Proteomics Analysis of Antitumor Activity of Agrimonia pilosa Ledeb. in Human Oral Squamous Cell Carcinoma Cells.

Oral cancer is a malignant neoplasm of oral cavity. It accounts for approximately 5% of all malignant tumors. Approximately 97% of all oral cancers are squamous cell carcinomas, followed by adenocarcinomas, and rarely malignant melanomas. It occurs particularly in males (twice as common in males than in females) of middle age (above 40 years). Agrimonia pilosa Ledeb. has traditionally been known for its effective antitumor activity and is currently used in China for cancer therapy. A. pilosa Ledeb. has been traditionally used for the treatment of abdominal pain, sore throat, headache, blood discharge, parasitic infections, and eczema in Korea and other Asian countries. Most studies on A. pilosa Ledeb. are related to the leaves and a few investigated the roots of the plant. However, detailed mechanisms of antitumor activity of A. pilosa Ledeb. have not been fully elucidated. Furthermore, to date, there have been no reports on the antitumor effect of A. pilosa Ledeb. in oral squamous cells. In this study, we used proteomic technology to observe changes in proteins related to anticancer activity of A. pilosa Ledeb. and identified target proteins among altered proteins to reveal the underlying mechanism of action.

Protective mechanisms of Agrimonia pilosa Ledeb in dextran sodium sulfate-induced colitis as determined by a network pharmacology approach.

Previous studies reported that Agrimonia pilosa (AP) Ledeb possessed diverse biological activities, including anti-inflammatory, antioxidant, and anti-tumor activities. However, the effect of AP on ulcerative colitis (UC) remains unclear. In this study, we investigated the therapeutic effect and mechanisms of AP on dextran sodium sulfate (DSS)-induced colitis. The potential constituents of AP were investigated by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). A total of 13 compounds were recognized by UPLC-Q-TOF/MS chromatogram. Furthermore, a network pharmacology approach revealed that there are 297 candidate targets of UC and 549 common targets for the 13 active ingredients of AP. GO enrichment and KEGG pathway analysis indicated that AP might have a protective effect on UC through the nuclear factor κB (NF-κB) and nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathways. Subsequent experimental validation in a DSS-induced colitis model revealed that AP alleviated the severity of DSS-induced colitis, reduced the production of proinflammatory factors, and protected against the loss of intestinal integrity. Moreover, AP inhibited the phosphorylation of NF-κB p65 and the activation of the NLRP3 inflammasome. In conclusion, AP ameliorated DSS-induced colitis through suppressing the activation of the NLRP3 inflammasome and NF-κB signaling pathways.

Agrimonia pilosa Ledeb., Cinnamomum cassia Blume, and Lonicera japonica Thunb. protect against cognitive dysfunction and energy and glucose dysregulation by reducing neuroinflammation and hippocampal insulin resistance in ß-amyloid-infused rats.

OBJECTIVES: The water extracts of Cinnamomum cassia Blume bark (CCB; Lauraceae), Lonicera japonica Thunb. flower (LJT; Caprifoliaceae), and Agrimonia pilosa Ledeb. leaves (APL; Rosaceae) prevented amyloid-ß (25-35)-induced cell death in PC12 cells in our preliminary study. We evaluated whether long-term oral consumption of CCB, LJT, and APL improves cognitive dysfunction and glucose homeostasis in rats with experimentally induced AD-type dementia. METHODS: Male rats received hippocampal CA1 infusions of amyloid-ß (25-35, AD) or amyloid-ß (35-25, non-plaque forming, normal-controls, Non-AD-CON), at a rate of 3.6 nmol/day for 14 days. AD rats were divided into four groups receiving either 2% lyophilized water extracts of CCB, LJT, or APL or 2% dextrin (AD-CON) in high-fat diets (43% energy as fat). RESULTS: Hippocampal amyloid-ß deposition, tau phosphorylation, and expressions of tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) (neruoinflammation markers) were increased, and insulin signaling decreased in AD-CON. CCB, LJT, and APL all prevented hippocampal amyloid-ß accumulation and enhanced hippocampal insulin signaling. CCB, LJT, and APL decreased TNF-α and iNOS in the hippocampus and especially APL exhibited the greatest decrease. AD-CON exhibited cognitive dysfunction in passive avoidance and water maze tests, whereas CCB, LJT, and APL protected against cognitive dysfunction, and APL was most effective and was similar to Non-AD-CON. AD-CON had less fat oxidation as an energy fuel, but it was reversed by CCB, LJT, and especially APL. APL-treated rats had less visceral fat than AD-CON rats. AD-CON rats exhibited impaired insulin sensitivity and increased insulin secretion during oral glucose tolerance test compared with Non-AD-CON, but CCB and APL prevented the impairment. DISCUSSION: These results supported that APL, LJT, and CCB effectively prevent the cognitive dysfunction and the impairment of energy and glucose homeostasis induced by amyloid-ß deposition by reducing neuroinflammation and enhancing insulin signaling. APL exhibited the greatest effectiveness for improving cognitive function.

