Inhalable Andrographolide-β-cyclodextrin Inclusion Complexes for

Apr 5, 2017 - ABSTRACT: Bacterial pneumonia is a serious disease with high mortality if no appropriate and immediate therapy is available...
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Inhalable Andrographolide-β-cyclodextrin Inclusion Complexes for Treatment of Staphylococcus aureus Pneumonia by Regulating Immune Responses Tongtong Zhang,†,‡ Lifei Zhu,†,‡ Miao Li,‡ Yuzhen Hu,†,‡ Erfeng Zhang,§ Qingcheng Jiang,⊥ Guang Han,§ and Yiguang Jin*,†,‡,§ †

Department of Graduates, Anhui Medical University, Hefei 230001, China Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing 100850, China § Pharmaceutical College of Henan University, Kaifeng 475004, China ⊥ The First People’s Hospital of Tancheng, Shandong 276199, China ‡

ABSTRACT: Bacterial pneumonia is a serious disease with high mortality if no appropriate and immediate therapy is available. Andrographolide (AG) is an anti-inflammatory agent extracted from a traditional Chinese herb andrographis paniculata. Oral AG tablets and pills are clinically applied for treatment of upper respiratory tract infections. However, the low solubility and bioavailability of AG lead to high doses and long-term therapy. Here we developed an andrographolide-βcyclodextrin inclusion complex (AG-β-CD) for inhalation therapy of Staphylococcus aureus pneumonia. AG-β-CD was identified with X-ray diffraction and FT-IR. Surprisingly, both AG-βCD and AG showed little in vitro anti-S. aureus activity. However, pulmonary delivery of AG, AG-β-CD, or penicillin had significant anti-S. aureus pneumonia effects. Leukocytes, neutrophils, white blood cells, total proteins, TNF-α, IL-6, NF-κB p65 expression, and bacterial colonies in the bronchoalveolar lavage fluids were detected. Pulmonary delivery of AG and AG-β-CD led to bacterial inhibition and inflammation alleviation by regulating immune responses, while penicillin only killed bacteria without significant immune regulation. Moreover, the antipneumonia activity of AG-β-CD was much higher than that of AG, probably resulting from locally accelerated AG dissolution due to β-CD inclusion. The aerodynamic diameter of AG-β-CD powders was 2.03 μm, suitable for pulmonary delivery. Inhalable AG-β-CD is a promising antibacterial and anti-inflammatory medicine for the treatment of S. aureus pneumonia by regulating immune responses, and the effect is enhanced by β-CD inclusion. AG and its formulations might be potent weapons against the resistant bacterial pneumonia due to their specific mechanism in the future. KEYWORDS: andrographolide, bacterial pneumonia, cyclodextrin, immune regulation, inclusion complex, penicillin, pulmonary delivery, Staphylococcus aureus

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agents,14 antimicrobial agents,15 and anticancer agents.16,17 Paclitaxel (PTX) is an paradigm originally isolated from the bark of the Pacific yew tree, Taxus brevifolia, with favorable efficacy in metastatic and early stage breast cancers.18 Andrographis paniculata (Burm. F.) Nees (Acanthaceae) is a traditional Chinese herb as well as a common food in China. Andrographolide (AG), a diterpenoid lactone, is the major bioactive ingredient.19 AG has multiple pharmacological activities including anti-inflammation,20 antibacterial activity,21 antioxidation,22 and anticancer effect.23,24 It is regarded as an NF-κB inhibitor that regulates immune responses.25,26 Furthermore, AG reduces the inflammatory reactions in macrophages by inhibiting COX-2, NF-κB activation, apoptotic

