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INTRODUCTION

Garcinia kola (bitter kola) is a species of flowering plant

belonging to the Guttiferae (Clusiaceae) family. It is a native plant grown in tropical climates of Western Africa. Compounds

isolated from the seed of Garcinia kola and other Garcinia species have been shown to have anti-oxidant, anti-bacterial,

anti-inflammatory and anti-viral properties. There are four main classes of compounds found in the Guttiferae family:

benzophenones, bioflavonoids, coumarins and xanthones. A series of HIV-inhibitory benzophenones, Guttiferones A-E, have

been previously reported in the Garcinia genera. Guttiferone F;

the compound responsible for the HIV-inhibiting properties reported in other Guttiferae family

This research investigates approaches to extract and confirm

Guttiferones, specifically Guttiferone F and Garcinol in Garcinia kola via solvent–solvent [S-S] extractions and supercritical

fluid extractions (SFE). Garcinol; a reported1 isobaric C30-epimer of Guttiferone F, is readily available and will be used as

a standard for confirmation. The samples will be analyzed by LC and UltraPerformance Convergence Chromatography (UPC2)

whereas the chromatographic analysis is coupled to mass

spectrometry. Statistical analysis will also be performed on the dataset interrogating similarities/dissimilarities to each of

the extraction techniques for future reference.

Solvent-Solvent Extraction Vs. Supercritical Fluid Extraction of Garcinia kola Nuts

Yasah Vezele1,2, David Worthen1, Michael D. Jones3,4, Giorgis Isaac3, and Norman Smith4 1Department of Biomedical and Pharmaceutical Sciences, & 2Department of Chemical Engineering, Colleges of Pharmacy and Engineering, University of

Rhode Island, Kingston, RI, 3Waters Corporation, Milford, MA, 4King’s College London, School of Biomedical and Health Sciences, London, UK

METHODS

Solvent-Solvent “[S-S]” Extraction: Garcinia kola nuts were purchased, peeled, dried, ground using a rotary mill and was subject to solvent-solvent extraction using methanol, hexane, chloroform,

and ethyl acetate. The methanol fraction was then subject the method employed by Fuller et. al. Briefly, a combined portion of

extracts was further partitioned and permeated through Sephadex LH-20.

HPLC: Samples were further examined by Hitachi Organizer Reverse Phase High Performance Liquid Chromatography (HPLC) using

Agilent Zorbax SB-C8 (4.6x250mm, 5µ ) column. The mobile phase consisted of 80:20 Acetonitrile:Water (.1% Acetic Acid). The flow rate was 1 mL/min and 20minute run time. Detection was set from

200nm to 400nm.

Supercritical Fluid Extraction: Samples were ground Garcinia kola seeds and underwent extraction by carbon dioxide (CO2) with

three different modifiers: methanol, ethanol and IPA (5%, 10% and 30%) using a Waters MV-10 SFE instrument.

UPC2 coupled to Q-Tof MS: The ACQUITY UPC2 instrumentation used a UPC2 BEH column with dimensions 3.0 x 100mm; 1.7um. MS

data acquisition was performed using Xevo G2 Q-ToF. Alternating high and low collision energy was performed during a single injection; known as MSE, allowing for precursor and product ion

determination.

Reference: 1. Fuller et al, Journal of Natural Products, 1999, Vol 62, No. 1

RESULTS

Solvent-Solvent Extraction

Uses organic solvents Organic toxic solvents

Time consuming Specific sample preparation

SFE Rational

Uses supercritical carbon dioxide

Controlled by temperature

and or pressure Highly selective

Faster diffusivity Automated process Non toxic; leaves no

solvent residue

Figure 2: Schematic of the

Supercritical Fluid Extraction (SFE).

Results for Garcinol standard indicate a broad UPC2

chromatographic peak eluting at approx. 4.0 minutes. The HPLC resulting RT was about 12.5 minutes.

Mass accuracy of the Garcinol standard by ESI(-) was measured to be 1.3 ppm.

Solvent – Solvent extractions and SFE samples indicated no presence of Garcinol in any extract when based on ESI(-).

Garcinol was not found in Garcina kola when following the Sephadex procedure mentoned in the Fuller publication.

