Purification of Danshensu from Salvia miltiorrhiza Extract Using

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Purification of danshensu from Salvia miltiorrhiza extract using graphene oxide based composite adsorbent Rong Peng, Qijiayu Wu, Xiaonong Chen, and Raja Ghosh Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b00661 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 14, 2017

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Industrial & Engineering Chemistry Research

Purification of danshensu from Salvia miltiorrhiza extract using graphene oxide based composite adsorbent Rong Penga, b, Qijiayu Wua, Xiaonong Chena*, Raja Ghoshb*

a

Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, China b

Department of Chemical Engineering, McMaster University

1280 Main Street West, Hamilton, Ontario L8S 4L7, Canada ABSTRACT: Danshensu has generated significant interest due to its therapeutic properties e.g., as cardiovascular, antitumor and neurology drug. Column chromatography techniques that are currently used for the separation of danshensu from Salvia miltiorrhiza extract have some drawbacks such as high pressure drop, slow separation and the usage of organic solvents. We discuss the use of a novel composite adsorbent consisting of graphene oxide (GO) modified cotton fiber for danshensu purification at high flow rate and low back pressure. This adsorbent was prepared by directly grafting GO onto the surface of cotton fiber. Danshensu was purified from aqueous Salvia miltiorrhiza extract based on hydrophobic interaction in a flow-through mode. The adsorbent could then be regenerated by elution of bound impurities using 0.04 M NaOH solution. HPLC analysis showed that the purity of danshensu increased from 24.8% in the

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feed solution (crude aqueous extract) to about 70~75% in the purified danshensu sample, the recovery being greater than 96.0%. Adsorbent fouling was minimal as indicated by the low back pressure at the end of the process.

KEYWORDS: Danshensu; hydrophobic interaction.

Salvia

miltiorrhiza;

graphene oxide

grafting;

adsorption;

1. INTRODUCTION Botanical drugs are used for a wide range of therapeutic applications such as the treatment of cancer,1,2 cardiovascular diseases,3 bacterial infections,4 diabetes,5 viral infections such as hepatitis,6 Parkinson’s disease7 and Alzheimer’s disease.8 Danshensu, extracted from the plant Salvia miltiorrhiza (Danshen), has attracted significant attention due to its value as a drug for the treatment of cardiovascular diseases,9-12 cancer,13 neurological disorders,14,15 and chronic kidney diseases.16 Danshensu is a naturally occurring hydrophilic phenolic acid, its chemical name being 3-(3,4-dihydroxyphenyl)-2-hydroxy-propanoic acid (see Figure 1). Danshensu and salvianolic acid B (also shown in Figure 1) are the main hydrophilic phenolic acids present in Salvia miltiorrhiza extract.17 Dried Salvia miltiorrhiza contains about 4-5 wt. % salvianolic acid B, and relatively lower amount of danshensu (ca. 1 wt. % based on the total mass).18 The danshensu content of Salvia miltiorrhiza extract could be increased by hydrolysis of salvianolic acid B.19,20 Danshensu is quite prone to oxidation due to its two active phenol hydroxyl groups. Therefore, any separation process for this compound has to be rapid in order to minimize its oxidation and thereby maintain its recovery and quality.


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Figure 1. Chemical structures of danshensu and salvianolic acid B.

Macroporous resin-based column chromatography is commonly used for separation of danshensu from Salvia miltiorrhiza extract.21-23 Danshensu was obtained using a separation process21 including the following steps: crude danshensu separation from Salvia miltiorrhiza extract by counter-current extraction using a mixture of ethyl acetate/n-hexane as extracting solvent; purification of crude danshensu thus obtained by anion exchange column chromatography, using 1 M NaOH solution as eluent. The eluate thus obtained was recrystallized and highly pure danshensu (90~97%) purity was obtained. Jin et al.22 obtained crude danshensu from an aqueous extract of Salvia miltiorrhiza using AB-8 macroporous resin column. The crude danshensu was then adsorbed on LS-18 resin column and eluted with 95% ethanol. This eluate was further purified by Sephadex LH-20 column chromatography using ethanol as eluent. The last eluate was concentrated and recrystallized to obtain highly pure danshensu. Yang et al.23 separated danshensu from Salvia miltiorrhiza extract by combining macroporous resin based adsorption, active carbon adsorption and recrystallization. Some limitations related to these currently used column chromatography steps include high back pressure, low flow rate (i.e. long


