Isolation of a novel catechin from Bergenia rhizomes that has pronounced lipase-inhibiting and antioxidative properties

Fitoterapia 82 (2011) 212–218

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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

Isolation of a novel catechin from Bergenia rhizomes that has pronounced lipase-inhibiting and antioxidative properties Sergey A. Ivanov a, Kenzo Nomura b, Ilya L. Malfanov a, Ilya V. Sklyar a, Leonid R. Ptitsyn a,⁎ a b

Ajinomoto-Genetika Research Institute, 1-st Dorozhny pr. 1-1, Moscow 117545, Russia Research Institute for Health Fundamentals, 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan

a r t i c l e

i n f o

Article history: Received 28 July 2010 Accepted in revised form 23 September 2010 Available online 12 October 2010 Keywords Human pancreatic lipase (HPL) Antioxidants Inhibition (+)-Catechin 3,5-di-O-gallate

a b s t r a c t An aqueous ethanol extract of Bergenia crassifolia rhizomes strongly inhibited human pancreatic lipase activity and increased scavenging of DPPH free radicals in vitro. Chromatographic separation of this extract led to isolation of the hydrolysable tannins (+)-catechin 3,5-di-O-gallate (1) and (+)-catechin 3-O-gallate (2). This is the first report of the isolation of compound 1 from plant material. This compound strongly inhibited human pancreatic lipase (with an IC50 value of 0.42 μg/ml) and exhibited a remarkable free radical-scavenging ability (with an SC50 value of 1.04 μg/ml). The chemical structures of 1 and 2 were elucidated using MS, NMR and chemical approaches. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The use of compounds that inhibit the digestion and absorption of nutrients is an important strategy in the treatment of obesity. These compounds help to reduce energy intake through gastrointestinal mechanisms [1–3]. Because dietary lipids represent a major source of unwanted calories, specifically inhibiting human pancreatic lipase (HPL), which is responsible for the hydrolysis of 50–70% of total dietary fats, is an effective approach to reduce fat absorption [1,4,5]. To discover biologically active anti-obesity agents from natural herbal resources, various plants have been screened for their anti-lipase activity. HPL inhibitors include substances belonging to various chemical classes, such as polyphenolics (green-tea and grape-seed polyphenols, flavones, flavonols, tannins, and chalcones), saponins, terpenes, and others [6]. In addition to obesity, disrupted lipid metabolism causes a variety of other serious diseases, such as hypertension, functional depression of certain organs, and atherosclerosis

⁎ Corresponding author. Tel.: + 7 495 315 23 30; fax: + 7 495 315 06 40. E-mail address: [email protected] (L.R. Ptitsyn). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.09.013

[6]. Uncontrolled lipid peroxidation also contributes to the development of atherosclerosis [7]. Natural dietary antioxidants, such as vitamin E [8] and plant polyphenolic compounds [9,10], diminish free-radical-dependent lipoperoxidation. Bergenia crassifolia (Siberian tea, elephant ear; Saxifragaceae) is widely used in Russian ethnomedicine [11]. Extracts from the roots and leaves of this plant include antidiarrheal [11,12], antiinflammatory [13], antioxidant [14,15], antiviral [16], antimicrobial [17], diuretic [18], and immunostimulating [19,20] compounds. A prominent feature of B. crassifolia is the simultaneous presence of both green (young) and black (wintered) leaves on a single plant. Buryats and Mongols use B. crassifolia old blackened wintered leaves as a tea. The tea is popular as a health beverage as it contains no harmful stimulants such as caffeine [11,21]. Black leaves which have passed two winters are used as the adaptogen [22]. The phytochemical constituents of this plant include (+)-catechin 3-O-gallate, polymeric proanthocyanidins [23], the pectic polysaccharide bergenan [19], arbutin [24], gallic acid, and significant amounts of other phenolic compounds, such as flavonoids, phenolic acids, phenols, coumarins, and tannins [25]. The phenolic content varies according to the type of leaf [15,18]. All of the compounds listed above, except for coumarin and bergenin, are capable of scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH·) free radicals. The antioxidant activity of the green-leaf extract is due to a

