Chinese Journal of Natural Medicines 2014, 12(2): 01080113
Chinese Journal of Natural Medicines
Comparison of anti-bacterial activity of three types of di-O-caffeoylquinic acids in Lonicera japonica flowers based on microcalorimetry HAN Jin1#, LV Qing-Yuan1#, JIN Shi-Ying1, ZHANG Tian-Tian1, JIN Shi-Xiao1, LI Xian-Yi2*, YUAN Hai-Long1* 1 2
302 Military Hospital of China, Beijing 100039, China; Institute for Drug and Instrument Control, Health Department, GLD of PLA, Beijing 100071, China Available online 20 Feb. 2014
[ABSTRACT] The anti-bacterial activities of three types of di-O-caffeoylquinic acids (diCQAs) in Lonicera japonica flowers, a traditional Chinese medicine (TCM), on Bacillus shigae growth were investigated and compared by microcalorimetry. The three types of diCQAs were 3, 4-di-O-caffeoylquinic acid (3, 4-diCQA), 3, 5-di-O-caffeoylquinic acid (3, 5-diCQA), and 4, 5-di-O-caffeoylquinic acid (4, 5-diCQA). Some qualitative and quantitative information of the effects of the three diCQAs on metabolic power–time curves, growth rate constant k, maximum heat-output power P m , and the generation time t G , total heat output Q t , and growth inhibitory ratio I of B. shigae were calculated. In accordance with a thermo-kinetic model, the corresponding quantitative relationships of k, P m , Qt, I and c were established. Also, the half-inhibitory concentrations of the drugs (IC 50 ) were obtained by quantitative analysis. Based on the quantity–activity relationships and the IC 50 values, the sequence of inhibitory activity was 3, 5-diCQA ! 4, 5-diCQA ! 3, 4-diCQA. The results illustrate the possibility that the caffeoyl ester group at C-5 is the principal group that has a higher affinity for the bacterial cell, and that the intramolecular distance of the two caffeoyl ester groups also has an important influence on the anti-bacterial activities of the diCQAs. [KEY WORDS] di-O-caffeoylquinic acids; Lonicera japonica; Bacillus shigae; Antibacterial activity; Microcalorimetry
[CLC Number] R285
[Document code] A
[Article ID] 2095-6975(2014)02-0108-06
Introduction Traditional Chinese medicine (TCM) has been attracting more and more attention in recent years because of its complementary therapeutic effects to Western medicines, and its ability to deal with many essential problems that have not yet been solved by advanced medical practices. Among the most commonly used herbal drugs in TCM, Lonicerae japonicae Flos, the flowers of Lonicera japonica Thunb. [Received on] 11-June-2012 [Research funding] This project was supported the National Natural Science Foundation of China (No. 81073069). [ Corresponding author] YUAN Hai-Long: Prof., Tel: 86-21-66933367, E-mail: [email protected]; LI Xian-Yi: Tel: 86-21-66949072, E-mail: [email protected]. # Co-first authors These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
(Caprifoliaceae), is officially listed in the Chinese Pharmacopoeia, with reported efficacy for the treatment of exopathogenic wind-heat, epidemic febrile diseases, sores, carbuncles, furuncles, and some infectious diseases. Caffeoyl quinic acid derivatives, as the major active components of the herb, are well-known as potential antioxidants [1-5] . Furthermore, they have also been reported to possess significant antitumor activity [6], antimicrobial activities [7], hepatoprotective activity [8-9], anti-inflammatory activity [10], and potential anti-human immunodeficiency virus (HIV) [11-13] activity, and so on. This category of compounds, with diverse and extensive distribution, are also important bioactive components in some other traditional Chinese medicines, such as Herba Artemisiae Scopariae, Propolis, and Cortex Eucommiae [14]. Structure–activity relationship studies showed that their antioxidant [5] , antitumor [6] , and hepatoprotective [9] activities increased in proportion to the number of caffeoyl groups. It was also concluded that the radical scavenging activity of natural dicaffeoylquinic acids
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in the biological aqueous system might depend on the positions of caffeoyl ester groups [15]. The microcalorimetry technique is used to track and record the evolution of energetic content. It has been demonstrated that calorimetric methods can be used for fundamental studies on bacterial growth. A flow of thermal effect is generated while the development of microbial activity is being affected by various substances. The flow of thermal energy is directly related to an increase or decrease in the energy released by a variety of sources [16-17]. This method only demands learning the initial and final energetic states, and is also independent of organisms and reaction pathways. In addition, slow reactions can be easily followed by a continuous recording of the signal for a long time without any disturbance to or from the system. One of the most prominent features of microbial progress is the production of heat. If the heat is monitored by a microcalorimeter, much useful information, both quantitative and qualitative, can be obtained. By analyzing the information, the activity and potency of drugs on microbial growth can be compared. Microcalorimetry provides a general analytical tool for the characterization of microbial growth progress, which has been used widely to investigate drug and the microbial cell interactions [18-22], but the investigation of caffeoyl quinic acid derivatives has not been reported. In this study, considering the similarity of their molecular structures, the inhibitory effects of three types of caffeoyl quinic acid derivatives from the flowers of Lonicera japonica (3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA), which belong to the di-O-caffeoylquinic acids (diCQAs, Fig. 1), were investigated and compared by means of microcalorimetry. Bacillus shigae was chosen for studying the effects of the three types of diCQAs, because of its sensitivity to L. japonica flowers [23]. The power–time curves (P–t curves) produced by B. shigae alone, and B. shigae under the action of the three diCQAs at different concentrations were determined. From the P–t curves, growth rate constant k, maximum power output P m , growth inhibitory ratio I, generation time t G , total heat output Q t , and the half-inbibitory concentration of the drugs (IC 50 value) were calculated. The experiments showed that the three diCQAs had different anti-bacterial activities on B. shigae.
Isothermal Calorimeter (Thermometric AB, Stockholm, Sweden) was used to measure the heat output of the metabolism of B. shigae. This isothermal microcalorimeter is an eight-channel twin instrument. In this calorimeter, each measuring cylinder normally contains a sample and a reference in separate measuring cups (twin system). The heat output flows from sample to a large heat sink (in close contact with the water bath) via the thermoelectric detector. A differential or twin detector system is used to minimize the systematic errors and disturbance effects. The detection limitation and baseline stability of the system are 2 and 6 μW (over a period of 24 h) [24-25]. Picolog software (Pico Technology Ltd.) was used to process data. Materials. The strain of B. shigae was provided by China Center of Type Culture Collection (Wuhan University, Wuhan, China). The lactose broth (LB) culture medium contained 5 gL1 ? L1 peptone, and 5 L1 yeast extract, and the LB culture medium used in experiment was freshly prepared. The sterilized LB culture medium did not display the phenomenon of releasing heat according to microcalorimetric monitoring, indicating that the oxidation of the culture medium did not take place in this case. Sample preparation. The diCQAs were extracted from Lonicera japonica flowers (authenticated by Professor XIAO Xiao-He (302 Military Hospital of China, Beijing)) using solvent extraction. Three types of diCQAs were purified utilizing preparative HPLC, and the structures were elucidated by UV, IR, MS, 1H, and 13C NMR in comparison with literature data [1, 26-27]. The purity of the tested compounds was determined to be more than 98% by normalization of the peak areas detected by UPLC with DAD, and they were stable in methanol solution at 4 °C. Their chemical structures are shown in Fig. 1. Experimental procedure. The metabolic thermogenic curves were drawn using a TAM Air Isothermal Calorimeter. Briefly, B. shigae suspension culture (1 mL) was added to LB culture medium (100 mL), and the bacterial suspension (10 mL) was placed in a glass ampoule (20 mL) which was precleaned and sterilized. The freshly prepared diCQAs aqueous solutions at different concentrations were added to the B. shigae suspension sequentially. The metabolic thermogenic curves of B. shigae growth in the presence of tested diCQAs were drawn by TAM automatically, which was used to monitor the process continuously until the recorder returned to the baseline. The temperature of the calorimeter system and the isothermal box were set at 37 °C.
