Enhanced extraction and preconcentration of main target saponins from Panax notoginseng root using green and efficient formulated surfactant aqueous systems

Journal of Cleaner Production 210 (2019) 1507e1516

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Enhanced extraction and preconcentration of main target saponins from Panax notoginseng root using green and efficient formulated surfactant aqueous systems Qi Cui a, 1, Juzhao Liu a, 1, Wenjing Xu a, Yu-Fei Kang a, Xinyu Wang a, Yanyan Li a, Yujie Fu a, b, * a b

Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Forestry University, 100083 Beijing, China

a r t i c l e i n f o

a b s t r a c t :

Article history: Received 12 June 2018 Received in revised form 17 October 2018 Accepted 15 November 2018 Available online 17 November 2018

In this study, the formulated surfactant aqueous system composed of 2% (w/v) Triton X-114 and 0.03% (w/ v) Gemini 16-5-16 coupled with ultrasonic-assisted technology has been selected as an effective method for the extraction and preconcentration of five main saponin compounds in Panax notoginseng root samples. Under the optimum parameters, the extraction yields of notoginsenoside R1, ginsenosides Rg1, Re, Rb1 and Rd could reach the values of 28.66 mg/g, 46.52 mg/g, 12.91 mg/g, 29.31 mg/g and 9.26 mg/g, respectively. And the preconcentration factor could be 11.5-fold. In addition, the proposed formulated surfactant system is also suitable for the subsequent HPLC analysis with excellent linearity correlation coefficient R2
0.99, high repeatability with relative standard deviation (RSD) of less than 4.45% and good recoveries within 97.12%e101.28%. Thus, the method gave higher extraction efficiencies, higher enhancement factors as well as excellent chromatographic performance than conventional solvents for the studied five main saponins in Panax notoginseng. In addition, the green and environmentally friendly formulated surfactant aqueous system which endows the safety and reliability of the extraction and preconcentration process. Moreover, this work is a one-step sample preparation procedure that integrated of extraction and preconcentration of target constituents. And the results indicated that the developed method possess the potential to large-scale extract and purify the bioactive components from plant materials. © 2018 Elsevier Ltd. All rights reserved.

Keywords: Formulated surfactant Ultrasonic-assisted technology Saponins Panax notoginseng root HPLC analysis

1. Introduction Panax notoginseng (Burk.) F.H. Chen is a well-known and valuable Chinese medicinal plant. It has been used in Chinese medical practice for centuries (Zhao et al., 2006). The root parts of Panax notoginseng is commonly considered as medicinal part, which have been used for treatment of hematological and cardiovascular diseases (Chan et al., 2003). It has extensive bioactivities, such as hepatoprotective, cardiotonic, antioxidant and antidepressant activities (Dong et al., 2003; Liu et al., 2009; Yoshikawa et al., 2003). And its prominent activities could largely be attributed to

* Corresponding author. Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China. E-mail address: [email protected] (Y. Fu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jclepro.2018.11.142 0959-6526/© 2018 Elsevier Ltd. All rights reserved.

drammarane triterpenoid saponins, including 20(S)-protopanaxatriol-type and 20(S)-protopanaxadiol-type saponins (Lee et al., 2017). Notoginsenoside R1, ginsenosides Rg1, Re, Rb1 and Rd are considered as five main saponins in Panax notoginseng (Wang et al., 2017a, b). Thus, it is of great significance to develop a fast and green technology to extract and monitor them from Panax notoginseng. Previously, many normal extraction techniques including liquid-liquid extraction, solid-phase extraction and supercritical fluid extraction have been employed for the extraction of bioactive components from different plants (Li et al., 2017; Long et al., 2016; Dong et al., 2016). However, these methods require large amount of organic solvents or special equipment, which are not feasible for routine analysis (Chan et al., 2011; Heng et al., 2013). Therefore, there is an effective need to develop an efficient and target compounds-oriented extraction and enrichment technologies, preferentially operating at a mild condition. According to the previous studies, surfactant extraction is a