INTRODUCTION Pneumonia is a common disease characterized by lung parenchyma inflammations and mainly caused by infections of diverse pathogens, involving bacteria,1,2 viruses,3 mycoplasma,4 fungi,5 and chlamydia.6 Bacterial pneumonia is dominating, which is usually combined with viral influenza.7 Antibiotic therapies are mostly used for bacterial pneumonia, only to result in a lot of antibiotic-resistant bacteria.8 Recently, super resistant bacteria to multiple antibiotics have emerged.9 The mechanisms of microbial resistance may involve permeability changes of bacterial outer membranes, changes of targets, enzymatic degradation of drugs, and efflux of drugs.10 Moreover, antibiotic therapies could lead to toxic reactions,11 allergic responses,12 and even dual infections following longterm treatment.13 Therefore, it is necessary to find efficient, nontoxic antipneumonia agents with low resistant possibility, wherein natural products are focused on by scientists. Plant drugs are widely adopted clinically, such as anti-inflammation © XXXX American Chemical Society

Received: December 25, 2016 Revised: March 25, 2017 Accepted: March 29, 2017

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DOI: 10.1021/acs.molpharmaceut.6b01162 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Identification of AG-β-CD. AG-β-CD was identified with the infrared (IR) spectrometry on a PerkinElmer Model Spectrum IR Spectrometer (USA) and X-ray powder diffraction (XRD) on a Rigaku Miniflex desktop X-ray diffract meter (Japan). In Vitro Release Study. The dissolution profiles of AG and AG-β-CD were examined. In brief, 5 mg of AG and 50 mg of AG-β-CD were gently added to 50 mL of simulated lung fluid (SLF) at 37 ± 0.5 °C using a rotating paddle (100 rpm), respectively. At the predetermined time intervals, the sample (1 mL) was withdrawn at 0, 0.5, 1, 1.5, 2, 3, 5, 18, and 24 h. The supernatant was filtered through a 0.45-μm filter, and the concentration of AG and AG-β-CD in the test solutions was analyzed by HPLC. The fresh SLF of an equal volume was supplemented. The experiments were performed in triplicates. In Vitro Antimicrobial Investigation. A Gram-positive bacterium, S. aureus (ATCC29213), was provided by the Institute of Disease Control and Prevention of PLA, China. The subjects, including AG powders, AG-β-CD, and penicillin, were suspended or dissolved in Luria−Bertain broth (LBB) to a series of concentrations. Aliquots (5 μL, 7.5 × 108 CFUs/mL) of proliferated bacterial liquids were mixed with the above drugcontained media (5 mL each sample), respectively. The mixtures were continually cultured for 24 h under 37 °C and 200 rpm vibration. Some of cultured media were spread on the plates with an inoculating ring followed by culture at 200 rpm for 24 h. The growth of colonies was observed. In Vivo Anti-Pneumonia Studies. Bacterial-pneumonic rat models were prepared with the same method as in our previous research.36 Briefly, the suspension containing S. aureus was directly sprayed into the lung of rats through tracheal routes with soft long plastic tubes. Healthy rats were administered with saline (0.2 mL) as controls. The pneumonic rats were divided into four groups with six rats in each group. They were administered with different medicines following bacterial inoculation for 6 h, including saline (0.2 mL), AG powders (10 mg AG), AG-β-CD (containing 1 mg AG), and penicillin sodium solutions (240 mg/mL, 0.2 mL). Administration was performed with an IA-1B intratracheal Micro Sprayer Aerosolizer (Penn-Century Inc., USA) once daily for 3 days (Figure 1). On the second day after the final administration,

