Sample collection of T. foliolosum plants and processing

T. foliolosum, a perennial herb, was obtained in September month from the forest areas of the Nanital district (2290-m asl), Kumaon region of Uttarakhand state in India (Fig. 1). Nanital is located at the GPS coordinates of 29° 22′ 49.0944'' N and 79° 27′ 48.8520'' E. This region has a humid subtropical climate with an average annual temperature of 17.1 °C (62.7 °F) and 1903 mm (74.9 in) of rainfall. The plants were identified by Dr. Harsh Singh (Taxonomist scholar) and a voucher specimen number 257717(LWG) was deposited at CSIR-National Botanical Research Institute (India). The collected plant materials (leaves, stem and roots) were initially carefully washed with saline water, then the samples were placed under running tap water for further washing. After some time, plant materials are rinsed again with autoclave water for half an hour and then put on blotting paper for 48 h to dry. The plant materials were blotted dry before being chopped into small pieces and dried in the shade for 15–20 days. Finally, the dried plant material was ground into a fine powder using an electric grinder (Mill CT 293 Cyclotec TM) and stored in sterilized glass bottles [5, 6].

Fig. 1

Thalictrum foliolosum plant parts a aerial parts (Leaf and stem), b Roots of T. folilosum

Chemicals

All the standards of BIQ alkaloids (Magnoflorine chloride, berberine chloride, palmatine chloride and thalicarpine) were procured from Sigma-Aldrich (USA). Solvent such as methanol, hexane, ethyl acetate, chloroform and acetonitrile and other chemicals were purchased from Merck (India).

Extraction of plant materials for HPLC analysis

For quantitative analysis, fine powders of different plant materials (1 g) were extracted with 25 ml of acidic (1% HCl) methanol using ultrasonication method (Aczet, Ultrasonic cleaner CUB) at room temperature for 40 min. After ultrasonication, plant materials in extracted solvents were kept at room temperature for overnight. The next day, all sonicated extracts were filtered through a Whatman No 1 filter paper in a round bottle flask, and fresh extracted solvent was added to the same plant materials for additional ultra-sonification. Three times, the same procedure was followed. Later all extracted materials were pooled in round bottom flask and evaporated at 50 °C using a rotary evaporator (Buchi, USA) under low pressure. Finally, the dried extracted plant materials were dissolved in methanol and filtered through a 0.45 µm filter and stored at 4 °C for subsequent HPLC analysis [6, 7]. The acidic-methanol extract of T. foliolosum had a yield of 27.42%, 21.10% and 16.24% of the extracted root, leaves and stem respectively. Reference standard solutions (i.e. berberine, palmatine, magnoflorine and thalicarpine) (Sigma-Aldrich) were prepared in methanol. All of the extracted solutions as well as the standard solutions were maintained at 4 °C in the refrigerator.

HPLC method

Plant samples were extracted and subjected to further analysis using High-Performance Liquid Chromatography (HPLC) with a gradient method for identifying bioactive compounds, following the protocol established by Mishra et al. [6, 7]. The analysis was performed using a Shimadzu (Japan) HPLC Prominence system comprising a 20 μL sample loop, a PDA SPD M 20 A photodiode array system, an LC-20AD dual pump system, and a SIL-20 AC Autoinjector with a cooler. Compounds were separated on a Shimadzu RP-C18 column (250 × 4.6, 5 μm pore size) with a guard column of the same packing material. A gradient mobile phase was employed consisting of component A (0.3% formic acid + 0.3% triethylamine) and component B (acetonitrile). The mobile phase gradient program was as follows: 0–25 min, 5–25% B; 25–35 min, 25%-35% B; 35–45 min, 35–45% B; 45–60 min, 45–100% B, at a flow rate of 1 mL/min. Data integration and compound identification were performed using Shimadzu Lab Solution software at a wavelength of 265 nm [6]. Results were compared against standards obtained from Sigma-Aldrich, USA. Quantitative analysis was conducted by averaging the results of three independent analyses of the same sample.

