Diabetic Effects of Euphorbia Tithymaloides Ethanol Extract

Indonesian J. Pharm. Vol. 29 No. 1 : 1 – 9 ISSN-p : 2338-9427 DOI: 10.14499/indonesianjpharm29iss1pp1 Research Article In vitro evaluation of anti-inflammatory and anti- diabetic effects of Euphorbia tithymaloides ethanol extract Theresia Galuh Wandita1, Najuma Joshi1, Joseph dela Cruz2, Seong Gu Hwang1* 1. Laboratory of Applied ABSTRACT Biochemistry, Department of Euphorbia tithymaloides L., a native plant of tropical and Animal Life and subtropical areas in Asian countries which has been known as Environmental Science, traditional medicine with a wide range of healing effects, such as Hankyong National anti-hemorrhagic, anti-diabetic, anti-bacterial, anti-inflammatory, University, South Korea, 327 and anti-tumor activity. The present study was orchestrated to Jungang-ro, Anseong-si, evaluate the potential anti-inflammatory and anti-diabetic effects Gyeonggi-do, 456-749, South of Euphorbia tithymaloides ethanol extract (ETE). The anti- Korea inflammatory and anti-diabetic activities were studied through 2. Department of Basic the treatment of RAW 264.7 murine macrophages cells and 3T3- Veterinary Sciences, College L1 adipocytes with various concentrations of ETE (50, 100, 200, of Veterinary Medicine, University of the Philippines and 400µg/mL). The results showed that ETE below 400µg/mL Los Banos, Philippines has no negative effect on RAW 264.7 cell proliferation. ETE decreased nitric oxide production in macrophages RAW 264.7 cell Submitted: 11-11-2017 line and reduced the protein expression of cyclooxygenase 2, Revised: 25-12-2017 interlukin-6, inducible nitric oxide synthase, tumor necrosis Accepted: 06-01-2018 factor-α and nuclear factor-kB in a dose-dependent manner. In 3T3-L1 preadipocytes, the increase of ETE concentration did not *Corresponding author affect cell viability, but significantly enhanced adipogenesis Seong Gu Hwang through increase in differentiation and the expression of peroxisome proliferator-activated receptor gamma, CEBPα, Email: glucose transporter type 4 and insulin receptor substrate 1. ETE [email protected] also stimulated 2-NBDG uptake through activation of the insulin signaling pathway through insulin-sensitizing PPARγ agonists in a dose-dependent manner. In conclusion, our findings that ETE has immunomodulatory activity and anti-diabetic effects, which can be used for treating inflammation and type II diabetes. Keywords: Anti-diabetic, Euphorbia tithymaloides, Glucose uptake, Immunomodulation, Nitric oxide INTRODUCTION Inflammation is the most common type Medicinal plants have been used since of response employed by the body as a defense ancient times. Owing to the side effects of mechanism against harmful stimuli originating some modern medicines, The World Health from the surrounding environment. The Organization Expert Committee has inflammatory response is characterized by the recommended the use of traditional medicinal coordinated activation of various signaling plants for the treatment of some diseases. pathways that regulate the expression of both Ethno-botanical knowledge suggests that pro- and anti-inflammatory mediators in approximately about 800 plants have resident tissue cells and leukocytes recruited immunomodulatory and anti-diabetic potential from the blood. Nuclear factor kappa B (NF- (Singh, 2011). Medicinal plants may provide a kB) plays an important role in the activation of good alternative for treating diabetes mellitus genes involved in the immune responses. This and inflammation, and counter the high cost, transcription factor increases many cytokines side effects, and poor availability of the gene expression, such as interleukin (IL-1β, IL- currently available drugs in developing 2, IL-6, IL-8, and IL-12), cyclooxygenase countries such as the Philippines. (COX)-2, and tumor necrosis factor (TNF)-α. Volume 29 Issue 1 (2018) 1 Potential effects of Euphorbia tithymaloides They possess NF-kB binding sites and have to evaluate the potential effects of E. been shown to be transcriptionally regulated by tithymaloides ethanol extract as an NF-kB. Moreover, NF-kB transcriptionally immunomodulatory and anti-diabetic herbal regulates the gene for inducible nitric oxide medicine. synthase (iNOS) (Ghosh et al., 2014). Diabetes mellitus (type II diabetes) is MATERIALS AND METHODS caused by defective insulin production or Reagents insulin resistance. The prevalence of diabetes RAW 264.7 macrophages cell line and has increased at an alarming rate in Asian 3T3-L1 mouse preadipocyte cells were obtained countries including the Philippines. In 2016, the from ATCC (Manassas, VA, USA). Dulbecco’s death of, 33.2 million people in the Philippines modified Eagle’s medium (DMEM), bovine was attributable to diabetes. Adipogenesis is calf serum (BCS), phosphate