New 14-Membered Cyclopeptide Alkaloids from Zizyphus oxyphylla Edgew

Two new 14-membered cyclopeptide alkaloids, Oxyphylline B (4) and Oxyphylline C (5), along with three known 13-membered cyclopeptide alkaloids, were isolated from stem and roots of Zizyphus oxyphylla Edgew. The compounds were tested for antibacterial activity. Oxyphylline B (4) showed comparatively better antibacterial activities against Escherichia coli (MIC, 5 μg/mL) than other compounds. This compound also exhibited weak antimicrobial activities against Staphylococcus aureus (MIC, 25 μg/mL), Pseudomonas aeruginosa (MIC, 50 μg/mL) and Salmonella typhi (MIC, 50 μg/mL).


Introduction
Zizyphus is a genus of about 40 species of spiny shrubs and small trees in the family Rhamnaceae, distributed in warm-temperate and subtropical regions throughout the world. Zizyphus oxyphylla Edgew is a small or medium sized tree growing in northern areas of Pakistan. It is used in these areas as a folk medicinal remedy in the treatment of inflammatory conditions, pain, especially of rheumatic origin, fever, microbial infections, allergy and diabetes [1]. Zizyphus oxyphylla has been shown to possess analgesic and antipyretic activities in rats and mice [1]. The analgesic and antipyretic activities of some other species of the genus Zizyphus have also been reported [1,2]. Cyclopeptide alkaloids are a large group of compounds, which are present in a large number of plant families. Some of the representative families are Asteraceae, Celastraceae, Euphorbiaceae, Menispermaceae, Pandaceae, Rubiaceae, Sterculiaceae and Urticaceae along with Rhamnaceae. These compounds may be defined as basic compounds having a structure 10 or 12 member peptide type bridge span that is attached to benzene at 1,3 or 1,4 positions. These alkaloids have been reported to have antibacterial, antifungal and sedative activity [3].
Plant-derived medicines have been a part of traditional healthcare in most parts of the world for thousands of years and there is increasing interest in plants as the source of agents to fight microbial diseases [4]. As part of our continuous studies on Zizyphus oxyphylla [2,[5][6][7], to authenticate its traditional usage, in this paper we report the isolation, the structure elucidation and the antibacterial activity of two new and three known cyclopeptide alkaloids from Z. oxyphylla.

Results and Discussion
Chloroform fraction of the methanolic extract of Z. oxyphylla stem was subjected to multiple chromatographic steps involving column chromatography and preparative TLC. This process yielded two new cyclopeptide alkaloids (3,4,5) and from the dichloromethane fraction of root, two cyclopeptide alkaloids (1 and 2) were isolated as white amorphous powders ( Figure 1). Structures of the known compounds (1-3) were elucidated by comparing their analytical data with literature values [6,[8][9][10][11].

General Procedures
UV spectra were recorded in MeOH on a Shimadzu UV-240 spectrometer. IR spectra were obtained on a JASCO A-302 spectrophotometer as KBr discs. 1 H and 13 C NMR (500 MHz and 125 MHz) were recorded in C 3 D 6 O on a Bruker Av 500 NMR instrument, with TMS as internal standard. HRESI-MS spectra were recorded on QSTAR XL LC MS MS applied bio systems spectrometer. Column chromatography was conducted on silica gel (Kiesegel 60; 70-230 mesh) and TLC was carried out by using pre-coated silica-gel F254 aluminum sheets (0.25 mm thickness). Compounds were detected by spraying with Dragendorff reagent.

Antibacterial Activity
All compounds were screened for antibacterial activity against Escherchia coli (ATCC 25922), Bacillus subtilis (ATCC 6633), Shigella flexeneri (clinical isolate), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Salmonella typhi (ATCC 19430), according to the method as reported previously [13][14][15]. The disc diffusion technique was used to determine the antibacterial activity of the test compounds. The compounds were dissolved in DMSO to obtain a 10 mg/mL solution. A known volume (10 mL) of the solution was applied on to the sterilized filter paper discs with the help of a micropipette. The discs were dried at room temperature overnight and stored in sterile dry containers. Discs, soaked with 10 mL of DMSO, and air dried at room temperature, were used as negative control. Bacterial cultures were grown in a nutrient broth medium at 37 °C overnight and spread on to solidified nutrient agar medium in Petri plates using sterilized cotton swabs in a standard microbiological working environment. Test and control discs were then applied to the solidified medium surface with the help of sterilized forceps. The plates were incubated at 37 °C for 12-15 h. The results were recorded by measuring the zone of inhibition in mm against each compound. Impenem was used as standard drug. MIC was calculated as previously reported [13][14][15]. Compounds were briefly dissolved in DMSO and serially diluted with sterile water in microplates in a laminar flow cabinet. The same volume of an actively rowing culture of the test bacteria was added to the different wells and cultures were grown overnight in 100% relative humidity at 37 °C. The next morning, tetrazolium violet was added to all the wells. Growth was indicated by a violet color of the culture. The lowest concentration of the test solution that led to an inhibition of growth was taken as the MIC. The negative control DMSO had no influence on the growth at the highest concentration used. Impenem was used as controls for comparison (See Table 1).

Conclusions
Overuse of antibiotics has become the major factor for the emergence and dissemination of multi-drug resistant strains of several groups of microorganisms. Thus, there is urgent need to develop new antimicrobial agents that are very effective with minimal unwanted side effects. As higher plants have proved to be a source of lead compounds against various infectious diseases, therefore, five cyclopeptide alkaloids, including 2 new cyclopeptide alkaloids isolated from Z. oxyphylla, were screened for antibacterial activity. Results indicated weak antimicrobial potential of all isolated cyclopeptide alkaloids.