hequercetin, and kaempferorhamnoides L.

Yan Xie a,,1, Huilin Luo b,1, JingzeTong Zhang b, Tao Wu c, Guang Jia Research Center for Health and Nutrition, Shanghai Unb Teaching and Experimental Center, Shanghai Universitc Institute of Chinese Materia Medica, Shanghai Universd Global Pharmaceutical Research and Development, Hoe ed Hosp

Accepted in revised form 31 December 2013

Results: The P of isorhamnetin, kaempferol, and quercetin was improved 2.03-, 1.69-, and

quercetin in TFH waselated to their enhancedmary, IP6 is a potentialive and safe.B.V. All rights reserved.

Fitoterapia 93 (2014) 216225

Contents lists available at ScienceDirect

Fitoter

j ourna l homepage: www.e ls1. Introduction Correspondence to: Y. Xie, Research Center for Health and Nutrition,

Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road,Shanghai 201203, China. Tel.: +86 21 51322440; fax: +86 21 51322407.Conclusions: The oral bioavailability of isorhamnetin, kaempferol, andenhanced by the co-administration of IP6. The main mechanisms are raqueous solubility and permeability in the presence of IP6. In sumabsorption enhancer for pharmaceutical formulations that is both effect

2014 Elsevierapp

2.11-fold in the presence of 333 g/mL of IP6, respectively. Water solubility was increased 22.75-,15.15-, and 12.86-fold for isorhamnetin, kaempferol, and quercetin, respectively, in the presenceof 20 mg/mL IP6. The lipophilicity of the three flavonoids was slightly decreased, but theirhydrophilicity was increased after the addition of IP6 in the water phase as the logP values ofisorhamnetin, kaempferol, and quercetin decreased from 2.38 0.12 to 1.64 0.02, from2.57 0.20 to 2.01 0.04, and from 2.39 0.12 to 1.15 0.01, respectively. The absorptionenhancement ratios were 3.21 for isorhamnetin, 2.98 for kaempferol, and 1.64 for quercetin withco-administration of IP6 (200 mg/kg) in rats. In addition, IP6 (200 mg/kg, oral) caused neithersignificant irritation to the rat intestines nor cytotoxicity (400 g/mL) in Caco-2 cells.

FlavonoidsAbsorption enhancerOral absorptionSolubilityPermeability Correspondence to: G. Li, Pharmacy Department, ShHospital, 184 Baoding Road, Shanghai 200082, Chi65556806.

E-mail addresses: rosexie_1996@hotmail.com (Y. Xlgwshutcm@163.com (G. Li).1 These authors contributed equally to this work.

0367-326X/$ see front matter 2014 Elsevier B.V.http://dx.doi.org/10.1016/j.tote.2014.01.013and kaempferol were determined with and without IP6, and mucosal epithelial damage resultingfrom IP6 addition was evaluated by MTT assays and morphology observations.Available online 21 January 2014

Keywords:Phytic acidoral absorption of isorhamnetin,l in total avones of Hippophae

Duan a,c, Chao Hong a, Ping Ma d, Guowen Li e,,c

iversity of Traditional Chinese Medicine, Shanghai 201203, Chinay of Traditional Chinese Medicine, Shanghai 201203, Chinaity of Traditional Chinese Medicine, Shanghai 201203, Chinaspira Inc., 1776 North Centennial Drive, McPherson, KS 67460, USAital, Shanghai 200082, China

a b s t r a c t

Aim: Total flavones ofHippophae rhamnoides L. (TFH) have a clinical use in the treatment of cardiacdisease. The pharmacological effects of TFH are attributed to its major flavonoid components,isorhamnetin, kaempferol, and quercetin. However, poor oral bioavailability of these flavonoidslimits the clinical applications of TFH. This study explores phytic acid (IP6) enhancement of the oralabsorption in rats of isorhamnetin, kaempferol, and quercetin in TFH.Methods: In vitro Caco-2 cell experiments and in vivo pharmacokinetic studies were performed toinvestigate the effects of IP6. The aqueous solubility and lipophilicity of isorhamnetin, quercetin,Pharmacy Department, Shanghai TCM-Integrat

a r t i c l e i n f o

Article history:Received 27 November 2013Phytic acid enhances tanghai TCM-integratedna. Tel./fax: +86 21

ie),

All rights reserved.apia

ev ie r .com/ locate / f i to teCardiovascular diseases (CVDs) are the number one causeof death in the world. In 2008, the World Health Organization(WHO) reported that approximately 17.3 million people diedfrom CVDs, representing 30% of global deaths with 80% ofthe deaths occurring in low- and middle-income countries.

This prevalence illustrates the urgent need for efficient CVDprevention and treatment. Various pharmacological prop-erties of total flavones of Hippophae rhamnoides L. (TFH),extracted from the unique and valuable sea buckthorn plant(Hippophae rhamnoides L., Elaeagnaceae), have been re-ported. These properties include antioxidant effects, anti-hypoglycemia effects, blood circulation improvement,plasma cholesterol level reduction, anti-hypertensive ac-tivity, and cardiac protection [16]. In China, TFH has beenclinically used recently for cardiac diseases and hyperpiesiain capsule and tablet forms for oral administration. TFH useis similar to the world-wide use of the herbal extract ofGinkgo biloba (GbE) for cerebrovascular disease indications,which is formulated as tablets, capsules, and injections.

