Cytotoxic, Nitric Oxide and Α-Glucosidase Inhibitory Activities of Biflavonoids from Garcinia Prainiana King 1

Advances in Applied Chemistry and Biochemistry ISSN: 2652-3175 10.33513/ACBC/1901-08 OCIMUM Jabit ML et al. Adv Appl Chem Biochem 2019(1): 55-67.

Research Article Cytotoxic, Nitric Oxide and α-Glucosidase Inhibitory Activities of Biflavonoids from Garcinia prainiana King 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 Mohd Lip Jabit1, Mohd Nazrul Hisham Daud1* extracts and tested these for cytotoxicity, Nitric Oxide (NO) inhibition in mouse and Shamsul Khamis2 macrophages (RAW 264.7) and α-glucosidase inhibition. Extract of G. prainiana leaf showed potent inhibition of NO production from lipopolysaccharide 1Malaysian Agriculture Research Institute, Serdang, stimulated RAW 264.7 cells, with IC of 17.55 ± 2.37 µg/mL. Bioactivity- Selangor, Malaysia 50 guided fractionation led to the isolation of four biflavonoids from the extract: 2School of Environmental and Natural Resource morelloflavone 1( ), amentoflavone 2( ), 4‴-methyl amentoflavone 8( ) and Sciences, National University of Malaysia, Bangi, 2″,3″-dihydromorelloflavone or GB-2a (9); and the glucosides of 1 and 9, Selangor, Malaysia morelloflavone-7″-O-β-glucoside (7) and 2″,3″-dihydromorelloflavone-7″- O-β-glucoside (10), respectively. The four biflavonoid compounds1, 2, 8 and inhibited NO production within the range of IC = 44.80 - 75.20 µM. The Received: 22 July 2019 9 50 α-glucosidase inhibition assay revealed variable activity from IC of 6.34 µM Accepted: 19 August 2019 50 (4‴-methyl amentoflavone ( )) to IC of 43.75 µM (morelloflavone glucoside Version of Record Online: 06 September 2019 8 50 (7)). This study highlights the potential for value-adding G. prainiana as an Citation anti-inflammatory and anti-diabetic food source. Jabit ML, Daud MNH, Khamis S (2019) Cytotoxic, 2. Keywords Nitric Oxide and α-Glucosidase Inhibitory Activities of Biflavonoids fromGarcinia prainiana King. Adv Anti-Diabetic; Anti-Inflammatory; Bioactivity Guided Isolation; Dereplication; Appl Chem Biochem 2019(1): 55-67. Flavonoid; Guttiferae

Correspondence should be addressed to Mohd Nazrul Hisham Daud, Malaysia 3. Introduction E-mail: [email protected] Conserving and promoting the potential of rare fruit offers opportunities for developing high-value new crops. This is one of the programmed activities Copyright of the Malaysian Agricultural Research and Development Institute, who has Copyright © 2019 Mohd Nazrul Hisham Daud collected more than 58 underutilised fruits of 32 different species from 21 et al. This is an open access article distributed genera from the Peninsular and East Malaysia for planting in a special plot under the Creative Commons Attribution License area. These fruits are known to be associated with many nutritional and which permits unrestricted use, distribution, and medicinal properties [1]. One of these rare fruits is Garcinia prainiana King reproduction in any medium, provided the original known as Button Mangosteen or Kechupu which is a small to moderate- author and work is properly cited. 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 ofG. 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].

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

Figure 1: Chemical structure of biflavonoids isolated fromG. prainiana King leaf.

Submit Manuscript Advances in Applied Chemistry and Biochemistry [ISSN: 2652-3175] .02. 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. DOI: 10.33513/ACBC/1901-08

