GARCINIA KOLA: PHYTOCHEMICAL, BIOLOGICAL AND FORMULATION STUDIES

The objective of this research was to search for an antiviral compound, garcinol (or guttiferone F) from the Garcinia kola nuts using two different extraction techniques and formulate a suitable liposome for control release using an antiviral compound. The two extraction techniques used were: liquid-liquid extraction (for simplicity purposes, the term solvent-solvent, S-S, will be used interchangeable) and Supercritical Fluid Extraction (SFE). Both techniques were used to profile compounds found in Garcinia kola and for statistical comparison purposes. Extracts from both techniques were collected and analyzed for separation using ReversedPhase High Performance Liquid Chromatography (RP-HPLC) using Ultraviolet Photodiode Array (UV-PDA) detection, determined physical and chemical properties by proton and phosphorus Nuclear Magnetic Resonance ( 1H NMR and 31 P-NMR), and quantify by Ultra Performance Convergence Chromatography (UPC 2 ) coupled to Quadrupole Time of Flight Mass Spectroscopy (Q-ToF/MS). The UPC 2 Q-ToF/MS data was processed using TransOmics Informatics for Metabolomics and Lipidomics (TOIML) and statistical analysis was performed interrogating similarities and dissimilarities of each extraction techniques. Biological assay were conducted on three of the S-S extracts and compared to know antiviral inhibiting drugs as controls. For the formulation study, Dynamic Light Scattering (DLS), Differential Scanning Calorimetry (DSC) and release studies were conducted in order to characterize the garcinol liposomes. Initial RP-HPLC results showed no compounds with photodiode array (PDA) fingerprints characteristic of garcinol were found in either of the extracts from both extraction techniques. NMR confirmed what was already observed: garcinol has the same molecular formula, weight and chemical structure as the previously reported guttiferone F and both names (garcinol and guttiferone F) are used interchangeable. Comparison of the different extraction techniques by UPC 2 QToF/MS resulted in the identification of 1291 chemical features and the features were grouped together statistically based on their relative extraction solvents and techniques. Of the 1291 detectable mass constituents, 43.81% can be extracted by S-S technique and 40.09% can be extracted by SFE technique, relatively similar extraction profile. Approximately 20.99% of the mass constituents were detected using both extraction techniques. Overall, results from the SFE showed the comprehensive profile of all mass constituents found within this natural product and each mass grouped statistically based on their relative extraction solvents to determine extraction coverage. In addition, these statistical analysis and extraction techniques have never been performed simultaneously and or compared on Garcinia kola seeds. Biological data revealed that extracts from the S-S technique was negative for the inhabitation of antiviral activities as compare to the controls. The results of the liposome release studies indicated initial rapid release of less than 1% of garcinol from the liposomes, followed by a sharp drop in concentration at 4 hours, suggesting precipitation in the dissolution medium with no further release as the concentration leveled off to 140 hours. DSC results indicated that the surface charge of the liposome was -10.5 mV and remained relatively constant for 7 days suggesting stability issues. DLS results indicated an increase in particle size and Polydispersity index over a 7 day period which also suggests stability issues. Overall, the formulation study showed garcinol released less than 1% and may interact significantly with the DPPC phosphate head groups, as indicated by the large increase in net negative surface charge. Although no antiviral activities were active in Garcinia kola, this edible plant could serve as an abundant source of naturally-occurring, bioactive compounds for further pharmaceutical development for other biological activities.