The major bioactive flavonoids in TFH are isorhamnetin,kaempferol, and quercetin (Fig. 1) [7,8], which are efficaciousagainst cardiac diseases. However, poor aqueous solubility,low lipophilicity, and extensive first-pass metabolism in thegut and liver contribute to the relatively low oral bioavail-ability of the three aglycons [9,10]. These properties limit theability of these compounds to traverse the lipid-rich biolog-

preparations is a practical and effective method to improvethe bioavailability of active components in phytomedicine.

Phytic acid (myo-inositol hexaphosphate, IP6) is a majorform of phosphorylated inositol present in food; it consti-tutes 15% by weight of most cereal, nuts, legumes, grains,and oilseeds [16] and is found in some edible vegetables [17].It is an important nutritional substance because it can releaseinorganic phosphate (Pi) [18], which could support growthduring the early stages of seedling development. Structurally,IP6 contains six phosphates, as shown in Fig. 1D. IP6 has beenreported to possess various significant health benefits includ-ing its potential as an antioxidant agent [19], anticancer agent[20], chelating agent [21], and heart disease prevention agent[22]. IP6 is not only found in many foods, such as infant flours[23], but is also used as a protective agent in apple juice [24],beef, and pork products [25]. Recently, Matsumoto et al. [26]reported that oral co-administration of IP6 and anthocyanins inrats and humans dramatically enhanced plasma concentrationsof anthocyanins and their excretion in the urine. However,whether IP6 could similarly enhance the oral absorption ofisorhamnetin, kaempferol, and quercetin in TFH is unknown.

A), kae

217Y. Xie et al. / Fitoterapia 93 (2014) 216225ical membranes, resulting in the poor oral absorption of TFH.Therefore, it is necessary to improve the oral absorption ofTFH to reach effective plasma concentrations and enhancepharmacologic effects after oral administration.

Currently, many pharmaceutical technologies, includingsolid lipid nanoparticles [11], solid dispersions [12], andself-emulsified preparations [13], have been utilized to im-prove the oral absorption of TFH. Unfortunately, the practicaluse of these pharmaceutical technologies in herbal extracts hasbeen limited because of low drug loading capacity, the highdose required, and poor patient compliance. Therefore, alter-native formulations that can improve the intestinal absorptionof active components in herbal extracts are highly desirable.Absorption enhancement is a technology aimed to increase thesolubility and permeability of chemicals and therefore improvethe oral bioavailability. In addition, most of absorptionenhancers are pharmaceutical adjuvants [14,15]. As a result,the addition of absorption enhancers in herbal extract

Fig. 1. The chemical structures of isorhamnetin (The objective of this study was to investigate the influenceof IP6 on the oral absorption of isorhamnetin, kaempferol, andquercetin in TFH, outline its preliminary mechanism and laya foundation for the potential use of TFH and IP6 in clinicalapplications.

2. Materials and methods

2.1. Chemicals

TFH powders were obtained from Sichuan Medco Pharma-ceutical Co. Ltd. (Sichuan, China), and contained isorhamnetin,quercetin, and kaempferol (10%, 3.13%, and 1.0% (w/w), res-pectively). Isorhamnetin, quercetin, kaempferol, and baicaleinwere purchased from the National Institute for the Control ofPharmaceutical and Biological Products (Beijing, China). IP6 waspurchased from Aladdin Chemistry Co. Ltd. (Shanghai, China).HPLC grademethanolwas purchased from SK Chemicals (Ulsan,

mpferol (B), quercetin (C), and phytic acid (D).

218 Y. Xie et al. / Fitoterapia 93 (2014) 216225Korea), and formic acid was obtained from Fluka (Burchs,Switzerland). Ultra-pure deionized water was generated froma Millipore Milli-Q Gradient system (Millipore CorporationBedford, MA, USA). All other reagents were of analytical grade.

2.2. Cell culture, cytotoxicity, and transport studies of isorhamnetin,kaempferol, and quercetin in TFH in a Caco-2 cell monolayer model

2.2.1. Cell culture of Caco-2 cellsCaco-2 cells were obtained from Shanghai Institutes for

Biological Sciences (SIBS, Shanghai, China). The cells weregrown in culture dishes (Corning Costar Corp, NY, USA) inhigh glucose Dulbecco's modified eagle's medium (DMEM,Thermo Fisher Scientific, Utah, USA) supplemented with10% (v/v) fetal bovine serum (Gibco BRL Life Technology, NY,USA), 1% (v/v) non-essential amino acid solution (GibcoBRL Life Technology, NY, USA), and 100,000 U/L antibioticantimycotic solution (Gibco BRL Life Technology, NY, USA).The cells were grown in an atmosphere of 5% CO2/95% O2 and90% relative humidity at 37 C. The medium was replacedevery 23 days after incubation. The cells were passagedand split 1:5 every 5 days using 0.25% trypsin with 0.02%ethylene diamine tetraacetic acid (EDTA), when the cellconfluence reached 70%80%. For the transport experiments,cells at passage 4045 were seeded at a density of 1 105 cells/cm2 onto permeable polycarbonate inserts (0.6 cm2,0.45 m pore size, Millipore Corp, MA, USA) in 24-well plasticplates. The media in the culture plates were changed everytwo days during the first week of post-seeding and thenreplaced daily. The integrity of cell monolayers was assessedby the measurement of transepithelial electrical resistance(TEER) using a Millicell-ERS-electrode (Millipore Corp, MA,USA). Caco-2 cells were used for the transport experiments2128 days after seeding, when the TEER values exceeded400 cm2 after resistance correction from the controlblank-well readings.