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high concentration of arbutin, whereas gallic acid is the primary bioactive compound in the black-leaf extract [15]. Fermented green leaves of B. crassifolia have adaptogenic effects associated with presence of arbutin and a biosynthesis of protocatechuic acid [26]. To our knowledge, no HPL-inhibitory compounds from this plant have been identified. The purpose of this study was to isolate HPL inhibitors from Bergenia rhizomes (BR) and elucidate their chemical structures. We also attempted to identify compounds with putative DPPH· radical-scavenging activity. The BR extract exhibited high levels of HPL inhibition and antioxidant activity. A hydrolysable tannin, (+)-catechin 3,5-di-O-gallate (1), was isolated for the first time. This compound possesses pronounced HPL-inhibitory and antioxidant properties.

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2.3. Chemicals Organic solvents used for extraction met ACS or Ph. Eur. quality standards (Fluka, USA). Ultra-gradient acetonitrile used in LPLC and HPLC was obtained from Lab-Scan (Poland). Reagents including 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, 4-methylumbelliferyl oleate (4-MUO), human pancreatic lipase (HPL), porcine pancreatic colipase (PPCL), tannase from Aspergillus ficuum, and other chemicals were purchased from Sigma-Aldrich Corp. (USA). Other reagents [(+)-catechin, (−)-catechin, (−)-catechin 3-O-gallate, and (−)-epicatechin 3-O-gallate] were purchased from Wako Co. (Japan). 2.4. Extraction and isolation of lipase inhibitor

2. Materials and methods 2.1. Plant material Dried rhizomes of B. crassifolia were purchased from the herbal company Khorst in March 2008. The rhizomes were collected from the Altai region of the Russian Federation (batch no. 080308). A voucher specimen (28-B) was deposited in our laboratory at Ajinomoto-Genetika Research Institute (AGRI).

2.2. General experimental procedures The dried BR material (30 g) was crushed in a mill and soaked in 70% aqueous ethanol (1:40, w/v) with stirring (200 rpm) overnight at room temperature. The suspension was filtered through a paper filter (Whatman no. 4), and the ethanol was removed using a rotary evaporator. After freezedrying, 12.2 g (yield 47% of dry weight) of crude extract was obtained. The crude extract was stored at − 20 °C in the dark until further analysis. The crude fractionation of the BR extract was performed using low-pressure liquid chromatography (LPLC) on an Aktaprime plus fractionation system (Amersham Biosciences) equipped with 280- and 365-nm filters. HPLC-guided isolation of individual compounds was achieved using a Waters chromatographic system equipped with a Waters 600E gradient pump and a 2996 PDA detector controlled by Empower software. UV–visible spectra were recorded on an Ultrospec 3300pro spectrophotometer (Amersham Biosciences). In the DPPH assay, the optical density was measured using a microtiter Multiskan Ascent plate reader (Thermo Electron Corp.). In the lipase-inhibition assay, fluorescence was measured a multi-well Genios FL fluorimeter (Tecan). The rotation angle [α]D was measured on a P-2200 polarimeter (JASCO). IR spectra were recorded on a Spectrum™ 100 spectrometer (Perkin Elmer) using the ATR spectrum mode. Mass spectra were obtained using a Quattro micro API mass spectrometer (Waters Corp.) and a Bruker MALDI-TOF mass spectrometer using a laser energy of 85% in reflectron mode, and dihydroxybenzoic acid was used as the matrix (the last analysis was performed at the Institute of Physical Chemistry and Electrochemistry of RAS, Moscow). A Bruker Avance-400 spectrometer (Bruker Co.) was used to obtain 1H, 13C, HSQC and HMBC spectra with TMS as internal standard.