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potential alternative to the traditional extraction methods due to the excellent superiority in the extraction of constituents including hydrophobic and hydrophilic molecules (Tan et al., 2017). Except for performing the function as a kind of extraction solvent, it could also enrich the target compounds through itself property of cloud point property. The aqueous surfactant solution will be separated into two phases when the temperature exceeds the cloud point or adds the appropriate salt (Do et al., 2014; Berthod and Garcia-AlvarezCoque, 2000; Bordbar and Hosseinzadeh, 2006; Bordbar et al., 2007; Pradhan and Bhattacharyya, 2017; Wang et al., 2017a, b). And the separation of two phases requires appropriate experimental conditions depending on the nature of the surfactant. Thus, temperature change results in two-phase separation of surfactant solutions, while other parameters are involved in two-phase separation process of surfactants. In the view of above, the surfactant solution system has prominent advantages, such as low dosage, high extraction efficiency, high enrichment effect, low cost and environment friendly. In addition, mixed surfactant solution system has been possible by the addition of a small amount of the ionic surfactant, such as cetyltrimethylammonium bromide (CTAB), into the original nonionic surfactant system (Wu et al., 2017; Sharma et al., 2015). The combination of two kinds of surfactants are in order to increase the ionic strength and also extend the range of compounds extracted (Vieira et al., 2017). And according to literatures, the mixed surfactant solution system has significant influence on the improvement of extraction efficiency (Gong et al., 2016; Wang et al., 2016; Ma et al., 2016).

On the basis of previous studies, we developed the mode of mixed surfactant system, in order to be more suitable for extraction of saponins in Panax notoginseng. The common ionic surfactant was replaced by the novel gemini surfactant. Gemini surfactant molecules are composed of two hydrophilic head groups and two hydrophobic chains covalently linked by a spacer group (Xiao et al., 2013). As compared with traditional single-chain surfactants, gemini surfactants possessed more superior performance, and the length of spacer chain has a significant influence on the interaction between target compounds and gemini surfactants (Phasukarratchai et al., 2017; Guo et al., 2017; Peng et al., 2016; Amiririgi and Abbasi, 2016). In the present study, we finally choose gemini Cm-s-Cm2Br with m being 16 and s being 5 as the synergetic surfactant through preliminary experiment, to selectively extract saponins in Panax notoginseng. Thus, the formulated surfactants, which were composed of nonionic surfactant and ionic surfactant were developed for extraction and enrichment of target compounds in the natural plant. In this paper, the formulated surfactants and ultrasonic-assisted technology were used together for the simultaneous extraction and preconcentration of five main saponins in Panax notoginseng. In addition, two-phase system was formed by adding ammonium sulfate as the electrolyte to achieve the enrichment of target compounds. It should be noted that although the unique properties

Extraction yield ðmg=gÞ ¼

of surfactants have been applied in various areas of analytical chemistry, the introduction of gemini surfactant to the extraction solution system has not been reported in the literature yet. 2. Materials and methods 2.1. Materials and reagents Panax notoginseng roots were obtained from Yunnan province, China, and identified by Professor Zi-Jun Mao from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, PR China. The plant materials were dried at room temperature under the absence of light in a well-ventilated room. The dried roots were crushed into powders (40 mesh) by a disintegrator. The standards of notoginsenoside R1, ginsenosides Rg1, Re, Rb1 and Rd were purchased from SigmaeAldrich (Steinheim, Germany), and their purities were all
98%. Their structures were shown in Fig. 1. N,N-dimethylhexadecylamine, 1,5dibromopentane, HPLC-grade methanol and acetonitrile were purchased from J & K Chemical Ltd. (China). Deionized water was prepared by a Milli-Q Water Purification system (Millipore, MA, USA). 2.2. Synthesis of cationic gemini surfactant 16-5-16 The gemini surfactant was synthesized by the following procedure as described by Alam and Mandal (2015):

