Background: The chemical characterization is essential to validate the pharmaceutical use of vegetable raw materials. Ultraviolet spectroscopy is an important technique to determine flavonoids, which are important active compounds from Ocimum basilicum. Objective: The objective of this work was to optimize a spectrophotometric method, based on flavonoid-aluminum chloride (AlCl ₃ ) complexation to determine the total flavonoid content (TFC) in leaves of O. basilicum (herbal material), using response surface methodology. Materials and Methods: The effects of (1) the herbal material: Solvent ratio (0.02, 0.03, 0.05, 0.07, and 0.08 g/mL), (2) stock solution volume (0.8, 2.3, 4.4, 6.5, and 8.0 mL) and (3) AlCl ₃ volume (0.8, 1.0, 1.2, 1.4, and 1.6 mL) on the TFC were evaluated. The analytical performance parameters precision, linearity and robustness of the method were tested. Results: The herbal material: Solvent ratio and stock solution volume showed an important influence on the method response. After choosing the optimized conditions, the method exhibited a precision (RSD%) lower than 6% for repeatability (RSD%) and lower than 8% for intermediate precision (on the order of literature values for biotechnological methods), coefficient of correlation of 0.9984, and no important influence could be observed for variations of the time of complexation with AlCl ₃ . However, the time and temperature of extraction were critical for TFC method and must be carefully controlled during the analysis. Conclusion: Thus, this study allowed the optimization of a simple, fast and precise method for the determination of the TFC in leaves of O. basilicum, which can be used to support the quality assessment of this herbal material.

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Background: Quercetin is universally distributed in the plant kingdom and is the most abundant flavonoid in the human diet. In a previous study, we have reported that quercetin stimulated glucose uptake in cultured C2C12 skeletal muscle through an insulin-independent mechanism involving adenosine monophosphate-activated protein kinase (AMPK). AMPK is a key regulator of the whole body-energy homeostasis. In skeletal muscle, activation of AMPK increases glucose uptake through the stimulation of the glucose transporter GLUT4 translocation to the plasma membrane. In liver, AMPK decreases glucose production mainly through the downregulation of the key gluconeogenesis enzymes such as phosphoenolpyruvate carboxylase (PEPCK) and Glucose -6-phosphate (G6Pase). Objective: To study the effect of quercetin on glucose homeostasis in muscle and liver. Materials and Methods: L6 skeletal muscle cells, murine H4IIE and human HepG2 hepatocytes were treated with quercetin (50 μM) for 18 h. Results: An 18 h treatment with quercetin (50 μM) stimulated AMPK and increased GLUT4 translocation and protein content in cultured rat L6 skeletal muscle cells. On the other hand, we report that quercetin induced hepatic AMPK activation and inhibited G6pase in H4IIE hepatocytes. Finally, we have observed that quercetin exhibited a mild tendency to increase the activity of glycogen synthase (GS), the rate-limiting enzyme of glycogen synthesis, in HepG2 hepatocytes. Conclusions: Overall, these data demonstrate that quercetin positively influences glucose metabolism in the liver and skeletal muscle, and therefore appear to be a promising therapeutic candidate for the treatment of in type 2 diabetes.

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Background: Diabetes mellitus is one of the leading chronic diseases worldwide. In patients with poor glycemic control, high blood glucose level may lead to other life-threatening complications. Pandanus amaryllifolius Roxb. (PA) leaves are used in traditional medicine for the treatment of diabetes. Objective: This study evaluated the effect of crude extract from PA leaves on blood glucose level and the hypoglycemic mechanisms. Materials and Methods: Thirty healthy volunteers were asked to drink PA tea (test-group) or hot water (control group) 15 min after glucose loading (75 g) in a standard oral glucose tolerance test. To study hypoglycemic mechanisms, PA leaves were extracted using two different methods. Method 1; dried PA leaves were extracted with distilled water at 90°C for 15 min, and method 2; dried PA leaves were extracted with 95% ethanol. Both PA extracts were tested for α-glucosidase enzyme inhibition, insulin stimulation, and glucose uptake stimulation. Results: The average of blood glucose level in the control group was 5.55 ± 0.98 mmol/l, while in PA treated group was 6.16 ± 0.79 mmol/l which were statistically different (P < 0.001). The results of antihyperglycemic mechanism showed that PA extracts, prepared both methods, could inhibit α-glucosidase enzyme and induce insulin production in rat pancreatic cell (RINm5F) in dose-dependent manner (P < 0.05). Conclusions: The knowledge gained from this research can be used as a basis for a new drug discovery for the treatment of diabetes.

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Background: Plants of the family Combretaceae are amongst the most widely used plants for traditional medicinal purposes in southern Africa. In particular, many species of Combretum and Terminalia are used for their antibacterial, antifungal, antiprotozoal, antiviral, antidiarrhoeal, analgesic, antimalarial, antioxidant, anti-inflammatory and anticancer activities, yet their antimicrobial potential has not been rigorously studied and compared. Materials and Methods: A survey of antimicrobial activity was undertaken on selected South African Combretum and Terminalia species. Sixteen extracts from 6 Combretum and 2 Terminalia plant species with a history of medicinal usage were investigated by disc diffusion assay against a panel of bacteria and fungi and their MIC values were determined. Toxicity was determined using the Artemia franciscana nauplii bioassay. Results: All extracts tested displayed broad spectrum antibacterial activity, inhibiting the growth of 12-16 (75-100%) of the bacteria tested, with Gram-positive and Gram-negative bacteria being approximately equally susceptible. Potent antibacterial activities (generally in the range 200-5000 μg/ml) were evident for all Combretaceae extracts against both Gram-positive and Gram-negative bacteria. Similarly, the extracts also displayed good antifungal activity, inhibiting the growth of 2-3 (66.7-100%) of the fungal species tested, with fungal growth inhibition activities generally in the range 200-4000 μg/ml. In general, the Terminalia extracts had better efficacies than the Combretum extracts. Furthermore, the methanol extracts were generally better antimicrobial agents than the water extracts. All extracts were also shown to be non-toxic in the Artemia nauplii bioassay. Conclusion: The lack of toxicity of these extracts and their inhibitory bioactivity against a panel of bacteria and fungi indicate their potential as medicinal agents and partially validate their usage in multiple South African traditional medicinal systems.

