Investigation of baicalin, baicalein and low-dose sulfasalazine for orocecal transit time determination in saliva and plasma

Abstract

The application of high-dose sulfasalazine (≥ 500 mg) followed by plasma or salivary sampling represents an established method for orocecal transit time determination. In the present study, we aimed to establish an alternative procedure. For this, we investigated.

whether the onset of baicalin or its aglycon baicalein in plasma or saliva after oral application correlated with the onset of sulfapyridine in plasma and.
whether the onset of sulfapyridine in saliva correlated with its onset in plasma.

following the oral ingestion of low–dose sulfasalazine (50 mg) and 400 mg baicalin. Furthermore, in a second study arm 400 mg baicalein were applied together with 50 mg sulfasalazine to enhance the understanding of the oral pharmacokinetics and conversion of baicalin and baicalein.

The oral application of baicalein led to an onset of plasma concentration that was substantially earlier than that of sulfapyridine. In contrast, the plasma appearance times of baicalin after oral baicalin administration was not statistically significant different from sulfapyridine plasma appearance time. However, baicalin was not secreted into saliva.

Low–dose sulfasalazine proved to be a reliable salivary marker for the determination of the orocecal transit time, like already reported for high dosages. The correlation between plasma and salivary concentrations and appearance times was excellent. However, there was a statistically significant difference between sulfapyridine plasma and saliva appearance time, mainly attributable to the lower salivary sulfapyridine concentrations taking slightly more time to reach the threshold defined as appearance. Hence, it is necessary to consider the generally lower salivary sulfapyridine concentrations in appearance time determination. As this procedure only uses minimal dosages (1/10 of the lowest therapeutic dosage), the risk for side effects is reduced. Taking into consideration that the possibility for saliva sampling enables a non–invasive procedure without the need for specialized personal, this approach represents an easy to use method for studies investigating orocecal transit times.

Introduction

The orocecal transit time is defined as the time period between oral ingestion of food, dosage forms or active pharmaceutical ingredient (API) and their arrival in the colon. It is of major importance for oral biopharmaceutics. For example, colon-targeted dosage forms are most often meant to release their drug content only after cecal arrival [1]. But also the pharmacokinetic/absorption characteristics of drug substances can be investigated and better understood by the determination of the orocecal transit time, as some drugs are still absorbed in the colon, whereas others are not [2]. Based on plasma concentration curves and simultaneously determined orocecal transit times, pharmacokinetic properties like absorption windows can be identified [3], [4].

The orocecal transit time can be determined by different ways [5]. Among imaging methods that require specialized and expensive facilities and equipment, like scintigraphy and magnetic resonance imaging (MRI) [6], there are several methods that utilize the abundance of microbiota in the colon to estimate the orocecal transit time. One example of this are telemetric capsules, such as the SmartPill®, which can be used to estimate the colon arrival of the device by recording the intraluminal pH. This is because the microbiota present in the colon produce acidic metabolites, such as short-chain fatty acids. Usually, a sharp decrease of the measured pH (e.g. by 0.5 or 1.0) after a predefined lag time following gastric emptying is considered to reflect the devices entry into the cecum [7], [8]. However, it has to be considered that the transit of large monolithic objects, like telemetric capsules, through the gastrointestinal tract differs from dispersed formulations. Other methods utilize the bacterial metabolization of orally applied tracers. A very common approach is the lactulose breath test. In the human body, only bacteria are able to produce hydrogen [9]. The colonic microbiota in the cecum produce hydrogen when they break down lactulose, which is not absorbed in the small intestine. This hydrogen can then be detected in the breath of the subject. A rise in breath hydrogen of a certain threshold (e.g. 3, 5 or 10 ppm) above baseline values indicates the orocecal transit time.

The sulfasalazine/sulfapyridine method represents another indirect way to determine the orocecal transit time [10], [11]. This procedure does not require specialized equipment, but only plasma or saliva samples. These are collected in pharmacokinetic studies anyways, which makes the sulfasalazine/sulfapyridine especially suitable for the simultaneous application in these studies. Under these conditions, it is a cost-effective alternative to the aforementioned methods. It could be shown, that gastrointestinal transit times determined by this method are in line with the results of other established methods (scintigraphy, lactulose-H2-breath-test) [10], [12]. Furthermore, sulfasalazine itself has no impact on the orocecal transit time [12]. In the vast majority of published pharmacokinetic studies employing the sulfalasalazine/sulfapyridine method, therapeutical doses of at least 500 mg sulfasalazine were applied [13]. However, sulfapyridine was shown to be measurable in plasma even after orally applied micro doses of 25 mg sulfasalazine [14]. Thus, such small sulfasalazine doses are also appropriate for orocecal transit time determination by plasma sampling. In contrast, the suitability of non–invasive saliva sampling also only has been shown for a high oral sulfasalazine dosage of 2000 mg [15], [16].