The obtained reaction products were recrystallized from petroleum ether/ethyl acetate mixed solution to obtain pure compounds. The product was identified and confirmed by infrared spectrum, 1H NMR and 13C NMR (shown in Electronic Supporting Information). The yield of the product is found to be 94.6%. 2.3. Ultrasonic-assisted surfactant extraction of five target saponins An accurately weighed amount (1.0 g) of Panax notoginseng root was put into an erlenmeyer flask and extracted with certain type of surfactant aqueous solution under certain condition. Then the erlenmeyer flask was placed in the ultrasound water bath, which is combined with surfactant system to jointly extract saponins in Panax notoginseng. After extraction, the solution was filtered and the surfactant aqueous solution containing Panax notoginseng extract was obtained. All the solutions were filtered through 0.45 mm nylon membranes before HPLC analysis. The parameters involved in the extraction process including surfactant type, surfactant concentration, solvent pH, liquid to solid ratio, ultrasonic power and time on the extraction yields of notoginsenoside R1, ginsenosides Rg1, Re, Rb1 and Rd were systematically investigated by single factor experiment. The extraction yield of each compound was calculated as follows:

Amount of the saponins after preconcentration Amount of the saponins before preconcentration

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Fig. 1. Chemical structure of the surfactants and saponin compounds used in this work.

Fig. 2. Schematic representation of the sample extraction and preconcentration.

2.4. Pre-concentration of saponins by cloud-point extraction After ultrasonic-assisted surfactant extraction and filtration, the supernatant sample was transferred into another centrifuge tube for preconcentration of the extracted bioactive components through phase separation of the aqueous surfactant solution. The schematic diagram was shown in Fig. 2. To investigate the preconcentration of five target saponins, ammonium sulfate was added to the centrifuge tube and the solution was vortex-dissolved

for 2 min. Then the extraction solvent was centrifuged at 5000 rpm for 10 min for the complete separation into two distinct phasesdthe upper phase was surfactant-rich phase with small volume and the lower phase was aqueous phase with large volume. After that, the aqueous phase was removed and the sticky surfactant-rich phase was left in the centrifuge tube. Methanol was added to lower the viscosity of the surfactant-rich phase in prior to HPLC analysis. The recovery of each compound was calculated as follows:

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Recovery yield ð%Þ ¼

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Amount of the saponins after preconcentration  100% Amount of the saponins before preconcentration

2.5. HPLC analysis Quantification of five saponin compounds in Panax notoginseng was conducted on an Agilent 1200 HPLC system using a HIQ Sil C18W reversed-phase column (250 mm  4.6 mm i.d.,5 mm). The Gradient elution was carried out with A (deionized water) and B (acetonitrile), and the elution process was 0e20 min, 79% (A); 20e45 min, 79-54% (A). The detection wavelength was 203 nm. The flow rate was 1.5 mL/min, the injection volume was 20 mL, and the column temperature was maintained at 30  C. The standard curves for notoginsenoside R1, ginsenosides Rg1, Re, Rb1 and Rd were Y ¼ 2037.2X þ 5.23 (R2 ¼ 0.999), Y ¼ 4015.7X - 16.45 (R2 ¼ 0.995), Y ¼ 2232.5X e 38.23 (R2 ¼ 0.996), Y ¼ 3217.9X þ 605.47 (R2 ¼ 0.996) and Y ¼ 3412.2X þ 64.83 (R2 ¼ 0.998), respectively. 2.6. Method validation In this section, the analytical determination of five target saponins in Panax notoginseng was validated through linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, and recovery. 2.6.1. Linearity, limit of detection (LOD) and limit of quantification (LOQ) The linearity of calibration curve was investigated by analyzing seven concentrations of saponin standards. The calibration curves of the five standards were constructed by plotting the integrated chromatography peak areas (Y) versus the corresponding concentrations of the injected standard solutions (X) using a least-squares linear regression model. LOD and LOQ for each compound were achieved at signal-to-noise ratios (S/N) of 3 and 10, respectively (Xiao et al., 2016; Kitamura et al., 2017). 2.6.2. Precision and recovery Intra- and inter-day variations were applied to determine the precision of the established method. The sample was analyzed six times within 24 h and in triplicate on six consecutive days for intraday and inter-day variations, respectively. Relative standard deviations (RSDs) were used to measure the precision. The recovery experiments were applied to examine the accuracy of the developed method by adding the saponin standards to the Panax notoginseng matrix. Then, the materials mixed with standards were prepared under the optimum extraction conditions, and analyzed by HPLC according to Section 2.5. 3. Results and discussion 3.1. Selection of surfactant formulations for saponin extraction In the present study, formulated surfactant system was aimed to be developed for selectively extraction of five main saponins in Panax notoginseng. Initially, SDBS, SDS, Tween series, Brij series, Triton X series were tried as extraction solvents. And then on the basis of previous studies, we improved co-surfactant in order to get better extraction efficiency. Finally, a novel Gemini Cm-s-Cm2Br surfactant (m being 16 and s being 5), which could be considered as the dimer of common cationic surfactant, was synthesized in our

