Ethno-botanical survey, in vitro antisickling and free radical scavenging activities of Garcinia punctata Oliv. (Clusiaceae)

Drepanocytosis is a genetic and neglected disease, endemic in some populations originated from tropical regions. One of the main characteristics of this pathology is the production of a large amount of free radicals, leading to a severe oxidative stress and the consumption of NO by free oxygen radicals, and/or by cell-free plasma heme. The present study evaluated the antisickling and radical scavenging activities of extracts from Garcinia punctata stem barks using Emmel’s test and the DPPH assay. G. punctacta was selected through a combination of ethno-pharmacological and zoo-pharmacognosy approaches. The results showed that methanolic, ethyl acetate and dichloromethane soluble fractions, anthocyanins and organic acids exhibited a significant antisickling and antioxidant activity compared to that of n-hexane soluble fraction, as revealed by both the observed normal biconcave form of sickle erythrocyte (normalization rate > 77%). Ethyl acetate exhibited a drastic decrease of radical DPPH absorbance (ED 50 = 0.603 ± 0.028 µg/mL). The chemical screening performed on the plant revealed the presence of anthocyanins and organic acids which were then extracted. Total anthocyanins and organic acids revealed interesting antisickling and antioxidant properties that could justify the integration of Garcinia punctata in Congolese pharmacopoeia for the management of sickle cell disease. Some extracts from this plant species could increase nitric oxide by scavenging free oxygen radicals. For the best our knowledge, Garcinia punctata has not been yet previously reported as antisickling plant in the traditional medicine database of Democratic Republic of the Congo.


INTRODUCTION
Drepanocytosis also known as Sickle cell disease (SCD) is a genetically inherited disease in which a single base substitution in the gene encoding the human β-globin subunit results in replacement of β6 glutamic acid by valine, leading to the devastating clinical manifestations of SCD [1,2]. This substitution causes drastic reduction in the solubility of sickle cell hemoglobin (Hb S) when deoxygenated. Under these conditions, the Hb S molecules polymerize to form long crystalline intracellular mass of fibers which cause the deformation of the biconcave disc shaped erythrocyte into a sickle shape [3]. Each year about 300,000 children are born with pathological hemoglobin of which 70% are affected by SCD. Most of them die before the age of five years when they do not receive regular medical care. In Democratic Republic of the Congo, almost 2% of the population is sicklers [3][4][5].
The first-line clinical management of SCD includes medullar transplantation, repeated blood transfusion to stabilize the patient's hemoglobin level, the use of hydroxyurea and anti-malarial prophylaxis. Unfortunately, all current proposed therapies are quite expensive and have attendant risk factors [6,7]. Therefore, there is a need to find alternative strategy with efficient and affordable novel drug agents. The new alternative therapy strategy use zoo-pharmacognosy approach for identifying effective antisickling plants [8].
The use of plants in self medication by the non human primates was reported to be an advantage in protecting them against parasitic and viral diseases [9]. Garcinia punctata Oliv.  [13][14][15][16][17][18][19][20]. As several other flavonoids, anthocyanins are powerful free radical scavengers and show antioxidant activity [21][22]. Our previous studies have also identified organic acids as antisickling agents [15,23]. During an ethno-botanical survey, it was reported that Garcinia punctata Oliv. known under the vernacular name of "Bosofo" [Lomongo name] is traditionally used by the local communities to treat malaria. Since great apes (bonobos) are infected by malaria parasite but they cannot develop malaria disease, it can therefore, be hypothesized that G. punctata could protect sickle erythrocyte against hemolysis by inhibiting the polymerization of sickle hemoglobin and radical oxygen species formation within sickle erythrocyte as it does for Plasmodium falciparum infected erythrocyte in bonobo. The present study was performed with the aim of evaluating the antisickling and antioxidant activities of different fractions, anthocyanins and organic acids extracts of G. punctata Oliv., a Congolese plant which has not yet been scientifically investigated for its antisickling and antioxidant properties. Studies on plant species used by great apes (bonobo) and human as medicinal food with the possibility of formulating them as important nutraceuticals will contribute to solve health problem. Such plants could potentially safe because of the closely phylogenetic relationship between bonobo and human.

