Background: Globally, research on identifying bioactive compounds from medicinal plants and characterizing their in vitro and in vivo pharmacological activities is progressing rapidly. Plants native to Mongolia have adapted to unique, extreme continental climatic conditions, accumulating distinct phytochemicals, and have historically been utilized in traditional medicine and dietary practices. Establishing the non-cytotoxic safety threshold of these wild and cultivated plant extracts in RBL-2H3 mast cells a universally accepted model for type I hypersensitivity and inflammation is a vital prerequisite. Determining these baseline non-toxic dose ranges provides the essential scientific foundation for subsequent cellular allergy models, animal studies, and the elucidation of molecular mechanisms underlying their therapeutic efficacy. Aim: To determine the cytotoxicity profile and establish the non-cytotoxic dose ranges of extracts from four selected Mongolian plant species (wild and cultivated) in RBL-2H3 cells. Materials and Methods: Plant samples including lingonberry (Vaccinium vitis-idaea), strawberry (Fragaria orientalis), black radish (Raphanus sativus), and blue-berried honeysuckle (Lonicera caerulea L.) were standardly collected, authenticated, and cataloged as herbarium specimens at the Botanic Garden and Research Institute, Mongolian Academy of Sciences. A total of 12 distinct extracts were prepared from plant leaves, stems, and fruits using water and ethanol extraction, followed by concentration via rotary evaporation and freeze-drying. Rat basophilic leukemia (RBL-2H3) cells were maintained in EMEM growth medium and utilized during the exponential growth phase. Cell viability and non-cytotoxic concentration thresholds were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Result: Ethanolic extracts of strawberry leaves, black radish, and honeysuckle leaves (≥6.25 µg/mL), as well as aqueous extracts of honeysuckle fruits (≥6.25 µg/mL) and stems (≥25 µg/mL), exhibited dose-dependent cytotoxicity. Conversely, aqueous extracts of lingonberry leaves (≤200 µg/mL), strawberry leaves (≥100 µg/mL), and black radish (≤200 µg/mL), alongside ethanolic extracts of lingonberry leaves (≥50 µg/mL), demonstrated non-cytotoxic profiles. Furthermore, ethanolic extracts of honeysuckle stems (≤300 µg/mL) and fruits (≥12.5 µg/mL), as well as the aqueous extract of honeysuckle leaves (≤50 µg/mL), significantly enhanced RBL-2H3 cell viability. Conclusion The tested plant extracts influenced RBL-2H3 cell viability in a solvent polarity and plant organ dependent manner. Ethanolic extracts of strawberry leaves, black radish, and honeysuckle leaves, along with aqueous extracts of honeysuckle fruits and stems, demonstrated relatively high cytotoxicity, reducing cell viability by >40% starting at concentrations of 12.5, 25, and 50 µg/mL. In contrast, aqueous extracts of strawberry leaves and black radish, as well as both aqueous and ethanolic extracts of lingonberry leaves, maintained low cytotoxicity at concentrations below 200 µg/mL. Notably, ethanolic extracts of honeysuckle stems and fruits (>12.5 µg/mL) and its aqueous leaf extract (<100 µg/mL) enhanced cell viability, exhibiting significant cell proliferation promoting activity.