Based on the traditional use, the inclusion of G. prainiana G. prainiana leaf was subjected to Vacuum Liquid Column leaves in the diet may help to maintain a safe blood glucose Chromatography (VLC). The column was packed with normal level, even with the high intake of carbohydrate. High blood phase silica, 200-425 mesh (Sigma-Aldrich) and eluted with glucose levels may otherwise facilitate the accumulation of following solvents accordingly, hexane (Merck, Analysis grade fat, so indirectly, the use of these plants could help people EMSURE®) , mixture of hexane-ethyl acetate (1:1), ethyl reduce fat in their body. The characterisation ofα- glucosidase acetate (Merck, Analysis grade EMSURE®), mixture of ethyl inhibitor from the G. prainiana leaves may help to rationalise acetate-methanol (1:1), and finally methanol(Merck, Analysis the usage of the leaves from this plant. grade EMSURE®). The VLC fractionation was repeated ten times to yield 120 g of hexane-ethyl acetate (1:1) fraction The main aim of this paper was to investigate the bioactive (GPL1), 60 g of ethyl acetate fraction (GPL2), 30 g of ethyl constituents from G. prainiana leaves. Bioactivity-guided acetate-methanol (1:1) fraction (GPL3) and 7.1 g of methanol fractionation was employed to ensure any active constituents fraction (GPL4). All fractions were then subject to bioactivity were not excluded from the extract of interest. Liquid assays (Sections 4.3 - 4.5) and chemical profiling (Section 4.6). Chromatography Mass Spectrometry (LCMS) profiling was used for chemical dereplication of known biflavonoids and 4.3. Cytotoxicity assay Nuclear Magnetic Resonance (NMR) spectroscopy was used for structural elucidation of novel compounds in this species. Cytotoxicity against RAW 264.7 murine leukemic monocyte NO and α-glucosidase inhibition assays were used to evaluate macrophages was assayed in 96-well plates using the ATPlite™ the bioactivity of the fractions and compounds. Here we report assay kit (PerkinElmer, Glen Waverley, Australia) with four new biflavonoids in this species and their associated chlorambucil (Sigma-Aldrich, C025) as a positive control. anti-inflammatory activity, along with particularly potent Cells were grown in clear 96-well plates. The growth medium α-glucosidase inhibitory activity in one biflavonoid glycoside. consisted of colour-free Dulbecco’s modified Eagle’s medium Further characterisation of these bioactive biflavonoids in containing 10% (v/v) fetal bovine serum (FBS; Interpath, G. prainiana leaves could help value-adding this species as a Heidelberg, Australia), L-glutamine (2 mM, Sigma-Aldrich, functional or medicinal food; i.e., a food with health properties G7513), sodium pyruvate (1 mM, Sigma-Aldrich, C8636), over and above the basic nutritional properties. penicillin (200 U/mL, Sigma-Aldrich, P4333) and streptomycin (200 μg/mL, all from Invitrogen, Mulgrave, Australia). RAW 4. Materials and Methods 264.7 cells were plated at a concentration of 30000 cells/well (90 μL of cell suspension/well), respectively. Samples and control 4.1. Plant materials compound were dissolved in DMSO at six concentrations and further diluted 20-fold in the medium. Then, samples Garcinia prainiana King leaves were obtained from an and control compound were added to the cell suspension at underutilised fruit plot of the Malaysian Agricultural Research 10 μL/well, and the plates were incubated at 37°C with 5% and Development Institute (MARDI) in Serdang, Selangor, CO2 for 24 h. Following incubation, cell lysates were assayed Malaysia and a voucher specimen SK 06/01 was deposited in for ATP with the ATPlite™ assay kit as per the manufacturer’s the Herbarium of Institute of Bioscience, University Putra instructions. The mammalian cell lysis solution (50μ L) was Malaysia. The plant raw material was cleaned and air-dried added to each well of the cell culture microplate. The plate at room temperature and ground to 1 mm particle size and was shaken on an orbital microplate shaker (500 rpm, 5 min), kept in dark coloured plastic bags. then the substrate solution (50 μL/well) was added, and the plate was further shaken (500 rpm, 5 min). The plate was 4.2. Preparation and chemical profiling of extracts dark adapted for 10 min, and the luminescence measured on a and semi-purified fractions Wallac 1450 Microbeta luminescence counter (Wallac, Turku, Finland). Half-maximal inhibitory concentration (IC ) values 3 kg dried and ground leaves were macerated in 5 L of methanol 50 (Merck, Analysis grade EMSURE®) for 3 days. Then, the were calculated using GraphPad Prism version 5 (La Jolla, CA, solvent was filtered with Whatman filter paper no.1 and the USA). The assay was repeated three times. filtrate was evaporated to dryness under reduced pressure (5.3 4.4. Nitrite (Griess) assay for NO inhibition kPa) to give a dark brown gum crude methanol extract. The solvent was replenished with fresh methanol and the procedure RAW 264.7 cells were cultivated as described above. Cell 6 was repeated 8 times to yield 200 g of crude methanol extract. suspensions (120 μL/well, 10 cells/mL) were added to the wells of a 96-well microplate and incubated for 20 h (37°C,

Approximately, 20 g of crude methanolic extract from 5% CO2), after which test compounds (dissolved in DMSO

