Antimicrobial, α-Glucosidase and Alkaline Phosphatase Inhibitory Ac- tivities of Bergenin, The Major Constituent of Cissus populnea Roots

Bergenin (1) was isolated as the major compound of the stem barks of Cissus populnea. Its structure was elucidated on the basis of spectral analysis, including 1D and 2D NMR experiments and Mass Spectrometry (MS) as well as by comparison with available data in the literature. Bergenin (1) was evaluated for its antimicrobial activity against four bacteria strains (Salmonella typhi ATCC6539, Salmonella typhi (isolate), Pseudomonas aeruginosa ATCC9721 and Escherichia coli (isolate)) and four yeasts (Candida albicans ATCC9002, Candida parapsilosis ATCC22019, Candida krusei (isolate) and Candida albicans (isolate)) using micro broth dilution method and for its enzymatic activities against α-glucosidase and Alkaline Phosphatase (h-TNAP and h-IAP). It exhibited significant to moderate antibacterial activities with the MIC ranking from 8 to 64 μg.mL-1, while it appeared to be inactive (MIC>125 μg.mL-1) against all the tested yeast. However, it was found to be high potent inhibitor of both h-TNAP and h-IAP alkaline phosphatase isoenzymes, with a selectivity for the h-TNAP. Daucosterol (4) and a mixture of sitosterol (2+3) were also obtained and were screening for the same bioactivities.


Introduction
Cissus populnea belongs to the Vitaceae family, a grape family of angiosperm with about 14 genera and 900 species principally distributed in tropical regions, with a few genera in temperate regions [1]. C. populnea is very little known in Cameroon and not well documented. However, it is an important medicinal plant in Nigeria where it is very famous for its uses in traditional medicine as well as like a spice. The plant is cited in the treatment of several infectious diseases including male infertility, urinary infections, skin diseases, indigestion, wounds, venereal diseases, gonorrhoea, ulcers, jaundice, icterus, trypanosomiasis [2]. C. populnea is also used against inflammations, colics, hypertension, hemorrhoids, fever, oedema, yellow fever, leprosy [3,4], malaria [5]. The roots of the plant are used in Adamawa region of Cameroon for the treatment of breast cancer [6]. C. populnea has been extensively studied biologically. Antimalarial activity [5,7], larvicidal activity [8], antimicrobial properties [9,10], antianaemic activities [11], spermatogenic potentials [12], hypoglycemic activity [13], as well as antiproliferative activities [6,14] of the plant parts have been reported. Previous qualitative phytochemical screening revealed that the aqueous extract of the stem bark contains flavonoids, steroids, saponins and tanins [12]. The stem barks were also found to contain carbohydrates, tannins, cyanogenic glycosides, saponins and anthraquinones [15,16]. The quantitative phytochemical analysis of the plant has also been reported [3,4]. However, to the best of our knowledge, no studies related to the isolation of compounds from this plant have been reported.
In the present investigation, the extraction and the isolation of the roots of C. populnea gave a brown extract from which Bergenin (1) was isolated. Bergenin (1) is a crystalline compound which has been previously isolated from Cissus pteroclada [17,18] and from several other plant species [19][20][21][22][23]. However, this paper reports for the first time its isolation from C. populnea. Furthermore, extraction of the roots of C. populnea gave in addition to Bergenin (1) a mixture of sitosterol (2 and 3), and Daucosterol (4). The isolated compounds were evaluated for their antimicrobial, α-glucosidase, and alkaline phosphatase inhibitory activities.
Alkaline Phosphatases (APs) are enzymes present in large quantities in the liver and bones. These enzymes can be used as markers in certain cancers, since an increase in their level may be related to bone cancer, liver cancer or others. However, this over expression of APs has been reported in patients with type 2 Diabetes for several years [24]. The use of α-glucosidase inhibitors is considered to be an effective strategy in the treatment of diabetes. Several α-glucosidase inhibitors, such as acarbose and voglibose obtained from natural sources have been used clinically in the treatment of diabetes mellitus [25], since they control effectively the blood glucose level after food intake. Unfortunately, only a few α-glucosidase inhibitors are commercially available and most of them are often associated with a number of serious side effects [26]. Screening of α-glucosidase and APs inhibitors from plants and synthetic sources is increasing. Inhibitors of these enzymes have been recently developed from natural sources [27,28].

