degen-An examination of the antimicrobial and anticancer properties of Garcinia cambogia fruit pericarp extracts

Background: Garcinia cambogia (synonym Garcinia gummi-gutta) is commonly known as brindleberry and Malabar tamarind. It has received considerable recent interest due to its potential in the prevention and treatment of obesity and obesity related diseases. The fruit pericarp also has traditional uses in the treatment of a wide variety of diseases and medical conditions, yet these have received relatively little investigation to date. Methods : Garcinia cambogia fruit extracts were investigated for their ability to inhibit the growth of a panel of bacteria of medicinal importance. The extracts were also tested for their ability to block the proliferation of the CaCo 2 and HeLa human carcinoma cell lines. Results: Garcinia cambogia methanolic, aqueous and ethyl acetate extracts displayed broad spectrum antimicrobial activity, inhibiting the growth of between 16 (64 %; ethyl acetate extract) and 22 (88 % aqueous extract) of the 25 bacterial species tested. Strong inhibitory activity was detected with minimum inhibitory concentration (MIC) values <1000 µg/ml against many bacteria. All extracts were effective against both Gram negative and Gram positive bacteria, although Gram negative bacteria were more sensitive. All extracts displayed anti-proliferative activity against CaCo 2 and HeLa carcinoma cells, yet were non-toxic in the Artemia franciscana bioassay, with LC50 values greatly in excess of 1000 µg/ml. Conclusion: The inhibitory bioactivity against a range of microbes, the anti-proliferative activity against CaCo 2 and HeLa cells, as well as the lack of toxicity, indicate the potential for G. cambogia in the discovery and development of new natural pharmaceuticals.


INTRODUCTION
Garcinia (Family Clusiaceae) is a large diverse genus of approximately 240 species.Several Garcinia spp.have been reported to have high antioxidant contents.Possibly the best known species is Garcinia mangostana (purple mangosteen).Recent studies have reported the therapeutic potential of G. mangostana fruit pericarp and linked these properties to its antioxidant capacity. 1 In particular, antibacterial and anticancer activities were reported.The West African species, Garcinia kola (bitter kola), is well known for its use in a variety of traditional medicine systems to treat bronchitis, throat infections, coughs and colds, as well being used as a general antiseptic, anti-parasitic and purgative. 2Laboratory studies have also demonstrated the high free radical scavenging activity 3 and Ebola inhibitory effects 2 of G. kola fruit.Several other Garcinia species also have high antioxidant capacities and free radical scavenging activities.Epidemiological studies have shown that a diet rich in antioxidants is associated with a decreased incidence of chronic diseases. 4High antioxidant levels have also been shown to act as a preventative against the development of degenerative diseases such as cancer, 5 cardiovascular diseases and neural degen-eration, 6 diabetes and obesity 7 .Phenolic compounds are generally strong antioxidants. 8Their primary action involves the protection of cell constituents against oxidative damage through the scavenging of free radicals, thereby averting their deleterious effects on nucleic acids, proteins, and lipids in cells 8 .Phenolics interact directly with receptors or enzymes involved in signal transduction 9 , clearly indicating that they play a specific role in human physiology.Therefore, examination of the therapeutic properties of other high antioxidant Garcinia species is warranted.Garcinia cambogia (Geartn.)Desr. is a slow-growing evergreen tree which grows in tropical areas of South Eastern and Eastern Asia, as well as in Western and Central Africa, although it is believed to have originated from the Indian subcontinent 10 .It is known by a variety of names including brindleberry, Malabar tamarind, pot tamarind and Garcinia gummi-gutta.The fruit is deeply lobed with thin green to pale yellow skin and is about the size of a grapefruit.The fruit consists of a soft white edible pulp surrounded by the pericarp.The pericarp may be used as a spice, or (in conjunction with salt) to dry and cure fish 11 .It is also sought after for its therapeutic properties.G cambogia has traditional uses for a diverse array of ailments, including use as a laxative and as a general antiseptic. 12ecent interest in the therapeutic potential of G. cambogia has focussed on its apparent hypocholesteremic and anti-obesity effects. 124][15] Despite this lack of definitive evidence, G cambogia has achieved notable recent public acclaim, at least in part due to its coverage as a weight loss aid on the popular American television show, Dr. Oz 6 and subsequent media coverage.Much more research is required to establish the potential of G cambogia in the prevention and treatment of obesity, diabetes and cholesteremic disorders.G cambogia contains many interesting phytochemicals, although much of the interest has focussed on hydroxyacetic acid (HCA) due to its functional inhibition of citrate lyase. 17Due to the role of citrate lyase in fatty acid biosynthesis, inhibition of this enzyme would be expected to block the synthesis and storage of triacylglycerols and result in fatty acid mobilisation from the adipose tissue.Several studies have reported this effect in rat models. 18However, similar trends have not been reported for human trials. 13,15spite the current interest in the therapeutic potential of G. cambogia, scientific research is focussed almost entirely on its potential antiobesity potential.Other therapeutic properties have not been adequately explored.This study was undertaken to examine several other therapeutic properties of G. cambogia.In particular, the antimicrobial properties of G. cambogia extracts were evaluated against an extended panel of bacteria.This study also aimed to examine the anticancer properties of G. cambogia by testing the extracts againstthe CaCo 2 colorectal and HeLa cervical cancer cell lines.The toxicity of the G. cambogia extracts was also examined to further determine their suitability for therapeutic usage.