Background: Evaluation of genotoxic potential is essential for reducing carcinogenic risk and for assessing the safety of newly developed pharmaceutical products, traditional herbal medicines, and health supplements. Such evaluation plays an important role in protecting human health. Therefore, in Mongolia, it is necessary to investigate the safety of domestically produced pharmaceutical preparations, natural products, and traditional medicines on a scientific basis in accordance with international standards; to develop and implement national policies and standards; to introduce and localize appropriate methodologies; to protect human health; to prevent cancer, hereditary diseases, and longterm adverse effects; and to promote the rational use of medicines. Aim: To investigate the mutagenic properties of an herbal combination preparation in vitro and its genotoxic effects in vivo. Materials and Methods: Extracts were prepared from Dasiphora fruticosa, Cynara scolymus, and Rosa acicularis, and a herbal combination preparation was formulated in specific proportions. Mutagenicity was evaluated according to OECD Guideline 471 (Bacterial Reverse Mutation Test) using the Salmonella typhimurium TA100 strain under both S9 (-) and S9 (+) conditions with the Muta-Chromoplate™ assay. The combination preparation was tested at concentrations of 5, 2.5, 1.25, 0.625, 0.3125, and 0.1562 mg/mL in 96 wells, and reverse-mutated bacterial colonies were assessed by counting the number of wells that changed color from purple to yellow. In addition, using the IPHASE Biosciences kit, the preparation was tested at doses of 5, 2.5, 1.25, 0.625, 0.3125, and 0.1562 mg/plate on Salmonella typhimurium TA100 and TA98 strains under both S9 (-) and S9 (+) conditions. After incubation, the number of bacterial colonies formed was counted and compared with that of the positive control group. Genotoxicity was assessed according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) by evaluating the numbers of polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs), and micronucleated polychromatic erythrocytes in the bone marrow of BALB/c mice. Result: In the study using the Muta-Chromoplate™ assay, bacterial colonies were observed at doses ranging from 5.0 to 0.1562 mg/mL of the herbal combination preparation. However, compared with the positive control group (aminoanthracene), the number of bacterial colonies was significantly lower (p<0.05). In the study using the IPHASE Biosciences kit, 30–154 bacterial colonies were observed in Salmonella typhimurium TA98 and TA100 strains under S9 (-/+) conditions at doses ranging from 5.0 to 0.156 mg/plate, which was approximately threefold lower than that observed in the positive control groups. In the positive control group treated with cyclophosphamide, the number of micronucleated polychromatic erythrocytes was increased. Conclusion The herbal combination preparation showed no mutagenic activity in either of the two mutation detection assays using Salmonella typhimurium TA98 and TA100 strains under S9 (-/+) conditions. In addition, it showed no genotoxic effect in the micronucleus assay.

Background: Stress, insomnia, and anxiety are among the most prevalent mental health disorders in society and are associated with negative consequences such as reduced work capacity, decreased quality of life, and an increased risk of chronic diseases worldwide. During stress, the sympathetic nervous system becomes activated, leading to physiological changes such as increased heart rate, vasoconstriction, and elevated arterial blood pressure. If prolonged, these changes may increase the risk of developing hypertension and cardiovascular diseases. In recent years, pharmacological research on medicinal plants with sedative, hypnotic, and anxiolytic effects has been intensively conducted. Therefore, there is a growing need to develop a combined herbal preparation from Valeriana officinalis L. and Leonurus sibiricus L., which have been used in traditional medicine for sedative, hypnotic, and anxiolytic effects, and to investigate their effects on arterial blood pressure. Aim: To investigate the effect of an herbal complex preparation on arterial blood pressure. Materials and Methods: An herbal complex preparation was formulated from 70% ethanol extracts of Valeriana officinalis L. and Leonurus sibiricus L. in a 30:70 ratio. Its effects on normal blood pressure and Angiotensin II-induced hypertension were evaluated in C57BL/6 mice. The preparation was administered orally at doses of 750 and 1000 mg/kg. Systolic and diastolic blood pressure were measured using a Softron BP-98AWU system and compared between groups. Results: At doses of 750 and 1000 mg/kg, the complex preparation reduced systolic blood pressure (SBP) by 6.4-12.0% and diastolic blood pressure (DBP) by 16.1-28.1% under normal conditions (p<0.05). In the intraperitoneal Angiotensin II-induced hypertension model, SBP decreased by 5.9-19.6% and DBP by 6.3-20.1% (p<0.05). In the osmotic pump-induced Angiotensin II hypertension model, the 750 mg/kg dose reduced SBP by 18.2-33% and DBP by 17.2-28.3% (p<0.001). Conclusion The herbal complex preparation at doses of 750 and 1000 mg/kg demonstrated a significant antihypertensive effect in both normal and Angiotensin II-induced hypertensive mice.

Background: In recent years, natural compounds have been shown to play an important role in cancer prevention and early-stage therapy. Flavanones, a class of polyphenolic compounds present in plants, vegetables, seeds, fruit peels, and flowers, have been identified to possess anticancer, antioxidant, anti-inflammatory, and antibacterial bioactivities. Cancer has become a major global economic and public health challenge. According to international statistics, one in four individuals (22.8%) die from non-communicable diseases, while one in six (16.8%) die from cancer. Aim: To investigate the effect of a flavanone (5,2’,3’-trihydroxy-6,7-methylenedioxyflavanone) isolated from Iris tenuifolia on the migration of liver cancer cells. Materials and Methods: The study was conducted at the Institute of Biomedicine, Mongolian National University of Medical Sciences. The effect of the flavanone (5,2’,3’-trihydroxy-6,7-methylenedioxyflavanone) on cancer cell migration was evaluated in vitro using the scratch assay. Human liver cancer cells (HepG2) were revived and stabilized before experiments were performed. Results: HepG2 cells were treated with a 2.5 μg/mL concentration of the flavanone for 24 hours. In the control group, an initial scratch width of 0.9 mm was reduced to 0.65 mm after 24 hours due to cell proliferation and migration into the denuded area. However, treatment with the 2.5 μg/mL flavanone concentration resulted in the complete inhibition of cell migration. Conclusion The flavanone isolated from Iris tenuifolia effectively suppresses the migration of liver cancer cells.

