http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(6): 706–711 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.865241
ORIGINAL ARTICLE
Isolation and biological activity of compounds from Garcinia preussii Bernadette Biloa Messi1,2, Raimana Ho2, Alain Meli Lannang3, Delphine Cressend2, Karl Perron4, Augustin Ephrem Nkengfack1, Pierre-Alain Carrupt2, Kurt Hostettmann2, and Muriel Cuendet2 Department of Organic Chemistry, University of Yaounde´ I, Yaounde´, Cameroon, 2School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland, 3Department of Chemistry, Higher Teachers’ Training College, University of Maroua, Maroua, Cameroon, and 4Department of Botany and Plant Biology, Microbiology Unit, University of Geneva, Geneva, Switzerland
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Abstract
Keywords
Context: Plants of the genus Garcinia (Clusiaceae) are traditionally used to relieve stomachaches, toothaches, and as a chew stick. Objective: In order to determine which compounds were responsible for these activities, a phytochemical investigation of the fruits and leaves of Garcinia preussii Engl. was pursued. Materials and methods: Plants were extracted by solvents of various polarities. Compounds isolation was then carried out using chromatography methods (medium- and high-pressure liquid chromatography, open column and thin-layer chromatography). The isolated compounds were identified and characterized by using 1D and 2D NMR spectroscopies. The antioxidant activity was evaluated using DPPH, ABTS, ALP, and ORAC assays. The antimicrobial activity was assayed against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis by determining the minimum inhibitory concentration (MIC) value. The cytotoxic activity of most of the isolated compounds was evaluated on a small panel of human cancer cell lines (DU145, HeLa, HT-29, and A431) using the XTT method. Results: The phytochemical investigation of G. preussii led to the isolation of eight known compounds, six benzophenones and two flavonoids. These compounds were tested for their biological activities. 1, 2, 3, 4, 7 and 8 demonstrated a high free radical scavenging activity with ER50 ranging from 0.1 to 0.7. The antimicrobial activity was shown only against Gram-positive bacteria for 1, 4, and 5. A moderate cytotoxic activity with IC50 ranging from 7 to 50 mM was observed, except for 6 which was not active. Conclusion: These results appear to support some of the properties reported for Garcinia species.
Antimicrobial, antioxidant, benzophenones, Clusiaceae, cytotoxicity
Introduction Plants of the Clusiaceae family are known to be very good sources of natural products with biological properties (Kuete et al., 2007b). The genus Garcinia belongs to this family and is distributed in tropical Asia, Africa, and Polynesia. It consists of 180 species, of which 21 species are found in Cameroon (Guedje et al., 2001). More specifically, Garcinia preussii Engl is traditionally used in Congo (Brazzaville) to treat stomachache (Bouquet, 1969). In Ivory Coast, the leaves are taken as a decoction to relieve tooth pain (Visser, 1975). Previous phytochemical studies indicated that mostly xanthones (Kuete et al., 2007a), benzophenones (Ahmad et al., 2010), triterpenes (Chung et al., 1998), flavonoids, and biflavonoids (Iwu & Igboko, 1982) were found in the genus Garcinia and they exhibited antioxidant, antimicrobial, antiinflammatory, and antitumor activities (Hay et al., 2004). Correspondence: Prof. Muriel Cuendet, School of Pharmaceutical Sciences, University of Geneva, Quai Ernest-Ansermet 30, CH-1211 Geneva, Switzerland. Tel: +41 22 379 33 86. Fax: +41 22 379 33 99. E-mail: [email protected]
History Received 22 August 2013 Revised 19 October 2013 Accepted 9 November 2013 Published online 6 February 2014
The course of many diseases such as cancer (Kruk & Aboul-Enein, 2007), liver disease (Cederbaum et al., 2009), Alzheimers’s disease (Cai & Yan, 2007), and inflammation (Kao et al., 2009) is influenced by oxidative stress. Also, Gram-positive pathogens are the most common and troublesome causes of nosocomial infections in neonatal intensive care units (Kocher et al., 2010). Owing to the increased incidence of infections caused by these pathogens, the development of novel agents with antibacterial activity is requisite. In order to reduce those health hazards, the study of medicinal plants for their antioxidant, antibacterial, and cytotoxic activities seems to be an interesting route to find treatment for such diseases and the scientific interest to find secondary metabolites remains intact. The aim of the present study was to further investigate compounds from the leaves of G. preussii (Biloa Messi et al., 2012), as well as study the chemical composition of the fruits, and to assess their biological properties, in order to explain some of the traditional uses of the plant.
DOI: 10.3109/13880209.2013.865241
Materials and methods Chemicals Chemicals used were 2,2-diphenyl-1-picrylhydrazine (DPPH), 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), alkaline phosphatase (ALP), 2,20 azobis(2-methylpropionamidine) dihydrochloride (AAPH), 4-methylumbelliferyl phosphate (MUP), p-iodonitrotetrazolium violet (INT), gentamicin, colchicine, and {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} (XTT). They were obtained from Sigma-Aldrich (St. Gallen, Switzerland).
