Chemical and biological assessment of Jujube (Ziziphus jujuba)-containing herbal decoctions: Induction of erythropoietin expression in cultures

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Chemical and biological assessment of Jujube (Ziziphus jujuba)-containing herbal decoctions: Induction of erythropoietin expression in cultures Candy T.W. Lam, Pui H. Chan, Pinky S.C. Lee, Kei M. Lau, Ava Y.Y. Kong, Amy G.W. Gong, Miranda L. Xu, Kelly Y.C. Lam, Tina T.X. Dong, Huangquan Lin, Karl W.K. Tsim ∗ Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Hong Kong, China

a r t i c l e

i n f o

Article history: Received 12 May 2015 Received in revised form 27 July 2015 Accepted 17 September 2015 Available online xxx Keywords: TCM Decoctions Erythropoietin Jujube LC–DAD–MS/MS

a b s t r a c t Jujubae Fructus, known as jujube or Chinese date, is the fruit of Ziziphus jujuba (Mill.), which not only serves as daily food, but acts as tonic medicine and health supplement for blood nourishment and sedation. According to Chinese medicine, jujube is commonly included in herbal mixtures, as to prolong, enhance and harmonize the efficiency of herbal decoction, as well as to minimize the toxicity. Here, we aim to compare the chemical and pharmacological properties of three commonly used jujube-containing decoctions, including Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and Zao Tang (ZOT). These decoctions share common herbs, i.e. Glycyrrhizae Radix et Rhizoma Praeparata cum Melle, Zingiberis Rhizoma Recens and Jujube, and they have the same proposed hematopoietic functions. The amount of twelve marker biomolecules deriving from different herbs in the decoctions were determined by LC–MS, and which served as parameters for chemical standardization. In general, three decoctions showed common chemical profiles but with variations in solubilities of known active ingredients. The chemical standardized decoctions were tested in cultured Hep3B cells. The herbal treatment stimulated the amount of mRNA and protein expressions of erythropoietin (EPO) via the activation of hypoxia response elements: the three herbal decoctions showed different activation. The results therefore demonstrated the hematopoietic function of decoctions and explained the enhancement of jujube function within a herbal mixture. © 2015 Elsevier B.V. All rights reserved.

1. Introduction JujubaFructus (JF), also known as jujube or Chinese date, is the fruit of Ziziphus jujuba (Mill.), and which is considered as one of five valuable fruits in China. Jujube serves as daily food, as well as being prescribed as a tonic medicine for blood nourishment and sedative effect. In recent studies, jujube was reported to have various functions, including anti-oxidation [1,2], neuro-protection

Abbreviations: ASR, Anelicae Sinensis Radix; CC, Cinnamomi Cortex; CR, Cinnamomi Ramulus; EPO, erythropoietin; GRRPM, Glycyrrhizae Radix et Rhizoma Praeparata cum Melle; HIF, hypoxia induced factor; GZT, Guizhi Tang; HRE, hypoxia response element; IS, internal standard; JF, JujubaFructus; NDT, Neibu Dangguijianzhong Tang; PAR, Paeoniae Alba Radix; ZOT, Zao Tang; ZRR, Zingiberis Rhizoma Recens. ∗ Corresponding author at: Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Clea Water Bay Road, Hong Kong, China. Fax: +852 2358 1559. E-mail address: [email protected] (K.W.K. Tsim).

[3,4], improving cardiovascular system [5], anti-microbial [6], antiinflammatory effect [7], anti-ulcer [8] and anti-cancer [9]. Several lines of evidence support the blood nourishing function of jujube [5,10]. According to Chinese medicine, blood nourishment includes blood formation (hematopoiesis), oxygenation and controlling factors for formation of red blood cells. Indeed, many herbal decoctions were demonstrated to facilitate blood formation, and different cell models were established to investigate this function [11]. Erythropoietin (EPO), a glycoprotein involving in erythropoiesis [12], is produced by embryonic liver and adult kidney cells. Under hypoxia, when the oxygen sensors in liver and kidney detect the low oxygen level, this will trigger the production of EPO, as to increase amount of red blood cells and hence to restore oxygen level [13,14]. This response is regulated by a hypoxia response element (HRE) signaling pathway. HRE is an enhancer sequence located on promoter region of EPO gene, and the activation of HRE initiates expression of EPO [15–17]. Jujube is frequently being included in a herbal formulated decoction, which is aiming to enhance the medicinal values, to facilitate

http://dx.doi.org/10.1016/j.jchromb.2015.09.021 1570-0232/© 2015 Elsevier B.V. All rights reserved.

