Preliminary in vitro antimicrobial screening of chemical constituents isolated from the root of Lepleae mayombensis ( Meliaceae )

a Institute of Medical Research and Medicinal Plants Studies P.O Box 6163 Yaoundé, Cameroon. b Department of Organic Chemistry, University of Yaoundé I, P.O Box 812, Yaoundé, Cameroon. c Sri Paramakalyani Centre of Excellence in Enviromental Sciences Manonmaniam Sundaranar University, Alwarkurichi -627412, Tamil Nadu, India. d Department of chemistry, Higher Teachers Training College, P.O Box 47, Yaoundé, Cameroon. e Department of Chemistry, Manonmaniam Sudaranar University, Tirunelveli 627012, Tamil Nadu, India. f Department of Biochemistry, University of Yaoundé I, P.O Box 812, Yaoundé, Cameroon. g Department of Pharmacy, Faculty of Medicine and Pharmaceutical Sciences, University of Douala, P.O Box: 2701 Douala, Cameroon. h TU Chemnitz, Institut Für Anorganische Chemie, Straße Der Nationen 62 0911 Chemnitz, Germany.


INTRODUCTION
Natural products are the most consistently successful source of drug leads.Natural products continue to provide greater structural diversity than standard combinatorial chemistry and so they offer major opportunities for finding novel low .molecular weight lead structures that are active against a wide range of assay targets (Harvey, 2000).Plants supply most of the active ingredients of traditional medicinal products, and plant extracts have long been used in screening programs in pharmaceutical companies and university institutes.It might be thought that most of the plant kingdom has been thoroughly examined in the search for biologically active molecules but only 10% of plants species existing around the world have been tested for some type of biological activity (Verpoorte, 1998).The genus Leplaea (meliaceae) has 8 species mainly widespread in tropical Africa.In Cameroon, 5 Leplaea species have been identified (Kouma et al., 1998).Significant chemical constituents of meliaceae family are triterpenoids and limonoids (Amit and Shailendra, 2006).Plants of meliaceae family are rich source of limonoids, which proves to be a class of bioactive natural products with highly oxygenated and modified triterpene skeleton (Li-Li et al., 2014).Limonoids are derivatives of terpenoids.meliaceous limonoids have attracted interest due to their diverse structures and biological activities, such as insect antifeedant, antimalarial, cytotoxic, and 11β-HSD1 inhibitory effect (Tsamo et al., 2013;Nugroho et al., 2013;Rao et al., 2012;Tan et al., 2011).
In continuation of investigations of medicinal plants with aim to search for potent antimicrobial agents, the present study was designed to isolate compounds from the root of Leplaea mayombensis, and study their antibacterial and antifungal activities.

General experimental technique
All organic solvents used were of analytical grade and Fractions were monitored by TLC and performed on precoated silica gel 60 F254 plates (Merck, Dramstadt, Germany).The spots were revealed using both ultra-violet light (254 nm and 366 nm) and 10 % H 2 SO 4 spray reagent.The structures of isolated compounds were elucidated by means of spectroscopic experiments mainly 1D and 2D NMR performed, on a 500 and 150 MHz Bruker Avance III-600 spectrometer equipped with a 5mm BBFO+ probe at 300K and ESIMS / HRESIMS analyses recorded on a SYNAPT G2 HDMS (Waters) mass spectrometer and by comparison with literature data.

Sample collection
L. mayombensis root was harvested in Yaoundé-Cameroon, (October, 2014) and identified by Mr. Victor Nana (Plant taxonomist) of the Cameroon National Herbarium (HNC), where a voucher specimens are deposited (46220/HNC).Then, root were collected, cut into small pecies, dried at room temperature and powdered.

Phytochemical screening of plant extract and fractions
Crude extract and fractions were subjected to phytochemical screening to detect the presence of alkaloids, tannins, saponins, triterpenoides, steroids, flavonoids, phenol, coumarins and antraquinones using protocols described by Harbone, 1973.

Stock solutions and disc preparation
For the antimicrobial activity, stock solutions of compounds were prepared at 0.02 mg/mL in DMSO 10 %.Amplicillin, Chloramphenicol, Tetracyclin, Fluconazole and Nystatin were prepared in the same conditions.For disc preparation, 10 μL of each stock solution was dropped onto sterilized paper disks (6 mm diameter) and dried at room temperature for a final concentration of 0.02 mg/disc.

Antimicrobial activity
In vitro antimicrobial activity was assess by disc diffusion method using Mueller Hinton (Bauer et al., 1966) Agar (MHA) and Sabouraud dextrose Agar (SDA) obtained from Mast Group Ltd.The Agar plates were prepared by pouring 15 mL of molten media into sterile plates (90 mm).The plates were allowed to solidify for 5 min and 0.1 mL of inoculum suspension was swabbed uniformly and the inoculum allowed drying for 5 minutes.
The different compounds and references drugs loaded at 0.02 mg /discs were placed on the surface of the medium and allowed to diffuse for 5 min.The plates were incubated at 35 °C for 24 hours for bacteria and for 48 hours for fungi.Negative control was prepared using 10 % DMSO.At the end of incubation, inhibition zones formed around the disc were measured with a Vernier Calliper in millimeter.Each experiment was performed in triplicate.

