Assessment of the DPPH and Α-Glucosidase Inhibitory Potential Of

Full Length Research Paper

Assessment of the DPPH and -glucosidase inhibitory potential of gambier and qualitative identification of major bioactive compound

F. B. Apea-Bah1*, M. Hanafi2, R. T. Dewi2, S. Fajriah2, A. Darwaman2, N. Artanti2, P. Lotulung2, P. Ngadymang2 and B. Minarti2

1Biotechonology and Nuclear Agriculture Research Institute, Ghana Atomic Energy Commission, P. O. Box LG 80, Legon, Ghana. 2Research Centre for Chemistry, Indonesian Institute of Sciences, Kawasan Puspiptek, Serpong, Tangerang, Indonesia.

Accepted 3 July, 2009

Gambir (Uncaria gambir) and other plants belonging to the genus Uncaria have been used in traditional medicine in southeastern Asia, Africa and South America and they have been studied widely over the past century. Gambier, the dried leaf extract from gambir is known to have antioxidant properties and some studies have attributed it to the presence of tannins and condensed tannins. The objective of this study was to investigate the potential of commercial gambier on the Indonesian market as a scavenger of reactive free radicals, evaluate its ability to inhibit -glucosidase and determine the bioactive compound responsible for these activities. An ethanolic extract of commercial gambier was extracted with ethyl acetate. The ethanol and ethyl acetate extracts as well as the aqueous extract after ethyl acetate extraction and residue from ethanol extraction were tested for free radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH). They were also tested for -glucosidase inhibitory activity. The extracts were then studied using reverse phase HPLC, LCMS and NMR to identify the bioactive compound. It was observed that all the extracts had high activity for DPPH inhibition but moderate activity for inhibiting -glucosidase in vitro. Apart from the aqueous extract, 92% DPPH inhibition by the extracts was achievable at 30 g/ml. The ethanol and ethyl acetate extracts had significantly higher (p < 0.01) DPPH inhibitory activity than the aqueous extract. IC50 of the organic extracts and residue ranged between 13.8 to 16.2 g/ml for DPPH inhibition while that of the aqueous extract was 27.4 g/ml. With regards to -glucosidase inhibition, however, IC50 range of 15.2 and 49.5 g/ml was recorded. Catechin was identified as the major bioactive compound present.

Key words: Uncaria gambir, gambier, DPPH, alpha-glucosidase, HPLC, LCMS, NMR

INTRODUCTION

Gambir (Uncaria gambir) is a vine belonging to the family processed and used for medicinal purposes (Ahmed et Rubiaceae and genus Uncaria (Heitzman et al., 2005). al., 1978; Das and Griffiths, 1967; Phillipson et al., 1978). Species of the genus Uncaria have many tradi-tional The aqueous extracts of leaves and young twigs of U. medicinal uses including treatments for wounds and gambir are used traditionally for the treatment of diarrhea ulcers, fevers, headaches, gastrointestinal illnesses and and dysentery. They may also be used as gargle for bacterial and fungal infections (Aquino et al., 1991; Cerri treatment of sore throat (Taniguchi et al., 2007). Dried et al., 1988; Chang et al., 1989; Lemaire et al., 1999; hooks of some Uncaria species have also been integral Rizzi et al., 1993; Wurm et al., 1998). In Malaysia, components in traditional oriental medicines, where they Singapore, Borneo and Sumatra where U. gambir is com- are used as spasmolytics, analgesics and sedatives for mon or indigenous, aqueous extracts of the leaves are symptoms associated with nervous disorders and in the treatment of hypertension (Heitzman et al., 2005). Gambier, the dried leaf extract from U. gambir is believed to have antioxidant properties which are attri- *Corresponding author. E-mail: [email protected]. buted to the presence of tannins and condensed tannins