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processing time), low purification factors, low recovery and usage of organic solvent (and thereby disposal issues). Alternative adsorbent formats such as membrane adsorbers have been used to overcome some of the above-limitations related to column chromatography. Membrane adsorption being convection based, is much fast than diffusion-limited packed-bed adsorption, and can be operated at high flow rate with low back pressure.24-26 This translates to lower equipment and energy cost. Membrane adsorption27 has been used to carry out affinity chromatography, ion exchange

chromatography,

hydrophobic

interaction

chromatography

and

multi-stage

separations.28-30 While membrane adsorption has been extensively used for purification of biomacromolecules, such as protein, DNA and virus,25 there are very few report in the literature related to the separation small molecules using this technique. In this study, we discuss the use of an adsorbent in a format similar to that used in membrane adsorption for separation of danshensu from Salvia miltiorrhiza extract. Graphene oxide (GO) has a two-dimensional structure, large specific surface area (2600 m2/g) and conjugated aromatic ring structure.31 GO membranes fabricated by stacking together GO sheets could reject 99% organic dye and 20~60% salt ions32 and these have great potential for application in water treatment, desalination and gas separation. However, such size-exclusion membranes have not be used for isolation and purification of organic compounds as these molecules are indiscriminately rejected, i.e. there is no selectivity.33 GO has a significant capacity to adsorb hydrophobic molecules containing aromatic ring structure through strong π-π and hydrophobic interactions. Moreover, there are active groups on the surface of GO (e.g., carboxyl, hydroxyl and epoxy), which allows the conjugation of GO to a suitable supporting substrate or modifier. For example, folic acid modified GO could be loaded with the anticancer


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drug doxorubicin, up to 67.5% of its own mass.34 PEGylated GO could adsorb 11.2% (wt.) of the drug paclitaxel through a combination of π-π conjugation and hydrophobic interaction.35 Functionalized GO composite has been used as adsorbent for dyes such as methyl orange (~300 mg/g)36 and methylene blue (~53.0 mg/g).37 Clearly, GO sheets could potentially be used as ligand of hydrophobic chromatography membrane. In a recent paper we have reported the preparation of hydrophobic interaction adsorbent by grafting GO onto cotton fiber through epoxy ring-opening reaction between the amino groups on modified cotton fiber surface and the epoxy moieties on GO sheet.38 Cotton fiber was chosen as adsorbent substrate due to wide and easy availability, low cost, high water permeability, bio-degradability and reasonably good physical stability. This paper discusses the use of a similarly prepared membrane-like adsorbent for separation of danshensu from Salvia miltiorrhiza extract in a flow-through mode. The factors that affect the separation performance were systematically investigated and are reported. 2. EXPERIMENTAL SECTION 2.1. Materials. Graphene oxide dispersion (purity > 98.5 wt.%; 2 mg/mL aqueous dispersion, pH 5-7; sheet size 0.1-3 µm; layer number 3-5 layers; oxygen content 46-49 wt.%) was purchased from Sinocarbon Company (Taiyuan, Shanxi Province, China). Cotton fiber (Batch No.2014.03.06) was obtained from Zhongbeibojian Company, Beijing, China. Salvia miltiorrhiza was purchased from Pharmacy of Traditional Medicine (YONG AN TANG, Beijing, China). HPLC grade acetonitrile was purchased from Caledon Laboratories Ltd., Georgetown, ON, Canada. HPLC grade n-octanol was obtained from J&K Chemical, Beijing, China. Sodium danshensu (98.0%), sodium phosphate monobasic (99.0%), sodium phosphate dibasic (99.0%) and phosphoric acid (85 wt. % in H2O) and sodium hydroxide pellets were purchased from Sigma Aldrich, Oakville, ON, Canada. Sodium bicarbonate and sodium carbonate anhydrous