A portion of the crude ethanol extract (5 g) was suspended in water (1:10, w/v) and centrifuged. The pellet was then suspended in 50% ethanol (1:2, w/v) and centrifuged again. Both supernatants were combined, loaded onto an Amberlite XAD4 column (2.5 × 10 cm), and eluted with distilled water followed by 80% isopropyl alcohol (iso-PrOH). The water-soluble fraction was partitioned on an XAD7 resin (1.6 × 32 cm) column by stepwise elution with water, 80% isoPrOH and pure methanol to yield three fractions (A–C). The B fraction (1.8 g) was separated on a LiChroprep RP-C18 column (40–63 μm, Merck, Germany) with an iso-PrOH:H2O (0:10 → 8:2) gradient; six fractions (B1–6) were obtained. Fraction B5 (100 mg) was dissolved in MeCN:H2O (1:9) containing 0.05% (v/v) TFA to obtain a 10-mg/ml loading solution, which was further purified using a Waters HPLC system equipped with a semi-preparative Spherisorb ODS2 C18 column (10 μm, 80 Å, 10 × 250 mm, flow rate 3 ml/min, 25 °C) and a PDA detector. Approximately 25 mg of fraction B5 was purified during each run by applying MeCN:H2O (1:9 → 8:2) supplemented with 0.05% (v/v) TFA as an eluent. After four chromatographic runs, the fractions from 28 to 30 (tR 28–30 min) were combined and subjected to re-chromatography on the same column to yield compound 1 (1.2 mg). 2.5. Extraction and isolation of DPPH-scavenging substances The crude ethanol BR extract (6 g) was suspended in water (1:20, w/v) with vigorous stirring in an ultrasound bath. Phytochemicals were extracted in a stepwise procedure using n-hexane (C6H14), chloroform (CHCl3), and ethyl acetate (EtOAc) (1:20, w/v) with intense shaking in a separating funnel (Table 1). The aqueous and organic phases were separated, evaporated, and freeze-dried, and the insoluble matter was removed. A portion (160 mg) of the EtOAc fraction (860 mg) was subjected to LiChroprep RP-C18 column chromatography with elution by a MeCN:H2O (1:9 → 8:2) gradient system in 0.03% (v/v) TFA. Four fractions (GI–IV) were resolved. Fractions GII–IV (130.8 mg) were separated using a Waters HPLC system equipped with a semipreparative Spherisorb ODS2 C18 column (10 μm, 80 Å, 10 × 250 mm, flow rate 3 ml/min, 25 °C) and a PDA detector. Approximately 20 mg of each fraction (4 mg/ml loading solution) was injected during each run. A linear gradient of MeCN:H2O (1:9 → 8:2) in 0.05% (v/v) TFA was used. Polishing was achieved on an analytical Delta-Pak C18 column (5 μm,

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Table 1 HPL inhibition and antioxidant capacity of the organic and aqueous fractions of an aqueous ethanol extract of B. crassifolia rhizomes.

Crude extract Hexane fraction Chloroform fraction Ethyl acetate fraction Water fraction a b

HPL inhibition, IC50 (μg/ml)

DPPH-scavenging, SC50 (μg/ml) a

3.4 ± 0.2 N10 3.4 ± 0.3 3.3 ± 0.2 3.6 ± 0.2

3.67 ± 0.12 ND b N 20 2.90 ± 0.08 6.11 ± 0.42

SC50 values represent the mean ± SD (n = 4). ND — not determined.

100 Å, 3.9 × 150 mm, flow rate 1 ml/min, 25 °C) by applying the same mobile phase. This procedure yielded compounds 1 (5.8 mg) and 2 (2.2 mg). 2.5.1. (+)-Catechin 3,5-di-O-gallate (1) Compound 1 was characterized as a pale-yellow amorphous powder; [α]20 D + 2.84º (c 0.5, MeOH); UVmax (80% EtOH, v/v): 282 (lg ε, 4.26); IR (KBr): 3319, 1684, 1604, 1530, 1445, 1313, 1192, 1136, 1025, 757 cm− 1; 1 H NMR and 13 C NMR, see Table 2; LC/MS ESI+: m/z 595.1046 [M + H]+; calc. for [C29H23O14]+: 595.1088. 2.5.2. (+)-Catechin 3-O-gallate (2) Compound 2 was characterized as a white amorphous powder; [α]20 D +44.2º (c 0.5, MeOH); UVmax (80% EtOH, v/v): Table 2 1 H (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectral data of the isolated compounds 1 and 2 and the reference compound (−)-catechin 3-Ogallate. (Chemical shifts (δ) are in ppm; coupling constants J (in parentheses) are in Hz.). Compound 1

