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Simultaneous extraction and separation of flavonoids and alkaloids from Crotalaria sessiliflora L. by microwave-assisted cloud-point extraction
Separation and Purification Technology 175 (2017) 266–273
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Simultaneous extraction and separation of flavonoids and alkaloids from Crotalaria sessiliflora L. by microwave-assisted cloud-point extraction Xunyou Tang a, Dan Zhu a,b, Wenbei Huai c, Wei Zhang c,⇑, Chujun Fu c, Xiujuan Xie a, Sisi Quan a, Huajun Fan a,⇑ a b c
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China Guiyang College of Traditional Chinese Medicine, Guiyang 550025, China School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006, China
a r t i c l e
i n f o
Article history: Received 12 June 2016 Received in revised form 5 November 2016 Accepted 12 November 2016 Available online 18 November 2016 Keywords: Microwave-assisted cloud point extraction Crotalaria sessiliflora L. Triton X-100 Flavonoids Alkaloids Orthogonal test
a b s t r a c t A novel method of simultaneous extraction and separation of flavonoids and alkaloids from Crotalaria sessiliflora L., was developed by microwave-assisted cloud-point extraction (MACPE) using Triton X100NaClHCl system as extractant. The optimum conditions including Triton X-100 concentration, NaCl concentration, HCl concentration, solvent-to-material ratio, extraction temperature and time were investigated by orthogonal test on the single factor experiment. The extraction yields of vitexin, isovitexin and monocrotaline reached respectively up to 601.4, 386.7 and 105.5 lg/g under the optimum conditions. Compared with heating reflux extraction (HRE) and ultrasonic-assisted extraction (UAE), MACPE exhibited a significantly higher extraction yields and took a shorter time (only 10 min). The MACPE mechanism was discussed based on phase’s shift and extraction property. As a result, flavonoids and alkaloids can be transferred respectively to bottom phase and top phase after simultaneous extraction. The proposed method integrated MAE with cloud-point extraction (CPE) in one-step procedure took both advantages. It provided a simple and efficient alternative to extraction and separation of active constituents from the herb materials. Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction Crotalaria sessiliflora L. is a Chinese herbal medicine as a leguminous plant, commonly used in the treatment of esophageal cancer, cervical cancer and skin cancer [1,2]. Its main active constituents were reported to be alkaloids, flavonoids and amino acids [3–7]. Monocrotaline is the most important pyrrolizidine alkaloid due to high antitumor effects. Due to strong liver toxicity, its derivatives were also synthesized through structural transformation to reduce toxicity and retain the anticancer effects [7–10]. However, monocrotaline as a drug is used widely in clinic research for pulmonary hypertension model [11–17]. Relatively, flavonoids present in plants have pharmacological effects of antioxidant, hepatoprotective, anticancer, anti-inflammatory and anti-diabetes [18–25]. Flavonoids like vitexin, isovitexin and analogues were found from Crotalaria sessiliflora L. especially for the treatment of cardiovascular diseases [3,6,26–29]. Therefore, multipharmacological effects (especially anticancer effects) of the
⇑ Corresponding authors. E-mail addresses: [email protected] (W. Zhang), [email protected] (H. Fan). http://dx.doi.org/10.1016/j.seppur.2016.11.038 1383-5866/Ó 2016 Elsevier B.V. All rights reserved.
extract from Crotalaria sessiliflora L. have attracted attention due to multi active constituents [2,8,30–32]. Moreover, vitexin have been used as primary ingredients in cardiovascular pharmaceutical preparations such as drop pills and dispersible tablets (named Yixintong), and the demands of monocrotaline in research uses are also increasing. Active constituents in Crotalaria sessiliflora L. are usually extracted by conventional methods such as heat reflux extraction and ultrasonic extraction [3,6,32–35], which are subject to remarkable shortcomings, including the lengthy process, the high cost of organic solvents, low recovery, and toxic solvent residuals in the products. In previous work, we extracted flavonoids and alkaloids respectively by ultrasonic-assisted extraction (UAE) and microwave-assisted extraction (MAE) using methanol and ethanol as extracting solvents [36,37]. However, the pretreatment of sample purification had to be conducted prior to chromatographic analysis due to containing many impurities and interferences in the extracts. Now there is an increasing realization that an efficient alternative to extraction of active constituents from the herb is necessary in their researches. In the past decades, novel extraction techniques attracted great attentions to extraction and enrichment of active
X. Tang et al. / Separation and Purification Technology 175 (2017) 266–273
compounds from natural products based on two-phase (or multiphase) separation caused by combination of water, organic solvents, ionic liquid, polymers, salt and others such as aqueous two phase extraction (ATPE), dispersive liquid-liquid extraction (DLLE), micellar extraction and cloud-point extraction (CPE) [38–42]. Among them, CPE has been widely applied in the extraction and preconcentration of constituents such as metal ions and hydrophilic proteins, especially small molecule organics as a promising, environmentally-friendly, cost-effective means for sample preparation [42–48]. As aqueous nonionic surfactant solutions split into two phases beyond a cloud-point temperature (CPT), a hydrophobic constituent with similar polarity is extracted into the surfactant-rich phase, while the hydrophilic material is left in the aqueous phase. Thus, CPE like ATPE exhibited many advantages such as non-volatility, non-flammability, less consumption of solvents, less impact on the environment, the simple procedure, the convenient to follow steps and protection of the material nature [48–51]. With the development of CPE technology, combining it with other techniques can greatly improve the extraction efficiency. Microwave-assisted extraction (MAE) combining with CPE, is an efficient alternative to extraction of active constituents from the herb plant for enrichment and purification [52–54]. However, the combination of both in fact doesn’t occur simultaneously, subjecting to diverse processes. In other words, MAE and CPE were conducted respectively in two-steps procedures using different extractants. To make full use of MAE advantages, a micellar solution as extractant can be formed by dispersing surfactant into an aqueous solution, and addition of inorganic salts can enhance the polarity of the solution, leading to be a good dielectric poverty to produce a strong coupling effect with the microwave. Moreover, this cloud point system (CPS) as homogeneous system can extract active constituents from the herb materials (unlike aqueous two phase system (ATPS) even under the presence of salt), and transferring target compounds into top or bottom phase when two-phase system was formed beyond lower CPT. Thus, microwave-assisted cloud point extraction (MACPE), combining CPE with microwave-assisted extraction (MAE), is a strategy for not only simultaneous extraction of active constituents from herb plants but also separation or purification of them with each other due to the formation of one phase and two phases. It is different from the conventional MAE prior to CPE (a nonionic surfactant was only used for preconcentration or enrichment of target constituents extracted by another solvent), resulting in not only take advantage of the extraction selectivity but also improve extraction efficiency through demixing effect of CPE and coupling effect of MAE. In this work, our aim is to integrate MAE with CPE for extraction and purification of active constituents from Crotalaria sessiliflora L. using a CPS of the nonionic surfactant Triton X-100. The technique that named MACPE, would improve demixing effect and field intensification for enrichment of desired constituents and enhancement of extraction yields. After screening of non-ionic surfactants, the experimental conditions of extraction of vitexin, isovitexin and monocrotaline (shown in Fig. 1), including the composition of the CPS, addition of the salt, extraction temperature, extraction time, solvent-to-material ratio, etc., were investigated and optimized by single-factor experiment and orthogonal experiment. To evaluate the extraction efficiency, the results of MACPE were compared with heat reflux extraction (HRE) and ultrasonic-assisted extraction (UAE). It was found that the results of MACPE were quite satisfying, and its operational process was simpler, easier and more efficient. To the best of our knowledge, this is the first application of a novel MACPE in the extraction of active constituents from Crotalaria sessiliflora L.
