Effect of Extraction Method on Antioxidant Activity, Anti-inflammation and Cytotoxicity of Curcuma aeruginosa Roxb. Rhizome
Candra Irawan, Imas Solihat*, Sellafiana Sunu Praptiwi, Miska Mufidah,
Elisa Listianti, Bulan Safitri
Department of Food Nanotechnology, Politeknik AKA Bogor, Jalan Pangeran Sogiri, Tanah Baru,
Bogor 16154, Indonesia.
*Corresponding Author E-mail: imaskhairani@gmail.com
ABSTRACT:
Temu Hitam (Curcuma aeruginosa Roxb) is recognized as one of the plants that has the potential to be utilized in the practice of traditional medicine. The selection of the extraction method is an essential initial step in the process of chemical component purification. In this particular investigation, the extraction process was carried out utilizing a variety of techniques, including hot maceration, cold maceration, and ultrasound-assisted extraction (UAE), in order to investigate the anti-inflammatory, antioxidant, and cytotoxicity capabilities of the subject material. The percent yield from the UAE method showed the highest amount of 51.94%, and the IC50 values from the cold method, hot method, and UAE using the FRAP method were 4.65, 6.22, and 5.57 ppm, respectively. The extract using the UAE method has higher cytotoxicity to Artemia salina Leach larvae. In addition, UAE showed better anti-inflammatory activity with an IC50 value of 2.96 ppm.
KEYWORDS: Curcuma aeruginosa Roxb, extraction.
INTRODUCTION:
Temu Hitam (Curcuma aeruginosa Roxb.) is a species of plant that belongs to the family Zingiberaceae1. Almost all the parts of the plant have been used in traditional medicine practices2. This plant is often used as a traditional herbal medicine3 because it contains many bioactive compounds such as flavonoids, saponins, triterpenoids, and polyphenols4, which are characterized by a distinctive aroma in the rhizomes and leaves. Curcuma aeruginosa Roxb.) has pharmacological functions such as good antimicrobial and antioxidant properties so that it can be used as a preservative in food and spices, medicines, and herbal therapy5, preventing fungal infections6, treating rheumatic diseases7, menstrual disorders8, gastric medicine9, and cough and asthma medicine10. The bioactive components contained in Curcuma aeruginosa Roxb were 70% terpenoids, 1.9% flavonoids, 3.5%, steroids, 2.3% aromatic compounds, and 19.3% other components11.
The process of removing the active components from a material can be done through the extraction method12. An important first step in medicinal plant research is the selection of the extraction method as a starting point for the isolation and purification of the chemical components present in the plant13. One method commonly used is the maceration technique14, either by a cold or hot method (the conventional method). Maceration is a simple extraction method that is carried out by soaking natural materials using certain solvents for some time. Selection of the right material is very important to be able to extract the desired volatile compounds. Conventional extraction techniques have a number of drawbacks, the most prominent of which is that they require a lot of time and large volumes of solvent15,16.
Various modern extraction methods can be used, such as microwave assisted extraction (MAE)17, ultrasound-assisted extraction (UAE), and supercritical liquid extraction, which are more efficient, faster, and solvent efficient than conventional techniques18. Based on the research of Luna19, the UAE method can increase the reaction rate, yield, and quality of extraction because the ultrasonic waves used cause a cavitation effect, which produces a mechanical effect, so that the contact surface area between the solid and liquid phases will increase because the solvent penetrates deeper into the sample matrix and the solute will diffuse rapidly from the solid phase to the solvent20.
In this investigation, extraction was performed on Curcuma aeruginosa Roxb. using both traditional methods (hot and cold methods) and contemporary methods (UAE), with the goal of determining how the extraction method influences the antioxidant, anti-inflammatory, and cytotoxicity effects of the compound.
Curcuma aeruginosa Roxb. fresh collected from Bogor market (West Java, Indonesia). The rhizomes were cleaned and pureed using a blender, then stored in an airtight package until ready to be analyzed. Aquades, ascorbic acid, 1% oxalic acid, 10% trichloroacetic acid, 0.1% FeCl3, phosphate buffer (0.2 M pH 6.6), Curcuma aeruginosa Roxb. rhizome, 96% ethanol, 1% potassium ferricyanide, paper filter, bovine serum albumin, tris buffer saline, Artemia salina, and seawater.
