Protein tyrosine phosphatase 1B inhibitory by dammaranes from Vietnamese Giao-Co-Lam tea

Journal of Ethnopharmacology 124 (2009) 240–245

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Protein tyrosine phosphatase 1B inhibitory by dammaranes from Vietnamese Giao-Co-Lam tea Tran Manh Hung a,b, Duc Manh Hoang b, Jin Cheol Kim c, Han-Su Jang d, Jong Seog Ahn b, Byung-Sun Min a,∗ a

College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea d Gyeongbuk Institute of Bio Industry, Gyeongbuk 760-380, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 1 December 2008 Received in revised form 1 April 2009 Accepted 17 April 2009 Available online 3 May 2009 Keywords: Gynostemma pentaphyllum Cucurbitaceae Dammarane Triterpene Protein tyrosine phosphatase 1B activity

a b s t r a c t Ethnopharmacological relevance: Gynostemma pentaphyllum (Thunb.) tea was used in Vietnamese folk medicine as anti-diabetic agent. Aim of the study: This study was aimed to investigate the inhibitory activities of fractions and constituents isolated from Gynostemma pentaphyllum on protein tyrosine phosphatase 1B (PTP1B) since it has been proposed as a treatment therapy for type 2 diabetes and obesity. Materials and methods: The 70% EtOH extract, CHCl3 fraction, EtOAc fraction, BuOH fraction, and seven isolated dammarane triterpenes were evaluated for their inhibitory activity in protein phosphatase enzymes (PTP1B and VHR). Results: CHCl3 -soluble fraction showed a dose-dependent inhibitory activity of the PTP1B enzyme with the IC50 value of 30.5 ␮g/mL. Among seven tested compounds, compounds 6 showed the most potent PTP1B inhibitory activity with IC50 value of 5.3 ± 0.4 ␮M compared to a range 15.7–28.5 ␮M for the other six compounds. The inhibition mode of 6 was competitive toward p-NPP with a Ki value of 2.8 ␮M. Conclusion: These study results suggested that the PTP1B inhibitory activity of these dammaranes may enable this plant to play an important role in the treatment of diabetes. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Diabetes mellitus is major diseases that has been becoming more common due to the ageing population, a lifestyle and diet that promotes obesity, a growing Hispanic community that has a particularly prevalence of diabetes. Many studies showed that insulin resistance is one of the characteristic pathogenic signs of type 2 diabetes, and several drugs that increase the insulin sensitivity are currently in clinical use (King et al., 1998). However, these drugs have a number of limitations, including adverse effects and high rates of secondary failure (Moller, 2001). Recently, protein tyrosine phosphatase 1B (PTP1B) has been shown to be a negative regulator of the insulin-signaling pathway, suggesting that inhibitors of this enzyme may be a promising therapeutic target in effective treatment of type 2 diabetes (Kennedy, 1999). PTP1B is a major nontransmembrane phosphotyrosine phosphatase in human tissues and was one of the earliest PTP identified. PTP1B inhibitors would increase insulin sensitivity by blocking the PTP1B-mediated nega-

∗ Corresponding author. Tel.: +82 53 8503613; fax: +82 53 8503602. E-mail address: [email protected] (B.-S. Min). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.04.027

tive insulin-signaling pathway and might be an attractive target in type 2 diabetes mellitus and obesity (Kennedy, 1999). During the screening effort, we found that the CHCl3 -soluble extract of aerial parts of Gynostemma pentaphyllum (Thunb.) Makino inhibited PTP1B activity (Hung et al., 2007). The genus Gynostemma belongs to the family Cucurbitaceae and the plants in this genus are known in mainland China as “xiancao”, or the “herb of immortality” (Lin et al., 1993). They have been used in folk medicine to lower cholesterol levels, regulate blood pressure, strengthen the immune system, and reduce inflammation (Li et al., 1993). Previous investigations of this genus shown that the dammarane-type glycosides called the gypenosides are structurally related to ginseng saponins (Cui et al., 1999). Of the 14 Gynostemma species currently used in traditional Chinese medicine, Gynostemma pentaphyllum has been widely investigated for its phytochemical constituents with various important biological activities (Megalli et al., 2005). In Vietnam, Gynostemma pentaphyllum is a traditional tea due to its sweetness, efficacy in the treatment of elevated cholesterol, anti-tumor, anti-oxidant, anti-diabetes, and hypoglycemic effects (Loi, 2001), as reported in a number of studies (Norberg et al., 2004; Hoa et al., 2007). However, the effect exerted by its components on PTP1B has not been studied. In the present work, we

