INTRODUCTION:

Plants provide humanity with many of its most essential resources like food and medicine. Many pharmaceutical products are derived from the molecular components of plants. This is due to the active compounds contained in plants, which have a wide spectrum of pharmacological activity^1,2,3.

Since these active chemical components are naturally involved in the physiological processes of plants, they are thought to be more compatible with the human body⁴. Among the diverse phenolic compounds found in plants, flavonoids are the most prevalent and widely distributed, demonstrating a variety of biological effects^5,6. Various classes of flavonoids exhibit multiple pharmacological activities, including antioxidant and hepatoprotective effects^7-11.

The Helichrysum genus, a member of the Asteraceae family, comprises approximately 600 species worldwide¹². Various in vivo and in vitro studies have demonstrated their antioxidant, antidiabetic, choleretic, antibacterial, antifungal, sedative, antiseptic, hepatoprotective, and other therapeutic properties^13-17.

Among this genus the species H. arenarium (Helichrysum arenarium (L.) Moench) chiefly contain 39 flavonoids with chalcone flavanones salipurposide, isosalipurposide, naringenin, and prunin as the primary constituents¹⁹.

H. arenarium is commercially available in three medicinal forms: dried flower heads (Helichrysi arenarii flos), fluid extract (Helichrysi arenarii extractum fluidum), and dried extract of flower heads (Extractum florum Helichrysi arenarii siccum). Helichrysi flos, derived from Helichrysum arenarium (L.) Moench, is a widely recognized herbal drug in traditional medicine. It is utilized as a cholagogue, choleretic, diuretic, mild spasmolytic, hepatoprotective agent, and for detoxification purposes²⁰.

In medical practice, the drug "Flamin" has been successfully used for more than fifty years, which has a pronounced choleretic activity. It is a mixture of flavonoid compounds: glycosides salipurposide, isosalipurposide and aglycones kaempferol, apigenin, naringenin^19,21,22.

Helichrysum italicum is another of the most well-studied Helichrysum species, and it has been used to treat ischemic heart disease, obesity, diabetes, lipid metabolism disorders, and atherosclerosis. Moreover, it was demonstrated that H. italicum infusion could indeed improve the inflammation-induced intestinal barrier dysfunction^17,23.

It is important to note that Armenia's flora is abundant in plant species, such as Helicrhrysum, which have bioactive compounds and can be used as raw materials to make new medications. However, they haven't been used in medical practice because of their inadequate research.

Many species Helicrhrysum have been included in various pharmacopoeias. Among the least studied is Helichrysum rubicundum, which is widely distributed in the flora of Armenia¹².

Based on the aforementioned arguments, we studied Helichrysum rubicundum (C. Koch.) to determine its flavonoid composition. Additionally, we evaluated the hepatoprotective properties of its extract by examining the effect on the histological structure of the liver in an experimental model of toxic cirrhosis.

MATERIALS AND METHODS:

Reagent and chemicals:

CCl₄, hematoxylin, eosin, ethyl alcohol, and other reagents were purchased from Sigma-Aldrich.

Plant material:

The dried extract of Helichrysum rubicundum (C. Koch.) flowers, which were grown in Armenia, was provided by the Laboratory of Medical Herbs Chemistry at STCOPC NAS RA. The dried powder is conditionally referred to as FLR^24,25.

Determination of flavonoids:

UHPLC ESI-QToF (Waters UHPLC Xevo G3 QToF) system was used to analyze flavonoids extracted from Helichrysum rubicundum (C.Koch.). For sample preparation, 1mL of a working solution containing a 50:50 (v/v) mixture of deionized water and acetonitrile was added to 50mg of FLR and vortexed for 2 minutes. The mixture underwent ultrasonication followed by centrifugation at 10,000rpm, with each step lasting 10 minutes. Subsequently, 20µL of the supernatant was diluted with 1mL of the working solution. The final injected concentration was 1µg/µL, while the injection volume was 5µL for both the working solution and the sample.

Chromatographic separation was performed by C18 column (1.7µm, 2.1 × 50mm) maintained at 30°C. The mobile phase included two elements: (A) deionized water and (B) acetonitrile. Details of the gradient program are provided in the supplementary materials. The analysis was carried out over 25 minutes with a flow rate of 0.3mL/min. The source and desolvation gas temperatures were set to 120°C and 450°C, respectively. A capillary voltage of 3.0kV was applied, with cone gas and desolvation gas flow rates of 50L/h and 900L/h. The analysis was performed in negative ion mode²⁶.

Raw data were acquired using Waters MassLynx software and processed with the open-source software MZmine.

Evaluation of hepatoprotective Activity:

Ethical considerations:

Throughout the animal handling process, ethical principles were followed in accordance with the approval of the Institutional Animal Care and Use Committee of the IMB, NAS RA. They were placed in a room at 21±2°C, and 12hours’ diurnal cycle with humidity levels 40–43%. Food and water were given ad libitum. Three animals of the same sex were placed in cages of 1600cm².