Results Fig. 1 Chemical structures of the investigated caffeoyl quinic acid derivatives (CQAs ) from L. japonica flowers
Materials and Methods Apparatus. The Thermal Activity Monitor (TAM) Air
The growth power–time curve of B. shigae at 37 °C is shown in Fig. 2. It was a typical growth curve for B. shigae, and could be divided into four phases: the exponential phase, the lag phase, the stationary phase, and the decline phase. The curve shows the total metabolic profile of B. shigae.
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at different concentrations of drugs show that the heights of the highest peaks all declined with the increase of concentration of the diCQAs. However, the shapes of growth metabolism curves were basically the same in the presence or absence of diCQAs, i.e., the four phases still existed. The exponential model of B. shigae metabolism can be used to describe the growth phase [28]: P t = P 0 exp(kt) or ln P t = lnP 0 + kt (1) where t is the incubation time, P 0 was the heat output power at time 0, P t was the heat output power at time t and k is the constant of cell growth rate. Using Eq. (1), the rate constant of cell growth k was calculated by analyzing the experimental data of P t and t obtained from the cell growth curves to a linear equation. The growth rate constant k of B. shigae with the corresponding RSD is shown in Table 1, which indicated good reproducibility of the experiments.
Fig. 2 Growth power–time curve of B. shigae cultured in a peptone culture medium and monitored by a microcalorimeter at 37 ºC
Correspondingly, the P–t curves of B. shigae growth at 37 °C affected by different concentrations of the three diCQAs (3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA) were recorded and are shown in Fig. 3. These curves of B. shigae
Fig. 3 Growth power–time curves of B. shigae in the presence of three diCQAs and B. shigae cultured in a peptone culture medium with different concentrations of the three diCQAs, and monitored by a microcalorimeter at 37 ºC. (A) 3, 4-diCQA , (B) 3, 5-diCQA, (C) 4, 5-diCQA; (a) 25 μgmL1, (b) 50 μgmL1, (c) 100 μgmL1, (d) 200 μgmL1, (e) 300 μgmL1, (f) 400 μgmL1, (g) 500 μgmL1
Table 1 Growth rate constants (k) of B. shigae at 37 ºC without drug No.
a
1
2
3
4
5
6
Mean value
RSDa /%
k1
0.030 29
0.026 22
0.026 76
0.033 27
0.029 46
0.033 75
0.029 958
1.28
Rb
0.995 8
0.994 4
0.998 3
0.995 9
0.999 2
0.993 0
0.9961
0.233 6
Relative standard deviation
b
Correlation coefficient
Based on the above Eq. (1), the growth rate constants k of all the other experiments were calculated, and are listed in Table 2. From Table 2, it was found that the rate constants k all decreased with increasing concentrations c of the three types of diCQAs. The growth inhibitory ratio I was defined as: I = [(k 0 k c )/k 0 ] × 100% (2) where k 0 was the growth rate constant at concentration 0, as was k c at concentration c. When the inhibitory rate is 50%, the corresponding concentration of inhibitor was designated as the half-inhibitory concentration, the IC 50 . The IC 50 value is one of the most important indexes in the evaluation of the anti-bacterial activity of drugs, and is used to represent the sensitivity of bacteria to drugs 29). In
order to obtain the IC 50 values of the three types of diCQAs, the relationship between I and c was calculated in Table 2 and fitted in Fig. 4. For further evaluating the activities and investigating the quantity-activity relationship of the three diCQA types on B. shigae growth, the relationships of the quantitative thermokinetic parameters in Table 2 and the concentration c were also established, as shown in Fig. 4. k–c Relationship. The relationships between the rate constants k and concentrations c of the three types of diCQAs (Fig. 4A) showed that the diCQAs all inhibited the growth of B. shigae. They could be expressed by the following equations using the linear regression method: For 3, 4-diCQA: k = 5.5 × 105c + 0.030 3, R = 0.996 8 (0500 μg·mL1)
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Table 2 Thermal kinetic parameters of B. shigae growth in the presence of the tested diCQAs at 37 ºC CQAs
c/(μgmL1)
k
t G /min
P m /mV
Q t /J
I/%
Control 3, 4-diCQA
0 25 50 100 200 300 400 500 25 50 100 200 300 400 500 25 50 100 200 300 400 500
0.030 14 0.029 31 0.028 10 0.023 84 0.020 35 0.012 45 0.008 82 0.003 19 0.023 37 0.021 54 0.016 36 0.011 59 0.006 74 0.003 23 0.001 13 0.027 02 0.026 01 0.020 37 0.014 74 0.011 33 0.003 82 0.001 23
23.00 23.65 24.67 29.07 34.06 55.67 78.59 217.29 23.60 25.17 31.00 39.41 54.41 111.26 613.40 23.48 24.33 30.31 40.21 50.12 109.68 253.90
0.506 1 0.499 5 0.477 8 0.460 6 0.456 1 0.424 0 0.361 9 0.305 8 0.504 7 0.498 9 0.476 4 0.456 9 0.334 5 0.278 5 0.255 0 0.412 7 0.412 4 0.376 5 0.367 6 0.356 4 0.322 2 0.298 6
0.120 7 0.116 9 0.112 7 0.105 6 0.082 2 0.067 9 0.063 4 0.060 6 0.115 7 0.105 8 0.082 2 0.055 4 0.048 7 0.043 1 0.041 7 0.119 3 0.116 0 0.091 6 0.085 1 0.059 7 0.056 2 0.055 4
0 2.754 6.768 20.90 32.48 58.69 70.74 89.42 22.46 28.53 45.72 61.55 77.64 89.28 96.25 10.35 13.70 32.42 51.09 62.41 87.33 95.92
3, 5-diCQA
4, 5-diCQA
IC 50 /(μgmL1) 278.27
159.91
222.45
Fig. 4 Relationships between some thermokinetic parameters and concentration c of 3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA. (A) Relationship between k and c of the three diCQAs, (B) Relationship between P m and c of the three diCQAs, (C) Relationship between Q t and c of the three diCQAs
For 3, 5-diCQA: k = 5.3 × 105 c + 0.024 8, R = 0.960 4 (0500 μg·mL1) For 4, 5-diCQA: k = 5.8 × 105 c + 0.028 2, R = 0.989 2 (0500 μg·mL1) The good linearity with R > 0.960 0 of the kc relationship for the three diCQAs showed that the k values were almost linearly decreased with the increase in the concentrations of 3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA. P m –c Relationship. The curves of B. shigae at different concentrations of drugs (Fig. 3) show that the heights of the highest peaks all declined with the increase of concentrations of diCQAs. The tendency could be reflected from the values of P m in Table 2, and the relationships in Fig. 4B. The relationships could be deduced by fitting P m and k to a linear equation: For 3, 4- diCQA: P m = 6.621 2c + 0.307 2, R = 0.967 9 (0500 μg·mL1) For 3, 5-diCQA: P m = 9.456 1c + 0.240 0, R = 0.969 3 (0500 μg·mL1)
