Cirsium Setidens Water Extracts Containing Linarin Block Estrogen Deprivation-Induced Bone Loss in Mice.

Osteoporosis is evident in postmenopausal women and is an osteolytic disease characterized by bone loss that further increases the susceptibility to bone fractures and frailty. The use of complementary therapies to alleviate postmenopausal osteoporosis is fairly widespread among women. Edible Cirsium setidens contains various polyphenols of linarin, pectolinarin, and apigenin with antioxidant and hepatoprotective effects. This study aimed to determine whether Cirsium setidens water extracts (CSEs), the component linarin, and its aglycone acacetin blocked ovariectomy (OVX)-induced bone loss. This study employed OVX C57BL/6 female mice as a model for postmenopausal osteoporosis. CSEs, acacetin, or linarin was orally administrated to OVX mice at a dose of 20 mg/kg for 8 weeks. Surgical estrogen loss in mice for 8 weeks reduced bone mineral density (BMD) of mouse femur and serum 17ß-estradiol level and enhanced the serum receptor activator of NF-κB ligand/osteoprotegerin ratio with uterine atrophy. CSEs and linarin reversed such adverse effects and enhanced femoral BMD in OVX mice. Oral administration of CSEs and linarin attenuated tartrate-resistant acid phosphate activity and the induction of αvß3 integrins and proton suppliers in resorption lacunae in femoral bone tissue of OVX mice. In addition, CSEs and linarin curtailed the bone levels of cathepsin K and matrix metalloproteinase-9 responsible for osteoclastic bone resorption. On the other hand, CSEs and linarin enhanced the formation of trabecular bones in estrogen-deficient femur with increased induction of osteocalcin and osteopontin. Further, treatment with CSEs and linarin enhanced the collagen formation-responsive propeptide levels in the circulation along with the increase in the tissue non-specific alkaline phosphatase level in bone exposed to OVX. Supplementing CSEs, acacetin, or linarin to OVX mice elevated the formation of collagen fibers in OVX trabecular bone, evidenced using Picrosirius red staining. Accordingly, CSEs and linarin were effective in retarding osteoclastic bone resorption and promoting osteoblastic bone matrix mineralization under OVX conditions. Therefore, linarin, which is abundant in CSEs, may be a natural compound for targeting postmenopausal osteoporosis and pathological osteoresorptive disorders.

The antihypertensive potential of flavonoids from Chinese Herbal Medicine: A review.

With the coming of the era of the aging population, hypertension has become a global health burden to be dealt with. Although there are multiple drugs and procedures to control the symptoms of hypertension, the management of it is still a long-term process, and the side effects of conventional drugs pose a burden on patients. Flavonoids, common compounds found in fruits and vegetables as secondary metabolites, are active components in Chinese Herbal Medicine. The flavonoids are proved to have cardiovascular benefits based on a plethora of animal experiments over the last decade. Thus, the flavonoids or flavonoid-rich plant extracts endowed with anti-hypertension activities and probable mechanisms were reviewed. It has been found that flavonoids may affect blood pressure in various ways. Moreover, despite the substantial evidence of the potential for flavonoids in the control of hypertension, it is not sufficient to support the clinical application of flavonoids as an adjuvant or core drug. So the synergistic effects of flavonoids with other drugs, pharmacokinetic studies, clinical trials and the safety of flavonoids are also incorporated in the discussion. It is believed that more breakthrough studies are needed. Overall, this review may shed some new light on the explicit recognition of the mechanisms of anti-hypertension actions of flavonoids, pointing out the limitations of relevant research at the current stage and the aspects that should be strengthened in future researches.

Linarin Protects the Kidney against Ischemia/Reperfusion Injury via the Inhibition of Bioactive ETS2/IL-12.