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General experimental procedures 1D and 2D NMR spectra (1H, 13C, COSY, HSQC, and HMBC) were recorded in acetone-d6 or methanol-d4 on a Varian Unity Inova 500 MHz spectrometer (Palo Alto, CA) with a CapNMR probe from Protasis/MRM and on a Bruker DRX 500 MHz 1H Larmor frequency using a 5 mm QNP direct detection probe. UHPLC/TOF-MS data were recorded on a Waters Micromass LCT-Premier mass spectrometer with an electrospray interface (ESI) coupled to an Acquity UHPLC system (Waters, Milford, MA) with an Acquity BEH C18 UHPLC column (1.7 mm, 100 2.1 mm i.d.; Waters). HRMS spectra were obtained using electrospray as the ion source, capillary voltage 2.8 kV, cone voltage 40 V, MCP detector voltage 2650 V, source temperature 120 C, desolvation temperature 250 C, cone gas flow 10 L/h, desolvation gas flow 550 L/h. HPLC-UV-DAD analysis was carried out on a HP 1100 system equipped with a photodiode array detector (Agilent technologies, Palo Alto, CA) with a symmetry C18 column (5 mm, 250 4.6 mm i.d.; Waters). The detection was performed at 210, 254, 280, and 360 nm. Medium pressure liquid chromatography (MPLC) was performed with a Bu¨chi 681 pump equipped with a Knauer UV detector using a LiChroprep RP-18 (40–63 mm, 460 50 mm i.d.; Merck, Darmstadt, Germany). The detection was performed at 254 nm. Column chromatography was carried out using LiChroprep RP-18 (15–25 mm, Merck) or silica gel 60 (20– 63 mm, Merck). Thin-layer chromatography was performed on silica gel 60 F254 A1 sheets (Merck). Plant material The fruits and leaves of G. preussii were collected in July 2008 at Ngoume´, located in the central part of Cameroon. The botanical identification was performed by Mr. Nana Victor, a botanist from the National Herbarium, Yaounde´, Cameroon, where a voucher specimen is conserved under the reference number 55520/HNC. Compound isolation from the fruits of G. preussii The air-dried powdered fruits (2 kg) were extracted by maceration with MeOH (3 3 L), for up to 48 h each at room temperature. After filtration, the MeOH extract was evaporated to dryness. The residue (310 g) was then dissolved in 800 mL of water and successively partitioned with hexane and EtOAc to give 90 g and 70 g, respectively. A portion of the hexane extract (50 g) was chromatographed over a silica gel column (20–63 mm, 5 60 cm) using
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an EtOAc/hexane step gradient (0:100–100:0 in 10% steps) to afford nine fractions (F1–F9). F1 (900 mg) was purified by semipreparative HPLC (MeOH/H2O 90:10 þ 0.1% FA, 10 mL/min) to afford garciniagifolone (1, 3 mg, tR ¼ 7.39 min), garcimultiflorone E (2, 5 mg, tR ¼ 7.29 min), and garcinialiptone B (3, 3 mg, tR ¼ 6.27 min). F2 (14 g) was separated by MPLC on LiChroprep RP-18 (40–63 mm, 5 46 cm), with a MeOH/H2O step gradient (60:40–100:0 in 10% steps, 3 mL/min) to afford five subfractions (F21– F25). This separation yielded garcinol (4, 7 g) from F22. Further purification of F25 was carried out over a reverse phase C18 column and separated with gradient mixtures of MeOH/H2O (80:20–100:0 in 10% steps) to afford nine fractions (F251–F259). F258 provided 7-epi-clusianone (5, 250 mg). Compound isolation from the leaves of G. preussii The air-dried leaves (5 kg) were extracted by a successive maceration with acetone (3 5 L) and then methanol (3 5 L) for 24 h each at room temperature. The methanol extract (50 g) was submitted to silica gel flash chromatography eluted with CH2Cl2/MeOH mixtures of increasing polarity (100:0, 2 L; 90:10, 2 L; 80:20, 2 L; 0:100, 1 L) which were collected into 300 mL fractions. Similar fractions were combined after TLC examination to provide four fractions (F1–F4). F4 (5 g) was applied to a Sephadex LH-20 column. Elution with MeOH (5 L) yielded seven fractions (F41–F47). F41 (30 mg) was repurified by column chromatography over Sephadex LH-20 (2 40 cm) using MeOH to afford eriodictyol (8, 4 mg). F43 (85.4 mg) was purified on silica gel column (20–63 mm, 2.5 60 cm) using CH2Cl2/MeOH 90:10 to give garcimangosone (6, 2 mg). F44 (38 mg) was also purified on a silica gel column (20–63 mm, 2.5 60 cm) using CH2Cl2/MeOH 90:10 to yield astragalin (7, 5 mg). Antioxidant assays DPPH assay The radical scavenging activity of the compounds was assessed spectrophotometrically in microplates, as described previously (Brand-Williams et al., 1995). ER50, the ratio of compound over radical concentration inducing a 50% decrease in absorbance after 90 min, was determined with a dose–response curve. ABTS assay The reducing activity of the compounds was assessed spectrophotometrically in microplates by following the decolorization of the ABTS radical (67 mM) (Re et al., 1999). ER50, the ratio of compound over radical concentration inducing a 50% decrease in absorbance after 90 min, was determined with a dose–response curve. ALP assay The antioxidant activity was assessed by the ability of a compound to preserve the catalytic effectiveness of the enzyme ALP (2 mU/mL in glycine buffer) despite the presence of peroxyl radicals generated by AAPH (5 mM). This was assessed by following the enzymatic
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dephosphorylation of MUP (20 mM) to the fluorescent 4-methyllumbelliferone. Enzymatic hydrolysis rates of MUP were determined by a continuous spectrofluorimetric assay (Bertolini et al., 2007). The percentage of ALP protection by tested compounds was calculated according to the following equation, after determining the hydrolytic activity of oxidized samples (hasample), oxidized controls (haox), and non-oxidized controls (hanon-ox): % ALP protection ¼ 100
ðhasample haox Þ ðhanon-ox haox Þ
