Advances in Applied Chemistry and Biochemistry

ISSN: 2652-3175

Research Article

Cytotoxic, Nitric Oxide and ?-Glucosidase Inhibitory Activities of Biflavonoids from Garcinia prainiana King

Mohd Lip Jabit1,Mohd Nazrul Hisham Daud1* and Shamsul Khamis2

1Malaysian Agriculture Research Institute, Serdang, Selangor, Malaysia

2School of Environmental and Natural Resource Sciences, National University of Malaysia, Bangi, Selangor, Malaysia

Received: 22 July 2019

Accepted: 19 August 2019

Version of Record Online: 06 September 2019

Citation

Jabit ML, Daud MNH, Khamis S (2019)Cytotoxic, Nitric Oxide and α-Glucosidase Inhibitory Activities of Biflavonoids from Garcinia prainiana King. Adv Appl Chem Biochem 2019(1): 55-67.

Correspondence should be addressed to
Mohd Nazrul Hisham Daud, Malaysia

E-mail: nazrul@mardi.gov.my
DOI: 
10.33513/ACBC/1901-08

Copyright

Copyright © 2019 Mohd Nazrul Hisham Daud et al. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and work is properly cited.

1. Abstract

Garcinia prainiana King is a rare fruit from South East Asia with known anti-inflammatory activity. In this study, we isolated biflavonoids from the leaf extracts and tested these for cytotoxicity, Nitric Oxide (NO) inhibition in mouse macrophages (RAW 264.7) and α-glucosidase inhibition. Extract of G. prainiana leaf showed potent inhibition of NO production from lipopolysaccharide stimulated RAW 264.7 cells, with IC50 of 17.55 ± 2.37 µg/mL. Bioactivity-guided fractionation led to the isolation of four biflavonoids from the extract: morelloflavone (1), amentoflavone (2), 4'''-methyl amentoflavone (8) and 2″,3″-dihydromorelloflavone or GB-2a (9); and the glucosides of 1 and 9, morelloflavone-7″-O-β-glucoside (7) and 2″,3″-dihydromorelloflavone-7″-O-β-glucoside (10), respectively. The four biflavonoid compounds 1, 2, 8 and 9 inhibited NO production within the range of IC50 = 44.80 - 75.20 µM. The α-glucosidase inhibition assay revealed variable activity from IC50 of 6.34 µM (4'''-methyl amentoflavone (8)) to IC50 of 43.75 µM (morelloflavoneglucoside(7)). This study highlights the potential for value-adding G. prainiana as an anti-inflammatory and anti-diabetic food source.

2. Keywords

Anti-Diabetic; Anti-Inflammatory; Bioactivity Guided Isolation; Dereplication; Flavonoid; Guttiferae

3. Introduction

Conserving and promoting the potential of rare fruit offers opportunities for developing high-value new crops. This is one of the programmed activities of the Malaysian Agricultural Research and Development Institute, who has collected more than 58 underutilised fruits of 32 different species from 21 genera from the Peninsular and East Malaysia for planting in a special plot area. These fruits are known to be associated with many nutritional and medicinal properties [1]. One of these rare fruits is Garcinia prainiana King known as Button Mangosteen or Kechupu which is a small to moderate-sized tree, commonly found in the south of Thailand and north of the Peninsular Malaysia. Local Malay people use the young fruit in cooking to provide a sour taste in dishes. The fruits and young leaves of G. prainiana are also eaten raw by the local Temuan tribe in Peninsular Malaysia [2]. The leaves are large and elliptical, ranging from 10-23 cm long, 4.5-11.5 cm wide, and as with other Garcinia species, this plant produces gummy white latex that is present mostly in the bark [3].

Previous studies on G. prainiana fruit extract have demonstrated a total phenolic content of 1668.15 ± 11.68 mg Garlic Acid Equivalent/100 g in the edible portion and 91.90% of antioxidant capacity, which are among the highest antioxidant activities reported from all tested Malaysian rare fruits[1]. Preliminary screening of several Garcinia species also suggests that G. prainiana leaf extracts have antioxidant activity, as these extracts inhibited Nitric Oxide production (NO) by Lipopolysaccharide (LPS) stimulated RAW264.7 cells with an IC50 of 11 µg/mL, without cytotoxicity to the cells [4]. Therefore, further phytochemical investigations of G. prainiana leaf are warranted in order to identify the secondary metabolites responsible for NO inhibition in the extract and to further characterise the bioactivity of these compounds. A previous study on the natural products in extracts from G. prainiana has identified six biflavonoids(Figure 1); (+)-morelloflavone (1), amentoflavone (2), prainianonide (3), (2S)-eriodictyol 7-O-β-D-glucuronide (4), naringenin 7-O-β-D-glucuronide (5) and (–)-GB-1a (6) [5]. Biflavonoids are known to have diverse bioactivitiessuch as anti-HIV [6], anti-inflammatory and immunomodulatoryactivities [7], anti-tumour [8], cytotoxicity [9], anti-microbial [10,11] and analgesic activities [12]. Some xanthones, biflavonoids and depsidones have also shown potential as inhibitors of the enzyme α-glucosidase [13-16]. Glycoside trimming enzymes are crucially important in a broad range of metabolite pathway. Amongst the large array of enzymes, glucosidases are postulated to be a powerful therapeutic target [17]. The inhibition of intestinal α-glucosidase could postpone digestion and absorption of carbohydrates and thus reduce postprandial hyperglycemia [18]. There has been an increasing amount of research on α-glucosidase inhibitors from plant extracts, particularly from Garcinia species [15,16,19,20]. Based on the traditional use, the inclusion of G. prainiana leaves in the diet may help to maintain a safe blood glucose level, even with the high intake of carbohydrate. High blood glucose levels may otherwise facilitate the accumulation of fat, so indirectly, the use of these plants could help people reduce fat in their body. The characterisation of α-glucosidase inhibitor from the G. prainiana leaves may help to rationalise the usage of the leaves from this plant.

Cytotoxic Nitric Oxide and Glucosidase Inhibitory Activities of Biflavonoids from Garcinia prainiana King

Figure 1: Chemical structure ofbiflavonoids isolated from G. prainiana King leaf.