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2. Experimental 2.1. Materials and reagents Dry samples of Crotalaria sessiliflora L. were purchased from Guangxi in China (Batch number 20120926). The samples were powdered, sieved and placed in a desiccator. Vitexin (purity P 98%) was purchased from National Institutes for Food and Drug Control (China); Isovitexin and monocrotaline (purities P 98%) was bought from Shanghai Forever Biotech Co., Ltd. (China). Triton X-100 of analytical grade was purchased from Shanghai Macklin Biochemical Co., Ltd. (China). Acetonitrile and methanol of HPLC grade were purchased from Merck Darmstadt Ltd. (Germany). Hydrochloric acid, NaCl, ammonia, ethanol and chloroform of analytical grade were obtained from Guangzhou Reagent factory (China). 2.2. Instruments and apparatus MACPE experiments were performed on an EXCEL microwave extraction system (PreeKem Scientific Instruments Co., Ltd., China) equipped with a digital timer, power and a temperature controller. HPLC analysis was carried out using Agilent 1200 Infinity chromatograph with a UV detector (Agilent Technologies Co., Ltd. USA). 2.3. Preparation of the CPS For preparation of Triton X-100–NaCl–HCl of the CPS, a certain amount of Triton X-100 was dissolved in deionized water, and then added a certain mass of sodium chloride and a certain volume of hydrochloric acid, respectively. After adding appropriate amount of deionized water, the mixture mixed well by a vortex stirrer. The CPS of Triton X-100 as an extractant was obtained at final concentrations of 0.5–5% (v/v) Triton X-100, 1.4% (w/v) sodium chloride and 0.1 mol/L hydrochloric acid, respectively. 2.4. Extraction strategy The strategy of MACPE combine MAE with CPE for extraction and purification of flavonoids and alkaloids from Crotalaria sessiliflora L. was planed according to the following scheme as shown in Fig. 2. In this way, flavonoids and alkaloids were together extracted from the herb material by MAE with the CPS of Triton X-100/sodium chloride, and then flavonoids and alkaloids were separated from each other by phase separation at a water bath beyond the CPT. 2.5. MACPE procedure 0.5 g of the herb powder and 50 mL of the CPS were put into an extraction vessel, which was then sealed and placed in the microwave extraction system at 80 °C under 780 W for 10 min. After extraction completed, the extract was transferred to a centrifuge tube for centrifugation at 3800 rpm for 1 min. The supernatant was placed in a water bath at 68 °C to stand for 30 min, then centrifuging at 3800 rpm for 5 min. The top phase was collected and dried in a rotary evaporator. The residue obtained and bottom phase were diluted respectively with methanol up to 5 mL for HPLC analysis. The extraction was assessed by the yield (Y) of target constituent according to the following formula.
Yield ðmg=gÞ ¼
me m0
where me is the total amount of the target constituent extracted from the plant material (m0).
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X. Tang et al. / Separation and Purification Technology 175 (2017) 266–273 OH
OH
HO
HO
HO OH O
O
O
HO
OH
HO HO
O
HO OH HO OH
O
O
O
O
O
OH
N
OH
O
Vitexin
Isovitexin
Monocrotaline
Fig. 1. The chemical structures of vitexin, isovitexin and monocrotaline.
Fig. 2. The scheme of extraction and separation of flavonoids and alkaloids from Crotalaria sessiliflora L. by MACPE.
2.6. HPLC analysis The extract of each phase was filtered through a 0.22 lm membrane, and HPLC analysis of flavonoids and alkaloids was carried out according to the following conditions: 10 lL of sample was injected into an Agilent 1200 Infinity chromatograph for separation on a Phenomenon Gemini C18 Column (4.6 mm 250 mm, 5 lm) with acetonitrile-0.5% phosphate solution (v/v, 14:86) for flavonoids and methanol-0.1% ammonia solution (v/v, 10:90) for alkaloids as the mobile phases at 1.0 mL/min of flow rate. Then flavonoids and alkaloids were detected by a UV detector at 340 nm and 220 nm, respectively.
3. Results and discussion 3.1. Single-factor experiment 3.1.1. The effect of the concentrations of Triton X-100 and NaCl For extraction of flavonoids and alkaloids from Crotalaria sessiliflora L., concentration of the surfactant determines not only extraction yield but also the phase volume ratio at phase separation. The addition of salt will improve microwave effect by increasing polarity of the extractant, and facilitating to reduce the CPT. So Triton X-100 and NaCl were used as extractant and addictive respectively, the effects of their concentrations on the extraction yields of several constituents were investigated in the range of 0.5–5%(v/v) and 0.6–1.4% (w/v), as shown in Fig. 4. The results demonstrated that the extraction yields achieved the maximum yields when Triton X-100 concentration and NaCl
concentration was increased to 3% (v/v) and 1.4% (w/v) respectively. The CPT was reduced with increase of NaCl concentration from 63 to 47 °C. Nevertheless, more than 1.4% (w/v) NaCl would make surfactant-rich phase converted into the top phase from the bottom phase, and demarcation between two phases separated was not clear. For the CPE process, 68 °C was determined as the most appropriate CPT at 1.4% (w/v) NaCl because of good demixing effect. From results, the selection of 3% (v/v) TritonX-100 and 1.4% (w/v) NaCl was suitable for next experiments. 3.1.2. The effect of temperature and time Extraction temperature and time are the key factors affecting the extraction yields. In the MAE process, extraction temperature was controlled by the microwave power given, and extraction time dictated maximum extraction yields. Hence, the effects on extraction yields of flavonoids and alkaloids from Crotalaria sessiliflora L. were carried out at 60–110 °C for 5–25 min so as to determine their proper parameters. The results obtained were shown in Fig. 5. From Fig. 5, the extraction yields of the desired constituents were enhanced when increasing extraction temperature and time. The maximums of vitexin and isovitexin were at 90 °C for 10 min, whereas that of monocrotaline was at 80 °C for 10 min. Because raising temperature can improve the interaction between solvent molecules, and accelerate the mass transfer. But too high temperature may lead to decomposition, even coking the sample. 3.1.3. Effect of HCl concentration Acidity of the extractant is closely related to the structural form of flavonoids and alkaloids, so the effect of acidity on the extraction yields was investigated by adjusting HCl concentration. The results
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50
(a)
45
Bottom phase Top phase Standard
1
(b)
45
2
Bottom phase Top phase Standard
40
35
35
30
30
25
25
mV
mV
40
50
1--Vitexin 2--Isovitexin
20
20
15
15
10
10
5
5
0
Monocrotaline
0 0
5
10
15
20
25
30
35
40
45
50
0
5
10
Time (min)
15
20
25
30
Time (min)
Fig. 3. HPLC chromatograms of flavonoids (a) and alkaloids (b) extracted from Crotalaria sessiliflora L. in top and bottom phases.