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The EC50 value, obtained by a dose–response curve, represents the sample concentration which protects 50% of ALP enzymatic activity from a peroxyl radical-induced oxidation after 90 min. Oxygen radical absorbance capacity (ORAC) assay The ORAC assay was carried out using the same conditions as the above assay, except that a fluorescein solution at 6 108 M was used instead of ALP. The fluorescence was read at lEx 485 20 nm and lEm 528 20 nm to obtain the fluorescence value of oxidized samples (fluosample) and oxidized controls (fluoFLox) as well as non-oxidized controls (fluoFLnon-ox). This last one was calculated by the following equation: ðfluosample fluoFLox Þ % remaining fluorescein ¼ 100 ðfluonon-ox fluoFLox Þ The EC50 value, obtained by a dose–response curve, represents the sample concentration which protects 50% of fluorescein from a peroxyl radical-induced oxidation after 90 min (Huang et al., 2002). Antimicrobial assay The bacterial reference strains used in this study, Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), and Staphylococcus aureus (ATCC 29213) were obtained from the HUG (Geneva University Hospital, Geneva, Switzerland). Mueller-Hinton broth (MHB, Oxoid, Cambridge, UK) and Mueller-Hinton agar (MHA, BioMe´rieux, Balmes-LesGrottes, France) were used as liquid and solid media. All strains were grown for 24 h at 37 C. The minimum inhibitory concentration (MIC) of the various compounds were determined by using the broth dilution method in 96-well microtiter plates as previously described (Wiegand et al., 2008). The MIC corresponds to the lowest concentration of the compounds that inhibits visible growth (visual turbidity). The growth was detected by the reduction of INT into formazan, a red-purple molecule. The highest dilution of a compound in which no red-purple color appears corresponds to its MIC. Gentamicin was used as positive control (Eloff, 1998). Cytotoxicity assay DU145 (human prostate carcinoma), HeLa (human cervical cancer), HT-29 (human colorectal cancer), and A431 (human epidermoid carcinoma) cell lines were purchased from the
American Type Culture Collection. All cell lines were cultured in minimum essential medium (MEM) alpha containing 10% of fetal bovine serum, 100 IU/mL of penicillin G, and 100 mg/mL of streptomycin and maintained at 37 C in humidified environment containing 5% of CO2. The cytotoxicity of compounds was determined with the above cell lines as described previously (Roehm et al., 1991). Briefly, cells (in log growth phase) were harvested by trypsinization, counted, diluted in media, and added to 96-well plates containing test compounds dissolved in DMSO in triplicate; the final DMSO concentration was 0.05%. The plates were incubated for 3 d. Following the incubation, cells were stained with the addition of 80 mL of XTT (1 mg/mL) per well. The absorbance was measured at 450 nm after 3 h incubation at 37 C. The growth of the compound-treated cells was compared with the growth of DMSO-treated controls. With each compound, two-fold serial dilutions were tested in duplicate, with concentrations ranging above and below the IC50 values. The duplicate tests were used to construct dose–response curves, and IC50 values (mM) were determined by linear regression analysis. Results are the means of at least three independent determinations SD. Colchicine was used as positive control.
Results Chemical investigation of the hexane-soluble extract of the fruits and methanol-soluble extract of the leaves of G. preussii resulted in the isolation and characterization of eight known compounds garciniagifolone (1) (Shan et al., 2012), garcimultiflorone E (2) (Liu et al., 2010), garcinialiptone B (3) (Zhang et al., 2010), garcinol (4) (Krishnamurthy et al., 1981), 7-epi-clusianone (5) (Piccinelli et al., 2005), garcimangosone (6) (Huang et al., 2001), astragalin (7) (Nakabayashi, 1955) and eriodictyol (8) (Hussain & Waterman, 1982) (Figure 1). Each constituent was identified on the basis of its 1H-NMR spectra and HR-MS analysis by comparison with the literature. The antioxidant activity of compounds 1–8 was evaluated and compared to quercetin (Table 1). The antibacterial activity of each isolated compound was evaluated against four bacterial strains and showed activity only against Gram-positive bacteria. The MIC is shown in Table 2. Benzophenones 1–6 were also screened for their cytotoxic activity in DU145, HeLa, HT-29, and A431 cell lines (Table 3).
Discussion Species of the genus Garcinia are frequently used in African and Asian traditional medicine. They contain high levels of phenolic compounds that are reported to possess exciting biological properties. Despite the number of published studies on various Garcinia species, some plants of this genus have not been extensively studied from the chemical and pharmacological point of view. In order to explain some of the traditional use of G. preussii extracts, their antioxidant, antibacterial, and cytotoxic activities were measured. Active extracts were then fractionated to give eight pure compounds that were submitted to the various tests. Compound 4 was as potent as quercetin at scavenging the DPPH and ABTS– radicals and was slightly less active than
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Figure 1. Structures of compounds 1–8. 32
33
35
7
30
38
O
OH
3
31
1 O 9 5 O O
23
20 22
O
H
O
OH O
OH
1
25
26
HO
HO
18
H
2
OH
OH
OH HO
O
HO
O
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O
O
OH
O
O 4
3
OH OH
HO
O O
OH
O O
O
5 O 6 OH HO
O O O
OH
OH HO
O
OH OH
HO OH
7
Table 1. Activities of compounds 1–8 in DPPH, ABTS, ALP, and ORAC assays.
Compounds 1 2 3 4 6 7 8 Quercetin
DPPH (ER50)
ABTS (ER50)
ALP (EC50 mM)
ORAC (EC50 mM)
0.20 0.03 0.21 0.02 0.45 0.11 0.11 0.01 41 41 0.62 0.03 0.09 0.01
0.19 0.02 0.20 0.01 0.29 0.02 0.09 0.01 41 0.70 0.03 0.65 0.03 0.07 0.01
9.72 2.40 8.23 1.23 5.22 1.72 nd 9.31 2.70 2.30 0.25 4.53 0.71 1.00 0.07
6.04 0.36 5.72 0.40 7.57 0.17 3.24 0.28 23.1 2.10 2.96 0.27 4.75 0.46 1.66 0.07
Results are the means of at least three independent determinations SD. ER50: concentration inducing a 50% decrease in absorbance after 90 min. nd, not determined.
the positive control in the ORAC assay. Its activity in the ALP assay could not be determined as it is an inhibitor of this phosphatase at concentrations higher than 105 M. The antioxidant activity of compounds 1, 2, and 3 was lower than that of quercetin, but these entities had no selectivity as they were active in the four assays used. The antioxidant
OH
HO
O
OH
OH
OH
O 8
Table 2. Minimum inhibitory concentration (MIC) of compounds 1–8. MIC (mg/mL) Compounds
S. aureus
E. faecalis
1 2 3 4 5 6 7 8 Gentamicin
64 4128 4128 16 2 4128 4128 4128 1
128 128 128 16 2 4128 4128 4128 16
Results are based on at least three independent determinations.
