Biological properties of Hertia cheirifolia L. flower extracts and effect of the nopol on α-glucosidase

Accepted Manuscript Title: Biological properties of Hertia cheirifolia L. flower extracts and effect of the nopol on ␣-glucosidase Author: Kaouther Majouli Mohamed Ali Mahjoub Fazal Rahim Assia Hamdi Abdul Wadood Malek Besbes Hlila Abderraouf Kenani PII: DOI: Reference:

S0141-8130(16)31389-7 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.12.008 BIOMAC 6822

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

27-8-2016 29-11-2016 3-12-2016

Please cite this article as: Kaouther Majouli, Mohamed Ali Mahjoub, Fazal Rahim, Assia Hamdi, Abdul Wadood, Malek Besbes Hlila, Abderraouf Kenani, Biological properties of Hertia cheirifolia L.flower extracts and effect of the nopol on ␣-glucosidase, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.12.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Biological properties of Hertia cheirifolia L. flower extracts and effect of the nopol on α-glucosidase

Kaouther Majoulia,*, Mohamed Ali Mahjoubb, Fazal Rahimc, Assia Hamdid, Abdul Wadoode, Malek Besbes Hlilaf, Abderraouf Kenania

Laboratory of Biochemistry, Research Unit: UR 12ES08 “Cell Signaling and Pathologies”. Faculty of

a

Medicine, University of Monastir, Tunisia b

Laboratory of Genome Diagnostics and Valorisation, ISBM, University of Monastir, Tunisia

c

Department of Chemistry, Hazara University, Mansehra-21120, Khyber Pukhtunkhwa, Pakistan

d

Laboratory of chemical, galenic and pharmacological development of drugs, Faculty of Pharmacy,

University of Monastir, Tunisia e

Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali

Khan University Mardan, Pakistan f

Laboratory of Transmissible Diseases and of Biologically Active Substances, MDT01, Faculty of

Pharmacy, University of Monastir, Tunisia

*

Corresponding author: E-Mail: [email protected]

1

Abstract In screening for antioxidant and α-glucosidase inhibitors from the extracts of Hertia cheirifolia L. flowers, the petroleum ether extract showed interesting antioxidant activity and inhibitory effect on the activity of α-glucosidase. The fractionation of this extract resulted in the isolation of a compound which is characterized by NMR and ESI-MS as a nopol. The nopol exhibited potent α-glucosidase inhibitory potential with IC50 value of 220 μM. The kinetic evaluation indicated that it acts as a non-competitive inhibitor. A molecular docking study proved that the nopol presented a strong affinity with amino acid residues of αglucosidase.

Keywords: Antioxidant activity; α-Glucosidase inhibition; Molecular docking.

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1. Introduction Oxidative stress is implicated as one of the main factors responsible for the induction of type 2 diabetes mellitus [1]. Diabetes mellitus is the most serious chronic metabolic disorder and is characterized by high blood glucose levels. Antioxidants are compounds that can slow down the process of oxidation of a compound. Since antioxidants can inhibit the formation of free radicals in its early stages, or interfere with the propagation reaction of free radical chain reactions. Thus antioxidants are widely used in the food industry as potential inhibitors of lipid peroxidation [2]. However, it has been reported that synthetic antioxidants can be accumulated in the human organism and possibly cause cancer or other potential damages. Accordingly, using natural antioxidants extracted from plants can reduce these adverse health effects [3]. One therapeutic approach for diabetes is to delay absorption of glucose through inhibition of carbohydrate-hydrolyzing enzymes, e.g., α-glucosidase in the digestive organs. There are reports of established α-glucosidase inhibitors from plants and its effects on blood glucose levels after food uptake [4]. These αglucosidase inhibitors exhibit high promise as therapeutic agents for the treatment of type 2 diabetes mellitus and hyperglycemia [5]. Acarbose is the most widely-used α- glucosidase inhibitor, but it also has gastrointestinal side effects [6]. Medicinal plants, rich with their secondary metabolites, offer a reservoir of preventive and therapeutic options. Through the efforts of ongoing scientific researches, increasing number of phytochemicals have been tested and developed into effective modern drugs [7,8]. A benefit of plant-derived bioactive compounds is the minimal side effect, compared to those of synthetic drugs [9,10]. In the present work, we investigated the antioxidant and α-glucosidase inhibitory activities of the Hertia cheirifolia L. flower extracts. As a part of the search for the bioactive constituents of H. cheirifolia we further recently reported the isolation and identification of nopol. 3