profile. Approximately 20.99% of the mass constituents were detected using both extraction techniques. Overall, results from the SFE showed the comprehensive profile of all mass constituents found within this natural product and each mass grouped statistically based on their relative extraction solvents to determine extraction coverage. In addition, these statistical analysis and extraction techniques have never been performed simultaneously and or compared on Garcinia kola seeds. Biological data revealed that extracts from the S-S technique was negative for the inhabitation of antiviral activities as compare to the controls. The results of the liposome release studies indicated initial rapid release of less than 1% of garcinol from the liposomes, followed by a sharp drop in concentration at 4 hours, suggesting precipitation in the dissolution medium with no further release as the concentration leveled off to 140 hours. DSC results indicated that the surface charge of the liposome was -10.5 mV and remained relatively constant for 7 days suggesting stability issues. DLS results indicated an increase in particle size and Polydispersity index over a 7 day period which also suggests stability issues. Overall, the formulation study showed garcinol released less than 1% and may interact significantly with the DPPC phosphate head groups, as indicated by the large increase in net negative surface charge. Although no antiviral activities were active in Garcinia kola, this edible plant could serve as an abundant source of naturally-occurring, bioactive compounds for further pharmaceutical development for other biological activities.     including Calophyllum, Hypericum, Allanblackia, and Garcinia. (Whitmore et al., 1973). Guttiferae is divided into three subfamilies: Kielmeyeroideae; Hypericoideae; and Clusioideae (Gustafsson et al., 2002). Garcinia belongs to the subfamily Clusioideae. There are well over fifty species of evergreen trees and shrubs belonging to the Garcinia genus, including Garcinia cambogia, Garcinia indica, Garcinia mangostana, Garcinia livingstonei, Garcinia candelliana and Garcinia kola. (Smith et. al., 2004). Known products containing Garcinia mangostana are commonly sold as dietary supplements in the United States under the trade name Hydroxycut TM .
This core structure is a common structural motif of the benzophenones which have been isolated from plants. The benzophenones are increased in hydrophobicity per number of prenyl functional groups attached.
2 Garcinia kola has a distinct bitter taste-hence its common name "bitter kola" and also its common name "male kola" because of its claimed aphrodisiac activity.
There is an average of four seeds contained in a fruit ( Figure 2). The seeds are extracted by breaking open the fruit. The seed contains fat, proteins, carbohydrates, xanthones, bioflavonoids, benzophenones, and other compounds. They are chewed for the relief of cough, colds, colic, hoarseness of voice, and throat infection (Ofokansi et al., 2008). The fruit pulp is used to treat jaundice (Uko et al., 2001). The sap is used for the treatment of parasitic skin diseases, while the latex is orally ingested for the treatment of gonorrhea (Ofokansi et al., 2008) and applied to wounds to assist in healing (Uko et al., 2001). The roots and stems are eaten as a snack food known as a chewing stick for dental care, to suppress appetite and to aid in the digestion process.
Compounds isolated from Garcinia kola and other members of the Garcinia genus have been shown to have useful therapeutic, bio-protective, and other biological activities. Garcinia kola is used for the treatment of liver disorders (Adaramoye et al., 2006). More recently, Garcinia kola has been assessed for its potential utility for fighting infectious viral diseases, such as Ebola, by preventing viral replication (BBC Health News, 1999). Despite its bitter taste, Garcinia kola has been traditionally consumed for cultural, social, and traditional ceremonies, and for the worship of ancestral gods. Garcinia kola has also been employed as a substitute for hops in tropical beer (Ajebesone et al, 2004). Paradoxically, it has been claimed to cause both nervous alertness and to induce insomnia (Uko et al., 2001). Thus, there is significant ethnobotanical and epidemiological evidence available suggesting that Garcinia kola is relatively non-toxic to humans when ingested or applied to the body.
The most abundant biologically active constituent in the seed of Garcinia kola is known as kolaviron ( Figure 3). Kolaviron is a mixed biflavoniod complex derived from the defatted alcoholic extract of the plant. The complex consists of GB-1, GB-2, and kolaflavanone) shown below in Figure 3 (Cotterill et al., 1978). The primary difference between GB-1 and GB-2 is the replacement of hydrogen (H) with a hydroxyl group (OH). Earlier work on the phytochemistry of Garcinia species resulted in the isolation of pharmacological compounds from several chemical classes including xanthones, bioflavonoids, triterpenes and benzophenones. Xanthones are polyphenols that arise from the biosynthetic pathways of shikimate and acetate. A number of xanthones are known for their anti-cancer and anti-obesity properties (Iwu et al., 1987). The fruit hull of Garcinia mangostana possesses anti-cancer activity and this biological effect has been attributed to xanthones. Xanthones are believed to act by blocking inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression (Wan et al., 2004). Xanthones also possess anti-inflammatory activities (Chen et al., 2008) by the reduction of iNOS in murine macrophages. Bioflavoniods are phenolics, a class of phytochemicals which primarily act as plant pigments and possess antioxidant activities. Bioflavonoids have been isolated from the Garcinia kola and are abundant in the seeds of the plant (Faromi et al., 2011). Triterpenes are compounds comprising six isoprene units which are also precursors of steroids.
Triterpenes are found in the root bark of Garcinia polyantha and they possess antimalarial properties (Lannang et al., 2008). Benzophenones are ketone molecules used in perfume and medicine. Kolanone is a prenylated (addition of a hydrophobic prenyl moiety) benzophenone isolated from Garcinia kola possessing antimicrobial properties (Hussain et al., 1982). These chemical classes are important and they may play a role in modulating various molecular metabolic pathways essential to human life. They also show the diverse functions that this natural product possesses, suggesting many potential therapeutic and biological applications for Garcinia kola.

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The central objective of this study is to compare two different extraction techniques for the isolation, identification and conduct a pharmaceutical formulation of a potential antiviral compound responsible for inhibiting viral replication from Garcinia kola. Although there is evidence that such isolation methods can be applied to isolate potential benzophenone antiviral compounds from related plant genera, these particular methods have not been applied to Garcinia kola. In order to accomplish this, the following hypotheses are made: 1. Utilize liquid-liquid extraction technique of Fuller et al.1999 for the isolation of antiviral compound Guttiferone F (also called Garcinol) found in the rootwood of Allanblackia stuhlmannii and applying the same extraction technique to Garcinia kola nuts could lead to the isolation of the same antiviral compound. In addition, profile the compounds found in this extraction technique.

Utilize a second extraction technique, Supercritical Fluid Extraction (SFE) on
Garcinia kola nuts for the isolation of antiviral compound Guttiferone F (also called Garcinol). In addition, profile the compounds found in this extraction technique and compare both extraction techniques.
3. Formulate a representative antiviral compound into suitable liposomes for controlled drug release.
The advantage of using both extraction techniques is to obtain a wide range of extracted compounds found in Garcinia kola and for comparison purposes.