MATERIALS AND METHODS
G. cambogia fruit pericarp powder was bought from One Stop Nutrition, Australia as an air dried ground powder.An amount of 1 g of plant material was weighed into each of five tubes and five different extracts were prepared by adding 50 ml of methanol, water, ethyl acetate, chloroformor hexane respectively.All solvents were obtained from Ajax and were AR grade.Powdered material was extracted into each solvent for 24 hours at 4 o C with gentle shaking.The extracts were filtered through filter paper (Whatman No. 54) under vacuum, followed by drying by rotary evaporation in an Eppendorf concentrator 5301.The resulting dry extracts were weighed and redissolved in 10 ml deionised water (containing 1% DMSO).

Evaluation of antimicrobial activity
3][24] Briefly, 100 µl of the test bacteria were grown at 37 o C in 10 ml of fresh nutrient broth until they reached a count of approximately 10 8 cells/ml (determined by direct microscopic counting).One hundred microliters of microbial suspension was spread onto the appropriate agar plates.The extracts were tested using 5 mm sterilised filter paper discs.Discs were impregnated with 10 µl of the test sample, allowed to dry and placed onto inoculated plates.The plates were allowed to stand at 4 o C for 2 hours before incubation with the test microbial agents.Plates were incubated at 37 o C for 24 hours and then the diameters of the inhibition zones were measured in millimetres.All measurements were to the closest whole millimetre.Each antimicrobial assay was performed in at least triplicate and mean values were determined.Standard discs of ampicillin (2 µg) were obtained from Oxoid Ltd. and served as positive controls.Filter discs impregnated with 10 µl of distilled water were used as negative controls.

Minimum inhibitory concentration determination
The minimum inhibitory concentrations (MIC) of the G. cambogia extracts were determined by the disc diffusion MIC method across a range of doses. 25,26Discs were impregnated with 10 µl of the test dilutions, allowed to dry and placed onto inoculated plates.The assay was performed as outlined above and graphs of the zone of inhibition versus concentration were plotted for each extract.Linear regression was used to calculate the MIC values.

Cancer cell lines
The CaCo 2 and HeLa carcinoma cell lines used in this study were obtained from American Type Culture Collection (Rockville, USA).The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies), supplemented with 20 mM HEPES, 10 mM sodium bicarbonate, 50 µg/ml streptomycin, 50 IU/ml penicillin, 2 mM glutamine and 10 % foetal calf serum (Life Technologies).The cells were maintained as monolayers in 75 ml flasks at 37 o C, 5 % CO 2 in a humidified atmosphere until approximately 80 % confluent.