activity of compounds 1, 2, 3 and 4 can be explained by the conjugated catechol moiety on the structure. Compounds 7 and 8 were protectors of the ALP protein and of fluorescein against peroxyl-radical-induced oxidation. The similar potency in the ALP and ORAC assays points out that these compounds do not interact with the protein. Unlike them,
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Table 3. Cytotoxicity (IC50 in mM) of isolated benzophenones (1–6) in human cancer cell lines. Compounds
DU145
HeLa
HT-29
A431
1 2 3 4 5 6 Colchicine
7.7 1.4 14.5 3.2 11.5 2.0 14.9 0.8 33.4 5.2 4150 0.0230 0.0008
14.6 7.2 21.0 5.9 13.0 3.7 16.4 4.0 49.5 15.1 4150 0.0060 0.0014
7.4 0.2 15.9 4.1 14.0 3.0 16.1 3.7 32.6 1.7 4150 0.0140 0.0021
9.9 2.0 17.1 3.5 11.0 3.5 9.9 1.0 38.2 1.1 4150 0.0060 0.0003
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Compounds were evaluated for cytotoxicity by standard procedures as described in the Experimental section. The following cultured human cell lines were employed: DU145 (prostate carcinoma), HeLa (cervical cancer), HT-29 (colorectal cancer), and A431 (epidermoid carcinoma). Results are shown as IC50 values (mM) and are the means of at least three independent determinations SD.
compound 6 exhibited a weak ALP-protective activity only. This capacity might be due to a protein binding because of the hydrogen atom donors and acceptors containing scaffold. Compound 5 did not show any antioxidant capacity in the four assays performed since its structure does not enable reducing activity. None of the compounds showed activity against the two Gram-negative bacteria tested, E. coli and P. aeruginosa. However, compounds 4 and 5 displayed a good antibacterial activity against the Gram-positive bacteria S. aureus and E. faecalis. Compound 4 is already known to fight methicillin-resistant S. aureus and might be an alternative for classical antibiotic treatments (Iinuma et al., 1996). The MIC value of 16 mg/mL for S. aureus was identical to the value previously obtained with compound 4 purified from Garcinia bancana (Rukachaisirikul et al., 2005). Compound 5 exhibited a strong antibacterial activity with a MIC of 2 mg/mL against both S. aureus and E. faecalis. This compound has also been previously shown to be a promising compound for the treatment of multidrug-resistant S. aureus infections (Xiao et al., 2007). In addition, compound 5 has recently been proposed for the prevention or treatment of cavities since it affects the biofilm formation and the acid tolerance to the cariogenic Gram-positive bacterium Streptococcus mutans (Almeida et al., 2008). The mechanism of action of these compounds on bacteria is not known. However, due to their specificity for Gram-positive bacteria, the effect seems to be related to the characteristics of the cell envelope. Grampositive bacteria include important pathogens, known to be a leading cause of infectious diseases. They represent the most common and troublesome causes of nosocomial infections in neonatal intensive care units (Kocher et al., 2010). The increasing occurrence of bacteria resistant to antibiotics represents a serious threat for the treatment of infectious diseases. The discovery and development of novel agents with antibacterial activity is, therefore, needed. The benzophenones 1–4 displayed almost the same cytotoxic activity in each cell line tested (DU145, HeLa, HT-29, and A431) with IC50 values around 10 mM. Compound 5 was about three-times less active. This might be due to the lack of a catechol group. Among the benzophenones tested, 6 did not show cytotoxic activity against any of the cell lines (IC504150 mM). It might be due to structural differences, as compound 6 does not have the bicyclo [3.2] nonane system, as the other isolated benzophenones have. Benzophenones with a bicyclo [3.2] nonane
system have been widely studied for their biological properties.
Conclusion Medical plants play an important role in the management of bacterial infections, oxidative stress, and cancer, especially in developing countries where resources are meager. The results obtained can justify some of the traditional uses of this plant. Additionally, this study shows that G. preussi is a natural potential source of antioxidant compounds. Finally, the isolated benzophenones are expected to be useful for the study of anticancer agents in the future. However, the safety and toxicity of these compounds need to be investigated. Furthermore, those results are consistent with previous reports on the antioxidant, antimicrobial, and cytotoxic activities of phenolic compounds isolated from Garcinia species (Arif et al., 2009; Matsumoto et al., 2003; Tanaka et al., 2000; Yamaguchi et al., 2000)
Acknowledgements The authors are grateful to Dr. L. Marcourt for her help with the NMR data. Bernadette Biloa Messi gratefully acknowledges the Ministry of Higher Education of Cameroon (MINESUP) and the Swiss Confederation for her fellowship. The authors would like to thank the Swiss National Science Foundation (Grant no. 200020-107775/1 to Prof. K. Hostettmann) and the Alfred and Alice Lachmann Nutrition Fund (Grant to Prof. M. Cuendet) for financial support of this work.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the manuscript.
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Kruk J, Aboul-Enein HY. (2007). Physical activity and cancer prevention: Updating the evidence. The role of oxidative stress in carcinogenesis. Curr Cancer Ther Rev 3:81–95. Kuete V, Komguem J, Beng VP, et al. (2007a). Antimicrobial components of the methanolic extract from the stem bark of Garcinia smeathmannii Oliver (Clusiaceae). S Afr J Bot 73: 347–54. Kuete V, Nguemeving JR, Beng VP, et al. (2007b). Antimicrobial activity of the methanolic extracts and compounds from Vismia laurentii De Wild (Guttiferae). J Ethnopharmacol 109:372–9. Liu X, Yu T, Gao X-M, et al. (2010). Apoptotic effects of polyprenylated benzoylphloroglucinol derivatives from the twigs of Garcinia multiflora. J Nat Prod 73:1355–9. Matsumoto K, Akao Y, Kobayashi E, et al. (2003). Cytotoxic benzophenone derivatives from Garcinia species display a strong apoptosis-inducing effect against human leukemia cell lines. Biol Pharm Bull 26:569–71. Nakabayashi T. (1955). Isolation of astragalin and isoquercitin from Bracken, Pteridium aquilinum. Bull Agr Chem Soc Jpn 19:104–9. Piccinelli AL, Cuesta-Rubio O, Chica MB, et al. (2005). Structural revision of clusianone and 7-epi-clusianone and anti-HIV activity of polyisoprenylated benzophenones. Tetrahedron 61:8206–11. Re R, Pellegrini N, Proteggente A, et al. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26:1231–7. Roehm NW, Rodgers GH, Hatfield SM, Glasebrook AL. (1991). An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Methods 142:257–65. Rukachaisirikul V, Naklue W, Sukpondma Y, Phongpaichit S. (2005). An antibacterial biphenyl derivative from Garcinia bancana MiQ. Chem Pharm Bull (Tokyo) 53:342–3. Shan W-G, Lin T-S, Yu H-N, et al. (2012). Polyprenylated xanthones and benzophenones from the bark of Garcinia oblongifolia. Helv Chim Acta 95:1442–8. Tanaka T, Kohno H, Shimada R, et al. (2000). Prevention of colonic aberrant crypt foci by dietary feeding of garcinol in male F344 rats. Carcinogenesis 21:1183–9. Visser LE. (1975). Plantes me´dicinales de la Coˆte d’Ivoire. Mededelingen Landbouwhogeschool Wageningen-Nederland, 79:54. Wiegand I, Hilpert K, Hancock REW. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3:163–75. Xiao ZY, Mu Q, Shiu WKP, et al. (2007). Polyisoprenylated benzoylphloroglucinol derivatives from Hypericum sampsonii. J Nat Prod 70:1779–82. Yamaguchi F, Ariga T, Yoshimura Y, Nakazawa H. (2000). Antioxidative and anti-glycation activity of garcinol from Garcinia indica fruit rind. J Agric Food Chem 48:180–5. Zhang LJ, Chiou CT, Cheng JJ, et al. (2010). Cytotoxic polyisoprenyl benzophenonoids from Garcinia subelliptica. J Nat Prod 73:557–62.
