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Chromatographic isolation of nanoparticles from Ma-Xing-Shi-Gan-Tang decoction and their characterization
Journal of Ethnopharmacology 151 (2014) 1116–1123
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Chromatographic isolation of nanoparticles from Ma-Xing-Shi-Gan-Tang decoction and their characterization Jianwu Zhou a,b, Guanzhen Gao a,b, Qiuping Chu b, Huiqin Wang a,b, Pingfan Rao a,b, Lijing Ke a,b,n a b
CAS.SIBS-Zhejiang Gongshang University Joint Centre for Food and Nutrition Sciences, Zhejiang Gongshang University, Hangzhou 310035, China Institute of Biotechnology, Fuzhou University, Fuzhou 350002, China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 June 2013 Received in revised form 9 December 2013 Accepted 18 December 2013 Available online 30 December 2013
Ethnopharmacological relevance: The herbal decoction is a complex dispersion system containing solutes, colloid, aggregates, emulsions and precipitates. In which phase bioactive phytochemicals are dispersed determines their delivery, action and metabolism. This study took ephedrine, a well-studied and widely used phytochemical, as an example to elucidate its exact distribution in the phases of Ma-Xing-ShiGan-Tang decoction (MXSGT), which is an Ephedra sinica Stapf. containing traditional Chinese medicinal formula, and the biological meaning of this distribution correspondingly. It may provide an important update to the safety and efficacy assessment of the herbal decoction and its active phytochemicals. Materials and methods: In this study, the decoction was fractionated with size-exclusion chromatography coupled with multi-angle laser light scattering detector. The morphology of fractionated nanoparticles was observed with AFM and SEM. The bioactivities of the decoction, the ephedrine alkaloids loaded NPs (prepared by chromatography isolation) and the synthetic ephedrine were assessed by cell proliferation tests using five cell lines, namely Caco-2, L-02, Hep-G2, NR-8383, and Hela-229. Results: Nanoparticles with radii of gyration ranged from 50 to 150 nm were isolated, in spherical shape. Further analysis of nanoparticles on the subsequent reversed phase chromatography revealed that the majority of ephedrine (99.7%) and pseudoephedrine (95.5%) were associated with these nanoparticles, rather than dispersed freely in the real solution. The addition of both the herbal decoction and the separated ephedrine-loaded nanoparticles reserved higher cell viability/proliferation than that of the sole synthetic ephedrine among the Caco-2, L-02, Hep-G2, and NR-8383 cells. In contrast, the nanoparticles reduced the proliferating power of ephedrine on Hela-229 cells. In general, the ephedrineloaded NPs conducted the intermediate influences on the cell viability, in either way. Conclusions: The colloidal nanoparticles were separated from the decoction. The association of ephedrine alkaloids with nanoparticles was demonstrated and may have changed the bioactivity of the alkaloids. The naturally occurred colloidal nanoparticles may play an important role in the pharmacological properties of both the decoction and its active phytochemicals, therefore warrant further studies. & 2013 Elsevier Ireland Ltd. All rights reserved.
Chemical compounds studied in this article: Ephedrine (PubChem CID: 9294) Pseudoephedrine (PubChem CID: 62946) Keywords: Nanoparticles Size-exclusion gel chromatography Ephedrine Pseudoephedrine Ma-Xing-Shi-Gan-Tang decoction Cell proliferation
1. Introduction The Ephedra sinica Stapf. stem, sundried (Herba Ephedrae, Ma-Huang, ephedra) is widely used in traditional Chinese medicines and Japanese Kampo medicines (Okamura et al., 1999) for the treatment of asthma, fever, coughs, lack of sweating, urination promotion and alleviating edema (Bensk et al., 2004). Ma-XingShi-Gan-Tang (MXSGT) decoction is a classic traditional Chinese herbal medicinal recipe of which ephedra is appointed as the n Correspondence to: No. 149, Jiaogong Road, Hangzhou, Zhejiang Province 310035, China. Tel.: þ 86 571 88071024; fax: þ 86 571 88056656. E-mail addresses: [email protected], [email protected], [email protected] (L. Ke).
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.12.029
principle ingredient. This formulae has more than two thousand years of medical application history, e.g. as a treatment for asthma, acute pneumonia in adults and juveniles (Yang et al., 2013). Besides the ephedra, there are three ingredients in the formulae: Radix Glycyrrhizae, Semen Armeniacae Amarum and gypsum. The ephedrine alkaloids are considered as the main active composition of ephedra, as amygdalin is for Semen Armeniacae Amarum and glycyrrhizin is for Radix Glycyrrhizae. The pharmacological studies indicate that ephedrine alkaloids are correlated to the increased blood pressure and circulation, central nervous system stimulation, which links to weight loss and enhanced physical performance (Schaneberg et al., 2003). Amygdalin showed anti-carcinogenic activities in vitro (Chen et al., 2013), but has been questioned about its clinical efficacy in cancer
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prevention and treatments. Glycyrrhizin showed hepato-protective (Ikeda et al., 2006), anti-inflammation (Akamatsu et al., 1991) and anti-carcinogenic activities (Niwa et al., 2007). As the key composition of principle ingredient of MXSGT decoction, ephedrine alkaloids provide pharmacological activities highly relevant to the therapeutic functions of decoction and therefore became the major subject of this study. Dietary supplements containing ephedra, ephedra extracts and ephedrine alkaloids were popularly used in the body weight loss and physical performance enhancement, until US FDA prohibited the sale in 2004 for its potential adverse effects (Pellati and Benvenuti, 2008), including cardiotoxic effects (Dunnick et al., 2007). In contrast, the use of Chinese herbal medicines containing ephedra (as it is in MXSGT decoction) rarely induced sever adverse effects, evidenced by two randomized double-blinded clinical trials (Boozer et al., 2002; Kim et al., 2013) reporting no significant adverse events from the ingestion of ephedra on healthy subjects during a six months study and an eight weeks study. The paradox remained in the safety assessment of ephedra and ephedrine warrants an explanation. As an aqueous herbal extraction, the decoction is a complex dispersion system containing molecules, colloids, aggregates, emulsions and precipitates (Zhuang et al., 2008). In what form and with whom the bioactive phytochemicals are dispersed determine their delivery, assimilation, acts and metabolism. For instance, when effectively modest ephedrine and caffeine are combined, the pharmacodynamics interactions produce significant cardiovascular, metabolic, and hormonal responses (Haller et al., 2004). Many analytical methods have been developed for the quantification of total ephedrine alkaloids in plant material and the aqueous extract of Ephedra sinica Stapf. (Okamura et al., 1999; Niemann and Gay, 2003; Schaneberg et al., 2003; Ganzera et al., 2005). In these studies, membrane filtration was often employed to remove precipitates from plant extracts. Even though, the filtrate remains a multi-dispersion system containing solutes and nano-scale colloids. However, little attention had been paid to the distribution pattern of ephedrine alkaloids in different dispersions, and thereafter the possible influence on their biological implications of these alkaloids. This study aims to analyze and elucidate the existing form of ephedrine in MXSGT decoction with liquid chromatographic approach. The bioactivities of full-body decoction and ephedrine in different dispersion were evaluated and compared on five cell lines in vitro.
2. Methods and materials 2.1. MXSGT decoction A daily MXSGT formulae is composed of 9 g Ephedra sinica Stapf. stem, sundried (Herba Ephedrae, Ma-Huang, ephedra), 6 g Glycyrrhiza uralensis Fisch., root and rhizome, honeyed (Radix Glycyrrhizae, Gan-Cao, licorice root), 9 g Prunus armeniaca L. var. ansu Maxim., seed, stir-fried (Semen Armeniacae Amarum, Ku-Xing-Ren, bitter apricot seed) and 24 g gypsum (Gypsum Fibrosum, Shi-Gao, CaSO4 U2 H2O), purchased from Beijing Shuangqiao Yanjing Medicinal Material Factory, Co., Ltd. and authenticated by Prof. Chengzi Yang from Fujian University of Traditional Chinese Medicine. Voucher specimens were deposited at the Museum of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, under the identification code: Herba Ephedrae (SQYJ-201005011NM1202), Radix Glycyrrhizae (SQYJ201106062NM1202), Semen Armeniacae Amarum (SQYJ-201111094HB 1202) and gypsum (SQYJ-201110044HB1202).
