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Microcalorimetric assay on the antimicrobial property of five hydroxyanthraquinone derivatives in rhubarb (Rheum palmatum L.) to Bifidobacterium adolescentis.

ABSTRACT

It was found that the intestinal bacteria balance would be deteriorated by rhubarb especially in long term treatment. Bifidobacteria is one of the most common species of probiotics in human intestine. The suppression of this particular probiotic, such as Bifidobacterium adolescentis, one of the dominant anaerobes in the intestines of humans, might lead to imbalance of intestinal flora and is considered to be potentially riskful for human health. Hence, the inhibitory effects of the five main components of hydroxyanthraquinones (HAQs) contained in rhubarb on B. adolescentis growth were investigated by microcalorimetry to discover the suppression potential of rhubarb) and the structure-function relationship of such HAQs. The value of the maximum power- output ([P.sub.max]) and slope (k) of the thermogenic growth curves of B. adolescentis were found of decrease in the presence of the five HAQs, while the peak time ([T.sub.p]) of the thermogenic curves were found to be delayed. The sequence of antimicrobial activity of the five HAQs is rhein > emodin > aloe-emodin > chrysophanol > physicion. The functional groups carboxyl, hydroxyl and hydroxylmethyl on phenyl ring in HAQs could improve the antimicrobial activity. The influence of substituent groups on anti- B. adolescentis activity might be related with the polarity and the sequence was carboxyl > hydroxyl > hydroxylmethyl > methyl and methoxyl.

ARTICLE INFO

Keywords: Hydroxyanthraquinone Antibacterial activity Bifidobacterium adolescentis Microcalorimetry Structure-function relationship

Introduction

Rheum palmatum L. is a traditional Chinese medicinal (TCM) herb, and is officially listed in Chinese, Japanese and European Pharmacopoeia (Pharmacopoeia of the People's Republic of China 2005a, 2005b; Japanese Pharmacopoeia 2006; European Pharmacopoeia 2001). The crude material of R. palmatum called rhubarb was widely used as laxative to treat constipation. Furthermore, it was usually used for body slimming based on its laxative effect in China and some orient countries, and the treatment cycle was sometimes beyond the recommended course. It was considered that the over long-time consumption of rhubarb might lead to imbalance of intestinal flora eliminating the beneficial flora (Li and Liu 1989; Xu et al. 2005; Ding et al. 2006). The ethanol extract of R. palmatum has strong antibacterial activities (Dictionary of Traditional Chinese Medicine 1997) and the major active components in it are considered to be five hydroxyanthraquinones (HAQs), namely aloe-emodin, rhein, emodin, chrysophanol and physcion (Pharmacopoeia of the People's Republic of China 2005a, 2005b).

Bifidobacteria is one of the most common species of probiotics in human intestine and play an important role of intestinal bacteria balance as the perdominant bacterial species of the human intestinal microflora (Gorbach and Nahas 1967). This particular probiotic is considered to be useful for human health, providing nutrients for intestinal cells, promoting absorption of nutrients, creating a healthy intestinal environment, and promoting a healthy immune system (Bauer et al. 2006; Czarnecki 2008). It has been shown to be effective in inhibiting LPS-induced inflammation, by blocking NF-kB activation (Riedel et al. 2006). It was also reported that bifidobacteria could suppresse enteritis in animal models of inflammatory bowel disease (Shiba et al, 2003; Matsumoto et al. 2001). Moreover, Bifidobacterium improves ulcerative colitis in patients (Ishikawa et al. 2003; Furrie et al 2005; Gibson and Roberfroid 1995). Imbalance in intestinal microflora constitution, i.e. lack of Bifidobacteria, could lead to increase of possibility of morbidity, such as inflammatory bowel disease (Hiromasa et al. 2008).

