Decrease of Blood TNF-α by Myrmecodia Pendans Ethanol Extract (Study on Sprague Dawley Rats with Pulpitis)

Pulpitis is a dental pulp tissue inflammation usually caused by bacteria invasion that stimulates the release of inflammation mediators...


Introduction
Inflammation is a normal respond protective of tissue toward physical traumatic, chemical injury or microbiology activity [1]. Inflammation divided into acute and chronic [2]. Pulpitis is inflammation of pulp tissue that commonly occurred and often found on dental practice. Pulpitis may due to variety of factors such as bacterial invasion, trauma and iatrogenic [3,4]. Pulp inflammation activates variety of biological system such non specific inflamed reaction mediated by histamine, bradykinin, and arachidonic acid [5]. The final stage of inflamed respond is to deliver the protein plasm and phagocyte into injured area for isolation, destruction or inactivation of incoming agent, clean the debris and prepare tissue for wound healing process [6]. Porphyromonas gingivalis (P.gingivalis) is an anaerobic Gram negative bacteria that expressed several virulent factors like fimbriae lekin-like adesina, polysaccharide capsule, lipopolysaccharide, hemagglutinin, hemolytic, vesicle membrane, and proteolytic enzymes that cause inflam-mation of gingiva, tissue destruction and tooth loss [7]. Lipopolysaccharide (LPS) is an important part of P.gingivalis that responded by inflammatory cells that release inflammatory mediators indeed inflamed reaction [8]. Tumor Necrosis Factor Alpha (TNF-α) is a derivative protein of monocyte that has pro inflammatory and immunomodulatory effect. This type of cytokine as inflammatory mediator will stimulate bone resorption, prostaglandin synthesis, and protease production by cells such as fibroblast and osteoblast [9].
TNF-α is also a main cytokine of acute inflamed respond towards negative Gram bacteria.
The severed infection may trigger a lot of TNF-α production resulted in systemic reaction. Lipopolysaccharide is a potent stimulant to TNF-α secretion by macrophage [10]. TNF-α is suspected as an important inflammatory mediator. Increase of TNF-α has toxic potential effect like hypersensitivity reaction [11,12]. Indonesia has a lot of potential herbal plants [13]. Medical world recently used traditional medicine through the usage of medicinal plants. One of these potential plants is Sarang semut (Myrmecodia pendans) that used empirically as anti tumor, anti-cancer and diabetes remedies [14]. Ethanol extract of this plant has anti inflammation [15]. The phytochemical content of this plant like flavonoid compound, triterpenoid/steroid and saponin [16,17]. Previous study revealed that the flavonoid content has anti inflammation activity [17,18].
Flavonoid has role in inactivation of carcinogenic and cells proliferation also inhibition of angiogenesis [19]. These activities have been proven by previous studies. However there is no specific study of this fact on hard tissue therefore the purpose of this in vivo study is to find out the anti-inflammation effect of ethanol extract M.pendans toward the blood TNF-α level of Sprague Dawley rats that has molar tooth induced pulpitis by P.gingivalis. The blood TNF-α level was measured by ELISA method.

Methods and Materials
This study was approved by the Animal Care and Use Committee

Pulpitis Treatment
On day 2 (48 hours), the temporary filling of group III and IV

The Blood Sample Collection
Subjects were general anesthetized. The blood samples were collected after the execution of subjects on day 2, 4, 7, 14 for 3 subjects of each group. As much as 4mL heart blood was collected that injected into the tube and centrifuged 1.000rpm for 10 minutes to filtrate the serum. Put the serum into tube and keep on -20 0 C before tested with ELISA method for the TNF-α level ( Figure 3).

Result and Discussion
The samples that were placed within well needs to be mapping at the ELISA kit ( Table 1). The plate of samples was measured with the ELISA reader (λ=450 nm) within 30 minutes to obtain the absorbance value ( Table 2). The results was then calculated with computer-based curve fitting software using a Four Parameter Logistics (4PL) curve fitting algorithm (Figure 8). Fit results was obtained from the software regression formula of four parameter logistic as followed:     Figure 9): From   production [22,23]. The high increase of TNF-α insult the systemic reaction [24]. In inflammation, TNF-α has role to increase the prothrombotic function, stimulate adhesion molecule of leukocyte, induct the endothelial cell, manage macrophage activity and immune responding within tissue by stimulate growth factor and other cytokines [24,25]. Today, medicinal plant widely used such as Myrmecodia pendans that has phytochemical compounds such as flavonoid, triterpenoid/steroid, and saponin [14,16,17]. Previous study revealed that flavonoid has anti inflammation, antioxidant, and antimicrobial, defend the neutrophil degranulation, leukocyte accumulation, inflammatory mediator releasing like histamine and prostaglandin [26,27].
Post Hoc LSD test showed that on day 4, there was significant It means that the inflammation phase of wound healing process has been decreased to go to later stage of healing process [2].

Conclusion
Ethanol extract of M.pendans has anti-inflammation effect that has been proven by the decrease of inflammation mediator TNF-α blood level after day 4. On day 7, the TNF-α blood level of Ca (OH) 2 group is lower than M.pendans groups. However, on day 14, the anti-inflammation effect of M.pendans equals to those of Ca(OH) 2 as a commercial product of inflamed pulp tooth.