Cardin is a partially characterized peptide-based preparation consisting of a mixture of biologically active peptide fractions derived from neonatal lamb heart tissue. The peptide fraction was obtained through homogenization in 0.9% NaCl, followed by thermal treatment at 80 °C, ammonium sulfate precipitation, and subsequent purification using gel filtration and ion-exchange chromatography.

The resulting preparation was formulated as a hydroalcoholic solution containing approximately 0.001% active peptide complex in 40% ethanol (v/v). Due to the multicomponent nature of the preparation, Cardin does not possess a single defined chemical structure and should be regarded as a heterogeneous peptide mixture. Its identity is therefore defined based on its source, standardized production protocol, and physicochemical handling conditions.

Preliminary characterization was performed using high-performance liquid chromatography (HPLC), which demonstrated multiple peptide fractions. Molecular weight analysis was conducted using Chip-HPLC-QTOF mass spectrometry, identifying five major peptide fractions with molecular weights of 7553.021, 5371.111, 7232.144, 9659.142, and 6847.232 Da. However, the complete primary amino acid sequences of these peptides were not fully elucidated.

For in vivo experiments, the Cardin solution was freshly prepared under sterile conditions prior to administration. The vehicle control group received an equivalent volume of 40% ethanol under identical experimental conditions.

A total of 586 laboratory animals were used, including mice (20 ± 2 g), rats (140 ± 10 g), guinea pigs, and rabbits of both sexes. Animals were acclimatized for at least 7 days and housed under standard laboratory conditions (22 ± 2 °C, 55 ± 10% humidity, 12 h light/dark cycle) with free access to food and water. All experimental procedures were approved by the Institutional Ethics Committee of Institute of Bioorganic Chemistry named after O. Sodikov Academy of Sciences of the Republic of Uzbekistan (protocol No. 1-2-1451-2-255 10/2025) and conducted in accordance with international ethical standards and ARRIVE 2.0 guidelines.

Animals were randomly assigned to experimental groups using a random number generation method. Each group consisted of 6–8 animals unless otherwise specified.

Outcome assessment was performed under blinded conditions where feasible; however, full blinding was not always possible due to the nature of the experimental procedures. Dose selection was based on preliminary screening studies and literature data on peptide-based biologically active compounds, taking into account safety margins.

Acute toxicity was evaluated in mice and rats following single oral and intraperitoneal administration of Cardin at graded dose levels expressed in mg/kg body weight. Animals (n = 6 per group) were observed continuously for the first 6 h and daily for 14 days for clinical signs of toxicity and mortality. Behavioral parameters, including locomotor activity, respiration, posture, and reflexes, were recorded. The median lethal dose (LD₅₀) was estimated using probit analysis. In the tested dose range, no mortality was observed, indicating low acute toxicity.

Cumulative toxicity was assessed in mice over 26 days with repeated oral administration of Cardin at fractionated doses relative to the maximum tested dose. The cumulative toxicity coefficient (Kc) was calculated as the ratio of LD₅₀ after repeated administration to that after a single dose.

Chronic toxicity was evaluated in rats and rabbits following daily oral administration of Cardin at doses of 10⁻⁴, 10⁻², and 1.0 mg/kg for 30 consecutive days (n = 6–8 per group). Blood samples were collected on days 10 and 30 for biochemical and hematological analysis. A subset of animals were observed for an additional 30-day recovery period. Biochemical parameters (ALT, AST, glucose, bilirubin, creatinine, urea, total protein) and hematological parameters (RBC, WBC, hemoglobin, hematocrit, platelet count) were measured using automated analyzers [12,13,14].

Hemostatic parameters were assessed using standard coagulation assays, including activated partial thromboplastin time (APTT), thrombin time (TT), and plasma recalcification time.

Antithrombotic activity was evaluated using a ferric chloride-induced arterial thrombosis model in rats. Thrombus weight was measured 24 h after induction.

Antihypoxic activity was evaluated using a sodium nitrite-induced hypoxia model in mice. Cardin was administered orally at doses of 10⁻⁴ and 10⁻² mg/kg prior to hypoxia induction. Survival time was recorded and compared to the control group.