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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. DOI: 10.33513/ACBC/1901-08 and further diluted 20-fold in the medium) were added to which have not previously been reported from this species for the cell suspension at 10 μL/ well. Following incubation for isolation, structure elucidation and bioactivity assessment. 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 The chemical profiling of extracts and fractions was performed was centrifuged (1500g, 3 min), and 90 μL of the supernatant using the Agilent 1200 LCMS system with Diode Array Detector transferred to a clear flat-bottom assay plate (PerkinElmer, Glen (DAD) coupled with a single quadruple mass spectrometer (APCI mode). The column used was a Phenomenex Luna C Waverley, VIC, Australia) and assayed immediately for nitrite. 18 column (5 μm, 250 mm x 4.6 mm i.d.). The mobile phase Nitrite standards (0, 3.13, 6.26, 12.5, 25, 50 and 100 used was Millipore deionised water with 0.005% trifluoroacetic μM) were prepared in the medium. Then 90 μL of each acid (A) and acetonitrile with 0.005% trifluoroacetic acid standard and cell supernatant was transferred to a flat-bottom (B). The gradient system used was: 0 - 2 min., 10% B; 7 - 12 microplate (Greiner Bio-One, Frickenhausen, Germany) with min., 50% B; 17 - 22 min., 95% B; 27 - 32 min., 10% B. The 90 μL of Griess reagent (0.1% N-1-naphthylethylenediamine flow rate used was 0.75 mL/min and sample injection volume dihydrochloride, 1% sulfanilic acid in 5% phosphoric acid) was 20 µL. The mass detector conditions were set as follows: added to each well, followed by incubation (23°C, 20 min) on APCI positive mode from 50 to 1000 m/z, capillary voltage an orbital plate shaker. Following incubation, the absorbance set at -4000 V, needle temperature set at 340°C and gas flow was read at 550 nm in a Wallac 1450 Microbeta plate reader rate was at 5 L/min. (Wallac, Turku, Finland). Samples and controls were assayed in triplicate. Standard calibration curves were calculated from Due to lower cytotoxicity and good NO inhibitory activity nitrite standard solution, and R2 values determined from the GPL2 fraction was the main focus of further investigation. the regression line of best fit to verify linearity. Mean and Based on chemical profiling using LC-APCI-MS (Figure 2), + standard deviations were calculated from the replicates. The NO four compounds with [M+1] at m/z 719, 721, 559, and 553, (measured as nitrite) production in sample wells was calculated were hitherto not known from G. prainiana. Therefore, further as a percentage of the production in solvent (DMSO) control isolation work was carried out to purify these compounds from wells. The assay was repeated on 3 different occasions. G. prainiana leaf. 4.5. α-Glucosidase assay 4.7. Isolation and structural elucidation of compounds from GPL2 Theα- glucosidase inhibitory activity was determined using a fluorescent method optimized by Payn [21]. The α-glucosidase Ten grams of GPL2 was subjected to C18 Preparative HPLC on enzyme and the substrate, 4-methylumbelliferyl- α-D- a Gilson 322 system with a UV/vis-155 detector, connected to glucopyranoside were purchased from Sigma. The initial an FC204 fraction collector and using a Phenomenex Luna C18 concentration of the substrate solution was 84 µM in sodium column (5 μm, 150 × 21.2 mm i.d.). The mobile phase used acetate buffer pH 5.5. The enzyme solution (45 µL/well) was was Millipore deionised water with 0.005% trifluoroacetic acid mixed with the sample or control (10 μL/well) in a black 96- (A) and methanol with 0.005% trifluoroacetic acid (B). The well microplate (flat bottom) and the reaction was started by gradient system used was: 0 - 5 min., 50% B; 10 -15 min., adding the substrate solution (45 µL/well). The solution was 95% B; 17 - 22 min., 50% B. incubated at 37°C for 30 min and the reaction stopped by In total, thirty fractions were collected and chemical profiling adding 100 mM glycine-sodium chloride solution pH 5.5. by LCMS revealed relatively pure compounds in fraction 22 To determine inhibition, the fluorescence of the solution was (compound 2, 30 mg, [M+1]+ at m/z = 539) and in fraction measured using a Perkin Elmer Wallac Victor 2 plate reader (λ ex 24 (compound 8, 40 mg, [M+1]+ at m/z = 553). The mass 355 nm, λ 460 nm). Half-maximal Inhibitory Concentration em spectra were found to match the chemical profile in figure 2, (IC ) values were calculated using GraphPad Prism version 5 50 showing similar retention times and [M+1]+ at 11.3 min, m/z (La Jolla, CA, USA). Samples were assayed in duplicate. The 539 and 14.3 min, m/z 553, respectively. Based on the chemical assay was repeated at least two times. profiling and dereplication, fractions 13-15 were combined, 4.6. Chemical profiling and dereplication then 50 mg of the combined fraction was subjected to another preparative HPLC fractionation using the same parameters Since some biflavonoids have already been reported from and solvent system above. This yielded compound 9 (23 mg, G. prainiana leaf extract [5], we used chemical profiling and [M+1]+ at m/z = 559) in subfraction 16 and compound 1 (5.7 dereplication procedures [22] to identify new biflavonoids mg, [M+1]+ at m/z = 557) in subfraction 19. Figure 3 shows from the extracts. This procedure prioritised the compounds a comparison of the chemical profile (UV chromatogram at

Submit Manuscript Advances in Applied Chemistry and Biochemistry [ISSN: 2652-3175] .04. 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. DOI: 10.33513/ACBC/1901-08

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

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( ).

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280 nm) of isolated compounds, GPL2 extracts and crude isolated four known biflavonoids that have never been reported extract (GPL). from this species: morelloflavone-7″-O-β-glucoside (7), 4‴-methyl amentoflavone 8( ), 2″,3″-dihydromorelloflavone Further isolation work was then carried out on GPL2 to isolate (9) and 2″,3″-dihydromorelloflavone-7″-O-β-glucoside the other components in the bioactive fraction. Forty grams of (10), along with two previously reported compounds GPL2 extract was fractionated using 200 g of MCI gel CHP20P from this plant; morelloflavone 1( ) and amentoflavone 2( ) (Supelco, Bellafonte, PA, USA) column Chromatography (Figure 1). Bioactivity-guided fractionation of the crude extracts (C1) and yielded 12 fractions. These fractions were combined and testing of the purified compounds revealed that these according to the LCMS chemical profile and yielded fraction biflavonoids were the primary bioactive compounds responsible A (1-4), fraction B (5-8) and faction C (9-12). Fraction A (32 for NO inhibition, cyctotoxicity and α-glucosidase activity in g) was subjected to further chromatography on another MCI G. prainiana leaf extracts (Table 1). column (C2) to yield 46 fractions. Then, fractions 1-7 (1 g) of C2 column chromatography were subjected to C18 (Sepra 5.1. Cytotoxicity and NO inhibition of G. prainiana C18-E, 50 μm, 65A; Phenomenex Torrance, CA, USA) open extracts and bioactivity-guided fractionation column chromatography to yield compound 7 (9.4 mg, [M+1]+ at m/z = 721) from subfractions 20 to 22 and compound 10 The crude leaf extract exhibited good NO inhibition with + (15 mg, [M+1] at m/z = 719) from subfractions 28 to 34. an IC50 of 17.55 ± 2.37 µg/mL, without showing cytotoxic activity against RAW 264.7 cells (Table 1). The crude extract All isolated compounds were dissolved in deuterated Dimethyl was fractionated using VLC column to yield four fractions Sulfoxide (DMSO-d ) or deuterated methanol (CD OD) 6 3 (GPL1 - 4), which were subjected to NO inhibition assays. solvent and structurally elucidated using 13C and 1H NMR GPL1 demonstrated the highest inhibitory activity with IC50 spectroscopy. NMR spectra were acquired on a Bruker of 22.58 µg/mL, but was also cytotoxic towards RAW264 AVANCE II 500 MHz spectrometer. The chemical structures cells (IC50 = 13.10 ± 7.40 µg/mL). The inhibitory activity of were elucidated and confirmed by comparison with previously GPL1 extract was mainly due to the cell death. GPL2 showed published data. moderately good NO inhibition (IC50 of 44.27 ± 4.27 µg/mL) without showing cytotoxicity towards RAW264 cells (Table 5. Results and Discussion 1). GPL3 also showed moderate NO inhibitory activity with IC of 51.10 ± 14.20 µg/mL without showing cytotoxicity This study confirms that G. prainiana leaf extracts, in addition 50 to the fruit, are rich in bioactive biflavonoids. We successfully towards RAW264 cells. GPL4 fraction did not show any