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Raw materials were soaked in distilled water (1:8, w/v) for 30 min at room temperature with stirring. The ephedra was boiled for 30 min prior to mixing with other three components. The whole mixture was subsequently boiled for another 30 min, cooled to room temperature and filtered through two layers of cotton gauze. 2.2. Size-Exclusion Gel Chromatography with Multi-Angle Laser Light Scattering (SEC-MALLS) The decoction was centrifuged at 400 g for 15 min. 2 mL of the supernatant was applied to a pre-equilibrated (0.02 M citrate buffer, pH 5.0) Sephacryl S-1000 column (1.0 cm 100 cm, Amersham Biosciences, USA) equipped with a HPLC system (BioCAD 700E, Applied Biosystems, US). The column was eluted with the citrate buffer at a flow rate of 0.34 mL/min. The eluates were continuously monitored with a UV detector and a multi-angle laser light scattering detector (MALLS, Dawn EOS, Wyatt Technology, CA) to obtain the absorbance at 280 nm and light scattering intensity at 690 nm. The fractions were collected every 20 min for further analysis. Fraction 11 and 12 were collected, freeze dried and reserved for the ephedrine content analysis and cell viability tests. Data obtained from MALLS were analyzed using Astra software (Version 5.3.4.20, Wyatt Technology, CA). The weight-average molecular mass (Mw) and z-average radius of gyration (Rz) were calculated using the following equations: Rθ ¼ M w PðθÞ 2A2 cM 2w P 2 ðθÞ Knc
ð1Þ
where Rθ is the excess Rayleigh ratio. P(θ) is the particle scattering factor, which is approximately equal to 1 2μ2 〈r 2 〉=3! þ :::, where μ ¼ ð4π =λÞ sin ðθ=2Þ. c is the concentration of the nanoparticles (g/mL). Mw is the weight-average molecular mass (g/mol). A2 is the second virial coefficient (mol mL/g2), and Kn is a constant. For vertically polarized incident light with a wavelength λ0 in vacuum, Kn is given by Kn ¼
4π 2 n20
λ40 NA
ðdn =dc Þ2
ð2Þ
where n0 is the refractive index of the solvent at wavelength λ0, NA is Avogadro0 s number and dn/dc is the specific refractive index increment of the fractions, which of 0.119 mL/g was used in this calculation. A plot of (Knc)/Rθ vs. sin2(θ/2) (Debye plot) was constructed at each retention time, which is the classical elaboration of light scattering data from which the molecular weight and RMS radius distributions are estimated. M can be directly determined from the intercept at zero angle, whereas, the slope yields the mean square radius. If the concentration is sufficiently small than A2c ¼0, and Eq. (1) is solved for A2 ¼ 0, than for the molecular weight and RMS radius one obtains the following equations: M¼
R0 Knc
and for〈r 2 〉1=2 pffiffiffiffiffiffiffiffi 3λ0 pffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 1=2 m0 M w 〈r 〉 ¼ 4π n0
ð3Þ
ð4Þ
2.3. Refractive index increment (dn/dc) of SEC fractions An interferometric refractometer (Optilab DSP, Wyatt Technology, USA) was used to determine the values of specific refractive index increment (dn/dc). The concentration of each fraction was calculated by subtracting the weight of buffering salt from the weight of freeze-
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dried fractions. The dn/dc values of SEC factions were determined by using serial concentrations (0, 0.322, 0.644, 0.970, 1.290, 1.610 and 1.930 mg/mL) in 0.02 M citrate buffer. The mean dn/dc values of four factions (fraction 11–14) was 0.119870.0015 mL/g. Consequently, 0.119 mL/g was used as a fixed dn/dc value for calculating the molar mass and radius of gyration (Rg) of particles. 2.4. Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM) AFM measurements were performed with the E-sweep/NanoNavi Station system (SII Nano Technology Inc.) in tapping mode. TEM measurements were performed according to the method described in (Bellare et al., 1988). 2.5. Reversed phase high performance liquid chromatograph (RP-HPLC) 20 μl of each fraction collected from SEC was subjected to a Daisogel-C18 column (15–20 μm, 300 Å, 46 250 mm, Vydac, Hesperia, CA) equipped in a HPLC system (BioCAD 700E, Applied Biosystems, US). The C18 column was equilibrated in buffer A (100% methanol) at 1.0 mL/min, eluted by 7.5% A þ92.5% B (0.01 mol/L KH2PO4, pH 2.5) for 10 min, followed by a 30 min linear gradient of buffer A from 7.5% to 100% (v/v). The absorbance of eluates was monitored at 207 nm. Ephedrine and pseudoephedrine (kindly provided by Fujian Institute for Drug Control, China) were weighed and dissolved in methanol–water solution (7.5% methanol þ92.5% 0.01 mol/L KH2PO4, pH 2.5) to serial concentrations (10.9, 21.9, 43.8, 87.5, 175.0 and 350.0 μg/mL). The standard curve was analyzed using
linear least-squares regression equation derived from the peak area. Concentrations of ephedrine and pseudoephedrine in samples were calculated from the regression analysis. Peaks were identified by comparing their retention time (tR) in UV spectra with those of standard compounds (ephedrine and pseudoephedrine). As a control, a corresponding amount (2 mL) of the MXSGT decoction was centrifuged at 400 g for 10 min, the supernatant was subjected to RP-HPLC analysis.
2.6. Cell culture, cytotoxicity and cellular viability Five cell lines with four different organ origins, i.e. human colon adenocarcinoma (Caco-2) cells, human hepatoblastoma (Hep-G2) cells, human cervical carcinoma (HeLa-229) cells, rat alveolar macrophage (NR8383) cells and human normal hepatocytes (L-02), were used to evaluate the influence of MXSGT decoction, ephedrine-associated NPs and ephedrine on the cellular viabilities. Among them, the first three are carcinoma cells while the other two are normal cells. The assayed sample concentrations were chosen according to the nontoxic dosages of the MXSGT decoction obtained in the earlier cytotoxicity tests, with respect to the realistic ephedrine content in the decoction. Samples were adjusted to the universal serial of ephedrine concentrations or its equivalence (6.37, 3.19, 1.59, 0.80, 0.40, 0.20, 0.10 μg/mL) to make results comparable. The highest equivalent concentration was well below the cytotoxic dosage of ephedrine reported (Lee et al., 2000). The lowest concentrations are close to the maximum plasma ephedrine concentrations of 63.5 ng/ml after the ingestion of dietary supplements containing ephedra alkaloids (Haller et al., 2002).