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were obtained from EMD Chemicals, Mississauga, ON, Canada. Ultrapure (18 MΩ-cm) water was obtained from a DiamondTM NANO pure water purification unit (Barnstead International, Dubuque, IA, USA). 2.2. Preparation process of graphene oxide grafted adsorbent. GO grafted membrane-like adsorbent was prepared as described previously.38 Cotton fiber was soaked in 14 g/L aqueous NaOH solution for 24 h and washed with pure water until the washings showed neutral pH. The cotton fiber was then dried at room temperature. Dried cotton fiber (25 g) was added to sodium periodate solution (30 mM, 2500 mL, pH 2) and stirred at 45oC for 5 h in the dark. Glycerol was added to this and stirring was continued for an additional 2 h. The resultant dialdehyde cotton fiber (DCF) was washed with pure water until the filtrate indicated neutral pH. The material thus obtained was freeze dried. Under nitrogen atmosphere, 7 g of DCF was dispersed in 700 mL of acetate buffer (pH 6) and kept for 15 min, followed by addition of hexanediamine (7 mL) and stirred at 50oC for 1 h to form Schiff-base. Then resultant fibrous material was reduced using sodium borohydride (2%) in an ice water bath (for 12 h). It was then washed with water and freeze dried to obtain aminated cotton fiber (ACF). ACF (3 g) was immersed in 600 mL of GO dispersion (50 mg/L, pH 8) and stirred at room temperature for 1 h, then heated to 80oC and maintained for an additional 2 h. GO-fiber reaction mixture was washed with water and freeze dried to obtain GO grafted cotton fiber. The grafting ratio of GO on the cotton fiber was approximately 0.4% (weight/weight) based on the UV absorbance measurements.38 GO modified cotton fiber was vigorously stirred in pure water using a magnetic stirrer for 2~3 h to obtain the GO grafted cotton fiber pulp. The pulp was then used to prepare the GO grafted cotton fiber membrane-like adsorbent by vacuum filtration.38 The morphology of the adsorbent was studied by scanning electron microscopy using a JSM-7500F Instrument (JEOL


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USA, Peabody, MA, USA) or S-4700 instrument (Hitachi, Japan). The adsorbent was housed in a membrane module. Surface area of sample was determined by BET multipoint method using surface area analyzer (Bei Shi De instrument, 3H-2000PS2, Beijing, China) at 77.3 K in the pressure (P/P0) range of 0.04 to 0.16. XPS analysis was conducted using an ESCA LAB 250 Spectrometer (monochromatic Al kalph 150 W source, Thermo Fisher Scientific), operating at 30 eV for high resolution scans.

2.3. Set-up for adsorption experiments. The membrane-like GO grafted cotton fiber adsorbent was housed inside a module having 18.5 mm effective diameter.38,39 The thickness of adsorbent was approximately 1 mm, with a dry weight of about 0.1 g. The set-up used for the adsorption experiments is shown in Figure 2. In addition to the module, it included a sample injector, two pumps (5966-Optos Pump ISM, Eldex, Napa, CA, USA), an on-line UV detector (Spectra/Chrom® UV monitor, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) and a pressure sensor (OMEGA, 0-50 PSID, Omega Engineering, Stamford, CT, USA). Appropriate elution gradients were generated two pumps controlled using a program written with Lab VIEWTM 2011 (National Instruments Canada, Toronto, ON, Canada). The UV absorbance of the module effluent (measured at 280 nm) and the module pressure-drop were monitored and data was logged using LabPro (Vernier Software, Beaverton, OR, USA).

Figure 2. Experimental set-up for danshensu purification.


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2.4 Extraction of danshensu from Salvia miltiorrhiza. Salvia miltiorrhiza root was first ground into a powder. The extraction process was conducted under nitrogen atmosphere with 15.0 g Salvia miltiorrhiza powder using 50 mL of 2 M NaOH solution, under shaken condition at 30o C for 3 h. Then, 100 mL pure water was added to the above mixture followed by ultrasonication for 30 min, and pH adjustment to 2~3 using 1 M HCl. The extract thus obtained was centrifuged and the supernatant was collected. This is subsequently referred to as Salvia miltiorrhiza crude extract. 2.5. Purification of danshensu using graphene oxide grafted adsorbent. The GO grafted adsorbent was first equilibrated with the binding phase. Then feed solution (Salvia miltiorrhiza crude extract) was then injected into the module, followed by washing using the binding phase until the UV absorbance reached the base line. Preliminary experiments indicated that danshensu was relatively more hydrophilic than the major impurities present in the extract. Therefore, danshensu was obtained in the flow-through while the impurities remained bound to the adsorbent. The adsorbent was then regenerated by eluting the bound impurities using 0.04 M NaOH solution. The samples corresponding to flow-through and elution peaks were collected and analyzed by HPLC. 2.6. HPLC analysis. Chromatograms were obtained using a 600 WatersTM HPLC system consisting of a WatersTM 600 controller, a WatersTM 600 pump, and a WatersTM 486 detector. A Sun Fire® C18 (5 µm, 4.6 × 250 mm) column was used for HPLC analysis. Mobile phase A consisted of 10% acetonitrile with 0.1% phosphoric acid while mobile phase B consisted of 90% acetonitrile with 0.1% phosphoric acid. A 20-µl sample loop was used for sample injection. Flow rate used for HPLC was 1.0 mL/min and absorbance was monitored at 280 nm. For gradient elution, mobile phase B increased linearly from 0% to 63% over 50 min. The calibration curve of