ORIGINAL ARTICLE
Isolation and biological activity of compounds from Garcinia preussii Bernadette Biloa Messi1,2, Raimana Ho2, Alain Meli Lannang3, Delphine Cressend2, Karl Perron4, Augustin Ephrem Nkengfack1, Pierre-Alain Carrupt2, Kurt Hostettmann2, and Muriel Cuendet2 Department of Organic Chemistry, University of Yaounde´ I, Yaounde´, Cameroon, 2School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland, 3Department of Chemistry, Higher Teachers’ Training College, University of Maroua, Maroua, Cameroon, and 4Department of Botany and Plant Biology, Microbiology Unit, University of Geneva, Geneva, Switzerland
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Abstract
Keywords
Context: Plants of the genus Garcinia (Clusiaceae) are traditionally used to relieve stomachaches, toothaches, and as a chew stick. Objective: In order to determine which compounds were responsible for these activities, a phytochemical investigation of the fruits and leaves of Garcinia preussii Engl. was pursued. Materials and methods: Plants were extracted by solvents of various polarities. Compounds isolation was then carried out using chromatography methods (medium- and high-pressure liquid chromatography, open column and thin-layer chromatography). The isolated compounds were identified and characterized by using 1D and 2D NMR spectroscopies. The antioxidant activity was evaluated using DPPH, ABTS, ALP, and ORAC assays. The antimicrobial activity was assayed against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis by determining the minimum inhibitory concentration (MIC) value. The cytotoxic activity of most of the isolated compounds was evaluated on a small panel of human cancer cell lines (DU145, HeLa, HT-29, and A431) using the XTT method. Results: The phytochemical investigation of G. preussii led to the isolation of eight known compounds, six benzophenones and two flavonoids. These compounds were tested for their biological activities. 1, 2, 3, 4, 7 and 8 demonstrated a high free radical scavenging activity with ER50 ranging from 0.1 to 0.7. The antimicrobial activity was shown only against Gram-positive bacteria for 1, 4, and 5. A moderate cytotoxic activity with IC50 ranging from 7 to 50 mM was observed, except for 6 which was not active. Conclusion: These results appear to support some of the properties reported for Garcinia species.
Antimicrobial, antioxidant, benzophenones, Clusiaceae, cytotoxicity
Introduction Plants of the Clusiaceae family are known to be very good sources of natural products with biological properties (Kuete et al., 2007b). The genus Garcinia belongs to this family and is distributed in tropical Asia, Africa, and Polynesia. It consists of 180 species, of which 21 species are found in Cameroon (Guedje et al., 2001). More specifically, Garcinia preussii Engl is traditionally used in Congo (Brazzaville) to treat stomachache (Bouquet, 1969). In Ivory Coast, the leaves are taken as a decoction to relieve tooth pain (Visser, 1975). Previous phytochemical studies indicated that mostly xanthones (Kuete et al., 2007a), benzophenones (Ahmad et al., 2010), triterpenes (Chung et al., 1998), flavonoids, and biflavonoids (Iwu & Igboko, 1982) were found in the genus Garcinia and they exhibited antioxidant, antimicrobial, antiinflammatory, and antitumor activities (Hay et al., 2004). Correspondence: Prof. Muriel Cuendet, School of Pharmaceutical Sciences, University of Geneva, Quai Ernest-Ansermet 30, CH-1211 Geneva, Switzerland. Tel: +41 22 379 33 86. Fax: +41 22 379 33 99. E-mail: [email protected]
History Received 22 August 2013 Revised 19 October 2013 Accepted 9 November 2013 Published online 6 February 2014
The course of many diseases such as cancer (Kruk & Aboul-Enein, 2007), liver disease (Cederbaum et al., 2009), Alzheimers’s disease (Cai & Yan, 2007), and inflammation (Kao et al., 2009) is influenced by oxidative stress. Also, Gram-positive pathogens are the most common and troublesome causes of nosocomial infections in neonatal intensive care units (Kocher et al., 2010). Owing to the increased incidence of infections caused by these pathogens, the development of novel agents with antibacterial activity is requisite. In order to reduce those health hazards, the study of medicinal plants for their antioxidant, antibacterial, and cytotoxic activities seems to be an interesting route to find treatment for such diseases and the scientific interest to find secondary metabolites remains intact. The aim of the present study was to further investigate compounds from the leaves of G. preussii (Biloa Messi et al., 2012), as well as study the chemical composition of the fruits, and to assess their biological properties, in order to explain some of the traditional uses of the plant.
