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Phenolic compounds from Nepenthes gracilis Korth : isolation, identification and antioxidant studies
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Type
Thesis
Author
Ma, Hnin Hnin Aung
Supervisor
Chia, Lian Sai
Abstract
One of the most remarkable features of medicinal plant research of the last decade is the enormously increasing interest being taken in the biological activities of phenolic compounds including flavonoids. Flavonoids and other phenolics have been suggested to play a preventative role of chronic diseases such as cancer and heart disease. The antioxidant activity of flavonoids is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers. In our research, we chose the tropical pitcher plant Nepenthes gracilis Korth. The objectives of this study are: (1) to isolate and identify the phenolics including flavonoids from Nepenthes gracilis Korth leaves and cups; (2) to study the antioxidant potency of the crude extracts and of the isolated phenolics including flavonoids from Nepenthes gracilis Korth by ESR assay and ABTS-WNIS assay; (3) to test the proanthocyanidin percentages of the extract from the pitcher plant, and (4) to investigate the free radical generation and the type of radicals in the pitcher fluid from different Nepenthes species. The time consuming work of isolation, purification and identification of phenolic compounds from Nepenthes gracilis Korth leaves and cups were done by HPLC online W-VIS assay for screening of component composition, preliminary separation by silica gel, polyamide and Sephadex column chromatography and further isolation using preparative H PLC. The structural elucidation o f isolated compounds was identified by various spectral methods, e.g. HPLC online UV-VIS spectrometry, NMR, MS, and X-ray crystallography. As a result, a total of twenty-three compounds, seven non-flavonoid phenolics and sixteen flavonoid compounds (including eight gallate glycosides, which are very rare in nature) were isolated and identified from N. gracilis leaves and cups. In addition, the crystal structure of avicularin was determined. To the best of our knowledge the determinations of this compound have not been reported so for.
In this study, evaluation of antioxidant potency of twenty-one flavonoids isolated compounds was measured by ESR assay in etoposide-HzOz-HRP system and TroIox equivalent antioxidant capacities (TEAC) assay with UV-VIS spectrometry. The quantative measurement of TEAC assay showed that the isolated flavonoids are more powerful than the Trolox (on molar basic). The main advantages of TEAC assay are that (1) it is convenient to use, (2) it can be used for both lipid and aqueous systems, (3) quantative measurements can be made. The order of TEAC values of flavonoids agreed well with that of the radical scavenging activities of these compounds, as determined by ESR assay. Among the isolated flavonoids, quercetin 3-0-a-(2"-galloylrhamnopyranoside), quercetin 3-0-a-(3"-0- galloylrhamnopyranoside), quercetin 3-0-P-(2"-0-galloylxylopyranoside), quercetin 3-0-P-(3"-0-galloylxylopyranoside), quercetin 3-0-a-(2"- galloylarabinofuranoside), quercetin 3-0-P-(6"-galloylglucopyranoside), quercetin 3-0-P-(6"-galloylgalatopyranoside), and epicatechin 3-0-gallate have higher antioxidant activities and percent yield than those of the flavonol glycoside compounds. Quercetin 3-0-P-(6"- galloylgalatopyranoside) was found to be a very powerful antioxidant in N. gracilis plant, while quercetin 3-0-a-(2"-galloylarabinofuranoside) was obtained in very high yield. The higher antioxidant activities of compounds with gallic acid should be of importance to plant biochemists and enzymologists.
The relationship between structures of flavonoids and the antioxidant activities was also studied. The quercetin derivatives (flavonol-type compounds) isolated, having an additional -OH at C-3, had stronger antioxidant activity than the other type (e.g. flavone). The C-3 linked sugar groups have the same structural backbone and hydroxyl groups, but differ in the types of sugars linked to the C-3 position. Their ability to scavenge radicals shows a descending pattern in this order: galactose > glucose. The data further indicate that the gallate glycosides at C-3 of quercetin enhance the TEAC values of flavonoids. In addition, gallate esterfication at C-6", C-3" and C-2" to quercetin glycosides and at C-3 to epicatechin (flavonol) can enhance the antioxidant activity of the conlpound. The order was found to be: C-6" > C-3" > C-2".