Phytochemical Analysis of Agrimonia pilosa Ledeb, Its Antioxidant Activity and Aldose Reductase Inhibitory Potential.

The aim of this study was to determine aldose reductase (AR) inhibitory activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of compounds from Agrimonia pilosa Ledeb (AP). We isolated agrimoniin (AM), four flavonoid glucosides and two flavonoid glucuronides from the n-butanol fraction of AP 50% methanol extract. In addition to isolated compounds, the AR-inhibitory activity and the DPPH free radical scavenging activity of catechin, 5-flavonoids, and 4-flavonoid glucosides (known components of AP) against rat lens AR (RLAR) and DPPH assay were measured. AM showed IC50 values of 1.6 and 13.0 µM against RLAR and DPPH scavenging activity, respectively. Additionally, AM, luteolin-7-O-glucuronide (LGN), quercitrin (QU), luteolin (LT) and afzelin (AZ) showed high inhibitory activity against AR and were first observed to decrease sorbitol accumulation in the rat lens under high-sorbitol conditions ex vivo with inhibitory values of 47.6%, 91.8%, 76.9%, 91.8% and 93.2%, respectively. Inhibition of recombinant human AR by AM, LGN and AZ exhibited a noncompetitive inhibition pattern. Based on our results, AP and its constituents may play partial roles in RLAR and oxidative radical inhibition. Our results suggest that AM, LGN, QU, LT and AZ may potentially be used as natural drugs for treating diabetic complications.

Flavonoids from Agrimonia pilosa Ledeb: Free Radical Scavenging and DNA Oxidative Damage Protection Activities and Analysis of Bioactivity-Structure Relationship Based on Molecular and Electronic Structures.

To clarify the substantial basis of the excellent antioxidant capacity of Agrimonia pilosa Ledeb. Fourteen flavonoids were isolated and identified from Agrimonia pilosa Ledeb, seven of which have notable DPPH radical scavenging activities, i.e., catechin, luteolin, quercetin, quercitrin, hyperoside, rutin, luteolin-7-O-ß-glucoside with IC50 values of 5.06, 7.29, 4.36, 7.12, 6.34, 6.36 and 8.12 µM, respectively. The DNA nicking assay showed that five flavonoids from Agrimonia pilosa Ledeb-taxifolin, catechin, hyperoside, quercitrin and rutin-have good protective activity against DNA oxidative damage. Further, we analyzed the bioactivity-structure relationship of these 14 flavonoids by applying quantum theory. According to their O-H bond dissociation enthalpy (BDE), C ring's spin density and stable molecular structure, the relationship between their structures and radical scavenging capacities was evaluated and clarified. We found that among flavonoid aglycones from Agrimonia pilosa Ledeb, the O-H BDE of quercetin is lowest with the values of 69.02 and the O-H BDE of apigenin is highest with the values of 79.77. It is interesting that the O-H BDE value of isovitexin (78.55) with glycoside at C-6 position is lower than that of its aglycone (79.77) and vitexin (99.20) with glycoside at C-8 position. Further analysis indicated that the glycosidation of flavonoids at C-6 in the A-ring makes a more uniform distribution of spin density and improves the stability of free radicals leading to the increase in antioxidant capacity. Flavonoids with good antioxidant capacity might contribute to the pharmacological effects of Agrimonia pilosa Ledeb.

