In order to overcome the downside of long conventional freeze-drying (CFD) process times for monoclonal antibody formulations, microwave-assisted freeze-drying (MFD) was introduced. Recently, the general applicability and potential shortening of drying times were shown. However, little is known about the storage stability of MFD products compared to CFD references. Additionally, batch homogeneity issues were seen within MFD in the past. In this study, we examined four different formulations of two different monoclonal antibodies using three different glass-forming excipients: sucrose, trehalose, and arginine phosphate. These formulations were freeze-dried with two different drying protocols (CFD and MFD), stored for 24 weeks, and analyzed for solid-state and protein-related quality attributes. Moreover, a new microwave generator setup was investigated for its potential to improve batch homogeneity. In all investigated formulations, comparable stability profiles were found, although the classical magnetron generator led to inferior batch homogeneity with respect to residual moisture distribution. In contrast, the new MFD setup indicated the potential to approximate batch homogeneity to the level of CFD. However, for future applications, there is an unabated need for new machine designs to comply with pharmaceutical manufacturing requirements.  Conventional freeze-drying (CFD), also referred to as lyophilization, is a gentle drying method to improve the long-term stability of pharmaceuticals, specifically of protein drugs . The metIn general, freeze-drying comprises three steps: freezing, primary drying (= sublimation drying), and secondary drying (= desorption drying). Typically, the sublimation step is widely described to be the most time-consuming, and conventional freeze-drying is associated with lengthy process times ,10,11,12The current study examines four different formulations of two different monoclonal antibodies in the presence of three different glass-forming excipients: sucrose, trehalose, and arginine phosphate. These formulations were freeze-dried with two different drying protocols, i.e., using conventional freeze-drying and microwave-assisted freeze-drying. Moreover, a new microwave setup equipped with a semiconductor solid-state microwave generator was used for one of the formulations. Samples were stored for 24 weeks at different temperatures (2–8 °C and 40 °C) and analyzed at fixed times for their solid-state and protein-related quality attributes. We hypothesize that, on the one hand, irrespective of the monoclonal antibody formulation, comparable stability profiles can be found for CFD and MFD. On the other hand, we anticipate the new microwave machinery setup to have a positive effect on batch homogeneity in microwave-assisted freeze-dried products. Two different IgG type 1 monoclonal antibodies (mAb) were investigated. mAb1 was kindly provided by Boehringer Ingelheim Pharma GmbH & Co. KG . mAb2 was an on stock at Ludwig-Maximilians-Universität München (LMU).® exp L-Arginine (Ph. Eur. certified), EMSURE® ortho-Phosphoric acid 85% and Ph. Eur. certified Tween 80® were obtained from Merck KGaA . For mAb2-formulation, EMPROVE® exp sucrose (Ph.Eur.-certified) purchased from Merck KGaA  was used.For mAb1-formulations, the following excipients were used: ACS certified D(+) Sucrose, which was purchased from Sigma-Aldrich , D(+) Trehalose dihydrate (min. 99% purity) was obtained from VWR International BVBA . EMPROVE® base BioXtra (>99.9%) and Trizma® hydrochloride BioXtra (>99.0%) were purchased from Sigma-Aldrich . Sodium chloride was obtained from Bernd Kraft . Sodium hydroxide was purchased from Merck KGaA .L-Histidine monohydrochloride monohydrate (min. 99% purity) and L-Histidine (Cell culture reagent) were purchased from Alfa Aesar . Di-sodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate were obtained from AppliChem . TrizmaFor the preparation of buffers and stock solutions, water for injection  was used. The four different formulations, F1–F4, which were dried either by microwave-assisted freeze-drying (MFD) or by conventional freeze-drying (CFD), were stored for 24 weeks at refrigerator temperature 2–8 °C, at 25 °C (F1), and at 40 °C . The low®  with two buffer exchanges. After dialysis, the concentration of mAb1 was measured with a NanoDrop™ 2000 UV photometer  at 280 nm using an extinction coefficient of ε0.1% = 1.49 g/100 mL−1 cm−1. mAb1 was dialyzed against 10 mM histidine buffer  or 10 mM arginine phosphate (F3) at pH 6.0 for 24 h using dialysis membranes Spectra/Por−1 cm−1 and a molecular weight of MW = 145.5 kDa.mAb2 (F4) was dialyzed and concentrated using a cross-flow filtration unit Minimate™ TFF capsule with omega polyethersulfone (PES) membrane  by adding a 10-fold excess of 10 mM histidine buffer (pH 5.5). After reaching the desired volume, the concentration of the mAb was measured with a NanoDrop™ 2000 UV photometer at 280 nm using an extinction coefficient of ε = 225,000 MFormulations were prepared according to the composition shown in ® RF vacuum filter unit . For each formulation, 2.3 mL was filled in 10R tubing vials  and semi-stoppered with lyophilization stoppers . The vial population for conventional freeze-drying was arranged on a lyophilization tray, and surrounded with at least one row of 10% (w/v) sucrose shielding vials.F1–F3 were filtered using 0.2 µm PES membrane syringe filters , whereas F4 was filtered using a 0.22 µm PES SartolabAll samples of a corresponding formulation were frozen in the same freezing step. The formulations F1–F3 were frozen in a Christ ε2-6D laboratory-scale freeze-dryer  with equilibration at −5 °C for 1 h, followed by ramping down the shelf with 1 K/min to a −60 °C set point.Formulation F4 was frozen in an FTS Systems LyoStar™ 3 freeze-dryer  with equilibration at 5 °C for 1 h, followed by ramping down the shelf with 1 K/min to a −50 °C set point.The frozen samples were subjected to one of the following drying protocols:The conventional freeze-drying cycles are summarized in Microwave-assisted freeze-drying was conducted on a modified laboratory-scale Püschner µWaveVac 0250fd vacuum dryer prototype . For the formulations F1–F3, the setup described previously  was usedIn contrast, F4 was processed with a partially modified setup comprising a semiconductor solid-state 500 W/2450 MHz microwave radiation source tunable from 5 W to 450 W, which directly emitted its radiation into the product chamber . MoreoveFor process monitoring, two glass fiber temperature measurement probes  were used. Stoppering of the samples was carried out externally in a glove bag flushed with dry nitrogen. The dried crimped samples were kept refrigerated until analysis.w/w). For verification of equipment performance, three aliquots of Apura® water standard oven 1% by Merck KGaA  were measured within a sequence.Karl Fischer titration, equipped with a headspace module, was used to determine residual water content after freeze-drying. Between samples, aliquots of 9 mg and 28 mg were prepared in a glove box filled with pressurized air with a relative humidity of less than 10%, placed into 2R vials, and stoppered. The samples were then placed in an oven with 100 °C to enable the fast extraction of water. The headspace moisture is transported into a coulometric Karl Fischer titrator , Germany). Results were calculated in relative water content  krypton gas adsorption in a liquid nitrogen bath at 77.3 K . Samples of 90–200 mg were gently crushed with a spatula and weighed into glass tubes. Prior to measurement, an outgassing step was performed for at least 6 h at room temperature. An 11-point gas adsorption curve was measured, covering a p/pTo determine the solid-state of the lyophilizates, an XRD 3000 TT diffractometer  was used. The device was equipped with a copper anode  and had a wavelength of 0.154178 nm. The scintillation detector voltage was 1000 V. The samples were placed on the copper sample holder and analyzed in the range of 5–45° 2-theta, with steps of 0.05°.The lyophilized cakes were reconstituted by the addition of WFI. The WFI volume for each formulation was calculated to match the volume of the water removed during freeze-drying. Reconstitution time was determined by recording the time between adding the respective formulation-specific volume of water for injection and obtaining a clear solution without visible matter. This observation was performed by manual visual inspection. Reconstitution was performed applying gentle swirling for 5 s directly after the addition of water.A Waters 2695 Separation module  equipped with a Waters 2487 Dual λ Absorbance Detector  at 214 and 280 nm was used. Isocratic elution with a 25 mM sodium phosphate running buffer containing 200 mM sodium chloride (pH 7.0) was performed. For mAb1-formulations (F1–F3), 10 µL of a reconstituted solution corresponding to a loading of 50 µg protein were loaded on a Tosoh TSKgel G3000SWxl, 7.8 × 300 mm, 5 µm column  and separated with a flow rate of 0.7 mL/min. For mAb2 (F4), samples were diluted with 10 mM histidine buffer (pH 5.5) to 1 g/L protein concentration, and 25 µL was injected, corresponding to a load of 25 µg protein. A YMC-Pack Diol-300, 300 × 8.0 mm, 5 µm column  with a flow rate of 0.8 mL/min was used for separation. Samples were measured in triplicates with three individual injections. Data integration of relative areas at 280 nm was performed using Chromeleon 6.80 , provided that every peak eluting before the monomer corresponded to high molecular weight (HMW) species. No peaks could be detected after the monomer. For verification of equipment performance, an internal standard of thawed mAb formulation was injected at the beginning and end of a sequence. A Waters 2695 Separation module  equipped with a Waters 2487 Dual λ Absorbance Detector  at 214 and 280 nm was used for weak cation exchange chromatography. A linear sodium chloride gradient of 0% to 20% solvent B in solvent A over 30 min was used for elution at a flow rate of 1 mL/min. For all cation exchange (CEX) analysis, a ProPac™ WCX-10G BioLC™ Analytical column 4 × 250 mm equipped with a ProPac™ WCX-10G BioLC™ guard column 4 × 50 mm  was used. For mAb1-formulations (F1–F3), the solvents were composed of A: 20 mM TRIS (pH 7.1) and B: 20 mM TRIS (pH 7.1) plus 300 mM sodium chloride. Reconstituted sample aliquots of 10 µL, corresponding to a loading of 50 µg protein, were loaded on the column. For mAb2 (F4), the solvents were composed of A: 20 mM TRIS (pH 7.5) and B: 20 mM TRIS (pH 7.5) plus 300 mM sodium chloride. Before analysis, samples were diluted with solvent A to 1 g/L protein concentration, and 50 µL was injected, corresponding to a load of 50 µg protein. Samples were measured as triplicates with two individual injections. Data integration of relative areas was performed using the Chromeleon 6.80 software , provided that every peak eluting before the main peak corresponded to acidic species and peaks eluting after the main peak corresponded to basic species. For verification of equipment performance, an internal standard of thawed mAb formulation was injected at the beginning and end of a sequence.One method used to determine subvisible particles of the formulation F1 was light obscuration. Therefore, a PAMAS SVSS-35 particle counter  equipped with an HCB-LD 25/25 sensor, which had a detection limit of approximately 120,000 particles ≥ 1 µm per mL, was used. The pre-rinsing volume was 0.4 mL and was followed by three measurements of 0.2 mL. The fill rate, emptying rate, and rinse rate of the syringe were set to 10 mL/min. Before and between samples, the system was rinsed with WFI until less than 30 particles/mL ≥ 1 µm and no particles larger than 10 µm were present. Data collection was done using PAMAS PMA software, and particle diameters in the range of ≥1 µm to 200 µm were determined. All results are given in cumulative particles per milliliter.