Ischemia/reperfusion injury (IRI), a participant in acute kidney injury (AKI), can occur as a series of pathological processes such as inflammation. Linarin (LIN) has been widely used for different diseases. To confirm the anti-inflammatory value and relevant mechanism of LIN during IRI, in vivo and vitro models were established. LIN or dissolvent was given, and histologic analysis, quantitative (q)RT-PCR, serum creatinine and blood urea nitrogen testing were used to evaluate kidney injury. Microarray analysis, protein-protein interaction (PPI) analysis and molecular docking were used to identify the target protein of LIN, and small interfering RNA (siRNA) transfection was applied to explore the crucial role of identified protein. First, we found that LIN inhibited kidney injury in an in vivo IRI model and decreased the expression of interleukin-12 (IL-12) p40 in vivo and in vitro IRI models. To explore the mechanism of LIN, we collected raw data from a public microarray database and identified E26 oncogene homolog 2 (ETS2) as a crucial protein of LIN according to microarray analysis and PPI. Meanwhile, qRT-PCR indicated that IL-12 p40 showed no significant difference between ETS2 knock down group and LIN treated ETS2 knock down group after hypoxia reoxygenation treatment. In addition, according to molecular docking the contact area is highly conserved and located on a PPI domain of ETS2 which indicates that LIN may alter the interaction with synergistic proteins in the regulation of IL-12 p40 expression. Our study demonstrated the anti-inflammatory effect of LIN during IRI-AKI, broadening the medicinal value of LIN and the therapeutic options for IRI-AKI.

Purification of linarin and hesperidin from Mentha haplocalyx by aqueous two-phase flotation coupled with preparative HPLC and evaluation of the neuroprotective effect of linarin.

The volatile oil of Mentha haplocalyx is widely used in medicine, food, and cosmetics. However, a large amount of its residue after steam extraction of volatile oil is abandoned, resulting in a waste of resources. The method of aqueous two-phase flotation coupled with preparative high-performance liquid chromatography was established for the separation and purification of nonvolatile active compounds from Mentha haplocalyx for the first time. The parameters of the two-phase aqueous flotation were optimized. Under the optimal conditions including flotation solvent PEG 1000 aqueous solution (1:1, w/w), pH 5, (NH4 )2 SO4 concentration of 350 g/L in aqueous phase, N2 flow rate of 20 mL/min, and flotation time of 20 min, the flotation efficiency of linarin, hesperidin, and didymin was 82.24, 76.38, and 89.33%, respectively. The linarin and hesperidin with the high purities of 95.8 and 97.2%, respectively, were obtained by using preparative high performance liquid chromatography. The neuroprotective effect of linarin against H2 O2 -induced oxidative stress in rat hippocampal neurons was investigated. The experimental result indicated that linarin could alleviate H2 O2 -induced oxidative stress. The work indicated that the combination of aqueous two-phase flotation and preparative high performance liquid chromatography is a feasible and practical method for the purification of nonvolatile active substances from Mentha haplocalyx, which would provide a reference process for the comprehensive utilization of M. haplocalyx. Especially, linarin might be used as a good source of natural neuroprotectants.

[Correlations between content of linarin in Chrysanthemum indicum and climatic factors in habitats].

Chrysanthemi Indici Flos(CIF), the capitulum of Chrysanthemum indicum, is widely used in proprietary Chinese medicine and daily chemical products. At present, CIF is mainly produced from wild resources and rarely cultivated. This study aims to reveal the correlations between linarin content in CIF and climatic factors in different habitats, and provide a theoretical basis for suitable zoning and rational production of medicinal materials. The content of linarin in CIF was determined by HPLC. Grey relational analysis and Pearson correlation analysis were carried out for linarin content with climatic factors. The results showed that the content of linarin in CIF was significantly different among different habitats. The grey relational degrees of climatic factors with linarin content was in an order of average annual precipitation>annual average sunshine hours>annual average temperature>longitude>annual frost-free period>latitude>altitude. Longitude, annual average temperature and average annual precipitation had significantly positive correlations with the content of linarin in CIF, whereas latitude and altitude showed negative correlations with it. The annual frost-free period and annual average sunshine hours had no significant correlation with the content of linarin in CIF. The content of linarin in CIF varied significantly in different habitats. High longitude, low latitude, low altitude, high annual average temperature and high annual average precipitation could be used as indicators for the habitats of high-quality Ch. indicum. This study provides a reference for selecting suitable producing areas of Ch. indicum and establishing artificial cultivation system.

Separation of acteoside and linarin from Buddlejae Flos by high-speed countercurrent chromatography and their anti-inflammatory activities.