IC50 (µg/mL* or µM†) Cytotoxic activity against Nitric oxide α – Glucosidase RAW 264.7 cells inhibition 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 fromG. prainiana leaf.

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

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

Submit Manuscript Advances in Applied Chemistry and Biochemistry [ISSN: 2652-3175] .06. 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. DOI: 10.33513/ACBC/1901-08 activity at the maximum test concentrations. 7 was obtained as a yellowish brown solid. The UV spectrum of 7 clearly indicated three prominent peaks at 210, 290 Based on these results we focused on GPL2 for further and 350 nm, which is similar to compound 1, but 7 showed fractionation. GPL1 was deemed too cytotoxic for accurate higher polarity than compound 1, eluting earlier in the C18 assessment of nitric oxide assay, whereas GPL4 was not active. HPLC chromatogram (Figure 3). The mass spectrum of 7 GPL3 was less active than GPL2 and chemical profiling revealed showed [M+1]+ fragment at m/z 719, suggesting the molecular overlap in the compound composition, suggesting the same weight is 718 a.m.u. The presence of the fragment ion at m/z bioactive compounds were likely to be present but some of the 557 suggests the loss of a sugar moiety (162 a.m.u.) from constituents in GPL2 appeared in greater amounts than GPL3 7, which indicates the presence of a pyranoside sugar in the extract. Therefore, isolation was prioritised on GPL2 extract. structure. This evidence suggests the structure of 7 is similar 5.2. Structural elucidation of biflavonoids from to morelloflavone but with the presence of the sugar moiety. G. prainiana leaf extract fraction GPL2 The 1H NMR spectrum of 7 (Table 2) showed three pairs of ortho coupling proton signals (H-2′ and H-3′; H-5′ and H-6′; Further investigation on GPL2 fraction led to the isolation and H-5‴ and H-6‴) and two pairs of meta coupling protons and identification of compounds (1, 2, 7, 8, 9 and 10). (H-6 and H-8; H-2‴ and H-6‴) in benzene ring substitution; However, only the structure elucidation for compounds 7-10 one oxygenated methine proton at δ 6.17 (H-2) coupling with are presented here in detail as they are being reported for the another methine proton at δ 4.84 (H-3) of which the signal is first time from this species. underneath the solvent peak, and two singlets corresponding 5.2.1. Morelloflavone-7″-O-β-glucoside (7): Compound 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 7.14, br d, J = 8.1 7.15, br d, J = 8.0 2′ 7.16, d, J = 8.6 Hz 129.61 8.12, d, J = 2.4 Hz 132.74 129.12 130.09 Hz Hz 6.72, br d, J = 8.4 6.70, br d, J = 8.0 3′ 6.38, d, J = 8.6 Hz 115.61 121.07 114.84 115.77 Hz Hz 4′ 158.59 160.49 157.74 158.65 6.72, br d, J = 8.4 6.70, br d, J = 8.0 5′ 6.38, d, J = 8.6 Hz 115.61 7.24, d, J = 8.7 Hz 117.73 114.84 115.77 Hz Hz 8.02, dd, J = 8.7, 7.14, br d, J = 8.1 7.15, br d, J = 8.0 6′ 7.16, d, J = 8.6 Hz) 129.61 128.92 129.12 130.09 2.4 Hz Hz Hz 5.46, br t, J = 11.0 5.66, br d, J = 2″ 166.46 164.87 78.52 83.92 Hz 12.0Hz 2.60, m 3″ 6.47, s 103.76 6.69, s 104.33 42.77 5.08, m 48.95 2.67, m

4″ 184.17 183.57 196.79 199.02

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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 6.81, br d, J = 7.85 6.82, br d, J = 8.0 5‴ 6.93, d, J = 8.4 Hz 117.14 6.92, d, J = 9.0 Hz 115.40 115.32 116.20 Hz Hz 7.33, dd, J = 8.4, 6.65, br d, J = 8.0 6‴ 120.89 7.72, d, J = 9.0 Hz 129.03 6.68, m 117.68 119.26 2.0 Hz Hz

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 - - - - 3.32d, 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 3.52, ddd, J = 9.0, 5‴′ 78.89 - - - - 3.50, m 78.43 2.3, 5.8 Hz 3.92, dd, J =12.3, 3.92, br d, J = 12.0 2.3 Hz 6‴′ 62.67 - - - - Hz 62.35 3.72, dd, J =12.3, 3.75, m 5.8 Hz Table 2: 1H and 13C NMR assignments for Compounds 7-10.

