Antimicrobial steroidal saponin and oleanane-type triterpenoid saponins from Paullinia pinnata

Background Paullinia pinnata L. (Sapindaceae) is an African woody vine, which is widely used in traditional medicine for the treatment of human malaria, erectile dysfunction and bacterial infections. A phytochemical investigation of its methanol leaf and stem extracts led to the isolation of seven compounds which were evaluated for their antimicrobial properties. Methods The extracts were fractionated and compounds were isolated by chromatographic methods. Their structures were elucidated from their spectroscopic data in conjunction with those reported in literature. The antimicrobial activities of the crude extracts, fractions and compounds were evaluated against bacteria, yeasts and dermatophytes using the broth micro-dilution technique. Results Seven compounds: 2-O-methyl-L-chiro-inositol (1), β-sitosterol (2), friedelin (3), 3β-(β-D-Glucopyranosyloxy) stigmast-5-ene (4), (3β)-3-O-(2′-Acetamido-2′-deoxy-β-D-glucopyranosyl) oleanolic acid (5), (3β,16α-hydroxy)-3-O-(2′-Acetamido-2′-deoxy-β-D-glucopyranosyl) echinocystic acid (6) and (3β)-3-O-[β-D-glucopyranosyl-(1″-3′)-2′-acetamido-2′-deoxy-β-D-galactopyranosyl]oleanolic acid (7) were isolated. Compounds 5 and 7 showed the best antibacterial and anti-yeast activities respectively (MIC value range of 0.78-6.25 and 1.56-6.25 μg/ml), while 6 exhibited the best anti-dermatophytic activity (MIC value range of 6.25-25 μg/ml). Conclusion The results of the present findings could be considered interesting, taking into account the global disease burden of these susceptible microorganisms, in conjunction with the search for alternative and complementary medicines.


Background
Paullinia pinnata L. (Sapindaceae), an African woody vine, whose leaves and roots are widely used in traditional medicine for the treatment of human malaria [1] and erectile dysfunction [2]. In the West Region of Cameroon, its leaf decoction is used for the treatment of bacterial infections like typhoid fever, syphilis, gonorrhea, diarrhea, and symptoms like stomach-ache and waist pain. In East Africa, the leaves are reported to be used in the treatment of gonorrhea, wounds and microbial infections [3]. Previous phytochemical investigations have shown the presence of triterpene saponins, cardiotonic catechol tannins [4,5], flavone glycosides [6], steroids, steroidal glycosides [7], cerebroside and ceramide [8], as well as fatty acids [1] in P. pinnata collected from different parts of Africa.
There is a very limited biological investigation of the chemical constituents from the title species P. pinnata. As a result of our interest in the chemical and biological investigation of this plant, a methylinositol (1), steroidal terpenoids (2 and 4), and oleanane triterpenoids (3,(5)(6)(7) were isolated from its leaf and stem methanol extracts. In our previous studies, the antityphoid and antioxidant properties of these compounds were evaluated [9]. The extracts, fractions and all the compounds were evaluated for their antimicrobial activities against eight bacteria, five yeasts and five dermatophytes species and the results are reported herein. Compounds 2 (β-sitosterol) and 4 (daucosterol) were formally isolated from P. pinnata leaves collected from Cameroon [7]. To the best of our knowledge, 1, 3 and 5-7 are isolated from P. pinnata for the first time, and the antimicrobial properties of 5-7 are presented here for the first time.

Plant material
The air-dried leaves and stems of P. pinnata were obtained from Dschang, West Region of Cameroon, in January 2009. The identification of plant specimens was done at the Cameroon National Herbarium in Yaounde by Mr Tadjouteu Fulbert, where a voucher specimen was deposited under the reference number 10702/SRFCam.

Extraction and isolation
The air-dried leaves (2.04 Kg) and stems (2.02 kg) of P. pinnata were powdered and extracted with MeOH (7 l × 2, 48 h each) at room temperature to give crude extracts (233.8 g and 152.17 g respectively) after concentration under reduced pressure. The leaf extract (230 g) was exhaustively and successively washed with n-hexane and acetone to afford the hexane (45.2 g), acetone (8 g) and methanol residue (156.8 g) fractions while the stem extract was partitioned into petroleum ether, ethyl acetate and water to obtain the PE fraction (8.08 g), EtOAc fraction (9.13 g) and aqueous residue fraction (109.89 g).

