High-performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma

A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of four active polyphenol components of hawthorn (Crataegus), chlorogenic acid, epicatechin, hyperoside and isoquercitrin, in rat plasma. Following extraction from the plasma samples with ethyl acetate–methanol (2:1, v/v), these four compounds were successfully separated using a C₁₈ column with a gradient elution of 5 and 25% acetonitrile in 25 mM phosphate buffer (pH 2.4). The flow-rate was set at 1 ml/min and the eluent was detected at 325 nm for chlorogenic acid, 278 nm for epicatechin, and 360 nm for both hyperoside and isoquercitrin. Narignin (0.82 μg) was used as the internal standard and was detected at 278 nm. The method is linear over the studied range of 0.16–40, 0.63–160, 0.13–32 and 0.13–30 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin, respectively. The correlation coefficient for each analyte was greater than 0.995. The intra-day and inter-day precision of the analysis was better than 4 and 7%, respectively. The extraction recoveries at low to high concentration were greater than 85% for both epicatechin and chlorogenic acid, and greater than 94% for both hyperoside and isoquercitrin. The detection limits were 0.04, 0.20, 0.03 and 0.03 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin. The developed method was used to analyze the plasma concentrations of the four analytes after the intravenous administration of hawthorn polyphenol extract to rats.     

Study on the intestinal permeability of lamivudine using Caco-2 cells monolayer and Single-pass intestinal perfusion

BackgroundThe aim of this work is to investigate the intestinal permeability of lamivudine and explore its absorption mechanism.MethodCaco-2 cells monolayer and single-pass intestinal perfusion (SPIP) were selected for the investigation of lamivudine under different conditions, such as different concentration, absorption time, bidirectional transportation, and transportation with efflux transporters inhibitor. The concentration of lamivudine both in Caco-2 cells monolayer samples and SPIP samples was detected by HPLC-UV. Then the permeability parameters were calculated.ResultsThe established HPLC-UV method reach the requirements for detection. There is no statistically difference between absorption parameters of lamivudine both in Caco-2 cells monolayer and SPIP (P > 0.05) under different dose groups. After transportation with efflux transporters inhibitor, the efflux rate of lamivudine in three dose groups was significantly decreased from 2.67, 2.59 and 2.59 to 1.78, 1.61, and 1.81 respectively. Lamivudine exhibits an absorption mechanism of passive diffusion.ConclusionThe absorption of lamivudine may be related to efflux transporters. In addition, lamivudine is a moderate-permeability drug in Biopharmaceutics Classification System.     

The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers

We determined the extent of Na⁺-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na⁺-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na⁺-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, -alanine, -aminoisobutyric acid (AIB), -methylaminoisobutyric acid (MeAIB), -amino-n-butyric acid and l-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both d-serine and d-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and d-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2,7-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (I _sc) in voltage-clamped Caco-2 cell monolayers in Na⁺-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, d-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pH_i and inward I _sc. In conclusion, Caco-2 cells express a Na⁺-independent, H⁺-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.This study was supported by a Wellcome Trust Fellowship (to DTT). Charlotte Ward, Maureen Sinclair and Ken Elliott provided excellent technical assistance.     

Comparative in situ study of the intestinal absorption of aluminum, manganese, nickel, and lead in rats

This comparative study of the intestinal absorption of four toxic metals (aluminum, manganese, nickel, and lead) carried out in rats using the in situ intestinal perfusion technique was able to measure the partition of each metal between the intestine (intestinal retention), the blood circulation, and target tissues after 1 h. The perfused metal solutions were at concentrations likely to occur during oral intoxication. It was found that aluminum (48 and 64 mM), even as a citrate complex, crossed the brush border with difficulty (0.4% of the perfused amount); about 60% of this was retained in the intestine and the remainder was found in target tissues (about 36%). Conversely, lead (4.8–48 μM) penetrated the intestine more easily (about 35% of the perfused amount), was slightly retained (about 12% of the input), and was soon found in the tissues (about 58% of the input) and to a lesser degree in circulation (about 29%). Within the same concentration range, nickel and manganese showed certain similarities, such as a reduced crossing of the brush border proportional to the increase in the concentration perfused (0.17–9.5 mM). There was similar intestinal retention and absorption (about 80% and 20% of the input, respectively). Manganese crossed the brush border more easily and was diffused more rapidly into tissues. Finally, the addition of equimolar amounts of iron (4.7 mM) produced opposite effects on the absorption of the two elements, inhibiting manganese and showing a trend to increase in nickel absorption. This could be the result of competition between Fe²⁺ and Mn²⁺ for the same transcellular transporters and the slight predominance of paracellular mechanism in the event of “Fe²⁺-Ni²⁺” association.     