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the absorption and/or to reduce the toxicity of individual herbs. According to Chinese medicinal theory, a herbal decoction normally comprises of four elements, including “Jun” (prime), “Chen” (minister), “Zuo” (assistant) and “Shi” (servant): the four elements are acting together to harmonize the therapeutic functions [18–20]. Jujube is widely included in numerous herbal decoctions serving as assistant or servant herb. Among these jujube-containing herbal mixtures, many of them are popularly used today for blood nourishing functions. Guizhi Tang (GZT), composed of Cinnamomi Ramulus (CR; Guizhi), Paeoniae Alba Radix (PAR; Baishao), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM; Zhigancao), Zingiberis Rhizoma Recens (ZRR; Shengjiang) and JujubaeFructus (JF; Zao), was prescribed in Shang Han Lun by Zhang Zhongjing in Han Dynasty (∼200 AD). Neibu Dangguijianzhong Tang (NDT), composed of Angelicae Sinensis Radix (ASR; Danggui), Cinnamomi Cortex (CC; Rougui), Paeoniae Alba Radix (PAR; Baishao), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM; Zhigancao), Zingiberis Rhizoma Recens (ZRR; Shengjiang) and JujubaeFructus (JF; Zao), was prescribed in Bei ji Qian jin Yao Fang by Sun Simiao in Tang Dynasty (652 AD). Zao Tang (ZOT), composed of Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM; Zhigancao), Zingiberis Rhizoma Recens (ZRR; Shengjiang) and JujubaeFructus (JF; jujube), was prescribed in Formulae of the Pharmacy Service for Great Peace and for the Benefit of the People by Official Bureau of Physicans (Taiyi ju) in Sung Dynasty (1078–1085 AD). Although the three decoctions are prescribed in different dynasties, they are considered as “Jia Jian Fang” of having modification by adding herbs on top of a basic formula, and thus the three jujube-containing decoctions are sharing herbal compatibility with similar functions. Here, we hypothesize that different decoctions should have a variation in hematopoietic function, i.e. stimulating EPO expression. Chemical parameters were established here to standardize the herbal decoctions, and subsequently they were tested functionally in cultured Hep3B liver cells to reveal mRNA and protein expressions of EPO, as well as the transcriptional activity of HRE.

2. Materials and methods 2.1. Materials The fruits of Z. jujuba cv. Jinsixiaozao (JF) from Hebei of China were collected in 2012. The dried stem bark and dried young branch of Cinnamomum cassia Presl (CC and CR) were collected from Guangxi China. The roots of Angelica sinensis (Oliv) Diels. (ASR), the processed root of Paeonia lactiflora Pall. (PAR), the root and rhizome of Glycyrrhiza uralensis Fisch. or Glycyrrhiza inflata Bat. under the method for stir-baking with honey (GRRPM), the rhizome of Zingiber officinale Roscoe (ZRR), were collected from Gansu, Anhui, Inner Mongolia and Guangdong of China. The raw materials were purchased from herbal markets. No specific permissions were required for collection of raw materials, and the location was also not privately-owned or protected. The plant materials were authenticated by Dr. Tina Dong based on their morphological characteristics and deposited in the Center for Chinese Medicine, The Hong Kong University of Science and Technology. The dried jujube was chopped into half, and ginger was chopped into slices according to ancient preparation method. cAMP (1) and cGMP (2) were purchased from Sigma–Aldrich (St. Louis, MO). Rutin (4) and astilbin (IS) were purchased from Tauto Biotech (Shanghai, China). Formononetin (5) and calycosin (6) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Paeoniflorin (3), cinnamic acid (7), glycyrrhizic acid (8), Z-ligustilide (10) and ferulic acid (12) was purchased from

The Testing Laboratory for Chinese Medicine (Hong Kong, China). 6-Gingerol (9) was purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Sichuan, China). Liquiritin (11) was purchased from Shanghai R&D Center for Standardization of Chinese Medicine (Shanghai, China). The purity of all marker biomolecules were determined to be over 98% by normalization of peak areas, as revealed by HPLC–DAD. MS-grade acetonitrile and water were purchased from Merck (Darmstadt, Germany). Ultra-pure water was prepared using a Mili-Q purification system (Molsheim, France) HPLC-grade methanol was purchased from Merck. Other reagents used here were of analytical grade.

2.2. Preparation of herbal extracts Total herbs (25 g) were weighted according to the formulation and were boiled together under moderate heating in 20 vol of water for 1 h, twice. The combined extracts were dried into powder under vacuum by lyophilizer at −80 ◦ C. Before the assessments, the powder was re-dissolved with water to a concentration of 10 mg/mL and 100 mg/mL, as stock solutions for chemical and biological assessments, respectively. For chemical assessment under LC–DAD–MS/MS and HPLC system, the extracts were filtered through a 0.45-␮m membrane filter before the analysis. For biological assessments, the extracts were filtered through a 0.22-␮m membrane filter before the cell treatment.

