Exploring the Potential Mechanisms of Melilotus officinalis (L.) Pall. in Chronic Muscle Repair Patterns Using Single Cell Receptor-Ligand Marker Analysis and Molecular Dynamics Simulations.

Information regarding the function of Melilotus officinalis (L.) Pall. in skeletal muscles is still unknown. In this study, we explored the possible regulatory targets of M. (L.) Pall. that affects the repair patterns in chronic muscle injury. We analyzed the potential target genes and chemical composition of M. (L.) Pall. and constructed a "drug-component-disease target genes" network analysis. Five active ingredients and 87 corresponding targets were obtained. Muscle-tendon junction (MTJ) cells were used to perform receptor-ligand marker analysis using the CellphoneDB algorithm. Targets of M. (L.) Pall. were screened further for the cellular ligand-receptor protein action on MTJs. Enrichment analysis suggests that those protein-associated ligand receptors may be associated with a range of intercellular signaling pathways. Molecular docking validation was then performed. Five proteins (CCL2, VEGFA, MMP2, MET, and EGFR) may be regulated by the active ingredient luteolin and scoparone. Finally, molecular dynamics simulations revealed that luteolin can stably target binding to MMP2. M. (L.) Pall. influences skeletal muscle repair patterns by affecting the fibroblast interactions in the muscle-tendon junctions through the active ingredients luteolin and scoparone.

Genome and systems biology of Melilotus albus provides insights into coumarins biosynthesis.

Melilotus species are used as green manure and rotation crops worldwide and contain abundant pharmacologically active coumarins. However, there is a paucity of information on its genome and coumarin production and function. Here, we reported a chromosome-scale assembly of Melilotus albus genome with 1.04 Gb in eight chromosomes, containing 71.42% repetitive elements. Long terminal repeat retrotransposon bursts coincided with declining of population sizes during the Quaternary glaciation. Resequencing of 94 accessions enabled insights into genetic diversity, population structure, and introgression. Melilotus officinalis had relatively larger genetic diversity than that of M. albus. The introgression existed between M. officinalis group and M. albus group, and gene flows was from M. albus to M. officinalis. Selection sweep analysis identified candidate genes associated with flower colour and coumarin biosynthesis. Combining genomics, BSA, transcriptomics, metabolomics, and biochemistry, we identified a ß-glucosidase (BGLU) gene cluster contributing to coumarin biosynthesis. MaBGLU1 function was verified by overexpression in M. albus, heterologous expression in Escherichia coli, and substrate feeding, revealing its role in scopoletin (coumarin derivative) production and showing that nonsynonymous variation drives BGLU enzyme activity divergence in Melilotus. Our work will accelerate the understanding of biologically active coumarins and their biosynthetic pathways, and contribute to genomics-enabled Melilotus breeding.

The impact of aqueous and N-hexane extracts of three Fabaceae species on seed germination and seedling growth of some broadleaved weed species.