2.2.2. Preparation of transport bufferHank's balanced salt solution (HBSS) was used as the

transport buffer for the transport studies in the Caco-2 cellmonolayermodel. The buffer pHwas adjusted to 7.5, 6.5, and 5.5by addition of 0.5 mol/L sodium hydroxide or hydrochloric acid.

2.2.3. Stability of isorhamnetin, quercetin, and kaempferol intransport buffer

The stability of isorhamnetin, kaempferol, and quercetinin TFH was investigated in HBSS. TFH was dissolved in DMSOand then diluted in HBSS with different pH values (pH 5.5,6.5, and 7.5) to a 1% (w/v) final DMSO concentration. Thefinal concentrations of isorhamnetin, quercetin, and kaempferolwere 17.95, 6.56, and 2.27 g/mL, respectively. All samples wereincubated in an atmosphere of 5% CO2/95% O2 and 90% relativehumidity at 37 C for 120 min. Next, 100 L of each sample wasremoved and immediately added to an equal volume ofacetonitrile. After shaking to mix uniformly, all samples werecentrifuged at 13,000 rpm for 15 min and supernatants wereanalyzed by HPLC.

2.2.4. Cytotoxicity studies of IP6 in Caco-2 cellsThe MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl

tetrazolium bromide) assay [27] was used to assess thepotential cytotoxicity of IP6 in Caco-2 cells. The Caco-2 cellswereseeded in 96-well plates at 1 105 cells/well in DMEM andcultured at 37 C for 24 h. Subsequently, the culture mediumwas replaced with 100 L of IP6 in DMEM culture mediumat the concentrations of 20, 50, 100, 200, and 400 g/mL.After incubation with IP6 for 24 h, the Caco-2 cells wereincubated with freshly prepared MTT solution (10 L per wellat 5 mg/mL). Following a 4 h incubation, the solution in eachwell was removed and the formazan dyes were dissolved withDMSO (150 L per well). The absorbance at 490 nm wasmeasured using a microtiter plate reader (Multiskan MK3,Thermo Fisher Scientific Inc., Shanghai, China). The DMEMculture medium was used for background subtraction, and theCaco-2 cells incubated with DMEM culture medium were usedas the reference for 100% cell viability. The cytotoxicity of IP6was estimated by the survival rate of Caco-2 cells at each drugconcentration.

2.2.5. Effects of IP6 on the transport of isorhamnetin, quercetin,and kaempferol in a Caco-2 cell monolayer model

Cell culture experiments were performed as describedabove. On the day of the transport experiment, the mono-layers were incubated with blank HBSS at 37 C for 30 min.Afterwards, 400 L of HBSS solution containing the threecompounds (isorhamnetin at 17.95 g/mL, quercetin at6.56 g/mL, and kaempferol at 2.27 g/mL) and varyingconcentrations of IP6 (0, 83.3, 166.5, and 333 g/mL) wereloaded onto the apical side, whereas 600 L of blank HBSSsolution was added to the basolateral side. At 0 and 80 min,100 L of samplewas drawn from the apical side and processedfor HPLC analysis. One hundred microliter (100 L) samplesfrom the basolateral side were taken every 20 min andreplenished with fresh pre-warmed HBSS at each time point.After that, 10 L of baicalein solution (7.5 g/mL, internalstandard) and 200 L ethyl acetate were added to the 100 Lsamples and vortexed for 2 min, followed by centrifugation at13,000 rpm for 10 min. The supernatant (organic layer) wascollected and evaporated to dryness under a streamof nitrogengas at 37 C. The residuewas dissolved in 100 Lmethanol andcentrifuged at 13,000 rpm for 10 min. The supernatant wasanalyzed by UPLCMS. At the end of the transport experiment,the integrity of the monolayer was monitored by TEER.

The apparent permeability coefficients (Papp) of isorhamnetin,quercetin, and kaempferol in the presence or absence of IP6were determined by Eq. (1):

Papp dQ=dt 1=AC0 1

where dQ / dt is equal to the rate of linear appearance of massin the receiver solution; A is the cross-sectional area of themembrane in cm2; and C0 is the initial drug concentration inthe donor compartment.

2.3. Effects of IP6 on the solubility of isorhamnetin, quercetin,and kaempferol in TFH

Solubility studies were performed using Milli-Q waterwith a shaker (Donglian Electronic & Technology Develop-ment Co. Ltd, Beijing, China) at 37 0.2 C. A known excessof TFH was added to 2 mL of IP6 solution at 0, 2, 5, 10,and 20 mg/mL, respectively. After shaking 48 h, the samples

219Y. Xie et al. / Fitoterapia 93 (2014) 216225were centrifuged at 13,000 rpm for 10 min, and the superna-tant was collected and filtered through a 0.22 m filter. Theconcentrations of isorhamnetin, quercetin, and kaempferol inthe filtrate were determined by HPLC analysis. Experimentswere performed in triplicate.

