Recovery Effect of a Rutin-Enriched Fraction Prepared From Forsythia koreana Flowers on Alloxan-Induced Pancreatic Islets in Zebrafish Larvae ( Danio rerio )

Abstract

A simple method to prepare a high-content rutin fraction from Forsythia koreana flowers (HRFK) is described. Rutin, isolated for the first time from the flowers, was identified from spectroscopic data including nuclear magnetic resonance, mass spectrometry, and infrared. As a result of HPLC quantitative analysis, the content of rutin was found to be 80.0 ± 0.02% in HRFK. Our previous study reported that F koreana MeOH extract (FK) significantly recovered alloxan-induced pancreatic islets in zebrafish. However, HRFK as well as rutin exhibited an enhanced anti-diabetic effect compared to FK in our latest experiments. In conclusion, HRFK, as well as rutin and FK, have potential as anti-diabetic agents.

Forsythia koreana NAKAI (Oleaceae), a perennial shrub, is distributed widely in Korea and China. The shrub grows up to 1 to 3 m high and has ovate-lanceolate and oblong leaves. ¹ The fruit of F koreana (Forsythiae fructus), known in Korea as “Yeon-Kyo,” has been used in oriental medicine as a detoxification and anti-pyretic agent. ² In our previous study, we reported the identification of phenylethanoids and lignans in the flowers of F koreana, as well as their pharmacological activities such as anti-diabetic, anti-inflammatory, and neuroprotective effects.^3–5 Our ongoing research aimed to find extract fractions to enhance the anti-diabetic effect. As a result, a high-content rutin fraction, which showed strong anti-diabetic activity, was isolated.^6,7 This article describes an easy method to prepare a high-rutin content fraction from F koreana (HRFK). Additionally, quantitative analysis of rutin in HRFK was conducted through an HPLC experiment. HRFK and F koreana (FK) MeOH extract were evaluated for their recovery effect on injured PI in the ZF model.

FK was fractionated into nonpolar, residue, and aqueous fractions by solvent fractionation based on polarity. Among these fractions, the residue fraction yielded rutin (1). The chemical structure of 1 was revealed based on spectroscopic analyses and was confirmed through comparison with literature values. ⁸

Quantification of 1 in HRFK was performed from the peak areas recorded at 254 nm compared with calibration curves (y = 5005.6x − 6908.9) obtained using standard solutions under the analysis conditions described in the experimental section. The correlation coefficient (r²) in the regression curve of 1 was .999, indicating this method was reliable. Rutin eluted at 22.96 min, and its content in the residue fraction was 80.0 ± 0.02% (Figure 1).

Figure 1.

Effects of rutin (1) (1 and 10 μg/mL), HRFK (1 μg/mL), and FK (1 μg/mL) on alloxan-induced pancreatic islet-damaged zebrafish. (A) Size change of pancreatic islets for each group. (B) Pancreatic islet images. (^###P < .001; compared to normal), (*P < .1, **P < .01, ***P < .001; compared to alloxan). Scale bar = 100 μm.

HRFK was evaluated for its recovery effect against AX-induced PI damage in ZF larvae. AX, a diabetogenic chemical reported to decrease β-cell mass in PI,^9,10 was used to damage PI of ZF. AX treatment decreased the PI size significantly by 51.2% (P < .0001) compared to the normal group (Figure 1). Moreover, to observe PI under a microscope, we used 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), a fluorescent dye derived from glucose modified with an amino group at the C-2 position, ¹¹ which is utilized widely in diabetes studies to measure the ability of cells to absorb glucose and has been validated using the ZF model for PI observation. ⁹ To evaluate HRFK, a positive control, for the recovery effect on PI damaged by AX in ZF, the size of PI was examined after treatment of samples. The size of glimepiride-treated PI increased by 91.0% (P < .001) compared to the AX-induced group. The PI size for the 1 and 10 μg/mL rutin-treated groups increased by 99.5% (P = .0004) and 88.4% (P = .0023), respectively, compared to the AX-induced group (Figure 1). The HRFK-treated group showed an increase in PI size of 93.6% (P = .0001), in contrast to the FK-treated group, in which PI size increased by 74.7% (P = .0266). These results showed an enhanced recovery effect of HRFK compared to FK (Figure 1).

Conclusion

In the present study, we established a simple method for producing HRFK (content of rutin: 80.0 ± 0.02%), yielding 8.6% relative to F koreana flower extract, and evaluated its anti-diabetic efficacy. As a result, HRFK, as well as rutin (1) and FK MeOH extract, were shown to be potential anti-diabetic agents, and further studies are warranted. Furthermore, since rutin is a well-known active compound with various activities reported, including anti-SARS-CoV-2, ¹² it is also necessary to study the interaction of HRFK with other diseases.

Experimental

Plant Materials and General Experimental Procedures

The plant materials and general experimental procedures used are as previously described.^3-5,13

Fractionation of High-Content Rutin Fraction From Forsythia Koreana Flowers

Dried F koreana flowers (1.8 kg) were extracted in 80% MeOH (90 L × 4) at room temperature for 24 h, filtered and concentrated in vacuo. The obtained MeOH extract (952 g) was poured into H₂O (4 L) and successively extracted with organic solvents (n-hexane: ethyl acetate = 1:1) (4 L × 4). As a result, it was divided 3 fractions (frs) (nonpolar, residue, and aqueous fr.). Each layer was concentrated under reduced pressure to obtain a nonpolar fr. (87 g) and aqueous fr. (783 g), along with the high-content rutin fr. (residue fr. [HRFK], 82 g).

The Quantitative Analysis of Rutin (1) in HRFK Through HPLC Experiment

One mg of rutin (Sigma, St. Louis) was accurately weighed and dissolved in MeOH to obtain a stock solution with 1.0 mg/mL concentration. Calibration curves were made from the standard solution at 11 different concentrations (0.97-1000 ppm). HRFK were filtered through a 0.22 μm membrane filter (Woongki Science) and evaporated under vacuum. A 10 μL aliquot of the fraction solution (1000 ppm) was injected into the HPLC system. Analysis was achieved using a Waters 600S series (Milford) with a Waters 2487 UV detector (254 nm). The column was a Shimpack Gist (250 × 4.6 mm; 3 μm); the mobile phase: 0.1% FA (H₂O, A), 0.1% (AcN, B); flow rate 1 mL/min; elution of B; 20% (0.01 min) → 70% (20 min) → 100% (25 min) → 100% (30 min). The analysis was replicated 3 times.

Evalution for Recovery Effect of HRFK on AX-Induced PI in ZF Larvae

The ZF larvae were divided into: normal group, AX-induced group (control group), and AX-induced group treated with HRFK. Wild-type ZF larvae 6 days postfertilization were placed into 24-well plates. The larvae were exposed to 600 μM AX for 3 h to induce PI damage, as previously described, with a few modifications. ⁹ To determine the recovery efficacy of the HRFK, the AX-induced larvae were treated with either 1 or 10 μg/mL compound or 1 μg/mL extracts for 12 h. Then, the larvae were stained for 30 min with 40 μM 2-NBDG, and rinsed with 0.03% sea salt solution for 20 min. After staining, PI was observed under a fluorescence microscope and analyzed using Focus Lite and Image J software.

Statistical Analysis

Statistical analysis was performed using Graphpad Prism Ver 5. Data were expressed as mean ± standard error of mean (SEM). The significance was determined using repeated one-way ANOVA followed by Tukey's test. The probability level of statistical significance was determined as P-value (P < .05, P < .01, P < .001).