Fig. 1. MXSGT decoction was fractioned by a Sephacryl S-1000 column (1.0 cm 100 cm) equilibrated with 0.02 M Citrate buffer (pH 5.0). The column was eluted with the same buffer at a flow rate of 0.34 mL/min. The purple curve shows the elution profile of MXSGT decoction at 280 nm, the black curve shows the elution profile of the mixture of blue glucosan (peak 1, MW 2,000,000 Da) and potassium dichromate (peak 2, MW 294.18 Da), the blue curve shows the light scattering intensities at 901 of MXSGT extract fractions by means of multi-angel laser light scattering. The MW and RMS radius distributions of the first peak of UV chromatogram were also shown in the inset. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
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In brief, samples were added to the cells pre-cultured in 96-well plates, 4 duplicates for each concentration, and cultured at 37 1C, 5% CO2 for 48 h. The cellular viability was then assessed by cell proliferation (MTT assay) (Loosdrecht et al., 1994), and presented as the percentage of viable cells (Means7 SD, n ¼4) given by Viability ¼ ðmeanO:D:570nm_sample =meanO:D:570nm_control Þ100% ð5Þ The significant levels were examined with the Student0 s t-test and ranked as P o0.05 or P o0.01. 3. Results and discussion 3.1. Separation of colloidal nanoparticles from MXSGT decoction It has been well demonstrated that the particles from environments including nanoparticles (Kirkland, 1979; Siebrands et al., 1993; Wilcoxon et al., 2000), viruses and even micrometer-scale particles, e.g. bacterial cells (Liu et al., 2010), could be separated by chromatographic approach, most popularly the size exclusion chromatography (Kirkland, 1979; Siebrands et al., 1993). The MALLS detector was commonly used to analyze the molar mass and RMS radius of nanoparticles (Wang and Lucey, 2003). For the first time, the colloidal nanoparticles from the aqueous herbal extracts were separated and characterized by this SECMALLS approach. As is shown in Fig. 1, three finely separated peaks of OD 280 nm were observed in the UV chromatogram of SEC. Simultaneously, only one light scattering peak was observed with MALLS, which partially overlapped the first peak in UV chromatogram (P1). The strong light scattering intensity indicates the occurrence of particles. It also shows the separation of particleassembly components to other high molar mass components in P1. Furthermore, the overlapped peaks imply the possible participation of protein in the particle assembly. For further investigation, P1 was collected from four fractions (fraction 11, 12, 13 and 14). 3.2. Morphology of the separated colloidal nanoparticles The Mw and Rz of these four fractions were calculated by constructing Debye plot of each fraction. Debye plot examples of fraction 11, 12 and 13 were given in Fig. 2. The Mw distribution and RMS radius distributions of P1 were also shown in the inset of Fig. 1. Shown in Table 1, the Rz of fraction 11, 12 and 13 were 138.1, 101.7 and 65.3 nm, whose Mw were 4.49 108, 2.55 107 and 5.86 106 separately. The Rz of fractions 14 could not be calculated due to the poor light scattering intensity. The morphologic observations, revealed by AFM and SEM, showed the nanoparticles from fraction 11, 12 and 13 are spherical (as shown in Fig. 3a and b). 3.3. Analysis of ephedrine alkaloids content and distribution All the SEC fractions were subsequently subjected to RP-HPLC for analyzing their contents of ephedrine and pseudoephedrine. Their contents were linear to their peak areas, in a range of 10– 350 μg/mL. As a control, there were 243.47 2.8 μg of ephedrine and 158.77 2.4 μg of pseudoephedrine in 2 mL of the MXSGT decoction. As the result, ephedrine alkaloids were only detected in fraction 11 and 12 Fig. 4. Shown in Table 2, fraction 11 contained 39.3 μg of ephedrine and 31.3 μg of pseudoephedrine. Faction 12 contained 203.3 μg of ephedrine and 120.2 μg of pseudoephedrine. The sum of ephedrine and of pseudoephedrine in fraction 11 and 12 took 99.7% and 95.5% of their total content in the whole extract, respectively. The results indicate that the majority of
Fig. 2. Debye plot examples of fraction 11, 12 and 13 from SEC separation.
Table 1 The Mw and Rz of fraction 11, 12, 13 and 14. Fractions
Rz (nm)
Mw (g/mol)
dn/dc (mL/g)
11 12 13 14
138.1 101.7 65.3 ND
4.49 108 2.55 107 5.86 106 ND
0.119 0.119 0.119 0.119
ND: Not Detected
ephedrine alkaloids are associated with the NPs containing fractions, i.e. fraction 11 and 12. Since the SEC0 s separating power relies upon the difference in molecular mass of separates, it is unusual to see the ephedrine and pseudoephedrine, both are small molecule with the same molecular mass of 165.23, being eluted together with thousands times bigger nanoparticles. A reasonable explanation to this abnormal chromatographic behavior lies on the association of ephedrine/ pseudoephedrine with some NPs from the decoction. Nanoparticles have been observed in 86 aqueous herbal extracts with dynamic light scattering method and electron microscopy (Zhuang et al., 2008), although their pharmacological nature and formation mechanism remain unknown. Like the selfassociation of black tea polyphenols theaflavins and caffeine (Charlton et al., 2000), the formation of nanoparticles from MXSGT decoction may count on the amphipathic molecules driven by multiple forces, i.e. hydrophobic interaction, hydrogen bond, electrostatic interaction or Van der Waals attraction. Both of ephedrine and pseudoephedrine are amphipathic molecules, which could interact with other molecules via hydrophobic or ionic force to form nanoparticles. During the RP-HPLC separation, the MXSGT nanoparticles remained intact when adsorbed to hydrophobic resin. But later on, while the hydrophobic force was diminished by the increasing methanol concentration in eluent, the ephedrine and pseudoephedrine were released from the
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Fig. 3. AFM topography image collected in tapping mode (a) and TEM image (b) of fraction 12 from SEC separation, both showed spherical nanoparticles. In TEM, the image of particles was magnified by 100,000 times.
Table 2 The contents and recoveries of ephedrine and pseudoephedrine.
Ephedrine
Pseudo-ephedrine
2 mL MXSGT decoction (n¼ 3)
Fraction 11 (n ¼3)
Fraction 12 (n¼ 3)
Content (μg)
243.472.8
203.3 7 1.9
Recovery (%)
–
39.3 7 1.6 242.6 73.5 16.2 7 0.7 99.7 7 1.4
Content (μg)
158.7 72.4
Recovery (%)
–
31.3 7 3.1 151.5 7 6.8 19.7 7 2.0 95.5 7 4.3
82.7 7 0.8 120.2 7 3.7 75.4 7 2.3
cluster of other particles components and appeared in the elutes. This hypothesis is diagramed as Fig. 5.
3.4. Bioactivity assessment of colloidal nanoparticles The MXSGT decoction, ephedrine alkaloids loaded NPs (prepared by SEC separation) and the pure synthetic ephedrine were assessed for their bioactivities. In general, the decoction and the SEC separated NPs demonstrated significantly different impacts on the cell viability, as compared to the pure synthetic ephedrine solution (as shown in Fig. 6). The former two reserved higher cell viability than that of sole synthetic ephedrine on four kinds of cells, except on HeLa-229 cells. The decoction marginally increased the growth of Caco-2 cells and NR8383 cells by about 10%, while significantly promoted the proliferation of L-02 (42 75% as maximum) and Hep-G2 (667 7% as maximum) at the equivalent ephedrine concentration of 0.4 μg/ mL and above (P o0.01 as compared to blank control). Similarly, the SEC separated NPs (contains ephedrine/pseudoephedrine) only slightly affected the cytoviability of Caco-2 cells and NR8383 cells at an insignificant level (P 40.05 as compared to blank control). One notable difference, as compared to the decoction, is the NPs slightly suppressed cytoviability of NR8383 cell at 6.4 μg/mL and 3.2 μg/mL (P o0.01). The cytoviabilities of the hepatic cells were promoted by the NPs (P o0.01 as compared to bland control). Unlike the decoction0 s preference to Hep-G2 cells,
Fig. 4. Reversed phase chromatograms of MXSGT decoction (A), nanoparticles containing fraction 12 (B) and standard samples of ephedrine and pseudoephedrine (C). The Daisogel-C18 column was equilibrated in buffer A (100% methanol), eluted by 7.5% A þ 92.5% B (0.01 mol/L KH2PO4, pH 2.5) for 10 min, then by a linear gradient from 7.5% A to 100% A (v/v) in 30 min. The flow rate was 1.0 mL/min, detector absorbance wavelength was set at 207 nm.
the maximum percent viability caused by the NPs was on L-02 cells (517 8%) at 0.4 μg/mL. The pure ephedrine did not affect the cytoviability of Caco-2 cells, while inhibited the proliferation of L-02, Hep-G2 and NR8383 cells by 18 74%, 32 71% and 27 73% separately (Po 0.01 as compared to blank control). This is significantly different from the
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Fig. 5. Diagram of possible mechanism of ephedrine detaching from MXSGT nanoparticles.
