Plant material and its extract preparation
Berberis aristata root bark was obtained locally from Amritsar, Punjab. The formal identification of the plant material was carried out by Dr. Amarjit Singh Soodan (Associate Professor), Department of Botanical and Environmental Sciences, Guru Nanak Dev University (Amritsar), Punjab and was thereby deposited in the above said department wide accession no. 766-768 Bot. & Env. Sc. dated 02/07/15. The plant name has been checked with http://www.theplantlist.org. The plant material was surface sterilized, air dried and powdered. An aqueous extract was then prepared by aseptically suspending a known amount of powdered plant material into a known volume of sterile distilled water and extracting it in a hot water bath at 40 °C for 20 min. The extracted material was then vacuum filtered using whatman filter paper No.1 and subjected to antimicrobial testing by agar well diffusion assay (ADA) (Arora and Sood 2017). The chemicals and antibiotics were procured from Hi-Media Pvt. Ltd., India.
Microorganisms used in the study
The reference strains of bacteria and yeast such as Enterococcus faecalis (MTCC 439), Staphylococcus aureus (MTCC 740), Staphylococcus epidermidis (MTCC 435), Escherichia coli (MTCC 119), Klebsiella pneumoniae 1 (MTCC 109), Klebsiella pneumoniae 2 (MTCC 530), Pseudomonas aeruginosa (MTCC 741), Salmonella typhimurium 1 (MTCC 98), Salmonella typhimurium 2 (MTCC 1251), Shigella flexneri (MTCC 1457) and yeast strains such as Candida albicans (MTCC 227) and Candida tropicalis (MTCC 230) were procured from microbial type culture collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh (Arora and Sood 2017). Besides these standard strains, a clinical isolate of MRSA was procured from Post Graduate Institute of Medical Education and Research, Chandigarh (India). All the cultures were preserved in the glycerol stock at − 80 °C.
Inoculum preparation and antimicrobial screening
The inoculum was prepared as per McFarland standard (Arora and Sood 2017). The 10% plant aqueous extract was initially screened for the presence of antimicrobial activity against the reference strains by agar well diffusion assay (Arora and Onsare 2014c). Three antibiotics, i.e., two antibacterial antibiotics (gentamicin, chloramphenicol) with different spectrum of inhibitory activity and one antifungal antibiotic (amphotericin B) were used to compare their antimicrobial activity, in terms of zone of inhibition, with that of the aqueous extract and phytoconstituents extracted from Berberis aristata.
Optimization of physiochemical parameters using one-factor-at-a-time approach
Parameters such as extract concentration, extraction temperature, extraction time, pH and filtration methods were optimized using classical approach as described previously in Arora and Sood (2017).
Statistical optimization of the parameters by response surface methodology (RSM) using Box–Behnken design
On the basis of results obtained from the one- factor-at- a-time method, various parameters such as extraction temperature, extraction time and extract concentration were taken as the independent variables so as to find out the effect of the variables’ interaction on the antimicrobial potential. Each variable was studied at three different levels (− 1, 0, + 1), which were 40 °C, 50 °C and 60 °C (extraction temperature); 20 min, 40 min and 60 min (extraction time), and 10%, 15% and 20% (concentration). The experimental design included 17 tubes with five replicates having all the variables at their central coded values, where the zone of inhibition (in mm) for two Gram positive (MRSA, S. aureus) and two Gram negative (E. coli, K. pneumoniae 1) bacteria was taken as the response G(1–4). The mathematical relationship of response G (for each parameter) and independent variable X (X1: concentration; X2: time; X3: temperature) was calculated by the following quadratic model equation:
$$G_{{\left( { 1{-} 4} \right)}} = \beta_{0} + \beta_{ 1} X_{ 1} + \beta_{ 2} X_{ 2} + \beta_{ 3} X_{ 3} + \beta_{ 1 1} X_{ 1}^{ 2} + \beta_{ 2 2} X_{ 2}^{ 2} + \beta_{ 3 3} X_{ 3}^{ 2} + \beta_{ 1 2} X_{ 1} X_{ 2} + \beta_{ 1 3} X_{ 1} X_{ 3} + \beta_{ 2 3} X_{ 2} X_{ 3} ,$$
where G is the predicted response; β0 intercept; β1, β2, and β3 linear coefficients; β11, β22, and β33 squared coefficient and β12, β13, and β23 interaction coefficients. The Analysis of the experimental data was done from the response surface graphs using MINITAB software version 11 (Box and Behnken 1960; Kaur et al. 2015).