For 4, 5-diCQA: P m = 5.339 6c + 0.280 7, R = 0.888 9 (0500 μg·mL1) The above equations showed that the relationships of P m c for 3, 4-diCQA and 3, 5-diCQA were satisfied with R > 0.967 0, while for 4, 5- diCQA it was not so good with R < 0.900 0. Q t -c Relationship. By analyzing the values of Qt in the Table 2 and the curves in Fig. 4C, it could be seen that the total heat output Qt of growth phase all decreased with increasing concentrations of 3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA. These data illustrated that the growth of B. shigae was inhibited and that the total heat output decreased accordingly. The Q t -c equations could be described as below: For 3, 4-diCQA: Qt = 4 × 104 c + 0.508 5,
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R = 0.974 2 (0500 μg·ml1) For 3, 5-diCQA: Qt = 6 × 104 c + 0.524 1, R = 0.977 8 (0500 μg·ml1) For 4, 5-diCQA: Qt = 3 × 104 c + 0.441 3, R = 0.883 2 (0500 μg·ml1)
HAN Jin, et al. / Chin J Nat Med, 2014, 12(2): 108113
The above equations showed that the relationships of Q t -c for 3, 4-diCQA and 3, 5-diCQA were satisfied with R > 0.974 0, while for 4, 5-diCQA it was not so good with R < 0.900 0. I-c Relationship and IC 50 value. Based on the above analysis, it was found that in the concentration range 25500 μg mL-1, with increasing concentration of the three diCQAs, the growth rate constant k, the maximum heat-out power P m , and the total heat output Q t decreased, suggesting that 3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA all had strong inhibitory activities on the growth of B. shigae. The relationships between I and c are shown in Fig. 5. The I-c equations and the values of IC 50 for the three diCQAs were obtained as follows: For 3, 4-diCQA: I = 0.181 6c0.532 7, R = 0.996 8 (0500 μg·mL1), IC 50 = 278.27μg·mL1 For 3, 5-diCQA: I = 0.156 9c + 24.910, R = 0.980 4 (0500 μg·mL1), IC 50 = 159.91 μg·mL 1 For 4, 5-diCQA: I = 0.183 9c + 9.090 6, R = 0.990 1 (0500 μg·mL1) , IC 50 = 222.45 μg·mL1 With R > 0.980 0, the relationships between I and c were all nearly linear, and the quantitative quantity–activity relationships (QQASs) of I and c were satisfied for the three diCQAs, showing that the inhibitory activities on the growth of B. shigae were enhanced with the increase of concentrations of 3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA. From the values of IC 50 , it could be seen that the sequence of inhibitory effects of the three diCQAs on B. shigae was: 3, 5-diCQA > 4, 5-diCQA > 3, 4-diCQA. 3, 5-diCQA had the lowest value of IC 50 (159.91 μg·mL1), while 3, 4-diCQA had the highest value of IC 50 (278.27 μg·mL1) among the three diCQAs. ˉ
Fig. 5
Relationship between I and c of the three diCQAs
From the results of this study, it could be seen that in comparison with control, the values of k, P m , and Q t were decreased, and t G and I prolonged and increased, suggesting that the anti-bacterial activity of the drugs was enhanced. From the P-t curves in Fig. 3 and the thermal kinetic parameters in Table 2, it could be seen that the anti-bacterial activities varied with different diCQAs, and that 3, 5-diCQA gave the best
anti-bacterial activity, while 3, 4-diCQA showed the weakest anti-bacterial activity on B. shigae growth.
Discussion This study shows that microcalorimetry is a powerful tool for monitoring the kinetics of bacterial growth and estimating the bioactivity of drugs, and thereby studying the quantitative quantity–activity relationships of these drugs. The growth power–time curves of B. shigae in the presence of the three types of diCQAs indicated that all of the tested compounds had inhibitory effects on the tested bacteria. The height of the highest peak of B. shigae growth declined with increasing concentrations of the three diCQAs, at the same time, the total heat output also decreased. Furthermore, the appearance time of the maximum peak of bacterial growth was longer with increasing concentrations of the diCQAs, indicating that the bacteria took a longer time to generate a detectable signal. These results probably were because the excess diCQAs inhibited the growth of B. shigae or killed the bacteria. Such information is significant for the study on Lonicerae japonicae Flos and other traditional Chinese medicines. Based on both the quantity-activity relationships and median inhibitory concentration (IC 50 ), the order sequence of inhibitory activity was: 3, 5-diCQA ˚ 4, 5-diCQA ˚ 3, 4- diCQA. The results indicated that the specific differences in the chemical structures of the diCQAs influenced the growth of B. shigae. The three drugs have isomeric molecular structures comprised of one quinic acid group and two caffeoyl groups, and the latter is considered to be the main and active moiety of caffeoyl quinic acid derivatives [14]. However, the different positions of the caffeoyl ester groups on the cyclohexane ring at C-3, C-4, and C-5 result in different anti-bacterial activities for these diCQAs. The presence of caffeoyl ester groups at both C-3 and C-5 on the cyclohexane ring improved the anti-bacterial activity more strongly than when it was at both C-4 and C-5. Placing the caffeoyl ester group at both C-3 and C-4 on the cyclohexane ring shows the poorest activity. These results illustrated the possibility that the caffeoyl ester group at C-5 is the principal group that has a higher affinity for the bacterial cell, and that the intramolecular distance between the two caffeoyl ester groups in the molecule has an important influence on the anti-bacterial activities of the diCQAs. The latter result was in agreement with the conclusions of the previous structure-activity relationship investigations [5-6, 9, 15, 30]. It was also established that microcalorimetry can be used as a method to elucidate the pharmacologic and pharmacodynamic properties of Lonicerae japonicae Flos and its ingredients. Further studies will be focused on the mechanism of action of these different inhibitory activities of the diCQAs on B. shigae.