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241

Fig. 1. A representative HPLC profile of isolated compounds (1–7) from CHCl3 fraction, detected at UV 205 nm.

therefore report on the isolation and PTP1B inhibitory activity of seven dammarane-type triterpenes. 2. Experimental 2.1. General procedures and reagents Optical rotations were measured with a JASCO DIP 1000 digital polarimeter. UV spectra were recorded on a JASCO V-530 spectrophotometer, and CD spectra were recorded on a JASCO J-715 CD/ORD spectropolarimeter. IR spectra were obtained on a JASCO FT/IR 300-E spectrometer. NMR experiments were conducted on a Varian Unity INOVA 400 spectrometer. 1 H and 13 C NMR spectra were recorded at 400 and 100 MHz, respectively, and tetramethylsilane was used as the internal standard. ESIMS and HRESIMS analyses were performed on a Micromass QTQF2 mass spectrometer. EIMS spectra were obtained on a JEOL JMS-SX102A spectrometer. TLC was carried out on Merck silica gel F254 precoated glass plates and RP-18 F254S plates. HPLC was performed on a Waters 600E multisolvent delivery system connected to a UV detector using Supelco Supelcosil LC-SI (5 ␮m, 10 mm × 250 mm) and Isco Allsphere ODS-2 (10 ␮m, 10 mm × 250 mm) semipreparative columns.

60:1 to 5:1), then EtOAc–MeOH (from 20:1 to 1:1), to yield five fractions (H1–H5) according to their TLC profiles. The PTP1B inhibitory activity was found to be concentrated in the H2 fraction (IC50 = 21.6 ␮g/mL, 0.8 g), which was chromatographed over silica gel column using a gradient of hexane–EtOAc (from 20:1 to 0:1), to yield three subfractions H2.1–H2.3. The H2.2 fraction was further purified by semipreparative HPLC [RS Tech Optima Pak C18 column (10 mm × 250 mm, 10 ␮m particle size); mobile phase ACN–H2 O (63:37); flow rate 2 mL/min; UV detection at 205 nm] resulted in the isolation of compound 1 (12.3 mg, tR = 34.9 min), compound 2 (6.1 mg, tR = 36.5 min), compound 3 (5.0 mg, tR = 47.3 min), compound 4 (7.6 mg, tR = 53.1 min), compound 5 (5.6 mg, tR = 58.0 min), compound 6 (4.8 mg, tR = 61.3 min), and compound 7 (14.1 mg, tR = 62.8 min) (Fig. 1). (20S)-3␤,20,23␰-Trihydroxydammarane-24-en-21-oic acid-21, ◦ 23 lactone (6): white powder, mp: 156–158 ◦ C; [␣]25 D + 11.5 (c 0.10, MeOH), IR (KBr) max : 3500, 3348, 2980, 2938, 2864, 1665 cm−1 ; ESIMS m/z 473 [M+H]+ ; HRESIMS m/z 473.3621 [M+H]+ (calcd. for C30 H48 O4 ); for 1 H and 13 C NMR spectral data, see Table 1. (20R)-3␤,20,23␰-Trihydroxydammarane-24-en-21-oic acid-21, ◦ 23 lactone (7): white powder, mp: 160–162 ◦ C; [␣]25 D − 18.5 (c 0.10, MeOH), IR (KBr) max : 3500, 2985, 2835, 2875 cm−1 , 1660; ESIMS m/z 473 [M+H]+ ; HRESIMS m/z 473.3643 [M+H]+ (calcd for C30 H48 O4 ); for 1 H and 13 C NMR spectral data, see Table 1.