Buddleja officinalis Maxim., a deciduous, flowering shrub, is used as a traditional Chinese medicine; the bioactivity of B. officinalis is primarily due to flavonoids and phenylethanoid glycosides. In the study, acteoside and linarin were successfully isolated from B. officinalis by high-speed countercurrent chromatography with a two-phase solvent system composed of ethyl acetate: n-butanol: water (5:0.8:5, v/v/v). The purities of acteoside and linarin were determined to be 97.3 and 98.2%, respectively, using one-step high-speed countercurrent chromatography separation. The chemical structures of the two compounds were identified by electrospray ionization-mass spectrometry and nuclear magnetic resonance. After separation, the anti-inflammatory effects of the two compounds were evaluated using lipopolysaccharide-induced human umbilical vein endothelial cells. Acteoside and linarin inhibited the expression of nitric oxide, tumor necrosis factor α and interleukin 1ß, which demonstrated that acteoside and linarin possessed anti-inflammatory activity.

LC-MS/MS Method for Simultaneous Determination of Linarin and Its Metabolites in Rat Plasma and Liver Tissue Samples: Application to Pharmacokinetic and Liver Tissue Distribution Study After Oral Administration of Linarin.

Linarin, a flavone glycoside, is considered to be a promising natural product due to its diverse pharmacological activities. Recently, it has been brought into focus for its potential to treat liver failure. In this study, a rapid and sensitive liquid chromatography electrospray-ionization tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of linarin and its three metabolites (acacetin, apigenin, and p-hydroxy benzaldehyde) in plasma and liver tissue samples of normal rats and rats with d-galactosamine (d-GalN)-induced liver injury. After liquid-liquid extraction (LLE) with ethyl acetate, chromatographic separation of the four analytes was achieved using an ACQUITY UPLC BEH-C18 (1.7 µm, 2.1 × 50 mm) with a mobile phase of 0.01% formic acid in methanol and 0.01% formic acid at a flow rate of 0.3 mL/min. The detection was accomplished on a tandem mass spectrometer via an electrospray ionization (ESI) source by multiple reaction monitoring (MRM) in the negative ionization mode. The method had a good linearity over the concentration range of 1.00-200 ng/mL for linarin and its metabolites. The validated method was successfully applied to the pharmacokinetic and liver tissue distribution study of linarin and its metabolites after a single oral administration of linarin (90 mg/kg) to rats.

Inhibitory activity of linarin on osteoclastogenesis through receptor activator of nuclear factor κB ligand-induced NF-κB pathway.

Linarin, a natural flavonoid glycoside widely found in plants, has been reported to possess anti-inflammation, neuroprotection and osteogenic properties. However, its impact on osteoclast remains unclear. In the present study, the effects of linarin on osteoclastogenesis and its underlying molecular mechanisms of action were investigated. Using the culture systems of osteoclasts derived from bone marrow macrophages (BMMs), we found that linarin dose-dependently inhibited osteoclasts formation and bone resorptive activity. The Cell Counting Kit-8 test displayed that the viability of cells was not influenced by linarin at doses up to 10 µg/mL. In addition, linarin downregulated osteoclast-related genes expression, including nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR) and c-Fos, as shown by quantitative real time polymerase chain reaction (RT-qPCR). Western blot analysis further showed that linarin inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced nuclear factor kappa B (NF-κB) p65 and NFATc1 activity. The present findings show that linarin exerted a potent inhibitory effect on osteoclastogenesis through RANKL-induced NF-κB signaling pathway. In conclusion, the results suggest that linarin has anti-osteoclastic effects and may serve as potential modulatory agents for the prevention and treatment of bone loss-associated diseases.

Characterization of the In Vivo and In Vitro Metabolites of Linarin in Rat Biosamples and Intestinal Flora Using Ultra-High Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Linarin, a flavone glycoside, is considered to be a promising natural product due to its diverse pharmacological activities, including analgesic, antipyretic, anti-inflammatory and hepatoprotective activities. In this research, the metabolites of linarin in rat intestinal flora and biosamples were characterized using ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS). Three ring cleavage metabolites (4-hydroxybenzoic acid, 4-hydroxy benzaldehyde and phloroglucinol) were detected after linarin was incubated with rat intestinal flora. A total of 17 metabolites, including one ring cleavage metabolite (phloroglucinol), were identified in rat biosamples after oral administration of linarin. These results indicate that linarin was able to undergo ring fission metabolism in intestinal flora and that hydrolysis, demethylation, glucuronidation, sulfation, glycosylation, methylation and ring cleavage were the major metabolic pathways. This study provides scientific support for the understanding of the metabolism of linarin and contributes to the further development of linarin as a drug candidate.