Background: Insects are an inexhaustible source of medicine, and it is essential to study the biologically active substances contained in insect bodies. Although there is information about various insects and beetles used in traditional Mongolian medicine treatment, there are few studies that have compiled and compared these at the source level. Aim: To study and determine from primary sources information about the composition of pheromone secretions contained in the bodies of various insects and worms used in “Entomotherapy (Insect therapy)” in traditional medicine, particularly rare beetle species, and information about what diseases and ailments these insects were used to treat. Materials and Methods: This study used primary source and historical research methods, analysis-synthesis methods, systematic analytical methods, and historical comparative methods. Information was extracted from traditional medicine source books about insect therapy, particularly identifying and compiling primary sources about the use of beetles in folk medical treatment. Results: According to primary sources, traditional Mongolian medicine used quite a variety of “therapeutic insects and worms” of animal origin, including representatives of arthropods such as scorpions, spiders, centipedes, wasps, bees, various types of beetles, and moths. Among these, stag beetles, dung beetles, and black beetles were used in traditional medicine for treating joint and muscle diseases, various women’s diseases, epilepsy and convulsions, headaches, and all 11 types of bile diseases, with specific prescriptions developed for each condition. Conclusion Traditional medicine treatment used various insects from the biological classification of the insect class, particularly many types of beetles from the Coleoptera order. The pheromone secretions contained in their bodies include many different types of chemical substances.

Background: HepG2 cells are widely used as an in vitro model for investigating alterations in hepatic cellular metabolism. Studies evaluating the biological activities of plant-derived compounds using HepG2 cells have also increased. Strawberry (Fragaria orientalis), lingonberry (Vaccinium vitis-idaea), radish (Raphanus sativus), and blue honeysuckle (Lonicera caerulea L.) contain polyphenols, flavonoids, anthocyanins, and other secondary metabolites. The biological activities of these plants may vary depending on species, plant tissue, growing conditions, and extraction methods. Prior to evaluating the biological activities of plant extracts, it is essential to assess their cytotoxic effects on cells and determine appropriate concentrations for subsequent experiments. Therefore, evaluating the effects of plant extracts on the viability of HepG2 cells, a human hepatocellular carcinoma cell line, is of considerable importance. Aim: To evaluate the effects of aqueous and ethanolic extracts of strawberry, lingonberry, radish, and cultivated and wild blue honeysuckle on HepG2 cell viability and to determine concentrations exhibiting cytotoxic effects. Materials and Methods: A total of 18 plant extracts were prepared using water and ethanol as extraction solvents. The extracts were obtained from strawberry leaves, lingonberry leaves, radish, and the fruits, stems, and leaves of cultivated and wild blue honeysuckle. The extracts were subsequently dried. HepG2 cells maintained in the Cell Biology Laboratory of the Institute of Biomedical Sciences were revived, stabilized, and used for the experiments. The plant extracts were prepared at concentrations of 6.25, 12.5, 25, 50, 100, 200, and 300 μg/mL and used to treat HepG2 cells. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. Cytotoxicity was defined as a reduction in cell viability of more than 30% compared with the control group. Results: The aqueous extract of cultivated blue honeysuckle and the ethanolic extracts of its fruits and stems maintained HepG2 cell viability above 70% across the concentration range of 6.25–300 μg/mL, indicating relatively low cytotoxicity. In contrast, the ethanolic extract of cultivated blue honeysuckle leaves reduced cell viability at higher concentrations. The aqueous and ethanolic extracts of fruits and stems of wild blue honeysuckle showed no apparent cytotoxic effects, whereas both aqueous and ethanolic extracts of its leaves reduced cell viability to approximately 65% at 300 μg/mL, indicating relatively higher cytotoxicity. Most aqueous and ethanolic extracts of strawberry, radish, and lingonberry maintained cell viability above 70% and showed no cytotoxic effects. The ethanolic extract of strawberry leaves reduced cell viability to approximately 63% even at the lowest concentration of 6.25 μg/mL; however, cell viability tended to increase with increasing extract concentration. Conclusion The effects of plant extracts on HepG2 cell viability varied depending on plant species, tissue type, extraction solvent, and concentration. Most extracts of strawberry, lingonberry, radish, and the fruits and stems of cultivated and wild blue honeysuckle exhibited relatively low cytotoxicity at concentrations ranging from 6.25 to 300 μg/mL. However, the ethanolic extract of cultivated blue honeysuckle leaves at 200 μg/mL and the aqueous and ethanolic extracts of wild blue honeysuckle leaves at 300 μg/mL reduced cell viability by more than 30%, indicating relatively higher cytotoxicity.

