2.1 Materials
White turmeric rhizomes aged 9–10 months were obtained from CV Windra Mekar. The analysis used various chemicals including distilled water, pure ethanol (Merck), BHT (Butylated Hydroxytoluene, Sigma), DPPH solution (2,2-diphenyl-1-picrylhydrazyl), Sigma-Aldric) 0,1 mM, pure Folin-cilocalteu (Merck), Na2CO3 (Merck, 20%), saturated Na2CO3, Na-CMC (Sodium Carboxymethyl Cellulose) suspension stimuno dan S. aureus 1,0x108 sel/mL.
2.2 Equipment
The chemical equipment used include measuring cups (pyrex Iwaki), beaker glass (pyrex Iwaki), test tubes (pyrex Iwaki), measuring pipettes (pyrex), volumetric flasks (pyrex Iwaki), dropper pipettes, stirring rods, micro pipettes (Acura 825 autoclavable), analytical scales (Ohaus Pioneer PA214), filter paper (Whatman no 42), vortex (Maxi Mix II type 37600) and UV-Vis spectrophotometer (Shimadzu UV mini 1240).
2.3 Measurement Procedure
Various phases in this research involve the conversion of white turmeric into powder with variations in the parts of the white turmeric rhizome (main rhizome and tiller) and the experiment includes altering the duration of steam blanching within a range of times (0, 2.5, 5, 7.5, and 10 min). and analysis of white turmeric powder in vitro and in vivo.
2.4 Preparation of White Turmeric Powder
White turmeric is sorted to separate the main rhizome and tillers. After sorting, white turmeric is peeled and washed. The washed white turmeric undergoes steam blanching treatment with durations of 0; 2.5; 5; 7.5; and 10 minutes. The blanched white turmeric is drained, sliced to a thickness of 1–2 mm, and dried using a modern cabinet dryer at 55 ºC for 11 hours. The dried white turmeric is finely blended and sieved through a 60-mesh sieve.
2.5 Method of antioxidant activity using DPPH (2,2-diphenyl-1-pikrilhidrazil) method [26]
The assessment of antioxidant activity utilizing the DPPH method was conducted by taking 0.2 ml of sample and then adding 0.1 mM DPPH solution as much as 3.8 ml, then vortexed for 1 minute and kept in the dark at room temperature for 30 minutes during the incubation period which is 28°C. Blank was made using ethanol. The spectrophotometer was utilized to gauge the absorbance at λ 517 nm. The activity of free radical scavenging was indicated as % Radical Scavenging Activity.
%RSA = \(\text{1-}\frac{\text{absorbance}\text{ sampl}\text{e}}{\text{absorbance}\text{ control}}\text{ ×100%}\) ........................................................................................................................(1)
2.6 Total Phenolic Content (TPC) Folin-Ciocalteu [27]
The extract's total phenolic content was quantified using the Folin-Ciocalteu reagent with minor modification. 50 µl of the sample were combined with 250 µl of Folin-Ciocalteu solution, left undisturbed for 1 minute, followed by the addition of 750 µl of 20% Na2CO3. After vortexing, distilled water was introduced to achieve a total volume of 5 ml. Subsequently, the incubation was conducted at ambient temperature for 2 hours, then measured with a spectrophotometer at λ 760 nm.
TPC (mg GAE/g wb)=\(\frac{\text{concentration ppm}\text{ ×100}}{\text{weight of sample}\text{ (g)}}\) ............................................................................................................(2)
TPC (mg GAE/g db)= \(\frac{\text{100}}{\text{(100 - }\text{moisture content)}}\text{×TPC }\text{wb}\) .............................................................................................(3)
2.7 Imonomodulator Assay in vivo
The measurement of SOD, IL-1, IL-6, IL-8, IgE, IgG, and IgM was applied to the blood of experimental rats. The categorization involves 24 male Wistar rats, segregated into 4 groups. Group 1 serves as the control with a standard feed, group 2 is a 0.5% Na-CMC suspension for negative control, group 3 is a suspension of stimuno 0.9 mg / 200 g b / b, group 4 is white kunir 16.2 mg / 200 g b / b. On the 8th day, blood was drawn using a blood glucose meter. On the 8th day, blood was drawn using a micropipette. IL-1, IL-6, and IL-8 were determined using the spectrophotometric method according to the kit employed. All samples from the treatment were adapted for 1 week, then given treatment according to the standard feed of each group. The treatment was conducted for 16 days, and on the 14th day, an injection of S. aureus at a concentration of 1.0x108 cells/mL was administered, with a volume of 0.1 mL intraperitoneally (directly injected into the peritoneal cavity).
SOD determination was carried out using 0.6 mL hepatic supernatant reacted with 2.70 mL of 50 mM Na2CO3 buffer containing 0.01 mM EDTA (pH 10), 0.06 mM, 10 mM of xanthine, 0.5% BSA 0.03 mM and 2.5 mM NBT as much as 0.03 mM. Subsequently, xanthine oxidase was introduced at a concentration of 0.04 units and left to stand. After 30 min, it was measured at λ 560 nm. Calculation of SOD levels as follows [28].
Activity SOD (%) = \(\left(\text{1-}\frac{\text{A}}{\text{B}}\right)\)× 100% ......................................................................................................................(4)
Description:
A = Absorbance of sample
B = Control absorbance
Determination of immunomodulatory effects on antibody values (IgE, IgG and IgM) in male rats is measured by the antibody titer method by agglutination [29]. Determination of the immunomodulatory effect observed on day 17th, blood samples of each rat were taken through a vein in the tail and tissue was taken.
2.8 Statistical Analysis
The research design employed is a Completely Randomized Block Design (CRBD) with 2 treatment factors, namely variation of white turmeric rhizome parts (main and tiller) and variation of blanching time using the steam blanching method (0; 2.5; 5; 7.5 and 10 min).