2.3. Chromatographic conditions and MS/MS analysis Agilent RRLC 1200 series system (Waldron, Germany) equipped with a degasser, a binary pump, an auto-sampler, a diode array detector (DAD) and a thermo-stated column compartment was adopted for establishment of fingerprint for herbal extracts. The mobile phase consisted of 0.03% phosphoric acid in water (A) and acetonitrile (B), respectively. The extracts were separated on an Agilent ZORBAX SB-Aq (4.6 × 250 mm, 5 ␮m) C18 column, and a wavelength of 210 nm were adopted for measurement. For MS analysis, the extracts were separated on an Agilent Eclipse Plus C18 RRHD (2.1 × 50 mm column, 1.8 ␮m) column by Agilent RRLC 1200. The mobile phases were composed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), respectively. A elution gradient was set up as follows: 0–2 min isocratic gradient 99% (A) with flow rate of 0.3 mL/min; 2–4 min, linear gradient 99 → 90% with flow rate of 0.3 mL/min; 4–8.75 min, linear gradient 90% → 80% (A) with flow rate of 0.3 mL/min; 8.75–10 min, isocratic gradient 80% (A) with flow rate of 0.3 mL/min; 10–17 min, linear gradient 80% → 70% (A) with flow rate of 0.3 mL/min; 17–22 min, linear gradient 70% → 65% (A) with flow rate of 0.3 mL/min; 22–30.75 min, linear gradient 65% → 30% (A) with flow rate of 0.3 mL/min; 30.75–38 min, isocratic gradient 30% (A) with flow rate changing from 0.3 to 0.2 mL/min. A pre-equilibration period of 3 min was used between each run. The column temperature was set to 25 ◦ C. The injection volume was 10 ␮L. A wavelength of 210 nm was employed for analysis. Agilent QQQ-MS/MS system equipped with an ESI ion source was operated in both positive and negative ion modes. The drying gas temperature was 325 ◦ C; drying gas flow: 10 L/min; nebulizer pressure: 35 psig; capillary voltage: 4000 V; delta electro multiplier voltage: 400 V. One to two suitable transition pairs was chosen for acquisition in multiple reactions monitoring (MRM) mode for selected markers and internal standard astilbin. The fragmentor voltage and collision energy values were optimized to obtain the highest abundance. Agilent MassHunter Workstation software version B.06.00 was used for data acquisition, processing and analysis.

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Table 1 Historical variation of different decoction formulae. Decoction

Record

Compositiona

GZT

Shang Han Lun written in ∼200 AD

NDT

Beiji Qian jin Yao Fang written in 652 AD

ZOT

Taiping Huimin Heji Jufang written in 1078–1085 AD

Cinnamomi Ramulus (CR; 6 g), Paeoniae Alba Radix (PAR; 6 g), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM; 4 g), Zingiberis Rhizoma Recens (ZRR; 6 g), JujubaeFructus (JF; 3 g) Angelicae Sinensis Radix (ASR; 4.5 g), Cinnamomi Cortex (CC; 3.5 g), Paeoniae Alba Radix (PAR; 6.8 g), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM; 2.25 g), Zingiberis Rhizoma Recens (ZRR; 6.8 g), JujubaeFructus (JF; 1.3 g) Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM; 8.3 g), Zingiberis Rhizoma Recens (ZRR; 16.9 g), JujubaeFructus (JF; 2.8 g)

a

The herbal composition was calculated according to the historical record in g.

2.4. Cell line and biochemical analysis

2.7. Western blot analysis

Human hepatocellular carcinoma Hep3B cells from American Type Culture Collection (ATCC; Manassas, VA) were cultured in DMEM supplemented with 100 IU/mL penicillin, 100 ␮g/mL streptomycin and 10% fetal bovine serum. For sub-culturing cells, cells were rinsed with 5 mL phosphate buffered saline containing 1 mM EDTA twice, and 1 mL trypsin/EDTA solution was applied to detach the cells. Reagents for cell cultures were purchased from Invitrogen Life Technologies (Invitrogen; Carlsbad, CA). Cobalt chloride (≥97%) were purchased from Sigma.