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danshensu based on HPLC peak area is y = 36064.03 + 20266.53x (R2 = 0.9998, range 0~1.4 mg/mL). 2.7 Component distribution of Salvia miltiorrhiza extract in n-octanol/water system. The effect of buffer pH (in the range of 3 to 8) on the distribution of components present in Salvia miltiorrhiza extract between n-octanol and water was studies.40 First, solution with the lowest pH (pH 3) was prepared using 1 mM HCl while the remaining solutions (pH range 4-8) were prepared using 20 mM citrate and phosphate buffers. Corresponding saturated n-octanol was prepared by saturating pure n-octanol with the respective aqueous solution. Crude Salvia miltiorrhiza extract (150 µL) was then added to 4 mL of saturated n-octanol with vigorous mixing, followed by addition of 4 mL of aqueous solution with continuous stirring at 120 rpm for 4 h. The mixture was then centrifuged and the lighter phase (n-octanol) was separated from aqueous phase and the components present in the two phases were determined by UV spectrophotometry (PerkinElmer Lambda 365, wavelength 190-900 nm). 3. RESULTS AND DISCUSSION 3.1 Morphology of graphene oxide grafted adsorbent. Figure 3 shows images of the GO grafted adsorbent. Figure 3a shows photographs of an unmodified cotton fiber pad (Before) and a GO grafted cotton fiber adsorbent pad (After) within the O-ring used in the membrane module. The diameter and thickness of adsorbent pad were 18.5 mm and 1 mm, respectively. Figure 3b shows the SEM image of GO modified cotton fiber where the GO sheet morphology is evident. Figures 3c and 3d show the morphology of the surface and the cross section of the GO grafted cotton fiber adsorbent. The fibers were stacked loosely, resulting in pores in the range of several microns to tens of micron. Low back pressure during separation could therefore be anticipated.


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Figure 3. Images of graphene oxide grafted absorbent. a) photographs of cotton fiber before (left) and after modification using graphene oxide (right); b) SEM image of graphene oxide modified cotton fiber, scale bar 5 µm; c) SEM image of top surface of adsorbent, scale bar 100 µm; d) SEM image of cross section of adsorbent, scale bar 10 µm)

The pore properties of samples were determined by BET analysis. BET method is a common method to determine specific surface area and pore volume. As shown in Figure S1(a) (Supporting Information), the adsorption/desorption isotherms had similar trend. The calculated specific area of cotton fiber was 1.03 m2/g while that of graphene oxide grafted adsorbent was 1.17 m2/g. This increase (0.14 m2/g) could be attributed to the grafted graphene oxide. As shown in Figure S1(b) (Supporting Information), the calculated pore volume for graphene oxide grafted adsorbent was 0.007 mL/g and the pore diameter was in the range of 2-3 nm. Therefore adsorption on the graphene oxide modified cotton fiber mainly occurred on the surface with the pores playing a very limit role. High resolution XPS spectra in C1s, N1s and O1s regions were obtained using an ESCA LAB 250 Spectrometer. Figure S2 (Supporting Information) shows the surface chemistry of graphene oxide grafted adsorbent and graphene oxide. The C1s spectra of GO could be deconvoluted into four peaks: C=C/C-C (284.8 eV), C-O (hydroxyl and epoxy, 286.7 eV), C=O (287.1) and O-C=O (288.6 eV). The appearance of C=O and O-C=O peaks in C1s spectra of GO grafted absorbent indicates the presence of graphene oxide. A C-N peak at


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285.7 eV confirmed the formation of C-N bonds due to the epoxy-opening reaction between GO and amino group. The N1s spectra of GO grafted adsorbent, i.e. the peak at 399.6 eV, further demonstrated a significant increase in nitrogen element amount compared with GO (very weak N1s signal appears in GO spectrum).

