Phytochemical Composition and Biological Activity of Faidherbia albida ( Mimosaceae ) Roots and Leaves

International Journal of Pharmaceutical Sciences Review and Research International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net ©Copyright protected. Unauthorised republication, reproduction, distribution, dissemination and copying of this document in whole or in part is strictly prohibited. Available online at www.globalresearchonline.net 124 Tchebemou Bakang Bruno, Soh Désiré, Nkwengoua Tchouboun Zondegoumba Ernestine*, Nganso Ditchou Yves Oscar, Emmanuel Mouafo Tekwu, Kerstin Andrea-Marobela, Nyasse Barthélemy * 1Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, Box 812 Yaoundé, Cameroon. 2 Department of Chemistry, Higher Teacher Training College, University of Bamenda, PO Box 39 Bambili, Cameroon /TWAS Research Unit (TRU) of The University of Bamenda, Bamenda, Cameroon. 3Department of Chemistry, Faculty of Science, University of Maroua, P.O Box 814, Maroua, Cameroon. 4Laboratory of Tuberculosis Research and Pharmacology, Biotechnology Centre, Nkolbisson, University of Yaoundé 1, Yaoundé, Cameroon. 5Department of Biological Sciences University of Botswana Block 235/Room 251 Private Bag 0022 Gaborone Botswana. *Corresponding author’s E-mail: ernestine.nkweng@gmail.com


INTRODUCTION
his work is a follow up of our previous efforts on the study of medicinal plants of Africa in general and in particular of the chemical constituents of Dissotis perkinsiae (Melastomaceae) so as to promote sources of compounds with potential antimicrobial activities 1 . These research axe could be explored in the development of new the new therapeutic natural product, which corrobores with the assumption that more than 60% of the African population make use of their traditional medicine with more implication of the medicinal plants 2 . The special interest on Faidherbia albida 3 , also known as Acacia albida 4 , a plant of Mimosaceae/Leguminosacae/Fabaceae family is based on the fact that the species grows in more arid zones, which confers its with anti-stress ability and a capacity to fix nitrogen, water and other useful nutrients from deep soils [5][6][7][8] . The bark and leaves extracts are prescribed in traditional medicine to treat respiratory infections, fertility disorders, and digestive ailments, backache, malaria, etc [9][10][11][12] . Moreover in vitro qualitative phytochemical studies shown this plant possess a great quantity of triterpenes, sterols, glycosides, and tannins [13][14] .
Pharmacological studies have indicated antiinflammatory, anti trypanosomal, antidiabetic, antimalarial, antifungal, Neumaticidal activities of their bark crude bark extracts [14][15][16][17][18][19][20][21] , providing scientific resources for their indigenous use. Previous phytochemical studies indicated that the seeds and fruits essential oil of were rich in fatty acids 22 and the GC-MS analysis of essential oils from the stem bark revealed the presence of 37 different constituents of which α-pinene was the major one 22 . In addition, the extraction and characterization of the fruits of the plant revealed the presence non polar constituents such as of stearic acid, oleic acid, linoleic acid, methyl ester and linolenic acid, methyl ester 23 . In our knowledge, there is unreported literature on their roots and leaves. The chemotaxanomic and ethnopharmacological significance of the genus Faidherbia prompted us to investigate the secondary metabolites of Faidherbia albida. Thus, this work reports on the isolation and identification of 14 constituents isolated from the leaves and roots of Faidherbia albida and on the biological activities observed on some extracts and isolated secondary metabolites.