2.4. Effects of IP6 on lipophilicity of isorhamnetin, quercetin, andkaempferol in TFH

The octanol/water partition coefficients were determinedby measuring the partition of isorhamnetin, quercetin, andkaempferol between n-octanol and water. Briefly, n-octanolandwater were pre-saturatedwith each other in amber bottlesfor 24 h. Next, a known excess of TFH was added to 50 mL ofwater-saturated n-octanol. After shaking 12 h, the sample wascentrifuged at 13,000 rpm for 10 min. Then, 1 mL of thesupernatant was collected and equilibrated with 9 mL phos-phate buffer (pH = 6.8) containing 0, 5, and 10 mg/mL IP6 for12 h in a shaker (Donglian Elactronic & Technology Develop-ment Co. Ltd, Beijing, China) at 37 0.2 C. The n-octanoland water layers of the samples were separated by centrifuga-tion, and the concentrations of isorhamnetin, quercetin, andkaempferol were analyzed by HPLC. Experiments were carriedout in triplicate.

2.5. Effects of IP6 on the oral bioavailability of isorhamnetin,quercetin, and kaempferol in TFH in rats

All animal experiments were obtained from ExperimentalAnimal Center at the Shanghai University of Traditional ChineseMedicine, and carried out according to the local institutionalguidelines for animal care approved by Shanghai University ofTraditional Chinese Medicine.

2.5.1. Toxicity of IP6 to rat intestinesA total of sixteen Sprague Dawley rats were orally

gavaged with IP6 at the doses of 50, 100, and 200 mg/kg.Normal saline (10 mL/kg) was orally administered as thecontrol. After 6 h, rats were sacrificed under ether anesthesia,and the duodenum, jejunum, ileum, and colon were harvestedand flushed with ice-cold normal saline. The intestinal seg-ments were then fixed in formalin (10%, v/v), embedded inparaffin wax and cut into 5-m-thick sections onto adhesive-coated slides. The sections were stained with hematoxylinand eosin Y solution. The stained samples were then examinedby light microscopy (OLYMPUS system microscope BX51,Olympus Corporation, Tokyo, Japan).

2.5.2. In vivo studiesTwenty four Sprague Dawley rats (300 10 g, male)

were grouped randomly into four groups and fasted for 12 hprior to oral administration, but given access to water adlibitum. The TFH solutions containing IP6 at the doses of 50,100, and 200 mg/kg, were administered to the rats by oralgavage at 60 mg/kg (equal to isorhamnetin at 6 mg/kg,quercetin at 1.88 mg/kg, and kaempferol at 0.6 mg/kg), andthe TFH solution (60 mg/kg) without IP6 was orally admin-istered in parallel as the control. At 0, 0.25, 0.5, 1, 2, 4, 8, 12,24, 48, and 96 h following oral administration, 0.25 mL ofblood was collected from the oculi chorioideae vein intoheparinized tubes, centrifuged at 4000 rpm for 10 min forplasma separation and stored at 80 C for further analysis.Plasma samples were processed and analyzed by UPLCMS aspreviously described [28]. To determine the pharmacokineticparameters of isorhamnetin, quercetin, and kaempferol inTFH, concentration-time data were analyzed by DAS software(ver.2.1.1, Mathematical Pharmacology Professional Com-mittee of China, Shanghai, China). The absorption enhance-ment ratios of isorhamnetin, quercetin, and kaempferol withor without IP6 were calculated as Eq. (2):

Absorption enhancement ratio AUCwith phytic acid=AUCcontrol without phytic acid :

2

2.6. Determination of isorhamnetin, quercetin, and kaempferol

Isorhamnetin, quercetin, and kaempferol were determinedby UPLCMS in plasma and transport buffer samples [28] andby HPLC methods from other in vitro samples as previouslydescribed [29].

2.7. Statistical analysis

Statistical significance of the Papp, pharmacokinetic param-eters, and toxicity index obtained from various treatments wasestimated by Student's t-test or one-way ANOVA. A p value ofless than 0.05 was considered statistically significant. All dataare expressed as the mean SD.

3. Results

3.1. Stability of isorhamnetin, kaempferol, and quercetin intransport buffer

As shown in Fig. 2, isorhamnetin, kaempferol, and quercetinwere stable in HBSS at pH 5.5 and 6.5, in whichmore than 90%remained after two hours, but unstable at pH 7.5, in which lessthan 90% of the isorhamnetin and quercetin remained after 2 h.In comparison, kaempferol was the most stable component(the recoverywas between 97.28 and 102.73%) in TFH in HBSS,while isorhamnetin and quercetin were the unstable compo-nents (the recovery of isorhamnetin and quercetin at pH 7.5was 87.72% and 89.91%, respectively). To mimic the in vivoconditions, transport experiments were performed at pH 6.5on both the apical and basolateral sides.

3.2. Effects of IP6 on the permeability of isorhamnetin, kaempferol,and quercetin in a Caco-2 cell monolayer model

As shown in Fig. 3, the apparent permeability values (Papp)of isorhamnetin, kaempferol, and quercetin were (0.60 0.18) 105 cm/s, (1.23 0.09) 105 cm/s, and (0.46 0.15) 105 cm/s, respectively. In the presence of IP6 atvarious concentrations (83.3, 166, and 333 g/mL), the Pappvalues of all three components were increased, but to differentextents. The greatest enhancementwas achieved in the presenceof IP6 at 333 g/mL, where the Papp values of isorhamnetin,kaempferol, and quercetin were increased significantly by203% ((1.22 0.33) 105 cm/s), 169% ((2.08 0.31) 105 cm/s), and 211% ((0.97 0.27) 105 cm/s), respectively,

the log P values of the three components decreased with

Fig. 2. Stability of isorhamnetin, kaempferol, and quercetin in TFH in HBSS at varywere 17.95, 6.56, and 2.27 g/mL, respectively. All samples were incubated at 37 C f(n = 3 experiments).