Hep3B cells cultured in 12-well plate were treated with herbal extracts for 24 h. The cultures were then collected in high salt lysis buffer (1 M NaCl, 10 mM HEPES, pH 7.5, 1 mM EDTA, 0.5% Triton ×-100), followed by centrifugation at 16,100 × g for 10 min at 4 ◦ C. Samples with equal amount of total protein were added with 2× lysis buffer (0.125 M HCl, pH 6.8, 4% SDS, 20% glycerol, 2% 2-meracptoethanol and 0.02% bromophenol blue) and heated under 95 ◦ C for 10 min before performing electrophoresis on a 9% gel. After electrophoresis, proteins were transferred to nitrocellulose membrane using a Mini Trans-Blot® cell at 40 V, 0.1A for 16 h in 1× transfer buffer containing 24 mM Tris, 192 mM glycine, 15% ethanol, 0.1% SDS. Complete transfer and equal protein loading of samples were confirmed by Ponceau-S staining. The nitrocellulose membrane was then blocked with 5% fat-free milk in TBS-T for 1 h and incubated with diluted primary antibody in 2.5% fat-free milk in TBS-T overnight at 4 ◦ C. The primary antibodies used were: rabbit anti-EPO (H162) from Santa Cruz Biotechnology (1:1000) (Dallas, CA) and GAPDH from Abcam Inc. (Cambridge, MA). Subsequently, the nitrocellulose membrane was rinsed with TBS-T for 1 h and incubated for another 2 h at room temperature with diluted HRP-conjugated anti-rabbit and anti-mouse secondary antibody (Invitrogen) in 2.5% fat-free milk in TBS-T. Again, the nitrocellulose membrane was rinsed with TBS-T for 1 h. Under intense washing with TBS-T, the immune complexes were visualized by ECL Western Blotting Kit (Sigma).

2.5. Quantitative real time PCR The cultures were treated with extracts or CoCl2 for 24 h. Total RNA was isolated by RNAzol reagent from Molecular Research Center Inc. (Cincinnati, OH), followed by reverse transcription into cDNA according to the manufacturer’s instruction (Invitrogen). Real-time PCR was conducted by using FastStart Universal SYBR Green Master (ROX) according to manufacturer’s instructions (Roche Diagnostics; Mannheim, Germany). The primers for human EPO gene were 5 -ACT TTC CGC AAA CTC TTC CG-3 and 5 -TGA ATG CTT CCT GCT CTG GG-3 (330 bp; NM 000799.2). 18S rRNA was used as internal control and reference gene. The sequence of 18S rRNA was: 5 -TGT GAT GCC CTT AGA TGT CC-3 and 5 -GAT AGT CAA GTT CGA CCG TC-3 (320 bp; NR 003286). SYBR green signal was detected by ABI 7500 fast real time PCR system (Applied Biosystems; Foster City, CA). Transcript levels were quantified by using DCt value method under Real time PCR 7500 software v2.0.6 in which the values of EPO genes were normalized first by 18S rRNA in same sample before comparison [21]. The PCR products were analyzed by gel electrophoresis, and the melting curve analysis was for confirmation of amplification [16]

2.6. DNA transfection and luciferase assay Cultured Hep3B cells were transfected with pBI-GL vector plasmid (BD Biosciences; San Jose, CA) containing six copies of HRE gene (5 -TCG AGG CCC TAC GTG CTG TCT CAC ACA GCC TGT CTG ACG-3 ) and a firefly luciferase reporter gene, i.e. pHRE-Luc. Lipofectamine 3000 (Invitrogen) was used for transfection according to the manufacturer’s instruction. Under this condition, over 40% cells were transfected as determined by another control plasmid having a ␤-galactosidase under a cytomegalovirus enhancer promoter. Herbal extracts were applied onto transfected Hep3B cells for 24 h, and luciferase assay was performed with a commercial kit (Applied Biosystems). The luminescence was quantified in a microplate luminometer (Promega; Madison, WI), and the luciferase was expressed as the absorbance (up to 560 nm) per mg of protein.

2.8. Other assays The concentration of total protein in samples were determined by Bradford method from Bio-Rad Laboratories (Bio-Rad; Hercules, CA). Individual data was expressed as mean ± SD. Statistical tests were performed with t-test (version 13.0, SPSS). Statistically significant changes were classified as significant (*) where p < 0.05, very significant (**) where p < 0.01, and highly significant (***) where p < 0.001. 3. Result 3.1. Chemical standardization of herbal extracts Three herbal decoctions were chosen to study here: GZT, NDT and ZOT. The reasons for this choice of herbal mixtures are: (i) they are historical recorded and still commonly used today for “Qi” stimulation; (ii) they share common herbs of GRRPM, ZRR and JF (Table 1). The herbal decoctions were prepared according to the historical recorded method. In order to standardize the decoctions, 12 biomolecules were selected for chemical analysis, which included: JF-derived cAMP (1), cGMP (2) and rutin (3); GRRP-derived calycosin (4), formononetin (5), glycyrrhizic acid (6), and liquiritin

