NF-κB; TNF-α; IKKβ; IL-6

Homoplantaginin exhibits an IC50 of 0.35 μg/mL for DPPH radical reduction. In addition to increasing glutathione, glutathione peroxidase, and superoxide dismutase in the supernatant of human hepatocyte HL-7702 cells exposed to H2O2, homoplantaginin (0.1-100μg/mL) significantly reduces lactate dehydrogenase leakage[1]. Toll-like receptor-4 expression induced by palmitic acid (100 μM) is reduced by homoplantaginin (0.1, 1, 10 μM) dose-dependently. By inhibiting the NLRP3 and caspase-1 proteins, which are reactive oxygen species-sensitive thioredoxin-interacting proteins, homoplantaginin tightly regulates palmitic acid-induced reactive oxygen species to prevent NLRP3 inflammasome activation[2]. Homoplantaginin pre-treatment significantly reduces palmitic acid-induced mRNA expression of TNF-α and IL-6 as well as IKKβ and NF-κB p65 phosphorylation in human umbilical vein endothelial cells. Homoplantaginin significantly alters IRS-1's Ser/Thr phosphorylation, enhances Akt and endothelial nitric oxide synthase phosphorylation, and boosts NO production in the presence of insulin[3].

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Antioxidative effect of Homoplantaginin [1]
To evaluate the direct scavenging effects of homoplantaginin on free radicals, we determined DPPH, a stable free radical, scavenging activity. As shown in Fig. 2, homoplantaginin reduced the levels of DPPH radical at the concentrations up to 1.6 μg/ml and showed the free radical scavenging effects with IC50 values of 0.35 μg/ml. l-Ascorbic acid showed the free radical scavenging effect with an IC50 value of 0.43 μg/ml.

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Cell viability exposed to Homoplantaginin in vitro [1]
HL-7702 cells were exposed to homoplantaginin (0.1, 1, 10, 50, 100, 200 μg/ml) for up to 72 h and then the rates of cell growth inhibition were evaluated based on the viable cell number as estimated by MTT assay. As shown in Fig. 3, homoplantaginin at each of these concentrations did not cause any apparent cytotoxicity (P > 0.05 vs untreated cells).

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Inhibition of LDH release [1]
The protective effect of Homoplantaginin on hepatocyte injury was evaluated by using the model of HL-7702 cells exposure to H2O2. Exposure to H2O2 for 18 h markedly increased LDH leakage into medium. As shown in Fig. 4, the level of LDH was 21 U/ml in supernatant of control cells, while it was increased to 122 U/ml under H2O2 stress. Pretreatment of HL-7702 cells with homoplantaginin significantly inhibited LDH leakage in a dose-dependent manner. At the concentrations of 0.1, 1, 10, 50, and 100 μg/ml, the levels of LDH were reduced to 73.5 ± 4.4, 65.2 ± 5.1, 58.4 ± 3.2, 52.1 ± 2.6, and 41.6 ± 4.3 U/ml, respectively (Fig. 4).

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Increase of GSH, GSH-Px, and SOD [1]
The activity of GSH, GSH-Px, and SOD were evaluated in H2O2-injured HL-7702 cells. Results were showed in Table 1. GSH level in H2O2-injured cells was decreased to 40.5 ± 5.7 nmol/mg protein as compared to a normal value of 58.4 ± 4.5 nmol/mg protein. Homoplantaginin prevented the depletion of GSH by H2O2. A dose-dependent increase of GSH was observed within the concentrations (0.1–100 μg/ml) after 24 h pretreatment (Table 1). We then examined the effects of Homoplantaginin on GSH-Px and SOD in hepatocytes. Exposure of HL-7702 cells to H2O2 decreased the activities of GSH-Px and SOD (Table 1). Homoplantaginin increased the activities of two antioxidant enzymes dose-dependently (Table 1).