proteins expression, and by regulating cytokines (TNF-α and IL-6).20,22 AG dripping pills and other oral dosage forms are marketed in China as OTC products and widely used for the treatment of sore throat caused by upper respiratory tract infection.20 However, the antipneumonia effect of AG has not been confirmed, and no detailed mechanisms are reported. In addition, AG is very poorly water-soluble and of low bioavailability, leading to limited pharmacological effect. β-Cyclodextrin (β-CD) is a macrocycle composed of seven α-D-glucopyranoside units. Hydrophobic drugs are entrapped into the cylinder space of β-CD to form inclusion complexes that can enhance drug solubility, chemical stability, bioavailability, and reduce toxicity of drugs.27,28 Recently, β-CD is not only regarded as an excipient of parenteral formulations, but also a component of inhalers for pulmonary delivery.29,30 Pulmonary drug delivery is a noninvasive administration method through the throat and branchillea.31 Inhalable therapy may be considered an efficient treatment of local lung diseases such as asthma, pneumonia, and chronic obstructive pulmonary disease (COPD).32 Dry powder inhalations (DPIs) can directly transport drugs into the deep sites of the lung.33 Compared to nebulizers and pressurized metered-dose inhalers (pMDIs), DPIs are the portable solid powders without propellants, and the stability of loaded drugs is generally improved.34,35 Here we developed inhalable andrographolide-β-cyclodextrin inclusion complexes (AG-β-CD) for the treatment of Staphylococcus aureus pneumonia. We found the therapeutic effect on the pneumonic rat models was markedly increased using AG-βCD compared to the nonformulated AG powders though they showed poor antibacterial effect in vitro. The anti-S. aureus pneumonia mechanism of AG was explored. The prospect of development of AG and AG-β-CD was discussed, especially in relation to the fight against resistant bacterial pneumonia.

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MATERIALS AND METHODS Materials. AG was provided by Sichuan Wenlong Pharmaceutical Co., Ltd., China. β-CD was from Shandong Xinda Chemical Industry Co., Ltd., China. Penicillin Sodium Injection was purchased from North China Pharmaceutical Co., Ltd., China. Luria−Bertani broth and powdered agar were purchased from by Beijing Aobox Biotechnology Ltd. Other reagents were of analytic grade. Pure water prepared with Heal Force Pure Water System was always used. Animals. Male Sprague−Dawley (SD) rats (190−200 g) were provided by Vital River Experimental Animal Technology Co., Ltd. (Beijing, China). The handling and surgical process of these animals complied with the Guidinglines for Laboratory Animals. Animal experiments were accomplished in Beijing Institute of Radiation Medicine (BIRM) and approved by the animal subject review committee. Before sacrifice of the animals, peripheral blood was collected via tail veins. Lung bronchoalveolar lavage fluids (BALFs) were collected after washing three times. The lung tissues were excised followed by hematoxylin and eosin (H&E) staining. Preparation of AG-β-CD. AG was dissolved in ethanol and β-CD was dissolved in water at the same molar concentration to AG solution. The two solutions of an equal volume were mixed followed by continuously grinding by hand at room temperature for 1 h. The suspension was freeze-dried for 24 h on a lyophilizer (LGJ-30F, Beijing Songyuan Huaxing Technology Develop Co., Ltd., China). The powder of AG/ β-CD (1:1, mol/mol) inclusion complexes was obtained after 180-mesh sieving.

Figure 1. Illustration of the pneumonic rat model and the antipneumonia study. B

DOI: 10.1021/acs.molpharmaceut.6b01162 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 2. Structure of AG (a), FT-IR spectra (b), and XRD curves (c) of AG, β-CD, AG-β-CD, and the physical mixture of AG and β-CD.