aCD3OD, 500 MHz b DMSO-d6 ,500 MHz cAnomeric proton dUnderneath solvent peak

The oxymethine protons of the sugar moiety H-3‴′,( H-5‴′) methines [δ 71.26 (C-4‴′), δ 75.05 (C-2‴′), δ 78.57 (C-3‴′), showed coupling constants of ca 9 Hz (Table 2) indicating δ 78.89(C-5‴′) and δ 101.77 (C-1‴′)] and one methylene [δ that the protons were in axial orientation that suggested a 62.67 (C-6‴′)] from the sugar moiety. glucose. The anomeric proton signal atδ 5.12 (d, H-1‴′) gave The chemical shift of the anomeric carbon (C-1‴′) at δ a coupling constant of 7.8 Hz that is indicative of an axial 101.77 indicated that the sugar moiety was on O-glucoside. orientation and was therefore deduced to be β-glucose. Thus, The connection of glucose to the aglycone was suggested by the interglycosidic linkage of glucose in 7 was confirmed as inspection of the HMBC spectrum of 7. The3 JCH correlation a β-linkage. of the anomeric proton signal (δH 5.12, H-1‴) to the carbon The 13C NMR data of 7 (Table 2) showed thirty six carbons signal at δ 162.33 (C-7″) confirmed the glucose was linked consisting of ten oxygenated benzene carbons, two carbonyl to this position. The structure of 7 was thus established as carbons , nine signals from methine benzene carbons, five morelloflavone-7″-O-β-glucoside with molecular formula quaternary carbons from benzene rings, one oxygenated of C36H30O16 (molecular weight = 718.62 g/mol; PubChem quaternary carbon [δ 166.46 (C-2″)], one oxygenated methine CID 101973939) and further confirmed by comparison with [δ 82.52 (C-2)] and two methine [δ 51.12 (C-3) and δ 103.76 published NMR data [23]. (C-3″)] from alicyclic rings. The rest of the signals are the five Compound 7, also known as fukugiside, was previously

Submit Manuscript Advances in Applied Chemistry and Biochemistry [ISSN: 2652-3175] .08. 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. DOI: 10.33513/ACBC/1901-08 found in a range of Garcinia species including G. multiflora CID 11467081), which has been reported from Garcinia heartwood, G. cymosa stem bark, G. spicata stem bark, G. subelliptica. This compound is reported for the first time from xanthocymus leaf, G. intermedia fruit, G. livingstonie fruit G. prainiana. Compound 9, also known as GB-2a, was also and G. brasiliensis fruit [23-27]. previously isolated from G. spicata and G. multiflora and G. perusii [25,34,35]. 5.2.2. 4‴-methyl amentoflavone (8): Compound 8 has an [M+1]+ ion at m/z 553, indicating the molecular weight of 5.2.4. 2″,3″-dihydromorelloflavone-7″-O-glucoside (10): 552 a.m.u (Figure 2). The UV spectrum showed a similar Compound 10 was obtained as a yellow gummy solid. The pattern to that of the UV spectrum of compound 2 with λmax UV spectrum showed similarity to 9 with the λmax of 198, absorption at 198, 270 and 332 nm. 226 and 288 nm. The mass spectrum (APCI, positive mode) showed an [M +1]+ fragment at m/z 721 (Figure 2) suggesting 1 13 The H NMR and C NMR signals (Table 2) were similar to the molecular mass is 720 a.m.u. The presence of a fragment amentoflavone 2( ) except for the presence of a methoxy signal at m/z 559 in the mass spectrum of 10 indicated the loss of at δ 3.79 and δ 55.50, respectively. The position of the methoxy 162 a.m.u from the molecular ion peak, which suggests the group in compound 8 was confirmed by the correlation of compound contains a pyranoside sugar. methoxy signal to δ 163.50 (C-4‴) in the HMBC spectrum. Therefore, the structure of compound8 was confirmed as The 1H NMR data (Table 2) contained four doublet aromatic

4‴-methyl amentoflavone with molecular formula of C31H20O10 signals [(δH 7.15, 2H, J = 8.0 Hz, H-2′ and H-6′); (δH 6.82,

(molecular weight = 552.69 g/mol; PubChem CID 53206444). 1H, J = 8.0 Hz, H-5‴); (δH 6.70, 2H, J = 8.0 Hz, H-3′ and