® 8100  equipped with a 10× magnification cell (81 µm × 700 µm). Prior to a measurement set, the cell was cleaned with a 1% Hellmanex III solution and WFI. For adjustment of the focus, the default autofocus procedure using 20 µm calibration beads was performed. Sample solution volumes of 150 µL were measured with a flow rate of 0.10 mL/min, at an image rate of 29 frames per second, and an estimated run time of 1.5 min. After each measurement, the flow cell was flushed with WFI. For particle identification, the following settings were used: 3 µm distance to the nearest neighbor, particle segmentation thresholds for dark pixels and light pixels of 13 and 10, respectively. The particle size was reported as the equivalent spherical diameter (ESD). Frames were collected with VisualSpreadsheet® 4.7.6 software and were evaluated for total particle counts of cumulative particles greater or equal to 1 µm, 10 µm and, 25 µm per mL.Due to the high transparency of protein particles, an orthogonal method for subvisible particle determination was introduced for formulations F2–F4. Flow-imaging microscopy was performed on a FlowCAM2/g ± 0.02 m2/g, and MFD, 0.66 m2/g ± 0.05 m2/g. Irrespective of the storage temperature and time point, neither differences nor relevant changes in specific surface areas were observed. With regard to residual moisture content, analysis showed low values for both CFD and MFD . These values changed only slightly over 24 weeks at all investigated storage temperatures. However, within the microwave-processed products, some samples exhibited higher variances represented by higher standard deviations, which were not found within the conventionally freeze-dried samples. The results for residual moisture (RM) and specific surface area (SSA) determination are presented in 2/g ± 0.01 m2/g and 1.22 m2/g ± 0.06 m2/g for CFD and MFD, respectively. In regards to residual moisture, MFD samples appeared to be moister than CFD samples (1.2% ± 0.8% vs. 0.3% ± 0.0%). These differences remained over the course of six months of storage irrespective of the storage temperature. Yet, the moisture content in CFD cakes doubled at 40°C (0.7% ± 0.0%), unlike MFD cakes. However, high variances within MFD samples may have masked such effects.Similar results were found for the low concentrated mAb formulation stabilized with trehalose b. No rel2/g ± 0.09 m2/g and 0.95 m2/g ± 0.04 m2/g for CFD and MFD, respectively. Slight, but non-significant changes over storage were observed. The initially different SSA values correlated inversely with the observed residual moisture mean values (CFD: 1.0% ± 0.1% and MFD: 2.8% ± 0.4%). A micro-collapse within MFD samples was assumed. Unlike the sucrose (F1) and trehalose (F2) formulations, low concentration mAb formulations with arginine phosphate (F3) exhibited differences with respect to specific surface area c. Initia2/g ± 0.15 m2/g and 0.89 m2/g ± 0.04 m2/g for CFD and MFD, respectively. F4, which was comprised of a 1:1-mixture (weight-wise) of sucrose and mAb2, was dried with a different microwave-setup. By this, high variances in residual moisture, which occasionally occurred before within MFD samples, were not observed anymore d. For coIn order to confirm the amorphicity of all formulations, XRD was used. The results for the two sucrose-based formulations, F1 and F4, are presented in Amorphous halos and the absence of typical peaks ,30 were An overall XRD-amorphicity, represented by an amorphous halo, was also found for the significantly moister arginine phosphate formulations . A referBefore liquid analysis, lyophilized products needed to be reconstituted. Within one formulation, no significant difference between the distinct drying protocols was seen. However, small differences with regard to other formulation were observed .The subvisible particle counts (SvP) obtained by light obscuration for F1 are presented in The SvP counts for the low concentration mAb formulation stabilized by arginine phosphate, F3, are shown in At accelerated storage conditions b, a modew/v), sucrose-stabilizer neither at refrigerator , data from CEX measurements is shown in For the arginine phosphate-formulation (F3), CEX data is shown in In the case of the high concentration mAb formulation stabilized with sucrose (F4), almost no differences were found between conventional and microwave-assisted freeze-dried products with regard to CEX results a,b. At rw/v) sucrose (F1) are displayed in The relative amount of monomeric and high molecular weight (HMW) species was assessed by high-performance size-exclusion chromatography. The results of the HP-SEC analysis for the low concentration mAb formulation with 10% , more changes were observed c. At refThe formulation with 50 g/L of mAb2 (F4) showed a higher monomeric SEC-purity of 99.9% ± 0.0%, compared to mAb1 formulations prior to freeze-drying c. At refIn the case of freeze-dried products, attributes like residual moisture content, solid-state, and specific surface area are critical to assess and to monitor over storage and shelf-life . With reIn contrast, in trehalose samples (F2), residual moisture levels differ already after the freeze-drying step, i.e., 0.3% ± 0.0% for conventionally dried and 1.2% ± 0.8% for microwave-assisted dried samples b. The CFAn arginine phosphate-based formulation (F3) was expected to be different. Firstly, because of the permanently charged character of arginine which causes high dielectric loss, i.e., the ability of a material to absorb electromagnetic energy , as repo2/g ± 0.04 m2/g for MFD, compared to 1.33 m2/g ± 0.09 m2/g for CFD, initially after application of the different drying protocols  produced by MFD was not observed anymore. Two possible reasons are: 1) The formulation, which consisted of roughly 50% monoclonal antibody, may have changed the matrix properties; and 2) the newly implemented microwave technical setup as described in the materials and method section. The authors assume that the increase in batch homogeneity within MFD samples was mainly due to the change in machinery setup. This is believed because: Firstly, similar formulations with the same mAb have been dried with the previously described MFD setup [The 50 g/L mAb2 formulation stabilized with sucrose (F4) appeared to be different with respect to residual moisture content d. The ocFD setup , and then = 3, potential batch inhomogeneities were observable. Thirdly, the solid-state semiconductor setup led to a more stable and better tuneable power input during the course of drying. Supportively, Bianchi et al. [Secondly, with typical sample sizes of i et al.  simulateWith regard to X-ray diffraction analysis, all formulations revealed an XRD-amorphous solid-state exhibiting an amorphous halo . By thatThe sucrose-based formulation with 5 g/L of mAb1 (F1) showed no clear trend in subvisible particles (LO) at 4 °C and 25 °C storage temperature a and no For the trehalose-based formulation (F2), the more sensitive flow-imaging microscopy technique was used for subvisible particle determination . Initialβ, “which may be regarded as proportional to the inverse of molecular mobility for global motions” [β). They found that an arginine phosphate formulation  revealed longer relaxation times than other arginine formulations. These relaxation times unexpectedly had an inversely proportional correlation to the observed aggregation constants, which suggested that increased molecular mobility had a positive effect on protein stability [Arginine phosphate, as discussed in motions” . Subsequtability . This matability , and thetability . Howevertability ,55, givitability , for parFor the high concentration mAb2 formulation (F4), no clear trend could be derived from subvisible particle analysis. Unexpectedly, particle counts at refrigerator temperature appeared to be higher or at similar levels compared to 40 °C after six months . With reIn the future, an improved prototype dryer with a sophisticated technical setup that provides the operator with standard pharmaceutical freeze-drying features, such as freezing and stoppering within the same machine, is needed. A combination of current pharmaceutical freeze-drying equipment with modern semiconductor solid-state microwave generators is imaginable, in the authors’ opinion. For future experiments, a look into relaxation behavior and potential differences between conventionally freeze-dried and microwave-assisted freeze-dried solids could be of interest. Thermal history is expected to be different. Moreover, a deeper look into potential chemical changes that may occur during MFD should be taken. For those analytical techniques focusing on structural changes like FT-IR and circular dichroism, but also methods like (peptide mapping) LC-MS, should be considered.Microwave-assisted freeze-drying is an emerging technique recently introduced to the field of pharmaceutical freeze-drying of biologicals ,24. Desp


Ocimum basilicum L.) and rosemary  essential oils against multidrug- resistant clinical strains of Escherichia coli. A detailed analysis was performed of the resistance of the drug to the strains and their sensitivity to the tested oils. The antibacterial activity of the oils was tested against standard strain Escherichia coli ATCC 25922 as well as 60 other clinical strains of Escherichia coli. The clinical strains were obtained from patients with infections of the respiratory tract, abdominal cavity, urinary tract, skin and from hospital equipment. The inhibition of microbial growth by both essential oils, presented as MIC values, were determined by agar dilution. Susceptibility testing to antibiotics was carried out using disc diffusion. The results showed that both tested essential oils are active against all of the clinical strains from Escherichia coli including extended-spectrum β-lactamase positive bacteria, but basil oil possesses a higher ability to inhibit growth. These studies may hasten the application of essential oils in the treatment and prevention of emergent resistant strains in nosocomial infections.The considerable therapeutical problems of persistent infections caused by multidrug-resistant bacterial strains constitute a continuing need to find effective antimicrobial agents. The aim of this study was to demonstrate the activities of basil ( Escherichia coli are responsible for opportunistic infections, including nosocomial ones, which are difficult to treat, especially in immunocompromised patients. E. coli is responsible for severe cases of urinary tract infection, meningitis in newborns, digestive system illnesses, and even pneumonia. In recent years, strains of Enterobacteriaceae producing an extended spectrum β-lactamase have become a concern in the antimicrobial treatment of persistent infections and control of infection in hospitals [Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa and Acinetobacter baumanii. Extended-spectrum β-lactamases are enzymes produced by Gram-negative bacilli that mediate resistance to penicillin, cephalosporins, and monobactams [The multidrug-resistant