Phytochemical Screening and Antioxidant Activity of Selected Estonian Galium Species.

The aim of the present study was to examine three different Galium species from the native population of Estonia, Galium verum, Galium aparine, and Galium mollugo, to characterise their non-volatile and volatile phytochemical composition and antioxidant activity. The main groups of bioactive compounds in the plants were quantified by colorimetric tests, showing high concentrations of polyphenols (up to 27.2 ± 1.5 mg GAE/g), flavonoids (up to 7.3 ± 0.5 mg QE/g) and iridoids (up to 40.8 ± 2.9 mg AE/g). The species were compared using HPLC-DAD-MS/MS, revealing some key differences in the phytochemical makeup of the extracts. The most abundant compound in the extracts of Galium verum blossoms and herb was found to be asperuloside, in Galium aparine herb, asperulosidic acid, and in Galium mollugo herb, chlorogenic acid. Additionally, the composition of volatile compounds was analysed by SPME-GC-MS. The degree of variability between the samples was high, but three volatiles, hexanal, anethole, and ß-caryophyllene, were quantified (≥1%) in all analysed samples. The antioxidative activity of all extracts was evaluated using the ORACFL method, demonstrating that the Galium species from Estonia all exhibit strong antioxidant capacity (up to 9.3 ± 1.2 mg TE/g). Out of the extracts studied, Galium verum blossoms contained the highest amounts of bioactives and had the strongest antioxidant capacity.

Immunomodulatory Activity and Phytochemical Profile of Infusions from Cleavers Herb.

Extracts from aerial parts of G. aparine (cleavers) constitute a herbal remedy with monography in British Herbal Pharmacopeia. On the European market, there are several drugs and food supplements consisting of Galium extracts. In folk medicine, cleavers was used topically in Europe, Asia, and the Americas to treat skin diseases. In several remedies, cleavers is also listed as an immunomodulatory active herb influencing the defense response of the human body. The aim of this study was to investigate the immunostimulatory activity and antioxidant potential in vitro of a raw infusion of cleavers and bioactive fractions. The functional activity of lymphocytes in the reaction of the lymphocyte blast transformation (RLBT) method was used for immunomodulatory activity assays and direct scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and hydrogen peroxide (H2O2) was chosen for the examination of antioxidant activity. It was shown that both the raw extract and fractions show significant immunostimulatory and scavenging activities. The obtained data partially justify the traditional use of cleavers as topical remedy for skin infections and for wounds.

Separation and purification and in vitro anti-proliferative activity of leukemia cell K562 of Galium aparine L. petroleum ether phase.

To explore material basis of in vitro anti-proliferative activity of leukemia cell K562 of petroleum ether phase of product resulting from Galium aparine L. 60% ethanol extraction, the experiment adopts column chromatography combined with thin layer preparation, isolates and purifies petroleum ether, conducts structural identification of obtained single compound and applies MTT method for viability assay of in vitro anti-proliferative activity of leukemia cell K562. Experimental results show that G. aparine L. petroleum ether contains mainly ß-sitosterol, daucosterol and dibutyl phthalate and other substances. Under experimental conditions, the three could inhibit the proliferation of leukemia cell K562 with dose-effect and time-effect relationship, of which dibutyl phthalate has strongest activity. Dibutyl phthalate with excellent activity, ß-sitosterol with rich content and moderate effect should be the main contributor to its biological activity.

Comparative study on mechanical properties and biomineralization of hooks in the diaspores of three epizoochorous plant species.

Most of the plants using epizoochory show adaptations to this diaspore dispersal strategy by having the diaspores covered by barbs, hooks, spines or viscid outgrowths, which allow diaspores to easily attach to an animal surface. Many previous studies have been mainly focused on the dispersal distances and efficiency, or effectiveness of diverse attachment structures depending on their size, anatomy, and morphology. However, the knowledge about the mechanical properties of these structures remains rather poor. In this study, we use a combination of scanning electron microscopy, energy dispersive X-ray element analysis and nanoindentation, to examine the microstructure, biomineralization and mechanical properties of single hooks in Arctium minus, Cynoglossum officinale and Galium aparine. Both the biomineralization and mechanical properties of the hooks strongly differ in examined plant species; mechanical properties depend on the biomineralization pattern, such as the accumulation of silicon and calcium. Elastic modulus and hardness decrease in the series C. officinaleG. aparineA. minus. Anisotropic mechanical properties are found between the radial and longitudinal directions in each single hook. By characterizing the mechanical properties and biomineralization of plant hooks, this paper contributes to the understanding of attachment biomechanics related to seed dispersal. STATEMENT OF SIGNIFICANCE: The dispersal of seeds is essential for plant survival. Many of the plants that use the outside surface of animals to transport the seeds show adaptations to this dispersal strategy by having the seeds covered with hooks. Although these hooks have various sizes, morphologies and anatomical structures, all of them provide mechanical interlocking to animal surfaces. To reduce the risk of interlocking failure, the hooks are usually reinforced by mineralization. However, the relationship between mineralization, mechanical properties and specialized function of plant hooks has been largely overlooked. Here we perform a characterization study on the hooks of three plant species. Our results deepen the current understanding of the mineralization-material-function relationship in specialized hooks of plant seeds.