Instrumentation:
The equipment used includes main and supporting equipment. The main equipment includes analytical balance, rotary evaporator, thermometer, hot plate dan UV-VIS spectrophotometer. The supporting eqipments includes 100 mL; 50 mL; 25 mL; 10 mL volumetric flask, dropper pipette, bulb, 5 mL serological type measuring pipette, 1000 and 100 μL micropipette, spray flask, 50 mL and sterile tube.
Preparation:
Conventional method extraction:
The conventional cold extraction method refers to Spigno's research with modifications21. The rhizome simplicial Curcuma aeruginosa Roxb. (10 g) was mixed with ethanol (96%) and left at room temperature for 48 hours. The hot method was carried out by adding distilled water (500 mL) to Curcuma aeruginosa Roxb. rhizome simplicia (10 g) and heating it at 40 °C with stirring for 1 hour and 30 minutes. The excess solvent was removed by rotary evaporator under low pressure.
Ultrasound-assisted extraction (UAE):
The method refers to Sukiman's research employing the UAE approach22. Seven grams of Curcuma aeruginosa Roxb. rhizome simplicia was weighed and placed in a 250 mL beaker. After adding a solvent consisting of technical ethanol at a concentration of 70%, the sample was agitated until it was completely submerged. Then the mixture was extracted with UAE for 30 minutes at an amplitude of 60 percent. In a weighted beaker, the liquid extract was filtered, and then the dregs were separated from the filtrate. In order to improve the overall yield, the ethanol solvent was evaporated out of the filtrate in an oven preheated to 40°C and left to do so until it was completely gone. The dry weight of the extract was used in the calculation of the yield percentage.
FRAP method antioxidant test:
Samples were prepared in a standard series from 40 mg/L to 160mg/L, then 0.4mL of 0.001M citric acid, 0.2 mL of 0.002 M Fe3+ solution, and 0.4mL of 0.2% o-phenanthroline were added. The solution was then measured with distilled water and homogenized. The standard series were then incubated for 35 minutes at 37 °C and measured with a UV-Vis spectrophotometer at 510nm. Gallic acid standard solution was used as a standard.
Anti-inflammation:
The anti-inflammatory test used the protein denaturation inhibition method with bovine serum albumin (BSA), which refers to Irawan's research with modifications23. Samples were prepared with standard series of 2, 4 and 8 mg/L and added 0.2% BSA solution in tris buffer saline (BTS). The solution was heated for 5 minutes in a water bath at 72°C, then cooled and measured with a UV-Vis spectrophotometer at 660 nm. The positive control was dichlorofenac sodium.
Cytotoxicity:
The cytotoxicity testing procedure refers to Parra's with modification24. In seawater, add shrimp larvae (Artemia salina) and aerate for 24 hours until the larvae hatch. An extract of the rhizome of Curcuma aeruginosa Roxb. was made in concentrations of 10, 100, 250, 500, and 100 ppm, and seawater was used as a control. Each series of extracts was added to the larvae and left for 24 hours at a temperature around 37 °C with constant oxigen supply25, and the IC50 calculation was carried out, which was obtained from linear regression calculations.
RESULTS:
Extraction method:
The solvents utilized in the extraction procedure are polar and environmentally safe distilled water and ethanol. The choice of solvent is critical in order for the secondary metabolites in the sample to be thoroughly dissolved. Based on research, Zohmachhuana extract (Curcuma aeruginosa Roxb.) from the methanol fraction has great antioxidant potential26. The yield of the extraction results from the rhizome of Curcuma aeruginosa Roxb. which was carried out in this study can be seen in Figure 1.
