β-Glucuronidase Inhibitory Effect of Phenolic Constituents from Phyllanthus amarus.

Abstract

The present study demonstrates the β-glucuronidase inhibitory action of methanol, 50% methanol, and aqueous extracts as well as isolated actives such as corilagin, brevifolin carboxylic acid, phyllanthin, and hypophyllanthin from Phyllanthus amarus. Schum and Thonn (Euphorbiaceae). The results revealed that 50% methanol and water extracts were highly active, and corilagin was found to be more potent as an isolated active. Brevifolin carboxylic acid was also found to have β-glucuronidase inhibitory action, whereas phyllanthin, hypophyllanthin, and methanol extracts were devoid of inhibitory action. These results suggest that phyllanthin and hypophyllanthin lignans isolated from the methanol extracts of Phyllanthus amarus. exhibit their hepatoprotective effect through a mechanism independent of β-glucuronidase inhibition. Corilagin, the phenolic principle isolated from the 50% methanol extract of Phyllanthus amarus., was found to be more potent at the concentration of 200 µg/ml, which is comparable with the standard drug silymarin.

Keywords:

• Brevifolin carboxylic acid
• corilagin
• β-glucuronidase
• Phyllanthus amarus

Introduction

Phyllanthus amarus. Schum and Thonn (Euphorbiaceae) is used in the Indian system of medicine for the treatment of a variety of ailments including hepatic disorders (Kirtikar & Basu, 1933). P. amarus. has been reported to exhibit marked anti-hepatitis B virus surface antigen both in vivo. (Thyagarajan et al., 1988) and in vitro. (Unander & Blumberg, 1981). Two major lignans, phyllanthin and hypophyllanthin, found in this plant have been reported to be hepatoprotective (Syamasunder et al., 1985). Certain known hepatoprotective plant extracts and phytoconstituents are proven to inhibit β-glucuronidase. For instance, silymarin is an inhibitor of β-glucuronidase (Kim et al., 1994). Due to this correlation, β-glucuronidase inhibitors are suggested as potential hepatoprotective agents (Shim et al., 2000). In this context, we evaluated methanol, 50% methanol, and aqueous extracts of P. amarus. along with phyllanthin, hypophyllanthin, corilagin, and brevifolin carboxylic acid for β-glucuronidase inhibition. This was done in order to verify the mechanism of hepatoprotection exhibited by P. amarus. and its active principles.

Materials and Methods

Plant material

Phyllanthus amarus.leaves were obtained from the herbal crude drug market, Chennai, India. The authenticated voucher specimen was deposited in the Herbarium of R&D, Amrutanjan Limited, Chennai, India.

Extraction and isolation

P. amarus. leaves (air-dried) were extracted with methanol, 50% methanol, and water separately (hot extraction). The filtrate was concentrated and dried under vacuum yielding 10.5, 18, and 16% (w/w), respectively. Isolation of phyllanthin and hypophyllanthin was done as per Somanabandhu et al. (1993).

Isolation of corilagin and brevifolin carboxylic acid

The powdered and dried material of Phyllanthus amarus. leaves (10 kg) was subjected to reflux with 50% aqueous methanol in the ratio 1:5, 1:3, and 1:3 volumes for a 2 h period each, and the aqueous methanol extract was partitioned with n.-butanol. The organic layer was further concentrated and dried under vacuum and dissolved in demineralized water in the ratio 1:1. This solution was subjected to CC with 200 g of cross-linked dextran gel (Sephadex G-25 F), three-times, using a 140 cm glass column and 50% aqueous acetone as the mobile phase. The column was developed under a head pressure of 15 psi giving a flow rate of 12–15 ml/min. Fractions were collected and activity was ascertained with the β-glucuronidase assay. The combined most active fractions were concentrated to a minimal volume and subjected to purification by CC using MCI (CQPO6) (Mitsubishi Chem Corporation) gel as stationary phase. A mixture of methanol:water (1:1) was used as an eluent to obtain fraction 1-corilagin (2.07 g) as a white powder and fraction 2-brevifolin carboxylic acid (0.27 g) as a yellow crystalline powder. Final purification of corilagin was done using preparative HPLC (Shimadzu LC-8A) delta-pak C₁₈ column (300 × 50 mm I.D, 100 Å) as RP stationary phase and 50% methanol in water as mobile phase (elution rate: 10 ml/min). Identification of corilagin and brevifolin carboxylic acid were confirmed by comparison with the standard reference spectra (Zhang et al., 2000).