Immuno-Enhancing Effects of Galium aparine L. in Cyclophosphamide-Induced Immunosuppressed Animal Models.

This study investigates the immunomodulatory potential of Galium aparine L. (GAE) in immunodeficient animals. In this study, animals were categorized into five groups: the normal group, CYP group (cyclophosphamide intraperitoneal injection), GA5 group (cyclophosphamide + 5 µg GAE), GA50 group (cyclophosphamide + 50 µg GAE), and GA500 group (cyclophosphamide + 500 µg GAE). The CYP group exhibited significantly reduced spleen weights compared to the normal group, while the groups obtaining GAE displayed a dose-dependent increase in spleen weight. Furthermore, the GAE demonstrated dose-dependent enhancement of splenocyte proliferating activity, with significant increases observed in both LPS and ConA-induced assays. NK cell activity significantly increased in the GA50 and GA500 groups compared to the CYP group. Cytokine analysis revealed a significant increase in IL-6, TNF-α, and IFN-γ levels in ConA-induced splenocytes treated with GAE. Gene expression analysis identified 2434 DEG genes in the extract groups. Notable genes, such as Entpd1, Pgf, Thdb, Syt7, Sqor, and Rsc1al, displayed substantial differences in individual gene expression levels, suggesting their potential as target genes for immune enhancement. In conclusion, Galium aparine L. extract exhibits immunomodulatory properties. The observed gene expression changes further support the potential of Galium aparine L. extract as a natural agent for immune augmentation.

Bringing back Galium aparine L. from forgotten corners of traditional wound treatment procedures: an antimicrobial, antioxidant, and in-vitro wound healing assay along with HPTLC fingerprinting study.

BACKGROUND: The wound healing process, restoring the functionality of the damaged tissue, can be accelerated by various compounds. The recent experimental analysis highlights the beneficial effects of phytochemicals in improving skin regeneration and wound healing. In traditional medicine, one of the widespread plants used for treating different injuries or skin afflictions is Galium aparine L. (GA). Besides, previously reported chemical compounds of GA suggested its therapeutic effects for the wound healing process, yet its regulatory effects on the cellular and molecular stages of the wound healing process have not been investigated. METHODS: In the present study, the phytochemical profile of the GA extract was analyzed using HPTLC fingerprinting, and further scientific evaluation of its phytochemicals was done. The wound-healing effects of GA extract were explored at the cellular and molecular levels while accounting for cell toxicity. The wound closure enhancing effect, antibacterial activity, and antioxidant activity were assessed. RESULTS: The HPTLC fingerprinting of the GA extract proved its previously reported phytochemical profile including phenols, flavonoids, tannins, plant acids, ergot alkaloids, flavonoids, anthraquinones, terpenoids, sterols, salicin, lipophilic compounds, saponins, iridoids, and heterocyclic nitrogen compounds. Antimicrobial assessment, of the extract, indicated the more susceptibility of S. aureus to the inhibitory effects of GA rather than E. coli and S. epidermidis. DPPH test results revealed the antioxidant property of GA extract, which was comparable to ascorbic acid. The results of the viability assay showed no cytotoxicity effects on human umbilical endothelial cell (HUVEC) and normal human dermal fibroblast (NHDF) cell lines treated with different concentrations of whole plant extract and cell viability increased in a dose-dependent manner. The results of the scratch assay showed improved cell migration and wound closure. CONCLUSIONS: This study shows the anti-oxidant, anti-microbial, and in vitro wound healing wound-healing effects of GA hydroalcoholic extract, which aligns with its use in traditional medicine. No cytotoxicity effects were shown. The results from this study can be the basis for further investigations such as animal models and phytochemical studies. Further evaluation of its effects on mechanisms and signaling pathways involved in the wound healing processes such as angiogenesis and cell proliferation can provide novel insights into the potential therapeutic effects of the GA extract.

Galium aparine L. protects against acetaminophen-induced hepatotoxicity in rats.

The toxicity of acetaminophen (N-acetyl-para-aminophenol (APAP)) is the most frequent cause of drug-induced liver damage. Galium aparine L. (GA) is traditionally used to treat jaundice. We aimed to investigate the hepatoprotective potential of GA in the APAP-induced hepatic encephalopathy (HE) rat model. Qualitative phytochemical characterization of GA was performed by LC/Q-TOF/MS analysis. Wistar rats were pretreated with GA (250 and 500 mg/kg b.wt. per oral) for five days. On the 6th day, the rats were exposed to APAP (1500 mg/kg b.wt. oral gavage) and behavioral tests (open field and passive avoidance tests) were applied on the 7th and 8th days. The animals were killed, and biochemical and histopathological parameters were assessed in blood and hepatic specimens. GA pretreated rats exhibited a significant reduction in APAP-induced liver damage, evidenced by the reduction in liver necrosis and alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin (BIL). GA demonstrated an anxiolytic effect, as seen in the acquisition trial and grooming behavior. The short-term memory performances of animals were not changed in all groups, suggesting that APAP intoxication did not affect hippocampal function. These results show that GA extract markedly exerts hepatoprotective activity, while its effect on hepatic encephalopathy was limited.