Antiviral properties found in the Garcinia family
There are various species in the Garcinia genus which are reported to possess antiviral properties. These species include Garcinia livingstonei, Garcinia ovalifolia (Gustafson et al., 1992), and Garcinia mangosteen (Chen et al., 1996), among others.
The compounds responsible for the antiviral properties have been identified as guttiferones. Guttiferones are polyisoprenylated benzophenone derivatives that inhibit by cytopathic effects the activity of the virus responsible for HIV infection (Gustafson et al., 1992). Most polyprenylated benzophenones isolated from these plants also contain carbonyl, hydroxyl and other polar groups.
Guttiferone and related benzophenone antiviral compounds have been reported to be isolated from Garcinia kola and other Garcinia spp. and they contain a number of valuable biological active compounds. However, relatively few studies have been reported describing the design and performance of Garcinia derived formulations and drug delivery systems on its proposed antiviral properties. Accordingly, another goal of this work is to develop a pharmaceutical formulation for the controlled delivery of a benzophenone antiviral compound.

Natural Product Development
According   tablets (Onunkwo et al., 2004). Briefly, in their method, a chemical assay was conducted on the dry, powdered seeds as well as the crude aqueous extract of the seeds. Next, the powdered material (50 mg) and an aliquot part of the crude extract (10 mg) were formulated into tablets using the wet granulation method. Extraction of the powdered seeds was performed using ethanol as a solvent and concentrated using a rotary evaporator, resulting in a dry mass. After evaluating those parameters listed above, the results indicated that the tablets had acceptable disintegration time, dissolution, hardness and friability profiles.
A more recent study conducted in January 2010 at the Lagos University from the large plant family Guttiferae or Clusiaceae (Gustafson et al., 1992). Fuller et al. isolated the first member of the guttiferone F (also called garcinol) class of polyprenylated benzophenones from the genus Allanblackia (also in the Clusiaceae family). In this study it is hypothesized that the same or similar antiviral compounds occur in, and may be isolated from, Garcinia kola since it is in the same genus. If the Garcinia genus contains guttiferones A-E, there is a possibility that Garcinia kola might also contain guttiferone F; which has yet to be isolated and identified in Garcinia kola, which is said to have antiviral properties. It is hypothesized that, by using the isolation methods of Fuller et al., the same or similar compounds may be isolated from Garcinia kola. These are core to be answered by this research.
One of the most important issues that arise when formulating natural products is poor aqueous solubility and stability. Many new chemical entities display poor aqueous solubility. However, there are many techniques used to possibly overcome solubility, such as nanosupensions, drug dispersion, and chemical modification.
Nanosuspensions are colloidal dispersion of particles of a drug that are stabilized by surfactants. Nanosupensions also increases dissolution rates due to a large surface area to unit volume ratio. Drug dispersion in carriers which include solid solutions (solid solute molecularly dispersed in solid solvent), eutectic mixtures (fusion melt of solute and solvent showing complete miscibility at or above all constituent melting points) and solid dispersion (dispersion of one or more compounds in an inert carrier in the solid state) (Gowthamarajan et al., 2010). To be more specific, solid dispersion is used to improve the solubility of poorly soluble compounds, as well as mask the taste of a drug substance, improve the disintegration of oral tablets, and reduce the formulation particle size and thus increase the dissolution rate. This technique is widely applied in the pharmaceutical industry. The last technique that will be discussed for the use to overcome poor solubility is chemical modification. Some examples of chemical modification are the formation of a salt complex and the use of a prodrug (Mohanachandran et al., 2010).
As written in the previous paragraphs, solubility is a challenge in the successful formulation of compounds such as the benzophenones found in the Garcinia family. An example of this is found in the work described by Bhaskar et al.

According to Bhaskar et al., Garcinia mangosatana is proven to be active against
Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acne, which are responsible for the outbreak of acne. Preformulation studies using a mixture of the aqueous extract of Garcinia mangosatana and with the excipient Aloe vera proved to overcome poor solubility and results shows Garcinia mangosatana to be a potential for drug delivery as a topical agent for the treatment of acne, which was the goal of the study. Another study of Garcinia mangostana (specifically α-Mangostin) sought to improve the solubility and bioavailability of poorly-soluble αmangostin using solid dispersion (Aisha et al., 2011). The poor solubility of αmangostin hinders its therapeutic application by limiting its oral absorption. The study 13 showed an enhancement in the solubility of α-mangostin using solid dispersion systems. Also, further research suggests the need for a new therapeutic strategy of HIV therapy due to the poor solubility of such compounds (Gupta et al., 2010).
Overall, the formulation of Garcinia mangostana and HIV therapies in general are often shown to have issues of poor solubility and the utilization of a dispersion system maybe a useful technique for enhancing solubility. Thus, it is hypothesized that if anti-viral Garcinia mangostana constituents derived from a plant from the same family as Garcinia kola, have poor solubility, than analogous materials derived from Garcinia kola could also display poor solubility and be amenable to solid dispersion or another solubility type enhancing formulation. A carrier formulation that may protect the plant constituents from oxidation or hydrolysis, but that may also be useful for oral or topical delivery, such as a physiologically-compatible liposome, may be a viable formulation alternative.
In summary, there are antiviral compounds found in the Garcinia spp. There is a need on the market for more antiviral therapeutic drugs. Research suggests that although there are many studies of Garcinia spp. which focus on formulation, the formulation of antiviral compound(s) from Garcinia kola is lacking. Now that we know what types of compounds are found within the Garcinia spp., it's important to also look at different extraction techniques used to extract these compounds from their natural source.