3.2. Extraction of danshensu from Salvia miltiorrhiza powder. The reported danshensu content of Salvia miltiorrhiza is approximately 1% of dry weight.18,41,42 As reported in the literature, danshensu content could be increased by the hydrolysis of other phenolic acids present in Salvia miltiorrhiza.19,20,41 A modified alkaline hydrolysis method41 was therefore used to extract danshensu from Salvia miltiorrhiza. The HPLC chromatograms obtained with standard danshensu and Salvia miltiorrhiza crude extract are shown in Figure 4.

Figure 4. HPLC chromatograms obtained with standard danshensu (0.1 mg/mL in water) and Salvia miltiorrhiza crude extract.

Based on the HPLC result, the amount of danshensu obtained in the crude extract was estimated to be about 3.1% of dry weight of the Salvia miltiorrhiza powder, which was significantly greater than that reported in the literature.18,41,42 Based on peak areas of UV absorbing species, the extract contained 24.8% danshensu, 59.7% major UV absorbing impurities (peak at 25 min) and 15.5% of minor UV absorbing impurities. The concentration of


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danshensu in the crude extract was about 2.2 mg/mL (calculated from HPLC chromatogram based on calibration curve). The retention time of danshensu was about 9.8 min (Figure 4) which indicated that it was relatively more hydrophilic than its impurities. Based on this, it was hypothesized that danshensu could be obtained in the flow-through while the impurities would remain bound to the GO grafted adsorbent. 3.3. Selection of binding phase for the purification of danshensu from Salvia miltiorrhiza crude extract. Figure 5a and b shows the chromatograms and the pressure profiles of the adsorption module obtained during experiments carried out to select appropriate binding phase for the separation of danshensu from Salvia miltiorrhiza crude extract. The binding phases examined included (A) pure water, (B) 1 mM HCl solution, (C) 20 mM sodium phosphate buffer, pH 6.0, (D) 20 mM sodium phosphate buffer, pH 7.0, (E) 20 mM sodium phosphate buffer, pH 8.0, and (F) 20 mM NaHCO3, pH 8.3. The first peak (flow- through or peak 1) in each of the chromatograms shown in Figure 5a corresponded to the non-retained UV-adsorbing compounds, while the second peak (eluate or peak 2) corresponded to reversibly adsorbed compounds. Peak 1 and 2 samples were collected and analyzed by HPLC (Figure 5c and d respectively). Figure 5b indicates that the back pressure was around 20 kPa at a flow rate of 1.5 mL/min and changed only very slightly, depending on binding and eluting phases. The low pressure-drop observed is consistent with that expected based on the morphology of GO grafted adsorbent shown in Figure 3. Paper-PEG-based membranes of comparable thickness and dimension used at same flow rate for protein fractionation showed similar pressure-drop (~22 kPa).43 These values are both considerably lower than that observed in equivalent column chromatography.24 A low pressuredrop implies that very high flow-rates could be used during separation, resulting in a short process time, high productivity, and reduce degradation of danshensu by oxidation during


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purification. Also, the almost constant pressure-drop during the separation processes is indicative of low or negligible membrane fouling.

Figure 5. Chromatograms (a) and pressure profiles (b) obtained during separation of danshensu using different binding phases, and HPLC chromatograms for Peak 1 (c) and Peak 2 (d) samples from the above experiments (Feed solution: 4 times-diluted crude extract (544 µg/mL danshensu); sample loop: 0.5 mL; elution: step change after 600 s to 0.04 M NaOH solution; flow rate: 1.5 mL/min)

Table 1. Purification of danshensu using different binding phases Binding phase

Purity a (%)

A (pure water)

38.2

B (1 mM HCl)

0

C (20 mM sodium phosphate buffer, pH 6.0)

73.2

D (20 mM sodium phosphate buffer, pH 7.0)

75.7


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E (20 mM sodium phosphate buffer, pH 8.0)

70.4

F (20 mM NaHCO3 pH 8.3)

0

a

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based on HPLC analysis (see Figure 5c).