General experimental procedures
Mass spectroscopic data [Electrospray ionization mass spectrometry (ESI-MS)] were measured on a Waters Synapt HDMS spectrometer. NMR spectra were recorded with a Varian spectrometer at 400 MHz. Chemical shifts (δ) were quoted in parts per million (ppm) from the internal standard tetramethylsilane (TMS). Deuterated solvents dimethyl sulfoxide (DMSO-d6), and chloroform (CDCl3) were used as solvents for the NMR experiments. Column chromatography was performed on silica gel 60 [(0.2-0.5 mm) and (0.2-0.063 mm)] mesh (Sigma-Aldrich, Germany). Pre-coated silica gel 60 F254 thin layer chromatography (TLC) plates (Merck, Germany) were used for monitoring fractions and spots were detected with UV light (254 and 365 nm) and then sprayed with 30% sulphuric acid (H2SO4) followed by heating to 110°C.

Collection of Plant and Preparation
Roots and leaves of Faidherbia albida were collected in Maroua-Cameroon, (June, 2013) by Dr Frumsia (Plant taxonomist) and identified at the Cameroon National Herbarium (HNC), where a voucher specimens N°58978 /SRF/Cam are deposited.

Extraction and isolation of Faidherbia albida roots and leaves
The roots and leaves were dried at room temperature and powdered. 1500g of powdered roots were extracted by maceration in 07 liters (7l) of dichloromethane/methanol (DCM/MeOH, 1:1, v/v) mixture at room temperature for 72 h. The filtrate was concentrated in rotavapor (Büchi R-200) under reduced pressure at 65°C to yield 240g of extract. The crude material was dissolved in water and successively extracted with hexane, dichloromethane, ethyl acetate and n-butanol. All these extracts collected were preserved for chemical analysis.
1240g of powdered leaves were extracted by maceration in seven liters (5l) of dichloromethane/methanol (DCM/ methanol (MeOH), 1:1, v/v) mixture at room temperature for 72 h. The filtrate was concentrated in rotavapor (Büchi R-200) under reduce pressure at 65°C to yield 143g of extract. Dried extract was dissolved in water and successively extracted with hexane, dichloromethane, ethyl acetate and n-butanol. All these extracts collected were preserved for chemical analysis.

Preparation of stock solution of plants crude extracts, fractions and compounds
The different stock solution of plant, fractions and compounds were prepared by dissolving 2 mg in 1 ml of DMSO 10 % for a final concentration of 2 mg/ml. Reference antibiotics were prepared in the same condition by dissolving 512 mg of Fluconazole (Sigma Aldrich) and 2mg of Chloramphenicol and Ampicillin (Sigma Aldrich) in 1 ml of DMSO 10 % to yield 512µg/ml and 2 mg/ml respectively. After preparation, the different stock solutions were

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Antioxidant assay
A sample of 20 ml of potassium persulphate (50 mg/ml) was added to the 1 ml of azino-bisethylbenzothiazolinesulphonic acid diammonium salt (ABTS, Fluka, Switzerland). The mixture was incubated for 1 h at room temperature until persulphate had oxidized ABTS to a green-black coloured product. The oxidized ABTS stock solution was then diluted 1:10 in distilled water and 200ml of diluted working solution was pipetted into each well of a 96well-microtiter plate. A solution of 60% ethanol in water was used as a negative control and ascorbic acid (1 mM) was used as appositive control. An amount of 10 ml of controls or compounds was added to duplicate wells of the plate and stirred gently using the pipette tip. Antioxidant activity was recorded for compounds which reduced ABTS from green to colourless.

α-Glucosidase and Trypsin Inhibition &Activity Assay Starch
Agar plates were prepared by boiling agar (10 g/lHi Media) with potato starch (15 g/l) in distilled water. The mixture was poured into petri dishes (Falcon) to solidify. α-Glucosidase was Isolated from 4-day old pea shoots by grinding fresh shoots with phosphate buffered saline (oxoid). An amount of 10 ml of each compound was spotted onto the agar along the edge of the plate. In the center of the plate, 10 ml of 60% ethanol (negative control) and 10ml of a carbose (4 mg/ml, SIGMA) were spotted side by side. Onto each spot, 10 ml of pea shoot extract (α-Glucosidase) was added and left to digest for 10 min; 1 ml of iodine solution (UniLAB/Saarch, South Africa) was then poured over the surface of the plate and the excess discarded. Areas where the starch was digested by α-Glucosidase appeared clear in colour, indicating no inhibition of enzymatic activity. Spots which appeared blue due to iodine dying the undigested starch indicated the inhibition of α-Glucosidase.