Extraction technique overview
Extraction is a broad term used in the pharmaceutical industry and it involves the separation of medicinally active compound(s) of plants and or animals from the 14 inactive components by using solvents from extraction procedures. One of the most universal forms of extraction is brewing coffee: low molecular weight/concentration molecule (caffeine) is removed from the high molecular weight/concentration material (coffee bean). Another example is brewing tea which uses the same principle. This is an example of solid/liquid extraction. On the other hand, liquid-liquid extraction is commonly used in organic laboratory to separate and purify the desire product from a mixture leaving behind a starting material and by-products. For example, the extracts from plants are fairly impure therefore standardized extraction procedures is needed for the final quality of the medicinal compound which can lead to a potential drug.
Both techniques are governed by distribution coefficient, a measure of how an organic compound distribute between aqueous and organic phase. Distribution coefficient is very important in drug delivery because a drug must be carried throughout the body to its targeted site. A drug must have enough water solubility to dissolve in the blood and organic solubility to get through the cell. Below are two extraction techniques used in the pharmaceutical industry as well as some advantages and disadvantages for their use.

Liquid-Liquid extraction
Liquid-liquid extraction/partitioning (the term solvent-solvent (S-S) extraction will be used interchangeable) is a method in which a desire compound is pulled from one solvent to another solvent only if the two solvents are immiscible (example oil and water, Figure 6). The most common method of liquid-liquid extraction is using a separatory funnel. Separatory funnels are used to extract a compound either from or into an aqueous layer. Depending on the density of the solvent(s) repetitive use of the 15 separatory funnel or another type of extractor is needed. Some advantage of using liquid-liquid extraction includes it being the simplest form of extraction every used for wide range of application in both chemical laboratories and pharmaceutical industries.
Other advantages include the ability to operate in a continuous mode, and the use of two solvent phases. One major disadvantage is the use and disposal of disposal of solvents used. In addition, this technique can be performed with simple equipment, and its selective separation is usually highly efficient.

Supercritical Fluid Extraction (SFE)
Supercritical Fluid Extraction (SFE) is a technique used to separate one component from another component using supercritical fluids as the extracting solvent. SFE is also an alternative sample preparation method generally used to reduce the use of organic solvents and increase sample throughput. SFEs are produced by heating a gas above its critical temperature or compressing a liquid above its critical pressure ( Figure 7). Because of its low critical parameters (31.1 ᵒ C, 73.8 bar), carbon dioxide (CO 2 ) is the most used supercritical fluid used and is at times modified by cosolvents such as methanol and ethanol in order to overcome polarity limitations. Advantage of using CO 2 includes its favorable physical properties, its inexpensive, relatively safe and in abundance.
A typical SFE system includes a CO 2 source, a pump to pressurize the gas, an oven containing the extraction vessel, a pressure regulator, an analyte collector and a detector ( Figure 8). Another advantage of using SFE is its extraction procedure having little to no solvent residue and extraction at very low temperatures. The rationale for the use of SFE was because of its shorter extraction time, and selective extraction by varying temperature, pressure and or modifier.
SFE has been used for various extraction and isolation of natural products including decaffeination of coffee, and extraction of hops, spices and tea because it is an eco-friendly alternative to chemicals and solvents used by chemist. This is because Chemist generate large amount of waste organic solvents which is growingly attributed in environmental pollution and hazardous to human health. However, the use of SFE in natural product extraction is limited to plant natural products.

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The goal of the SFE technique was to search for garcinol by performing liquidliquid extractions, and SFE extractions with CO 2 , modifier which included methanol, ethanol, and isopropanol, and analyze via UPC 2 coupled with Q-Tof-MS. The second goal was to perform principal component analysis (PCA) of the SFE and S-S extracts in order to find correlations to streamline sample prep approach, exploit TransOmics workflows to aid visualization of data, targeted processing for garcinol, and comprehensive processing for feature differences. Dynamic Light Scattering (DLS). Lastly, the rate of release and extent of release was conducted using dissolution tests. The follow discuss the materials and methodology used.

Extraction techniques
The following sections include the extraction methodology employed on Garcinia kola using the two extraction techniques mentioned above.