pathogenic strains of ospitals ,2,3,4,5.obactams ,7,8,9. TA worrying development is the fast spread of resistant clones of these bacteria on a global scale ,11,12. TOcimum L. (basil) and Rosmarinus L. (rosemary) genera belong to the family Lamiaceae. Among the plants known for medicinal value, basil and rosemary plants are highly regarded for their therapeutic potentials. Ocimum basilicum L. and Rosmarinus officinalis L. essential oils offer promise as biologically active constituents, in that they confer antibacterial, antifungal and antioxidant properties. Basil and rosemary oils have long been used for treating, among other things, colds, fever, cough, asthma, sinusitis and rheumatism, as well as accelerating the process of wound healing [Ocimum basilicum L. and rosemary oil from Rosmarinus officinalis L. against standard and clinical strains of Escherichia coli isolated from patients and from hospital equipment.The  healing ,26,27,28Ocimum basilicum L., the main ones being estragole (86.4%), 1,8-cineole (4.9%) and trans-α-bergamotene (3.0%). The chemical composition of the basil oil is presented in Rosmarinus officinalis L. contains thirty-seven components: the main ones being 1,8 cineole (46.4%), camphor (11.4%), α-pinene (11.0%), β-pinene (9.2%) and camphene (5.2%). The chemical composition of the rosemary oil is presented in Chemical analysis showed the presence of forty-eight constituents within basil oil from Escherichia coli clinical strains was detected for strains from the abdominal cavity (n = 4), bronchia (n = 4), wounds (n = 4), urine (n = 4) and for strains isolated from blood (n = 3) and catheters (n = 3). The results are shown in Escherichia coli (ESBL+) were generally resistant to β-lactams, aminoglycosides and quinolones recommended for susceptibility testing. Most of them were resistant to cephalosporins and β-lactam antibiotics with such inhibitors as clavulanic acid, sulbactam and tazobactam.Extended spectrum β-lactamase production for the tested Escherichia coli ESBL negative strains, characterized by a much lower resistance to β-lactam antibiotics, were resistant mainly to ampicillin, piperacillin, tikarcillin and also to ticarcillin/clavulanic acid. Most of them were resistant to aminoglycosides  and quinolones (ciprofloksacin) and tetracycline. The results are shown in Escherichia coli were found to demonstrate significant resistance to the reference antibiotics in the susceptibility tests. In our tests, all twenty-two of the tested clinical isolates of ESBL positive E. coli were resistant to AMC , CZ , CXM , GM , AM , PIP , TIC , TIM , FEP , CIP , AN , NET , C , TE  and SXT . The thirty-eight tested ESBL negative E. coli strains were generally resistant to AMC , GM , PIP , TIC , TIM , CIP , AN , C , TE  and SXT .The (ESBL+) and (ESBL−) E. coli strains were between 8.0 µL/mL to 11.5 µL/mL. The basil oil showed inhibitory activity against E. coli ATCC 25922 standard strain at 8.0 µL/mL.The MIC values for sixty tested E. coli ESBL+ strains isolated from the abdominal cavity (n = 4) and from the bronchia (n = 4) were sensitive to basil oil at a concentration range from 8.25 µL/mL to 9.0 µL/mL. For (ESBL+) clinical strains from wounds (n = 4), MIC values were between 8.5 µL/mL to 9.25 µL/mL. The growth inhibition concentrations for bacteria isolated from blood were 8.75 µL/mL (n = 1) and 9.25 µL/mL (n = 2). Basil oil at a concentration of 8.75 µL/mL inhibited the growth of one of the tested clinical strains isolated from urine, while 9.0 µL/mL inhibited the growth of three. The MIC values for strains from catheters were 8.75 µL/mL (n = 2) and 9.25 µL/mL (n = 1). The susceptibility of Escherichia coli (ESBL+) strains to basil oil is shown in Most Escherichia coli (ESBL−) strains at a concentration range from 8.25 µL/mL to 11.5 µL/mL. The most tested strains isolated from the abdominal cavity (n = 4) were inhibited by concentrations of 11.0–11.5 µL/mL. Clinical strains from the bronchia (n = 5) were sensitive to basil oil at concentrations of 10.25–11.5 µL/mL. The MIC values for most strains from wounds (n = 4) were from 10.0 µL/mL to 11.5 µL/mL. Most Escherichia coli (ESBL−) strains isolated from blood and urine were inhibited by basil oil at concentrations ranging from 9.5 µL/mL to 11.5 µL/mL. The MIC values for bacteria from the catheters (n = 6) were 10.0–11.5 µL/mL. The susceptibility of Escherichia coli (ESBL−) strains to basil oil is demonstrated in The basil oil was active against Escherichia coli clinical strains obtained from the diverse clinical materials and the hospital equipment. The MIC values were between 18.0 and 20.0 µL/mL. The standard strain E. coli ATCC 25922 was sensitive to rosemary oil at a concentration of 18.5 µL/mL.The rosemary oil was less active against the sixty tested Escherichia coli (ESBL+) strains isolated from the abdominal cavity (n = 3) were sensitive to rosemary oil at concentrations from 18.0 µL/mL to 18.5 µL/mL, and (n = 1) at 19.25 µL/mL concentration. The MIC values for bacterial strains isolated from the bronchia (n = 4) and wounds (n = 4) were 18.25–19.0 µL/mL and 18.5–19.25 µL/mL, respectively. E. coli (ESBL+) strains isolated from blood were sensitive to rosemary oil at concentrations between 18.75 and 19.75 µL/mL. All strains from urine (n = 4) were inhibited in the concentration ranges 18.0 µL/mL to 18.75 µL/mL, while those from catheters (n = 3) were inhibited from 18.25 µL/mL to 18.75 µL/mL. The results of the tests detailing the susceptibility of Escherichia coli (ESBL+) strains to rosemary oil are presented in Most Escherichia coli (ESBL−) strains isolated from wounds. For the blood (n = 7) and urine (n = 6) isolates, the MIC values were in the range 18.25-19.75 µL/mL. Finally, similar MIC values were found (18.25–19.75 µL/mL) against E. coli (ESBL−) strains isolated from catheters (n = 7). The susceptibility of the Escherichia coli (ESBL−) strains to rosemary oil is shown in Rosemary oil was found to have MIC values from 18.5 µL/mL to 19.75 µL/mL for isolates from the abdominal cavity (n = 6). Growth inhibition was found to occur at concentrations of 19.0–20.0 µL/mL for clinical bacterial strains obtained from the bronchia (n = 6). Rosemary oil at concentrations ranging from 18.25 µL/mL to 20.0 µL/mL inhibited the growth of all tested Escherichia coli were found to be sensitive to basil (Ocimum basilicum L.) and rosemary  essential oils, irrespective of the clinical conditions they were obtained under or the pattern of antibiotic resistance they demonstrated, but basil oil was more active against the tested bacteria. Out of the sixty clinical strains of E. coli, twenty-two strains were extended-spectrum beta-lactamase positive (ESBL+). Concentrations of basil oil ranging from 8.25 µL/mL to 9.25 µL/mL were seen to inhibit the growth of eighteen Escherichia coli (ESBL+) strains. For the extended-spectrum beta-lactamase negative strains, seventeen out of the thirty-eight were sensitive to basil oil at concentrations between 11.25–11.50 µL/mL. The MIC values for the other twenty-one Escherichia coli (ESBL−) strains ranged from 8.0 µL/mL to 11.0 µL/mL.In our investigation, all clinical strains of E. coli (ESBL+), rosemary oil concentrations ranging from 18.0 µL/mL to 19.0 µL/mL were effective for eighteen of them. Eleven of the Escherichia coli (ESBL−) strains out of the thirty-eight obtained under various conditions were inhibited by 19.75 µL/mL of rosemary oil. MIC values ranging from 18.25 µL/mL to 19.5 µL/mL for comparable numbers of strains was obtained.The rosemary oil demonstrated significantly lower activity. No apparent differences in activity of the essential oil were found against extended-spectrum β-lactamase-positive and negative strains. Out of the twenty-two clinical strains of Escherichia coli found in different clinical conditions. It is also significant that extended-spectrum β-lactamase (ESBL)− producing clinical strains of E. coli are sensitive to these oils. Studies Orhan at al [Foeniculum sp., Mentha sp., Ocimum sp., Origanum sp. and Satureja sp. (Lamiaceae family) possess strong antibacterial activity against extended-spectrum beta-lactamase (ESBL) positive strains of Klebsiella pneumoniae isolated from food. The obtained MIC values ranged from 32 to 64 μg/ml for a number of strains resistant to trimetoprime-sulfametoxazole, sulbactam-ampicilin, clavulonate-amoxicilin, ceftriaxon, cefepime, imipenem, ceftazidime, tobramicine, gentamisine, ofloxacin, and ciprofloxacin. According our investigations, the Escherichia coli (ESBL+) responsible for human infectious diseases were significantly more resistant to the basil and rosemary essential oils.The results of our tests clearly demonstrate that basil and rosemary essential oils can be widely used to eliminate clinical strains of an at al , confirmOcimum basilicum L., is as given in the ISO-11043 standard. The composition of the tested basil oil corresponded to required standards, according to which the content of estragole must be higher than 75.0%. The composition of the essential oil derived from Rosmarinus officinalis L. was found to meet the requirements of the European Pharmacopoeia 6 [p-cymene, camphor, bornyl acetate, α-terpineol and borneol.The antimicrobial properties of essential oils are strictly connected with their chemical composition. The usefulness of essential oils as effective antimicrobial agents can be evaluated only by analysing their individual components. Therefore, a thorough GC-FID-MS analysis of the tested basil and rosemary essential oils was conducted. The composition of the essential oil obtained from opoeia 6  and of topoeia 6  for the Lamiaceae family [Satureja hortensis L. demonstrates high levels of activity. Mihajilov-Kristev et al. [Escherichia coli, with MIC values from 0.025 µL/mL to 0.78 µL/mL according to the broth microdilution method. In our study for basil and rosemary essential oils, we obtained significantly higher MIC values. This high activity demonstrated by Satureja hortensis L. essential oil is certainly related to the high content of carvacrol, which is one of the most potent antimicrobial compounds.The highest antibacterial activity is demonstrated by phenolic compounds such as carvacrol, thymol and eugenol. Another effective group of active compounds are alcohols: terpinen-4-ol, γ-terpineol, geraniol, cytronellol, menthol and linalol. Many of them are synthesized by plants from the e family . For insv et al. , showed et al. [Ocimum basilicum essential oil possesses antibacterial activity against the human pathogenic bacteria Bacillus subtilis, Enterobacter cloacae, Escherichia coli O157:H7, Micrococcus flavus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidis, S. typhimurium, Staphylococcus epidermidis and S. aureus. According to the authors, basil essential oil containing 69.3% linalool as a main component possesses antibacterial properties against Escherichia coli O157:H7 with MIC and MBC values of 6.0 µg/mL when assessed by microdilution. The multidrug clinical strains of E. coli tested in this study were more resistant to basil essential oil containing mainly estragole (86.4%).The literature reports that basil oil, which contains mainly estragole and linalool, also possesses antibacterial agents which are effective against a variety of Gram-positive and Gram-negative bacteria . Accordiet al. , Ocimum et al. [Ocimum basilicum containing mainly linallol 32.6% and eugenol 28.1%, and oil from Ocimum gratissimum containing 93.9% eugenol, have