Therefore, the inhibitory effects of the five HAQs from R. palmatum on Bifidobacteria growth were investigated by microcalorimetry to discover the suppression potential of rhubarb and the structure-function relationship of such HAQs. The species of Bifidobacteria tested in this study was paradigmaticly chosen as Bifidobacterium adolescentis, one of the dominant anaerobes in the intestines of humans typically reaching densities greater than [10.sup.10] cells per g of feces (Evison and James 1973; Resnick and Levin 1981).

Microcalorimetry has been used successfully to evaluate the effects of various substances on microbial metabolism (Kong et al. 2008a, 2009a; Maniewska et al. 2009). During the growth of microbe along with affection by various substances, a flow of thermal effect is generated, which is directly related to an increase or decrease in the energy release. To follow the evolution of energetic content, the microcalorimetry technique showed to be a useful tool (Kong et al. 2009b). The slow reactions along with the growth of microbe can be easily followed by microcalorimetry through continuous recording of the signal, such as heat flow power (HFP), for a long time without any disturbance of the system. And the temporal details of microbe growth are not observable by other techniques (Chardin et al. 2002; Leng et al. 2007). It has recently been demonstrated that calorimetric methods can be used for fundamental studies of bacterial growth (Kwon et al. 2008; Ah et al. 2008). By analyzing the heat flow power - time curves of microbial metabolism, kinetic parameters such as rate constant for bacterial growth, peak power for microbial activity can be obtained, and the effects of compounds and drugs on these microbes can be evaluated (Kong et al. 2008b; Granqvist et al. 2009).

Materials and methods

Strain, substrates and chemicals

B. adolescentis (AS.1.2190) was purchased from the Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China. B. adolescentis was grown in a peptone culture medium, which contained 10 g of trypticase, 5 g of peptone, 2.5 g of yeast extract, 0.15 g of Ca[Cl.sub.2], 5g of glucose, 2g of [K.sub.2]HP[O.sub.4], 0.5 g of Mg[Cl.sub.2], 0.5 g of cysteine hydrochloride, 0.25 g of Zn[SO.sub.4], and 1 ml of tween-80, all of which were dissolved in 1000 ml of distilled water. Medium pH was adjusted to 6.5 with 1 mol/1 NaOH and 1 mol/1 HC1 before autoclaving at 121 [degrees]C for 30 minutes. R. palmatum was collected in Gansu province, China. The ethanol extract of R. palmatum was prepared by refluxing with 250 ml ethanol to 1.5 g rhubarb powder for 60 min and the solution was evaporated to dryness under reduced pressure. The chemical contents of the five HAQs were determined by HPLC using the method in Chinese Pharmacopoeia (Pharmacopoeia of the People's Republic of China 2005a, 2005b). The five HAQs, aloe- emodin, rhein, emodin, chrysophanol and physcion, were extracted and isolated from R. palmatum and the degree of purity of all were over 98% through HPLC analysis with a UV detector at 256 nm. Three benzoic acid derivatives, namely salicylic acid, o-aminobenzoic acid and gallic acid, with the similar [-(C=O)-C-(C-X)-, X=OH or [NH.sub.2]] structural feature to the five HAQs were also tested to further the understanding of the structure- function relationship. The structures of the five HAQs and analogues are given in Fig. 1.

Preparation of the samples

At the beginning of the experiments, B. adolescentis was inoculated into the peptone medium, with 2 X [10.sup.6] cells per ml. Cells were suspended in the peptone culture medium and the freshly prepared HAQs solutions (solved in 0.5% [Na.sub.2][CO.sub.3] solution) with different concentrations were added. The culture medium and samples were filled in glass ampoules and then sealed to ensure the anaerobic environment just before the microcalorimetric analysis. The final concentration of five HAQs in culture medium to compare the antibacterial effect was 7 [micro]mol [l.sup.-1]. The final concentration of rhein in culture medium to investigate the dose-effect relationship was in the range of 0.175 to 7 [micro]mol [l.sup.-1]. The ethanol extract of R. palmatum and the three benzoic acid derivatives were also solved in 0.5% [Na.sub.2][CO.sub.3] solution freshly. The final concentration of R. palmatum was in the range of 5 to 100 mg [l.sup.-1] (counted with the quantity of crude material of R. palmatum). The final concentration of the three benzoic acid derivatives was 150 [micro]mol [l.sup.-1].