220 Y. Xie et al. / Fitoterapia 93 (2014) 216225compared to the Papp values without IP6 (p b 0.05). Theseresults indicated that the permeability of the cells toisorhamnetin, kaempferol, and quercetin in TFH was signif-icantly enhanced by IP6.

3.3. Effects of IP6 on the solubility of isorhamnetin, kaempferol,and quercetin in TFH

As shown in Fig. 4, the aqueous solubility of isorhamnetin,kaempferol, and quercetin in TFH was lower than 9 g/mL. Incontrast, their solubility in TFH increased as the IP6 concentra-tion increased. Specifically, the solubility of isorhamnetin,kaempferol, and quercetin was increased 1.21- to 22.75-fold,1.21- to 15.15-fold, and 1.26- to 12.86-fold, respectively. Thesolubility enhancement of isorhamnetin was the most signif-icant, notably in the presence of 20 mg/mL IP6 solution(22.75-fold for isorhamnetin vs. 15.15-fold and 12.86-foldfor kaempferol and quercetin, respectively). The correlationcoefficients between the solubility of isorhamnetin, kaempferol,

and quercetin in TFH and the concentration of IP6 were 0.9999,

Fig. 3. Effects of phytic acid on the apparent permeability coefficients (Papp)of isorhamnetin, kaempferol, and quercetin transport across Caco-2 cellmonolayers. The permeability experiments were performed in the apical tobasal direction. Asterisks (*) indicate a statistically significant differencecompared to the control group (no addition) (p b 0.05). Data are expressedas the mean SD of three independent experiments.increasing IP6 concentrations in aqueous buffers, suggestingthat their lipophilicity decreased with the addition of IP6.Conversely, their hydrophilicity was enhanced by IP6. Notably,quercetin hydrophilicity enhancement by IP6 was greater than0.9980, and 0.9991, respectively, indicating that the solubility ofisorhamnetin, kaempferol, and quercetin in TFH was dependenton IP6 concentration.

3.4. Effects of IP6 on the lipophilicity of isorhamnetin, kaempferol,and quercetin in TFH

The effects of IP6 on the lipophilicity of isorhamnetin,kaempferol, and quercetin in TFHwere evaluated bymeasuringits partition between n-octanol and aqueous buffers at pH of6.8. As shown in Table 1, the log P values were 2.38 0.12,2.57 0.20, and 2.39 0.12 at 37 C for isorhamnetin,kaempferol, and quercetin, respectively. In the presence of IP6,

ing pH. The final concentrations of isorhamnetin, quercetin, and kaempferolor 120 min and then analyzed by HPLC. Data are expressed as the mean SDfor the other two components. The log P of quercetin decreasedby nearly 50% (from 2.39 0.12 to 1.15 0.01) and waslower than the other components (0.69- and 0.78-fold forisorhamnetin and kaempferol, respectively).

Fig. 4. Effects of phytic acid on the solubility of isorhamnetin, kaempferol,and quercetin in TFH (n = 3).

IP6 are shown in Fig. 7. The morphology of the duodenum,

Fig. 5. Plasma concentration-time profiles of isorhamnetin (A), kaempferol(B), and quercetin (C) in TFH with phytic acid after oral administration inrats. TFH solutions at 60 mg/kg (equal to isorhamnetin at 6 mg/kg, quercetinat 1.88 mg/kg, and kaempferol at 0.6 mg/kg), containing phytic acid at dosesof 50, 100, and 200 mg/kg, were administered to the rats by oral gavage. TheTFH solution (60 mg/kg) without IP6 was orally administered in parallel asthe control. Data are expressed as the mean SD (n = 6).

221Y. Xie et al. / Fitoterapia 93 (2014) 2162253.5. Effects of IP6 on the bioavailability in rats of isorhamnetin,quercetin, and kaempferol in TFH

As shown in Fig. 5, a double peak phenomenon wasobserved for isorhamnetin, kaempferol, and quercetin in theconcentration-time curves in the presence of IP6, but not inthe control group. In addition, all three components wereabsorbed faster in the presence of IP6 than in the controlgroup because their maximum plasma concentrations wereobtained immediately after oral administration. This sug-gested that the oral absorption properties of isorhamnetin,kaempferol, and quercetin in TFH were changed whenco-administered with IP6.