(−)-Catechin 3-O-gallate a

Compound 2

Proton δH

Carbon

δC

Proton δH

δH

2 3 4a 4b 6 8 2′ 5′,6′ 2″,6″ 2‴,6‴

2 3 4 5 6 7 8 9 10 1′ 2′ 3′,4′

79.8 70.8 24.4 152.2 104.2 158.8 102.1 156.9 105.6 131.5 114.6 146.8 146.9 116.8 119.4

2 3 4a 4b 6 8 2′ 5′,6′ 2″,6″

5.05, d 5.37, q 2.65, dd 2.79, dd 5.93, d 5.95, d 6.83, s 6.71, m 6.95, s

5.18, d (5.2) 5.38, q (5.4) 2.66, dd 2.75, dd 6.26, d (2.4) 6.35, d (2.4) 6.82, s 6.72, m 6.94, s 7.15, s

5′ 6′ 3-O-galloyl 1″ 2″,6″ 3″,5″ 4″ C″OO 5-O-galloyl 1‴ 2‴,6‴ 3‴,5‴ 4‴ C‴OO a

Wako Co, Japan.

121.6 110.6 146.9 140.4 167.9 120.6 111.0 147.1 141.0 166.8

5.06, d 5.36, q 2.71, dd 2.81, dd 5.93, d 5.95, d 6.83, s 6.71, d 6.96, s

279 (lg ε, 4.30); IR (KBr): 3121, 1672, 1606, 1518, 1473, 1434, 1400, 1244, 1143, 1119, 1064, 1036, 996, 946, 819, 766, 710 cm− 1; 1H NMR and 13C NMR, see Table 2; LC/MS ESI+: m/z 443.03 [M + H]+; calc. for [C22H19O10]+: 443.10. 2.6. Lipase-inhibition assay The activity of HPL (Sigma-Aldrich Corp., L9780) was evaluated using 4-MUO as a substrate. The sample solution (25 μl) was dissolved in water, and 50 μl of the 0.1 mM 4-MUO solution was dissolved in a buffer consisting of 66 mM Tris–HCl (pH 7.4), 7 mM NaCl, 3 mM CaCl2, and 2 mM sodium taurodeoxycholate. These solutions were mixed in a 96-well microtiter plate. Next, 25 μl of the lipase solution (0.24 μg, 0.12 U), containing 0.12 μg of colipase in 66 mM Tris–HCl (pH 7.4), was added to initiate the enzymatic reaction. After incubation at 37 °C for 60 min, the amount of 4-methylumbelliferone released by lipase was measured on a fluorometrical microplate reader (Genios FL, Tecan) at λex 360 nm and λem 460 nm. The halfmaximal inhibitory concentration (IC50) values were calculated from a dose–response curve as the dependence of lipaseinhibition efficacy (%) on the concentration of the inhibitor. 2.7. DPPH radical-scavenging assay The DPPH assay was performed as described in a previous publication [27]. Approximately 10 mg of BR extract or 1 mg of an individual compound was dissolved with vigorous stirring in an ultrasound bath in appropriate amount of 80% EtOH to obtain a 10 mg/ml stock solution. Dilutions were obtained by adding 80% EtOH to the stock solution. A 2.5 mM DPPH free-radical stock solution was prepared in 80% EtOH using sonication. Gallic acid, (+)-catechin, and 2,6-di-tertbutyl-4-methylphenol (BHT) were used as positive controls. The reaction mixtures contained Tris–HCl buffer (10 mM, pH 7.4), sample (0.1–10 μg/ml), and DPPH (0.1 mM) at a final volume of 200 μl (adjusted with 80% EtOH as needed). The reaction proceeded for 30 min at 25 °C on a 96-well microplate. The absorbance was then read at 520 nm, and mean values were obtained from duplicate readings. The scavenging ratio (in %) was determined from the following equation: [1 − (Asample − Ablank sample) / (Acontrol − Ablank control)] × 100, where Ablank sample and Ablank control represented corresponding mixtures without DPPH. Antioxidative activity was expressed as the half-scavenging concentration (SC50) value, that is, the concentration of a sample required to scavenge 50% of DPPH free radicals. SC50 values were determined from the dose– response curves. 2.8. Hydrolysis of (+)-catechin gallates (+)-Catechin 3,5-di-O-gallate (1) and (+)-catechin 3-Ogallate (2) (95 and 70 μg, respectively; 0.16 μmol) were dissolved in H2O (0.5 ml) and incubated with tannase (10 μg) at 40 °C for 1 h. The reaction mixture was dried, and the residue was suspended in MeOH. To identify gallic acid and (+)-catechin, the methanol-soluble portion was subjected to chromatography on an Inertis ODS3 column (5 μm, 4.6 × 250 mm) using MeCN:H2O (15:85, v/v) in 0.05% (v/v) TFA as the mobile phase. The spectral data (UV and CD) were

