T . Kisnanto et al .: Radioprotection 2024 , 59 ( 2 ), 144 – 151 145 Radioprotectors can protect against radiation because of their ability to bind radicals resulting from the radiolysis of water . Furthermore , antioxidants can act as radioprotectors due to the similarities between oxidative stress and radiation injury . Therefore , an ideal radioprotector should offer significant protection against the long-term effects of radiation exposure ( Caro et al ., 2012 ; Obrador et al ., 2020 ). At this time , the use of natural compounds to protect human health is increasing . The choice of radioprotector is more emphasized in plant products ( Obrador et al ., 2020 ; Javadi et al ., 2022 ; Jit et al ., 2022 ).
Several studies have proven that many traditional plants have antioxidant capacity and act as radical scavengers . Temulawak ( Curcuma xanthorrhiza Roxb .) is a plant from Indonesia that is useful for medicine . The most active components are curcumin , which results from the secondary metabolism of temulawak and is considered to have a strong antioxidant capacity ( Atun et al ., 2020 ; Rosidi , 2020 ; Shabeeb et al ., 2020 ). Garlic ( Allium sativum L .) is scientifically proven to have antioxidant capacities . Its antioxidant activity is in water-soluble organosulfur compounds , such as S-allilcysteine and S-allylmercaptocysteine . ( Caro et al ., 2012 ; Bertrand et al ., 2016 ). N-acetylcysteine ( NAC ) is a derivative of cysteine , a thiol-reducing agent found in vegetables such as garlic , onions , and peppers . NAC is commonly used as a standard antioxidant because of its ability to inhibit oxidative stress and suppress DNA damage ( Mercantepe et al ., 1977 ; Kilciksiz et al ., 2008 ).
The present study focused on exploring the ability of temulawak and garlic extract to suppress oxidative damage caused by 6 Gy TBI in Wistar rats .
2 Materials and methods
2.1 Materials and extract preparation
Male Wistar rats , 8 – 12 weeks old , weighing 180 – 220 g , were used in the present study . The animals were purchased from iRATco Veterinary Laboratory Service , Bogor , Indonesia . The animals were housed and acclimatized in temperature conditions ( 21 – 24 ° C ) and optimum lighting ( 12 h dark / light cycle ) in the Integrated Animal Laboratory , National Research and Innovation Agency ( BRIN ), Indonesia . The animals were fed a pelleted rodent diet ( iRATco ), water ad libitum , and weighed regularly once a week .
Temulawak and garlic were obtained from Indonesian Farmers Store , while NAC was purchased fromSigma-Aldrich ( product number A7250 ).
Temulawak extract was made at the Indonesian Medicinal and Aromatic Crops Research Institute ( IMACRI ) using the phytochemical method . The dose of temulawak extract was used 100 mg per kilogram body weight ( mg / kg BW ) ( Liju et al ., 2020 ). At the same time , the garlic extract was done by following the previously published method ( Kisnanto et al ., 2020 ).
2.2 Animal treatment , irradiation , and sample collection
Twenty male Wistar rats were classified into five groups ( N = 4 each ): control ( non-irradiation ), 6 Gy , temulawak extract + 6 Gy , garlic extract + 6 Gy , and NAC + 6 Gy . The extracts were administered gavage orally for seven consecutive days .
Six Gy with a dose rate of 1 Gy / min TBI was given on the eighth day using a gamma ray cobalt-60 IRPASENA device at the Research Center for Radiation Process Technology , BRIN , Indonesia . After 24 h post-irradiation , all rats were anaesthetized and euthanized with ketamine-xylazine ( 75 – 100 mg / KgBW ) intraperitoneally . Surgery was performed by opening the thoracic cage , and the blood was removed directly using a 10 mL syringe . The blood was placed into the EDTA tube for lymphocyte isolation . After blood collection , the liver and the spleen were immediately removed and preserved at �80 ° C for further use .
2.3 Lymphocyte isolation and tissue homogenate
Hasan Basri et al . ( 2017 ) determined the lymphocyte isolation procedure with slight modification . Anticoagulated blood samples were mixed with 3 mL of Phosphate Buffered Saline ( PBS ) pH 7.6 in a centrifiigation tube . The blood cells were carefully mixed through the walls of the tube containing 3 mL of lymphocyte-separating medium ( Histopaque 1077 ), followed by centrifugation for 30 min at 1500 rpm . The layer of lymphocyte cells looks slightly greyish white , located between the blood plasma and Histopaque , and then transferred to a centrifuge tube containing 5 mL of PBS ( pH 7.6 ), followed by centrifugation for 15 min at 1000 rpm . The lymphocyte cells were washed three times with PBS ( pH 7.6 ). The supernatants were removed , while the pellets were resuspended by adding RPMI-1640I medium ( Sigma- Aldrich ), stored at �80 ° C .
The liver and the spleen tissue ( each 100 mg ) were dissolved in 0.5 mL of 0.01 MPBS ( pH 7.4 ) and then homogenized using a homogenizer machine . The mixture was centrifuged for 10 min at 3500 rpm . The supernatant was taken and stored at �80 ° C .
2.4 Measurement of MDA level
The MDA levels were measured based on the method of Wills ( 1971 ) with some modifications . The MDA level was assayed using spectrophotometry ( Genesys 20 ), and absorbance was read at a wavelength of 530 nm . The MDA level was expressed in nmol / mg tissue .
2.5 Measurement of total protein concentration
Determination of the total protein concentration in the liver and spleen tissue was based on the method by Mercantepe et al . ( 1977 ) with some modifications .
2.6 Measurement of GSH level
The GSH concentration was assayed based on the method of Ellman ( 1959 ) with modifications . The GSH concentration was assayed using spectrophotometry ( Genesys 20 ), and absorbance was read at a wavelength of 412 nm . The GSH