800 700 600
600
500
500
400 300
(b)
Vitexin Isovitexin Monocrotaline
700
Yield (µg/g)
Yield (µg/g)
800
Vitexin Isovitexin Monocrotaline
(a)
400 300
200
200
100
100 0
0 0
1
2
3
4
0.6
5
0.8
1.0
1.2
1.4
CNaCl (%, w/v)
CTriton X-100 (%, v/v)
Fig. 4. The effects of the concentrations of Triton X-100 (a) and NaCl (b) on the extraction yields.
700
800
(a)
Vitexin Isovitexin Monocrotaline
700
600
600
Yield (µg/g)
500
Yield (µg/g)
Vitexin Isovitexin Monocrotaline
(b)
400 300
500 400 300
200
200
100
100
0
0 60
70
80
90
100
110
4
8
12
16
20
24
28
Time (min)
Temperature ( ) Fig. 5. The effect of temperature (a) and time (b) on the extraction yields.
shown in Fig. 6 indicated that the maximum yield of vitexin, monocrotaline and isovitexin were at 0.05 mol/L, 0.05 mol/L and
0.10 mol/L, respectively. The reason is that acidity would structurally change the greater polarity transformation of target
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800
Vitexin Isovitexin Monocrotaline
700
Yield (µg/g)
600 500 400 300 200 100 0 0.00
0.05
0.10
0.15
0.20
0.25
CHCl (mol/L) Fig. 6. The effect of HCl concentration on extraction the yields.
constituents, leading to their repartition in two phases. It also revealed that monocrotaline in cationic form would be easy to be extracted in an acidic top phase whereas two flavonoids in molecular forms were transferred into the bottom phase. Thus, 0.1 mol/L HCl was used in next experiment. 3.1.4. Effect of solvent-to-material ratio The effect of solvent-to-material ratio on the extraction yields was also investigated on the basis of other parameters determined above. i.e. According to the CPS composition of 3% Triton X-100 (v/ v) and 1.4% (w/v) NaCl, the extraction experiment was performed at 90 °C for 10 min under presence of 0.1 mol/L HCl. The results as shown in Fig. 7 gave relatively stable yields while solvent-tomaterial ratio was over 90:1. The maximum yields of vitexin, isovitexin and monocrotaline were found at 100:1. In fact, raising solvent-to-material ratio meant to increase solvent volume, which would be helpful to dissolve more targets constituents until their saturation. So the solvent-to-material ratio was determined as 100:1 in the following experiment. 3.2. Optimization of MACPE conditions with orthogonal test Considering the limitation of single factor experiment, especially interrelation among single factors, further optimization is 800
Vitexin Isovitexin Monocrotaline
700
Yield (µg/g)
600 500 400 300 200 100 0 60
70
80
90
100
110
Ratio of solvent to material Fig. 7. The effect of solvent-to-material ratio on the extraction yields.
necessary for applicable use. On the basis the results of single factor experiment, several key factors such as Triton X-100 concentration, extraction temperature and time, solvent-to-material ratio, were investigated with orthogonal test by evaluating the yields of vitexin, isovitexin and monocrotaline. Thus, these four factors were arranged to 9 experiments according to an orthogonal test design of L9 (34) as shown in Table 1. Data of every experiment in triplicate were obtained from a 9-run-experiment by HPLC analysis. The experimental results and variance analysis also listed in Tables 2 and 3, respectively. Seen from the results obtained above, it can be found that Triton X-100 concentration has more significant effect on the extraction yield of three constituents among factors. The factors could be ranked by importance for flavonoids and the alkaloid as follows: A (Triton X-100 concentration) > C (solvent-to-material ratio) > B (extraction temperature) > D (extraction time). The results of range analysis also gave the optimum levels for vitexin and isovitexin were A3B2C2D2 while that for monocrotaline was A3B1C2D2. In other words, the effects of these factors on their extraction yields were not always consistent. Relatively, monocrotaline is susceptible to high temperatures due to chemical stability. Thus, the optimum conditions of FMATPE were selected as Triton X-100 concentration of 4% (v/v), extraction temperature of 80 °C, solvent-to-material ratio of 100:1, extraction time of 10 min. 3.3. Validation of optimum conditions To confirm feasibility of MACPE, the validation of experiments were conducted under the optimum conditions. The mean value of triplicate results follows as 601.4 lg/g for vitexin, 386.7 lg/g for isovitexin and 105.5 lg/g for monocrotaline, respectively. Their relative standard deviations (RSDs) were less than 4.3%. The validation results demonstrated that adapting L9 (34) orthogonal array scheme could optimize MACPE conditions with higher yields for target constituents. The fact was proved that MACPE is an efficient alternative to simultaneous extraction and separation of flavonoids and alkaloids from the herb material. 3.4. Comparison with different extraction methods In order to assess MACPE approach, a comparative study was conducted using the identical CPS consisting of Triton X-100– NaCl–HCl as extractant with the conventional methods such as heating reflux extraction (HRE) and ultrasonic-assisted extraction (UAE). In view of the above, the experiments of extraction of vitexin, isovitexin and monocrotaline from the herb material could be respectively accomplished under the same conditions of 4% Triton X-100 concentration, 1.4% NaCl concentration, extraction temperature of 80 °C and solvent-to-material ratio of 100, in addition to extraction time of 10 min for MACPE, 90 min for HRE and 60 min for UAE. The obtained results were listed in Table 4. As shown in Table 4, the results exhibited that MACPE possessed the greatest yields and took the shortest extraction time while compared with that of HRE and UAE. The conventional HRE also had relatively low yield by contrast to UAE. For example, MACPE had the outstanding extraction efficiency up to 601.4 lg/g for vitexin, which was found to be superior to others we used methanol and ethanol [35–37]. In microwave field, the CPS consisted of Triton X-100, was proved to be a good extractant with stronger extraction capacity owing to its dielectric property. Moreover, its hydrophilic and hydrophobic groups also provided a specific function to further improve the extraction selectivity through demixing effect when temperature was over the CPT. As a results, flavonoids would be transferred to the rich-surfactant bottom phase, whereas alkaloids could be migrated to the aqueous top phase after phase separation. Therefore, MAE combined with CPE
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X. Tang et al. / Separation and Purification Technology 175 (2017) 266–273 Table 1 L9 (34) orthogonal test design for extraction of flavonoids and alkaloids. Level
Factor
1 2 3
A (Triton X-100) (%)
B (Temperature) (°C)
C (Solvent-to-material ratio) (mL: g)
D (Extraction time) (min)
2 3 4
80 90 100
90:1 100:1 110:1
5 10 15
Table 2 The results of the orthogonal test by L9 (33). No.
