Ethics statement

All experimental procedures were carried out according to the principles and guidelines of the Ethics Committee of Faculty of Medicine, Assiut University, Egypt, for biological studies, with an approval number 17101912.

General phytochemical experimental procedures

The 1H, APT, and 2D NMR experiments were run at 400 and 100 MHz using Bruker Avance III spectrometer, Faculty of Science, Zagazig University. The HR-ESI-MS analysis was performed using a Bruker Bioapex-FTMS with electrospray ionization (USA). Chromatographic adsorbents include silica gel G60 (60–120 mesh, Merck, Darmstadt, Germany), C18-RP silica gel (230–400 mesh, Merck, Darmstadt, Germany), MN-polyamide-SC-6 (50–160 μm, Sorbent Technologies, Norcross, GA, USA), Sephadex® LH-20 (Mitsubishi Kagaku, Tokyo, Japan), Florosil (60–100 mesh, Carlo Erba Reagents, France) and Diaion® HP-20 (Sorbent Technologies, Norcross, GA, USA). TLC was conducted on pre-coated silica 60 F254 aluminum sheets, 0.25 mm and RP-18 F254, 0.25 mm (E-Merck), Darmstadt, Germany). Solvents used for extraction and isolation were of analytical grade, purchased from (Adwic - El Nasr Pharmaceutical Co., Cairo, Egypt). Deuterated solvents for NMR spectral analysis: DMSO-d6, CD3OD, CDCl3, C5D5N (Cambridge Isotope Laboratories, Inc., MA).

Plant material

Livid amaranth (Amaranthus blitum L.) is an annual herbaceous vegetable belonging to the Amaranthaceae family (https://www.worldfloraonline.org/taxon/wfo-0000530085). The aerial parts of AB were collected during the flowering season in September 2020 from The Medicinal Plants Station, Pharmacognosy Department, Assiut University, Assiut, Egypt. The plant was authenticated by Dr. Mostafa Aboelela, Associate Professor of Plant Taxonomy, Botany Department, Faculty of Science, Assiut University. A voucher herbarium specimen (No. 0002017) was deposited in the Pharmacognosy Department Museum, Faculty of Pharmacy, Assiut University. The collected materials were air-dried, powdered, and kept until subsequent research.

Extraction and fractionation

The powdered plant material (3.6 kg) was macerated in 70% ethanol (5 × 6 L). The combined extracts were vacuum concentrated at 40 °C to give a residue of 248 g. This residue was suspended in distilled water (600 mL) and partitioned with n-hexane (8 × 1 L). The combined n-hexane extracts were vacuum concentrated to obtain the n-hexane fraction (104 g). The remaining aqueous layer was concentrated to a dried residue (140 g). The dried aqueous residue was subjected to Diaion-HP20 (500 g) column chromatography (CC), eluted with 100% H2O (3 L), followed by 100% MeOH (3 L). These eluents were vacuum concentrated to yield two fractions of 125 g and 10 g, respectively. The latter fraction, weighing 10 g, will be referred to as the polar fraction. An illustrative scheme was provided to depict the process (Supplementary scheme S1).

Isolation of compounds 1–6 from n-hexane fraction

A portion of n-hexane fraction (90 g) was fractionated into five fractions labelled as (F1–F5), using vacuum liquid chromatography. The elution was carried out using a combination of n-hexane (n-Hex) and acetone (Ace) as solvent systems, with a gradient of increasing proportions of acetone. The step-by-step procedures for isolating compounds 1–6 from these fractions, along with an illustrative scheme of the process, can be found in (Supplementary S1 and scheme S2).

Isolation of compounds 7–22 from the polar fraction

The polar fraction (10 g) was fractionated into 12 subfractions (F-I to F-XII), using polyamide column chromatography (250 g). The elution was carried out using water (H2O) initially, followed by gradient solvent systems of H2O and methanol (MeOH). The details for isolating compounds 7–22 from these subfractions, along with corresponding illustrative schemes of the process, can be found in (Supplementary S2, Scheme S3, and Fig. 1).

Fig. 1

Isolation and purification of compounds 16–22 from the polar fraction of Amaranthus blitum L.

In vitro anthelmintic effects

The anthelmintic efficacy of AB extracts and selected pure isolates were evaluated, for the first time, against Trichinella spiralis muscular larvae, in comparison with albendazole (ABZ) as a standard. The larvae were recovered from experimental Trichinella spiralis-infected albino mice and prepared using the procedure described by [11]. This investigation included three extracts (crude ethanolic extract, n-hexane fraction, and the polar fraction) at two concentrations of 0.5 and 1 mg/mL, and four compounds (2 and 18–20), at concentrations of (6.25, 12.5, 25, and 50 μg/mL), compared with ABZ (20 μg/mL). The treated larvae were inspected under a light microscope (Leica®, Germany) to monitor the viability rates at successive time points. Besides, scanning electron microscope (JEOL model, JEM-100 CXII, Tokyo, Japan) was used to examine the morphological alterations associated with different treatments.