2. Materials and methods 2.1. General reagents Petroleum ether (PE), ethyl acetate (EtOAc), butanol (n-BuOH), methanol (MeOH), 2,2diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS), β-carotene, linoleic acid, butylated hydroxytoluene (BHT), hydrochloric acid (HCl), Sodium hydroxide (NaOH), Folin-Ciocalteu, sodium carbonate (Na2CO3), aluminum chloride (AlCl3),

α-glucosidase

(isolated

from

Aspergillus

niger),

4-p-nitrophenyl-α-D-

glucopyranoside (4-pNPG) were purchased from Sigma-Aldrich. Silica gel 60 F254 (thin layer chromatography plates), silica gel (60-120 Mesh) were purchased from Merck (Darmstadt, Germany). 2.2. Extraction and Isolation The flowers of H. cheirifolia were air-dried at room temperature for 15 days and reduced to coarse powder. The powdered plant material was extracted by maceration in the methanol and water (MeOH/H2O 80:20 (v/v)) for 72 hours at room temperature. The hydro-methanolic extraction was carried out in triplicate. After filtration, the obtained extract was further subjected to a successive extraction using petroleum ether (PE), ethyl acetate (EtOAc) and butanol (BuOH) to yield dried fractions. The extracts were maintained at 4 °C before analysis. The active PE extract (8 g) was fractionated on a chromatographic column (column length 110 cm, diameter 6 cm) over silica gel (300 g) eluted with a step gradient of petroleum ether in EtOAc. Eleven fractions were collected from this PE extract and then they were combined according to their similarity on the Thin Layer Chromatography (TLC) to give four subfractions. The repeated chromatographic column separations of the third subfraction (145 mg) yielded the nopol (18 mg) in pure form. 2.3. Bioassay-guided fractionation of petroleum ether extract

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According to the results of the inhibitory activity of α-glucosidase, the PE extract (8 g) was successively permeated through a Sephadex LH-20 column previously equilibrated with PE:EtOAc (2:1). Eleven fractions of 50 ml each were obtained. After TLC comparison using PE:EtOAc, as mobile phase, fractions with similar TLC patterns were combined together and tested in inhibitory activity of α-glucosidase (Table 4). The third subfraction that revealed the highest activity was applied to a silica column chromatography (column length 70 cm, diameter 5 cm, containing 250 g silica gel, PE:EtOAc, 8:2) in order to give the nopol. 2.4. General experimental procedures 1

H and 13C nuclear magnetic resonance (NMR) spectra were obtained with Bruker WP 300

spectrometer at 300 MHz for 1H NMR and 75 MHz for 13C NMR. Measurements were made in deuterated chloroform (CDCl3) at 27°C The resonance of residual solvent were used as internal references. Thus, the electrospray ionisation mass spectrometry (ESI-MS) was used. 2.5. Screening of antioxidant activity The in vitro antioxidant activity of the extracts of H. cheirifolia was evaluated by the following experiments: the radical scavenging effects of 2,2-diphenyl-1-picrylhydrazyl (DPPH) [11], of 2,2-azino-bis-3-ethylenebenzothiozoline-6-sulfonic acid (ABTS.+) [12] and the inhibition of superoxide radical [13]. 2.6. Colorimetric quantification of antioxidants 2.6.1. Determination of total phenolic content (TPC) The total phenolic amount was determined by using Folin-Ciocalteu reagent [14]. A total of 100 µL of extract was transferred into a test tube and 0.75 mL of Folin-Ciocalteu reagent (previously diluted 10-fold with deionized water) was added and mixed. The mixture was kept to stand at a temperature of 25 °C for 5 min. 0.75 mL of saturated sodium carbonate solution was added to the mixture and then mixed gently. After 25 °C for 90 min, the absorbance was recorded at 765 nm with an UV−vis spectrometer. The amount of total