a broad spectrum of antibacterial activity against reference strains of Gram-positive, Gram-negative bacteria and Candida albicans. According to the authors, essential oils obtained from these two genera of Ocimum were active against Escherichia coli CCT0547 standard strain with a MIC value of >2 mg/mL according to the microplate method. These results also confirm that oils with active constituents such as eugenol tend to have high antibacterial properties. Our results are slightly higher than those obtained by Sartoratto et al. The MIC values were in the range from 7.92 mg/mL to 11.04 mg/mL for clinical strains of Escherichia coli which were both positive and negative for extended-spectrum beta-lactamase activity.Results obtained by Sartoratto et al. , show thet al. [Ocimum gratissimum, with eugenol as its main constituent, possesses antibacterial activity against clinical strains of Escherichia coli ATCC 25922 with MIC—6 mg/mL. The MICs of this essential oil against a group of other Gram-negative bacteria comprising Shigella flexneri, Salmonella enteritidis, Klebsiella sp. and Proteus mirabilis were found to be from 0.3 to 12 mg/mL. The MIC values given in our study were slightly higher than those given by Nakamura et al. Pereira et al. [Ocimum gratissimum possesses antibacterial activity against clinical Escherichia coli strains isolated from urinary tract infections. The essential oil was found to be active against about 70% of tested E. coli clinical isolates. The results of the present study demonstrate that a chemotype containing mainly 1,8-cineole, eugenol, methyleugenol, thymol, p-cimene, cis-ocimene and cis-caryophyllene has lower activity.Nakamura et al. , demonsta et al. , in theiOcimum basilicum, containing mainly estragole (86.4%), inhibited the growth all strains isolated from various clinical materials. Among them were bacteria isolated from urine, which were also extended-spectrum beta-lactamase positive. Our studies confirm that antibacterial activity is possessed by not only basil oil chemotypes with linalool or eugenol as their main components, but also that of Ocimum basilicum, containing mainly estragole. Our research showed that basil essential oil was significantly more effective against all clinical isolates than rosemary essential oil. Similar results were obtained by Hammer et al [Acinetobacter baumanii, Aeromonas veronii biogroup sobria, Candida albicans, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhimurium, Serratia marcescens and Staphylococcus aureus, using an agar dilution method. The authors confirmed that basil (Ocimum basilicum) oil is more active against Escherichia coli than rosemary . The MIC values were 0.5 and 1.0% (v/v), respectively.In our tests, basil oil obtained from er et al , who stuet al. [Ocimum basilicum and Rosmarinus officinalis have an antibacterial potential against the Gram-positive bacteria Staphylococcus aureus, Enterococcus faecalis and Listeria monocytogenes and against Gram-negative bacteria Escherichia coli, Yersinia enterocolitica, Salmonella choleraesuis and Pseudomonas aeruginosa as foodborne bacterial strains. The authors present a detailed analysis of the tested oils and their ability to inhibit the growth of bacteria. Their basil and rosemary essential oils were of a similar composition to the essential oils in our investigations. The main component of the basil essential oil was estragole—82% ± 1.2%, while the rosemary essential oil contains 1.8-cineole—48% ± 9.1%, camphor—17% ± 4.0, β-pinene—4.8% ± 0.9% and β-caryophyllene—6.8% ± 3.7%. The authors confirm that the basil oil is more effective at inhibiting the growth of Escherichia coli strains.According to Lopez et al. , the oilet al [Escherichia coli ATCC 25922. Although the basil oil was also seen to demonstrate low activity against the tested bacteria, no data was given about the constituents of the essential oils. The minimal inhibitory concentration for rosemary oil against E. coli was >6.4 mg/mL. The MIC values obtained by the present study were higher and ranged from 16.02 mg/mL to 17.35 mg/mL against E. coli clinical strains. Fabio et al [Streptococcus pyogenes, S. agalactiae, S. pneumoniae and Klebsiella pneumoniae, Staphylococcus aureus and Stenotrophomonas maltophilia. In our tests, rosemary oil was also found to demonstrate antibacterial activity against Escherichia coli strains with different patterns of resistance, including extended-spectrum β-lactamase positive strains isolated from various clinical materials. The rosemary essential oil used in the present study obtained from Rosmarinus officinalis contains mainly 1,8-cineole (46.4%), camphor (11.4%) and α-pinene (11.0%). The composition of the rosemary essential oil used by Jiang et al. [Staphylococcus epidermidis, Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria  and fungi . Bendeddouche et al [Rosmarinus tournefortii De Noé growing wild in the occidental region of Algeria possesses antimicrobial activity also against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) pathogenic bacteria. The main constituents of the tested essential oil were camphor (37.6%), 1,8-cineole (10.0%), p-cymene-7-ol (7.8%) and borneol (5.4%).Probuseenivasan et al , confirmio et al , report g et al. , was simhe et al , showed A number of studies show that essential oils and their constituents possess useful properties concerning human health. Many of them may be applied in anticancer therapy, cardiovascular and nervous system disorders to reduce the level of cholesterol, to regulate the glucose level or to stimulate hormone production . They alE. coli ATCC 25922, was obtained from the collection of the Medical and Sanitary Microbiology Department, Medical University of Lodz. The clinical strains of Escherichia coli were collected in 2011 and 2012 from a range of clinical materials recovered from patients and from the hospital equipment in various wards from one of the Medical University hospitals in Lodz: internal medicine, surgery, urology and the intensive care unit. The tested bacterial strains were isolated from the abdominal cavity (n = 10), bronchia (n = 10), wounds (n = 10), blood (n = 10), urine (n = 10) and from catheters (n = 10).The standard strain, E. coli strains were cultured on Columbia Agar  and on Mac Conkey Agar (bioMerieux). They were identified to the species by using API 20 E tests (bioMerieux). The bacteria were incubated at 37 °C for 24 h.Ocimum basilicum L. and rosemary—Rosmarinus officinalis L. were purchased from the manufacturer  and analyzed by GC-FID-MS in the Institute of General Food Chemistry, Lodz University of Technology, using a Trace GC Ultra apparatus  MS DSQ II detectors and FID-MS splitter (SGE). Operating conditions: apolar capillary column Rtx-1ms , 60 m × 0.25 mm i.d., film thickness 0.25 µm; temperature program, 50–300 °C at 4 °C/min; SSL injector temperature 280 °C; FID temperature 300 °C; split ratio 1:20; carrier gas helium at regular pressure 200 kPa.; FID temperature 260 °C; carrier gas, helium; 0.5 mL/min; split ratio 1:20. Mass spectra were acquired over the mass range 30–400 Da, ionization voltage 70 eV; ion source temperature 200 °C. The analysis of the constituents of the oils were performed two times independently.Commercial essential oils from basil—8-C26). A quantitative analysis, expressed as percentages of each component, was carried out by peak area normalization measurements without correction factors.Identification of components was based on the comparison of their MS spectra with those of the laboratory-made MS library, commercial libraries  and with literature data ,48 along8 cells/mL, equal to an optical density of 0.5 on the Mc Farland scale, by a bioMerieux densitometer. The agar dilution method was employed for the screening of antimicrobial activities of the essential oils [v/v of oils. Although the tested essential oils dissolve well in ethanol, only minimum amounts were used, as it can inhibit the growth of the tested bacteria. This solution was mixed with a culture medium to obtain concentrations from 7.25 µL/mL to 11.75 µL/mL for basil oil and 17.75 µL/mL to 20.25 µL/mL for rosemary oil and poured into sterile Petri dishes. An inoculum containing 1–2 × 108 cells/mL (0.1 mL) per spot was seeded upon the surface of the agar with various oil concentrations, as well as on agar with no oil added (acting as a control for strain growth). The Minimal Inhibitory Concentration, MIC, was determined after 24 h of incubation at 37 °C under aerobic conditions. The MIC was considered the lowest concentration of the sample at which no visible growth was observed. The analysis of the antibacterial activity of the oil was performed three times independently. Control media containing only alcohol at concentrations used in the dilutions of tested essential oils did not inhibit the growth of bacterial strains.The standard and clinical strains were cultivated in Columbia Agar medium and incubated at 37 °C for 48 h in aerobic conditions. The microbial suspension was standardized to a cell density of 1–2∙× 10ial oils ,49,50,51Becton Dickinson) were used for susceptibility testing of Escherichia coli strains : AM—ampicillin (10 µg) , AMC—amoxicillin/clavulanic acid (20 µg/10 µg) , CF—cefalotin (30 µg) , CZ—cefazoline (30 µg) , CXM—cefuroxime (30µg) , GM—gentamicin (10 µg) , TE—tetracycline (30 µg) , NOR—norfloxacin (10 µg)  (only for the isolates from urine), FTN—nitrofurantoin (300 µg)  (as above), FOS—fosfomycin (200 µg)  (as above), STX—trimethoprim/sulfamethoxazole (1.25 µg/23.75 µg) , PIP—piperacillin (100 µg) , TIC—tikarcillin (75 µg) , TZP—piperacyllin/tazobaktam (100/10 µg) , TIM—ticarcillin/clavulanic acid (75 µg/10 µg) , FOX—cefoxitin (30 µg) , CTX—cefotaxim (30 µg) , CAZ—ceftazidime (30 µg) , FEP—cefepim (30 µg) , ATM—aztreonam (30 µg) , IMP—imipenem (10 µg) , MEM—meropenem (10 µg) , ETP—ertapenem 10 µg) , DOR—doripenem (10 µg) , CIP—ciprofloxacin (5 µg) , AN—amikacin (30 µg) , NET—netilmicin (30 µg) , TOB—tobramycin (10 µg) , C—chloramphenicol (30 µg) , TGC—tigecyclin (15 µg) .The following antibiotics . Cultures were incubated at 37 °C for 16–18 h. The results were interpreted according to EUCAST guidelines .E. coli ATCC 25922 was used as a positive control [The double-disk synergy test and combination disk method were used to determine ESBL production. The sensitivity of the DDST can be improved by reducing the distance between the disks of cephalosporins and clavulanate. The disk approximation method was performed on a Muller-Hinton agar plate inoculated with the clinical bacterial strain, by placing disks containing CAZ—ceftazidime (30 µg), CTX—cefotaxime (30 µg) and ATM—aztreonam (30 µg) 20 mm (edge to edge) from a disk of AMC—amoxicillin/clavulanic acid (20/10 µg). Following incubation for 16–18 h at 37 °C, any enhancement of the zone of inhibition between a cephalosporin and monobactam-aztreonam disk from the amoxicillin/clavulanic acid disk, was indicative of the presence of an ESBL.  control ,53.(1)Escherichia coli clinical strains, and also against extended-spectrum β-lactamase positive bacteria.The results of these experiments indicate the potential use of basil and rosemary essential oils against resistant (2)Escherichia coli clinical strains.The tested basil oil was more active against all (3)The action of essential oils against bacteria exhibiting different mechanisms ofresistance may beuseful, not only in treating but alsopreventingthe spreadof resistantstrains.


