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Study on anti-inflammatory efficacy and correlative ingredients with pharmacodynamics detected in acute inflammation rat model serum from Caulis Lonicerae japonicae.

ABSTRACT

Background: Caulis Lonicerae japonicae (CLJ) is often used for the treatment of inflammation such as acute fever, headache, respiratory infection and epidemic diseases. Nevertheless, domestic and foreign researches simply fail to focus on reports of CLJ, especially its anti-inflammatory effects and correlative components.

Purpose: In this study, we investigated anti-inflammatory effects and serum components of Caulis Lonicerae japonicae (CLJ) in models of acute inflammation and correlative analysis between anti-inflammatory effects and serum components of CLJ in rat serum to analyze the changes in the relative contents of components in serum with time and in the corresponding values to characterize active fractions of CLJ and identify the major active components of CLJ in rats.

Study design: Active fractions of CLJ were screened using xylene-induced ear oedema mice model. Anti-inflammatory effects were evaluated using carrageenan-induced paw oedema rat model, and then correlative components in rat serum were demonstrated with HPLC-QTOF/MS/MS method explored.

Methods: HPLC-QTOF/MS/MS was developed to analyze the components absorbed in rat serum after oral administration of CLJ.

Results: Ethyl acetate extracts (ECLJ) and n-butanol extracts (BCLJ) of CLJ were preliminarily screened as active fractions of CLJ (EBCLJ) using xylene-induced ear oedema mice model, and effectively inhibited edema and values of interleukin-1 (IL-1), nuclear factor-kappaB (NF-[kappa]B), tumor necrosis factor-[alpha] (TNF- [alpha]) activities and prostaglandinE2 (PG[E.sub.2]) productions using carrageenan-induced paw oedema rat model. 33 peaks were found in total ion current chromatograms of EBCLJ, and 27 components were observed in EBCLJ-treated rat serum, only 11 of which were correlated with anti-inflammatory effects.

Conclusions: The present study contributes to the study on the pharmacodynamic basis of CLJ and provides potent evidence for developing CLJ as a safe and promising natural drug in inflammation treatment.

Keywords:

Caulis Lonicerae japonicae

Active fractions

Anti-inflammatory effect

Serum components

Correlation analysis between

anti-inflammatory effects and serum components

Introduction

Lonicera japonica Thunb. (LJT) is a commonly used medicinal plant that plays an important role in the clinical treatment of Traditional Chinese medicine (TCM). Caulis Lonicerae japonicae (CLJ), Rendongteng which is derived from the dried caulis of LJT, has long been used for the treatment of acute fever, headache, respiratory infection and epidemic diseases (Chinese Pharmacopoeia Committee, 2010). Meanwhile, CLJ for external use could clinically cure hemorrhoids, anal oedema and prostatitis, while CLJ juice for external use could treat dose-fractured limbs, eliminate oedema and promote bone recovery significantly (Lu, 2012).

Flos Lonicerae japonicae (FLJ), Jinyinhua which is derived from the dried flower buds of LJT, has also been used for the treatment of exopathogenic wind-heat or epidemic febrile diseases, sores and furuncles for centuries. Also they are specially considered two totally different medicines in Chinese pharmacopoeia after 2000. Phytochemical investigations on CLJ and FLJ, different parts of the same plant, have discovered that phenolic acids and iridoid glycosides were the common components in both parts, like chlorogenic acid, loganin, sweroside, etc., while their relative contents revealed large differences (Zhang et al., 2015) and flavonoids were hardly detected in CLJ (Ye et al., 2014). Although they are partly similar in chemical properties, they exhibit different pharmacological effects. Modern pharmacological studies have elucidated that FLJ possesses extensive pharmacological effects, such as anti-bacterial, anti-inflammatory, anti-viral, hepatoprotective, anti-tumor, anti-hyperlipidemic, anti-thrombotic, etc. (Lu, 2012). At present, domestic and foreign research mainly focused on reports of FLJ while no research was conducted on CLJ singly, especially anti-inflammatory effects and correlative components.

In the present study, the CLJ extract was tested for novel pharmacological activity. HPLC-QTOF/MS/MS was developed to analyze the components absorbed in rat serum after the administration of CLJ. Based on the values of IL-1, NF-[kappa]B, TNF-[alpha] and PG[E.sub.2] and paw oedema degree (inhibition rate of oedema) in carrageenan-induced rats at different points in time, we analyzed the changes in the relative contents of the serum components with time and in the corresponding values to identify the effective components. These experiments enabled us to identify the major components of CLJ for reducing IL-1, NF-[kappa]B, TNF-[alpha] and PG[E.sub.2] levels in rats. As a consequence, the present study contributes to the study on the pharmacodynamic basis of CLJ and lays a foundation for the development of new drugs in inflammation treatment.

Material and methods

Plant material

CLJ (Caulis Lonicerae japonicae) was obtained in Hunan Province, China in December 2014 (No. 111207). CLJ was identified and authenticated by Prof. Wu-De Kang of the School of Pharmacy of the Nanjing University of Chinese Medicine. Voucher specimens were deposited in the Nanjing University of Chinese Medicine, Nanjing 210038, PR China

Chemicals

Ethanol; ether; petroleum ether; ethyl acetate; n-butanol (Chemical pure)-, Methanol and acetonitrile (HPLC grade) were supplied by 30 Tedia Company Inc. (Fairfield, USA). HPLC-grade acetic acid was purchased from Merck Company (Darmstadt, Germany). 4, 5-dicaffeoylquinic acid ([C.sub.25][H.sub.24][O.sub.12], MW 1/4 516.46, No. 110773-201012) with a purity of 98.0% as determined by HPLC was purchased from the National Institute for Food and Drug Control (Beijing, China). Interleukin-1 (IL-1), tumor necrosis factor-[alpha] (TNF-[alpha]), nuclear factor-kB (NF-[kappa]B) and prostaglandin[E.sub.2] (PG[E.sub.2]) kits were obtained from Roche Diagnostics(Mannheim, Germany). All other reagents were of analytical grade, and ultrapure water (Wahaha, Hangzhou, China) was used throughout the experiment.

Extraction and isolation

The dried CLJ (600 g) was turned into powder form and extracted with 10 times the amount of 70% ethanol (three times x 1.5 h). The extractive solutions were filtered, concentrated under vacuum and dried, obtaining CLJ total extracts (TCLJ) (117 g, with respect to 600 g of the plant material). Then TCLJ (116 g, with respect to 592 g of the plant material) were submitted to sequential extraction with ethyl acetate, and n-BuOH by extensive maceration (five times), obtaining ethyl acetate extracts (ECLJ) (41 g, with respect to 592 g of the plant material), n-BuOH extracts (BCLJ) (47 g, with respect to 592 g of the plant material) and [H.sub.2]O extracts(HCLJ) (27 g, with respect to 592 g of the plant material). The ECLJ (40 g) was combined with the BCLJ (46 g) to generate active fractions of CLJ (EBCLJ). All extracts were used for animal experiments and stored at 4[degrees]C.

The EBCLJ (0.01 g), filled in conical flask with stopper (100 ml), were extracted with 25 ml of 50% methanol (v/v) for 30 min by ultrasonication and cooled to room temperature. 50% methanol was added to compensate for the lost weight. After the methanol solution was filtered, the filtrate was stored at 6[degrees]C and then the solution was subjected to HPLC-MS analysis after centrifugation at 10,000 rpm for 10 min, obtaining samples of EBCLJ. An aliquot of 10 [micro]l solution was injected for HPLC analysis.

4, 5-O-dicaffeoylquinic acid was prepared by dissolving them in 50% methanol at concentrations of 0.58 mg/ml with a purity of 98.0% as determined by HPLC.

Carrageenan was prepared by dissolving them in physiological saline at concentrations of 0.01 g/ml as inflammatory-induced reagents.