References
– 112 –
[1]
Hung TM, Na MK, Thuong PT, et al. Antioxidant activity of caffeoyl quinic acid derivatives from the roots of Dipsacus
HAN Jin, et al. / Chin J Nat Med, 2014, 12(2): 108113
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
asper Wall [J]. J Ethnopharmacol, 2006, 108 (2): 188-192. Iwai K, Kishimoto N, Kakino Y, et al. In vitro antioxidative effects and tyrosinase inhibitory activities of seven hydroxycinnamoyl derivatives in green coffee beans [J]. J Agric Food Chem, 2004, 52 (15): 4893-4898. Góngora L, Máñez S, Gíner RM, et al. Inhibition of xanthine oxidase by phenolic conjugates of methylated quinic acid [J]. Planta Med, 2003, 69 (5): 396-401. Nakatani N, Kayano S, Kikuzaki H, et al. Identification, quantitative determination, and antioxidative activities of chlorogenic acid isomers in prune (Prunus domestica L.) [J]. J Agric Food Chem, 2006, 48 (11): 5512-5516. Maruta Y, Kawabata J, Niki R. Antioxidative caffeoylquinic acid derivatives in the roots of Burk (Arctium lappa L.) [J]. J Agric Food Chem, 1995, 43 (10): 2592-2595. Mishima S, Inoh Y, Narita Y, et al. Identification of caffeoylquinic acid derivatives from Brazilian propolis as constituents involved in induction of granulocytic differentiation of HL-60 cells [J]. Bioorg Med Chem, 2005, 13 (20): 5814-5818. Zhu X, Zhang H, Lo R. Phenolic compounds from the leaf extracto f artichoke (Cynara scolymus L.) and their antimicrobial activities [J]. J Agric Food Chem, 2004, 52 (10): 7272-7278. Basnet P, Matsushige K, Hase K, et al. Four di-O-caffeoyl quinic acid derivatives from propolis. Potent hepatoprotective activity in experimental liver injury models [J]. Biol Pharm Bull, 1996, 19 (11): 1479-1484. Xiang T, Xiong QB, Ketut AI, et al. Studies on the hepatocyte protective activity and the structure-activity relationships of quinic acid and caffeic acid derivatives from the flower buds of Lonicera bournei [J]. Planta Med, 2001, 67 (4): 322-325. Góngora L, Máñez S, Gíner RM, et al. Inhibition of xanthine oxidase by phenolic conjugates of methylated quinic acid [J]. Planta Med, 2003, 69 (5): 396-401. Robinson WE Jr, Cordeiro M, Abdel-Malek S, et al. Dicaffeoylquinic acid inhibitors of human immunodeficiency virus integrase: inhibition of the core catalytic domain of human immunodeficiency virus integrase [J]. Mol Pharmacol, 1996, 50 (4): 846-855. Zhu K, Cordeiro ML, Atienza J, et al. Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids [J]. J Virol, 1999, 73 (4): 3309-3316. Yang B, Meng ZY, Dong JX, et al. Metabolic profile of 1,5-dicaffeoylquinic acid in rats, an in vivo and in vitro study [J]. Drug Metab Dispos, 2005, 33 (7): 930-936. Tang D, Li HJ, Li P, et al. Interaction of bioactive components caffeoylquinic acid derivatives in Chinese medicines with bovine serum albumin [J]. Chem Pharm Bull, 2008, 56 (3): 360-365. Saito S, Kurakane S, Seki M, et al. Radical scavenging activity of dicaffeoyloxycyclohexanes: Contribution of an intramolecular interaction of two caffeoyl residues [J]. Bioorg Med Chem, 2005, 13 (13): 4191-4199. Liu P, Liu Y, Xie ZX, et al. Microcalorimetric studies of the
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
toxic action of La3+ on Halobacterium halobium R1 growth [J]. Chin J Chem, 2003, 21 (6): 693-697. Kong WJ, Zhao YL, Shan LM, et al. Investigation of the effect of four organic acids in Radix Isatidis on E. coli growth by microcalorimetry [J]. Chin J Chem, 2008, 26 (1): 113-115. Tang HY, Yan D, Zhang QZ, et al. Microcalorimetric investigation of four bioactive extracts from Cornu Cervi Pantotrichum on intestinal diagnostic flora growth [J]. Acta Phys-Chim Sin, 2010, 26 (5): 1442-1447. Han YM, Yan D, Zhao YL, et al. Toxic effects of protoberberine alkaloids from Rhizoma Coptidis on Tetrahymena thermophila BF5 growth based on microcalorimetry [J]. J Therm Anal Calorim, 2012, 108 (1): 341-346. Dai CM, Wang JB, Kong WJ, et al. Investigation of anti-microbial activity of catechin on Escherichia coli growth by microcalorimetry [J]. Environ Toxicol Pharmacol, 2010, 30 (3): 284-288. Kong WJ, Zhao YL, Xiao XH, et al. Investigation of the anti-fungal activity of coptisine on Candida albicans growth by microcalorimetry combined with principal component analysis [J]. J Appl Microbiol, 2009, 107 (4): 1072-1080. Kong WJ, Zhao YL, Wang JB, et al. Investigation on the anti-Candida albicans effect of palmatine hydrochloride based on microcalorimetry and principal component analysis [J]. Acta Chim Sin, 2009, 67 (21): 2511-2516. Fu SS, Zhang TT, Lv JL, et al. Comparison of microcalorimetric fingerprint profiles of Lonicerae japonicae Flos and Lonicerae Flos [J]. Acta Pharm Sin, 2011, 46 (10): 1251-1256. Kong WJ, Zhao YL, Shan LM, et al. Thermochemical studies on the quantity-antibacterial effect relationship of four organic acids from Radix Isatidis on Escherichia coli growth [J]. Biol Pharm Bull, 2008, 31 (7): 1301-1305. Wadso I. Microcalorimetric techniques for characterization of living cellular systems. Will there be any important practical applications [J] ? Thermochim Acta, 1995, 269-270 (20): 337- 350. Li YL, Paul PHB, Vincent ECO, et al. Antiviral activity and mode of action of caffeoylquinic acids from Schefflera heptaphylla (L.) Frodin [J]. Antiviral Res, 2005, 68 (1): 1-9. Dos Santos MD, Gobbo-Neto L, Albarella L, et al. Analgesic activity of di-caffeoylquinic acids from roots of Lychnophora ericoides (Arnica da Serra) [J]. J Ethnopharmacol, 2005, 96 (3): 545-549. Li X, Liu Y, Wu J, et al. Microcalorimetric study of Staphylococcus aureus growth affected by selenium compounds [J]. Thermochim Acta, 2002, 387 (1): 57-61. Sen XS, Liu Y, Zhou CP, et al. Thermochemical studies on the quantity-antibacterial effect relationship of fluoroquinolones [J]. Acta Chim Sin, 2000, 58 (11), 1463-1468. Yoshimoto M, Yahara S, Okuno S, et al. Antimutagenicity of mono-, di-, and tricaffeoylquinic acid derivatives isolated from sweet potato (Ipomoea batatas L.) leaf [J]. Biosci Biotechnol Biochem, 2002, 66 (11): 2336-2341.
