Extraction of nettle (Urtica dioica L.) toxins under natural biting conditions

Experimental site

The experimental site was located at latitude 36 ° 68 ′ N, longitude 48 ° 38 ′ E, and an altitude of 1579 m with a cold climate, a mean annual precipitation of 295 mm, and a mean annual temperature of 10 ° C. The average minimum temperature in the coldest month is “Bahman to -7.5”, and the average maximum temperature in the warmest month is “August, 32.1”. During the year, the temperature drops below zero for 118 days, with January and February ranking first with 27 days. The wettest month is May with 52.5 mm, and the driest month is September with 3.5 mm.

The maximum recorded wind speed of 27 m per second was “97 km per hour”. The prevailing wind in Zanjan in most months of the year was in the east, and the average wind speed of 3 m per second was “11 km per hour”.

Plant material

The academic permission for collecting and researching medicinal plants was obtained from Head of Biotechnology, Department Research Institute of Modern Biological Techniques, University of Zanjan, Zanjan, Iran. The study complies with all relevant guidelines. All experiments were performed on 20 nettle ecotypes in the National Live Collection of Iranian Nettle (LCINs), located at the Research Institute of Modern Biological Techniques, University of Zanjan, Zanjan, Iran. A total of 4 nettle ecotypes (4 different provinces of Iran), similar in age and growth morphological conditions, were randomly selected and further used to collect nettle bites and venom. These nettle ecotypes were collected from different nettle habitats in Iran from different provinces in Iran, including Mazandaran, Gilan, Mashhad and Zanjan. These collections were cultivated in both greenhouse and field conditions and were raised and maintained (2013–2022) (Fig. 1).

Figure 1

Schematic diagram of the structure and arrangement of circular surfaces collecting nettle venom. S1, S2 and S3 are three types of cylindrical circular surfaces: styrofoam, nanofabric and sponge.

Morphology study of explants

All stages of hair penetration in to 3 different surfaces were analyzed during the research process, and sample stems, leaves and hairs were documented with a stereoscopic microscope (Nikon SMZ1, Tokyo, Japan). Photographs were taken with an Olympus microscope (Olympus CX31, Tokyo, Japan) at 40 and 400 magnifications.

Collection of poison

Three small porous circular plates with a length of 15 cm and a diameter of 4 cm in three types of styrofoam, nanofabric and sponge of the same length (15 cm) and same diameter (4 cm) were embedded on a light rod and so that they could be rotated on the rod in accordance with the rod axis.

GCMS analysis

GC – MS analysis of the extract was performed via an Agilent 7890B (USA) gas chromatograph equipped with an HP5-MS (5% phenyl / 95% dimethylpolysiloxane) capillary column (60 m × 0.25 mm 1D × 0.25 μm). This was coupled with a 5977A mass spectrometer in the electron impact (EI) ionization mode with an ionizing energy of 70 eV. . Helium gas (99.999%) was used as the carrier gas at a constant flow rate of 1 ml / min, and an injection volume of 1 μl was employed (split ratio of 1:20) under the following conditions: injector temperature 280 ° C, ion-source temperature 230 ° C and MS transfer line of 300 (^ circ )C. The oven temperature was programmed from 70 ° C (isothermal for 5 min.), With an increase from 15 ° C / min to 200 ° C and then 8 ° C / min going up to 280 ° C, and held for 10 my. A full scan interval of 0.5 s and fragments from 45 to 600 amu (Da) was programmed in the mass detector part15. A relative% amount of each component was calculated by comparing its average peak area to the total peak areas. The interpretation on mass spectrum GC – MS was conducted using the database of National Institute Standard and Technology (NIST) and Wiley Registry of Mass Spectral Data, 6th Edition (Wiley Interscience, New York) with more than 140,000 patterns.

Statistical analysis

The experimental design was employed with 4 × 3 factorial treatments based on a randomized complete block design (RCBD) having three replications. The ecotype in 4 levels (Mashhad, Mazandaran, Gilan and Zanjan) and the circular plate in three levels of Styrofoam, Nanofabric and Sponge were used in this study. The analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) were used to analyze the data, which was performed by SAS software (SAS Institute, 2003).

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