Antimicrobial and antioxidant activity of kaempferol rhamnoside derivatives from Bryophyllum pinnatum

Background Bryophyllum pinnatum (Lank.) Oken (Crassulaceae) is a perennial succulent herb widely used in traditional medicine to treat many ailments. Its wide range of uses in folk medicine justifies its being called "life plant" or "resurrection plant", prompting researchers' interest. We describe here the isolation and structure elucidation of antimicrobial and/or antioxidant components from the EtOAc extract of B. pinnatum. Results The methanol extract displayed both antimicrobial activities with minimum inhibitory concentration (MIC) values ranging from 32 to 512 μg/ml and antioxidant property with an IC50 value of 52.48 μg/ml. Its partition enhanced the antimicrobial activity in EtOAc extract (MIC = 16-128 μg/ml) and reduced it in hexane extract (MIC = 256-1024 μg/ml). In addition, this process reduced the antioxidant activity in EtOAc and hexane extracts with IC50 values of 78.11 and 90.04 μg/ml respectively. Fractionation of EtOAc extract gave seven kaempferol rhamnosides, including; kaempferitrin (1), kaempferol 3-O-α-L-(2-acetyl)rhamnopyranoside-7-O-α-L-rhamnopyranoside (2), kaempferol 3-O-α-L-(3-acetyl)rhamnopyranoside-7-O-α-L-rhamnopyranoside (3), kaempferol 3-O-α-L-(4-acetyl)rhamnopyranoside-7-O-α-L-rhamnopyranoside (4), kaempferol 3-O-α-D- glucopyranoside-7-O-α-L-rhamnopyranoside (5), afzelin (6) and α-rhamnoisorobin (7). All these compounds, except 6 were isolated from this plant for the first time. Compound 7 was the most active, with MIC values ranging from 1 to 2 μg/ml and its antioxidant activity (IC50 = 0.71 μg/ml) was higher than that of the reference drug (IC50 = 0.96 μg/ml). Conclusion These findings demonstrate that Bryophyllum pinnatum and some of its isolated compounds have interesting antimicrobial and antioxidant properties, and therefore confirming the traditional use of B. pinnatum in the treatment of infectious and free radical damages.


Background
Bryophyllum pinnatum (Lank.) Oken, syn. B. calucinum or Kalanchoe pinnata (Crassulaceae) is a perennial succulent herb which grows in Africa and Asia [1]. An ethnobotanical survey of plants used in the treatment of infectious diseases in Mbouda subdivision (Cameroon) indicated that B. pinnatum was one of the most used medicinal plants in the area. Therefore information collected directly from traditional healers and herbal sellers in this area indicated that, leaves or the whole plant are used as analgesic and to treat blennorrhoea, syphilis, jaundice, candidiasis, dysmenorrhoea, external ulcers, burns and convulsions. B. pinnatum is also used elsewhere for treatment of ear infections, cough and dysentery [2]. This wide range of traditional uses justifies its being called "life plant", "resurrection plant" or "goodluck" [3,4]. Previous phytochemical studies revealed the presence of terpenoids [1], cytotoxic bufadienolides [5] and antileishmanial flavonoids [6] in this plant. In addition, 60% methanolic extract of leaves of B.
pinnatum have shown antimicrobial activity [2], while water extracts have shown anti-ulcer, antinociceptive, anti-inflammatory, antidiabetic, neurosedative and muscle relaxant activities [3,4,7], and ethanolic extract have exhibited hepatoprotective activity [8]. In the course of our search for bioactive components from Cameroonian medicinal plants, phytochemical and biological investigations of B. pinnatum were carried out. We describe here the isolation and structure elucidation, antimicrobial and antioxidant properties of seven kaempferol rhamnosides (1-7) from B. pinnatum which may account for some of the ethnomedicinal uses of leaves or the whole plant.

Antimicrobial activity
The MeOH, EtOAc and hexane extracts as well as isolated compounds were tested for their antimicrobial activity and the results obtained are presented in Table  1. The crude extracts have shown both antibacterial and antifungal activities, on the set of germs tested with MIC values ranging from 16 to 1024 μg/ml. The EtOAc extract was more active (MIC = 16-128 μg/ml) than the MeOH extract (MIC = 32-512 μg/ml) and the hexane extract (MIC = 128-1024 μg/ml), meaning that the active principles might be more concentrated in the EtOAc extract. Compound 7 isolated from the EtOAc extract was more active (MIC = 1-2 μg/ml) than all the other tested compounds. This is followed in decreasing order by compounds 5, 6, 1, 3 and 4. The antimicrobial activity of compound 7 was in some cases equal to those of the positive controls (ciprofloxacin and nystatin). Nevertheless, the antibacterial activity of this compound (MIC = 1 μg/ml) was higher than that of the positive control ciprofloxacin (MIC = 2 μg/ml) against Pseudomonas aeruginosa.

Antioxidant activity
The results of the antioxidant activities of the MeOH, EtOAc and hexane extracts as well as their isolated compounds are presented in Table 2. It appeared that the MeOH extract (IC 50 = 52.48 μg/ml) was more active than the EtOAc extract (IC 50 = 78.11 μg/ml) and hexane extract (IC 50 = 90.04 μg/ml). The antioxidant activity of compound 7 (IC 50 = 0.71 μg/ml) was higher than that of the reference drug (IC 50 = 0.96 μg/ml). No biological test was done with compound 2 since it was obtained as a mixture with compound 3.