Cite this article as: HAN Jin, LV Qing-Yuan, JIN Shi-Ying, ZHANG Tian-Tian, JIN Shi-Xiao, LI Xian-Yi, YUAN Hai-Long. Comparison of anti-bacterial activity of three types of di-O-caffeoylquinic acids in Lonicera japonica flowers based on microcalorimetry [J]. Chinese Journal of Natural Medicines, 2014, 12(2): 108-113
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Chinese Journal of Natural Medicines
Comparison of anti-bacterial activity of three types of di-O-caffeoylquinic acids in Lonicera japonica flowers based on microcalorimetry HAN Jin1#, LV Qing-Yuan1#, JIN Shi-Ying1, ZHANG Tian-Tian1, JIN Shi-Xiao1, LI Xian-Yi2*, YUAN Hai-Long1* 1 2
302 Military Hospital of China, Beijing 100039, China; Institute for Drug and Instrument Control, Health Department, GLD of PLA, Beijing 100071, China Available online 20 Feb. 2014
[ABSTRACT] The anti-bacterial activities of three types of di-O-caffeoylquinic acids (diCQAs) in Lonicera japonica flowers, a traditional Chinese medicine (TCM), on Bacillus shigae growth were investigated and compared by microcalorimetry. The three types of diCQAs were 3, 4-di-O-caffeoylquinic acid (3, 4-diCQA), 3, 5-di-O-caffeoylquinic acid (3, 5-diCQA), and 4, 5-di-O-caffeoylquinic acid (4, 5-diCQA). Some qualitative and quantitative information of the effects of the three diCQAs on metabolic power–time curves, growth rate constant k, maximum heat-output power P m , and the generation time t G , total heat output Q t , and growth inhibitory ratio I of B. shigae were calculated. In accordance with a thermo-kinetic model, the corresponding quantitative relationships of k, P m , Qt, I and c were established. Also, the half-inhibitory concentrations of the drugs (IC 50 ) were obtained by quantitative analysis. Based on the quantity–activity relationships and the IC 50 values, the sequence of inhibitory activity was 3, 5-diCQA ! 4, 5-diCQA ! 3, 4-diCQA. The results illustrate the possibility that the caffeoyl ester group at C-5 is the principal group that has a higher affinity for the bacterial cell, and that the intramolecular distance of the two caffeoyl ester groups also has an important influence on the anti-bacterial activities of the diCQAs. [KEY WORDS] di-O-caffeoylquinic acids; Lonicera japonica; Bacillus shigae; Antibacterial activity; Microcalorimetry
[CLC Number] R285
[Document code] A
[Article ID] 2095-6975(2014)02-0108-06
Introduction Traditional Chinese medicine (TCM) has been attracting more and more attention in recent years because of its complementary therapeutic effects to Western medicines, and its ability to deal with many essential problems that have not yet been solved by advanced medical practices. Among the most commonly used herbal drugs in TCM, Lonicerae japonicae Flos, the flowers of Lonicera japonica Thunb. [Received on] 11-June-2012 [Research funding] This project was supported the National Natural Science Foundation of China (No. 81073069). [ Corresponding author] YUAN Hai-Long: Prof., Tel: 86-21-66933367, E-mail: [email protected]; LI Xian-Yi: Tel: 86-21-66949072, E-mail: [email protected]. # Co-first authors These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
(Caprifoliaceae), is officially listed in the Chinese Pharmacopoeia, with reported efficacy for the treatment of exopathogenic wind-heat, epidemic febrile diseases, sores, carbuncles, furuncles, and some infectious diseases. Caffeoyl quinic acid derivatives, as the major active components of the herb, are well-known as potential antioxidants [1-5] . Furthermore, they have also been reported to possess significant antitumor activity [6], antimicrobial activities [7], hepatoprotective activity [8-9], anti-inflammatory activity [10], and potential anti-human immunodeficiency virus (HIV) [11-13] activity, and so on. This category of compounds, with diverse and extensive distribution, are also important bioactive components in some other traditional Chinese medicines, such as Herba Artemisiae Scopariae, Propolis, and Cortex Eucommiae [14]. Structure–activity relationship studies showed that their antioxidant [5] , antitumor [6] , and hepatoprotective [9] activities increased in proportion to the number of caffeoyl groups. It was also concluded that the radical scavenging activity of natural dicaffeoylquinic acids
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in the biological aqueous system might depend on the positions of caffeoyl ester groups [15]. The microcalorimetry technique is used to track and record the evolution of energetic content. It has been demonstrated that calorimetric methods can be used for fundamental studies on bacterial growth. A flow of thermal effect is generated while the development of microbial activity is being affected by various substances. The flow of thermal energy is directly related to an increase or decrease in the energy released by a variety of sources [16-17]. This method only demands learning the initial and final energetic states, and is also independent of organisms and reaction pathways. In addition, slow reactions can be easily followed by a continuous recording of the signal for a long time without any disturbance to or from the system. One of the most prominent features of microbial progress is the production of heat. If the heat is monitored by a microcalorimeter, much useful information, both quantitative and qualitative, can be obtained. By analyzing the information, the activity and potency of drugs on microbial growth can be compared. Microcalorimetry provides a general analytical tool for the characterization of microbial growth progress, which has been used widely to investigate drug and the microbial cell interactions [18-22], but the investigation of caffeoyl quinic acid derivatives has not been reported. In this study, considering the similarity of their molecular structures, the inhibitory effects of three types of caffeoyl quinic acid derivatives from the flowers of Lonicera japonica (3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA), which belong to the di-O-caffeoylquinic acids (diCQAs, Fig. 1), were investigated and compared by means of microcalorimetry. Bacillus shigae was chosen for studying the effects of the three types of diCQAs, because of its sensitivity to L. japonica flowers [23]. The power–time curves (P–t curves) produced by B. shigae alone, and B. shigae under the action of the three diCQAs at different concentrations were determined. From the P–t curves, growth rate constant k, maximum power output P m , growth inhibitory ratio I, generation time t G , total heat output Q t , and the half-inbibitory concentration of the drugs (IC 50 value) were calculated. The experiments showed that the three diCQAs had different anti-bacterial activities on B. shigae.
Isothermal Calorimeter (Thermometric AB, Stockholm, Sweden) was used to measure the heat output of the metabolism of B. shigae. This isothermal microcalorimeter is an eight-channel twin instrument. In this calorimeter, each measuring cylinder normally contains a sample and a reference in separate measuring cups (twin system). The heat output flows from sample to a large heat sink (in close contact with the water bath) via the thermoelectric detector. A differential or twin detector system is used to minimize the systematic errors and disturbance effects. The detection limitation and baseline stability of the system are 2 and 6 μW (over a period of 24 h) [24-25]. Picolog software (Pico Technology Ltd.) was used to process data. Materials. The strain of B. shigae was provided by China Center of Type Culture Collection (Wuhan University, Wuhan, China). The lactose broth (LB) culture medium contained 5 gL1 ? L1 peptone, and 5 L1 yeast extract, and the LB culture medium used in experiment was freshly prepared. The sterilized LB culture medium did not display the phenomenon of releasing heat according to microcalorimetric monitoring, indicating that the oxidation of the culture medium did not take place in this case. Sample preparation. The diCQAs were extracted from Lonicera japonica flowers (authenticated by Professor XIAO Xiao-He (302 Military Hospital of China, Beijing)) using solvent extraction. Three types of diCQAs were purified utilizing preparative HPLC, and the structures were elucidated by UV, IR, MS, 1H, and 13C NMR in comparison with literature data [1, 26-27]. The purity of the tested compounds was determined to be more than 98% by normalization of the peak areas detected by UPLC with DAD, and they were stable in methanol solution at 4 °C. Their chemical structures are shown in Fig. 1. Experimental procedure. The metabolic thermogenic curves were drawn using a TAM Air Isothermal Calorimeter. Briefly, B. shigae suspension culture (1 mL) was added to LB culture medium (100 mL), and the bacterial suspension (10 mL) was placed in a glass ampoule (20 mL) which was precleaned and sterilized. The freshly prepared diCQAs aqueous solutions at different concentrations were added to the B. shigae suspension sequentially. The metabolic thermogenic curves of B. shigae growth in the presence of tested diCQAs were drawn by TAM automatically, which was used to monitor the process continuously until the recorder returned to the baseline. The temperature of the calorimeter system and the isothermal box were set at 37 °C.
