Herbal Materials
Twenty (20) kinds of dried Taiwanese folk medicinal plants with antitoxic ability were purchased from the crude drug market in Taipei, Taiwan: Amaranthus viridis Linn., Angelica dahurica var. formosana Yen, Bidens pilosa L. var. minor (blume) Sherff, Broussonetia papyrifera (Linn) L'Herit. Ex Vent., Centella asiatica L., Equisetum ramosissimum subsp..debile (Roxb.) Hauke., Euphorbia hirta L., Euphorbia thymifolia L., Eupatorium formosanum Hay., Kyllingia brevifolia Rottb., Litsea cubeba (Lour.) Pers., Persoon Actinodaphne citrata (Blume) Hayata, Lygodium japonicum (Thunb.) Sw., Pinellia ternate (Thunb.) Breit., Plantago asiatica L., Polygonum perfoliuatum Linn., Portulaca oleracea var. sativa DC., Pteris multifida Poir., Rhus semialata var. roxburghiana DC., Serissa japonica (Thunb.) Thunb., Urena lobata L.. All were identified by Prof. Ling-Ling Yang and the specimens were stored at the Department of Pharmacognosy, Taipei Medical University.
Microorganisms
Twenty-eight (28) kinds of standard microorganism were purchased from Food Industry Research and Development Institute (FIRDI) in Taiwan. Standard strains include Klebsiella Pneumoniae ATCC 23856, (ESBL) ATCC 700603, Escherichia coli ATCC 25922, ATCC 35218, Staphylococcus. aureus (MSSA) ATCC 29213, (MRSA) 85/2082, (hVISA) Mu3, (VISA) Mu50, Staphylococcus epidermidis ATCC 12228, Group B streptococcus ATCC 12401, Enterococcus faecalis ATCC 29212, ATCC 29212, Pseudomonas aeruginosa ATCC 14207, ATCC 27853, Samonella enteric Typhimurium ATCC 13076, Samonella multivorum ATCC 35656, Samonella typhimurium ATCC 13311, Samonella paratyphi (A) ATCC 9150, Samonella anatum (E) ATCC 9270, Samonella choleraesusis ATCC 10744, ATCC 115462, Shigella boydii ATCC 9207, Shigella dysenteriae ATCC 13983, Shigella flexneri ATCC 10772, Shigella sonnei ATCC 15965, ATCC 25931, Candida albicans ATCC 90018, Candida parapsilosis ATCC 22019. While the thirty-three (33) clinical strains were isolated and collected from Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan, which labeled as Klebsiella Pneumoniae (ESBL) 1520914, 1527226, 1527920, 1520160-1, 1521656, 1522657, 1523941, 1524581, 1526783, 1543407, 1541487, 1542299, 1545756, 80, 81, 91, 98, and Escherichia coli (ESBL) 1526829, 1522652, 1530404, 1521949, 1521624, 1521569, 1530090, 1520331, 1520641, 1522991, 1522953, 1519678, 1520264, 1527958, 1528051, 1527082).
Chemicals and reagents
All chemicals and reagents were analytical grade including dimethyl sulfoxide (DMSO), gallic acid, Folin-Ciocalteu’s reagent, rutin, aluminum chloride, epicatechin, ferrous chloride (FeCl2), trolox, 2-thiobarbituric acid (TBA), 1,1,3,3-tetraethoxypropane (TEP), bovine serum albumin, phosphoric acid, methanol, sodium hydroxide (NaOH) and 4-dimethylaminocinnamaldehyde (DMACA) were purchased from Sigma Chemical (St. Louis, MO).
Animals
Imprinting Control Region (ICR) mice were purchased from BioLASCO Taiwan Co., Ltd., housed in plastic cages in a temperature and humidity-controlled environment and bred at the Experimental Animal Center of Taipei Medical University. All experiments were approved and performed in accordance with the guidelines for Experiments Animal Center of Taipei Medical University and the Laboratory Animal Ethics Committee of Taipei Medical University with the guiding principles for the care and use of laboratory animals approved by the Chinese Society of Laboratory Animal Sciences, Taiwan (No. LAC980119). All efforts were made to minimize animal suffering and to reduce the number of animals used.
Extraction and sample solution preparation
Dried plants were washed continuously with tap water to remove impurities, cut into small pieces and then dried in the air circulating oven at 60℃ for 48 hr. Each 100 g of dried material was extracted twice with 1000 mL of ethanol, filtered, and combined the filtrates, then concentration and removed the solvent in vacuo by vacuum rotary evaporator (Eyela CCA-1111) to 50mL. The concentrated extract solutions were dried by lyophilizer (Eyela FDU-1200) to obtain the extract. The preliminary antimicrobial activity to standard strains of Kp and E. coli ATCC bacteria of each extract was identified to find RSR the most potent extract from the 20 Taiwanese endemic plants. Therefore, RSR was further extracted by n-hexane, ethyl acetate, acetone, ethanol and water, respectively. Each crude extract (20 mg) was dissolved in 1 mL DMSO as a stock solution and serial dilution with PBS for the following assays. ( DMSO <0.05%).