Apea-Bah et al. 737

(Desmarchelier et al., 1997; Wirth and Wagner, 1997). phosphate buffer pH 7.0, analytical balance (Mettler Toledo AT400), Gambir was formerly cultivated extensively in Malaysia rotary evaporator (Buchi Rotavapor R-124), column chromatograph, and Indonesia as a commercial source of tanning mate- thin layer chromatograph plates, forced convection oven (Memmert 854 Schwabach, Germany), UV-Visible spectrophotometer (Hitachi rials (Das and Griffiths, 1967) and catechin is the most U-2000, Japan), HPLC (Shimadzu, Japan), liquid chromatograph- abundant polyphenolic constituent in the plant (Taniguchi mass spectrometer (LC: Hitachi L 6200; Mariner Biospectrometry, et al., 2007; Das and Griffiths, 1967). Other polyphenols Electrospray Ionisation System), micropipettes, glassware, water extracted and identified in gambir include gambiriins bath, stopwatch and other supplies. which are chalcane-flavan dimers, (+)-epicatechin and dimeric proanthocyanidins (Taniguchi et al., 2007). The Sample preparation presence of catechin in green tea and fermented tea has been associated with health protective and cancer pre- A dried leaf extract of gambir was purchased from a local market in ventive properties in animal models due to their antioxi- Serpong, a surburb of Tangerang on the Java Island of Indonesia. dant activity (Sang et al., 2002). Of particular importance 10 g of the gambier (dried leaf extract of gambir) was dispersed in 100ml of 95% aqueous ethanol and stirred. It was then filtered in cancer prevention is the ability of catechin to scavenge through Whatmann No.1 filter paper into a conical flask. The resi- reactive oxygen species that play a role in carcinogenesis due from the ethanol filtration was dried for further analysis while a (Sang et al., 2002). portion of the ethanol filtrate was evaporated on a rotary evaporator Diabetes mellitus is a serious chronic metabolic disor- (50°C, 90 -100 rpm) to obtain the ethanol extract. The other portion der characterized by high blood glucose levels (Corry and of the ethanol filtrate was further extracted with ethyl acetate, Tuck, 2000). Worldwide, the number of patients is rapidly resulting in ethereal and aqueous layers which were separated using a separating funnel. The ethereal and aqueous layers were growing with increasing obesity (King et al., 1998) and concentrated using rotary evaporator (45°C, 90 - 100 rpm for ways to control postprandial hyperglycemia involve medi- ethereal layer and 53°C, 90 – 100 rpm for aqueous fraction) to cation with dietary restriction and body exercise (Goke obtain the ethyl acetate extract and the aqueous extract (after ethyl and Herrmann-Rinke, 1998). Among the several thera- acetate extraction). The samples were dried in an oven at peutic approaches is retarding glucose absorption using temperature of about 45°C overnight (24 h). inhibitors of carbohydrate-hydrolyzing enzymes such as -amylase and -glucosidase (Holman et al., 1999; Saito DPPH radical scavenging activity et al., 1998; Toeller, 1994). Although Acarbose (Schmidit et al., 1977), miglitol (Standl et al., 1999), voglibose The DPPH radical scavenging activity was determined using the (Saito et al., 1998) and nojirimycin (Hansawasdic et al., method of Hatano et al. (1988). 4 mg of each sample was weighed 2000) are known to be potent inhibitors of -glucosidase, and added to 4 ml of methanol. 4 concentrations, 10, 50, 100 and further screening for -glucosidase inhibitors from natural 200 g/ml, were prepared from each sample solution using methanol as solvent and 500 l DPPH was added giving a total sources such as plants and microorganisms will help in volume of 2.5 ml per sample concentration. Standards were also reducing overdependence on synthetic drugs. Pine bark prepared at similar concentrations using standard catechin while extract which is rich in polyphenols such as catechin, the blank was prepared using methanol and DPPH without any quercetin, dihyhydroquercetin, taxifolin and phenolic sample. They were then incubated in a water bath at 37°C for 30 acids (Markham and Porter, 1973; Packer et al., 1999), is min after which absorbance of samples and standards were read reportedly effective in suppressing postprandial hyper- against the blank at 517 nm. The percent inhibition was estimated as % inhibition = [(C - S)/C] x 100%, where S is the sample absor- glycemia in diabetics (Kim et al., 2005). Even though bance and C is absorbance of the blank. Scatter diagrams were gambir is also rich in polyphenols, it is not used plotted and linear regression estimated using the equation y = ax+b, traditionally in treating diabetes mellitus. There is the where y is % inhibition and x is concentration (g/ml). IC50 was need to study its potential as an inhibitor of -glucosidase. calculated as the concentration that caused 50% inhibition of DPPH. In the present study, we investigated the free radical scavenging and -glucosidase inhibitory potential of -Glucosidase inhibitory activity commercial gambier sold in a local Indonesian market in Tangerang. The major compound responsible for the The -glucosidase inhibitory activity was determined using the bioactivity was qualitatively identified using reversed- method described by Sutedja (2005). The enzyme solution was phase HPLC, LCMS and NMR. prepared by dissolving 0.5 mg -glucosidase in 10 ml phosphate buffer (pH 7.0) containing 20 mg bovine serum albumin. It was diluted further to 1:10 with phosphate buffer just before use. Sam- MATERIALS AND METHODS ple solutions were prepared by dissolving 4 mg sample extract in 400 l DMSO. 3 concentrations; 6.25, 12.5 and 25 g/ml were Materials prepared and 5 l each of the sample solutions or DMSO (sample blank) was then added to 250 l of 20 mM p-nitrophenyl--D- Distilled water, methanol, deuterated methanol (CD3OD), ethanol, glucopyranoside and 495 l of 100 mM phosphate buffer (pH 7.0). It ethyl acetate, hexane, chloroform, sulphuric acid in methanol, 2,2- was pre-incubated at 37°C for 5 min and the reaction started by diphenyl-1-picrylhydrazyl, catechin standard (Sigma-Aldrich Chemi- addition of 250 l of the enzyme solution, after which it was cal Co.), -glucosidase (Sigma-Aldrich Chemical Co.), p- incubated at 37°C for exactly 15 min. 250 l of phosphate buffer nitrophenyl--D-glucopyranoside (Sigma-Aldrich Chemical Co.), was added instead of enzyme for blank. The reaction was then Na2CO3 (E. Merck), Na2HPO4.2H2O (E.Merck), KH2PO4 (E.Merck), stopped by addition of 1000 l of 200 mM Na2CO3 solution and the bovine serum albumin (E.Merck), dimethyl sulphoxide (E. Merck), amount of p-nitrophenol released was measured by reading the

738 J. Med. Plant. Res.

absorbance of sample against sample blank (containing DMSO with 120 no sample) at 400 nm using UV-visible spectrophotometer. Stan- dard solutions of same concentrations were also prepared using ‘koji’, an extract from Aspergillus terreus. Scatter diagrams were 100 plotted and linear regression estimated. IC50 was calculated as the concentration that caused 50% inhibition of enzyme activity. 80

o i

t 60 HPLC analysis i b i h n

The samples were analyzed using reverse phase HPLC to I 40 y = 21.41Ln(x) - 9.2035

ascertain presence of catechin. Wavelength for maximum absorp- % 2 tion of catechin was determined using the UV-visible spectropho- R = 0.8046 20 tometer. The standards and samples were then analyzed using HPLC at 210 nm. Water and methanol (30:70) was used as the eluent and was set to a flow rate of 1 ml/min and elution time of 20 0 min. 0 50 100 150 200 250 Concentration (ug/ml)