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compared directly with those obtained from standard samples (0.08 μmol of (+)-catechin and (−)-catechin). 3. Results and discussion 3.1. In vitro effects of Bergenia rhizome extract The crude aqueous ethanol extract of BR inhibited HPL (IC50 3.4 μg/ml) and scavenged DPPH free radicals (SC50 3.7 μg/ml) in a dose-dependent manner in vitro (Table 1). Rough fractionation of the BR extract was achieved with water and organic solvents in a stepwise manner; producing water-, hexane-, chloroform- and ethyl acetate-soluble fractions. HPL inhibition and DPPH-scavenging activity were evaluated (Table 1). The ethyl acetate fraction was the most potent DPPH scavenger, whereas neither the water nor the organic fraction showed enhanced HPL inhibition. Further investigation using bioassay-guided fractionation was performed to isolate the constituents responsible for the observed effects of the BR. 3.2. Isolation and structural elucidation of the HPL-inhibitory compound The HPL inhibitor was isolated directly from the BR ethanol extract through several steps of low-pressure and high-pressure RP chromatography. HPLC-UV/PDA profiles of fractions B4 and B5 taken during the final semi-preparative chromatographic step (see Materials and methods) displayed varying chemical compositions (Fig. 1). On the basis of a single band near 270–280 nm in the UV spectra [28], we attributed several peaks to compounds with non-conjugated aromatic rings, which are peculiar to plant phenolics, such as derivatives of catechin. Compounds corresponding to peaks A and C and several peaks in a broad region of the gradient (denoted as B on Fig. 1A), as well as the poorly resolved region B and peak C on Fig. 1B, revealed noticeable HPL inhibition (IC50 of 0.5–0.8 μg/ml). The mass analysis of substances from peak A revealed a molecular ion [M+H]+ of m/z 595. Structural analysis indicated that peak A corresponded to compound 1, and this compound was designated as (+)-catechin 3,5-di-O-gallate (Fig. 2). Compounds from peak C yielded several molecular ions that we attributed to polymers of irregular monomeric units, most likely protonated oligomeric proanthocyanidins [29,30]. The fractionation of proanthocyanidins is complicated [29] and was beyond the scope of this study. We hypothesize that substances from the broad hump-like region B belong to the same class of oligomeric phenolics due to the similarity of their UV spectra (data not shown). Compound 1 was a pale-yellow amorphous powder. Mass-spectroscopic analysis (LC TOF/MS ESI+) revealed a molecular ion of m/z 595.1046 [M+H]+, consistent with the molecular formula C29H23O14 (calc. for [C29H23O14]+: m/z 595.1088). The 400-MHz 1 H NMR spectrum showed a singlet signal of double intensity at δ 6.94 ppm that was similar to the same signal from (−)-catechin 3-O-gallate (δ 6.96), indicating the presence of a 3-O-galloyl residue in compound 1 (Table 2). An identical signal at δ 7.15 ppm with an up-field shift of the H-6 (δ 6.26) and H-8 (δ 6.35) aromatic protons denoted the presence of a second galloyl residue at C-5 [31]. A