lab. Consequently, the cationic ion-pair reagent, Gemini 16-5-16, was used to form the ion-pair of saponin-Gemini 16-5-15 before surfactant extraction. And the saponin-Gemini 16-5-15 ion-pair could transfer effectively into the aggregates of optimized surfactant compared to its original forms, leading to the significant increase of extraction efficiency. Thus, the type of surfactant and the concentration of surfactant and co-surfactant were the first parameters to investigate. 3.1.1. Effect of surfactant type on the extraction efficiency of saponins Effective selection of suitable type of surfactant is one of the most important parameters to acquire the target components efficiently in surfactant extraction. Eight kinds of surfactants including ionic surfactants SDBS and SDS, nonionic surfactants Tween-20, Tween-80, Brij-35, Brij-58, Triton X-100 and Triton X114 were tested. And their properties were shown in Table 1. The physico-chemical properties of surfactants vary distinctly above and below the CMC (critical micelle concentration) value (Schramm et al., 2003; Cheng et al., 2017). As shown in Fig. 3, nonionic surfactants exhibited better extraction efficiencies, especially Triton X series. And this may be due to there are many hydroxyl groups with high electron cloud density on target saponins, which are more easily captured by Triton X series (Kukusamude et al., 2012). So the nonionic surfactants Triton X series are favorable for saponins extraction than other surfactants. Meanwhile, compared with Triton X-100, Triton X-114 exhibited better properties such as higher extraction yields, lower CMC and CMT (critical micelle temperature). And it is relatively cheap and non-toxic. Besides, Triton-114 has no absorption at the wavelength of 203 nm and will not interfere with the determination of target compounds. Thus, Triton X-114 was chosen for the following experiments. 3.1.2. Effect of surfactant and co-surfactant concentration on the extraction efficiency of saponins As the main aim of this study was increasing saponin extraction efficiency applying the formulated surfactant system, proper concentrations of surfactant and co-surfactant were imperative to successful enhancement of extraction efficiency. Firstly, the concentration of surfactant TX-114 was optimized, and the results were shown in Fig. 4A. It could be seen that the content of saponins extracted was quite similar between water alone and TX-114 aqueous solution with the concentration of 0.01%, which does not reach the critical micelle concentration (CMC) of TX-114 (data shown in Table 1). Nevertheless, when the concentration of surfactant used exceeded the CMC, the increasing extraction yields of saponins appeared to be positively correlated with the increase of surfactant concentration. And the extraction yields of five target compounds reached maximum at the TX-114 concentration of 2%. However, it is interesting to note that the extraction yields decreased when the TX-114 concentration exceeded 2%. That may be because the solution became too sticky, which is hard for mass transfer. According to the experimental results, 2% TX-114 was used as an appropriate concentration for the following experiments. And then Gemini 16-5-16 in the concentration range of 0.01e0.05% (w/v) was successively added into 2% TX-114 aqueous solution. As the results in Fig. 4B, it illustrated clearly that the

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Table 1 Chemical and physical properties of the studied surfactants. Surfactant family Ionic surfactant Nonionic surfactant

a b

Surfactant name

Molecular Weight (g mol1)

