Biochemical characterisation and evaluation of anti-cancer properties of medicinally significant Jujube cultivars

Author: Kishore Madderla

Madderla, Kishore, 2022 Biochemical characterisation and evaluation of anti-cancer properties of medicinally significant Jujube cultivars, Flinders University, College of Medicine and Public Health

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Jujube (Ziziphus jujuba), commonly known as red dates, is used in Traditional Chinese Medicine (TCM). Jujube is rich in phenolics, flavonoids, triterpenoids and have high antioxidant activity. The South Australia Jujube Growers Association (established in 2019) aimed to investigate the biochemical composition of the fruit, leaves, and seeds of five Jujube cultivars, Li 2, Chico, Shanxi Li, Sihong, and Honeyjar, to quantify the content of phenolics, flavonoids, and to assess antioxidant activity and anticancer activity. The aim of this study was to biochemically characterize the different Jujube bioresources and to determine activity against human colon cancer cells (HCT116) of crude ethanolic extracts of the leaves, fruit and seeds. Ultrasound-assisted extraction was used to extract protein and carbohydrates using water as a solvent, while extraction of phenolics, flavonoids, triterpenoids used 80% ethanol as a solvent. UPLC-MS analyses were carried out to identify bioactive compounds such as flavonoids (Rutin, Quercetin), triterpenoids (Ursolic acid) and alkaloids (Quinine), some of which were quantifiable. Antioxidant activity of these ethanolic extracts was quantified using the FRAP and cell viability was assessed in dose-response MTT assays of crude ethanolic extracts of Jujube cultivar leaves, seeds and fruit. To investigate the mineral contents (Fe, Mg, Ca, K), ICP-OES was performed by Flinders Analytical. Total dietary fibre was analysed by CSIRO (Adelaide, SA). Total protein content was highest in leaves (0.607-2.02 g/100 g DW), while total carbohydrate content (g/ 100 g DW) was highest in fruit (ranging from 33.44 (Sihong) to 58.45 (Li 2)). Of the minerals (Fe, Mg, Ca, K) investigated, Ca contents were highest in Jujube leaves (39.84 to 46.61 mg/g DW), followed by K (13.71 to 17.33 mg/g DW), Mg (3.51 to 4.13 mg/g DW), and Fe (ranged from 0.14 to 0.48 mg/g DW). Total dietary fibre was highest in seeds (56.4 to 85.5 g/ 100 g DW). Total polyphenol, total flavonoid, and total triterpenoid contents (g/ 100 g DE) were highest in leaves, 16.77 (Honeyjar) to 18.57 (Sihong), 4.28 (Honeyjar) to 5.13 (Li 2), and 190.73±3.58 (Chico) to 365.75±9.63, respectively. Antioxidant activity (mmol FeSO4/ 100 g DE) of crude ethanolic extracts of leaves had 10 to 20-times higher activities (90.876±8.29 (Li 2) to 229.53±5.95 (Shanxi Li)) compared to fruit and seeds. The quantification of bioactive compounds by UPLC-MS showed that leaves are a promising source of Rutin (16.07 (Chico) to 50.98 (Shanxi Li) mg/ g DE), Quercetin (0.42 (Honeyjar) to 1.01 (Sihong) mg/ g DE), but amounts were bordering detection limit in fruit and seeds. Ursolic acid content varied with cultivar but were highest in leaves (0.24 (Honeyjar) to 3.15 (Sihong) mg/ g DE), bordering detection limits in fruit, whilst highly variable in seeds for most cultivars. LC-MS analysis confirmed the presence of quinine, but contents were below the quantification limit. 24 h dose-response tests of ethanolic crude extracts of Jujube leaves, seeds and fruit showed that viability of HCT116 cells (human colon cancer cells) decreased at higher concentrations of leaf extracts of Li 2, Chico, Shanxi Li, while fruit and seed extracts had no effect. In conclusion, the leaves of SA Jujube cultivars are the richest source of minerals (Ca, Fe, Mg) and bio-active compounds. Leaf extracts of all Jujube cultivars may also find applications in cosmetic formulations, as they show high antioxidant activities. As crude ethanolic leaf extracts adversely affected viability of HCT116 cells, there could be potential in cancer treatments, but this requires further studies that include normal colon cells to demonstrate that such extracts do not indiscriminately reduce cell viability.

Keywords: Jujube, Red Dates, Chinese dates, South Australia Growers Association, Traditional Chinese Medicine, Total Phenolics, Total Flavonoids, Minerals, Ultrasound assisted extraction, Bioactives

Subject: Biotechnology thesis

Thesis type: Masters
Completed: 2022
School: College of Medicine and Public Health
Supervisor: Kirsten Heimann