HPLC-QTOF/MS/MS analysis

For HPLC-QTOF/MS/MS analysis, a LC-20a Shimadzu HPLC system was coupled to the orthogonal AB SCIEX Triple TOFTM 5600 mass spectrometry equipped with electronic spray ionization (ESI) source. The chromatographic separation was performed on a Thermo C]g ODS HYPERSIL column (4.6 mm x 150 mm, 5 [micro]m) at 35[degrees]C. A mixture of solvent A (1.5% glacial acetic acid; v/v) and solvent B (acetonitrile) was used as the mobile phase at a flow rate of 0.8 ml [min.sup.-1]. The gradient elution program was: 0-15 min, 5-10% B; 15-20 min, 10% B; 20-35 min, 10-16% B; 35-55 min, 16-20% B; 55-60 min, 20-80% B; 60-70 min, 80% B; 70-75 min, 80-5% B. Then this was followed by 15 min equilibrium period prior to the injection of next sample. The injection volume was 10 [micro]l. ESIMS spectra were acquired in positive and negative ion mode for the full-scan MS analysis, with negative ion mode better, and the spectra was recorded in the range of m/z 50-1500. The conditions of MS analysis were designed as follows: capillary voltage, 2800 V; the source temperature 100[degrees]C; the cone voltage, 20 V; data collected mode, dynamic background deduction and information dependent acquisition; MCR detection voltage, 2100V; collision energy, 10 V; spray voltage, 20 V; nebulizer, 55 psi; aux gas pressure, 60 psi; curtain gas, 40 psi; IS 20 V; gas flow 450 (L/HR); desolvation temp 250[degrees]C; injection volume, 10[micro]l; detector, time of flight mass spectrometer; declisting potential, -70 V.

Experimental animals

Male ICR mice (18-22 g, specific pathogen free) were purchased from Comparative Medicine Center of Yangzhou University (Yangzhou, Jiangsu, China) with animal license: SCXK (Su) 20070001. Adult male SD rats (200-230g, SPF grade) were purchased from the Shanghai Jiesijie Experimental Animal Center (Shanghai, China) with animal license: SCXK (Hu) 20130006. They were maintained in laboratory standard conditions of temperature (25[degrees]C), humidity (70%), and light (12 h light/dark cycles). The rats had free access to laboratory rodent food and water. All animals were acclimated in the laboratory for at least 1 week prior to the experiment. The rats were fasted and given free access to water for 12 h prior to the experiment. The experiment was approved by the Animal Ethics Committee of our university and was in accordance with the international guidelines for laboratory animal use and care found in the European Community Guidelines.

Anti-inflammatory activity

Xylene-induced ear oedema test in mice

In order to investigate the anti-inflammatory activity of CLJ, 70 male ICR mice approximately 7 weeks of age were randomly divided into seven groups, including blank group, model group, Indo group (0.025 g/kg), TCLJ group (0.72 g, with respect to 3.7 g of the plant material, 1.2 g/kg), ECLJ group (0.26 g, with respect to 3.7 g of the plant material, 0.42 g/kg), BCLJ group (0.3 g, with respect to 3.7 g of the plant material, 0.48 g/kg), HCLJ group (0.2 g, with respect to 3.7 g of the plant material, 0.3 g/kg), orally administered to mice. 45 min later, each animal received 25 [micro]l of xylene on the anterior and posterior surfaces of the right ear lobe, and the left ear was considered as control without treatment. One hour later, the mice were anaesthetized by ether and both ears were removed. Ear biopsies of 9.0 mm in diameter were punched out and weighed. The degree of ear oedema was calculated based on the weight difference in the right and left biopsies of the same animal.

Carrageenan -induced paw oedema test in rats

To assess the anti-inflammatory activity of active fractions of CLJ, 60 male SD rats approximately 7-8 weeks of age were randomly divided into six groups, including blank group, model group, Indo group (0.025 g/kg), EBCLJ group (0.5 g/kg, 1.0g/kg and 2.0 g/kg), orally administered to rats for 3 days. The rats received a subplantar injection of 100 ul of a 1 % (w/v) suspension of carrageenan lambda in the left hind paw. The volume of the paw was measured by a Plethysmometer immediately prior to carrageenan injection and then at 30 min after that. Moreover, blood was withdrawn at 30 min for measurement of IL-1, TNF-[alpha], NF-kB and PG[E.sub.2], and 60 min for components analysis from the vena ophthalmica after rats were anesthetized with ketamine hydrochloride. The data were expressed as the variation in the paw volume (ml) and were compared to pre-injection values (basic volume).

Paw oedema (ml)

= Paw volume at each time point - Basic volume Inhibition rate of oedema %

= Volume (Model group - Druggroup)/Model group x 100%.

Carrageenan--induced paw oedema test in rats at different time points

To further evaluate the anti-inflammatory activity and study the relative contents and metabolic changes of serum components in EBCLJ at different time points, 20 male SD rats approximately 7-8 weeks of age were randomly divided into two groups, including model group and EBCLJ group (1.0 g/kg), orally administered to rats per day for 3 days. The rats received a subplantar injection of 100 ul of a 1% (w/v) suspension of carrageenan lambda in the left hind paw. The volume of the paw was measured by a Plethysmometer immediately prior to carrageenan injection and then at 0.5,1.5, 2.5, 3.5, 4.5 and 6.0 h after that. Moreover, blood was withdrawn from the vena ophthalmica at specified time points (0.5, 1.5, 2.5, 3.5, 4.5 and 6.0 h) for measurement of PG[E.sub.2] after rats were anesthetized with ketamine hydrochloride for component-efficacy correlation analysis. The data were expressed as the variation in the paw volume (ml) and were compared to pre-injection values (basic volume).

Paw oedema (ml)

= Paw volume at each time point - Basic volume

Inhibition rate of oedema %

= Volume (Model group - Drug group)/Model group x 100%.

Preparation of serum samples

After administration, blood was withdrawn at specified time points (0.5, 1.5, 2.5, 3.5, 4.5 and 6.0 h) from the vena ophthalmica described above. The blood samples were placed in a water bath for 15 min at 37[degrees]C, and then centrifuged at 4000 rpm for 10 min to obtain the serum (supernatant). Each serum sample was divided into two portions: one portion was developed for evaluation of the anti-inflammatory activity, whereas the other was used for LC-MS analysis. All samples were frozen at -80[degrees]C until the assay.

Methanol (600 [micro]l) was added to the serum (200 [micro]l), and the solution was vortexed for approximately 1 min. The suspension was centrifuged at 10,000 rpm for 10 min to obtain the supernatant and dried using nitrogen gas at 35[degrees]C. The residues were redissolved in 100 p.1 of methanol and centrifuged at 10,000 rpm for 10 min. The supernatant was subjected to HPLC-QTOF/MS/MS analysis.

4, 5-O-dicaffeoylquinic acid (20 [micro]l) as internal standard was added to 500 [micro]l of the supernatant. Also, methanol (1.5 ml) was added to the serum (500 pi), and the solution was vortexed for approximately 1 min. The suspension was centrifuged at 10,000 rpm for 10 min to obtain the supernatant and dried using nitrogen gas at 35[degrees]C. The residues were redissolved in 100 [micro]l of methanol and centrifuged at 10,000 rpm for 10 min. The supernatant was subjected to LC-MS analysis.

Measurement of serum IL-1 NF-[kappa]B, TNF-[alpha], and PG[E.sub.2] values

Interleukin-1, NF-kB, TNF-[alpha], and prostaglandin [E.sub.2] (PG[E.sub.2]) activities were measured in rat serum using enzyme linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Statistical analysis

Statistical calculations were performed using SPSS 16.0 for Windows software package (Statistica). Data were expressed as the mean 7 S.E.M. of six independent experiments and were analyzed using one-way analysis of variance. The results were considered as significant if the probability of deviation was < 5%. Bivariate correlation analysis was used to analyze the relationship of all indexes.