MATERIALS AND METHODS Ethno-botanical survey
Ethno-botanical information about the plant species selected for this study was obtained by interviewing traditional healers during field work which was conducted in the villages surrounding the Salonga National Park (Equateur, Democratic Republic of the Congo). Surveys were conducted from January to March 2011. A total of 10 traditional healers were interviewed. Informants were selected for their authentic knowledge on the utilization of medicinal plants and bonobo (Pan paniscus, Schwarz 1929) plant foods. Lingala, the national language of Equateur was used during anthropological interviews. Traditional healers were interviewed on a voluntary basis. The study followed principles laid out in the Declaration of Helsinki as previously reported [24]. The questionnaires were divided into three sections: (i) personal information such as name, age, sex, marital status and studies level; (ii) traditional medicine practice (including knowledge of diseases and symptoms); (iii) plant vernacular names, plant part used, preparation methods, and administration route of remedies. Informed consent was obtained from both the provincial Government of Equateur to collect plant samples and to conduct non-commercial research on Congolese medicinal plants and the respondents to divulge information. A benefit-sharing agreement on mutually agreed terms was also established between University of Kinshasa and Malagasy Institute of Applied Research according to the principles laid out in the Nagoya protocol [25][26][27].

Selection and plant material collection
The tested plant material used in this study were collected from Garcinia punctata Oliv. by Professor K.N. Ngbolua during a field work in the Salonga National Park

Extraction and chemical screening
The dried and powdered plant material (stem bark, 10 g) was repeatedly extracted by cold percolation with 95% ethanol (EtOH) and water (100 mL x 1) for 48 hours. Chemical screening was done in aqueous and organic extract according to a well known protocol as previously reported [28]. Fractions were filtered and concentrated to dryness under reduced pressure using a rotary evaporator. Extraction of anthocyanins was then done using 100 g of dried powdered plant material with acidified methanol (1% HCl) following an established protocol [13][14][15][16][17][18][19][20]. Anthocyanins extract was then defatted by nhexane. Organic acids were extracted according to the protocol of Ouattara et al. with minor modification [29]. Briefly, the powdered stem barks of G. punctata (50 g) were macerated with 100 mL of methanol-H 2 O (50/50) and then percolated with 400 mL of the same solvent at room temperature. The extract was concentrated under reduced pressure until 100 mL. The aqueous solution was basified to pH 9 with Na 2 CO 3 and repeatedly extracted with ether. The aqueous solution was then acidified with 4% acetic acid. The resulting acidic (pH 3) solution was repeatedly extracted by ethyl acetate. The solution were dried over Na 2 SO 4 and concentrated to give organic acids crude extract.

Preparation of methanol extract and increasing polarity extracts
Plant powder (100 g) was macerated in methanol 80% (1L x 2) for 48 hours. After filtering the mixture, the aqueous-methanolic filtrate was concentrated under reduced pressure using a rotary evaporator. The methanolic extract was suspended in distilled water and sequentially partitioned with n-hexane, dichoromethane, ethyl acetate, ethanol, and methanol (1:1, v/v) three times at room temperature. The resulting fractions were evaporated to dryness on an evaporator apparatus. All extracts were stored at +4 °C.

Biological material
Blood samples used to evaluate the antisickling activity of the plant extracts in this study were taken from known drepanocitary adolescent patients attending the "Centre de Médecine Mixte et d'Anémie SS" and "Centre Hospitalier Monkole", both located in Kinshasa area, D. R. Congo. None of the patients had been transfused recently with Hb AA blood. All antisickling experiments were carried out with freshly collected blood. In order to confirm their SS nature, the above-mentioned blood samples were first characterized by Haemoglobin electrophoresis on cellulose acetate gel, as previously reported [5]. They were found to be SS blood and were then stored at ± 4 °C in a refrigerator. An informed consent was obtained from all the patients participating in the study. All the research procedures have received the approval of Department of Biology Ethics Committee.