Fatty acid analysis using gas liquid chromatography

500 mg dry weight of plant samples (leaf, stem and roots) were used for FAME preparation. Fatty acid methyl esters (FAMES) were prepared as per method followed by Bureau of Indian Standards IS: 548 (Part-III), 1976 reaffirmed 1994. Prepared FAME dissolved in hexane were analyzed using GC system (7890B GC System, Agilent Technologies) equipped with a flame ionization detector (FID). FAMEs were separated on DB-225 column (30 m × 0.25 mm ID × 0.25 µm film thickness). Nitrogen was used as carrier gas and hydrogen and air as ignition gases. The conditions used for GC analysis were: injector temperature of 230 °C, detector (FID) temperature of 260 °C with split mode injection (1:20). The oven temperature program was started from 90 (2 min hold) to 130 °C at ramping rate of 3 °C min−1. Temperature from 130° to 230° (5 min hold) was achieved with ramp of 2 °C min−1. The peaks were identified by comparing with standards and the results are presented as mean of triplicates. Data were integrated by Open LAB CDS Chem Station Edition software. Reference standards of methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, methyl arachidate and methyl behenate, methyl linoleate were procured by M/s Sigma-Aldrich.

Extracts preparation for antimicrobial assay

For antimicrobial activity various plant parts materials (20 g) were extracted in aqueous methanol (50%) by sonication method. Then extracted solvents were further fractionated by various organic solvents, such as ethyl acetate, chloroform and hexane [5]. All extracted samples were filtered using Whatman filter paper No. 1. The filtrate was then dried using a low-pressure rotary evaporator (Buchi, USA) and lyophilized. All samples were stored at 4 °C in an airtight tube until further analysis was completed.

Microorganism used in study

In vitro antimicrobial activity was determined against four different human pathogenic bacteria i.e. E. coli (ATCC 25922), S. mutans (ATCC 25175), S. aureus (ATCC 4944) and P. aeruginosa (MTCC 424). E. coli is a gram-negative, rod-shaped bacterium that is commonly found in the lower intestine and most of these strains are harmless. However, pathogenic E. coli varieties cause serious food poisoning, septic shock, meningitis, or urinary tract infections in humans [16]. S. mutans is a gram-positive coccus bacterium, primarily found in mouth, pharynx, and intestine and significantly involved in tooth decay [17]. Staphylococcus aureus is a gram positive, round shape, facultative anaerobe bacterium. S. aureus are frequently found in the upper respiratory tract and on the skin and being a common cause of respiratory infections including skin infections [18]. P. aeruginosa is encapsulated rod-shaped, gram-negative bacterium. The important characteristic of this bacteria is multidrug resistance. Usually, P. aeruginosa found in medical equipment’s hence these bacteria associated hospital acquired infections, i.e. ventilator associated pneumonia and other sepsis syndrome the general symptoms of these bacteria are inflammation and sepsis but if their colonization occurs in lungs, kidneys and urinary tract it may be fatal [19].

Screening of antimicrobial activity

In vitro antimicrobial activity was determined against four different human pathogenic bacteria using disk-diffusion method with slight modification [20]. In short, 18–24 h old bacteria culture (0.5 OD600 nm) was spreading over the entire surface of nutrient agar plates (90 mm size) using autoclaved steel spreader and sterile paper disks (approx. 6 mm diameter) were placed on them. Then different concentrations of test samples (25, 50 and 100 µg) were loaded on disks. Streptomycin (25 µg) was utilized as a positive control and respective solvents to which extract dissolved was used as a negative control. Then, plates were allowed to incubate at 37 ± 1 °C for 18–24 h. The diameter of the inhibition zone (ZOI) was measured after 24 h of incubation to assess antibacterial activity (measured in mm including disk size). All experiments were carried out in triplicate, and the observed ZOI values are expressed as a mean with standard error of the mean (SEM).

Minimum inhibitory concentration (MIC)

MIC was determined of various extract of leaves, stems and roots parts of T. foliolosum against the different pathogenic bacteria by using the previously reported serial dilution method using 96-well microtiter plates. In brief, all pathogenic bacteria were grown in nutrient broth for 6 h before being inoculated, followed by 106 cells/mL bacterial culture was inoculated in 200 μL nutrient broth containing tube. Now each extract (leaf, stem, and root) has been added separately in bacterial broth with concentrations ranging from 100 to 800 μg/ml. All tubes were incubated at 37 °C for 24 h and further examined for visible turbidity. The minimum inhibitory concentration (MIC) was determined to be the lowest concentration that inhibited visible growth of the tested bacteria. MIC of berberine, magnoflorine and streptomycin were determined with similar procedure.