Phytochemical content of crude extract and fractions
The preliminary phytochemical study (Table 1) showed that extract from root of L. mayombensis contain phenols, steroids, terpenoids, flavonoids, coumarins, alkaloids, saponins and tannins.The same chemical composition was found with methanol fraction.Only alkaloids were absent in ethyl acetate fraction of this extract.Whereas, hexane fraction contains only terpenoids, steroids and coumarins.

Antimicrobial activities
The bacterial inhibition zone diameters of crude extracts, fractions and compounds are summarized in table 2. The results below indicate that the inhibition zone diameters vary from 0.00 ± 0.00 to 29.00 ± 0.00 mm on both bacteria and fungi.This inhibition was found to depend to compounds and microorganism tested.
All the compounds tested have showed antimicrobial activities on at least one microorganism.This activity was better than those exhibits by referents antibiotics.The preliminary phytochemical screening revealed the presence of phenols, steroids, terpenoids, flavonoids, coumarins, alkaloids, saponins, tannins and the difference of secondary metabolites composition of fractions compared to crude extracts.This result can be explained by the fact that during fractionation, compounds are separated according to their affinity and solubility with extraction solvent (Cowan et al., 1999).However, the increasement of quantity of alkaloids and anthraquinones in methanol fraction compared to the loss of some classes of secondary metabolites in hexane and ethyl acetate fractions can be explain by the fact that fractionation could concentrated some classes of secondary metabolites and reduce others (Harbone, 1984;Bolou et al., 2011).The presence of all these classes of secondary metabolites in extract of root of L. mayombensis is an indication of its pharmacological importance.In fact, these classes of metabolites are reported to possess many importance biological activities including, antimicrobial, anticancer, antioxydant (Shariff, 2001;Ghoghari and Rajani, 2006;Hadacek, 2002;Panda and Kar, 2007).The fractionation of root extracts was lead to isolation and identification of 8 compounds and their antimicrobial activity was determined by disc diffusion method.The results showed that activity is closely related to structure of compounds tested.β-sitosterol and stigmasterol, respectively, two compounds of the same classes of steroids were found to target the same group of microorganisms.The antimicrobial activities of stigmasterol were found to be moderate as compared the reference drugs.This observation is in good agreement with previous reports that showed low antimicrobial (or antibacterial) activities of stigmasterol against Acetobacter sp., E. coli, S. aureus, and Streptococcus sp, P. aeruginosa (Ahamed et al., 2002;Tamokou et al., 2011).On the other hand, antibacterial activity of βsitosterol was also found to be consistent with literature reports that discuss low/moderate antibacterial activity of β-sitosterol against several bacterial species that include S. aureus, E. coli, and P. aeruginosa (Beltrame et al., 2002).: (8S,9R,10R,11S,13R,17R)-6, 4,8,10,12,13,15,16,phenanthrene-3,7(8H,9H,10H,14H)-dione (1) was the less active compounds studied.This can be explain the steric congestion around the hydroxyl group limiting his reactivity.The inhibitory activity of stigmasterol 3-O-β-D-glucopyranoside (8) was better than compounds 1, 6 and 7.This activity was due to the substitution of the single and free hydroxyl on the carbon skeleton by the glucopyranoside group which enhance the activity of this stigmasterol.In fact, this glucopyranoside group is known as responsible for antimicrobial activity due to their ability to complex with bacteria cell wall, (Cowan, 1999) and therefore, inhibiting the microbial growth.The best and high spectrum antimicrobial activity was observed with 9,10-cyclopropyl-((3S,5R,8R,13R,14S,17R) )-4,4,13,14,17-pentamethyl-l7-((2R,5S,6R)-5,6,7-trihydroxy-6-methylheptan-2-yl)hexadecahydro-1H-cyclopentane[α]phenanthrèn-3-ol) (5) which is a cycloartane triterpenoïds.In fact, terpenoïds are involve in membrane disruption by the lipophilic compounds which lead to the death of microorganisms (Mendoza et al., 1997).The antimicrobial activities varied with the bacterial and fungal species.These variations may be due to genetic differences between the microorganisms.The findings of the present study showed that there were differences between the antimicrobial activities of isolated compounds.This suggests that the root extract of L. mayombensis contains several antifungal and antibacterial principles with different polarities as shown by the phytochemical study.Therefore, it is clear that limoinds and cycloartane are the active constituents of meliaceae family plant.

Table 1 :
Phytochemical composition of extracts and fractions of L. mayombensis