Department of Pharmacognosy, Cumhuriyet University Faculty of Pharmacy, Sivas, Turkey; Department of Pharmacognosy, Selcuk University Faculty of Pharmacy, Konya, Turkey.
Department of Pharmaceutical Toxicology, Cumhuriyet University Faculty of Pharmacy, Sivas, Turkey.
Phytomedicine. 2019 Feb;53:234-242. doi: 10.1016/j.phymed.2018.09.016. Epub 2018 Sep 5.
Morus alba and Morus nigra leaves which have been widely used as herbal teas in Anatolian region of Turkey, were extracted twice by 50 mM HCI solution, derivatized with 9-fluorenylmethyl chloroformate and analyzed by reversed phase HPLC equipped with a fluorescence detector.
HYPOTHESIS/PURPOSE: This study was performed to determine the main antidiabetic active compounds 1-deoxynojirimycin by HPLC method and evaluate the in-vitro antioxidant and antidiabetic activity of ethanol extracts prepared from Morus alba L. and Morus nigra leaves.
A reliable simple, and rapid high-performance liquid chromatographic (HPLC) method for the determination of 1-deoxynojirimycin in M. alba L. and M. nigra leaves with fluorimetric detection after pre-column derivatization with 9-fluorenylmethyl chloroformate was developed. In addition, the chemical composition of ethanol extract of mulberry leaves was analyzed with GC-MS.
Separation and quantitation were performed on C18, 250 × 4.6 mm, 5 µm analytical column. Mobile phase consisted of acetonitrile and 0.1% acetic acid solution (1:1, v/v) was performed applied to the column 1.0 ml/min flow rate at 26 °C. Potential antioxidant activity of ethanol extract of different mulberry varieties were evaluated by DPPH, and ABTS radical scavenging assay as well as total phenol and flavonoid content were determined. In addition, α-amylase and α-glucosidase activity was determined by 96-well plate method to evaluate the probable antidiabetic potential use of Turkish mulberry leaves.
The isocratic HPLC method showed excellent correlation coefficient (r= 0.9985) between 0.3 and 30 µg/ml calibration points. The method was specific and sensitive with detection and quantification limits of 1.07 and 3.27 ng/ml, respectively. Intraday and interday method precision (n = 5) were < 7.3 (RSD%). Intraday and interday method accuracy (n = 5) were between 3.77 and (-8.35) (RE%). The average method recovery (n = 3) was 102.5%. The results showed that the content of 1-deoxynojirimycin in leaves of Morus alba L. was 0.103% (n = 3), and in leaves of M. nigra L. was 0.102%. 2-hexadecen-1-ol, oleamide, 2-propenoic acid, and cyclododecane were identified as the major compounds by GC-MS in the ethanol extract of mulberry leaves.
The obtained robustness values from emission and excitation detection, mobile phase ingredients and flow rates changes showed that method was very strong. This work contributes to the knowledge of antioxidant and antidiabetic properties of Morus species, thus may be provide useful data in evaluation of food products and pharmaceutical preparations produced from Morus species.
在土耳其安纳托利亚地区，桑白皮和黑桑白皮被广泛用作草药茶，本研究采用 50mM HCI 溶液对其进行两次提取，用 9-芴甲基氯甲酸酯衍生化，并用配备荧光检测器的反相高效液相色谱法进行分析。
假说/目的：本研究旨在通过 HPLC 法测定 1-脱氧野尻霉素，评价桑白皮和黑桑白皮乙醇提取物的体外抗氧化和抗糖尿病活性。
建立了一种可靠、简单、快速的高效液相色谱法（HPLC），用于测定桑白皮 L.和黑桑白皮中 1-脱氧野尻霉素，并用 9-芴甲基氯甲酸酯衍生化后进行荧光检测。此外，还通过 GC-MS 分析了桑树叶乙醇提取物的化学成分。
分离和定量在 C18、250×4.6mm、5µm 分析柱上进行。流动相由乙腈和 0.1%乙酸溶液（1:1，v/v）组成，以 1.0ml/min 的流速在 26°C 下应用于柱上。通过 DPPH 和 ABTS 自由基清除测定法以及总酚和类黄酮含量测定，评估不同桑品种乙醇提取物的潜在抗氧化活性。此外，通过 96 孔板法测定α-淀粉酶和α-葡萄糖苷酶活性，以评估土耳其桑树叶的可能抗糖尿病潜力。
该等度 HPLC 法在 0.3 至 30μg/ml 校准点之间显示出极好的相关系数（r=0.9985）。该方法具有特异性和灵敏度，检测限和定量限分别为 1.07 和 3.27ng/ml。日内和日间方法精密度（n=5）均<7.3（RSD%）。日内和日间方法准确度（n=5）分别为 3.77 至（-8.35）（RE%）。平均方法回收率（n=3）为 102.5%。结果表明，桑白皮 L.叶片中 1-脱氧野尻霉素的含量为 0.103%（n=3），黑桑白皮 L.叶片中为 0.102%。GC-MS 鉴定出乙醇提取物中的主要化合物为 2-十六碳烯-1-醇、油酰胺、2-丙烯酸和环十二烷。
从发射和激发检测、流动相成分和流速变化中得到的稳健值表明，该方法非常强大。这项工作有助于了解桑属植物的抗氧化和抗糖尿病特性，因此可能为评价桑属植物来源的食品产品和药物制剂提供有用的数据。