Results and discussion

Screening of active fractions of CLJ on anti-inflammatory activity

To characterize active fractions of CLJ total extracts (TCLJ) of CLJ and their several extracts such as ethyl acetate extracts (ECLJ), n-BuOH extracts (BCLJ), and [H.sub.2]O extracts (HCLJ) of CLJ in the amounts of 0.72 g 0.26 g 0.3 g and 0.2 g (19.6%, 7.0%, 8.0% and 4.6% with respect to 3.7 g of the plant material, respectively) were submitted to the xylene ear test on xylene-induced ear oedema mouse model for a preliminary screening of their anti-inflammatory activity. Compared to the model group, TCLJ and ECLJ on anti-inflammatory effects had difference (P<0.05), and BCLJ and the non steroidal anti-inflammatory drug (NSAID) indomethacin reduced the oedema to about 50%, thus revealed large difference (P < 0.01),which indicated better anti-inflammatory effects than TCLJ, while HCLJ demonstrated no difference. Therefore, ECLJ and BCLJ could be considered as active fractions of CLJ on anti-inflammatory effects (Fig. 1).

The components of CLJ included phenolic acids, flavonoids, and iridoid glycosides (Peng et al., 2000), which were dissolved in ethyl acetate and n-butanol easily. With the use of the polar solvent extraction method, TCLJ were divided into ethyl acetate, n-butanol and [H.sub.2]O sections as screening of the active fractions of CLJ on anti-inflammatory activities. Therefore, ethyl acetate and n-butanol fractions of CLJ (EBCLJ), which significantly decreased the acute xylene-induced acute inflammatory changes in vivo, were screened as the active fractions of CLJ for further study on components and activity evaluation.

Evaluation of anti-inflammatory activity of active fractions of CLJ

To evaluate the anti-inflammatory activity of EBCLJ, carrageenan-induced paw edema model in rats was administered to observe paw oedema and levels of IL-1, NF-kB, TNF-[alpha] and PG[E.sub.2] at 30 min, and the results were shown in Figs. 2 and 3. The paw oedema degrees and levels of IL-1, NF-kB, TNF-[alpha] and PG[E.sub.2] in the model group were significantly higher than those in the blank group (P < 0.001). Compared to the model group, EBCLJ dose-dependently reduced the carrageenan-induced paw oedema ([P.sub.0.5g/kg] <0.05, [P.sub.1.0g/kg] < 0-01 and [P.sub.2.0g/kg] < 0.001) and the release of IL-1, NF-kB, and TNF-[alpha], and the activities of PG[E.sub.2](P < 0.001), suggesting that EBCLJ could effectively decrease the carrageenan-induced acute inflammatory changes in vivo. Especially, 2.0g/kg of EBCLJ significantly inhibited the levels of IL-1, NF-kB, TNF-[alpha] and PG[E.sub.2], similar to the indomethacin group at oral doses of 0.025 g/kg on anti-inflammatory effects.

According to Chinese Pharmacopoeia, CLJ was a traditional antipyretic and anti-inflammatory medicine and was clinically used for the treatment of hot sera dysentery, carbuncles sore, rheumatic fever, joint pain, and oedema, but the evaluation on anti-inflammatory activity had not been reported. In order to evaluate anti-inflammatory effects of EBCLJ, carrageenan-induced rat paw oedema model was explored. As shown in Fig. 2, EBCLJ exerted an inhibitory influence on carrageenan-induced paw oedema.IL-1, an important pro-inflammatory cytokine and immune factor, and inflammation initiation factor, played a central role in local and systemic inflammatory responses, hence induced other inflammatory cytokines (such as IL-6, TNF-[alpha]), chemokines, adhesion molecules, synthesis of acute phase protein and tissue remodeling enzymes. As shown in Fig. 3A, the levels of IL-1 were significantly lower (P < 0.001) than those in the model group, indicating EBCLJ could effectively suppress the release of inflammatory cytokines IL-1 using carrageenan-induced rat paw oedema model, whose possible therapeutic target for inflammation was IL-1 receptor-associated kinase (IRAK). Previous studies demonstrated that Phosphatidylinositol3 (PI 3)-kinase signaling pathway, JAK-STAT signal transduction pathways and ion channels may also participate in regulating the immune and inflammatory response. Besides, a growing body of evidence indicated that NF-kB playing an important role in gene expression of pro-inflammatory cytokines (e.g., IL 1, IL 8 and IL 6, etc.) is a nuclear transcription factor in various kinds of cells, as a result of a number of viral and bacterial infections. In this study, the levels of NF-kB in each dose group were dramatically lower (P < 0.001) than those in the model group, suggesting that EBCLJ could effectively inhibit activity of NF-kB on carrageenan-induced rat paw edema (Fig. 3B). Accumulated evidence had indicated that anti-inflammatory activity of the CLJ was related to inhibition of NF-kB activation through reducing I-kappaBalpha (IkB) degradation (Lee et al. 2001), and IkB protein phosphorylation or proteasome degradation which depended on IKK[beta] and IKK[gamma], as well asp100 precursor protein processing adjustment depending on IKK[alpha] kinase and NF-kB induction (NIK) (Hayden and Ghosh 2008). Additionally, TNF-[alpha], an important initiation factor and inflammatory peptide neurotransmitter secreted by macrophage, can cause fever and inflammation, and release a series of cytokine production. Also, continuing high levels of TNF-[alpha] suggested potential to develop into multiple organ dysfunction. The experimental results revealed that the levels of NF-kB after being treated with EBCLJ were significantly lower (P < 0.001) than those in the model group (Fig. 3C), indicating that EBCLJ could effectively inhibit the expression of TNF-[alpha]-induced cytokine and media on carrageenan-induced rat paw edema. In addition, cyclooxygenase isoenzyme-generated PG[E.sub.2], with protection of gastric mucosa, could increase renal blood flow, dilate blood vessels, and inhibit inflammation. In the present study (Fig. 3D), we confirmed that the levels of PG[E.sub.2] after EBCLJ treatment were significantly lower (P < 0.001) than those in the model group, which indicated that EBCLJ showed anti-inflammatory effects on the inhibition of prostaglandin activity and PG[E.sub.2] expression on carrageenan-induced rat paw edema.

Indomethacin, a positive drug, exerted anti-inflammatory activity mainly via the inhibition of prostaglandin synthesis, which might regulate p38pathway to inhibit NF-kB activation (Zielinsky et al., 2014). On the other hand, it could cause an allergic reaction, and also had adverse reactions of the digestive system, blood system, urinary system and the nervous system. Moreover, it seriously resulted in coma after the oral administration. Compared to indomethacins, CLJ, an antipyretic and anti-inflammatory, was mainly a traditional Chinese medicine, clinically safe and effective, which may be considered as one of useful drugs for the development of anti-inflammatory agents.

In summary, this study demonstrates that EBCLJ shows significant anti-inflammatory effects on carrageenan-induced paw oedema rats. The possible mechanisms of CLJ may attenuate the activation of NF-kB and the subsequent production of proinflammatory mediators such as IL-1, TNF-[alpha] and PG[E.sub.2].

Analysis on components of active fractions of CLJ

In order to accurately analyze active components of EBCLJ, HPLC-QTOF/MS/MS method was used for comparative analysis of total ion current chromatograms of (Fig. 4a) EBCLJ, (Fig. 4b) the serum sample after oral administration of EBCLJ and (Fig. 4c) the model rat serum at 60 min. A total of 33 peaks were found in total ion current chromatograms of EBCLJ, including 16 iridoid glycosides, 8 phenolic acids, 4 flavonoids. Within 6 h, 27 peaks were found in serum total ion current chromatograms (Fig. 6), 13 prototype components and 10 metabolic components initially identified in blood, including 9 iridoid glycosides and 13 phenolic acids (Table 1). Based on fragmentation rules of the fragment peaks, metabolic pathways and literatures, their structures (Fig. 5) were analyzed and speculated as follows:

Prototype components

Phenolic acids. Compound 2 exhibited a [[M - H].sup.-] ion at m/z 191 and its M[S.sup.2] fragmentation gave fragment ions at m/z 173[M - [[H.sub.2]O - H].sup.-], 129[[M - [H.sub.2]O - C[O.sub.2] - H].sup.-], 111[[M - [H.sub.2]O - C[O.sub.2] - [H.sub.2]O - H].sup.-]. Thus compound 2 was deduced to be quinic acid (Gouveia and Castilho 2009).Compound 12a showed a [[M - H].sup.-] ion at m/z 341, which dissociated to give ion at m/z 179 by losing a hexose sugar. As a consequence, compound 12a was acknowledged as caffeic acid hexoside (Sandhu and Gu 2010).

