Antidiabetic, Antisickling and Antibacterial Activities of Anacardium occidentale L. (Anacardiaceae) and Zanthoxylum rubescens Planch. Ex Hook (Rutaceae) from DRC

It was recently reported a rare association of two genetic diseases notably sickle cell anemia and diabetes in one patient in the Democratic Republic of the Congo. Both diseases constitute a serious public health problem and have a common denominator that is to make patients susceptible to bacterial infections. Given the difficult and limited management of these diseases, the use of medicinal plants is considered as an effective alternative. Leaves of Anacardium occidentale and Zanthoxylum rubescens collected in the surroundings of University of Kinshasa and Gbadolite in Kinshasa and Nord Ubangi provinces respectively and these plants were selected through a chemo-taxonomic approach while a phytochemical screening was performed using a qualitative approach. Bacterial strains used to assess the antibacterial activity were Staphylococcus aureus and Escherichia coli and mice were used for the antidiabetic activity. The phytochemical screening revealed the presence of tannins, anthocyanins, leucoanthocyanins, flavonoids, bound quinones as well saponins and alkaloids. The aqueous extracts of A. occidentale and Z. rubescens showed an antisickling activity. Only S. aureus was sensitive to A. occidentale where petroleum ether extract (MIC = 125 μg.mL -1 ) showed a good activity than other extracts and no activity was observed on E. coli. Meanwhile, Z. rubescens showed no antibacterial activity on both strains (MIC = > 500 μg.mL -1 ). The mean values of blood glucose after 120 minutes in untreated and treated mice were 99.5 ± 7.77mg.dL -1 (0.9% NaCl), 41.6 ± 10.07mg.dL -1 (Glibenclamide 10mg.Kg -1 ) and 64 ± 13.98mg.dL -1 (methanolic extract of A. occidentale 500mg.Kg -1 ). These findings show that A. occidentale plant possess an antihyperglycemic activity. To our knowledge, it is for the first time that the antisickling activity of A. occidentale and Z. rubescens is reported thus validating the chemotaxonomic approach used as a criterion of selection of these two plants. It is also for the first time that antidiabetic activity of A. occidentale is reported.


Introduction
It was recently reported a rare association of two genetic diseases notably sickle cell anemia and diabetes in one patient in the Democratic Republic of the Congo (DRC). Both diseases constitute a serious public health problem worldwide as well in DRC [1]. Sickle cell anemia is characterized by the presence of hemoglobin S in the blood and its clinical manifestations are vaso-occlusion and heamolytic anemia [2]. On the other hand, Diabetes is characterized by a hyperglycemia (sugar level > 1.2 g.dL -1 of fasting), a hyperketonemia (keto acid level > 3 mmol.L -1 ), acid-base and hydro-electrolytic imbalance [3]. Considering the difficult and limited management of sickle cell disease and diabetes in the DRC, the use of traditional medicine and medicinal plants can be as an effective alternative in the management of these two diseases in emergency situations.
The World Health Organization (WHO) reported that more than 80% of African populations in poor regions use medicinal plants for their primary care [4,5]. Several scientific studies carried out both in DRC and elsewhere highlight the antisickling and antidiabetic properties of medicinal plants as well as their antimicrobial activity [6][7][8][9]. It has to be noted that Anacardium occidentale used in the current study is well known to traditional healers for its antidiabetic potential. [10], while some species of the genus Zanthoxylum are cited in the literature as having an antisickling potential but both plants possess as well the antibacterial properties [11][12][13].
The main goal of the current research was to verify whether A. occidentale and Z. rubescens possess antisickling, antibacterial and anti-hyperglycemic activities.
To this end, it should be expected that a plant having an antisickling property has also an antidiabetic property i.e. biochemically these two diseases lead to a disruption of glucose metabolism. Moreover, it is well established that the sickling of disease red blood cells is an inhibitory factor of glycolysis (inhibition of key enzymes by tactoids). The non-sequestered hemoglobin (Hb) fraction can then undergo non-enzymatic glycation to form glycated Hb generating oxygen free radicals [14]. Glycated Hb is a biomarker of diabetes while the formation of glycated Hb depends upon ambient glucose concentrations in which erythrocytes circulate as well the duration of exposure. A whole blood sample for glycated Hb is sufficient regardless of prandial state and clinical setting [3]. However, both diseases share the common denominator which is to make patients susceptible to infections [2,15]. In addition, it should be expected that Z. rubescens possess an antisickling activity using a chemo-taxonomic approach. The staple objective of this research was to provide a scientific evidence that the ethnomedical use of these plants (A. occidentale and Z. rubescens) is based thru phytochemical screening and the assessment of their antisickling, antibacterial and antidiabetic activities of these plant extracts.
The significance of the current research is to promote the use of medicinal plants in the treatment of sickle cell disease and diabetes in countries with limited health care. To our knowledge, it is for the first time that the antisickling activity of A. occidentale and Z. rubescens as well the validation of the hypothesis according to which an antidiabetic plant could potentially be antisickling is reported.