Cryptotympana pustulata Fabricius, has been used in traditional medicine for its many clinical pharmacological effects, including the treatment of psychological symptoms in PD. However, scientific evidence for the use of CP in neurodegenerative diseases, including PD, is lacking. Here, we investigated the protective effects of CP on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP-) induced PD in mice and explored the underlying mechanisms of action, focusing on Nurr1. CP increased the expression levels of Nurr1, tyrosine hydroxylase, DOPA decarboxylase, dopamine transporter, and vesicular monoamine transporter 2 via extracellular signal-regulated kinase phosphorylation in differentiated PC12 cells and the mouse SNPC. In MPTP-induced PD, CP promoted recovery from movement impairments. CP prevented dopamine depletion and protected against dopaminergic neuronal degradation via mitochondria-mediated apoptotic proteins such as B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X, cytochrome c, and cleaved caspase-9 and caspase-3 by inhibiting MPTP-induced neuroinflammatory cytokines, inducible nitric oxide synthase, cyclooxygenase 2, and glial/microglial activation. Moreover, CP inhibited lipopolysaccharide-induced neuroinflammatory cytokines and response levels and glial/microglial activation in BV2 microglia and the mouse brain. Our findings suggest that CP might contribute to neuroprotective signaling by regulating neurotrophic factors primarily via Nurr1 signaling, neuroinflammation, and mitochondria-mediated apoptosis.Parkinson's disease (PD) is characterized by dopaminergic neuronal loss in the substantia nigra pars compacta (SNPC) and the striatum. Nuclear receptor-related 1 protein (Nurr1) is a nuclear hormone receptor implicated in limiting mitochondrial dysfunction, apoptosis, and inflammation in the central nervous system and protecting dopaminergic neurons and a promising therapeutic target for PD. Cicadidae Periostracum (CP), the cast-off skin of  Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by bradykinesia, resting tremor, postural instability, and rigidity . The disNuclear receptor-related 1 protein (Nurr1) is a transcription factor that regulates the expression of genes that are critical for the development, maintenance, and survival of dopaminergic neurons , 5. In pCryptotympana pustulata Fabricius , was originally described in the Chung-bu category of Dongui Bogam, an ancient Korean medical book [Cicadidae Periostracum (CP), the cast-off skin of cal book , 16. In cal book . Since tcal book , 19. Reccal book –23. HoweCP was purchased from Kwong-Mung-dang Company  and authenticated by Dr. Goya Choi , and a voucher specimen (3-18-0038) was deposited at the Herbal Medicine Resources Research Center, Korea Institute of Oriental Medicine. Briefly, CP was extracted in distilled water for 3 h under reflux (100 ± 2°C). Then, the extract was filtered, evaporated on a rotary vacuum evaporator, and lyophilized . The powder was kept at 4°C until use.Male C57BL/6 mice  were purchased from Doo Yeol Biotech  and maintained under temperature- and light-controlled conditions  with food and water provided ad libitum. All animals were acclimatized for 7 days prior to drug administration. The experimental protocol was approved by the institutional animal care committee of KIOM (KIOM-18-056) and performed according to the guidelines of the Animal Care and Use Committee at KIOM.n = 11), (2) MPTP (n = 11), (3) MPTP+CP 1 mg/kg/day (n = 11), (4) MPTP+CP 10 mg/kg/day (n = 11), (5) MPTP+CP 25 mg/kg/day (n = 11), (6) MPTP+ropinirole 1 mg/kg/day (n = 11), (7) CP 5 mg/kg/day (n = 5), (8) CP 25 mg/kg/day (n = 5), (9) control (n = 7), (10) lipopolysaccharide , and (11) LPS+CP 25 mg/kg/day (n = 7). CP, dissolved in normal saline, was administered for 5 days consecutively. The control group received an equal volume of normal saline for the same duration. MPTP  or LPS (Sigma-Aldrich) were administered acutely as described previously [Mice were assigned to 1 of 11 groups: (1) control (eviously –29. On deviously : (1) we The rotarod test and pole test were assessed according to previously published methods , 29, 31.The pole test is a useful method to measure bradykinesia in a mouse model of PD . We perfμm thick coronal sections were cut on a freezing microtome  and stored in a cryoprotectant , 25% glycerol (Sigma-Aldrich), and 0.05 M phosphate buffer at 4°C until use for immunohistochemistry (IHC) study. For western blotting mRNA analysis, and kit-based analyses, in brief, the SNPC and ST were rapidly dissected, homogenized, and centrifuged using standard laboratory techniques. The final supernatant was stored at 70°C until use [On days one and seven after MPTP treatment or at 3 h after LPS treatment, mice were anesthetized immediately and perfused transcardially with 0.05 M phosphate-buffered saline , followed by cold 4% paraformaldehyde  in 0.1 M phosphate buffer. Brains were removed and postfixed in 0.1 M phosphate buffer (Sigma-Aldrich) containing 4% PFA overnight at 4°C and then immersed in a solution containing 30% sucrose in 0.05 M PBS for cryoprotection. Serial 15 or 30 ntil use .For IHC analysis, the assessment of dopaminergic neurons in the SNPC was made by analyzing coronal sections at approximately 3.28 mm behind the bregma . Brain sμL) was transferred to a fresh tube into which 0.5 mL of 2-propanol was also added. After incubation for 10 min at 4°C, the mixture was centrifuged again at 14,000 rpm for 10 min at 4°C. After separation, the supernatant was removed, washed with 1 mL of 75% ethanol, and centrifuged again at 10,000 rpm for 5 min at 4°C. The resulting RNA pellet was then dried, and the purified RNA was dissolved in diethyl pyrocarbonate- (DEPC-) distilled water. Equal amounts of RNA (200 ng) were reverse transcribed (RT) into cDNA using an iScript cDNA synthesis kit  according to the manufacturer's protocol. Real-time RT polymerase chain reaction (PCR) analysis was performed for selected genes using the CFX96 real-time PCR system (Bio-Rad) and the SYBR green fluorescence quantification system (Bio-Rad) to quantify the amplicons. The PCR conditions were 50 cycles of 95°C (30 s) and 58°C (30 s) and a standard denaturation curve. The primer sequences are listed in the 5′ to 3′ orientation in Supplementary μL of cDNA and 9.5 μL of PCR master mix, which contained 2× SYBR Green, 10 pmol each of the forward and reverse primers, and 4.5 μL of DEPC-distilled water in a final volume of 15 μL. To normalize the cDNA content of the samples, we used the comparative threshold (CT) cycle method, which includes normalization of the number of target gene copies vs. the endogenous reference gene glyceraldehyde 3-phosphate dehydrogenase. The CT is defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe passes a fixed threshold baseline when amplification of the PCR products is first detected.Homogenization of SNPC tissue was conducted using TRIzol reagent . After homogenization, 0.2 mL of chloroform was added to each sample. The tubes were shaken vigorously by hand for 15 s and then incubated at room temperature for 3 min. Next, the mixture was centrifuged at 14,000 rpm for 15 min at 4°C, after which the resulting upper aqueous phase (400 2 at 37°C. The PC12 cells were seeded on 96-well plates or 6-well plates at a density of 1 or 2 × 105 cells/mL and treated with CP (0–1000 μg/mL) for 24 h (μg/mL) for 1 h. Then, they were stimulated with MPP+ (100 μM) for an additional 23 h . PC12 cells were maintained in Roswell Park Memorial Institute medium  supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco) in an atmosphere of 95% air and 5% COfor 24 h . For difnal 23 h .2 at 37°C. The BV2 cells were seeded on 24-well plates at a density of 1.5 × 105 cells/mL and treated with CP (0–1000 μg/mL) for 24 h  supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin in an atmosphere of 95% air and 5% COfor 24 h .Living cells were evaluated using the Cell Counting Kit  according to the manufacturer's protocol. In brief, cells were seeded on 96-well plates and treated with CP. The CCK-8 reagent was added to each well, and the mixture was incubated for 4 h. The absorbance was read at 450 nm using the SpectraMax i3 Multi-Mode Detection Platform . Cell viability was calculated using the following equation: %of living cells left after CP treatment = (mean absorbance in CP − treated cells/mean absorbance in untreated controls) × 100.ψm was monitored with the lipophilic cationic probe tetraethylbenzimidazolylcarbocyanine iodide (JC-1) reagent, supplied with the Δψm detection kit, according to the manufacturer's instructions. Differentiated PC12 cells were seeded on coverslips in 96-well plates and treated simultaneously with CP at concentrations of 1, 10, or 50 μg/mL and 100 μM MPP+ for 24 h. Then, the cells were rinsed with PBS and incubated with the diluted lipophilic cationic probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1) at 37°C for 30 min, after which the cells were washed and transferred to 96-well plates. The ratio of red (585/590 nm) and green (510/527 nm) fluorescence was determined using a fluorescence plate reader.Western blotting was performed according to previously published methods , 35, 36.μL) was mixed with 40 μM siRNA solution (an equimolar mix of both Nurr1 siRNA and scrambled siRNA) and 2.5 mL of Opti-MEM (Gibco). After 30 min at room temperature, 300 μL of the mix was added to 300 μL of serum-free RPMI in each dish and incubated for 24 h.Differentiated PC12 cells were used at a confluence of 80–85% in 100 mm dishes. Cells were transfected with stealth small interfering RNA (siRNA) using Lipofectamine 2000 (Invitrogen). Lipofectamine 2000 . Moreover, cytokine proteins were determined using a cytokine membrane array kit following the manufacturer's instructions .p value < 0.05 was considered to be statistically significant.All statistical parameters were calculated using the GraphPad Prism 7.0 software . Values are expressed as means ± standard error of the mean (S.E.M.). Statistical comparisons between the different treatments were performed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison posttest. A To examine the effect of CP on MPTP-induced poor motor coordination and postural balance, a rotarod test was performed. We found that MPTP significantly decreased the retention time to 13.71 ± 2.06 s on day 1, compared with the control. However, retention times were significantly increased in the MPTP+1–25 mg/kg/day CP and ropinirole groups from 27.14 ± 4.31 to 56.86 ± 13.99 s and 55.86 ± 14.81 s, respectively, on day 1 . In addiμM) inhibited MAO-B activity in a dose-dependent manner; in contrast, CP had no effect on MAO-B activity  Figures . Moreoveactivity .