Weed infestation is a persistent problem for centuries and continues to be major yield reducing issue in modern agriculture. Chemical weed control through herbicides results in numerous ecological, environmental, and health-related issues. Moreover, numerous herbicides have evolved resistance against available herbicides. Plant extracts are regarded as an alternative to herbicides and a good weed management option. The use of plant extracts is environmentally safe and could solve the problem of herbicide resistance. Therefore, laboratory and wire house experiments were conducted to evaluate the phytotoxic potential of three Fabaceae species, i.e., Cassia occidentalis L. (Coffee senna), Sesbania sesban (L.) Merr. (Common sesban) and Melilotus alba Medik. (White sweetclover) against seed germination and seedling growth of some broadleaved weed species. Firstly, N-hexane and aqueous extracts of these species were assessed for their phytotoxic effect against lettuce (Lactuca sativa L.). The extracts found more potent were further tested against germination and seedling growth of four broadleaved weed species, i.e., Parthenium hysterophorus L. (Santa-Maria), Trianthema portulacastrum L. (Pigweed), Melilotus indica L (Indian sweetclover). and Rumex dentatus L. (Toothed dock) in Petri dish and pot experiments. Aqueous extracts of all species were more toxic than their N-hexane forms for seed germination and seedling growth of lettuce; therefore, aqueous extracts were assessed for their phytotoxic potential against four broadleaved weed species. Aqueous extracts of all species proved phytotoxic against T. portulacastrum, P. hysterophorus, M. indica and R. dentatus and retarder their germination by 57, 90, 100 and 58%, respectively. Nevertheless, foliar spray of C. occidentalis extract was the most effective against T. portulacastrum as it reduced its dry biomass by 72%, while M. alba was effective against P. hysterophorus, R. dentatus and M. indica and reduced their dry biomass by 55, 68 and 81%, respectively. It is concluded that aqueous extracts of M. alba, S. sesban and C. occidentalis could be used to retard seed germination of T. portulacastrum, P. hysterophorus, M. indica and R. dentatus. Similarly, aqueous extracts of C. occidentalis can be used to suppress dry biomass of T. portulacastrum, and those of M. alba against P. hysterophorus, R. dentatus. However, use of these extracts needs their thorough testing under field conditions.

Antioxidant and Antiinflammatory Effects of Epilobium parviflorum, Melilotus officinalis and Cardiospermum halicacabum Plant Extracts in Macrophage and Microglial Cells.

BACKGROUND: We investigated the phenolic content characterizing different plant extracts from Epilobium parviflorum, Cardiospermum halicacabum, and Melilotus officinalis, their antioxidant, antiinflammatory effects, and their mechanism of action. METHODS: plant samples were macerated in 40% ethanol or hot/ cold glycerate and assessed for polyphenols content. The antioxidant activity was investigated by DPPH radical scavenging assay and H2DCFDA test in LPS-stimulated RAW264.7 macrophages and N9 microglial cells. MTS experiments and antiinflammatory properties verified cellular toxicity through NO assay. Interaction with A2A adenosine receptors was evaluated through binding assays using [3H]ZM241385 radioligand. RESULTS: Polyphenols were present in 40% ethanol plant extract, which at 0.1-10 µg/µL achieved good antioxidant effects, with a DPPH radical scavenging rate of about 90%. In LPS-stimulated cells, these plant extracts, at 1µg/µL, did not affect cell vitality, displayed significant inhibition of H2DCFDA and NO production, and inhibited ZM 241385 binding in CHO cells transfected with A2A receptors. RAW 264.7 and N9 cells presented a density of them quantified in 60 ± 9 and 45 ± 5 fmol/mg of protein, respectively. CONCLUSION: Epilobium parviflorum, Cardiospermum halicacabum, and Melilotus officinalis extracts may be considered a source of agents for treating disorders related to oxidative stress and inflammation.

Step-by-step design of poly (ε-caprolactone) /chitosan/Melilotus officinalis extract electrospun nanofibers for wound dressing applications.

Composition of polymers and choosing the type of solvents in electrospinning systems is of great importance to achieve a mat with optimal properties. In this work, with emphasizing the influence of a novel solvent system, an electrospun wound dressing was designed in four steps. Firstly, to study the effect of polymer-solvent interactions and electrospinning distance, a constant amount of polycaprolactone (PCL) was dissolved at different compositions of formic acid (FA)/dichloromethane (DCM) and was electrospun at different distances. The composition of 80FA/20DCM and distance of 15 cm were selected as optimal conditions by lowest average diameter of fibers and simultaneously good surface uniformity. In the second step, the concentration of PCL was considered variable to achieve the lowest diameter of fibers. Finally, in the third and fourth steps, different concentrations of chitosan (CN) and constant dosage of Melilotus officinalis (MO) extract were added to the solution. The extract contained fibers had a mean diameter of 275 ± 41 nm which is in the required condition for wound caring. Eventually, the optimized PCL/CN and PCL/CN/MO specimens were evaluated by FTIR, DSC, Tensile, water contact angle, antibacterial assays, cell viability, and drug release analysis for determining their function and properties.