Antisickling assay
Sickle cell blood was diluted with 150 mM phosphate buffered saline (NaH 2 PO 4 30 mM, Na 2 HPO 4 120 mM, NaCl 150 mM) and mixed with an equivalent volume of 2% sodium metabisulfite. A drop from the mixture was spotted on a microscope slide in the presence or absence of ethyl acetate, methanolic or anthocyanins extracts and covered with a cover slip. Paraffin was applied to seal the edges of the cover completely to exclude air (Hypoxia). Duplicate analyses were run for each extract. The RBCs were analyzed by measuring various parameters including the area, perimeter and the radius of each RBC using a computer assisted image analysis system (Motic Images 2000, version 1.3; Motic Chine Group Co LTD) and statistical data analysis were processed using Microcal Origin 6.1 package software.
The radical scavenging activity of extracts for DPPH free radical was measured on the principle that antioxidants reduce the DPPH radical to a yellow-coloured compound (diphenylpicrylhydrazin) and the extent of the reaction will depend on the hydrogen donating ability of the antioxidant. Briefly, a 100 µM solution of DPPH radical in methanol was prepared. 3,5 mL of this solution added to 0,5 mL solution of each extract in methanol at concentrations ranging from 0,1 to 1 mg/mL, thus obtaining the desired final concentrations in the reaction mixture. The mixture was shaken vigorously and incubated in the dark at room temperature for 30 min. The absorbance was measured at 517 nm using a spectrophotometer SP-1105 Brand model. Methanol was used as a blank. The control solution consist of 0,5 mL of methanol and 3,5 mL of DPPH radical solution. The antiradical activity of a sample (calculated by the following formula) is given as percentage of reduced DPPH free radical: %I = [(OD control -OD sample)/OD control] ×100. The IC 50 value (μg/mL) is the effective concentration at which DPPH radicals were scavenged by 50%. L-ascorbic acid was used as positive control. Duplicate analyses were run for each extract.

RESULTS AND DISCUSSION Ethno-botanical survey
During ethno-botanical survey, ten traditional healers were interviewed about medicinal plants used both in folk medicine and eaten by great apes. The most cited plant was Garcinia punctata Oliv. with the use value and informant consensus factor of 0.51 and 0.33 respectively.

Extraction yields
Extraction yields of G. punctate Oliv. stem barks are given in Figure 2. According to these results, it is clearly shown that the unpolar solvents have fewer yields than polar ones. This reveals that the abundant metabolites in the stem barks of G. punctata Oliv. are those which pass easily through the polar solvents. The yield of extraction of the anthocyanins and organic acids on stem barks powders of the plant arerespectively 19.664 % (9.95 g) and 5.116 % (1.55 g).In comparison with some other results, e.g. Dicliptera colorata C. (7.04%) and Euphorbia hirta L. (2.54%), G. punctata Oliv. exhibited a high content of anthocyanins. The chemical screening performed on the Anthocyanins aqueous and alcoholic extracts of G. punctata revealed the presence of phenolic compounds, alkaloids, quinones, terpenoids and organic acids. Antisickling activity of different fractions from Garcinia punctata Oliv. stem barks Figures 3, 4a, 4b, 4c and 4d show respectively the micrographies of SS blood alone in a NaCl 0.9% solution (control, fig. 3) and the SS blood incubated with the n-hexane ( fig. 4a), dichloromethane (fig. 4b) ethyl acetate (fig. 4c) and methanolic (Fig. 4d) soluble fractions of Garcinia punctata Oliv. stem barks.       (Fig. 4, a-d), the majority of erythrocytes are reversed normal-shape. Normalization of sickle red cells is much better with dichloromethane, ethyl acetate and methanolic fractions than with n-hexane soluble fraction. This indicates that Garcinia punctata Oliv. stem barks have antisickling effects, thus justifying the use of this plant in Congolese traditional medicine. A similar result was already obtained for some medicinal plant species used for the management of SCD by Congolese traditional healers. This activity could be due to compounds easily extracted by the used polar solvents such as anthocyanins or to phenolic or triterpenoic acids as previously reported [13-20, 15, 23]. The treated SS RBCs demonstrated a remarkable similarity to normal blood values. The maximal normalization rate or minimal concentration of normalization (MCN) of potential fractions showing 70% of normalized red cells were determined. Figure 5 shows the dose dependent antisickling activity of dichloromethane, ethyl acetate and methanolic soluble fractions of Garcinia punctata Oliv. The curves show that, the normalization of drepanocytes increases with the extract concentration and reach a maximum and constant value at 25 μg/mL. This minimal concentration corresponding to the maximal normalization rate is called minimal concentration of normalization (MCN). This corresponds to a normalization rate of 78.73% for the dichloromethane fraction, 87.14% for ethyl acetate fraction, and 88.58% for the methanolic extract. The antisickling activity is dose dependent. These results show that the methanolic extract and ethyl acetate fraction are more active than the dichloromethane. Antisickling activity of anthocyanin and organic acids extracts from Garcinia punctata Oliv. stem barks Figure 6a and 6b give the optical micrograph phenotypes of SS blood treated with anthocyanin and organic acid crude extracts from Garcinia punctata Oliv. stem barks.  Figure 6a and 6b clearly show that in the presence of both anthocyanins and organic acid extracts, the majority of sickle-shaped erythrocytes in SS blood (Fig. 3) reversed into normal biconcave form. This indicates that anthocyanins and organic acids are the major antisickling agents of Garcinia punctata Oliv. stem barks.
These results confirm those already given by our research team with anthocyanins and organic acids such as betulinic acid, maslinic acid and lunilaric acid from other plants used in traditional medicine for the management of sickle cell anaemia [15,23]. In fact, it is known that anthocyanins have the ability to interact with proteins [28]. Interaction of these pigments with hemoglobin S could compete with the polymerization of this abnormal hemoglobin and prevent the sickling of sickle cells. In addition, anthocyanins (for which intestinal catabolism gives phenolic acids), also known for their antioxidant properties, could affect the Fe 3+ /Fe 2+ higher ratio in sickle cells and the stability of erythrocytes membranes by preventing the oxidation of membranes phospholipids [31]. As SCD is a chronic disease, using anthocyanins as medicinal foods or nutraceuticals would be a good approach instead of giving pharmaceutical products to sicklers during all their life. Figure 7 shows the evolution of normalization rate of the anthocyanins and organic acids extracts on drepanocytes. The normalization of sickled cells with the anthocyanins and organic acids extracts increase with the extract concentration and reached a maximum and constant value at 50 μg/mL (MCN). This corresponds to a normalization rate of 78.65 % for anthocyanins extracts and 86.12 % for organic acids extracts. Therefore, the antisickling activity of extracts is dose dependent.