μg/mL, but not at 1–50 μg/mL, increased neuronal shrinkage and damage and decreased dendritic length of PC12 and differentiated PC12 cells 24 h after treatment   Figures .To evaluate the effects of CP on Nurr1 expression, we performed Nurr1-specific immunofluorescence staining and western blotting. MPTP significantly decreased Nurr1 (by 71.49 ± 1.27%) in shrunken dopaminergic neurons (by 33.40 ± 3.17%) compared with the control, and treatment with 5–25 mg/kg CP reduced MPTP-induced Nurr1 (by 76.08 ± 1.15% to 82.67 ± 1.30%) and Nurr1 in dopaminergic neurons further (by 30.06 ± 2.40% to 95.06 ± 7.22%) Figures . MoreoveTo evaluate the effects of CP on MPTP-induced expression of Nurr1 and its regulating proteins, we assessed the levels of TH, DDC, DAT, and VMAT2 in the mouse SNPC by western blotting. MPTP significantly decreased TH (by 49.87 ± 5.21%), DDC (by 36.73 ± 8.28%), DAT (by 36.17 ± 0.97%), and VMAT2 (by 69.12 ± 10.67%) levels, compared with the control, while treatment with 1–25 mg/kg CP reduced MPTP-induced expression of TH (by 46.31 ± 3.94% to 95.71 ± 10.59%), DDC (by 53.45 ± 16.29% to 140.27 ± 15.04%), DAT (by 34.98 ± 7.3% to 60.85 ± 1.08%), and VMAT2 (by 42.14 ± 9.55% to 149.34 ± 43.42%), compared with the control group Figures . In addiψm) and red fluorescence  indicate Δψm depolarization. MPP+-induced toxicity decreased Δψm (by 4.64 ± 0.08 ratio), whereas CP pretreatment prevented depolarization of the mitochondrial membrane (by 1.99 ± 0.05 to 1.42 ± 0.07 ratio)  polymerase (PARP), cleaved caspase-9, and cleaved caspase-3 in the mouse SNPC. MPTP caused a significant decrease in Bcl-2 and an increase in Bax, Cyt-c, PARP, cleaved caspase-9, and cleaved caspase-3, while 1, 5, or 25 mg/kg CP increased Bcl-2 and decreased Bax, Cyt-c, PARP, cleaved caspase-9, and cleaved caspase-3 . Green f Figures .To investigate whether CP affects dopaminergic production, we assessed the levels of GDNF and Ret in the mouse SNPC. MPTP significantly decreased the expression of GDNF and Ret, while 5 or 25 mg/kg CP treatment increased the levels of GDNF and Ret . Using aμg/mL CP, as assessed by the MTT assay at 24 h after treatment. However, 125–1000 μg/mL CP increased cytotoxicity (data not shown). Thus, all further experiments were performed with CP at 1–62.5 μg/mL. Cytokine array kits revealed that the levels of intercellular adhesion molecule 1 (ICAM-1), interleukin-6 (IL-6), KC, MCP-5, and RANTES were significantly increased  in LPS-treated microglial BV2 cells. In contrast, CP suppressed the increase in ICAM-1, IL-6, KC, monocyte chemotactic protein 5 (MCP-5), and RANTES   in the SNPC of the MPTP-treated group. In contrast, CP suppressed this decrease in GFAP, Iba-1, iNOS, and Cox-2  Figures . Represe Figures . The lev Figures .Many intrinsic signals and extrinsic transcription factors have been identified to play critical roles in dopaminergic neuronal development in the midbrain in PD. This process depends on two major signaling pathways: (i) Sonic hedgehog/FoxA2 and (ii) wingless-type MMTV integration site family, member 1/Lmx1a, and their downstream signaling molecules , 38. TheDongui Bogam) and identified one hit drug. A previous report showed that Nurr1 promotes dopaminergic neuronal development by inducing the expression of neurotrophic factors, such as TH, DDC, DAT, and VMAT2 [According to the WHO, as much as 80% of the world's population relies primarily on animal- and plant-based medicines –45. Animnd VMAT2 . Dopaminnd VMAT2 , 51. It nd VMAT2 . In the nd VMAT2 . The Nurnd VMAT2 . It has nd VMAT2 –54. In tnd VMAT2 . To confnd VMAT2 , 55. Howα-synuclein accumulation [The above-described findings prompted us to test whether CP can ameliorate motor behavior deficits in a mouse model of MPTP-induced PD. This animal model is used widely because dopaminergic neuronal loss in the SNPC and ST is associated with the onset of motor symptoms, and there is a direct relationship between the extent of dopamine loss and motor dysfunction , 56. Themulation . Reducedmulation . Thus, tκB via modulating reactive oxygen species induced by ultraviolet B irradiation on keratinocyte HaCaT cells [α) was increased more than 2-fold. Remarkably, CP reduced the expression of ICAM-1, KC, IL-6, MCP-5, and RANTES (>30%). However, further studies using Nurr1 transgenic mice will be required to confirm our findings. Next, we analyzed protein-protein interactions using the STRING database and potential molecular mechanisms using the KEGG database  in vitro and in vivo . Further


Camelus dromedarius is well-adapted to survive in extreme desert climate and it has GSTs, for which limited information is available. This study investigated the structure-function and thermodynamic properties of a mu-class camel GST (CdGSTM1) at different pH. Recombinant CdGSTM1 (25.7 kDa) was expressed in E. coli and purified to homogeneity. Dimeric CdGSTM1 dissociated into stable but inactive monomeric subunits at low pH. Conformational and thermodynamic changes during the thermal unfolding pathway of dimeric and monomeric CdGSTM1 were characterised via a thermal shift assay and dynamic multimode spectroscopy (DMS). The thermal shift assay based on intrinsic tryptophan fluorescence revealed that CdGSTM1 underwent a two-state unfolding pathway at pH 1.0–10.0. Its Tm value varied with varying pH. Another orthogonal technique based on far-UV CD also exhibited two-state unfolding in the dimeric and monomeric states. Generally, proteins tend to lose structural integrity and stability at low pH; however, monomeric CdGSTM1 at pH 2.0 was thermally more stable and unfolded with lower van't Hoff enthalpy. The present findings provide essential information regarding the structural, functional, and thermodynamic properties of CdGSTM1 at pH 1.0–10.0.Glutathione S‒transferases (GSTs) are multifunctional enzymes that play an important role in detoxification, cellular signalling, and the stress response.  Glutathione S-transferase GST; EC 2.5.1.18) is one of three major groups of enzymes that carry out detoxification  and is found in virtually all organisms .5.1.18 i–5. GSTs GST class μ  is a homodimeric protein , 20. EacPart of the active site is located on the dimer interface; therefore, GST dimerization is essential to for its activation , 24. MorCamelus dromedarius is adapted to extreme desert climate including elevated solar radiation, temperature, dryness, low nutrition, and scarcity of water  containing 300 mM NaCl. Elution of bound CdGSTM1 was achieved with 50 mM Tris-HCl buffer [pH 8.0] and 10 mM of GSH.Recombinant CdGSTM1was estimated at spectrophotometrically at 280 nm, with a molar extinction coefficient of 39,880 M−1cm−1.Active fractions were pooled and subjected to size-exclusion chromatography to eliminate minor impurities and reduced glutathione. Superdex 75 16/600 GL prepacked column was equilibrated with 50 mM phosphate buffer containing 150 mM NaCl at pH 7.5. The active fraction of GSH-agarose elute was pooled and loaded on a column using a 5 mL superloop on an AKTA purification system. The purity of the gel filtration peak was analysed with a 4–15% mini-PROTEAN pre-casted TGX gel (Bio-rad) and stained with Coomassie Brilliant Blue G-250. The concentration of purified CdGSTM1 was calculated using a Superdex 200 increase 10/300 GL Prepacked Tricorn Column, calibrated with five proteins: thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (44 kDa). Purified CdGSTM1 (2.5 mg mL-1) in 50 mM Phosphate buffer containing 150 mM NaCl at pH 7.0 was applied on the column using a 500 μL superloop. To determine the quaternary structure at different pH, 0.25 mg CdGSTM1 at different pH  were loaded on the column pre-equilibrated with buffer containing 150 mM NaCl at the aforementioned three values of pH.The molecular weight of the CdGSTM1 activity was determined spectrophotometrically based on the increase in UV absorbance at 340 nm at 25°C owing to the conjugation of GSH with 1-chloro-2,4 dinitrobenzene (CDNB) in accordance with a previously reported method [-1 cm-1. The reaction was carried out in 1 mL of 0.1 M potassium phosphate buffer (pH 6.5), 1.0 mM GSH, and 1.0 mM CDNB dissolved in ethanol and the enzyme preparation. One unit of CdGSTM1 activity was defined as the amount of GST that catalyses the formation of 1.0 μmole of thioether per min.d method , with anCdGSTM1 (0.2 mg mL-1) was equilibrated overnight with buffers pH 1.0 to 10.0 at 22°C. The following buffers (50 mM each) were used: KCl-HCl (pH 1.0); Gly-HCl (pH 2.0 and 3.0); acetate buffer (pH 4.0 and 5.0); phosphate buffer (pH 6.0 and 7.0); Tris-HCl (pH 8.0); Gly-HCl (pH 9.0 and 10).CdGSTM1 (0.1 mg mL-1) at different pH (1.0–10.0) in a 10-mm path-length cuvette was excited at 295 nm  for recording total and tryptophan fluorescence emission spectra, respectively. For aggregation studies of CdGSTM1at different pH, the Rayleigh light scattering (RLS) method was used. CdGSTM1 concentration at different pH was maintained at 0.1 mg mL-1. The CdGSTM1 sample was excited at 500 nm, and emission was collected from 450–550 nm. The bandwidth of excitation and emission slit was 1.5 nm. Fluorescence intensity at 500 nm was plotted against different pH. The change in surface hydrophobicity with respect to pH was measured using 8-anilinonaphthalene-1-sulfonic acid (ANS) extrinsic fluorophore. Fifty-molar excess of ANS was added in the CdGSTM1 samples at different pH and incubated at room temperature for 30 min. Subsequently, samples were excited at 380 nm (2.5 nm bandwidth), and emission of ANS-bound proteins was measured at 400–600 nm (5-nm bandwidth).Total and tryptophan fluorescence emission spectra were measured at 25°C in a Cary Eclipse Fluorescence Spectrophotometer , coupled with Peltier temperature controller. -1) samples. The quencher was added to the protein samples till saturation was achieved (0–0.12 M). The Trp residues of protein sample were excited at 295 nm, and the emission spectrum was recorded at 300–400 nm. Reduction in fluorescence intensity at wavelength maximum was analyzed using the Stern–Volmer equation.Stepwise two μl of acrylamide quencher was added from the stock of 5.0 M into incubated protein  and data were collected at 330, 340, and 350 nm (10-nm bandwidth).Thermal stability of CdGSTM1 concentration of 0.2 mg mL-1 with a 1-mm path length cell at 25°C. Three spectra of each sample were obtained at 200–260 nm. Air and buffer blank were subtracted from each spectrum. Percent secondary structure content was calculated using CDNN software.Far-UV CD measurements were obtained using a Chirascan Plus spectropolarimeter , coupled with a Peltier temperature controller. The instrument was calibrated with (1S)-(+)-10-camphorsulphonic acid. The far-UVCD spectra were measured at a CdGSTM1 at pH 2.0 and 7.0 was performed with a Chirascan-Plus spectrophotometer. Protein samples (0.2 mg mL-1) equilibrated overnight at pH 2.0 and 7.0 were gradually heated at 1°C min-1, and far-UV CD spectra were recorded at 200–250 nm. The actual sample temperature was monitored using an internal temperature probe. Thermal melting curves were analysed using Chirascan’s Global 3 software, Applied Photophysics Ltd, UK.Dynamic multi-mode spectroscopy of CdGSTM1 (0.5 mg mL-1) at pH 2.0, 4.0, and 7.0 were treated with 0.25% glutaraldehyde at room temperature for 15 min. Subsequently, cross-linking was quenched by addition of 0.2 M sodium borohydride and incubated for 20 min at room temperature. Thereafter, 0.005 mL of 10% sodium deoxycholate was added. The cross-linked protein was precipitated by decreasing the pH to 2.0–2.5 via addition of orthophosphoric acid. The precipitated protein was separated via centrifugation at 13,000 rpm for 10 min at 4°C. The pellet was dissolved in SDS-loading dye.Glutaraldehyde cross-linking was performed in accordance with a previously reported method  with sli