2.2. Plant material 2.4. Protein tyrosine phosphatase 1B inhibitory activity The whole plant of Gynostemma pentaphyllum were collected from Hoa Binh province, north of Vietnam, in July 2006 and identified by Professor Pham Thanh Ky, Department of Pharmacognosy, Hanoi College of Pharmacy. A voucher specimen (HN-0152) was deposited in the herbarium of the Hanoi College of Pharmacy. 2.3. Extraction and isolation The dried aerial parts (0.5 kg) were extracted with 2 L of 70% ethanol, three times. The 70% EtOH extract was combined and concentrated to yield a residue, which was suspended in water and then successively partitioned with CHCl3 , EtOAc, and BuOH to afford CHCl3 -, EtOAc-, and BuOH-soluble fractions. The CHCl3 fraction (IC50 = 37.5 ␮g/mL, 5.3 g) was separated by silica gel column chromatography using a gradient of hexane–EtOAc (from

Protein tyrosine phosphatase 1B (PTP1B, human, recombinant) was purchased from BIOMOL® International LP (USA) and the enzyme activity was measured using p-nitrophenyl phosphate (pNPP) as a substrate (Cui et al., 2006). To each 96-well (final volume: 200 ␮L) were added 2 mM p-NPP and PTP1B (0.05–0.1 ␮g) in a buffer containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol (DTT) with or without test compounds. Following incubation at 37 ◦ C for 30 min, the reaction was terminated with 10 M NaOH. The amount of produced p-nitrophenol was estimated by measuring the absorbance at 405 nm. The nonenzymatic hydrolysis of 2 mM p-NPP was corrected by measuring the increase in absorbance at 405 nm obtained in the absence of PTP1B enzyme. Ursolic acid, corosolic acid, and RK-682 were used as positive controls (Hamaguchi et al., 1995; Na et al., 2006).

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Table 1 1 H (400 MHz) and 13 C (100 MHz) NMR data (in CDCl3 ) of compounds 6 and 7. Position

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

6

7

ıH (mult, J in Hz)

ıC

ıH (mult, J in Hz)

ıC

1.76 (m), 1.07 (m) 2.01 (m), 1.52 (m) 3.20 (dd, 4.8, 11.2)

37.9 26.6 75.4 40.5 56.0 18.4 35.4 39.1 50.8 37.3 21.5 25.8 45.9 50.1 31.5 27.5 44.5 16.2 16.4 81.5 177.6 39.1

1.70 (m), 0.92 (m) 2.03 (m), 1.54 (m) 3.20 (dd, 5.2, 11.2)

39.2 26.6 79.1 40.6 56.0 18.4 35.4 39.1 50.9 37.3 21.5 25.3 42.4 50.7 31.5 27.6 45.8 16.6 16.4 78.4 179.0 40.8

1.15 (m) 2.18 (m), 2.05 (m) 1.68 (m), 1.28 (m) 1.68 (m) 1.65 (m), 1.42 (m) 2.10 (m), 2.03 (m) 2.03 (m) 1.60 (m), 1.12 (m) 1.90 (m), 1.14 (m) 1.40 (m) 0.77 (s) 0.88 (s)

2.30 (dd, 6.6, 13.2, Hb) 2.14 (ddd, 5.6, 13.2, Ha) 5.34 (ddd, 5.6, 8.4, 8.8) 5.19 (d, 8.8) 1.79 (s) 1.76 (s) 0.93 (s) 0.94(s) 0.97 (s)

2.5. VHR dual-specificity protein tyrosine phosphatase activity Dual-specificity PTPase was assayed with the GST–VHR fusion enzyme, over expressed in Escherichia coli (Sodeoka et al., 2001). The reaction mixture containing GST–VHR fusion enzyme, 10 mM pNPP and assay buffer (50 mM succinate, 1 mM EDTA, 140 mM NaCl, 0.05% Tween 20, pH 6.0) was incubated at 30 ◦ C for 1 h. The reaction was terminated by the addition of 1 N NaOH, and the dephosphorylation activity was measured at 410 nm (Zhou et al., 1994). 2.6. Kinetic analysis The reaction mixture consisted of different concentration of pNPP as a PTP1B substrate in the absence or presence of compound. The Michaelis constant (Km ) and maximum velocity (Vmax ) of PTP1B were determined by a Lineweaver–Burk plot. 2.7. Statistical analysis All the results were expressed as mean ± S.D. of three determinations at each concentration for each sample. The inhibitory concentration 50% (IC50 ) was calculated using Microsoft Excel program. Statistical significance was calculated by one-way analysis of variance (ANOVA), followed by Dunnett’s test. 3. Results and discussion 3.1. Isolation of active compounds The whole plant of Gynostemma pentaphyllum was air dried, ground to a powder and extracted with 70% EtOH at room temperature. The obtained alcoholic extract was partitioned into CHCl3 , EtOAc, BuOH and aqueous fractions. In the primary study, we tested the PTP1B inhibitory activity of the 70% EtOH extract and fractions