CMC(mM)

CMC(wt%)

CMT( C)

SDBSa SDSb

348.48 288.38

1.6 8.2

0.06 0.24

e e

Tween-20 Tween-80 Brij-35 Brij-58 Triton X-100 Triton X-114

1227.50 1310 1199.57 1123.49 646.86 558.75

0.05 0.01 0.06 0.006 0.24 0.27

0.006 0.001 0.007 0.0007 0.016 0.015

95 >100 >100 63 64 23

SDBS: sodium dodecyl benzene sulfonate. SDS: sodium dodecyl sulfate.

Fig. 3. Effect of different surfactant extraction systems on the extraction yields of five target compounds.

Fig. 4. Effect of A) TX-114, B) 16-5-16 surfactant concentration on extraction yields of five target compounds.

concentration of Gemini 16-5-16 significantly affects the extraction efficiency. The contents of five saponins increased much with increasing Gemini 16-5-16 concentration up to 0.03% (w/v). This behavior may be due to the formation of the ion-pair of saponinGemini 16-5-15, which is conducive to be extracted by TX-114 surfactant system. Beyond the concentration of 0.03%, the contents of five saponins were only slightly affected by the Gemini 165-16 concentration, so the 0.03% (w/v) of Gemini 16-5-16 was chosen for the following experiments.

3.2. Optimization of surfactant extraction conditions As the type of surfactant and the concentration of surfactant and co-surfactant were fixed, the extraction yields of five saponins were still influenced by many other factors, such as pH of sample

solution, liquid to solid ratio, ultrasonic power and ultrasonic time. In consequence, the impact of these parameters on the extraction yields of notoginsenoside R1, ginsenosides Rg1, Re, Rb1 and Rd in Panax notoginseng was studied.

3.2.1. Effect of pH of sample solution on the extraction efficiency of saponins The effect of pH on the target compounds extraction efficiency was investigated in the range of 5e9, using 10 mmol/L phosphate buffer solution. The results in Fig. 5A showed that the pH of solution had a noteworthy influence on the extraction efficiency of saponins. The extraction yield of saponins reached to the maximum when pH was 8. The cause of this phenomenon maybe due to the formation of ion-pairs between the target compounds and Gemini 16-5-16 at pH 8. Therefore, to ensure the extraction of the target saponins with

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Fig. 5. Effect of A) pH, B) liquid to solid ratio (mL/g), C) ultrasonic power (W) and D) extraction time (min) on extraction yields of five target compounds.

highest yields, the pH of 8 was selected for further studies. 3.2.2. Effect of liquid to solid ratio on the extraction efficiency of saponins Liquid to solid ratio is also a quite important factor in practical production and application. Therefore, the effect of liquid to solid ratio on the extraction efficiencies of the target compounds were considered in this experiment. If a large number of solvent were used, the complexity of the extraction process will be increased and unnecessary waste will be caused. However, a small number of solvent may not be able to effectively extract the target compounds. Fig. 5B showed that the extraction yields of the target compounds increased significantly with the increase of the solvent volume. The extraction yields reached the maximum when the liquid to solid ratio reached 30:1 and no longer increase continuously with the increase of solvent volume. In hence, from the economic point of view, the liquid to solid ration of 30:1 (mL/g) was sufficient for the extraction of target saponin compounds. 3.2.3. Effect of ultrasonic power and time on the extraction efficiency of saponins Ultrasonication is used to improve the extraction yields of five target saponins from Panax notoginseng. The effect of ultrasonic power on the extraction yields were performed on the range of 100 We250 W. In Fig. 5C, the results showed that the extraction efficiency increased with the ultrasonic power increasing from 100 W to 250 W, which was attributed to the cavitation effect of the extraction solvent under the effect of ultrasonic wave, which is beneficial to the extraction of target compounds (Liu et al., 2015). Higher ultrasonic power can provide stronger cavitation to extract target compounds in a shorter time. Thus, ultrasonic power 250 W was fit for the extraction. Another factor influencing the ultrasonic extraction of target compounds was ultrasonic time. So the effect of ultrasonic time on