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Background: Clausena harmandiana Pierre. (CH) contains various bioactive analytes with pharmacological benefits. Most researches were focused on carbazole analytes determined by isocratic high-performance liquid chromatography (HPLC), only few were focused on coumarin analytes and harvested location. Objective: To develop and validate gradient HPLC method to analyze the variance of nine target analytes contained in roots of CH grown naturally in four different provinces of Thailand. Materials and Methods: The analytical method was undertaken by gradient HPLC with 3% tetrahydrofuran in acetonitrile, and 0.05% phosphoric acid in water as mobile phases, on Hypersil ODS column (4.0 × 250 mm, 5 μm), at flow rate 1.0 mL/min and detected at wavelength 280 nm. The method was validated for system linearity, limit of detection, limit of quantitation, precision and accuracy. Results: The new-developed method was able to detect the nine target analytes in CH root. The validation showed the reliability of the method. All system suitability parameters were within the satisfied limits. The linear responses of method were observed at r² ≥ 0.999 for all analytes. The obtained amount of nine analytes showed the biodiversity of contents in different provinces. Of the nine target analytes, the level of nordentatin and dentatin in coumarin groups were considerably high in plants collected from one specific province of Thailand. Conclusion: This study has shown that the new-developed method is reliable, precise, accurate and sensitive to determine and quantify the nine target analytes in CH. Nordentatin and dentatin obviously show the higher level in one specific province of Thailand.

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Background: Extraction process of dried flowers of formula particles should be investigated from lab investigation to pilot-scale because of good water absorbing capacity and obscure active pharmaceutical ingredients (API) dissolution. Objective: Reliable analysis of on-line near-infrared (NIR) technique and novel application in fascinating modern, traditional Chinese medicine production (formula particles) was proved. Materials and Methods: The extraction process of Sophora japonica L. (formula particles) was used as an example, the rutin was regarded as API. On-line NIR technology was used to monitor the variation of rutin in the extraction process. High-performance liquid chromatography (HPLC) was used as a reference method to determine the content of rutin during the extraction process. The sample set was selected by Kennard-Stone (KS) algorithm. Different pretreatment methods were compared. The synergy interval partial least square (SiPLS) algorithm was applied. Chemometrics indicators and multivariate detection limits method were mutually used to assess the model. Results: According to both errors α (0.05) and β (0.05), rutin content could be detected by on-line NIR, which was more than 0.181 mg/mL. Conclusions: This work demonstrated the feasibility of NIR for on-line determination of rutin in the pilot-scale extraction process of S. japonica. L. It provided technical support for the NIR application in the extraction process of formula particles.

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The present study aimed at characterizing the oil extracted from Bertholletia excelsa H.B.K. almond, a native species from the Amazon region. Analytical methods used for oils and fats were employed through pharmacopoeia assays, AOCS (American Oil Chemists Society) standard methods as well as those recommended by ANVISA (National Health Surveillance Agency) such as acidity, peroxide value, saponification index, iodine value and refractive index, pH and relative density, and also thermoanalytical analyses (thermogravimetry, differential thermogravimetry and differential thermal analysis) as well as chromatographic analysis (gas chromatography). The characterization assessments of B. excelsa oil showed results indicating that the oil contains polyunsaturated fatty acids in large proportion. The termoanalytical tests indicated that B.excelsa oil showed thermal stability up to 220 °C, These results showed that the oil extracted from B. excelsa has acceptable characteristics and is of good quality.

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Background : The standardized liquid extract of Brosimum gaudichaudii Trécul is an alternative for the treatment of vitiligo. There is a shortage of solid oral dosage forms developed from standardized extracts of this plant specie. Objective: This study is aimed to obtain pellets with a standardized liquid extract of B. gaudichaudii. Results: The standardized liquid extract of B. gaudichaudii was obtained through maceration and percolation with a 55% ethanol-water solution (v/v). Pellets were obtained through a mixture of extract of 500 g of B. gaudichaudii standardized extract, 500 g of microcrystalline cellulose PH101 and 10 g of hydroxypropyl methylcellulose K100. The pellets obtained presented a homogeneity yield of 92%, aspect ratio of 1.16 ± 0.65, shape fator e_R of 0.35 ± 0.09 and Feret diammeter of 0.87 ± 0.27. These pellets were coated with a suspension composed of titanium dioxide, aluminum red lacquer, ethyl cellulose, talc and magnesium stearate. Before the photostability test, the uncoated pellets showed psoralen content equal to 0.13 ± 0.01% and to the 5-MOP was 1.40 ± 0.27%. After exposure to one level (3 J.cm ^-2 ) of UVB irradiation the uncoated pellets presented a degradation of 2.16% of psoralen and 8.1% of 5-MOP. After exposure to three levels (10, 20 and 30 J.cm ^-2 ) of UVA irradiation the uncoated pellets exhibited photodegradation of 9.78, 17.64, 24.21% of psoralen and 18.95, 23.68, 28.48% for 5-MOP. The coated pellets where unaffected after photostability test. Conclusion: Pellets were obtained with the standardized liquid extract of B. gaudichaudii and coating is a technological alternative to ensure the stability of the formula.