Discussion
The findings of the present study showed differences between the antimicrobial activities of crude MeOH extract and extracts from partition. This suggests that B. pinnatum contains several antifungal and antibacterial principles with different polarities as shown by the phytochemical study. The partition of the MeOH extract enhanced its antimicrobial activity in EtOAc fraction, and reduced that of hexane fraction. This indicates that the active principles might be more concentrated in EtOAc fraction and more diluted in hexane fraction. All the isolated compounds showed antimicrobial activities on at least one microorganism. Such a finding supports the traditional use of this plant in the treatment of infectious diseases. The result of the antimicrobial activity of MeOH extract from the whole plant of B. Pinnatum corroborates that of Akinpelu [2]. The antimicrobial activities varied with the bacterial and fungal species. These variations may be due to genetic differences between the microorganisms. A keen look at the results of MIC and minimum microbicidal concentration (MMC) ( Table 1), showed that the MIC values obtained are in most cases equal to the MMC values on the corresponding (sensitive) microorganisms, confirming the microbicidal effects of the tried samples [13]. This is interesting in view of the perspective of developing new antibacterial drugs from natural products.
The antimicrobial activity was more concentrated in EtOAc fraction. In contrast, the antioxidant activity is more concentrated in MeOH extract. This indicates that partition of the crude extract did not enhance the antioxidant activity of its fractions. Flavonoid compounds such as compounds 1-7 are known to be potential antioxidant due to their ability to scavenge free radicals and active oxygen species such as singlet oxygen, superoxide anion radical and hydroxyl radicals [14,15]. The antimicrobial activity of flavonoids (compounds 1-7) might be due to their ability to complex with bacteria cell wall and therefore, inhibiting the microbial growth [16]. The presence of these compounds could explain the antioxidant activity found in the crude extract.
Compounds 1, 3-7 displayed both antibacterial and antifungal activities. However, this is the first report concerning the antimicrobial and antioxidant activities of these compounds.
The overall results of this study can be considered as very promising in the perspective of new drugs discovery from plant sources, when considering the medical importance of tested microorganisms. Staphylococcus aureus is a major cause of community and hospital-associated infection with an estimated mortality of around 7-10% [17]. About 77% of immune-deficient patients' death is attributed to microscopic fungi, such as Candida species and Cryptococcus neoformans [18]. Also, Candida albicans has been reported to account for 50-70% cases of invasive candidiasis [19]. Alarmingly, the incidence of nosocomial candidemia has risen sharply in the last decade [20]. All this has resulted in severe consequences including increased cost of medicines and mortality of patients. Typhoid fever caused by Salmonella typhi continues to be a serious public health problem in developing countries in general and in Sub-Saharan Africa in particular [17]. Generally, these pathogens were found to be sensitive to extracts and isolated compounds.

Conclusion
These findings demonstrated that methanol and ethyl acetate extracts of B. pinnatum and the isolated compounds exhibited interesting antimicrobial and antioxidant properties, justifying the traditional uses of the plant in the treatment of infectious diseases and free radical damages. However, further pharmacological and toxicological studies need to be done in order to confirm or infirm this hypothesis.

Plant material
The whole plant of B. pinnatum was collected in Mbouda subdivision, West region of Cameroon in August 2009. It was identified by Mr. Nana at the National Herbarium, Yaoundé where a voucher specimen 33394 HNC describing the plant is deposited.

Extraction and isolation
Dried and ground whole plant of B. pinnatum [1] (1.50 Kg) was extracted by percolation with methanol (10 L) at room temperature. Filtration and evaporation of solvent under reduced pressure gave a brown residue (148 g). This extract was successively partitioned with n-hexane and ethyl acetate to yield 38.03 and 34.03 g of extracts respectively [21]. All extracts were kept in the refrigerator at around 4°C.
The EtOAc extract (34.03 g) was subjected to silica gel column chromatography (6 × 30 cm, 300 g) eluted with a gradient system of hexane-EtOAc-MeOH gradients.    (preliminary analyses with 1% (v/v) DMSO do not inhibit the growth of the test organisms). The negative control well consisted of 195 μl of appropriate medium (MHB for bacteria and SDB for yeasts) and 5 μl of the standard inoculum. The plates were covered with the sterile lid, then agitated to mix the contents of the wells using a plate shaker and incubated at 35°C for 24 h (for bacteria) or for 48 h (for yeasts). The assay was repeated thrice. The MICs of samples were determined by adding 50 μl of a 0.2 mg/ml p-iodonitrotetrazolium violet solution followed by incubation at 35°C for 30 min. Viable micro-organisms reduced the yellow dye to a pink color. MICs were defined as the lowest sample concentrations that prevented this change in color indicating a complete inhibition of microbial growth. For the determination of MMCs, a portion of liquid (5 μl) from each well that showed no growth of microorganism was placed on Mueller Hinton Agar or Sabouraud Dextrose Agar and incubated at 35°C for 24 h (for bacteria) or 35°C for 48 h (for yeasts). The lowest concentrations that yielded no growth after this sub-culturing were taken as the MMCs [22]. Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) and nystatin (Merck, Darmstadt, Germany) for bacteria and yeasts, respectively, were used as positive controls.