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Fig. 1. The structure of marker biomolecules. The chemical structure of 12 marker biomolecules, including JujubaFructus (JF)-derived cAMP(1), cGMP (2), rutin (3); Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM)-derived calycosin (4), formononetin (5), glycyrrhizic acid (6), liquiritin (7); Zingiberis Rhizoma Recens (ZRR)-derived 6-gingerol (8); Cinnamomi Ramulus/Cinnamomi Cortex (CR/CC)-derived cinnamic acid (9); Paeoniae Alba Radix (PAR)-derived paeoniflorin (10); Angelicae Sinensis Radix (ASR)-derived ferulic acid (11), Z-ligustilide (12); and astilbin (internal standard) were shown.

(7); ZRR-derived 6-gingerol (8); CR/CC-derived cinnamic acid (9); PAR-derived paeoniflorin (10); ASR-derived ferulic acid (11) and Z-ligustilide (12) (Fig. 1). As a first parameter for quality control, a typical HPLC fingerprint at absorbance of 210 nm was employed to standardize the decoctions (Fig. 2). The most abundant constituent found in three decoctions was liquiritin, which was used as a reference standard in fingerprints. The peaks corresponding to glycyrrhizic acid and 6-gingerol were identified (Fig. 2). A rapid LC–MS/MS method was employed to quantify the amount of marker biomolecules in decoctions. Both positive and negative modes of MA analysis, as well as 210 nm absorbance, were employed. Fig. 3A shows the HPLC profile of different herbal decoctions and marker biomolecules at 210 nm, and Fig. 3B and C show the MRM profiles from MS analysis. The fragmentor voltage and collision energy were optimized to obtain the highest value by Optimizer program in the system (Table A.1 & A.2). Astilbin (IS), an internal standard, was spiked into the samples as reference in analysis. From the three decoctions, 10 chosen markers were detected under MS positive mode (Fig. 3B), except ferulic acid (Fig. 3C). In addition to GZT, NDT and ZOT, the extracts from individual herbs were also calibrated similarity (Fig. A.1). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2015.09. 021. The results were used to establish quality control for three decoctions. To validate the analytic method, linearity, sensitivity, precision, repeatability and accuracy of analytes were taken into account. For the linearity, calibration curves were constructed using a range of concentrations of working standards in which at least eight different concentrations were used (Table A.3). The square values of the correlation coefficient (r2 ) of calibration curve were higher than 0.99. The limit of detection (LOD) and limit of quantity (LOQ) were employed to evaluate the sensitivity. The LOD was estimated with a signal-to-noise ratio (S/N) of 3; while LOQ was determined with S/N of 10 (Table A.3). The relative standard deviation (RSD) was taken as a measurement of precision, repeatability and recovery. The assay precision was determined by intra-day and inter-day variations analysis (Table A.4). Repeatability test was conducted by analyzing five independent samples prepared from the

Fig. 2. Typical HPLC fingerprint of decoctions extract at 210 nm wavelength. Ten mg/mL herbal extract was subjected to HPLC analysis. The chemical fingerprint was revealed by a DAD detector at 210 nm. The elution gradient at a flow rate of 0.5 mL/min was set up as follows: 0–10 min, isocratic gradient 99% (A); 10–20 min, linear gradient 99% → 90% (A); 20–35 min, isocratic gradient 90% (A); 35–40 min, linear gradient 90% → 80% (A); 40–60 min, linear gradient 80% → 70% (A); 60–90 min, linear gradient 70% → 50% (A). A pre-equilibration period of 10 min was used between each run. The injection volume was 20 ␮L. Liquiritin at 56 min, glycyrrhizic acid at 78 min and 6-gingerol at 83 min were identified in the HPLC fingerprint. Representative chromatograms are shown, n = 5.