Agrimol B suppresses adipogenesis through modulation of SIRT1-PPAR gamma signal pathway.

Studies of human genetics have implicated the role of SIRT1 in regulating obesity, insulin resistance, and longevity. These researches motivated the identification of novel SIRT1 activators. The current study aimed to investigate the potential efficacy of agrimol B, a polyphenol derived from Agrimonia pilosa Ledeb., on mediating SIRT1 activity and fat metabolism. Results showed that agrimol B significantly induced cytoplasm-to-nucleus shuttle of SIRT1. Furthermore, we confirmed that agrimol B dramatically inhibited 3T3-L1 adipocyte differentiation by reducing PPARγ, C/EBPα, FAS, UCP-1, and apoE expression. Consequently, adipogenesis was blocked by treatment of agrimol B at the early stage of differentiation in a dose-dependent manner, the IC50 value was determined as 3.35 ± 0.32 µM. Taken together, our data suggest a therapeutic potential of agrimol B on alleviating obesity, through modulation of SIRT1-PPARγ signal pathway.

Development of a liquid chromatography-mass spectrometry method for determination of agrimol B in rat plasma: application to preclinical pharmacokinetics.

A sensitive and accurate liquid chromatography coupled with mass spectrometry (LC-MS) method was developed for the determination of agrimol B, a main active ingredient isolated from Agrimonia pilosa Ledeb., in rat plasma. Chromatographic separation was achieved on a Zorbax CN column (150 × 4.6 mm, 5 µm), with isocratic elution consisting of acetonitrile and water (15:85, v/v) at a flow rate of 0.6 mL/min. Agrimol B and dryocrassin ABBA, an internal standard (IS), were analyzed by selected ion monitoring at m/z transitions of 681.3 and 819.4, respectively. This assay exhibited a good linearity with a correlation coefficient >0.99 and showed no endogenous interference with the analyte and IS. The limit of quantification of agrimol B was 8.025 ng/mL with acceptable precision and accuracy. The method was successfully applied in the pharmacokinetic study of agrimol B in rats after intravenous (1 mg/kg) and oral (2, 5 and 10 mg/kg) doses of agrimol B. The absolute bioavailability of agrimol B was 16.4-18.0% in rat. Our study clarifies the pharmacokinetic behavior of agrimol B in animals.

The anti-colorectal cancer effect and metabolites of Agrimonia pilosa Ledeb.