Microcalorimetric analysis

The 3114/3115 TAM air isothermal microcalorimeter (Thermometric AB, Sweden) was used to determine the metabolic HFP-time curves of B. adolescentis cells. This microcalorimeter was an eight-channel twin instrument and thermostated at the range of 5-60[degrees]C, with a limit of detectibility of 2 [micro]W. The software supplied to TAM air was used to monitor and record the heat flow over the Peltier module when the baseline drift was less than 20 [micro]W over 24 h. For more details of the instrument, see the report of Wadso (2002). Cultivation was performed at 37[degrees]C in an anaerobic incubator containing an atmosphere of 90% nitrogen and 10% carbon dioxide.

Results

HFP-time curves of B. adolescentis growth

The thermogenic curve of B. adolescentis growth at 37 C without any substance was shown in Fig. 2. It was a typical HFP-time curve of 6. adolescentis and could be divided into three stages: stage I is a balance phase of the instrument, stage II is the quick growth phase and stage III is a decline phase. In stage III, there was a shoulder peak but it might disappear under inhibition of HAQs.

The corresponding HFP-time curves of B. adolescentis growth affected by different HAQs were shown in Fig. 3. As could be seen from the profiles of these curves, the growth of B. adolescentis was influenced by these compounds. The five HAQs except rhein at same concentration of 7 [micro]mol[l.sup.-1] had similar profiles but different peak-heights shown in Fig. 3a. Physcion and chrysophanol had no evident influence on the growth curve of B. adolescentis, which demonstrated poor antibacterial activity of the two compounds. The peak time ([T.sub.p]) of the curves affected by emodin and aloe-emodin were prolonged significantly, while the maximum power-output ([P.sub.max)] was degraded evidently compared to the control. The curve of B. adolescentis growth in the presence of rhein at concentration of 7 [micro]mol [l.sup.-1] was fully restrained and no visible growth was observed in the whole course, which demonstrated the strong antibacterial activity.

[FIGURE 3 OMITTED]

Quantitative thermokinetic parameters for B. adolescentis growth

As shown in Figs. 2 and 3, there was a characteristic peak of the thermogenic curves. The peak time ([T.sub.p]) of thermogenic curve of B. adolescentis was demonstrative to the growth of microbes. Generally, the peak time would be suspended when the microbe growth was restrained. The longer the peak times are, the stronger antibacterial activity the drugs possess. Other quantitative thermokinetic parameters such as the maximum power- output ([P.sub.max]), the slope (k) of the growth curve in stage II and the heat output in the whole course (Q) obtained from the HFP-time curves of B. adolescentis growth affected by different HAQs solutions were shown in Table 1. The general results from Fig. 3a and Table 1 indicated that the five HAQs inhibited the growth of B. adolescentis. But, with the differences of k, [P.sub.max] and Q values, the five HAQs had different antibacterial activities. Rhein, emodin and aloe- emodin suppressed the growth of B. adolescentis more than chrysophanol and physcion. Rhein completely inhibit growth of B. adolescentis at 7 [micro]mol [l.sup.-1]. The different inhibitory action of five compounds could be clearly demonstrated by the plots shown in Fig. 3a.

[FIGURE 2 OMITTED]

Relationship between quantitative thermokinetic parameters and concentration of rhein

The curves shown in Fig. 3b demonstrated that the peak time prolonged with the increasing concentrations of rhein compared to the control. The results in Table 1 showed that the values of k and [P.sub.max] decreased with the increasing concentration of rhein compared to the control. The relationship between quantitative thermokinetic parameters and logarithmic concentration of rhein were of good linearity shown in Fig. 4. The peak time ([T.sup.p]) was of positive correlation (Fig. 4a), while the maximum power-output ([P.sub.max]) and the growth rate constant (k) were of negative correlation (Fig. 4b, c). The correlation coefficient of [P.sub.max]-Ln C was over 0.99 in the range of 0.175 to 3.5 [micro]mol [l.sup.-1]. However, the growth of B. adolescentis was fully restrained when the concentration of rhein was 7 [micro]mol [l.sup.-1].