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phenolics in the extract was calculated and expressed in milligrams of gallic acid equivalents (GAE) per gram of sample on a dry weight basis (mg GAE/g DW). 2.6.2. Determination of total flavonoid content (TFC) The content of total flavonoids was determined using the aluminium chloride (AlCl3) [15]. A volume of 1.5 mL (1 mg/mL) of extract was added to an equal volume of a 2% AlCl3-6H2O solution. The mixture was vigorously shaken, and the absorbance was recorded at 367 nm after 10 min of incubation with an UV−vis spectrometer. The TFC was expressed in milligrams of quercetin per gram of sample on a dry weight basis (mg QE/g DW). 2.7. α-Glucosidase inhibition assay The α-glucosidase inhibition assay was performed according to the method of Tao et al. (2013) with some modifications. The α-glucosidase reaction mixture, contained 2.5 mM 4-pnitrophenyl-α-D-glucopyranoside (4-pNPG), 250 μL of extract (varying concentrations) in dimethyl sulfoxide (DMSO) and 0.3 U/mL α-glucosidase in phosphate buffer pH 6.9, was incubated in a water bath at 37 °C for 15 min. Control tubes contained only DMSO, enzyme and substrate (4-pNPG), while in positive controls Acarbose replaced the plant extracts. Absorbance of the resulting p-nitrophenol (pNP) was determined at 405 nm and was considered directly proportional to the activity of the enzyme. Each sample was performed in triplicate. Percentage inhibition by extracts and acarbose (I%) were calculated using the following equation: (I%) = (1− (ΔDO sample/ΔDO control)) × 100. The IC50, which is the concentration of the sample required to inhibit 50% of the enzyme and that was determined for each sample. 2.8. Kinetics study of α-glucosidase inhibitor Lineweaver-Burk plot analysis was performed to determine the inhibition mode of the nopol, and kinetic studies of inhibitory activity against α-glucosidase were measured using

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increasing concentrations of 4-pNPG as a substrate in the presence of stable concentration of the nopol. The reaction mixture at 37 °C for 15 min performed the enzyme reaction. 2.9. Molecular docking To understand the binding interactions of the nopol in the active site of α-glucosidase, molecular docking was performed using MOE-Dock program. The crystal structure of αglucosidase is not available yet, so, we developed a homology model using the same protocol as described by Taha et al. (2015). The energy of the modelled protein molecule was minimized after the 3D protonation using the default parameters of MOE energy minimization algorithm (gradient: 0.05, Force Field: Amber 99). The three dimensional coordinates of the nopol was constructed using MOE. Then, the molecule was energy minimized using the default parameters of MOE energy minimization algorithm (gradient: 0.05, Force Field: Amber99). The minimized molecule was saved in the mdb file format as input file. The binding pocket of the enzyme was selected by the site finder, implemented in MOE software. The compound was docked into the binding pocket of α-glucosidase enzyme using default parameters of MOE-Dock. 2.10. Statistical analysis The results were given as the average ± SE for at least three replicates for each sample. The data were subjected to one-way ANOVA, and Duncan’s multiple range tests was used to compare means. Statistical analyses were performed with the SPSS statistical software program (SPSS v.16). p values <0.05 were regarded as significant. 3. Results and discussion 3.1. Antioxidant Activity As the bioactivities of medicinal plant extracts were often linked to their phytochemical contents, we determined the TPC and the TFC of H. cheirifolia extracts. The highest TPC were detected in the EtOAc extract (75.69 ± 8.00 mg GAE/g dry matter). The lowest amounts