the extraction efficiency was investigated by varying the time 30e60 min. The results in Fig. 5D indicated that the extraction equilibrium was reached when the ultrasonic time was 40 min, and further increase of time couldn't affect the extraction yields much. That may be because 40 min surfactant extraction time was sufficient to achieve extraction equilibrium and a good extraction efficiency. Therefore, the surfactant extraction was carried out at 250 W for 40 min. In summary, an optimal extraction yields with the value of notoginsenoside R1, 28.66 mg/g, ginsenosides Rg1 46.52 mg/g, ginsenosides Re 12.91 mg/g, ginsenosides Rb1 29.31 mg/g and ginsenosides Rd 9.26 mg/g were obtained in the ultrasonic-assisted formulated surfactant extraction process at surfactant type and concentration: 2% Triton-114/0.03% Gemini 16-5-16, extraction pH: pH ¼ 8, liquid/solid ratio: 30:1 mL/g, ultrasonic power: 250 W and ultrasonic time: 40 min.

Fig. 6. Effect of different salt concentrations on the recovery yields of five target compounds.

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3.3. Preconcentration of saponins by cloud-point phenomenon After the use of formulated surfactants for the extraction of saponins, the preconcentration of the bioactive compounds by cloud-point phenomenon was studied. According to the literatures and our previous work, it can be found that adding electrolyte into the solution and then centrifugation for few minutes will facilitate the separation of the two phases. In our work, we used ammonium sulfate as the electrolyte and then optimized its concentration. The study of the influence of salt concentration on the extraction recovery was performed by adding ammonium sulfate with different concentrations from 10 to 30% (w/v). The results showed that the addition of salt would induce the extraction solution to be separated into two phases, that were the surfactant-rich phase and the aqueous phase. Moreover, with the increase of salt concentration, the size and aggregation number of micelle will increase, which make the solubility of analytes in the aqueous phase become less soluble, while the CMC value remains unchanged (Huang et al., 2015). The results in Fig. 6 indicated that the salt concentration of

Fig. 7. Comparation of different solvent systems.

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20% (w/v) gave the maximum extraction recovery with the values of 81.45%, 88.28%, 84.94%, 86.65%, 89.38% for notoginsenoside R1, ginsenosides Rg1, Re, Rb1 and Rd. When the concentration exceeded 20% (w/v), the extraction solution could not achieve the separation of phase, so 20% (w/v) was optimum concentration of added salt to achieve the phase separation. Based on the results described above, under the optimal extraction and preconcentration conditions, a surfactant-rich volume (2.6 mL) and enrichment factor for original sample solution (11.5) were obtained. The final volume of the surfactant-rich phase obtained by adding methanol to lower the viscosity was 5 mL and the final enrichment factor was 6.

3.4. Comparison of the extraction efficiency of formulated surfactants with conventional solvents In order to evaluate and compare the extraction efficiency of target compounds among formulated surfactants and other extraction solvents, identical experimental conditions were used: 1 g sample was extracted with 30 mL extraction solvent at 250 W for 40 min. Fig. 7 showed that the extraction efficiency of formulated surfactant was prominently higher than those of water, methanol and ethanol under uniform experimental conditions. It can be speculated that the formulated surfactants can promote the transfer of bioactive components from plant matrix to water, thus improving the mass transfer coefficient. Besides, as compared with the results of previous literatures, it was found that the extraction yields of five target saponins obtained by using ultrasonic-assisted formulated surfactant extraction were significantly higher in a shorter time as shown in Table 2. Moreover, surfactant aqueous solution was safer, cheaper and greener than organic solvents, and was an excellent alternative solvent for the extraction and preconcentration of bioactive ingredients from herb materials. Therefore, the developed ultrasonic-assisted formulated surfactant extraction method was suitable for extracting of saponins in Panax

Table 2 Comparison of different extraction methods on the extraction yields of five active compounds. Extraction method

Extraction yield (mg/g)