[FIGURE 4 OMITTED]
Table 1

Quantitative thermokinetic parameters for B. adolescentis growth at 37
[degrees]C affected by different HAQs from R. Palmatum.

Experiment  C ([micro]mol  [T.sub.p]  [P.sub.max]  k ([micro]W    Q (J)
            [1.sup-1])     (min)      (mW)         [s.sup-1])

Control        0           456.7      2.365        0.1916      66.71

Rhein         0.175        523.3       1.931         0.2150      47.00

              0.35         616.7       1.775         0.1575      68.78

              1.05         696.7       1.511         0.1349      59.75

              2.1          796.7       1.319         0.1218      58.69

              3.5          923.3       1.290         0.0995      62.30

              7            -           -             -           10.16

Emodin        7            603.3       1.826         0.1244      58.92

Chrysophanol  7            483.3       2.133         0.2454      48.13

Physicion     7            483.3       2.217         0.2445      57.10

Aloe-emodin   7            556.7      1.967         0.1475       62.95


Restraining effect of R. palmatum on B. adolescentis growth

As shown in Fig. 5, there was a dose- depended restraining effect of R. palmatum on B. adolescentis growth and the growth was fully restrained at the concentration of 100 mg [l.sup.-1] counted with the quantity of crude material of R. palmatum. The contents of the five HAQs in the ethanol extract of R. palmatum were 0.413, 2.107, 0.360, 0.607 and 0.360 % (counted with the quantity of crude material) for aloe- emodin, rhein, emodin, chrysophanol and physcion respectively. Hence, the concentration of rhein in the sample solution of R. palmatum at 100 mg [l.sup.-1] was 2.107 mg [l.sup.-1] or 7.413 [micro]mol [l.sup.-1], which was nearly equal to the fully restraining concentration (7 [micro]mol [l.sup.-1]) of rhein found in Fig. 3. So it could be induced that rhein played the most effective role in R. palmatum to restrain the growth of B. adolescentis.

[FIGURE 5 OMITTED]

Restraining effect of benzoic acid derivatives on B. adolescentis growth

The restraining effect of HAQs to the growth of the micro-organism relates on the -(C=O)-C-(C-OH)- structural feature. To assess the structure- function relationship, three benzoic acid derivatives of a ketone group neighbored with a polar group like hydroxyl group or amino group (Fig. 1) were determined and the results were shown in Fig. 6. It was found that the tested benzoic acid derivatives revealed restraining effect on the growth of B. adolescentis at the concentration of 150 [micro]mol [l.sup.-1], which was significantly higher than that of the HAQs, especially rhein. In the structure of the HAQs, there is a rigid planar framework of ketone and hydroxyl groups restricted by anthracene nucleus; however, the tested benzoic acid derivatives do not have this framework. So we could induce that the rigid planar structure of ketone and hydroxyl groups in neighborhood was important to the antimicrobial ability of these compounds. To compare the restraining ability of the three benzoic acid derivatives, the sequence was salicylic acid > gallic acid > o-aminobenzoic acid. Based on this result, a hydroxyl group might be much more important than an amino group in neighborhood of the ketone group and a proper spacing between the hydroxyl group and ketone group was necessary for the restraining effect.

[FIGURE 6 OMITTED]

Discussion

Because the five HAQs are not soluble in water but in alkaline liquor, we got the solutions dissolved in 0.5% [Na.sub.2][CO.sub.3] solution. In the preliminary experiment, the antibacterial effect of [Na.sub.2][CO.sub.3] on B. adolescentis growth was firstly investigated to eliminate the influence of this solvent on this study. The repeated results showed that there was no visible anti-B. adolescentis activity when 30 [micro]l of 0.5% [Na.sub.2][CO.sub.3] solution was added into the ampoule with the cell suspension of B. adolescentis. So, the final added volume of HAQ solution that was dissolved in 0.5% [Na.sub.2][CO.sub.3] solution was 30 [micro]l.