Analyzing the Carotenoid Composition of Melilot (Melilotus officinalis (L.) Pall.) Extracts and the Effects of Isolated (All-E)-lutein-5,6-epoxide on Primary Sensory Neurons and Macrophages.

Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-ß-cyclodextrin) was investigated on Ca2+-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1ß release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1ß release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.

Investigation of in vivo anti-inflammatory and anti-angiogenic attributes of coumarin-rich ethanolic extract of Melilotus indicus.

Inflammation and angiogenesis are two major contributors to tumourigenesis. Melilotus indicus is traditionally used as an anti-inflammatory agent. The current study was designed to investigate the anti-inflammatory and anti-angiogenic properties of ethanolic extract of M. indicus (Miet) whole plant and its marker compound (coumarin) using a series of in vivo methods. Extraction by maceration was adopted to prepare ethanolic extract. Phytochemical compounds present in Miet were investigated using both qualitative and quantitative methods. In vivo safety profile of Miet was investigated in behavioural studies. Four acute oedema models such as carrageenan, serotonin, histamine-induced paw oedema and xylene-induced ear oedema, and chronic formaldehyde-induced paw oedema model were employed to explore the anti-inflammatory potential of Miet. Chorioallantoic chick membrane assay (CAM) was performed to explore anti-angiogenic potential of Miet. Histopathological evaluations were conducted to access improvement in skin texture of paws. TNF-α ELISA kit was used to study effects of treatment on serum levels of TNF-α. Extraction by maceration resulted in formation of greenish coloured semisolid extract with a high coumarin content. In vivo toxicological studies revealed LD50 of Miet was greater than 8000 mg/kg. Data of acute inflammatory models depicted significant (p < 0.05) inhibition of oedema in Miet, coumarin and standard (piroxicam/indomethacin) treated groups. 750 mg/kg of Miet induced comparable (p > 0.05) anti-inflammatory effects to that of standard-treated groups. Coumarin showed better anti-inflammatory effects in carrageenan-induced paw oedema model as compared with histamine- and serotonin-induced oedema models. Data of chronic inflammatory models also depicted dose-dependent anti-inflammatory attributes of Miet which were comparable with standard treated groups. Significant (p > 0.05) downregulation of TNF-α in serum samples of animals treated with Miet and piroxicam was observed as compared with control group. Furthermore, Miet significantly halted blood vessels formation in CAM assay. Overall, data of the current study highlight that M. indicus has anti-inflammatory and anti-angiogenic potentials, and, thus, can potentially be used as an adjuvant therapy in solid tumours management.

A pharmacological review of dicoumarol: An old natural anticoagulant agent.

Dicoumarol is an oral anticoagulant agent prescribed in clinical for decades. It is a natural hydroxycoumarin discovered from the spoilage of Melilotus officinalis (L.) Pall and is originally discovered as a rodenticide. Due to its structural similarity to that of vitamin K, it significantly inhibits vitamin K epoxide reductase and acts as a vitamin K antagonist. Dicoumarol is mainly used as an anticoagulant to prevent thrombogenesis and to cure vascular thrombosis. Other biological activities besides anticoagulants such as anticancer, antimicrobial, antiviral, etc., have also been documented. The side effects of dicoumarol raise safety concerns for clinical application. In this review, the physicochemical property, the pharmacological activities, the side effects, and the pharmacokinetics of dicoumarol were summarized, aiming to provide a whole picture of the "old" anticoagulant.

The regression of endometriosis with glycosylated flavonoids isolated from Melilotus officinalis (L.) Pall. in an endometriosis rat model.