Table 2 shows the pharmacokinetic parameters of Cmax,Tmax, T1/2, and AUC, obtained by non-compartmental analysisof the plasma concentration-time curves of isorhamnetin,kaempferol, and quercetin. Notably, the Cmax and AUC ofisorhamnetin and kaempferol with IP6 were higher thanthose from the control group, especially for the IP6 doses at100 and 200 mg/kg (p b 0.01). However, the Tmax and T1/2 ofisorhamnetin and kaempferol did not change in the presenceof IP6, except for T1/2 of kaempferol at 200 mg/kg of IP6,which was significantly decreased compared to the control(p b 0.05). Furthermore, the absorption enhancement ratiosof isorhamnetin and kaempferol were in the range of 1.253.21 and 1.132.98, respectively. As for quercetin, only in thepresence of 200 mg/kg IP6, Cmax and AUC of quercetin weresignificantly increased compared to the control group(p b 0.01). Interestingly, Tmax of quercetin in the presenceof IP6 was significantly decreased compared to the controls(p b 0.01), which indicated faster absorption was achieved.For quercetin, the absorption enhancement ratios were 1.10and 1.64 with 100 and 200 mg/kg IP6, respectively. Thesefindings indicated that IP6 effectively improved the absorp-tion properties of isorhamnetin, kaempferol, and quercetin inTFH. IP6 may be utilized in the 100200 mg/kg dose range asan absorption enhancer to improve TFH flavonoid bioavail-ability after oral administration in rats.

3.6. Effects of IP6 on intestinal membrane toxicity

Table 1Effects of phytic acid on octanol/water partition coefficient (logP) ofisorhamnetin, kaempferol, and quercetin in TFH (mean SD, n = 3).

Components Concentration of phytic acid (mg/mL)

0 5 10

Isorhamnetin 2.38 0.12 2.22 0.03 1.64 0.02Kaempferol 2.57 0.20 2.40 0.04 2.01 0.04Quercetin 2.39 0.12 2.15 0.01 1.15 0.013.6.1. In vitro cytotoxicityFig. 6 shows the Caco-2 cell viability after incubation with

varying concentrations of IP6 for 24 h. For all the concentra-tions of IP6 tested, no significant decrease in cell viability wasobserved. These results suggest that at concentrations up to400 g/mL, IP6 was not toxic to the Caco-2 cells.

3.6.2. Toxicity of IP6 in rat intestinesMorphological observations of the intestinal mucosa from

each intestinal segment after oral administration of saline andjejunum, ileum, and colon in each IP6-treated group showed nosignificant difference compared to that of the control group.The mucosal epithelial cells and villi remained intact withoutnecrosis and abscission. No sign of atrophy, hyperplasia ormetaplasia was observed in submucosal intrinsic glands,and no significant inflammatory cell infiltration, extension, orhyperemia was observed in blood vessels of the interstitialconnective tissues. Muscle layers maintained intact struc-ture and there were no inflammatory exudates in the serousmembranes.

Table 2Effects of phytic acid on the absorption of isorhamnetin, kaempferol, and quercetin

Components Parameters TFH TFH and P(low dose

Isorhamnetin AUC(096 h) (g/mL h) 8.96 3.58 11.18 1T1/2 (h) 27.67 1.18 28.17 1Tmax (h) 7.20 1.79 6.40 2.1Cmax (g/mL) 0.246 0.099 0.316 0Enhancement ratio 1.25

Kaempferol AUC(096 h) (g/mL h) 2.58 0.85 2.92 0.6T1/2 (h) 28.28 9.61 28.53 7Tmax (h) 7.20 1.79 7.20 1.7Cmax (g/mL) 0.084 0.020 0.090 0Enhancement ratio 1.13

Quercetin AUC(096 h) (g/mL h) 0.49 0.15 0.36 0.0T1/2 (h) 15.23 2.66 17.70 8Tmax (h) 1.90 1.92 0.25 0

1 0

222 Y. Xie et al. / Fitoterapia 93 (2014) 2162254. Discussion

The objective of this studywas to investigate the absorptionenhancement of IP6 on isorhamnetin, kaempferol, and querce-tin in TFH in rats. The underlying mechanisms were investi-gated from two directions: 1) the effects of IP6 on theirphysicochemical properties and membrane permeability wereevaluated, and 2) the in vitro IP6 cytotoxicity and in vivo intes-tinal membrane toxicity were also investigated.

As shown in Table 2 and Fig. 5, the plasma concentrationprofile of isorhamnetin, kaempferol, and quercetin followingTFH oral administration is altered with IP6 co-administrationwith the appearance of a clear double peak. IP6 facilitated oralabsorption of isorhamnetin, kaempferol, and quercetin inTFH in a dose-dependent manner. This finding implies thatthe oral absorption of the three flavonoids in TFH is improvedby IP6 co-administration. Biphasic pharmacokinetic behavior

Cmax (g/mL) 0.029 0.014 0.02Enhancement ratio 0.73

Data are expressed as mean SD (n = 6). p b 0.05 as compared to TFH group.

p b 0.01 as compared to TFH group.is commonly observedwith flavonoids in herb extracts becauseof their glucuronidation, enteric circulation, and enterohepaticcirculation [30,31]. Interestingly, the double peak phenome-non of flavonoids in TFH was not observed in this study, whichwas consistent with our previous reports [28], but wasobserved again with IP6 co-administration. The first peaks of

Fig. 6. Caco-2 cell viability after exposure to various concentrations of phyticacid for 24 h. The values represent the means of three independent experiments.isorhamnetin, kaempferol, and quercetin were observed at0.5 h, 0.5 h, and 0.25 h, respectively, demonstrating that theirin vivo absorption rate was accelerated. The second peaks ofisorhamnetin, kaempferol, and quercetin occurred at 8 h, 8 h,and 4 h, respectively, and were the same as their correspond-ing peaks in the control group, except for the peak of quercetinat 0.5 h. A possible explanation for these phenomena is thatIP6 strengthened the enteric circulation or the enterohepaticrecirculation of the three flavonoids; however, further studiesare needed for confirmation of this mechanism.