1 2 3 4 5 6 7 8 9 K1 K2 K3 R K1 K2 K3 R K1 K2 K3 R
Yield (lg/g)
Factor A
B
C
D
Vitexin
Isovitexin
Monocrotaline
1 1 1 2 2 2 3 3 3 1612.88 1709.46 1760.2 147.32 1030.18 1089.8 1129.53 99.35 192.5 241.71 274.38 81.88
1 2 3 1 2 3 1 2 3 1701.73 1703.12 1677.69 25.43 1084.79 1091.19 1073.53 17.98 240.34 239.01 219.24 21.1
1 2 3 2 3 1 3 1 2 1637.67 1744.16 1700.71 106.49 1045.45 1123.47 1080.59 78.02 210.44 263.52 234.63 53.08
1 2 3 3 1 2 2 3 1 1687.63 1707.06 1687.65 19.41 1073.6 1091.3 1084.61 17.7 233.56 246.62 228.41 18.21
519.19 561.56 532.13 586.82 572.86 549.78 595.72 568.7 595.78
328.17 362.21 339.8 377.72 361.89 350.19 378.9 367.09 383.54
55.64 78.13 58.73 88.02 80.55 73.14 95.35 81.66 97.37
Table 3 The results of variance analysis for orthogonal test. Factor
A B C D Error *
Vitexin
Isovitexin
Monocrotaline
Sum of squares of deviations
F
P
Sum of squares of deviations
F
P
Sum of squares of deviations
F
P
3733.94 136.28 1911.34 82.95 82.96
45.01 1.64 23.04 1.00
<0.05* >0.05 <0.05* >0.05
1667.05 53.29 1017.85 53.25 53.25
31.31 1.00 19.11 1.00
<0.05* >0.05 <0.05* >0.05
1132.59 24.49 470.81 58.74 24.49
46.24 1.00 19.22 2.40
<0.05* >0.05 <0.05* >0.05
F0.05(2, 2) = 19.00, significant.
Table 4 The results of the extraction of vitexin, isovitexin and monocrotaline by different methods (n = 3). Method
MACPE HRE UAE
Vitexin
Isovitexin
Monocrotaline
Yield (lg/g)
RSD (%)
Yield (lg/g)
RSD (%)
Yield (lg/g)
RSD (%)
601.4 381.1 413.4
1.9 2.4 2.2
386.7 243.17 269. 49
1.1 2.2 1.9
105.5 70.01 73.15
2.2 2.1 2.0
using Triton X-100, not only the extraction efficiency is improved, but also the separation of flavonoids and alkaloids was achieved. It is a simple, rapid and efficient alternative to extraction and enrichment of active constituents of the herb materials. 3.5. Mechanism of MACPE Based on the facts and evidences observed in our study, MACPE extraction of flavonoids and alkaloids from Crotalaria sessiliflora L.
was photographed as shown in Fig. 8. The fact illustrated that MACPE process experienced two procedures due to physicochemical properties of the CPS with hydrophilic and hydrophobic groups. First, Triton X-100 can be dispersed into aqueous solution to form a homogeneously micellar system. This micellar CPS may interact with microwave in MAE process, facilitating to more effectively release targets constituents from the herb material. Second, the aggregation of surfactant molecules spontaneously occur in the CPE process beyond CPT (63 °C), resulting in demixing effect. This
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Fig. 8. The photographs of extracting flavonoids and alkaloids from Crotalaria sessiliflora L. in the MACPE process.
phase separation was further accelerated by centrifugation. Furthermore, addition of NaCl not only increased the polarity of the CPS to intensify extraction process, but also lowered the CPT to enhance phase separation. As a result, mono-phase micelle was helpful to mass transfer, and both phases could be selectively recovered or concentrated target constituents according to the difference of their polarity or dielectric poverty. From the results of HPLC analysis (see Fig. 3), the fact explained extraction selectivity of flavonoids and alkaloids in the MACPE process. In acidic medium, alkaloids turned into cations were readily dissolved in the aqueous top phase, whereas flavonoids presented in molecular forms was preferentially extracted to the weaker polar bottom phase. It is worth mentioning that the CPS of Triton X-100 as micellar extractant played diverse roles in MAE and CPE processes based on characteristics of hydrophilic and hydrophobic groups. Therefore, exploiting the functions of nonionic surfactant, MACPE integrated MAE with CPE is a successful combination for simultaneous extraction and separation (or purification) of flavonoids and alkaloids from Crotalaria sessiliflora L. with higher extraction efficiency. 4. Conclusion In this study, MACPE integrating MAE with CPE was developed for simultaneous extraction and separation of flavonoids and alkaloids with a CPS of Triton X-100NaClHCl system. The MACPE optimum conditions were investigated by orthogonal test and single factor experiment. Under the optimum conditions, the extraction yields of vitexin, isovitexin and monocrotaline respectively achieved to 601.4, 386.7 and 105.5 lg/g, which were significantly superior to conventional methods such as HRE and UAE. Based on the experimental results and facts observed, we can find out about the different functions of the CPS of Triton X-100 in MAE and CPE processes. The formation of the homogeneous micellar system with hydrophilic groups contributed to extraction performance for high yields, and the aggregation of surfactant molecules with the hydrophobic groups led to the two-phase separation for high recoveries. After extracted from Crotalaria sessiliflora L. by MAE, alkaloids and flavonoids could be separated and transferred to the top and bottom phases, respectively. Thus, the results revealed that MACPE integrated MAE with CPE had both advantages as exploiting the physicochemical properties of nonionic surfactant. It was proved that MACPE is a simple and efficient for the selective extraction and separation of active constituents from Crotalaria sessiliflora L. Acknowledgements The authors would like to thank the Science and Technology Planning Project of Guangdong Province (2016A040403119) and