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Background: Alpha-pinene (α-pinene) is a monoterpene commonly found in essential oils with gastroprotective activity obtained from diverse medicinal plants, including Hyptis species. The genus Hyptis (lamiaceae) consists of almost 400 species widespread in tropical and temperate regions of America. In the north and northeastern Brazil, some Hyptis species are used in traditional medicine to treat gastrointestinal disturbances. Objective: The present study has investigated the gastoprotective effect of purified α-pinene in experimental gastric ulcer induced by ethanol and indomethacin in mice. Materials and Methods: Gastric ulcers were induced in male Swiss mice (20-30 g) by oral administration of absolute ethanol or indomethacin 45 min after oral pretreatment with vehicle, standard control drugs or α-pinene (10, 30, and 100 mg/kg). One hour after the ulcerative challenges, the stomach were removed, and gastric lesions areas measured. The effects of α-pinene on the gastric juice acidity were determined by pylorus ligation model. The gastrointestinal motility and mucus depletion were determined by measuring the gastric levels of phenol red and alcian blue, respectively. Hematoxylin and eosin stained sections of gastric mucosa of the experimental groups were used for histology analysis. Results: α-pinene pretreatment inhibited ethanol-induced gastric lesions, reduced volume and acidity of the gastric juice and increased gastric wall mucus (P < 0.05). Furthermore, we showed an interesting correlation between concentration of α-pinene and gastroprotective effect of Hyptis species (P Pearson = 0.98). Conclusion: Our data showed that the α-pinene exhibited significant antiulcerogenic activity and a great correlation between concentration of α-pinene and gastroprotective effect of Hyptis species was also observed.

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Background: The present study was designed to investigate the effect of Saraca indica (SI) flowers extract and different bioactive fraction on in vitro aldose reductase (AR) inhibitory activity, high glucose-induced cataract in goat lens and in vivo streptozotocin (STZ; 45 mg/kg, i.p) induced cataract in rats. Methods: Extract of flowers of SI tested for inhibition against rat lens AR. Furthermore, bioactive fraction was investigated against high glucose-induced opacification of the lens in vitro lens culture and STZ induced diabetic cataract in rats. Identification of the bioactive component was attempted through high-performance thin-layer chromatography, high-performance liquid chromatography and liquid chromatography-mass spectrometry analysis. Results: Ethyl acetate fraction of S. indica (EASI) produced maximum inhibition that may be due to high phenolic content. Goat lenses in media containing glucose developed a distinctly opaque ring in 72 h and treatment with EASI fraction lowered lens opacity in 72 h. Prolonged treatment with EASI to STZ-induced diabetic rats inhibited the AR activity and delayed cataract progression in a dose dependent manner. Conclusion: Ethyl acetate fraction of S. indica fraction has potential to inhibit rat lens AR enzyme and prevent cataractogenesis not only in goat lens model (in vitro), but also in STZ induced diabetic rats (in vivo). This study is suggestive of the anticataract activity of EASI fraction that could be attributed to the phytoconstituents present in the same.

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Background: Fructus Sophorae pill, one of the traditional Chinese medicine, was widely used for hemorrhoids, hypertension and odontalgia. This paper describes a sensitive and specific assay for the determination of the 15 active constituents (sophoricoside, genistin, genistein, rutin, quercetin, kaempferol, baicalein, baicalin, naringin, naringenin, hesperidin, neohesperidin, wogonin and cimifugin, prim-O-glucosylcimifugin) in Fructus Sophorae pill. Materials and Methods: Chromatographic separation was performed on a C ₁₈ column with acidified aqueous methanol gradients at a flow rate of 0.8 mL/min. The identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was applied to quantification with switching electrospray ion source polarity between positive and negative modes. Results: The proposed method was used to analyze 40 batches of samples with good linearity (r, 0.9990-0.9999), intraday precisions (RSD, 0.14-2.55%), interday precisions (RSD, 0.51-2.81%), stability (RSD, 0.31-2.65%), and recovery (RSD, 1.29-2.95%) of the 15 compounds. In addition, the hierarchical cluster analysis, including a method called furthest neighbor and nearest neighbor, was employed to classify samples according to characteristics of the 15 constituents. Conclusion: The results indicated that the analytical method was rapid, reliable, simple and suitable for the quality evaluation of Fructus Sophorae pill.

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Background: Bupleurum L. (Aspiaceae) species are used as herbal remedy in Chinese traditional medicine. Objective: The aim was to investigate the flavonoids in three annual European Bupleurum species, including B. baldense, B. affine and B. flavum, and to test their antioxidant and possible hepatoprotective effects. Materials and Methods: Flavonoids from the methanol-aqueous extracts were quantified by solid-phase extraction-high-performance liquid chromatography. Bupleurum extracts (1-220 mg/ml) were tested for their antioxidant activity in DPPH and ABTS assays, as well as on isolated liver rat microsomes. In vitro hepatoprotective activity of B. flavum flavonoid (BFF) mixture and rutin, and narcissin, isolated from the same mixture, were evaluated on carbon tetrachloride (CCl ₄ ) and tert-butyl hydroperoxide (t-BuOOH) toxicity models in isolated rat hepatocytes. Results: Narcissin was the dominant flavonol glycoside in B. flavum being present at 24.21 ± 0.19 mg/g, whilst the highest content of rutin (28.63 ± 1.57 mg/g) was found in B. baldense. B. flavum possessed the strongest DPPH (IC ₅₀ 22.12 μg/ml) and ABTS (IC ₅₀ 118.15 μg/ml) activity. At a concentration 1 mg/ml of BFF (rutin 197.58 mg/g, narcissin 75.74 mg/g), a stronger antioxidant effect in microsomes was evidenced in comparison with silymarin, rutin and narcissin. The hepatoprotective effect of BFF significantly reduced the elevated levels of lactate dehydrogenase and malondialdehyde, and ameliorated glutathione, being most active in t-BuOOH-induced injury model when compared with CCl ₄ toxicity (P < 0.001). Conclusion: In BFF, synergism of rutin and narcissin could be responsible for stronger protection against mitochondrial induced oxidative stress.

