Tuesday, 26 April 2016

Re: Adjusting Critical Parameters of HPLC-CUPRAC Assay Optimizes Measurements of Ginkgo Components' Antioxidant Activities

  • Ginkgo (Ginkgo biloba, Ginkgoaceae)
  • HPLC-CUPRAC Method
  • Antioxidant Activity
Date: 04-15-2016HC# 101562-542

Rimkiene L, Ivanauskas L, Kubiliene A, Vitkevicius K, Kiliuviene G, Jakstas V. Optimization of a CUPRAC-based HPLC postcolumn assay and its applications for Ginkgo biloba L. extracts. J Anal Methods Chem. July 5,2015;2015:280167. doi: 10.1155/2015/280167.

Excess oxidative stress has been reported as an important factor in the development of a myriad of diseases. Ongoing research investigates the potential of plant-based antioxidants to restore the balance of reactive oxygen species. Streamlined methodology for the detection of antioxidants in botanicals is necessary to assess the activities of these compounds. Techniques, such as high-performance liquid chromatography (HPLC) paired with a postcolumn bioactivity assay, may be useful to this end. Ginkgo (Ginkgo biloba, Ginkgoaceae) leaves are reported to have antioxidant activity. This basic research study investigated the HPLC-CUPRAC (cupric ion reducing antioxidant capacity) method of assessing antioxidant activity of the compounds 
quercetin, kaempferol, and isorhamnetin in a hydrolysed ginkgo leaf extract.

Ginkgo leaves were collected from the seven following sites: Šiauliai University Botanical Garden, Klaipėda University Botanical Garden, and Kaunas Botanical Garden, located in Šiauliai, Klaipėda, and Kaunas, Lithuania, respectively, and Lazdijai, Kazlų Rūda, Šilutė, and Utena, Lithuania. Leaves (2.5 g) were extracted in 50 ml of 60% aqueous acetone and heated two times under reflux conditions for 30 minutes each. Acetone was evaporated, and the material was diluted with 30 ml methanol and 4.4 ml hydrochloric acid, and filled to volume (50 ml) with water. The extract was subsequently placed in a heated water bath for 25 min, filtered, and run on an HPLC. After separation, the eluent was reacted with CUPRAC reagent solution, which, when reduced, results in a yellow color change that can be measured with an ultraviolet/visible (UV/Vis) detector. Sample compounds were assessed in parallel with the Trolox (a vitamin E analog) equivalent antioxidant capacity (TEAC) assay, a commonly used method to gauge antioxidant activity.
The authors report problems with sediment clogging the HPLC instrumentation and conclude that optimization of the flow rate, reaction loop, and temperature is necessary for applying this methodology. Keeping a stable temperature of 25°C resulted in the best performance (higher temperatures apparently increased the back pressure), as did a flow rate of 0.2 ml/min. Also, a reaction loop length between 5-10 meters was optimal to allow a sufficiently long reaction time among the compounds and CUPRAC reagent and minimize scattering of results. Quercetin, kaempferol, isorhamnetin, and Trolox were used to validate the method.  
The flavonols quercetin, kaempferol, and isorhamnetin were found to be responsible for the antioxidant activity detected by CUPRAC. The higher antioxidant activity of quercetin in CUPRAC, as compared to the other two compounds, was confirmed with the TEAC calculation (60-81% of total leaf antioxidant activity). Kaempferol and isorhamnetin had descending antioxidant capacity, according to the CUPRAC method, also confirmed with the TEAC values (17-33% and 0-7%, respectively, of the total). The authors suggest that this is due to the relative concentrations of these compounds in the extract flavone glycosides. However, no evaluation of the antioxidant activity among the three flavonols at equal concentration was carried out, nor did the authors discuss previously published data comparing the antioxidant activities of the compounds. The leaves collected from Kazlų Rūda showed the best reducing activity (25.99 ± 0.42 µmol/g), while the lowest was seen in material from Kaunas (4.74 ± 0.08 µmol/g).
This study suggests that the CUPRAC method may be very useful in not only phytochemical fingerprinting of botanical extracts, but in detecting bioactivity in various compounds during the HPLC technique used in acquiring the complex fingerprint. Those wishing to use this methodology would do well to optimize their instrumentations with attention to flow rate, temperature, and reaction loop length. Ideally, the HPLC-CUPRAC method will aid in characterizing botanicals for antioxidant bioactivity.
Amy C. Keller, PhD
Peer Review Comments:
A key problem with the study is that the authors worked on a hydrolysed ginkgo extract. A hydrolysed extract is not representative of genuine, unadulterated ginkgo extracts on the market.
The hydrolysis of the ginkgo flavonol glycosides would have stemmed from the use of hydrochloric acid in the extraction process, resulting in substantial amounts of the flavonol aglycones quercetin, kaempferol, and isorhamnetin [clearly evident in Fig. 1(b)]. It is clear from the recent literature that ginkgo leaves and unadulterated, unhydrolysed extract contain very low or undetectable levels of these free aglycones—virtually all of the ginkgo flavonols occur in glycoside form.1,2 This fact is being somewhat obscured by pharmacopoeial methods that assay the aglycones following acid hydrolysis.
While it could be argued that the free aglycones may well form in the digestive tract as a result of acid hydrolysis in the stomach, it is important not to perpetuate the misconception that unhydrolysed and unadulterated ginkgo extracts contain free quercetin, kaempferol, or isorhamnetin.
The structure-antioxidant activity relationship among flavonols has been detailed in many papers.3 It is well known that a catechol moiety, as in quercetin, will lead to higher antioxidant activity. As such, the question arises as to how the authors conclude that the activity is only concentration-dependent and does not relate to the chemical structure of the flavonols.
1Avula B, Sagi S, Gafner S, et al. Identification of Ginkgo biloba supplements adulteration using high performance thin layer chromatography and ultra high performance liquid chromatography-diode array detector-quadrupole time of flight-mass spectrometry.Anal Bioanal Chem. 2015;407(25):7733-7746.
2Wohlmuth H, Savage K, Dowell A, Mouatt P. Adulteration of Ginkgo biloba products and a simple method to improve its detection.Phytomedicine. 2014;21(6):912-918.
3Heim KE, Tagliaferro AR, Bobilya DJ. Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. J Nutr Biochem. 2002;13(10):572-584.