Constituent removing and chemical identification

A preparative thin layer chromatography plate (PTLCP) was used to achieve constituent removal. Each band on the plate corresponded to one constituent and was regarded as a positive sample (namely targeted constituent marked by letter M+). The rest of bands was defined as the corresponding negative sample (marked by letter M−).

Next, we used Ultra-performance liquid chromatography (UPLC) and quadrupole time-of-flight mass spectrometry (Q-TOF MS) to identify the constituent of each band and to determine whether there were residual compounds in the corresponding negative sample. Using this method, the constituents in the positive samples were preliminarily identified (Supplementary Fig. S4) as berberine (BER), palmatine (PAL), coptisine (COP), epiberberine (EPI), jateorrhizine (JAT) and columbamine (COL), respectively. The levels of the removed constituents in the negative samples were nearly undetectable (Supplementary Fig. S4). Furthermore, the molecular structures of the removed constituents were not damaged by the removal procedure (Supplementary Fig. S5).

Identification of the active constituents

The growth and metabolism of living organisms are accompanied by heat/energy production, which can be affected by pathological changes or the action of drugs. Therefore, it is possible to evaluate changes in microbial heat production in the presence or absence of different drugs using microcalorimetry. Accordingly, we determined the bioactivity of R. coptidis extract, removed samples and added samples in terms of bacteriostasis.

The normal growth thermogenic curve for S. dysenteriae at 37°C is shown in Fig. 2a. The heat flow power-time (HFP-t) curve showed that the S. dysenteriae metabolic profile included two main stages (stages 1 and 2) and five phases, (lag phase [a–b], the first exponential growth phase [b–c], transition phase [c–d], the second exponential growth phase [d–e] and the decline phase [e–f]). The quantitative thermokinetic parameters of the HFP-t curve for S. dysenteriae growth could be delineated using the equation (1):

where P 0 and P t represent the heat flow power at time 0 or time (min), respectively. To test the reliability of the microcalorimetry, we repeated the experiment on eight occasions in untreated bacteria and obtained good reproducibility. We then quantified the following thermokinetic parameters from the HFP-t curves in the presence of difference concentrations of the samples: p 1 , p 2 , t 1 and t 2 (Table 1). PCA revealed that k 2 and t 2 explained 87% of the variation of samples, including the R. coptidis extract, removed samples and added samples. Therefore, we focused on parameters k 2 and t 2 in this study.

Table 1 Thermokinetic characteristics of R. coptidis (200 μg/mL) and samples containing different concentrations of BER on S. dysenteriae growth at 37°C (n = 3, Mean ± SD) Full size table

Figure 2 Results of identification of the active constituents. (a) Heat flow power-time (HFP-t) curve for control S. dysenteriae cultured in L.B. culture medium alone. (b, c) Effects of the R. coptidis extract, removed constituents and negative samples on HFP-t curves of S. dysenteriae growth. (d) Contributions of the removed constituents and their corresponding negative samples to the bacteriostatic activity of R. coptidis. (b–d) Control: S. dysenteriae alone; reference: R. coptidis extract (0.8 mg/mL); removed samples: BER+, COP+, EPI+, PAL+ and (JAT + COL)+; negative samples: R. coptidis extract lacking COP (COP−), EPI (EPI−), PAL (PAL−) and JAT + COL combined (JAT + COL)−. The measurements of relative inhibition ratio were performed in triplicate and error bars represent standard error of the mean. P value compared to R. coptidis extract determined by two-way ANOVA. Full size image

Figures 2b, c illustrate the effects of R. coptidis (0.8 mg/mL) extract, removed constituents and corresponding negative samples on the HFP-t curves of S. dysenteriae. We found that, compared with the control and the R. coptidis extract, the kinetics of the removed and negative samples showed marked variation (Supplementary Table S1).