在体单向肠灌流模型研究白芷中三种成分的吸收特性

目的:研究白芷中有效成分欧前胡素、异欧前胡素和花椒毒酚在大鼠小肠各段的吸收特征。方法:采用大鼠在体单向肠灌流模型结合HPLC法同时测定肠灌流液中欧前胡素、异欧前胡素和花椒毒酚含量的变化,考察高、中、低三组剂量下三种成分在十二指肠、空肠和回肠的吸收特性。结果:三种成分随着剂量的增大,吸收速率常数(Ka)和有效渗透系数(Peff)逐渐增大。三种成分相同剂量下各肠段间吸收均无显著性差异,其吸收大小顺序为:欧前胡素异欧前胡素花椒毒酚。结论:白芷三种主要成分在各肠段均吸收良好,吸收大小呈剂量相关性。     

Kainate responses of leech Retzius neurons in situ and in vitro

Responses to the ionotropic glutamate receptor agonist kainate were measured in Retzius cells (RCs) of intact segmental ganglia (in situ), acutely isolated RCs, and cultured RCs (in vitro) of the leech Hirudo medicinalis. RCs in intact ganglia responded to kainate (5–20 μM) with depolarizations up to 30 mV or with an inward current under voltage-clamp that reversed near -10 mV. The membrane conductance increased by a factor of 2.5 at a holding potential of -70 mV in the presence of 20 μM kainate. In RCs in situ the membrane responses to 5 μM kainate increased when applied repeatedly 3-5 times. After this potentiation, the amplitude and time course of the membrane responses to 5 μM kainate were similar to the membrane response to 20 μM kainate. In current-clamp experiments kainate evoked an increase in intracellular calcium concentrations ([Ca²⁺]¡) only when the membrane depolarized beyond -40 mV. In voltage-clamped RCs at a holding potential of -70 mV, kainate caused no significant rise in [Ca²⁺]¡, indicating that the Ca²⁺ permeability of these kainate-gated ion channels appears to be negligible. The potentiation of the kainate-induced responses in RCs in situ was also present in voltage-clamped cells, where no or only small changes in [Ca²⁺]¡ occurred, suggesting that the underlying mechanism seemed to be independent of intracellular Ca²⁺ changes. In addition, the potentiation of the kainate-induced membrane responses was unaffected by cyclothiazide (100 μM), concanavalin A (0.5 mg/mL), and in the presence of extracellular low-Ca²⁺ and high-Mg²⁺ concentrations to suppress synaptic transmission in the ganglion. During whole-cell patch-clamp recordings (up to 50 min) potentiation remained the same indicating that small intracellular messenger molecules, which would be expected to dissipate, were not likely to be involved in mediating this potentiation. In acutely isolated RCs kainate induced no or only very small voltage responses. A potentiation of the kainate response was never observed in acutely isolated RCs. In cultured RCs (2–7 days in vitro) kainate evoked membrane responses with no apparent potentiation. Cultured RCs also responded with Ca²⁺ transients only when depolarized beyond -40 mV. The results show that RCs respond differently to kainate when kept isolated in culture compared to RCs in intact ganglia. The mechanism underlying the potentiation of the kainate response of RCs in situ, however, could not yet be identified. © 1996 John Wiley & Sons, Inc.     