When pure water (A) was used as binding phase, danshensu peak could be observed in the HPLC chromatograms obtained with both peaks 1 and 2 samples (see Figures 5c and d), indicating that danshensu was distributed in both flow through and eluate. When 1 mM HCl solution was used as binding phase, no danshensu peak was observed in peak 1, while danshensu along with the main impurities were observed in peak 2, indicating very poor selectivity. Clearly, both pure water and 1 mM HCl were not suitable as binding phase for danshensu purification. When sodium phosphate buffers of different pH values (i.e. 6, 7 and 8) were used as binding phases, danshensu was obtained in peak 1, while the corresponding peak 2 samples were virtually free of this compound. On the other hand, most of the impurities bound to the adsorbent and were obtained in the eluate (peak 2). Quite clearly, sodium phosphate buffers of these pH values seemed promising as binding phase, and were examined further. Sodium bi-carbonate solution on the other hand proved to be totally unsuitable for the above danshensu purification. The purity of danshensu obtained in the above experiments are summarized in Table. 1. When a sodium phosphate buffer (pH 6, 7 or 8) was used as binding phase, the purity of danshensu obtained in the flow-through was in the 70%~76% range. Hence, danshensu could be purified from Salvia miltiorrhiza extract in a flow-through mode based on its greater hydrophilicity compared to its impurities, which bound on the adsorbent and could be subsequently be eluted using 0.04 M NaOH solution. The most likely mechanism for impurity binding onto GO grafted adsorbent was hydrophobic interaction.44 The manner of desorption of the impurities employed in the above experiment, i.e. by increasing pH of the mobile phase is similar to that discussed in the literature.44-46


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Control experiments were carried out using unmodified cotton fiber to demonstrate that the danshensu separation described in the above paragraphs could be attributed to the GO, and not to the supporting cotton fiber. For this, an adsorbent module, identical to that used for the GO grafted adsorbent was packed with cotton fiber, using an identical protocol. Danshensu purification experiments were then carried out with Salvia miltiorrhiza crude extract using 20 mM sodium phosphate buffer (pH 6.0) as binding phase with both devices (i.e. containing GO grafted adsorbent and cotton fiber). The results thus obtained are shown in Figure 6. The flowthrough (peak 1) and the eluate (peak 2) were collected from both experiments and analyzed by HPLC (see Figures 6 b and c). Very negligible amounts of UV absorbing solutes bound to the cotton fiber. Almost everything that was injected to the cotton fiber containing module appeared in the flow through, the HLPC chromatogram obtained with it being almost identical to that obtained with Salvia miltiorrhiza crude extract (see Figure 4). By contrast, the flow through obtained with the GO grafted adsorbent contained primarily danshensu while its eluate consisted of impurities. Based on these results, it may be inferred that the adsorbent property and profile obtained with the GO grafted adsorbent was entirely due to hydrophobic interaction and π-π conjugation between GO and impurities, the supporting cotton fiber had virtually no role to play in the impurity binding. 3.4. Effect of the pH of sodium phosphate buffer on the purification of danshensu. The effect of the pH of sodium phosphate buffer (binding phase) on the purification of danshensu was studied at pH 6, 7 or 8 by injecting 2 mL of crude extract containing 4352 µg danshensu into the adsorption module. The results obtained are shown in Figures 7a and b. The purity and recovery of danshensu in the above separation experiments are summarized in Table 2. These results indicate that the pH of the sodium phosphate buffer used as binding phase had a limited


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impact on danshensu purification in the range examined, i.e. 6-8. It has been reported that danshensu is more stable in an acidic environment.47 Based on this criteria, sodium phosphate buffer (pH 6) was deemed as appropriate binding buffer for danshensu purification.

Figure 6. Comparison of danshensu purification using unmodified cotton fiber sheet and graphene oxide grafted fiber absorbent. (a) Chromatograms obtained from separation processes, (b) HPLC chromatogram of Peak 1 and (c) Peak 2. (Feed solution: 4 times-diluted crude extract (544 µg/mL danshensu); sample loop: 0.5 mL; binding solution: 20 mM sodium phosphate buffer with pH 6; eluting solution: step change after 600 s to 0.04 M NaOH solution; flow rate: 1.5 mL/min)


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Figure 7. Effect of the pH of sodium phosphate buffer on purification of danshensu. HPLC chromatograms for (a) flow-through, and (b) eluate.

Table. 2. Purification of danshensu by using buffers with different pH values. pH of buffer

Purity a

Recovery b

6

61.2

96.3

7

57.9

97.1

8

60.8

98.3

a

b

based on the peak area ratio of danshensu (see Figure 7a). (danshensu in flow through, mg) / (danshensu in feed solution, mg) × 100%

To verify that the above separation was indeed based on hydrophobic interaction we investigated the hydrophobicity of the components present in Salvia miltiorrhiza extract by studying their partitioning behavior between n-octanol and water using a shake-flask method40, 48 (see Figure 8). UV/VIS spectroscopy