Materials
Plant materials used in the current study were leaves of A. occidentale L. (Anacardiaceae) and Z. rubescens PLANCH (Rutaceae) collected in the surroundings of University of Kinshasa in Kinshasa and Gbadolite in Nord Ubangi provinces respectively. A. occidentale was identified at the Herbariun of Faculty of Sciences, University of Kinshasa while Z. rubescens was identified at "Centre de Surveillance de la Biodiversité", University of Kisangani in Kisangani city. Blood samples used to assess the antisickling activity of plant extracts were collected from a sickle cell adolescent patient from the "Centre de Médecine Mixte et d'Anémie SS" of Kinshasa. Bacterial strains used were provided by the Laboratory of Microbiology, Faculty of Pharmaceutical Sciences (University of Kinshasa) and mice used for the assessment of the antidiabetic activity were provided by the Institut National des Recherches Biomédicales (INRB), Kinshasa, DRC.

Collection and Conditioning of Plant Material
Plant samples were collected in 2014 in Kinshasa and Gbadolite respectively. After collection, leaves were washed with tap water and dried under shade for one month, and then crushed separately in a grinder (Moulinex brand). The powders obtained were sieved in order to obtain fine powders for further analyses.

Extraction with Solvent of Increasing Polarity
Fifty grams of powder of each species were macerated in petroleum ether, ethyl acetate, and methanol (1:10, w/v) respectively for 48 hours. After filtration, various extracts were evaporated to dryness using a rotary evaporator device at 37°C.

Phytochemical Screening
The phytochemical screening is a chemical screening that includes a number of qualitative analysis that allows the identification of secondary metabolites present in a certain sample. The detection of these chemical groups is performed through color and precipitation reactions occurring with the addition of specific reagents [15][16][17][18]. The phytochemical screening was carried out according to the standard protocol as described by Bongo et al. [18] and it can be performed in aqueous or organic phases [19].

Biological Experiments (i) Emmel Test
The hemoglobin S becomes insoluble and crystallizes when the oxygen content decreases (hypoxia); it follows that, in case of sickle cell disease, the erythrocytes or red corpuscles containing hemoglobin S lose their normal circular shape, to assume a characteristic sickle shape. The evaluation of antisickling activity consists in seeing the effect of the drug on the sickling of red blood cells.
Four to eight drops of saline solution were used for the dilution of total SS blood (NaCl 0.9%) and on a slide was placed a drop of diluted blood, a drop of the drug/extract along with a drop of Na 2 S 2 O 5 2%. The mixture obtained constitutes the microscopic preparation covered with a coverslip which is super-cooled with paraffin on the edges of the slide. The solutions of our extracts (petroleum ether, ethyl acetate and methanol) were prepared by dissolving a few mg of these extracts into the saline solution. Different preparations obtained were observed using an optical microscope (brand Olympus) at 10X and 40X magnifications after 24 hours [20][21][22][23].

(ii) Antibacterial Assay
The antibacterial activity was evaluated using the microdilution method in a liquid medium.