General experimental procedures
Column chromatography (CC) was performed on silica gel 60 (Merck, 63-200 µm) with a gradient of n-hexane-EtOAc then, EtOAc-MeOH for the elution. For analytical Thin Layer Chromatography (TLC), silica gel precoated aluminium sheets F 254 Merck (20 × 20 cm) were used. The TLC plates after development were visualized under UV light (254 and 365 nm) and then sprayed with 15% sulfuric acid followed by heating at 105°C for 15 min. Melting points of the compounds were measured in open capillaries using Stuart melting point apparatus (SMP-3) and are uncorrected. 1 H (500 MHz) and 13 C (125 MHz) NMR spectra were recorded on a Bruker Avance AV-500 spectrometer, with chemical shifts given in ppm. The spectra were run using CDCl 3 or DMSO-d 6 as solvents and TMS as internal standard. ESI-MS spectra were measured on a Q-TOF Ultima spectrometer (Waters). Ciprofloxacin (Sigma-Aldrich, St Quentin Fallavier, France), and Ketoconazole (European Pharmacopoeia, Sigma-Aldrich) were used as reference standards respectively for antibacterial and antifungal assay. All organic solvents used for the tests were upgrade. Water used was distilled. All solutions were used on the day of preparation [29][30][31].

Plant material
The

Extraction and isolation
Air-dried powder of roots (300 g) were extracted by maceration (2 L) resulting after evaporation to dryness under reduced pressure to a dark brown extract (20.72 g) which was then refluxed in EtOAc (750 mL) three rounds of four hours each. All the EtOAc extracts were combined and then evaporated to dryness under reduced pressure at 40°C to a minimum volume and allowed to stand resulting in the precipitation of a white solid. This precipitate was washed several times using hexane, EtOAc, and acetone then recrystallized in MeOH. After recrystallization, the MeOH soluble portion was passed through a column chromatography of sephadex LH-20 using a mixture of EtOAc/MeOH 8:2 as solvent of elution leading to compound 1 as a white powder (510 mg). The remaining EtOAc fraction (7.8 g) was chromatographed on a silica gel column and eluted with a gradient of increasing polarity using Hexane/EtOAc (1:0-0:1, v/v) and EtOAc/ MeOH (1:0-0:1, v/v) as solvent. From the main column, in addition of 1, compounds 2 and 3 were obtained as a mixture in a ratio (2.4:1) and compounds 4 as a pure compound.

In vitro antimicrobial assays
Microorganisms and culture media: Eight microorganisms including four bacterial strains (Salmonella typhi ATCC6539, Salmonella typhimurium (isolate), Pseudomonas aeruginosa ATCC9721, Escherichia coli (isolate)) and four yeasts (Candida albicans ATCC9002, Candida parapsilosis ATCC22019, Candida krusei (isolate) and Candida albicans (isolate)), were used for the assays. E. coli was isolated from Ayub Theacthing Hospital of Abbottabad (Pakistan), S. typhimurium, C. krusei and C. albicans were isolates from the Laboratory of Bacteriology and Mycology of the "Centre Pasteur" of Yaounde, Cameroon while the reference strains were obtained from American Type Culture Collection (ATCC). Microorganisms were maintained on agar slant in refrigerator at 4°C. Mueller Hinton Agar (MHA) was used for the activation of tested Gram-negative bacteria while Mueller Hinton Broth (MHB) was used for the in vitro antibacterial assay as culture media.

Antibacterial assay: The Minimal Inhibitory Concentrations and
Minimal Bactericidal Concentrations (MICs and MBCs) of the samples and reference antibiotic were determined by the microdilution method using the MTT colorimetric assay as previously described by Nyemb et al. [27].
Anticandidal assay: For the anticandidal activity of all samples, the broth microdilution method previously described by Dzoyem et al. [32] was used for the determination of MICs and Minimum Fungicidal Concentrations (MFCs).

α-Glucosidase inhibitory activity:
The α-glucosidase inhibition was evaluated according to the spectrophotometric method reported by Nyemb et al. [27], in which α-glucosidase from Saccharomyces cerevisiae (baker's yeast) was used. Baker's yeast is widely used for enzymatic assays due to its availability and ease of handling [27,28,32,33].
Alkaline phosphatase inhibition assay: The luminescence method using CDP-Star as substrate for the determination of enzyme inhibition of samples on human tissue non-specific alkaline phosphatase (h-TNAP) and human intestine alkaline phosphatase (h-IAP) enzymes, was used as previously reported by Nyemb et al. [27].