Evaluation of cancer cell antiproliferative activity
Antiproliferation activity of the extracts was assessed as previously described. 27,28Briefly, 1 ml of trypsin (Sigma) was added to the culture flasks and incubated at 37 o C, 5% CO 2 for 15 min to dislodge the cancer cells.The cell suspensions were then transferred to a 10 ml centrifuge tube and sedimented by centrifugation.The supernatant was discarded and the cells were resuspended in 9 ml of fresh media.Aliquots of the resuspended cells (70 µl, containing approximately 5000 cells) were added to the wells of a 96 well plate.A volume of 30 µl of the test extracts or cell media (for the negative control) was added to individual wells and the plates were incubated at 37 o C, 5% CO 2 for 12 hours in a humidified atmosphere.A volume of 20 µl of Cell Titre 96 Aqueous One solution (Promega) was subsequently added to each well and the plates were incubated for a further 3 hours.Absorbances were recorded at 490 nm using a Molecular Devices, Spectra Max M3 plate reader.All tests were performed in at least triplicate and triplicate controls were included on each plate.The antiproliferative activity of each test was calculated as a percentage of the negative control using the following formula:

Proliferation (% untreated control) = (A ct /A cc ) x 100
A ct is the corrected absorbance for the test extract (calculated by subtracting the absorbance of the test extract in media without cells from the extract cell test combination) and A cc is the corrected untreated control (calculated by subtracting the absorbance of the untreated control in media without cells from the untreated cell media combination).

Toxicity screening
Reference toxin for biological screening Potassium dichromate (K 2 Cr 2 O 7 ) (AR grade, Chem-Supply, Australia) was prepared as a 2 mg/ml solution in distilled water and was serially diluted in synthetic seawater for use in the A.franciscana nauplii bioassay.

Artemia franciscana nauplii toxicity screening
0][31] Briefly, A. franciscana cysts were obtained from North American Brine Shrimp, LLC, USA (harvested from the Great Salt Lake, Utah).Synthetic seawater was prepared using Reef Salt, AZOO Co., USA.Seawater solutions at 34 g/l distilled water were prepared prior to use.An amount of 1 g of A. franciscana cysts was incubated in 500 ml synthetic seawater under artificial light at 25 o C, 2000 Lux with continuous aeration.Hatching commenced within 16-18 h of incubation.Newly hatched A. franciscana (nauplii) were used within 10 h of hatching.Nauplii were separated from the shells and remaining cysts and were concentrated to a suitable density by placing an artificial light at one end of their incubation vessel and the nauplii-rich water closest to the light was removed for biological assays.The extracts and positive control were serially diluted in artificial seawater for LC 50 determination.A volume of 400µl of seawater containing approximately 43 (mean 43.2, SD 13.8, n=80)nauplii were added to wells of a 48 well plate and immediately used for bioassay.The plant extracts were diluted to 4 mg/ml in seawater for toxicity testing, resulting in a 2 mg/ml concentration in the bioassay.A volume of 400 µl of diluted plant extract or the reference toxin was transferred to the wells and incubated at 25 ± 1 o C under artificial light (1000 Lux).A negative control (400 µl seawater) was run in at least triplicate for each plate and all treatments were performed in at least triplicate.The wells were checked at regular intervals and the number of dead counted.The nauplii were considered moribund if no movement of the appendages was observed within 10 sec.After 48h all nauplii were sacrificed and counted to determine the total number per well.The LC 50 with 95 % confidence limits for each treatment was calculated using probit analysis.

Statistical analysis
Data are expressed as the mean ± SEM of at least three independent experiments.One way ANOVA was used to calculate differences between control and treated groups with a P value < 0.05 considered to be significant.