Results Fig. 1 Chemical structures of the investigated caffeoyl quinic acid derivatives (CQAs ) from L. japonica flowers
Materials and Methods Apparatus. The Thermal Activity Monitor (TAM) Air
The growth power–time curve of B. shigae at 37 °C is shown in Fig. 2. It was a typical growth curve for B. shigae, and could be divided into four phases: the exponential phase, the lag phase, the stationary phase, and the decline phase. The curve shows the total metabolic profile of B. shigae.
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at different concentrations of drugs show that the heights of the highest peaks all declined with the increase of concentration of the diCQAs. However, the shapes of growth metabolism curves were basically the same in the presence or absence of diCQAs, i.e., the four phases still existed. The exponential model of B. shigae metabolism can be used to describe the growth phase [28]: P t = P 0 exp(kt) or ln P t = lnP 0 + kt (1) where t is the incubation time, P 0 was the heat output power at time 0, P t was the heat output power at time t and k is the constant of cell growth rate. Using Eq. (1), the rate constant of cell growth k was calculated by analyzing the experimental data of P t and t obtained from the cell growth curves to a linear equation. The growth rate constant k of B. shigae with the corresponding RSD is shown in Table 1, which indicated good reproducibility of the experiments.
Fig. 2 Growth power–time curve of B. shigae cultured in a peptone culture medium and monitored by a microcalorimeter at 37 ºC
Correspondingly, the P–t curves of B. shigae growth at 37 °C affected by different concentrations of the three diCQAs (3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA) were recorded and are shown in Fig. 3. These curves of B. shigae
Fig. 3 Growth power–time curves of B. shigae in the presence of three diCQAs and B. shigae cultured in a peptone culture medium with different concentrations of the three diCQAs, and monitored by a microcalorimeter at 37 ºC. (A) 3, 4-diCQA , (B) 3, 5-diCQA, (C) 4, 5-diCQA; (a) 25 μgmL1, (b) 50 μgmL1, (c) 100 μgmL1, (d) 200 μgmL1, (e) 300 μgmL1, (f) 400 μgmL1, (g) 500 μgmL1
Table 1 Growth rate constants (k) of B. shigae at 37 ºC without drug No.
a
1
2
3
4
5
6
Mean value
RSDa /%
k1
0.030 29
0.026 22
0.026 76
0.033 27
0.029 46
0.033 75
0.029 958
1.28
Rb
0.995 8
0.994 4
0.998 3
0.995 9
0.999 2
0.993 0
0.9961
0.233 6
Relative standard deviation
b
Correlation coefficient
Based on the above Eq. (1), the growth rate constants k of all the other experiments were calculated, and are listed in Table 2. From Table 2, it was found that the rate constants k all decreased with increasing concentrations c of the three types of diCQAs. The growth inhibitory ratio I was defined as: I = [(k 0 k c )/k 0 ] × 100% (2) where k 0 was the growth rate constant at concentration 0, as was k c at concentration c. When the inhibitory rate is 50%, the corresponding concentration of inhibitor was designated as the half-inhibitory concentration, the IC 50 . The IC 50 value is one of the most important indexes in the evaluation of the anti-bacterial activity of drugs, and is used to represent the sensitivity of bacteria to drugs 29). In
order to obtain the IC 50 values of the three types of diCQAs, the relationship between I and c was calculated in Table 2 and fitted in Fig. 4. For further evaluating the activities and investigating the quantity-activity relationship of the three diCQA types on B. shigae growth, the relationships of the quantitative thermokinetic parameters in Table 2 and the concentration c were also established, as shown in Fig. 4. k–c Relationship. The relationships between the rate constants k and concentrations c of the three types of diCQAs (Fig. 4A) showed that the diCQAs all inhibited the growth of B. shigae. They could be expressed by the following equations using the linear regression method: For 3, 4-diCQA: k = 5.5 × 105c + 0.030 3, R = 0.996 8 (0500 μg·mL1)
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Table 2 Thermal kinetic parameters of B. shigae growth in the presence of the tested diCQAs at 37 ºC CQAs
c/(μgmL1)
k
t G /min
P m /mV
Q t /J
I/%
Control 3, 4-diCQA
0 25 50 100 200 300 400 500 25 50 100 200 300 400 500 25 50 100 200 300 400 500
0.030 14 0.029 31 0.028 10 0.023 84 0.020 35 0.012 45 0.008 82 0.003 19 0.023 37 0.021 54 0.016 36 0.011 59 0.006 74 0.003 23 0.001 13 0.027 02 0.026 01 0.020 37 0.014 74 0.011 33 0.003 82 0.001 23
23.00 23.65 24.67 29.07 34.06 55.67 78.59 217.29 23.60 25.17 31.00 39.41 54.41 111.26 613.40 23.48 24.33 30.31 40.21 50.12 109.68 253.90
0.506 1 0.499 5 0.477 8 0.460 6 0.456 1 0.424 0 0.361 9 0.305 8 0.504 7 0.498 9 0.476 4 0.456 9 0.334 5 0.278 5 0.255 0 0.412 7 0.412 4 0.376 5 0.367 6 0.356 4 0.322 2 0.298 6
0.120 7 0.116 9 0.112 7 0.105 6 0.082 2 0.067 9 0.063 4 0.060 6 0.115 7 0.105 8 0.082 2 0.055 4 0.048 7 0.043 1 0.041 7 0.119 3 0.116 0 0.091 6 0.085 1 0.059 7 0.056 2 0.055 4
0 2.754 6.768 20.90 32.48 58.69 70.74 89.42 22.46 28.53 45.72 61.55 77.64 89.28 96.25 10.35 13.70 32.42 51.09 62.41 87.33 95.92
3, 5-diCQA
4, 5-diCQA
IC 50 /(μgmL1) 278.27
159.91
222.45
Fig. 4 Relationships between some thermokinetic parameters and concentration c of 3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA. (A) Relationship between k and c of the three diCQAs, (B) Relationship between P m and c of the three diCQAs, (C) Relationship between Q t and c of the three diCQAs
For 3, 5-diCQA: k = 5.3 × 105 c + 0.024 8, R = 0.960 4 (0500 μg·mL1) For 4, 5-diCQA: k = 5.8 × 105 c + 0.028 2, R = 0.989 2 (0500 μg·mL1) The good linearity with R > 0.960 0 of the kc relationship for the three diCQAs showed that the k values were almost linearly decreased with the increase in the concentrations of 3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA. P m –c Relationship. The curves of B. shigae at different concentrations of drugs (Fig. 3) show that the heights of the highest peaks all declined with the increase of concentrations of diCQAs. The tendency could be reflected from the values of P m in Table 2, and the relationships in Fig. 4B. The relationships could be deduced by fitting P m and k to a linear equation: For 3, 4- diCQA: P m = 6.621 2c + 0.307 2, R = 0.967 9 (0500 μg·mL1) For 3, 5-diCQA: P m = 9.456 1c + 0.240 0, R = 0.969 3 (0500 μg·mL1)