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characteristic interaction of H-6/H-8 protons with a coupling constant of J = 2.4 Hz corresponded to a flavan-3-ol moiety with hydroxyl groups attached to C-5 and C-7 of ring A (Fig. 2) [31]. Strong interaction of H-2/H-3 protons (J = 5.2–5.4 Hz) was consistent with trans orientation of the substituents at the C-2/C-3 linkage [32]. A doublet signal from H-2 (δ 5.18) also suggested an a–a or e–e orientation (that is, trans location of the H-2/H-3 protons) [31,33]. These data suggest that compound 1 has a (+/−)-catechin-like core structure. To define the absolute configuration of compound 1, this compound was treated with tannase followed by LC/CD analysis using (+)-catechin and (−)-catechin as reference compounds [34]. The core structure of compound 1 was identified as (+)-catechin. Detailed analysis of the HMBC spectrum (Fig. 2A) revealed a correlation between H-3 (δ 5.38) and the adjacent galloyl-CO (δ 167.9). Proton H-2′ (δ 6.82) interacted with the proximal carbon C-6′ (δ 119.4) of the same ring C and C-2 (δ 79.8) and with distal carbons C-3″/C-5″ (δ 146.9) of the neighboring galloyl group on the C-3 of ring B. High-distant cross peaks of low intensity were observed between H-5′/H-6′ (δ 6.72) and C-3‴/C-5‴ (δ 147.1) of the remote galloyl group on C-5 of ring A. On the basis of the mass-spectroscopic, NMR and CD data, compound 1 was identified as (+)-catechin 3,5-di-O-gallate. To our knowledge, this is a novel plant phenolic compound. 3.3. Isolation and structural elucidation of antioxidant compounds The ethyl acetate fraction with the most pronounced antioxidant activity (Table 1) was subjected to guided repetitive fractionation using reversed-phase HPLC, resulting in the isolation of two compounds (1 and 2). Compound 1 was identical to (+)-catechin 3,5-di-O-gallate. The mass spectrum (LC/MS ESI+) of compound 2 showed a molecularion peak of m/z 443.03 [M+H]+. The 1H NMR spectrum and optical rotation of compound 2 were compared to those of the reference compound (−)-catechin 3-O-gallate (Table 2). Compound 2 was identified as (+)-catechin 3-O-gallate (Fig. 2B). 3.4. HPL-inhibitory activity of the isolated compounds Compound 1 showed strong HPL-inhibitory activity (IC50 0.42 μg/ml) (Table 3). This compound was more active than (+)-catechin 3-O-gallate (2) (IC50 2.0 μg/ml) or (−)-catechin 3-O-gallate and (−)-epicatechin 3-O-gallate (IC50 2.4 and 3.2 μg/ml, respectively). According to Nakai et al. [35], the IC50 values of these compounds are 0.54 μM (0.24 μg/ml) for (−)catechin 3-O-gallate, 0.45 μM (0.20 μg/ml) for (−)-epicatechin 3-O-gallate, and 0.1 μM (0.06 μg/ml) for (−)-epigallocatechin 3,5-di-O-gallate for the inhibition of lipase from porcine pancreas (type VI-S, Sigma) (1.25 U, ≤0.06 μg per assay). These data, in combination with our findings, indicate that the specific activity of mammalian lipase inhibitors varies with the origin of the enzyme. 3.5. Antioxidant properties of the isolated compounds Both isolated compounds, (+)-catechin 3,5-di-O-gallate (1) and (+)-catechin 3-O-gallate (2), possessed strong

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Fig. 1. Semi-preparative HPLC of fractions B5 (A) and B4 (B). Peaks with noticeable HPL-inhibitory activity (A, B, C) are shown. UV spectra and MS data for peaks A and C of the gradient profile of fraction B5 are shown (A).