buffer saline regulated by a complex gene expression such as (PBS), fetal bovine serum (FBS), and 2-(N-(7- peroxisome proliferator-activated receptor nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2- gamma and CCAAT/enhancer binding protein deoxyglucose (2-NBDG) were purchased from (CEBPα). It is considered as a nuclear receptor GIBCO/Invitrogen (Carlsbad, CA, USA). in adipocytes (Winrow et al., 1996; Rosen et al., Lipopolysaccharide (LPS), bovine serum 2000; Vergara et al., 2015). Adipocytes are albumin (BSA), isobutyl-3-methyl-xanthine turning up as a potential medicine for (IBMX), insulin (INS), and dexamethasone treating diabetes mellitus (Hassan et al., 2007). (DEX), were acquired from Sigma-Aldrich (St. The mechanism involves insulin-triggered Louis, MO, USA). Primers used in these relocalization of a glucose transporter (GLUT4), experiments were purchased from Macrogen which is highly expressed, and insulin-stimulated (South Korea). The antibodies used in these glucose uptake in adipocytes by the rapid experiments were obtained from Cell Signaling translocation of GLUT4 from intracellular Technology (Danver, Massachusetts, USA). stores to the plasma membrane (Choi et al., 2009; Manaharan et al., 2013). Plant material Euphorbia tithymaloides belongs to the The dried whole plant of E. tithymaloides family Euphorbiaceae. It is a perennial succulent was powdered and successively extracted in 80% spurge that has been used to treat inflammation of ethanol for 24h. The ethanol extract of this and cancer in Asian countries, especially the plant (ETE) was filtered, and evaporated under Philippines. The root of this plant has been vacuum. The freeze-dried sample was shown to be effective against intestinal worms powdered and stored at -80ºC until use. and to reduce inflammation when ingested (Jain Hereafter, the extract was mixed with the and Srivastava, 2005; Heinrich et al., 1992; Adzu medium. et al., 2008). The extracts of the leaves have been used to treat conditions such as asthma, Cell culture and cell viability assay laryngitis,and persistent coughing. In an earlier RAW 264.7 macrophages cell line and study, various bio-active compounds were 3T3-L1 preadipocytes cells were grown in found in this plant such as octacosanol, beta- DMEM with 10% FBS and DMEM with 10% sitosterol, cycloartenone, and oxime. One of BCS, respectively. To activate macrophages, these, octacosanol, significantly decreased the 10µL/mL of lipopolysaccharide was added to pro-inflammatory cytokines genes expression the culture medium. To induce the in in-vitro and in-vivo studies (Oliveira et al., 2012; differentiation of 3T3-L1, the cells were Castaldo and Capasso, 2002; Njamen et al., cultured with fresh induction of 1µM 2003). Galactose-specific lectin, also contained dexamethasone, 0.5mM isobutyl-3-methyl- in this plant might be useful for the treatment xanthine, and 10µL/mL insulin. of diabetes mellitus as mentioned by Joseph Cell viability was determined by using and Priya (2011). Furthermore, there have been cell counting kit (CCK)-8 assay (Dojindo no reports on the anti-inflammatory and anti- Laboratories, Japan). The cells, RAW 264.7 diabetic activities of this plant in macrophages macrophages cells (1x105 cell/well) and 3T3-L1 and 3T3-L1 cell. Thus, our present study aimed preadipocytes cells (1x104 cell/well) were 2 Volume 29 Issue 1 (2018) Theresia Galuh Wandita seeded and treated with various concentrations fluorescent material in the wells, the cells were of ETE (50, 100, 200, 400µg/mL) for 24h. washed twice with PBS after incubation. The CCK solution (1µg/mL) was then added to fluorescence retained in the cell monolayers at each well. The absorbance was quantified at 540nm using an ELISA microplate reader. 450nm by using an ELISA microplate reader. Negative control (NC) and positive control (PC) Six replicates were performed for each assay. were designed to identify the effect of ETE in 3T3-L1 preadipocytes cells. The addition of Anti-inflammatory activity insulin was considered as positive control Anti-inflammatory activity was which helps to enhance glucose uptake to the determined from the nitrite oxide production cells. and the expression of pro-inflammatory To measure the accumulation of cytokines. Nitric oxide production was triglycerides, adipocyte differentiation was determined as the nitrite concentration in induced at the end of the treatment period, culture medium by using a colorimetric assay. which is a common measurement of PPARγ The cells were seeded in a 96-well plate and agonist activity. The differentiated adipocytes treated with different concentration of ETE. cells were fixed with formaldehyde and washed Negative control (NC) and positive control with distilled water. Oil-Red-O solution (0.5% (PC) were designed to identify the effect

Load more