aliquots (20 μL) of blood were withdrawn via tail veins, and then the leukocytes and neutrophils were detected. The rats were anesthetized with isoflurane using an ABS mini-anesthetic machine (Yuyan, Shanghai, China). The tracheas were exposed. The left lung was ligated using a hemostatic clamp. A tracheal cannula was inserted into the right lung, and the 4 °C cold saline was flushed to the right lung with 2 mL once three times. The acquired BALFs were centrifuged at 3000 rpm for 10 min, and the supernatant was collected and stored in a −80 °C refrigerator. The precipitated cells were resuspended using saline (6 mL) to count white blood cells (WBC). Leukocyte and Neutrophil Measurement. Aliquots (20 μL) of blood from the infected and treated rats were withdrawn via tail veins into the EDTA-coated tubes containing the isotonic diluents (2 mL, ISOTONAC-3) 12 h after the final administration. The samples were analyzed with an automated hematology analyzer (MEK-7222K, Nihon Kohden, Japan) for leukocyte and neutrophil measurement. Lung Homogenates for Counting Bacteria. The upper lobe of the left lung was homogenated with saline (1 mL, 4 °C) using a glass tissue homogenizer. After appropriate dilution, the bacteria in the homogenates were counted following inoculation and culture at 37 °C for 20 h. Histopathological Examination. The middle lobe of the left lung was immersed in 10% formalin solutions and then embedded in paraffin. The 5 μm-thick pathological sections were obtained and H&E stained. The sections were observed under a microscope. Immunohistochemistry. The lower lobe of the left lung was processed as mentioned above. The tissues embedded in paraffin were deparaffined in xylene and rehydrated with ethanol. They were immersed in the EDTA antigen retrieval solutions (pH 8.0), and the antigens were removed after microwave heating for 15 min. The sample was washed with water for 5 min and processed with hydrogen peroxide solutions (3%, 30 μL) to remove the endogenous peroxidases. The primary antibody of NF-κB p65 diluted with PBS (pH 7.4) was applied and incubated for 30 min at room temperature. The secondary antibody of the primary antibody was applied for 30 min at room temperature with interval PBS washing. Immunohistochemical detection was performed according to

the kit instruction. The stained sections were observed under a fluorescent microscope (Nikon Eclipse, Japan). Measurement of TNF-α, IL-6, and Total Protein. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and total protein (TP) are the important markers of inflammatory immune responses.37 The concentrations of TNF-α, IL-6, and TP in the supernatants of BALFs were measured with the ELISA kits (Neobioscience Technology Co., Ltd., China) according to the instructions. Statistical Analysis. Data were presented as mean ± standard deviation (SD) and processed using the Excel software. Student’s t-test was used to evaluate statistical differences with p < 0.05 or p < 0.01. Characterization of AG-β-CD for Pulmonary Delivery. The particle morphology was investigated using a 5 kV, S-4800 scanning electron microscope (SEM, Hitachi, Japan). The tap and bulk density, and angle of repose of AG-β-CD powders were measured with similar methods as those mentioned the report.38 The volume diameters of AG-β-CD were acquired using a laser particle size analyzer (BT2001, Dandong Bettersize, China). The theoretical mass mean aerodynamic diameter (MMAD) of AG-β-CD was calculated with eq 1: MMAD = d(ρ /ρ0 X )1/2

(1)

where d was the geometric mean diameter, ρ0 was a reference density of 1 g/mL, X was the dynamic shape factor that was 1 for a sphere, and ρ was the tapped density (also the bulk density), and the aerodynamic diameter was calculated from D50 (the D50 value was obtained from the measurement of volume diameters). The powder in vitro deposition was measured using the next generation impactor (NGI, Copley, British).39 The fine particle fraction (FPF), respirable fraction (RF), and emitted dose (ED) were calculated with eqs 2−4. fine particle fraction(FPF) = fine particle dose/initial particle mass loaded into capsules × 100% C