Fig. 2. Key HMBC correlations of compound 1 (A) and structures of phenolic compounds 1 and 2 from B. crassifolia (B).

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217

Table 3 Lipase inhibitory activity of catechins.

IC50, μg/ml a b

b

(+)-Catechin 3,5-di-O-gallate (1)

(+)-Catechin 3-O-gallate (2)

(−)-Catechin 3-O-gallate

0.42 ± 0.03

2.02 ± 0.11

2.45 ± 0.23

Compound

SC50 (μg/ml)

1, (+)-Catechin 3,5-di-O-gallate 2, (+)-Catechin 3-O-gallate Gallic acid (+)-Catechin hydrate BHT

1.04 ± 0.06 1.33 ± 0.05 0.59 ± 0.06 1.97 ± 0.23 5.90 ± 0.74

b

(−)-Epicatechin 3-O-gallate

a

3.22 ± 0.25

Wako Co, Japan. IC50 values represent the mean ± SD (n = 4).

Table 4 Antioxidant activity of compounds 1 and 2 from B. crassifolia rhizome and reference substances.

a

a

a

SC50 (μM)

a,b

1.75 ± 0.10 3.01 ± 0.11 3.47 ± 0.35 6.79 ± 0.79 26.8 ± 3.4

SC50 values represent the mean ± SD (n = 6). The values were calculated for anhydrous compounds.

antioxidant properties that were comparable to those of known antioxidants, such as gallic acid, (+)-catechin, and BHT [36,37] (Table 4). Antioxidant activity increased (i.e., SC50 decreased) from compound 2 (1.33 μg/ml) to compound 1 (1.04 μg/ml) as the number of galloyl residues in the molecule increased, consistent with published data [36] and our experimental data indicating that gallic acid is a stronger antioxidant (SC50 0.59 μg/ml) than (+)-catechin hydrate (SC50 1.97 μg/ml) (Table 4). A novel phytochemical, (+)-catechin 3,5-di-O-gallate (1), and a previously identified chemical, (+)-catechin 3-Ogallate (2) [23], were isolated from an aqueous ethanol extract of B. crassifolia. Compound 1 had strong lipase inhibitory activity in vitro that was comparable to that of oolong-tea catechins [35]. In addition, compound 1 and compound 2 were strong antioxidants in vitro; their antioxidant activity was comparable to that of established natural antioxidants, such as gallic acid and (+)-catechin. Although the crude aqueous ethanol extract of BR displayed strong anti-lipase and antioxidant activity, the isolated compounds may not be exclusively responsible for the medicinal properties of B. crassifolia; a vast variety of phytochemicals may also contribute to the overall activity. In future studies, it will be interesting to determine whether (+)-catechin 3,5-di-O-gallate (1) and (+)-catechin 3-O-gallate (2) can reduce postprandial triglyceride levels and free-radicalinduced lipoperoxidation in mammals. Acknowledgements The authors thank Dr. K. Ishii and Dr. T. Miwa for stimulating discussions. References [1] Foster-Schubert KE, Cummings DE. Emerging therapeutic strategies for obesity. Endocr Rev 2006;27:779–93. [2] Shi Y, Burn P. Lipid metabolic enzymes: emerging drug targets for the treatment of obesity. Nat Rev Drug Discov 2004;3:695–710. [3] Strader CD, Hwa JJ, Van Heek M, Parker EM. Novel molecular targets for the treatment of obesity. Drug Discov Today 1998;3:250–6.

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