ETHNOPHARMACOLOGICAL RELEVANCE: Agrimonia pilosa Ledeb. (Rosaceae, A. pilosa) has been used in traditional medicine in China, Japan, Korea, and other Asian countries for treatment of acute and chronic enteritis and diarrhea. Secondary metabolites have been isolated and tested for biological activities. It remains unclear in terms of its potential components of anti-colorectal cancer properties. AIM OF THE STUDY: The study aimed to how extracts from A. pilosa and their components influenced tumor microenvironment and the colorectal tumor growth in vivo on AOM/DSS induced colorectal cancer mice, the metabolites of A. pilosa was also been studied. MATERIALS AND METHODS: Different methods have been used to extract different parts of A. pilosa. And the anti-proliferation effect of these extracts on colon cancer cells have been tested. The components of A. pilosa and its metabolites in vivo were analyzed by UPLC-QTOF-MS/MS. The anti-colorectal cancer (CRC) effects of A. pilosa and its components in vivo were studied on AOM/DSS induced CRC mice. The effects of constituents of A. pilosa on the composition of immune cells in tumor microenvironment (TME) were analyzed by flow cytometry. 16 S rDNA technology was used to analyze the effect of administration on the composition of intestinal microflora. Pathological section staining was used to compare the morphological changes and molecular expression of intestinal tissue in different groups. RESULTS: The constituent exists in root of A. pilosa showed the strongest anti-proliferation ability on colon cancer cells in vitro. The extract from the root of A. pilosa could attenuate the occurrence of colorectal tumors induced by AOM/DSS in a concentration-dependent manner. Administration of the extract from the root of A. pilosa could affect the proportion of γδT cells, tumor associated macrophages and myeloid derived suppressor cells in TME, increasing the proportion of anti-tumor immune cells and decrease the immunosuppressive cells in the TME to promote the anti-tumor immune response. The administration of the extract adjusted the composition of gut microbiota and its components Agrimoniin and Agrimonolide-6-o-glucoside showed the strongest anti-CRC effect in vivo with adjusting the gut microbiota differently. CONCLUSIONS: The extract from root of A. pilosa showed anti-colorectal cancer effects in vivo and in vitro, affecting the composition of gut microbiota and the anti-tumor immune response. Within all components of A. pilosa, Agrimoniin and Agrimonolide-6-o-glucoside showed remarkable anti-CRC efficiency in vivo and in vitro. Besides, the metabolites of extract from root of A. pilosa in gastrointestinal tract mainly composed of two parts: Agrimonolide-related metabolites and Urolithins. The extract from root of A. pilosa could contribute to potential drugs for assisting clinical anti-colon cancer therapy.

Current Progress and Outlook for Agrimonolide: A Promising Bioactive Compound from Agrimonia pilosa Ledeb.

Agrimonolide (AM), which is a derivative of isocoumarins, is found mainly in the herb Agrimonia pilosa Ledeb. This compound is highly lipophilic and readily crosses the blood-brain barrier. In recent years, interest has grown in the use of AM as a multitarget natural treatment for various diseases, such as cancer, inflammation, hepatic injury, myocardial damage, and diabetes mellitus. The potential mechanisms of these pharmacological effects have been clarified at cellular and molecular levels. AM shows no cytotoxicity over a range of concentrations in different types of cells, providing evidence for its good safety profile in vitro. These findings indicate that AM is a promising medicinal agent. However, most studies on AM's pharmacological activities, mechanisms of action, and safety lack substantial animal or human data. Additionally, the pharmacokinetics, metabolism, and disposition of this compound have received little attention. This review highlights the status of current information regarding the sources, properties, pharmacological effects, and safety of AM. Furthermore, potential strategies to resolve problematic issues identified in previous studies are fully discussed. This summary and analysis of the research progress of AM may inspire deeper investigations and more extensive applications of AM in the future.

Flavonoid compound from Agrimonia pilosa Ledeb improves adipose insulin resistance by alleviating oxidative stress and inflammation.

BACKGROUND: Researches and practice of traditional Chinese medicine indicated that Agrimonia pilosa Ledeb could improve insulin resistance (IR) and treat type 2 diabetes (T2DM). To reveal its underling mechanisms, we isolated Flavonoid component (FC) from Agrimonia pilosa Ledeb and elucidated its effects on glucose metabolism to improve IR by suppressing oxidative stress and inflammation. METHODS: Adipocytes or mice IR model was established with overdosed glucose and insulin or high-fat diet. The uptake of 2-NBDG and glucose consumption were measured to verify insulin sensitivity in vitro and vivo. Reactive oxidative species (ROS) were detected by flow cytometry, and superoxide dismutase (SOD) activity as well as the malondialdehyde (MDA) content were also measured. Meanwhile, factors associated with insulin signal pathway including PPARγ, insulin receptor substrate-1 (IRS-1), GLUT4, and oxidative stress including NF-E2-related factor 2 (Nrf2), as well as the related inflammatory cytokines such as NF-κB, IL-1ß, IL-6 and TNF-α were tested. Furthermore, the JNK/PI3K/Akt signal pathway was also explored. RESULTS: FC extracted from Agrimonia pilosa Ledeb ameliorated the impaired glucose metabolism significantly. Further study indicated that FC could regulate the insulin signal pathway to improve insulin resistance. Moreover, it could upregulate PPARγ with the similar efficacy as pioglitazone (Piog) straightway. FC also decreased the endogenous ROS and MDA content, increased SOD activity and Nrf2 expression to facilitate oxidative homeostasis. It attenuated expression and secretion of inflammatory cytokines obviously. At last, our results indicated JNK/PI3K/Akt pathway was regulated by FC in adipocytes and adipose tissue. CONCLUSION: FC could ameliorate glucose metabolism and improve IR. It exerted these effects by suppressing oxidative stress and inflammation. FC from Agrimonia pilosa Ledeb has a good prospect to be drugs or functional foods for IR and T2DM.

