The Pharmacokinetic Drug-Drug Interactions of Andrographis paniculata and Ibuprofen in the Plasma of Healthy Oryctolagus cuniculus Rabbits

An HPLC method was developed and validated for the pharmacokinetic drug-drug interaction between Andrographis paniculata and ibuprofen in the plasma of Oryctolagus cuniculus rabbits after a single oral administration of the mixture. Nine healthy rabbits (6 males and 3 females, weight 1.68-2.42 kg) were acclimatized for 7 days and were randomly divided into 3 groups. At day-8th the rabbits were group (1) treated with a single oral administration of ibuprofen (dose of 28 mg/kg BW); group (2) treated with a single oral administration of Andrographis paniculata infusion (7.04 mL/kg BW); group (3) treated with a single oral administration of a mixture of Andrographis paniculata (7.04 mL/kg BW) infusion and ibuprofen (dose of 28 mg/kg BW). Plasma samples were prepared by collecting the blood from the marginal ear vein at 0, 30, 60, 90, and 120 minutes after the mixture administration, followed by centrifuging it for 30 minutes 3000 rpm. Chromatographic separation was performed on a LiChrosorb RP-18 with methanol and double-distilled water (70:30) as the mobile phase, flow rate 1 mL/minute. UV detection was set at 227 nm. The absorption and distribution of ibuprofen were fast (Tmax = 30 min; Cmax = 4.02962 mcg/mL), however, interestingly this drug could improve the absorption and distribution of andrographolide in Oryctolagus cuniculus rabbits


Introduction
The bioavailability of a drug is defined as its amount and rate in the systemic circulation. Once entering the blood, the concentration of a drug will denote its total concentration in the circulatory system. 1 The bioavailability of a drug can be interfered with by another drug when both drugs are administered at the same time due to the occurrence of pharmacokinetic drug-drug interactions (DDIs). Many pharmacokinetic DDIs studies in humans have been reported doi: 10.15416/pcpr.v4i3.27508 underlying the mechanisms for DDIs of different degrees. [2][3][4][5][6] Andrographolide has been reported in possessing anti-inflammatory activity among its other pharmacological activities. 7-11 A previous bioavailability study of sambiloto (Andrographis paniculata) infusion, determined as andrographolide ( Figure  1a), reported that this lactone diterpene compound was absorbed and quantified in the plasma of New Zealand male rabbits at 1.5h (Cmax = 0.2136 µg/mL) after a single oral administration. 12 A bioavailability of andrographolide in a mixture of ethanolic extracts of sambiloto and turmeric in rabbits has been reported. In this study, andrographolide was absorbed and distributed in blood within 60-90 minutes (Cmax = 3.06 -4.41 ppm). 13 However, there is a very limited report of its pharmacokinetic DDIs study with NSAIDs. This work aimed to study the pharmacokinetic DDIs of Andrographis paniculata infusion and ibuprofen ( Figure  1b) in the plasma of healthy Oryctolagus cuniculus rabbits.

Chemicals and Plants
Andrographolide 98% 500 mg CAS 5508-58-7 for R & D use (Aldrich) (Figure 1a Optimization of the HPLC System 20 mcg/mL of andrographolide solution was injected into a C-18 column with a mixture  The rabbits were randomly divided into 3 groups and were acclimatized for 7 days. Standard pellet feeds (@ 75 g/ rabbit) were given twice at 08.00 and 16.00 daily.

The pharmacokinetic DDIs of Andrographis paniculata infusion and ibuprofen in the plasma of healthy Oryctolagus cuniculus rabbits
At day-8th the rabbits were observed for their heart rate (HR), respiration rate, body temperature, and body weight at day-8th (Table 1). At the same day, the rabbits were hematology-analyzed (Table 1) and were treated with: group (1) a single oral administration of ibuprofen (dose of 28 mg/kg BW); group (2) a single oral administration of Andrographis paniculata infusion (7.04 mL/ kg BW); group (3) a single oral administration of a mixture of Andrographis paniculata (7.04 mL/kg BW) infusion and ibuprofen (dose of 28 mg/kg BW). Plasma samples were prepared by collecting the blood from the marginal ear vein ( Figure 5) at 0, 30, 60, 90, and 120 minutes after the mixture administration, followed by centrifuging it for 30 minutes 3000 rpm. Chromatographic separation was performed on a LiChrosorb RP-18 with methanol and double-distilled water (70:30) as the mobile phase, flow rate 1 mL/minute. UV detection was set at 227 nm.

Infrared and Ultraviolet Spectrophotometry
The ultraviolet spectrum of andrographolide showed one maximum at 227 nm, which is caused by the pi -> pi* transition of two double bonds (C8-C9 and C11-C12). The peak of ibuprofen occurs at 225 nm due to the aromatic transition.

Optimization of the HPLC System
The optimization of the HPLC system indicated that methanol: double distilled water (70:30) is the best condition (resolution 1.39; retention factor 0.94; tailing factor 1.14; retention time 1.58 minutes). The chromatogram of andrographolide ( Figure  4a) indicates a distinct peak at 1.58 minutes, while ibuprofen elutes at 6.88 minutes.

The pharmacokinetic DDIs of Andrographis paniculata infusion and ibuprofen in the plasma of healthy Oryctolagus cuniculus rabbits
During 7 days of acclimatization, all rabbits were in healthy condition as proven by their physiological status (Table 1) and biochemical parameters (Table 2).
Rabbits are defined as suffering an abnormal condition when its heart rate exceeds 300 ibuprofen as proven by its occurrence in the rabbit's blood at 60 minutes ( Figure 6: green curve). These data confirmed that there is a pharmacokinetic DDIs between Andrographis paniculata (measured as andrographolide) and ibuprofen when these compounds are taken together at the same time.

Conclusion
A pharmacokinetic DDIs between Andrographis paniculata (measured as andrographolide) and ibuprofen was observed in the plasma of healthy rabbits. Interestingly, our data indicated that the oral administration of ibuprofen together with Andrographis paniculata could improve the absorption and distribution of the latter in Oryctolagus cuniculus rabbits. According to Melillo (2007) and Özkan (2012), the normal value of leucocyte and hematocrit ranges 33.10-10.57 %. 16,17 The blood sampling from the marginal ear vein of the rabbit was carried out as described by Parasuraman et al. (2010) 14 ( Figure 5).
The pharmacokinetic profile of Andrographis paniculata infusion (determined as andrographolide) in the plasma of healthy Oryctolagus cuniculus rabbits compared to those of ibuprofen alone and the mixture of ibuprofen and Andrographis paniculata infusion is presented in Figure 6.  Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism(s) involved in its