Computational toxicity prediction was performed using the ProTox-II web platform. Predicted endpoints included hepatotoxicity, neurotoxicity, immunotoxicity, receptor interactions, and cytochrome P450-related metabolism [10,11,15,16].

Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Differences were considered statistically significant at p < 0.05.

The peptide complex Cardin was successfully isolated from lamb (Ovis aries) heart tissue using sequential extraction, thermal denaturation, ammonium sulfate precipitation, and chromatographic purification (gel filtration and ion-exchange chromatography). The overall yield of the peptide fraction was approximately 0.00015% (w/w).

Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis revealed that Cardin is a multicomponent peptide mixture, consisting of five major (K-1–K-5) and four minor peptide fractions. The optimal chromatographic separation was achieved on a ZORBAX Eclipse C18 column (4.6 × 250 mm, 5 µm) with an acetonitrile–0.1% trifluoroacetic acid gradient, enabling reproducible peptide mapping within 15 min.

As shown in Figure 1, reverse-phase high-performance liquid chromatography (RP-HPLC) analysis demonstrated that Cardin is a multicomponent peptide mixture consisting of five major (K-1–K-5) and four minor peptide fractions. Mass spectrometry (Chip-HPLC-Q-TOF, Agilent 6530B Agilent Technologies, Santa Clara, CA, USA) identified the molecular masses of the major peptides as follows: K-1: 7553.0 Da, K-2: 5371.1 Da, K-3: 7232.1 Da, K-4: 9659.1 Da, K-5: 6847.2 Da. Amino acid analysis demonstrated that all fractions are composed predominantly of hydrophobic and neutral amino acids (Leu, Val, Gly, Ala), with variable content of charged residues (Lys, Arg, Asp/Glu), suggesting potential interactions with coagulation-related proteins [8,9].

Fragmentation analysis (LC–MS/MS) revealed multiple peptide fragments; however, full primary structures could not be reconstructed using available databases, confirming the novel and heterogeneous nature of the Cardin peptide complex. The detailed mass spectrometric characteristics of the identified peptides are presented in Table 1.

Cardin and its peptide fractions significantly reduced thrombus formation in rats: thrombus weight decreased by 40% (Cardin) and 60% (K-3, K-4), and clot inhibition rate increased by up to 63.5% (K-4).

The antithrombotic activity of Cardin and its peptide fractions is presented in Table 3. These results demonstrate a strong dose-dependent antithrombotic effect, with fractions K-3 and K-4 showing the highest activity.

In an FeCl₃-induced carotid artery thrombosis model in rats, thrombus weight decreased: control: 1.9 ± 0.2 mg; Cardin: 0.8 ± 0.06 mg (58% reduction). Coagulation parameters also improved: APTT increased by 40%, and fibrinogen decreased by 40%. The results are summarized in Table 4.

The effects of Cardin were comparable to or stronger than those of nebivolol (0.46 mg/kg), particularly in reducing fibrinogen levels.

In a sodium nitrite-induced hemic hypoxia model, Cardin (1 × 10⁻⁴ mg/kg) increased survival latency by 42–56% compared to the control. A higher dose (1 × 10⁻² mg/kg) showed maximal effect at 24 h (57% increase), followed by reduced activity at 48 h. These findings indicate moderate but significant antihypoxic activity, potentially related to improved microcirculation [15,17].

Across all experimental conditions:

These findings suggest a favorable safety profile within the studied dose range, despite in silico predictions indicating potential toxicity risks.

The results demonstrate that Cardin exerts pronounced anticoagulant effects, primarily through modulation of the intrinsic pathway of coagulation. This is supported by significant prolongation of activated partial thromboplastin time (APTT), thrombin time (TT), and plasma recalcification time (PRT), along with a reduction in fibrinogen levels, while prothrombin time (PT) remained largely unaffected.