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percentages between AG and AG-β-CD in different hours of 0.5, 5, and 24 h were 5.41%, 36.65%, 39.66% and 6.03%, 70.95%, 75.02%, respectively. β-CD or its derivatives can improve the dissolution of hydrophobic drugs after the formation of complexes.27 In this study, AG-β-CD showed faster drug release and a higher release level than AG powders because of the higher dissolution of the complexes than AG. Furthermore, AG-β-CD may have improved in vivo stability and bioavailability of AG according to the in vitro result. Poor In Vitro Antimicrobial Effect of AG-β-CD. Penicillin showed strong in vitro anti-S. aurerus effect at the minimal inhibitory concentration (MIC) of 12.5 μg/mL as in other reports (Figure 4c).40,41 However, both of AG and AG-βCD had little antibacterial activity even at a high level of 40 and 120.23 mg/mL (Figure 4a,b). AG-β-CD Facilitated Bacterial Clearance from the Lung. In the previous section, we mentioned that AG and AG-β-CD had no antibacterial activity in vitro. However, we found they showed antipneumonia action in our preliminary in vivo study. In this study, we detected bacteria in the pneumonic rat lungs with or without pulmonary administration of medicines. Surprisingly, all the medicines including AG, AGβ-CD, and penicillin showed low colony forming unit (CFU) levels in the lungs after 24 h postinfection (Figure 5G). More importantly, the effect of AG-β-CD was significantly lower than that of AG, and the AG dose of AG-β-CD was only 1/10 of administered AG. Therefore, there is definitely an uncovered mechanism by which AG-β-CD facilitates bacterial clearance from the lung besides improvement of AG release due to β-CD inclusion. Furthermore, there was statistically significant difference (p = 0.035) between the CFUs of the AG-β-CD and penicillin groups, i.e., the in vivo anti-S. aureus effect of AGβ-CD was lower than that of penicillin though the effects were close. This result may be predicated because penicillin is a potent anti-S. aureus agent. In contrast, AG had antibacterial effect mainly by regulating immune responses. However, the anti-inflammatory effect of AG-β-CD was higher than that of penicillin. AG-β-CD Regulated Immune Responses in Pneumonic Lungs. Bacterial infection generally initiates immune responses, leading to increase of inflammatory cytokines. The immune system kills or eliminates the bacteria with phagocytosis, etc. However, overexpression of immune responses will damage the normal functional cells. Therefore, the appropriate regulation of immune responses may eliminate bacteria, reduce damages, and facilitate the recovery of normal functions for the treatment of bacterial pneumonia.42−44 In this

respirable fraction(RF) = mass of particle deposited on stages 2−7 /total particle mass on all stages × 100%

(3)

emitted dose(ED) = (initial mass in capsules − final mass remaining in capsules)

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/initial mass in capsules × 100%

(4)

RESULTS AND DISCUSSION Characteristics of AG-β-CD. AD-β-CD was identified by comparing the FT-IR spectra of the complex and AG, where the 1727 cm−1 peak of carbonyl and the 2958, 2979 cm−1 peaks of methylene disappeared after inclusion. The result suggested that the guest AG molecules and the host β-CD molecules formed AD-β-CD based on the intermolecular force between AG and β-CD (Figure 2b). The XRD further demonstrated the formation of inclusion complexes (Figure 2c). Two 2θ peaks at 5.2° and 10.3° disappeared in the XRD graph of inclusion complexes compared to AG and the AG/β-CD physical mixtures. Rapid and High in Vitro Release of AG from AG-β-CD. Cyclodextrin complexes can improve the dissolution of hardwater-soluble drugs.27 We investigated AG release from AG powders and AG-β-CD in the SLF. Both of them showed rapid AG release within 3 h (Figure 3). At 5 h, the released AG from

Figure 3. Release profiles of AG from AG powders and AG-β-CD powders in the SLF.

AG-β-CD increased to 72%, whereas the value of AG powders was only 39%. Furthermore, variation of cumulative release

Figure 4. In vitro anti-S. aureus effects of AG (a), AG-β-CD (b), and penicillin (c) with a series of gradient concentrations. D

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Figure 5. Effects of different medicines on the leukocytes (A), neutrophils (B), white blood cells (WBC, C), total protein (TP, D), TNF-α (E), IL-6 (F), and bacterial colonies (CFU, G) of S. aureus pneumonic rats. The data are present as mean ± SD (n = 6). *, p < 0.05; **, p < 0.01.

Figure 6. Histopathological pictures (400×) of the lungs (H&E) and the expression of NF-κB p65 (F&I). Healthy rats (a); the S. aureus pneumonic rats (b); the pneumonic rats treated with AG (10 mg) (c); the pneumonic rats treated with AG-β-CD (eq to 1 mg of AG) (d); the pneumonic rats treated with 48 mg of penicillin (e). The red arrows indicate the expression of NF-κB p65.