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Palmitic acid (PA)-induced vascular endothelial inflammation plays a pivotal role in the occurrence and development of vascular diseases. The present study was conducted to examine the effect of Homoplantaginin, a main flavonoid from a traditional Chinese medicine Salvia plebeia R. Br., on PA-treated human umbilical vein endothelial cells inflammation and the underlying molecular mechanism. Firstly, we found that homoplantaginin (0.1, 1, 10 μM) dose-dependently reduced expression of toll-like receptor-4 evoked by PA (100 μM). The inhibitory effect of homoplantaginin was further confirmed under lipopolysaccharide challenge. In addition, downstream adapted proteins including myeloid differentiation primary response gene 88, toll/interleukin-1 receptor-domain containing adaptor-inducing interferon-β and tumor necrosis factors receptor associated factor-6 were successfully inhibited by homoplantaginin under PA treatment. Also, we found that homoplantaginin tightly controlled PA-induced reactive oxygen species to prevent nucleotide-binding domain-like receptor 3 (NLRP3) inflammasome activation by suppressing reactive oxygen species-sensitive thioredoxin-interacting protein, NLRP3, and caspase-1. Meanwhile, protein and mRNA levels of inflammatory mediators (interleukin-1β, intercellular cell adhesion molecule-1, and monocyte chemotactic protein-1) were decreased by homoplantaginin. Furthermore, homoplantaginin restored PA-impaired nitric oxide generation. Taken together, these results indicated that homoplantaginin protected endothelial cells from ameliorating PA-induced endothelial inflammation via suppressing toll-like receptor-4 and NLRP3 pathways, and restoring nitric oxide generation, suggesting it may be a potential candidate for further development in the prevention and treatment of vascular diseases.[2]

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Recent data have indicated that inflammation plays an important role in the development of insulin resistance. The present study aims at examining the activity of Homoplantaginin, a flavonoid from a traditional Chinese medicine Salvia plebeia R. BR., on palmitic acid (PA)-induced insulin sensitivity and the underlying mechanisms of its anti-infammatory properties in the endothelial cells. Pre-treatment of homoplantaginin on human umbilical vein endothelial cells (HUVECs) significantly inhibited PA induced tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA expression, and inhibitory κB kinase beta (IKKβ) and nuclear factor-κB (NF-κB) p65 phosphorylation. To the PA-impaired insulin-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and decrease in nitric oxide (NO) production, pretreatment of homoplantaginin could effectively reverse the effects of PA. Additionally, homoplantaginin significantly modulated the Ser/Thr phosphorylation of IRS-1, improved phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), and increased NO production in the presence of insulin. Taken together, our results demonstrated that homoplantaginin ameliorates endothelial insulin resistance by inhibiting inflammation and modulating cell signalling via the IKKβ/IRS-1/pAkt/peNOS pathway, suggesting it may be used for the prevention and treatment of endothelial dysfunction associated with insulin resistance. [3]

Homoplantaginin (25-100mg/kg) significantly lowers the rise in serum alanine and aspartate aminotransferase levels and lowers TNF-α and IL-1 levels. The same procedure also increases the levels of GSH, GSH-Px, and SOD in hepatic homogenate and decreases the content of thiobarbituric acid-reactive substances[1]. Homoplantaginin absorbs quickly (Tmax=16.00±8.94min), and its mean Cmax ranges between 0.77 and 1.27 nmol/mL. It is estimated that only 0.75% percent of the oral bioavailability is absolute.

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Therapeutic effects of Homoplantaginin on ILI induced by BCG/LPS in mice [1]
The therapeutic effects of homoplantaginin were evaluated using ILI induced by BCG/LPS in mice. In this model, the mice manifested severe liver damage with an elevation of serum aminotransferase (ALT, AST), proinflammatory cytokines such as TNF-α and IL-1, and decrease of many antioxidant enzymes such as GSH, GSH-Px and SOD (Table 2, Table 3). Homoplantaginin evidently prevented the development of ILI. As shown in Table 2, the levels of ALT and AST in serum of mice were significantly decreased after 10 d administration with homoplantaginin. The decrease of ALT and AST was dose-dependently (25, 50, 100 mg/kg/d). On the other hand, a significant weight loss was not observed during the continuous administration (data not shown). Similarly, bifendate (100 mg/kg) reduced the levels of ALT and AST as opposed to BCG plus LPS-treated mice (Table 2).