Liquid-liquid extraction
Two kilograms of Garcinia kola nuts were spread on a plastic bag and dried in a Robertshaw Drying Oven at 135 ᵒ F for five days. Upon exhaustive drying, the nuts were peeled and the peelings were kept separately for future evaluation. The nuts were ground using a coffee grinder yielding a total of 1071 grams of ground material.
One kilogram of the ground nuts was weighed and transferred to a large glass chromatography extraction column with a mechanical mixer. Three liters of hexane, then three liters acetone, then three liters of methanol, and then with one liter of a combined 1:1 methylene chloride-methanol was then introduced to the nuts respectfully. It is important to note that one liter of solvent was added at a time, mixed for 12 hours to 24 hours, and then removed. This entire process was monitored for roughly six days. The seeds were also subject to other solvents including ethyl acetate and chloroform (<250 mL each) solvent extraction scale. The various solvent systems served as a removal vehicle of compounds found in Garcinia kola nuts. After each successive extraction, the solvents were removed by rotary evaporation, and the 20 resulting residues were labeled, sealed, covered and stored. The hexane extract was bright yellow, and the acetone and methanol extracts reddish-brown and brownish-red, respectively.
Next, the method described by Fuller et al., comprising solvent-solvent extraction (hence the use of the term S-S because the rotary evaporated samples, mentioned above were re-introduced to solvent), was applied to the initial extract residues by combining a 5 g portion of the 1:1 methylene chloride-methanol and methanol extracts (2.5g each). This combination was further separated by solventsolvent partitioning into hexane (200 mL), methyl-tert-butyl ether (MTB, 10 mL), ethyl acetate, and water. The purpose of partitioning was to ensure that the compounds were separated based on their solubilities into two different immiscible liquids. In short, after the portioning fraction was added to the separating funnel with the 1:1 methylene chloride-methanol and methanol extracts combined, the funnel was closed, shaken abruptly which lead to one phase (hexane for instance) being separated out from the 1:1 methylene chloride-methanol and methanol extracts phase and slowly opening the tap of the funnel and collecting each immiscible fractions. The partitioned hexane, MTB, ethyl acetate and water fractions were labeled, sealed with aluminum foil and Parafilm® around the neck of the beaker, and placed in the laboratory refrigerator in Fogarty 41.
The MTB fraction was passed through Sephadex LH-20 loaded into a glass chromatography column using 1:1 methylene chloride-methanol as the mobile phase, yielding 8 fractions. The third and fourth fractions were collected are noted as (Sephadex fraction 3/4). An illustration of this protocol is further depicted in Figure 9, 21 all of which were analyzed by analytical techniques in section 3.3.

Supercritical fluid extraction (SFE)
Sample preparation for the SFE experiment was performed by weighing out 1 gram of Garcinia kola nuts which were placed in empty white tea bags, the bags were folded and stapled shut. The tea bags were placed in a metal vessel and sealed tightly.
The vessels were then loaded into the oven portion of the SFE. Valves were connected, the instrument set with 100% carbon dioxide (CO 2 ), and organic solvent modifiers, including methanol (MeOH), ethanol (EtOH) and isopropyl alcohol (IPA) 5%, 10% and 30% each by w/w. Each sample collected was subjected to various analytical methods also mentioned in section 3.3.

Analytical analysis
The following sections include the analytical analysis of samples extracted and collected with both extraction techniques mentioned previously. isocratic method on each of the separation process found in Figure 9. Another type of chromatography used is Ultra Performance Convergence Chromatography (UPC 2 ). UPC 2 is also an analytical technique that incorporates the re-design of a chromatographic system using liquid carbon dioxide (CO 2 ) as the primary mobile phase. UPC 2 uses both gas and liquid as mobile phases; hence, the term convergence. A co-solvent or modifier, typically methanol, is used as an elution solvent. The technique follows the principles and selectivity of normal phase chromatography (hydrophilic stationary phase). UPC 2 also uses stationary phases consisting of smaller particle size (< 2 µm) therefore providing greater efficiencies that results in a greater resolving power capable of separating more analytes per unit time. UPC 2 works great for improving the productivity, efficiency, and throughput of samples. It reduces retention times, and thus processing cost, and drastically reduces solvent usage. Alternating high and low collision energy was performed during a single injection, a technique known as MS E , which uses two different steps of mass spectroscopy, allowing for precursor and product ion determination. This method is used for finding out related substances of the compound of interest. The alternating scanning provides accurate mass measurement for all detectable ions for the system and provides faster speed and resolution. By applying a collision energy field, MS E can be applied in an aim to measures the molecule's distinct fragmentation pattern which provides structural information. Note that Base Peak Intensity (BPI) was used 25 as a trace that subtracts the background noise in the spectra. UPC 2 may also be coupled to quadrupole and time-of-flight mass analyzers (Q-Tof MS) in order to target a broad range of compounds in complex samples. And this technology is used in order to comprehensively acquire data, locate and identify known compounds, identify unknown peaks of interest, and elucidate structures of unknown.

Reverse Phase High Performance
The workflow employed for the chemical profiling of Garcinia kola is described in Figure 10. Extracts from the samples listed in Figure 9, were analyzed using Waters ACQUITY UPC 2 instrumentation. An ACQUITY UPC 2 BEH column with dimensions 3.0 x 100mm; 1.7um was observed as providing the best separation with the greatest number of peaks. Ethylene Bridged Hybrid (BEH) columns are used in small molecule to large biopharmaceutical analysis. MS data acquisition was performed using Waters Xevo G2 Q-ToF. Alternating high and low collision energies, 5V and 30V, respectfully, allowed for precursor and product ion determination as stated above.