Observation of muscular larvae viability using light microscopy

Using a sterile 6-well plates, three wells were dedicated for each concentration. In accordance with the described protocol [11], T. spiralis muscular larvae (ML) was diluted in Rapid Prototyping and Manufacturing Institute (RPMI)-1640 medium. A larval suspension (50 μL) of approximately 100 larvae was added to 1 mL of each sample. The 6 well plates were then sealed and incubated at 37 °C in an atmosphere containing 5% CO2 at different time points of 1, 4, 24, 48, and 72 h. At the end of the incubation periods, the number of viable ML, those that survive the applied treatment, was determined by direct observation under the light microscope, estimated by their motility. The results were expressed as the viability percentage. Besides, phenotypic changes induced by different treatments were observed at different time-points using light microscope.

Scanning electron microscopy (SEM)

To thoroughly detect any change in morphological features, an SEM examination of exposed larvae was undertaken. Certain specimens, that induced noteworthy phenotypic changes when examined under a light microscope, were chosen including, the three extracts at 0.5 mg/mL, compounds 2 and 18 at 50 μg/mL, compound 19 at 25 μg/mL, and compound 20 at 12.5 μg/mL. A typical protocol was followed for preparing parasite specimens [11]. After being properly washed in PBS, larvae were fixed for 2 h with 2.5% glutaric dialdehyde. The larvae were rewashed in PBS, post-fixed in a 2% osmium tetroxide in sodium cacodylate buffer for 1 h. Afterward, dehydration of postfixed worms was done in alcohol series of increasing concentrations, followed by air-drying, mounting on a stub, and gold-coating. Eventually, high resolution graphs were generated for parasites using a scanning electron microscope.

Evaluation of the in vitro hematological activity

Blood samples were obtained from freshly donated blood bags at Assiut university hospital, central blood bank. In addition to the typical procedures for selection of donors applied at the blood bank, meticulous drug history was obtained from the donors to exclude any possibility of test results alteration due to drugs. CPDA-1 (Citrate phosphate dextrose adenine) was the anticoagulant utilized in blood bags. Fifteen mL from each bag were withdrawn into plain plastic tubes without any additional anticoagulant.

Preparation of platelet rich plasma (PRP) and platelet poor plasma (PPP)

At the beginning of the procedure, blood samples were placed in a centrifuge and spun at 1000 rpm (RPM) for 10 minutes. This step aimed to obtain a three-layered solution. The upper layer, known as plasma, was carefully examined for any remaining erythrocytes. If necessary, the samples underwent an additional centrifugation for 5 minutes at the same speed. During this process, the superficial layer containing platelets was extracted with caution, ensuring no disturbance to the intermediate buffy layer or underlying erythrocytes. This extracted layer is referred to as Platelet-Rich Plasma (PRP). To prepare Platelet-Poor Plasma (PPP), the PRP obtained previously underwent a second centrifugation at 2500 RPM for 20 minutes. This step caused the platelets to precipitate as pellets. The resulting supernatant, known as PPP, was carefully examined for hemolysis and then transferred into sterile tubes. By gently shaking the platelet pellets with a small amount of plasma, PRP is formed [12].

Light transmission aggregometry

The effect of AB extracts and compounds 2, 19, and 20 on platelet aggregation in PRP samples was evaluated, within 1 h of blood samples withdrawal, using a light transmission aggregometry. This technique relies on measuring the passage of light through turbid PRP, which contains uniformly circulating platelets. The addition of an agonist causes platelets to aggregate, resulting in a clearer, less light-absorbing solution. We used two common platelet agonists, ADP and AA, to activate the platelets. The light transmittance is detected using a photocell. The method involves passing an infrared beam through two cuvettes: one filled with platelet-poor plasma (PPP) as a reference and the other containing the PRP sample being analyzed. The resulting signals represent the continuous difference in light transmittance between the analyzed and reference samples. Prior to measurement, the PRP samples (495 μL) were pre-incubated with the examined plant extracts (5 μL) for 15 minutes at 37 °C. Subsequently, the samples were transferred into aggregometer cuvettes. Platelet aggregation was measured after stimulation with ADP or AA using concentrations of 10 μmol/L for ADP and 0.5 mg/mL for AA. Stock solutions of the AB extracts and compounds were prepared using 30% DMSO. The level of aggregation was expressed as a percentage of aggregation [13].

Measurement of the haemostatic parameters

In this assay, we measured the blood clotting times using automated Sysmex coagulation analyzers. The clotting times tested included prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT). Before conducting the assay, plasma samples were pre-incubated with the tested extracts and compounds at 37 °C for 20 minutes. The final concentrations used for the crude ethanolic extract, n-hexane fraction, and polar fraction were 50 μg/mL. For compounds 2, 19, 20, and dabigatran (reference anticoagulant drug), the concentrations used were 5 μg/mL [14].

Statistical analysis

SPSS (Statistical Package for the Social Science, version 20, IBM, and Armonk, New York) was used for statistical analysis. Data were gathered and expressed as the mean ± SD. Level of confidence was kept at 95% and hence, *p value ≤ 0.05 was considered statistically significant while **p value ≤ 0.01 was considered highly statistically significant. For significant difference between dependent multiple groups at different time points, Kruskal-Wallis test was used. For significant difference between the mean values of independent groups compared with the control group, multiple t-test and student t-test were applied.