The proanthocyanidins analysis of ethyl acetate and aqueous layers shows that the aqueous layer possesses high relative percent of proanthocyanidins and also is higher in total antioxidant activity. The results of the present study suggest that while the flavonoids in pitcher plant exhibit good antioxidant potential, they are not presenting sufficient amounts to be considered as the main source of antioxidants. This is an important observation as there is a general perception that quercetin and related flavonols may be major nutritional antioxidants. Other phenolic compounds of the phenylpropanoid biosynthesis pathway, such as proanthocyanidins and epicatechin, may contribute to a greater degree to the antioxidant properties of N. gracilis Korth.
Finally, regarding the interesting findings of the insects digested inside the pitcher fluid, our experimental results demonstrate that free radicals not only oxidize proteins, but also can kill or paralyze the insects. It appears that the presence of free radicals in pitcher fluid will facilitate the digestion process. The Nepenthes pitcher plants get their nutrient via digestion of insects by using free radicals.
In this study, evaluation of antioxidant potency of twenty-one flavonoids isolated compounds was measured by ESR assay in etoposide-HzOz-HRP system and TroIox equivalent antioxidant capacities (TEAC) assay with UV-VIS spectrometry. The quantative measurement of TEAC assay showed that the isolated flavonoids are more powerful than the Trolox (on molar basic). The main advantages of TEAC assay are that (1) it is convenient to use, (2) it can be used for both lipid and aqueous systems, (3) quantative measurements can be made. The order of TEAC values of flavonoids agreed well with that of the radical scavenging activities of these compounds, as determined by ESR assay. Among the isolated flavonoids, quercetin 3-0-a-(2"-galloylrhamnopyranoside), quercetin 3-0-a-(3"-0- galloylrhamnopyranoside), quercetin 3-0-P-(2"-0-galloylxylopyranoside), quercetin 3-0-P-(3"-0-galloylxylopyranoside), quercetin 3-0-a-(2"- galloylarabinofuranoside), quercetin 3-0-P-(6"-galloylglucopyranoside), quercetin 3-0-P-(6"-galloylgalatopyranoside), and epicatechin 3-0-gallate have higher antioxidant activities and percent yield than those of the flavonol glycoside compounds. Quercetin 3-0-P-(6"- galloylgalatopyranoside) was found to be a very powerful antioxidant in N. gracilis plant, while quercetin 3-0-a-(2"-galloylarabinofuranoside) was obtained in very high yield. The higher antioxidant activities of compounds with gallic acid should be of importance to plant biochemists and enzymologists.
The relationship between structures of flavonoids and the antioxidant activities was also studied. The quercetin derivatives (flavonol-type compounds) isolated, having an additional -OH at C-3, had stronger antioxidant activity than the other type (e.g. flavone). The C-3 linked sugar groups have the same structural backbone and hydroxyl groups, but differ in the types of sugars linked to the C-3 position. Their ability to scavenge radicals shows a descending pattern in this order: galactose > glucose. The data further indicate that the gallate glycosides at C-3 of quercetin enhance the TEAC values of flavonoids. In addition, gallate esterfication at C-6", C-3" and C-2" to quercetin glycosides and at C-3 to epicatechin (flavonol) can enhance the antioxidant activity of the conlpound. The order was found to be: C-6" > C-3" > C-2".
The proanthocyanidins analysis of ethyl acetate and aqueous layers shows that the aqueous layer possesses high relative percent of proanthocyanidins and also is higher in total antioxidant activity. The results of the present study suggest that while the flavonoids in pitcher plant exhibit good antioxidant potential, they are not presenting sufficient amounts to be considered as the main source of antioxidants. This is an important observation as there is a general perception that quercetin and related flavonols may be major nutritional antioxidants. Other phenolic compounds of the phenylpropanoid biosynthesis pathway, such as proanthocyanidins and epicatechin, may contribute to a greater degree to the antioxidant properties of N. gracilis Korth.
Finally, regarding the interesting findings of the insects digested inside the pitcher fluid, our experimental results demonstrate that free radicals not only oxidize proteins, but also can kill or paralyze the insects. It appears that the presence of free radicals in pitcher fluid will facilitate the digestion process. The Nepenthes pitcher plants get their nutrient via digestion of insects by using free radicals.
Date Issued
2004
Call Number
QK898.F5 Ma
Date Submitted
2004