Thermostability studies
Fifteen percent aqueous extract of the plant was exposed to a temperature range of 60–100 °C for 1 h and any loss in activity was worked out by comparing it with that of untreated plant extract.
Determination of the best organic solvent
The 15% aqueous extract (100 mL) was extracted thrice with an equal volume (100 mL) of the organic solvents, i.e., chloroform, hexane, ethyl acetate and butanol (s d fine chem-limited). After every extraction cycle, the organic layers were collected, pooled and then concentrated at 45 °C using a rotary evaporator (Buchi Rotavapor R-210). The concentrates for each solvent were finally reconstituted in 30% DMSO (s d fine chem-limited) to give the respective stock solutions of 40.15 mg/mL, 38.82 mg/mL, 42.33 mg/mL and 44.03 mg/mL. Their antimicrobial potency was thus ascertained by the agar well diffusion assay (Arora and Sood 2017).
Qualitative and quantitative profiling of phytoconstituents
The powdered plant material was qualitatively analyzed for the presence/absence of the major groups of phytoconstituents by suspending it in a particular solvent as per the protocol used for each particular phytoconstituent (protocols mentioned in Additional file 1). They were then isolated using standard quantitative techniques, dissolved in a known volume of 30% DMSO and then tested for their antimicrobial activity (Kaur and Arora 2009; Ezeonu and Ejikeme 2016; Arora and Sood 2017). The qualitative and quantitative estimation of the phytoconstituents was carried out using standard protocols as mentioned in Additional file 1. The results obtained were then compared to the standard antibiotics like gentamicin, chloramphenicol (for bacteria) and amphotericin B (for yeast).
Minimum inhibitory concentration (MIC)
The MIC of all the test extracts was assessed by the agar dilution method (Arora and Sood 2017). The various concentrations, ranging from 0.5 to 25 mg/mL (aqueous extract), 0.01–5 mg/mL (organic extract) and 0.01–10 mg/mL (phytoconstituents), were prepared to determine their MIC values. The MICs were compared to that of standard antibiotics (gentamicin, chloramphenicol and amphotericin B). The diluent (30% DMSO) without the test compound was considered as the negative control.
Viable cell count (VCC) studies
To work out the kill time, the stock solutions (as described previously) of different test extracts were prepared and the assay was performed as per protocol followed in Sood et al. (2015).
Determination of the post antibiotic effect of the ethyl acetate extract, flavonoids and diterpenes
The PAE of the ethyl acetate extract, flavonoids and diterpenes was performed according to Babakhani et al. (2011) and Raja et al. (2011). The test compounds were mixed in equal ratio with the suspension of respective test organism and incubated at 37 °C for 2 h, after which the drug activity was halted by diluting the mixture. From this diluted suspension, samples (0.1 mL) were taken and plated at an interval of 2 h up to 24 h so as to obtain the CFU count. The culture without the test compound was taken as positive. The values was worked out as PAE = T–C, as T (denotes test) is the time required for the CFU count to increase by 1 log10CFU/mL whereas C represents the control.
Biosafety evaluation of Berberis aristata root bark by Ames test and MTT assay
The test extracts were subjected to Ames mutagenicity test (by plate incorporation method) and MTT toxicity assay as described earlier in Arora and Sood (2017). In Ames Mutagenicity test, the aqueous extract, ethyl acetate extract and the phytoconstituents (at their MIC concentrations) were mixed with an equal volume of the diluted (10−3) culture of Salmonella typhimurium (MTCC 1251, IMTECH, Chandigarh) in a top agar medium containing 0.5 mM histidine–biotin mixture (1:1 ratio). Here, Sodium azide (5 µL of 17.2 mg/mL) was used as a positive control.