Liquid extraction yields and qualitative phytochemical screening
Extraction of 1 g quantities of dried plant material with various solvents yielded dried plant extracts ranging from 157 mg to 398 mg (Table 1).Methanol and water gave relatively high yields of dried extracted material (398 and 305 mg for the methanolic and aqueous extracts respectively), whilst ethyl acetate, chloroform and hexane extracted lower masses (260, 269 and 157 mg, respectively).The dried extracts were resuspended in 10 ml of deionised water (containing 1% DMSO), resulting in the extract concentrations shown in Table 1.
BEMS Reports, Vol 2, Issue 2, Jul-Dec, 2016   Phytochemical studies (Table 1) show that methanol and water extracted the widest range and largest amount of phytochemicals in this study.Both extracts showed moderate to high levels of total phenolics and insoluble phenolics (water soluble phenolics were below the threshold of detection), as well as low to moderate levels of flavonoids.Similar classes of phytochemicals were detected in the ethyl acetate and chloroform extracts, although at substantially lower levels.Alkaloids were not detected in any of the extracts.None of the phytochemical classes were detected in the hexane extract.

Antibacterial activity
Aliquots (10 µl) of each extract were tested in the disc diffusion assay against panels of clinical isolated bacterial strains (Figure 1) and reference bacterial strains (Figure 2).The methanolic, aqueous and ethyl acetate extracts displayed broad spectrum inhibitory activity against the clinical bacteria strains (Figure 1).Both Gram positive and Gram negative bacteria were susceptible.The number of susceptible Gram negative clinical isolates inhibited was higher for the aqueous extract (11 out of 14 Gram negative bacterial species tested; 79 %) than in the methanol extract (10 out of 14 Gram negative bacteria tested; 71%).Similarly, the ethyl actetate extract was also an effective at inhibiting growth of the bacterial clinical isolates, inhibiting the growth of 9 of the 14 Gram negative bacteria tested (64 %).The methanolic, aqueous and ethyl acetate extracts were also effective inhibitors of the Gram positive clinical isolates, each inhibiting 2 of the 5 Gram negative clinical isolates tested (40 %).The growth of none of the clinical isolates was inhibited by the chloroform or hexane extracts.The extracts were similarly effective at inhibiting the growth of the reference bacterial strains (Figure 2).The methanolic and aqueous extracts inhibited the growth of all Gram positive and Gram negative reference strains tested (100 %).The ethyl acetate extract also displayed broad spectrum antibacterial activity against the reference bacterial strains, inhibiting the growth of 5 of the 7 species tested (71 %).Both Gram posi-  tive and Gram negative reference bacterial strains were inhibited by the ethyl acetate extract to approximately the same extent (4 out of 7 Gram negative bacterial species tested (57 %), compared to 1 of the 2 reference Gram positive bacterial species (50 %)).
The relative level of antimicrobial activity was further evaluated by determining the MIC values (Table 2) for each extract against the bacterial species which were shown to be susceptible by disc diffusion assays.Most of the extracts were effective at inhibiting microbial growth at low concentrations, with MIC values against the bacterial species that they inhibited often < 1000 µg/ml (< 10 µg impregnated in the disc), indicating the potent antimicrobial activity of these extracts.These MIC values compare favourably with the dosages of the pure standard ampicillin (2 µg per disc).The ethyl acetate extract was particularly potent, achieving MIC values as low as 76 µg/ml (0.8 µg per disc against C. cloaceae).
Several MIC values below100 µg/ml were also noted for the ethyl acetate extract against other bacterial species.The methanolic and aqueous extracts also had good antibacterial efficacies,with MIC values < 1000 µg/ml for many bacteria.The G. cambogia fruit extracts generally displayed lower efficacies towards Gram positive bacteria than Gram negative bacteria.Indeed, MIC values against Gram positive bacteria were generally > 1000 µg/ml (Table 2).Of the Gram positive bacterial species tested, only S. epidermidis was highly susceptible to the G. cambogia extracts, with MIC values <1000µg/ml for the methanolic, aqueous and ethyl acetate extracts.

