Protective Effects of Nephrolepis exaltata Mask against Blood Glucose Level (A Study of Wistar Rats with Chronic Exposure to Nanosilica)

Diponegoro University, Semarang City,Central Java, Indonesia; evanwidiono@yahoo.co.id Department of Clinical Pathology, Diponegoro University, Semarang City, Central Java, Indonesia; setyadi85@yahoo.co.id Department of ENT, Diponegoro University, Semarang City, Central Java, Indonesia; awalpras@yahoo.com Department of Anatomical Pathology Diponegoro University, Semarang City, Central Java, Indonesia; udadisadhana@yahoo.com Department of Physics, Diponegoro University, Semarang City, Central Java, Indonesia; agus_fadhil@yahoo.com


Introduction
Diabetes Mellitus (DM) is a cluster of metabolic disorder with high blood glucose level. According to World Health Organization (WHO) diabetes mellitus is the 6th most common death cause 1 . Health is often discussed in urban areas because urban residents have higher risk factors of developing diseases despite having sufficient health care.
Particulate Matter (PM) is a term describing solid or liquid particle found in air both in large or small size. Urban area is one of the areas with high PM concentration, which affects respiratory tracts 2

. Research showed
Protective Effects of Nephrolepsis exaltata Mask against Blood Glucose Level (A Study of Wistar Rats with Chronic Exposure to Nanosilica) that as PM concentration in air increases the prevalence of DM increases proportionally 3 .
Oxidative stress is an imbalance between free radicals and antioxidants in the body 4 . Oxidative stress can happen from PM getting inside the body. A research has shown that systemic oxidative stress associates with insulin resistance 5 .
Silica is a PM component which is sized 0,02 µm thus can be inhaled into respiratory tracts. It can induce inflammation and scar tissue formation in respiratory tracts 2,4 . Utilization of nano sized particles is gaining fame in various fields, among them is nanosilica as basic ingredient of fertilizer 6 .
Indonesian farmers with constant exposure to nanosilica fertilizer can develop serious health problems including elevated blood glucose level 7 . In light of this situation, we aim to figure out the effect of nanosilica particle against blood glucose level elevation compared to streptozotocin induced blood glucose level elevation.
Wearing mask is an attempt to reduce risks of developing diseases and to prevent the exposure of pollutant. Nephrolepis exaltata (N. exaltata) mask is highly effective in reducing risks of developing disease 8 . In an earlier research N. exaltata was found to be able to absorb formaldehyde, xylene, tricholoethylene, and carbon monoxide from air which are the causative agents of oxidative stress. Given this situation, we aim to study the effect of wearing N. exaltata mask against oxidative stress due to chronic nanosilica exposure by evaluating the blood glucose levels in rats 9 .

Samples and Tested Variables
This study is a true experimental study with pretest-post test only design, with 28 male Wistar rats as the studied samples. Tested variables were given for 28 days. The samples were divided into 4 experimental groups (Table 1), each group had 7 samples meeting these criterias: Prior to variable testing all Wistar rats were acclimatized to the cage and standard feeding ad libitum for 1 week. The rats were then given the tested variables in accordance to grouping for 28 days. Blood glucose level were measured before and after variable testing by taking 1mL of blood sample from tail vein with anesthesia 10 . Obtained blood sample were tested with blood glucose strip connected to a glucose meter.

Data Analysis
The data of Wistar rats' blood glucose levels were put under Shapiro-Wilk normality test. If the data were normally distributed the analysis would proceed with Oneway Anova, else ways data will be transformed to normalize the distribution. If data remained abnormally distributed analysis proceeded with Mann-Whitney test. P-value under 0,05 determined significance of difference between the groups.
Significant difference was found after Anova test (Table 2), analysis proceeded with Post Hoc test ( Table 4). The test found significant difference in group P3 and P4 (p<0,05). Difference was insignificant between group P1 and P2, as well as between group P1 and P3, group P1 and P4, group P2 and P3, group P2 and P4.

Discussion
Sample of this study was a group of 28 male Wistar rats meeting inclusion criterias. Samples were randomly divided into 4 groups. Rats were made acclimatized to the cage and standard feeding during the experiment.
Samples were divided into 4 groups for testing different variables: a control group induced with streptozotocin, experimental group exposed to 175 ml/L of nanosilica fertilizer inhalation, experimental group exposed to nanosilica fertilizer inhalation and given regular mask, experimental group exposed to nanosilica fertilizer inhalation and given N. exaltata mask. The variables were tested for 28 days. Rats' blood glucose levels were tested before and after variable testing using glucose meter.
Nanosilica, a 100 nm sized nano-particle can induce ROS formation inside the body 11 . High ROS concentration in body causes oxidative stress. Oxidative stress caused by ROS elevation is cytotoxic to cells of human body 12 . One of the affected components of the body is insulin receptor transduction signal in peripheral tissue which can cause insulin resistance in type II diabetes mellitus 13 .
In this study the average blood glucose level deviation was elevated after nanosilica inhalation, which indicated that nanosilica inhalation causes blood glucose elevation in rats. The elevation of blood glucose caused by nano-silica inhalation was not significantly different from the elevation of blood glucose caused by streptozotocin induction. This suggests that elevated blood glucose caused by nanosilica inhalation can cause similar effect as the case with streptozotocin.
Regular mask used in this study is fabric mask that filters only large sized particles, it can't filter micro or nano sized particles 14 . This allows nano-particles to go through the mask.
In this study the average blood glucose level in Wistar rats exposed to nanosilica inhalation wearing regular mask was different from the Wistar rats exposed to nanosilica inhalation wearing N. exaltata herbal mask. Blood glucose level elevation after nanosilica inhalation in rats wearing herbal mask lowered while it elevated in rats wearing regular mask. Herbal mask nanosilica had shown to lower blood glucose elevation caused by nanosilica particles, although statistically the difference between the two groups was insignificant.
Researches show that N. exaltata herbal mask contains xanthone and mangifer in with anti inflammation and antioxidant effects 15 . Mangifer in is found to inhibit phagocytosis activity of macrophage and ROS formation 16,17 . Elevation of ROS concentration causes oxidative stress impairing cells in the body including insulin receptor cells 12 .

Conclusions
In this study nanosilica inhalation and streptozotocin induction had shown to cause blood glucose elevation in Wistar rats and wearing N. exaltata inhibited blood glucose elevation in Wistar rats exposed to chronic nanosilica inhalation.
Research on human with high frequency of direct exposure to nanosilica such as farmers needs to be done to evaluate blood glucose level elevation. Future research on the effect of wearing N. exaltata mask against blood glucose level in Wistar rats that are chronically exposed to nanosilica needs to be done with higher frequency and longer duration of exposure.