DOI: 10.3109/13880209.2013.865241
Materials and methods Chemicals Chemicals used were 2,2-diphenyl-1-picrylhydrazine (DPPH), 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), alkaline phosphatase (ALP), 2,20 azobis(2-methylpropionamidine) dihydrochloride (AAPH), 4-methylumbelliferyl phosphate (MUP), p-iodonitrotetrazolium violet (INT), gentamicin, colchicine, and {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} (XTT). They were obtained from Sigma-Aldrich (St. Gallen, Switzerland).
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General experimental procedures 1D and 2D NMR spectra (1H, 13C, COSY, HSQC, and HMBC) were recorded in acetone-d6 or methanol-d4 on a Varian Unity Inova 500 MHz spectrometer (Palo Alto, CA) with a CapNMR probe from Protasis/MRM and on a Bruker DRX 500 MHz 1H Larmor frequency using a 5 mm QNP direct detection probe. UHPLC/TOF-MS data were recorded on a Waters Micromass LCT-Premier mass spectrometer with an electrospray interface (ESI) coupled to an Acquity UHPLC system (Waters, Milford, MA) with an Acquity BEH C18 UHPLC column (1.7 mm, 100 2.1 mm i.d.; Waters). HRMS spectra were obtained using electrospray as the ion source, capillary voltage 2.8 kV, cone voltage 40 V, MCP detector voltage 2650 V, source temperature 120 C, desolvation temperature 250 C, cone gas flow 10 L/h, desolvation gas flow 550 L/h. HPLC-UV-DAD analysis was carried out on a HP 1100 system equipped with a photodiode array detector (Agilent technologies, Palo Alto, CA) with a symmetry C18 column (5 mm, 250 4.6 mm i.d.; Waters). The detection was performed at 210, 254, 280, and 360 nm. Medium pressure liquid chromatography (MPLC) was performed with a Bu¨chi 681 pump equipped with a Knauer UV detector using a LiChroprep RP-18 (40–63 mm, 460 50 mm i.d.; Merck, Darmstadt, Germany). The detection was performed at 254 nm. Column chromatography was carried out using LiChroprep RP-18 (15–25 mm, Merck) or silica gel 60 (20– 63 mm, Merck). Thin-layer chromatography was performed on silica gel 60 F254 A1 sheets (Merck). Plant material The fruits and leaves of G. preussii were collected in July 2008 at Ngoume´, located in the central part of Cameroon. The botanical identification was performed by Mr. Nana Victor, a botanist from the National Herbarium, Yaounde´, Cameroon, where a voucher specimen is conserved under the reference number 55520/HNC. Compound isolation from the fruits of G. preussii The air-dried powdered fruits (2 kg) were extracted by maceration with MeOH (3 3 L), for up to 48 h each at room temperature. After filtration, the MeOH extract was evaporated to dryness. The residue (310 g) was then dissolved in 800 mL of water and successively partitioned with hexane and EtOAc to give 90 g and 70 g, respectively. A portion of the hexane extract (50 g) was chromatographed over a silica gel column (20–63 mm, 5 60 cm) using
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an EtOAc/hexane step gradient (0:100–100:0 in 10% steps) to afford nine fractions (F1–F9). F1 (900 mg) was purified by semipreparative HPLC (MeOH/H2O 90:10 þ 0.1% FA, 10 mL/min) to afford garciniagifolone (1, 3 mg, tR ¼ 7.39 min), garcimultiflorone E (2, 5 mg, tR ¼ 7.29 min), and garcinialiptone B (3, 3 mg, tR ¼ 6.27 min). F2 (14 g) was separated by MPLC on LiChroprep RP-18 (40–63 mm, 5 46 cm), with a MeOH/H2O step gradient (60:40–100:0 in 10% steps, 3 mL/min) to afford five subfractions (F21– F25). This separation yielded garcinol (4, 7 g) from F22. Further purification of F25 was carried out over a reverse phase C18 column and separated with gradient mixtures of MeOH/H2O (80:20–100:0 in 10% steps) to afford nine fractions (F251–F259). F258 provided 7-epi-clusianone (5, 250 mg). Compound isolation from the leaves of G. preussii The air-dried leaves (5 kg) were extracted by a successive maceration with acetone (3 5 L) and then methanol (3 5 L) for 24 h each at room temperature. The methanol extract (50 g) was submitted to silica gel flash chromatography eluted with CH2Cl2/MeOH mixtures of increasing polarity (100:0, 2 L; 90:10, 2 L; 80:20, 2 L; 0:100, 1 L) which were collected into 300 mL fractions. Similar fractions were combined after TLC examination to provide four fractions (F1–F4). F4 (5 g) was applied to a Sephadex LH-20 column. Elution with MeOH (5 L) yielded seven fractions (F41–F47). F41 (30 mg) was repurified by column chromatography over Sephadex LH-20 (2 40 cm) using MeOH to afford eriodictyol (8, 4 mg). F43 (85.4 mg) was purified on silica gel column (20–63 mm, 2.5 60 cm) using CH2Cl2/MeOH 90:10 to give garcimangosone (6, 2 mg). F44 (38 mg) was also purified on a silica gel column (20–63 mm, 2.5 60 cm) using CH2Cl2/MeOH 90:10 to yield astragalin (7, 5 mg). Antioxidant assays DPPH assay The radical scavenging activity of the compounds was assessed spectrophotometrically in microplates, as described previously (Brand-Williams et al., 1995). ER50, the ratio of compound over radical concentration inducing a 50% decrease in absorbance after 90 min, was determined with a dose–response curve. ABTS assay The reducing activity of the compounds was assessed spectrophotometrically in microplates by following the decolorization of the ABTS radical (67 mM) (Re et al., 1999). ER50, the ratio of compound over radical concentration inducing a 50% decrease in absorbance after 90 min, was determined with a dose–response curve. ALP assay The antioxidant activity was assessed by the ability of a compound to preserve the catalytic effectiveness of the enzyme ALP (2 mU/mL in glycine buffer) despite the presence of peroxyl radicals generated by AAPH (5 mM). This was assessed by following the enzymatic
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dephosphorylation of MUP (20 mM) to the fluorescent 4-methyllumbelliferone. Enzymatic hydrolysis rates of MUP were determined by a continuous spectrofluorimetric assay (Bertolini et al., 2007). The percentage of ALP protection by tested compounds was calculated according to the following equation, after determining the hydrolytic activity of oxidized samples (hasample), oxidized controls (haox), and non-oxidized controls (hanon-ox): % ALP protection ¼ 100