Identification and structure elucidation of active component Figure 1. Percent inhibition of DPPH against catechin A sample of the ethyl acetate extract was purified using column concentration. chromatography with hexane, ethyl acetate and methanol in different proportions as eluent. The purified extract and other samples were analyzed using liquid chromatograph-mass spectrometer 120 (LC: Hitachi L 6200; Mariner Biospectrometry; Electrospray Ionisation System) to determine the molecular weights (m/z values) 100 of the major compounds. 20 l of the samples were injected and run at a flow rate of 1 ml/min using acetonitrile: water (70:30) as n 80 o i eluent and C18 column (Supelco: 150 mm x 2 mm x 5 m). Nuclear t i b magnetic resonance spectroscopy (JEOL) with radio frequency of i 60 h

500 MHz was then used to elucidate the structure of the active n

I y = 21.614Ln(x) - 11.089 compound in the purified sample which was prepared using 40 2 % R = 0.7863 deuterated methanol (CD3OD) as solvent. One and two dimensional NMR spectra were developed and analyzed. 20

0 Data analysis 0 50 100 150 200 250

Two way analysis of variance (Montgomery, 1991) was used to Concentration (ug/ml) determine the effects of concentration and type of extraction on the DPPH and -glucosidase inhibitory activities. Where significant differences existed (p < 0.05), least significant difference (LSD) was Figure 2. Percent inhibition of DPPH against concentration of used for mean comparison. Statgraphics centurion XV (Statgra- ethanol extract phics, 2005) statistical software was used for all statistical analysis.

RESULTS AND DISCUSSION 120

DPPH Radical scavenging activity 100

Figures 1 to 4 show the linear regression curves of 80

concentration of catechin standard and gambier extracts n o i plotted against percent inhibition of DPPH. The regres- t 2 i 60 b sion coefficient (R ) of the logarithmic trend line obtained i h n

for the extracts and standard was between 0.79 - 0.80, I 40 implying that 80% of the variability in DPPH inhibition can % y = 26.941Ln(x) - 35.809

be attributed to the concentration of the extracts. Analysis 2 R = 0.7981 of variance (ANOVA) showed concentration to signifi- 20 cantly (p < 0.01) affect percent inhibition. From the mean comparison using least significant difference, it was 0 observed that inhibition of DPPH was significantly low at 0 50 100 150 200 250 10 g/ml concentration (Table 1) compared to all the Concentration (ug/ml) other concentrations. The extracts from gambier were not significantly different (p > 0.05) from the catechin stan- Figure 3. Percent inhibition of DPPH against concentration dard with regards to DPPH inhibition. The IC50 for the of Residue after Ethanol extraction.

Apea-Bah et al. 739

Table 1. Concentration of Catechin and extracts from Gambier and their IC50 for DPPH inhibition.

% Inhibition Concentration Catechin Ethyl acetate Ethanol Residue from *Mean (g/ml) standard extract extract ethanol extraction 200 92.41 91.39 90.67 91.61 91.52a 100 92.26 92.33 91.75 92.19 92.13a 50 92.48 92.62 92.48 92.48 92.51a 10 31.14 21.59 29.11 14.72 24.14b

IC50 (g/ml) 15.88 20.70 16.88 24.17

*Means in a column with same superscript are not significantly different from each other (p<0.01)

120 to 10) from the logarithmic trend line. The IC50 of the ethyl acetate extract was least and closest to that of catechin 100 standard while that of the aqueous extract was highest 80 (Table 2) indicating that the active compound is very

n soluble both in ethanol and ethyl acetate. o i t i From the ANOVA, both sample type or extract and b i 60

h concentration significantly affected (p < 0.01) percent n I y = 24.495Ln(x) - 24.218 DPPH inhibition. Concentrations of 30 to 50 g/ml were

% 40

2 not significantly different from each other but resulted in

R =0.794

significantly higher DPPH inhibition than 5 to 20 g/ml 20 (Table 2). The lower concentrations of 5, 10 and 20 g/ml were all significantly different from each other. This 0 means therefore that for optimum free radical scavenging 0 50 100 150 200 250 using organic extracts of commercial gambier, a Concentration (ug/ml) concentration of 30 g/ml is adequate.