OBJECTIVE: Melilotus officinalis (L.) Pall. is commonly used for treating bronchitis, painful menstruation, hemorrhoids, kidney stones, ulcers of the eyes, earache, and hardening and swelling of uterus. The European Medicines Agency reported the use of M. officinalis orally against stomach ache, gastric ulcer, and disorders of the liver and uterus in folk medicine. The present study aimed to appraise the activity of M. (L.) Pall. aerial parts in endometriosis rat model. MATERIALS AND METHODS: The endometriosis rat model was used to evaluate the potential activity of M. officinalis aerial parts based on its folkloric usage. The aerial parts of M. officinalis were extracted with n-hexane, ethyl acetate (EtOAc), and methanol (MeOH), respectively. The adhesion scores, endometrial foci areas, and cytokine levels were measured in all treated groups. After the biological activity studies, phytochemical studies were performed on the active extract and the fractions obtained from the active extract. RESULTS: The MeOH extract significantly decreased the endometrial foci areas and cytokine levels in rats with endometriosis. Fractionation was performed on the MeOH extract to achieve bioactive molecules. Following the fractionation, the fractions obtained from the MeOH extract were tested. Fraction C showed the highest activity in the rat endometriosis model. Phytochemical investigation of the active fraction (Fraction C) resulted in isolation and elucidation of some quercetin and kaempferol glucoside derivatives. CONCLUSION: Fraction C obtained from the MeOH extract of M. officinalis showed the highest activity, yielding four glycosylated flavonoids.

Investigating In Vitro Antibacterial Activities of Medicinal Plants Having Folkloric Repute in Ethiopian Traditional Medicine.

Medicinal plants are targeted in the search for new antimicrobial agents. Nowadays, there is an alarmingly increasing antimicrobial resistance to available agents with a very slow development of new antimicrobials. It is, therefore, necessary to extensively search for new agents based on the traditional use of herbal medicines as potential source. The antibacterial activity of 80% methanol extracts of the leaves of Verbena officinalis (Vo-80ME), Myrtus communis (Mc-80ME), and Melilotus elegans (Me-80ME) was tested against 6 bacterial isolates using agar well diffusion technique. In each extract, 3 concentrations of 10, 20, and 40 mg/well were tested for each bacterium. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were also determined. Vo-80ME and Mc-80ME exhibited promising antibacterial activity against Staphylococcus aureus with the highest zone of inhibition being 18.67 and 26.16 mm, respectively at concentration of 40 mg/well. Regarding gram-negative bacteria, Vo-80ME exhibited an appreciable activity against Escherichia coli and Salmonella typhi. Mc-80ME displayed remarkable activity against all isolates including Pseudomonas aeruginosa with the maximum zone of inhibition being 22.83 mm. Me-80ME exhibited better antibacterial activity against E coli, but its secondary metabolites had little or no activity against other gram-negative isolates. The MIC values of Vo-80ME ranged from 0.16 to 4.00 mg/mL. The lowest MIC was observed in Mc-80ME, with the value being 0.032 mg/mL. Mc-80ME had bactericidal activity against all tested bacterial isolates. Mc-80ME showed remarkable zone of inhibitions in all tested bacterial isolates. Besides, Vo-80ME showed good antibacterial activity against S aureus, E coli, and S typhi. Conversely, Me-80ME has shown good activity against E coli only. Generally, M communis L and V officinalis have good MIC and MBC results.

A new isoflavane-4-ol derivative from Melilotus officinalis (L.) Pall.

A new isoflavane derivative, melilofficinaside together with seven other metabolites including coumarin, uridine, methyl-α-d-fructofuranoside, and flavonoid glucosides were isolated from the aerial parts of Melilotus officinalis (L.) Pall.

Melilotus, Rutin and Bromelain in primary and secondary lymphedema.

