Revista Brasileira de Farmacognosia 27 (2017) 105–111 ww w.elsevier.com/locate/bjp Original Article Metabolic profile and ␤-glucuronidase inhibitory property of three species of Swertia ∗ Swagata Karak, Gargi Nag, Bratati De Phytochemistry and Pharmacognosy Research Laboratory, Department of Botany, University of Calcutta, Kolkata, India a a b s t r a c t r t i c l e i n f o Article history: ␤-Glucuronidase inhibitors are suggested as potential hepatoprotective agents. Swertia chirayita (Roxb.) Received 29 February 2016 Buch.-Ham. ex C.B. Clarke, Gentianaceae, is known for its hepatoprotective and anti-hepatotoxic activ- Accepted 18 July 2016 ity in Ayurvedic system of medicine for ages. This plant is substituted by other species like S. decussata Available online 3 October 2016 Nimmo ex C.B. Clarke and S. bimaculata (Siebold & Zucc.) Hook. f. & Thomson ex C.B. Clarke. The aim of the study was to compare metabolite profile and ␤-glucuronidase inhibitory activity of these three important Keywords: species of Swertia and to identify the active constituents. S. chirayita (IC50 210.97 ␮g/ml) and S. decussata Swertia (IC 269.7 ␮g/ml) showed ␤-glucuronidase inhibitory activity significantly higher than that of silymarin, ␤-Glucuronidase 50 the known inhibitor of the enzyme. The activity of S. bimaculata was low. The metabolites present in the Xanthones Hepatoprotective three species were analyzed by HPLC and GC-MS based metabolomics approach. Five amino acids, twenty one organic acids, one inorganic acid, eight fatty acids, twenty one phenols including xanthones, eight sugars, seven sugar alcohols, five terpenoids and amarogentin were identified. Activities of the xan- thones mangiferin (IC50 16.06 ␮g/ml), swerchirin (IC50 162.84 ␮g/ml), decussatin (IC50 195.11 ␮g/ml), 1-hydroxy-3,5,8-trimethoxy xanthone (IC50 245.97 ␮g/ml), bellidifolin (IC50 390.26 ␮g/ml) were signifi- cantly higher than that of silymarin (IC50 794.62 ␮g/ml). Quinic acid (IC50 2.91 mg/ml), O-acetylsalicylic acid (IC50 48.4 mg/ml), citric acid (IC50 1.77 mg/ml), d-malic acid (IC50 14.82 mg/ml) and succinic acid (IC50 38.86 mg/ml) also inhibited the enzyme ␤-glucuronidase. The findings suggest that constituents, in addition to the xanthones, probably also contribute to the bioactivity of different Swertia species by synergistic effect. Further in vivo study is required to support the claim. © 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction Liver damage causes an increase in the level of ␤-glucuronidase in blood (Pineda et al., 1959), and liver cancer could be related to this Liver disease has become a major health issue globally (Byass, enzyme (Mills and Smith, 1951). ␤ 2014). The liver is a vital organ that is involved in mainte- -Glucuronidase inhibitors reduce the carcinogenic potential of nance of metabolic functions and helps in the detoxification toxic compounds normally excreted in bile after glucuronidation process by countering several exogenous and endogenous chal- (Walaszek et al., 1984). Due to this correlation, ␤-glucuronidase lenges (Kshirsagar et al., 2011). Glucuronidation is a major pathway inhibitors are suggested as potential hepatoprotective agents (Shim of phase II xenobiotic biotransformation (de Graaf et al., 2002). et al., 2000). Certain hepatoprotective plant extracts and their Conjugation of toxins with glucuronic acid deactivates potentially constituents are known to inhibit the enzyme, ␤-glucuronidase damaging compounds and subsequently eliminates them from the (Joshi and Sanmugapriya, 2007). Silymarin (a mixture of flavono- body. However, this process becomes limited by the rate of deglu- lignans), the commercial plant derived ␤-glucuronidase inhibitor curonidation by ␤-glucuronidase. Hydrolysis of the glucuronide (Kim et al., 1994), is used to treat liver disorders and also certain moiety can be carried out by ␤-glucuronidase present in most of cancers (Dixit et al., 2007). But it has poor bioavailability (Dixit et al., the tissues, in endocrine and reproductive organs (Dutton, 1980). 2007). Silymarin has certain other limitations related to gastroin- testinal tract like bloating, dyspepsia, nausea, irregular stool and diarrhoea. It also produced pruritus, headache, exanthema, malaise, asthenia, and vertigo (Pradhan and Girish, 2006). Hence, search for glucuronidase inhibitory compounds from medicinally important ∗ traditional plants that are earlier reported to be hepatoprotective Corresponding author. is necessary. E-mail: [email protected] (B. De). http://dx.doi.org/10.1016/j.bjp.2016.07.007 0102-695X/© 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 106 S. Karak et al. / Revista Brasileira de Farmacognosia 27 (2017) 105–111 Plants of the genus Swertia, Gentianaceae, are well recognized in used for studying the enzyme inhibition activity in vitro as well as literature as important medicinal herb having an array of biologi- for HPLC and GC/MS analysis. cal and therapeutic properties (Negi et al., 2011). Hepatoprotective and anti-hepatotoxic activity of Swertia sp. have already been estab- Assay for ˇ-glucuronidase inhibition lished in Ayurvedic medical system and validated scientifically in animal system (Mukherjee et al., 1997; Karan et al., 1999; Reen ␤-Glucuronidase inhibition assay was carried out as per the et al., 2001). Swertia chirayita (Roxb.) Buch.