ðhasample haox Þ ðhanon-ox haox Þ
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The EC50 value, obtained by a dose–response curve, represents the sample concentration which protects 50% of ALP enzymatic activity from a peroxyl radical-induced oxidation after 90 min. Oxygen radical absorbance capacity (ORAC) assay The ORAC assay was carried out using the same conditions as the above assay, except that a fluorescein solution at 6 108 M was used instead of ALP. The fluorescence was read at lEx 485 20 nm and lEm 528 20 nm to obtain the fluorescence value of oxidized samples (fluosample) and oxidized controls (fluoFLox) as well as non-oxidized controls (fluoFLnon-ox). This last one was calculated by the following equation: ðfluosample fluoFLox Þ % remaining fluorescein ¼ 100 ðfluonon-ox fluoFLox Þ The EC50 value, obtained by a dose–response curve, represents the sample concentration which protects 50% of fluorescein from a peroxyl radical-induced oxidation after 90 min (Huang et al., 2002). Antimicrobial assay The bacterial reference strains used in this study, Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), and Staphylococcus aureus (ATCC 29213) were obtained from the HUG (Geneva University Hospital, Geneva, Switzerland). Mueller-Hinton broth (MHB, Oxoid, Cambridge, UK) and Mueller-Hinton agar (MHA, BioMe´rieux, Balmes-LesGrottes, France) were used as liquid and solid media. All strains were grown for 24 h at 37 C. The minimum inhibitory concentration (MIC) of the various compounds were determined by using the broth dilution method in 96-well microtiter plates as previously described (Wiegand et al., 2008). The MIC corresponds to the lowest concentration of the compounds that inhibits visible growth (visual turbidity). The growth was detected by the reduction of INT into formazan, a red-purple molecule. The highest dilution of a compound in which no red-purple color appears corresponds to its MIC. Gentamicin was used as positive control (Eloff, 1998). Cytotoxicity assay DU145 (human prostate carcinoma), HeLa (human cervical cancer), HT-29 (human colorectal cancer), and A431 (human epidermoid carcinoma) cell lines were purchased from the
American Type Culture Collection. All cell lines were cultured in minimum essential medium (MEM) alpha containing 10% of fetal bovine serum, 100 IU/mL of penicillin G, and 100 mg/mL of streptomycin and maintained at 37 C in humidified environment containing 5% of CO2. The cytotoxicity of compounds was determined with the above cell lines as described previously (Roehm et al., 1991). Briefly, cells (in log growth phase) were harvested by trypsinization, counted, diluted in media, and added to 96-well plates containing test compounds dissolved in DMSO in triplicate; the final DMSO concentration was 0.05%. The plates were incubated for 3 d. Following the incubation, cells were stained with the addition of 80 mL of XTT (1 mg/mL) per well. The absorbance was measured at 450 nm after 3 h incubation at 37 C. The growth of the compound-treated cells was compared with the growth of DMSO-treated controls. With each compound, two-fold serial dilutions were tested in duplicate, with concentrations ranging above and below the IC50 values. The duplicate tests were used to construct dose–response curves, and IC50 values (mM) were determined by linear regression analysis. Results are the means of at least three independent determinations SD. Colchicine was used as positive control.
Results Chemical investigation of the hexane-soluble extract of the fruits and methanol-soluble extract of the leaves of G. preussii resulted in the isolation and characterization of eight known compounds garciniagifolone (1) (Shan et al., 2012), garcimultiflorone E (2) (Liu et al., 2010), garcinialiptone B (3) (Zhang et al., 2010), garcinol (4) (Krishnamurthy et al., 1981), 7-epi-clusianone (5) (Piccinelli et al., 2005), garcimangosone (6) (Huang et al., 2001), astragalin (7) (Nakabayashi, 1955) and eriodictyol (8) (Hussain & Waterman, 1982) (Figure 1). Each constituent was identified on the basis of its 1H-NMR spectra and HR-MS analysis by comparison with the literature. The antioxidant activity of compounds 1–8 was evaluated and compared to quercetin (Table 1). The antibacterial activity of each isolated compound was evaluated against four bacterial strains and showed activity only against Gram-positive bacteria. The MIC is shown in Table 2. Benzophenones 1–6 were also screened for their cytotoxic activity in DU145, HeLa, HT-29, and A431 cell lines (Table 3).
Discussion Species of the genus Garcinia are frequently used in African and Asian traditional medicine. They contain high levels of phenolic compounds that are reported to possess exciting biological properties. Despite the number of published studies on various Garcinia species, some plants of this genus have not been extensively studied from the chemical and pharmacological point of view. In order to explain some of the traditional use of G. preussii extracts, their antioxidant, antibacterial, and cytotoxic activities were measured. Active extracts were then fractionated to give eight pure compounds that were submitted to the various tests. Compound 4 was as potent as quercetin at scavenging the DPPH and ABTS– radicals and was slightly less active than
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DOI: 10.3109/13880209.2013.865241
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Figure 1. Structures of compounds 1–8. 32
33
35
7
30
38
O
OH
3
31
1 O 9 5 O O
23
20 22
O
H
O
OH O
OH
1
25
26
HO
HO
18
H
2
OH
OH
OH HO
O
HO
O
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O
O
OH
O
O 4
3
OH OH
HO
O O
OH
O O
O
5 O 6 OH HO
O O O
OH
OH HO
O
OH OH
HO OH
7
Table 1. Activities of compounds 1–8 in DPPH, ABTS, ALP, and ORAC assays.