Compounds 8b, 15 and 17 gave a [[M - H].sup.-] ion at m/z 353. Meanwhile, their M[S.sup.2] spectrums showed a similar fragment ion at m/z 191 as base peak. Among monocaffeoylquinic acids, compound 15 was definitely identified as chlorogenic acid (3-CQA) by comparison with commercial standard. Based on their characteristic fragment ions and the reference, compound 8b and 17 were characterized as 5-CQA and 4-CQA respectively (Zhang et al., 2013).Compound 10a and 25e displayed a molecular ion at m/z 515[[M - H].sup.-] and m/z 353 and 191 in the M[S.sup.2] spectra, hence they were inferred as 1, 3-O-dicaffeoylquinic acid and 3, 5-O-dicaffeoylquinic acid, respectively (Zhang et al., 2013). Compound 25b presented a molecular ion at m/z 677[[M - H].sup.-] with the molecular formula [C.sub.34] [H.sub.30] [O.sub.15] and m/z 515, 353 and 191 in the M[S.sup.2] spectra (Zhang et al., 2013). Thus, compound 25b was inferred as 3, 4, 5-tricaffeoylquinic acid.

Iridoid glycosides. Compound 8a yielded a molecule [[M - H].sup.-] at m/z 389, and produced fragment ions at m/z 227 [[M - glc - H].sup.-], 183[[M - glc - C[O.sub.2] - H].sup.-], 165[[M - glc - C[O.sub.2] - [H.sub.2]O - H].sup.-]. Based on their characteristic fragment ions and the literature report, compound 8a was characterized as scandoside (Li et al., 2008). Compound 22a, 23a and 23b provided a [[M - H].sup.-] ion at m/z 387, whose molecular formula was determined to be [C.sub.17] [H.sub.24] [O.sub.10], and revealed a fragment ion at m/z 225 [[M - glc - H].sup.-]. According to literatures (Kakuda et al., 2000 and Li et al. 2009), compounds 22a and 23a were identified as vogeloside and epi-vogeioside, respectively. Since compound 23b had a fragment ion [[M - glc - C[H.sub.3]OH - H].sup.-] at m/z 179, it was indicated to be secologanin (Zhang et al., 2015).

The MS2 spectrum of compound 16 gave several fragment ions at m/z 221,193 and 149, quite similar to the report (Qi et al., 2009). Hence compound 16 was ascribed as secologanic acid. Compound

23 showed the quasi-molecular ion peak [[M - H].sup.-] at m/z 4[O.sub.3]. The fragment ions at m/z 371 and 223 were correspondent with [[M C[O.sub.2] - H].sup.-] and [[M - glc - [H.sub.2]O - H].sup.-]. Fragment ion at m/z 165 was formed by a loss of COOC[H.sub.2] at C-4 from [[M - glc - [H.sub.2]O - H].sup.-]. Also, the highest abundance fragment ion at m/z 121 was confirmed by a follow-up loss of C[O.sub.2] from the fragment at m/z 165. Thus compound 23 by comparison with commercial standard was identified as secoxyloganin. Additionally, compound 21 yielded a [[M - H].sup.-] ion at m/z 357, and its molecular formula was determined to be [C.sub.16][H.sub.22][O.sub.9] by accurate elemental composition from LCMS. Neutral loss of a glucose unit (162 Da) generated the aglycone ion [[M - glc - H].sup.-] at m/z 195. The fragment ion at 125, as base peak in product ion scan, proved to be originated from RDA cleavage. Therefore, compound 21 was unambiguously characterized as sweroside (Li et al., 2009). Moreover, compound 14 and compound 22 were definitely assigned as loganic acid and loganin by comparison with commercial standard. Meanwhile, compound 11 showed the same [[M - H].sup.-] ion with compound 14 at m/z 375, too. The M[S.sup.2] spectrum gave some fragment ions at m/z 113, 119, 151, 169 and 213, which were similar with loganic acid. Compound 11 was therefore deduced to be s8-epi-loganic acid (Li et al., 2013).

Compound 26c exhibited a [[M - H].sup.-] ion at m/z 609, and the fragmentation of this compound yielded the fragment ions at m/z 356[[M - glc - [H.sub.2]O - H].sup.-] and 167[[M - glc - [H.sub.2]O - C[O.sub.2] - H].sup.-]. Thus, compound 26c was identified as L-phenylalaninosecologanin (Kakuda et al., 2000). Moreover, compound 27 gave a [[M - H].sup.-] ion at m/z 537, and fragment ion [[M - glc - H].sup.-] at m/z 375 was characterized by the loss of a neutral glucose unit of mass 162 Da in its MS/MS spectrum. Other fragment ions were detected at m/z 179[[M - glc - [C.sub.10] [H.sub.10] [O.sub.4] - H].sup.-], 161 [[M - glc - [C.sub.10][H.sub.10][O.sub.4] - [H.sub.2]O H].sup.-]

Compound 20 gave high-abundant acetic acid adduct [[M + HAc - H].sup.-] at m/z 447, suggesting their molecular formula might be [C.sub.17][H.sub.24][O.sub.10], with its molecular weight at 388. Thus, compound 20 was considered 7-ketologanin. Compound 16a exhibited a [[M - H].sup.-]ion at m/z 507 whose fragment ions were at m/z 357, 327 and 283. According to the literature (Zhang et al., 2015), compound 16a was deduced to be syringetin hexoside. Moreover, compound 25 exhibited a [[M - H].sup.-] ion at m/z 687, which yielded the fragment ions at m/z 525, 329 and 167. The first fragment ion was extracted after the neutral loss of [C.sub.6][H.sub.12][O.sub.5] corresponding to the glucityl group, while fragment ion at m/z 167 was attributed to isovanillic acid moiety and ion at m/z 329 was corresponding to the fragment caused by cleavage at C-6. Consequently, compound 25 was authorized as 7-0-(4[beta]-D-glucopyranosyloxy-3-methoxylbenzoyl)secologanolic acid. Compound 26a showed a [[M - H].sup.-]ion at m/z 757, and the fragmentation of this compound yielded the fragment ions at m/z 595 and 525, corresponding to the loss of the hexosyl moiety and a RDA cleavage within the glycone respectively. Thus, compound 26a was considered aldosecologanin (Zhang et al., 2015).

Flavonoids

Compound 26b generated a [[M + HAc - H].sup.-] ion at m/z 495 and its M[S.sup.2] fragmentation gave an fragment ion at m/z 273 which was assigned to an adduct of the phloretin aglycon [[M - glc H].sup.-]. Thus, compound 26b was identified as phloridzin (Hilt et al., 20[O.sub.3]). Compound 27a exhibited a [[M - H].sup.-] ion at m/z 285 and its M[S.sup.2] fragmentation gave fragment ions at m/z 151 and 133. Also, compound 27b showed a [[M - H].sup.-] ion at m/z 301 and its M[S.sup.2] fragmentation gave fragment ions at m/z 179, 151 and 121. By comparison with standards and based on their characteristic fragment ions and the reference (Li et al., 2009), compounds 27a and 27b were definitely identified as luteolin and quercetin respectively. Besides, compound 25a gave a [[M - H].sup.-] ion at m/z 609 with the molecular formula [C.sub.27][H.sub.30][O.sub.16] and in the M[S.sup.2] spectra m/z 301 [[M - glc - rha].sup.-]. Thus, compound 25a was identified as rutin (Mario et al., 2012).