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Increase of antioxidant enzymes [1]
The levels of GSH, GSH-Px, and SOD in liver tissue of mice were also measured. As shown in Table 3, Homoplantaginin (25, 50, 100 mg/kg) increased the activities of GSH and GSH-Px markedly (P < 0.01 vs untreated model), and weakly elevated the activity of SOD (25 and 50 mg/kg, P > 0.05 vs untreated model. 100 mg/kg, P < 0.05 vs untreated model, Table 3).

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Decrease of TBARS [1]
Injection of LPS after BCG priming in mice caused a significant elevation in TBARS levels in liver tissue (Table 3). Homoplantaginin significantly reduced the elevation of TBARS (25 mg/kg, P > 0.05 vs untreated model; 50 and 100 mg/kg, P < 0.05 vs untreated model, Table 3).

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Decrease of TNF-α and IL-1 [1]
As shown in Table 4, the levels of TNF-α and IL-1 in serum were significantly higher in BCG/LPS-induced mice than in control mice. Homoplantaginin obviously counteracted the increase of TNF-α and IL-1 levels in sera of ILI mice (25 mg/kg, P < 0.05 vs untreated model; 50 and 100 mg/kg, P < 0.01 vs untreated model, Table 4).

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Effect of Homoplantaginin on liver histology [1]
Liver histopathologic examination showed no histological abnormalities in normal mice (Fig. 5A). In LPS injection in BCG-primed mice, congestion in liver sinusoids with scattered infiltration of inflammatory cells, spotty necrosis, piecemeal necrosis, bridging necrosis could be seen, and inflammatory cells were arranged around the necrotic tissue (Fig. 5B). In homoplantaginin (25, 50, 100 mg/kg/d) treated mice, the area and extent of necrosis were decreased and the immigration of inflammatory cells was reduced (Fig. 5D–F). Table 2 showed the category of liver damage in various groups of mice. The grade of liver injury was significantly ameliorated in homoplantaginin treatment groups (50, 100 mg/kg/d, P < 0.05 vs untreated model Table 2).

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The purpose of the present paper was to study the pharmacokinetic characteristics of Homoplantaginin, a major active ingredient of Salvia plebeia R.Br. In this study, the effective partition coefficient, in situ absorption in rat intestinal segments and in vitro biotransformation of homoplantaginin by rat intestinal bacteria were determined. In addition, homoplantaginin was administered to rats by intravenous, peritoneal injection and oral administration. The concentrations of homoplantaginin and hispidulin, a metabolite of homoplantaginin, were determined by a validated highperformance liquid chromatographic (HPLC) assay. After intravenous, peritoneal injection, the concentration of hispidulin could not be determined. In contrast, after oral administration, hispidulin and homoplantaginin were simultaneous quantified, homoplantaginin was rapidly absorbed (Tmax=16.00±8.94min), reaching a mean Cmax between 0.77 and 1.27nmol/mL. The absolute oral bioavailability was calculated to be only 0.75%, and the area under curve (AUC) of hispidulin was about 5.4 times than that of homoplantaginin. The poor oral bioavailability may be attributed to the biotransformation of homoplantaginin by rat intestinal bacteria. [4]

Antioxidative activity [1]
The antioxidative activity of Homoplantaginin was evaluated using DPPH-scavenging assay (Watjen et al., 2007, Yang et al., 2005). Hundred and sixty microliters of reaction mixtures containing homoplantaginin (test compound) and 40 μl of DPPH (1,1-diphenyl-2-picrylhydrazyl radical dissolved in MeOH) were plated in 96-well plates and incubated in dark for 30 min. The control used distilled water in place of test compound solution. After reaction, the remaining DPPH was determined colorimetrically at 517 nm. The absorbance of homoplantaginin alone was subtracted as the blank from that of the reaction mixture. The radical scavenging activity was calculated as 100 × (ODcontrol − ODtest)/ODcontrol. The IC50 value was defined as the concentration of the test compound required to inhibit the formation of DPPH radical by 50%. l-Ascorbic acid, a well-known antioxidant, was used as a positive control (Yang et al., 2005).

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The biotransformation of Homoplantaginin in vitro [4]
A complete incubation system was employed for intestinal bacteria incubation, which contained rat intestinal flora and GAM solution (1:9, v:v) containing 2.16 μM of homoplantaginin. The mixture was then incubated with anaerobic culture at 37 °C. At designed times (5, 10, 20, 30, 45, 60, 90, 120, 180, 300, 480, 720 min), the reaction mixture was taken out and extracted with methanol(1:1, v:v). The mixture was vortexed for 3 min and subsequently centrifuged at 12,000 rpm for 10 min. Finally, 20 μL of the supernatant was injected into HPLC system for analysis. The conditions are the same as 2.3.

