Protective effects of Scutellariae Radix Carbonisata-derived carbon dots on blood-heat and hemorrhage rats

Abstract

As the charcoal processing product of Scutellariae Radix (SR), SR Carbonisata (SRC) has been clinically used as a cooling blood and hemostatic agent for thousands of years. However, the underlying active ingredients and mechanism of SRC still remained unspecified. In this study, SRC derived carbon dots (SRC-CDs) were extracted and purified from the aqueous solution of SRC, followed by physicochemical property assessment by series of technologies. The cooling blood and hemostatic effects of SRC-CDs were further evaluated via a blood-heat and hemorrhage (BHH) rat model. Results showed that the diameters of obtained fluorescent SRC-CDs ranged from 5.0 nm to 10.0 nm and possessed functional group-rich surfaces. Additionally, the as-prepared SRC-CDs showed remarkable cooling blood and hemostasis effects in BHH model, mainly manifested by significant improvement of elevated rectal temperature, inflammatory cytokines (TNF-α, IL-6, and IL-1β) levels, as well as protein expressions of myD88 and NF-κB p65, abnormal coagulation parameters (elevated APTT and FIB), hemogram parameters (RBC, HGB, and HCT), and histopathological changes in lung and gastric tissues. This study, for the first time, demonstrated that SRC-CDs were the cooling blood and hemostatic active components of SRC, which could inhibit the release of inflammatory cytokines by regulating myD88/NF-κB signaling pathway, and activating the fibrin system and endogenous coagulation pathway. These results not only provide a new perspective for the study of active ingredients of carbonized herbs represented by SRC, but also lay an experimental foundation for the development of next-generation nanomedicines.

Keywords: Scutellariae Radix Carbonisata-derived carbon dots (SRC-CDs); active ingredients; anti-inflammation; cooling blood and hemostatic effects; hemostasis.

FIGURE 1

(A) Flow diagram of preparing process of Scutellariae Radix Carbonisata—derived carbon dots (SRC-CDs) and the solution outside the dialysis membrane of SRC (SRC-OD). (B) HPLC fingerprint of SRC and SRC-CDs. (C) Tail bleeding time of mice (n = 6 per group) treated with normal saline (NS), Yunnan Baiyao (positive), Scutellariae Radix (SR), SRC, SRC-CDs and SRC-OD. Data are represented as mean ± SD. *p < 0.05 vs. NS group or SR group and **p < 0.01 vs. NS group.

FIGURE 2

The morphology characterization of Scutellariae Radix Carbonisata—derived carbon dots (SRC-CDs). (A,B) High-resolution transmission electron microscopy (HRTEM) images. Inset: magnification figure. (C) Histogram depicting particle size distribution. (D) Line profiles of the corresponding HRTEM images and (E) X-ray diffraction pattern (XRD) of SRC-CDs.

FIGURE 3

The optical characterization of Scutellariae Radix Carbonisata—derived carbon dots (SRC-CDs). (A) Ultraviolet-visible spectrum (UV-Vis), (B) fluorescence spectrum and (C) Fourier transform infrared spectrum (FTIR).

FIGURE 4

The X-ray photoelectron spectroscopy (XPS) spectra of Scutellariae Radix Carbonisata—derived carbon dots (SRC-CDs). (A) Full survey spectrum. (B) C 1s spectrum, (C) O 1s spectrum and (D) N 1s spectrum.

FIGURE 5

Histopathology of (A) lung tissues and (B) gastric tissues (×100). Sprague Dawley rats were assigned into six groups (n = 10): control (NS), model, positive and Scutellariae Radix Carbonisata—derived carbon dots (SRC-CDs) at high- (25.00 mg/kg), medium- (12.50 mg/kg) and low (6.25 mg/kg) doses groups. The arrows represents local bleeding point, while “△(red)” represents inflammatory cells infiltration.

FIGURE 6

Effects of Scutellariae Radix Carbonisata—derived carbon dots (SRC-CDs) on coagulation parameters. (A) Prothrombin time (PT), (B) activated partial thromboplastin time (APTT), (C) thrombin (TT) and (D) fibrinogen (FIB). Sprague Dawley rats were assigned into six groups (n = 10): control, model, positive and SRC-CDs at high- (25.00 mg/kg), medium- (12.50 mg/kg) and low (6.25 mg/kg) doses. *p < 0.05 and **p < 0.01 vs. model group or control group.

FIGURE 7

Effects of Scutellariae Radix Carbonisata—derived carbon dots (SRC-CDs) on the levels of inflammatory cytokines. The levels of (A) TNF-α in plasma, (B) TNF-α, (D) IL-1β and (F) IL-6 in lung tissues, (C) TNF-α, (E) IL-1β and (G) IL-6 in gastric tissues. Sprague Dawley rats were assigned into six groups (n = 10): control, model, positive and SRC-CDs at high- (25.00 mg/kg), medium- (12.50 mg/kg) and low (6.25 mg/kg) doses. **p < 0.01 and *p < 0.05 vs. control group or model group.

FIGURE 8

Effects of Scutellariae Radix Carbonisata—derived carbon dots (SRC-CDs) on the expression of NF-κB p65 and myD88 proteins in both (A) lung and (B) gastric tissues. (A), (a) and (B), (a) Representative immunoblot analysis and densitometry. (A), (b) and (B), (b) Quantification of NF-κB p 65 protein and myD88 protein. Sprague Dawley rats were assigned into five groups (n = 10): control, model and SRC-CDs at high- (25.00 mg/kg), medium- (12.50 mg/kg) and low (6.25 mg/kg) doses. *p < 0.05 and **p < 0.01 vs. control group or model group.