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The aim of this work was to increase the solubility and oral bioavailability of isorhamnetin, kaempferol, and quercetin in the total flavones of Hippophae rhamnoides L. (TFH) by preparing their nanosuspensions (NSs) and an inclusion complex. Based on the particle size and zeta potential, P407, Soluplus, SDS, PEG-6000, and HP-β-CD were selected as stabilizers. TFH NSs and a TFH/HP-β-CD inclusion complex were prepared, and their morphology, crystallinity, molecular interactions, and cytotoxicity were investigated. Furthermore, the saturation solubility, dissolution, and pharmacokinetics of the three flavonoids in the TFH, TFH NSs, and TFH/HP-β-CD inclusion complex were compared. The five obtained TFH NSs were physically stable, and their particle sizes were all below 200 nm. The solubility and dissolution of the three active components were obviously enhanced by the formation of the TFH NSs and TFH/HP-β-CD inclusion complex. Correspondingly, the oral bioavailability of isorhamnetin, kaempferol, and quercetin increased up to 4.11-, 3.85-, and 6.73-fold, respectively, in the TFH NSs and 2.89-, 3.71-, and 9.51-fold, respectively, in the TFH/HP-β-CD inclusion complexes compared to those in the raw TFH. In brief, both NSs and inclusion complexes can improve the oral bioavailability of the three flavonoids in TFH. Taking the drug loading and the stable ratio of the multiple components into consideration, the NSs is a more promising strategy than the inclusion complex for increasing the oral bioavailability of multiple water-insoluble components in herbal extracts. Graphical abstract.

In this study, we examined the mechanism of Flavone of Hippophae (H-flavone) in regulating macrophage foaming and atherosclerosis (AS) plaque formation. H-flavone treatment increased the secretion of C1q/tumor necrosis factor-related proteins 6 (CTRP6) in Ox-LDL-treated mouse peripheral blood macrophage cells (PBMC) and significantly reduced the percentage of cholesteryl ester (CE) in PBMC. Additionally, H-flavone suppressed Ox-LDL-induced cell foaming and the production of inflammatory cytokines through upregulating CTPR6 expression. Next, we further validated the inhibitory effect of H-flavone on plaque formation and inflammation in a mouse AS model. A substantial reduction in the secretion of inflammatory cytokines was observed in apoE -/- mice by H-flavone. Immunohistochemistry and Oil Red O staining results showed that H-flavone suppressed macrophage infiltration and the development of AS plaque. These effects were more pronounced in early administration. Our results suggest that H-flavone effectively inhibits macrophage foaming, inflammation and vascular plaque formation by upregulating CTRP6 and may be used to reduce AS risk.

In this study, a valid method was established for the isolation and purification of flavone glycosides from Hippophae rhamnoides L. seed residues using high-speed counter-current chromatography in one step, with a solvent system of ethyl acetate-methanol-n-butyl alcohol-water (9:1:0.5:9, v/v/v/v). A total of 28.8 mg compound I and 57.3 mg compound II were obtained from 200 mg of flavone H-glycosides rich extract, with purities of 98.3 and 96.4%, respectively. The structures of two compounds were identified by MS and NMR. 3-O-β-D-Sophorosylkaempferol-7-O-{3-O-[2(E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoyl]}-α-L-rhamnoside is compound I and compound II named hippophanone is a new compound were identified by MS and NMR. The method was efficient and convenient, which could be used for the preparative separation of flavone glycosides from H. rhamnoides L. seed residues.