Metabolic components

Compound 4 produced a [M - H]" ion at m/z 247, and the fragmentation of this compound generated the fragment ions at m/z 167[[M - S[O.sub.3] - H].sup.-] and 123[[M - S[O.sub.3] - C[O.sub.2] - H].sup.-]. Thus, compound 4 was authenticated as vanillic acid sulfate (Gao et al., 2012). In the M[S.sup.2] spectra compounds 3 and 13 showed the same ions at m/z 179 and 135, which were characteristic fragmentations of caffeic acid. Compound 3 presented MS base peak at m/z 259 and m/z 179 and 135 in the M[S.sup.2] spectra, which exhibited a fragmentation pathway that corresponds to the loss of sulfate acid of mass 80 Da and carboxyl of mass 44 Da. Hence, compound 3 was inferred as caffeic acid sulfate. Compound 13 exhibited m/z 355 [[M - H].sup.-] in the MS spectra and m/z 179 and 135 in the M[S.sup.2] spectra, corresponding to the loss of glucuronic acid of mass 176 Da and carboxyl of mass 44 Da, respectively. So compound 13 was identified as caffeic acid glucuronide. Additionally, compound 5 had a [[M - H].sup.-] ion at m/z 261 and its M[S.sup.2] fragmentation patterns produced ions at m/z 137[[M - [C.sub.6] [H.sub.8] [O.sub.6] - C[O.sub.2] - H].sup.-] and 181 [[M - [C.sub.6] [H.sub.8] [O.sub.6] - H].sub.-]. Thus compound 5 was authenticated as dihydrocaffeic acid glucuronide. Compound 24 generated a molecular ion at m/z 193[[M - H].sup.-], whose fragmentation yielded the fragment ions at m/z 178[[M - C[H.sub.3] - H].sup.-], 149[[M - C[O.sub.2] - H].sup.-] and 134[[M - C[O.sub.2] - C[H.sub.3] - H].sup.-]. Therefore, compound 24 was undoubtedly inferred as ferulic acid, which was not identified from EBCLJ in vitro. Meanwhile, compounds 8 and 12 showed the same ions at m/z 178, 149 and 134, which were characteristic fragmentations of ferulic acid. Compound 8 exhibited a [[M - H].sup.-] ion at m/z 273 and m/z 193, 178 and 134 in the M[S.sup.2] spectra, which exhibited a fragmentation pathway that corresponds to the loss of sulfate acid of mass 80 Da, methyl of mass 15 Da and carboxyl of mass 44 Da, respectively. Thus, compound 8 was inferred as ferulic acid sulfate. Similarly, compound 12 was acknowledged as ferulic acid glucuronide by its ions at m/z 369[[M - H].sup.-] in the MS spectra and m/z 193, 178 and 134 in the M[S.sup.2] spectra, corresponding to the loss of glucuronic acid of mass 176 Da, methyl of mass 15 Da and carboxyl of mass 44 Da, respectively. In addition, a molecular ion at m/z 179[[M - H].sup.-] of compound 18 was observed in the MS spectra. This ion dissociated in the M[S.sup.2] spectra to generate several ions at m/z 135[[M - H - C[O.sub.2]].sup.-] and 161 [[M - H - [H.sub.2]O].sup.-]. Thus, compound 18 was identified as caffeic acid (Angelique et al., 2014). Compound 26 gave a [[M - H].sup.-] ion at m/z 193 and its M[S.sup.2] fragmentation had fragment ions [[M - COOC[H.sub.3] - H].sup.-] at m/z 133 and [[M - C[H.sub.2] - H].sup.-] at m/z 179. As a result, compound 26 was authenticated as methyl caffeate which was not identified from EBCLJ in vitro (Chen et al., 2010). Similarly, compound 19 was inferred as methyl caffeate glucuronide by its ions at m/z 369[[M-H].sup.-] in the MS spectra and m/z 193 and 133 in the M[S.sup.2] spectra.

In addition, compound 1 exhibited a molecular ion at m/z 133[[M - H].sup.-] and in the M[S.sup.2] spectra m/z 115[[M - H - [H.sub.2]O].sup.-] and 71 [[M - H - [H.sub.2]O - C[O.sub.2]].sup.-] (Zhang et al., 2015). Therefore, compound 1 was inferred as malic acid.

Correlative analysis between serum components and anti-inflammatory activity in EBCLJ

Bioassay of EBCLJ for anti-inflammatory activity at different time points

In order to evaluate whether the 27 serum components of EBCLJ had anti-inflammatory activities, PG[E.sub.2] values in serum samples and inhibition rates of paw oedema at different time points were observed after exploring carrageenan-induced rat paw edema model rat treated with EBCLJ (Tables 2 and 3). In previous study, there was a statistical difference in anti-inflammatory effects of EBCLJ between 0.5 h and 6 h. Compared to the blank group, the relative PG[E.sub.2] values of the rats in the model group were significantly higher within 6.0 h, which revealed a statistically significant difference (P < 0.001). Compared to the model group, values of PG[E.sub.2] and inhibition rate of paw oedema in EBCLJ-treated model rats were dramatically lower within 6.0 h, and the difference was statistically significant (P < 0.01, P < 0.001).

Serum chromatograms of active fractions of CL] at different time points

To study relative contents and metabolic changes of serum components in EBCLJ at different time points, serum component total ion current chromatograms at different time points were shown in Fig. 6 and Table 1. Peak S is 4, 5-O-dicaffeoylquinic acid as internal standard. Each chromatographic peak at 0.5 - 6.0 h was shown in Table 1.

Correlative analysis between relative contents of components detected in serum and values of PG[E.sub.2] and inhibition rates of oedema in EBCLJ

The ratio of relative peak intensity of each peak and the internal standard was considered as an index, describing the relative content of each serum component of EBCLJ at different time points (Fig. 6). SPSS 16.0 was used to analyze correlation between relative content of each peak in total ion current chromatograms and values of PG[E.sub.2] and inhibition rates of oedema at different time points respectively, and then the values of the correlation coefficient was calculated. The results are shown in Table 4. Based on the results in Table 1 on analysis and identification of serum components of EBCLJ, peaks 1, 7, 9, 10, 13, 14, 15, 16, 17, 18, 20, 21, 22 and 27 whose correlation coefficients were above 0.5 were positively correlated with the values of PG[E.sub.2], same as inhibition rates of paw oedema, including malic acid, caffeic acid glucuronide, loganic acid, chloroenic acid, secologanic acid, 4-O-caffeoylquinic acid, caffeic acid, 7-ketologanin, sweroside, loganin, and grandifloroside, consistent with correlation results of inhibition rates of oedema. However, correlation coefficients of peaks 2, 4, 6, 11, 19 and 24 with values of PG[E.sub.2] were different with inhibition rates of oedema, and correlation coefficients of peak 19 with inhibition rates of oedema were above 0.5 while correlation coefficients of peaks 2,4, 6, 11, 24 with inhibition rates of oedema were below 0.5. In addition, peaks 3, 8 and 23 were negatively correlated, respectively including caffeic acid sulfate, ferulic acid sulfate and secoxyloganin. Therefore, we speculated that the components positively correlated may initially be considered as active components on anti-inflammatory effects of EBCLJ.

Meanwhile, peaks 1, 7, 9, 10, 13, 14, 15, 16, 17, 18, 20, 21, 22, 27 were positively correlated with PG[E.sub.2] and inhibition rates of oedema in serum chromatograms in Fig. 7A and B. The relative peak intensities of each component were considered as the left ordinate, and levels of PG[E.sub.2] and inhibition rates of oedema of EBCLJ were considered as the right ordinate in a coordinate system with time as the abscissa to analyze the effective components from EBCLJ in carrageenan-induced paw oedema rats.