The mixture of Agrimonia pilosa Ledeb. and Salvia miltiorrhiza Bunge. extract produces analgesic and anti-inflammatory effects in a collagen-induced arthritis mouse model.

Pain and inflammation typically manifest in patients with arthritis. It is now widely known that Agrimonia pilosa Ledeb (AP) and Salvia miltiorrhiza Bunge (SM) exert anti-inflammatory and antinociceptive effects. We have previously reported that the mixture extract (ME) from AP and SM produces antinociceptive and anti-inflammatory effects in gout arthritis and monoiodoacetate (MIA)-induced arthritis models. In the present study, we assessed the antinociceptive and anti-inflammatory effects on the collagen-induced arthritis (CIA) model. The antinociceptive effects in mice were measured using the von Frey test. ME administered once or for one week (once per day) once, and one-week reduced the pain in a dose-dependent manner (from 50 to 100 mg/kg) in the CIA-induced osteoarthritis (OA) model. ME treatment also reduced tumor necrosis factor (TNF)-α and C-reactive protein (CRP) levels in plasma and ankle tissues. Furthermore, COX-1, COX-2, NF-κB, TNF-α, and IL-6 expressions were attenuated after ME treatment. In most experiments, the antinociceptive and anti-inflammatory effects induced by ME treatment were almost equal to or slightly better than those induced by Perna canaliculus (PC) treatment, which was used as a positive control. Our results suggest that ME possesses antinociceptive and anti-inflammatory effects, indicating its potential as a therapeutic agent for arthritis treatment.

Agrimonia pilosa Ledeb.: A review of its traditional uses, botany, phytochemistry, pharmacology, and toxicology.

Ethnopharmacological relevance: Agrimonia pilosa Ledeb. is the dried above-ground part of dragon's tooth grass, a plant of the Rosaceae family, which is widely distributed in China, Korea, and Japan. Agrimonia pilosa Ledeb. is a herbal medicine with great scope for development and use. It is astringent and hemostatic, and it is used for treating malaria, preventing dysentery, detoxification, and as a tonic for deficiency. Aim of the review: We summarize the traditional uses, botanical and chemical composition, extraction methods, and pharmacological and toxicological progress of Agrimonia pilosa Ledeb. and discuss the future research trends and development prospects of this plant. Materials and methods: Information on Agrimonia pilosa Ledeb. was gathered via the Internet (China National Knowledge Infrastructure, Google Scholar, PubMed, Web of Science, SpringerLink, Wiley, Wanfang Data, and Baidu Academic). Additional information was obtained from books (Ben Cao Tu Jing, A Textual Research on the Name and Reality of Plants, Modern Practical Chinese Medicine, Zhen Nan Ben Cao) and PhD and MS dissertations. Results: Phytochemical studies have identified more than 252 compounds from Agrimonia pilosa Ledeb., including flavonoids, volatile oils, tannins, phenols, m-benzotrienols, pentacyclic triterpenoids, isocoumarins, lignans, organic acids, and other chemical constituents. The compounds and extracts isolated from Agrimonia pilosa Ledeb. show various pharmacological activities, including anti-inflammatory, anticancer, antitumor effects, antioxidant, analgesic effects, and other pharmacological effects. Conclusion: This review highlights the botany, phytochemistry, pharmacology, toxicology, and traditional uses of Agrimonia pilosa Ledeb., providing a basis for future research and clinical applications. Agrimonia pilosa Ledeb. has shown remarkable effectiveness in the treatment of various diseases, especially enteritis, gastric ulcers, and gastrointestinal bleeding. Most prescriptions for Agrimonia pilosa Ledeb. are empirical and lack rigorous clinical observation. For these reasons, the toxicology, standardized clinical studies, nature of active ingredients, pharmacokinetics, mechanism, and metabolism of Agrimonia pilosa Ledeb. should be deepened, especially through clinical trials, to ensure the clinical safety of its use for further research.