-Ham ex C.B. Clarke, method of Kim et al. (1999) with modification. In brief, 100 ␮l of considered to be the most important species of Swertia reported ␤-glucuronidase (986.4 units/ml in 0.1 M phosphate buffer, pH 7.0) from India, for its medicinal properties, has been considered as crit- and 340 ␮l of test solution/reference standard of various concentra- ◦ ically endangered plant (Pant et al., 2000; Joshi and Dhawan, 2005; tions in 0.1 M phosphate buffer (pH 7.0) were pre-incubated at 37 C Bhargava et al., 2009). This plant is substituted by other species for 15 min. Following the pre-incubation, 60 ␮l of p-nitrophenyl-␤- like S. decussata Nimmo ex C.B. Clarke and S. bimaculata (Siebold & d-glucuronide (3.15 mg/ml in 0.1 M phosphate buffer, pH 7.0) was ◦ Zucc.) Hook. f. & Thomson ex C.B. Clarke (Chopra et al., 1956; Phoboo added and incubated at 37 C for 50 min. The colour developed was et al., 2010). Metabolites such as terpenoids, flavonoids, iridoid read at 405 nm in spectrophotometer. Controls were devoid of test glycosides and xanthones are considered as active constituents of samples. The percent inhibition was calculated as follows: Swertia sp., xanthones being the main active secondary metabolite − (Brahmachari et al., 2004; Nag et al., 2015). Control OD Sample OD Inhibition (%) = × 100. In a previous study antioxidant, anti-glycosidase and anti- Control OD acetylcholinesterase properties of S. chirayita and the two substitutes were reported from the laboratory (Nag et al., 2015). HPLC analysis Although the hepatoprotective property of S. chirayita is well known, the mode of action for hepatoprotection has not yet been The HPLC analysis was performed on an Agilent 1260 (Agilent studied. The active principles for hepatoprotection are also not ␤ Technologies, USA) HPLC system consisting of a quaternary pump, known. -Glucuronidase inhibitory properties of the extracts of a column temperature controller and a diode-array detector (DAD). these plants would further validate their hepatoprotective prop- The analytical column (Agilent Eclipse plus C18, 100 × 4.6 mm, erty. So, the aim of the study was to compare metabolite profile 3.5 ␮m) was used for the analysis. The mobile phase was composed and ␤-glucuronidase inhibitory activity of three important species of solvent A (acetonitrile) and solvent B (0.1%formic acid aqueous, of Swertia i.e. S. chirayita, S. decussata and S. bimaculata in order to v/v). The linear gradient programme followed was: 10% A at 0 min, identify the active constituents. 30% A at 20 min, 60% A at 35 min and 80% A at 45 min (Du et al., 2012). The flow rate was 0.7 ml/min. 20 ␮l aliquots were injected. Materials and methods UV spectra of the peaks were recorded from 190–400 nm over a range of 8 different UV wavelengths (210, 214, 230, 250, 254, 260, Plant material 273, and 280 nm respectively). Leafy shoots of three species of Swertia, Gentianaceae, namely GC/MS analysis Swertia chirayita (Roxb.) Buch.-Ham ex C.B. Clarke (Voucher no. Bot 332S-1), S. bimaculata (Siebold & Zucc.) Hook. f. & Thomson GC–MS analysis was performed using Agilent 7890 A GC [soft- ex C.B. Clarke (Voucher No. Bot 332S-2) were collected from Dar- ware driver version 4.01 (054)] equipped with 5795C inert MSD jeeling Himalayas. The third species S. decussata Nimmo ex C.B. with Triple Axis Detector. The column used for quantification anal- Clarke (Voucher No. Bot 332S-3) was collected from the Western ysis was HP-5MS capillary column [Agilent J & W; GC Columns Ghats, India. Voucher specimens are available in the Department (USA)] of dimensions 30 m × 0.25 mm × 0.25 ␮m. The method of of Botany, University of Calcutta. The two names S. chirayita and Kind et al. (2009) was followed after modification (Das et al., S. decussata are unresolved as per IPNI (International Plant Names 2016). The analysis was performed under the following oven tem- Index). ◦ ◦ perature programme: oven ramp 60 C (1 min hold), to 325 C at ◦ 10 C/min, held for 10 min before cool-down producing a run time Chemicals and reagents ◦ of 37.5 min. The injection temperature was set at 250 C, the MSD ◦ ◦ transfer line at 290 C and the ion source at 230 C. Helium was ␤-Glucuronidase (ex.