Cytokine Responses of Intestinal Epithelial-Like Caco-2 Cells to Non-Pathogenic and Opportunistic Pathogenic Yeasts in the Presence of Butyric Acid

Candida albicans, Saccharomyces cerevisiae and their cell wall components, zymosan and glucan, have been shown to stimulate interleukin-8 (IL-8/CXCL-8) production by intestinal epithelial cell-like Caco-2 cells pre-cultured with 10 mM butyric acid. We examined in this study whether these yeasts also altered the production of other cytokines and cyclooxygenases (COXs) by Caco-2 cells. Culturing Caco-2 cells with 10 mM butyric acid and 15% FBS for 4 days enhanced the basal levels of mRNA encoding IL-6, IL-8, IL-18, monocyte chemoattractant protein (MCP)-1, stem cell factor, transforming growth factor (TGF)-β1, TGF-β3, tumor necrosis factor (TNF)-α, COX-1, and COX-2, but not of granulocyte-macrophage colony-stimulating factor (GM-CSF) and TGF-β2. The inclusion of live S. cerevisiae or C. albicans further enhanced the production of IL-8, but not of the other cytokines and COXs. The non-pathogenic yeasts, C. kefyr, C. utilis, C. versatilis, Kluyveromyces lactis, K. marxianus, Schizosaccharomyces pombe and Zygosaccharomyces rouxii, used for the production of fermented foods and probiotics, and the opportunistic pathogens, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis, isolated from human tissue samples also enhanced IL-8 secretion by Caco-2 cells.     

In vitro stability and ex vivo absorption of thymol monoglucosides in the porcine gut

Thymol α-D-glucopyranoside (TαG) and thymol β-D-glucopyranoside (TβG) are believed to have different kinetic behaviours in the porcine gut than its parent aglycon thymol. However, recently, it was shown that concentrations of both glucosides decreased rapidly in the stomach and proximal small intestine following oral supplementation to piglets as did thymol. Yet, the stability of thymol glucosides in gut contents and their absorption route remains obscure. Therefore, a series of in vitro incubations were performed, simulating the impact of pH, digestive enzymes, bacterial activity and mucosal extracts on stability of these glucosides. Their absorption mechanisms were investigated using the Ussing chamber model in the presence or the absence of inhibitors of sodium-dependent glucose linked transporter 1 and lactase phlorizin hydrolase. Both glucosides remained intact at physiological pH levels in the presence of digestive enzymes. Recoveries from TαG and TβG were below 90% when incubated with small intestinal homogenates from the distal jejunum or from all sampled sites, respectively. However, no aglycon could be detected in these samples. Bacterial inoculum of the small intestine, on the other hand, hydrolysed TβG quickly with up to 44% of free aglycon appearing. TαG proved more resistant to porcine gastro-intestinal bacterial glucosidases with only trace amounts (<1%) of free thymol at the end of the incubations. Electrophysiological measurements in Ussing chambers did not suggest active transport of the glucosides. Mucosal TαG and TβG concentrations were unchanged between start and end of the absorption measurements. Additionally, no TαG and only a very limited amount of TβG were retrieved from the serosal side. Tissue associated concentrations, although marginal (<1% of luminal concentration), were mainly as intact glucoside or as aglycon for TαG and TβG, respectively. Addition of both inhibitors significantly increased the amount of intact glucosides retrieved from the mucosal tissues as compared to controls. In conclusion, bacterial hydrolysis was identified as the most important source of TβG loss, whereas TαG seemed less prone to degradation or absorption in these in vitro and ex vivo models.     

Preparation,in vitro release,in vivo absorption and biocompatibility studies of insulin-loaded microspheres in rabbits

The purpose of this study was to develop a single-dose insulin delivery system based on poly (lactide-co-glycolide) (PLGA) microspheres to provide basal insulin level for a prolonged period. Insulin-loaded PLGA microspheres were prepared by water-in-oil-in-water double emulsion (batch A) and solid-in-oil-in-water emulsion (batch B) methods. Microspheres were characterized for physical characteristics and in vitro release. In vivo absorption of insulin and biocompatibility of insulin-loaded PLGA microspheres were performed in diabetic New Zealand white rabbits. Light and transmission electron microscopy were performed on the skin tissues excised from microspheres injected sites in order to study the biocompatibility. The burst release of insulin was high (47%) from batch B and low (5%) from batch A. Therefore, we mixed microspheres of batch A and B in ratio of 3:1 w/w, which produced desirable in vitro release profile. In vivo absorption study showed that insulin-loaded microspheres provided a serum insulin level of 20-40 microU/ml up to 40 days. Biocompatibility study provided evidence of normal inflammatory and foreign body reactions, which were characterized by the presence of macrophages, fibroblasts and foreign body giant cells. Neither necrosis nor tissue damage was identified. At the end of 12 weeks, no distinct histological differences were observed in comparison to the control tissue samples. In conclusion, insulin-loaded PLGA microspheres controlled the in vivo absorption of insulin to maintain the basal insulin level for longer period and the delivery system was biocompatible.     