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Objective: In the present study, we investigated the efficacy of a methanol extract from Impatiens balsamina L. (MEIB) against HSC-2 human oral cancer cells. Materials and Methods: The anti-cancer efficacies of MEIB were performed by methanethiosulfonate assay, phospho-kinase array, Western blot, 4'- 6-diamidino-2-phenylindole staining, trypan blue exclusion assay and 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide assay. Results: MEIB decreased the cell viability of HSC-2 cells. According to phospho-kinase arrays, MEIB markedly activated AMP-activated protein kinase (AMPK) signaling, but inactivated mammalian target of rapamycin signaling. MEIB induced apoptosis as evidenced by activation of caspase-3, poly (ADP-ribose) polymerase cleavage and nuclear condensation. In addition, AMPK activation by two known activators (5-aminoimidazole-4-carboxamide-1-β-ribofuranoside and metformin) decreased cell viability and induced apoptosis. Moreover, MEIB increased the expression levels of mitochondria-related proteins (t-Bid, Bak and Bad), which contributed to the disruption of mitochondrial membrane potential, cytochrome C release and activation of caspase-9. Metformin also increased t-Bid expression and the subsequent release of cytochrome C into the cytosol. Conclusion: These results suggest that MEIB may be of therapeutic value for treating oral cancer and that its mechanism of action occurs through AMPK and t-Bid.

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Background: Saussurea lappa is one of the popular Ayurvedic herb; costunolide and dehydrocostus lactones are well-known sesquiterpene lactones contained in many plants used as popular herbs, such as S. lappa, and have been considered as potential candidates for the treatment of various types of tumor. Objective: The present study was used for the quantification of costunolide and dehydrocostus lactone in S. lappa and its polyherbal formulations, stability studies of markers and characterization of their degradants. Materials and Methods: HPLC analysis was performed on Waters NOVAPAK HR C ₁₈ column (300 mm Χ 3.9 mm i.d., 6 μm) using isocratic elution with acetonitrile and water (60:40% v/v). Results: The calibration curves of both analytes showed good linearity within the established range 5-100 μg/ml. The limits of detection (LOD) and quantification (LOQ) were 1.5 and 4.6 μg/ml for costunolide and 1.3 and 4.0 μg/ml for dehydrocostus lactone, respectively. Good results were achieved with respect to repeatability (%RSD < 2.0) and recovery (99.3-101.8%). Conclusion: The method was found to be precise, accurate, specific, and was successfully used for analyzing costunolide and dehydrocostus lactone in S. lappa and its polyherbal formulations. The developed method was found to be suitable for stability studies of markers and characterization of their degradation products.

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Background: Lavatera cretica L. was used in folk medicine as anti-inflammatory among other applications. As inflammation is many times associated with oxidative processes, the aim of the present work was to evaluate the ability of aqueous extracts obtained from different parts of L. cretica to prevent oxidation or inflammation using several methods in vitro. Materials and Methods: The capacity of samples for preventing lipid peroxidation, scavenging free radicals, chelating metal ions, reducing power, and inhibiting lipoxygenase activity was investigated. This last assay also permits to evaluate the anti-inflammatory activity. The quantification of total phenols was performed using Folin-Chiocalteu reagent. Results: The highest concentrations of total polyphenols and flavonoids were found in the leaf extract (254.62 ± 6.50 mg gallic acid equivalent/gram; dry weight). Leaf and flower extracts were the most active for scavenging 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt free radicals [Inhibition concentration (IC 50 = 2.88 ± 0.54 and IC50 = 4.37 ± 0.54 ΅g/mL, respectively)], and leaf extract was also the best for scavenging hydroxyl radicals (IC ₅₀ = 0.81 ± 0.05 μg/mL). Bract plus sepal extract possessed the best capacity for preventing lipid peroxidation when lecithin liposome was the lipid substrate (IC ₅₀ = 0.19 ± 0.03 μg/mL) and scavenging superoxide anion radicals (IC ₅₀ = 1.13 ± 0.48 μg/mL). Leaf and flower extracts were the best lipoxygenase inhibitors (IC ₅₀ = 0.013 ± 0.0034 μg/mL in both extracts). Conclusions: L. cretica extracts were able to scavenge free radicals, inhibit lipid peroxidation and lipoxygenase activity. With these attributes, this plant can have an important role in the treatment of neurodegenerative disorders.

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Background: In the last decade, a growing interest particularly in determining the cardiovascular effects of herbal extracts took place among researchers. Objective: Herein, we aimed to investigate the microvascular and blood pressure lowering effects of two differently processed extracts of the same herb, Alchemilla vulgaris (Rosaceaea), which was revealed to contain high levels of vasoactive compounds. Materials and Methods: For the purpose, endothelium intact rat mesenteric arteries were mounted in a myograph system and contracted with prostaglandin F _2α (PGF _2α: 3 × 10⁻⁵ M) or potassium chloride (K ⁺ : 40 mM). Then, aqueous and methanol extracts were added at 0.01-10 mg/ml concentrations in a cumulative manner. Results: Both extracts produced relaxations in PGF _2α (3 × 10⁻⁵ M) precontracted arteries which were insensitive to the inhibitors of endothelium derived vasoactive substances namely, L ^G -nitro-L-arginine (10⁻⁴ M), ODQ (10⁻⁵ M) and indomethacin (10⁻⁵ M) or removal of endothelium. Opposite vascular effects were observed when extracts were applied in K ⁺ precontracted arteries. In addition, oral administration of the methanol extract of Alchemilla vulgaris, but not the aqueous extract, reduced blood pressure significantly in L-NAME hypertensive rats. Conclusion: Our results demonstrated that the methanol extract of Alchemilla vulgaris has more prominent and favourable vascular effects in normal and experimental hypertensive conditions reinforcing its traditional use in cardiovascular disorders, in particular hypertension. These results most likely give rise to further studies to reveal its mechanism of action and clinical value of this herb.