Fig. 6. Influence of MXSGT decoction (◆), SEC isolated NPs, (■) and ephedrine (▴) an on the cellular viabilities of five cell lines. Cell lines: Caco-2, L-02, Hep-G2, NR-8383, Hela-229. All samples were adjusted to the same ephedrine concentrations or their equivalence. The data presented is the average of 4 duplicates (n¼4). The significant levels are indicated as “Δ” or “n” meaning significantly different (Po 0.05) as compared to the MXSGT decoction or ephedrine, respectively, when “ΔΔ” or “nn” (Po 0.01) meaning highly significant.
proliferation promoting effects of the NPs, particularly on L-02 cells and Hep-G2 cells (Fig. 6, P o0.01). Differed from the other four cell lines, some reverse effects were observed on HeLa-229 cells. The pure ephedrine promoted cell proliferation by about 80% to 90% at 0.4 μg/mL, 0.8 μg/mL and 1.6 μg/mL (P o0.01), while the decoction slightly but significantly inhibited the proliferation by about 15% (P o0.01). Interestingly, the ephedrine alkaloids-associated NPs significantly increased the
Hela-229 proliferation by 757 7% (P o0.01 as compared to blank control) at the equivalent ephedrine concentration of 1.6 μg/mL. When being diluted, NPs lost their proliferating activity significantly quicker than the pure ephedrine (P o0.01). Generally, the ephedrine/pseudoephedrine-associated NPs affected the cytoviability differently from the pure ephedrine, in a diminished (i.e. in L-02 and Hep-G2) even reversed manner (i.e. in HeLa-229). The pseudoephedrine, which is simultaneously
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associated with the NPs and accounts for about 40% of total ephedrine alkaloids content, may not cause the difference due to its similar bioactivity with ephedrine. If pseudoephedrine was the reason, it would have amplified the effects of ephedrine rather than diminished them. On the other hand, many other active phytochemicals (e.g. amygdalin and glycyrrhizin) have been kept away from ephedrine-associated NPs by SEC separation, which granted ephedrine alkaloids as the major actives. Combining these two reasons together, it implies the association of ephedrine alkaloids to NPs may have altered their native bioactivity, in likelihood with their chromatographic behavior changes in the SEC separation. Though the exact influences of other compositions of NPs would only be fully understood till every composition of the NPs have been identified and characterized. A similar inference was made on the tea polyphenols: Casein proteins (from bovine milk) completely suppressed the cardiovascular protective effects of tea, indicating the formation of polyphenol–protein complex reduced the antioxidant activity of polyphenols and affected their bioavailability (Lorenz et al., 2007; Bandyopadhyay et al., 2012). The unexpected profound proliferating activity of ephedrine on the human cervical carcinoma HeLa cells was rather a rare discovery, while other studies conducted on ephedrine-type alkaloids are mostly about their cytotoxicity, stimulant and metabolic effects, often at several orders of magnitude higher concentrations (Lee et al., 2000; Haller et al., 2004). Nevertheless, its growth-promoting effect on carcinoma cells in vitro does not necessarily indicate the carcinogenesis in vivo, evidenced by an animal feeding study found no carcinogenic effects of ephedrine sulfate (NTP, 1986). 3.5. Active component: single molecule or complex? To date, phytochemical and pharmacological studies on herbal extracts have led to the discovery of hundreds of bioactive compounds (Lee, 1999; Gong and Sucher, 1999), most of which emphasized on the individual molecules, e.g. ephedrine (Schaneberg et al., 2003). In contrast, the assembly or polymerization of active compounds was merely investigated, and their corresponding pharmacological impacts remained unidentified. Besides the MXSGT decoction, the naturally occurred nanoparticles or aggregates have been discovered in other Chinese herbal decoctions (Zhuang et al., 2008) and culinary soups (Ke et al., 2011). Some primary tests were performed for its biological activities (Hu et al., 2009). A paradox remains in the herbal medicine research is that the extracts/decoction of herb somehow demonstrate the significantly superior efficacy and safety to the purified single compound (Williamson, 2001). The simultaneous interactions among coexisting actives may be a reason behind, while the integrating of active compounds with nanoparticles and the subsequent changes on their pharmacological characteristics provide another promising approach to solve the puzzle. Furthermore, some herbal formula was far more effective than the constituent herb used alone (Scholey and Kennedy, 2002). Again, the key to the explanation may lie in the nanoparticle structures. More versatile properties can be expected from the nanostructures assembled by compositions from multiple ingredients than from a single one. From the nano-materials point of view, in comparison to the conventional NPs synthesized with metallic molecules or carbon, the MXSGT NPs have the relatively greater size and wider radius distribution, which is in a range of a few dozen to a few hundred nanometers. This is similar to the NPs assembled with polysaccharides or polypeptides (Tang et al., 2013). Due to the complex nature of decoction, MXSGT NPs are probably the assemblies of more than one kind of compounds rather than the homogenous composition of conventional NPs, e.g. gold (Daniel and Astruc,
2004), carbon (Magrez et al., 2006) or chitosan derivatives (Liu et al., 2008). Last but not the least, the NPs from Chinese herbal decoction are expected to be more stable and easier for medicinal applications than their synthetic counterparts while attracting much less safety concerns, because they survived the boiling process of decoction at the first place and have been consumed as medicine or even food for hundreds of years. Further work is warranted to elucidate the therapeutic significance of ephedrine alkaloids-associated nanoparticles. As indicated by the cytoviability tests, it is reasonable to predict some very different pharmacological and toxicological properties of ephedrine alkaloids in association with nanoparticles. Any efficacy and safety evaluations of ephedrine alkaloids focused only on their total content would be incomplete, or even misleading. Much more work is warranted to understand pharmacological implications of the self-assembled, phytochemical-associated nanostructures formed in the boiling process of herbal tonic, which may add the essential elements to the better understanding of complex systems like herbal medicines.
4. Conclusions In conclusion, the natural occurred colloidal NPs were successfully separated from the decoction of a classic Chinese herbal medicine (Ma-Xing-Shi-Gan-Tang) with liquid chromatographic approach. Ephedrine and pseudoephedrine have been demonstrated to be associated with the NPs, which may have conducted profound influences on the assimilation, delivery and bioactivities of the alkaloids. Our approaches and findings may inspire the studies on the interaction between active phytochemicals and colloidal nanoparticles in herbal decoctions, and the corresponding pharmacological implications.
Acknowledgments This research was supported by the National Basic Research Program of China (Grant no. 2010CB530605) and the Natural Science Foundation of Fujian Province of China (Nos. 2012J05057 and 2012J01133). References Akamatsu, H., Komura, J., Asada, Y., Niwa, Y., 1991. Mechanism of anti-inflammatory action of glycyrrhizin: effect on neutrophil functions including reactive oxygen species generation. Planta Med. 57, 119–121. Bandyopadhyay, P., Ghosh, A.K., Ghosh, C., 2012. Recent developments on polyphenol–protein interactions: effects on tea and coffee taste, antioxidant properties and the digestive system. Food Funct. 3, 592–605. Bellare, J.R., Davis, H.T., Scriven, L.E., Talmon, Y., 1988. Controlled environment vitrification system: an improved sample preparation technique. J. Electron Microsc. Tech. 10, 87–111. Bensk, D., Clavey, S., Erich, S., Andrew, G., Bensky, L.L., 2004. Chinese Herbal Medicine: Materia Medica, 3rd ed. Eastland Press, Washington. Boozer, C.N., Daly, P.A., Homel, P., Solomon, J.L., Blanchard, D., Nasser, J.A., Strauss, R., Meredith, T., 2002. Herbal ephedra/caffeine for weight loss: a 6-month randomized safety and efficacy trial. Int. J. Obes., 593–604. Charlton, A.J., Davis, A.L., Jones, D.P., Lewis, J.R., Davies, A.P., Haslam, E., Williamson, M.P., 2000. The self-association of the black tea polyphenol theaflavin and its complexation with caffeine. J. Chem. Soc. Perkin Trans. 2, 317–322. Chen, Y., Ma, J., Wang, F., Hu, J., Cui, A., Wei, C., Yang, Q., Li, F., 2013. Amygdalin induces apoptosis in human cervical cancer cell line HeLa cells. Immunopharmacol. Immunotoxicol. 35, 43–51. Daniel, M.-C., Astruc, D., 2004. Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem. Rev. 104, 293–346. Dunnick, J.K., Kissling, G., Gerken, D.K., Vallant, M.A., Nyska, A., 2007. Cardiotoxicity of Ma Huang/caffeine or ephedrine/caffeine in a rodent model system. Toxicol. Pathol. 35, 657–664. Ganzera, M., Lanser, C., Stuppner, H., 2005. Simultaneous determination of Ephedra sinica and Citrus aurantium var. amara alkaloids by ion-pair chromatography. Talanta 66, 889–894.
