Structure activity relationship of bergenin, p-hydroxybenzoyl bergenin, 11-O-galloylbergenin as potent antioxidant and urease inhibitor isolated from Bergenia ligulata

Ethanol extract of the aerial parts of Bergenia ligulata was subjected to solvent–solvent separation followed by various chromatographic techniques that lead to isolation of bergenine (1), p-hydroxybenzoyl bergenin (2), 11-O-galloylbergenin (3) and methyl gallate (4) as major constituents. Ethyl acetate fraction showed a dose-dependent urease inhibitory pattern with IC50 value of 54μg/mL. Structures of compounds 1 and 3 were established by XRD and 2, 4 by NMR. All these compounds were subjected to DPPH scavenging activity, reducing power assay and urease inhibitory activity. The EC50 7.45 ± 0.2 μg/mL and 5.39 ± 0.28 μg/mL values in terms of antioxidant and reducing power, respectively, were less for 3. Compounds 1–3 showed moderate to significant urease inhibitory potential with IC50 57.1 ± 0.7, IC50 48.4 ± 0.3 and 38.6 ± 1.5. Antioxidant activities and urease inhibitory potential were investigated and compound 3 was found to be the most active.


Introduction
Accumulation of reactive oxygen species (ROS) in a living cell results in the lipid peroxidation, causes oxidative damage to proteins and denaturation of DNA and also can cause diseases such as cancer, diabetes, aging and cardiovascular dysfunction (Halliwell et al. 1992, Knight 1995Finkel & Holbrook 2000). Antioxidant compounds are used to protect and overcome the damages induced by ROS. Plants are rich sources of antioxidant compounds of diverse chemical nature with minimal side effects or no side effect at all (Mokbel & Hashinaga 2006).
Bergenin (1) is a bioactive molecule and reported to have neuroprotective (Kogen et al. 1999), antitussive (Takahashi 2003), hypolipidaemic (Piegen 1980), anti-HIV, antiarrhythmic (Jahromi et al. 1992), antiinflammatory (Piacente et al. 1996), PTP1B inhibitory (Pu et al. 2002) and gastroprotective (Swarnalakshmi et al. 1984) activities. The exact molecular mechanism is still not clear for these activities. Other important pharmacological activities include an inhibitory effect on platelet aggregation (Li et al. 2005) and an inhibitory effect on bovine adrenal tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of catecholamine (Goel et al. 1997). Its antioxidant activities are well established, and interestingly in one study it was found to be a potent antioxidant as ascorbic acid or quercetin (Nazir et al. 2007). Its hepatoprotective activity is studied in vitro and in vivo in various models such as carbon tetrachloride-intoxicated rats (Lee et al. 2005) and D-galactosamine-induced hepatotoxicity in rats (Zhang et al. 2003). The antifungal activities of bergenin against seven pathogenic plant species were reported (Lim et al. 2000). However, for 11-O-galloylbergenin (2), only the antiinflammatory and analgesic activities have been reported.
Wide spectrum of the pharmacological profile of bergenin (1) still permits the investigations for its new therapeutic targets and the associated molecular mechanisms. In the current study, the structure -activity relation with respect to antioxidant and urease inhibitory potential of bergenin (1), para-hydroxybergenin (2) and 11-O-galloylbergenin (3) isolated from Bergenia ligulata was studied.

Results and discussion
The bergenin (1) and 11-O-Galloylbergenin (3) were transparent crystals and characterised by Single Crystal X-ray Diffraction ( Figure S1) and also 2 and 3 were hitherto unreported form the genus Bergenia and 3 was isolated for the first time in a crystalline form as a natural product. Compounds 2 and 4 ( Figure S2) were isolated as an amorphous powder and their structures were established by 1 H NMR, 13 C NMR and by comparing their spectral data with those of the reported compounds from the literature (Yoshida et al. 1982).
From all the in vitro models of antioxidant activities, it is evident that 11-O-galloylbergenin (3) is a potent and effective antioxidant. Furthermore, it is also established that benzoyl moiety along with three hydroxyl groups (two meta and one para) has important structural features for the scavenging of free radicals that are appreciably involved in interaction with the free radicals, and the inhibitory potential decreases if OH groups are removed from the benzoyl moiety as phydroxybenzoyl bergenin has EC 50 of 64.16^0.071 and 0.824^0.0007 mg/mL in terms of DPPH radical scavenging and reducing power, respectively, higher than 11-O-galloylbergenin (3).

Urease Inhibitory Activity SAR
The bergenin and its natural derivatives (2, 3) displayed moderate to significant urease inhibitory potential and 11-O-galloylbergenin is the most potent urease inhibitor with IC 50 value of 23.1^0.7 mM as compared to Thiourea (Table S3). The methyl gallate did not display any significant inhibitory effect, i.e. IC 50 . 100 mM, but it significantly increases the inhibitory potential of bergenin when attached to it through ester linkage, i.e. IC 50 value of bergenin is 37.2^1.5 mM while that of 11-O-galloylbergenin IC 50 is 23.1^0.7 mM. Furthermore, IC 50 value of p-hydroxybenzoyl bergenin is 32.4^0.4 mM suggesting that benzoyl function along with the three hydroxyl groups are effectively involved in interaction with enzyme. Based on this study, it is therefore suggested that substitution of another galloyl moiety in compound 3 will sufficiently increase the inhibitory potential.
The bergenin and its natural derivatives (3 and 4) proved to have no significant cytotoxicity allowing them to be used as a lead compound for the synthesis of new therapeutic agent, which could be more effective and specific for the treatment of various kinds of ulcers with no deleterious side effects (Takahashi et al. 2003).

Conclusion
It is concluded from the present study that bergenine and its natural derivatives possess urease inhibitory potential and that substitution of a galloyl moiety remarkably increases the inhibitory potential.

Supplementary material
Experimental details relating to this paper are available online, alongside Figures S1 and S2 and Tables S1 -S17.