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Tissue-specific variations of piperine in ten populations of Piper longum L.: bioactivities and toxicological profile – Scientific Reports

Plant materials

Different plant parts (leaf, stem, fruit) of P. longum were collected from 10 different regions of West Bengal, India (Fig. S2) including both local cultivated and medicinal plant garden. Fresh plant parts were collected and bought to the laboratory. Dr. Avinash Mundhra, Assistant Professor, Department of Botany, Rishi Bankim Chandra College, West Bengal, India, identified the plant specimens. For reference in the future, a voucher specimen (PL01) was stored at the departmental herbarium in the Department of Life Sciences at Presidency University.

Ethical approval

Authors confirm that the use of plants in the present study complies with international, national and/or institutional guidelines. We gained appropriate permissions for collection of plant specimens from respective garden authorities and/or owners.

Chemicals and reagents

Piperine analytical standard, DPPH (1,1 diphenyl-2-picrylhydrazyl), ABTS (2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] were purchased from Sigma-Aldrich, India. Ascorbic acid, potassium persulphate was bought from Himedia laboratories.

Plant extract and standard solution preparation

All the plant parts were shed dried and powdered used grinder separately, passed through a 80-mesh sieve. For extraction 1-g dried powder of each sample were weighed accurately and macerated with methanol for 24 h in a shaker (Lab X incubator shaker) in room temperature. The extracts were filtered via Whatman filter paper (grade-1, 90 mm), and a rotary evaporator was used to evaporate the solvent. Each final extract was weighed, redissolved in methanol (10 mg/mL), and kept in a refrigerator at 4 C for further HPTLC analysis. A precise 5 mg of reference standard piperine (Sigma Aldrich, India) was weighed out and dissolved in 5 mL of methanol to create the standard piperine solution.

Chromatographic condition

Chromatography was carried out on HPTLC plates of 10 cm × 20 cm that were precoated with 0.25 mm layers of silica gel 60 F254 (Merck, Germany). The HPTLC system CAMAG (Muttenz, Switzerland) was composed of a CAMAG TLC scanner-3 with software (Win CATS 3) and an Linomat-V automatic sample applicator. For calibration curve, piperine standard solution (2, 4, 6, 8, 10 µl) were applied to the plates in 6 mm wide bands using the automatic sample applicator Linomat-V [“Linomat5200109” S/N 200,109 (1.00.13)] (with nitrogen flow) that was supplied with a Hamilton syringe (100 µL) with a delivery rate set at 150 nl/s. The plant samples were also applied to the plates in 6 mm wide bands of 10 ml at a delivery rate of 150 nl/s. The plates were developed at a distance of 8.0 cm in in linear ascending condition in a CAMAG twin-trough glass chamber (20 × 10 × 4 cm) chamber pre-saturated with mobile phase vapour using toluene:ethyl acetate:diethyl ether (6:3:1 v/v/v) as the mobile phase at room temperature 22 ˚C (± 2 °C) and relative humidity of 50%. Plates were dried using a hot air drier after development up to 75 mm, and then clear bands were seen under UV (UV cabinet with dual wavelength UV lamp) at λ = 254 nm. The plates were scanned at 254 nm (the maximum wavelength for piperine) using WinCats 3 software [“Scanner_200208” S/N 200208 (2.01.02)] and a CAMAG TLC scanner 3. A graph was created by plotting the average peak area versus the amount of piperine after this procedure was repeated five times. The parameters for densitometric scanning were adjusted to a 4.00 × 0.30 mm slit size, 20 mm/s scanning speed, and 100 µm/step data resolution.

Preparation of standard curve

Using peak areas of the standard piperine (2, 4, 6, 8, 10 µl) versus its’ pertinent concentrations, a calibration curve was prepared (Fig. S1). In all of the plant samples that were put to the test, the yield of the reference compound was calculated using a regression equation and the associated peak area.

Validation

In accordance with ICH (International Conference on Harmonization council) (ICH, 2005) recommendations, the method was validated by evaluating the peak purity, precision, specificity limit of detection (LOD), limit of quantitation (LOQ), instrument precision, repeatability, reproducibility and piperine’s percentage recovery from samples.

Precision

To test the instrument’s precision, the same spot for piperine (100 ng/spot) with n = 5 was employed. Three different concentrations of the reference substances were tested for repeatability on the same day (intra-day) and reproducibility (inter-day) three consecutive days, respectively. The findings of these seven different analyses are represented as mean % RSD (Relative Standard Deviation).

Limit of detection and limit of quantification

LOD and LOQ values were assessed for the signal-to-noise (S/N) ratio using various concentrations of reference substances and methanol as a blank. The difference between LOD and LOQ was measured as 3,1 (SD/S) and 10,1 (SD/S), respectively. Here, S denotes slope and SD represents standard deviation of the Y-intercept from the regression line.

Specificity

For more specificity, the peak purity of the standard compound and the plant samples were matched at the peak’s beginning, at the peak maxima, and at the peak end. In addition, marker compound and the overlay spectra of the isolated bands of the plant samples were compared. The accompanying Rf values were verified using the separated band of the standard chemical with plant samples in their scanning densitometric chromatograms.