Ultrasonic-assisted formulated surfactant extraction, 40 min, 250 W Ethanol-reflux extraction, 60% ethanol, 1.51 h, 80  C, three times Microwave-assisted extraction, 120 s Ultrasonic extraction, 100 min Soxhlet extraction, 8 h Cold-maceration, 36 h Ethanol-reflux extraction, 95  C, 70% ethanol, 2 h Methanol-reflux extraction, 80  C, 2 h Ultrasonic extraction, 300 W, 1 h Pressurized liquid extraction, 150  C, 15 min, 6895 KPa Pressurized liquid extraction, 150  C, 15 min, 6895 KPa Maceration, 12e36 h a b

Ref.

NG-R1a

G-Rg1b

G-Reb

G-Rb1b

G-Rdb

28.66 1.12e5.63 9.10 8.68 9.20 7.51 11.6 0.71 0.78 5.26e7.32 5.3e7.2 0.4e1.2

46.52 10.52e23.35 37.29 35.60 41.53 33.90 41.0 2.94 3.00 26.94e39.10 29.6e39.1 2e6

12.91 1.13e3.43 e e e e e

29.31 14.09e18.85 21.90 20.27 23.93 17.93 36.1 1.84 1.72 25.28e38.05 26.7e32.4 2e6

9.26 5.15e6.76 e e e e 7.8 0.44 0.43 5.58e9.10 5.7e8.4 2e4

3.38e5.14 3.8e5.1 e

This study Hu et al. (2018) Dong et al. (2009)

Gong et al. (2015) Qu et al. (2012) Wan et al. (2006a), b Wan et al. (2006a), b Dong et al. (2005)

NG, Notoginsenoside. G, Ginsenoside.

Table 3 Calibration data and precision for five active compounds. Analyte

Notoginsenoside R1 Ginsenoside Rg1 Ginsenoside Re Ginsenoside Rb1 Ginsenoside Rd a

The unit is mg/mL.

Linearity rangea

31.25e1000 125e2000 15.63e500 31.25e1000 15.63e500

LODa

0.23 0.38 0.26 0.22 0.24

LOQa

0.76 1.27 0.88 0.73 0.79

Intra-day RSD

Inter-day RSD

Rt (%)

Pa (%)

Rt (%)

Pa (%)

0.29 0.21 0.18 0.16 0.25

4.04 2.33 3.55 3.73 4.67

0.34 0.31 0.25 0.24 0.38

4.45 2.43 3.67 3.79 4.88

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notoginseng root, which possessed the advantages such as shorter extraction time, lower energy consumption, operation at normal temperature and pressure, and excellent extraction efficiency. 3.5. Method validation In order to verify the reliability of the proposed analysis method, the validation tests were performed by taking notoginsenoside R1, Table 4 Recovery of five active compounds. Analyte

Amount added (mg)

Recovery (%)

RSD (%)

Notoginsenoside R1

40 80 40 80 40 80 40 80 40 80

99.65 101.28 98.32 99.34 97.12 99.91 98.85 99.24 98.83 100.15

2.26 2.71 3.13 3.65 2.59 3.02 2.93 3.25 2.83 3.06

Ginsenoside Rg1 Ginsenoside Re Ginsenoside Rb1 Ginsenoside Rd

ginsenosides Rg1, Re, Rb1 and Rd, which are the main components in Panax notoginseng root. The linearity of the standard was good for all analytes (15.63e1000 mg/mL) as proved by the satisfactory correlation coefficients being more than 0.99. The LOD and LOQ values of five target compounds were less than 0.38 and 1.27 mg/mL (Table 3), which indicated the established HPLC method was of excellent sensitivity. As shown in Table 3, the RSDs of five target compounds peak area (Pa) and retention time (Rt) were less than 4.88% and 0.38%, indicating good precision and repeatability. The recovery was examined by using spiked Panax notoginseng samples. In Table 4, the mean recoveries of five target saponins varied between 97.12% and 101.28% with RSD values less than 3.65%. The above results indicated that the established HPLC method was appropriate to analyze and determine saponins in Panax notoginseng. 3.6. Application of optimized method From the results above, the coupling of formulated surfactant system with HPLC is proved to be a relatively well-established method in both terms of pretreatment and analysis. And then the

Fig. 8. HPLC chromatographic profiles of A) surfactant solution, B) samples of Panax notoginseng extracts and C) standard mixtures: (1) Notoginsenoside R1, (2) Ginsenoside Rg1, (3) Ginsenoside Re, (4) Ginsenoside Rb1, (5) Ginsenoside Rd.