[Determination of 3 components in Xinjiang Ziziphora bungeana by HPLC].

This study aimed to develop an HPLC method for simultaneous determination of the 3 components in Ziziphora bungeana. The optimum HPLC condition was as follows：ZORBAX SB-C18 column（4.6 mm×250 mm, 5.0 µm）with gradient elution of methanol (A)-0.2% glacial acetic acid (B), detection wavelength 340 nm,column temperature 30 °C, flow rate 1 mL·min⁻¹. There were good linearity between peak areas and injection quantity of caffeic acid, rosmarinic acid, and linarin in the range of 2-40 mg·L⁻¹ï¼ˆr=0.999 9）, 3-60 mg·L⁻¹ï¼ˆr=1）, 7-140 mg·L⁻¹ï¼ˆr=0.999 9）, respectively. The average recoveries were 100.9%（RSD 1.3%）,98.25%（RSD 2.0%）,and 98.73%（RSD 1.5%）, respectively. The HPLC method was stable and accurate, which could be used to detect caffeic acid,rosmarinic acid, and linarin in Ziziphora bungeana.

Optimization of extraction of linarin from Flos chrysanthemi indici by response surface methodology and artificial neural network.

The extraction of linarin from Flos chrysanthemi indici by ethanol was investigated. Two modeling techniques, response surface methodology and artificial neural network, were adopted to optimize the process parameters, such as, ethanol concentration, extraction period, extraction frequency, and solvent to material ratio. We showed that both methods provided good predictions, but artificial neural network provided a better and more accurate result. The optimum process parameters include, ethanol concentration of 74%, extraction period of 2 h, extraction three times, solvent to material ratio of 12 mL/g. The experiment yield of linarin was 90.5% that deviated less than 1.6% from that obtained by predicted result.

The Phenolic Fraction of Mentha haplocalyx and Its Constituent Linarin Ameliorate Inflammatory Response through Inactivation of NF-κB and MAPKs in Lipopolysaccharide-Induced RAW264.7 Cells.

Mentha haplocalyx has been widely used for its flavoring and medicinal properties and as a traditional Chinese medicine with its anti-inflammation properties. The present study was designed to investigate the anti-inflammatory effects and potential molecular mechanisms of the phenolic fraction of M. haplocalyx (MHP) and its constituent linarin in lipopolysaccharide (LPS)-induced RAW264.7 cells. The high-performance liquid chromatography coupled with linear ion trap-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap MS) was used to analyze the chemical composition of MHP. Using the enzyme-linked immunosorbent assay (ELISA) and quantitative realtime polymerase chain reaction (qRT-PCR), the expression of pro-inflammatory meditators and cytokines was measured at the transcriptional and translational levels. Western blot analysis was used to further investigate changes in the nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and Akt signaling pathways. Fourteen phenolic constituents were identified from MHP based on the data of the mass spectrometry (MS)/MS analysis. MHP and linarin decreased the production of NO, tumor necrosis factor-α (TNF-α), interlenkin-1ß (IL-1ß), and IL-6. The messenger ribonucleic acid (mRNA) expression levels of inducible NO synthase (iNOS), TNF-α, IL-1ß, and IL-6 were also suppressed by MHP and linarin. Further investigation showed that MHP and linarin down-regulated LPS-induced phosphorylation content of NF-κB p65, inhibitor kappa B α (IκBα), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. However, MHP and linarin showed no inhibitory effect on the phosphorylated Akt. These results suggested that MHP and linarin exerted a potent inhibitory effect on pro-inflammatory meditator and cytokines production via the inactivation of NF-κB and MAPKs, and they may serve as potential modulatory agents for the prevention and treatment of inflammatory diseases.

Anti-DHAV-1 reproduction and immuno-regulatory effects of a flavonoid prescription on duck virus hepatitis.