Compared to FLJ, the representative components of CLJ included iridoid glycosides (loganin and sweroside) and caffeoylquinic acids (chlorogenic acid and 4-O-caffeoylquinic acid) (Zhang et al., 2015). In these anti-inflammatory-related serum components, iridoid glycosides (e.g. loganic acid, sweroside, 7-ketologanin, grandifloroside, secologanic acid and loganin) were strongly confirmed as active components which produced anti-inflammatory activity (Hyun et al., 2013 and Wei et al., 2012). In caffeoylquinic acids components and their metabolites, chlorogenic acid showed in-vivo anti-inflammatory activity on lipopolysaccharide-induced inflammation (Jin et al., 2006). Besides, caffeic acid was considered one of correlative components with anti-inflammatory activity (Mehrotra et al., 2011). 4-O-caffeoylquinic acid showed anti-inflammatory activity via inhibiting histamine release (Wang et al., 2006). In addition, malic acid exhibited anti-inflammatory activity on inhibiting oedema (Pasquale et al., 2013; Ruiz et al., 1998 and Roberg et al., 1999). Therefore, it was suggested that these components contribute to the in-vivo anti-inflammatory activity.

Conclusions

CLJ, a traditional medicine of antipyretic and anti-inflammatory, is widely used in the clinical treatment of Chinese medicine for the treatment of carbuncles sore, rheumatic fever, joint pain, oedema and so on, while their anti-inflammatory effects and components have not been studied systematically. In order to characterize active fractions of CLJ, the anti-inflammation experiment of xylene-induced mice ear oedema was conducted. Moreover, active components of CLJ on anti-inflammatory effects were studied systematically and ear oedema degree was considered as the index to evaluate anti-inflammatory activity of CLJ. Among various extracts, ethyl acetate extracts and n-butanol extracts of CLJ (EBCLJ) were characterized as active fractions of CLJ. To further evaluate the anti-inflammatory activity of EBCLJ, carrageenan-induced paw edema model in rats was administered to observe paw oedema and levels of IL-1, NF-kB, TNF-[alpha] and PG[E.sub.2], and the results were demonstrated that EBCLJ showed significant anti-inflammatory effects on carrageenan-induced paw oedema rats, significantly and dose-dependently attenuating carrageenan-induced paw oedema and the activation of NF-kB and the subsequent production of proinflammatory mediators such as IL-1, TNF-[alpha] and PG[E.sub.2].

In current studies, HPLC-QTOF/MS/MS was used to identify components of EBCLJ, namely, iridoid glycosides compounds e.g., scandoside, 8-epiLoganic acid, loganic acid, secologanic acid, 7-ketologanin, sweroside, loganin, secoxyloganin, grandifloroside, aldosecologanin, 7-0-(4[beta]-D-glucopyranosyloxy-3-methoxylbenzoyl) secologanolic acid, flavonoid compounds (e.g., quercetin and luteolin), and phenolic acids (e.g., chlorogenic acid and its two isomers, 4-O-caffeoylquinic acid and 5-O-caffeoylquinic acid). Meanwhile the components absorbed in the serum of rats were identified. Compared to vitro total ion current chromatogram of EBCLJ, 13 prototype components and 10 metabolic components, including 9 iridoid glycosides and 13 phenolic acids, were found. As a consequence, 11 of these serum components, namely, malic acid, caffeic acid glucuronide, loganic acid, chlorogenic acid, secologanic acid, 4-O-caffeoylquinic acid, caffeic acid, 7-ketologanin, sweroside, loganin and grandifloroside, which were correlated positively with anti-inflammatory effects, were screened as anti-inflammatory substances of CLJ. Further research needs to be conducted to evaluate these metabolites of CLJ for anti-inflammatory activity.

http://dx.doi.org/10.1016/j.phymed.2016.01.016

ARTICLE INFO

Article history:

Received 9 September 2015

Revised 9 December 2015

Accepted 23 January 2016

Abbreviation: CLJ, Caulis Lonicerae japonicae; ECLJ, Ethyl acetateextracts of CLJ; BCLJ, n-butanol extracts of CLJ; EBCLJ, ethyl acetate extracts and n-butanol extracts of CLJ; 1H, interleukin-1; NF-[kappa]B, nuclear factor-kappaB; TNF-[alpha], tumor necrosis factor-[alpha]; PG[E.sub.2], prostaglandinE2: LJT, Lonicera japonica Thunb; TCM, Traditional Chinese medicine; FLJ, Flos Lonicerae japonicae; ESI, electronic spray ionization; ELISA, enzyme linked immunosorbent assay; TCLJ, total extracts of CLJ; HCLJ, [H.sub.2]O extracts of CLJ; NSAID, non steroidal anti-inflammatory drug; IRAK, IL-1 receptor-associated kinase; PI 3, Phosphatidylinositol3; IkB, 1-kappaBalpha; NIK, IKKa kinase and NF-[kappa]B induction; Indo, Indomethacin; RPI, relative peak intensities; IRO, inhibition rate of oedema.

Conflict of interest

All authors have no financial or scientific conflict of interest with regard to the research described in this manuscript.

Acknowledgments

This study is supported by Natural Science Foundation of Jiangsu Province (no. BK2002033), Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China and Research Foundation of Nanjing University of Traditional Chinese Medicine, China.

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Yuanlin Tang, Lian Yin *, Yida Zhang, Xi Huang, Fangli Zhao, Xiaobing Cui, Le Shi, Li Xu

College of Pharmacy, Nanjing University of Chinese Medicine, 210023 Nanjing, China

* Corresponding author. Tel.: +86 13813971246; fax: +86 25 85811524.

E-mail address: njxlyl2002@163.com (L. Yin).

Table 1
Identified results of the constituents of active fractions of
Caulis Lonicerae japonicae.

No    Rt      [ESI.sup.-] m/z
      (min)
              MS                             MS/MS

1     3.58    133 [[M-H].sup.-]              71, 115, 133
2     3.75    191 [[M-H].sup.-]              111, 129, 173
3     3.83    259 [[M-H].sup.-]              135, 179
4     4.38    247 [[M-H].sup.-]              108, 123, 152, 167
5     5.24    261 [[M-H].sup.-]              137, 181
6     6.38    476 [[M-[H.sub.2]O-H].sup.-]   148, 230, 312, 394
7     6.39    608 [[M+Na-2H].sup.-]          198, 280, 362, 444
8     7.58    273 [[M-H].sup.-]              134, 149, 178, 193
8a    7.90    389 [[M-H].sup.-]              165, 183
8b    11.05   353 [[M-H].sup.-]              179, 191
9     11.34   445 [[M-H].sup.-]              195, 227, 245, 327, 409
10    11.39   327 [[M+HAc-H].sup.-]          133, 151, 169, 195, 215,
                                               227
10a   12.01   515 [[M-H].sup.-]              173, 179, 191, 353
11    15.49   375 [[M-H].sup.-]              113, 119, 151, 169, 213
12    15.91   369 [[M-H].sup.-]              134, 149, 178, 193
12a   16.85   401 [[M+HAc-H].sup.-]          161, 179
13    16.89   355 [[M-H].sup.-]              135, 179, 311
14    17.35   375 [[M-H].sup.-]              119, 151, 195
15    18.53   353 [[M-H].sup.-]              135, 161, 173, 191
16    19.28   373 [[M-H].sup.-]              113, 193, 221
16a   21.23   507 [[M-H].sup.-]              283, 327, 357
17    21.73   353 [[M-H].sup.-]              191
18    21.98   179 [[M-H].sup.-]              135, 161
19    24.28   369 [[M-H].sup.-]              113, 133, 193
20    25.27   447 [[M+HAc-H].sup.-]          101, 123, 201, 225
21    26.44   357 [[M-H].sup.-]              125, 151, 195
22    27.23   403 [[M-H].sup.-]              101, 127, 227
22a   31.92   387 [[M-H].sup.-]              155, 225
23    32.81   403 [[M-H].sup.-]              121, 165, 179, 223, 371
23a   33.93   387 [[M-H].sup.-]              111, 155, 225
23b   34.78   387 [[M-H].sup.-]              123, 155, 179, 225
23c   37.23   581 [[M-H].sup.-]              153, 419
24    38.44   193 [[M-H].sup.-]              134, 149, 178
24a   41.79   581 [[M+HAc-H].sup.-]          329, 341, 359
25    42.31   687 [[M-H].sup.-]              167, 329, 525
25a   43.42   609 [[M-H].sup.-]              271, 301
25b   43.88   677 [[M-H].sup.-]              191, 353, 515
25c   44.91   521 [[M+HAc-H].sup.-]          461, 521
25d   47.65   717 [[M-H].sup.-]              197, 359, 555
25e   49.58   515 [[M-H].sup.-]              179, 191, 353
26    50.56   193 [[M-H].sup.-]              133, 179
26a   54.68   757 [[M-H].sup.-]              493, 525, 595
26b   55.93   495 [[M+HAc-H].sup.-]          167, 179, 273
26c   57.33   536 [[M-H].sup.-]              164, 272, 312, 356
27    60.37   537 [[M-H].sup.-]              135, 161, 179, 201, 335,
                                               375
27a   60.95   285 [[M-H].sup.-]              133, 151
27b   61.01   301 [[M-H].sup.-]              121, 151, 179