Differences in the gut bacterial flora of healthy and milk-hypersensitive adults, as measured by fluorescence in situ hybridization

We enumerated the predominant gut genera from fecal samples of nine healthy and eight milk-hypersensitive adults both before and after 4 weeks Lactobacillus rhamnosus GG (LGG) supplementation. The anaerobic intestinal microflora of milk-hypersensitive adults was found to resemble that of healthy adults. LGG-consumption resulted in a significant increase in the number of bifidobacteria in healthy but not in milk-hypersensitive subjects, as well as a general increase in bacterial numbers in all other bacterial genera tested in both groups. In conclusion, the composition of the gut microbiota in milk-hypersensitive adults appears to be normal. LGG may have potential in reinforcing the endogenous flora.     

Dynamics of DNA-demethylation in early mouse and rat embryos developed in vivo and in vitro

Virtually all mammalian species including mouse, rat, pig, cow, and human, but not sheep and rabbit, undergo genome-wide epigenetic reprogramming by demethylation of the male pronucleus in early preimplantation development. In this study, we have investigated and compared the dynamics of DNA demethylation in preimplantation mouse and rat embryos by immunofluorescence staining with an antibody against 5-methylcytosine. We performed for the first time a detailed analysis of demethylation kinetics of early rat preimplantation embryos and have shown that active demethylation of the male pronucleus in rat zygotes proceeds with a slower kinetic than that in mouse embryos. Using dated mating we found that equally methylated male and female pronuclei were observed at 3 hr after copulation for mouse and 6 hr for rat embryos. However, a difference in methylation levels between male and female pronuclei could be observed already at 8 hr after copulation in mouse and 10 hr in rat. At 10 hr after copulation, mouse male pronuclei were completely demethylated, whereas rat zygotes at 16 hr after copulation still exhibited detectable methylation of the male pronucleus. In addition in both species, a higher DNA methylation level was found in embryos developed in vitro compared to in vivo, which may be one of the possible reasons for the described aberrations in embryonic gene expression after in vitro embryo manipulation and culture.     

Intestinal histopathology and in situ postures of Gymnophalloides seoi in experimentally infected mice

The intestinal histopathology and in situ postures of Gymnophalloides seoi (Digenea: Gymnophallidae) were studied using C3H/HeN and C57BL/6 mice as experimental hosts; the effects of immunosuppression were also observed. The metacercariae isolated from naturally infected oysters, 300 or 1,000 in number, were infected orally to each mouse, and the mice were killed at days 3-21 post-infection (PI). In immunocompetent (IC) mice, only a small number of flukes were found in the mucosa of the duodenum and jejunum during days 3-7 PI, with their large oral suckers pinching and sucking the root of villi. The intestinal mucosa showed mild villous atrophy, crypt hyperplasia, and inflammations in the villous stroma and crypt, with remarkable goblet cell hyperplasia. These mucosal changes were almost restored after days 14-21 PI. In immunosuppressed (IS) mice, displacement as well as complete loss of villi adjacent to the flukes was frequently encountered, otherwise the histopathology was generally mild, with minimal goblet cell hyperplasia. In these mice, numerous flukes were found, and it seemed that they were actively moving and rotating in situ. Several flukes were found to have invaded into the submucosa, almost facing the serosa. These results indicate that in IC mice the intestinal histopathology caused by G. seoi is generally mild, and the flukes do not penetrate beyond the mucosa, however, in IS mice, the flukes can cause severe destruction of neighboring villi, and some of them invade into the submucosa.     