Design of Experiment:

The hepatoprotective activity of the FLR extract was evaluated using a CCl₄-induced hepatic damage model. The experiment was conducted on 18 outbreed male albino rats. The animals weighing 120-150g underwent 10-day quarantine before the experiment. The animals were split into three groups, each containing six rats. Group 1 consisted of normal untreated animals, Groups 2 and 3 received intraperitoneal injections of 1ml/kg of 10% CCl₄ in olive oil three times weekly over 12 weeks to induce liver cirrhosis^27,28. Additionally, Group 3 was given 300mg/kg of FLR in olive oil orally in parallel. On the final day of the experiments, the animals were sacrificed (intraperitoneal injection of 40mg/kg Nembutal Sodium) and the livers were excised, followed by fixation in 10% buffered formalin.

For histopathological investigations after the fixation liver tissues were processed, sectioned at 3–5μm with a microtome, and stained with H&E²⁹. Photomicrographs were obtained at 100 x, 400x magnification via AmScope MU500 5MP USB2.0 Microscope Digital Camera & Software (USA)^30,31.

To assess the effects of FLR on CCl₄-intoxicated rats, various liver tissue parameters were graded. These included hepatocyte necrosis, fatty changes, fibrosis, portal tract inflammation, portal vein congestion, and sinusoidal dilatation. Grading was performed using the following scale: 0–1 for no or minimal changes, 2–3 for moderate changes, and 4–5 for severe changes^31,32.

Statistical Analysis:

Data were analyzed statistically using IBM SPSS, applying one-way analysis of variance (ANOVA) with Bonferroni test for multiple comparisons.

RESULTS:

Determination of flavonoids:

This study focused on assessing the composition of the dried extract (FLR) from Helichrysum rubicundum (C. Koch.) flowers, one of the less explored subspecies of the Helichrysum genus. Research has identified 22 polyphenolic compounds in the dried extract of Helichrysum rubicundum flowers, as shown in Figure 1 and Table 1 (Supplementary Material https://www.ebi.ac.uk/biostudies/studies/S-BSST2134). Our findings indicate that, apart from alpha-pyrone and chalcone derivatives, the extract contains nearly all classes of flavonoids (Table 1).

Figure 1. UHPLC ESI- QTOF MS spectrum: A. Blanc working solution chromatogram and B. Total Ion Chromatogram of FLR.

Table 1. Determination of flavonoids components in FLR.

S. No	Flavonoid	Chemical formula	Theoretical M/z (negative mode)	Found M/z (negative mode)	Retention time (minute)
1	Diosmin	C28H32O15	607.1663	607.1714	1.12
2	Myricetin	C15H10O8	317.0298	317.0320	8.96
3	Salipurposide	C21H22O10	433.1135	433.1149	9.39
4	Myricitrin	C21H20O12	463.0876	463.0889	9.53
5	Taxifolin	C15H12O7	303.0505	303.0533	9.79
6	Rutin	C27H30O16	609.1456	609.1446	9.82
7	Eriodictyol	C15H12O6	287.0556	287.0582	10.45
8	Scopoletin	C10H8O4	191.0344	191.0380	10.80
9	Isosalipurposide	C21H22O10	433.1135	433.1150	11.39
10	Quercetin	C15H10O7	301.0348	301.0371	12.05
11	Tiliroside	C30H26O13	593.1295	593.1286	12.31
12	Rhoifolin	C27H30O14	577.1557	577.1555	12.46
13	Hesperetin	C16H14O6	301.0712	301.0732	13.06
14	Diosmetin	C16H12O6	299.0556	299.0582	13.09
15	Kaempferol	C15H10O6	285.0399	285.0427	13.09
16	Apigenin	C15H10O5	269.0450	269.0480	13.14
17	Naringenin	C15H12O5	271.0606	271.0638	13.20
18	Rhamnetin	C16H12O7	315.0505	315.0532	13.24
19	Tangeretin	C20H20O7	371.1131	371.1146	13.33
20	Chrysin	C15H10O4	253.0501	253.0526	14.53
21	Pinocembrin	C15H12O4	255.0657	255.0688	14.75
22	Arzanol	C22H26O7	401.1600	401.1616	19.48

DISCUSSION:

Plants are raw materials for the development of new natural products and their derivatives, offering a vast range of possibilities for drug discovery. Among that, flavonoids are natural substances synthesized in several parts of plants that exhibit a high antioxidant capacity.

Structurally, flavonoids are divided into six major groups: flavan-3-ols, flavones, flavonols, flavanones, isoflavones, and anthocyanins. Their wide range of biological and pharmacological effects has made them a focal point of study for scientists in various disciplines³³. Research has demonstrated that the biological effects of flavonoids stem from their ability to regulate redox processes, stabilize cell membranes, and modulate enzyme and receptor activity^7,34.