No    MW    MF                               Name

1     134   [C.sub.4][H.sub.6][O.sub.5]      Malic acid
2     192   [C.sub.7][H.sub.12][O.sub.6]     Quinic acid
3     260   [C.sub.9][H.sub.8][O.sub.7]S     Caffeic acid sulfate
4     248   [C.sub.8][H.sub.8][O.sub.7]S     Vanillic acid sulfate
5     262   [C.sub.9][H.sub.10][O.sub.7]S    Dihydrocaffeic acid
                                               sulfate
6     457   Unidentified                     Unidentified
7     587   Unidentified                     Unidentified
8     274   [C.sub.10][H.sub.10][O.sub.7]S   Ferulic acid sulfate
8a    390   [C.sub.17][H.sub.26][O.sub.10]   Scandoside
8b    354   [C.sub.16][H.sub.26][O.sub.9]    5-O-Caffeoylquinic acid
9     446   [C.sub.24][H.sub.14][O.sub.9]    Unidentified
10    268   [C.sub.9][H.sub.16][O.sub.7]S    Unidentified
10a   516   [C.sub.25][H.sub.24][O.sub.12]   1,3-O-Dicaffeoylquinic
                                               acid
11    376   [C.sub.16][H.sub.24][O.sub.10]   8-epiLoganic acid
12    370   [C.sub.16][H.sub.18][O.sub.10]   Ferulic acid glucuronide
12a   342   [C.sub.16][H.sub.22][O.sub.8]    Caffeic acid hexoside
13    356   [C.sub.15][H.sub.16][O.sub.10]   Caffeic acid glucuronide
14    376   [C.sub.16][H.sub.24][O.sub.10]   Loganic acid
15    354   [C.sub.16][H.sub.18][O.sub.9]    Chlorogenic acid
16    374   [C.sub.16][H.sub.22][O.sub.10]   Secologanic acid
16a   508   [C.sub.28][H.sub.28][O.sub.9]    Syringetin hexoside
17    354   [C.sub.16][H.sub.18][O.sub.9]    4-O-Caffeoylquinic acid
18    180   [C.sub.9][H.sub.8][O.sub.4]      Caffeic acid
19    370   [C.sub.16][H.sub.18][O.sub.10]   Methyl caffeate
                                               glucuronide
20    388   [C.sub.17][H.sub.24][O.sub.10]   7-Ketologanin
21    358   [C.sub.16][H.sub.22][O.sub.9]    Sweroside
22    404   [C.sub.17][H.sub.26][O.sub.10]   Loganin
22a   388   [C.sub.17][H.sub.24][O.sub.10]   Vogeloside
23    404   [C.sub.17][H.sub.24][O.sub.11]   Secoxyloganin
23a   388   [C.sub.17][H.sub.24][O.sub.10]   epi-Vogeloside
23b   388   [C.sub.17][H.sub.24][O.sub.10]   Secologanin
23c   582   [C.sub.35][H.sub.34][O.sub.8]    Unidentified
24    194   [C.sub.10][H.sub.10][O.sub.4]    Ferulic acid
24a   522   [C.sub.24][H.sub.26][O.sub.13]   Unidentified
25    688                                    7-0-(4[beta]-D-
                                               Glucopyranosyloxy-3-
                                               methoxylbenzoyl)
                                               secologanolic acid
                                               ologanolic acid
25a   610   [C.sub.27][H.sub.30][O.sub.16]   Rutin
25b   678   [C.sub.34][H.sub.30][O.sub.15]   3,4,5-Tricaffeoylquinic
                                               acid
25c   462   [C.sub.28][H.sub.30][O.sub.6]    Unidentified
25d   718   [C.sub.36][H.sub.30][O.sub.16]   Unidentified
25e   516   [C.sub.25][H.sub.24][O.sub.12]   3,5-O-Dicaffeoylquinic
                                               acid
26    194   [C.sub.10][H.sub.10][O.sub.4]    Methyl caffeate
26a   758   [C.sub.34][H.sub.46][O.sub.19]   Aldosecologanin
26b   436   [C.sub.21][H.sub.24][O.sub.10]   Phloridzin
26c   537   [C.sub.26][H.sub.35]N            L-phenylalaninosecologanin
              [O.sub.11]
27    538   [C.sub.25][H.sub.30][O.sub.13]   Grandifloroside
27a   286   [C.sub.15][H.sub.10][O.sub.6]    Luteolin
27b   302   [C.sub.15][H.sub.10][O.sub.7]    Quercetin

No    Observed (h)                   Detected

                                     Serum   Vitro

1     0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       +
2     0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       +
3     0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       -
4     0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       -
5     0.5;1.5;2.5;3.5;4.5,6.0        +       -
6     0.5:1.5; 4.5                   +       -
7     0.5; 1.5; 2.5                  +       -
8     0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       -
8a    --                             -       +
8b    --                             -       +
9     0.5; 1.5; 2.5; 3.5; 4.5        +       -
10    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       -
10a   --                             -       +
11    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       +
12    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       -
12a   --                             -       +
13    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       -
14    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       +
15    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       +
16    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       +
16a   --                             -       +
17    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       +
18    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       -
19    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       -
20    0.5; 1.5                       +       +
21    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       +
22    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       +
22a   --                             -       +
23    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       +
23a   --                             -       +
23b   --                             -       +
23c   --                             -       +
24    3.5; 4.5; 6.0                  +       -
24a   --                             -       +
25    0.5; 1.5                       +       +
25a   --                             -       +
25b   --                             -       +
25c   --                             -       +
25d   --                             -       +
25e   --                             -       +
26    0.5; 1.5; 2.5; 3.5; 4.5; 6.0   +       -
26a   --                             -       +
26b   --                             -       +
26c   --                             -       +
27    0.5; 1.5; 2.5; 3.5; 4.5        +       +
27a   --                             -       +
27b   --                             -       +

Table 2
Results of the anti-inflammation effects of active fractions of
Caulis Lonicerae japonicae on PG[E.sub.2] levels at different points
in serum on carrageenan-induced paw oedema rats model ([bar.X].
[+ or -] S) (n = 10)

Data show the mean [+ or -] S.E.M. (#) P < 0.05, (##) P < 0.01, (###)
P < 0.001 at the analysis of variance, as compared to blank group. *
P < 0.05, ** P < 0.01, *** P < 0.001 at the analysis of variance, as
compared to model group. Note: Indo: indomethacin; EBCLJ: ethyl
acetate and n-butanol extracts of Caulis Lonicerae japonicae. Model
group: rats were given hypodermic injection of carrageenan (1%, 0.1
ml). EBCLJ group: rats were given hypodermic injection of carrageenan
(1%, 0.1 mi), and given EBCLJ (1.0g/kg).Indo group: rats were given
hypodermic injection of carrageenan (1%, 0.1 ml), and given
Indomethacin (0.025 g/kg).