Background: In recent years, natural compounds have been shown to play an important role in cancer prevention and early-stage therapy. Flavanones, a class of polyphenolic compounds present in plants, vegetables, seeds, fruit peels, and flowers, have been identified to possess anticancer, antioxidant, anti-inflammatory, and antibacterial bioactivities. Cancer has become a major global economic and public health challenge. According to international statistics, one in four individuals (22.8%) die from non-communicable diseases, while one in six (16.8%) die from cancer. Aim: To investigate the effect of flavanone (5,2’,3’-trihydroxy-6,7-methylenedioxyflavanone) isolated from Iris tenuifolia on the migration of lung cancer cells. Materials and Methods: The study was conducted at the Institute of Biomedicine, MNUMS. The effect of flavanone (5,2’,3’-trihydroxy-6,7-methylenedioxyflavanone) on cancer cell migration was evaluated in vitro using the scratch assay. Human lung cancer cells (A549) were revived and stabilized before experiments were performed. Results: We treated A549 cancer cells with different concentrations of flavanone (1.25 μg/ml, 2.5 μg/ml) for 24 hours and analyzed them using the scratch assay. A cell-free gap of 0.9 mm in width was created, and after 24 hours, A549 cells migrated and proliferated into the gap, reducing its width to 0.25 mm. Treatment with 2.5 μg/ml flavanone completely inhibited cell migration. Conclusion Flavanone isolated from Iris tenuifolia inhibits lung cancer cell migration in a doseand time-dependent manner.

Background: In recent years, scientists have found that certain natural compounds have significant potential in cancer prevention and early-stage cancer treatment. Flavanones, a class of polyphenolic compounds found in plants, vegetables, seeds, fruit peels, and flowers, have been identified to possess anticancer, antioxidant, anti- inflammatory, and antibacterial bioactivities. Cancer has become a major global challenge in terms of both economic and public health concerns. Global statistics indicate that 22.8% of deaths are attributed to non-communicable diseases, and 16.8% are caused by cancer, accounting for one in four and one in six deaths, respectively. Aim : To investigate anticancer effects of Iris Tenuifolia-derived flavanone on cancer cell lines. Materials and Methods : The study was conducted at the Bio-Medical Research Institute of the Mongolian National Uni versity of Medical Sciences, investigating the effect of flavanones on cancer cell viability under in vitro conditions using the MTT assay. In the study, colon, liver, and lung cancer cells were cultured, stabilized, and used for the experiments. Colorectal cancer cells (MC38), liver cancer cells (HepG2), and lung cancer cells (A549) were revived, cultured, and stabilized for use in the experimental procedures. Statistical analysis of the results was performed using Microsoft Excel 2010, and graphs were generated using GraphPad Prism 8. Differences between groups were analyzed using Student’s t-test, and a p-value of <0.05 was considered statistically significant. Results : We treated MC38, HepG2, and A549 cancer cells with different concentrations of flavanone (2.5 µM, 5 µM, and 10 µM) for 24 to 48 hours to evaluate cell viability. Flavanone inhibited A549 cell viability by 2.5 μM-10%, 5 μM-25%, and 10 μM-38%, respectively. For HepG2 cells, flavanone treatment at concentrations of 5-10 µM reduced cell viability by 28–58%. No statistically significant effect on the viability of MC38 cells was observed following treatment with flavanone at concentrations ranging from 2.5 to 10 µM. Additionally, although MC38 inhibited cell viability in a dose-de pendent manner in cell cultures, it had a statistically significant effect at higher concentrations of 30-200 μM (p<0.01). Conclusion Flavanone inhibits the cancer cell viability in a dose and time dependent manner