The main aim of this paper was to investigate the bioactive constituents from G. prainiana leaves.Bioactivity-guided fractionation was employed to ensure any active constituents were not excluded from the extract of interest. Liquid Chromatography Mass Spectrometry (LCMS) profiling was used for chemical dereplication of known biflavonoids and Nuclear Magnetic Resonance (NMR) spectroscopy was used for structural elucidation of novel compounds in this species.NO and α-glucosidase inhibition assays were used to evaluate the bioactivity of the fractions and compounds. Here we report four new biflavonoids in this species and their associated anti-inflammatory activity, along with particularly potent α-glucosidase inhibitory activity in one biflavonoid glycoside. Further characterisation of these bioactive biflavonoids in G. prainiana leaves could help value-adding this species as a functional or medicinal food; i.e., a food with health properties over and above the basic nutritional properties.

4. Materials and Methods

4.1. Plant materials

Garcinia prainiana King leaves were obtained from an underutilised fruit plot of the Malaysian Agricultural Research and Development Institute (MARDI) in Serdang, Selangor, Malaysia and a voucher specimen SK 06/01 was deposited in the Herbarium of Institute of Bioscience, University Putra Malaysia. The plant raw material was cleaned and air-dried at room temperature and ground to 1 mm particle size and kept in dark coloured plastic bags.

4.2. Preparation and chemical profiling of extracts and semi-purified fractions

3 kg dried and ground leaves were macerated in 5 L of methanol (Merck, Analysis grade EMSURE®) for 3 days. Then, the solvent was filtered with Whatman filter paper no.1 and the filtrate was evaporated to dryness under reduced pressure (5.3 kPa) to give a dark brown gum crude methanol extract. The solvent was replenished with fresh methanol and the procedure was repeated 8 times to yield 200 g of crude methanol extract.

Approximately, 20 g of crude methanolic extract from G. prainiana leaf was subjected to Vacuum Liquid Column Chromatography (VLC). The column was packed with normal phase silica, 200-425 mesh (Sigma-Aldrich) and eluted with following solvents accordingly, hexane (Merck, Analysis grade EMSURE®) , mixture of hexane-ethyl acetate (1:1), ethyl acetate (Merck, Analysis grade EMSURE®), mixture of ethyl acetate-methanol (1:1), and finally methanol(Merck, Analysis grade EMSURE®). The VLC fractionation was repeated ten times to yield 120 g of hexane-ethyl acetate (1:1) fraction (GPL1), 60 g of ethyl acetate fraction (GPL2), 30 g of ethyl acetate-methanol (1:1) fraction (GPL3) and 7.1 g of methanol fraction (GPL4). All fractions were then subject to bioactivity assays (Sections 4.3 - 4.5) and chemical profiling (Section 4.6).

4.3. Cytotoxicity assay

Cytotoxicity against RAW 264.7 murine leukemic monocyte macrophages was assayed in 96-well plates using the ATPlite™ assay kit (PerkinElmer, Glen Waverley, Australia) with chlorambucil (Sigma-Aldrich, C025) as a positive control.Cells were grown in clear 96-well plates.The growth medium consisted of colour-free Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal bovine serum (FBS; Interpath, Heidelberg, Australia), L-glutamine (2 mM, Sigma-Aldrich, G7513), sodium pyruvate (1 mM, Sigma-Aldrich, C8636), penicillin (200 U/mL, Sigma-Aldrich, P4333) and streptomycin (200 μg/mL, all from Invitrogen, Mulgrave, Australia). RAW 264.7 cells were plated at a concentration of 30000 cells/well (90 μLof cell suspension/well), respectively. Samples and control compound were dissolved in DMSO at six concentrations and further diluted 20-fold in the medium.Then, samples and control compound were added to the cell suspension at 10 μL/well, and the plates were incubated at 37°C with 5% CO2 for 24 h. Following incubation, cell lysates were assayed for ATP with the ATPlite™ assay kit as per the manufacturer’s instructions. The mammalian cell lysis solution (50 μL) was added to each well of the cell culture microplate. The plate was shaken on an orbital microplate shaker (500 rpm, 5 min), then the substrate solution (50 μL/well) was added, and the plate was further shaken (500 rpm, 5 min). The plate was dark adapted for 10 min, and the luminescence measured on a Wallac 1450 Microbeta luminescence counter (Wallac, Turku, Finland). Half-maximal inhibitory concentration (IC50) values were calculated using GraphPad Prism version 5 (La Jolla, CA, USA). The assay was repeated three times.

4.4. Nitrite (Griess) assay for NO inhibition

RAW 264.7 cells were cultivated as described above. Cell suspensions (120 μL/well, 106cells/mL) were added to the wells of a 96-well microplate and incubated for 20 h (37°C, 5% CO2), after which test compounds (dissolved in DMSO and further diluted 20-fold in the medium) were added to the cell suspension at 10 μL/ well. Following incubation for 1 h, LPS solution (10 μL/well, 10 μg/ mL) was added and the plate incubated for a further 20 h. After incubation, the plate was centrifuged (1500g, 3 min), and 90 μL of the supernatant transferred to a clear flat-bottom assay plate (PerkinElmer, Glen Waverley, VIC, Australia) and assayed immediately for nitrite.

Nitrite standards (0, 3.13, 6.26, 12.5, 25, 50 and 100 μM) were prepared in the medium.Then 90 μL of each standard and cell supernatant was transferred to a flat-bottom microplate (Greiner Bio-One, Frickenhausen, Germany) with 90 μL of Griess reagent (0.1% N-1-naphthylethylenediamine dihydrochloride, 1% sulfanilic acid in 5% phosphoric acid) added to each well, followed by incubation (23°C, 20 min) on an orbital plate shaker. Following incubation, the absorbance was read at 550 nm in a Wallac 1450 Microbeta plate reader (Wallac, Turku, Finland).Samples and controls were assayed in triplicate. Standard calibration curves were calculated from nitrite standard solution, and R2 values determined from the regression line of best fit to verify linearity.Mean and standard deviations were calculated from the replicates.The NO (measured as nitrite) production in sample wells was calculated as a percentage of the production in solvent (DMSO) control wells.The assay was repeated on 3 different occasions.