Free radical scavenging activity
The radical scavenging activity of different fractions is given in Table 1.
As it can be seen in Table 1, ethyl acetate soluble fraction possess the lowest ED 50 value, compared to the positive control (high antioxidant activity), followed by the methanolic extract, anthocyanin extracts, organic acids extracts and dichloromethane soluble fraction respectively. Previous work on Garcinia xanthochymus has revealed that the high antioxidant activity of ethyl acetate could be correlated to the high content of total phenols [32]. In fact, ethyl acetate seems to Normalization rate (%) Concentration (ug/ml)

Anthocyanins extracts
Organic acids extracts be the solvent that concentrates best phenolic substances of intermediate polarity [33]. Therefore, the antisickling activity of extracts from Garcinia punctata Oliv. could be due to the presence of polyphenols known for their antioxidant properties. Increasing evidence accumulated over the last decades indicates that reactive oxygen species (ROS) play a key role in the pathophysiology of various ischemic diseases including SCD. The oxidative stress in SCD is likely the result of intravascular sickling and transient vaso-occlusive event leading to the decrease of nitric oxide (NO) probably due to consumption of NO by free oxygen radicals, and/or by cell-free plasma heme as a result of hemolysis [34]. The results outlined in this paper, indicate the antisickling and scavenging effects of Garcinia punctata, as attractive potential candidate for SCD therapy for improving the quality life of sicklers. As reducing agent, Garcinia punctata could prevent in vivo oxidative reactions, often by scavenging ROS before they can damage cells.

CONCLUSION
The present study evaluated the phytochemical screening and the in vitro antioxidant and antisickling activity of Garcinia punctata Oliv. stem barks. This plant species displayed promising antisickling and radical scavenging effects in vitro. The ability of anthocyanins and organic acids extracts to display such pharmacological properties may represent a rational explanation for the use of Bonobo plant food species by the traditional healers to treat SCD. The combination of ethno-pharmacological and zoo-pharmacognosy approaches as new tool has permit us to detect antisickling activity in Garcinia punctata, a plant no previously reported as antisickling plant in the traditional medicine knowledge of Democratic Republic of the Congo. Further studies involving the metabolomic profiling of the active fractions are in progress.