With regards to the samples, however, ethanol and Figure 4. Percent inhibition of DPPH against ethyl acetate extracts were statistically not different (P < concentration of ethyl acetate extract. 0.01) from the catechin standard. They were also not significantly different from the residue from ethanol extraction. They all, however, showed significantly higher potential for DPPH inhibition than the aqueous extract catechin standard was 15.9 g/ml while it was in the (Table 2). This suggests that the major bioactive range of 16.9 to 24.2 g/ml for the extracts (Table 1). compound has lower solubility in water than in ethanol Since IC50 was lowest for ethanol extract and highest for and ethyl acetate. residue, it implies that the ethanol extract had the highest free radical scavenging activity at concentrations between 10 and 200 g/ml, while that of the residue was least. This suggests that the compounds present in -Glucosidase inhibitory activity gambier that are respon-sible for scavenging DPPH free radicals are very soluble in ethanol. It is worth noting from Figures 11 to 16 show the linear regression curves of the linear regression curves (Figures 1 to 4) that concentration of koji extract and gambier extracts plotted against percent inhibition of -glucosidase. The regres- increasing sample concen-tration above 50 g/ml did not 2 cause a corresponding increase in percent inhibition of sion coefficient (R ) of the trend lines obtained for the DPPH. There was there-fore the need to study extracts and standard was between 0.94 - 0.99, implying concentrations between 10 and 50 g/ml. that 94 to 99% of the variability in -glucosidase inhibition It was observed from studies using lower concentra- can be attributed to the concentration of the extracts. tions of standard catechin and gambier extracts that Since koji, an extract from Aspergillus terreus, is known maximum DPPH inhibition was measured at 30 g/ml to have high activity for -glucosidase inhibition, the (Table 2), except for the aqueous extract obtained after gambier extracts were compared to it for -glucosidase ethyl acetate extraction of gambier, which showed per- inhibitory activity. Three concentrations, 6.25, 12.5 and 25 cent DPPH inhibition to increase correspondingly with g/ml, were studied. For all extracts, percent -glucosi- concentration until 50 g/ml. At the lower concentrations, dase inhibition increased with increasing concentration. a coefficient of regression (R2) of 0.87 for catechin and Koji extract showed a mean of 74% inhibition while 0.94 - 0.97 for gambier extracts were obtained (Figures 5 ethanol, ethyl acetate and the aqueous extraction caused

740 J. Med. Plant. Res.

Table 2. Percent DPPH inhibition by catechin and Gambier Extracts at lower concentrations.

% Inhibition Concentration Catechin Ethanol Ethyl acetate Residue after Aqueous (g/ml) Mean Standard Extract extract ethanol extraction extract 50 91.89 91.77 92.63 92.24 92.44 92.20h 40 92.48 91.50 92.44 92.63 73.28 88.47h 30 92.99 91.93 92.56 92.36 67.80 87.53h 20 92.60 68.07 70.66 54.24 33.01 63.71i 10 35.71 27.68 33.32 17.22 14.16 25.62j 5 14.32 5.90 6.72 4.45 0.61 6.40k Mean 70.00m 62.81mn 64.72mn 58.86n 46.88p

IC50 (g/ml) 11.62 14.54 13.78 16.20 27.35

*Means with same superscript are not significantly different from each other (p < 0.01).

100 100 90 90

80 80

n n o 70 70 i o t i i t

i 60 b 60 i

y = 37.549Ln(x) - 42.087 b i h 50 50 h n 2 I n

40 R = .8690 I 40 y = 41.554Ln(x) - 61.231 %

% 30 30 2

R .9571 =

20 20

10 10

0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Concentration (ug/ml) Concentration (ug/ml)

Figure 5. Linear regression curve of percent DPPH Figure 7. Linear regression curve of percent DPPH inhibition at lower concentration of catechin standard. inhibition at lower concentration of ethanol extract.

100 90 100 80 90

70 Catechin 80 n

o 60 70

t EtOH ext n i 60

50 o b i i t

EtOAc ext i h 40 50

b n i

I y = 44.23Ln(x) - 73.169

30 Residue h 40 n I %

30 20 2

Water ext R = .9393 % 20

10 0 0 10 20 30 40 50 60 0 0 10 20 30 40 50 60 Concentration (ug/ml) Concentration (ug/ml)

Figure 6. Linear Regression curve of percent DPPH inhibition lower concentrations of catechin standard and gambier Figure 8. Linear Regression Curve of Percent DPPH extracts. inhibition at lower concentration of residue from ethanol extraction.

extracts caused below 50% inhibition of -glucosidase moderate -glucosidase inhibitory activity in vitro. 2 way activity (Table 3). This suggests that the extracts have ANOVA showed both the extracts and their concentrations

Apea-Bah et al. 741

100 100 90 90 80 80 70 n 70 o i

t 60 i 60 b n i 50 y = 40.723Ln(x) - 56.835 o h i

t 50 n

I 40 b 2 R = .9607 i 40

h % 30

30 y = 1.1567x + 32.46

20 2

R = .9953

0 10

0 10 20 30 40 50 60 0 Concentration (ug/ml) 0 5 10 15 20 25 30

Concentration (ug/ml) Figure 9. Linear regression curve of percent DPPH inhibition at lower concentration of ethyl acetate extract. Figure 12. Linear regression curve of percent - glucosidase inhibition at lower concentration of ethanol extract.

100

90 80 100 70 90 n 60 o i

t y = 2.0549x - 6.2024 80 i 50 b i 2 70 h 40 R = .9709 n

I y = 15.416Ln(x) - 26.974

30 n 60 o

i 2 % t

20 i 50 R = 0.9981

10 h 40

n I 0 30 0 10 20 30 40 50 60 % 20 Concentration (ug/ml) 10

0 Figure 10. Linear regression curve of percent DPPH inhibition at lower concentration of water extract. 0 5 10 15 20 25 30 concentration (ug/ml)

100 Figure 13. Linear Regression curve of percent - glucosidase inhibition at lower concentration of 90 residue of ethanol extract 80

70 n o i

t 60 i b i 50 to significantly (p < 0.01) affect percent inhibition. h

n y = 22.368Ln(x) + 18.546

i Inhibition of -glucosidase was significantly highest for 40 2 koji and lowest for residue after ethanol extraction. The %

30 R = .9383

other solvent extracts did not differ significantly from each

20 other. Percent inhibition was also significantly highest at 10 25 g/ml concentration compared to the lower 0 concentrations of 6.25 and 12.5 g/ml, which did not 0 5 10 15 20 25 30 significantly differ from each other (Table 3). Increasing the concentrations of the extracts up to 50 concentration (ug/ml) g/ml did not improve their -glucosidase inhibitory Figure 11. Linear regression curve of percent -glucosidase activity (Figure 17). The IC50 for koji extract was 4.08 inhibition at lower concentration of ‘koji’ extract from Aspergillus g/ml while it was in the range of 15.16 g/ml to 49.48 terreus extract. g/ml for the extracts (Table 3). Since IC50 was lowest for

742 J. Med. Plant. Res.

Table 3. Concentration of Koji and Extracts from Gambier and their IC50 for -glucosidase inhibition.