75.4 122.6 140.6 26.0 18.6 28.2 15.5 15.6

1.12 (m) 2.15 (m), 2.08 (m) 1.67 (m), 1.30 (m) 1.65 (m) 1.65 (m), 1.40 (m) 2.15 (m), 2.07 (m) 2.02 (m) 1.78 (m), 1.20 (m) 1.83 (m), 1.16 (m) 1.40 (m) 0.78 (s) 0.88 (s)

2.47 (dd, 6.4, 13.2, Hb) 2.04 (dd, 7.6, 13.2, Ha) 5.12 (dd, 8.4, 15.6) 5.27 (d, 8.8) 1.77 (s) 1.75 (s) 0.93 (s) 0.94(s) 0.97 (s)

74.3 123.4 140.2 25.3 18.6 28.2 15.6 15.5

(CHCl3 -, EtOAc-, BuOH-soluble). The results showed that the CHCl3 and EtOAc-soluble fractions inhibited PTP1B with IC50 values of 35.5 and 82.1 ␮g/mL, respectively (Table 1). As CHCl3 -soluble fraction demonstrated the greatest potency, it was selected for the isolation of active constituents. 3.2. Structure elucidation of isolated compounds Chromatographic purification of the CHCl3 -soluble fraction led to the isolation of seven compounds (1–7). The structures of isolated compounds were identified on the basis of spectroscopic analysis including 1 H, 13 C NMR data, electrospray ionization mass spectrometry (ESI-MS), and by comparing with those published in literature. It is evident that the chemical structures of some isolates are similar with aglycone moiety of some triterpene glycosides, which were previously isolated from Gynostemma pentaphyllum. Fig. 1 shows a representative HPLC profile during the isolation of those triterpenes from CHCl3 fraction. These compounds included (23S)-3␤,20␨,21␨-trihydroxy-19-oxo-21,23-epoxydammarane-24ene (1), (20S,24S)-3␤,12␤,20,24-epoxydammarane-25-triol (2), (20S,24S)-3␤,12␤,20,24-epoxy-12,25-dihydroxydammarane (3), (20S),3␤,21-trihydroxy-25-methoxydammarane-23-ene (4), and (20S)-3␤,12␤,12,25-dihydroxydammar-23-ene (5) (Razmovski-Naumovski et al., 2005) (Fig. 2). Compound 6 was obtained as amorphous powder. The molecular formula was assigned as C30 H48 O4 (from the positive mode HRESIMS quasi molecular ion at m/z 473.3621 [M+H]+ . The IR spectrum of 6 showed a hydroxyl group (max 3500 and 3348 cm−1 ). The 1 H NMR spectra of 6 exhibited seven tertiary methyl groups at ı 0.77–1.79, two of which were diagnostic for methyl linked to sp2 carbon atom (ı 1.76 and ı 1.79), nine methylenes including a methylene group of a lactone ring [ı 2.14 (1H, ddd, J = 2.3, 5.6, 13.2 Hz, H1 -22), 2.30 (dd, J = 6.6, 13.2 Hz, H2 -22)], and seven sp3 methines including one oxymethine at ı 3.20 (1H, dd, J = 4.8, 11.2 Hz, H-3).

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243

Fig. 2. Chemical structure of isolated compounds 1–7.