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of polyphenols were detected in the BuOH (11.18 ± 2.00 mg GAE/g dry matter) and the PE extracts (2.84 ± 0.50 mg GAE/g dry matter). Similar trend was observed in the TFC, with the highest levels detected in the EtOAc extract (65.33 ± 4.00 mg QE/g dry matter) (Table 1). Because of the complex nature of phytochemicals, the antioxidant activities of plant extracts cannot be evaluated using a single method [18]. Indeed, many available methods are used to evaluate plant extracts for their antioxidant activity. Therefore, three complementary test methods (DPPH, ABTS and β-carotene/linoleic acid) were used to evaluate the antioxidant capacities of the extracts of H. cheirifolia flowers. As shown in Table 2, statistical analysis showed that DPPH radical scavenging method has been proven to be the most important with significant IC50 values (p<0.05). The EtOAc is the best solvent for extracting DPPH free radical scavenging constituents from H. cheirifolia. Besides the EtOAc extract inhibited the free radicals with the lowest concentration (IC 50= 0.041 ± 0.02 mg/mL). The important activity indicated by this extract can be associated with its high TPC. Amongst the tested samples on the ABTS radical scavenging activity, PE extract showed an outstanding scavenging activity with an IC50 value of 0.390 ± 0.01 mg/mL. While EtOAc and BuOH extracts showed a moderate activity (IC50 = 0.460 ± 0.02 mg/mL and 0.570 ± 0.03 mg/mL, respectively) compared with BHT (synthetic antioxidant used as reference). The effect of the BuOH extract (IC50 = 0.190 ± 0.02 mg/mL) in the inhibition of the βcarotene bleaching assay was stronger than those of EtOAc and PE extracts (p<0.05). In fact, β-carotene/linoleic acid method is a commonly used model to analyze the antioxidant activity of the plant extracts because β-carotene is extremely sensitive to free radical mediated oxidation of linoleic acid [19].

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The recent experiments have uncovered new insights into the role of oxidative stress in diabetic complications and these reports have demonstrated innovative approaches to employ natural, antioxidant therapies [20]. Various plant species have been evaluated for antioxidant activities which have as well shown to exhibit antidiabetic activity [21]. This is the first antioxidant evaluation using H. cheirifolia flowers because previous antioxidant studies of this plant were focused on vegetative part. 3.2. Analysis of inhibitor 3.2.1. α-Glucosidase inhibition assay The screening of the α-glucosidase inhibitory activity showed that the PE fraction was more potent than the reference product (Acarbose) and exhibited the highest inhibitory activity with a significant value (p<0.05) of IC50 = 0.242 ± 0.02 mg/mL (Table 3). A bioassay guided fractionation of this fraction via Sephadex LH-20 gave four subfractions, which the third subfraction revealed the highest activity (Table 4). The fractionation of this subtraction afforded the nopol in pure form. Nopol is an optically active bicyclic primary alcohol used in the soaps’ factories and the agrochemical industries to produce pesticides, detergents, polishes and other household products [22]. It had a hypotensive effects and caused vasodilation [23]. Our results showed that this compound has a significant inhibitory activity against αglucosidase enzyme. Furthermore, EtOAc extract with higher phenolic contents had significantly lower αglucosidase inhibitory effect compared to PT extract with lower phenolic contents. Thus the inhibitory effect of an extract cannot be explained only on the basis of its phenolic content but, also requires its proper characterization of the other compounds. The TPC did not include

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all the possible inhibitors [24]. These results demonstrated the inhibitory potency of H. cheirifolia extracts is not limited to the phenolic content. 3.2.2. Mode of α-glucosidase inhibition by the nopol The Lineweaver-Burk plots indicated that the nopol inhibited α-glucosidase in a noncompetitive mode (Fig. 1). Indeed, the addition of this compound to the reaction medium led to a change in the maximum velocity (Vmax) of α-glucosidase while keeping the same value of Michaelis-Menten constant (Km). At the same concentration of the nopol, the Vmax decreased from 0.25 ΔDO/min in the absence of any inhibitor to 0.058 ΔDO/min in the presence of the nopol, while Km remained constant with value of 1.73 mM. According to this type of inhibition, we can suggest that the nopol binds to a site other than the active site of the enzyme, without competing with the substrate, to retard the substrate conversion. 3.3. Identification of the nopol Nopol, oil (Fig. 2): ESI-MS, m/z 167 [M+H]+. (C11H18O+H); 189 [M+Na]+ (C11H18O+Na); 149 [M+H- H2O]+. 1H NMR (300 MHz, CDCl3 (7.27 ppm)), δ = 5.34 (m, 1H, H-3); 3.60 (t, 2H, H-11); 2.39 (ddd, 1H, H-7b); 2.25 (m, 2H, H-10); 2.21 (m, 2H, H-4); 2.15 (m, 1H, H-5); 2.05 (dd, 1H, H-1); 1.27 (s, 3H, H-9); 1.15 (d, J= 6.1, 1H, H-7a); 0.84 (s, 3H, H-8). 13C NMR (75 MHz, CDCl3 (77.00 ppm)), δ = 144.6 (C-2); 119.2 (C-3); 59.9 (C-11); 45.5 (C-1); 40.6 (C-5); 40.1 (C-10); 37.8 (C-6); 31.7 (C-7); 31.3 (C-4); 26.1 (C-9); 21.1 (C-8). 3.4. Molecular docking The molecular docking was performed to find the binding interaction between αglucosidase enzyme and the nopol compound. The docking result showed that the compound was accommodated very well in the enzyme. From the docking conformation of the compound, it was observed that this compound formed two polar interactions with the Asp 68 and Arg 439. The compound has biological activity with the IC50 of 220 μM and the corresponding docking score of -7.3023. The hydroxyl moiety of the compound was observed