Nuclear Magnetic Resonance ( H NMR and 31 P-NMR)
Nuclear Magnetic Resonance (NMR) is a spectroscopic technique used to study nuclei with non-zero nuclear spins. All nuclei contain protons hence they have charge and spin. Many nuclear spin values are possible. The most important are those with spin ½ such as 1 H and 31 P that are relevant here. When these non-zero nuclear spin nuclei are placed in a magnetic field, two spin states form; one of higher energy and a more abundant lower energy state. NMR is detected by observing the spins moving between these energy states after an electromagnetic pulse. NMR can be used to investigate molecular structure using proton 1H NMR as well as investigate lipid bilayer structure packing with phosphorus 31 P NMR.
The result of an NMR experiments is reported as a chemical shift (δ, delta) from a reference compound and is measured in parts per million (ppm

Biological Assay
Antiviral and cytotoxicity evaluation of three selected extracts of Garcinia kola (Table 1 and

Preparation of liposome formulations
Liposomes are hollow bi-lamellar or multi-lamellar spheres (vesicles) typically comprised of physiologically compatible lipids. These membrane-like structures have performed after sonication of the hydrated lipid film to confirm the particle size and its distribution. Finally, the liposome formulation was subject to release studies in order to assess the rate and extent of garcinol release.

Sonication
After the liposomes were make, its particle size was very large. To improve and decrease the size of the liposomes, they were carefully sonicated for 2 minutes at 50% amplitude.

nano-Differential Scanning Calorimetry (nano-DSC)
Differential Scanning Calorimetry (DSC) is used to measure melting temperature, heat of fusion, latent heat of melting, reaction energy and temperature, glass transition temperature, and precipitation energy and temperature, denaturation temperatures, and specific heat or heat capacity. DSC measures the amount of energy absorbed or released by a sample when it is heated or cooled, providing quantitative and qualitative data on endothermic and exothermic processes. DSC also measures the degree of crystallinity as well as the drug release profile which can be used to optimize formulation of suspensions/emulsions and predict long term stability. This is done by the change in the heat that flows through the sample and a reference at a certain temperature is observed as the test temperature changes. An empty aluminum pan serves as the reference sample. Data is measured as a function of temperature, which is typically varied over a period of several minutes The liposome 1, 2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in Figure   11 which is a neutral phospholipid containing two palmitic acid (16-carbon saturated chains) esterified to glycerol with a phosphate group and choline attached at the polar end was used. The phase transition temperature of DPPC from gel to liquid crystal is 42 °C, higher than human body. DPPC is surface active and fervently forms liposomes, with the molecule actively comprising a high compaction capacity as a result of the free rotation and attraction of the palmitoyl tails to each other.

Figure 11: 1, 2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (courtesy of Google image)
Analysis of the DPPC liposome was performed using a DSC Q100 from TA Universal Instruments. Here, 300µL of the sample was weighed and placed in an aluminum pan with a cover. The heating rate was 10 °C/min from 0 ᵒ C to 180 °C under 40 cm 3 /min nitrogen flow. The DSC apparatus was set equilibrated at 20 °C.
Run time was roughly 2 hours. Upon the conclusion of the sample run, 1 L of deionized water was used to clean the DSC apparatus to ensure that there were no liposomes left stuck in the apparatus. Data was further assessed on the instruments software.
The same theory discussed in the previous paragraph is applicable to nano-DSC but on a nano scale (nanometers, nm, in size). Nano-DSC was performed using a TA Instruments Nano DSC (New Castle, DE, USA). Samples at a concentration of 0.1 mM lipid were degassed under vacuum for 30 min before loading into a 0.6 mL capillary cell. The cell was then pressurized with nitrogen to 1 atm and equilibrated at 25 °C. The sample was scanned at 1 °C min -1 over a range of 25 °C to 60 °C.

Zeta potential
The fundamental states of matter are solids, liquids and gases. A colloidal system occurs when one of the states of matter is finely dispersed in another. This effect is evident in measuring thermal analysis by observing a particles' zeta potential. Zeta potential, Greek letter (ζ), ζ-potential, measures a fluid containing particle (in this case the dispersion of the garcinol liposome) in a colloidal system. It measure the electrical potential difference between the double layer of a particle at the slipping plane with respect to the bulk liquid away from the interface as seen in Figure 12. This measurement provides detailed information about the stability of the colloidal system.
Particles within the dispersion with a zeta potential tends to move toward the electrode of opposite charge with a velocity relative to the amount of zeta potential. This velocity is observed when the particle mobility is captured by the laser which is then converted to zeta potential. A smaller molecule that has a high zeta potential (let's say ±60 mV) means that the molecules in solution resist aggregation (electrically stable) hence exposing charged phosphate groups. However, coagulation occurs at lower zeta potential (±5 mV).
There is no direct way to measure zeta potentials but there have been various theoretical models and experimental techniques used to calculate it. A known theoretical model used to describe the stability of a colloidal system is the DVLO theory; named after its discoverers Derjaguin, Landau, Verwey and Overbeek. DVLO theory states that the stability of a particle in solution depends on its total potential energy (V T = V S + V A + V R ) which consists of the total energy (V T ) produced by the 33 solvent (V S ), total attractive forces (V A ) and the total repulsive force (V R ). In order to obtain stability in colloidal systems, there should be minimal attractive force and repulsion in order to resist the flocculation of particles. One experimental technique used to estimate zeta potential of a particle in other words measure colloidal stability is electrophoresis.
A small aliquot part of each formulation (17 mM DPPC) was diluted with 137 mM PBS to give a final lipid concentration of 1 mM. Zeta potential values were then determined using a laser doppler procedure with a Malvern Instruments Zetasizer Nano ZS at 25 °C. Air drop interference was eliminated before measuring the zeta potential.