The purpose of this work was to improve the oral bioavailability of a poorly soluble functional food ingredient, the total flavones of Hippophaë rhamnoides L. (TFH). A self-microemulsion drug delivery system (SMEDDS) was developed to overcome the problems of poor absorption of TFH in vivo. The optimal SMEDDS significantly enhanced the solubility of TFH up to 530 times compared to that in water. The mean droplet size was 61.76 nm with uniform distribution. And the loaded system was stable at 25 °C for 3 mo with transparent appearance. The in vitro release of TFH from SMEDDS was faster and more complete than that from suspension. After oral administration of TFH-SMEDDS in rats, the relative bioavailability of TFH was dramatically improved for 3.09 times compared with the unencapsulated form. The investigation indicated the potential application of SMEDDS as a vehicle to improve the oral bioavailability of TFH., Practical Application: The lipid-based nanotechnology, namely self-microemulsion drug delivery system (SMEDDS) was used to improve the bioavailability and oral delivery of total flavones of Hippophaë rhamnoides L. (TFH). The relevant bioavailability of TFH could be remarkably 3-fold improved by the optimized SMEDDS. The SMEDDS produced via a simple one-step process for poorly soluble TFH to achieve a significant improvement in the bioavailability, may endorse the promising utilization of TFH in functional foods as well as pharmaceutical fields with an enhanced absorption in vivo., (© 2017 Institute of Food Technologists®.)

Pharmacokinetic properties of isorhamnetin, quercetin, and kaempferol in three different total flavones of Hippophae rhamnoides (TFH) preparations were compared after oral administration to beagle dogs by a UPLC-MS method. The pharmacokinetic results showed that C max of isorhamnetin and quercetin in TFH solid dispersion (TFH-SD) and TFH self-emulsifying (TFH-SE) preparations was significantly enhanced than that in TFH preparations (p < 0.05). The AUCs of isorhamnetin and quercetin in TFH-SD were 5.9- and 3.1-fold higher than that of TFH, while the AUCs of isorhamnetin and quercetin in TFH-SE were 3.4- and 2.4-fold higher than that of TFH. These findings suggested that the oral bioavailability of isorhamnetin and quercetin in beagle dogs can be significantly increased in TFH-SD and TFH-SE preparations compared to TFH preparations, which was helpful to explore the new forms for oral administration TFH and explain their in vivo processes.

Sea buckthorn (Hippophae rhamnoides L.) flavones have been used as candidate functional food ingredients because of their bioactivities, such as treating cardiovascular disorders, lowering plasma cholesterol level, and regulating immune function. However, the protective effects of sea buckthorn flavones against retinal degeneration remain unclear to date. This study investigated the protective effects of total flavones from H. rhamnoides (TFH) against visible light-induced retinal damage and explored the related mechanisms in pigmented rabbits. Rabbits were treated with TFH (250 and 500 mg/kg) for 2 weeks pre-illumination and 1 week post-illumination until sacrifice. Retinal function was quantified by performing electroretinography 1 day before and 1, 3, and 7 days after light exposure (18000 lx for 2 h). Retinal degeneration was evaluated by measuring the thickness of the outer nuclear layer (ONL) and performing the TUNEL assay 7 days after light exposure. Enzyme-linked immunosorbent assay, Western blot analysis, and immunohistochemistry were used to explore the antioxidant, anti-inflammatory, and anti-apoptotic mechanisms of TFH during visible light-induced retinal degeneration. Light exposure produced a degenerative effect primarily on the ONL, inner nuclear layer (INL), and ganglion cell layer (GCL). TFH significantly attenuated the destruction of electroretinograms caused by light damage, maintained ONL thickness, and decreased the number of TUNEL-positive cells in the INL and GCL. TFH ameliorated the retinal oxidative stress (GSH-Px, CAT, T-AOC, and MDA), inflammation (IL-1β and IL-6), angiogenesis (VEGF), and apoptosis (Bax, Bcl2, and caspase-3) induced by light exposure. Therefore, TFH exhibited protective effects against light-induced retinal degeneration by increasing the antioxidant defense mechanisms, suppressing pro-inflammatory and angiogenic cytokines, and inhibiting retinal cell apoptosis.