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A

5

B

C

Fig. 3. Typical RRLC-DAD-QQQ MS/MS chromatograms of 12 marker biomolecules in water extracts of decoctions and jujube. The LC condition was described in Section 2. (A) The identification of (7) liquiritin was determined by DAD at 210 nm. (B) The identification of cAMP (1), cGMP (2), rutin (3), calycosin (4), formononetin (5), glycyrrhizic acid (6), 6-gingerol (8), cinnamic acid (9), paeoniflorin (10) and Z-ligustilide (12) were determined under MS positive mode system. (C) The identification of ferulic acid (11) was determined under MS negative mode system. Astilbin was served as internal standard (IS) in both positive and negative mode. The chemical markers were numbered according to Fig. 1.

same herbal extracts. The accuracy was evaluated as percentage recovery of analytes in the spiked samples. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2015.09. 021. By aforementioned analytic method, the amount of biomolecules (in mg/kg of dried extract) in herbal extracts were determined, as listed in Table 2. This chemical composition, together with HPLC fingerprint, served as parameters for quality control, which ensured the repeatability of analyses on cell cultures. 3.2. Herb–herb interaction in chemical solubility Having a mixture of various herbs within a formulated decoction, this is believed that the extraction efficacy of active ingredients

could be increased, while toxic biomolecules could be reduced. In order to study herb-to-herb interaction, the chemical solubilities of various marker biomolecules from herbal extracts were calculated (Table 3). Among the three decoctions, the solubilities of chosen biomolecules deriving from GRRPM, ZRR and JF were compared. The solubilities of calycosin, formononetin, glycyrrhizic acid, liquiritin and 6-gingerol were significant higher than that in single herbal extract (Fig. 4). The JF-derived biomolecules, i.e. cAMP, cGMP and rutin, did not show much difference as compared to single herbal extract. The robust increase of chemical solubilites was revealed in all GRRPM-derived biomolecules: the increase could be at ∼13 folds for calycosin in ZOT, or ∼6 folds in GZT (Fig. 4). The ZRR-derived 6gingerol was increased by ∼2 folds in ZOT as well. In summary, the herbal mixtures in general could provide more biomolecules achieved within the decoctions. This suggest that the boiling of

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Table 2 Assessments of 12 marker chemicals in three decoctions and single herbal extracts. Chemical markersGZT

NDT

ZOT

JF

GRRPM

ZRR

CC

CR

PAR

ASR

cAMP 106.0 ± 1.7 53.1 ± 3.5 103.8 ± 1.2 305.8 ± 20.9 – – – – – – 146.3 ± 12.2 121.3 ± 19.0 112.6 ± 10.6 4447.8 ± 16.9– – – – – – cGMP Rutin 13.9 ± 3.2 7.6 ± 0.3 14.4 ± 1.4 31.8 ± 1.0 – – – – – – Calycosin 2.4 ± 0.5 0.5 ± 0.1 5.9 ± 1.2 – 10.3 ± 2.4 – – – – – 66.5 ± 2.4 156.2 ± 29.2 – 445.6 ± 73.0 – – – – Formononetin 93.3 ± 20.1 15085.1 ± 2240.2– – – – – Glycyrrhizic acid 5131.7 ± 153.8 3390.2 ± 716.25290.3 ± 509.1 – 14225.3 ± 324.47610.3 ± 947.516774.4 ± 784.0– 50666.5 ± 2540.1– – – – – Liquiritin 507.7 ± 115.7 525.1 ± 142.0 3362.4 ± 388.1 – – 22728.0 ± 1399.1– – – – 6-Gingerol Cinnamic acid 140.8 ± 12.3 167.2 ± 1.3 – – – – 12825.8 ± 2484.77109.6 ± 277.5– – Paeoniflorin 154.0 ± 27.0 384.8 ± 42.5 – – – – – 1354.7 ± 256.2– Ferulic acid – 235.0 ± 43.4 – – – – – – 0. ± 0.0 – 9.9 ± 0.2 – – – – – – 1438.8 ± 98.8 Z-ligustilide Values are expressed in mg/kg of dried extract. Mean ± SD, n = 5. Table 3 The solubility of marker chemicals in three decoctions and single herbal extracts. Chemicals

GZT

NDT

ZOT

JF

GRRPM

ZRR

CC

CR

PAR

ASR

cAMP cGMP Rutin Calycosin Formononetin Glycyrrhizic acid Liquiritin 6-Gingerol Cinnamic acid Paeoniflorin Ferulic acid Z-ligustilide

144.2 ± 51.1 260.6 ± 0.3 12.9 ± 1.0 2.1 ± 0.3 183.6 ± 31.3 6494.0 ± 113.4 14004. ± 356.8 279.1 ± 25.1 120.5 ± 11.0 243.2. ± 27.0 – –

119.9 ± 4.5 331.2 ± 48.6 23.4 ± 2.1 0.8 ± 0.1 110.8 ± 37.2 4139.1 ± 714.8 15529.7 ± 1020.1 408.5 ± 22.2 189.8 ± 26.5 285.9 ± 70.3 285.7 ± 62.9 8.1 ± 0.2

199.4 ± 17.4 213.2 ± 26.2 11.5 ± 3.3 3.9 ± 0.8 117.0 ± 18.4 3970.0 ± 396.0 20481.5 ± 3908.8 1447.6 ± 28.4 – – – –