In MTT toxicity assay, 100 µL of the diluted blood cell suspension was added in each well and incubated at 37 °C for overnight, followed by addition of 200 µL of the extract (aqueous extract, ethyl acetate extract and the phytoconstituents) and incubation for 24 h. Then 20 µL MTT solution (5 mg/mL) was added to each well and incubated further for 3.5 h at 37 °C on orbital shaker at 60 rpm. After incubation, the supernatant was removed carefully and 50 µL DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 590 nm using an automated microplate reader (Biorad 680-XR, Japan). The wells with untreated cells served as control.
In vitro cytotoxicity testing using RD, L20B and Hep2 cell lines by MTT assay
The cytotoxic effect of the most active phytoconstituent (diterpenes) of Berberis aristata root bark was studied against three cell lines [RD (Human Rhabdomyosarcoma), L20B (Diploid mouse lung cell line) and Hep 2 (Human larynx epidermoid carcinoma)] by MTT assay as described earlier in (Al-Asady et al. 2014; Das and Devi 2015) with slight modifications, as per protocol given in Additional file 2. The cell lines were procured from Central Research Institute (C.R.I), Kasauli, Himachal Pradesh, India. Twofold serial dilutions ranging from 10 to 0.039 mg/mL were used in the experimentation. The IC50 values were calculated from the dose–response curve generated for each cell line.
Acute oral toxicity study of Berberis aristata diterpenes in Swiss albino mice
In order to validate the non-toxicity of the diterpenes in animal models, acute oral toxicity was studied as described previously (Jothy et al. 2011; Ping et al. 2013) with slight modifications. The experiment was conducted at Central Research Institute, Kasauli, Himachal Pradesh, India. For the study, healthy Swiss albino mice (males and females), weighing between 25 and 35 g and aged 8 to 10 weeks, were obtained from the animal house, Central Research Institute, Kasauli (H.P.). The mice were divided into 4 groups: 6 males in the test male group; 6 females in the test female group, 6 males in the control male group and 6 females in the control female group. The treatment or test group was given a single dose (5000 mg/kg) of the diterpenes (dissolved in normal saline) and the control group was given normal saline lacking the diterpenes. Prior to dosage by oral route, the mice were fasted overnight but were allowed free access to water. Following the fasting period, body weight of the mice was determined and a single dose was calculated in reference to the body weight, as the volume of the compound (diterpenes) to be given to the mice is 10 mL/kg. The mice were kept under continuous observation for any signs of toxicity and mortality, firstly at 4 h and 24 h interval, and then daily for a period of 14 days. During this period the surviving animals were weighed and visually observed for changes in behavioral pattern, physical appearance, injury and any signs of illness. On the 15th day, the final weights of mice were noted and they were subsequently anaesthetized using xylaxine and ketamine (5 mg/kg and 2.5 mg/kg b.wt. respectively). For biochemical analysis, the blood samples were collected (from both treated and control group) via cardiac puncture in non-heparinized tubes. Subsequently, the serum was separated and analyzed for alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (TBIL), urea and creatinine levels. Following the blood collection, all the animals were sacrificed by an overdose of anesthesia. The vital organs (mainly liver, kidney and heart) were removed, cleaned with saline and macroscopically examined for any lesions, followed by their histopathological examination. The experimental procedure was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (No. CPCSEA/IAEC/CRI/14-114-2016), which was performed according to the Organization of Economic Co-operation and Development (OECD) guideline 420 used for the testing of chemicals (see Additional file 3).
Data analysis
The experiments were done in duplicate sets and repeated three times. The values in most experiments were compared with standard antibiotics. All the statistical analysis was done by using IBM SPSS Statistics Data editor Version 20. The differences of means between the groups were analyzed using non-parametric test (Post hoc Tukey’s t test). A two sided p value of < 0.05 was considered as statistically significant.