The discovery of berberine erythrocyte-hemoglobin self-assembly delivery system: a neglected carrier underlying its pharmacokinetics

Figures & data

Figure 1. The blood concentration-time profile of free BBR. (A) Oral administration of BBR (400 mg/kg), (B) Intravenous administration of BBR (2.5 mg/kg).

Table 1. Pharmacokinetic parameters of BBR after oral and intravenous administration in rats ($\overline{x}$ ± $s_{\overline{x}}$) (n = 8).

Parameters	Oral administration (400 mg/kg)	Intravenous administration (2.5 mg/kg)
AUC0–t (μg/mL*h)	0.681 ± 0.093	6.422 ± 0.685
AUC0–∞ (μg/mL*h)	0.685 ± 0.093	7.438 ± 0.548
MRT (h)	4.525 ± 0.515	5.600 ± 0.561
t1/2z (h)	3.136 ± 0.357	3.885 ± 0.389
Tmax (h)	1.000 ± 0.000	–
Clz/F (L/h/kg)	253.89 ± 10.510	131.430 ± 2.513
Cmax/C0 (μg/mL)	0.260 ± 0.032	2.833 ± 0.132

Figure 2. Effects of BBR on contents of WBC, erythrocyte, PLT and Hb (n = 8). WBC: white blood cell, PLT: platelet, Hb: hemoglobin. *p < .05, **p < .01 vs Normal 5 min; ^#p < .05, ^##p < .01 vs Normal 30 min; ^△p < .05, ^△△p < .01 vs Normal 120 min.

Table 2. C_e/p of BBR determined in rats following intraperitoneal injection at the dose of 2.5 mg/kg.

Time (min)	Rats no.	Ce (μM)	Cp (μM)	Ce/p
5	1	1.313	0.000	∞
	2	0.039	0.489	0.081
	3	0.000	0.000	–
	4	0.375	0.233	1.610
	5	0.862	0.233	3.700
	6	0.584	0.416	1.405
30	1	0.539	0.616	0.875
	2	0.044	0.000	∞
	3	0.000	0.000	∞
	4	0.212	0.000	∞
	5	0.026	0.282	0.090
	6	0.176	0.197	0.897
120	1	0.225	0.000	∞
	2	–	1.243	–
	3	0.000	0.000	∞
	4	0.000	0.000	∞
	5	0.000	0.000	∞
	6	0.132	0.000	∞

C_e: Drug concentration in erythrocytes, C_p: Drug concentration in plasma, C_e/p: Erythrocytes/plasma partition coefficient. ∞: Infinity.

Figure 3. The hemolysis rate of BBR at different concentrations for 2% erythrocytes.

Figure 4. Effect of BBR concentration on 30% erythrocytes at 37 °C for 2 h.

Table 3. Effect of BBR concentration on 30% erythrocytes at 37 °C for 2 h ($\overline{x}$ ± $s_{\overline{x}}$) (n = 3).

The BBR concentration in BBR-erythrocyte system (μg/mL)	Extracellular BBR concentration after incubation (μg/mL)	Loading percentage (%)
185.000	181.843 ± 10.611	29.070 ± 4.141
18.500	2.288 ± 0.105	88.772 ± 0.410
1.850	0.115 ± 0.072	95.528 ± 2.622
0.185	0 ± 0.051	100 ± 19.84

Figure 5. Transmission electron microscopy of BBR-loaded erythrocytes by endocytosis. Control erythrocytes (A–D) were typical biconcavediscs with high electron density, and BBR-loaded erythrocytes (E–H) were close to the circular with several discrete vacuoles. Magnification was ×1,100 and ×10,400, respectively.

Figure 6. Stern-Volmer plots for the quenching of BHB by BBR (A) at different temperatures (corrected). Conditions: BHB: 2 × 10⁻⁶mol/L; pH = 7.4.

Table 4. Stern-Volmer quenching constants for the interaction of BBR with BHB at three different temperatures.

Sample	T (K)	Ksv (×10^4 L mol^−1)	kq (×10^12 L mol^−1 s^−1)	R^a
BBR	293	4.1272	4.1272	0.99808
	298	3.8445	3.8445	0.99670
	310	3.0957	3.0957	0.97000

^aR is the correlation coefficient.

Table 5. The relative thermodynamic parameters of the BBR–BHB systems.

Sample	T (K)	ΔH° (kJ mol^−1)	ΔS° (J mol^−1K^−1)	ΔG° (kJ mol^−1)
BBR	293	−4.7495	72.113	−25.900
	298		72.114	−26.261
	310		71.724	−26.984

Figure 7. Absorption spectral titration of BHB (curve a) with increasing amounts of BBR (curves b–j). Conditions: BHB: 1 × 10⁻⁵mol/L; BBR/(10⁻⁵mol/L): (a) 0, (b) 0.2, (c) 0.4, (d) 0.6, (e) 0.8, (f) 1.0, (g) 1.2, (h) 1.4, (a) 1.6 and (j) 1.8. The concentration of BBR alone is 1.0 × 10⁻⁵mol/L; pH = 7.4 and T = 298 K.

Figure 8. Synchronous fluorescence spectra of BHB with increasing amounts of BBR (curves b–g) (corrected): (A) Δλ = 15 nm; (B) Δλ = 60 nm. Conditions: BHB: 2 × 10⁻⁶mol/L; BBR (×10⁻⁵): (a) 0, (b) 0.25, (c) 0.5, (d) 0.75, (e) 1, (f) 1.25 and (g) 1.5; pH = 7.4 and T = 298 K.

Figure 9. CD spectra of BHB and BHB-BBR systems. Conditions: BHB: 5.0 × 10⁻⁸mol/L; (a) 0, (b) 1 × 10⁻⁷mol/L, (c) 2 × 10⁻⁷mol/L and (d) 4 × 10⁻⁷mol/L; pH = 7.4 and T = 298 K.

Figure 10. Raman spectrum of BHB (a) and BHB-BBR (b) system.

Figure 11. Docking conformations of BBR (A) in complex with BHB. (B) The rmsd value of BBR-BHB complex, (C) The protein-ligand conformation before (color: yellow) and after (color: orange) MD simulation. BBR was displayed in balls and sticks, yellow dotted lines indicated hydrogen bond (PDB ID: 1G09).