This compound is also known as podocarpusflavone A [28-30]. H-5′); and (δH 6.65, 1H, J = 8.0 Hz, H-6‴)] from benzene methines, four singlet signals from benzene methines [(δ 6.81 The 1H NMR and 13C NMR data of compound 8 were H (H2‴); (δH 6.39, H-6); (δH 6.25, H-6″); and (δH 5.91, H-8)], consistent with that reported previously for podocarpusflavone three doublet signals from alicyclic methine [(δ 5.72, H-2); A from Ouratea multiflora and Podocarpus neriifolius D. Don H (δH 5.66, H-2″); and (δH 4.67, H-3], one multiplet signal at δH [31,32]. This compound is reported for the first time from 5.08 (H-3″) and seven oxymethine proton signals from a sugar Garcinia prainiana. moiety [δ 5.05 (H-1‴′ (anomeric proton)), δ 3.92 (H-6‴′a), 5.2.3. 2″,3″-dihydromorelloflavone (9): Compound 9 was δ 3.75 (m, H-6‴′b), δ 3.50 (H-5‴′), δ 3.48 (H-3‴′), δ 3.39 obtained as a pale brown gum and the mass spectrum (APCI, (H-4‴′) and δ 3.32 (H-2‴′ (underneath the solvent peak)). 13 positive mode) showed an [M+1]+ ion at m/z 559, indicating The C NMR data (Table 2) showed thirty six carbons signals a molecular weight of 558 a.m.u (Figure 2). Its UV spectrum from 10, consisting of nine oxygenated quaternary carbons from benzene rings [δ 165.58 (C-5), δ 164.88 (C-7), δ 162.09 showed absorption bands at λmax 198, 226 and 290 nm. The 1H NMR data (Table 2) contained signals from two hydroxyl (C-9), δ 158.65 (C-4′), δ 165.36 (C-5″), δ 164.09 (C-7″), δ protons at δ 12.13 (5-OH) and δ 12.17 (5″-OH), five doublet 161.10 (C-9″), δ 146.42 (C-3‴) and δ 146.82 (C-4‴)], ten signals (H-2; H-3; H-2′ and H-6′; H-3′ and H-5′; and H-5‴), methine carbon signals from benzene rings [δ 96.77 (C-6), three singlet signals from benzene methines (H-8; H-6; and δ 97.48 (C-8), δ 97.27 (C-6″), two carbons at δ 130.09 H-6″ and H-2‴), one broad triplet signal from alicyclic methine (C-2′ and C-6′), two carbons at δ 115.77 (C-3′ and C-5′), δ (δ H 5.46, J = 10.95 Hz, H-2″) and three multiplet signals 114.31 (C-2‴), δ 116.20 (C-5‴) and δ 119.26 (C-6‴)], two representing the methylene protons H-3″ and one of the carbonyl carbons at δ 198.59 (C-4) and δ 199.02 (C-4″), five methylene protons of H-6‴. 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 The 13C NMR data showed thirty carbons signals (Table 2) 131.61 (C-1‴)], three methine carbon signals from alicyclic consisting of nine oxygenated quaternary carbon from benzene rings [δ 50.10 (C-3), δ 83.92 (C-2″) and δ 82.23 (C-2)], one rings, ten methine carbon signals from benzene rings, two methylene carbon signal from an alicyclic ring at δ 48.95 (C-3″) carbonyl carbons at δ 196.79, five quaternary carbon signals and six carbon signals from a sugar moiety [five oxygenated from benzene rings, three methine carbon signals from alicyclic methines, (δ 71.09 (C-4‴′), δ 75.15 (C-2‴′), δ 78.17 (C-3‴′), rings (C-2, C-3 and C-2″), and one methylene carbon signal δ 78.43(C-5‴′) and δ 101.35 (C-1‴′)) and one oxygenated from an alicyclic ring at δ 42.77 (C-3″). methylene (δ 62.35 (C-6‴′))]. Comparison of carbon signals from compound 9 with The location of the sugar substituent was confirmed at C-7 by published data [33], supported that the structure of compound the correlation of proton signal of H-1‴′ (anomeric proton) 9 is 2″,3″-dihydromorelloflavone with molecular formula of with carbon signal at δc 164.88 (C-7) in HMBC spectrum.

C30H22O11 (molecular weight = 558.50 g/mol; PubChem

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The 1H NMR and 13C NMR data were similar to 9 except 5.3. Nitric oxide inhibition and cytotoxicity for the presence of 6 carbon signals and 7 proton signals studies of the isolated biflavonoids from the sugar moiety. Through the comparison of 13C NMR data of compound 10 and 9, the structure was confirmed as Compounds (1, 2, 7, 8, 9 and 10) were subjected to NO 2″,3″-dihydromorelloflavone-7″-O-glucoside with molecular inhibition and cytotoxicity assays (Table 1). Amentoflavone (2) formula of C36H32O16 (molecular weight = 720.63 g/mol; demonstrated the highest NO inhibition activity with an IC50 HMDB30609), which is also known as xanthochymuside. of 44.80 ± 12.20 µM, followed by 2″,3″-dihydromorelloflavone Xanthochymuside has been isolated from G. multiflora (9), 4‴-methyl amentoflavone (8), and morelloflavone (1) with

