Chromatogram profiles of andrographolide in A 23187-induced New Zealand rabbit ’ s urine and faeces

Article history: Received on: 03/11/2016 Revised on: 20/11/2016 Accepted on: 10/12/2016 Available online: 31/01/2017 Andrographolide is the main bioactive component of Andrographis paniculata (Burm. F.) Nees) which has been traditionally used as pain reducer in Indonesia. Our previous study showed that boiled water of Andrographis paniculata herbs, calculated as andrographolide, administered in healthy rabbits, was fastly absorbed from the stomach (tmax 1 hour), distributed in the circulation system (tmax 1.5 hours) and metabolized in the liver (tmax 2 hours), in subsequent process. In this work, we investigated the chromatogram profile of andrographolide in A23187 induced-New Zealand rabbits’ urine and faeces. Prior to be treated, the animals were given orally 40 mg of andrographolide. Urine and faeces were collected during 2 x 24 hours then were extracted using a mixture of ethyl acetate-water (1:1). The water extract was further analyzed using reversed-phase HPLC with methanolwater (55:45) as mobile phase, flow rate 1 mL/minute for faeces and 0.5 mL/minute for urine. Detection was set at λ = 227 nm. HPLC chromatogram showed that andrographolide was not detected in the urine while compounds with higher polarity were observed at 3.0 to 6.0 minutes of elution. Andrographolide was still detected in faeces along with a more nonpolar compound. It could be concluded that andrographolide showed a good bioavailability in rabbit. This compound was metabolized and excreted in the form of more polar compounds in urine and a more nonpolar compound in faeces.


INTRODUCTION
Andrographolide is the main bioactive component of Andrographis paniculata (Burm.F.) Nees) which has been traditionally used as pain reducer in Indonesia.This compound has been proven in exerting anti-inflammatory activity (Levita et al, 2010;Lim et al, 2012), hepato-protector (Maiti et al, 2009), anti-hyperglycemia (Zhang et al, 2009), anti-diare (Hui et al, 2013), anti-oxidant (Guan et al, 2013), anti-HIV (Reddy et al, 2005), and anti-cancer (Song et al, 2015).Andrographolide and its derivates reduced the production of inflammation mediator due to the inhibitory of NF-kB signaling pathway (Lim et al, 2012).Furthermore, Tao Zhu and colleagues concluded that there was a decreasing of inflammation in LPSinduced rats due to inhibition of NF-kB activity (Zhu et al, 2013).A few investigations of the pharmacokinetics have been reported after oral administration of andrographolide.Six new andrographolide metabolites were isolated from urine, faeces, and small intestine contents of rats, as reported by He and colleagues (He et al, 2003), while four new metabolites in healthy human urine were reported by Cui, who concluded that four of them were characterized as sulfate and one was cysteine-S-conjugate (Cui et al, 2005).A comparative investigation of andrographolide's in vitro metabolic pathways in human, dog, and rat liver microsomes was carried out by Zhao and teamwork.They concluded that significant species differences indicated a more cautious strategy for further pharmacokinetics research of andrographolide in animal models (Zhao et al, 2013).
Our previous study showed that boiled water of Andrographis paniculata herbs, calculated as andrographolide, administered in healthy rabbits, was fastly absorbed from the stomach (C max 0.5549 µg/mL; AUC 1.7451 µg.h/mL; t max 1 h), distributed in the circulation system (C max 0.2136 µg/mL; AUC 0.434 µg.h/mL; t max 1.5 h), and metabolized in the liver (C max 0.0051 µg/mL; 0.0038 µg.h/mL; t max 2 h), in subsequent process.Andrographis paniculata herbs infusion showed good bioavailability in rabbit (Levita et al, 2014).
In this work, we investigated the chromatogram profile of andrographolide in A23187 induced-New Zealand rabbit's urine and faeces.A23187 was chosen as inducer, as it was reported by Rao and colleagues that this compound could affect the changes of intracellular calcium and induced inflammation in rats (Rao, et al, 1994).Other researchers concluded that A23187 stimulated prostaglandin synthase type 2 (PTGS2) and suppressed 15hydroxyprostaglandin dehydrogenase (PGDH) expression (Casciani et al, 2008).A23187 also activated IkappaB kinase 2 in mast cells and NF-kB as reported by Hosokawa, hence this compound played a role in degranulation and cytokine production in activated mast cells through independent mechanism of PKCβ (Hosokawa et al, 2013).
Animals used in this project were New Zealand strain rabbits (Oryctolagus cuniculus) (4 males and 2 females), purchased from Roemah Kelinci Sumedang, West Java, Indonesia.