Such a pattern suggests that Cardin may interfere with intrinsic coagulation factors (e.g., factors XII, XI, IX, and VIII) or enhance endogenous anticoagulant mechanisms rather than affect the extrinsic pathway. The observed decrease in fibrinogen further indicates a potential inhibition of fibrin formation and/or promotion of fibrinolytic processes.

Importantly, the reduction in thrombus formation and increased clot inhibition observed in vivo are consistent with the anticoagulant effects and indicate that Cardin can suppress pathological thrombosis without completely disrupting physiological hemostasis. Among the peptide fractions, K-3 and K-4 demonstrated the most pronounced activity, suggesting that specific components within the mixture may be primarily responsible for the observed pharmacological effects [2,4,6,9].

Cardin is a heterogeneous mixture of medium-molecular-weight peptides (approximately 5–10 kDa), distinguishing it from conventional low-molecular-weight anticoagulants. The amino acid composition of these peptides, enriched in hydrophobic residues (Leu, Val, Ala) and containing charged residues (Lys, Arg, Asp, Glu), may facilitate interactions with proteins involved in coagulation cascades, platelet function, or endothelial regulation. Such structural diversity may contribute to the multimodal biological activity observed in this study, potentially involving synergistic interactions among different peptide fractions rather than the effect of a single active compound [5,10,11].

A notable finding of this study is the discrepancy between in silico toxicity predictions and in vivo experimental results. Computational models predicted potential hepatotoxicity, neurotoxicity, and immunotoxicity, whereas no corresponding toxic effects were observed in animal studies at the tested doses. This discrepancy may be attributed to several factors. First, in silico models are largely based on predefined chemical structures and known toxicophores; however, Cardin represents a complex mixture of partially characterized peptides, which may not be accurately represented in such databases. Second, the administered doses in vivo were relatively low, which may fall below toxicity thresholds. Third, peptides are subject to enzymatic degradation and limited bioavailability, reducing systemic exposure. Finally, biological systems possess adaptive detoxification and compensatory mechanisms that are not accounted for in computational models. Thus, the observed differences highlight the limitations of predictive toxicity tools when applied to heterogeneous biological preparations and emphasize the importance of experimental validation [12,13,18,19,20].

Cardin demonstrated moderate but significant antihypoxic activity in a sodium nitrite-induced hemic hypoxia model. The increased survival time observed may be indirectly related to improvements in blood rheology and microcirculatory function.

By reducing thrombosis and potentially improving blood flow, Cardin may enhance oxygen delivery to tissues under hypoxic conditions. Additionally, modulation of endothelial function and nitric oxide-related pathways may contribute to the observed effects, although these mechanisms require further investigation [17,18,19,21,22,23,24,25,26].

In response to concerns regarding reproducibility and experimental rigor, this study provides detailed descriptions of animal models, dosing regimens, analytical methods, and experimental conditions. Randomization was applied during group allocation, and observations were conducted under partially blinded conditions. However, further standardization of blinding and randomization procedures in future studies would strengthen methodological robustness and compliance with international guidelines such as ARRIVE 2.0.

Several limitations should be acknowledged. First, the structural characterization of Cardin remains incomplete, as the full amino acid sequences of individual peptide fractions were not fully elucidated. Second, the multicomponent nature of the preparation makes it difficult to attribute specific biological effects to individual peptides. Third, the current study primarily focuses on acute and subchronic toxicity, while long-term toxicological and immunogenic assessments remain limited. Finally, pharmacokinetic parameters, including absorption, distribution, metabolism, and excretion (ADME), were not evaluated. These limitations should be addressed in future studies through advanced proteomic techniques, isolation of individual active fractions, and comprehensive pharmacokinetic and toxicological profiling [9].

The findings of this study suggest that Cardin is a promising peptide-based candidate with anticoagulant, antithrombotic, and antihypoxic properties at relatively low doses. Its high biological activity and favorable safety profile distinguish it from conventional anticoagulant agents. Future research should focus on full structural elucidation using advanced analytical methods, identification and isolation of the most active peptide fractions (particularly K-3 and K-4), detailed mechanistic studies at the molecular level, and comprehensive pharmacokinetic and long-term safety evaluations [2,6,12].