Bauhinia variegata L. and to study them for their antioxidant and cytotoxic activities. The bark was extracted with methanol, followed by column chromatography and thus isolating kaempferol, stigmasterol, protocatechuic acid-methyl ester (PCA-ME) and protocatechuic acid (PCA). 2,2-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and 2, 2’-diphenyl-1-picrylhydrazyl radical (DPPH) radical scavenging assays were utilized for assessment of antioxidant activity, and 3--2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay was used to determine cytotoxic activity against C-6 glioma rat brain, MCF-7 breast cancer, and HCT-15 colon cancer cell lines. The compounds were found to have significant antioxidant and cytotoxic activity. Since there is a considerable increase in characterizing novel chemical compounds from plant parts, the present study might be helpful for chemotaxonomic determinations, for understanding of medicinal properties as well as for the quality assessment of herbal supplements containing B. variegata bark, thus establishing its use in traditional medicine.Plants have been the basis of traditional medicine since the dawn of civilizations. Different plant parts possess various phytochemicals, playing important roles in preventing and curing diseases. Scientists, through extensive experimental studies, are playing an important part in establishing the use of phytochemicals in medicine. However, there are still a large number of medicinal plants which need to be studied for their phytochemical profile. In this study, the objective was to isolate phytochemicals from bark of  Environmental factors like polluted water, air, soil, UV radiations, intake of pesticides by food consumption, all might result in production of reactive oxygen species (ROS) ,2,3,4. IAmong other classes of health promoting compounds, polyphenols from fruits, vegetables and medicinal plants represent a peerless class of antioxidants which can counteract ROS-triggered damages. The cancer preventive activities of various phytochemicals lie in their ability to affect cellular defenses like detoxification, scavenging ROS, as well as induction of antitumor and anti-inflammatory responses. Thus, antioxidants from medicinal plants present themselves as the safer option to chemically formulated anticancer drugs ,12,13. APrevention is better than cure, and cancer, though being a major cause of deaths worldwide, is a disease which can largely be prevented by modulating dietary supplements . Among tBauhinia belongs to family Fabaceae and it includes an approximate of 350 shrubs, lianas and small tree species which are recognized easily with their bilobed leaves as well as palmate venation. Due to its bilobed leaves, the genus was named Bauhinia by Linnaeus to honor Swiss Botanists, Johann and Gaspard, who were identical twins + which indicated the molecular formula C15H10O6. Thus on the basis of above spectral data and comparison with previously known spectral values + , indicating the molecular formula C29H48O. The above spectral data is in comparison with previously known spectral values of stigmaserol [The IR absorption spectrum presented absorption peaks at 2960.53 cmgmaserol ,33,34, t1H NMR spectrum revealed signals at δ 7.42 for C-5 and C-6 protons each and at δ 6.80, suggesting a 1,3,4-trisubstituted benzene ring. The slightly down fielded signal at δ 3.84 indicated methoxy group in the structure. The 13C NMR spectrum revealed seven carbons with a signal at δ 167.8 assignable to carboxyl carbon (-COO). The signals at δ 122.5, 121.5, 116.3, and 114.8 corresponded to C-6, C-1, C-2, and C-5 carbons respectively. Similarly, signals at δ 145.1 and 150.6 represented C-3 and C-4 aromatic carbons bearing hydroxyl groups. The methoxy group at C-1’ yielded a signal at δ 51.2 (The fraction BV3 was obtained as a brown solid mass. The t δ 51.2 . −1 (O-H group), 1300–1000 cm−1 (C-O group). A C-H stretching of sp3 carbon was corresponded to the bands at 2954.74 cm−1 and 2925.81 cm−1. The peak at 1697.24 cm−1 corresponded to C=O and the peaks at 1604.66 cm−1 and 1456.16 cm−1 suggested C=C of aromatic ring. Thus, fraction BV3 was recognized as 3,4-dihydroxybenzoic acid methylester (protocatechuic acid methyl ester) with molecular formula C8H8O4, based on the spectral data.The IR absorption spectrum showed peaks mainly in the region of 3600–3200 cm1H NMR spectrum showed signals at δ 7.43, 7.43 and 6.81 indicating a 1,3,4-trisubstituted benzene ring. 13C NMR spectrum revealed seven carbons with a signal at δ 169.2 assignable to carboxyl carbon (COOH). Similarly, four signals at δ 122.9, 122.0, 116.7, and 114.7 corresponded to C-6, C-1, C-2, and C-5 carbons respectively. C-3 and C-4 aromatic carbons bearing hydroxyl groups corresponded to the signals at δ 145.0 and 150.5 (The fraction BV4 was a yellowish-brown powder. The nd 150.5 .−1 region (O-H group of the phenyl ring as well as of the carboxylic acid) and in the 1300-1000 cm−1 region (C-O group). There was C-H stretching of sp3 carbon revealed by the bands at 2954.74 cm−1, 2929.67 cm−1. A peak at 1701.10 cm−1 suggested C=O and a peak at 1608.52 cm−1 correspond to C=C of aromatic ring. The C-O stretching vibration for acid appeared in the region of 1292–1230 cm−1. The peak at 943.13 cm−1 suggested hydrogen bonded OH out of plane bending vibration.The IR spectrum showed peaks in 3600–3200 cm7H6O4, based on data that matched with the reported spectral data [Thus, fraction BV4 was recognized as 3,4-dihydroxybenzoic acid (protocatechuic acid) with molecular formula Cral data .50, it was tested at a concentration range of 20 µg/mL to 200 µg/mL. In both ABTS and DPPH radical scavenging assays, the compounds isolated from B. variegata exhibited a lower IC50 than BHT.It was found that BV3 was able to scavenge 87.38% ABTS radicals followed by BV4 (83.82% scavenging) and BV1 (69.73% scavenging) at the maximum tested concentration (20 µg/mL) respectively. At the concentrations ranging from 2 µg/mL to 20 µg/mL, BV2 did not show any notable ABTS radical scavenging activity . The fra50 values. The fraction BV4 was found to have the maximum GI50 in case of C6 cell line , liquid chromatography mass spectroscopy (LC-MS), etc. . Drug diBauhinia shows a high proportion of flavonoids, terpenes, steroids and alkaloids. Isolation of Lupeol, 5, 7-dimethoxyflavonone-4–O–α– L–rhamnopyranosyl-β-D-glucopyranoside, β-sitosterol, kaempferol-3- glucoside from the stem of the have been reported [The chemical composition of the plants belonging to genus reported . In the 50 of 220µg/mL against DPPH radicals.Kaempferol has been reported to have very significant antioxidant activity . It is a50 of 8.76 ± 1.23 µg/mL as compared to PCA having an IC50 of 9.39 ± 0.45 µg/mL. Similar was the case with DPPH assay where the IC50 values were 4.46 ± 0.21 µg/mL and 7.28 ± 1.64 µg/mL for PCA-ME and PCA respectively.Protocatechuic acid has wide natural distribution and shares structural similarity with caffeic acid, vanillic acid, gallic acid, syringic acid which are well acknowledged antioxidants . In the -O-rhamnoside hinders the proliferation of MCF-7 cells dose-dependent mode and also promotes apoptosis. Among A549 and MCF-7 cells, induction of MAPK (mitogen-activated protein kinase) has been considered to be an essential factor in kaempferol initiated apoptosis. Also, the MAPK activation mediated by kaempferol may avoid impairment to DNA leading to cell transformation. Kaempferol was further found to increase expression of haeme oxygenase (HO)-1 gene, resulting in a surge in the antioxidant capacity of cells [50 33.6 μM) for NF-κB activity [Literature reports also suggest lower radical scavenging activity of PCA compared to PCA-ME in alcoholic solvents. In an experiment carried out by Saito and Kwabata , it was of cells . In HeLaof cells . A cytosof cells . Luo et activity . Similaractivity  reportedactivity . Imran eactivity  reported50 values of 173.97 ± 0.81 µg/mL, 132.01 ± 0.55 µg/mL, 151.20 ± 0.74 µg/mL for C-6, MCF-7 and HCT-15 cell lines respectively. Malikova et al. [In case of stigmasterol, when evaluated for cytotoxic potential, it was observed that it showed moderate activity against the cell lines under investigation showing GIa et al.  establisa et al. , a reduc50 49.14 ± 0.23 µg/mL), while PCA-ME have shown significant cytotoxic activity against MCF-7 (GI50 54.18 ± 0.16 µg/mL) as well as against HCT-15 cell line (GI50 92.17 ± 1.17 µg/mL). The mechanism of cytotoxic action of PCA has been proposed to be associated with antioxidant activity. It can influence metabolism phases 1 and 2 of various carcinogens thus blocking specific binding sites of the carcinogens with the DNA. This leads to the prevention of adduct formation that could have resulted into mutations and neoplastic transformations [PCA have shown considerable cytotoxic potential against MCF-7 cell line  in the bark. It is concluded that B. variegata L. can be a potential addition in pharmaceutical products for the improvement of human health by contributing in the antioxidant defense system fighting against the production of free radicals.A number of higher plant species are used in traditional medicine have not yet screened for the presence of powerful bioactive compounds. Different plant parts of


Annona squamosa. One of these alkaloids, 6, 7-dimethoxy-1-(α-hydroxy-4-methoxybenzyl)-2-methyl-1, 2, 3, 4-tetrahydroisoquinoline, was isolated for the first time from natural sources, while, the alkaloid, Coclaurine was known in Annona squamosa L. Moreover, the isolated alkaloids tested for the anti-cancer activities on various cell lines . Two alkaloids, belonging to benzylisoquinoline alkaloids, were isolated from The online version of this article (10.1186/s13065-019-0536-4) contains supplementary material, which is available to authorized users. Annona L. belongs to the family Annonaceae which is a large family comprising about 135 genera and more than 2500 species distributed mainly in tropical and sub-tropical regions [Annona genus includes approximately 162 species of trees, shrubs and, rarely lianas [Annona are of economic importance because of their edible fruits and medicinal properties, like Annona squamosa (sugar apple), Annona muricata (soursop), Annona reticulata (custard-apple) and Annona cherimola (cherimoya) [Annona genus Annona squamosa was reported in Saudi Arabia [Annona squamosa small tree, 2–3 m tall. Leaves without stipules, petiolate, alternate, lanceolate to elliptic-oblong, 8–13 × 3–6 cm, entire. Flowers solitary or in clusters of 2–4, arising opposite the leaves, borne on a recurved pedicel. Perianth segments in 3 s. Narrowly triangular, green to yellowish brown. Stamens were numerous. Carpels united into a fleshy mass in fruit. Seeds brown, surrounded by white, sweet pulp [y lianas . Some spi Arabia . Annona A. squamosa are used by different ethnic communities for the treatment of various chronic diseases such as cancerous