(iii) Preparation of the Stock Solution and Bacterial
Suspension In a sterile test tube, place 0.020 g of each extract diluted in 250 µL of DMSO then stir for 10 minutes and add five mL of Mueller Hinton culture medium using a pipette and mix.
Afterwards, place two mL of the saline solution into two sterile test tubes. Using a sterile platinum loop, two isolated colonies of the two strains to be tested namely Escherichia coli ATCC 27195 and Staphylococcus aureus ATCC 33591 were collected and a colony of each strain was placed in the saline solution into both tubes. This bacterial suspension was diluted in the appropriate culture medium at a rate of 9 mL.

(iv) Dilution of Extracts and Inoculation of the Microplate
A 96-well sterile polystyrene microplate (8 rows A-H. x 12 columns) with a round bottom was used, whereby each well was filled with 100 µL of culture medium as follows: from A 2 to A 8 , B 2 to B 8 , C 2 to C 8 , D 2 to D 8 , E 2 to E 8 and F 2 to F 8 while the 11 th and 12 th wells served as controls. Using a micropipette, 200 µL of the stock solution of extract 1 was inoculated in A 1 and B 1 wells, 200 µL of the stock solution of extract 2 in C 1 and D 1 wells while 200 µL of the stock solution of extract 3 in E 1 and F 1 (i.e. 2 wells were used for each extract) respectively. Meanwhile, 100 µL of each stock or control solution were collected and serial dilutions of 2 by 2 were performed afterwards. Moreover, 100 µL of A 1 B 1 , C 1 D 1 and E 1 F 1 wells were collected and transferred to A 3 B 3 , C 3 D 3 and E 3 F 3 wells, then from the previous wells 100 µL were collected and transferred to A 4 B 4 , C 4 D 4 , E 4 F 4 wells. These solutions were thoroughly mixed and the same procedure continued till we reached A 8 B 8 , C 8 D 8 and E 8 F 8 wells. The last 100 µL taken from these wells were removed and thrown away [24][25].
Aseptically, 15 µL of the inoculum was collected and transferred to all wells of the microplate except for the wells of the 11 th column (control of sterility of culture medium). Wells of the 12 th column served as a positive control (inoculum and culture medium). The microplate was incubated in an oven at 37°C for 24 hours. Later, five µL of TCC 2% was added to each well and the observation occurred 15 min later.

(v) Determination of the Minimum Inhibitory Concentration
The principle of this method is based on the ability of living cells to reduce the tetrazolium salt to a red precipitate or formazan. The growth observed in different wells containing extracts or controls were compared to that in the bacterial growth control well (well-having inoculum without extracts or antibiotics). For a test to be considered valid, an acceptable growth has to be observed in the control wells. If the growth is insufficient in these wells, there is a need to re-inoculate the microplate and after the addition of 5 µL of TCC 2% (2, 3, 5triphenyltetrazoliumchloride) to the control wells then the minimum inhibitory concentration can be read. The minimum inhibitory concentration was then read from the first wells showing no bacterial growth after 48 hours [24][25].

(vi) Oral Glucose Tolerance Test
The assessment of the antidiabetic activity was carried out using an experimental design on an animal model consisting of 10 NMRI mice (male and female) subjected to temporary hyperglycemia by gavage of a glucose solution (200mg.mL -1 ) as per the protocol described by Williamson et al. [26]. These 10 mice were divided into three groups as follows: a first group of two mice as a negative control (saline solution), a second group of three mice as a positive control (Glibenclamide 10mg.Kg -1 ) and the third group of five mice to be tested with the ethyl acetate extract (500mg.Kg -1 ). Blood glucose testing was performed using a contour TS blood glucose meter from the tail.
The animal was immobilized by keeping its head raised and its mouth opened where a syringe loaded with the product along with the gastro-esophageal probe was introduced to the stomach, then by pushing the plunger of the syringe, the drug was administered to the animal as shown in figure 1 below. Before the administration of the drug, the extract was resolubilized in an appropriated solvent. For lipophilic extracts, the detergent such as DMSO was used.