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Background: Agrimonia Pilosa Ledeb (APL), a traditional Chinese medicine, has been reported a variety of biological activities, including treating T2DM. Objective: Triterpenoid compound (TC) was collected from APL. The aim of this study was to investigate the effects of TC on 3T3-L1 preadipocytes differentiation and genes related to differentiation and IR. Materials and Methods: Column chromatography was used to collect TC from ALP. 3T3-L1 cell differentiation was induced typically in the presence of various concentrations of TC or pioglitazone. Oil red O staining and measurement of intracellular TG content were performed on the seventh day of differentiation. Then quantitative polymerase chain reaction (Q-PCR) was used to test the expressions of three transcription factors ͳ͸PPARγ, CCAAT enhancer binding protein-α (C/EBP-α), and sterol regulatory element-binding protein 1 (SREBP-1)ͳ͹ and the target genes of PPARγ including glucose transporter (GLUT4), lipoprotein lipase (LPL), fat acid binding protein (AP2), and adiponectin in 3T3-L1 cells. Results: At the concentration of 5, 25 and 125 μg/mL, TC significantly promoted triglyceride accumulation. Further study showed that TC could promote the expression of PPARγ, C/EBPα and ADD1/SREBP1 significantly at 125 μg/mL. As for downstream genes controlled by PPARγ, TC at 25 and 125 μg/mL could significantly promote the expression of GLUT4 and adiponectin. However, the expression of aP2 related to lipid metabolism and adiposity in the TC group was significantly lower than that in the pioglitazone group. Conclusion: TC could promote preadipocytes differentiation through activating PPARγ and downstream controlled genes. TC has the ideal insulin sensitization with lower adipogenic action than classical TZDs in vitro. So TC from Agrimonia Pilosa Ledeb has a good prospect as a natural drug for IR and T2DM.

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Objective: To determine the free amino acid contents of chamomile flowers using reverse-phase high-performance column chromatography preceded by pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), and to determine the reliability of this method. Materials and Methods: Derivatization with reconstituted AQC was used to prepare the samples and standards for injection into the chromatography column. The peaks were analyzed by fluorescence detection (λ excitation, 250 nm; λ emission, 395 nm. Results: Alanine, proline, and leucine were the most abundant amino acids, whereas tyrosine and methionine were the least abundant. The linearity of the method was found to be good with amino acid concentrations of 0.012-0.36 μM. The precision was 0.05-1.36%; average recovery, 91.12-129.41%; and limit of detection, 0.006-0.058 μM. Conclusion: The method is reliable for determining the free amino acid content of different types of chamomile flowers.

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Background: To extract, purify and identify the active constituents in ethanol extract of Radix Salviae Miltiorrhizae, and to analyze the protective effects of tanshinone IIA on cerebral ischemia-reperfusion injury in rats. Materials and Methods: Radix Salviae Miltiorrhizae was extracted by ultrasonic extraction, effective parts were extracted by extraction method, compounds were isolated by preparative TLC and preparative HPLC, and structures of compounds were identified by ¹ H NMR and ¹³ C NMR; the effects of tanshinone IIA on cerebral ischemia-reperfusion injury in rats were determined by establishing rat model of middle cerebral artery occlusion (MCAO). Results: The experimental data show four compounds were isolated, namely tanshinone IIB, hydroxymethylene tanshinone, salvianolic acid B and 9"'-methyl lithospermate B. Tanshinone IIA could alleviate the symptoms of neurological deficit in rats, the neurological deficit alleviating effect became more obvious with the increase of dose; tanshinone IIA experimental groups could reduce the cerebral infarction size and brain water content in rats, different concentrations of tanshinone IIA could decrease the SOD content and increase the MDA content in the frontal and parietal cortices of ischemic hemisphere in the ischemia reperfusion group, the differences were statistically significant compared with the ischemia reperfusion group. Conclusion: Radix Salviae Miltiorrhizae has the protective effects on cerebral ischemia reperfusion injury in rats.

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Background: Ethanol causes hepatic cellular damage by alterations in biological functions. This study evaluated the hepatoprotective potential of the methanolic extract originating from Firmiana simplex (Sterculiaceae) stem bark against the ethanol-induced hepatotoxicity in rat primary hepatocytes. Materials and Methods: The extract of F. simplex stem bark was successively fractionated into n-hexane, chloroform, ethyl acetate (EtOAc), and n-butanol. Column chromatography with silica gel and sephadex LH-20 was used to isolate the EtOAc fraction. Rat primary hepatocytes were cultured to study the hepatoprotective activity of isolated substances against ethanol-induced toxicity. Intracellular reactive oxygen species (ROS) levels, the antioxidant activities of glutathione reductase (GR) and glutathione peroxidase (GSH-P _X ) enzymes, and the GSH content were measured to examine the antioxidative property of the isolated compounds. Results: Two flavonoid glycosides, quercitrin (1) and tamarixetin 3-O-rhamnopyranoside (2), were isolated from the active EtOAc fraction. Compound 1 significantly protected rat primary hepatocytes against ethanol-induced oxidative stress by reducing the intracellular ROS level and preserving antioxidative defense systems such as GR, GSH-P _X , and total GSH. Conclusion: This is the first report on the hepatoprotective activities of the extract of F. simplex. The EtOAc fraction of F. simplex stem bark and its major constituent quercitrin (1) could function as hepatoprotective agents to attenuate the development of alcoholic liver disease.

