IN VIVO EVALUATION OF THE ANTI-INFLAMMATORY AND IMMUNOMODULATORY EFFECTS OF A 3:1 COMBINATION OF Hypericum scabrum AND Calendula officinalis

ABSTRACT

Inflammatory disorders remain a significant medical challenge, highlighting the need for safe plant-based therapeutics. This study evaluated the anti-inflammatory and immunomodulatory effects of a 3:1 combination of Hypericum scabrum L. (grown in Uzbekistan) and Calendula officinalis in a dextran sulfate sodium (DSS)–induced in vivo inflammation model. Clinical signs, hematological indices, leukocyte profiles, and pro-inflammatory cytokines were assessed and compared with control groups.

The herbal mixture reduced inflammatory manifestations and promoted normalization of hematological and immunological parameters. These results indicate that the combined extract may suppress inflammatory activity and support immune regulation. Therefore, the 3:1 formulation represents a promising phytotherapeutic candidate with potential use as a supportive approach alongside conventional treatment or for individuals seeking natural health-restoring strategies. Further studies are needed to elucidate its molecular mechanisms of action.

АННОТАЦИЯ

Воспалительные заболевания остаются серьезной медицинской проблемой, подчеркивая необходимость безопасных терапевтических средств на растительной основе. В данном исследовании оценивались противовоспалительные и иммуномодулирующие эффекты комбинации Hypericum scabrum L. (выращенного в Узбекистане) и Calendula officinalis в соотношении 3:1 на модели воспаления in vivo, индуцированного сульфатом декстрана натрия (DSS). Были оценены и сравнены с контрольными группами клинические признаки, гематологические показатели, профили лейкоцитов и провоспалительные цитокины.

Растительная смесь уменьшила воспалительные проявления и способствовала нормализации гематологических и иммунологических параметров. Эти результаты показывают, что комбинированный экстракт может подавлять воспалительную активность и поддерживать иммунную регуляцию. Таким образом, формула 3:1 представляет собой перспективный фитотерапевтический кандидат с потенциальным применением в качестве вспомогательного подхода наряду с традиционным лечением или для лиц, ищущих стратегии восстановления здоровья с помощью натуральных средств. Необходимы дальнейшие исследования для выяснения молекулярных механизмов его действия.

Keywords: dextran sulfate sodium; in vivo study; anti-inflammatory activity; immunomodulation; cytokines; hematological parameters; Uzbekistan flora.

Ключевые слова: сульфат декстрана натрия; исследование in vivo; противовоспалительная активность; иммуномодуляция; цитокины; гематологические показатели; флора Узбекистана.

Introduction

Inflammation is a protective response to harmful stimuli, but chronic inflammation contributes to diseases such as autoimmune, gastrointestinal, and metabolic disorders [1]. Although synthetic anti-inflammatory drugs are effective, their long-term use may cause adverse effects, increasing interest in plant-based alternatives [2].

Medicinal plants are rich sources of bioactive compounds with anti-inflammatory and immunomodulatory activity. H. scabrum L., native to Central Asia, contains flavonoids, phenolic acids, and naphthodianthrones that regulate inflammatory mediators [3,4], while C. officinalis L. possesses triterpenoids, flavonoids, and carotenoids that reduce cytokines and promote tissue repair [5,6].

Dextran sulfate sodium (DSS)–induced inflammation is a reliable in vivo model for studying immune-mediated inflammatory processes [7]. Considering the pharmacological potential of both plants, their combination may produce synergistic effects. However, data on their combined activity remain limited. Therefore, this study investigated the anti-inflammatory and immunomodulatory effects of a 3:1 mixture of H. scabrum and C. officinalis in a DSS-induced model.

Materials and Methods

This study evaluated the anti-inflammatory and immunomodulatory effects of a 3:1 combination of H. scabrum L. and C. officinalis L. using a dextran sulfate sodium (DSS)–induced inflammation model. The protocol followed internationally accepted guidelines for preclinical pharmacological research [8].

Animals were randomly assigned to groups: intact control, DSS-induced pathology, and treatment groups receiving the herbal combination after inflammation induction. Randomization and standardized housing conditions were maintained to minimize environmental bias [9].

Aerial parts of H. scabrum L., collected from ecologically clean regions of Uzbekistan during flowering, were authenticated by a botanist, while C. officinalis flowers were obtained from certified sources. The plant materials were shade-dried, powdered, and mixed in a 3:1 ratio (H. scabrum: C. officinalis).