Quercetin and Quercitrin from Agrimonia pilosa Ledeb Inhibit the Migration and Invasion of Colon Cancer Cells through the JNK Signaling Pathway.

Considering the high metastatic potential of colorectal cancer (CRC), the inhibition of metastasis is important for anti-CRC therapy. Agrimonia pilosa Ledeb (A. pilosa) is a perennial herbaceous plant that is widely distributed in Asia. The extracts of A. pilosa have shown diverse pharmacological properties, such as antimicrobial, anti-inflammatory, and antitumor activities. In the present study, the antimetastatic activity of A. pilosa was evaluated. Methanol extraction from the roots of A. pilosa was performed by high-performance liquid chromatography (HPLC) and 12 fractions were obtained. Among these, fraction 4 showed the most potent inhibitory effect on the migration of colon cancer cells. Using LC-HR MS analysis, quercetin and quercitrin were identified as flavonoids contained in fraction 4. Like fraction 4, quercetin and quercitrin effectively inhibited the migration and invasion of RKO cells. While the level of E-cadherin was increased, the levels of N-cadherin and vimentin were decreased by the same agents. Although they all activate the p38, JNK, and ERK signaling pathways, only SP600125, an inhibitor of the JNK pathway, specifically inhibited the effect of fraction 4, quercetin, and quercitrin on cell migration. An in vivo experiment also confirmed the antitumor activity of quercetin and quercitrin. Collectively, these results suggest that A. pilosa and its two flavonoids, quercetin and quercitrin, are candidates for the antimetastatic treatment of CRC.

Secondary metabolites isolated from Agrimonia pilosa Ledeb.

A pilot study on the ethanol extracts of Agrimonia pilosa found to have anti-α-glucosidase and anti-inflammatory activities. Subsequent chemical study afforded a new phenylethyl isocoumarin glycoside (1) and eight known compounds (2-9). The structure of 1 was elucidated by comprehensive spectroscopic analysis and chemical transformations. All compounds showed modest α-glucosidase inhibitory activity (IC50 values ranging from 36.8 to 210.7 µM), which was lower than that of the positive control acarbose (IC50=301.9 µM). Those compounds except inactive compounds 3 and 6 showed weak anti-inflammatory activity.[Formula: see text].

Oxidative Stress-Mediated Antibacterial Activity of the Total Flavonoid Extracted from the Agrimonia pilosa Ledeb. in Methicillin-Resistant Staphylococcusaureus (MRSA).