the Fund Project of Guangdong Pharmaceutical University (No. 52104109) for finically supporting this work. We would also like to acknowledge Professor James Z. Tang from University of Wolverhampton for his help. References [1] Nanjing University of Traditional Chinese Medicine, Great Dictionary of Chinese Medicine, second ed., Shanghai Scientific and Technical Publishers, Shanghai, 2001, pp. 925–928. [2] Y. Li, J.Y. Sun, Q.Q. Yao, Research progress on Crotalaria sessiliflora L., Food Drug 17 (2015) 147–151. [3] H.S. Yoo, J.S. Lee, C.Y. Kim, J.W. Kim, Flavonoids of Crotalaria sessiliflora, Arch Pharm. Res. 27 (2004) 544–546. [4] Y.Q. Jing, H.S. Li, R.H. Chen, Studies on composition of amino acids in Crotalaria sessiliflora L., Bull. Chin. Mater. Med. 12 (1987) 41–42, 63. [5] E. Röder, X.T. Liang, K.J. Kabus, Pyrrolizidine alkaloids from the seeds of Crotalaria sessiliflora, Planta Med. 58 (1992) 283. [6] A. Mun’im, O. Negishi, T. Ozawa, Antioxidative compounds from Crotalaria sessiliflora, Biosci. Biotechnol. Biochem. 67 (2003) 410–414. [7] C.F. Yu, Study on bioactive and pharmacodynamic about pyrrolizidine alkaloids, Chin. Pharm. J. 24 (1989) 585–587. [8] L. Huang, K.M. Wu, Z. Wu, J.C. Chen, L.Z. Xu, S.P. Xu, Y.G. Xi, The isolation of antitumor active principle of Crotalaria sessiliflora and synthesis of its derivatives, Chin. Pharm. J. 15 (1980) 278–283. [9] C.F. Yu, Synthesis of the derivatives of the alkaloids of Crotalaria sessiliflora L., J. Beijing Med. Univ. 19 (1987), 182, 204. [10] X.J. Wu, B.S. Luo, Z.B. Tu, Crystal structure of monocrotaline, Nat. Prod. Res. Dev. 10 (1998) 39–42. [11] J. Tang, M. Hattori, Pyrrolizidine alkaloids-containing Chinese medicines in the Chinese Pharmacopoeia and related safety concerns, Acta Pharm. Sinica 46 (2011) 762–772. [12] J.G. Gao, C.H. Wang, Y. Li, Z.T. Wang, Advance on pharmacologic actions, toxicity and pharmacokinetics of pyrrolizidine alkaloids, China J. Chin. Mater. Med. 34 (2009) 506–511. [13] Y. Tanino, Monocrotaline-induced pulmonary hypertension in animals, Nihon Rinsho 59 (2001) 1076–1080. [14] D. Zhu, Z.Z. Jiang, L.Y. Zhang, Research progress on the mechanism of pulmonary arterial hypertension about monocrotaline, Central South Pharm. 9 (2011) 456–459. [15] J.P. Silva-Neto, R.A. Barreto, B.P.S. Pitanga, C. Souza, V. Silva, A.R. Silva, et al., Genotoxicity and morphological changes induced by the alkaloid monocrotaline, extracted from Crotalaria retusa, in a model of glial cells, Toxicon 55 (2010) 105–117. [16] R. Nogueira-Ferreira, R. Vitorino, R. Ferreira, T. Henriques-Coelho, Exploring the monocrotaline animal model for the study of pulmonary arterial hypertension: a network approach, Pulm. Pharmacol. Ther. 35 (2015) 8–16. [17] J.G. Gomez-Arroyo, L. Farkas, A.A. Alhussaini, D. Farkas, D. Kraskauskas, N.F. Voelkel, H.J. Bogaard, The monocrotaline model of pulmonary hypertension in perspective, Am. J. Physiol. Lung Cell. Mol. Physiol. 302 (2012) L363–L369. [18] A. Dhiman, A. Nanda, S. Ahmad, A quest for staunch effects of flavonoids: utopian protection against hepatic ailments, Arab. J. Chem. 86 (2012) 1702– 1711. [19] J.A. Yáñez, P.K. Andrews, N.M. Davies, Methods of analysis and separation of chiral flavonoids, J. Chromatogr. B 848 (2007) 159–181. [20] D. Barron, R.K. Ibrahim, Isoprenylated flavonoids – a survey, Phytochemistry 43 (1996) 921–982. [21] A.D. Villiers, P. Venter, H. Pasch, Recent advances and trends in the liquidchromatography-mass spectrometry analysis of flavonoids, J. Chromatogr. A 1430 (2016) 16–78. [22] P. Zeng, Y. Zhang, C. Pan, Q. Jia, F.J. Guo, Y.M. Li, W.L. Zhu, K.X. Chen, Advances in studying of the pharmacological activities and structure–activity
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Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Simultaneous extraction and separation of flavonoids and alkaloids from Crotalaria sessiliflora L. by microwave-assisted cloud-point extraction Xunyou Tang a, Dan Zhu a,b, Wenbei Huai c, Wei Zhang c,⇑, Chujun Fu c, Xiujuan Xie a, Sisi Quan a, Huajun Fan a,⇑ a b c
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China Guiyang College of Traditional Chinese Medicine, Guiyang 550025, China School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006, China
a r t i c l e
i n f o
Article history: Received 12 June 2016 Received in revised form 5 November 2016 Accepted 12 November 2016 Available online 18 November 2016 Keywords: Microwave-assisted cloud point extraction Crotalaria sessiliflora L. Triton X-100 Flavonoids Alkaloids Orthogonal test
a b s t r a c t A novel method of simultaneous extraction and separation of flavonoids and alkaloids from Crotalaria sessiliflora L., was developed by microwave-assisted cloud-point extraction (MACPE) using Triton X100NaClHCl system as extractant. The optimum conditions including Triton X-100 concentration, NaCl concentration, HCl concentration, solvent-to-material ratio, extraction temperature and time were investigated by orthogonal test on the single factor experiment. The extraction yields of vitexin, isovitexin and monocrotaline reached respectively up to 601.4, 386.7 and 105.5 lg/g under the optimum conditions. Compared with heating reflux extraction (HRE) and ultrasonic-assisted extraction (UAE), MACPE exhibited a significantly higher extraction yields and took a shorter time (only 10 min). The MACPE mechanism was discussed based on phase’s shift and extraction property. As a result, flavonoids and alkaloids can be transferred respectively to bottom phase and top phase after simultaneous extraction. The proposed method integrated MAE with cloud-point extraction (CPE) in one-step procedure took both advantages. It provided a simple and efficient alternative to extraction and separation of active constituents from the herb materials. Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction Crotalaria sessiliflora L. is a Chinese herbal medicine as a leguminous plant, commonly used in the treatment of esophageal cancer, cervical cancer and skin cancer [1,2]. Its main active constituents were reported to be alkaloids, flavonoids and amino acids [3–7]. Monocrotaline is the most important pyrrolizidine alkaloid due to high antitumor effects. Due to strong liver toxicity, its derivatives were also synthesized through structural transformation to reduce toxicity and retain the anticancer effects [7–10]. However, monocrotaline as a drug is used widely in clinic research for pulmonary hypertension model [11–17]. Relatively, flavonoids present in plants have pharmacological effects of antioxidant, hepatoprotective, anticancer, anti-inflammatory and anti-diabetes [18–25]. Flavonoids like vitexin, isovitexin and analogues were found from Crotalaria sessiliflora L. especially for the treatment of cardiovascular diseases [3,6,26–29]. Therefore, multipharmacological effects (especially anticancer effects) of the
⇑ Corresponding authors. E-mail addresses: [email protected] (W. Zhang), [email protected] (H. Fan). http://dx.doi.org/10.1016/j.seppur.2016.11.038 1383-5866/Ó 2016 Elsevier B.V. All rights reserved.
