Anti-bacterial effect of phytoconstituents isolated from Alimatis rhizoma

Five compounds including three triterpenoids and two sesquiterpenes were isolated from Alimatis rhizoma. Their chemical structures were determined to be alisol B 23-acetate (1), alisol C 23-acetate (2), alisol B (3), alismol (4) and alismoxide (5) by various spectroscopic analysis, including 1H-NMR, 13C-NMR, HMBC and MS spectra. Compounds 1–5 were evaluated for their antibacterial potential against 6 strains of bacteria including three drug-resistant bacteria (one methicillin-resistant Staphylococcus aureus strain CCARM 3506, two quinolone-resistant Staphylococcus aureus strains CCARM 3505 and CCARM 3519), two G+ bacteria (Streptococcus mutans KCTC 3289 and Staphylococcus aureus KCTC 209) and one G− bacterium (Escherichia coli KCTC 1924). Compounds 1–5 showed strong antibacterial effect against S. mutans KCTC 3289, their MIC values were 2, 64, 16, 32 and 32 μg/mL, respectively. The antibacterial activity results of compounds 1–5 against these bacteria were reported for the first time. The results indicate that Alimatis rhizoma are potential sources of new antibacterial material.


Introduction
Since the advent of antibiotics, it has cured countless infected patients around the world, but with the passage of time and people's abuse of antibiotics, it has led to the emergence of drug-resistant strains. The original strong antibacterial effect is getting worse, even some antibiotics have been loss of function which seriously threatens human life [1]. Today the drug resistance crisis is becoming more and more serious, and has became one of the biggest threats facing the global society. If the trend of bacterial resistance is not effectively controlled, human will fall into the crisis of no drugs available, so it is urgent to separate active compounds with good antibacterial activity from natural medicines.
In this research, we demonstrated the isolation and structural characterization of three protostane-type triterpenoids (1-3) and two guaiane-type sesquiterpenes (4, 5) from the methanolic extract of A. rhizoma. All compounds were explored for antibacterial activity test.
A voucher specimen (YBU-R012) was stored at the Medicinal Chemistry of Natural Products Laboratory, YanBian University, YanJi, China.
Alisol B 23-acetate (1) (KCTC 1924). The bacteria were cultured in Mueller-Hinton Broth (MHB) and grown to mid-log phase, and then diluted 1000 times in the medium under the same environment. Under aseptic conditions, the cells were inoculated into MHB broth, and then evenly distributed into 96-well microtiter plates for further cultivation. The antibacterial activity of the compounds was tested by serial dilution to determine the minimum inhibitory concentration (MICs). A microtiter ELISA reader was used to measure the absorbance at 650 nm to determine bacterial growth.
Compound 2 showed very similar 13 C-NMR spectrum with that of 1, except for one carbonyl carbon at δ c 208.1 (C-16) in place of one methylene carbon. In comparison with chemical shifts of C-13, C-14 and C-15, downfield shift at C-13 (δ c 177.2) and C-15 (δ c 45.7), upfield shift at C-14 (δ c 49.8) were observed in 2. Compound 2 was determined to be alisol C 23-acetate by comparison with previously reported data [14].
Compound 3 showed very similar NMR signals with that of 1, except that acetyl group of C-23 was converted to hydroxyl group in 3. By comparison with previously reported data, 3 was identified as alisol B [14].
Compound 4, colorless oil. The molecular formula was determined to be C 15  . From the NMR spectroscopic data, suggested that 4 is bicyclic sesquiterpenes, which is derivative of pseudoguaiane-type sesquiterpene. Thus, the structure of 4 was identified as alismol [15].
The molecular weight of 5 was determined to be 238 from the molecular ion peak at m/z 220 [M-H 2 O] + in EI-MS, indicating that the structure of 5 had one more hydroxyl group than 4. NMR signals of 5 were similar to those of 4, with the exception of the signals from exomethylene double bond of 4 converted into methyl group (δ H 1.27, δ c 21.3) and tertiary alcohol group (δ c 75.3) in 5. Consequently, compound 5 was determined as alismoxide [15].
mL. This observation suggested that the absence of acetyl group at C-23 or the presence of ketone group at C-16 in protostane-type resulted in the loss of antibacterial activity against S. mutans 3289. In addition, compounds 4 and 5 exhibited the same antibacterial activity with MICs of 32 µg/mL, indicating that the presence of exomethylene group at C-10 of guaiane-type sesquiterpene had not effect on antibacterial activity. Moreover, the minimum bacterial concentration (MBC) values of five compounds were lower than 4 × MIC (Table 3).
In conclusion, five compounds including three triterpenoids and two sesquiterpenes were isolated from A. rhizoma. All compounds showed strong antibacterial activities against S. mutans 3289 with MIC values of 2-64 µg/mL. Among them, compound 1 had a good potential for use antibacterial agents. In addition, from the structure effective relationship, it found that acetyl group at C-23 and/or ketone group at C-16 of protostane-type is probably active group. It is the first report on antibacterial activity of all isolated compounds against six bacteria strains. Taken together, these results could provide potential sources of antibacterial compounds for A. rhizoma.