Compounds 1 2 3 4 6 7 8 Quercetin
DPPH (ER50)
ABTS (ER50)
ALP (EC50 mM)
ORAC (EC50 mM)
0.20 0.03 0.21 0.02 0.45 0.11 0.11 0.01 41 41 0.62 0.03 0.09 0.01
0.19 0.02 0.20 0.01 0.29 0.02 0.09 0.01 41 0.70 0.03 0.65 0.03 0.07 0.01
9.72 2.40 8.23 1.23 5.22 1.72 nd 9.31 2.70 2.30 0.25 4.53 0.71 1.00 0.07
6.04 0.36 5.72 0.40 7.57 0.17 3.24 0.28 23.1 2.10 2.96 0.27 4.75 0.46 1.66 0.07
Results are the means of at least three independent determinations SD. ER50: concentration inducing a 50% decrease in absorbance after 90 min. nd, not determined.
the positive control in the ORAC assay. Its activity in the ALP assay could not be determined as it is an inhibitor of this phosphatase at concentrations higher than 105 M. The antioxidant activity of compounds 1, 2, and 3 was lower than that of quercetin, but these entities had no selectivity as they were active in the four assays used. The antioxidant
OH
HO
O
OH
OH
OH
O 8
Table 2. Minimum inhibitory concentration (MIC) of compounds 1–8. MIC (mg/mL) Compounds
S. aureus
E. faecalis
1 2 3 4 5 6 7 8 Gentamicin
64 4128 4128 16 2 4128 4128 4128 1
128 128 128 16 2 4128 4128 4128 16
Results are based on at least three independent determinations.
activity of compounds 1, 2, 3 and 4 can be explained by the conjugated catechol moiety on the structure. Compounds 7 and 8 were protectors of the ALP protein and of fluorescein against peroxyl-radical-induced oxidation. The similar potency in the ALP and ORAC assays points out that these compounds do not interact with the protein. Unlike them,
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Table 3. Cytotoxicity (IC50 in mM) of isolated benzophenones (1–6) in human cancer cell lines. Compounds
DU145
HeLa
HT-29
A431
1 2 3 4 5 6 Colchicine
7.7 1.4 14.5 3.2 11.5 2.0 14.9 0.8 33.4 5.2 4150 0.0230 0.0008
14.6 7.2 21.0 5.9 13.0 3.7 16.4 4.0 49.5 15.1 4150 0.0060 0.0014
7.4 0.2 15.9 4.1 14.0 3.0 16.1 3.7 32.6 1.7 4150 0.0140 0.0021
9.9 2.0 17.1 3.5 11.0 3.5 9.9 1.0 38.2 1.1 4150 0.0060 0.0003
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Compounds were evaluated for cytotoxicity by standard procedures as described in the Experimental section. The following cultured human cell lines were employed: DU145 (prostate carcinoma), HeLa (cervical cancer), HT-29 (colorectal cancer), and A431 (epidermoid carcinoma). Results are shown as IC50 values (mM) and are the means of at least three independent determinations SD.
compound 6 exhibited a weak ALP-protective activity only. This capacity might be due to a protein binding because of the hydrogen atom donors and acceptors containing scaffold. Compound 5 did not show any antioxidant capacity in the four assays performed since its structure does not enable reducing activity. None of the compounds showed activity against the two Gram-negative bacteria tested, E. coli and P. aeruginosa. However, compounds 4 and 5 displayed a good antibacterial activity against the Gram-positive bacteria S. aureus and E. faecalis. Compound 4 is already known to fight methicillin-resistant S. aureus and might be an alternative for classical antibiotic treatments (Iinuma et al., 1996). The MIC value of 16 mg/mL for S. aureus was identical to the value previously obtained with compound 4 purified from Garcinia bancana (Rukachaisirikul et al., 2005). Compound 5 exhibited a strong antibacterial activity with a MIC of 2 mg/mL against both S. aureus and E. faecalis. This compound has also been previously shown to be a promising compound for the treatment of multidrug-resistant S. aureus infections (Xiao et al., 2007). In addition, compound 5 has recently been proposed for the prevention or treatment of cavities since it affects the biofilm formation and the acid tolerance to the cariogenic Gram-positive bacterium Streptococcus mutans (Almeida et al., 2008). The mechanism of action of these compounds on bacteria is not known. However, due to their specificity for Gram-positive bacteria, the effect seems to be related to the characteristics of the cell envelope. Grampositive bacteria include important pathogens, known to be a leading cause of infectious diseases. They represent the most common and troublesome causes of nosocomial infections in neonatal intensive care units (Kocher et al., 2010). The increasing occurrence of bacteria resistant to antibiotics represents a serious threat for the treatment of infectious diseases. The discovery and development of novel agents with antibacterial activity is, therefore, needed. The benzophenones 1–4 displayed almost the same cytotoxic activity in each cell line tested (DU145, HeLa, HT-29, and A431) with IC50 values around 10 mM. Compound 5 was about three-times less active. This might be due to the lack of a catechol group. Among the benzophenones tested, 6 did not show cytotoxic activity against any of the cell lines (IC504150 mM). It might be due to structural differences, as compound 6 does not have the bicyclo [3.2] nonane system, as the other isolated benzophenones have. Benzophenones with a bicyclo [3.2] nonane
system have been widely studied for their biological properties.
Conclusion Medical plants play an important role in the management of bacterial infections, oxidative stress, and cancer, especially in developing countries where resources are meager. The results obtained can justify some of the traditional uses of this plant. Additionally, this study shows that G. preussi is a natural potential source of antioxidant compounds. Finally, the isolated benzophenones are expected to be useful for the study of anticancer agents in the future. However, the safety and toxicity of these compounds need to be investigated. Furthermore, those results are consistent with previous reports on the antioxidant, antimicrobial, and cytotoxic activities of phenolic compounds isolated from Garcinia species (Arif et al., 2009; Matsumoto et al., 2003; Tanaka et al., 2000; Yamaguchi et al., 2000)
Acknowledgements The authors are grateful to Dr. L. Marcourt for her help with the NMR data. Bernadette Biloa Messi gratefully acknowledges the Ministry of Higher Education of Cameroon (MINESUP) and the Swiss Confederation for her fellowship. The authors would like to thank the Swiss National Science Foundation (Grant no. 200020-107775/1 to Prof. K. Hostettmann) and the Alfred and Alice Lachmann Nutrition Fund (Grant to Prof. M. Cuendet) for financial support of this work.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the manuscript.
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DOI: 10.3109/13880209.2013.865241
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