The Carbon Dots from Seabuckthorn (Hippophae rhamnoides L.) Leaves: Recycle the Herbal Waste Products for a Nano-Formulation in Delivering Bioactive Compounds

Abstract

Carbon dots have emerged as promising nanocarriers for drug delivery due to their unique physicochemical properties and biocompatibilities. Here, the potential of leaf-derived carbon dots (named as SBL_CD), derived from Seabuckthorn (Hippophae rhamnoides L.), was illustrated as a novel nano-formulation for bioactive compound delivery. Seabuckthorn leaves, rich in flavonoids, are the waste product during the production of Seabuckthorn fruits. The wasted leaves were utilized to synthesize carbon dots via a hydrothermal method. The resulting SBL_CD, characterized by TEM, FT-IR and Raman spectroscopy, exhibited a diameter of ~5 nm in both amorphous and quasi-crystalline forms. Applications of SBL_CD in cultures demonstrated robust properties of anti-inflammation and inducing neuronal cell differentiation. Furthermore, SBL_CD was able to encapsulate luteolin, a bioactive flavonoid. The enhanced delivery efficiency translated to superior biological activity, with SBL_CD-luteolin requiring only 1.50 μg/mL in achieving the EC₅₀ efficacy, as compared to 6.82 μg/mL for free luteolin in pNF200-Luc expression assays. This approach not only valorizes Seabuckthorn leaf by-products but also potentially improves the efficacy of encapsulated flavonoids. The development of SBL_CD as a multifunctional platform for flavonoid delivery represents a promising strategy in enhancing the efficacy of neuroactive compounds, combining anti-inflammatory effects (>70% cytokine suppression) with enhanced cellular uptake (4.5-fold increase).

Keywords: Seabuckthorn; bioavailability enhancement; carbon dot; drug encapsulation; flavonoid.

Figure 1

Preparation of carbon dots from Seabuckthorn leaves (SBL_CD). Dried powder of Seabuckthorn leaf was dispersed in deionized water and hydrothermally treated at 180 °C for 5 h in a Teflon-lined autoclave. After natural cooling, the suspension was centrifuged and filtered to remove insoluble material, dialyzed against deionized water for 24 h (MWCO 500 Da) and freeze-dried to obtain SBL_CD powder for subsequent experiments.

Figure 2

Structural and optical characterization of SBL_CD. (A) Transmission electron microscopy (TEM) revealed uniformly dispersed nanoparticles with a mean diameter of ~5 nm (a, with white arrow indicating typical SBL_CD nanoparticles), and high-resolution TEM imaging shows lattice fringes with d-spacing ~0.3 nm (b). (B) FT-IR spectrum displayed O-H stretching (3250 cm⁻¹), aliphatic C-H stretching (2931 cm⁻¹), carbonyl/aromatic C=C stretching (1612 cm⁻¹), C-N vibration (1330 cm⁻¹) and C-O stretching (1028 cm⁻¹). (C) XPS indicated elemental composition of C 64.02%, O 29.51% and N 6.11% (a), with high-resolution C1s peaks for sp² carbon (284.7 eV, green line), C-N/C-O (286 eV, red line) and carbonyl carbon (288.6 eV, orange line) (b); O1s peaks for ether/hydroxyl (533 eV, blue line) and carbonyl O (531.9 eV, orange line) (c); and N1s peaks for graphitic (400.9 eV, blue line) and pyrrolic (399.3 eV, orange line) nitrogen (d). (D) Raman spectrum showed the D-band at 1388 cm⁻¹ (defects) and G-band at 1613 cm⁻¹ (graphitic domains). (E) UV-visible absorption exhibited a main peak at 270 nm and a shoulder near 400 nm, characteristic of π-π* transitions in aromatic domains. (F) Photoluminescence spectra at 320–640 nm excitation displayed excitation-dependent emission, reaching a maximum emission intensity at 460 nm when excited at 380 nm.

Figure 3

Comparative biological activities of SBL_CD against SBL. (A) MTT assay shows cell viability in cultured PC12 and BV2 cells after 0–400 μg/mL SBL_CD treatment. (B) BV2 cells pretreated with SBL_CD (2 h) exhibited dose-dependent suppression on LPS (20 ng/mL)-induced (16 h) IL-1β, IL-6 and TNF-α expressions. Cytokine mRNA levels were quantified by qRT-PCR and expressed as fold change (× basal) relative to the untreated control. Dexamethasone (10 μM) serves as a positive control. (C) In NF200-Luc DNA transfected PC12 cells, the activity of pNF200-Luc reporter was induced by 0–120 μg/mL SBL_CD in a dose-dependent manner. (D) At equivalent flavonoid concentrations (0–6 μg/mL), SBL_CD elicits greater induction of pNF200-Luc activity than that of Seabuckthorn leaf (SBL) extract in pNF200-Luc transfected PC12 cells. (E) Under the same flavonoid equivalents, SBL_CD shows stronger inhibition of LPS-induced cytokines in BV2 cells than SBL extract. Experimental procedure was performed as in (B). Values are presented as the percentage of control (%), the percentage of change (%) or the fold of change (× basal) to the control, in mean ± SEM, n = 4. (*) or (**) means the significance of changes between the control group and the SBL_CD- or SBL- treated group. (*) p < 0.05, (**) p < 0.01.

Figure 4

Luteolin loading capacity of SBL_CD and enhancement of cellular delivery. (A) Loading with of luteolin per 1 mg/mL SBL_CD reached a loading plateau of ~2.8 μg/mg. (B) Confocal images of PC12 cells after incubation with free luteolin or SBL_CD-lutelin showed DPBA-labeled flavonoids (green), mitochondria (MitoTracker Red, red) and nuclei (DAPI, blue); arrowheads mark enhanced cytoplasmic accumulation in the SBL_CD group. (C) Fluorescence quantification confirmed higher intracellular luteolin with SBL_CD carriers than with free luteolin. (D) HPLC-MS/MS analysis showed greater luteolin uptake with SBL_CD delivery, normalized to cellular protein. (E) pNF200-Luc assay revealed a dose-dependent neurite promotion by free luteolin, SBL_CD-luteolin and an SBL_CD + luteolin physical mixture; the complex outperforms both controls at all doses. Values are presented as the percentage of control (%) or the percentage of change (%), in mean ± SEM, n = 4. (*) or (**) means the significance of changes between the control group and the drug-treated group. (*) p < 0.05, (**) p < 0.01.