4.5. α-Glucosidase assay

The α-glucosidase inhibitory activity was determined using a fluorescent method optimized by Payn [21]. The α-glucosidase enzyme and the substrate, 4-methylumbelliferyl- α-D-glucopyranoside were purchased from Sigma. The initial concentration of the substrate solution was 84 µM in sodium acetate buffer pH 5.5. The enzyme solution (45 µL/well) was mixed with the sample or control (10μL/well) in a black 96-well microplate (flat bottom) and the reaction was started by adding the substrate solution (45 µL/well). The solution was incubated at 37°C for 30 min and the reaction stopped by adding 100 mM glycine-sodium chloride solution pH 5.5. To determine inhibition, the fluorescence of the solution was measured using a Perkin Elmer Wallac Victor 2 plate reader (λex 355 nm, λem 460 nm). Half-maximal Inhibitory Concentration (IC50) values were calculated using GraphPad Prism version 5 (La Jolla, CA, USA). Samples were assayed in duplicate. The assay was repeated at least two times.

4.6. Chemical profiling and dereplication

Since some biflavonoids have already been reported from G. prainiana leaf extract [5], we used chemical profiling and dereplication procedures [22] to identify new biflavonoids from the extracts. This procedure prioritised the compounds which have not previously been reported from this species for isolation, structure elucidation and bioactivity assessment.

The chemical profiling of extracts and fractions was performed using the Agilent 1200 LCMS system with Diode Array Detector (DAD) coupled with a single quadruple mass spectrometer (APCI mode). The column used was a Phenomenex Luna C18 column (5 μm, 250 mm x 4.6 mm i.d.). The mobile phase used was Millipore deionised water with 0.005% trifluoroacetic acid (A) and acetonitrile with 0.005% trifluoroacetic acid (B). The gradient system used was: 0 - 2 min., 10% B; 7 - 12 min., 50% B; 17 - 22 min., 95% B; 27 - 32 min., 10% B.The flow rate used was 0.75 mL/min and sample injection volume was 20 µL. The mass detector conditions were set as follows: APCI positive mode from 50 to 1000 m/z, capillary voltage set at -4000V, needle temperature set at 340°C and gas flow rate was at 5 L/min.

Due to lower cytotoxicity and good NO inhibitory activity the GPL2 fraction was the main focus of further investigation. Based on chemical profiling using LC-APCI-MS (Figure 2), four compounds with [M+1]+ at m/z 719, 721, 559, and 553, were hitherto not known from G. prainiana. Therefore, further isolation work was carried out to purify these compounds from G. prainiana leaf.

Cytotoxic Nitric Oxide and Glucosidase Inhibitory Activities of Biflavonoids from Garcinia prainiana King

Figure 2: Chromatographic profile of crude methanol extract of G. prainiana leaf at 360 nm, showing the presence of biflavonoids, 4'''- methylamentoflavone (8), 2″,3″-dihydromorelloflavone (9), morelloflavone (1), amentoflavone (2), morelloflavone-7″-O-β-glucoside (7) and 2″,3″-dihydromorelloflavone-7″-O-glucoside(10).

4.7. Isolation and structural elucidation of compounds from GPL2

Ten grams of GPL2 was subjected to C18 Preparative HPLC on a Gilson 322 system with a UV/vis-155 detector, connected to an FC204 fraction collector and using a Phenomenex Luna C18 column (5 μm, 150 × 21.2 mm i.d.). The mobile phase used was Millipore deionised water with 0.005% trifluoroacetic acid (A) and methanol with 0.005% trifluoroacetic acid (B). The gradient system used was: 0 - 5 min., 50% B; 10 -15 min., 95% B; 17 - 22 min., 50% B.

In total, thirty fractions were collected and chemical profiling by LCMS revealed relatively pure compounds in fraction 22 (compound 2, 30 mg, [M+1]+ at m/z = 539) and in fraction 24 (compound 8, 40 mg, [M+1]+ at m/z = 553).The mass spectra were found to match the chemical profile in figure 2, showing similar retention times and [M+1]+ at 11.3 min, m/z 539 and 14.3 min, m/z 553, respectively. Based on the chemical profiling and dereplication, fractions 13-15 were combined, then 50 mg of the combined fraction was subjected to another preparative HPLC fractionation using the same parameters and solvent system above.This yielded compound 9 (23 mg, [M+1]+at m/z = 559) in subfraction 16 and compound 1 (5.7 mg, [M+1]+at m/z = 557) in subfraction 19. Figure 3 shows a comparison of the chemical profile (UV chromatogram at 280 nm) of isolated compounds, GPL2 extracts and crude extract (GPL).

Cytotoxic Nitric Oxide and Glucosidase Inhibitory Activities of Biflavonoids from Garcinia prainiana King

Figure 3: Comparison of chemical profiles (UV chromatogram at 280 nm) of from crude extracts (GPL), fraction GPL2 and isolated compounds, 4?- methyl amentoflavone (8), 2″,3″- dihydromorelloflavone (9), morelloflavone (1) and amentoflavone (2), 2″,3″- dihydromorelloflavone-7″-O-β-glucoside (10) and morelloflavone-7″-O-β-glucoside (7).

Further isolation work was then carried out on GPL2 to isolate the other components in the bioactive fraction. Forty grams of GPL2 extract was fractionated using 200 g of MCI gel CHP20P (Supelco, Bellafonte, PA, USA) column Chromatography (C1) and yielded 12 fractions. These fractions were combined according to the LCMS chemical profile and yielded fraction A (1-4), fraction B (5-8) and faction C (9-12). Fraction A (32g) was subjected to further chromatography on another MCI column (C2) to yield 46 fractions. Then, fractions 1-7 (1 g) of C2 column chromatography were subjected to C18 (Sepra C18-E, 50 μm, 65A; Phenomenex Torrance, CA, USA) open column chromatography to yield compound 7 (9.4 mg, [M+1]+ at m/z = 721) from subfractions 20 to 22 and compound 10 (15 mg, [M+1]+ at m/z = 719) from subfractions 28 to 34.