Concentration Ethyl acetate Residue after Ethanol Aqueous Koji extract Ethanol extract *Mean (g/ml) extract extraction extract 25.0 88.71 61.09 45.77 22.38 66.73 56.94c 12.5 80.85 47.78 43.15 12.50 38.51 44.56d 6.25 53.43 39.11 40.73 1.01 35.08 33.87d *Mean 74.33e 49.33g 43.21g 11.96f 46.77g IC50 (g/ml) 4.08 15.16 40.65 49.48 16.41

*Means with same superscripts are not significantly different from each other (p>0.01). ¥Koji is an extract from Aspergillus terreus.

100 100 90 90 Koji 80 80 70 Residue EtOH 70 ext n 60

o Water ext i

n 60 t i 50

o i b i t ext EtOH i 50 h 40 b n i I

h 40 30 EtOAC ext n % I

30 20

y = 3.6358Ln(x) + 34.029 % 10 20 R2 = 0.9995 0 10 0 10 20 30 0 Concentration (ug/ml) 0 5 10 15 20 25 30

Concentration (ug/ml) Figure 16. Linear regression curve of percent - glucosidase inhibition at lower concentrations of gambier extracts. Figure 14. Linear regression curve of percent - glucosidase inhibition at lower concentration of ethyl acetate extract 100 90 80 100

70 Pure Gambier 90 n o 60 i EtOAc ext t i

80 b 50 i Residue h 40 70 n I EtOH ext 30

60 % Water ext

50 o i 10 t i b i 40 0 h

n y = 1.7696x + 20.968

I 30 0 10 20 30 40 50 60

% R = 0.9459 20 Concentration (ug/ml)

Figure 17. Linear regression curve of percent -glucosidase 0 inhibition at higher concentrations of gambier extracts. 0 5 10 15 20 25 30

Concentration (ug/ml)

ethanol extract and highest for residue from ethanol Figure 15. Linear regression curve of percent - extract, it implies that the ethanol extract had the highest glucosidase inhibition at lower concentration of water -glucosidase inhibitory activity in vitro while that of the extract. residue from ethanol extraction was least.

Apea-Bah et al. 743

TIC=>NR(2.00) 100 T1.5 2.8E+4 T1.3

T1.9

y 90 t i s n

e T6.5 t n I

% 80

70 0 2.4 4.8 7.2 9.6 12.0

Retention time (min)

Figure 18. LCMS spectrum for ethanol extract showing three peaks at T1.3, T1.5, T1.9.

Mariner Spec /34:34 (T /1.26:1.26) -28:28 (T -1.26:1.26) ASC=>MC=>NR(2.00)[BP = 145.6, 143] 145.63 100 142.8

70 y t

i 60

s 329.10 n e

t 50 n I

% 40 147.62 30 330.13 20 619.67

10 185.64 626.60 114.80 331.10 187.59 260.33 403.10 478.36 551.16 775.39 911.93 0 0 108.0 298.2 488.4 678.6 868.8 1059.0 Mass (m/z)

Figure 19. m/z spectral lines of compounds present in peak T1.3 for ethanol extract.

HPLC analysis At sample concentrations of 500 and 1000 g/ml, for all the extracts, retention times were similar (Table 5) to that Preliminary work on the wavelength for maximum absorp- of standard catechin indicating that the bioactive tion using the UV-visible spectrophotometer resulted in 5 compound could be catechin. ppm standard catechin giving best results at 209.4 nm. All HPLC analyses were therefore done at 210 nm wavelength. Catechin standard gave maximum peak at reten- LCMS analysis tion time range of 3.258 to 3.271 min (Table 4) for the various concentrations studied. The extracts upon analysis using liquid chromatograph-

744 J. Med. Plant. Res.

Table 4. Retention time of standard catechin at 210 nm using HPLC.

Retention time (min) Concentration (g/ml) Area 3.266 5 1650628 3.263 10 3495729 3.264 25 7766748 3.271 50 15226023 3.266 100 28116464 3.263 500 70398534 3.258 1000 83927104

Table 5. HPLC Retention time of Samples at higher concentration at 210nm

Sample Concentration (g/ml) Retention time Area 500 3.299 60923005 Ethyl Acetate 1000 3.275 70239090 500 3.274 62442591 Ethanol 1000 3.266 75125886 500 3.256 65592871 Water 1000 3.227 92219895 500 3.277 79735339 Residue 1000 3.265 92755082

Mariner Spec /34:34 (T /1.26:1.26) -28:28 (T -1.26:1.26) ASC=>MC=>NR(2.00)[BP = 145.6, 143] 145.63 100 142.8

70 y t

i 60

s 329.10 n e

t 50 n I

% 40 147.62 30 330.13 20 619.67

10 185.64 626.60 114.80 331.10 187.59 260.33 403.10 478.36 551.16 775.39 911.93 0 0 108.0 298.2 488.4 678.6 868.8 1059.0 Mass (m/z)

Figure 19. m/z spectral lines of compounds present in peak T1.3 for ethanol extract.