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Glossary MXSGT: Ma-Xing-Shi-Gan-Tang decoction; SEC-MALLS: Size-Exclusion Chromatography with Multi-angle Laser Light Scattering; AFM: Atomic Force Microscopy; TEM: Transmission Electron Microscope; SEM: Scanning Electron Microscope; NPs: Nanoparticles.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Chromatographic isolation of nanoparticles from Ma-Xing-Shi-Gan-Tang decoction and their characterization Jianwu Zhou a,b, Guanzhen Gao a,b, Qiuping Chu b, Huiqin Wang a,b, Pingfan Rao a,b, Lijing Ke a,b,n a b
CAS.SIBS-Zhejiang Gongshang University Joint Centre for Food and Nutrition Sciences, Zhejiang Gongshang University, Hangzhou 310035, China Institute of Biotechnology, Fuzhou University, Fuzhou 350002, China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 June 2013 Received in revised form 9 December 2013 Accepted 18 December 2013 Available online 30 December 2013
Ethnopharmacological relevance: The herbal decoction is a complex dispersion system containing solutes, colloid, aggregates, emulsions and precipitates. In which phase bioactive phytochemicals are dispersed determines their delivery, action and metabolism. This study took ephedrine, a well-studied and widely used phytochemical, as an example to elucidate its exact distribution in the phases of Ma-Xing-ShiGan-Tang decoction (MXSGT), which is an Ephedra sinica Stapf. containing traditional Chinese medicinal formula, and the biological meaning of this distribution correspondingly. It may provide an important update to the safety and efficacy assessment of the herbal decoction and its active phytochemicals. Materials and methods: In this study, the decoction was fractionated with size-exclusion chromatography coupled with multi-angle laser light scattering detector. The morphology of fractionated nanoparticles was observed with AFM and SEM. The bioactivities of the decoction, the ephedrine alkaloids loaded NPs (prepared by chromatography isolation) and the synthetic ephedrine were assessed by cell proliferation tests using five cell lines, namely Caco-2, L-02, Hep-G2, NR-8383, and Hela-229. Results: Nanoparticles with radii of gyration ranged from 50 to 150 nm were isolated, in spherical shape. Further analysis of nanoparticles on the subsequent reversed phase chromatography revealed that the majority of ephedrine (99.7%) and pseudoephedrine (95.5%) were associated with these nanoparticles, rather than dispersed freely in the real solution. The addition of both the herbal decoction and the separated ephedrine-loaded nanoparticles reserved higher cell viability/proliferation than that of the sole synthetic ephedrine among the Caco-2, L-02, Hep-G2, and NR-8383 cells. In contrast, the nanoparticles reduced the proliferating power of ephedrine on Hela-229 cells. In general, the ephedrineloaded NPs conducted the intermediate influences on the cell viability, in either way. Conclusions: The colloidal nanoparticles were separated from the decoction. The association of ephedrine alkaloids with nanoparticles was demonstrated and may have changed the bioactivity of the alkaloids. The naturally occurred colloidal nanoparticles may play an important role in the pharmacological properties of both the decoction and its active phytochemicals, therefore warrant further studies. & 2013 Elsevier Ireland Ltd. All rights reserved.
Chemical compounds studied in this article: Ephedrine (PubChem CID: 9294) Pseudoephedrine (PubChem CID: 62946) Keywords: Nanoparticles Size-exclusion gel chromatography Ephedrine Pseudoephedrine Ma-Xing-Shi-Gan-Tang decoction Cell proliferation
1. Introduction The Ephedra sinica Stapf. stem, sundried (Herba Ephedrae, Ma-Huang, ephedra) is widely used in traditional Chinese medicines and Japanese Kampo medicines (Okamura et al., 1999) for the treatment of asthma, fever, coughs, lack of sweating, urination promotion and alleviating edema (Bensk et al., 2004). Ma-XingShi-Gan-Tang (MXSGT) decoction is a classic traditional Chinese herbal medicinal recipe of which ephedra is appointed as the n Correspondence to: No. 149, Jiaogong Road, Hangzhou, Zhejiang Province 310035, China. Tel.: þ 86 571 88071024; fax: þ 86 571 88056656. E-mail addresses: [email protected], [email protected], [email protected] (L. Ke).
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.12.029
principle ingredient. This formulae has more than two thousand years of medical application history, e.g. as a treatment for asthma, acute pneumonia in adults and juveniles (Yang et al., 2013). Besides the ephedra, there are three ingredients in the formulae: Radix Glycyrrhizae, Semen Armeniacae Amarum and gypsum. The ephedrine alkaloids are considered as the main active composition of ephedra, as amygdalin is for Semen Armeniacae Amarum and glycyrrhizin is for Radix Glycyrrhizae. The pharmacological studies indicate that ephedrine alkaloids are correlated to the increased blood pressure and circulation, central nervous system stimulation, which links to weight loss and enhanced physical performance (Schaneberg et al., 2003). Amygdalin showed anti-carcinogenic activities in vitro (Chen et al., 2013), but has been questioned about its clinical efficacy in cancer
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prevention and treatments. Glycyrrhizin showed hepato-protective (Ikeda et al., 2006), anti-inflammation (Akamatsu et al., 1991) and anti-carcinogenic activities (Niwa et al., 2007). As the key composition of principle ingredient of MXSGT decoction, ephedrine alkaloids provide pharmacological activities highly relevant to the therapeutic functions of decoction and therefore became the major subject of this study. Dietary supplements containing ephedra, ephedra extracts and ephedrine alkaloids were popularly used in the body weight loss and physical performance enhancement, until US FDA prohibited the sale in 2004 for its potential adverse effects (Pellati and Benvenuti, 2008), including cardiotoxic effects (Dunnick et al., 2007). In contrast, the use of Chinese herbal medicines containing ephedra (as it is in MXSGT decoction) rarely induced sever adverse effects, evidenced by two randomized double-blinded clinical trials (Boozer et al., 2002; Kim et al., 2013) reporting no significant adverse events from the ingestion of ephedra on healthy subjects during a six months study and an eight weeks study. The paradox remained in the safety assessment of ephedra and ephedrine warrants an explanation. As an aqueous herbal extraction, the decoction is a complex dispersion system containing molecules, colloids, aggregates, emulsions and precipitates (Zhuang et al., 2008). In what form and with whom the bioactive phytochemicals are dispersed determine their delivery, assimilation, acts and metabolism. For instance, when effectively modest ephedrine and caffeine are combined, the pharmacodynamics interactions produce significant cardiovascular, metabolic, and hormonal responses (Haller et al., 2004). Many analytical methods have been developed for the quantification of total ephedrine alkaloids in plant material and the aqueous extract of Ephedra sinica Stapf. (Okamura et al., 1999; Niemann and Gay, 2003; Schaneberg et al., 2003; Ganzera et al., 2005). In these studies, membrane filtration was often employed to remove precipitates from plant extracts. Even though, the filtrate remains a multi-dispersion system containing solutes and nano-scale colloids. However, little attention had been paid to the distribution pattern of ephedrine alkaloids in different dispersions, and thereafter the possible influence on their biological implications of these alkaloids. This study aims to analyze and elucidate the existing form of ephedrine in MXSGT decoction with liquid chromatographic approach. The bioactivities of full-body decoction and ephedrine in different dispersion were evaluated and compared on five cell lines in vitro.