Antimicrobial activity
All the isolated compounds were screened for their antimicrobial activity using the microbroth dilution method against eight microorganisms including four Gram (-) bacteria and four yeasts.
Regarding the results of the antibacterial activity (Table 1), it appears that Bergenin (1) was active against all the four selected bacteria including ATCC strains and clinical phenotypes, with MICs ranging from 8 μg/mL to 64 μg/mL. Bergenin (1) exhibited a significant antibacterial activity against P. aeruginosa ATCC9721 (8 μg/mL), and moderate activities against the others bacterial strains [35,36]. The mechanism of antibiosis of Bergenin (1) determined by the MIC index (MBC/MIC values) suggested its bactericidal effect (MBC/MIC ≤ 4) on all the tested Gram-negative bacteria [36][37][38]. These results suggest that Bergenin (1) could be able to easily cross the complex and multilayered lipopolysaccharide cell walls of the tested Gram-negative bacterial strains [39]. The significant bactericidal activity (MIC<10 µg.mL -1 and MBC/MIC ≤ 4) of Bergenin (1) against P. aeruginosa recorded in this study is very important since P. aeruginosa is considered as an important nosocomial pathogen highly resistant to clinically used antibiotics, causing a wide spectrum of infections and leading to substantial morbidity and mortality [40]. However, the activity of Bergenin (1) against all the tested Gram-negative bacteria was lower comparing to the reference antibiotic compound Ciprofloxacine (0.5 µg.mL -1 ). Among the other compounds, only Daucosterol (4) exhibited a moderate activity (32 µg.mL -1 ) against E. coli while its activity against P. aeruginosa was low (128 µg.mL -1 ) [35,36].
No compound exhibited any antifungal activity (MIC>128 µg.mL -1 ) at the tested concentration against all the tested yeasts. This could probably explain why C. populnea is not used in traditional medicine to treat fungal infections. The inactivity of this compound towards all strain of tested yeast could be probably due to the peculiar structure of the external layers of yeasts, as for example C. albicans presents a thick cell wall where β-glucans and chitin are abundant [41].

Enzymatic activity
The enzymatic activity of the isolated compounds was also evaluated in vitro against α-glucosidase and alkaline phosphatase (h-TNAP and h-IAP) ( Table 2). All the tested compounds were weakly active against the α-glucosidase enzyme at the tested concentration. The glucosidase activity recorded here for Bergenin (1) is not surprising since Kashima et al. [44] also reported its low inhibitory activity (22.7 ± 0.6% at 300 µM) against the same α-glucosidase enzyme from baker's yeast but at the concentration of 0.2 U/mL. β-Sitosterol (2) and Daucosterol (4) also showed activities against yeast and bacterial α-glucosidase enzyme [34,45]. However, the α-glucosidase inhibitory activity of the same compound can be different in different reports, due to the fact that the enzyme inhibition is dependent on the concentration of the substrate, the concentration of the enzyme, the concentration and the pH of the buffer solution and the duration of incubation with the enzyme.
Bergenin (1) was active against the two alkaline phosphatase isoenzymes, h-TNAP and h-IAP used in this study. It exhibited high potency against h-TNAP and h-IAP with the IC 50 values of 1.62 ± 0.01 μM and 12.86 ± 0.03 μM, respectively. However, this activity of Bergenin (1) was ~8 fold more selective for h-TNAP over h-IAP and was respectively ~12 and ~6 fold higher compared to the positive controls levamisole and L-phenylalanine. Daucosterol (4) was also evaluated for its alkaline phosphatase inhibitory activities and exhibited good inhibitory activities against both isoenzymes but with a slight specificity for h-TNAP.

Conclusion
As far as we are aware, this is the first report regarding the isolation of compounds from the roots of C. populnea. This first phytochemical study of the roots of C. populnea led to the isolation of Bergenin (1) as the major compound along with Daucosterol (4) and a mixture (2:1) of β-Sitosterol and Stigmasterol. The alkaline phosphatase inhibitory activity of Bergenin (1) and Daucosterol (4) was also evaluated for the first time in this study and both compounds were found to have potent activities against the two isoenzymes TNAP and IAP used. With a broad spectrum of antimicrobial activity against the tested Gramnegative bacteria, Bergenin (1) might be the active constituent at least in part, responsible for the previous antibacterial activities observed for this plant. The results showed that C. populnea could be a good source of antibacterials and APs inhibitors.