extract from Crotalaria sessiliflora L. have attracted attention due to multi active constituents [2,8,30–32]. Moreover, vitexin have been used as primary ingredients in cardiovascular pharmaceutical preparations such as drop pills and dispersible tablets (named Yixintong), and the demands of monocrotaline in research uses are also increasing. Active constituents in Crotalaria sessiliflora L. are usually extracted by conventional methods such as heat reflux extraction and ultrasonic extraction [3,6,32–35], which are subject to remarkable shortcomings, including the lengthy process, the high cost of organic solvents, low recovery, and toxic solvent residuals in the products. In previous work, we extracted flavonoids and alkaloids respectively by ultrasonic-assisted extraction (UAE) and microwave-assisted extraction (MAE) using methanol and ethanol as extracting solvents [36,37]. However, the pretreatment of sample purification had to be conducted prior to chromatographic analysis due to containing many impurities and interferences in the extracts. Now there is an increasing realization that an efficient alternative to extraction of active constituents from the herb is necessary in their researches. In the past decades, novel extraction techniques attracted great attentions to extraction and enrichment of active
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compounds from natural products based on two-phase (or multiphase) separation caused by combination of water, organic solvents, ionic liquid, polymers, salt and others such as aqueous two phase extraction (ATPE), dispersive liquid-liquid extraction (DLLE), micellar extraction and cloud-point extraction (CPE) [38–42]. Among them, CPE has been widely applied in the extraction and preconcentration of constituents such as metal ions and hydrophilic proteins, especially small molecule organics as a promising, environmentally-friendly, cost-effective means for sample preparation [42–48]. As aqueous nonionic surfactant solutions split into two phases beyond a cloud-point temperature (CPT), a hydrophobic constituent with similar polarity is extracted into the surfactant-rich phase, while the hydrophilic material is left in the aqueous phase. Thus, CPE like ATPE exhibited many advantages such as non-volatility, non-flammability, less consumption of solvents, less impact on the environment, the simple procedure, the convenient to follow steps and protection of the material nature [48–51]. With the development of CPE technology, combining it with other techniques can greatly improve the extraction efficiency. Microwave-assisted extraction (MAE) combining with CPE, is an efficient alternative to extraction of active constituents from the herb plant for enrichment and purification [52–54]. However, the combination of both in fact doesn’t occur simultaneously, subjecting to diverse processes. In other words, MAE and CPE were conducted respectively in two-steps procedures using different extractants. To make full use of MAE advantages, a micellar solution as extractant can be formed by dispersing surfactant into an aqueous solution, and addition of inorganic salts can enhance the polarity of the solution, leading to be a good dielectric poverty to produce a strong coupling effect with the microwave. Moreover, this cloud point system (CPS) as homogeneous system can extract active constituents from the herb materials (unlike aqueous two phase system (ATPS) even under the presence of salt), and transferring target compounds into top or bottom phase when two-phase system was formed beyond lower CPT. Thus, microwave-assisted cloud point extraction (MACPE), combining CPE with microwave-assisted extraction (MAE), is a strategy for not only simultaneous extraction of active constituents from herb plants but also separation or purification of them with each other due to the formation of one phase and two phases. It is different from the conventional MAE prior to CPE (a nonionic surfactant was only used for preconcentration or enrichment of target constituents extracted by another solvent), resulting in not only take advantage of the extraction selectivity but also improve extraction efficiency through demixing effect of CPE and coupling effect of MAE. In this work, our aim is to integrate MAE with CPE for extraction and purification of active constituents from Crotalaria sessiliflora L. using a CPS of the nonionic surfactant Triton X-100. The technique that named MACPE, would improve demixing effect and field intensification for enrichment of desired constituents and enhancement of extraction yields. After screening of non-ionic surfactants, the experimental conditions of extraction of vitexin, isovitexin and monocrotaline (shown in Fig. 1), including the composition of the CPS, addition of the salt, extraction temperature, extraction time, solvent-to-material ratio, etc., were investigated and optimized by single-factor experiment and orthogonal experiment. To evaluate the extraction efficiency, the results of MACPE were compared with heat reflux extraction (HRE) and ultrasonic-assisted extraction (UAE). It was found that the results of MACPE were quite satisfying, and its operational process was simpler, easier and more efficient. To the best of our knowledge, this is the first application of a novel MACPE in the extraction of active constituents from Crotalaria sessiliflora L.
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2. Experimental 2.1. Materials and reagents Dry samples of Crotalaria sessiliflora L. were purchased from Guangxi in China (Batch number 20120926). The samples were powdered, sieved and placed in a desiccator. Vitexin (purity P 98%) was purchased from National Institutes for Food and Drug Control (China); Isovitexin and monocrotaline (purities P 98%) was bought from Shanghai Forever Biotech Co., Ltd. (China). Triton X-100 of analytical grade was purchased from Shanghai Macklin Biochemical Co., Ltd. (China). Acetonitrile and methanol of HPLC grade were purchased from Merck Darmstadt Ltd. (Germany). Hydrochloric acid, NaCl, ammonia, ethanol and chloroform of analytical grade were obtained from Guangzhou Reagent factory (China). 2.2. Instruments and apparatus MACPE experiments were performed on an EXCEL microwave extraction system (PreeKem Scientific Instruments Co., Ltd., China) equipped with a digital timer, power and a temperature controller. HPLC analysis was carried out using Agilent 1200 Infinity chromatograph with a UV detector (Agilent Technologies Co., Ltd. USA). 2.3. Preparation of the CPS For preparation of Triton X-100–NaCl–HCl of the CPS, a certain amount of Triton X-100 was dissolved in deionized water, and then added a certain mass of sodium chloride and a certain volume of hydrochloric acid, respectively. After adding appropriate amount of deionized water, the mixture mixed well by a vortex stirrer. The CPS of Triton X-100 as an extractant was obtained at final concentrations of 0.5–5% (v/v) Triton X-100, 1.4% (w/v) sodium chloride and 0.1 mol/L hydrochloric acid, respectively. 2.4. Extraction strategy The strategy of MACPE combine MAE with CPE for extraction and purification of flavonoids and alkaloids from Crotalaria sessiliflora L. was planed according to the following scheme as shown in Fig. 2. In this way, flavonoids and alkaloids were together extracted from the herb material by MAE with the CPS of Triton X-100/sodium chloride, and then flavonoids and alkaloids were separated from each other by phase separation at a water bath beyond the CPT. 2.5. MACPE procedure 0.5 g of the herb powder and 50 mL of the CPS were put into an extraction vessel, which was then sealed and placed in the microwave extraction system at 80 °C under 780 W for 10 min. After extraction completed, the extract was transferred to a centrifuge tube for centrifugation at 3800 rpm for 1 min. The supernatant was placed in a water bath at 68 °C to stand for 30 min, then centrifuging at 3800 rpm for 5 min. The top phase was collected and dried in a rotary evaporator. The residue obtained and bottom phase were diluted respectively with methanol up to 5 mL for HPLC analysis. The extraction was assessed by the yield (Y) of target constituent according to the following formula.
Yield ðmg=gÞ ¼
me m0
where me is the total amount of the target constituent extracted from the plant material (m0).
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OH
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Isovitexin
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Fig. 1. The chemical structures of vitexin, isovitexin and monocrotaline.
Fig. 2. The scheme of extraction and separation of flavonoids and alkaloids from Crotalaria sessiliflora L. by MACPE.
2.6. HPLC analysis The extract of each phase was filtered through a 0.22 lm membrane, and HPLC analysis of flavonoids and alkaloids was carried out according to the following conditions: 10 lL of sample was injected into an Agilent 1200 Infinity chromatograph for separation on a Phenomenon Gemini C18 Column (4.6 mm 250 mm, 5 lm) with acetonitrile-0.5% phosphate solution (v/v, 14:86) for flavonoids and methanol-0.1% ammonia solution (v/v, 10:90) for alkaloids as the mobile phases at 1.0 mL/min of flow rate. Then flavonoids and alkaloids were detected by a UV detector at 340 nm and 220 nm, respectively.