Figure 1: Percentage yield of Curcuma aeruginosa Roxb extract
Antioxidant FRAP method:
Testing for antioxidant activity with the FRAP method is a simple procedure to determine a compound that functions as an antioxidant. Antioxidants in the rhizome of Curcuma aeruginosa Roxb. are included in the category of strong antioxidants because the IC50 value is less than 50 ppm29. Antioxidant content in the rhizome extract of Curcuma aeruginosa Roxb. can be seen in Table 1.
Table 1: Antioxidant activity test result of the FRAP method
|
Sample |
Mean inhibiton (%)±SD |
IC50±SD |
|
Gallic acid |
32.1569±0.50 57.5881±0.20 74.5088±0.07 |
0.36±0.003
|
|
Cold method
Hot method
|
31.9867±0.40 43.8446±0.30 60.3448±0.30
3.0861±0.80 29.6121±0.20 51.1809±0.40 |
4.65±0.02
6.22±0.02 |
|
UAE |
11.4905±0.80 35.1469±0.40 55.5041±0.30 |
5.57±0.40 |
Cytotoxicity test:
One of the cytotoxicity test methods is the Brine Shrimp Lethality Test (BSLT), which can be used as a pre-screening of compounds thought to be efficacious in the field of medicine. The bioindicator used in the toxicity test was Artemia salina Leach shrimp larvae to determine the lethal concentration (IC)31. This is because Artemia salina is cheap and does not require ethical code approval for its use. The results of the cytotoxicity test can be seen in Figure 2.
Figure 2: Percent mortality of Artemia salina
Based on Figure 2, the IC50 obtained in the cold, hot, and UAE methods was 40.26 mg/L, 645.67 mg/L, and 22.31 mg/L, respectively. The cytotoxicity test results supported the use of Curcuma aeruginosa Roxb. rhizome extract. The conventional method (hot and cold) and the UAE method are classified as toxic (IC50 < 1000 ppm)32, and the UAE extract has the highest toxicity with an IC50 value of 22.31 ppm.
Toxic effects were related to the content of bioactive compounds contained in the extract33. Bioactive compounds will affect the structure of the cytoplasm and can cause leakage of macromolecules and cellular lysis, so that organisms will experience death34.
Anti-inflammatory test:
Inflammation is the response of living tissue to damage whether due to physical, chemical or microbiological35. The whole plant is used for anti-inflammatory, immuno stimulating effect and in piles36. In this study, the highest denaturation inhibition was found in UAE extract with an IC50 value of 2.96 ppm, as shown in Figure 3. This is because the UAE method of extracting secondary metabolites from Curcuma aeruginosa Roxb. rhizome extract had more than other methods. The ability to inhibit is influenced by the content of phytochemical compounds37.
Figure 3: Anti-inflammatory activity of Curcuma aeruginosa Roxb. rhizome extract
According to Oanh, the main constituents of the rhizome extract of Curcuma aeruginosa Roxb. are the terpenoids (sesquiterpenes, monoterpenes, esters, and steroids)38. Terpenoid compounds have been reported to inhibit the release of prostaglandins from source cells, so that the formation of histamine, prostaglandins, and other chemical mediators that result in inflammation can be inhibited and provide analgesic effects39. Flavonoids will directly inhibit the lipoxygenase pathway and activate free radicals, which can attract various inflammatory mediators. In addition, Ko and his friends reported in their research that triterpenoid compounds have the ability to stabilize the membrane, so that the rhizome of Curcuma aeruginosa Roxb. has the potential to be used as an anti-inflammatory drug40.
CONCLUSION:
The ultrasound-assisted extraction method (UAE) provides more optimal results compared to the cold and hot methods. Extraction with UAE results in a higher percent yield with strong antioxidant properties, high toxicity, and high anti-inflammatory activity (through protein denaturation inhibition).
CONFLICT OF INTEREST:
The authors who wrote this study said that they have no other interests that could be seen as competing with this one.
ACKNOWLEDGEMENT:
The conclusions section should come in this section at the end of the article, before the acknowledgements.
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Received on 31.01.2023 Modified on 19.05.2023
Accepted on 22.07.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(5):2180-2184.
DOI: 10.52711/0974-360X.2024.00343