146.7 ± 6.8 216.8 ± 32.3 14.2 ± 0.9 – – – – – –

– – – 0.3 ± 0.0 68.6 ± 3.9 2472.1 ± 526.0 8965.9 ± 316.0 – – – – –

– – – – – – – 442.1 ± 47.0 – – – –

– – – – – – – – 666.5 ± 28.0 – – –

– – – – – – – – 609.1 ± 48.4 – – –

– – – – – – – – – 336.9 ± 13.6 – –

– – – – – – – – – – 570.2 ± 2.8 3.2 ± 0.7

– –

Values are expressed in mg/kg of dried extract. Mean ± SD, n = 5.

ical variation might account for the difference in pharmacological properties among the three decoctions and jujube extract. 3.3. Three decoctions induce the expression of erythropoietin (EPO)

Fig. 4. The solubilities of marker biomolecules in herbal extracts. The chemical markers including glycyrrhizic acid, liquiritin, formononetin, calycosin and 6-gingerol were determined in decoctions and single herb extracts. Values are expressed in % of change by comparing with single herbal extract as mean ± SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.

multiple herbs together could enhance the extraction efficiency of certain biomolecules from the herb, which therefore could improve the nutrition and pharmacological effects. In addition, this chem-

The herbal extracts reached the minimal chemical standardized parameters as described previously, which guaranteed the repeatability of following biological results. In order to compare the hematopoietic function among the herbal decoctions and jujube extracts at molecular level, the extracts were applied onto cultured Hep3B cells for 24 h. The concentrations (from 0.5 to 4 mg/mL) used in this experiment did not show cytotoxicity, as revealed by MTT assay (Fig. A.2). The mRNA expression of EPO was revealed. Under the application of herbal extracts, the mRNA of EPO was induced in a dose-dependent manner (Fig. 5A). 50 ␮M CoCl2 was a positive control in this experiment for preventing HIF 1␣ degradation, giving 12–18 folds of increase in EPO mRNA expression [22,23]. All the decoctions induced the mRNA expression of EPO in greater extent when compare to jujube extract, as well as in a dose-dependent manner. The maximal induction was achieved by the extract from 1 to 2 mg/mL, giving ∼100% of increase (Fig. 5A). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2015.09. 021. HRE plays a very important role in activating the expression of EPO mRNA [24,25]. To reveal the underlying working mechanism of decoctions on mRNA expression of EPO, luciferase reporter constructs carrying six repeats of HRE and a luciferase gene were transfected into Hep3B cells for 24 h. The transfected cells were then subjected to herbal extract treatment for 24 h. Finally, luciferase assay was employed to quantify the transcriptional activity of HRE. The authentication of pHRE-Luc was confirmed by the activation under treatment of CoCl2 [10]. Result demonstrated that

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A

B

Fig. 6. Decoctions and jujube extract induce protein expression of EPO in cultured Hep3B cells. (A) Cultured Hep3B cells were treated with extracts (0.5–4 mg/mL) for 24 h. The cell lysates were collected to determine the protein expression of EPO (∼34 kDa) using specific antibody. CoCl2 (50 ␮M CoCl2 ) served as the positive control. GAPDH (∼38 kDa) served as loading control. (B) The EPO proteins were quantified from the blots by a calibrated densitometry. Values are expressed as the percentage of increase to basal reading (untreated culture), mean ± SD, n = 4; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 5. Herbal extracts induce mRNA expression of EPO in cultured Hep3B cells. (A) Cultured Hep3B cells (2 × 105 cells/mL) were treated with different extracts at various concentrations (0.5–4.0 mg/mL) for 24 h. The level of EPO mRNA was revealed by real-time PCR, while 18S rRNA was used as an internal control for normalization. CoCl2 (50 ␮M) served as a positive control. (B) Decoctions and JF extract stimulate hypoxia response element (HRE)-mediated transcriptional activity. A luciferase-reporter containing six repeats of HREs and a downstream luciferase-reporter gene, named as pHRE-Luc, was used as a study tool (upper panel). Cultured Hep3B cells transfected with pHRE-Luc were treated with decoctions or JF extracts (0.5–4.0 mg/mL) for 24 h. CoCl2 (50 ␮M) served as a positive control. The cell lysates were subjected to luciferase assay. Values are expressed as the percentage of increase to basal reading (untreated culture) and as mean ± SD, where n = 4, each with triplicate samples. Statistical comparison was made with jujube extract; *p < 0.05; **p < 0.01; ***p < 0.001.