3. Results and discussion 3.1. Single-factor experiment 3.1.1. The effect of the concentrations of Triton X-100 and NaCl For extraction of flavonoids and alkaloids from Crotalaria sessiliflora L., concentration of the surfactant determines not only extraction yield but also the phase volume ratio at phase separation. The addition of salt will improve microwave effect by increasing polarity of the extractant, and facilitating to reduce the CPT. So Triton X-100 and NaCl were used as extractant and addictive respectively, the effects of their concentrations on the extraction yields of several constituents were investigated in the range of 0.5–5%(v/v) and 0.6–1.4% (w/v), as shown in Fig. 4. The results demonstrated that the extraction yields achieved the maximum yields when Triton X-100 concentration and NaCl
concentration was increased to 3% (v/v) and 1.4% (w/v) respectively. The CPT was reduced with increase of NaCl concentration from 63 to 47 °C. Nevertheless, more than 1.4% (w/v) NaCl would make surfactant-rich phase converted into the top phase from the bottom phase, and demarcation between two phases separated was not clear. For the CPE process, 68 °C was determined as the most appropriate CPT at 1.4% (w/v) NaCl because of good demixing effect. From results, the selection of 3% (v/v) TritonX-100 and 1.4% (w/v) NaCl was suitable for next experiments. 3.1.2. The effect of temperature and time Extraction temperature and time are the key factors affecting the extraction yields. In the MAE process, extraction temperature was controlled by the microwave power given, and extraction time dictated maximum extraction yields. Hence, the effects on extraction yields of flavonoids and alkaloids from Crotalaria sessiliflora L. were carried out at 60–110 °C for 5–25 min so as to determine their proper parameters. The results obtained were shown in Fig. 5. From Fig. 5, the extraction yields of the desired constituents were enhanced when increasing extraction temperature and time. The maximums of vitexin and isovitexin were at 90 °C for 10 min, whereas that of monocrotaline was at 80 °C for 10 min. Because raising temperature can improve the interaction between solvent molecules, and accelerate the mass transfer. But too high temperature may lead to decomposition, even coking the sample. 3.1.3. Effect of HCl concentration Acidity of the extractant is closely related to the structural form of flavonoids and alkaloids, so the effect of acidity on the extraction yields was investigated by adjusting HCl concentration. The results
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CNaCl (%, w/v)
CTriton X-100 (%, v/v)
Fig. 4. The effects of the concentrations of Triton X-100 (a) and NaCl (b) on the extraction yields.
700
800
(a)
Vitexin Isovitexin Monocrotaline
700
600
600
Yield (µg/g)
500
Yield (µg/g)
Vitexin Isovitexin Monocrotaline
(b)
400 300
500 400 300
200
200
100
100
0
0 60
70
80
90
100
110
4
8
12
16
20
24
28
Time (min)
Temperature ( ) Fig. 5. The effect of temperature (a) and time (b) on the extraction yields.
shown in Fig. 6 indicated that the maximum yield of vitexin, monocrotaline and isovitexin were at 0.05 mol/L, 0.05 mol/L and
0.10 mol/L, respectively. The reason is that acidity would structurally change the greater polarity transformation of target
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800
Vitexin Isovitexin Monocrotaline
700
Yield (µg/g)
600 500 400 300 200 100 0 0.00
0.05
0.10
0.15
0.20
0.25
CHCl (mol/L) Fig. 6. The effect of HCl concentration on extraction the yields.
constituents, leading to their repartition in two phases. It also revealed that monocrotaline in cationic form would be easy to be extracted in an acidic top phase whereas two flavonoids in molecular forms were transferred into the bottom phase. Thus, 0.1 mol/L HCl was used in next experiment. 3.1.4. Effect of solvent-to-material ratio The effect of solvent-to-material ratio on the extraction yields was also investigated on the basis of other parameters determined above. i.e. According to the CPS composition of 3% Triton X-100 (v/ v) and 1.4% (w/v) NaCl, the extraction experiment was performed at 90 °C for 10 min under presence of 0.1 mol/L HCl. The results as shown in Fig. 7 gave relatively stable yields while solvent-tomaterial ratio was over 90:1. The maximum yields of vitexin, isovitexin and monocrotaline were found at 100:1. In fact, raising solvent-to-material ratio meant to increase solvent volume, which would be helpful to dissolve more targets constituents until their saturation. So the solvent-to-material ratio was determined as 100:1 in the following experiment. 3.2. Optimization of MACPE conditions with orthogonal test Considering the limitation of single factor experiment, especially interrelation among single factors, further optimization is 800
Vitexin Isovitexin Monocrotaline
700
Yield (µg/g)
600 500 400 300 200 100 0 60
70
80
90
100
110
Ratio of solvent to material Fig. 7. The effect of solvent-to-material ratio on the extraction yields.
necessary for applicable use. On the basis the results of single factor experiment, several key factors such as Triton X-100 concentration, extraction temperature and time, solvent-to-material ratio, were investigated with orthogonal test by evaluating the yields of vitexin, isovitexin and monocrotaline. Thus, these four factors were arranged to 9 experiments according to an orthogonal test design of L9 (34) as shown in Table 1. Data of every experiment in triplicate were obtained from a 9-run-experiment by HPLC analysis. The experimental results and variance analysis also listed in Tables 2 and 3, respectively. Seen from the results obtained above, it can be found that Triton X-100 concentration has more significant effect on the extraction yield of three constituents among factors. The factors could be ranked by importance for flavonoids and the alkaloid as follows: A (Triton X-100 concentration) > C (solvent-to-material ratio) > B (extraction temperature) > D (extraction time). The results of range analysis also gave the optimum levels for vitexin and isovitexin were A3B2C2D2 while that for monocrotaline was A3B1C2D2. In other words, the effects of these factors on their extraction yields were not always consistent. Relatively, monocrotaline is susceptible to high temperatures due to chemical stability. Thus, the optimum conditions of FMATPE were selected as Triton X-100 concentration of 4% (v/v), extraction temperature of 80 °C, solvent-to-material ratio of 100:1, extraction time of 10 min. 3.3. Validation of optimum conditions To confirm feasibility of MACPE, the validation of experiments were conducted under the optimum conditions. The mean value of triplicate results follows as 601.4 lg/g for vitexin, 386.7 lg/g for isovitexin and 105.5 lg/g for monocrotaline, respectively. Their relative standard deviations (RSDs) were less than 4.3%. The validation results demonstrated that adapting L9 (34) orthogonal array scheme could optimize MACPE conditions with higher yields for target constituents. The fact was proved that MACPE is an efficient alternative to simultaneous extraction and separation of flavonoids and alkaloids from the herb material. 3.4. Comparison with different extraction methods In order to assess MACPE approach, a comparative study was conducted using the identical CPS consisting of Triton X-100– NaCl–HCl as extractant with the conventional methods such as heating reflux extraction (HRE) and ultrasonic-assisted extraction (UAE). In view of the above, the experiments of extraction of vitexin, isovitexin and monocrotaline from the herb material could be respectively accomplished under the same conditions of 4% Triton X-100 concentration, 1.4% NaCl concentration, extraction temperature of 80 °C and solvent-to-material ratio of 100, in addition to extraction time of 10 min for MACPE, 90 min for HRE and 60 min for UAE. The obtained results were listed in Table 4. As shown in Table 4, the results exhibited that MACPE possessed the greatest yields and took the shortest extraction time while compared with that of HRE and UAE. The conventional HRE also had relatively low yield by contrast to UAE. For example, MACPE had the outstanding extraction efficiency up to 601.4 lg/g for vitexin, which was found to be superior to others we used methanol and ethanol [35–37]. In microwave field, the CPS consisted of Triton X-100, was proved to be a good extractant with stronger extraction capacity owing to its dielectric property. Moreover, its hydrophilic and hydrophobic groups also provided a specific function to further improve the extraction selectivity through demixing effect when temperature was over the CPT. As a results, flavonoids would be transferred to the rich-surfactant bottom phase, whereas alkaloids could be migrated to the aqueous top phase after phase separation. Therefore, MAE combined with CPE
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X. Tang et al. / Separation and Purification Technology 175 (2017) 266–273 Table 1 L9 (34) orthogonal test design for extraction of flavonoids and alkaloids. Level
Factor
1 2 3
A (Triton X-100) (%)
B (Temperature) (°C)
C (Solvent-to-material ratio) (mL: g)
D (Extraction time) (min)
2 3 4
80 90 100
90:1 100:1 110:1
5 10 15
Table 2 The results of the orthogonal test by L9 (33). No.