The MTT assay is used to assess the viability of cultured cells. Homoplantaginin is applied to human umbilical vein endothelial cells for 48 hours at varying concentrations (0.1, 1, 3, 10, 30, 100 M). Then, each well receives 20 μL of MTT (5 mg/mL) for an additional 4 hours at 37°C. After removing the supernatant, DMSO is added to help dissolve the formazan crystals. At 540 nm[3], the optical absorbance is measured.

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Cell culture and viability [1]
Human hepatocyte line HL-7702 was maintained in RPMI-1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillin–streptomycin (100 IU/ml–100 μg/ml), 2 mM of glutamine, and 10 mM of Hepes buffer at 37 °C in a humid atmosphere (5% CO2, 95% air). Cell growth and viability were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay as described elsewhere (Zhang et al., 2008a).

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Oxidative stress induced by H2O2 [1]
HL-7702 cells were cultured in 6-well plates at a density of 3 × 105 per ml and allowed to grow to the desired confluence. The cells were pretreated with various concentrations (0.1–100 μg/ml) of Homoplantaginin for 24 h, and then exposed to 750 μM H2O2 for another 18 h (Wang et al., 2005a). Non-H2O2-treated cells were incubated under the same conditions as those used in the experimental protocols with H2O2.

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Measurement of Intracellular ROS Levels [2]
HUVECs (5 × 104 cells/well) were seeded in 6-well plates and maintained at 37°C in a 5% CO2 incubator for 48 hours. Then the culture medium was replaced with serum-free medium to starve cells for 2.5 hours. Cells were treated with PA (100 μM) for 1.5, 3, and 6 hours respectively, or pretreated with different concentrations of Homoplantaginin (0.1, 1, and 10 μM) or Sal (500 μM) for 0.5 hours before PA (100 μM) treatment for 3 hours. Then the cells were harvested and incubated in dark with 10 μM DCFH-DA at 37 °C for 20 minutes. After washing twice with cold phosphate buffered saline, cells were analyzed with FACS Calibur flow cytometry at an excitation wave length of 488 nm and an emission wavelength of 525 nm.24 Dicholorofluorescein (DCF) fluorescence distributions were calculated by Cell Quest software.

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Western Blotting [2]
HUVECs (5 × 104 cells/well) were seeded in 6-well plates and maintained at 37 °C in a 5% CO2 incubator for 48 hours. Then cultured medium was replaced with serum-free medium to starve cells for 2.5 hours. Cells were pretreated with different concentrations of Homoplantaginin (0.1, 1, 10 μM) or Sal (500 μM) for 0.5 hours before PA (100 μM) treatment for 3 hours. In addition, HUVECs were seeded and starved as mentioned above. Then, cells were pretreated with homoplantaginin (10 μM) or salicylate (500 μM) for 0.5 hours followed by addition of lipopolysaccharide (LPS) (1 μg/mL) for another 1 hour. Associated protein lysates were obtained according to manufacturer's suggested protocol of Membrane and Cytosol Protein Extraction Kit, and concentration of proteins were detected by BCA Protein Assay Kit. Equal amounts of proteins (30 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently electro-transferred onto polyvinylidenedifluoride membranes. After being blocked in Tris-buffered saline containing 5% skimmed milk and 0.05% Tween 20 at 37 °C for 2 hours, the membranes were incubated with the indicated antibodies. Protein bands were visualized using the ChemiDoc XRS system and analyzed with Image J software (National Institutes of Health).

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ELISA Assay for Inflammatory Mediators [2]
HUVECs (5 × 104 cells/well) were seeded in 6-well plates and maintained at 37 °C in a 5% CO2 incubator for 48 hours. Then cultured medium was replaced with serum-free medium to starve cells for 2.5 hours. Cells were pretreated with different concentrations of Homoplantaginin (0.1, 1, and 10 μM) or Sal (500 μM) for 0.5 hours before PA (100 μM) treatment for 3 hours. Concentrations of IL-1β, ICAM-1, and MCP-1 in cell supernatants were quantified by corresponding ELISA kits following the manufacturer's instructions.