Summary of the 1291 features:

50.74% can be extracted by SFE and 55.46% can be

extracted by [S-S].

23% of the abundant features are extracted by both SFE

and [S-S].

Of the 23% extracted by SFE, the majority was extracted by the use of 5% EtOH modifier.

Common ESI(-) m/z found in all extracts were m/z=425.26 and m/z=365.17

Both extraction techniques are necessary to achieve a comprehensive profile of constituents found within natural

products

CONCLUSIONS Figure 5: Breakdown of

features found in S-S vs. SFE determined by a

normalized abundance > 1% based on the TOIML

dendrogram data set.

Figure 6: SFE breakdown of

the 23% overlapped [S-S] and SFE features.

Overall Analysis Workflow

Statistical Analysis

The data set was processed using TransOmics Informatics for Metabolomics and Lipidomics (TOIML). The extractions were

grouped various ways to statistically determine trends of similarity and dissimilarity. Discriminate features were determined for each of the dissimilar extraction groupings. Features are defined by

mass/charge (m/z) and retention time pairs, whereas 1291 features were found in the entire extraction analysis dataset. Plots

generated by TOIML facilitated visualization of these trends. Figure 4 shows a dendrogram where each of the 1291 features can be reviewed by standardized normalized abundance within each of

the extraction procedures. This should account for variations in concentration. In addition, the dendrogram can show origins for a

grouping of features the further the path is traced up the lattice.

Figure 7: Trend plot as determined by principle component analysis

(PCA)

HPLC Analysis of Solvent– Solvent Extractions

All the [S-S] extractions were analyzed by HPLC with diode array detection. Garcinol was not detected in any of the extracts overlaid

in figure 2. Most interestingly, it was not detected in the Sephadex extract as it was found in a similar Garcinia genera.

Garcinol STD

Figure 8: HPLC overlaid Chromatograms of each of the [S-S]

extractions and the injection of the Garcinol standard.

WORK FLOW OVERVIEW

Figure 2: UPC2/MSE ES– BPI MS traces of the [S-S] extractions.

Garcinol was not observed to be present

Figure 3: UPC2/MSE ES– BPI MS traces of the IPA SFE and 100%

CO2 extractions. Garcinol was not observed to be present in these

or any of the other SFE extractions with MeOH and EtOH

UPC2/MSE Analysis

Figure 1: UPC2/MSE ES– BPI MS trace of the Garcinol Standard.

Spectral analysis confirms a mass accuracy calculated to be 1.3 ppm for the elemental composition of Garcinol (C38 H49 O6). The

retention time was observed to be 4.0 minutes. The MSE data provided precursor and product ion data with the single injection. The major fragment ions (above) were recorded and used to

confirm Garcinol presence in the extraction samples

Garcinol

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80

%

0

100

03112013_Garcinia_Neg002 1: TOF MS ES- BPI

4.02e4

3.88109.0285

3.77109.02813.72

109.0280

3.68109.0284

1.86455.3522

1.72585.3586

0.98273.1490

0.96273.1486

1.01273.1492

1.13;331.1552

3.61109.0282

1.89455.3520

2.03585.3569 2.86

109.0290

3.89109.0281 4.13

109.0284

4.24109.0278

4.36109.02794.46;109.0283

4.55;109.0283

03112013_Garcinia_Neg002 2: TOF MS ES- BPI

1.32e5

3.89601.3528

3.78601.3530

3.69601.3529

3.67601.3535

1.86455.3514

1.72585.35890.98

465.3365 1.13523.3439 1.56

211.0970

1.92585.3517 3.14

197.8069

4.06601.3533

4.40601.3516

4.76601.3531

High CE

Low CE Garcinol

Chloroform

Sephadex

Rest of SFE,

Ethyl Acetate

and Hexane

extracts

MBTE

5% EtOH SFE

Figure 4: TOIML dendrogram and abundance plot, whereas a single

feature (RT 1.05min;m/z=499.2848) was found in the Sephadex and 5% EtOH SFE extractions

The overlap of features found in the solvent-solvent

extractions and the SFE extractions accounted for about 23% of coverage for both extraction techniques.

Figure 1: Solvent-

Solvent [S-S]

apparatus.