Background: Cancer incidence and mortality are steadily increasing both globally and in Mongolia. As these rates rise, traditional Mongolian medicine has long utilized herbal formulas for the treatment of gastric and esophageal cancers and precancerous conditions. One such formulation—Hot Natured 3 Herbs (HN3H)—comprises three species from the Ranunculaceae family: Atragene sibirica L., Ranunculus repens L., and Pulsatilla bungeana L.. However, scientific validation of its anti-tumor effects is essential. This study aimed to investigate the effect of HN3H in a tumor-induced animal model. Aim: To identify the biologically active compounds of HN3H and evaluate their effect in an experimentally induced tumor model in animals. Materials and Methods: The three herbs comprising HN3H—Atragene sibirica L., Ranunculus repens L., and Pulsatilla bungeana L.—were collected during their flowering stage (May–June) in Khishig-Undur, Bulgan province, and dried according to official procedures. Extraction was carried out by maceration in 96% ethanol at a 1:10 ratio. The concentrated extract was suspended in water (1:1) and successively fractionated with dichloromethane, ethyl acetate, butanol, chloroform, and n-hexane. The study was approved by the Research Ethics Committee of the Mongolian National University of Medical Sciences (Protocol №2020/03-04). A colorectal cancer model was established by subcutaneous injection of MC-38 cells (Kerafast, USA) into C57BL/6 mice. Immunohistochemistry was performed using CK20, CDX2, Ki67, and p53 antibodies at 1:100 and 1:200 dilutions. Results: The ethanol extract of HN3H contained 2.98±0.04% total phenolics and 2.16±0.05% total flavonoids. Body weight and tumor volume were measured daily with three repetitions. All groups showed a time-dependent increase in body weight. Mice in groups 1A and 1B received ethanol extract at 50 and 100 mg/kg doses; groups 2A and 2B received dichloromethane extract at the same doses. The negative control group was administered 0.5 mg/kg PBS orally, while the positive control group received intraperitoneal injections of 5-fluorouracil (5FU) at 10 mg/kg twice a week. Tumor growth increased in a time-dependent manner across groups. Compared to the negative control, tumor volumes in four treatment groups showed statistically significant reduction (p˂0.05), while no significant difference was observed when compared to the positive control (p=0.08). Histological analysis revealed necrosis in all groups, with variation in extent. Conclusion The ethanol extract of HN3H exhibited moderate levels of phenolic compounds and a high concentration of flavonoids. HN3H extract inhibited tumor progression and activated lymphocyte-predominant inflammation in tumor tissues, indicating potential anti-tumor activity (p˂0.05).

Background: According to the World Health Organization (WHO), approximately 70-95% of developing countries rely on traditional medicine, which includes around 365 plant, animal, and mineral-based preparations. Natural products consist of numerous biologically active compounds that exert effects against pathogens, making up about 25% of modern pharmaceuticals derived from plants. Since plants are a combination of various metabolites, they can have therapeutic effects, side effects, and toxicity in the human body. Based on the traditional use of medicinal herbs in Mongolian and Tibetan medicine for their sedative properties, we selected the medicinal herbs Valeriana officinalis L. and Leonurus sibiricus L., The objective is to develop new medicinal preparations that can be utilized in modern medical practice to treat, prevent, or supplement the treatment of depression and anxiety. Consequently, it is necessary to prepare a herbal complex from these selected plants and conduct studies to investigate their subchronic toxicity and sedative activities. Aim: To study subchronic and sedative activity of herbal complex preparations. Materials and Methods: The herbal complex preparation was prepared from the 70% ethanol extract of the roots of Valeriana officinalis L. and the aerial parts of Leonurus sibiricus L., and a compound preparation was made in a 30:70 ratio. Subchronic toxicity study was conducted on Wistar rats weighing 180-250 g according to the OECD-407 guidelines. The sedative activity of herbal complex preparation was studied on C57BL/6 and BALB/c mice using the dark/light transition test according to Takao K., and the hole-board test according to Hiroshi Takeda. Results: In the sub-chronic toxicity study of the herbal complex preparation, biochemical analysis of the serum (including ALT, AST, creatinine, and urea) and histopathological examination of the liver, kidney, and heart showed no statistically significant changes when comparing the experimental groups to the control group. The herbal complex preparation at a dose of 1000 mg/kg increased the time spent in the dark area, decreased the time spent in the light area, and the number of transitions between the two areas of mice in the dark/light transition test, and reduced the number of head-dipping into the holes of mice in the hole-board test. Conclusion The herbal complex preparation exhibited low toxicity at doses of 1000 mg/kg and 1500 mg/kg based on biochemical and histopathological examinations in the subchronic toxicity study. Furthermore, the preparation demonstrated sedative effects at a dose of 1000 mg/kg.