40

was employed to determine the distribution of the

components between the two phases. As shown in Figure 8a, the UV absorption peak of danshensu corresponded to 283 nm. Absorption peaks (300-400 nm) that also appeared in the buffered aqueous phases were due to the impurities present in the Salvia miltiorrhiza extract. At all pH values investigated, the danshensu peak was much more significant than the impurity peaks. By contrast, the danshensu peaks had significantly lower intensity in the spectra obtained with the n-octanol phase samples (Figure 8b). The above results clearly demonstrated that


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danshensu was less hydrophobic than its impurities. Interestingly, when the pH of the buffer increased from 3 to 8, the impurity peaks in the n-octanol phase decreased (Figure 8b), indicating that the impurities could be eluted by switching to mobile phase having higher pH value, for instance, 0.04 M NaOH solution.

Figure 8. Effect of buffer/solution pH on the distribution of danshensu and impurities in n-octanol/water system. (a) UV spectra of Salvia miltiorrhiza extract components present in buffered aqueous phase, and (b) UV spectra of Salvia miltiorrhiza extract components present in n-octanol phase. (pH 3: 1 mM HCl; pH 4 and 5: 20 mM citrate buffer with pH 4, 5 respectively; pH 6, 7 and 8: 20 mM phosphate buffer with pH 6, 7 and 8 respectively)

3.5 Effect of concentration of sodium phosphate buffer on the purification of danshensu. The effect buffer concentration on the purification of danshensu was examined at the following concentrations: 5 mM, 10 mM and 20 mM, with the pH value being kept fixed at 6. There experiments were performed using 2 mL of Salvia miltiorrhiza crude extract containing 4352 µg danshensu as feed solution. The results obtained are shown in Figures 9a and b. The purity of danshensu obtained were 59.2%, 58.2% and 60.8% respectively, for 20 mM, 10 mM and 5 mM sodium phosphate buffer. The purity obtained with the three buffer concentrations were fairly similar. However, considering that less buffer salts would be consumed with 5 mM sodium phosphate buffer, it selected for all further experiments in this investigation.


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Figure 9. Effect of the concentration of sodium phosphate buffer on the purification of danshensu. HPLC of samples corresponding to (a) flow-through, and (b) eluate.

3.6. Purification of danshensu from Salvia miltiorrhiza extract using two modules in series. In order to increase the efficiency of danshensu separation, experiments were carried out using two adsorber modules in series, i.e. in the form of a two-stage cascade. Flow through and eluate samples obtained from such a system were compared with those obtained from a single stage system. Figure 10 shows a comparison of the results thus obtained. The purity of danshensu in the flow through obtained from the one-module and two-module experiments were 63.4% and 74.1%, respectively. The purity of danshensu obtained using the two-module separation was higher than that reported in the literature for column based separation.21 Moreover, the absence of a danshensu peak in the HPLC chromatogram obtained with the eluate suggest that its recovery in the flow through was very high (>96%). These results clearly demonstrate that separation efficiency using the two-module separation process was very high.


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Figure 10. HPLC chromatograms obtained with (a) flow-through, and (b) eluate obtained using one-module mode and twomodule purification processes (Feed solution: crude extract (2176 µg/mL danshensu); sample loop: 1 mL; binding solution: 5 mM sodium phosphate buffer with pH 6; eluting solution: 0.04 M NaOH solution)

4．．CONCLUSION Composite graphene oxide (GO) grafted cotton fiber adsorbent was found to be suitable for purifying danshensu from Salvia miltiorrhiza crude extract. Danshensu being more hydrophilic than its impurities was obtained in the flow-through, while the impurities remained bound to the adsorbent. The interaction of the impurities with the adsorbent took place through a combination of hydrophobic interaction and π-π conjugation. The bound impurities could be eluted using sodium hydroxide solution and the adsorbent could thereby be regenerated. Unlike other chromatographic method for danshensu purification, this method did not require any organic solvents, and was therefore relatively more environment-friendly. The optimized binding phase consisted of 5 mM sodium phosphate buffer (pH 6) while 0.04M NaOH solution was found to be a suitable eluting phase. The efficiency of the separation process could be improved by using two adsorber modules in series. Under the optimized conditions, danshensu purity obtained in the flow through was about 70~75%, which was higher than that reported in the literature for column


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chromatography based danshensu purification. The recovery of this hydrophilic phenolic acid was recovery was found to be about 96.0%. Adsorbent fouling was found to be negligible.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. XPS spectra and BET measurement of graphene oxide and graphene oxide grafted adsorbent.