All isolated compounds were dissolved in deuterated Dimethyl Sulfoxide (DMSO-d6) or deuterated methanol (CD3OD) solvent and structurally elucidated using 13C and 1H NMR spectroscopy. NMR spectra were acquired on a Bruker AVANCE II 500 MHz spectrometer. The chemical structures were elucidated and confirmed by comparison with previously published data.

5. Results and Discussion

This study confirms that G. prainiana leaf extracts, in addition to the fruit, are rich in bioactive biflavonoids. We successfully isolated four known biflavonoids that have never been reported from this species: morelloflavone-7″-O-β-glucoside (7), 4'''-methylamentoflavone (8), 2″, 3″-dihydromorelloflavone (9) and 2″, 3″-dihydromorelloflavone-7″-O-β-glucoside (10), along with two previously reported compounds from this plant; morelloflavone (1) and amentoflavone (2) (Figure 1). Bioactivity-guided fractionation of the crude extracts and testing of the purified compounds revealed that these biflavonoids were the primary bioactive compounds responsible for NO inhibition, cyctotoxicity and α-glucosidase activity in G. prainiana leaf extracts (Table 1).

 

IC50 (µg/mL* or µM)

Cytotoxic activity against

RAW264.7 cells

Nitric oxide

inhibition

α – Glucosidase

inhibition

Crude extract*

>100

17.55± 2.37

29.70± 2.10

Fraction GPL1*

13.10 ± 7.40

22.58 ± 2.61

111.80± 40.90

Fraction GPL2*

>100

44.27± 4.27

15.70± 1.80

Fraction GPL3*

>100

51.14 ± 14.21

>200

Fraction GPL4*

>100

> 71.40

>200

Compound 1

>179

75.20±1.10

16.16± 4.49

Compound 2

>185

44.80 ± 12.20

8.64 ± 1.77

Compound 7

>200

>200

10.39± 4.23

Compound 8

>181

58.60± 0.40

6.34 ± 0.04

Compound 9

>179

46.00± 0.70

14.40± 4.56

Compound 10

>200

>200

43.75± 0.83

Fucoidan*

Not used in cytotoxic and Nitric oxide assay

0.8± 0.2

Table 1: Cytotoxic activity against RAW 264.7 cells, Nitric Oxide (NO) inhibition in RAW 264.7 cells and α - glucosidase inhibiting activity of extracts, fractions and purified compounds from G. prainiana leaf. 

*Inhibitory Concentration (IC50) is in µg/mL for extracts and fractions,

Inhibitory Concentration (IC50) is converted to μM for purified compounds.

5.1. Cytotoxicity and NO inhibition of G. prainiana extracts and bioactivity-guided fractionation

The crude leaf extract exhibited good NO inhibition with an IC50 of 17.55 ± 2.37 µg/mL, without showing cytotoxic activity against RAW 264.7 cells (Table 1). The crude extract was fractionated using VLC column to yield four fractions (GPL1 - 4), which were subjected to NO inhibition assays. GPL1 demonstrated the highest inhibitory activity with IC50 of 22.58 µg/mL, but was also cytotoxic towards RAW264 cells (IC50 = 13.10 ± 7.40 µg/mL). The inhibitory activity of GPL1 extract was mainly due to the cell death.GPL2 showed moderately good NO inhibition (IC50 of 44.27 ± 4.27 µg/mL) without showing cytotoxicity towards RAW264 cells (Table 1). GPL3 also showed moderate NO inhibitory activity with IC50 of 51.10 ± 14.20 µg/mL without showing cytotoxicity towards RAW264 cells. GPL4 fraction did not show any activity at the maximum test concentrations.

Based on these results we focused on GPL2 for further fractionation. GPL1 was deemed too cytotoxic for accurate assessment of nitric oxide assay, whereas GPL4 was not active. GPL3 was less active than GPL2 and chemical profiling revealed overlap in the compound composition, suggesting the same bioactive compounds were likely to be present but some of the constituents in GPL2 appeared in greater amounts than GPL3 extract. Therefore, isolation was prioritised on GPL2 extract.

5.2. Structural elucidation of biflavonoids from G. prainiana leaf extract fraction GPL2

Further investigation on GPL2 fraction led to the isolation and identification of compounds (1, 2, 7, 8, 9 and 10). However, only the structure elucidation for compounds 7-10 are presented here in detail as they are being reported for the first time from this species.

5.2.1. Morelloflavone-7″-O-β-glucoside (7): Compound 7 was obtained as a yellowish brown solid. The UV spectrum of 7 clearly indicated three prominent peaks at 210, 290 and 350 nm, which is similar to compound 1, but 7 showed higher polarity than compound 1, eluting earlier in the C18 HPLC chromatogram (Figure 3). The mass spectrum of 7 showed [M+1]fragment at m/z 719, suggesting the molecular weight is 718 a.m.u. The presence of the fragment ion at m/z 557 suggests the loss of a sugar moiety (162 a.m.u.) from 7, which indicates the presence of a pyranoside sugar in the structure. This evidence suggests the structure of 7 is similar to morelloflavone but with the presence of the sugar moiety.

The 1HNMR spectrum of (Table 2) showed three pairs of ortho coupling proton signals (H-2′ and H-3′; H-5′ and H-6′; and H-5'''and H-6''') and two pairs of meta coupling protons (H-6 and H-8; H-2''' and H-6''') in benzene ring substitution; one oxygenated methine proton at δ 6.17 (H-2) coupling with another methine proton at δ 4.84 (H-3) of which the signal is underneath the solvent peak, and two singlets corresponding to olefinic protons at δ 6.47 (H-3″) and δ 6.64 (H-6″). The rest of 1H signals came from the sugar moiety.