Extraction was performed with a hydroethanolic solvent under controlled temperature, followed by filtration, concentration under reduced pressure, and storage at +4 °C. The extraction method and solvent were selected according to established protocols to maximize flavonoid and phenolic compound yield [10].

Induction of Experimental Inflammation

Inflammation was induced using dextran sulfate sodium (DSS), a widely accepted agent for modeling immune-mediated inflammatory pathology [11]. DSS was administered according to standardized protocols to produce an acute inflammatory response. Clinical signs, including behavioral and physiological parameters, body weight, and general condition, were monitored daily.

Treatment Protocol

After inflammation induction, treatment groups received the 3:1 herbal combination at a body weight–adjusted therapeutic dose during the active inflammatory phase. The pathology control group received DSS without treatment, while the intact group received neither DSS nor the herbal preparation.

Hematological and Immunological Assessment

At the end of the experiment, biological samples were collected for analysis. Hematological parameters—total leukocyte count, lymphocytes, neutrophils, monocytes, and eosinophils—were measured using an automated analyzer according to standard procedures [12]. Pro-inflammatory cytokine levels, particularly interleukin-6 (IL-6), were determined by ELISA following the manufacturer’s protocol [13].

Statistical Analysis

Results were expressed as mean ± standard deviation (SD). Statistical comparisons were conducted using appropriate parametric tests after assessing normality, with p < 0.05 considered statistically significant.

Experimental Groups

Table 1.

Experimental design and group distribution

The table presents four experimental groups used to assess the anti-inflammatory and immunomodulatory effects of a 3:1 combination of H. scabrum and C. officinalis in DSS-induced inflammation. Group I served as the intact control, Group II received DSS as a negative control, Group III received DSS followed by the herbal treatment to evaluate efficacy, and Group IV received only the herbal mixture to assess safety. This design enables comparison among normal, pathological, and treated conditions, ensuring reliable evaluation of both therapeutic effect and safety.

Results

Clinical Observations

Dextran sulfate sodium (DSS) induced evident inflammatory signs in the pathology group, including reduced activity, ruffled fur, and mild weight loss. Animals treated with the 3:1 H. scabrum and C. officinalis combination showed improved behavior and general condition, indicating reduced inflammation.

Hematological Parameters

DSS caused a significant increase in total leukocytes, neutrophils, and monocytes, along with decreased lymphocyte levels, reflecting systemic inflammation. Treatment with the herbal mixture restored leukocyte profiles toward normal values, demonstrating its immunomodulatory effect.

Table 2.

Hematological parameters (mean ± SD)

Table 2 shows baseline hematological values in the intact control group. DSS administration significantly increased WBC and neutrophil counts while decreasing lymphocyte and eosinophil percentages, indicating systemic inflammation. Treatment with the 3:1 H. scabrum and C. officinalis combination restored most parameters toward control levels, demonstrating notable anti-inflammatory and immunomodulatory activity and confirming the mixture’s therapeutic potential in regulating immune cell distribution.

Immunological Markers

Pro-inflammatory cytokine IL-6 was markedly elevated in the DSS group compared with intact controls. Herbal treatment reduced IL-6 levels toward normal ranges, indicating suppression of inflammatory signaling pathways.

Discussion

The 3:1 combination of H. scabrum and C. officinalis demonstrated notable anti-inflammatory and immunomodulatory activity in a DSS-induced in vivo model, which reflects human inflammatory pathology. The normalization of hematological indices and reduction of IL-6 suggest synergistic action of their bioactive compounds, including flavonoids, phenolic acids, triterpenoids, and carotenoids. Previous studies report that H. scabrum regulates cytokines and oxidative stress, while C. officinalis supports tissue repair and immune balance.

The combined formulation showed greater efficacy than individual extracts in earlier reports, indicating synergism and supporting multi-component phytotherapy concepts. These findings suggest that the mixture may serve as a supportive adjunct to conventional anti-inflammatory therapy and merits further clinical investigation.

Conclusion

This study evaluated the anti-inflammatory and immunomodulatory effects of a 3:1 combination of Uzbekistan-grown H. scabrum and C. officinalis in a DSS-induced in vivo model. The herbal mixture improved clinical signs of inflammation, normalized hematological parameters, and reduced pro-inflammatory cytokines, particularly IL-6, confirming the study objectives. These findings indicate that the combination may be a promising phytotherapeutic agent with potential use as a supportive approach alongside conventional therapy or as a natural strategy for maintaining health. Further studies are needed to clarify molecular mechanisms and assess clinical applicability, but the present results provide a solid basis for developing standardized therapeutic herbal formulations.

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