AUTHOR INFORMATION Corresponding authors *Tel.: +86-010-64419060. E-mail: [email protected] (X. Chen) *Tel.: +1 905 525 9140x27415. E-mail: [email protected] (R. Ghosh). NOTES The authors declare no competing financial interest. ACKNOWLEDGEMENT This work was funded by the China Scholarship Council (NSCIS No. 201406880014) and the Natural Science and Engineering Research Council (NSERC) of Canada. The authors thank Paul Gatt (Department of Chemical Engineering, McMaster University) for fabricating the membrane module used in the experiment, Lei Lei and Si Pan (Department of Chemical Engineering, McMaster University) for help with running the HPLC experiments, and Dan Wright (Department of Chemical Engineering, McMaster University) for writing a program for controlling the pumps used in our experiments.


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Table of Contents graphic

We discuss the use of a novel composite graphene oxide (GO)-cotton fiber adsorbent for purifying danshensu form Salvia miltiorrhiza extract.


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Figure 1. Chemical structures of danshensu and salvianolic acid B. 82x148mm (96 x 96 DPI)


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Figure 2. Experimental set-up for danshensu purification. 144x76mm (96 x 96 DPI)



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Figure 3. Images of graphene oxide grafted absorbent. a) photographs of cotton fiber before (left) and after modification using graphene oxide (right); b) SEM image of graphene oxide modified cotton fiber, scale bar 5 µm; c) SEM image of top surface of adsorbent, scale bar 100 µm; d) SEM image of cross section of adsorbent, scale bar 10 µm) 155x116mm (96 x 96 DPI)


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Figure 4. HPLC chromatograms obtained with standard danshensu (0.1 mg/mL in water) and Salvia miltiorrhiza crude extract. 139x140mm (96 x 96 DPI)



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Figure 5. Chromatograms (a) and pressure profiles (b) obtained during separation of danshensu using different binding phases, and HPLC chromatograms for Peak 1 (c) and Peak 2 (d) samples from the above experiments (Feed solution: 4 times-diluted crude extract (544 µg/mL danshensu); sample loop: 0.5 mL; elution: step change after 600 s to 0.04 M NaOH solution; flow rate: 1.5 mL/min) 146x190mm (96 x 96 DPI)


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Figure 6. Comparison of danshensu purification using unmodified cotton fiber sheet and graphene oxide grafted fiber absorbent. (a) Chromatograms obtained from separation processes, (b) HPLC chromatogram of Peak 1 and (c) Peak 2. (Feed solution: 4 times-diluted crude extract (544 µg/mL danshensu); sample loop: 0.5 mL; binding solution: 20 mM sodium phosphate buffer with pH 6; eluting solution: step change after 600 s to 0.04 M NaOH solution; flow rate: 1.5 mL/min) 158x162mm (96 x 96 DPI)



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Figure 7. Effect of the pH of sodium phosphate buffer on purification of danshensu. HPLC chromatograms for (a) flow-through, and (b) eluate. 212x108mm (96 x 96 DPI)


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Figure 8. Effect of buffer/solution pH on the distribution of danshensu and impurities in n-octanol/water system. (a) UV spectra of Salvia miltiorrhiza extract components present in buffered aqueous phase, and (b) UV spectra of Salvia miltiorrhiza extract components present in n-octanol phase. (pH 3: 1 mM HCl; pH 4 and 5: 20 mM citrate buffer with pH 4, 5 respectively; pH 6, 7 and 8: 20 mM phosphate buffer with pH 6, 7 and 8 respectively) 154x69mm (96 x 96 DPI)



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Figure 9. Effect of the concentration of sodium phosphate buffer on the purification of danshensu. HPLC of samples corresponding to (a) flow-through, and (b) eluate. 209x107mm (96 x 96 DPI)


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Figure 10. HPLC chromatograms obtained with (a) flow-through, and (b) eluate obtained using one-module mode and two-module purification processes (Feed solution: crude extract (2176 µg/mL danshensu); sample loop: 1 mL; binding solution: 5 mM sodium phosphate buffer with pH 6; eluting solution: 0.04 M NaOH solution) 217x112mm (96 x 96 DPI)



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We discuss the use of a novel composite graphene oxide (GO)-cotton fiber adsorbent for purifying danshensu form Salvia miltiorrhiza extract. 223x70mm (96 x 96 DPI)


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Purification of Danshensu from Salvia miltiorrhiza Extract Using

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