Compound

7a

 

8b

 

9b

 

10a

Position

δ (1H)

δ (13C)

 

δ (1H)

δ (13C)

 

δ (1H)

δ (13C)

 

δ (1H)

δ (13C)

2

6.17, d, J = 11.6 Hz

82.52

 

 

164.99

 

5.72, d, J = 12.1 Hz

81.45

 

5.72, d, J = 12.0 Hz

82.23

3

4.84d

51.12

 

6.71, s

104.47

 

4.56, d, J = 12.1 Hz

47.33

 

4.67, d, J = 12.0 Hz

50.10

4

 

197.54

 

 

183.20

 

 

196.79

 

 

198.59

5

 

165.87

 

 

163.73

 

 

163.66

 

 

165.58

6

5.96, d, J = 2.0 Hz

96.99

 

6.24, d, J = 2.0 Hz

99.83

 

5.91, br s

96.20

 

6.39, s

96.77

7

 

165.29

 

 

164.94

 

 

166.30

 

 

164.88

8

5.94, d, J = 2.0 Hz

97.72

 

6.50, d, J = 2.0 Hz

94.90

 

5.89, br s

95.50

 

5.91, br s

97.48

9

 

168.0

 

 

158.97

 

 

162.10

 

 

162.09

10

 

103.14

 

 

105.48

 

 

101.23

 

 

104.42

5-OH

 

 

 

 

 

 

12.13, s

 

 

 

 

1′

 

130.81

 

 

123.49

 

 

127.60

 

 

130.59

2′

7.16, d, J = 8.6 Hz

129.61

 

8.12, d, J = 2.4 Hz

132.74

 

7.14, br d, J = 8.1 Hz

129.12

 

7.15, br d, J = 8.0 Hz

130.09

3′

6.38,d, J = 8.6 Hz

115.61

 

 

121.07

 

6.72, br d, J = 8.4 Hz

114.84

 

6.70, br d, J = 8.0 Hz

115.77

4′

 

158.59

 

 

160.49

 

 

157.74

 

 

158.65

5′

6.38, d, J = 8.6 Hz

115.61

 

7.24, d, J = 8.7 Hz

117.73

 

6.72, br d, J = 8.4 Hz

114.84

 

6.70, br d, J = 8.0 Hz

115.77

6′

7.16, d, J = 8.6 Hz)

129.61

 

8.02, dd, J = 8.7, 2.4 Hz

128.92

 

7.14, br d, J = 8.1 Hz

129.12

 

7.15, br d, J = 8.0 Hz

130.09

2″

 

166.46

 

 

164.87

 

5.46, br t, J = 11.0 Hz

78.52

 

5.66, br d, J = 12.0Hz

83.92

3″

6.47, s

103.76

 

6.69, s

104.33

 

2.60, m

2.67, m

42.77

 

5.08, m

48.95

4″

 

184.17

 

 

183.57

 

 

196.79

 

 

199.02

5″

 

163.05

 

 

162.60

 

 

162.46

 

 

165.36

6″

6.64, s

99.31

 

6.45, s

100.40

 

5.91, br s

96.20

 

6.25, s

97.27

7″

 

162.33

 

 

162.92

 

 

160.69

 

 

164.09

8″

 

104.10

 

 

104.66

 

 

101.65

 

 

104.18

9″

 

156.78

 

 

163.04

 

 

163.76

 

 

161.10

10″

 

106.50

 

 

105.50

 

 

101.14

 

 

104.80

5″-OH

-

-

 

-

-

 

12.17, s

-

 

-

-

1'''

 

123.29

 

 

124.46

 

 

129.80

 

 

131.61

2'''

7.38, d, J = 2.0 Hz

114.35

 

7.72, d, J = 9.0 Hz

129.02

 

6.88, br s

113.46

 

6.81, br s

114.31

3'''

 

147.19

 

6.92, d, J = 9.0 Hz

115.40

 

 

145.28

 

 

146.42

4'''

 

151.60

 

 

163.50

 

 

145.69

 

 

146.82

5'''

6.93, d, J = 8.4 Hz

117.14

 

6.92, d, J = 9.0 Hz

115.40

 

6.81, br d, J = 7.85 Hz

115.32

 

6.82, br d, J = 8.0 Hz

116.20

6'''

7.33, dd, J = 8.4, 2.0 Hz

120.89

 

7.72, d, J = 9.0 Hz

129.03

 

6.68, m

117.68

 

6.65, br d, J = 8.0 Hz

119.26

4'''-OCH3

-

-

 

3.79, s

55.50

 

-

-

 

-

-

1′′′′

5.12c, d, J = 7.8 Hz

101.77

 

-

-

 

-

-

 

5.05c, m

101.35

2′′′′

3.29d, m

75.05

 

-

-

 

-

-

 

5.05d,m

75.15

3′′′′

3.46, t, J = 9.0 Hz

78.57

 

-

-

 

-

-

 

3.48, m

78.17

4′′′′

3.37, md

71.26

 

-

-

 

-

-

 

3.39, m

71.09

5′′′′

3.52, ddd, J = 9.0, 2.3, 5.8 Hz

78.89

 

-

-

 

-

-

 

3.50, m

78.43

6′′′′

3.92, dd, J =12.3, 2.3 Hz

3.72, dd, J =12.3, 5.8 Hz

62.67

 

-

-

 

-

-

 

3.92, br d, J = 12.0 Hz

3.75,m

62.35

Table 2: 1H and 13C NMR assignments for Compounds 7-10.

aCD3OD, 500 MHz

bDMSO-d6,500 MHz

cAnomeric proton

dUnderneath solvent peak

The oxymethine protons of the sugar moiety (H-3′′′′, H-5′′′′) showed coupling constants of ca 9 Hz (Table 2) indicating that the protons were in axial orientation that suggested a glucose. The anomeric proton signal at δ 5.12 (d, H-1′′′′) gave a coupling constant of 7.8 Hz that is indicative of an axial orientation and was therefore deduced to be β-glucose. Thus, the interglycosidic linkage of glucose in 7 was confirmed as a β-linkage.