The signals at ı 5.19 (1H, d, J = 8.8 Hz, H-24) and ı 5.34 (1H, ddd, J = 5.6, 8.4, 8.8 Hz, H-23) were indicated for the occurrence of an olefinic proton and a –CHOH– proton. The 13 C and DEPT NMR spectra gave 30 signals that could be assigned to a triterpene bearing a dammarane skeleton with an unusual side chain. The chemical shifts at ı 75.4 (C-3), 56.5 (C-5), 50.8 (C-9), 45.9 (C-13), 44.5 (C-17), 75.4 (C-23), and 122.6 (C-24) were signaled for seven sp3 methine carbons. Further featured signals at ı 40.5 (C-4), 39.1 (C-8), 37.3 (C10), 50.1 (C-14) were assigned for four quarternary carbons, signal at ı 81.5 ppm was established for an oxygenated carbon (C-20), ı 177.6 ppm indicated the occurrence of a carboxylic carbon (C-21), and ı 140.0 ppm indicated for a tetrasubstituted carbon (C-25). The planer structure of 6 was determined by 2D NMR spectrum. The HMBC spectrum of 6 indicated a long-range correlation between H17 (ıH 0.88) and C-13, C-16, and C-20; Me-18 (ıH 0.77) and C-8, C-9, and C-14; between Me-19 (ıH 0.88) and C-1, C-9, and C-10; between Me-26 (ıH 1.79)/Me-27 (ıH 1.76) and C-24, C-25; between Me-28 (ıH 0.93)/Me-29 (ıH 0.94) and C-3, C-4 and C-5; and between Me-30 (ıH 0.97) and C-7, C-14, and C-15. In the 1 H–1 H COSY spectrum, H-22 (ıH 2.30, 2.14) correlated with H-23 (ıH 5.34) and H-23 correlated with H-24 (ıH 5.19), which could be assigned for spin-decoupling of –CH2 –CHOH–CH C sequence. The signals at ıC 81.5 (C-20), 177.6 (C-21), 39.1 (C-22), 75.4 (C-23), and 122.6 (C-24) with those of corresponding protons at ıH 2.14, 2.30, 5.34 and 5.19 suggested the side chain with a lactone ring bearing a carboxylic group. A 3␤hydroxy substituent was evident from both the chemical shifts and the J values of the proton ascribable to H-3␣ (3.20, 1H, dd, J = 4.8, 11.2 Hz). Compound 7 was also obtained as white powder. It showed a HRESIMS fragmentation pattern super-imposable on that of 6. The comparisons of 1 H, 13 C, and 2D NMR spectra of the two compounds indicated their structural similarity. The main differences were the chemical shifts of H-22 (ı 2.14 and 2.30 in 6 versus ı 2.04 and 2.47 in 7) in 1 H NMR spectrum. In the comparison with some dammarane compounds prompted us to hypothesize that the difference between the two should be confined to the stereochemistry at C-20. The 13 C NMR resonances of C-13 (ı 45.9 in 6 versus ı 42.4 in 7), C-17 (ı 44.5 in 6 versus ı 45.8 in 7), C-20 (ı 81.5 in 6 versus ı 78.4 in 7), C-21 (ı 177.6 in 6 versus ı 179.0 in 7), C-22 (ı 39.1 in 6 versus ı 40.8 in 7), C-23 (ı 75.4 in 6 versus ı 74.3 in 7), and C-24 (ı 122.6 in 6 versus ı 123.4 in 7) were in good agreement with those of published data for 20R epimer of 6 and 20S epimer of 7 (Piacente et al., 1995). On the basis of the

