A., Bowyer, M.C., Scarlett, C.J., 2015. Microwave-assisted extraction of Eucalyptus robusta leaf for the optimal yield of total phenolic compounds. Ind. Crops Prod. 69, 290–299. Chavan, S.P., Pasupathy, K., Sivappa, R., Venkatraman, M.S., 2004. Facile syntheses of ABC ring skeleton of camptothecin and related alkaloids. Syn. Commun. 34, 3099–3110. Chen, Y., Gu, X., Huang, S., quan Li, J., Wang, X., Tang, J., 2010. Optimization of ultrasonic/microwave assisted extraction (UMAE) of polysaccharides from Inonotus obliquus and evaluation of its anti-tumor activities. Int. J. Biol. Macromol. 46, 429–435. Dahmoune, F., Spigno, G., Moussi, K., Remini, H., Cherbal, A., Madani, K., 2014. Pistacia lentiscus leaves as a source of phenolic compounds: microwave-assisted extraction optimized and compared with ultrasound-assisted and conventional solvent extraction. Ind. Crops Prod. 61, 31–40. Deng, C., Mao, Y., Yao, N., Zhang, X., 2006. Development of microwave-assisted extraction followed by headspace solid-phase microextraction and gas chromatography-mass spectrometry for quantification of camphor and borneol in Flos Chrysanthemi Indici. Anal. Chim. Acta 575, 120–125. Dong, Z., Gu, F., Xu, F., Wang, Q., 2014. Comparison of four kinds of extraction techniques and kinetics of microwave-assisted extraction of vanillin from Vanilla planifolia. Food Chem. 149, 54–61. Du, W., 2003. Towards new anticancer drugs: a decade of advances in synthesis of camptothecins and related alkaloids. Tetrahedron 59, 8649–8687. Fulzele, D.P., Satdive, R.K., 2005. Comparison of techniques for the extraction of the anti-cancer drug camptothecin from Nothapodytes foetida. J. Chromatogr. A 1063, 9–13. González-Centeno, M.R., Comas-Serra, F., Femenia, A., Rosselló, C., Simal, S., 2015. Effect of power ultrasound application on aqueous extraction of phenolic

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