Variation in contents of active components and antibacterial activity in different parts of Lonicera japonica Thunb

Of note, 2 g powder of different parts of L. japonica Thunb was refluxed three times at 80°C using 100 mL of 80% aqueous methanol (v/v) for 3 h. The extracting solution was merged for vacuum rotatory evaporation at 35°C and completely dried in an atmospheric oven. Then the crude extract solutions were obtained and diluted with methanol to 50 mL. All extracts were stored in refrigerator and filtered before analysis.

Total flavonoids were determined according to the methods in Pharmacopoeia of the People's Republic of China, using rutin as the standard [25]. In a 10-mL Eppendorf tube, 0.2 mL of extract, 0.5 mL of 5% NaNO₂, 0.5 mL of 10% AlCl₃, 4.0 mL 4% NaOH, and 4.8 mL distilled water were added sequentially every 5 min. The absorbance was measured at 510 nm. Accuracy was evaluated by analyzing the standard solution six times. Then, the relative standard deviation (RSD) of the absorbance of the standard compounds was calculated. To confirm the repeatability, five different sample solutions prepared from the same sample were analyzed and RSD was used to indicate the change. For stability studies, each of the above sample solutions was analyzed at 10, 20, 30, 40, 50, and 60 min, respectively. Recovery test was used to evaluate the accuracy of the method. The ratio of detection quantity (actual) to scalar quantity (theoretical) was used to calculate the recovery rate.

One gram of powder was immersed with ether for 12 h to obtain the crude extract solutions. Then the extracts were stored at 4°C until use.

Using helium as a carrier gas, the components were separated at a constant flow rate of 1.0 mL/min for 45 min. The 5 μL sample extract was injected. The initial temperature was set at 100°C, whereas the injector temperature was set at 280°C. Throughout the process, temperature flow was set at the speed of increasing 10°C/min.

The mass ionization was carried out using electron ionization mode at +70 eV and acquired m/z from 30 to 650.

The samples prepared as 2.3 were filtered through a Millipore filter (0.45 μm) before analysis. The nine analytes were measured according to our previous work [31]. A mixture of solvent A (0.5% aqueous acetic acid; v/v) and solvent B (acetonitrile) was used as the mobile phase at a flow rate of 1.0 mL/min. The gradient elution program was 0–10 min, 12–20% B; 10–20 min, 20% B; and 20–40 min, 20–30% B. The column temperature was kept at 25°C; the detection wavelength was 327 nm; and the injection volume was 10 μL.

The antimicrobial activity was tested by agar well diffusion method against both gram-positive and gram-negative bacteria. Each strain was grown in sterile nutrient broth at 37°C for 24 h and stored at 4°C before use. Twenty milliliters of sterilized agar medium were poured into each petri dish. Four holes were drilled in the agar medium orderly of each dish after medium was solidified. Plant extracts (50 μL) dissolved in sterile water at 1 g/mL concentration were added in the holes, and the same volume (50 μL) of sterile water was used as a negative control. Approximately 50 μL of suspension (10⁶ cfu/mL) of the test microorganisms was smeared uniformly onto the medium. The inoculated plates were incubated for 24 h at 37°C. Each sample was assayed in triplicate (1 disk/plate). After incubation, the diameters (mm) of the inhibition zone were measured.

The total flavonoids concentration was calculated from a calibration curve using rutin as the standard. A series of rutin standard solutions were prepared in methanol in the range of 10–70 μg/mL to obtain the linear response (Y = 0.00329 + 0.0122X, r = 0.9995, where Y is the absorbance at 510 nm and X is the concentration of total flavonoids [μg/mL]). The precision, repeatability, and stability were presented as RSD of 1.42, 2.13, and 1.79%, respectively. The recovery of the total flavonoids was 98.32%. All data demonstrated that the established method is accurate enough for the determination of total flavonoids in L. japonica Thunb LE, GB, and FL.

Qualitative evaluations were performed on L. japonica Thunb samples of LE, GB, and FL, which are shown in Table 1. The contents of total flavonoids in LE, GB, and FL were 6.68, 7.38, and 6.35%, respectively. It can be seen that the content of total flavonoids in LE of L. japonica Thunb is lower than that in GB; however, it is comparable with that in FL, which is consistent with the reports in literatures [28].