The 13C NMR data of 7 (Table 2) showed thirty six carbons consisting of ten oxygenated benzene carbons, two carbonyl carbons , nine signals from methine benzene carbons, five quaternary carbons from benzene rings, one oxygenated quaternary carbon [δ 166.46 (C-2″)], one oxygenated methine [δ 82.52 (C-2)] and two methine [δ 51.12 (C-3) and δ 103.76 (C-3″)] from alicyclic rings. The rest of the signals are the five methines [δ 71.26 (C-4′′′′), δ 75.05 (C-2′′′′), δ 78.57 (C-3′′′′), δ 78.89 (C-5′′′′) and δ 101.77 (C-1′′′′)] and one methylene [δ 62.67 (C-6′′′′)] from the sugar moiety.

The chemical shift of the anomeric carbon (C-1′′′′) at δ 101.77 indicated that the sugar moiety was on O-glucoside. The connection of glucose to the aglycone was suggested by inspection of the HMBC spectrum of 7. The 3JCH correlation of the anomeric proton signal (δH 5.12, H-1′′′) to the carbon signal at δ 162.33 (C-7″) confirmed the glucose was linked to this position. The structure of 7 was thus established as morelloflavone-7″-O-β-glucoside with molecular formula of C36H30O16 (molecular weight = 718.62 g/mol; PubChem CID 101973939) and further confirmed by comparison with published NMR data [23].

Compound 7, also known as fukugiside, was previously found in a range of Garcinia species including G. multiflora heartwood, G. cymosa stem bark, G. spicata stem bark, G. xanthocymus leaf, G. intermedia fruit, G. livingstonie fruit and G. brasiliensis fruit [23-27].

5.2.2. 4′′′-methyl amentoflavone (8): Compound 8 has an [M+1]ion at m/z 553, indicating the molecular weight of 552 a.m.u (Figure 2). The UV spectrum showed a similar pattern to that of the UV spectrum of compound 2 with λmax absorption at 198, 270 and 332 nm.

The 1H NMR and 13C NMR signals (Table 2) were similar to amentoflavone (2) except for the presence of a methoxy signal at δ 3.79 and δ 55.50, respectively. The position of the methoxy group in compound 8 was confirmed by the correlation of methoxy signal to δ 163.50 (C-4′′′) in the HMBC spectrum. Therefore, the structure of compound 8 was confirmed as 4′′′-methyl amentoflavone with molecular formula of C31H20O10 (molecular weight = 552.69 g/mol; PubChem CID 53206444). This compound is also known as podocarpusflavone A [28-30].

The 1H NMR and 13C NMR data of compound 8 were consistent with that reported previously for podocarpusflavone A from Ouratea multiflora and Podocarpus neriifolius D. Don [31,32]. This compound is reported for the first time from Garcinia prainiana.

5.2.3. 2″,3″-dihydromorelloflavone (9): Compound 9 was obtained as a pale brown gum and the mass spectrum (APCI, positive mode) showed an [M+1]+ ion at m/z 559, indicating a molecular weight of 558 a.m.u (Figure 2). Its UV spectrum showed absorption bands at λmax 198, 226 and 290 nm. The 1H NMR data (Table 2) contained signals from two hydroxyl protons at δ 12.13 (5-OH) and δ 12.17 (5″-OH), five doublet signals (H-2; H-3; H-2′ and H-6′; H-3′ and H-5′; and H-5′′′), three singlet signals from benzene methines (H-8; H-6; and H-6″ and H-2′′′), one broad triplet signal from alicyclic methine (δ H 5.46, J = 10.95 Hz, H-2″) and three multiplet signals representing the methylene protons H-3″ and one of the methylene protons of H-6′′′.

The 13C NMR data showed thirty carbons signals (Table 2) consisting of nine oxygenated quaternary carbon from benzene rings, ten methine carbon signals from benzene rings, two carbonyl carbons at δ 196.79, five quaternary carbon signals from benzene rings, three methine carbon signals from alicyclic rings (C-2, C-3 and C-2″), and one methylene carbon signal from an alicyclic ring at δ 42.77 (C-3″).

Comparison of carbon signals from compound 9 with published data [33], supported that the structure of compound 9 is 2″,3″-dihydromorelloflavone with molecular formula of C30H22O11 (molecular weight = 558.50 g/mol; PubChem CID 11467081), which has been reported from Garcinia subelliptica. This compound is reported for the first time from G. prainiana. Compound 9, also known as GB-2a, was also previously isolated from G. spicata and G. multiflora and G. perusii [25,34,35].

5.2.4. 2″,3″-dihydromorelloflavone-7″-O-glucoside (10): Compound 10 was obtained as a yellow gummy solid. The UV spectrum showed similarity to 9 with the λmax of 198, 226 and 288 nm. The mass spectrum (APCI, positive mode) showed an [M +1]+ fragment at m/z 721 (Figure 2) suggesting the molecular mass is 720 a.m.u. The presence of a fragment at m/z 559 in the mass spectrum of 10 indicated the loss of 162 a.m.u from the molecular ion peak, which suggests the compound contains a pyranoside sugar.