mass spectrometer (LCMS) showed 2 or 3 board peaks NMR analysis (Figures 18 to 37) each comprising different compounds and hence different m/z ratios. Notable among the m/z Plates 1 to 5 show the 1H, 13C, DEPT, HMQC and HMBC values was 291.23 with its major peak, which is indicative spectra generated from the NMR analysis at 500MHz. of catechin. NMR was used to then elucidate and confirm From the 1H NMR spectra (Plate 1), 2 double doublet the structure of the major bioactive compound. peaks each with one proton (1H) are observed at chemi-

Apea-Bah et al. 745

Mariner Spec /39:40 (T /1.45:1.49) -35:36 (T -1.45:1.49) ASC=>MC=>NR(2.00)[BP = 291.2, 104] 291.23 100 103.5

70 y t

i 60 s n e

t 50 n I

% 40

30 292.24 20

10 333.12 214.47 293.18 391.04 448.45 532.14 579.88 619.15 667.95 0 0 214.0 319.4 424.8 530.2 635.6 741.0 Mass (m/z)

Figure 20. m/z spectral lines present in peak T1.5 for ethanol extract

Mariner Spec /49:51 (T /1.84:1.91) -44:46 (T -1.84:1.91) ASC=>MC=>NR(2.00)[BP = 291.2, 24] 291.23 100 23.7

70 y t

i 60 s n e

t 50 n I

% 40

30 292.19 20

10 241.40 293.29 390.98 242.12 351.20 438.36 479.34 522.21 577.88 620.97 661.98 700.86 0 0 214.0 319.4 424.8 530.2 635.6 741.0 Mass (m/z)

Figure 21. m/z spectral lines present in peak T1.9 for ethanol extract.

cal shifts (), 2.48 - 2.53 ppm (J-coupling, 7.95Hz) and The 1Hs are in axial and equatorial positions at the 2.82 - 2.87 ppm (J-coupling 5.50 Hz). These peaks repre- bonded site (Plate 1). At 3.98 - 4.0ppm, a quartet peak sent an aliphatic group having a single near 1H and (J-coupling, 7.95Hz, 2.45 Hz) is observed indicating 3 another longer range 1H coupling per each double doublet. neighboring 1H-atoms. At 4.45 - 4.58 ppm, there is a

746 J. Med. Plant. Res.

TIC=>NR(2.00) T1.5 100 2.6E+4

T1.9 y t i s n e

t 90 n I T23.6 %

80 0 6 12 18 24 30 Retention Time (Min)

Figure 22. LCMS spectrum for ethyl acetate extract showing three peaks at T1.2, T1.5, T1.9.

Mariner Spec /33:34 (T /1.22:1.26) -26:27 (T -1.22:1.26) ASC=>MC=>NR(3.00)[BP = 619.7, 30] 619.67 100 30.2

80 145.64 70 y t

i 60

s 620.65 n

e 329.11

t 50 n I

% 40 625.65

30 147.63 333.12 20 641.60 163.77 330.07 623.68 10 334.03 624.65 114.83 164.80 214.51 486.82 0 0 109.0 244.8 380.6 516.4 652.2 788.0 Mass (m/z)

Figure 23. m/z spectral lines present in peak T1.2 for ethyl acetate extract.

doublet peak (J-coupling, 7.95 Hz) with one 1H indicating doublets, each with one 1H, having meta coupling (J- only one neighboring 1H and there is likelihood of a coupling 2.45 Hz) with each other in an aromatic ring. At neighboring O-atom at this value. Both the quartet and 6.71 - 6.73 ppm, there is a double doublet (J-coupling, doublet having J-coupling of 7.95 Hz are in long range 8.8 Hz, 1.85 Hz) representing 2 neighboring 1Hs in meta coupling with the double doublet at 2.48 - 2.53 ppm. and ortho positions in an aromatic ring. The neighboring 1 The deuterated methanol (CD3OD) produced its peak at ortho H-atom produced a doublet at 6.76 - 6.78 ppm (J- 3.31 ppm and another peak for water produced at 4.67 coupling, 8.55 Hz) while the neighboring meta 1H (J- - 4.95ppm (Plate 1). At 5.86 and 5.94 ppm, there are 2 coupling, 1.85 Hz) produced a doublet at 6.84 ppm (Plate 2).

Apea-Bah et al. 747

Mariner Spec /39:39 (T /1.45:1.45) -35:35 (T -1.45:1.45) ASC=>MC=>NR(2.00)[BP = 291.2, 127] 291.23 100 127.0

70 y t

i 60 s n e

t 50 n I

% 40

30 292.23 20

10 292.98 109.82 163.91 214.47 391.07 467.80 524.93 579.78 629.69 706.45 0 0 109.0 244.8 380.6 516.4 652.2 788.0

Mass (m/z)

Figure 24. m/z spectral lines present in peak T1.5 for ethyl acetate extract.

Mariner Spec /50:52 (T /1.87:1.95) -26:27 (T -1.87:1.95) ASC=>MC=>NR(2.00)[BP = 291.2, 98] 291.23 100 98.0

70 y t

i 60 s n e

t 50 n I

% 40

20 205.49 10 207.48 292.66 579.84 113.71 391.19 474.44 743.69 864.75 981.17 0 0 99.0 319.2 539.4 759.6 979.8 1200.0 Mass (m/z)

Figure 25. m/z spectral lines present in peak T1.9 for ethyl acetate extract

Comparing the 13C and DEPT spectra (Plate 3) reveal 156.91, 157.57 and 157.78 ppm. values above 100 13 CH2 as inverted DEPT peak at 28.56 ppm and quaternary ppm in the C spectra indicated aromatic C-atoms while C-atoms, being present in 13C spectra but absent in values of about 150 ppm indicated aromatic C-atoms DEPT spectra, at 100.94, 132.19, 146.25 ppm, 146.28, bonded to O-atom. It is therefore likely that C-atoms at