TNF-α is an important mediator of inflammation and emerges early during inflammatory development.46 IL-6 is also an inflammatory factor that emerges with inflammatory reactions.47 In this study, AG and AG-β-CD led to the lower total protein levels than penicillin in the pneumonic rats with statistical significance (Figure 5D), and the level in the case of AG-β-CD was lowest. The S. aurerus pneumonia led to very high TNF-α and IL-6 levels in the rat lungs, but they decreased following treatments (Figure 5E,F). The AD-β-CD group also had the lowest levels of TNF-α and IL-6 in all the groups. NF-κB participates in immunoreactions, inflammatory reactions, apoptosis, and tumor genesis by regulating multigenes expression. NF-κB p65 is a dipolymer combining the competent with NF-κB.48 We found that bacterial pneumonia markedly promoted NF-κB p65 phosphorylation. The lung

study, we detected the capability of the medicines to regulate the immune responses in the lung following bacterial infection. The amounts of leukocytes, neutrophil, and white blood cells (WBC) in the lung rapidly increased after S. aurerus infection (Figure 5A−C). The levels of these immune cells were downregulated by AG, AD-β-CD, and penicillin. However, the levels of immune cells were higher for AG and AD-β-CD than penicillin. Therefore, the local immune ability in the infected lungs after administration of AG and AG-β-CD remained at a level high enough to kill bacteria without strong immune damage. AG may be an efficient immunomodulatory agent. AG-β-CD Suppressed Inflammatory Reactions in Pneumonic Lungs. Inflammatory reactions are the major results of bacterial pneumonia.45 Generally, the amount of total proteins remarkably increases in the inflammatory exudates. E

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Figure 7. In vitro lung deposition (a) and SEM images (b,c) of AG-β-CD powders. Moc: micro-orifice collector.

Here, we showed the solid evidence of AG against S. aureus pneumonia. More importantly, a special pulmonary delivery route was applied rather than oral or injection routes. The comparison between the in vitro and in vivo results demonstrated that the mechanism of AG as an efficient antiS. aureus pneumonia agent is its strong ability to regulate immune responses. More importantly, the function is not related to the subject-bacteria. Therefore, AG and its formulations can be used for treating the infections of resistant bacteria, which will also be investigated in the future in our lab. Moreover, the multiple pharmacological activities of AG are definitely related to its immunoregulation, including the antiviral,49 antioxidation,50 and anticancer functions.51,52 Pneumonia is a commonly seen lung disease so that an inhalation therapy may be an optimal selection if no other syndromes occur. Inclusion complexes of β-CD could improve the dissolution and bioavailability of water-hard-soluble AG. Therefore, immunoregulation of AG, solubility improvement of β-CD, and local lung infections of S. aureus pneumonia allow the inhalable AG-β-CD powders to wonderfully treat S. aureus pneumonia with good inhalable behavior, low dose of drugs, and sufficient safety.

immunohistochemical results of NF-κB p65 showed that AG-βCD and AG significantly reduced the expression of NF-κB p65 seen from a little more dark brown stains than the bacterialpneumonic lung (Figure 6, F&I). AG-β-CD Attenuated Bacteria-Induced Lung Injury. Pneumonia definitely leads to lung injury that is the major cause of death. Therefore, the attenuation of lung injury is important for pneumonia therapy. In this study, the S. aureus pneumonia led to a lot of exudates and heavy hemorrhage in the lung (Figure 6, H&E-b). AG alone did not show significant therapeutic effect with hemorrhage and infiltration of inflammation cells (Figure 6, H&E-c). However, AG-β-CD remarkably attenuated the lung injury merely with a little local hemorrhage (Figure 6, H&E-d). Penicillin also attenuated the lung injury as AG-β-CD did (Figure 6, H&E-e). Pulmonary Delivery Properties of AG-β-CD. The repose and tapped density of AG-β-CD powders were 33.78 ± 1.43° and 0.523 ± 0.006 g/cm3, indicating the powders were flowable and loose. The D50 of AG-β-CD was 2.55 ± 0.08 μm (n = 3), and the particle of aerodynamic diameter was 2.03 ± 0.06 μm (n = 3), suitable for pulmonary inhalation. The inclusion efficiency (IE) and drug loads of AG-β-CD were 63.92 ± 3.98% and 9.61 ± 1.99%. The fine particle fraction (FPF), respirable fraction (RF), and emitted dose (ED) of AG-β-CD powders were 81.83 ± 2.01%, 31.81 ± 0.27%, 96.08 ± 0.3%, respectively. These values are relatively high, suggesting high lung deposition (Figure 7a). Particle morphology and sizes were visualized using SEM (Figure 7b,c). The powders were composed of grouped bricks, and the sizes of particles were less than 5 μm. Mechanism and Perspective of AG as an Efficient Antibacterial Pneumonia Agent. Andrographis paniculata is a traditional herb and usually used as a vegetable on the food table of the Chinese. Its pharmacological function has been recognized by the Chinese people for several thousand years. AG is also evidenced as the major active ingredient of this herb.