Phytochemical Profiles and In Vitro Immunomodulatory Activity of Ethanolic Extracts from Galium aparine L.

Galium aparine L., family Rubiaceae, is a widely spread species in the Galium genus. The herb of G. aparine is part of folk remedies and dietary supplements. In this study, we analyzed the chemical composition and immunomodulatory activities of G. aparine herb ethanolic extracts obtained from the plant material by maceration with 20%, 60% or 96% ethanol. The contents of hydroxycinnamic acid derivatives, flavonoids and polyphenols were determined spectrophotometrically, with extractives and polysaccharides quantified gravimetrically. The qualitative composition was studied using UHPLC-DAD-MS/MS analysis; isolation not previously described in G. aparine quercetin rhamnoglucoside was carried out through column chromatography, and the immunomodulatory activity of extracts was determined in the reaction of lymphocyte blast transformation. Major constitutes of extracts were iridoids, i.e., monotropein, 10-desacetylasperulosidic acid and asperulosidic acid; p-hydroxybenzoic acid; hydroxycinnamic acid derivatives, i.e., 3-O-caffeoylquinic, 5-O-caffeoylquinic, 3,4-O-dicaffeoylquinic, 3,5-O-dicaffeoylquinic, 4,5-O-dicaffeoylquinic acids and caffeic acid derivatives; flavonoids, i.e., rutin, quercetin 3-O-rhamnoglucoside-7-O-glucoside, and isorhamnetin 3-O-glucorhamnoside. Significantly, quercetin 3-O-rhamnoglucoside-7-O-glucoside was first isolated and identified in Galium species so far investigated. All G. aparine herb ethanolic extracts stimulate the transformational activity of immunocompetent blood cells, with 96% ethanolic extract being the most active. The data obtained necessitate further research into the mechanisms of immunomodulatory activity of extracts from G. aparine herb.

Effects of Galium aparine extract on the cell viability, cell cycle and cell death in breast cancer cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Galium species have been traditionally used for its anti-cancer, antioxidant, anti-inflammatory, antimicrobial and cardioprotective effects in the folk medicine. Galium aparine (GA) is a typical climbing plant growing widespread in Anatolia. AIM OF THE STUDY: To investigate the potential anti-proliferative and apoptotic effect of GA methanol (MeOH) extract on MCF-7 and MDA-MB-231 human breast cancer cells and MCF-10A untransformed breast epithelial cells. MATERIALS AND METHODS: First, the extract was characterized by both liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS) and gas chromatography-mass spectrometry (GC-MS) analyses. Then, cell viability and cell cycle distribution were investigated by XTT assay and PI staining by flow cytometry, respectively. Cell death was determined by Annexin V FITC/7-AAD staining. RESULTS: A total of 14 major phytochemicals were identified by LC/Q-TOF/MS and 34 volatile compounds were determined by GC-MS. The extract was cytotoxic in both breast cancer cell lines in a concentration and time dependent manner and showed G1 block after 72h extract treatment. However, it was not cytotoxic to MCF-10A breast epithelial cells. Flow cytometry analyses revealed that apoptosis was induced in MDA-MB-231 cells; however, necrosis was induced in MCF-7 cells. CONCLUSION: Our study suggests that GA MeOH extract may have potential anti-cancer effects against breast cancer cells without impairing normal breast epithelial cells. Ability to induction of non-apoptotic cell death besides apoptotic cell death by this complex plant-derived mixture may enable the killing of apoptosis resistant breast cancer cells but further studies should be conducted to investigate the bioavailability and metabolism of it in vivo.

Use of herbal supplements for overactive bladder.

Anticholinergics, specifically antimuscarinic agents, are the most common medications prescribed for overactive bladder (OAB). The most common side effects of these agents are dry mouth and constipation, although other more concerning effects include changes in blood pressure, pulse rate, or heart rhythm when treatment is initiated. Herbal treatments are an increasingly popular alternative for treating OAB. A 2002 survey of US adults aged ≥ 18 years conducted by the Centers for Disease Control and Prevention indicated that 74.6% of those with OAB had used some form of complementary and alternative medicine. The World Health Organization estimates that 80% of the world's population presently uses herbal medicine for some aspect of primary health care. Women were more likely than men to use complementary and alternative medicine. The authors review the most commonly used herbal medications for OAB.