Adhesion of Lactobacillus plantarum 423 and Lactobacillus salivarius 241 to the intestinal tract of piglets, as recorded with fluorescent in situ hybridization (FISH), and production of plantaricin 423 by cells colonized to the ileum

AIMS: To determine which intestinal section of pre and postweaned piglets are colonized by Lactobacillus plantarum 423 and Lactobacillus salivarius 241, and follow production of plantaricin 423 in a gastro-intestinal model. METHODS AND RESULTS: Lactobacillus plantarum 423 and Lact. salivarius 241, single or in combination, were administered to 1-, 14- and 28-day-old (postweaned) piglets. According to results obtained by fluorescent in situ hybridization (FISH), Lact. plantarum 423 adhered strongly to the ileum and posterior colon and Lact. salivarius 241 to the duodenum in preweaned piglets. High numbers of strain 241 were recorded in the duodenum and posterior colon of postweaned piglets, whereas strain 423 remained localized to the ileum. Lowering in Enterococcus faecalis cell numbers were recorded when preweaned piglets were challenged with strain 241. Plantaricin 423 was produced for 96 h in the ileum section of a gastro-intestinal model. CONCLUSIONS: Lactobacillus plantarum 423 and Lact. salivarius 241 adhere to different sections of the intestinal tract, depending on the piglet's age. Ent. faecalis were inhibited in vivo, probably by plantaricin 423. SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescent in situ hybridization proved valuable in the detection of probiotic bacteria adhered to the intestine. This is the first report of bacteriocin production in a model simulating the porcine gastro-intestinal tract.     

Intestinal absorption and metabolism of retinoyl beta-glucuronide in humans, and of 15-[14C]-retinoyl beta-glucuronide in rats of different vitamin A status

In order to prove the hypothesis that humans and animals with adequate vitamin A status do not absorb and metabolize orally administered all-trans retinoyl β-glucuronide, unlabeled retinoyl glucuronide (0.1 mmol) was orally dosed to fasting well-nourished young men. Neither retinoyl glucuronide nor retinoic acid, a possible metabolite, appeared in the blood within 12 h after ingestion. Next, radiolabeled all-trans 15-[¹⁴C]-retinoyl β-glucuronide was chemically synthesized by a new procedure, and fed orally to rats of different vitamin A status. Analysis of blood and other tissues 5 or 24 h after the dose, showed the presence of radioactivity ( 0.5%) in the blood of vitamin A deficient rats, but not in sufficient rats. Livers of all rats contained small, but detectable amounts (0.3 to 1.1% of the dose) of radioactivity. The accumulation of radioactivity in the liver was highest in deficient rats. Analysis of the retinoids showed that the radioactivity in serum and liver was due to retinoic acid formed from retinoyl glucuronide. Within 24 h after the dose, 31 to 40% of the administered radioactivity was excreted in the feces, and 2 to 4.7% of the dose was excreted in the urine. Results of the present studies show that oral administration of retinoyl β-glucuronide did not give rise to detectable changes in blood retinoyl glucuronide and/or retinoic acid concentrations in humans or rats with adequate vitamin A status.     

Circular dichroism,optical rotatory dispersion,and absorption studies on the conformation of bovine rhodopsin in situ and solubilized with detergent

Circular dichroism, optical rotatory dispersion and absorption of rhodopsin, the visual pigment of bovine rod outer segment membranes, were studied in situ and in membranes solubilized with various detergents. The -helical content of the membrane protein is approximately 30%. The membrane protein possesses little -structure. Solubilization of the membrane by the detergents, Emulphogene BC-720 and cetyltrimethylammonium salts, results in loss of protein helical structure and perturbation of aromatic residues. These effects are not observed on digitonin solubilization.In regard to the structural stability of the membrane during bleaching, the following conclusions were reached: (1) Delocalized conformational changes of rhodopsin in situ involving secondary and/or tertiary structure are very unlikely. (2) Localized conformational changes of rhodopsin in situ involving secondary structure must be limited to the involvement of no more than three amino acid residues and localized conformational changes involving tertiary structure must be limited to very short segments of the protein chain containing, at the most, only a few aromatic residues. (3) Large changes in the interaction of lipid and protein moieties of the membrane are unlikely. (4) The detergents, Emulphogene, cetyltrimethylammonium salts, and digitonin, significantly decrease the conformational stability of rhodopsin as compared to the in situ conditions. The effect is smaller with digitonin.Evidence is presented against a proposed mechanism by which optical activity of the prosthetic group, retinal, is induced by resonance coupling of the transition dipoles of retinal and the lowest energy transitions of the aromatic groups of the apoprotein, opsin. A mechanism in which atropisomers of retinal are preferentially bound by opsin is consistent with the present results. The optical activity of the prosthetic group is markedly changed upon solubilization of the membrane by detergent. This change in optical activity is probably coupled to changes in conformation of the protein moiety induced by solubilization.This work is based in part upon a Ph.D. dissertation submitted by C.N.R. to The Ohio State University (1974). A preliminary report of this work was presented at the sixteenth annual meeting of the Biophysical Society, Toronto, Canada, February, 1972, Abstracts SaPM-H8 and SaPM-H9     