From the above results, it can be seen that there was difference in total flavonoids in different part of L. japonica Thunb. However, it is not clear which flavonoid or component plays the determinative role. Therefore, nine analytes that commonly appeared in this plant were used for further comparison. The nine bioactive components including chlorogenic acid, neochlorogenic acid, isochlorogenic acid C, caffeic acid, luteolin, galuteolin, hyperoside, rutin, and quercetin were determined in different parts of L. japonica Thunb. The typical chromatography of standards and leaf sample are shown in Figure 1. Their contents are summarized in Table 2. First, the content of isochlorogenic acid C was much higher than other components in each part of L. japonica Thunb. Furthermore, the results indicated that the contents of galuteolin, luteolin, rutin, and isochlorogenic acid C were obviously higher in LE than those in FL and GB, and neochlorogenic acid content was similar in LE and FL. The contents of caffeic acid and chlorogenic acid detected in GB were the highest. Quercetin was not detected in all the samples even with sample concentration or using different sample disposal method. It can be concluded that quercetin does not exist in this kind of plant that we investigated. Interestingly, hyperoside was not detected in FL, GB, and LE which were collected from April to September. However, it can be found in other 6 months. It may relate to the temperature or the plant metabolism. The specific reason remains unknown at present and needs further research.

Figure 1

The volatile components in different sections of L. japonica Thunb were determined by GC-MS. Their content percentage is expressed by the area normalization method of total ion current (Figure 2). As shown in Table 3, the contents of 45 volatile components in samples were listed in details. There were 25 types of compounds detected in GB, compared with 21 in LE and 20 in FL. The results showed that there were no significant differences of the main compositions among FL, LE, and GB, and most of the contents were hydrocarbons with 66.07% in LE, 70.75% in FL, and 67.94% in GB, respectively. Apart from hydrocarbons, the content of terpenoids was the highest in LE (15.36%), which was higher than that of GB (10.69%). Interestingly, no terpenoid was found in FL. In addition, the content of (3β)-9,19-cyclolanost-24-en-3-ol was the highest in LE (13.51%), and 17 and 26 methyl carbonate were found only in FL and GB.

Figure 2

SA belongs to gram-positive bacteria and is one of the most common pathogens. It can cause systemic infection such as sepsis, pneumonia, and pseudomembranous enteritis, and it is commonly found on the surface of skin and upper respiratory tract mucosa. EC is a gram-negative bacterium, which can cause neonatal meningitis, urinary tract and intestinal tract infection, and other diseases, and it is commonly found in human and animal intestines. Based on these points, SA and EC were selected as the tested strains to study the antibacterial effect of different parts of honeysuckle. The bacteriostatic results of inhibition zone are shown in Figure 3 and Table 4. As can be seen, all the samples had antibacterial effect on SA and ESBL-SA. However, there was no obvious effect on EC and ESBL-EC. The cell wall of gram-positive bacteria mainly composed of the peptidoglycan, and flavonoids and organic acids in L. japonica Thunb can solubilize peptidoglycan components which can dissolve the cell wall. The cell wall of gram-negative bacteria is mainly composed of the lipid composition, and the antibacterial ingredient in L. japonica Thunb is difficult to dissolve, thus the inhibitory effect on gram-negative-bacteria is relatively poor [32]. Results showed that the LE had better inhibitory effect on SA and ESBL-SA (bacteriostatic ring was 13 and 14 mm, respectively) than GB (bacteriostatic ring was 12 and 11 mm, respectively) and FL (bacteriostatic ring was 11 and 13 mm, respectively) at the same picking period. From Table 2, the results indicated that the contents of galuteolin, luteolin, rutin, and isochlorogenic acid C were obviously higher in LE than those in FL and GB. Therefore, the extract of LE more easily dissolves the cell wall of SA and ESBL-SA. As a result, L. japonica Thunb LE had the best antibacterial activity that was even better than FL and GB.

Figure 3