2. Methods and materials 2.1. MXSGT decoction A daily MXSGT formulae is composed of 9 g Ephedra sinica Stapf. stem, sundried (Herba Ephedrae, Ma-Huang, ephedra), 6 g Glycyrrhiza uralensis Fisch., root and rhizome, honeyed (Radix Glycyrrhizae, Gan-Cao, licorice root), 9 g Prunus armeniaca L. var. ansu Maxim., seed, stir-fried (Semen Armeniacae Amarum, Ku-Xing-Ren, bitter apricot seed) and 24 g gypsum (Gypsum Fibrosum, Shi-Gao, CaSO4 U2 H2O), purchased from Beijing Shuangqiao Yanjing Medicinal Material Factory, Co., Ltd. and authenticated by Prof. Chengzi Yang from Fujian University of Traditional Chinese Medicine. Voucher specimens were deposited at the Museum of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, under the identification code: Herba Ephedrae (SQYJ-201005011NM1202), Radix Glycyrrhizae (SQYJ201106062NM1202), Semen Armeniacae Amarum (SQYJ-201111094HB 1202) and gypsum (SQYJ-201110044HB1202).
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Raw materials were soaked in distilled water (1:8, w/v) for 30 min at room temperature with stirring. The ephedra was boiled for 30 min prior to mixing with other three components. The whole mixture was subsequently boiled for another 30 min, cooled to room temperature and filtered through two layers of cotton gauze. 2.2. Size-Exclusion Gel Chromatography with Multi-Angle Laser Light Scattering (SEC-MALLS) The decoction was centrifuged at 400 g for 15 min. 2 mL of the supernatant was applied to a pre-equilibrated (0.02 M citrate buffer, pH 5.0) Sephacryl S-1000 column (1.0 cm 100 cm, Amersham Biosciences, USA) equipped with a HPLC system (BioCAD 700E, Applied Biosystems, US). The column was eluted with the citrate buffer at a flow rate of 0.34 mL/min. The eluates were continuously monitored with a UV detector and a multi-angle laser light scattering detector (MALLS, Dawn EOS, Wyatt Technology, CA) to obtain the absorbance at 280 nm and light scattering intensity at 690 nm. The fractions were collected every 20 min for further analysis. Fraction 11 and 12 were collected, freeze dried and reserved for the ephedrine content analysis and cell viability tests. Data obtained from MALLS were analyzed using Astra software (Version 5.3.4.20, Wyatt Technology, CA). The weight-average molecular mass (Mw) and z-average radius of gyration (Rz) were calculated using the following equations: Rθ ¼ M w PðθÞ 2A2 cM 2w P 2 ðθÞ Knc
ð1Þ
where Rθ is the excess Rayleigh ratio. P(θ) is the particle scattering factor, which is approximately equal to 1 2μ2 〈r 2 〉=3! þ :::, where μ ¼ ð4π =λÞ sin ðθ=2Þ. c is the concentration of the nanoparticles (g/mL). Mw is the weight-average molecular mass (g/mol). A2 is the second virial coefficient (mol mL/g2), and Kn is a constant. For vertically polarized incident light with a wavelength λ0 in vacuum, Kn is given by Kn ¼
4π 2 n20
λ40 NA
ðdn =dc Þ2
ð2Þ
where n0 is the refractive index of the solvent at wavelength λ0, NA is Avogadro0 s number and dn/dc is the specific refractive index increment of the fractions, which of 0.119 mL/g was used in this calculation. A plot of (Knc)/Rθ vs. sin2(θ/2) (Debye plot) was constructed at each retention time, which is the classical elaboration of light scattering data from which the molecular weight and RMS radius distributions are estimated. M can be directly determined from the intercept at zero angle, whereas, the slope yields the mean square radius. If the concentration is sufficiently small than A2c ¼0, and Eq. (1) is solved for A2 ¼ 0, than for the molecular weight and RMS radius one obtains the following equations: M¼
R0 Knc
and for〈r 2 〉1=2 pffiffiffiffiffiffiffiffi 3λ0 pffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 1=2 m0 M w 〈r 〉 ¼ 4π n0
ð3Þ
ð4Þ
2.3. Refractive index increment (dn/dc) of SEC fractions An interferometric refractometer (Optilab DSP, Wyatt Technology, USA) was used to determine the values of specific refractive index increment (dn/dc). The concentration of each fraction was calculated by subtracting the weight of buffering salt from the weight of freeze-
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dried fractions. The dn/dc values of SEC factions were determined by using serial concentrations (0, 0.322, 0.644, 0.970, 1.290, 1.610 and 1.930 mg/mL) in 0.02 M citrate buffer. The mean dn/dc values of four factions (fraction 11–14) was 0.119870.0015 mL/g. Consequently, 0.119 mL/g was used as a fixed dn/dc value for calculating the molar mass and radius of gyration (Rg) of particles. 2.4. Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM) AFM measurements were performed with the E-sweep/NanoNavi Station system (SII Nano Technology Inc.) in tapping mode. TEM measurements were performed according to the method described in (Bellare et al., 1988). 2.5. Reversed phase high performance liquid chromatograph (RP-HPLC) 20 μl of each fraction collected from SEC was subjected to a Daisogel-C18 column (15–20 μm, 300 Å, 46 250 mm, Vydac, Hesperia, CA) equipped in a HPLC system (BioCAD 700E, Applied Biosystems, US). The C18 column was equilibrated in buffer A (100% methanol) at 1.0 mL/min, eluted by 7.5% A þ92.5% B (0.01 mol/L KH2PO4, pH 2.5) for 10 min, followed by a 30 min linear gradient of buffer A from 7.5% to 100% (v/v). The absorbance of eluates was monitored at 207 nm. Ephedrine and pseudoephedrine (kindly provided by Fujian Institute for Drug Control, China) were weighed and dissolved in methanol–water solution (7.5% methanol þ92.5% 0.01 mol/L KH2PO4, pH 2.5) to serial concentrations (10.9, 21.9, 43.8, 87.5, 175.0 and 350.0 μg/mL). The standard curve was analyzed using
linear least-squares regression equation derived from the peak area. Concentrations of ephedrine and pseudoephedrine in samples were calculated from the regression analysis. Peaks were identified by comparing their retention time (tR) in UV spectra with those of standard compounds (ephedrine and pseudoephedrine). As a control, a corresponding amount (2 mL) of the MXSGT decoction was centrifuged at 400 g for 10 min, the supernatant was subjected to RP-HPLC analysis.
2.6. Cell culture, cytotoxicity and cellular viability Five cell lines with four different organ origins, i.e. human colon adenocarcinoma (Caco-2) cells, human hepatoblastoma (Hep-G2) cells, human cervical carcinoma (HeLa-229) cells, rat alveolar macrophage (NR8383) cells and human normal hepatocytes (L-02), were used to evaluate the influence of MXSGT decoction, ephedrine-associated NPs and ephedrine on the cellular viabilities. Among them, the first three are carcinoma cells while the other two are normal cells. The assayed sample concentrations were chosen according to the nontoxic dosages of the MXSGT decoction obtained in the earlier cytotoxicity tests, with respect to the realistic ephedrine content in the decoction. Samples were adjusted to the universal serial of ephedrine concentrations or its equivalence (6.37, 3.19, 1.59, 0.80, 0.40, 0.20, 0.10 μg/mL) to make results comparable. The highest equivalent concentration was well below the cytotoxic dosage of ephedrine reported (Lee et al., 2000). The lowest concentrations are close to the maximum plasma ephedrine concentrations of 63.5 ng/ml after the ingestion of dietary supplements containing ephedra alkaloids (Haller et al., 2002).
