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]. Advances in Applied Chemistry and Biochemistry [ISSN: 2652-3175] Submit Manuscript .01. 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 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