The 1H NMR data (Table 2) contained four doublet aromatic signals [(δ H 7.15, 2H, J = 8.0 Hz, H-2′ and H-6′); (δ H 6.82, 1H, J = 8.0 Hz, H-5′′′); (δ H 6.70, 2H, J = 8.0 Hz, H-3′ and H-5′); and (δ H 6.65, 1H, J = 8.0 Hz, H-6′′′)] from benzene methines, four singlet signals from benzene methines [(δ H 6.81 (H2′′′); (δ H6.39, H-6); (δ H 6.25, H-6″); and (δ H 5.91, H-8)], three doublet signals from alicyclic methine [(δ H 5.72, H-2); (δ H 5.66, H-2″); and (δ H4.67, H-3], one multiplet signal at δ H 5.08 (H-3″) and seven oxymethine proton signals from a sugar moiety [δ 5.05 (H-1′′′′ (anomeric proton)), δ 3.92 (H-6′′′′a), δ 3.75 (m, H-6′′′′b), δ 3.50 (H-5′′′′), δ 3.48 (H-3′′′′), δ 3.39 (H-4′′′′) and δ 3.32 (H-2′′′′ (underneath the solvent peak)). The 13C NMR data (Table2) showed thirty six carbons signals from 10, consisting of nine oxygenated quaternary carbons from benzene rings [δ 165.58 (C-5), δ 164.88 (C-7), δ 162.09 (C-9), δ 158.65 (C-4′), δ 165.36 (C-5″), δ 164.09 (C-7″), δ 161.10 (C-9″), δ 146.42 (C-3′′′) and δ 146.82 (C-4′′′)], ten methine carbon signals from benzene rings [δ 96.77 (C-6), δ 97.48 (C-8), δ 97.27 (C-6″), two carbons at δ 130.09 (C-2′ and C-6′), two carbons at δ 115.77 (C-3′ and C-5′), δ 114.31 (C-2′′′), δ 116.20 (C-5′′′) and δ 119.26 (C-6′′′)], two carbonyl carbons at δ 198.59 (C-4) and δ 199.02 (C-4″), five quaternary carbon signals from benzene rings [104.18 (C-8″), δ 104.42 (C-10), δ 104.80 (C-10″), δ 130.59 (C-1′) and δ 131.61 (C-1′′′)], three methine carbon signals from alicyclic rings [δ 50.10 (C-3), δ 83.92 (C-2″) and δ 82.23 (C-2)], one methylene carbon signal from an alicyclic ring at δ 48.95 (C-3″) and six carbon signals from a sugar moiety [five oxygenated methines, (δ 71.09 (C-4′′′′), δ 75.15 (C-2′′′′), δ 78.17 (C-3′′′′), δ 78.43(C-5′′′′) and δ 101.35 (C-1′′′′)) and one oxygenated methylene (δ 62.35 (C-6′′′′))].

The location of the sugar substituent was confirmed at C-7 by the correlation of proton signal of H-1′′′′ (anomeric proton) with carbon signal at δ c164.88 (C-7) in HMBC spectrum.

The 1H NMR and 13C NMR data were similar to 9 except for the presence of 6 carbon signals and 7 proton signals from the sugar moiety. Through the comparison of 13C NMR data of compound 10 and 9, the structure was confirmed as 2″,3″-dihydromorelloflavone-7″-O-glucoside with molecular formula of C36H32O16 (molecular weight = 720.63 g/mol; HMDB30609), which is also known as xanthochymuside. Xanthochymuside has been isolated from G. multiflora [25]. However, there are no published NMR data available. Therefore, this is the first report of the 13C NMR and 1H NMR assignments of xanthochymuside.

5.2.5. Morelloflavone (1): The mass spectrum of 1 showed an [M+1]ion at m/z 557 (Figure 2), which indicated the molecular weight was 556 a.m.u. The NMR spectral data from 1 were consistent with published data for morelloflavone from Garcinia densivenia [36] with molecular formula of C30H20O11 (molecular weight = 556.48 g/mol; PubChem CID 5464454).

1HNMR data (CD3OD): δ 6.18 (1H, d, J =2.05, H-6 ), δ 6.38 (1H,s, H-6″), δ 6.42 ( 1H, d, J = 2.0, H-8), δ 6.59(1H, H-3), δ 6.61 (1H,s, H-3″ ), δ 6.72 (2H, d, J = 8.6, H-3′′′and H-5′′′), δ 7.12 (1H, d, J = 8.6, H-5′), δ 7.52(2H, d, J = 8.8, H-2′′′and H-6′′′), δ 7.89 (1H, dd, J= 8.6, 2.25, H-6′), δ 7.93 (1H, d, J = 2.25,H-2′).

13CNMR data (CD 3OD): δ 184.4 (C-4), δ 183.98 (C-4″), δ 166.22 (C-7), δ 166.34 (C-2″), δ 166.08 ( C-2), δ 163.31 (C-5), δ 162.69 (C-5″), δ 162.98 (C-4′′′), δ 162.65( C-7″), δ 161.04 (C-9), δ 161.04 (C-4′), δ 156.63 (C-9″), δ 132.94 (C-2′), δ 129.45 (C-6′′′), δ 129.45 (C-2′′′), δ 129.11 (C-6′), δ 123.43 (C-1′), δ 123.37 (C-1′′′), δ 121.64 (C-3′), δ 117.45 (C-5′), δ 116.98 (C-5′′′), δ 116.98 (C-3′′′), δ 105.48 (C-10″), δ 105.42 (C-8″), δ 105.28 (C-10), δ 104.17 (C-3″), δ 103.54 (C-3), δ 100.06 (C-6″), δ 100.3 (C-6), δ 95.28 (C-8)

5.2.6. Amentoflavone (2): The mass spectrum of 2 showed an [M+1]+ ion at m/z 539, which indicated the molecular weight was 538 a.m.u. The 1H NMR and 13C NMR data of 2 were compared to the literature and found to be identical to published data for amentoflavone from Ouratea multiflora [31] with molecular formula of C30H18O10 (molecular weight = 538.46 g/mol; PubChem CID 5281600).

1H NMR data (CD3OD): δ 6.18 (1H, d, J =2.05, H-6 ), δ 6.38 (1H,s, H-6″), δ 6.42 ( 1H, d, J = 2.0, H-8), δ 6.59 (1H, H-3), δ 6.61 (1H,s, H-3″ ), δ 6.72 (2H, d, J = 8.6, H-3? and H-5?), δ 7.12 (1H, d, J = 8.6, H-5′), δ 7.52 (2H, d, J = 8.8, H-2''' and H-6'''), δ 7.89 (1H, dd, J= 8.6, 2.25, H-6′), δ 7.93 (1H, d, J = 2.25,H-2′).

13C NMR data (CD 3OD): δ 184.4 (C-4), δ 183.98 (C-4″), δ 166.22 (C-7), δ 166.34 (C-2″), δ 166.08 ( C-2), δ 163.31 (C-5), δ 162.69 (C-5″), δ 162.98 (C-4′′′), δ 162.65 ( C-7″), δ 161.04 (C-9), δ 161.04 (C-4′), δ 156.63 (C-9″), δ 132.94 (C-2′), δ 129.45 (C-6′′′), δ 129.45 (C-2′′′), δ 129.11 (C-6′), δ 123.43 (C-1′), δ 123.37 (C-1′′′), δ 121.64 (C-3′), δ 117.45 (C-5′), δ 116.98 (C-5′′′), δ 116.98 (C-3′′′), δ 105.48 (C-10″), δ 105.42 (C-8″), δ 105.28 (C-10), δ 104.17 (C-3″), δ 103.54 (C-3), δ 100.06 (C-6″), δ 100.3 (C-6), δ 95.28 (C-8).