In vitro studies with S. mansoni stock and working plant extract solutions
In vitro schistosomicidal evaluation was conducted on cercariae evolutionary form. Stock solutions of extract, fractions and isolated compounds were prepared according to the procedure described previously 24 .

Preparation of Cercariae Suspension
Schistosome cercariae's were obtained from experimentally infected B. pfeifferis nails as previously described 24 .

Microorganisms and growth conditions
C. albicans (Donated by Mr. M. Morobe, Department of Biological Sciences of the University of Botswana) were used as test organisms 25 : C. albicans Microorganisms was grown in nutrient Sabouraud Dextrose Broth (HiMedia, South Africa) at 32°C for 48 h. Microorganisms were maintained on agar plates at 4°C or stored as glycerol stocks at 80°C.

Antimicrobial assays
Sterile molten agar medium (400 mL) was pipetted into each well of a sterile 24-well culture plate (Corning) and left to solidify.
Yeast (C. albicans) were grown overnight in the irrespective media to a density of 1-106 cfu/ml. 10 ml of culture were pipettes onto each well of the agar plates. EtOH (60% aqueous solution, B&M, Scientific, Cape Town, South Africa) was the negative control across all antimicrobial assays. The positive controls were and penicillin/Streptomycin/fungizone (PSF, 100x; Highveld Biological, South Africa). 10 ml of EtOH, antibiotic, or compound were pipetted onto the surface of the agar in duplicate wells. The plates were later inverted and incubated in a moist chamber. Inhibition was indicated by a clear area on the agar surface, while visible growth of colonies indicated inhibition, poor/very poor/no inhibition.

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Available online at www.globalresearchonline.net ©Copyright protected. Unauthorised republication, reproduction, distribution, dissemination and copying of this document in whole or in part is strictly prohibited. Among these phytochemicals, Trans-Tiliroside (14) and 3β-Friedelinol (1) were found to be the majority compounds.
The first, a Triterpenes type is found in both ethyl acetate fractions, but approximately two times more present in roots than in leaves; whereas, the second, Flavonoids were found only in n-hexane fraction of roots. The result corresponds to the report in the literature that different solvent extraction and geographical area may result in obtaining different phytochemical constituents 36 .
In an attempt to characterize their bioactivities, isolated compounds and various extracts, fractions were investigated for their antioxidant potential, anthelmintic and antifungal effects against Schistosoma mansoni and Candida albicans respectively, α-Glucosidal and Trypsin activity.