CONTEXT: The flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) has a curative effect on duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1). However, the mechanism of this curative effect is not understood. OBJECTIVE: This study investigates the mechanism of the curative effect of BLIN on DVH caused by DHAV-1. We analyzed the anti-DHAV-1 reproduction mechanism and immuno-regulatory effect of BLIN. MATERIALS AND METHODS: The anti-DHAV-1 reproduction effects of BLIN at 20, 10, 5 and 2.5 µg/mL in vitro, as well as the influence of BLIN at 20 µg/mL on DHAV-1 adsorption, replication and release were tested using the qRT-PCR method. The promotion abilities of BLIN at 20, 10, 5 and 2.5 µg/mL on T- and B-lymphocyte proliferation were investigated by the MTT method. IL-2 and IFN-γ levels and total anti-DHAV-1 antibody secretion after treatment with DHAV-1 for 4, 8 and 54 h were determined by ELISA. RESULTS: BLIN showed a dose-dependent DHAV-1 reproduction inhibitory effect. The inhibitory effect was highest at 20 µg/mL, where DHAV-1 adsorption and release were significantly lower. Meanwhile, BLIN at 5 µg/mL significantly increased T and B lymphocyte proliferation. BLIN stimulated total anti-DHAV-1 antibody secretion in ducklings at the dosage of 4 mg per duckling, but did not stimulate IL-2 and IFN-γ secretion significantly. CONCLUSIONS: BLIN inhibits DHAV-1 reproduction by suppressing its adsorption and release. Additionally, BLIN promoted the duckling antiviral response.

Treatment effect of a flavonoid prescription on duck virus hepatitis by its hepatoprotective and antioxidative ability.

CONTEXT: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is an acute and lethal disease of young ducklings. However, there is still no effective drug to treat DVH. OBJECTIVE: This study assessed the curative effect on DVH of a flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) as well as the hepatoprotective and antioxidative effects of BLIN. MATERIALS AND METHODS: MTT method was used to test the anti-DHAV-1 ability of BLIN in vitro. We then treated ducklings by BLIN (3 mg per duckling, once a day for 5 days) to evaluate the in vivo efficacy. To study the hepatoprotective and antioxidative roles of BLIN in its curative effect on DVH, we investigated the hepatic injury evaluation biomarkers and the oxidative stress evaluation indices of the ducklings. RESULTS: On duck embryonic hepatocytes, DHAV-1 inhibitory rate of BLIN at 20 µg/mL was 69.3%. The survival rate of ducklings treated by BLIN was about 35.5%, which was significantly higher than that of virus control (0.0%). After the treatment of BLIN, both the hepatic injury and the oxidative stress of infected ducklings alleviated. At the same time, a significant positive correlation (p < 0.05) existed between the hepatic injury indices and the oxidative stress indices. CONCLUSIONS: BLIN showed a significant curative effect on DVH. The antioxidative and hepatoprotective effects of BLIN made great contributions to the treatment of DVH. Furthermore, BLIN is expected to be exploited as a new drug for the clinical treatment of DVH.

Highly selective and efficient biotransformation of linarin to produce tilianin by naringinase.

OBJECTIVES: To develop a practical method to prepare tilianin by highly selective and efficient hydrolysis of the C-7 rhamnosyl group from linarin. RESULTS: Naringinase was utilized to selectively catalyze the formation of tilianin using linarin as the starting material. The reaction conditions, including temperature, pH, metal ions, substrate concentration and enzyme concentration, were optimized. At 60 °C, naringinase showed enhanced α-L-rhamnosidase activity while the ß-D-glucosidase activity was abrogated. The addition of Mg(2+), Fe(2+) and Co(2+) was also beneficial for selective biotransformation of linarin to tilianin. Under the optimized conditions (pH 7.0 at 60 °C), linarin could be nearly completely transformed to tilianin with excellent selectivity (>98.9 %), while that of the by-product acacetin was less than 1.1 %. In addition, the structure of target product tilianin was fully characterized by HR-MS and (1)H-NMR. CONCLUSION: A highly selective and efficient biotransformation of linarin to tilianin was developed by the proper control of incubation temperature, which enhanced the α-L-rhamnosidase activity of naringinase and blocked its ß-D-glucosidase activity.

A rapid and sensitive LC-MS/MS method for the determination of linarin in small-volume rat plasma and tissue samples and its application to pharmacokinetic and tissue distribution study.