As reported in the literature, benzopyrones (alpha and gamma) have important effects on the microcirculation through various mechanisms. Coumarins are an alpha-benzopyrone as derivatives of Melilotus Officinalis, while bioflavonoids are a gamma-benzopyrone and include Rutin. Alpha-benzopyrones have two fundamental pharmacological effects: they have pro-lymphokinetic action by activating contractility of lymphangions; and the activation of macrophages to provide a proteolytic effect. Gamma-benzopyrones, such as Rutin, have an important anti-exuding and membrane stabilizing effect. Bromelain is known for its anti-inflammatory effect. The present study enrolled 52 patients with primary and/or secondary lymphedema in clinical stages I or II (according to the ISL classification) with 31 cases involving the lower limbs and 21 cases involving the upper limbs. All subjects were given for six months a natural compound consisting of 100 mg of natural Melilotus, that contains 20 grams of Coumarin, 300 mg of Rutin and 100 mg of Bromelain. The following parameters were studied at zero time (T0), after three months (T1), and after six months of treatment (T2): pitting, Stemmer's sign, measurement of limb circumferences, measurement of superficial tissue thickness in the affected limbs using ultrasound, and blood tests to evaluate hepatic function (ALT, AST, GGT, total and fractional bilirubin). At the end of the treatment (T2), the following results were observed: disappearance of pitting in 72% of the cases; unchanged Stemmer's sign; average decrease in limb circumferences of 4.2 cm; and average reduction of the superficial thickness of 29%. There was no variation in the liver function parameters examined. The combination of natural compounds (Melilotus, Rutin, and Bromelain) has been shown to be a valuable aid in the clinical control of both primary and secondary lymphedema of clinical stages I and II as well as in control of inflammatory phenomena related to chronic stasis. There were no side effects and no alteration of liver function parameters found.

In vitro pharmacological effects of Astragalus eremophilus and Melilotus parviflora.

Traditional medicines are composed of herbal formulations and their active ingredients and constituents which play a crucial role in the treatment of various human ailments. Astragalus eremophilus and Melilotus indicus (L.) All. (syn. Melilotus parviflora Desf.) are used traditionally as antiperspirant, tonic, diuretic, laxative and narcotic agents. The current study was designed to investigate the Astragalus eremophilus and Melilotus indicus (L.) All. (syn. Melilotus parviflora Desf.) methanol extracts for their antioxidant, antibacterial and antifungal activities. Fine powder of A. eremophilus and M. parviflora was extracted with 70% methanol to get crude methanol extract. Extract was characterized for antioxidant, antibacterial and antifungal activities. Antioxidant activity of various concentrations (3 mg/ml, 1.5 mg/ ml, 0.75 mg/ml, and 0.38 mg/ml) of both plant extracts was analyzed using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical. Salmonella typhemorium, Klebsiella pneumoniae (gram-negative) and Staphylococcus aureus, Enterococcus faecalis (gram-positive) bacterial strains were used for assessment of antibacterial activities. Antifungal activities of 7.5 mg/ml, 5.0 mg/ml, 2.5 mg/ml (A. eremophilus and M. parviflora) were conducted using Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus and Candida albicons. At high concentration (3 mg/ml), all the tested fractions of A. eremophilus and M. parviflora methanol extracts showed potent antioxidant activities, ranging between 83.8 and 63.33%. Antibacterial activities revealed that A. eremophilus showed a maximum zone of inhibition (8.1 ± 0.1) on Salmonella typhenorium followed by Enterococcus faecalis (7.2 ± 0.1), Klebsellesa pneumonia (6.1 ± 0.6), and Staphylococcus aureus (5.1 ± 0.4), and at highest concentration (7.5 mg/ml), however, maximum zone of inhibition of Melilotus parviflora was at 7.5 mg/ml followed by 5.0 mg/ml and 2.5 mg/ml against Klebsiella pneumonia, Staphylococcus aureus, Salmonella typhemorium and Enterococcus faecalis. Antifungal assessment of both plant extracts showed that the higher concentration (7.5 mg/ml) has significant inhibitory effect as compared to control. The results can lead to the conclusion that A. eremophilus and M. parviflora methanol extracts are indeed sources of potential therapeutic compounds against antibacterial, antifungal and free radical associated disorders.

Isolation of Tricin as a Xanthine Oxidase Inhibitor from Sweet White Clover (Melilotus albus) and Its Distribution in Selected Gramineae Species.