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Measurement of Intracellular Nitric Oxide Levels [2]
HUVECs (2 × 104 cells/well) were seeded in 48-well plates and maintained at 37 °C in a 5% CO2 incubator for 48 hours. Then the culture medium was replaced with serum-free medium to starve cells for 2.5 hours. Cells were pretreated with different concentrations of Homoplantaginin (0.1, 1, and 10 μM) or Sal (500 μM) for 0.5 hours before PA (100 μM) treatment for 3 hours. Then the cells were washed twice with PBS and incubated in dark with 5 μM, DAF-FM DA at 37 °C for 25 minutes. After being washed twice with PBS, cells were analyzed with Olympus fluorescence microscope at an excitation wavelength of 495 nm and an emission wavelength of 515 nm. Fluorescence intensity was analyzed with Image J software.

Rats: Homoplantaginin is dissolved in a solution of DMSO, PEG 400, ethanol, and normal saline (2:2:3:3, v/v/v/v) in rats at a concentration of 10 mg/mL. To administer oral administration (150 mg/kg), tail vein injection (15 mg/kg), and peritoneal injection (15 mg/kg) to the rats, the groups are randomly divided into three. At 5, 10, 20, 30, 45, 60, 90, 120, and 180 minutes after administration, blood samples (roughly 0.5 mL) are drawn from the retro-orbital plexus into heparinized microfugetubes. The plasma samples were extracted from the blood samples by centrifuging them at 10,000 rpm[4].
Mice: Homoplantaginin is dissolved in 5% amylum. Homoplantaginin is administered orally by gastric intubation during the experimental period at doses of 25, 50, 100 mg/kg/d, respectively. The mice are put to sleep with ether and blood samples are drawn by exsanguination from the inferior vein eight hours after LPS injection. To facilitate histological examination, the liver is taken out and fixed in formalin[1].

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Acute liver injury model and Homoplantaginin treatment [1]
The hepatoprotective effects of homoplantaginin were evaluated in Bacille-Calmette–Guérin and lipopolysaccharide (BCG/LPS)-induced immunological liver injury (ILI) in mice. Male Balb/c mice (20 ± 2 g) were housed in plastic cages with free access to food and water. BCG, 2.5 mg suspended in 0.2 ml saline, was injected through the tail vein in mice, and 10 d later they were injected with 7.5 μg lipopolysaccharide dissolved in 0.2 ml saline (LPS). Homoplantaginin was administered orally by gastric intubation during the experimental period at doses of 25, 50, 100 mg/kg/d, respectively. Bifendate, which is now clinically used for the treatment of hepatitis, was administered orally as a positive control (Pan et al., 2006). Eight hours after injection of LPS, the mice were anesthetized with ether and blood samples were collected by exsanguination from the inferior vein. The liver was removed and fixed in formalin for histological analysis (Lu et al., 2008, Wang et al., 2005b).

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Determination of apparent partition coefficient [4]
The apparent partition coefficient of Homoplantaginin and hispidulin was measured by shake-flask method. Homoplantaginin and hispidulin was dissolved in different pH solutions. The resulted homoplantaginin and hispidulin solutions was mixed with n-octanol (presaturated with water) in equal volume, shaken, and incubated in a water bath at 37 °C for 24 h. After centrifugation, the concentrations of homoplantaginin and hispidulin in the aqueous phase and the octanol phase were determined by HPLC. Then the apparent partition coefficient was obtained by the ratio of the homoplantaginin and hispidulin concentrations in octanol phase to that in aqueous phase respectively.