foregoing data, the structures of compounds 6 and 7 were proposed to be (20S)-3␤,20,23␰-trihydroxydammarane-24-en-21-oic acid-21,23 lactone and (20R)-3␤,20,23␰-trihydroxydammarane24-en-21-oic acid-21,23 lactone, respectively. By comparison of 1 H and 13 C NMR and other data of 6 and 7 with those of known dammarane-type triterpenes, it is evident that the chemical structures of 6 and 7 are similar with aglycone moiety of some triterpene glycosides, which were previously isolated from Gynostemma pentaphyllum (Piacente et al., 1995). However, this is the first time the former structures was isolated. 3.3. PTP inhibitory capacities PTP1B (human, recombinant) was purchased from BIOMOL® International LP (USA) and the enzyme activity was measured using p-nitrophenyl phosphate (p-NPP) as described previously (Cui et al., 2006). Seven isolated dammaranes were dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution of 5 mM, and appropriate dilutions were made before the enzyme assay (final DMSO concentration < 3%, the activity was not affected by this concentration). The PTP1B inhibitory activity of the isolates was tested in vitro and the results are presented in Table 2. All the isolated compounds inhibited PTP1B activity dose dependently. Compound 6 inhibited the hydrolysis of the p-NPP catalyzed by PTP1B with an IC50 values of 5.3 ± 0.5 ␮M, which was comparable to that of corosolic acid (IC50 = 7.5 ± 0.6 ␮M), ursolic acid (IC50 = 3.6 ± 0.2 ␮M) and RK-682 (3-hexadecanoyl-5hydroxymethyl tetronic acid, IC50 = 4.5 ± 0.3 ␮M) (Hamaguchi et al., 1995; Na et al., 2006). Compound 7 (IC50 = 15.7 ± 1.4 ␮M), which was identified as a 20S epimer of 6, had a lower activity than 6. Compounds 4 and 5, which possess a side chain as 20S,23-ene, displayed decreased inhibitory activity with IC50 values of 28.5 ± 1.8 and 25.1 ± 2.3 ␮M, respectively. Compounds 1–3, which possess epoxy groups, also displayed decreased inhibitory activity compared with compound 6 and those of controls. In addition, the isolated compounds were tested for their inhibitory effects on vaccinia H1-related phosphatase (VHR), another type of protein phosphatase. Compounds 1–7 showed no inhibitory effects toward a dual-specificity PTP with IC50 values of more than 100 ␮M. Therefore, we considered that compounds 1–7 have a certain degree of specific inhibitory effect on PTP1B. To investigate the inhibition mode of compounds 6, which showed the most potent PTP1B inhibitory activity, kinetic analysis was conducted at

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Fig. 3. Inhibition kinetic of compound 6. Lineweaver–Burk plot of inhibitory effect of compound 6 on PTP1B-catalyzed hydrolysis of p-NPP with the concentration of 0 ␮M (䊉), 1.0 ␮M (), 3.0 ␮M (), and 5.0 ␮M ().

various concentrations of 6 and p-NPP. As shown in Fig. 3, compound 6 increased the Km value, but did not alter the Vmax value of PTP1B, suggesting that the inhibition mode of 6 was competitive toward p-NPP with a Ki value of 2.8 ␮M. PTP1B is known to have several binding sites, such as those electrostatic, hydrophobic, and hydrogen-bond binding, and to have several N-terminals that are capable of binding to an acidic site. As with the insulin-signaling pathway, the leptin-signaling pathway can be attenuated by PTPs and compelling evidences implies the involvement of PTP1B in this process (Kennedy, 1999; Moller, 2001). Therefore, is has been suggested that compounds that reduce PTP1B activity or expression levels could be used for treating both type 2 diabetes and obesity. Of the triterpenes, the compounds with ursan-type have been reported to possess a wide range of

Table 2 Inhibitory activities of fractions and isolated compounds. Extract/compounds

b

70% EtOH extract CHCl3 exb EtOAc exb BuOH ex 1 2 3 4 5 6 7 Corosolic acidc Ursolic acidc RK-682c

IC50 (␮M)a PTP1B

VHR

>100 30.5 ± 3.8 82.1 ± 6.7 >100 19.3 ± 1.4 20.4 ± 1.6 21.7 ± 2.3 25.1 ± 2.3 28.5 ± 1.8 5.3 ± 0.5 15.7 ± 1.4 7.5 ± 0.6 3.6 ± 0.2 4.5 ± 0.3

ND ND ND ND >100 >100 >100 >100 >100 >100 >100 >100 >100 10.2 ± 0.3

ND: not determined. a IC50 values were determined by regression analyses and expressed as means ± S.D. of three replicates. b ␮g/mL. c Positive controls.