Xanthine oxidase, an enzyme present in significant levels in the intestine and liver, metabolizes hypoxanthine to xanthine and xanthine to uric acid in the purine catabolic pathway. An inhibitory compound acting against xanthine oxidase was isolated from sweet white clover (Melilotus albus) by bioassay and high-performance liquid chromatography guided separation. It was identified as tricin by spectroscopic analysis. Tricin possessed a potent xanthine oxidase inhibitory activity with an IC50 value of 4.13 µM. Further inhibition kinetics data indicated it to be a mixed-type inhibitor and Ki and KI values were determined to be 0.47 µM and 4.41 µM. To find a rich source of tricin, the distribution of tricin in seven different tissues from four Gramineae species was investigated by high-performance liquid chromatography analysis. The highest amount (1925.05 mg/kg dry materials) was found in the straw of wheat, which is considered as a potentially valuable source of natural tricin.

Analysis of miRNAs and their target genes in five Melilotus albus NILs with different coumarin content.

MicroRNAs (miRNAs) exhibit diverse and important roles in regulation of various biological processes at the post-transcriptional level in plants. In this study, Melilotus albus miRNA and their target genes were elucidated from five M. albus near-isogenic lines which differ in coumarin content to construct small RNA libraries through high-throughput sequencing. A total of 417 known miRNAs and 76 novel miRNAs were identified in M. albus. In addition, 4155 different target genes for 114 known miRNA families and 14 target genes for 2 novel miRNAs were identified in M. albus. Moreover, mtr-miR5248 and mtr-miR7701-5p target c35498_g3 and gma-miR396a-3p target c37211_g1 involved in coumarin biosynthesis were identified by using the differential expression of the miRNAs and their target genes correlation analysis. The abundance of miRNAs and potential target genes were validated by qRT-PCR analysis. We also found that there were both positive and negative expression changing patterns between miRNAs and their related target genes. Our first and preliminary study of miRNAs will contribute to our understanding of the functions and molecular regulatory mechanisms of miRNAs and their target genes, and provide information on regulating the complex coumarin pathway in M. albus for future research.

Determination of dicoumarol in Melilotus officinalis L. by using molecularly imprinted polymer solid-phase extraction coupled with high performance liquid chromatography.

In this work, a method for the extraction of dicoumarol from plant sample utilizing selective adsorbent based on molecularly imprinted polymer was developed. Molecularly imprinted polymer (MIP) was synthesized by bulk polymerization, and on the surface of magnetic particles using dicoumarol as a template, methacrylic acid as functional monomer, chloroform as a porogen, and ethyleneglycol dimethacrylate as a cross-linker. Prepared polymeric materials were evaluated on the base of capacity, selectivity, and morphology. The maximal specific adsoption capacities of the sorbents were 45 µg (for bulk MIP) and 36 µg (for MIP on the magnetic particles) of dicoumarol per 1 g of polymer. An efficiencies of sorption processes of dicoumarol on prepared sorbents were evaluated under various conditions (type of sample solvent, pH, types of washing and elution solvents). The MIP based sorbents were used for solid phase extraction of dicoumarol. Applicability of MIP-SPE coupled to HPLC-DAD was tested for the selective extraction of dicoumarol from sample of sweet clover. The method was linear over concentration range from 1 to 100 µg mL-1 (the correlation coefficient 0.9984) with limit of detection 0.2 µg mL-1. Accuracy of the method was assessed for spiked sample at three concentration levels and recovery values were higher than 84% with relative standard deviation lower than 3.2%.

Chemical Constituents and Antioxidant, Anti-Inflammatory and Anti-Tumor Activities of Melilotus officinalis (Linn.) Pall.

