AchE Inhibition by AHCs
The AchE inhibitory effect of the tested 4 AHCs was determined as shown in Figure 1 from which the respective IC50 values were obtained as summarized in Table 2. Berberine hemisulfate exhibited the strongest inhibition with an IC50 value (12.16 μmol/L) an order of magnitude lower than that of matrine (262.41 μmol/L) or osthole (233.21 μmol/L). No toxicity for trigonelline chloride was detected in the tested range.
It was worth to mention that osthole was dispersed in the form of emulsion due to it is slightly soluble in water. When testing a suspension of osthole in water instead of emulsion, at most 24% AchE activity loss was obtained at a content of 30 μmol/L. The activity loss was maintained at about 20% within a wide range of osthole content in aqueous suspension up to 244 μmol/L (Figure S1). This inhibition limit probably corresponds to the dissolution ratio of osthole in water, and only those dissolved part non-productively binds with AchE.
The binding of AHCs in AchE was simulated using Autodock and the results were shown in Figure 2. Berberine, matrine and osthole preferentially bound with AchE at the catalytic channel which is also anchored by the regular substrate, acetylcholine. The binding energies were -9.11, -9.41 and -7.99 kcal/mol, respectively (Table 2). The absolute values were much higher than that of acetylcholine (-4.75 kcal/mol), suggesting that the three AHCs formed more stable complex conformations with AchE than acetylcholine. Trigonelline was significantly different from them. It took up a favorite binding site away from the catalytic channel, suggesting there is no AchE inhibition effect of trigonelline. This was in accordance with the data as illustrated in Figure 1.
The amino acids interacted with AHC ligands were summarized in Table 2. Trp84, Phe330 and His440 were identical binding sites for both substrate (acetylcholine) and inhibitor (matrine, berberine or osthole). In previous papers, amino acids Trp84, Tyr130, Phe330 and Phe331 are responsible for the molecular recognition of quaternary ammonium ligands and also the catalytic anionic sites (Lee et al. 2015; Takomthong et al. 2021). Therefore, the three AHCs probably inhibit AchE through competitively occupation of the active catalytic channel. Berberine and matrine were stronger inhibitors than osthole probably due to that their higher binding energies (Table 2). Trigonelline preferably anchored the hydrophilic valley at the N-terminal of AchE, showing no competition with acetylcholine and thus no inhibition. It can be further deduced that trigonelline is probably not the active component in Ttigonella foenum graecum seed which has been considered as a kind of antiscolic herb.
Detoxicification of AHCs by Bacillus
B. subtilis Ruizhen@ was added into the AchE inhibition test system together with berberine, matrine and osthole at their respective IC50 concentration. The total live cell number of Ruizhen@ was 1×106 CFU/mL. These bacteria at this content level did not show any impact on the AchE activity (Figure 3a). After incubation at 30°C for 2 h, both berberine and matrine caused half loss of the AchE activity in presence of Ruizhen@ (Figure 3a). Interestingly, in the test of osthole incubated with Ruizhen@ at 106 CFU/mL level, the AchE activity loss was decreased to 66.57%. The results clearly indicate a detoxification of osthole with the addition of Ruizhen@.
At a higher content of live cells, i.e. 107 and 108 CFU/mL, B. subtilis Ruizhen@ independently depressed AchE activity by about 15% and 22%, respectively. These inhibition data were much lower than the alarming line (50% loss) and were considered to be safe in general (Printes et al. 2004). After incubating Ruizhen@ and the three AHCs at 30°C for 2 h, only berberine kept the same toxicity level to AchE. The measured AchE loss by combination of Ruizhen@ and berberine was almost exact half of the value of reference. In another word, there was no detoxification effect on berberine by incubation with bacillus Ruizhen@.
However, the toxicities of matrine and osthole as represented by AchE inhibition were obviously weakened after incubated with Ruizhen@ (107 or 108 CFU/mL). Their retained AchE activities were all above the expected half-loss line as shown in Figure 3a. The antagonistic effect suggests that Ruizhen@ is not suitable for mixed use together with biopesticides containing matrine and osthole and can somehow detoxify these two biopesticides residues.