Cercaricidal activity of crude roots and Leaves extracts, fractions and the compounds (13)
Schistosomiasis is a chronic and debilitating disease caused by a trematode of the genus Schistosoma and affects over 207 million people 36 . Chemotherapy is the only immediate recourse for minimizing the prevalence of this disease and involves predominately the administration of a single drug, praziquantel (PZQ). Although PZQ has proven efficacy, there is a recognized need to develop new drugs as schistosomicidal since studies have shown that repeated use of this drug in areas of endemicity may cause a temporary reduction in susceptibility in isolates of Schistosoma mansoni 36 .
The kinetics of mortality of the cercariae of S. mansoni after exposure to the crude extract, fractions and the compounds from Faidherbia albida are presented in figure  2: A-F.    The results indicate that cercariae death occur in a dosedependent manner for all extracts. The beginning of death begins after 15 min of exposition of the cercariae (Figure 2: A, D, E, F). With the concentration of 31.25 µg/ml of the extracts codes 5, 6 and 7, all the cercariae dead after 30, 60 and 90 min for extracts codes C, B and A respectively (Figure 2: A, B, C). On the other hand, one notes a death rate of 100% after 30 min when cercariae are exposed to each sample at 1000 µg/ml. In low concentrations (31.25, 62.5, and 125 µg/ml) some cercariae survived after respective time's intervals of 90 and 120 min, respectively (Figures 2:  D, E, F). An increase in the mortality rate of the cercariae for 15, 30, 60, 90, 120 and 150 min (Figures 2: A, B, C, D, F) were observed.
Roots crude extract's caused 100% cercariae death after 90 min at low concentration 31.25 µg/ml, less than 50 µg/ml, while leaves EtOAc fraction's, although was more active than leaves crude extract's, however none of them caused 100 % death. Comparing the effect of different concentrations of the crude extracts, fractions and isolated compounds on the S. mansoni cercariae mortality clearly shows that leaves CH2CL2 fraction's is the most active, seconded by the isolated compound 13 from roots. This might strongly indicate and justify synergistic effects of the chemical constituent's presents in these samples.
Previous works done by Gouveia in 2018 showed that Flavonoids were able to form complexes with cholesterol and decreased its level in plasma and increased cholinesterase activity or may be decreased the frequency of cardiac contraction and additionally, they are thus regarded as privileged structures used for a broad spectrum of activities and are potential candidates for sources of new drug prototypes 37 . Here, the Flavonoid compound (13) is the most active component, making Faidherbia albida a potential source of drugs for the fight against schistosomiasis. The exposure of the S. mansoni cercariae to crude extract, fractions and compounds showed that the plant have larvicidal activities against S. mansoni larvae.

Antioxidant, α-glucosidase and trypsin activities, αglucosidase and trypsin inhibiton and antifungal activities against candida albicans strain of crude roots and leaves extracts, fractions and the compounds (13)
The results of antioxidant, α-Glucosidase and trypsin activities, antifungal activities against Candida albicans strain, as well as of α-Glucosidase and Trypsin inhibition of compounds, extracts and fractions of roots and leaves of Faidherbia albida, are reported in Table 1.  25 , in comparison, ascorbic acid, which was used as reference drug. However, All the extracts (roots and leaves), as well as the ethyl acetate fraction of the roots were shown exhibited both anti-oxidant and α-Glucosidase activities respectively, while the ethyl acetate leave fraction exhibited only anti-oxidant activity. Interestingly, the compound Quercetin-3-O-α-rhamnoside (13) is active at the same time on α-Glucosidase inhibition, Trypsin inhibition and cercariae of Schistosoma mansoni, clarifying a possible candidate for multitarget drugs, although his antischistosomal against adult's worm were revealed by Xiao et al. in 2014 44 .
Susceptibilities of Candida albicans choose amongst Mycobacterium sp., which are the two major opportunistic pathogens occurring in an HIV/AIDS context, against extracts fractions and compounds 13 were also determined these samples were investigated. Up to 90% of HIVinfected individuals suffer from at least one episode of candidiasis, which is commonly characterized by oral thrush 45 . Although we could show that 2 mediates fungicidal activity of both fractions, their values are rather high for both extract, none of isolated compounds were active against yeasts. Therefore, the antifungal activity can be considered as weak.

CONCLUSION
Faidherbia albida is a Cameroonian medicinal plants used for the treatment of skin diseases, wounds, fever, rheumatism, malaria and/or infectious diseases. This plant species is rich sources of compounds that can play an important role in the human health, in the broader context, in the treatment of various diseases, such as helminthes, diabetes type 2, schistosomiasis and antioxidant-related ailments and The synergic effect of the extract and fraction, and also their triple of Quercetin-3-O-α-rhamnoside (13) could classified Faidherbia albida among the potential candidate for the development of multitarget drugs. This could reduce the combination of multiple biological active agent (multitherapy) therapy in favor of multitarget drug strategies.