the herbal extracts induced HRE transcriptional activity in a dosedependent manner. The maximal induction was achieved by ZOT at 2 mg/mL, giving ∼200% of increase (Fig. 5B). To further validate the hematopoietic function, EPO protein level was also revealed [24]. The herbal extracts induced the protein expression of EPO

protein at ∼34 kDa in a dose-dependent manner (Fig. 6A). The maximal induction was achieved by ZOT at 2 mg/mL, giving ∼100% of increase (Fig. 6B). 4. Discussion Under the idea of “Homology of Food and Medicine”, many people start to recognize the potential healing power of natural prescriptions. Herbal decoction, being one of the effective clinical practices in healing, has advantages when compared to single herbal treatment, which are better to enhance the medicinal values, to facilitate the absorption and to reduce the toxicity of individual herbs. In general, a herbal mixture is believed to improve greatly the efficacy of single herb [26]. Here, we aimed to disclose the underlying rationale for herbal compatibility by comparing the chemical constitutions and solubilities of herbs among three chosen decoctions. Three decoctions in our studies are written from different dynasties; in which they have minor modification but having similar pharmacological functions. The herbal compatibility of GRRPM, ZRR and JF was first introduced and routinely recorded in Shang

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Han Lun by Zhang Zhongjing in Han Dynasty (∼200 AD). One-third of prescriptions recorded in this ancient book were made up by this herbal compatibility, i.e. having GRRPM, ZRR and JF together. Hundreds of years later in Sung dynasty (1078–1085 AD), one-tenth of prescriptions were still made up of this compatibility. The consistent usage of GRRPM, ZRR and JF in a herbal mixture suggests that this herbal compatibility might be important in the health healing. The establishment of chemical control of decoctions was to ensure the decoctions with repeatable pharmacological functions. Here, we have demonstrated that the addition of one or more herbs within a herbal mixture could greatly promote the extraction efficacy of bio-active biomolecules, e.g. calycosin, formononetin, glycyrrhizic acid, liquritin and 6-gingerol. As shown here, the extraction efficacy of calycosin was enhanced up to ∼6 folds in GZT and ∼12 folds in ZOT, as compared to the extraction from GRRPM. In previous studies, calycosin and formononetin were reported to possess erythropoietic effects [27]. Thus, the improvement in extraction efficacy of calycosin and formononetin in herbal decoctions could account for better erythropoietic effects, as comparing to jujube extract alone [28,29]. The increase of active ingredients being soluble in water extract of a herbal mixture is a common phenomenon when several herbs being boiled together. Two possible reasons have been proposed: (i) increase solubility of chemical from the herb; and (ii) increase stability of chemical in a mixed herbal extract [27–29]. Interstitial fibroblasts in adult kidney cells and embryonic liver cells are the primarily production site of EPO. Renal production is predominant during adulthood. However, a switch of EPO production site from kidney to liver was reported in anemia patients when EPO production in kidney was impaired [30]. Here, we employed Hep3B liver cell line to evaluate and compare hematopoietic functions of herbal extracts. The regulation of EPO gene expression occurs primarily at the mRNA level under control of both transcriptional and post-transcriptional factors [31,32]. Our current results demonstrated that the herbal extracts described here could induce the mRNA and protein expression of EPO in dose-dependent manner via the activation of HRE signaling pathway. The expression of EPO under the treatment of decoctions was much higher as compared to jujube extract, suggesting better hematopoietic functions. Among the three decoctions, GZT and ZOT exhibited stronger effect in triggering EPO production. 5. Conclusion The result suggested that the herbal decoctions induced the expression EPO via activation of HRE signaling pathway. The hematopoietic function of decoctions was much higher than that of jujube extract, suggesting that a herbal mixture may serve better hematopoietic function than single herbal extract. Three decoctions, described here, could serve as potential natural alternative medicine and health food supplement for anemia patients. Acknowledgments Supported by Hong Kong Research Grants Council Theme-based Research Scheme (T13-607/12R), ITF (UIM/254), GRF (661110, 662911, 660411, 663012, 662713, M-HKUST604/13), TUYF12SC02, TUYF12SC03, TUYF15SC01, The Hong Kong Jockey Club Charities Trust (HKJCCT12SC01) and Foundation of The Awareness of Nature (TAON12SC01) to Karl Tsim. Candy Lam received a scholarship from Healthworks (Herbal Tea) Co., Ltd. References [1] J.G. Jiang, X.J. Huang, J. Chen, Q.S. Lin, Comparison of the sedative and hypnotic effects of flavonoids, saponins, and polysaccharides extracted from semen Ziziphus Jujube, Nat. Prod. Res. 21 (2007) 310–320.

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