Fig. 1. MXSGT decoction was fractioned by a Sephacryl S-1000 column (1.0 cm 100 cm) equilibrated with 0.02 M Citrate buffer (pH 5.0). The column was eluted with the same buffer at a flow rate of 0.34 mL/min. The purple curve shows the elution profile of MXSGT decoction at 280 nm, the black curve shows the elution profile of the mixture of blue glucosan (peak 1, MW 2,000,000 Da) and potassium dichromate (peak 2, MW 294.18 Da), the blue curve shows the light scattering intensities at 901 of MXSGT extract fractions by means of multi-angel laser light scattering. The MW and RMS radius distributions of the first peak of UV chromatogram were also shown in the inset. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
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In brief, samples were added to the cells pre-cultured in 96-well plates, 4 duplicates for each concentration, and cultured at 37 1C, 5% CO2 for 48 h. The cellular viability was then assessed by cell proliferation (MTT assay) (Loosdrecht et al., 1994), and presented as the percentage of viable cells (Means7 SD, n ¼4) given by Viability ¼ ðmeanO:D:570nm_sample =meanO:D:570nm_control Þ100% ð5Þ The significant levels were examined with the Student0 s t-test and ranked as P o0.05 or P o0.01. 3. Results and discussion 3.1. Separation of colloidal nanoparticles from MXSGT decoction It has been well demonstrated that the particles from environments including nanoparticles (Kirkland, 1979; Siebrands et al., 1993; Wilcoxon et al., 2000), viruses and even micrometer-scale particles, e.g. bacterial cells (Liu et al., 2010), could be separated by chromatographic approach, most popularly the size exclusion chromatography (Kirkland, 1979; Siebrands et al., 1993). The MALLS detector was commonly used to analyze the molar mass and RMS radius of nanoparticles (Wang and Lucey, 2003). For the first time, the colloidal nanoparticles from the aqueous herbal extracts were separated and characterized by this SECMALLS approach. As is shown in Fig. 1, three finely separated peaks of OD 280 nm were observed in the UV chromatogram of SEC. Simultaneously, only one light scattering peak was observed with MALLS, which partially overlapped the first peak in UV chromatogram (P1). The strong light scattering intensity indicates the occurrence of particles. It also shows the separation of particleassembly components to other high molar mass components in P1. Furthermore, the overlapped peaks imply the possible participation of protein in the particle assembly. For further investigation, P1 was collected from four fractions (fraction 11, 12, 13 and 14). 3.2. Morphology of the separated colloidal nanoparticles The Mw and Rz of these four fractions were calculated by constructing Debye plot of each fraction. Debye plot examples of fraction 11, 12 and 13 were given in Fig. 2. The Mw distribution and RMS radius distributions of P1 were also shown in the inset of Fig. 1. Shown in Table 1, the Rz of fraction 11, 12 and 13 were 138.1, 101.7 and 65.3 nm, whose Mw were 4.49 108, 2.55 107 and 5.86 106 separately. The Rz of fractions 14 could not be calculated due to the poor light scattering intensity. The morphologic observations, revealed by AFM and SEM, showed the nanoparticles from fraction 11, 12 and 13 are spherical (as shown in Fig. 3a and b). 3.3. Analysis of ephedrine alkaloids content and distribution All the SEC fractions were subsequently subjected to RP-HPLC for analyzing their contents of ephedrine and pseudoephedrine. Their contents were linear to their peak areas, in a range of 10– 350 μg/mL. As a control, there were 243.47 2.8 μg of ephedrine and 158.77 2.4 μg of pseudoephedrine in 2 mL of the MXSGT decoction. As the result, ephedrine alkaloids were only detected in fraction 11 and 12 Fig. 4. Shown in Table 2, fraction 11 contained 39.3 μg of ephedrine and 31.3 μg of pseudoephedrine. Faction 12 contained 203.3 μg of ephedrine and 120.2 μg of pseudoephedrine. The sum of ephedrine and of pseudoephedrine in fraction 11 and 12 took 99.7% and 95.5% of their total content in the whole extract, respectively. The results indicate that the majority of
Fig. 2. Debye plot examples of fraction 11, 12 and 13 from SEC separation.
Table 1 The Mw and Rz of fraction 11, 12, 13 and 14. Fractions
Rz (nm)
Mw (g/mol)
dn/dc (mL/g)
11 12 13 14
138.1 101.7 65.3 ND
4.49 108 2.55 107 5.86 106 ND
0.119 0.119 0.119 0.119
ND: Not Detected
ephedrine alkaloids are associated with the NPs containing fractions, i.e. fraction 11 and 12. Since the SEC0 s separating power relies upon the difference in molecular mass of separates, it is unusual to see the ephedrine and pseudoephedrine, both are small molecule with the same molecular mass of 165.23, being eluted together with thousands times bigger nanoparticles. A reasonable explanation to this abnormal chromatographic behavior lies on the association of ephedrine/ pseudoephedrine with some NPs from the decoction. Nanoparticles have been observed in 86 aqueous herbal extracts with dynamic light scattering method and electron microscopy (Zhuang et al., 2008), although their pharmacological nature and formation mechanism remain unknown. Like the selfassociation of black tea polyphenols theaflavins and caffeine (Charlton et al., 2000), the formation of nanoparticles from MXSGT decoction may count on the amphipathic molecules driven by multiple forces, i.e. hydrophobic interaction, hydrogen bond, electrostatic interaction or Van der Waals attraction. Both of ephedrine and pseudoephedrine are amphipathic molecules, which could interact with other molecules via hydrophobic or ionic force to form nanoparticles. During the RP-HPLC separation, the MXSGT nanoparticles remained intact when adsorbed to hydrophobic resin. But later on, while the hydrophobic force was diminished by the increasing methanol concentration in eluent, the ephedrine and pseudoephedrine were released from the
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Fig. 3. AFM topography image collected in tapping mode (a) and TEM image (b) of fraction 12 from SEC separation, both showed spherical nanoparticles. In TEM, the image of particles was magnified by 100,000 times.
Table 2 The contents and recoveries of ephedrine and pseudoephedrine.
Ephedrine
Pseudo-ephedrine
2 mL MXSGT decoction (n¼ 3)
Fraction 11 (n ¼3)
Fraction 12 (n¼ 3)
Content (μg)
243.472.8
203.3 7 1.9
Recovery (%)
–
39.3 7 1.6 242.6 73.5 16.2 7 0.7 99.7 7 1.4
Content (μg)
158.7 72.4
Recovery (%)
–
31.3 7 3.1 151.5 7 6.8 19.7 7 2.0 95.5 7 4.3
82.7 7 0.8 120.2 7 3.7 75.4 7 2.3
cluster of other particles components and appeared in the elutes. This hypothesis is diagramed as Fig. 5.