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CONCLUSION Oral AG tablets and pills have been clinically applied for treatment of upper respiratory tract infections. However, the detailed mechanism is not well-known. In this study, a β-CD inclusion complex of AG, AG-β-CD, was prepared. We demonstrate that the major mechanism of AG against the bacterial pneumonia induced by S. aureus is its immunoregulation after comparing the in vitro antibacterial effect and the in vivo antipneumonia effect of AG and AG-β-CD. The agents have no in vitro anti-S. aureus activity but show high anti-S. aureus pneumonia ability. Moreover, AG-β-CD highly enhances the local dissolution of AG in the lung and shows stronger antipneumonia effect than AG and penicillin. AG-β-CD is a F

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(12) Jamali, H.; Radmehr, B.; Ismail, S. Short communication: Prevalence and antibiotic resistance of staphylococcus aureus isolated from bovine clinical mastitis. J. Dairy Sci. 2014, 97, 2226−2230. (13) Song, K.; Han, C.; Dash, S.; Balart, L. A.; Wu, T. Mir-122 in hepatitis b virus and hepatitis c virus dual infection. Word J. Hepatol. 2015, 7, 498−506. (14) Dzoyem, J. P.; Eloff, J. N. Anti-inflammatory, anticholinesterase and antioxidant activity of leaf extracts of twelve plants used traditionally to alleviate pain and inflammation in south africa. J. Ethnopharmacol. 2015, 160, 194−201. (15) Gandhi, G. R.; Barreto, P. G.; Lima, B.; Quintans, J. d. S. S.; Araújo, A. A. d. S.; Narain, N.; Quintans-Júnior, L. J.; Gurgel, R. Q. Medicinal plants and natural molecules with in vitro and in vivo activity against rotavirus: A systematic review. Phytomedicine 2016, 23, 1830−1842. (16) Khazir, J.; Mir, B. A.; Pilcher, L.; Riley, D. L. Role of plants in anticancer drug discovery. Phytochem. Lett. 2014, 7, 173. (17) Martino, E.; Della Volpe, S.; Terribile, E.; Benetti, E.; Sakaj, M.; Centamore, A.; Sala, A.; Collina, S. The long story of camptothecin: From traditional medicine to drugs. Bioorg. Med. Chem. Lett. 2017, 27, 701−707. (18) Cai, J.; Chen, S.; Zhang, W.; Hu, S.; Lu, J.; Xing, J.; Dong, Y. Paeonol reverses paclitaxel resistance in human breast cancer cells byregulating the expression of transgelin 2. Phytomedicine 2014, 21, 984−991. (19) Chen, L.-G.; Yu, A.-M.; Zhuang, X.-D.; Zhang, K.; Wang, X.-P.; Ding, L.; Zhang, H.-Q. Determination of andrographolide and dehydroandrographolide in rabbit plasma by on-line solid phase extraction of high-performance liquid chromatography. Talanta 2007, 74, 146−152. (20) Lee, K.-C.; Chang, H.-H.; Chung, Y.-H.; Lee, T.-y. Andrographolide acts as an anti-inflammatory agent in lps-stimulated raw264.7 macrophages by inhibiting stat3-mediated suppression of the nf- κb pathway. J. Ethnopharmacol. 