Additionally, the solubility of isorhamnetin, kaempferol, andquercetin in TFH was 3.04 0.36 g/mL, 2.29 0.17 g/mL,and 8.28 0.54 g/mL (Fig. 4), indicating that each flavonoidis an insoluble substance according to the United StatesPharmacopoeia (USP 35), which might be a reason for theslow and poor absorption in rats [32,33]. Fortunately, thesolubility of the three components was significantly increased

in TFH by in vivo pharmacokinetic study.

hytic acid, 50 mg/kg)

TFH and Phytic acid(middle dose, 100 mg/kg)

TFH and Phytic acid(high dose, 200 mg/kg)

.61 15.84 2.14 28.79 2.88

.47 29.88 4.19 27.63 0.439 6.40 2.19 8.00 0.044 0.462 0.043 0.872 0.136

1.77 3.212 4.71 0.94 7.70 1.68

.73 28.76 5.91 18.57 3.66

9 7.20 1.79 8.00 0.016 0.150 0.027 0.245 0.058

1.83 2.988 0.54 0.15 0.809 0.059

.77 13.00 1.00 13.61 0.46 0.25 0 0.25 0

.008 0.045 0.017 0.077 0.024

1.10 1.65in a concentration-dependent manner with the addition ofIP6. For those substances classified as insoluble, the use ofsolubilizers or effective formulations for solubility enhance-ment in the gastrointestinal milieu resulted in oral absorptionimprovement [34]. For example, Sun et al. reported AUC ofquercetin was increased approximately 15-fold as a result of a70-fold increase in solubility [35]. Therefore, the oral absorp-tion improvement of isorhamnetin, kaempferol, and quercetinin TFH with co-administration of IP6 was mainly ascribed totheir significant enhancement in solubility.

Regarding the lipophilicity of the three flavonoids, all ofthem slightly decreased in the presence of IP6. In contrast, thehydrophilicity of the three flavonoids increased in the waterphase with the addition of IP6, consistent with the IP6-dependent increase in solubility. Generally, solubilizers couldbe used to induce lipophilic molecules to form water-solubleinclusion complexes with cyclodextrin [36,37] or to formmicelles with surfactants at concentrations above the criticalmicelle concentration [38,39] and to consequently enhancehydrophilicity and solubility. Presumably, intermolecular in-teractions such as hydrogen bonds were formed between thecarbonyl groups in flavonoids and the phosphate groups in IP6,

223Y. Xie et al. / Fitoterapia 93 (2014) 216225according to their chemical structure (Fig. 1). As a result, thelipophilic group of flavonoid molecules was shielded and theflavonoids exhibited increased hydrophilicity in the presenceof IP6.

Another possible explanation for the increased oral absorp-tion of isorhamnetin, kaempferol, and quercetin in vivo wasthe IP6-enhanced permeability of cells. The increase in drugabsorption across epithelial barriers by absorption enhancerscan occur via transcellular and paracellular routes. In this study,tight junction integrity and epithelial barrier properties werenot affected by IP6, as the TEER values stayed constantthroughout the course of the experiments (data not shown),indicating that the absorption enhancement was not through

Fig. 7. Histopathological comparison between treatment and control. Light microg2. jejunum, 3. ileum, 4. colon) after oral administration of phytic acid of different doscross-sections of intestinal tissues.the paracellular route. Pacheco et al. reported that IP6 couldrestore the decrease in TEER caused by deoxynivalenol [40].Therefore, the enhanced absorption of the three flavonoids byIP6wasmainly through the transcellular pathway. One possiblemechanism for IP6-dependent improvement in transcellularpermeability would be the increase in the membrane fluidity,which is supported by reports that several absorption en-hancers are able to enhance membrane fluidity [41,42]. It iswell known that phytic acid has a strong tendency to chelatemetal ions atweakly acidic to neutral pH [43], and this propertymight influence the membrane fluidity during the transport ofthe three flavonoids. In addition, flavonoids were reported assubstrates for efflux transporters such as P-gp, MRPs, and BCRP

raphs (scale bar = 100 m) of rat intestinal tissue sections (1. duodenum,es (A. control, B. 50 mg/kg, C. 100 mg/kg, D. 200 mg/kg). All panels represent

acid modulates in vitro IL-8 and IL-6 release from colonic epithelial cellsstimulated with LPS and IL-1beta. Dig Dis Sci 2007;52:93102.

224 Y. Xie et al. / Fitoterapia 93 (2014) 216225[4446]. Their increased transport was presumably the resultof the IP6 inhibition of efflux transporters, but the exactmechanisms need to be investigated further.

When absorption enhancers are applied in clinical applica-tions, their potential toxicity should always be considered. Todate, the in vitro or in vivo toxicity of IP6 as an absorptionenhancer has not been reported. In the current study, the in vitrocytotoxicity was directly measured using the MTT test. Theresults demonstrated that IP6 was not cytotoxic at concentra-tions from 20 to 400 g/mL, because cell viability in IP6experiments was greater than 90%. In addition, the examina-tion of gastrointestinal mucosa in the presence or absence ofIP6 at different doses (Fig. 7) clearly shows that there wasno significant change in the duodenum, jejunum, ileum, andcolon with IP6 at different doses. Collectively, our study hasdemonstrated that oral administration of IP6 at a dose of200 mg/kg did not result in significant irritation to the ratintestines and the addition of IP6 up to 400 g/mL in Caco-2cells had no cytotoxic effects. Therefore, IP6 was a safe oralabsorption enhancer in our studies.