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making hydrogen bonds with the Asp 68 and Arg 439 active residues of the binding pocket of the enzyme as shown in the Fig. 3. The compound has shown good interactions, it might be due to the presence of the chemically active hydroxyl group. 4. Conclusion The enzyme has numerous specific sites for various glucose residues. However, this study revealed H. cheirifolia extract as a significant source of α-glucosidase inhibitors and as effective agent to control the hyperglycemia. We have isolated a potent inhibitor from this important plant. The nopol which was characterized on the basis of NMR and ESI-MS, manifested non-competitive against α-glucosidase. To develop a pharmacophore model, we explored the binding mode of the nopol with α-glucosidase based on its important interaction with amino acids in the enzyme. Declaration of interest The authors declare that there are no conflicts of interest. Acknowledgements We are very grateful to Dr. Jean Michel Brunel (Laboratory of Synthesis and study of Natural Substances of biological activity). References [1] N. Houstis, E.D. Rosen, E.S. Lander, Nature 440 (2006) 944–948. [2] R. Scherer, H.T. Godoy, Food Chem. 112 (2009) 654–658. [3] J. Deng, W. Cheng, G. Yang, Food Chem. 125 (2011) 1430–1435. [4] S. Kumar, S. Narwal, O. Prakash, Pharmacogn. Rev. 9 (2011) 19–29 [5] A.D. Baron, Diabetes Res. Clin. Pr. 40 (1998) S51–S55. [6] R.F. Coniff, J.A. Shapiro, T.B. Seaton, G.A. Bray, Am. J. Med. 98 (1995) 443–451. [7] P.J. Facchini, V. De Luca, Plant J. 54 (2008) 763–784. [8] A. Bhanot, R. Sharma, M.N. Noolvi, Int. J. Phytomed. 3 (2011) 09–26.

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[9] E. Mannucci, N. Monami, G. Masotti, N. Marchionni, Diabetes Metab. Res. Rev. 20 (2004) 40–47. [10] H. Ping, G. Zhang, G. Ren, Food Chem. Toxicol. 48 (2010) 2344–2349. [11] T. Hatano, H. Kagawa, T. Yasuhara, T. Okuda, Chem. Pharm. Bull. 36 (1988) 2090– 2097. [12] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, J. Free Radic. Biol. Med. 26 (1999) 1231–1237. [13] E.H.K. Ikram, K.H. Eng, A.M.M. Jalil, A. Ismail, S. Idris, A. Azlan, J. Food Comp. Anal. 22 (2009) 388–393. [14] Y.S. Velioglu, G. Mazza, L. Gao, B.D. Oomah, Agric. Food Chem. 46 (1998) 4113– 4117. [15] I. Kosalec, M. Bakmaz, S. Pepeljnjak, E.I.C.S. Vladimir-Knez, Acta Pharm. 54 (2004) 65-72. [16] Y. Tao, Y. Zhang, Y. Cheng, Y. Wang, Biomed. Chromatogr. 27 (2013) 148–155. [17] M. Taha, N.H. Ismail, S. Imran, A. Wadood, F. Rahim, M. Alie, A. Ur Rehman, Med. Chem. Commun. 6 (2015) 1826. [18] E. Gioti, Y. Fiamegos, D. Skalkos, C. Stalikas, Food Chem. 117 (2009) 398–404. [19] I.I. Koleva, T.A. Van Beek, J.P.H. Linssen, A. de Groot, L.N. Evstatieva, Phytochem. Anal. 13 (2002) 8–17. [20] L. Bartosikova, J. Necas, V. Suchy, I.R. Kubinova, D. Vesela, L. Benes, Acta Vet. Brno. 72 (2003) 87–94. [21] G.B. Kavishankar, N. Lakshmidevi, S. Mahadeva Murthy, Int. J. Pharm. Biomed. Sci. 2 (2011) 65–80. [22] S.V. Jadhav, K.M. Jinka, H.C. Bajaj, Catal. Today 198 (2012) 98–105. [23] B. J. Northover, J. Verghese, J. scient. ind. Res. 21 (1962) 342.

