Two new proanthocyanidin trimers isolated from Cistus incanus L. demonstrate potent anti-inflammatory activity and selectivity to cyclooxygenase isoenzymes inhibition

Abstract Two new proanthocyanidin trimers have been isolated from Cistus incanus herb; gallocatechin-(4α→6)-gallocatechin-(4α→8)-gallocatechin (compound 1) and epigallocatechin-3-O-gallate-(4ß→8)-epigallocatechin-3-O-gallate-(4ß→8)-gallocatechin (compound 2). The structures were determined on the basis of 1D- and 2D-NMR (HSQC, HMBC) of their peracetylated derivatives, MALDI-TOF-MS and by acid-catalysed degradation with phloroglucinol. A more abundant proanthocyanidin oligomer was also isolated, purified and its chemical constitution studied by 13C-NMR and phloroglucinol degradation. The mean molecular weight of the polymer was estimated to be about 7 to 8 flavan-3-ol-units with a ratio of procyanidin : prodelphinidin units at 1:5, some of which are derivatised by gallic acid. Water extract and higher oligomeric proanthocyanidin fractions of C. incanus significantly inhibited TPA-induced oedema when applied topically at doses of 0.5 and 1 mg/ear in mice. Furthermore, the extracts and the pure compounds inhibited COX-1 and COX-2 activities. In addition, compound 2 exhibited an IC50 of 4.5 μM against COX-2 indicating its high selectivity towards COX-2. Graphical abstract


Introduction
The genus Cistus (Cistaceae) consists of a group of about 20 shrub species found in wide areas throughout the whole Mediterranean region (Comandini et al. 2006). As general remedies in folk medicine, Cistus sp. are used in treating various skin diseases, diarrhoea and inflammation (Attaguile et al. 2000). In Jordan, Cistus incanus and Cistus salvifolius are traditionally used for the treatment of gout (Al-Khalil 1995). Furthermore, ehrhardt et al. (2007) and  described the antiviral activity of a C. incanus plant extract (CYSTUS052) against influenza A virus infections. One controlled observational study was performed on patients, who suffered from painful infection in mouth and throat area and demonstrated the efficacy of Cistus extract (Kalus et al. 2010). In another randomised, placebo-controlled double blind clinical trial, it was shown that Cistus was more effective in reducing the average duration and severity of symptoms in patients with infection of the upper respiratory tract than placebo (Kalus et al. 2009).
Due to the biodiversity and chemical variability in addition to the chemotaxonomic differences between plants, in general and specifically the genus 'Cistus' , many scientists have studied the phytochemical composition of different Cistus sp. (Toniolo et al. 2014). The major characteristic compounds in Cistus sp. were found to be flavonoids with different grades of O-methylation, diterpenes and cinnamic esters of borneol Venditti et al. 2015). Furthermore, several flavan-3-ols and oligomeric proanthocyanidins were isolated and characterised from the air-dried herb material of C. incanus (Petereit et al. 1991;Danne et al. 1993). Among the flavan-3-ols, catechin, catechin-3-O-α-L-rhamnoside, gallocatechin and gallocatechin-3-O-gallate were isolated. The presence of the oligomeric proanthocyanidins, such as the procyanidins B1 and B3, gallocatechin-(4α→8)-catechin, gallocatechin-(4α→8)-gallocatechin, gallocatechin-(4α→8)-gallocatechin-(4α→8)-catechin and gallocatechin-(4α→8)-gallocatechin-(4α→8)-gallocatechin was also reported. By considering the ethnopharmacologically described actions of C. incanus and as a part of our continuing studies to search for COX inhibitory compounds from medicinal plants (Qa'dan et al. 2011), we aimed to investigate the anti-inflammatory, COX-1 and COX-2 inhibitory activities of the title plant. In this study, we describe the isolation and characterisation of two new trimeric proanthocyanidins and an oligomeric proanthocyanidin fraction (OPF) from the crude water extract in addition to their biological activity evaluation.

Bioactivity evaluation
Since no information is available yet on the kinetics and dynamics of the active ingredients of our extract or the OPF, their bioactivity was tested following topical administration on TPA-induced acute inflammation model (Hernandez et al. 2001;Ukiya et al. 2007). TPA application on a mouse ear resulted in significant oedema and thickness of the ear and this inflammatory pattern reached its maximum at 4 h post-TPA administration. This TPA-induced inflammation was significantly reduced when We or OPF was applied simultaneously with TPA (p < 0.05). The per cent reductions were dose-dependent and ranged from 62-74% to 72-82% for We and OPF, respectively at 0.5 and 1 mg of each (Figure 2). In comparison to indomethacin, 1 mg OPF resulted in a similar anti-inflammatory effect as 0.5 mg of indomethacin ( Figure 2).
Since it has been found that C. incanus reduces the symptoms of upper respiratory infections as well as skin inflammation presented herein, it can be suggested that OPF plays a major role in decreasing the signs and symptoms of both inflammatory conditions (Kalus et al. 2009;Badr et al. 2014). The latter anti-inflammatory properties of OPF may be attributed to decreasing the activity or production of inflammatory cytokines and/or increasing the activity or production of anti-inflammatory cytokines (Matalka et al. 2012;Badr et al. 2014). In addition, other than OPF, the anti-inflammatory We of C. incanus may be attributed to the presence of caffeic acid derivatives, polysaccharides and flavonoids next to proanthocyanidins (Okoye & Osadebe 2010).  Figure 2. the per cent reduction of tPa-induced ear oedema in mice. tPa and extracts/compounds were applied simultaneously to the right ear. Indomethacin at a dose of 0.5 mg/ear was used as a positive control. the We and oPF showed a dose-dependent oedema reduction (n = 6). As for COX inhibition assays, COX-1 inhibition by We (IC 50 63.2 μM) was more than by the OPF fraction (IC 50 93.7 μM), whereas the OPF fraction (IC 50 23.1 μM) exhibited greater COX-2 inhibition than the We (IC 50 44.7 μM). The effects of the extracts were compared with acetyl salicylic acid (ASA) (Table 1). Furthermore, COX-1 and COX-2 inhibitory activities by the pure compounds 1 and 2 were tested and among both tested compounds, 2 exhibited stronger inhibitory effect on COX-2 (IC 50 , 4.5 μM) than the positive control ASA (17.4 μM).
Flavan-3-ols and tannins have been reported previously to exhibit anti-inflammatory activity in many pharmacological studies. Therefore, the low molecular proanthocyanidin-like compounds 1 and 2 in addition to the higher OPF present in the water extract might be partially responsible for the anti-inflammatory, COX-1 and COX-2 inhibitory effects of C. incanus. Accordingly, the plant extract may serve as an important anti-inflammatory drug for both, topical and even systemic use. Moreover, understanding the link between compound 2 and the anti-inflammatory COX-2 inhibitory effect might help in developing more selective future drugs. Further studies, however, are necessary to explore in depth the anti-inflammatory potency and safety of compound 2.

Conclusions
The present study showed that the higher OPF of C. incanus has anti-inflammatory activity and has a great similarity in the chemical structure to the higher oligomeric proanthocyanidins isolated from the air-dried herb of Cistus albidus (Qa'dan et al. 2003). The similarity is in the predominance of 2,3-cis-configuration, 3′,4′,5′-trihydroxylated B-rings and the occurrence of galloylated units. Furthermore and to the best of our knowledge, compounds 1 and 2 as well as the NMR data of their peracetate derivatives are described for the first time. The presence of two galloylated moieties, and three trihydroxylated B-rings in the isolated compound 2 and its relative higher molecular weight might be responsible for the potent inhibitory activity against COX-2.