Two new p-hydroxybenzoic acid glycosides, namely p-hydroxybenzoic acid-4-O-α-d-manopyranosyl-(1 → 3)-α-l-rhamnopyranoside (compound 1) and 4-O-α-l-rhamnopyran-osyl-(1 → 6)-α-d-manopyranosyl-(1 → 3)-α-l-rhamnopyranoside (compound 2), and seven known compounds, compound 3, 6, 7 (acid components), compound 8, 9 (flavonoids), compound 4 (a coumarin) and compound 5 (an alkaloid), were isolated from the 70% ethanol aqueous extract of the aerial parts of Melilotus officinalis (Linn.) Pall. The structures of all compounds were elucidated by use of extensive spectroscopic methods Infrared Spectroscopy (IR), High resolution electrospray ionization mass spectrometry (HR-ESI-MS), and ¹H and 13C-NMR). Sugar residues obtained after acid hydrolysis were identified by high-performance liquid chromatography (HPLC). The antioxidant activity of all the compounds was evaluated by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) and 1,1-diphenyl-2-picrylhydrazyl (DPPH). The anti-inflammatory effects of the compounds were also evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. All compounds were shown to inhibit LPS-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2) production by suppressing the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively, in LPS-stimulated RAW 264.7 cells. The inhibitory effect of all the compounds on MCF-7 cells was determined by Cell Counting Kit-8 (CCK-8) method. The results showed that compounds 1, 2, 7, 8, 9 exhibited better antioxidant activity compared to the other compounds. compounds 1-9 had different inhibitory effects on the release of NO, TNF-α and IL-6 in LPS-stimulated RAW264.7 cells by LPS, of which compound 7 was the most effective against inflammatory factors. compounds 1 and 2 have better antitumor activity compared to other compounds. Further research to elucidate the chemical composition and pharmacological effects of Melilotus officinalis (Linn.) Pall is of major importance towards the development and foundation of clinical application of the species.

Effect of Melilotus officinalis extract on the apoptosis of brain tissues by altering cerebral thrombosis and inflammatory mediators in acute cerebral ischemia.

Present investigation evaluates the protective effect of Melilotus officinalis (MO) extract on the brain tissues in acute cerebral ischemia. Acute cerebral ischemia was induced by occlusion of carotid artery and rats with cerebral ischemia were treated with MO (100, 250 & 500mg/kg) for the duration of three days. Cerebral ischemia was confirmed by estimating infract volume and neurological deficit score. Moreover biochemical parameters in plasma such as 6-keto-PGF1α and TXB2 and concentration of cytokine, oxidative stress, apoptosis ratio and protein expressions of Bcl2 & Bax were estimated in the brain tissues. It was observed that treatment with MO significantly (p<0.01) decreases the infract volume and neurological deficit score than negative control group. There was significant decrease (p<0.01) in the oxidative stress and cytokine in the brain tissues and increase in the plasma concentration of 6-keto-PGF1α in MO treated group of rats compared to negative control group. Plasma concentration of TXB 2 was significantly enhanced in MO treated group compared to negative control group of rats. It was also found that treatment with MO ameliorates the apoptosis induced by cerebral ischemia. Present study concludes that MO ameliorates apoptosis of brain tissues in cerebral ischemic rats by decreasing cerebral thrombosis, oxidative stress and inflammatory mediators.

EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF MELILOTUS OFFICINALIS L. AGAINST PARACETAMOL AND CARBON TETRACHLORIDE INDUCED HEPATIC INJURY IN MICE.

Hepatic diseases are becoming common day by day and pose serious health threats to the life of humans. In order to treat these diseases, the attention of man is diverting towards herbal drugs, which are much safer and cost effective than synthetic drugs. The aim of present study was to investigate hepatoprotective activity of methanolic extract of Melilomus officinalis against paracetamol and carbon tetrachloride induced hepatic damage. Melilotus officinalis at selected oral doses of 50 mg/kg and 100 mg/kg showed significant hepatoprotective effects by decreasing the levels of serum marker enzymes such as total bilirubin, SGOT, SGPT, ALP, albumin and total protein, when compared with standard drug (silymarin) and negative control. Similarly, histopathological studies also supported biochemical estimations. It was concluded that extract of Melilotus officinali has strong hepatoprotective activity against paracetamol and carbon tetrachloride induced hepatotoxicity, which might be due to free radical scavenging mechanisms exhibited by flavonoids and phenolics, thus affirming its traditional therapeutic role in liver injury.