748 J. Med. Plant. Res.

TIC=>NR(2.00) T1.5 100 3.6E+4

90 y t i s n e

t 80 n I

70 T11.9 T2.8 T7.0

60 0 4 8 12 16 20 Retention Time (Min)

Figure 26. LCMS spectrum for residue from ethanol extraction showing broad peak at T1.5 and small peak at T2.8

Mariner Spec /31:35 (T /1.15:1.30) -20:25 (T -1.15:1.30) ASC=>NR(2.00)[BP = 328.5, 44] 328.45 100 44.3

70 y t

i 60 s

n 618.76 e

t 50

n 187.04 I

% 40 185.16 624.72 30

20 268.64 620.74 10 147.19 359.32 485.56 908.93 643.62 154.35 236.86 332.40 505.36 730.17 832.01 926.85 1020.06 0 0 99.0 319.2 539.4 759.6 979.8 1200.0 Mass (m/z)

Figure 27. m/z spectral lines present in peak T1.3 for residue from ethanol extraction.

values from 146.25 to 157.78 ppm are bonded to an O- 4.45 - 4.58 ppm was bonded to the C-atom at 82.86 ppm atom. while the 1H-atoms with 2 doublets at 5.86 and 5.94 ppm, From the HMQC spectra (Plate 4), it was observed that were bonded to C-atoms at 95.60 and 96.41 ppm. The the two 1H-atoms with double doublet peaks at 2.48 – double doublet 1H at 6.71 - 6.73 ppm was bonded to the 2.53 ppm (Plate 1) were bonded to the C-atom at 28.56 C at 120.16 ppm while the doublet 1H at 6.76 - 6.78 ppm ppm while the quartet 1H at 3.98 - 4.0ppm was bonded to was bonded to the C at 116.23 ppm. The doublet 1H at the C-atom at 68.81 ppm. The 1H with doublet peak at 6.84 ppm was bonded to the C-atom at 115.34 ppm.

Apea-Bah et al. 749

Mariner Spec /40:42 (T /1.49:1.57) -23:27 (T -1.49:1.57) ASC=>MC=>NR(2.00)[BP = 291.2, 191] 291.2347 100 190.8

70 y t

i 60 s n e

t 50 n I

% 40 329.1065 30

10 165.6618 619.6706 292.5783 139.7067 454.4737 577.8330 735.6859 879.6150 1024.0349 0 0 99.0 319.2 539.4 759.6 979.8 1200.0 Mass (m/z)

Figure 28. m/z spectral lines present in peak T1.5 for residue from ethanol extraction.

Mariner Spec /73:74 (T /2.76:2.80) -66:68 (T -2.76:2.80) ASC=>NR(2.00)[BP = 225.1, 72] 225.06 100 72.4

70 y t

i 60 s n e

t 50 n I

% 40

20 226.06

10 163.29 448.03 208.91 140.22 246.90 290.80 348.37 390.38 456.25 491.71 572.08 0 0 138 232 326 420 514 608 Mass (m/z)

Figure 29. m/z spectral lines present in peak T2.8 for residue from ethanol extraction.

The HMBC spectra (Plate 5) show the long range doublet 1H-atom at 6.72 ppm was coupled to 115.34, coupling between the 1H-atoms and the C-atoms. The 1H- 116.23 and 146 ppm. Its ortho-coupled neighbor at 6.77 atom at 4.57 ppm was coupled to the C-atoms at 28.56, ppm had similar C-atom coupling in addition to the C- 68.81, 115.34, 116.23, 132.20 and 156.91 ppm. The 1H- atom at 132.20 ppm while the meta-coupled neighbor at atoms at 5.86 and 5.94 ppm were coupled to C-atoms at 6.84 ppm had similar C- atom coupling in addition to the 100.94, 95.60, 96.41 and 157 ppm while the double C-atom at 120.16ppm.

750 J. Med. Plant. Res.

TIC=>NR(2.00) T1.5 100 3.1E+4

90 y t i s n e t n I

% 80 T6.6 T9.7

70 0 2.2 4.4 6.6 8.8 11.0 Retention Time (Min)

Figure 30. LCMS spectrum for aqueous extract showing broad peak at T1.5.

Mariner Spec /34:36 (T /1.26:1.34) -23:25 (T -1.26:1.34) ASC=>NR(2.00)[BP = 328.5, 130] 328.45 100 129.6

70 y t

i 60 s n e

t 50 n I

% 40

30 640.69 145.20 20 624.79 147.19 10 329.87 618.76 529.35 109.24 201.09 447.83 614.76 741.66 908.90 1005.23 0 0 99.0 319.2 539.4 759.6 979.8 1200.0 Mass (m/z)

Figure 31. m/z spectral lines present in peak T1.3 for aqueous extract.

From the NMR analyses, the major bioactive compound in the purified sample was identified as (+)-catechin (Figure 38).

Apea-Bah et al. 751

Plate 1. 1H NMR of Purified Ethyl acetate extract of Gambier using CD3OD at 500 MHz showing chemical shifts of 2.4ppm to 5.1 ppm.

Mariner Spec /38:40 (T /1.42:1.49) -23:25 (T -1.42:1.49) ASC=>MC=>NR(2.00)[BP = 329.1, 91] 329.11 100 90.6

70 y t

i 60

s 291.24 n e

t 50 n I

% 40

30 118.79 330.10 20 145.64 292.23 10 573.93 147.63 331.07 641.57 258.41 406.71 486.38 579.78 723.27 0 0 102.0 250.8 399.6 548.4 697.2 846.0 Mass (m/z)

Figure 32. m/z spectral lines present in peak T1.5 for aqueous extract.