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Background: One of the biological activities of agar wood (Aquilaria sinensis Lour., Thymelaeaceae), is anti-hyperglycemic activity. The methanolic extract (ME) was proven to possess the fasting blood glucose activity in rat and glucose uptake transportation by rat adipocytes. Objective: To determine the decreasing fasting blood glucose level of constituents affordable for in vivo test. If the test was positive, the mechanism which is positive to the ME, glucose transportation, will be performed. Materials and Methods: The ME was separated by column chromatography and identified by spectroscopic methods. Mice was used as an animal model (in vivo), and rat adipocytes were used for the glucose transportation activity (in vitro). Result: Iriflophenone 3-C-β-glucoside (IPG) was the main constituent, 3.17%, and tested for the activities. Insulin and the ME were used as positive controls. The ME, IPG and insulin lowered blood glucose levels by 40.3, 46.4 and 41.5%, respectively, and enhanced glucose uptake by 152, 153, and 183%, respectively. Conclusion: These findings suggest that IPG is active in lowering fasting blood glucose with potency comparable to that of insulin.

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Background: Schisandrol A, a lignan with anticancer effects, is one of the representative components that identifies Schisandra chinensis. Objective: A method for purifying schisandrol A from the stems of S. chinensis was established using an octadecylsilyl (ODS) column combined with preparative high-performance liquid chromatography (HPLC). Materials and Methods: Crude extracts obtained from the stems of S. chinensis using 70% ethanol were separated on an AB-8 macroporous resin column and then eluted with a graded ethanol series. After 70% methanol was used in an ODS column separation, preparative HPLC was used for subsequent purification. The structure was identified by electrospray ionization-mass spectrometry, ¹ H-nuclear magnetic resonance, and ¹³ C-nuclear magnetic resonance. HepG2 and Bel-7402 hepatocellular carcinoma cell lines were used for toxicological evaluation. Results: 21.4 mg of schisandrol A with a purity of 95.2% were collected. The cytotoxicity of the ODS-purified sample and schisandrol A were significantly stronger than that of a resin-purified sample. Conclusion: Schisandrol A was successfully extracted from the stems of S. chinensis and separated with an ODS column combined with preparative HPLC. The samples obtained during the purification process showed different levels of cytotoxicity on the HepG2 and Bel-7402 hepatocellular carcinoma cell lines.

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Background: Hirudo nipponica Whitman and Poecilobdella manillensis Lesson fall into the family of Hirudinidae Whitman, both of them are sanguinovorous leeches and used a anticoagulant medicines in China. Their medicinal parts are the dried bodies. However, the peptides in the dried body of the two leeches have not been very clear up to now. Objective: To analyze the peptides from two sanguinovorous leeches, H. nipponica and P. manillensis. Materials and Methods: In this article it is reported that the peptides were obtained from anticoagulant active extracted parts of dried bodies of the two leeches and their molecular weights were analyzed by ultra-performance liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry mass spectrometric detector online. Results: Three peptide components were identified from H. nipponica with their molecular weight separately 14998, 15988, and 15956, six peptide components were identified from P. manillensis with molecular weight 9590, 13642, 14998, 17631, 15988, and 16567. Two of peptides from P. manillensis have the same molecular weight 14998 and 15988 as that in H. nipponica. Conclusion: And the two peptides are the main peaks in the base peak ion chromatogram because they occupied a large ratio of total base peak area. Hence the composition of the extracted active part of the two leeches are very close, difference is in that the extract of P. manillensis has more small peptide peaks, but the extract of H. nipponica has not. Furthermore, the tryptic digestion hydrolysates of the extracted active part of each sample were analyzed and the results showed that there were four peaks which only exist in P. manillensis, but not in Hirudo nipponia. They may be the identified peak between the two leeches. This work support the viewpoint that P. manillensis can be used as a medicinal leech as H. nipponia and these peptide components of dried bodies of the two species leeches are a basis for their chemical identification and further investigations in active action.

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Background : Yuanhu Zhitong prescription (YZP) is a famous traditional Chinese medicine formula, which is officially recorded in Chinese Pharmacopoeia for the treatment of stomach pain, hypochondriac pain, headache and dysmenorrhea caused by qi-stagnancy and blood stasis. It is the first report for the simultaneous determination of 12 active components in YZP. Objective: A newly, simple, accurate and reliable method for the separation and determination of 12 active components (protopine, α-allocryptopine, coptisine, xanthotol, palmatine, dehydrocorydaline, glaucine, tetrahydropalmatine, tetrahydroberberine, imperatorin, corydaline, isoimperatorin) in YZP was developed and validated using HPLC-PAD. Materials and Methods: The analytes were performed on a Phenomenex Luna-C ₁₈ (2) column (250×4.6 mm, 5.0 μm) with a gradient elution program using a mixture of acetonitrile and 0.1% phosphoric acid water solution (adjusted with triethylamine to pH 5.6) as mobile phase. Analytes were performed at 30°C with a flow rate of 1.0 mL/min. Results: The validated method was applied to analyze four major dosage forms of YZP coming from different manufacturers with good linearity (r ² , 0.9981~0.9999), precision (RSD, 0.24~2.89%), repeatability (RSD, 0.15~3.34%), stability (RSD, 0.14~3.35%), recovery (91.13~110.81%) of the 12 components. Conclusion: The proposed method enables the separation and determination of 12 active components in a single run for the quality control of YZP.