Enhancing the in vitro and in vivo detection of aneuploidy by fluorescence in situ hybridization with the use of bromodeoxyuridine as a proliferation marker

Aneuploidy is associated with spontaneous abortions, birth defects, and many types of human cancers. Currently there are few assays developed for the efficient detection of aneuploidy in vivo. However, with the recent availability of chromosome-specific DNA probes for the rat, fluorescence in situ hybridization (FISH) techniques could be used for the rapid and sensitive detection of aneuploidy in different tissue and cell types. In order to develop a system that can detect alterations in chromosome number in rat cells in vitro, we treated cultured rat lymphocytes with three aneugens-noscapine hydrochloride (0–150 μM) and vincristine and vinblastine sulfate (0–0.06 μM). 5-Bromo-2-deoxyuridine (BrdU; 1 μM) was added to the culture medium to allow proliferating and non-proliferating cells to be distinguished. To test this assay under in vivo conditions, 21-day-old male Sprague–Dawley rats were subcutaneously implanted with osmotic pumps that delivered BrdU (12 mg/kg per day) continuously. These rats were administered vinblastine sulfate (0, 0.5 and 1 mg/kg) by intraperitoneal injection. The rat lymphocytes and hepatocytes incorporating BrdU were detected by immuno-fluorescent labeling, and FISH with a rat chromosome 4 probe was performed on the labeled and unlabeled cells. Highly significant increases in hyperdiploidy were seen in the replicating rat lymphocytes treated with noscapine, vincristine or vinblastine in vitro and in the rat hepatocytes treated with vinblastine in vivo. In contrast, no significant increase in hyperdiploidy was observed in the non-replicating cells. These results demonstrate that this BrdU-enhanced FISH assay with chromosome-specific rat probes can be used to efficiently detect numerical chromosomal aberrations in vitro and in vivo in slowly or moderately replicating rat tissues. The combination of BrdU-labeling and FISH allows the scoring of hyperdiploidy to be focused on the actively replicating cells, thereby increasing the sensitivity of the FISH technique.     

Transport of sennosides and sennidines from Cassia angustifolia and Cassia senna across Caco-2 monolayers – an in vitro model for intestinal absorption

Laxative effects of Senna preparations are mainly mediated by rheinanthrone, a metabolite formed in the intestinal flora from dianthrones. Nevertheless, it was not clear whether dianthrones are bioavailable at all and contribute to the overall effects of this important medicinal plant. Using the Caco-2 human colonic cell line as an in vitro model of the human intestinal mucosal barrier, the bioavailability of dianthrones was studied in apical to basolateral (absorptive) and basolateral to apical (secretive) direction. Permeability coefficients (P_c) and percent transport were calculated based on quantitations by HPLC. From the data obtained it was concluded that sennosides A and B, as well as their aglycones sennidine A and B are transported through the Caco-2 monolayers in a concentration-dependent manner and their transport was linear with time. The absorption in apical to basolateral direction was poor and P_c values were comparable to mannitol. The transport was higher in the secretory direction, indicating a significant efflux (e.g. by efflux pumps) of the (poorly) absorbed compounds in the intestinal lumen again. Our findings support the general understanding that the laxative effects of Senna are explainable mainly by metabolites and not by the natively present dianthrones.