Total flavones of Hippophae rhamnoides L. (TFH) are extracted from the widely distributed thorny bush Sea buckthorn (Hippophae rhamnoides L.). Isorhamnetin (IS) is one of the representative ingredients in TFH. In this study, the absorption properties of IS in TFH and its pure form were compared through transepithelial transport and cellular uptake experiments in a Caco-2 cell model. Our results show that the absorption properties of IS in TFH and its pure form were remarkably different: (1) Both PappAB and PappBA of IS in TFH were dramatically increased compared with those of IS pure form; consequently, its Pratio was 2.3-fold higher than that of IS; (2) Both the accumulation and efflux of IS in TFH were significantly enhanced compared with the single compound. One likely reason for these differences is that the multiple components in TFH significantly down regulated the mRNA expression level of MRP2, which lead to a decrease in the protein level of MRP2, based on western blotting and RT-PCR assays. This study highlights the significant differences in the absorption properties of flavonoid components in different forms and the potential multi-component interactions in TFH., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the effects of solid dispersions (SD) and self-emulsifying (SE) formulations on the solubility and absorption properties of active components in total flavones of Hippophae rhamnoides L. (TFH). The solubility, dissolution rate, permeability and pharmacokinetics of isorhamnetin, quercetin and kaempferol in TFH SD/SE formulations and TFH were compared. The results showed that the solubility and dissolution rate of isorhamnetin, quercetin and kaempferol in SD/SE formulations were significantly enhanced compared to those in TFH, however, their intestinal permeability was comparable. The bioavailability of isorhamnetin, quercetin and kaempferol in rats remarkably increased after oral administration of TFH SD formulations compared to TFH, but there was no significant increase after oral administration of TFH SE formulations. The results of this study indicated the SD formulations on the improvement of pharmacokinetic properties of isorhamnetin, quercetin and kaempferol in TFH were much better than those of SE formulations. The improvement of pharmacokinetic properties of isorhamnetin, quercetin and kaempferol in TFH by SD formulations was probably ascribed to the enhancement of the solubility and dissolution of the three components, but was not relevant to the intestinal permeability. Therefore, as for herb extracts containing multiple components, especially for their major components with poor water solubility, solid dispersion formulations might have the better potential to enhance their bioavailability.

The goal of the study is to evaluate the self-microemulsifying drug delivery system (SMEDDS) which enhances the oral bioavailability of the poorly water-soluble drug, total flavones of Hippophae rhamnoides (TFH). It is orally administered for the protection of human cardiovascular system. Self-microemulsifying time, particle size, polydispersity index (PDI), morphological characterization, in vitro dispersity, stability, in situ intestinal absorption and relative bioavailability were investigated in detail. The TFH-SMEDDS rapidly formed fine oil-in-water microemulsions with 0.1 mol x L(-1) hydrochloride solution, with average size of which was less than 40 nm, PDI was below 0.2, and the particles of which were observed round-shaped under transmission electron microscope. Almost 90% of TFH (expressed with quercetin) was released from SMEDDS within 20 min, which was remarkably higher than that from common capsules. The stability test showed the TFH-SMEDDS maintained stable in 6 months under accelerated condition. In situ absorption study demonstrated the absorption rate constant of TFH-SMEDDS (expressed with quercetin) was significantly higher than that of TFH in ethanolic solution (P < 0.05). The absorption of TFH from SMEDDS showed a 4.18-fold increase in relative bioavailability (expressed with quercetin) compared with that of the suspension. The results suggest that SMEDDS is a promising drug delivery system to increase the oral bioavailability of TFH.