A rapid and sensitive LC-MS/MS method was developed for the determination of linarin in small-volume rat plasma and tissue sample. Sample preparation was employed by the combination of protein precipitation (PPT) and liquid-liquid extraction (LLE) to allow measurement over a 5-order-of-magnitude concentration range. Fast chromatographic separation was achieved on a Hypersil Gold column (100 × 2.1 mm i.d., 5 µm). Mass spectrometric detection was achieved using a triple-quadrupole mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. Quantification was performed using selected reaction monitoring of precursor-product ion transitions at m/z 593 → 285 for linarin and m/z 447 → 271 for baicalin (internal standard). The total run time was only 2.8 min per sample. The calibration curves were linear over the concentration range of 0.4-200 µg/mL for PPT and 0.001-1.0 µg/mL for LLE. A lower limit of quantification of 1.0 ng/mL was achieved using only 20 µL of plasma or tissue homogenate. The intra- and inter-day precisions in all samples were ≤14.7%, while the accuracy was within ±5.2% of nominal values. The validated method has been successfully applied to pharmacokinetic and tissue distribution study of linarin.

Simultaneous Ultra Performance Liquid Chromatography Determination and Antioxidant Activity of Linarin, Luteolin, Chlorogenic Acid and Apigenin in Different Parts of Compositae Species.

Linarin (LA), luteolin (LE), chlorogenic acid (CA) and apigenin (AP) are four major flavonoids with various promising bioactivities found in Compositae (COP) species. A reliable, reproducible and accurate method for the simultaneous and quantitative determination of these four major flavonoids by Ultra Performance Liquid Chromatography (UPLC) analysis was developed. This method should be appropriate for the quality assurance of COP. The UPLC separation was carried out using an octadecylsilane (ODS) Hypersil (2.1 mm × 250 mm, 1.9 µm) and a mobile phase composed of acetonitrile and 0.1% formic acid in water at a flow rate 0.44 mL/min and ultraviolet (UV) detection 254 nm. Gradient elution was employed. The method was precise, with relative standard deviation below 3.0% and showed excellent linearity (R² > 0.999). The recoveries for the four flavonoids in COP were between 95.49%-106.23%. The average contents of LA, LE, CA and AP in different parts (flower, leave and stem) of COP were between 0.64-1.47 g/100 g, 0.66-0.89 g/100 g, 0.32-0.52 g/100 g and 0.16-0.18 g/100 g, respectively. The method was accurate and reproducible and it can provide a quantitative basis for quality control of COP.

[Modeling of thermal degradation of linarin during concentration process].

Ganmaoling granule, with annual sale of over one billion yuan, is the first brand of domestic cold medicine sales. As the only traditonal Chinese medicine（TCM） quality control indicator of Ganmaoling granule, linarin is thermally unstable. Its content will be changed significantly during the production process, which would then affect the quality of the finished product. In this paper, the law of degradation of linarin was investigated. The experimental results showed that degradation reaction of linarin belongs to the first reaction characteristics. The effective methods to reduce the loss of linarin would be realized fortunately by strictly controlling the heating temperature or shortening the heating time.

[Study on balance of process of alcohol precipitation of ganmaoling granules].

Ganmaoling granule is the first brand of domestic cold medicine sales, but its preparation method and process control parameters are relatively rough. Therefore it is urgent to upgrade the technologies of large varieties of traditional Chinese medicine (TCM). This paper focused on the balance between the remove of impurity and the retention of linarin during the process of alcohol precipitation of Ganmaoling granules. The effects of four factors on the process were investigated via single factor experiments. The results showed that the precipitating period, the initial ethanol concentration and the final ethanol concentration had a great effect on retention of linarin while the initial density of the extract has not. Similarly, the initial ethanol concentration, the final ethanol concentration and the initial extract density have a great effect on the yield of dry extract while the time of alcohol precipitation has not. The parameters of alcohol precipitation of Ganmaoling granules were optimized as 16 h of precipitating period, 95% ethanol as the initial reagent, 70% of the final ethanol concentration, and 1.10 of the initial extract density.

Determination and pharmacokinetics of linarin in rat plasma after intramuscular administration of linarin solution and Yejuhua injection by HPLC.

A simple and sensitive HPLC method was developed and validated for the determination of linarin in rat plasma. Plasma samples were carried out by protein precipitation with acetonitrile and separated on a Shim-pack VP-ODS analytical column (250 × 4.6 mm, 5 µm). The calibration curve was linear in the measured range of 20.8-4160 ng/mL and the lower limit of quantification was 20.8 ng/mL for linarin. The validated method was successfully applied to the pharmacokinetic study of linarin in rat plasma after intramuscular administration of monomeric compound and traditional Chinese medicinal preparation - Yejuhua injection.