[25]. However, there are no published NMR data available. an IC50 of 46.00 ± 0.70, 58.60 ± 0.40 and 75.20 ± 1.10 µM, Therefore, this is the first report of the13 C NMR and 1H NMR respectively. None of these compounds showed significant assignments of xanthochymuside. cytotoxic activity at the maximum test concentrations (Table 1). The high NO inhibition of amentoflavone2 ( ) compared to 5.2.5. Morelloflavone (1): The mass spectrum of 1 showed 4‴-methyl amentoflavone (8) could be due to reduced binding + an [M+1] ion at m/z 557 (Figure 2), which indicated the of hydroxyl group and the presence of the methoxy group at molecular weight was 556 a.m.u. The NMR spectral data from 4‴ position, while the loss of a double bond at positions 2″ 1 were consistent with published data for morelloflavone from and 3″ in compound 9 resulted in a the two fold increase in Garcinia densivenia [36] with molecular formula of C30H20O11 NO inhibition activity compared to morelloflavone. (molecular weight = 556.48 g/mol; PubChem CID 5464454). Morelloflavone 1( ) and amentoflavone 2( ) are well known 5.2.6. Amentoflavone (2): The mass spectrum of2 showed compounds and these compounds have been previously + an [M+1] ion at m/z 539, which indicated the molecular reported from G. prainiana leaves by Klaiklay et al., [5]. 1 13 weight was 538 a.m.u. The H NMR and C NMR data of Morelloflavone has been previously reported to inhibit 2 were compared to the literature and found to be identical the HMG CoA reductase, the rate limiting enzyme of the to published data for amentoflavone from Ouratea multiflora cholesterol biosynthetic pathway in vitro [37]. This compound [31] with molecular formula of C30H18O10 (molecular weight was also reported to inhibit vascular smooth muscle cell = 538.46 g/mol; PubChem CID 5281600). 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 1 H NMR data (DMSO-d6 ): δ 5.72 (1H, d, J = 12.00 Hz, H-2), δ 4.86 and anti-inflammatory agents. It is a good inhibitor of human (1H, d, J = 12.00 Hz, H-3), δ 5.96 (1H,s, H-6), δ 5.96 (1H,s, H-8), δ 7.14 ( 1H, d, J = 8.45 Hz, H-2′), δ 6.38 (1H, d, J = 8.45 Hz, H-3′), δ 6.38 (1H, Cathepsin B, a cysteine protease implicated in the pathology of d, J = 8.45 Hz, H-5′), δ 7.14 ( 1H, d, J = 8.45 Hz, H-6′), 6.54 (1H, s, some inflammatory diseases and cancer [39]. Consistent with H-3″), δ 6.21(1H, s, H-6″), δ 7.40 (1H, s, H-2‴), δ 6.90 (1H, d, J = 8.95 our study, this compound also was reported as an inhibitor Hz, H-5‴), δ 7.41 (1H, d, J= 8.95 Hz, H-6‴), δ 12.26 (1H, s, 5″-OH), δ of nitric oxide synthase through the inhibition of NF-κB 13.05 (1H, s, 5-OH) 13C NMR data (DMSO-d6 ): δ 80.89 (C-2), δ 48.28 (C-3), δ 196.19 activation in RAW 264.7 cells [40]. Previously, amentoflavone (C-4), δ 163.83 (C-5), δ 98.68 (C-6), δ 162.82 (C-7), δ 95.27 (C-8), δ was also reported to inhibit cAMP-phosphodiesterase which 166.60 (C-9), δ 102.80 (C-10), δ 128.18 (C-1′), δ 128.39 (C-2′), δ 114.4 could reduce inflammation in adipose tissues [41]. Our results (C-3′), δ 157.30 (C-4′), δ 114.4 (C-5′), δ 128.39 (C-6′), δ 163.37 (C-2″), expand the current knowledge on the anti-inflammatory and δ 102.15 (C-3″), δ 181.51 (C-4″), δ 160.48 (C-5″), δ 96.19 (C-6″), δ cytotoxic properties of 4‴-methyl amentoflavone (8) and 155.25 (C-7″), δ 100.59 (C-8″), δ 162.05 (C-9″), δ 102.80 (C-10″), δ 121.07 (C-1‴), δ 113.23 (C-2‴), δ 145.63 (C-3‴), δ 149.67 (C-4‴), δ 2″,3″-dihydromorelloflavone 9( ). 116.12 (C-5‴), δ 119.21 (C-6‴). 5.4. α-Glucosidase inhibitory activities of extracts, 1H NMR data (CD3OD): δ 6.18 (1H, d, J =2.05, H-6 ), δ 6.38 (1H,s, fractions and compounds from G. prainiana King 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, Type 2 diabetes is a chronic metabolic disorder that results from H-5′), δ 7.52 (2H, d, J = 8.8, H-2‴ and H-6‴), δ 7.89 (1H, dd, J= 8.6, α 2.25, H-6′), δ 7.93 (1H, d, J = 2.25,H-2′). a high blood glucose level [42]. Administration of -glucosidase 13C NMR data (CD 3OD): δ 184.4 (C-4), δ 183.98 (C-4″), δ 166.22 inhibitor has been proposed as treatment for type 2 diabetes, (C-7), δ 166.34 (C-2″), δ 166.08 ( C-2), δ 163.31 (C-5), δ 162.69 (C- since it works by preventing the digestion of carbohydrates [43]. 5″), δ 162.98 (C-4‴), δ 162.65 ( C-7″), δ 161.04 (C-9), δ 161.04 (C-4′), Therefore, postprandial blood glucose needs to be controlled δ 156.63 (C-9″), δ 132.94 (C-2′), δ 129.45 (C-6‴), δ 129.45 (C-2‴), in the early treatment of diabetes. 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 In the screening of a crude extract of G. prainiana leaf, 105.42 (C-8″), δ 105.28 (C-10), δ 104.17 (C-3″), δ 103.54 (C-3), δ the extract demonstrated moderately potent α-glucosidase 100.06 (C-6″), δ 100.3 (C-6), δ 95.28 (C-8). inhibitory with IC50 of 29.70 µg/mL (Table 1). Further

Submit Manuscript Advances in Applied Chemistry and Biochemistry [ISSN: 2652-3175] .10. 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. DOI: 10.33513/ACBC/1901-08 investigation on fractions, GPL1 to GPL4 revealed that ingredient with health benefits in soups, salads and other