2011, 135, 678−684. (21) Guo, X.; Zhang, L.-Y.; Wu, S.-C.; Xia, F.; Fu, Y.-X.; Wu, Y.-L.; Leng, C.-Q.; Yi, P.-F.; Shen, H.-Q.; Wei, X.-B.; Fu, B.-D. Andrographolide interferes quorum sensing to reduce cell damage caused by avian pathogenic escherichia coli. Vet. Microbiol. 2014, 174, 496−503. (22) Lu, C.-Y.; Yang, Y.-C.; Li, C.-C.; Liu, K.-L.; Lii, C.-K.; Chen, H.W. Andrographolide inhibits tnfα-induced icam-1 expression via suppression of nadph oxidase activation and induction of ho-1 and gclm expression through the pi3k/akt/nrf2 and pi3k/akt/ap-1 pathways in human endothelial cells. Biochem. Pharmacol. 2014, 91, 40−50. (23) Manoharan, S.; Singh, A. K.; Suresh, K.; Vasudevan, K.; Subhasini, R.; Baskaran, N. Anti-tumor initiating potential of andrographolide in 7,12-dimethylbenz[a]anthracene induced hamster buccal pouch carcinogenesis. Asian Pac. J. Cancer P. 2011, 13, 5701− 5708. (24) Lin, H.-H.; Tsai, C.-W.; Chou, F.-P.; Wang, C.-J.; Hsuan, S.-W.; Wang, C.-K.; Chen, J.-H. Andrographolide down-regulates hypoxiainducible factor-1α in human non-small cell lung cancer a549 cells. Toxicol. Appl. Pharmacol. 2011, 250, 336−345. (25) Lee, W.-R.; Chung, C.-L.; Hsiao, C.-J.; Chou, Y.-C.; Hsueh, P.J.; Yang, P.-C.; Janc, J.-S.; Cheng, Y.-W.; Hsiao, G. Suppression of matrix metalloproteinase-9 expression by andrographolide in human monocytic thp-1 cells via inhibition of nf-kb activation. Phytomedicine 2012, 19, 270−277. (26) Guo, W.-J.; Liu, W.; Chen, G.; Hong, S.-C.; Qian, C.; Xie, N.; Yang, X.-L.; Sun, Y.; Xu, Q. Water-soluble andrographolide sulfonate exerts anti-sepsis action in mice through down-regulating p38 mapk, stat3 and nf-κb pathways. Int. Immunopharmacol. 2012, 14, 613−619. (27) Liao, Y.; Zhang, X.; Li, C.; Huang, Y.; Lei, M.; Yan, M.; Zhou, Y.; Zhao, C. Inclusion complexes of hp-β-cyclodextrin with agomelatine: Preparation, characterization, mechanism study and in vivo evaluation. Carbohydr. Polym. 2016, 147, 415−425. (28) Ramos, A. I.; Braga, T. M.; Fernandes, J. A.; Silva, P.; RibeiroClaro, P. J.; Almeida Paz, F. A.; de Fatima Silva Lopes, M.; Braga, S. S.

promising pulmonary delivery medicine for inhalation therapy of bacterial pneumonia. Furthermore, AG and its formulations might be new weapons against the resistant bacterial pneumonia based on its immunoregulation function.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 10 88215159. Fax: +86 10 68214653. E-mail: jinyg@ sina.com. ORCID

Yiguang Jin: 0000-0002-3528-1397 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Prof. Li Han of the Institute of Disease Control & Prevention of PLA, China, for providing the bacteria, and Prof. Xiansheng Lu for his carefully proofreading.

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DOI: 10.1021/acs.molpharmaceut.6b01162 Mol. Pharmaceutics XXXX, XXX, XXX−XXX