Next, we calculated the inhibition ratio I as equation (2) and the relative inhibition ratio RI as equation (3) to quantify the contributions of the individual constituent to the bacteriostatic activity of R. coptidis extract. As shown in Fig. 2d, BER, COP, EPI, PAL and JAT + COL removed inhibited the growth and metabolism of S. dysenteriae; of which BER and COP had the greatest effects. The bacteriostatic activities of BER− and COP− were significantly lower than that of R. coptidis extract (P < 0.01). Furthermore, the bacteriostatic activities of EPI−, PAL− and (JAT + COL)− were not significantly different to that of R. coptidis extract. Thus, BER and COP appear to be the main bacteriostatic constituents of R. coptidis extract with contributions to the bacteriostatic activity of 54.10% and 39.75%, respectively (Fig. 2d).

where K 2c is the growth rate constant of the second exponential growth phase of S. dysenteriae in the culture medium alone; k 2s is the growth rate constant of the second exponential growth phase of S. dysenteriae exposed to the test samples; I s is the inhibition ratio of samples under evaluation; I e is the inhibition ratio of R. coptidis extract (reference); and RI is the relative inhibition ratio.

Constituent adding and chemical identification

After confirming that BER and COP were the main bioactive constituents of R. coptidis, we added BER to its negative sample (BER−) to final concentrations of 0, 15, 45, 60, 80 and 120 μg/mL, which was equivalent to the relative content of BER in R. coptidis extract was (14.52%, based on the no-show detection process). HPLC was used to confirm the chemical compositions of the added samples. We also prepared added samples of COP at concentrations of 0, 8, 16, 32, 64 and 128 μg/mL, similar to the COP content in R. coptidis extract (5.3%, based on the no-show detection method). The HPLC profiles are shown in Fig. 3a, b and Supplementary Fig. S6.

Figure 3 HPLC chromatograms following the adding of BER or COP and results of the bioassays. (a) HPLC profiles following BER added to the negative sample lacking constituent BER to final concentrations of 0, 15, 45, 60, 80 and 120 μg/mL. (b) HPLC profiles following COP added to the negative sample lacking constituent COP to final concentrations of 0, 8, 16, 32, 64 and 128 μg/mL. (c, d) heat flow power-time (HFP-t) curves of S. dysenteriae at 37°C exposed to R. coptidis extract or samples containing different concentrations of BER (c) or COP (d). S. dysenteriae in culture medium alone was used as the blank control. The concentration of R. coptidis extract was 200 μg/mL. The final concentrations of BER were 0, 15, 45, 60, 80 and 120 μg/mL. The final concentrations of COP were 0, 8, 16, 32, 64 and 128 μg/mL. The negative samples (BER− and COP−) were prepared by removing BER or COP from the R. coptidis extract. Full size image

Levels of the active constituents

The bacteriostatic activity of samples adding BER or COP was assessed by microcalorimetry (Fig. 3c,d) and I and RI were calculated (Tables 1 and 2). The relationship between RI and BER or COP concentrations are shown in Fig. 4. To determine the correlation between the concentrations of active constituents in added samples and I, we calculated the change in I induced by the each concentration of active constituents (P) using equation (4):

where I n is the inhibition ratio of the negative sample lacking the targeted constituent; I i is the inhibition ratio of the negative sample following the targeted constituent added; and W is the corresponding total concentration of the targeted constituent causing I i .

Table 2 Thermokinetic characteristics of R. coptidis (200 μg/mL) and samples containing different concentrations of COP on S. dysenteriae growth at 37°C (n = 3, Mean ± SD) Full size table

Figure 4 Relationship between the concentration of added BER/COP and I and the potency of BER/COP on inhibiting the growth of S. dysenteriae at 37°C. The measurements of potency per unit weight and inhibition ratio were performed in triplicate and error bars represent standard error of the mean. Full size image

As illustrated in Fig. 4, the relative potencies of COP (i.e., P COP ) and BER (i.e., P BER ) were similar (red curves in Fig. 4). For both constituents, their relative potencies increased with increasing weight. However, the potency started to decrease when the weight passed a threshold level. Therefore, the peak values in the P COP and P BER curves (e and f in Fig. 4) are likely to show the greatest potencies. Therefore, the concentrations of COP and BER that showed the greatest efficiency were 32 μg/mL and 80 μg/mL, respectively. We calculated that the greatest efficiency of COP and BER is at the relative concentrations 14.45% and 31.92%, respectively.