Flavonoids are a large group of phenolic compounds com-posed of a basic structure of A and B benzene rings and aheterocyclic ring C. Their solubility and permeability are differentaccording to the substitution patterns in the A and B benzenerings. Isorhamnetin, kaempferol, and quercetin are three typicalflavonoids, which can be categorized as BiopharmaceuticalClassification System (BCS) II compounds because they aresparingly soluble in water, but have comparatively high perme-ability [4749]. This was confirmed by our solubility andpermeability experiments in the current studies. In terms oforal bioavailability improvement for the BCS II compounds, theenhancement of aqueous solubility is more important than theincrease in intestinal permeation. As mentioned above, the oralbioavailability of isorhamnetin, kaempferol, and quercetin in TFHwas enhanced by IP6 mainly due to their increased solubility inwater and partially due to their increased permeability at highIP6 concentrations, consistent with other BCS II compounds[50,51]. In addition to solubility and permeability, other factors,including stability at physiological pH [47] and the extensivephase IImetabolism in intestine or liver [52,53],would also resultin overall low systemic bioavailability of flavonoids followingoral administration. Therefore, the effects of IP6 on the stabilityand metabolism of the three flavonoids and on specificmechanisms should be further investigated.

5. Conclusions

In conclusion, IP6 is an effective absorption enhancer toincrease the oral bioavailability of isorhamnetin, kaempferol,and quercetin in TFH. The aqueous solubility, hydrophilicity,and membrane permeability of the three flavonoid compo-nents were improved in a concentration-dependent mannerby the addition of IP6. In addition, the low cytotoxicity andbiological safety indicated that IP6 is a safe pharmaceuticaladditive. In summary, IP6 is a potential absorption enhancerin pharmaceutical formulations.

Conict of interest

The authors declare no conflict of interest.[18] Tran TT, Hatti-Kaul R, Dalsgaard S, Yu S. A simple and fast kinetic assayfor phytases using phytic acid-protein complex as substrate. AnalBiochem 2011;410:17784.

[19] Kunyanga CN, Imungi JK, Okoth MW, Biesalski HK, Vadivel V. Antioxidantand type 2 diabetes related functional properties of phytic acid extractfrom Kenyan local food ingredients: effects of traditional processingmethods. Ecol Food Nutr 2011;50:45271.

[20] Shamsuddin AM, Vucenik I, Cole KE. IP6: a novel anti-cancer agent. LifeSci 1997;61:34354.

[21] Jariwalla RJ. Rice-bran products: phytonutrients with potential applicationsin preventive and clinical medicine. Drugs Exp Clin Res 2001;27:1726.Acknowledgments

This study was sponsored by the National Science Founda-tion of China (81303304), the Shanghai Rising-Star Program(12QB1405100), the Innovation Program of the ShanghaiMunicipal Education Commission (12YZ061, 14YZ057), andthe Nano-Specific Project of the Shanghai Science andTechnology Commission (12nm0502400).

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Phytic acid enhances the oral absorption of isorhamnetin,quercetin, and kaempferol in total flavones of Hippophaerhamnoides L.1. Introduction2. Materials and methods2.1. Chemicals2.2. Cell culture, cytotoxicity, and transport studies of isorhamnetin,kaempferol, and quercetin in TFH in a Caco-2 cell monolayer model2.2.1. Cell culture of Caco-2 cells2.2.2. Preparation of transport buffer2.2.3. Stability of isorhamnetin, quercetin, and kaempferol in transport buffer2.2.4. Cytotoxicity studies of IP6 in Caco-2 cells2.2.5. Effects of IP6 on the transport of isorhamnetin, quercetin, and kaempferol in a Caco-2 cell monolayer model

2.3. Effects of IP6 on the solubility of isorhamnetin, quercetin, and kaempferol in TFH2.4. Effects of IP6 on lipophilicity of isorhamnetin, quercetin, and kaempferol in TFH2.5. Effects of IP6 on the oral bioavailability of isorhamnetin, quercetin, and kaempferol in TFH in rats2.5.1. Toxicity of IP6 to rat intestines2.5.2. In vivo studies

2.6. Determination of isorhamnetin, quercetin, and kaempferol2.7. Statistical analysis

3. Results3.1. Stability of isorhamnetin, kaempferol, and quercetin in transport buffer3.2. Effects of IP6 on the permeability of isorhamnetin, kaempferol, and quercetin in a Caco-2 cell monolayer model3.3. Effects of IP6 on the solubility of isorhamnetin, kaempferol, and quercetin in TFH3.4. Effects of IP6 on the lipophilicity of isorhamnetin, kaempferol, and quercetin in TFH3.5. Effects of IP6 on the bioavailability in rats of isorhamnetin, quercetin, and kaempferol in TFH3.6. Effects of IP6 on intestinal membrane toxicity3.6.1. In vitro cytotoxicity3.6.2. Toxicity of IP6 in rat intestines

4. Discussion5. ConclusionsConflict of interestAcknowledgmentsReferences