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In situ single-pass intestinal perfusion(SPIP) studies in rats [4]
The perfusion buffer consists of 1.4 g/L glucose, 20 mg/L phenol red, 7.8 g/L NaCl, 0.35 g/L KCl, 1.37 g/L NaHCO3, 0.32 g/L NaH2PO4, 0.02 g/L MgCl, 0.37 g/L CaCl2. Primary standard stock solution of Homoplantaginin was prepared in dimethylsulfoxide at 10 g/L. Working standard solutions of homoplantaginin were prepared by subsequent diluting primary standard stock solution with intestinal blank perfusion buffer.
Briefly, male SD rats were fasted overnight for 16–18 h with free access to water and anaesthetized using an intra-peritoneal injection of 10% chloral hydrate(3.4 mL/kg) and placed on a heated pad to keep normal body temperature. Upon verification of the loss of pain reflex, a midline longitudinal abdominal incision was made, a 10 cm section of rat duodenum, jejunum, ileum, and colon were respectively located gently rinsed with saline pre-warmed to 37 °C and then attached to the perfusion assemblye. The entire surgical area was then covered with Parafilm to reduce evaporation. Blank perfusion buffer was infused for 30 min by a syringe pump followed by working standard solutions of Homoplantaginin at a constant flow rate of 0.2 mL/min for 120 min. The outlet perfusate samples was collected every 15 min in microtubes. At the end, the length of segment was measured without stretching and finally the animal was euthanatized [12]. Study samples were stored at −20 °C until analysis. Samples generated from the permeability study were analysed using validated method. Samples were centrifuged at 12,000 rpm for 10 min and the supernatant was directly injected onto HPLC column and required no sample preparation prior to analysis. Along with the permeability samples, QC samples were distributed in the analytical run. The data was accepted based on performance of QCs prepared using rat intestinal perfusion blank.

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Pharmacokinetic study [4]
Homoplantaginin was dissolved in a solution consisting of dimethyl sulfoxide, PEG 400, ethanol and normal saline (2:2:3:3, v/v/v/v) at a concentration of 10 mg/mL. The rats were randomly divided into three groups to receive oral administration(150 mg/kg), tail vein injection (15 mg/kg) and peritoneal injectionv (15 mg/kg). Blood samples (approximately 0.5 mL) were collected from the retro-orbital plexus into heparinized microfuge tubes at 5, 10, 20, 30, 45, 60, 90, 120, and 180 min after administration. The plasma samples, separated by centrifuging the blood samples at 10,000 rpm for 1 min at 4 °C, were stored at −20 °C until further analysis. The following pharmacokinetic parameters were calculated by Phoenix_1.1 pharmacokinetic software according to non-compartmental model: the half-life (T1/2), area under the concentration-time curve (AUC), mean residence time (MRT), clearance (Cl), and the apparent volume of distribution at steady state (Vss). The data are presented in mean ± S.D. The maximum concentration (Cmax) and time of reaching maximum concentration (Tmax) were obtained from a concentration–time profile. The oral bioavailability (F) and intraperitoneal injection (Fi.p.) of bioavailability were calculated using the following equations:

Determination of apparent partition coefficient [4]
Hispidulin and Homoplantaginin were stable from pH 1.2 to pH 8.0 because the total concentration of the drug in both oil and water phase after 24 h incubation was not changed, suggesting that there are no degradation of hispidulin and homoplantaginin in gastrointestinal (GI) pH conditions. The results of apparent partition coefficient of hispidulin and homoplantaginin in different pH solutions are shown in Fig. 2. It can be observed that the apparent partition coefficients of hispidulin and homoplantaginin were ranged from 2.4 to 2.8 and 1.0–1.2. The results implied that hispidulin and homoplantaginin were not ionized in stomach or intestinal pH conditions and the absorption sites were stomach and intestine. It was also indicated by the results that the stomach and intestinal absorption of hispidulin and homoplantaginin independent of pH.

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In situ absorption of Homoplantaginin in rat intestinal segments [4]
It has been reported that phenol red could interfere with the absorption and transport of poor water-soluble drugs in rat intestinal perfusion due to its partial absorption in the intestine [13]. Gravimetric method was used to calibrate the volume change of the perfusate. Effective permeabilities of homoplantaginin in four intestinal segments at three concentrations are presented in Table 1. The absorption of homoplantaginin depends on the intestinal site which could be found between duodenum, jejunum, ileum, and colon at each tested concentration. The effective permeabilities at three concentrations shared the same rank order of ileum > duodenum > jejunum > colon. The minimum and maximum Peff in rat intestinal segments were determined to be 0.259 × 10−4 (colon) and 0.687 × 10−4 cm/s (ileum). The permeability coefficients of homoplantaginin obtained from single-pass intestinal perfusion study were comparable to the reported values of propranolol (0.30–0.75 × 10−4 cm/s) [14], [15], [16], which was often used as a reference compound of passive absorption via transcellular pathway. Furthermore, there was no statistical difference of Peffs in four intestinal segments at each homoplantaginin concentration, suggesting the transport mechanism of homoplantaginin in four intestinal segments might be primarily passive transport. The absorption rate (Ka) of homoplantaginin showed the highest in jejunum, obviously decreased in duodenum and ileum, and then significantly reduced in icolon.