Inhibition of cancer cell proliferation
G. cambogia fruit extracts were serially diluted to test their ability to inhibit CaCo 2 colorectal carcinoma cell growth (Figure3).All extracts displayed potent inhibitory activity when screened undiluted,with cell proliferation inhibited to as low as 3.8 ± 1.2% of the untreated control cell growth for the methanolic extract.The aqueous, ethyl acetate and chloroform extracts were very effective at inhibiting CaCo 2 proliferation (to <10% of untreated cell proliferation).The hexane extract also significantly inhibited CaCo 2 cell proliferation, albeit to a lesser extent (to approximately 40% of the untreated cell proliferation).Inhibition of proliferation was dose dependent withthe anti-proliferative activity decreasing at lower concentrations (Table 3).The G. cambogia extracts were similarly effective inhibitors of HeLa cervical carcinoma cell growth (Figure 4).All extracts displayed potent inhibitory activity when screened undiluted.Indeed, the methanolic and aqueous extracts inhibited 100% of the untreated control cell growth.The ethyl acetate, chloroform and hexane extracts were also was effective at inhibiting CaCo 2 proliferation (to <65 % of untreated cell proliferation).As for the CaCo 2 screening, inhibition of HeLa proliferation was dose dependent (Table 3).The level of anti-proliferative activity was further evaluated by determining the IC 50 values (Table 3) for each extract.The methanolic, aqueous and ethyl acetate extracts were particularly good inhibitors of both CaCo 2 and HeLa cell proliferation with an IC 50 generally < 100µg/ml, indicating their potential for cancer therapeutic development.The HeLa cells were most susceptible, with IC 50 values as low as 43.1 µg/ml (against the methanolic extract).

Quantification of toxicity
G. cambogia fruit extracts were diluted to 4000 µg/ml (to give a bioassay concentration of 2000 µg/ml) in artificial seawater for toxicity testing in the Artemia nauplii lethality bioassay.For comparison, the reference toxin potassium dichromate was also tested in the bioassay.Potassium dichromate was rapid in its induction of mortality, with mortality evident within 4 hours of exposure (unpublished results).The G. cambogia extracts were slower at inducing mortality, with ≥ 12 hours needed for mortality induction.Despite the slower onset of mortality, methanol,  water and ethyl acetate extracts all induced mortality significantly above that of the artificial seawater control (Figure 5).In contrast, the chloroform and hexane extracts were non-toxic, with mortality levels similar to those seen for the seawater control.