Antioxidant activity

DPPH radical scavenging assay

To assess the antioxidant capacity of the P. longum plant extracts a modified DPPH assay, as published by Chavan et al. 201448, was used. DPPH free radical solution was obtained by mixing 0.1 mM DPPH with methanol using a magnetic stirrer for 12 h at 25 °C. An extract dilution series with the concentration range 250–50 µg/mL was prepared using extracts of Pop3 (Chemotypes PL7, PL8, and PL9) as this population contains highest amount of Piperine. The DPPH radical solution (1 mL) was added to 3 mL of the extracts and standard solution (50, 100, 150, 200 and 250 µg/ml), separately and left to incubation in dark for 30 min at 25 °C. The absorbance was measured at 517 nm. Decreasing absorbance demonstrates DPPH free radical scavenging capacity.

The control consists of 3 ml of methanol and 1 ml of DPPH solution. The positive control is represented by a solution of standard antioxidants, ascorbic acid, whose absorbance is measured under the same conditions as the sample tested.

The percentage inhibition of DPPH is calculated according to the following formula:

Percentage of radical scavenging activity = (frac{Abs, of, control-Abs, of, sample}{Abs, of, control}times 100) where, Abs = Absorbance (Brand-Williams et al., 1995)49.

ABTS assay

Using the ABTS cation decolorization assay, the ABTS radical scavenging capacity was assessed according to the method described by Tuyen et al., 201750 with some modifications 0.7 mM ABTS and 2.45 mM K2S2O8 (Potassium persulfate) solution were reacted for 12 h in dark to procure experimental solution of ABTS cation radical. With the help of 0.1 M phosphate buffer (pH 7.4) dilation, the absorbance of the control solution was adjusted to 0.7 ± 0.1 at 734 nm. An extract dilution series with the concentration range 250–50 µg/mL was prepared using extracts of Pop3 (Chemotypes PL7, PL8, and PL9) as this population contains highest amount of Piperine. 30 µl of each sample and standard ascorbic acid at different concentrations (50 to 250 μg/ml) was added to 1 ml of the ABTS solution and mixed vigorously. The sample volume was increased to 4 mL by adding buffer solution. The absorbance at 734 nm was measured after the reaction mixture was allowed to stand at room temperature for 6 min. The reduction in sample absorption was used to calculate the ABTS· + radical scavenging activity. The ABTS scavenging effect was calculated as per the equation:

Percentage of ABTS scavenging effect =(frac{Abs, of ,control-Abs, of, sample}{Abs ,of, control}times 100) where, Abs = absorbance.

Genotoxicity assessment

Preparation of the aqueous extracts of P. longum plants

To prepare a stock solution (10%), 20 gm of the powdered sample was added to 200 mL of distilled water, stirred thoroughly, and then heated for 10 min in a beaker with a lid. After then, the solution was cooled down at room temperature. The extract was filtered using Whatman No. 1 filter paper. Workable solutions (5, 10 and 20 mg/mL) were created by gradually diluting the stock. At the outset of each trial, all of the extracts were freshly produced.

Allium cepa root tip assay

To investigate the genotoxicity of the high piperine-yielding P. longum population chemotype (PL9), the methodology described by as As¸kinÇelik and Aslantürk, 201051 was used, with a few minor modifications. Distilled water was used as a negative control, and ethyl methane sulfonate (EMS, 2 × 10–2 M) was used as a positive control. After 6 h incubation in the controls and the water extract (5, 10 and 20 mg/mL) of P. longum plants, the root tips of A. cepa were cut off from the onion bulbs and fixed at 4 °C in a solution of ethanol: glacial acetic acid (1:3 v/v) for 16 h. The roots were then placed in a solution of 70% (v/v) ethanol and kept in the refrigerator for further analysis. Following the hydrolysis of the root tips using 1 N HCl for 2 min, and staining the root tips in aceto-orcein [2% (w/v)], slides were prepared using the squashing technique. Each slide was observed under 40 × 10 magnification of a compound microscope (Olympus) with (n = 5 in each set). Mitotic-index (MI), which is the percentage-based comparison of the total number of dividing cells to the total number of cells in a given microscopic field, was used to measure genotoxicity. Different chromosomal aberrations such as anaphase bridges, multipolarity, binucleate cell, multi-nucleolar nucleus, micronucleus etc. were counted and the MI for the same are mentioned in Tables 4 and 5.

Statistical analysis

All the results procured from the HPTLC screening of the phytochemical and antioxidant assays were run three times with 19 samples in triplicate for each experimental set. Fisher’s least significant difference test was used to determine the significance of differences between means. Mean values are denoted as mean SE. Three times, calculations were done using mean values and standard error. The data is shown as mean (pm) SE. Student’s t-test usage offered statistical significance. P < 0.05 values were regarded as significant. Analysis of variance (ANOVA) was carried out for antioxidant assay as well as Allium cepa root tip assay using SPSS software. The tables were made with Microsoft Excel and graphs were made by Graphpad Prism software.