1 2 3 4 5 6 7 8 9 K1 K2 K3 R K1 K2 K3 R K1 K2 K3 R
Yield (lg/g)
Factor A
B
C
D
Vitexin
Isovitexin
Monocrotaline
1 1 1 2 2 2 3 3 3 1612.88 1709.46 1760.2 147.32 1030.18 1089.8 1129.53 99.35 192.5 241.71 274.38 81.88
1 2 3 1 2 3 1 2 3 1701.73 1703.12 1677.69 25.43 1084.79 1091.19 1073.53 17.98 240.34 239.01 219.24 21.1
1 2 3 2 3 1 3 1 2 1637.67 1744.16 1700.71 106.49 1045.45 1123.47 1080.59 78.02 210.44 263.52 234.63 53.08
1 2 3 3 1 2 2 3 1 1687.63 1707.06 1687.65 19.41 1073.6 1091.3 1084.61 17.7 233.56 246.62 228.41 18.21
519.19 561.56 532.13 586.82 572.86 549.78 595.72 568.7 595.78
328.17 362.21 339.8 377.72 361.89 350.19 378.9 367.09 383.54
55.64 78.13 58.73 88.02 80.55 73.14 95.35 81.66 97.37
Table 3 The results of variance analysis for orthogonal test. Factor
A B C D Error *
Vitexin
Isovitexin
Monocrotaline
Sum of squares of deviations
F
P
Sum of squares of deviations
F
P
Sum of squares of deviations
F
P
3733.94 136.28 1911.34 82.95 82.96
45.01 1.64 23.04 1.00
<0.05* >0.05 <0.05* >0.05
1667.05 53.29 1017.85 53.25 53.25
31.31 1.00 19.11 1.00
<0.05* >0.05 <0.05* >0.05
1132.59 24.49 470.81 58.74 24.49
46.24 1.00 19.22 2.40
<0.05* >0.05 <0.05* >0.05
F0.05(2, 2) = 19.00, significant.
Table 4 The results of the extraction of vitexin, isovitexin and monocrotaline by different methods (n = 3). Method
MACPE HRE UAE
Vitexin
Isovitexin
Monocrotaline
Yield (lg/g)
RSD (%)
Yield (lg/g)
RSD (%)
Yield (lg/g)
RSD (%)
601.4 381.1 413.4
1.9 2.4 2.2
386.7 243.17 269. 49
1.1 2.2 1.9
105.5 70.01 73.15
2.2 2.1 2.0
using Triton X-100, not only the extraction efficiency is improved, but also the separation of flavonoids and alkaloids was achieved. It is a simple, rapid and efficient alternative to extraction and enrichment of active constituents of the herb materials. 3.5. Mechanism of MACPE Based on the facts and evidences observed in our study, MACPE extraction of flavonoids and alkaloids from Crotalaria sessiliflora L.
was photographed as shown in Fig. 8. The fact illustrated that MACPE process experienced two procedures due to physicochemical properties of the CPS with hydrophilic and hydrophobic groups. First, Triton X-100 can be dispersed into aqueous solution to form a homogeneously micellar system. This micellar CPS may interact with microwave in MAE process, facilitating to more effectively release targets constituents from the herb material. Second, the aggregation of surfactant molecules spontaneously occur in the CPE process beyond CPT (63 °C), resulting in demixing effect. This
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Fig. 8. The photographs of extracting flavonoids and alkaloids from Crotalaria sessiliflora L. in the MACPE process.
phase separation was further accelerated by centrifugation. Furthermore, addition of NaCl not only increased the polarity of the CPS to intensify extraction process, but also lowered the CPT to enhance phase separation. As a result, mono-phase micelle was helpful to mass transfer, and both phases could be selectively recovered or concentrated target constituents according to the difference of their polarity or dielectric poverty. From the results of HPLC analysis (see Fig. 3), the fact explained extraction selectivity of flavonoids and alkaloids in the MACPE process. In acidic medium, alkaloids turned into cations were readily dissolved in the aqueous top phase, whereas flavonoids presented in molecular forms was preferentially extracted to the weaker polar bottom phase. It is worth mentioning that the CPS of Triton X-100 as micellar extractant played diverse roles in MAE and CPE processes based on characteristics of hydrophilic and hydrophobic groups. Therefore, exploiting the functions of nonionic surfactant, MACPE integrated MAE with CPE is a successful combination for simultaneous extraction and separation (or purification) of flavonoids and alkaloids from Crotalaria sessiliflora L. with higher extraction efficiency. 4. Conclusion In this study, MACPE integrating MAE with CPE was developed for simultaneous extraction and separation of flavonoids and alkaloids with a CPS of Triton X-100NaClHCl system. The MACPE optimum conditions were investigated by orthogonal test and single factor experiment. Under the optimum conditions, the extraction yields of vitexin, isovitexin and monocrotaline respectively achieved to 601.4, 386.7 and 105.5 lg/g, which were significantly superior to conventional methods such as HRE and UAE. Based on the experimental results and facts observed, we can find out about the different functions of the CPS of Triton X-100 in MAE and CPE processes. The formation of the homogeneous micellar system with hydrophilic groups contributed to extraction performance for high yields, and the aggregation of surfactant molecules with the hydrophobic groups led to the two-phase separation for high recoveries. After extracted from Crotalaria sessiliflora L. by MAE, alkaloids and flavonoids could be separated and transferred to the top and bottom phases, respectively. Thus, the results revealed that MACPE integrated MAE with CPE had both advantages as exploiting the physicochemical properties of nonionic surfactant. It was proved that MACPE is a simple and efficient for the selective extraction and separation of active constituents from Crotalaria sessiliflora L. Acknowledgements The authors would like to thank the Science and Technology Planning Project of Guangdong Province (2016A040403119) and
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