3.4. Bioactivity assessment of colloidal nanoparticles The MXSGT decoction, ephedrine alkaloids loaded NPs (prepared by SEC separation) and the pure synthetic ephedrine were assessed for their bioactivities. In general, the decoction and the SEC separated NPs demonstrated significantly different impacts on the cell viability, as compared to the pure synthetic ephedrine solution (as shown in Fig. 6). The former two reserved higher cell viability than that of sole synthetic ephedrine on four kinds of cells, except on HeLa-229 cells. The decoction marginally increased the growth of Caco-2 cells and NR8383 cells by about 10%, while significantly promoted the proliferation of L-02 (42 75% as maximum) and Hep-G2 (667 7% as maximum) at the equivalent ephedrine concentration of 0.4 μg/ mL and above (P o0.01 as compared to blank control). Similarly, the SEC separated NPs (contains ephedrine/pseudoephedrine) only slightly affected the cytoviability of Caco-2 cells and NR8383 cells at an insignificant level (P 40.05 as compared to blank control). One notable difference, as compared to the decoction, is the NPs slightly suppressed cytoviability of NR8383 cell at 6.4 μg/mL and 3.2 μg/mL (P o0.01). The cytoviabilities of the hepatic cells were promoted by the NPs (P o0.01 as compared to bland control). Unlike the decoction0 s preference to Hep-G2 cells,
Fig. 4. Reversed phase chromatograms of MXSGT decoction (A), nanoparticles containing fraction 12 (B) and standard samples of ephedrine and pseudoephedrine (C). The Daisogel-C18 column was equilibrated in buffer A (100% methanol), eluted by 7.5% A þ 92.5% B (0.01 mol/L KH2PO4, pH 2.5) for 10 min, then by a linear gradient from 7.5% A to 100% A (v/v) in 30 min. The flow rate was 1.0 mL/min, detector absorbance wavelength was set at 207 nm.
the maximum percent viability caused by the NPs was on L-02 cells (517 8%) at 0.4 μg/mL. The pure ephedrine did not affect the cytoviability of Caco-2 cells, while inhibited the proliferation of L-02, Hep-G2 and NR8383 cells by 18 74%, 32 71% and 27 73% separately (Po 0.01 as compared to blank control). This is significantly different from the
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Fig. 5. Diagram of possible mechanism of ephedrine detaching from MXSGT nanoparticles.
Fig. 6. Influence of MXSGT decoction (◆), SEC isolated NPs, (■) and ephedrine (▴) an on the cellular viabilities of five cell lines. Cell lines: Caco-2, L-02, Hep-G2, NR-8383, Hela-229. All samples were adjusted to the same ephedrine concentrations or their equivalence. The data presented is the average of 4 duplicates (n¼4). The significant levels are indicated as “Δ” or “n” meaning significantly different (Po 0.05) as compared to the MXSGT decoction or ephedrine, respectively, when “ΔΔ” or “nn” (Po 0.01) meaning highly significant.
proliferation promoting effects of the NPs, particularly on L-02 cells and Hep-G2 cells (Fig. 6, P o0.01). Differed from the other four cell lines, some reverse effects were observed on HeLa-229 cells. The pure ephedrine promoted cell proliferation by about 80% to 90% at 0.4 μg/mL, 0.8 μg/mL and 1.6 μg/mL (P o0.01), while the decoction slightly but significantly inhibited the proliferation by about 15% (P o0.01). Interestingly, the ephedrine alkaloids-associated NPs significantly increased the
Hela-229 proliferation by 757 7% (P o0.01 as compared to blank control) at the equivalent ephedrine concentration of 1.6 μg/mL. When being diluted, NPs lost their proliferating activity significantly quicker than the pure ephedrine (P o0.01). Generally, the ephedrine/pseudoephedrine-associated NPs affected the cytoviability differently from the pure ephedrine, in a diminished (i.e. in L-02 and Hep-G2) even reversed manner (i.e. in HeLa-229). The pseudoephedrine, which is simultaneously
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associated with the NPs and accounts for about 40% of total ephedrine alkaloids content, may not cause the difference due to its similar bioactivity with ephedrine. If pseudoephedrine was the reason, it would have amplified the effects of ephedrine rather than diminished them. On the other hand, many other active phytochemicals (e.g. amygdalin and glycyrrhizin) have been kept away from ephedrine-associated NPs by SEC separation, which granted ephedrine alkaloids as the major actives. Combining these two reasons together, it implies the association of ephedrine alkaloids to NPs may have altered their native bioactivity, in likelihood with their chromatographic behavior changes in the SEC separation. Though the exact influences of other compositions of NPs would only be fully understood till every composition of the NPs have been identified and characterized. A similar inference was made on the tea polyphenols: Casein proteins (from bovine milk) completely suppressed the cardiovascular protective effects of tea, indicating the formation of polyphenol–protein complex reduced the antioxidant activity of polyphenols and affected their bioavailability (Lorenz et al., 2007; Bandyopadhyay et al., 2012). The unexpected profound proliferating activity of ephedrine on the human cervical carcinoma HeLa cells was rather a rare discovery, while other studies conducted on ephedrine-type alkaloids are mostly about their cytotoxicity, stimulant and metabolic effects, often at several orders of magnitude higher concentrations (Lee et al., 2000; Haller et al., 2004). Nevertheless, its growth-promoting effect on carcinoma cells in vitro does not necessarily indicate the carcinogenesis in vivo, evidenced by an animal feeding study found no carcinogenic effects of ephedrine sulfate (NTP, 1986). 3.5. Active component: single molecule or complex? To date, phytochemical and pharmacological studies on herbal extracts have led to the discovery of hundreds of bioactive compounds (Lee, 1999; Gong and Sucher, 1999), most of which emphasized on the individual molecules, e.g. ephedrine (Schaneberg et al., 2003). In contrast, the assembly or polymerization of active compounds was merely investigated, and their corresponding pharmacological impacts remained unidentified. Besides the MXSGT decoction, the naturally occurred nanoparticles or aggregates have been discovered in other Chinese herbal decoctions (Zhuang et al., 2008) and culinary soups (Ke et al., 2011). Some primary tests were performed for its biological activities (Hu et al., 2009). A paradox remains in the herbal medicine research is that the extracts/decoction of herb somehow demonstrate the significantly superior efficacy and safety to the purified single compound (Williamson, 2001). The simultaneous interactions among coexisting actives may be a reason behind, while the integrating of active compounds with nanoparticles and the subsequent changes on their pharmacological characteristics provide another promising approach to solve the puzzle. Furthermore, some herbal formula was far more effective than the constituent herb used alone (Scholey and Kennedy, 2002). Again, the key to the explanation may lie in the nanoparticle structures. More versatile properties can be expected from the nanostructures assembled by compositions from multiple ingredients than from a single one. From the nano-materials point of view, in comparison to the conventional NPs synthesized with metallic molecules or carbon, the MXSGT NPs have the relatively greater size and wider radius distribution, which is in a range of a few dozen to a few hundred nanometers. This is similar to the NPs assembled with polysaccharides or polypeptides (Tang et al., 2013). Due to the complex nature of decoction, MXSGT NPs are probably the assemblies of more than one kind of compounds rather than the homogenous composition of conventional NPs, e.g. gold (Daniel and Astruc,
2004), carbon (Magrez et al., 2006) or chitosan derivatives (Liu et al., 2008). Last but not the least, the NPs from Chinese herbal decoction are expected to be more stable and easier for medicinal applications than their synthetic counterparts while attracting much less safety concerns, because they survived the boiling process of decoction at the first place and have been consumed as medicine or even food for hundreds of years. Further work is warranted to elucidate the therapeutic significance of ephedrine alkaloids-associated nanoparticles. As indicated by the cytoviability tests, it is reasonable to predict some very different pharmacological and toxicological properties of ephedrine alkaloids in association with nanoparticles. Any efficacy and safety evaluations of ephedrine alkaloids focused only on their total content would be incomplete, or even misleading. Much more work is warranted to understand pharmacological implications of the self-assembled, phytochemical-associated nanostructures formed in the boiling process of herbal tonic, which may add the essential elements to the better understanding of complex systems like herbal medicines.
4. Conclusions In conclusion, the natural occurred colloidal NPs were successfully separated from the decoction of a classic Chinese herbal medicine (Ma-Xing-Shi-Gan-Tang) with liquid chromatographic approach. Ephedrine and pseudoephedrine have been demonstrated to be associated with the NPs, which may have conducted profound influences on the assimilation, delivery and bioactivities of the alkaloids. Our approaches and findings may inspire the studies on the interaction between active phytochemicals and colloidal nanoparticles in herbal decoctions, and the corresponding pharmacological implications.
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Glossary MXSGT: Ma-Xing-Shi-Gan-Tang decoction; SEC-MALLS: Size-Exclusion Chromatography with Multi-angle Laser Light Scattering; AFM: Atomic Force Microscopy; TEM: Transmission Electron Microscope; SEM: Scanning Electron Microscope; NPs: Nanoparticles.