5.3. Nitric oxide inhibition and cytotoxicity studies of the isolated biflavonoids

Compounds (1, 2, 7, 8, 9 and 10) were subjected to NO inhibition and cytotoxicity assays (Table 1). Amentoflavone (2) demonstrated the highest NO inhibition activity with an IC50 of 44.80 ± 12.20 µM, followed by 2″,3″-dihydromorelloflavone (9), 4′′′-methyl amentoflavone (8), and morelloflavone (1) with an IC50 of 46.00 ± 0.70, 58.60 ± 0.40 and 75.20 ± 1.10 µM, respectively. None of these compounds showed significant cytotoxic activity at the maximum test concentrations (Table 1). The high NO inhibition of amentoflavone (2) compared to 4′′′-methyl amentoflavone (8) could be due to reduced binding of hydroxyl group and the presence of the methoxy group at 4′′′ position, while the loss of a double bond at positions 2″ and 3″ in compound 9 resulted in a the two-fold increase in NO inhibition activity compared to morelloflavone.

Morelloflavone (1) and amentoflavone (2) are well known compounds and these compounds have been previously reported from G. prainiana leaves by Klaiklay et al., [5]. Morelloflavone has been previously reported to inhibit the HMG CoA reductase, the rate limiting enzyme of the cholesterol biosynthetic pathway in vitro [37]. This compound was also reported to inhibit vascular smooth muscle cell migration into coronary stents, potentially relevant in cases of coronary artery bypass [38]. Amentoflavone (2) was also found to be bioactive in several previous studies on anticancer and anti-inflammatory agents. It is a good inhibitor of human Cathepsin B, a cysteine protease implicated in the pathology of some inflammatory diseases and cancer [39]. Consistent with our study, this compound also was reported as an inhibitor of nitric oxide synthase through the inhibition of NF-κB activation in RAW 264.7 cells [40]. Previously, amentoflavone was also reported to inhibit cAMP-phosphodiesterase which could reduce inflammation in adipose tissues [41]. Our results expand the current knowledge on the anti-inflammatory and cytotoxic properties of 4′′′-methyl amentoflavone (8) and 2″,3″-dihydromorelloflavone (9).

5.4. α-Glucosidase inhibitory activities of extracts, fractions and compounds from G. prainiana King

Type 2 diabetes is a chronic metabolic disorder that results from a high blood glucose level [42]. Administration of α-glucosidase inhibitor has been proposed as treatment for type 2 diabetes, since it works by preventing the digestion of carbohydrates [43]. Therefore, postprandial blood glucose needs to be controlled in the early treatment of diabetes.

In the screening of a crude extract of G. prainiana leaf, the extract demonstrated moderately potent α-glucosidase inhibitory with IC50 of 29.70 µg/mL (Table 1). Further investigation on fractions, GPL1 to GPL4 revealed that GPL2 showed the highest activity with IC50 of 15.70 µg/mL, follow by GPL1 with an IC50 nearly ten times lower (Table 1). The other two fractions did not show any activity. The six isolated compounds from GPL-2 were then subjected to the α-glucosidase assay. 4′′′-methyl amentoflavone (8) exhibited the highest α-glucosidase inhibition with an IC50 of 6.34 ± 0.04 µM follow by amentoflavone (2), morelloflavone-7″-O-glucoside (7), 2″,3″-dihydromorelloflavone (9), morelloflavone (1), and 2″,3″-dihydromorelloflavone-7″-O- -glucoside (10), (Table 1).Amentoflavone (2) showed lower activity than 4′′′-methyl amentoflavone suggesting that the loss of methoxy group at position 4′′′ effected α-glycosidase inhibitory activity. Previously, Kim et al.,[44] demonstrated the α-glucosidase inhibitory of amentoflavone in a study of twenty-one naturally occurring flavonoids [44]. Amentoflavone was reported to show one of the strongest inhibitory activities in the study. However, this is the first report of the 4′′′-methyl amentoflavone activity against α-glucosidase.

The significant loss of activity in 2″,3″-dihydromorelloflavone-7″-O-glucoside (10) as compared to morelloflavone-7″-O-glucoside (7) pointed out the importance of the double bond at position 2″ and 3″ in maintaining the α-glucosidase inhibitory activity of morelloflavone.A similar pattern also can be seen between 2″,3″-dihydromorelloflavone (9) and morelloflavone (1). It seems likely that the presence of the glycoside moiety does improve the α-glucosidase inhibition. The glycoside moiety in 7″ enhanced the inhibition of α-glucosidase, possibly through the improvement of solubility.

The presence of some biflavonoids with anti-glucosidase activity in the leaf extract of G. prainiana King suggests that this species has potential for further development as an anti-diabetic food. Further structure-activity relationship studies would be worthwhile on the biflavanoid class of compounds more generally to optimise the development of anti-diabetic drugs.

6. Conclusion

Our work provides scientific evidence for the potential to value-adding G. prainiana King, an underutilised plant, as anti-inflammatory and anti-diabetic food source, as well as a good source of anti-oxidant constituents. Phytochemical investigations revealed that this plant is a rich source of biflavonoids such as amentoflavone (2), 4′′′-methyl amentoflavone (8), morelloflavone (1), morelloflavone-7″-O-glucoside (7), 2″,3″-dihydromorelloflavone (9) and 2″,3″-dihydromorelloflavone-7″-O-glucoside (10), with potential to be developed for human health applications. Considering our data and information on local and traditional food uses of the species, the inclusion of the leaves as an ingredient with health benefits in soups, salads and other culinary products should be encouraged, provided that it can be demonstrated to be safe.

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