The thermogenic curves of B. adolescentis growth affected by various HAQs from R. palmatum indicated that all tested drugs had inhibitory activity on the tested bacteria. The peak time of bacterial growth prolonged with the presence of the test HAQs and also with increasing concentrations of rhein, indicating that the five HAQs all have the capacity of inhibiting the growth of B. adolescentis and the inhibitory potency varied with different HAQs. The inhibitory effects of these HAQs might be related with the type and number of substituent groups on the molecular structure, and the stability and solubility of the particular compound. All of these HAQs have a same hydroxyanthraquinone nucleus composed of two ketone groups at C9 and C10, two hydroxyl groups at C1 and C8, while the different groups are substituted at C3 and C6 of phenyl ring (Fig. 1). It was reported that the aromatic hydroxyls on this anthraquinone ring were probably important moieties for antibacterial effect (Tian et al. 2003). The polar substituent groups of carboxyl at C3 in rhein and hydroxyl at C6 in emodin might offer improvements on the anti-B. adolescentis activity, while the apolar substituent groups of methyl and methoxyl in chrysophanol and physicion might play a weakening role on antibacterial activity. So the influence of substituent groups on anti-B. adolescentis activity might be related with the polarity and the sequence was carboxyl > hydroxyl > hydroxylmethyl > methyl and methoxyl, which was almost consistent to the assay result on Staphylococcus aureus (Wu et al. 2005). Furthermore, the literature reported that the structural feature of a ketone and a hydroxyl group in neighborhood on the anthraquinone nucleus was important to the antimicrobial effect, i.e. l,3,8-trihydroxy-9,10-dihydroanthracene (Fig. 1) having the similar structure to emodin but without a ketone group revealed poor restraining effect and 9,10-anthraquinone (Fig. 1) without hydroxyl group revealed no restraining effect at all (Chen et al. 1962). And through comparison between the three benzoic acid derivatives and the HAQs, we found that the rigid planar structure of ketone and hydroxyl groups in neighborhood was also important to the antimicrobial ability of these compounds.

[FIGURE 1 OMITTED]

In summary, the inhibition effects of the five HAQs from R. palmatum on B. adolescentis growth were firstly evaluated by microcalorimetry in this study. All the five hydroxyanthraqui-nones revealed inhibitory effect on B. adolescentis growth, which might lead to imbalance of intestinal flora restraining the growth of B. adolescentis. The structure- antibacterial effect of the HAQs provides more references and insights for studying the action mechanism of these five natural components in R. palmatum. Based on comparison at same concentration, it was found that rhein was the most effective component in R. palmatum to restrain the growth of B. adolescentis.

New methods and approaches were needed in biological activity studies of drugs and other compounds and for the development of biological activity test systems. The data presented in this study indicated the potential applicability and development prospect of microcalorimetry for determining the influence of drugs and other compounds on different microbes.

Acknowledgement

The study was supported by grants from the National Natural Science Foundation of China (No. 30625042 and 30973947) and the National Basic Research Program of China (2007CB512607).

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J. Wang (a), H. Zhao (a), (b), W. Kong (a), (b), C. Jin (a), Y. Zhao (a), Y. Qu (a), X. Xiao (a), *

(a) China Military institute of Chinese Materia Medico, 302 Military Hospital, Beijing 100039, PR China

(b) Chengdu University of Traditional Chinese Medicine, Chengdu 610075, PR China

* Corresponding author. Tel.: +86 10 66933322; fax: +86 10 63879915.

E-mail addresses: pharmacy302@126.com. pharm_sci@126.com (X. Xiao).

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doi: 10.1016/j.phymed.2009.10.009
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Author:Wang, J.; Zhao, H.; Kong, W.; Jin, C.; Zhao, Y.; Qu, Y.; Xiao, X.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jul 1, 2010
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