(1) Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a zoonotic pathogen that causes endocarditis, pneumonia, and skin diseases in humans and livestock. (2) Methods: The antibacterial effect of the total flavonoid against MRSA (ATCC43300) extracted from the Agrimonia pilosa Ledeb. (A. pilosa Ledeb) was evaluated by the microdilution method. The oxidative stresses in MRSA were evaluated by the levels of intracellular hydrogen peroxide (H2O2), reactive oxygen species (ROS), and oxidative stress-related genes. The DNA oxidative damage was tested by the 8-hydroxy-2'-deoxyguanosine (8-OHdG) and DNA gel electrophoresis. The differentially expressed proteins were determined by the method of SDS-PAGE and NanoLC-ESI-MS/MS, while the mRNAs of differential proteins were determined by Real-Time PCR. The changes of ultra-structures in MRSA were observed by Transmission Electron Microscope (TEM). (3) Results: The minimum inhibitory concentration (MIC) of the total flavonoid against MRSA was recorded as 62.5 µg/mL. After treatment with the total flavonoid, the levels of intracellular H2O2 and ROS were increased and the gene expressions against oxidative stress (SodA, katA, TrxB) were decreased (p < 0.01), while the gene expression for oxidative stress (PerR) was increased (p < 0.01). The level of intracellular 8-OHdG in MRSA was increased (p < 0.01) and the DNA was damaged. The results of TEM also showed that the total flavonoid could destroy the ultra-structures in the bacteria. (4) Conclusions: The total flavonoid extracted from the A. pilosa Ledeb can induce the oxidative stress that disturbed the energy metabolism and protein synthesis in MRSA.

The Effects of Agrimonia pilosa Ledeb, Anemone chinensis Bunge, and Smilax glabra Roxb on Broiler Performance, Nutrient Digestibility, and Gastrointestinal Tract Microorganisms.

Poultry farming is growing globally, particularly in developing countries, to meet the demands of growing populations for poultry meat and eggs. This is likely to lead to an increase in the use of antibiotics in poultry feed, thus contributing to the development and spread of antibiotic resistance which, poses a serious threat to human and animal health worldwide. One way of reducing this threat is to reduce the use of antibiotics in poultry production by finding effective and sustainable antibiotic alternatives that can be used to support poultry health and productivity. Therefore, this study evaluates the incorporation of three medicinal plants, Anemone chinensis Bunge, Smilax glabra Roxb, and Agrimonia pilosa Ledeb, in poultry feed on production performance, nutrient digestibility, and bacteria in the chicken caecum in a 35-day performance trial with 420-day-old male Ross 308 broilers. Groups of randomly selected chicks received one of six dietary treatments. These included five experimental diets of reduced nutrient specifications as a negative control (NC); with amoxicillin as a positive antibiotic control (PC1); with A. pilosa Ledeb (NC1); with A. chinensis Bunge (NC2); and with S. glabra Roxb (NC3). One other positive control diet contained the recommended nutrient specification (PC2). Weight gain and feed intake were measured weekly and used to calculate the feed conversion ratio as performance parameters. Bacteria were enumerated from chicken caecum using a traditional plating method and selective agar. S. glabra Roxb and A. chinensis Bunge showed comparable effects to amoxicillin with significantly increased weight gain in birds offered these diets, compared to those offered the negative control from days 0 to 35 (p < 0.001). S. glabra Roxb exhibited effects similar to the amoxicillin control group with an improved feed conversion ratio (p < 0.001). In addition, S. glabra Roxb decreased numbers of E. coli and Campylobacter spp. on days 21 (p < 0.05) and 35 (p < 0.01) and increased numbers of lactic acid bacteria comparable to the antibiotic group on days 14 (p < 0.001) and 35 (p < 0.01). The findings of this in vivo trial highlight the potential of S. glabra Roxb and A. chinensis Bunge as beneficial feed material to promote poultry health and productivity in the absence of antibiotics.

The Anti-Inflammatory and the Antinociceptive Effects of Mixed Agrimonia pilosa Ledeb. and Salvia miltiorrhiza Bunge Extract.