For 4, 5-diCQA: P m = 5.339 6c + 0.280 7, R = 0.888 9 (0500 μg·mL1) The above equations showed that the relationships of P m c for 3, 4-diCQA and 3, 5-diCQA were satisfied with R > 0.967 0, while for 4, 5- diCQA it was not so good with R < 0.900 0. Q t -c Relationship. By analyzing the values of Qt in the Table 2 and the curves in Fig. 4C, it could be seen that the total heat output Qt of growth phase all decreased with increasing concentrations of 3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA. These data illustrated that the growth of B. shigae was inhibited and that the total heat output decreased accordingly. The Q t -c equations could be described as below: For 3, 4-diCQA: Qt = 4 × 104 c + 0.508 5,
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R = 0.974 2 (0500 μg·ml1) For 3, 5-diCQA: Qt = 6 × 104 c + 0.524 1, R = 0.977 8 (0500 μg·ml1) For 4, 5-diCQA: Qt = 3 × 104 c + 0.441 3, R = 0.883 2 (0500 μg·ml1)
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The above equations showed that the relationships of Q t -c for 3, 4-diCQA and 3, 5-diCQA were satisfied with R > 0.974 0, while for 4, 5-diCQA it was not so good with R < 0.900 0. I-c Relationship and IC 50 value. Based on the above analysis, it was found that in the concentration range 25500 μg mL-1, with increasing concentration of the three diCQAs, the growth rate constant k, the maximum heat-out power P m , and the total heat output Q t decreased, suggesting that 3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA all had strong inhibitory activities on the growth of B. shigae. The relationships between I and c are shown in Fig. 5. The I-c equations and the values of IC 50 for the three diCQAs were obtained as follows: For 3, 4-diCQA: I = 0.181 6c0.532 7, R = 0.996 8 (0500 μg·mL1), IC 50 = 278.27μg·mL1 For 3, 5-diCQA: I = 0.156 9c + 24.910, R = 0.980 4 (0500 μg·mL1), IC 50 = 159.91 μg·mL 1 For 4, 5-diCQA: I = 0.183 9c + 9.090 6, R = 0.990 1 (0500 μg·mL1) , IC 50 = 222.45 μg·mL1 With R > 0.980 0, the relationships between I and c were all nearly linear, and the quantitative quantity–activity relationships (QQASs) of I and c were satisfied for the three diCQAs, showing that the inhibitory activities on the growth of B. shigae were enhanced with the increase of concentrations of 3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA. From the values of IC 50 , it could be seen that the sequence of inhibitory effects of the three diCQAs on B. shigae was: 3, 5-diCQA > 4, 5-diCQA > 3, 4-diCQA. 3, 5-diCQA had the lowest value of IC 50 (159.91 μg·mL1), while 3, 4-diCQA had the highest value of IC 50 (278.27 μg·mL1) among the three diCQAs. ˉ
Fig. 5
Relationship between I and c of the three diCQAs
From the results of this study, it could be seen that in comparison with control, the values of k, P m , and Q t were decreased, and t G and I prolonged and increased, suggesting that the anti-bacterial activity of the drugs was enhanced. From the P-t curves in Fig. 3 and the thermal kinetic parameters in Table 2, it could be seen that the anti-bacterial activities varied with different diCQAs, and that 3, 5-diCQA gave the best
anti-bacterial activity, while 3, 4-diCQA showed the weakest anti-bacterial activity on B. shigae growth.
Discussion This study shows that microcalorimetry is a powerful tool for monitoring the kinetics of bacterial growth and estimating the bioactivity of drugs, and thereby studying the quantitative quantity–activity relationships of these drugs. The growth power–time curves of B. shigae in the presence of the three types of diCQAs indicated that all of the tested compounds had inhibitory effects on the tested bacteria. The height of the highest peak of B. shigae growth declined with increasing concentrations of the three diCQAs, at the same time, the total heat output also decreased. Furthermore, the appearance time of the maximum peak of bacterial growth was longer with increasing concentrations of the diCQAs, indicating that the bacteria took a longer time to generate a detectable signal. These results probably were because the excess diCQAs inhibited the growth of B. shigae or killed the bacteria. Such information is significant for the study on Lonicerae japonicae Flos and other traditional Chinese medicines. Based on both the quantity-activity relationships and median inhibitory concentration (IC 50 ), the order sequence of inhibitory activity was: 3, 5-diCQA ˚ 4, 5-diCQA ˚ 3, 4- diCQA. The results indicated that the specific differences in the chemical structures of the diCQAs influenced the growth of B. shigae. The three drugs have isomeric molecular structures comprised of one quinic acid group and two caffeoyl groups, and the latter is considered to be the main and active moiety of caffeoyl quinic acid derivatives [14]. However, the different positions of the caffeoyl ester groups on the cyclohexane ring at C-3, C-4, and C-5 result in different anti-bacterial activities for these diCQAs. The presence of caffeoyl ester groups at both C-3 and C-5 on the cyclohexane ring improved the anti-bacterial activity more strongly than when it was at both C-4 and C-5. Placing the caffeoyl ester group at both C-3 and C-4 on the cyclohexane ring shows the poorest activity. These results illustrated the possibility that the caffeoyl ester group at C-5 is the principal group that has a higher affinity for the bacterial cell, and that the intramolecular distance between the two caffeoyl ester groups in the molecule has an important influence on the anti-bacterial activities of the diCQAs. The latter result was in agreement with the conclusions of the previous structure-activity relationship investigations [5-6, 9, 15, 30]. It was also established that microcalorimetry can be used as a method to elucidate the pharmacologic and pharmacodynamic properties of Lonicerae japonicae Flos and its ingredients. Further studies will be focused on the mechanism of action of these different inhibitory activities of the diCQAs on B. shigae.