(vii) Testing of Normal Blood Sugar
Mice were given a 24-hour pre-feed and then administered the extracts. Blood glucose was measured at T 0 , T 1 , T 2 , T 3 , and T 4 .

(viii) Anti-Hyperglyceamic Activity
In this research, a temporary hyperglycemia was caused in mice by oral administration of glucose (diluted to 10% in distilled water) at a dose of 4 g.Kg -1 of body weight and the basic blood glucose of mice was first detected. The basal glucose level of mice was determined after 24 hours of fasting (Baseline) and then glucose was administered to the mice. After 30 minutes of glucose overload, the blood glucose was determined in order to note the temporary hyperglycemia (transient hyperglycemia should reach the maximal value 30 min after administration of glucose). And then three batches of mice according to sex, weight and especially temporary hyperglycemia were formed. Different batches of mice were treated as follows: A control batch treated with a saline solution at a dose of 0.9%. A reference batch treated with Glibenclamide at 10mg.Kg -1 of body weight and a test batch treated with ethyl acetate extract at a dose of 500 mg.Kg -1 of body weight.

Phytochemical Screening
The phytochemical screening of the aqueous phase of A. occidentale and of Z. rubescens leaves is presented in table 1. As shown in the above table, the leaves of A. occidentale and Z. rubescens are enriched in polyphenols namely tannins, anthocyanins (phytochemical markers of the antisickling activity), leucoanthocyanins, flavonoids, bound quinones as well saponins and alkaloids. The presence of these secondary metabolites could confer on both plants the antisickling, antibacterial and antidiabetic properties.

Antisickling Activity
The phenotype of untreated (a) and treated (b) sickle cells respectively is presented in Figure 2 below. The rate of normalization of red blood cells of our patient in presence of both plant extracts under hypoxic conditions (Nacl 0.9%; Na 2 S 2 O 5 2%) is presented in the table below. Legend: -, Inactive; +, 10 < RN < 50% (low activity); ++, 50 < RN < 70% (higher activity); +++, RN > 70% (the highest activity) Table 2 shows that in the presence of organic extracts (petroleum ether, ethyl acetate and methanol) of A. occidentale and Z. rubescens, the sickle cells are normalized under conditions of hypoxia. This normalization of SS erythrocytes in hypoxia conditions is a partial scientific evidence which can justify the integration of these two plants on the list of antisickling plants. The normalization of SS blood erythrocytes treated with extracts from these plants results in the reappearance of the circular form of sickle cells. These results are therefore in perfect agreement with those of previous studies [27]. However, it should be noted that the extract with petroleum ether of A. occidentale has shown a weak antisickling activity with a normalization rate between 10 and 50%.
As shown in the above table, A. occidentale extracts displayed no activity against E. coli ATCC 27195 but active against S. aureus. The best activity is obtained with the petroleum ether extract (EEP, MIC = 125 µg.mL -1 ) followed by methanol (MeOH, MIC = 250 µg.mL -1 ) and ethyl acetate (EAE, MIC = 500 µg.mL -1 ). From the above table, it is clearly shown that the extracts of Z. rubescens did not display any activity against the two bacterial strains tested because their MIC> 500 µg.mL -1 .

Antidiabetic Activity
The hyperglycemic test carried out with methanolic extract of A. occidentale is presented in the table below. From the above table, it is displayed that the mean values of blood glucose after 120 minutes in untreated and treated mice were 99.5 ± 7.77mg.dL-1 (0.9% NaCl), 41.6 ± 10.07mg.dL-1 (Glibenclamide 10mg.Kg-1) and 64 ± 13.98mg.dL-1 (methanolic extract of A. occidentale 500mg.Kg-1). These results show that A. occidentale plant possess an antihyperglycemic (hypoglycemic) activity. In fact, the rate of reduction of glycemia is 36% for the extract versus 58% for Glibenclamide respectively.