Arthritis is a common condition that causes pain and inflammation in a joint. Previously, we reported that the mixture extract (ME) from Agrimonia pilosa Ledeb. (AP) and Salvia miltiorrhiza Bunge (SM) could ameliorate gout arthritis. In the present study, we aimed to investigate the potential anti-inflammatory and antinociceptive effects of ME and characterize the mechanism. We compared the anti-inflammatory and antinociceptive effects of a positive control, Perna canaliculus powder (PC). The results showed that one-off and one-week treatment of ME reduced the pain threshold in a dose-dependent manner (from 10 to 100 mg/kg) in the mono-iodoacetate (MIA)-induced osteoarthritis (OA) model. ME also reduced the plasma TNF-α, IL-6, and CRP levels. In LPS-stimulated RAW 264.7 cells, ME inhibited the release of NO, PGE2, LTB4, and IL-6, increased the phosphorylation of PPAR-γ protein, and downregulated TNF-α and MAPKs proteins expression in a concentration-dependent (from 1 to 100 µg/mL) manner. Furthermore, ME ameliorated the progression of ear edema in mice. In most of the experiments, ME-induced effects were almost equal to, or were higher than, PC-induced effects. Conclusions: The data presented here suggest that ME shows anti-inflammatory and antinociceptive activities, indicating ME may be a potential therapeutic for arthritis treatment.

Anti-Nociceptive Effect and Standardization from Mixture of Agrimonia pilosa Ledeb and Salvia miltiorrhiza Bunge Extracts.

Agrimonia pilosa Ledeb has been previously reported to produce an anti-nociceptive effect in ICR mice in both tail-flick and hot-plate tests. Studies have shown that Salvia miltiorrhiza Bunge, also renowned in traditional Chinese medicine, is an effective anti-inflammatory treatment. Among the extraction solvents investigated, a 50% ethanol (EtOH) extract of A. pilosa produced the highest anti-nociceptive effect in monosodium uric acid-induced gout pain models and the greatest yield. The 80% EtOH extract of S. miltiorrhiza moderately inhibited lipopolysaccharide-induced nitric oxide release from RAW 264.7 murine macrophages and exhibited outstanding yield. The mixture of optimized A. pilosa and S. miltiorrhiza extracts were evaluated for enhanced anti-nociceptive effects in gout arthritis treatment. To control extract quality, four marker compounds were determined using an HPLC-DAD method. A 1:1 mixture of A. pilosa 50 and S. miltiorrhiza 80% EtOH extracts of produced better results than when the extracts were administered individually.

Opposite effects of Agrimonia pilosa Ledeb aqueous extracts on blood coagulation function.

BACKGROUND: Agrimonia pilosa Ledeb (APL) has showed anticoagulant and antithrombotic activities in some studies, whereas its actual effects on blood coagulation are still unclear. This study was designed to observe the in vitro effects of APL aqueous extracts on blood coagulation, as well as to investigate the underlying mechanisms. METHODS: Studies were divided into four groups: 0, 4, 20, and 80 g/L of APL aqueous extracts mixed with plasma or whole blood samples. Clotting time of whole blood, plasma coagulation tests, activities of plasma coagulation factors, plasma calcium ion, platelet aggregation test, and platelet fibrinogen receptor as well as the blood viscosity were measured. RESULTS: It was observed that the APL aqueous extracts in 4 g/L significantly prolonged the whole blood clotting time and activated partial thromboplastin time, shortened prothrombin time, decreased activities of coagulation factor VIII, IX and XI, and levels of platelet aggregation and fibrinogen receptor expression. However, coagulation factor VII activity, and blood viscosity were increased after the extracts treatment. And the effects of APL extracts were in a concentration-dependent manner (0-80 g/L). CONCLUSIONS: The results suggest that APL aqueous extracts have a total anticoagulant activity, whereas they exhibit opposite effects of greater anticoagulant activity than pro-coagulant activity.

Chemical constituents from Agrimonia pilosa Ledeb. and their chemotaxonomic significance.

Phytochemical investigation of the ethanol extract from the whole plant of Agrimonia pilosa led to the isolation of 31 compounds, including 16 flavonoids (1-16), 5 triterpenes (17-21), 1 isocoumarin (22), 5 phenolic acids (23-27), 1 ceramide (28), 2 agrimols (29-30) and 1 fatty acid (31). Their structures were determined by various spectroscopic analyses. Compounds 5, 7 and 20 were firstly isolated from the genus Agrimonia, and compounds 6, 10-11, 15, 26, 28 and 31 were isolated from the family Rosaceae for the first time. Moreover, the chemotaxonomic significance of these compounds was summarised.