Fractionation of Averrhoa bilimbi hexane extract corresponding to brown adipocytes stimulation

2.1 Plant extractions and fractions with Flash Column Chromatography

Bilimbi leaves were sampled from an orchard with the owner’s permission. The botanical authentication of the specimen was conducted by botanists of the School of Biological Sciences, Universiti Sains Malaysia (USM) and a voucher specimen 11738 was deposited in the herbarium of USM. Fresh leaves were dried in an oven at 45°C until the percentage yield of moisture content value was less than 10%. Dried leaves were then ground to a fine powder using a grinder then weighed, recorded and kept in a plastic bag. The powder was soaked in ethanol (EtOH) for 3 days at room temperature. After filtration with Whatman No. 1 filter paper, the solvent was removed under reduced pressure at 40°C using a rotary evaporator (Buchi, Switzerland) to obtain the crude ethanolic extract. The weight and percentage yield were recorded, stored in an amber bottle, labelled and kept refrigerated until further use. Next, the crude was sequentially extracted with organic solvents, namely n-hexane (n-Hex), ethyl acetate (EtOAc), and n-butanol (n-BuOH) with increasing polarity respectively. The filtrate was then concentrated and dried under reduced pressure at 40°C using a Buchi rotary evaporator. The remaining aqueous layer was evaporated and lyophilised in a freeze-dryer. The weight and percentage of each yield was recorded and kept refrigerated. A flow diagram showing the step-by-step extraction and partitioning processes is depicted in Figure 1.

Figure 1. Representation of continuous procedure of extraction, partitioning and fractionation of Averrhoa bilimbi.

After the bioactivity of these extracts was confirmed in cell-based assays, 200 g of dried leaves was soaked in 15 L of n-Hex to yield 12 g of extract. Fractionation was conducted with normal phase silica gel flash column chromatography. Mobile phases in the separation consisted of gradient of n-Hex, EtOAc, and methanol.

2.2 High-Performance Thin Layer Chromatography (HPTLC)

HPTLC has advanced separation efficiency and detection limits compared to conventional TLC Chromatography. The pre-coated silica gel Merck, TLC silica gel plates 60 F 254, 200 × 100 mm were used. 2 μl of the sample solution was applied on the plate using a CamagLinomat V automatic sampler applicator in the form of bands (length: 5 mm, width: 1 mm, distance between two bands: 10 mm) by using a 100 μl Camag Microlitre Syringe (Hamilton, Bonaduz, Switzerland). A constant application rate of 150 nl/s with mobile phase of n-hexane: ethyl acetate (80:20, v/v) was adopted. The plate was then placed in the mobile phase, and ascending development was performed to a distance of 8.5 cm, the plate was then air dried and performed densitometry scanning at 250 nm. Images of the TLC plates were captured using the Camag’s TLC Visualizer documentation system. The system provided illumination with white light (remission, transmission or a combination of both), where the exposure of RT white was optimised at 1.482 s, R 254 at 0.185 s, and R 366 at 0.877 s.

2.3 Cell culture and adipocyte differentiation

C2C12 myoblast was purchased from the American Type Culture Collection (Manassas, VA, USA) and was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Adipocyte differentiation in the myoblast was initiated after the cells reached confluent by adding 0.5 mM isobutyl methylxanthine (IBMX), 1 μM dexamethasone (DEX), 1 μg/ml insulin, and 1 μM rosiglitazone (ROSI). Fresh differentiation medium containing DMEM, 10% FBS, 1 μg/ml insulin and 1 μM ROSI was replaced two days later. These cells were then stabilised in DMEM containing 10% FBS for another two days until the formation of oil droplets was observed.⁸

2.4 Adipocyte determination by fluorescent dye staining

Cells were washed once with PBS and fixed with 4% paraformaldehyde for ten minutes. Lipid droplets produced by adipocytes were stained by Nile Red solution and were visualised using the IN Cell 2200 Analyzer High Content Screening System (GE Healthcare, PA, USA) at an excitation wavelength of 460 nm.¹⁰

2.5 GC/MS analyses and identification of components

The GC/MS analyses of non-polar hexane partition extract and active fractions were carried out using the Agilent 5977A Series GC/MSD system with an HP-5ms ultra inert column (30 m × 0.25 mm id, 0.25 μm film thicknesses). Spectroscopic detection by CG/MS involved an electron ionisation system which utilised high energy electrons (70 eV). Pure helium gas (99.995%) was used as the carrier gas with a flow rate of 1 ml/min. The initial GC oven temperature was set as 110°C for two minutes and programmed with increasing rate of 10°C/min to 200°C in five minutes. Finally, the temperature was increased to 250°C at 10°C/min in five minutes and held at that temperature for 13 minutes. The split ratio was 1:25 with a scan range of 40 to 550 amu.¹¹ Hexane fractions (1 μl, 1 mg/ml) and partition extract (3 μl, 10 mg/ml) diluted in n-Hex were injected into the GC/MS via an auto-injector. Compounds present in the partition extract and active fractions were analysed by the mass fragmentation pattern. The National Institute of Standards and Technology (NIST) 14 and Wiley 10 library (W1ON14.L) were used for the identification of compounds with spectral similarity score higher than 60%.¹²

3.1 Biological Assay-Guided Fractionation of A. bilimbi Extract

Four partitioned extracts were in the yield: n-Hex, EtOAc, n-BuOH and H₂O. The yield of each component is indicated in Figure 1.

