الفهرس 
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Abstract
Dextrin is a polysaccharide but is small and of low complexity. Naringin is a flavonoid widely known for its pharmacological properties. The present study designed to evolve a new formula of naringin-dextrin nanoformula (NDN), to improve bioavailability and therapeutic efficiency of naringin against carcinogenisty and followed by investigation the safety and anti-cancer efficacy of free naringin and NDN using in vitro and in vivo models.
Firstly, preparation and characterization of NDN formula using TEM, particle size distribution, entrapment efficiency, XRD, and FTIR were used to characterize NDN. In vitro cytotoxicity study on HepG2 cell line was conducted to evaluate the safety of formula. Other biological activity of NDN was evaluated such as cell migration assay, ROS generation, DNA fragmentation, and cell cycle arrest, proinflammatory factors, antiapoptotic, proapoptotic expressions and some cell signals.
Next, experimental model of carcinogenicity rats were divided to four groups ten for each group and were subjected to the following treatments:
group I (Normal group): The included rats of this group received, by intraperitoneal (i.p.) route, the equivalent volume of saline (0.9% NaCl) at the beginning of experiment for 2 weeks, two times per one week. Starting from the 3rd week, the rats received the equivalent volume of 1% tween 80, by oral gavage, four times per week for three weeks. This group was also orally given the equivalent volume 1% CMC (1 % w/v) as vehicle every other day till the end of the experiment.
group II (DEN/2AAF-administered control group): The animals within this group were given twice, i.p., doses of DEN (150 mg/kg b.w.) dissolved in 0.9% saline at the beginning of experiment. One week after the last injection of DEN, 2AAF (20 mg/kg b.w.), in 1 % tween 80, was administrated orally by gavage four days per week for three weeks (De Lujan Alvarez et al., 2002). The rats of this group were also orally administered the equivalent volume of 1% CMC (1 % w/v) daily for 20 weeks.
group III (DEN/2AAF-administered group treated with naringin): This group was given DEN/2AAF as group II and was orally treated with naringin at a dose of (10 mg/kg b.w.) dissolved in 1% CMC (1 % w/v), every other day till the end of the experiment.
group IV (DEN/2AAF-administered group treated with NDN): This group was given DEN/2AAF as group II and received a dose of (10 mg/kg b.w.) NDN dissolved in 1% CMC (1 % w/v) by oral gavage every other day until the end of the experiment.
At the end of the experiment, the rats were anesthetized by diethyl ether and blood samples were obtained from jugular vein and after decapitation and dissection, tissue samples, liver and lung were excised for biochemical analysis, histopathological investigation and molecular assay.
In the present study, the animals treated with DEN/2AAF showed hepatocellular carcinoma and lung cancer.
Hepatocellular carcinoma is elicited by the increased levels of serum enzymes ALT, AST, ALP activities and total bilirubin level as well as lowered albumin levels. The treatment of DEN/2AAF-administered rats with naringin and NDN led to marked improvements in these liver function parameters in serum. Moreover, NDN was more effective than naringin
The obtained data also revealed a significant increase of AFP, CEA and CA 19.9 levels in DEN/2AAF-administered rats. The treatment of DEN/2AAF-administered animals with naringin and NDN successfully improved these changes.
Concerning liver oxidative stress and antioxidant defense system, the increase of LPO and the depleted GSH content as well as the decreased GPx and SOD activities of DEN/2AAF-administered rats. these responses as well were significantly reversed by naringin or NDN.
Naringin and NDN suppressed DEN-induced upregulation of the proinflammatory factors TNF-α, IL-1β, NF-κB, and IL-8, and directly upregulated NRF2. Moreover, naringin and NDN significantly reduced the hepatic expression of antiapoptotic Bcl-2, and increased proapoptotic Bax, P53, and PDCD5 expressions. Naringin and NDN also reduced expression of IQGAP1, IQGAP3, Ras signaling and Ki-67, while increasing expression of IQGAP2, changes reversed by subsequent naringin or NDN treatment.
On the other hand, liver sections of DEN/2AAF-adminstered rats showed disrupted hepatic lobular structure. Further, well-differentiated tumor cells resembling hepatocytes were observed forming trabeculae, cords, and nests. There were also growing septa between the hepatic lobules. In regions with disruption of the normal hepatic lobular structure, the tumor cells formed glandular or lamellar structures. Dilated central veins, some with a signet ring appearance, binucleated hepatocytes, and hepatocytic steatosis. Clear hepatocyte foci, vacuolated hepatocytes, deeply eosinophilic foci of hepatocytes, and dysplastic hepatocytes with mitotic figures were also found. There was also an increase in fibrous tissue foci containing inflammatory cells, hepatocytes with dark shrunken nuclei, activated Kupffer cells, binucleated hepatocytes, and hepatocytes with large hyperchromatic nuclei and a prominent enlarged nucleolus or multiple nucleoli. The treatment of these animals with naringin and NDN successfully prevented most of these biochemical and histological alterations.
Lung cancer-induced by DEN/2AAF was evidenced by elevations of LPO and the depleted GSH content as well as the decreased GPx and SOD activities of DEN/2AAF-administered rats and these responses as well were significantly reversed by naringin or NDN.
In addition, rats receiving DEN/2AAF presented lung cancerous injuries with aggregation of tumor cells and diffuse thickening in interstitial tissues, noticed hemorrhage and congested blood vessels. Within the alveoli, tumor cells grow following lepidic and papillary growth patterns. Additionally, macrophages and other myeloid cells invaded adjacent alveolar air spaces. Tumor cells also displayed an acinar pattern of invasive adenocarcinoma. A vessel was seen being invaded by a tumor, diffuse thickening in interstitial tissues, Interstitial hemorrhage, sloughing of cell into alveoli and mitotic figure. The treatment with naringin and NDN successfully improvement the histological alterations.
Naringin and NDN exerted antitumor effects by downregulating the levels of anti-inflammatory biomarkers and stimulating the expression of pro-inflammatory biomarkers. Naringin and NDN also significantly reduced Bcl-2 expression and increased Bax and P53 expression. Moreover, naringin and NDN induced a decrease in Ki-67 expression
In conclusion, the present results clearly evidenced the loading of naringin on dextrin nano and formation of the novel NDN with sustained release properties and high loading efficiency. Moreover, the in vitro cytotoxicity results revealed the safety of formula for in vivo applications. The in vivo results displayed that NDN exhibited better anticancer efficacy relative to free naringin that could be attributed to prolonged-release properties, improved absorption, and increased bioavailability.
The actions of NDN were mediated by decreasing oxidative stress, improving antioxidant status, supressing inflammation and cell proliferation, and enhancing the expression levels of apoptotic genes.
Hence, the present study illustrated that NDN is safe and biocompatible and displayed better bioavailability and efficiency against liver and lung cancer comparing to free naringin. Thus, this formula could be suggested as a potential candidate for management of cancer, also for further in vivo and clinical investigations.