Chemicals

β-Glucuronidase (type B-1 from bovine liver, G-0251, Lot # 041K70359, EC No. 3.2.1.31, Sigma, USA), p.-nitrophenyl-β-glucuronide (Biochemika, 73677, Lot & Filling code # 449947/1 30609106, Fluka, USA), silymarin (S0292, Lot # 74H0415, Sigma, USA), sodium dihydrogen orthophosphate (RM1255, B.No. 8-0432, Himedia Laboratories, Mumbai, India), di-sodium hydrogen phosphate (RM257, B.No 3-0279, Himedia Laboratories, Mumbai, India).

β-Glucuronidase inhibition assay

β-Glucuronidase inhibition assay was carried out as per the method of Kim et al. (1999). In brief, 25 µl of β-glucuronidase (6666.6 units/ml in 0.1 M phosphate buffer, pH 7.0) and 210 µl of test solution/reference standard of various concentrations in 0.1 M phosphate buffer, pH 7.0, were pre-incubated at 37°C for 15 min. Following the pre-incubation, 15 µl of p.-nitrophenyl-β-glucuronide (3.15 mg/ml in 0.1 M phosphate buffer, pH 7.0) were added and incubated at 37°C for 50 min. The color developed was read at 405 nm in a microplate reader (Molecular Devices, Sunnyvale, USA). Controls, which were devoid of test samples, were run. The percent inhibition was calculated as follows: Inhibition (%) = (Control OD − Sample OD)/Control OD × 100. IC₅₀ values (µg/ml) were calculated using log-Probit analysis. Silymarin was used as the reference standard.

Results and Discussion

Methanol (50%), aqueous extract, corilagin, and brevifolin carboxylic acid showed potent β-glucuronidase inhibition with IC₅₀ values of 139.15, 218.85, 120.06, and 209.07 µg/ml, respectively. The IC₅₀ of silymarin was found to be 199.25 µg/ml (Table 1). β-Glucuronidase has been found in animals, plants, and bacteria (Stahl & Fishman, 1983). This enzyme catalyzes the hydrolysis of β-glucuronide conjugates of endogeneous and exogenous compounds in the body (Shim et al., 2000). Liver damage causes an increase in blood β-glucuronidase level (Pineda et al., 1959), and liver cancer could be related to this enzyme (Mills & Smith, 1951). Silymarin, a known hepatoprotective compound, has been shown to be a potent inhibitor of β-glucuronidase in both in vivo. and in vitro. systems. It is also proposed that silymarin exhibits its hepatoprotective effect through the mechanism of β-glucuronidase inhibition (Kim et al., 1999). Tectorigenin, a β-glucuronidase inhibitor from Pueraria thunbergiana. was shown to be hepatoprotective in CCl₄-treated rats (Lee et al., 2003). Similarly, the ether fraction of Ganoderma lucidum., which had potent β-glucuronidase inhibitory activity, protected against CCl₄ induced injury in animals (Kim et al., 1999). 18-β-Glycyrrhetinic acid, which was found to be active against CCl₄-induced toxicity in rats was found to have a β-glucuronidase inhibitory effect (Pineda et al., 1959). On account of these observations, it is suggested that β-glucuronidase should be closely related to liver injury, which could be prevented by an inhibitor of β-glucuronidase (Kim et al., 1999). In the present study, aqueous and 50% methanol extracts of P. amarus. and corilagin, a phenolic priniciple, showed potent β-glucuronidase inhibition, and their activity was comparable to that of silymarin. Methanol extract, phyllanthin, and hypophyllanthin failed to exhibit β-glucuronidase inhibition, whereas corilagin, a phenolic constituent, has shown potent β-glucuronidase inhibitory action.

Table 1.. β-Glucuronidase inhibitory effect of extracts and isolated actives from Phyllanthus amarus..

Tested material	Conc (µg/ml)	% Inhibition±SEM (n = 3)	IC50 (µg/ml)
Phyllanthus amarus. H2O extract	100	33.80±3.02
	200	49.27±1.53
	400	60.88±2.60	218.85
	600	69.71±1.87
Phyllanthus amarus. 50% MeOH ext	100	47.01±1.49
	200	53.13±4.42
	400	65.20±0.72	139.15
	600	71.14±3.06
Phyllanthus amarus. MeOH ext	250	23.36±0.23
	500	32.89±2.34	–
Corilagin	50	12.19±0.56
	100	30.19±3.26
	200	55.68±2.59	120.06
	300	69.32±0.89
Brevifolin carboxylic acid	50	5.68±0.26
	100	18.36±2.11
	200	42.11±1.56	209.07
	300	51.29±2.58
Phyllanthin	200	Not active	–
Hypophyllanthin	200	Not active	–
Silymarin^a.	50	8.18±5.20
	100	21.93±2.00
	200	50.24±4.24	199.25
	300	66.26±1.38

^a.Silymarine, Reference standard, purchased from M/s Sigma, USA.