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[24] I. Khacheba, A. Djeridane, A. Kameli, M. Yousf, Curr. Enzym. Inhib. 10 (2014) 58–67.

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Figure captions

Fig.1. Lineweaver-Burk plots of α-glucosidase inhibition at different 4-pNPG concentrations in absence (♦) or presence (■) of the inhibitor: the nopol. Fig. 2. Structure of the nopol. Fig. 3. The 3D binding mode and molecular interactions of the nopol with the Asp 68 and Arg 439 of α- glucosidase enzyme.

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Table 1 Total phenolic and flavonoid contents of H. cheirifolia flower extracts Extracts

Phenols (mg GAE/g)

Flavonoids (mg QE/g)

PE extract

2.84 ± 0.50a

1.06 ± 0.30a

EtOAc extract

75.69 ± 8.00b

65.33 ± 4.00b

n-BuOH extract

11.18 ± 2.00a

6.04 ± 1.00a

Values were expressed as mean ± SE (n= 3). The different letters indicate a significant difference between the extracts (p < 0.05).

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Table 2 Antioxidant activity of H. cheirifolia extracts by DPPH, ABTS and β-carotene/linoleic acid tests IC50 (mg/mL)

IC50 (mg/mL)

IC50 (mg/mL) of β-

of DPPH

of ABTS

carotene/linoleic acid

PE extract

0.270 ± 0.02d

0.390 ± 0.01b

0.229 ± 0.01c

EtOAc extract

0.041 ± 0.02b

0.460 ± 0.02c

0.300 ± 0.01d

n-BuOH extract

0.210 ± 0.01c

0.570 ± 0.02d

0.190 ± 0.02b

BHT

0.018 ± 0.001a

0.050 ± 0.001a

0.040 ± 0.001a

Extracts

Values were expressed as mean ± SE (n= 3). The different letters indicate a significant difference between the extracts (p < 0.05). The IC50 (mg/mL) value, which is the sample concentration providing 50% inhibition, was determined by plotting the inhibition percentage versus extract concentrations. BHT: butylated hydroxytoluene.

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Table 3 α-Glucosidase inhibition by H. cheirifolia flower extracts Extracts

IC50 (mg/mL)

PE extract

0.242 ± 0.02a

EtOAc extract

0.437 ± 0.03d

n-BuOH extract

0.421 ± 0.01c

Acarbose

0.280 ± 0.01b

Values were expressed as mean ± SE (n= 3). The different letters indicate a significant difference between the extracts (p < 0.05).

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Table 4 α-Glucosidase inhibition by H. cheirifolia PE subfractions Subfractions

IC50 (mg/mL)

Subfraction 1

0.296 ± 0.03c

Subfraction 2

0.319 ± 0.05e

Subfraction 3

0.235 ± 0.01a

Subfraction 4

0.309 ± 0.02d

Acarbose

0.280 ± 0.01b

Values were expressed as mean ± SE (n= 3). The different letters indicate a significant difference between the extracts (p < 0.05).

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1/Vi (∆DO/min)

Fig. 1. 120 100

Without inhibitor

80

With inhibitor

60 40 20 0

-1

-20

0

1

2

3

4

1/[4-pNPG] mM

19

Fig. 2. 8 9 H

6

5

4

7

1 H

3

2 10

11 HO

20

Fig. 3.

21