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Panax notoginseng root was first extracted and then enriched using formulated surfactant system. Fig. 8 showed that all of the five saponins could be reliably identified and quantified, without interfered by the solvent. 4. Conclusions In this study, the results obtained indicated that ultrasonicassisted extraction using formulated surfactant system possessed excellent performance on the extraction and preconcentration of notoginsenoside R1, ginsenosides Rg1, Re, Rb1 and Rd from Panax notoginseng without using expensive and potentially toxic organic solvents. What's more, the proposed formulated surfactant has no absorption at the wavelength of 203 nm and will not interfere with the determination of target compounds, which can be directly injected into the HPLC system for analysis. From the economical and effective point of view, the cloud-point property of non-ionic surfactants were applied to realize the extraction and preconcentration of different polar components in one-step so as to distribute them in a relatively small volume of surfactant-rich phase. The proposed extraction method using formulated surfactant aqueous system was efficient, fast, green and cheap for the extraction and preconcentration of target compounds from plant materials. It could be developed as a promising alternative to the traditional organic solvent for the extraction and separation procedures and used in large-scale application. Conflicts of interest There are no conflicts of interest to declare. Acknowledgements The authors gratefully acknowledge the financial supports by National Key R & D Program of China (2016YFD0600805), Fundamental Research Funds for the Central Universities (2572017ET03, 2572017EA03, 2572017DA04, 2572015EA04, 2572016AA48, 2572018AA17). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jclepro.2018.11.142. References Alam, M.S., Mandal, A.B., 2015. The clouding phenomena of mixed surfactant (nonionic Triton X-114 þ cationic gemini 16-5-16) solutions: influence of inorganic and organic additives on the cloud point. J. Mol. Liq. 212, 237e244. Amiririgi, A., Abbasi, S., 2016. Microemulsion-based lycopene extraction: effect of surfactants, co-surfactants and pretreatments. Food Chem. 197 (Pt A), 1002e1007. Berthod, A., Garcia-Alvarez-Coque, M.C., 2000. Micellar Liquid Chromatography. Chromatographic Science Series, first ed., vol. 83. Marcel Dekker, Inc, New York, p. 603. Bordbar, A.K., Hosseinzadeh, R., 2006. Binding of cetylpyridinum chloride to glucose oxidase. Colloids Surf., B 53, 288e295. Bordbar, A.K., Hosseinzadeh, R., Norozi, M.H., 2007. Interaction of a homologous series of n -alkyl trimethyl ammonium bromides with eggwhite lysozyme. J. Therm. Anal. Calorim. 87, 453e456. Chan, R.Y., Chen, W.F., Dong, A., Wong, M.S., 2003. Estrogen-like activity of ginsenoside Rg1 derived from Panax notoginseng. J. Clin. Endocrinol. Metab. 87, 3691e3695. Chan, C.H., Yusoff, R., Ngoh, G.C., Kung, F.W., 2011. Microwave-assisted extractions of active ingredients from plants. J. Chromatogr. A 1218, 6213e6225. Cheng, M., Zeng, G.M., Huang, D.L., Yang, C.P., Lai, C., Zhang, C., Liu, Y., 2017. Advantages and challenges of Tween 80 surfactant-enhanced technologies for the remediation of soils contaminated with hydrophobic organic compounds. Chem. Eng. J. 314, 98e113. Do, L.D., Stevens, T.L., Kibbey, T.C.G., Sabatini, D.A., 2014. Preliminary formulation

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