Detoxification effects were also found when incubating AHCs with other bacilli species (Figure 3b). Such effect on osthole was more obvious than on matrine. At the IC50 dosages, the addition of Bacillus subtilis (107 CFU/mL) or several other bacilli species detoxified osthole from 50% AchE inhibition to around 30%. There was still no obvious change found in the case of berberine. On the other hand, P. polymyxa gave stronger AchE toxicity than B. subtilis. Mixing these two probiotics (107 CFU/mL) with berberine (IC50 concentration) resulted in remarkable AchE inhibition degree up to 67%.
Growth Inhibition of Bacilli by AHCs
The antimicrobial activity of AHCs were tested with B. stibilis Ruizhen@ and the results were demonstrated in Figures 4 and 5. Separate green round colonies occurred after 24-h incubation of diluted cell suspensions of Ruizhen@ spread on CBCA plates. Numbers of colonies were decreased due to the addition of AHCs. There was a narrow tolerance range for Ruizhen@ to berberine (0~0.012 mmol/L), matrine (0~4.2 mmol/L) and osthole (0~0.53 mmol/L). Within the respective tolerant range, the loss of living cells was less than 15%. Berberine, matrine and osthole completely inhibited the growth of B. stibilis (4.2×107 CFU/mL) at a concentration of 0.43, 3.37 and 9.22 mmol/L, respectively.
Compared with Ruizhen@, the other two B. stibilis strains, BHQX and Y1336, showed much lower resistance to berberine, matrine and osthole. They were completely inhibited by berberine at 0.1 mmol/L and by matrine at 2.25 mmol/L. Osthole was also very toxic to them, inhibited 94.4% and 82.2% of live cells at a content of 0.5 mmol/L. As a comparison, no significant inhibition occurred in the test of Ruizhen@. This indicates the importance of training bacterium strains to be tolerant to certain AHCs before applying them to herbal plants.
Unlike B. stibilis, the other three bacilli species generally showed higher tolerance when exposed to berberine, matrine and osthole, following the order: B. mucilaginosus > B. laterosporus ~ P. polymyxa (Table 3). The only exception is that B. mucilaginosus was more sensitive to matrine and osthole than B. stibilis Ruizhen@. Among all the tested strains, B. laterosporus had the highest tolerance to AHCs. Little inhibitory effect was detected in the presence of herbal compounds within the tested concentration ranges.
Results also suggests the importance of training probiotic microbes that tolorate the anti-microbial activity of AHCs if they are used for detoxification purpose. The studied three AHCs could completely inhibited the growth of B. subtilis following an order of berberine (0.15 mmol/L) > Matrine (9 mmol/L) > osthole (1.5 mmol/L). In lit-erature, the potential of berberine as an antimicrobial agent against mixed microorganisms containing E. coli and B. subtilis has been reported by Kong et al. (2012). They found a low concentration (20 μg/mL) of berberine began to inhibit the growth of E. coli and mixed microorganisms, while promoting the growth of B. subtilis (Kong et al. 2012). Deep mechanism study revealed that berberine can inhibit not only B. subtilis cell growth, but also spore outgrowth, including inhibiting protein synthesis during this period (Wang et al. 2015). The presence of L-valine prevented the effect of berberine (200 μg/mL) on B.subtilis spore germination (Wang et al. 2015). Impressively, our data in Table 3 showed berberine blocked the growth of B.subtilis strains but more compatible with B.mucilaginosus, B.laterosporus and P. polymyxa. In another word, B.mucilaginosus and B.laterosporus were less sensitive to berberine (0.1 mmol/L) than B. subtilis strains. There might be metabolites by these two species that play a role in detoxification of berberine. The combination of P. polymyxa and berberine could be further explored through insecticide for developing conjugated bio-insectsides.
Antimicrobial properties of matrine are associated with glycerophospholipid metabolism and the sphingolipid metabolic signaling pathways (Huang et al. 2016). Previous authors reported matrine enhanced the pathogenicity of Beauveria brongniartii against agricultural insect pests (Wu et al. 2019). Osthole kill both Gram positive and negative bacteria by inhibiting the absorption of Ca2+, which affects the growth and spore germination of the bacteria and inhibits chitin deposition of the bacterial cell wall (Sun et al. 2021). Supplemental of soluble calcium salts in decomposition of osthole-containing herbal residues by Bacillus may help preserve the living cells.