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Time course of the biotransformation of Homoplantaginin by rat intestinal bacteria in vitro [4]
Fig. 3 shows the mean time-concentration curves of homoplantaginin in the biotransformation matrix of normal rat intestinal bacteria. As indicated in Fig. 3, homoplantaginin was metabolized fast, and almost completely biotransformation within 180 min, and 75% was transformed to hispidulin approximately at last. So, we know that in the case of the presence of intestinal bacteria, homoplantaginin can easily be converted into hispidulin, thus affecting its oral bioavailability.

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Pharmacokinetic results [4]
The validated method was successfully applied to a pharmacokinetic study of Homoplantaginin. Fig. 4 shows the mean plasma concentration–time profiles of homoplantaginin after intravenous and peritoneal administration. Hispidulin and homoplantaginin were simultaneous quantified after oral administration in this study. Fig. 5 shows the mean plasma concentration–time profiles of homoplantaginin and hispidulin after oral administration. Based on the homoplantaginin and hispidulin plasma concentrations, the basic pharmacokinetic parameters were calculated and summarized in Table 2.
As we can see after intravenous administration, the concentration of Homoplantaginin in plasma rapidly eliminated within 50 min. After peritoneal administration, it can be clearly seen that the drug concentration in plasma rapidly reached the peak value within 20 min with a mean Cmax between 15.45 and 21.99 nmol/mL and rapidly decreased during the next 100 min. The Fi.p.was calculated to be 102.85%. This indicated that homoplantaginin was absorbed completely in intestine as mentioned in determination of effective partition coefficient and in situ absorption in rat intestinal segments studies. Through the oral gavage, the Cmax of homoplantaginin was quickly reached among plasma after dosing due to fast absorption, with mean Tmax ranging from 7 to 25 min. The absolute oral bioavailability was calculated to be 0.75%. In comparison, hispidulin was rapidly absorbed (Tmax = 5–23 min), reaching a mean Cmax between 4.73 and 6.87 nmol/mL, the absolute bioavailability was 4.02%, which was about 5 times that of homoplantaginin. It was demonstrated that the majority of homoplantaginin were transformed to hispidulin by rat intestinal bacteria. As shown in Fig. 5, a double peak was observed in the concentration-time profile of oral administration. Several mechanisms might account for this phenomenon, such as enterohepatic circulation, fractionated gastric emptying, and separated “absorption windows”. Enterohepatic circulation may be an explanation for this because the double-peak was not observed in the IV concentration-time profile. Furthermore, the metabolites hispidulin presented the same phenomenon. [4]

[1]. Protective effects of Salvia plebeia compound homoplantaginin on hepatocyte injury. Food Chem Toxicol. 2009 Jul;47(7):1710-5.

[2]. Homoplantaginin Inhibits Palmitic Acid-induced Endothelial Cells Inflammation by Suppressing TLR4 and NLRP3 Inflammasome. J Cardiovasc Pharmacol. 2016 Jan;67(1):93-101.

[3]. Homoplantaginin modulates insulin sensitivity in endothelial cells by inhibiting inflammation. Biol Pharm Bull. 2012;35(7):1171-7.

[4]. Pharmacokinetics of homoplantaginin in rats following intravenous, peritoneal injection and oral administration. J Pharm Biomed Anal. 2016 Sep 10;129:405-9.

Homoplantaginin is a glycoside and a member of flavonoids.
Homoplantaginin has been reported in Salvia plebeia, Salvia officinalis, and other organisms with data available.