              Dose     PG[E.sub.2] (ng/l)
              (g/kg)
                                  0.5 h

Blank group   --       253.595 [+ or -] 15.8
Model group   --       329.121 [+ or -] 12.2 (###)
EBCLJ group   1.0      267.812 [+ or -] 13.3 ***
Indo group    0.025    255.290 [+ or -] 15.8 ***

              PG[E.sub.2] (ng/l)

                         1.5 h                         2.5 h

Blank group   253.595 [+ or -] 15.8         253.595 [+ or -] 15.8
Model group   335.581 [+ or -] 14.3 (###)   345.543 [+ or -] 11.6 (###)
EBCLJ group   275.068 [+ or -] 16.2 ***     287.146 [+ or -] 20.1 ***
Indo group    268.170 [+ or -] 12.3 ***     277.290 [+ or -] 11.8 ***

              PG[E.sub.2] (ng/l)

                         3.5 h                         4.5 h

Blank group   253.595 [+ or -] 15.8         253.595 [+ or -] 15.8
Model group   359.330 [+ or -] 12.7 (###)   348.720 [+ or -] 10.3 (###)
EBCLJ group   293.558 [+ or -] 9.2 ***      283.182 [+ or -] 11.7 ***
Indo group    285.090 [+ or -] 10.1 ***     275.120 [+ or -] 11.3 ***

              PG[E.sub.2] (ng/l)

                         6.0 h

Blank group   253.595 [+ or -] 15.8
Model group   346.660 [+ or -] 13.1 (###)
EBCLJ group   291.718 [+ or -] 15.0 ***
Indo group    287.030 [+ or -] 13.5 ***

Table 3
Results of the anti-inflammation effects of active fractions of
Caulis Lonicerae japonicae at different points on paw volume on
carrageenan-induced paw oedema rats model ([bar.X] [+ or -] S)
(n = 10)

Data show the mean [+ or -] S.E.M. (#) P < 0.05, (##) P < 0.01, (###)
P < 0.001 at the analysis of variance, as compared to blank group. *
P < 0.05, ** P < 0.01, *** P < 0.001 at the analysis of variance, as
compared to model group. Oedema (ml) = Paw volume at each time
point--Basic volume. Inflammation inhibition rate (%) = (Model
group-Drug group)/Model group x 100%. Note: Indo: indomethacin;
EBCLJ: ethyl acetate and n-butanol extracts of Caulis Lonicerae
japonicae. Model group: rats were given hypodermic injection of
carrageenan (1%, 0.1 ml) EBCLJ group: rats were given hypodermic
injection of carrageenan (1%, 0.1 ml), and given EBCLJ (1.0 g/kg)
indo group: rats were given hypodermic injection of carrageenan
(1%, 0.1 ml), and given Indomethacin (0.025 g/kg).

              Dose     Paw oedema value (ml)
              (g/kg)
                       0 h

Blank group   --       2.05 [+ or -] 0.05
Model group   --       2.17 [+ or -] 0.06
EBCL] group   1.0      2.10 [+ or -] 0.05
Indo group    0.025    2.07 [+ or -] 0.06
                       inhibition rate of CLJ (%)
EBCLJ group   --       --
Indo group    --       --

              Paw oedema value (ml)

              0.5 h                      1.5 h

Blank group   2.07 [+ or -] 0.06         2.06 [+ or -] 0.05
Model group   2.83 [+ or -] 0.13 (###)   2.96 [+ or -] 0.12 (###)
EBCL] group   2.63 [+ or -] 0.06 **      2.69 [+ or -] 0.07 **
Indo group    2.41 [+ or -] 0.05 ***     2.47 [+ or -] 0.11 ***

EBCLJ group   7.07                       9.12
Indo group    14.84                      16.55

              Paw oedema value (ml)

              2.5 h                      3.5 h

Blank group   2.06 [+ or -] 0.06         2.07 [+ or -] 0.04
Model group   3.19 [+ or -] 0.15 (###)   2.91 [+ or -] 0.12 (###)
EBCL] group   2.77 [+ or -] 0.05 ***     2.52 [+ or -] 0.10 ***
Indo group    2.68 [+ or -] 0.08 ***     2.53 [+ or -] 0.15 ***

EBCLJ group   13.16                      13.40
Indo group    12.98                      13.06

              Paw oedema value (ml)

              4.5 h                      6.0 h

Blank group   2.06 [+ or -] 0.0.7        2.07 [+ or -] 0.02
Model group   2.71 [+ or -] 0.09 (###)   2.51 [+ or -] 0.1 (###)
EBCL] group   2.46 [+ or -] 0.13 ***     2.22 [+ or -] 0.05 ***
Indo group    2.37 [+ or -] 0.06 ***     2.23 [+ or -] 0.14 ***

EBCLJ group   9.23                       11.55
Indo group    15.36                      13.23

Table 4
Correlation coefficients between relative intensities of components
detected in serum and values of PG[E.sub.2] or inhibition rates of
oedema at different time points.

No.    Relative intensities (cps)

       0.5 h    1.5 h    2.5 h    3.5 h     4.5 h    6.0 h

1      0.975    0.975    1.570    2.680     2.182    1.604
2      0.285    0.285    1.570    0.848     0.423    5.906
3      0.975    0.975    1.483    1.963     2.182    5.906
4      3.141    3.141    1.483    0.848     2.182    5.906
5      0.050    0.047    0.061    0.137     0.131    0.055
6      0.005    0.005    1.384    2.593     0.457    8.053
7      0.005    0.005    1.384    1.187     0.458    2.805
8      1.915    2.104    2.284    0.470     1.434    3.297
9      0.007    0.012    0.087    0.134     0.006    0.038
10     0.007    0.007    0.087    0.133     0.009    0.039
11     0.008    0.009    0.012    0.010     0.007    0.026
12     0.007    0.008    0.011    0.005     0.004    0.028
13     0.009    0.009    0.011    0.058     0.014    0.087
14     0.012    0.012    0.060    0.065     0.052    0.062
15     0.027    0.027    0.099    0.036     0.030    0.126
16     0.002    0.002    0.004    0.004     0.004    0.005
17     0.002    0.002    0.005    0.012     0.006    0.012
18     0.001    0.002    0.010    0.0059    0.004    0.006
19     0.010    0.02     0.193    0.058     0.058    0.033
20     0.001    0.001    0.004    0.003     0.003    0.006
21     0.001    0.002    0.140    0.045     0.076    0.185
22     0.058    0.085    0.141    0.113     0.043    0.252
23     0.107    0.147    0.004    0.005     0.005    0.131
24     0.001    0.001    0.003    0.004     0.005    0.015
25     0.135    0.236    0.264    0.121     0.227    0.211
26     0.261    0.341    0.323    0.2670    0.120    0.374
27     0.063    0.071    0.977    0.332     0.593    0.472

No.    Correlation coefficients

       PG[E.sub.2]   Inhibition rate
       (ng/l)        of oedema (%)

1       0.7696        0.6233
2       0.5940        0.3065
3      -0.0786       -0.3009
4       0.5465        0.3661
5       0.4905        0.3217
6       0.6637        0.4608
7       0.7915        0.6637
8      -0.0698       -0.1191
9       0.7233        0.8892
10      0.7340        0.8896
11      0.5406        0.3973
12      0.3892        0.2481
13      0.6731        0.4563
14      0.9530        0.8370
15      0.5708        0.5488
16      0.8915        0.7325
17      0.9038        0.7163
18      0.7156        0.8945
19      0.4155        0.6490
20      0.8134        0.7314
21      0.7160        0.6040
22      0.6251        0.5599
23     -0.4672       -0.5163
24      0.5873        0.2976
25      0.0801        0.1250
26      0.1481        0.3228
27      0.5821        0.6338
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Author:Tang, Yuanlin; Yin, Lian; Zhang, Yida; Huang, Xi; Zhao, Fangli; Cui, Xiaobing; Shi, Le; Xu, Li
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jun 1, 2016
Words:10825
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