Discussion
From the results obtained, we can observe that organic extracts (EEP, EAE, MeOH) of A. occidentale and Z. rubescens leaves normalize the sickled cells under hypoxia conditions created by the addition of Na 2 S 2 O 5 2%. Given that all extractions were performed on increasing polarity, the active principles of these plants are probably polar compounds. The antisickling activity of polar compounds such as phenylalanine and anthocyanins was reported in the literature by several authors [27][28][29][30][31][32][33][34]. The phytochemical screening carried out on the leaves of A. occidentale and Z. rubescens revealed the presence of polyphenols notably anthocyanins. The presence of anthocyanins in these two plants may partially justify the observed antisickling activity.
Moreover, Mpiana and his collaborators from University of Kinshasa in DRC have recently demonstrated that the antisickling activity of the majority of Congolese medicinal plants was attributed to this chemical group [27][28][29][30][31][32][33].
However, other authors reported that phenylalanine, phydroxybenzoic acid and its derivatives as well as maslinic, oleanolic and betulinic acids are responsible of the antisickling activity of plant extracts [34][35]. It is not excluded that these compounds are present in A. occidentale and Z. rubescens. To our knowledge, it is for the first time that antisickling activity of A. occidentale and Z. rubescens has been reported. The present study also revealed that only the extract with petroleum ether has a MIC < 500 µg.mL -1 and therefore active against S. aureus as indicated by Rios and Recio [36], the plant extract can be considered active when its MIC is less than or equal to 500 µg.mL -1 . This antiinfective activity (S. aureus) can be attributed to the alkaloids and polyphenols that are present in both plants [37]. The difference in activity observed is due to the presence in E. coli, a gram-negative bacterium, of an additional membrane layer impervious to the secondary metabolites contained in the various extracts. The results of this study are therefore similar to previous work. However, Ngbolua et al. [21][22][23] reported E. coli is less sensitive to plant extracts than S. aureus. (Anacardiaceae) and Zanthoxylum rubescens Planch. Ex Hook (Rutaceae) from DRC: A Pharmacological Screening The anti-hyperglycemic activity observed in mice treated with methanolic extract of A. occidentale can be attributed to polyphenols of which antidiabetic properties are well established scientifically [38]. These results confirm those obtained in previous studies indicating that the values of the glycemic index and load of the whole fruit of R. gentiliana particularly low, justifying the use of this fruit as hypoglycemic by the Congolese population [2]. Regarding the mechanisms of action of polyphenols in general and tannins in particular on glucose, in vitro studies carried out on the membrane vesicles of the brush border of the intestines of small rabbits showed that Catechin green tea extract had hypoglycemic activity by a mechanism of prevention of glucose uptake. The results on animal models showed that plant extracts could act through various mechanisms to lower blood glucose levels, thus reinforcing our findings. To our knowledge, it is for the first time that the antisickling activity of A. occidentale and Z. rubescens is reported, thus validating the chemo-taxonomic approach used as a criterion for selecting these two plants while it is also for the first time that the anti-hyperglycaemic activity of A. occidentale is reported. Henceforth, this validates the hypothesis that an antidiabetic plant would potentially possess an antisickling activity.

Conclusion
The main aim of the current research was to assess the antisickling, antibacterial and antihyperglycemic properties of two Congolese medicinal plants namely A. occidentale and Z. rubescens. The findings of the current study showed that both plants possess secondary metabolites that may confer to them the antisickling, antibacterial, and antihyperglycemic activities. These secondary metabolites are polyphenols, anthocyanins, tannins, alkaloids, flavonoids and saponins. Indeed, both plants displayed a strong in vitro antisickling activity as well a good antibacterial activity on S. aureus which was sensitive to petroleum ether exract of A. occidentale and the methanolic extract of A. occidentale is endowed with anti-hyperglycemic properties. It would therefore be desirable for a toxicological study to be carried out on both plants in order to include them in the Congolese antisickling traditional pharmacopoeia control. A thorough phytochemical study on the methanolic extract of A. occidentale has to be conducted in order to isolate the bioactive molecule responsible of this anti-hyperglycemic activity.