Abstract
There are more than 200 asparagus species worldwide. Green asparagus (Asparagus officinalis L.) is the only one commonly consumed commercially as food and it has an important industrial production value. The productive life of green asparagus is usually about 10 to 15 years, after which it must be replaced with new asparagus seedlings to improve productivity. When A. officinalis is harvested and processed, large quantities of by-products remain in the field. Asparagus roots (15%) may add value to the economics of production and profitability of asparagus production if a functional use is established. A. officinalis L. roots have been reported to contain several bioactive compounds (rutin, steroidal saponins, inosine and quercetin, caffeic acid, fumaric acid, isoferulic acid, ferulic acid, citric acid and asparagusic acid); however, the total phenolic compounds contributing to antioxidant properties of cultivars A. officinalis L roots from different regions have not been comprehensively investigated.
The aim of this thesis was to extract and identify the bioactive compounds from Asparagus officinalis L roots (AR) cultivars and their biological activities. The main focus was on examining and optimizing the extraction of bioactive compounds using conventional and novel extraction methods to provide suitable options for different commercial uptake. The specific objectives of the thesis were:
Firstly, to investigate and compare the efficiency of different extraction solvents (ethanol, methanol, chloroform, n-hexane, water, acetone, ethyl acetate, petroleum ether and isopropyl alcohol) in relation to the extraction of bioactive compounds from various AR cultivars (green and purple from New Zealand and green, purple, white and yellow from China). The total antioxidant activity, content of bioactive compounds and cultivar effect on the composition of AR extracts revealed that ethanol and methanol were the most efficient extraction solvents and were used in subsequent studies. The bioactive compounds varied among the varieties and were highest in yellow and green AR varieties.
The extraction parameters were optimized using conventional and modern extraction methods, i.e. microwave assistant extraction (MAE) and ultrasonic assistant extraction (UAE), in order to enhance the yield of bioactive compounds from AR cultivars (Chinese green, purple, white and yellow AR, and New Zealand green and purple AR). The bioactive compounds contents evaluated in this research included total phenolic content (TPC), total flavonoid content (TFC) and total saponin content (TSC). Antioxidant methods used in this thesis included ferric reducing-antioxidant power (FRAP), 2, 2-diphenyl-1-picryhydrazyl (DPPH), free radical scavenging, 2, 2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS), superoxide anion radical scavenging activity (O2-), β-carotene bleaching activity, oxygen radical absorbance capacity (ORAC) and angiotensin-converting enzyme (Grace) inhibitory assay (ACE). The optimal conditions for conventional extraction were at 50℃ for 75 min with 75.23% of ethanol at 1:50 solid: liquid ratio, and the Chinese yellow AR had the highest bioactive compounds (including inosine at 6.0 mg/g dry weight). Modern extraction methods, MAE under optimal conditions (56 s, 62.6% ethanol, 450 W and at solid to liquid ratio of 1:58.3) resulted in a 2% increase in bioactive compounds and a 2.5% increase in antioxidant compared to conventional extraction methods. Green New Zealand AR was higher (1.69 mg/g) than Chinese green AR (0.72 mg/g). The UAE processing at low power (360 W), extraction time (80 min), concentration of solvent (50%) and solid to liquid ratio (1:40) was effective in extracting more bioactive compounds from the AR cultivars and improving their bioactive capacities (1% to 3%) than MAE and conventional extraction methods.
In order to identify the bioactive compounds from AR cultivars, solid-phase extraction (SPE) was used as an efficient method for sample preparation. UAE-SPE-UPLC-MS/MS (Ultra-performance liquid chromatography-tandem mass spectrometry) has been used for the separation and purification of the bioactive compounds caffeic acid, ferulic acid, quercetin, apigenin, bacalein and kaempferol from AR cultivars. Of all the AR cultivars, caffeic acid had the highest content (2.05-5.97 mg/g) and apigenin had the lowest (0.13-0.42 mg/g).
Given the high antioxidant activity of the extracts, the potential use of AR extract to alleviate oxidative stress in cell models was studied. Their bioactive capacities were evaluated based on the health and integrity of the human liver cancer cell (Hep G2) and mouse fibroblasts (L929) by using 3-(4,5-dimethyl-2-yl)- 5-(3-carboxy methoxyphenyl) -2-(4- sulfophenyl) 2H-tetrazolium, inner salt; MTS cell viability; and lactate dehydrogenase leakage (LDH), against hydrogen peroxide induced oxidative stress, as markers for cell integrity. Chinese yellow AR delivered a better bio-protective capacity with the high concentration of extract (1 mg/ml), in which the membrane integrity of Hep G2 and L929 cells was completely protected from H2O2-induced oxidative damage. The antimicrobial activity of the extracts was evaluated against six gram-negative pathogenic bacteria (Escherichia coli, Campylobacter jejuni, Shigella flexneri, Listeria monocytogenes and Salmonella typhimurium) and three gram-positive pathogenic bacteria (Staphylococcus aureus, White staphylococcus and Bacillus subtilis). Chinese yellow AR possessed the highest antibacterial activity with a value of EC50 from 0.11-0.30 mg/ml in the eight bacterial strain lines.
This thesis demonstrated that UAE at a low power is a very promising method for extracting the bioactive compounds from AR. Evaluation of bioprotective capacities using Hep G2 and L929 cell assay as well as against antibacterial activities makes a unique contribution to the current knowledge regarding the health-promoting properties of AR extracts and supports further in vivo trials. The present study identified that AR extracts from different varieties had different activities and that yellow and green varieties were the most promising for commercial utilisation.