References
– 112 –
[1]
Hung TM, Na MK, Thuong PT, et al. Antioxidant activity of caffeoyl quinic acid derivatives from the roots of Dipsacus
HAN Jin, et al. / Chin J Nat Med, 2014, 12(2): 108113
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
asper Wall [J]. J Ethnopharmacol, 2006, 108 (2): 188-192. Iwai K, Kishimoto N, Kakino Y, et al. In vitro antioxidative effects and tyrosinase inhibitory activities of seven hydroxycinnamoyl derivatives in green coffee beans [J]. J Agric Food Chem, 2004, 52 (15): 4893-4898. Góngora L, Máñez S, Gíner RM, et al. Inhibition of xanthine oxidase by phenolic conjugates of methylated quinic acid [J]. Planta Med, 2003, 69 (5): 396-401. Nakatani N, Kayano S, Kikuzaki H, et al. Identification, quantitative determination, and antioxidative activities of chlorogenic acid isomers in prune (Prunus domestica L.) [J]. J Agric Food Chem, 2006, 48 (11): 5512-5516. Maruta Y, Kawabata J, Niki R. Antioxidative caffeoylquinic acid derivatives in the roots of Burk (Arctium lappa L.) [J]. J Agric Food Chem, 1995, 43 (10): 2592-2595. Mishima S, Inoh Y, Narita Y, et al. Identification of caffeoylquinic acid derivatives from Brazilian propolis as constituents involved in induction of granulocytic differentiation of HL-60 cells [J]. Bioorg Med Chem, 2005, 13 (20): 5814-5818. Zhu X, Zhang H, Lo R. Phenolic compounds from the leaf extracto f artichoke (Cynara scolymus L.) and their antimicrobial activities [J]. J Agric Food Chem, 2004, 52 (10): 7272-7278. Basnet P, Matsushige K, Hase K, et al. Four di-O-caffeoyl quinic acid derivatives from propolis. Potent hepatoprotective activity in experimental liver injury models [J]. Biol Pharm Bull, 1996, 19 (11): 1479-1484. Xiang T, Xiong QB, Ketut AI, et al. Studies on the hepatocyte protective activity and the structure-activity relationships of quinic acid and caffeic acid derivatives from the flower buds of Lonicera bournei [J]. Planta Med, 2001, 67 (4): 322-325. Góngora L, Máñez S, Gíner RM, et al. Inhibition of xanthine oxidase by phenolic conjugates of methylated quinic acid [J]. Planta Med, 2003, 69 (5): 396-401. Robinson WE Jr, Cordeiro M, Abdel-Malek S, et al. Dicaffeoylquinic acid inhibitors of human immunodeficiency virus integrase: inhibition of the core catalytic domain of human immunodeficiency virus integrase [J]. Mol Pharmacol, 1996, 50 (4): 846-855. Zhu K, Cordeiro ML, Atienza J, et al. Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids [J]. J Virol, 1999, 73 (4): 3309-3316. Yang B, Meng ZY, Dong JX, et al. Metabolic profile of 1,5-dicaffeoylquinic acid in rats, an in vivo and in vitro study [J]. Drug Metab Dispos, 2005, 33 (7): 930-936. Tang D, Li HJ, Li P, et al. Interaction of bioactive components caffeoylquinic acid derivatives in Chinese medicines with bovine serum albumin [J]. Chem Pharm Bull, 2008, 56 (3): 360-365. Saito S, Kurakane S, Seki M, et al. Radical scavenging activity of dicaffeoyloxycyclohexanes: Contribution of an intramolecular interaction of two caffeoyl residues [J]. Bioorg Med Chem, 2005, 13 (13): 4191-4199. Liu P, Liu Y, Xie ZX, et al. Microcalorimetric studies of the
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
toxic action of La3+ on Halobacterium halobium R1 growth [J]. Chin J Chem, 2003, 21 (6): 693-697. Kong WJ, Zhao YL, Shan LM, et al. Investigation of the effect of four organic acids in Radix Isatidis on E. coli growth by microcalorimetry [J]. Chin J Chem, 2008, 26 (1): 113-115. Tang HY, Yan D, Zhang QZ, et al. Microcalorimetric investigation of four bioactive extracts from Cornu Cervi Pantotrichum on intestinal diagnostic flora growth [J]. Acta Phys-Chim Sin, 2010, 26 (5): 1442-1447. Han YM, Yan D, Zhao YL, et al. Toxic effects of protoberberine alkaloids from Rhizoma Coptidis on Tetrahymena thermophila BF5 growth based on microcalorimetry [J]. J Therm Anal Calorim, 2012, 108 (1): 341-346. Dai CM, Wang JB, Kong WJ, et al. Investigation of anti-microbial activity of catechin on Escherichia coli growth by microcalorimetry [J]. Environ Toxicol Pharmacol, 2010, 30 (3): 284-288. Kong WJ, Zhao YL, Xiao XH, et al. Investigation of the anti-fungal activity of coptisine on Candida albicans growth by microcalorimetry combined with principal component analysis [J]. J Appl Microbiol, 2009, 107 (4): 1072-1080. Kong WJ, Zhao YL, Wang JB, et al. Investigation on the anti-Candida albicans effect of palmatine hydrochloride based on microcalorimetry and principal component analysis [J]. Acta Chim Sin, 2009, 67 (21): 2511-2516. Fu SS, Zhang TT, Lv JL, et al. Comparison of microcalorimetric fingerprint profiles of Lonicerae japonicae Flos and Lonicerae Flos [J]. Acta Pharm Sin, 2011, 46 (10): 1251-1256. Kong WJ, Zhao YL, Shan LM, et al. Thermochemical studies on the quantity-antibacterial effect relationship of four organic acids from Radix Isatidis on Escherichia coli growth [J]. Biol Pharm Bull, 2008, 31 (7): 1301-1305. Wadso I. Microcalorimetric techniques for characterization of living cellular systems. Will there be any important practical applications [J] ? Thermochim Acta, 1995, 269-270 (20): 337- 350. Li YL, Paul PHB, Vincent ECO, et al. Antiviral activity and mode of action of caffeoylquinic acids from Schefflera heptaphylla (L.) Frodin [J]. Antiviral Res, 2005, 68 (1): 1-9. Dos Santos MD, Gobbo-Neto L, Albarella L, et al. Analgesic activity of di-caffeoylquinic acids from roots of Lychnophora ericoides (Arnica da Serra) [J]. J Ethnopharmacol, 2005, 96 (3): 545-549. Li X, Liu Y, Wu J, et al. Microcalorimetric study of Staphylococcus aureus growth affected by selenium compounds [J]. Thermochim Acta, 2002, 387 (1): 57-61. Sen XS, Liu Y, Zhou CP, et al. Thermochemical studies on the quantity-antibacterial effect relationship of fluoroquinolones [J]. Acta Chim Sin, 2000, 58 (11), 1463-1468. Yoshimoto M, Yahara S, Okuno S, et al. Antimutagenicity of mono-, di-, and tricaffeoylquinic acid derivatives isolated from sweet potato (Ipomoea batatas L.) leaf [J]. Biosci Biotechnol Biochem, 2002, 66 (11): 2336-2341.
Cite this article as: HAN Jin, LV Qing-Yuan, JIN Shi-Ying, ZHANG Tian-Tian, JIN Shi-Xiao, LI Xian-Yi, YUAN Hai-Long. Comparison of anti-bacterial activity of three types of di-O-caffeoylquinic acids in Lonicera japonica flowers based on microcalorimetry [J]. Chinese Journal of Natural Medicines, 2014, 12(2): 108-113
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