Differential Light Scattering (DLS)
Differential Light Scattering (DLS) determines the size distribution profiles of particles in suspension. This occurs by a light (often a laser) hitting a particle (in this case the garcinol liposome), producing an electronic distortion that is emitted in all directions. An oscillating dipole in the electron cloud results. As the dipole changes, energy is scattered in all directions and a fluctuating (or dynamic) intensity which captures the size of the particle in solution is obtained. Smaller particles undergo Brownian Motion, random motion of particles in a liquid or gas resulting from collision molecules in the gas or liquid. And the distance between scattering changes consistently with time.
DLS was carried out after sonication of the hydrated lipid film to confirm the liposome particle size and its distribution. DLS measurements were performed using a Malvern Instruments Zetasizer Nano ZS with a backscattering detector angle of 173° and a 4 mW, 633 nm He-Ne laser (Worcestershire, UK). For size distribution studies, 1 ml of the liposome formulations was analyzed in an optical grade polystyrene cuvette at 37 °C. Before analysis, the samples were stored at 37 °C and then evaluated after 24 hours.

Dialysis
During the film rehydration stage of liposome (vesicle) preparation, a certain amount of PPB that was not trapped in the vesicles had dissolved garcinol in it.
Consider also that all of the garcinol might not have been entrapped and some may still be present in the untrapped PPB. To overcome these potential barriers, the garcinol liposomes were subjected to dialysis studies in order to remove any untrapped drugs so that only the release of drugs from the vesicles could be accounted for. The dialysis experiment was carried out until a constant drug concentration was obtained and that equilibrium had been achieved in order to ensure no unentrapped garcinol was present in the sample.
Dialysis experiments were conducted at room temperature (25 ± 0.5 °C) using MWCO 3500 kD cellulose membranes for 24 hrs in 50 mM potassium phosphate buffer solution with constant stirring so that any potential unencapsulated garcinol was removed.

Dissolution
Extensively used in the pharmaceutical industry, dissolution studies provide information about drug release for various dosage forms. Dissolution studies were conducted in order to determine the garcinol release profile from the liposome formulation. Dissolution studies are importanin in predicting the bioavailability of 35 various drug formulations. Dissolution was performed by transferring the dialysis tube into a smaller beaker of 50 mM potassium phosphate buffer (37 ± 0.5 °C) solution at pH of 7.4 with a magnetic stirrer at 75 rpm. As 1 ml buffer samples were collected for analysis of 24 hrs period, the buffer was replaced with new buffer in order to maintain a sink condition. Dissolution samples were collected at serial time points and were analyzed for garcinol content by HPLC in order to quantify release over time. The results obtained from the HPLC analyses are represented in the HPLC chromatogram displayed in Figure 13.
Results in Figure 13 shows the S-S samples were overlaid and compared to minutes. This suggests that the antiviral compound of interest, garcinol was either not present in any of the S-S extracts or was present at a concentration below the limits of detection of the HPLC assay.

Supercritical fluid extraction (SFE) results
The goal of the SFE technique was to search for guttiferone F. via 100% CO 2 , 5%, 10%, 30% modifier extractions which included methanol, ethanol, and isopropanol, and analyze via UPC 2 coupled with Q-Tof-MS. The second goal was to perform principle component analysis (PCA) of the SFE and S-S extracts in order to find correlations to streamline sample prep approach, exploit TransOmics workflows to aid visualization of data, targeted processing for garcinol, and comprehensive processing for feature differences found in the next section.
Results shown in Figure 14 are of the HPLC overlaid chromatograms for each of the SFE extractions and are compared to an injection of garcinol standard.
Synonymous to the previous section, results indicate none of the extracts contained detectable analytes with the same retention time as that of garcinol, which has a retention time of roughly 12.5 minutes. This also suggest that the antiviral compound of interest, garcinol was either not present in any of the SFE extracts or was present at a concentration below the limits of detection of the HPLC assay.

Comparison of both extraction techniques
After compiling the HPLC overlay as in Figure 13 and Figure   In order to look more in detail at the SFE fraction, quantitative analysis was performed using mass spectroscopy. Figure

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The results of the 1291 features in Graph 1, 20.67% are neglected. Of the remaining detectable compounds indicated, 50.74% of the extracted compounds can be extracted The observation of these common components could further provide a more insight into the extracts within Garcinia kola.
Next we examined garcinol at high and low collision energy as well as molecular fragmentation of garcinol. Figure 17 and Figure 18 represents that data.

