The isolation of glycoprotein from tongkat ali putih (eurycoma longifolia) for the elevation of testosterone hormone in mice / Sharifah Aminah Binti Tuan Said

Faculty of Industrial Sciences and Technology

Thesis (Doctor of Philosophy) -- Universiti Malaysia Pahang – 2024

Includes bibliographical references

Eurycoma longifolia (EL), Polyathia bullata (PB), and Stema tuberosa (ST) are three species of plants sharing the synonym of "Tongkat Ali" and commonly known as Tongkat Ali Putih, Tongkat Ali Hitam, and Tongkat Ali Merah, respectively. This study has been carried out to determine the yield of extracts and characterize the glycoprotein of the Tongkat Ali plants. The dried root powder of the plant was extracted using water under reflux. The protein and carbohydrate were characterized using Bradford Assay, SDSPAGE, and DNS Assay. The extraction of EL, ST, and PB yielded 7.433%, 5.715%, and 3.120% w/w and 0.750%, 0.450%, and 0.350% w/w of total protein, respectively. The SDS PAGE analysis showed a single band for each of the three Tongkat Ali plants at approximately between 10 - 15 kDa, and the DNS Assay showed the presence of reducing sugar in all three Tongkat Ali, with EL being the highest amount of 0.610 mg/mL, followed by PB with 0.594 mg/mL and the lowest, ST with 0.586 mg/mL. Next, lectin affinity chromatography (LAC) was used to separate specifically the glycoprotein and non-glycoprotein fractions using the elution buffer of 0.5M methyl-α-Dmannopyranoside in all three Tongkat Ali plants. Glycoproteins of varying concentrations were found in the plants, with EL being the highest total glycoprotein yield at 0.648%, followed by PB at 0.193%, and the lowest is ST at 0.177% w/w. Lastly, to isolate in bulk of the glycoprotein from the most popular Tongkat Ali plant followed by in vitro and in vivo testing. Only EL’s glycoprotein was further collected in bulk and freeze-dried for further testing due to its highest glycoprotein content compared to the other two Tongkat Ali. For in vitro and in vivo testing, the extracts of EL, PB, ST, and the glycoprotein of EL were treated with TM-3 Leydig cells (50 µg/mL) for 72 hours and with mice (6 mg/mL for extracts and 2.5 mg/mL for glycoprotein) twice/day for 20 days. For in vitro, the ELISA showed that the EL isolated glycoprotein had the highest testosterone concentration at 380.334 nmol/L, which is 226.969% of testosterone release in comparison to untreated control cells. This was followed by EL extract with 194.260 nmol/L (68.153%), ST extract with 140.120 nmol/L (21.289%), and the lowest, PB extract with 137.092 nmol/L (18.668%). For in vivo, the ELISA test showed that the EL isolated glycoprotein also had the highest testosterone concentration at 23.715 nmol/L which is 263.894% of testosterone release in comparison to untreated control mice. This was followed by EL extract with 15.015 nmol/L (132.502%), ST extracts with 7.526 nmol/L (16.538%), and the lowest, PB extract with only 6.825 nmol/L (5.683%). In conclusion, the outcome showed the glycoprotein and the crude extracts of EL, PB, and ST were able to elevate testosterone concentrations in vitro and in vivo in the order of glycoprotein> EL>ST>PB, all treated at similar dosing.