3.2 Flash Column Chromatography and High-Performance Thin Layer Chromatography (HPTLC)

Flash column chromatography was conducted on the partition extract with an equilibration solvent ratio of n-Hex and EtOAc (95:5). A Hi Flash Column with a size of 3 L (46 × 130 mm silica gel) was used. With a flow rate of 60 ml/min, a total of 80 fractions were collected and spotted on a plate, employing the HPTLC. Plate visualisation was done under short (254 nm) and long wavelength (366 nm) ultraviolet lights, as well as white light illumination. Fractions with similar separation profiles were pooled. A total of ten fractions were collected with respective TLC profiles as shown in Figure 2.

Figure 2. Thin layer chromatography plates showing separation of compounds of f1 – f10 using mobile phase n-Hex:EtOAc (80:20, v/v).

Two batches (1 & 2) of fractionation samples were collected and run on the same plates (A) TLC plate image viewed under UV 254 nm. (B) TLC plate image viewed under UV 366 nm. (C) TLC plate image viewed under white light illumination.

3.3 Differentiation of adipocytes in Myf5 lineage precursor cells

The partitioned extracts and fractions corresponded to the growth stimulation of brown adipocyte were investigated via cell-based study. The myf5-positive characteristics of C2C12 murine myoblast allow the co-development of mature myotubes and brown adipocytes upon appropriate stimulations. ROSI was served as the positive control. Adipogenesis was observed on day four after treatment with 1μM ROSI. A similar phenomenon was found in 200 μg/ml EtOH and 100 μg/ml n-Hex treated cells. Myotube development was observed in all treated cells. All positively treated cells showed intracellular lipid droplet production after 7 days of incubation. These lipid droplets were captured by Nile Red staining and imaged at 20× magnification (Figure 3). Among the four partitioned extracts tested, n-Hex exhibited the highest amount of lipid droplet production.

Figure 3. Differentiation of Myf5 lineage precursor cells upon stimulants.

Lipid droplets formation upon adipogenesis upon treatment by (A) 200 μg/ml EtOH, (B) 1 μM ROSI, (C) 100 μg/ml n-Hex, (D) 100 μg/ml EtOAc, (E) 100 μg/ml n-BuOH and (F) H₂O were stained by a Nile Red solution. Treatment of myoblasts with adipogenesis stimulants facilitated myocytes and adipocytes co-differentiation.

A bioassay guided fractionation technique was used to identify the active components present, as well as to evaluate the corresponding phytocompounds in brown adipocyte activation.

The cell-based study was then repeated on ten n-Hex fractions yielded from the HPTLC fractionation. Cell morphology and lipid droplet accumulation was observed for seven days with treatments by ten fractions. Myotube development was observed in all treated cells. Adipocyte differentiation was also observed in f5, f6, f8, f9 and f10 treated cells, with lipid droplet formation as depicted in Figure 4. Massive death was observed in f7 treated cells on day 3, and cell death was also found in f6 treatment from day 5 onwards. DMSO was used to replace bilimbi and ROSI in non-treated cells. These negative control cells were supplemented with adipogenesis culturing media and were fully developed into myotubes.

Figure 4. Differentiation of Myf5 lineage precursor cells upon stimulants.

Lipid droplet formation upon treatment by n-Hex fractions (f1 – f10) and 1 μM ROSI were stained by a Nile Red solution. n-Hex fractions with adipogenesis stimulating components facilitated myocyte and adipocyte co-differentiation. Cells treated with 0.5% DMSO served as negative control (-ve) and demonstrated a complete process of myogenesis.

3.4 Gas-Chromatography/Mass-Spectrometry (GC/MS)

A GC/MS based qualitative analysis was performed on n-Hex and subsequently three active fractions, namely f8, f9, and f10, to determine the types of compounds present (Figure 5-8). Major constituents identified are presented in Tables 1 & 2. Studies on f6 & f7 were discontinued after the demonstration of cytotoxic effect in these treated cells, as shown in Figure 4.

Figure 5. GC Chromatogram of A. bilimbi n-Hex.

Figure 6. GC Chromatogram of A. bilimbi n-Hex fraction f8.

Figure 7. GC Chromatogram of A. bilimbi n-Hex fraction f9.

Figure 8. GC Chromatogram of A. bilimbi n-Hex fraction f10.

The database information search via the MassHunter Library WION 14.L revealed the presence of nine predominant compounds in f8, ten in f9, and five in f10. (1.alpha., 4.alpha., 4a.alpha., 10a.alpha.) -1, 4, 4a, 5, 6, 7, 8, 9, 10, 10a -decahydro- 1, 4, 11, 11-tetramethyl-1, 4-methanocycloocta [d] pyridazine and i-propyl 3- (phenylamino) -2- (phenylseleno) -3- (phenyl) propanoate were detected in all the three fractions whereas nonanal and hexadecanoic acid were commonly present in f9 and f10. The high abundance compounds include 3-nonen-2-one; 2(4H)-Benzofuranone, 5, 6, 7, 7a-tetrahydro -6- hydroxy-4, 4, 7a-trimethyl-, (6S-trans)-; hexadecanoic acid; (12R)-(9Z)-12-hydroxy-9-octadecenoic acid; spiro [2.4] heptanes-5-carboxaldehyde; and i-propyl 3 -(phenylamino) -2- (phenylseleno) -3- (phenyl) propanoate.