Protocol of an antimicrobial agent development from natural plants source procedure
Five (5) steps were carried out to determine the most potent extract with antibacterial capability against Kp and E. coli and renal damage protective effect (Figure 1). Agar-dilution method was used to preliminary screening assess the antibacterial activity of crude extracts from 20 Taiwanese plants against standard strains of Kp and E. coli ATCC bacteria (E. coli ATCC 25922, ATCC 35218 and Kp ATCC 23856, ATCC 700603). RSR crude extract exhibited the maximum inhibitory effects and then subjected to further extract by n-hexane, ethyl acetate, acetone, ethanol and water, respectively. Each of these extracts was prepared as mentioned previously prior to antimicrobial assay against the same 4 bacteria by agar-dilution method.
The most potential extract of RSR (acetone extract) was chosen to evaluate the MIC50 and MIC90 against clinical Kp and E. coli. Time kill assay against Kp (ATCC) 23856, (ATCC) 700603, E. coli (ATCC) 25922, (ATCC) 35218, and clinical ESBL-Kp 1520914 and ESBL- E. coli 1526829 were also conducted.
To assess the antibacterial spectral range of the extract, agar-dilution method was performed against 24 kinds of microbes including Gram positive, gram negative bacteria and fungi. Finally, the renal protective capability was measured by ferrous ion-induced lipid peroxidation (LPO) on mice mitochondria.
Agar-dilution method
Agar-dilution method described by Nauman and Arshad (2011) was used with some modification. Briefly, extracts were added in appropriate amounts of 20 mL Mueller-Hinton (MH) agar to yield a two-fold serial dilution. The bacteria were plated onto the MH-agar surface (104 CFU per 0.3 μL per spot) by an A400 Multipoint Inoculators (Jencons Co. UK) and incubated at 37°C for 24 hours. MIC value was evaluated as the lowest concentration (µg/mL) of antimicrobial agent with no visible growth. MIC50 and MIC90 were detected with the concentration of antibacterial agent preventing 50% and 90% of colony formation of the strains tested.
Time-kill assay
Method as described by Darah et al. (2013) was used with some modification. Briefly, exponential-phase ESBL-producing Kp (ATCC) 23856, (ATCC) 700603, E. coli (ATCC) 25922, (ATCC) 35218, and clinical ESBL-Kp 1520914 as well as ESBL- E. coli 1526829 were diluted to 4×104 to 1×105 CUF/ mL and then exposed to RSR acetone extract (1, 2 and 4 x MIC), 1 mL DMSO, polymyxin B sulfate salt (PB) (0.5 and 1 x MIC) and 1 x MIC RSR acetone extract combined with 0.5 x MIC PB, respectively, incubated for 48 hours period in a total volume of 2 mL of MH media. Viable cell count was determined by agar plating (onto MH agar). Bactericidal activity was defined as > 3 log decrease in cell counts. For all strains in the present report, time kill assays were performed at least twice independently with similar results.
Inhibitory capability of LPO on mice kidney mitochondria
Preparation of mice kidney mitochondria and protein content quantitation
Method described by Lin et al. (2013) was used with some modification. Male ICR mice (4~6 weeks) were sacrificed by carbon dioxide and the kidneys were removed as soon as possible and perfuse with ice-cold PBS (0.1 M, pH 7.4) prior to homogenizing in a Potter Elvehjem homogenizer. The homogenate was suspended in PBS and centrifuged at 2000 rpm for 10 min at 4°C to separate the nuclear debris. The clear suspensions were re-centrifuged at 13000 rpm for 10 min at 4°C to obtain mitochondrial fraction and then suspended in PBS. Different concentrations of mitochondrial suspended solution were pipetted into 1.5 mL eppendorf tubes, and total volume was adjusted to 50 μL with PBS. Protein reagent was added and mixed with a vortex-mixer before measurement of absorbance at 595 nm by ELISA spectrophotometer (Synergy H4 Hybrid Reader). Quantitative analysis of the protein content was determined from bovine serum albumin standard curve.
Inhibitory capability of RSR (Rhus semialata var. roxburghiana) acetone extract on ferrous chloride induced LPO production in mice kidney mitochondria
The LPO inhibitory activity was determined by thiobarbituric acid reactive substances (TBARS) assay by quantitatively measure the MDA(TBA)2 products using ELISA reader (Synergy H4 Hybrid Reader). The reaction mixture solution (total volume of 500 μL) containing 100 μL of kidney mitochondria, 200 μL of PBS buffer, 100 μL of an FeCl2 solution (4 mM), and 100 μL of RSR acetone extract or positive control (trolox or gallic acid) was incubated at 37 °C for 1 hour, then centrifuged at 4000 rpm for 10 min. A 375 μL of H3PO4, 200 μL of distilled water, and 125 μL of TBA were added and the mixture was further incubated at 90 °C for 66 min prior to place in an ice bath to stop the reaction. Then, 350 μL of methanol-NaOH (9.1: 0.9 v/v) was added, mixed and absorbance of MDA(TBA)2 product was measured at 532 nm. The data was recorded and the inhibition of LPO calculated according to the following formula:
Inhibition percentage = [(A control (532 nm) - A sample (532 nm)) / (A control (532 nm) - A blank (532 nm))] × 100% while IC50 was calculated by a linear regression analysis, and results were expressed as mean ± SD.