DISCUSSION
The current study reports on the antimicrobial and anticancer activities of various G. cambogia fruit extracts, and on their toxicity.Bacterial growth was inhibited in both Gram positive and Gram negative bacteria by the G. cambogia fruit extracts, although the Gram negative bacteria were generally more susceptible.The ability of plant extracts to inhibit the growth of both Gram positive and Gram negative bacteria has been previously reported for other plants that have a history of medicinal usage for the treatment of microbial diseases.The antiseptic properties of the Eucalypt spp. 32Leptospermum spp. 33,34and Syzygium spp, 26,35,36 have been studied extensively and shown to inhibit the growth of a wide variety of bacteria.However, the greater susceptibility of the Gram negative bacterial species towards the G. cambogia fruit extracts is not worthy.This is in contrast toother previous studies which have reported a greater susceptibility of Gram positive bacteria towards solvent extracts for South American, 37 African. 38,39and Australian 34 plant extracts, although other examples of plants having a greater effect on Gram negative bacteria have also been reported. 40,41otent anti-proliferative activity against CaCo 2 and HeLa carcinoma cells was noted for all of the G. cambogia fruit pericarp extracts screened in this study,with IC 50 values generally substantially<200µg/ml for the methanol, water, ethyl acetate and chloroform extracts.Despite its wide array of traditional therapeutic uses 12 , the anticancer properties of G. cambogia were previously unreported.However, recent studies have examined the anti-proliferative properties of the taxonomically related species G. mangostana 1,42 and G. atroviridis 43 and reported similar anticancer efficacies.
Whilst the antimicrobial and anticancer components of G. cambogia are yet to be identified, pure xanthones isolated from the related species G. mangostana show promising therapeutic properties.α-Mangostin and its derivatives have been particularly well studied and have been shown to have potent inhibitory activity against Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus typhimurium 44 .In contrast, the same study reported only low inhibitory activity towards Escherichia coli, Klebsiella spp and Proteus spp.More recent studies have reported Mycobacterium tuberculosis 45 , S. aureus (including MRSA) 46 , Propionibacterium acnes and S. epidermidis 47 inhibitory activities of mangosteen xanthones.Numerous studies have also documented the anticancer effects of xanthones isolated from G. mangostana fruit pericarp.Several xanthones including α, β and γ mangostin, mangostinone, 2-iosprenyl-1,7-dihydroxy-3-methoxy xanthone and garcinone E exhibit anti-proliferative [48] and apoptotic properties 49 against human leukemia cell line HL60.Garcinone E has also been reported to have potent cytotoxic effects on HCC36, TONG, HA22T, Hep3B, HEpG2 and SK-Hep-1 hepatocarcinoma cell lines 50 .The same study demonstrated that garcinone E was also effective against NCI-Hut 125, CH27 LC-1, H2891 and Calu-1 lung carcinoma cells, as well as against AZ521, NUGC-3, KATO-III and AGS gastric carcinoma cell lines.Whilst it has yet to be established whether similar compounds are present in the G. cambogia fruit extracts, if they are subsequently detected, they may be responsible (at least in part) for the antibacterial and anticancer properties reported here.
The results of this study indicate that the G. cambogia fruit extracts examined in this report are worthy of further study due to their antibacterial activity and ability to block cancer cell proliferation.Furthermore, as extracts with LC 50 values greater than 1000 µg/ml in the Artemia nauplii bioassay have been defined as being non-toxic, 29,31 all the G. cambogia fruit extracts were determined to be nontoxic.Whilst the results of this study are encouraging, further studies to purify and identify the bioactive components are needed.Furthermore, studies are also required to examine the mechanisms of action of these agents.Whilst the extracts examined in this report have potential as antimicrobial and anticancer agents, caution is needed before these compounds can be applied to medicinal purposes.In particular, further toxicity studies using human cell lines are needed to determine the suitability of these extracts for these purposes.

CONCLUSION
The results of this study partially validate the traditional usage of G. cambogia fruit pericarp extracts in multiple traditional medicinal systems to treat bacterial diseases and cancer, indicating that they are worthy of further study.Bioactivity-driven purifications of the active components and examination of the mechanisms of action of these agents is required.

Table 1 :
The mass of dried extracted material, the concentration of extracts after resuspension in deionised water and the qualitative phytochemical screenings of solvent extractions.a large response; ++ indicates a moderate response; + indicates a minor response; indicates no response in the assay.

Figure 3 :
Figure 3: Anti-proliferative activity of G. cambogia fruit extracts against CaCo2 cancer cell lines measured as percentages of the untreated control cells.NC = untreated control; M = methanolic extract; W = aqueous extract; E = ethyl acetate extract; C = chloroform extract; H = hexane extract.Results are expressed as mean percentages ± SEM of at least triplicate determinations.* indicates values that are significantly different to the untreated control (P< 0.05).

Figure 4 :
Figure 4:Anti-proliferative activity of G. cambogia fruit extracts against HeLa cancer cell lines measured as percentages of the untreated control cells.NC = untreated control; M = methanolic extract; W = aqueous extract; E = ethyl acetate extract; C = chloroform extract; H = hexane extract.Results are expressed as mean percentages ± SEM of at least triplicate determinations.* indicates values that are significantly different to the untreated control (P< 0.05).

Table 3 : The IC50 (µg/ml) of CaCo2 and HeLa cells exposed to G. cambogia fruit extracts.
29,31 4shows the extract and control toxin concentrations required to achieve 50% mortality (LC 50 ) at various times.As toxicity of crude plant extracts has been defined as 24 h LC 50 values < 1000 µg/ml,29,31the measured LC 50 values indicate that all G. cambogia fruit pericarp extracts were non-toxic.