Lyophilized aqueous extracts of Mori Fructus and Mori Ramulus protect Mesenchymal stem cells from •OH-treated damage: bioassay and antioxidant mechanism

Abstract

Background: Mori Fructus and Mori Ramulus are two traditional Chinese herbal medicines from mulberries. The present work explores their beneficial effects on •OH-treated mesenchymal stem cells (MSCs) and discusses possible mechanisms.

Methods: Lyophilized aqueous extracts of Mori Fructus (LAMF) and Mori Ramulus (LAMR) were prepared and analyzed using HPLC. LAMF and LAMR (along with morin) were further investigated for their effects on •OH-treated MSCs using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The direct antioxidation mechanisms were studied using 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO•)-scavenging, 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS⁺•)-scavenging and 1,1-diphenyl-2-picryl-hydrazl (DPPH•)-scavenging, as well as Cu²⁺-reducing and Fe³⁺-reducing antioxidant power. Finally, the indirect antioxidant mechanism was investigated based on the UV-vis spectra of Fe²⁺-chelation.

Results: In each LAMF and LAMR, seven phytophenols were successfully measured by HPLC, including five flavonoids (morin, rutin, astragalin, isoquercitrin and luteolin) and two non-flavonoids (chlorogenic acid and maclurin). MTT assays revealed that LAMF, LAMR and morin could effectively increase the survival of •OH-treated MSCs at 10-100 μg/mL, and could effectively scavenge PTIO• (IC ₅₀ 6609.7 ± 756.6, 4286.9 ± 84.9 and 103.4 ± 0.9 μg/mL, respectively), DPPH• (IC ₅₀ 208.7 ± 3.0, 97.3 ± 3.1 and 8.2 ± 0.7 μg/mL, respectively) and ABTS⁺• (IC ₅₀ 73.5 ± 5.8, 34.4 ± 0.1 and 4.2 ± 0.2 μg/mL, respectively), and reduce Cu²⁺ (IC ₅₀ 212.5 ± 7.0, 123.2 ± 0.9 and 14.1 ± 0.04 μg/mL, respectively) & Fe³⁺ (IC ₅₀ 277.0 ± 3.1, 191.9 ± 5.2 and 5.0 ± 0.2 μg/mL, respectively). In the Fe²⁺-chelating assay, the five flavonoids produced much stronger shoulder-peaks than the two non-flavonoids within 420-850 nm.

Conclusion: Mori Fructus and Mori Ramulus, can protect MSCs from •OH-induced damage. Such beneficial effects can mainly be attributed to the antioxidant action of phytophenols, which occurs via direct (ROS-scavenging) and indirect mechanism (Fe²⁺-chelating). The ROS-scavenging mechanism, however, include at least a H⁺-transfer and an electron-transfer (ET), and possibly includes a hydrogen-atom-transfer (HAT). In the Fe²⁺-chelating, flavonoids are more effective than non-flavonoids. This can be attributed to several adjacent planar chelating-sites between the 3-OH and 4-C = O, between the 4-C = O and 5-OH, or between the 3'-OH and 4'-OH in flavonoids. Such multiple-Fe²⁺-chelating reactions cause overlap in the UV-vis absorptions to deepen the complex color, enhance the peak strength, and form shoulder-peaks. By comparison, two non-flavonoids with catechol moiety produce only a weak single peak.

Keywords: Antioxidant; Electron transfer; Fe-chelating; Flavonoids; H+-transfer; Herbal medicine; Mesenchymal stem cells; Mori Fructus; Mori Ramulus.

Fig. 1

Images of Morus alba L. (a), Mori Fructus ( b) and Mori Ramulus ( c)

Fig. 2

Preparation of lyophilized aqueous extracts of Mori Fructus (LAMF) and Mori Ramulus (LAMR)

Fig. 3

HPLC profiles and chemical contents of LAMA (a) and LAMR (b), and structures of relevant phytophenols (c)

Fig. 4

LAMF (a), LAMR (b), and morin (c) protect MSCs against •OH-induced apoptosis. Cell viability was assessed using the MTT method. Experiments were performed with 3 different batches of cells, and each batch was tested in triplicate. Data are the mean ± SD values. (*) p < 0. 05, compared with MSCs damage following FeCl₂ plus H₂O₂. LAMF, Lyophilized aqueous extract of Mori Fructus; LAMR, lyophilized aqueous extract of Mori Ramulus

Fig. 5

UV spectra of chelation products of LAMF, LAMR and relevant phytophenols with excess Fe²⁺ (The inset figures are the appearances of chelation products. The Fe²⁺-chelating ability of isoquercitrin was reported in our previous paper [15])

Fig. 6

The preferential conformation-based ball-stick models of chlorogenic acid (a) and morin (b). (The models were created by ChemOffice 2004, CambridgeSoft)

Fig. 7

The proposed reaction of Fe²⁺-chelating with chlorogenic acid (a), and with morin (b)