752 J. Med. Plant. Res.

Plate 2. 1H NMR of purified ethyl acetate extract of Gambier using CD3OD at 500 MHz showing chemical shifts of 5.8ppm to 7.0 ppm.

Plate 3. 13C NMR and dept of purified ethyl acetate extract of gambier using CD3OD at 500 MHz.

Apea-Bah et al. 753

Plate 4. HMQC NMR of purified ethyl acetate extract of Gambier using CD3OD at 500 MHz.

Mariner Spec /48:50 (T /1.80:1.88) -22:26 (T -1.80:1.88) ASC=>MC=>NR(4.00)[BP = 291.2, 43] 291.23 100 43.2

70 y t

i 60 s n e

t 50 n I

% 40

30 329.11 20

10 118.79 163.75 207.47 330.10 577.82 116.71 254.51 391.24 474.48 525.94 620.65 0 0 105.0 221.4 337.8 454.2 570.6 687.0 Mass (m/z)

Figure 33. m/z spectral lines present in peak T1.9 for aqueous extract.

754 J. Med. Plant. Res.

Plate 5. HMBC NMR of purified ethyl acetate extract of gambier using CD3OD at 500 MHz.

TIC=>NR(2.00) T1.5 100 3.5E+4 T1.3

90 y t i s n e

t 80 n I

% T2.0

70 T5.4

60 0 2 4 6 8 10 Retention Time (Min)

Figure 34. LCMS spectrum for purified extract from ethyl acetate fraction showing three peaks at T1.3, T1.5 and T2.0.

Apea-Bah et al. 755

Mariner Spec /35:36 (T /1.30:1.34) -26:27 (T -1.30:1.34) ASC=>MC=>NR(2.00)[BP = 187.5, 243] 187.53 100 243.4

70 y

t 269.24

i 60 s n e

t 50 n I

% 40 350.88 30 185.66 20 432.48 105.71 514.04 10 270.23 595.59 188.52 351.86 433.45 677.11 121.66 516.26 758.68 840.18 0 0 101.0 261.6 422.2 582.8 743.4 904.0 Mass (m/z)

Figure 35. m/z spectral lines present in peak T1.3 for purified extract from ethyl acetate fraction.

Mariner Spec /39:40 (T /1.45:1.49) -37:37 (T -1.45:1.49) ASC=>MC=>NR(3.00)[BP = 291.3, 69] 291.24 100 68.6

70 y t

i 60 s n e

t 50 n I

% 40

30 145.63 292.22 20 185.65 10 147.64 601.72 148.34 197.68 268.96 350.42 412.84 504.86 565.31 623.58 676.76 0 0 107.0 238.4 369.8 501.2 632.6 764.0 Mass (m/z)

Figure 36. m/z spectral lines present in peak T1.5 for the purified extract.

Conclusion in vitro free radical scavenging using organic extracts of commercial gambier, 30 g/ml is adequate. The study From the study it is concluded that ethanolic and ethyl has shown that the ethanolic extract of gambier and acetate extracts of commercial gambier, as well as their aqueous residue after ethyl acetate extraction, have residues on extraction have high ability to scavenge moderate in vitro -glucosidase inhibitory activity. reactive free radicals such as is produced by DPPH and Catechin was identified and confirmed as the major hence, have high antioxidant activity in vitro. For optimal bioactive compound in gambier.

756 J. Med. Plant. Res.

Mariner Spec /51:53 (T /1.91:1.99) -44:47 (T -1.91:1.99) ASC=>MC=>NR(3.00)[BP = 291.2, 34] 291.21 100 34.1

70 y t

i 60 s n e t 50 205.47 n I

% 40 163.73

30 207.47 20 292.17 209.46 10 390.92 140.65 214.54 293.19 454.96 498.39 601.60 0 0 115.0 232.8 350.6 468.4 586.2 704.0 Mass (m/z)

Figure 37. m/z spectral lines present in peak T2.0 for the purified extract.

OH 146.28 (6.83,d) 115.34 146.25

(5.86, d) 132.20 116.23 HO O (6.77,d) 120.16 95.60 (4.57) 157.78 156.91 82.86 (6.72,d)

96.41 100.94 68.81 (5.94, d) 157.57 28.56 (3.98, q) (2.52) (2.83) OH

Figure 38. Structure of catechin [2-(3,4-dihydroxyphenyl)chroma-3,5,7-triol] showing 1H and 13C chemical shifts as elucidated using NMR, 500MHz. Exact mass: 290.08, Molecular weight: 290.27. m/z: 290.08(100.0%). 3291.08(16.5%), 292.09(1.3%), 292.08(1.2%)

ACKNOWLEDGEMENTS Agriculture Research Institute of the Ghana Atomic Energy Commission (BNARI/GAEC) is acknowledged for The research team is grateful to WAITRO, LIPI and its support towards the success of the study. ISESCO for funding the research through organization of the WAITRO research fellowship programme 2008. The Research Centre for Chemistry of the Indonesian Institute REFERENCES of Sciences (RCChem/LIPI) is gratefully acknowledged Ahmed FR, Ng AS, Fallis AG (1978). 7a-Acetoxydihydronomilin: isola- for hosting the study. The Biotechnology and Nuclear tion, spectra, and crystal structure. Can. J. Chem. 56: 1020-1025.

Apea-Bah et al. 757

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