Identification of compounds
Optical rotations were measured with a JASCO P-1020 digital polarimeter. UV spectra were obtained using a Shimadzu UV-2401 PC spectrophotometer. IR spectra were recorded on a Bruker Tensor-27 infrared spectrophotometer using KBr pellets. 1 day and 2D NMR spectra were performed on Bruker AM-400 and DRX-500 spectrometers (BrukerBioSpinGmBH, Rheinstetten, Germany) with TMS as the internal standard. ESIMS spectra were recorded on a Bruker HTC/Esquire spectrometer. HREIMS was recorded on a Waters AutoSpec Premier P776 spectrometer. Column Chromatography (CC) was performed on silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Rp-18 (40-63 μm, Merk). Fractions were monitored by TLC (GF254, Qingdao Marine Chemical Ltd., Qingdao, China), and by heating silica gel plates sprayed with 10% H 2 SO 4 in ethanol. GC analysis was performed on an HP5890 gas chromatograph equipped with a H 2 flame ionization detector.

Acidic hydrolysis of 4-7, and GC analysis
Compounds 4-7 (2 mg) were each refluxed with 2 M HCl (1,4-dioxane/H 2 O 1:1, 2 ml) on water bath for 2 h. After cooling, the reaction mixture was extracted with CHCl 3 (3 × 5 ml). The aqueous layer was evaporated to dryness with MeOH until neutral. The dried residue (sugar) was dissolved in 1 ml anhydrous pyridine and treated with L-cysteine methyl ester hydrochloride (1.5 mg), and stirred at 60°C for 1 h. Trimethylsilylimidazole (1.0 ml) was added to the reaction mixture, and this was kept at 60°C for 30 min. The resulting supernatant (4 μl) was analyzed by GC under the following conditions: H 2 flame ionization detector; Column: 30QC2/ AC-5 quartz capillary column (30 m × 0.32 mm); Column temperature: 180-280°C at the rate of 3°C/min;carrier gas: N 2 (1 ml/min); injector temperature: 250°C; split ratio: 1/50. The configurations of D-glucose and Dgalactose for compounds 4-7 were determined by comparison of the retention times of their corresponding derivatives with those of standard D-glucose and Dgalactose giving a single peak at 10.669 and 10.969 min, respectively.

Antimicrobial assays Microorganisms and culture media
The microorganisms used in this study were obtained from the American Type Culture Collection (ATCC), "EcoleNationaleVétérinaired' Alfort" (E), "centre Pasteur" of Yaounde-Cameroon and "Institut Pasteur" of Paris-France (IP). They includedeight bacteria strains: The culture media, Nutrient Agar (NA, Conda) and Sabouraud Dextrose Agar (SDA, Conda), were used for culturing bacteria and fungi respectively, while Mueller Hinton Broth (MHB, Conda), and Sabouraud Dextrose Broth (SDB, Conda) were used for the determination of minimum inhibitory and minimum microbicidal concentrations.

Preparation of microbial inocula
The inocula of bacteria and yeasts were prepared from 24 h and 48 h old agar cultures respectively. The absorbance was read at 600 nm (Jenway 6105 UV/Vis spectrophotometer-50 Hz/60 Hz) and adjusted with sterile physiological solution to match that of a 0.5 McFarland standard solution. From the prepared microbial solutions, other dilutions with sterile physiological solution were prepared to give a final concentration of 10 6 colony-forming units (CFU) per milliliter for bacteria and 2 × 10 5 spores per milliliter for yeasts [10].
Conidia suspensions of dermatophyte species were prepared from 10 days old cultures. The number of conidia was determined using a spectrophotometer and adjusted with sterile saline (NaCl) solution (0.9%) to an absorbance of 0.600 at 450 nm,corresponding to a final concentration of about 1 × 10 5 spores/ml [11].