tumors, insect bites and other skin complaints [Traditionally, all parts of mplaints –8. Howevmplaints , 9. The mplaints –15. It hmplaints . Also, implaints .Annona squamosa have chemical compounds approximately belongs to all natural products compounds steroid, terpenoids, glycoside, alkaloid, flavonoid saponin and phenolic compounds [Constituents of ompounds , 9, 16.A. squamosa were dealing with the crude extract that contain all chemical constituents of A. squamosa or with non-alkaloidal parts especially acetogenin [A. squamosa, so in this research, we isolate some pure alkaloids from this plant also, we study the anti-cancer activity of the isolated alkaloids.All previous study done on the anti-cancer activity of etogenin , 19. Becetogenin –22 and nA. squamosa from Saudi origin resulted in the isolation of two benzylisoquinoline alkaloids, namely6, 7-dimethoxy-1-(α-hydroxy-4-methoxybenzyl)-2-methyl-1, 2, 3, 4-tetrahydroisoquinolin.1 and Coclaurine 2   , Human Brest cancer cells (MCF-7) and Human Liver cancer cells (HEPG-2). Table Anti-cancer activity of lls HEPG-. Table 2A. squamosa, afforded two alkaloids belong to simple benzylisoquinoline alkaloid class. One of them is reported from natural sources for the first time.Chemical analysis of the alkaloidal part of Isolated alkaloids gave an excellent activity on Colon cancer cells (HCT116) and Human Brest cancer cells (MCF-7), which is confident with the reported structure–activity relationship of activity of benzylisoquinoline alkaloids on a cancer cell.This result supports using the plant in folk medicine to treat cancer.Authors recommended to make total synthesis of the isolated alkaloids.2 atmosphere at 37 °C.All chemicals were purchased from Sigma-Aldrich. cell culture vessels were supplemented from Nunc Co. . Human colon (HCT 116), Human liver (HepG-2) and Human breast (MCF-7) cancer cell lines were purchased from Vacsera . Cells were maintained routinely in RPMI 1640 cell culture media supplemented with 1 mM sodium pyruvate, 2 mM L. glutamine, 100 units/mL penicillin–streptomycin and 10% fetal bovine serum. Cells were incubated in a humidified, 5% CO1H-NMR spectra were recorded on a Bruker DPX-500 MHz spectrometer with TMS as an internal standard. 13C–NMR spectra were recorded at 125.8 MHz using the same instrument.A. squamosa, Annonaceae, were collected from Jizan region in Kingdome of Saudi Arabia in January 2017. The plant material was identified in biology department of King Khalid University.The aerial parts Preparation of plant material:The aerial parts of the plant were dried in the shade for 15 days then ground to get 6 kg fine powder. The powder was soaked in petroleum ether of 10 days for defatting then extracted thoroughly with ethanol four times each time needs 7 days later the ethanol was evaporated to get a 650 g residue.The ethanol residue was dissolved in 5% HCl until the PH = 2 of the solution and filtered, the precipitate which contains neutral material was kept for further fractionation, and the filtrate which provides the basic material was basify using NH4OH solution, and the PH of the solution becomes around 8. After that, the solution was extracted with chloroform 500 mL three times the chloroform layer was evaporated to get 7.35 g of crude alkaloids which represent 0.123% of the dry plant.The crude alkaloids were subjected to silica gel column chromatography using a column packed in chloroform and polarity increasing using methanol till pure methanol was used. The fractions collected  were grouped according to their TLC behavior into six groups. Fraction III gave upon treatment of methanol a yellowish amorphous solid I (30 mg). Fraction V gave a dark brown amorphous solid when treated with methanol this solid were recrystallize by methanol to provide a solid white II (50 mg).Compound I: Yellowish amorphous solid, IR (KBr) νmax (cm−1) : 3393, 2927, 2852, 1612, 1514. 1H-NMR and 13C-NMR data in Table  νmax cm− : 3393, Coclaurine: Compound II was crystallised from MeOH as white powder, m.p. 254–256d  °C, 1H-NMR and 13C-NMR data in Table 0.01 g of each pure compounds was diluted in 1 mL of (DMSO) dimethyl sulfoxide as a stock solution.2 humidified incubator at 37 °C for 72 h. Doxorubicin was used as a positive control. Cells were treated with the extracts for 72 h then; they were fixed with TCA (10%) for 1 h at 4 °C. To remove TCA cells were washed many times, then 0.4% SRB solution was used to stain cells in a dark place for 10 min. Stained cells were washed with 1% glacial acetic acid. Finally, to dissolve SRB-stained cells, Tris–HCl was used. After drying overnight, the color intensity of remained cells was measured at 540 nm by Elisa.In the present study, SulphoRhodamine-B (SRB) assay had been chosen to detect the anticancer activity of isolated alkaloids. The anticancer activity of isolated alkaloids was tested against Human breast (MCF-7), Human colon (HCT 116), and Human liver (HepG-2) cancer cell lines. Cancer cells were exposed to a range of concentrations (0.01 to 100 µg/mL) of alkaloids and incubated in 5% CO50 calculation was performed using Sigma Plot version 12.0The ICAdditional file 1: Figure S1.13C-NMR spectra of 6, 7-dimethoxy-1-(α-hydroxy-4-methoxybenzyl)-2-methyl-1, 2, 3, 4-tetrahydroisoquinoline. Figure S2.13C-DEPT90 spectra of 6, 7-dimethoxy-1-(α-hydroxy-4-methoxybenzyl)-2-methyl-1, 2, 3, 4-tetrahydroisoquinoline. Figure S3.13C-DEPT135spectra of 6, 7-dimethoxy-1-(α-hydroxy-4-methoxybenzyl)-2-methyl-1, 2, 3, 4-tetrahydroisoquinoline. Figure S4. 1H-NMR spectra of 6, 7-dimethoxy-1-(α-hydroxy-4-methoxybenzyl)-2-methyl-1, 2, 3, 4-tetrahydroisoquinoline. Figure S5.13C-NMR spectra of Coclaurine. Figure S6.13C-DEPT 90 spectra of Coclaurine. Figure S7.13C-DEPT 135 spectra of Coclaurine. Figure S8.1H-NMR spectra of Coclaurine


Harpagophytum procumbens (Burch.) DC ex Meisn, commonly known as devil’s claw, are mainly used in modern medicine to relieve joint pain and inflammation in patients suffering from rheumatic and arthritic disorders. This paper describes for the first time the chemical profile of a commercial spagyric tincture (named 019) prepared from the roots of the plant. For comparison purposes, a commercial not-spagyric devil’s claw tincture (NST) was also analyzed. Chemical investigation of the content of specialized metabolites in the three samples indicated that harpagoside was the main compound, followed by the two isomers acteoside and isoacteoside. Compositional consistence over time was obtained by the chemical fingerprinting of another spagyric tincture (named 014) from the same producer that was already expired according to the recommendation on the label of the product. The two spagyric preparations did not show significant compositional differences as revealed by HPLC and MS analyses, except for a decrease in harpagide content in the expired 014 tincture. Moreover, their antioxidant capacities as assessed by 2,2’-di-phenyl-1-picrylhydrazyl (DPPH) and 2.2’-azin-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) methods resulted in very similar IC50 values. The expired 014 tincture showed instead a lower IC50 value compared to the 019 and NST tinctures with the ferric reducing antioxidant potential (FRAP) assay, indicating a higher ferric-reducing antioxidant ability. Overall, these results indicated that the two preparations could generally maintain good stability and biological activity at least for the four years from the production to the expiration date.Preparations from the dried tubers of  Harpagophytum procumbens (Burch.) DC ex Meisn, commonly known as devil’s claw, is a plant native to southern Africa, where it is traditionally used as a bitter tonic and a stomachic and also for the treatment of fever, gout, myalgia, and arthritis +, [m/z 517.2 (100) M + Na]+, +, [m/z 351.0 (44) (M + Na)-148-18]+, +) were in fact unequivocally consistent with the identification of harpagoside −, −, −, −); peak 2, isoacteoside, (Rt = 26.20 min), UV 217.7, 248.5 (sh), 292.3 (sh), and 326.9 (max) nm, fragmentation pattern ([m/z 623.0 (100) M − H]−, −, −, −); peak 3, 8-p-coumaroyl-harpagide, (Rt = 28.87 min), UV 224.8, 311.4 (max) nm, fragmentation pattern ([m/z 533.1 (100) M + Na]+, +, [m/z 351.0 (44) (M + Na)-164-18]+, +); peak 4, pagoside, (Rt = 33.60 min), UV 224.8 and 311.4 (max) nm, fragmentation pattern ([m/z 491.0 (100) M − H]−, −, −); peak 6, 8-cinnamoylmyoporoside (Rt = 41.64 min), UV 217.7 (max) and 278.1 nm, fragmentation pattern ([m/z 501.0 (100) M + Na]+, +, [m/z 335.2 (69) (M + Na)-148-18]+, +, +). The presence of harpagide (lacking absorbance at 270 nm), already detected by TLC, was confirmed by HPLC-ELSD (evaporative light scattering detector) analysis (m/z 365.0 (100) M + H]+, [m/z 347.1 (36) (M + H)-18]+, [m/z 305.1 (75) (M + H)-42-18]+, [m/z 275.1 (41) (M + H)-90]+, [m/z 203.0 (100) (M + H)-162]+, [m/z 185.0 (68) (M + H)-162-18]+). The identification of constituents of the two spagyric tinctures was also supported by the available literature  x 100, where Ablank is the absorbance of the blank solution at t = 0, and Asample is the absorbance of the test sample at t = 30 min. The IC50 concentration is the amount of drug necessary to give 50% inhibition of the DPPH radicals. Acteoside, quercetin, and harpagoside were used as reference compounds.The scavenging activity for DPPH  free radicals was measured according to Brand–Williams (1995), with some modifications. To 2.9 mL of a 10 × 10s-triazine) solution in MeOH; and c) a 20-mM FeCl3·6 H2O solution. The FRAP working solution was prepared by mixing TPTZ and FeCl3·6 H2O and acetate buffer in a ratio of 1:1:10. Test samples and reference compounds (100 μL) were added to 2.6 mL of FRAP reagent, along with 300 μL of H2O, and allowed to react for 30 min at 37 °C in dark conditions. Absorbance readings (ferrous tripyridyltriazine complex) were taken at 595 nm. A blank solution consisted of FRAP solutions above and in water. The standard curve was  using a concentration range of FeSO4 between 0.42 µg/mL and 20 µg/mL.The following stock solutions were prepared for the bioassay according to Benzie and Strain (1996): a) a 300-mM acetate buffer solution, pH 3.6; b) a 10-mM TPTZ  with 2.5 mM of potassium persulfate, K2S2O8. This solution was allowed to stand at room temperature in the dark for 12–16 h before use. For the analyses, the ABTS+· solution was diluted with water to an absorbance of 0.7 (±0.02) at 734 nm. For the UV determinations, 2.9 mL of the ABTS+· solution and 100 μL of each of the test samples were mixed, and an absorbance reading was taken at 734 nm, 2–6 min after mixing. Appropriate ABTS blanks were run in each assay. All solutions were freshly prepared for the daily experiments. The scavenging activity of each test sample was compared to that of Trolox  as the positive control. A linear calibration curve  was constructed using Trolox in a range from 4 μM to 26 μM.Radical scavenging capacity was also studied based on the reduction of ABTS+· radicals by the test samples. An ABTS+· radical cation was obtained by mixing an ABTS stock solution  of at least three replicates for each experiment. Microsoft Office Excel 2017 was used for processing the data.