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Salvia plebeia R. Br is a traditional Chinese herb which has been considered as an inflammatory mediator used for treatment of many infectious diseases including hepatitis. Previously, the compound homoplantaginin was isolated in our group. Hence, we evaluated the protective effects of homoplantaginin on hepatocyte injury. Homoplantaginindisplayed an antioxidant property in a cell-free system and showed IC(50) of reduction level of DPPH radical at 0.35 microg/ml. In human hepatocyte HL-7702 cells exposed to H(2)O(2), the addition of 0.1-100 microg/ml of homoplantaginin, which did not have a toxic effect on cell viability, significantly reduced lactate dehydrogenase (LDH) leakage, and increased glutathione (GSH), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in supernatant. In vivo assay, we employed the model of Bacillus Calmette-Guérin (BCG)/lipopolysaccharide (LPS)-induced hepatic injury mice to evaluate efficacy of homoplantaginin. Homoplantaginin (25-100mg/kg) significantly reduced the increase in serum alanine aminotranseferase (ALT) and aspartate aminotransferase (AST), decreased the levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1). The same treatment also reduced the content of thiobarbituric acid-reactive substances (TBARS), elevated the levels of GSH, GSH-Px and SOD in hepatic homogenate. The histopathological analysis showed that the grade of liver injury was ameliorated with reduction of inflammatory cells and necrosis of liver cells in homoplantaginin treatment mice. These results suggest that homoplantaginin has a protective and therapeutic effect on hepatocyte injury, which might be associated with its antioxidant properties. [1]

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As a vasodilator and vascular homeostatic molecule, NO plays a vital role in regulating physiological endothelial function. However, bioactivity and generation of NO are usually impaired in inflamed endothelial cells.49 Our data indicated an impairment of NO production under PA challenge. As expected, we found that homoplantaginin restored the production of NO (Fig. 8). It indicated that Homoplantaginin could protect endothelial cells from PA insult by restoring impaired NO generation. CONCLUSION: In summary, our study elucidates that Homoplantaginin down-regulates TLR4 and NLRP3 inflammasome pathways, subsequently inhibits related inflammatory mediators, then restores impaired NO generation from PA-triggered endothelial cells. These findings suggest that homoplantaginin may potentially be a candidate agent for further development in the prevention and treatment of vascular diseases. [2]

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In summary, this study first report the pharmacokinetic of Homoplantaginin in rats after intravenous, peritoneal injection, and oral administration. Hispidulin and homoplantaginin were simultaneous quantified after oral administration for first time. The apparent partition coefficient of hispidulin and homoplantaginin were stable from pH 1.2 to pH 8.0 and homoplantaginin in intestinal segments might be primarily passive transport. The intestine is the best absorption site of homoplantaginin but it was almost completely biotransformation intermediate by rat intestinal bacteria. After oral administration, homoplantaginin was rapidly absorbed and the absolute oral bioavailability was 0.75%.[4]

C22H22O11

462.4035

462.116

17680-84-1

17680-84-1

5318083

Light yellow to yellow solid

1.6±0.1 g/cm3

798.1±60.0 °C at 760 mmHg

256-258℃

279.7±26.4 °C

0.0±3.0 mmHg at 25°C

1.695

-0.97

6

11

5

33

721

5

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])O[H])O[H])OC1C([H])=C2C(C(C([H])=C(C3C([H])=C([H])C(=C([H])C=3[H])O[H])O2)=O)=C(C=1OC([H])([H])[H])O[H]

GCLAFEGUXXHIFT-IWLDQSELSA-N

InChI=1S/C22H22O11/c1-30-21-14(32-22-20(29)19(28)17(26)15(8-23)33-22)7-13-16(18(21)27)11(25)6-12(31-13)9-2-4-10(24)5-3-9/h2-7,15,17,19-20,22-24,26-

5-hydroxy-2-(4-hydroxyphenyl)-6-methoxy-7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one

Homoplantaginin; 17680-84-1; HISPIDULOSIDE; Dinatin 7-glucoside; (-)-Homoplantaginin; hispidulin-7-glucoside; 4H-1-Benzopyran-4-one, 7-(beta-D-glucopyranosyloxy)-5-hydroxy-2-(4-hydroxyphenyl)-6-methoxy-; 6-Methoxyapigenin 7-O-glucoside;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50~92 mg/mL (108.1~199.0 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.08 mg/mL (4.50 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)