Baicalein (5,6,7-trihydroxyflavone) “B” and baicalin (baicalein 7-O-glucuronide) “B-Gluc” are flavonoids that can be extracted from Scutellaria baicalensis Georgi, which has been used in Traditional Chinese medicine for centuries [17]. B-Gluc and B are being investigated for therapeutic potential, because of their antioxidant, anticancer, anti-inflammatory, antimicrobial, cardio-protective, hepatoprotective, renal protective and neuroprotective effects [18], [19], [20]. B-Gluc represents a glucuronic acid conjugate of B. B can be metabolized to B-Gluc by uridine 5’–diphospho (UDP)-glucuronosyltransferases (UGT). While there are no human gut enzymes that can hydrolyze glucuronic acid conjugates, the human microbiota of the gastrointestinal tract are capable of this metabolization reaction [21], [22], [23]. Thus, after oral application of B-Gluc or B, there is a mutual conversion between both flavonoids by human and bacterial enzymes. Following oral administration, B-Gluc is poorly absorbed from the gastrointestinal tract. Only after bacterial hydrolyzation to B and glucuronic acid, the resulting aglycon can be absorbed readily [24]. However, B represents the minor compound in plasma after resorption, as an extensive first pass metabolism causes the formation of several methylate, sulfate and glucuronic acid conjugates, B–Gluc among them [25], [26]. While B is well absorbed from the intestinal lumen, its oral bioavailability is poor due to these processes. Among the aforementioned extensive first pass metabolism, the excretion of the resulting metabolites into the intestinal lumen and the bile can be held responsible for this. These excreted B conjugates are poorly absorbed from the intestines and are transported through the gut. Upon colon arrival, the release of B by bacterial metabolization of its conjugates initiates another absorption phase of B. Overall, the bacterial metabolization, the extensive first pass metabolism, the excretion of B-conjugates and the enterohepatic circulation lead to complex pharmacokinetics, often with multiple plasma concentration peaks of B and its metabolites following oral application [27], [28], [29], [30], [31], [32], [33].

In the present study, we aimed to establish an alternative method for orocecal transit time determination to the application of sulfasalazine followed by plasma sampling. For this, the plasma pharmacokinetics of sulfapyridine following the oral application of 50 mg sulfasalazine served as reference data. As mentioned before, the sulfasalazine/sulfapyridine method has only been applied with saliva samples together with high sulfasalazine doses. In order to investigate the eligibility of saliva sampling for low–dose sulfasalazine, saliva samples were collected alongside plasma samples. Furthermore, B-Gluc was investigated as possible alternative marker for orocecal arrival due to the reported involvement of the colonic microbiota in its oral pharmacokinetics and its availability as dietary supplement – possibly simplifying a broader application of the procedure in contrast to the authorized drug sulfasalazine. Moreover, a second study arm with the oral application of B was conducted to further understand the oral pharmacokinetics of B-Gluc.

Download the full article as PDF here Investigation of baicalin, baicalein and low-dose sulfasalazine for orocecal transit time determination in saliva and plasma

or continue reading here

Materials

The dietary supplement containing 200 mg B-Gluc per capsule (Scutellaria baicalensis Georgi extract 85%) was from Medverita (Krakow, Poland). B capsules containing 200 mg came from Sunday Natural Products GmbH (Berlin, Germany). The contents of the dietary supplements had been confirmed by a high performance liquid chromatography (HPLC) method. Azulfidine® tablets (PFIZER PHARMA GmbH, Berlin, Germany) containing 500 mg sulfasalazine were purchased from the hospital pharmacy in Greifswald. ¹³C₃-caffeine was obtained from Eurisotop, a Cambridge Isotope Laboratories Company (Tewksbury, Massachusetts, USA). Caffeine (API) came from Caesar & Loretz GmbH (Hilden, Germany). ¹³C₃–caffeine was applied for investigating its saliva–plasma ratio (s/p-ratio) only in one study arm, while natural caffeine was applied in the other study arm to compensate for possible drug-drug interactions.

The capsules containing ¹³C₃-caffeine or natural caffeine and sulfasalazine were prepared manually. For this, an excess of Azulfidine® tablets was grinded by a tablets mortar (“melipul® Tablettenmörser”, Helmut Schwarz GmbH, Isny, Germany). An amount corresponding to 50 mg sulfasalazine was weighed into the capsule bottom part on an analytical balance (Sartorius MC1 Analytic AC 120 S, Sartorius, Göttingen, Germany). Subsequently, 25 mg ¹³C₃-caffeine or natural caffeine were added and the capsules were closed. As the dietary supplements also had different capsule shells (Baicalin medverita: hard gelatine; Baicalein Sunday: hydroxypropyl methylcellulose (HPMC)), hard gelatine capsules (WEPA Apothekenbedarf GmbH & Co KG, Hillscheid, Germany) were used for the capsules containing ¹³C₃-caffeine and sulfasalazine for study arm A, while HPMC capsules (Capsugel, Bornem, Belgium) were chosen for study arm B.

Stefan Senekowitsch, Nikolaus Alexander Link, Toni Wildgrube, Philipp Schick, Stefan Engeli, Werner Weitschies, Michael Grimm, Investigation of baicalin, baicalein and low-dose sulfasalazine for orocecal transit time determination in saliva and plasma, European Journal of Pharmaceutics and Biopharmaceutics, Volume 223, 2026, 115064, ISSN 0939-6411, https://doi.org/10.1016/j.ejpb.2026.115064.