An ultra performance liquid chromatography-mass spectrometric (UPLC-MS) method was developed to investigate the pharmacokinetic properties of isorhamnetin, kaempferol and quercetin from a total flavone extract of Hippophae rhamnoides L. (TFH) after single dose oral administration. Rat plasma samples were pretreated using liquid-liquid extraction, and chromatographic separation was performed on a C(18) column using a linear gradient of methanol and formic acid (0.1%). The pharmacokinetic parameters of isorhamnetin, kaempferol and quercetin from TFH in rats were quantitatively determined by UPLC with photodiode array detection (PDA). The qualitative detection of the three flavones was accomplished by selected ion monitoring in negative ion mode ESI-MS. Results of the pharmacokinetic study indicate that the three flavones in TFH were absorbed by passive diffusion in rats, and no "double-peak" phenomenon was observed in C-t curves of the three flavones from TFH except for quercetin. Results of this study indicate that the pharmacokinetic behaviors of isorhamnetin, kaempferol and quercetin when administered together in a complex herbal extract might be different than the individual behaviors of the same compounds administered in their pure forms. Results of this study also demonstrate that UPLC-MS is a rapid and practical method to determine the pharmacokinetic parameters of flavones present in an herbal extract., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Purpose: The objective of this study was to investigate the absorption behavior of total flavones of Hippophae rhamnoides L. (TFH) (the sum of isorhamnetin and quercetin as the index component) in the rat intestine using in situ circulation method., Methods: The accumulated TFH absorption and related absorption parameters were calculated. Furthermore, the influences of Cremophor ELP and the P-glycoprotein inhibitor, verapamil, on the intestinal absorption of TFH were studied using the in situ circulation model., Results and Discussion: The results showed that the absorption of TFH increased linearly with its concentration, indicating that a passive diffusion process was dominated. There were no significant differences in the absorption of TFH in three small intestine segments of duodenum, jejunum, and ileum and at different concentrations of Cremophor ELP ranging from 0.25% to 1% (P > 0.05). With the presence of P-gp inhibitor, verapamil, in the circulation fluid, the accumulated absorption of TFH did not increase significantly (P > 0.05). Further studies on the solubility and permeability enhancement of TFH should be investigated to develop new TFH products with high bioavailability.

A lack of adequate or accepted research methodology has been a major obstacle to study herbal medicines. In this study, instead of the prevalent hyphenated chromatographies, common high performance liquid chromatography equipped with ultraviolet detector (HPLC-UV) and multivariate statistical analysis were utilized to assess the qualities of total flavones of sea buckthorn (TFS), an 85% ethanol extract of the sea buckthorn berries. Two complementary HPLC-UV methods were developed, validated and combined to comprehensively determine the ingredients in TFS. Principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) of the combined analytical data showed that the six batches of TFS could be well differentiated. Hierarchical cluster analysis (HCA) using Ward's minimum variance method of the PLS-DA loading matrix demonstrated the known ingredients (quercetin, kaempferol, isorhamnetin, oleanolic acid and ursolic acid) and three unknown ingredients in TFS significantly contributed to the quality differences. A PLS regression model indicated that the results of the present method correlated well with the content of total flavones, which is now the quality control approach of TFS. Results from this study indicated that the proposed method is reliable for the quality reassessment of some widely used herbal extracts., (Crown Copyright 2009. Published by Elsevier B.V. All rights reserved.)

This study was designed to determine the cytoprotective activity of flavones of seabuckthorn (Hippophae rhamnoides L.) against tert-butyl hydroperoxide (tert-BOOH), used as an oxidant to induce oxidative damage, with lymphocytes as the model system. Addition of tert-BOOH (250 microM) to the cells resulted in enhanced cytotoxicity and free radical production. The intracellular calcium levels, caspase activity, and apoptosis were significantly increased following tert-BOOH treatment. Seabuckthorn flavones at the concentration of 100 microg/mL significantly inhibited tert-BOOH-induced cytotoxicity and free radical production and also restored the antioxidant status to that of control cells. Seabuckthorn flavones also significantly restricted tert-BOOH-induced apoptosis by decreasing intracellular calcium levels and caspase activity. The extract also decreased tert-BOOH-induced formation of DNA breaks by 30%. These observations suggest that the flavones of seabuckthorn have marked cytoprotective properties, which could be attributed to the antioxidant activity.

Objective: To develop self-microemulsifying preparations of total flavones of Hippophae Rhamnoides L. (TFH) and the determination method of dissolution., Method: The equilibrium solubility of TFH in different compositions of oils, emulsifier and assistant emulsifier was investigated. The self-microemulsion formula was optimized by constructing the pseudo-ternary phase diagrams of blank SMEDDS determining the self-microemulsifying efficiency and the stability of the SMEDDS. The 2 hours dissolution curve of TFH self-microemulsifying preparations was established. The optimal self-microemulsion formula was composed of MIGLYOL 812 N, Cremophor EL and 1,2-Propylene glycol., Result: The ratio of them was 0.5:5.7:3.8. The average particle size was 12.1 nm. The dissolution rate at 10 minutes of TFH self-microemulsifying preparation was 131% higher than that of Xinda kang tablets., Conclusion: The acquired microemulsion with small particle size is stable. The dissolution rate is evidently improved compared with market dosage forms.