Determination of minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC)
The MICs of the samples were determined by the broth microdilution method in 96-well micro-titre plates as described in the literature elsewhere [12]. The 96-well plates were prepared by dispensing into each well 100 μl of Mueller Hinton broth for bacteria and Sabouraud Dextrose broth for fungi. The test substances were initially prepared in 10% DMSO in broth medium and 100 μl of each test sample was added into the first wells of the micro-titre plate (whose wells were previously loaded with 100 μl of broth medium). Serial two-fold dilutions of the test samples were made and 100 μl of inoculum were then added into each well. This gave final concentration ranges of 12500 to 6.10 μg/ml for extract and fractions, 100 to 0.78 μg/ml for the compounds and 12.5 to 0.09 μg/ml for reference substances. The plates were sealed with parafilm, then agitated with a plate shaker to mix their contents and incubated at 35°C for 24 h for bacteria, 48 h for yeast and at 28°C for 7 days for dermatophytes.
For bacteria, MICs were determined upon addition of 50 μl (0.2 mg/ml) p-iodonitrotetrazolium chloride (INT, Sigma-Aldrich, South Africa). Viable bacteria reduced the yellow dye to a pink color. For yeasts and dermatophytes, MICs were determined by visualizing the turbidity of the wells. The MIC corresponded to the lowest well concentration where no color/turbidity change was observed, indicating no growth of microorganism. All tests were performed in triplicates.
Minimum microbicidal concentrations were determined by adding 50 μl (for bacteria and yeasts) or 5 μl (for dermatophytes) aliquots of the preparations (without INT for bacteria), which did not show any visible color/turbidity change after incubation during MIC assays, into 150 or 195 μl of sample-free broth. These preparations were further incubated and revealed as above to obtain the MMCs. Gentamycin, nystatin and griseofulvin were used as positive controls for bacteria, yeast and dermatophytes respectively.
The antimicrobial properties of the extracts, fractions and isolated compounds of the leaves and stems of P. pinnata are presented in Tables 1 and 2 respectively. In general, the crude extract of the leaves presented a better antibacterial activity compared to the stem extract; both of whose activities were comparable and not significant on fungi especially on dermatophytes. The results show that Compound 5 and 7 exhibited significant antibacterial and anti-yeast activities respectively (MIC value range of 0.78-6.25 and 1.56-6.25 μg/ml), while 6 exhibited the best anti-dermatophytic activity (MIC value range of 6.25-25 μg/ml). 1 was the most active antibacterial compound from the leaves, but was less active compared to 5. No compound from the leaves exhibited antifungal activity at the tested concentration. This probably explains why P. pinnata is not used by the local population in the treatment of fungal infections, since the leaf is the part locally used. β-sitosterol (2) was formally isolated from Citrus grandis fruits and shown to possess activity against gram-positive (Bacillus cereus, Bacillus subtilis and Staphylococcus aureus) and gramnegative (Escherichia coli and Salmonella enteritidis) bacteria, with MIC value of 300 μg/ml [26]. Friedelin (3), isolated from the stem bark of Vismia rubescens demonstrated antibacterial activity against Salmonella typhi, Staphylococcus aureus, Pseudomonas aeruginosa with MIC values of 25-200 μg/ml [27]. Besides, daucosterol (4) is a known antibacterial compound [28] In general, the antifungal activities of isolated compounds were relatively lower than their antibacterial activities. However 7 exhibited significant anti-yeast activity (MIC value range of 0.78-6.25 μg/ml), while 6 exhibited the best anti-dermatophytic activity (MIC value range of 6.25-25 μg/ml). Compounds 6 and 7 showed a wide spectrum of action on all the three types of microorganisms tested. Compounds 1, 3 and 5-7 are isolated from P. pinnata for the first time and the antimicrobial activities of 5-7 are reported herein for the first time. Though the best antibacterial compound, 5 did not show good antifungal activities. The structure-activity relationship of compound 5 and its analogue 6, shows that the introduction of an -OH group at C-16 in compound 6 considerably reduced its antibacterial activity, and increased its antifungal activities against the above mentioned microbial strains. The structures of 5 and 7 are similar, 7 showed more antifungal activity compared to 5 against the tested fungal species. This suggests that the introduction of a β-D-galactopyranose group at C-3′ of the sugar moiety of C-3 increased the antifungal activity. In addition, comparing the MIC and MMC values of compounds 2 and 4, it is seen that the presence of the C-3-β-D-glucopyranose group in 4 considerably increased the antimicrobial activity of the latter. Thus, the presence or absence of C-16-OH, C-3′-or C-3-β-D-hexopyranose groups play a critical role in determining the specific antimicrobial property of these types of oleanane triterpenoids and steroidal terpenes. The ratio MMC/MIC of compounds 4-7 was generally ≤ 4 with respect to all the microorganisms studied, indicative of a possible bactericidal nature of these compounds [29]. Moreso, the activities of compounds 6 and 7 were comparable to or better than the reference antibiotics on a considerable number of the tested microorganisms.

Conclusion
The results of the present findings could be considered interesting, taking into account the global disease burden of these susceptible microorganisms, in conjunction with the search for alternative and complementary medicines. They also show that an antifungal formulation could henceforth be envisaged from the stems of P. pinnata.