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Background: The whole herb of Lagochilus ilicifolius has been used as a folk medicine for treating hemostatic, inflammation and ulcer in China. There were only limited reports on its chemical constituents, and no reports on its pharmacology study. Objective: To isolate compounds from the whole herb of L. ilicifolius and evaluate their cytotoxic activity. Materials and Methods: The column chromatographic techniques were used for separating the constituents of the n-butanol-soluble fraction of the 95% ethanol extract from the whole plant of L. ilicifolius. The structures of one new lignan and two known lignans were elucidated on the basis of spectroscopic analyses and comparison with literature data. The cytotoxic activities of these three lignans were evaluated using the MTT-assay against PC12 cell line derived from rat adrenal pheochromocytoma. Results: The new lignan was identified as erythro-1-[(4-O-β-D-glucopyranosyl-3-methoxyl)- phenyl]-2-[(5'-methoxyl)-pinoresinol]-propane-1,3-diol (1), and two known lignans were identified as tortoside C (2) and sisymbrifolin (3). The new lignan exhibited significant cytotoxic activity against PC12 cell line with IC ₅₀ value of 1.22 ± 0.03 μmol/L. Conclusions: A new lignan, erythro-1-[(4-O-β-D-glucopyranosyl -3-methoxyl)-phenyl]-2-[(5'-methoxyl)-pinoresinol]-propane-1,3-diol and two known lignans were isolated from the whole herbs of L. ilicifolius. The two known lignans were reported for the first time in the genus Lagochilus. Three lignans were evaluated for in vitro cytotoxic activity. The new lignan showed relatively strong cytotoxicity against PC12 cell line, while sisymbrifolin and tortoside C exibited no cytotoxicity.

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Background: Lippia nodiflora (LN) Linn is a small herb distributed throughout the world. The plant extracts of LN is used traditionally as an analgesic, anti-inflammatory, antioxidant, antibacterial, antimicrobial, antipyretic, antitumor, antidiabetic, and possess hepatoprotective properties. Materials and Methods: To study the antibacterial and hepatoprotective effect of LN, we used methanolic extracts of leaves on HepG2 cells. Lipopolysaccharides (LPS) is a well-characterized hepatotoxin, so toxicity was induced on liver cells using LPS. Up-regulation of inflammation genes were quantified. Results and Conclusions: In our present study, we have showed that LN reduced reactive oxygen species (ROS) production against LPS induced toxicity on HepG2 cells, and ther by decreased the apoptotic gene expression and protect the liver cells against toxicity.

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Background: Ligularia kangtingensis, a species from the genus Ligularia (Compositae), is an indigenous plant in Southwest China and more than 20 species in this genus have been used as folk medicines in China. Objective: The chemical constituents of the whole plant of L. kangtingensis were studied. Materials and Methods: The dried whole plants were extracted with ethanol. Its chemical constituents were mainly isolated and purified by silica gel and Sephadex LH-20 column chromatography and their structures were identified on the basis of spectral analysis. Results: Twelve known terpenoids, including two monoterpenoids, five sesquiterpenoids and five triterpenoids, were isolated and identified from the whole plant of L. kangtingensis. Conclusion: All of the 12 known compounds were isolated for the first time from L. kangtingensis.

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Aims: Given the high-effectiveness and low-toxicity of abnormal savda munziq (ASMQ), its herbal formulation has long been used in traditional Uyghur medicine to treat complex diseases, such as cancer, diabetes, and cardiovascular diseases. Settings and Design: ASMQ decoction by reversed-phase high-performance liquid chromatography coupled with a diode array detector was successfully developed for the simultaneous quality assessment of gallic acid, protocatechuic acid, caffeic acid, rutin, rosmarinic acid, and luteolin. The six phenolic compounds were separated on an Agilent TC-C18 reversed-phase analytical column (4.6 × 250 mm, 5 μm) by gradient elution using 0.3% aqueous formic acid (v/v) and 0.3% methanol formic acid (v/v) at 1.0 mL/min. Materials and Methods: The plant material was separately ground and mixed at the following ratios (10): Cordia dichotoma (10.6), Anchusa italic (10.6), Euphorbia humifusa (4.9), Adiantum capillus-veneris (4.9), Ziziphus jujube (4.9), Glycyrrhiza uralensis (7.1), Foeniculum vulgare (4.9), Lavandula angustifolia (4.9), Dracocephalum moldavica L. (4.9), and Alhagi pseudoalhagi (42.3). Statistical Analysis Used: The precisions of all six compounds were <0.60%, and the average recoveries ranged from 99.39% to 104.85%. Highly significant linear correlations were found between component concentrations and specific chromatographic peak areas (R² > 0.999). Results: The proposed method was successfully applied to determine the levels of six active components in ASMQ. Conclusions: Given the simplicity, precision, specificity, and sensitivity of the method, it can be utilized as a quality control approach to simultaneously determining the six phenolic compounds in AMSQ.

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Background: The Antarctic moss Sanionia uncinata (Hedw.) Loeske has shown high ultraviolet (UV)-absorbers content after exposition to high levels of UV-B radiation and can be an important source of antioxidants. Objective: The aim was to investigate photoprotection and mutagenicity by the aqueous extract (AE) and hydroethanolic extract (HE) from the Antarctic moss S. uncinata. Materials and Methods: Photoprotective activities were determined through survival curves of Escherichia coli strains, after UV irradiation in an aqueous solution of thymine and in vitro sun protection factor (SPF). The Salmonella/microsome assays were applied to assess the mutagenicity. Results: Both extracts induced photoprotection against UV-C radiation. The AE showed a higher protection than the hydroethanolic one against UV-induced thymine dimerization. The SPFs were low in both extracts. In association to benzophenone-3 a significant increase in the SPF was detected for the AE, and a significant decrease was induced by the HE. No mutagenicity was found in the both extracts. Furthermore, it was observed absence of cytotoxicity. Conclusion: Water-extractable compounds seem to contribute on photoprotection of this Antarctic moss.