Helichrysum has been used medicinally since ancient times, and it contains a range of flavonoids that are responsible for its various biological activities. Extensive literature exists detailing the flavonoid composition and biological effects of various Helichrysum species ^20,23,35-37.

It can be assumed that studying the flavonoid composition of new Helichrysum species opens up a wide range of possibilities for both novel herbal medicine sources and synthetic or semi-synthetic products.

This study evaluated the composition of the dried extract (FLR) obtained from the flowers of Helichrysum rubicundum (C. Koch.), one of the least researched subspecies of Helichrysum. Our results confirm that the extract contains a wide range of flavonoids (Fig. 3), including their glycosides: rhoifolin, tiliroside, diosmin, rutin, salipurposide, myricetin, and isosalipurposide.

Figure 3. Different subclasses of flavonoids identified in FLR extracted from H. Rubicundum

The liver, as the primary organ for metabolism and excretion, is continuously responsible for detoxifying xenobiotics, environmental pollutants, and chemotherapeutic agents. As a result, liver-related disorders are numerous and varied in nature³⁸. Liver diseases represent a major worldwide health concern, highlighting the need for new medicines to counteract or prevent their progression. Consequently, research efforts have focused on identifying natural and synthetic compounds with hepatoprotective properties³⁹. Various medicines are available for treating liver diseases; however, they often lead to complications and unwanted side effects. To mitigate these issues, herbal medicines derived from plants offer a more effective alternative with minimal or no adverse effects⁴⁰. Numerous plants and compounds have been recognized for their hepatoprotective properties. The development of plant-based hepatoprotective drugs has gained significant importance in the global market⁴¹.

Animal models are frequently utilized to replicate and study the mechanisms of human diseases⁴². One of the most commonly used models for inducing toxic cirrhosis in animals is the CCl₄ model. It has well-established effects on the liver, such as severe inflammation and periportal and septal fibrosis^43,44. This model is frequently employed to evaluate the hepatoprotective potential of plant-derived extracts^45,46.

In this study, we also used carbon tetrachloride to simulate toxic liver cirrhosis. The toxin altered histological structure of the liver, causing pathological features characteristic of cirrhosis: there were regenerative nodules surrounded by fibrous connective tissue which formed bridges connecting the portal tracts. In the group treated with FLR simultaneously with intoxication, the observed pathological changes were mild or absent (Fig. 2).

Our study suggests that the preservation of liver tissue structure and the lower morphometric parameter values in the FLR group, compared to the cirrhotic group, indicate that FLR mitigates the toxic effects of carbon tetrachloride (Fig. 2, Table 2). The obtained data allow us to conclude that the antifibrotic, anti-inflammatory, and prominent hepatoprotective activities of Helichrysum rubicundum are related to its diverse flavonoid content, which includes both aglycones and glycosides (Fig. 3).

Research increasingly indicates that free radicals play a key role in hepatic fibrosis by acting on multiple cell types and signaling pathways. This has led to the recognition of flavonoids, naturally present in plant-based sources, as effective antifibrotic agents⁴⁷. Flavanoids antiradical properties inhibit chronic inflammatory processes and prevent the development of fibrosis^48,49. Studies have shown that although the antioxidant activity of aglycones is much higher compared to their glycosides, their unstable structures reduce bioavailability. In contrast, glycosylated flavonoids improve stability, helps maintain antioxidant activity after digestion, reduces toxic side effects, and enhances specific targeting^50,51.

Thus, antifibrotic effect may be attributed not only to the active aglycones of flavonoids contained in FLR but also to the diverse glycosylated flavonoids.

CONCLUSION:

Helichrysum rubicundum (C.Koch.) specie of genus Helichrysum has anti-inflammatory, antifibrotic and strong hepatoprotective properties due to its rich flavonoid composition. Also, it can be a raw material for the discovery of new hepatoprotectors, which require more in-depth and comprehensive studies.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS:

The authors acknowledge Dr. Hrach Ananikyan for the extraction and provision of the dried extract of Helichrysum rubicundum (C. Koch.).

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Received on 15.02.2025 Revised on 04.04.2025

Accepted on 16.08.2025 Published on 10.02.2026

Available online from February 16, 2026

Research J. Pharmacy and Technology. 2026;19(2):631-637.

DOI: 10.52711/0974-360X.2026.00092

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

	Hepatocytes necrosis	Fatty changes	Inflammation in portal tract	Fibrosis	Sinusoidal dilation	Congestion of central veins
Intact	0.00 ± 0.000	0.80 ±0.447	0.00 ± 0.000	0.00 ± 0.000	0.40 ± 0,548	0.40 ±0.548
CCl₄	4.40 ± 0.548	4.60 ± 0.548	4.00 ± 0.707	4.00 ± 0.707	3.40 ± 0.894	3.80 ± 0.837
CCl₄+ FLR	2.20 ± 0.837^*	3.00 ± 0.707^*	2.40 ± 0.548^*	1.20 ± 0.447^*	1.40 ± 0.548^*	2.00 ± 1.000*