Biological Study
The results for the cell-based, MTT anti-viral assay are listed in Table 2. The three Garcinia kola extracted samples tested were completely inactive against both HIV-1 (strain III B ) and HIV-2 (strain ROD). The extracted samples have a high cytotoxicity CC 50 values >60 µg/ml as compared to the control lower CC 50 values >20 µg/ml. These data are consistent with the HPLC and MS data which also suggest that garcinol is not present in any of the extracted compounds (S-S and or SFE); if indeed garcinol is an active in vitro antiviral agent.

Garcinol formulaiton study
The following are the results represent the antiviral formulation studying where Garcinol acts as the active pharmaceutical ingredient.

Zeta potential
The surface charge was measured through Zeta potential measurements and the results predicting the stability of the colloidal system below in Graph 7.

Nuclear magnetic resonance ( 1H NMR and 31 P-NMR) results
The results in Graph 11 shows a proton NMR spectrum of Garcinol and

Formulation study
In order to select the appropriate biological buffer for producing DPPC liposomes wherein garcinol would be stable, an HPLC stability study was conducted using garcinol in the presence of various biological buffers. This was conducted in order to select the best, non degrading buffer solution that garcinol would be stable in.
The biological buffers used were: 137 mM phosphate buffer saline (PBS); 0.9% sodium chloride (NaCl); 5 mM sodium phosphate; 5 mM sodium chloride; 5 mM Tris (hydroxymethyl aminomethane); 5 mM sodium citrate; and 50 mM potassium phosphate (PPB) all at pH=7.4 while using the previously developed acetonitrile:water isocratic HPLC method for analysis. The HPLC chromatograms for each buffer system were as shown in Figure 19 to Figure 30.     Figure 19 to Figure 29 show the injection of garcinol has a retention time of roughly 12.5 minutes. However, a number of degradants increased in size and were observed in the various buffer systems at about 7-9 minutes. The concentrations also increased over time in several buffer systems. Accordingly, potassium phosphate buffer solution was selected as the suitable liposome hydration medium for formulating garcinol in DPPC liposomes.

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The results of liposomal garcinol formulation are summarized in Graph 13 and 14 below.

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Graph 15 shows the garcinol HPLC calibration curve indicating a 97.26% linear trend line of concentration versus area under the curve. In order to study the release of the garcinol formulation, a dialysis study was conducted as seen in Graph 14. Over the 24 hour period, there was an increasing trend in concentration of the dialysis study.
Garcinol released over time during the 24 hours was proportional to time. In the dissolution study, results in graph 15 indicate a fast release follow by a sharp (65%) drop in concentration after 4 hrs, perhaps due to saturation and precipitation of garcinol in solution. Given the high affinity of garcinol for DPPC lipid, a significant amount of garcinol might still be present in the bilayers. Although garcinol was not present in both the extraction techniques mentioned above, the aim of this research was to formulate an antiviral compound. garcinol, available commercially and found in other Garcinia spp., was used as a reference standard for comparative means. Based upon the methods and techniques employed, it was determined that garcinol was not isolated from Garcinia kola in detectable amounts. Garcinol was used as a model compound for a representative liposomal formulation which was prepared and characterized. Preformulation experiments were conducted in order to identify an appropriate physiological buffer for preparing the liposomes, as it was found that garcinol was unstable in several representative buffer systems as well as release studies.
Garcinol liposome were made using DPPC which is widely used to study liposomes. H NMR and 31 P-NMR analysis were performed for particle size separation as well as to observe the amount of hydrogen and phosphorus, in the garcinol compound. The proton NMR confirmed that garcinol and guttiferone F is relatively the same compound and both names are used interchangeable. Phosphorous NMR observed any shielding effect of the garcinol liposomes. Nano-DSC studies were performed for the transition temperature of the liposome lipid component (DPPC) blank and to predict the transition temperature of the garcinol liposomes. Results indicated a lower transition temperature in the garcinol-DPPC formulation. DLS studies were performed in order to characterize the size and size distribution of the garcinol-DPPC liposome. The results indicate a uniform shape after sonication as well as an increase in size over time (7 days) which could be an indicator of stability issue.
Zeta potential was conducted for the characterization of the surface charge of the 64 vesicles. The results showed that garcinol had a strong interaction with the phosphate head groups in the DPPC vesicles which may cause stability issues of the garcinol formulation. Both dialysis and dissolution study show garcinol was released in the DPPC liposome although in very small quantities.
In conclusion, most of the applied techniques mentioned above including SFE, nano-DSC, UPC 2 coupled to Q-Tof MS, 31 P-NMR and liposomal formulation have not previously been performed on Garcinia kola preparations or garcinol. This research is original, innovative and multidimensional. Although Garcinia kola fractions did not inhibit HIV virus as compared to the controls, the techniques used in this research is trendsetting in the investigation of antiviral compounds found in Garcinia kola.