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Background: Nothapodytes nimmoniana (Grah.) Mabb. is a high valued medicinal plant endemic to Western Ghats of India, distributed in fragmented populations. The plant is valued for potent anticancer drug camptothecin (CPT). Objective: The study compares and expounds variation in CPT content from leaves and stems of N. nimmoniana obtained from three populations of Western Ghats, India. The study also describes a method for categorizing these populations using content range chart (CRC) method for percent yield of CPT. Materials and Methods: A total of 60 samples were investigated including ten each of leaves and stems from three localities. Micro-extraction method was implemented to extract CPT. reversed phase ultra-performance liquid chromatography photo diode array technique was used to quantify CPT. Results: Leaf samples of an individual collected from Joida, yielded lowest CPT content (0.002 ± 0.000 g/100 g), whereas a stem sample from Amgaon, yielded highest CPT content (0.123 ± 0.006 g/100 g). The findings suggest great variation in individuals producing and accumulating CPT. Using this data along with earlier published work, five categories of CPT yielding plants were made viz. I: Very low: <0.020, II: Low: 0.021-0.039, III: Moderate: 0.040-0.059, IV: High: 0.060-0.079 and V: Very high: >0.080. Based on CPT content in leaves, majority of individuals were under very low category (I ^st ) and on the other hand stem samples were in 'II' category. Besides, very few individuals were observed in category 'V'. Conclusion: The study expounds use of CRC method for identifying elite population and suggests the need for its conservation.

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Background: Acanthaceae is one of the advanced and specialized families with conventionally used medicinal plants. Simple sequence repeats (SSRs) play a major role as molecular markers for genome analysis and plant breeding. The microsatellites existing in the complete genome sequences would help to attain a direct role in the genome organization, recombination, gene regulation, quantitative genetic variation, and evolution of genes. Objective: The current study reports the frequency of microsatellites and appropriate markers for the Acanthaceae family genome sequences. Materials and Methods: The whole nucleotide sequences of Acanthaceae species were obtained from National Center for Biotechnology Information database and screened for the presence of SSRs. SSR Locator tool was used to predict the microsatellites and inbuilt Primer3 module was used for primer designing. Results: Totally 110 repeats from 108 sequences of Acanthaceae family plant genomes were identified, and the occurrence of dinucleotide repeats was found to be abundant in the genome sequences. The essential amino acid isoleucine was found rich in all the sequences. We also designed the SSR-based primers/markers for 59 sequences of this family that contains microsatellite repeats in their genome. Conclusion: The identified microsatellites and primers might be useful for breeding and genetic studies of plants that belong to Acanthaceae family in the future.

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Instruction: Despite the increased use of Brosimum gaudichaudii roots as raw material on medicine to treatment of vitiligo, there are not studies that showing the impact of unit operations on the quality and standardized of the extract of B. gaudichaudii. The quality of the herbal extract is essential to ensure the safety and efficacy of pharmaceutical product. Due the medical and commercial importance, this study aimed to evaluate the impact of the extraction method (ultrasound or percolation) on the quality of herbal extract and optimize the extraction of psoralen and 8-methoxypsoralen (8-MOP) from B. gaudichaudii. Materials and Methods: The extraction recovery was evaluate by high-performance liquid chromatography (C8 reverse phase column and acetonitrile: Water 45:55 and flow rate 0.6 mL/min). The extraction was performed by ultrasound-assisted extraction (UEA) or percolation using a Box-Behnken design. Results: From both chemical markers (psoralen and bergapten), the optimal conditions for the UEA were an extraction time of 25 min, the mean particle size of 100 μm, and an ethanol: Water ratio of 55:45 (v/v). Conclusion: The extraction by percolation revealed that ethanol 55% was more efficient than ethanol 80% to extract psoralen and bergapten.

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Background: Liver diseases are on rise and remain a serious health problem. Olax subscorpioidea and Distemonanthus benthamianus are two plants used in Ivorian traditional medicine in the treatment of many diseases including jaundice and hepatitis. Objective: The present study was carried out to assess the hepatoprotective and in vivo antioxidant potentials of the hydro-ethanolic leaf extracts of these plants in carbon tetrachloride (CCl ₄ )-induced hepatotoxicity in rats. Materials and Methods: The plant extracts at doses of 25 and 100 mg/kg bw, and silymarin (25 mg/kg bw) were intraperitoneally (i.p.) injected once daily for 7 days to different groups of rats. Hepatotoxicity was induced on the 7 ^th day in all the group animals except control. Rats were sacrificed on the 8 ^th day and blood was collected. Serum biochemical parameters and antioxidant activity were measured using standard procedures. Histopathological examinations of liver rats were also performed. Results and Discussion: Hepatotoxicity induced with CCl ₄ was well manifested by significant increase in serum activities of GOT, GPT, ALP and GGT, and enhancement of total bilirubin and TBARS levels. On the other hand, the level of total protein, albumin, α₁ -globulin significantly decreased, and DPPH-free radical scavenging activity and TAP were lowered. Pretreatment with plant extracts and silymarin prevent the toxic effects of CCl ₄ by decreasing serum enzyme activities, total bilirubin and TBARS levels and improving serum TAP and DPPH-free radical scavenging potential. Histopathological observations showed almost normal hepatic cells with a mild degree of inflammation, lesser fatty infiltration and absence of necrosis among the rats treated at 100 mg/kg of extracts of both the plants. Conclusion: The results suggest that the hydro-ethanolic leaf extracts of O. subscorpioidea and D. benthamianus possesses hepatoprotective and in vivo antioxidant activity.

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Objective: Investigation of possible effects of Silybum marianum extract (SME) on adenosine deaminase (ADA) activity in cancerous and noncancerous human gastric and colon tissues to obtain information about possible mechanism of anticancer action of S. marianum. Materials and Methods: Cancerous and noncancerous human gastric and colon tissues removed from patients by surgical operations were used in the studies. The extract was prepared in distilled water. Before and after treatment with the extract, ADA activities in the samples were measured. Results: ADA activity was found to be lowered significantly in cancerous gastric tissues but not in noncancerous gastric tissues after treatment with the SME. In the colon tissues, ADA activities were however found to increase after the treatment of SME. Conclusion: Our results suggest that the aqueous extract from S. marianum inhibits ADA activity in cancerous gastric tissues significantly. It is suggested that in addition to other proposed mechanisms, accumulated adenosine due to the inhibition of ADA might also play a part in the anticancer properties of the S. marianum.

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