The purpose of this study was to enhance the dissolution of total flavones of Hippophae rhamnoides L. (TFH) by solid dispersions consisting of the drug and a polymeric carrier, poloxamer 188 (PXM). The solvent evaporation method was used to prepare solid dispersions. A 3(2) full-factorial design approach was used for optimization wherein the amount of solvent (X(1)) and the drug-to-polymer ratio (X(2)) were selected as independent variables and the percentage of TFH dissolved in 10 min (Q(10)) was selected as the dependent variable. Multiple linear regression analysis revealed that a suitable level of (1) and X(2) was required for obtaining higher dissolution of TFH from PXM solid dispersions. Solid dispersions were characterized by differential scanning calorimetry, X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, and dissolution tests. Characterization studies revealed that solid dispersion of TFH-PXM showed enhancement of TFH dissolution due to the conversion of TFH into a less crystalline and/or amorphous form. In conclusion, dissolution enhancement of TFH was obtained by preparing its solid dispersions in PXM using solvent method.

Objective: To study the mechanisms of the anti-myocardial ischemia of total flavones of Hippophae rhamnoides (TFH) at the level of proteome., Method: Surface enhanced laser desorption/ionization (SELDI) mass spectrometry with protein chip IMAC3, SAX2 and NP20 was performed to compare the differentially expressed protein in myocardial ischemia in the TFH-treated groups with the 0.9% sodium chloride groups. Protein chips were examined in PBS II - C protein chip reader (ciphergen ciosystem inc) and the protein profiling was analyzed by Proteinchip Software 3. 0. 2., Result: The revealed six peaks had significant difference between the TFH-treated groups and the control groups, one of which were up-regulated in the TFH-treated groups, and the other were down-regulated. And in these six distinct proteins, there were four proteins on the IMAC3 chips and one protein on the SAX2 chips., Conclusion: The TFH could prevent the myocardium from ischemia via regulating expression of different proteins.

Solid lipid nanoparticles (SLNs) containing total flavones of Hippophae rhamnoides (TFH) were prepared by high-pressure homogenization (HPH), by both hot HPH and cold HPH. The influence of process parameters (lipid matrix, lipid concentration, carbohydrate type and its concentration) on the SLN size distribution, zeta potential, entrapment efficiency, crystal form, and in vitro release profile was investigated. The highest entrapment efficiency for TFH, at around 93%, was found for SLNs composed of TFH/Compritol 888 ATO in a 1:30 molar ratio and made by cold HPH. The advantages of TFH SLNs are the improved oral bioavailability of TFH and the prolonged mean retention time and drug release time.

The present investigation was undertaken to determine the efficacy of topical administration of flavone of sea buckthorn (Hippophae rhamnoides L.) on cutaneous wound healing in rats. Four full-thickness excision wounds were created on the back of rat and 1.0% w/v flavone prepared in propylene glycol was applied topically. Control animals received the vehicle alone in an identical manner. The healing of the wound was assessed by the rate of wound contraction, period of epithelialization, hydroxyproline, hexosamine, antioxidants estimation and histopathology of the granulation tissue. The sea buckthorn flavone promoted the wound healing activity as indicated by improved rate of wound contraction, decreased time taken for epithelialization (16.3 days versus 24.8 days in controls) and significant increase in hydroxyproline (26.0%) and hexosamine (30.0%) content. These findings were also confirmed by histopathological examinations. In addition, it was observed that sea buckthorn flavone possesses potent antioxidant properties as evidenced by significant increase in reduced glutathione (55.0%), vitamin C (70.0%) and catalase (20.0%) activities in wound granulation tissue. The flavone treatment also resulted in significant decrease in lipid peroxide levels (39.0%). The results suggest that the sea buckthorn flavone promotes wound healing activity.