pharmacological activities including anti-HIV, anti-inflammatory, hepatoprotective, anti-ulcer, antimicrobial, anti-tumor, antiangiogenic, immunomodulatory and antiproliferative activities. Recent reports demonstrated that a hydroxyl group at C-3 and a carbonyl group at C-28 or C-27 of the ursan-type triterpenes is an essential structure related to the PTP1B inhibitory activity (Na et al., 2006). In addition, the pentacyclic triterpenoids with acetoxyl group at the C-3 position can exhibit PTP1B activity (Kwon et al., 2008). In our results, although the structure–activity relationships of these compounds were not thoroughly investigated, due to the presence of a ␤-hydroxyl group at the C-3 position of both of the isolated compounds, the difference in the side chain from the C-20 to C-25 positions was responsible for the change of activity. In the case of compounds 6 and 7, even though they are epimers of each other, their difference of stereochemistry at C-20 might have affected the inhibitory activity of PTP1B. Na et al. suggested that the substitution and the number of hydroxy groups on the ursan-type can be potential PTP1B inhibitors (Na et al., 2006). In agreement with this suggestion, compounds 6 and 7 possess two hydroxy groups that showed potential inhibitory activity greater than that of other compounds with three (1, 2, and 4) or four (3 and 5) hydroxy groups. This result was attributed to the increased polarity of the compounds induced by the presence of hydroxyl groups, which may have decreased the affinity for a hydrophobic site of the enzyme. The extracts of Gynostemma pentaphyllum and/or isolated gypenosides have been reported to exert a variety of effects, such as the inhibition of inflammation (Lin et al., 1993), and lipidlowering effects (la Cour et al., 1995). The supplementation with a high dose of Gynostemma pentaphyllum ethanol extract has an antihyperglycemia effect in the type 2 diabetic animal model, by enhancing insulin secretion and its sensitivity and hepatic glucose utilization (Jang et al., 2001; Yeo et al., 2008). In previous investigations of Gynostemma pentaphyllum, Norberg et al. (2004) demonstrated that ethanol extract and an isolated dammaranetype saponin, phanoside, strongly stimulated insulin secretion from isolated rat pancreatic islets. Phanoside stimulated insulin secretion from Wand GK rat islets through the K-ATP channels and

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L-type Ca2C channels, which is on the exocytotic machinery of the B-cells, but the effect of phanoside is not glucose-dependent (Hoa et al., 2007). In those published experiments, it is possible to note that the saponin fraction and/or isolated individually compound may play important role for these biological actions. However, in the present study, the CHCl3 -soluble fraction potentially inhibited PTP1B enzyme with IC50 value higher 2 and 3-folder than EtOAc and BuOH-soluble fractions. The containing negligible amount of sugar compounds in CHCl3 -soluble fraction may manifest inhibitory PTP1B activity, which was confirmed by the inhibitory effect of those of isolated dammarane-type triterpenes. The EtOAc and BuOH-soluble fractions, which content saponin compounds, exhibited weak inhibitory activity. Thus, the different of aglycone and their glycoside may play a key role in the PTP1B inhibitory activity. To the best of our knowledge, we have here reported the PTP1B inhibitory activity of dammarane-type triterpenes is now being reported for the first time. The study results suggest that Gynostemma pentaphyllum can be used in folk medicine to lower cholesterol levels, regulate blood pressure, and exert anti-diabetes, and that the dammarane-type triterpenes and other constituents may enable this plant be useful for the treatment of diabetes. Acknowledgement This research was supported by Korea Research Foundation Grant (KRF-2007-331-E00331). References Cui, J., Eneroth, P., Bruhn, J.G., 1999. Gynostemma pentaphyllum: identification of major sapogenins and differentiation from Panax species. European Journal of Pharmaceutical Sciences 8, 187–191. Cui, L., Na, M., Oh, H., Bae, E.Y., Jeong, D.G., Ryu, S.E., Kim, S., Kim, B.Y., Oh, W.K., Ahn, J.S., 2006. Protein tyrosine phosphatase 1B inhibitors from Morus root bark. Bioorganic Medicinal Chemistry Letter 16, 1426–1429. Hamaguchi, T., Sudo, T., Osada, H., 1995. RK-682, a potent inhibitor of tyrosine phosphatase, arrested the mammalian cell cycle progression at G1phase. FEBS Letter 372, 54–58. Hoa, N.K., Norberg, A., Sillard, R., Van Phan, D., Thuan, N.D., Dzung, D.T., Jörnvall, H., Ostenson, C.G., 2007. The possible mechanisms by which phanoside stimulates insulin secretion from rat islets. Journal of Endocrinology 192, 389–394.

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