Methods
The New Zealand rabbits (four males and two females; 2-2.5 kg of body weight) were acclimatized for 7 days and treated according to the ethical standards of Universitas Padjadjaran Health Research Ethics Committee No. 771/UN6.C1.3.2/KEPK/PN/2015.Standard feed (a mixture of soy bean curd, spinach, and carrot) and drink were given ad libitum.
No vitamins were added in their feed.At the 8 th day, the rabbits were inflammation-induced by injecting 0.5 mL of A23187 (10 mg of A23187 in 10 mL of 0.5 M phosphate buffer pH 7.4) into their ear marginal vein 30 minutes prior to be treated with andrographolide dose 40 mg/rabbit regardless its body weight.Urine and faeces were collected during 2 x 24 hours then were extracted using a mixture of ethyl acetate-water (1:1).Validation of the method was performed according to ICH Q2A only for the LOD and LOQ, by using 10 mL of rabbit plasma, with the following procedure: Into 10 centrifuge tubes was put @ 1 mL of plasma and added by 5 mL of methanol.The mixture was vortexshaken for 2 minutes, followed by centrifugation for 30 minutes at 3000 rpm, and was used to dilute standard solutions of andrographolide until various concentrations of 0.5; 1.0; 2.0; 4.0; 8.0; 10.0 μg/mL were obtained.The solutions were Milliporefiltered.200 µL of the filtrates was injected into Acclaim ® PolarAdvantage C18 column, 250 mm in length, 5-μm particle size, and 120 Å pore size.A mixture of methanol-water (55:45) was used as mobile phase, whereas flow rate was set at 1 mL/min.Detection was set at λ = 227 nm which is the wavelength where maxima of andrographolide occurs.This step was repeated 3x for LOD and LOQ calculation.200 µL water extract was further analyzed using RP-HPLC with the same condition, that were methanol-water (55:45) as mobile phase, flow rate 0.5 mL/minute for urine and 1.0 mL/min for faeces.

RESULTS AND DISCUSSION
The rabbits were examined their health prior treatment and the result is given in Table 1.Inducing the rabbits with A23187 resulted in a slight elevation of the temperature, whereas andrographolide treatment lowered it (but not all, only 66.67%, all in male animals), as showed in Fig. 1.A fever was observed in animals No.5 and No.6 which both were females, which probably due to hormonal factors.Rao and colleagues observed that intraperitoneal injection of A23187 (20 μg/mouse) to mice could elicite marked and transient increases in immunoreactive levels of 6-ketoprostaglandin-F2 alpha, leukotrienes B4, C4, D4, E4, and F4 (Rao et al, 1993).LOD and LOQ calculations were showed in Table 2.  Overlayed HPLC chromatograms of urine extract and andrographolide standard with the same condition (flow rate 0.5 mL/minute) (Fig. 2) showed that andrographolide was not detected in the urine (t R = 11.4 minutes), while compounds with higher polarity (blue peaks) were observed at 3.0 to 6.0 minutes.Andrographolide standard, in various concentrations (darker peaks), was eluted at 11.4 minutes.Polar metabolites of andrographolide isolated in urine had been reported (Cui et al, 2004;Cui et al, 2005;Zhao et al, 2013).Different metabolic profiles of human, dog, and rat, were also reported (Zhao et al, 2013).Overlayed HPLC chromatograms of faeces extract and andrographolide standard with the same condition (flow rate 1 mL/minute) (Fig. 3) showed that andrographolide was still detected in the faeces (t R = 5.5 minutes), while one peak of compound with lower polarity (blue peak) was observed at 10 minutes.Andrographolide standard, in various concentrations (colourful peaks), was eluted at 5.5 minutes.

IV. CONCLUSION
In six A23187 induced-New Zealand strain rabbits (Oryctolagus cuniculus) (4 males and 2 females), bred in Roemah Kelinci Sumedang, West Java, Indonesia, which were orally treated with 40 mg of andrographolide, this compound was metabolized and excreted in the form of more polar compounds in the urine and a more nonpolar compound in the faeces.

Table 1 :
Baseline data of the rabbits.

Tabel 2 :
AUC measurements for LOD and LOQ calculations.