GPL2 showed the highest activity with IC50 of 15.70 µg/ culinary products should be encouraged, provided that it can mL, follow by GPL1 with an IC50 nearly ten times lower be demonstrated to be safe. (Table 1). The other two fractions did not show any activity. The six isolated compounds from GPL-2 were then subjected to References the α-glucosidase assay. 4‴-methyl amentoflavone 8( ) exhibited 1. Ikram EHK, Eng KH, Jalil AMM, Ismail A, Idris S, et al. (2009) α- the highest glucosidase inhibition with an IC50 of 6.34 ± Antioxidant capacity and total phenolic content of Malaysian 0.04 µM follow by amentoflavone 2( ), morelloflavone-7″-O- underutilized fruits. Journal of Food Composition and Analysis glucoside (7), 2″,3″-dihydromorelloflavone (9), morelloflavone 22: 388-393. (1), and 2″,3″-dihydromorelloflavone-7″-O- -glucoside (10), 2. Ong H-C, Chua S, Milow P (2011) Traditional knowledge of (Table 1). Amentoflavone 2( ) showed lower activity than edible plants among the Temuan villagers in Kampung Jeram 4‴-methyl amentoflavone suggesting that the loss of methoxy Kedah, Negeri Sembilan, Malaysia. Scientific Research and group at position 4‴ effected α-glycosidase inhibitory activity. Essays 6: 694-697. Previously, Kim et al., [44] demonstrated the α-glucosidase 3. Osman MB, Milan AR (2006) Fruits for the future 9: Mangosteen inhibitory of amentoflavone in a study of twenty-one naturally Garcinia mangostana. Southampton Centre for Underutilised occurring flavonoids [44]. Amentoflavone was reported to show Crops, University of Southampton, Southampton, UK. one of the strongest inhibitory activities in the study. However, 4. Jabit ML, Wahyuni FS, Khalid R, Israf DA, Shaari K, et al. this is the first report of the 4‴-methyl amentoflavone activity (2009) Cytotoxic and nitric oxide inhibitory activities of methanol against α-glucosidase. extracts of Garcinia species. Pharmaceutical Biology 47: 1019-1026. The significant loss of activity in2″,3″- dihydromorelloflavone- 7″-O-glucoside (10) as compared to morelloflavone-7″-O- 5. Klaiklay S, Sukpondma Y, Rukachaisirikul V, Towatana NH, glucoside (7) pointed out the importance of the double bond at Chareonrat K (2011) Flavanone glucuronides from the leaves ″ ″ α of Garcinia prainiana. Canadian Journal of Chemistry 89: position 2 and 3 in maintaining the -glucosidase inhibitory 461-464. activity of morelloflavone. A similar pattern also can be seen between 2″,3″-dihydromorelloflavone 9( ) and morelloflavone 6. Lin YM, Anderson H, Flavin MT, Pai YH, Mata-Greenwood E, (1). It seems likely that the presence of the glycoside moiety does et al. (1997) In vitro anti-HIV activity of biflavonoids isolated from Rhus succedanea and Garcinia multiflora. Journal of improve the α-glucosidase inhibition. The glycoside moiety in Natural Products 60: 884-888. 7″ enhanced the inhibition of α-glucosidase, possibly through the improvement of solubility. 7. Suh SJ, Chung TW, Son MJ, Kim SH, Moon TC, et al. (2006) The naturally occurring biflavonoid, ochnaflavone, inhibits The presence of some biflavonoids with anti-glucosidase activity LPS-induced iNOS expression, which is mediated by ERK1/2 via NF-kappaB regulation, in RAW264.7 cells. Archives of in the leaf extract of G. prainiana King suggests that this species Biochemistry and Biophysics 447: 136-146. has potential for further development as an anti-diabetic food. Further structure-activity relationship studies would 8. Pang X, Yi T, Yi Z, Cho SG, Qu W, et al. (2009) Morelloflavone, be worthwhile on the biflavanoid class of compounds more a biflavonoid, inhibits tumor angiogenesis by targeting rho GTPases and extracellular signal-regulated kinase signalling generally to optimise the development of anti-diabetic drugs. pathways. Cancer Research 69: 518-525. 6. Conclusion 9. Lin LC, Kuo YC, Chou CJ (2000) Cytotoxic biflavonoids from Selaginella delicatula. Journal of Natural Products 63: 627-630.

Our work provides scientific evidence for the potential to 10. Kaikabo AA, Eloff JN (2011) Antibacterial activity of two value-adding G. prainiana King, an underutilised plant, as biflavonoids from Garcinia livingstonei leaves against anti-inflammatory and anti-diabetic food source, as well as Mycobacterium smegmatis. Journal of Ethnopharmacology a good source of anti-oxidant constituents. Phytochemical 138: 253-255. investigations revealed that this plant is a rich source 11. Kaikabo AA, Samuel BB, Eloff JN (2009) Isolation and activity of biflavonoids such as amentoflavone (2), 4‴-methyl of two antibacterial biflavonoids from leaf extracts of Garcinia amentoflavone (8), morelloflavone (1), morelloflavone- livingstonei (Clusiaceae). Natural Product Communications 7″-O-glucoside (7), 2″,3″-dihydromorelloflavone (9) and 4: 1363-1366. 2″,3″-dihydromorelloflavone-7″-O-glucoside (10), with 12. Luzzi R, Guimarães C, Verdi L, Simionatto E, Delle Monache potential to be developed for human health applications. F, et al. (1997) Isolation of biflavonoids with analgesic activity Considering our data and information on local and traditional from Rheedia gardneriana leaves. Phytomedicine 4: 141-144. food uses of the species, the inclusion of the leaves as an 13. Antia BS, Pansanit A, Ekpa OD, Ekpe UJ, Mahidol C, et al.

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