AIP Conference Proceedings 2193, 030014 (2019); https://doi.org/10.1063/1.5139351 2193, 030014
© 2019 Author(s).
Uric acid and glucose level in high fructose high cholesterol induced Sprague-Dawley rats after therapy with Acalypha indica Linn.
ethanol extract
Cite as: AIP Conference Proceedings 2193, 030014 (2019); https://doi.org/10.1063/1.5139351 Published Online: 10 December 2019
Desak Gede Budi Krisnamurti, Fira Alyssa Gabriella Sinuraya, Tamara Ey Firsty, Rani Wardani Hakim, and Erni H. Purwaningsih
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Uric Acid and Glucose Level in High Fructose High Cholesterol Induced Sprague-Dawley Rats after Therapy
with Acalypha indica Linn. Ethanol Extract
Desak Gede Budi Krisnamurti
1,Fira Alyssa Gabriella Sinuraya
2, Tamara Ey Firsty
2, Rani Wardani Hakim
1, Erni H. Purwaningsih
1,a)1Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6,Central Jakarta 10430 Indonesia
2Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia.
a)Corresponding author: erniepoerwa@yahoo.com
Abstract. Diet with high fructose and cholesterol (HFHC) plays a role in the increasing incidence of diabetes mellitus and metabolic syndrome. Ethanol extract from Acalypha indica has been known to alleviate hyperglycemia and hyperuricemic conditions in rats induced by substances that destroy beta cells. This research aimed to evaluate its effect in rats induced by diet. Male Sprague-Dawley rats were divided into seven groups, six of which are given HFHC diet for 1,5 months. In the following month, rats were given therapy while diet continued. Therapy consisted of 250 mg/kg BW/day of Acalypha indica Linn. root’s ethanol extract, 100 mg/kgBW/day of metformin, 30mg/kg BW/day of allopurinol, or combination. Lowest blood glucose value was found in group receiving both AI and metformin. No significant difference was found between pre- and post- therapy blood glucose in groups treated with AI (p=0,831), metformin (p=0,056), or both (p=0,908). Uric acid level was increased in all groups, with highest rate found in group receiving both allopurinol and AI. The difference in uric acid level between treatment group was 0.331. Whilst insignificant, ethanolic extract of Acalypha indica Linn. was observed to lower blood glucose in rats. Group treated with AI showed similar rise in uric acid level, with combination therapy showed highest rise. Further research with longer duration of induction and therapy will be required to better understand the hypoglycemic and antihyperuricemic effects exerted by AI.
Keywords: Acalypha indica Linn., high-fructose-high-cholesterol, hyperuricemia, hyperglycemic, metformin, allopurinol.
INTRODUCTION
Epidemiologic data showed a shifting of diseases pattern from communicable to noncommunicable diseases, one of which being type 2 diabetes mellitus (T2DM) [1]. Data showed the prevalence of T2DM is 8.5% in 2014 and was projected to double by 2030 [2,3]. Central obesity, hyperglycemia, insulin resistance, increase of blood triglyceride, LDL level, and blood pressure; as well as low HDL level, or collectively called metabolic syndrome, is a risk factor for T2DM. Uncontrolled hyperglycemia can also cause macrovascular complications such as coronary heart disease and peripheral heart disease, as well as microvascular complications such as blindness and kidney failure. Thus, one prevention strategy in facing metabolic syndrome was regulation of blood glucose level [1-3]. Some newer researches showed the role of uric acid regulation in management of metabolic syndrome. An increase of 1 mg/dL in blood uric acid level correlated to twice the risk of having metabolic syndrome [4].
Increased in endogen uric acid production also induced insulin resistance and adipocyte dysfunction [5,6]. In the complicated conditions, hyperinsulinemia caused further uric acid increase by lowering its excretion via kidneys [7,8]. Therefore, metabolic syndrome management should consist of blood glucose and uric acid level regulation.
The first line pharmacological therapies for the regulation of blood glucose and uric acid level are metformin and allopurinol [9]. Metformin doesn’t induce body weight increment and when compared to other hypoglycemic agents has lower risk of causing hypoglycemic shock [10]. However, long-term use may hamper vitamin B12 absorption, hence potentially causes neuropathy and megaloblastic anemia [11]. Allopurinol is relatively safe yet may cause severe cutaneous adverse reaction (SCAR), a disease with 32% mortality rate when it is used by
The 4th Biomedical Engineering’s Recent Progress in Biomaterials, Drugs Development, Health, and Medical Devices AIP Conf. Proc. 2193, 030014-1–030014-6; https://doi.org/10.1063/1.5139351
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patients with kidney failure [12]. Usage of metformin in patients with kidney failure may risk lactic acidosis [13]., Kidney failure as a common complication in patients with metabolic syndrome should be the most consideration [14]. Thus, safer antidiabetic and antihyperuricemic agents are necessary for this patient group.
An alternative safer approach to lower blood glucose and uric acid level is by using herbal, especially those containing flavonoid which is known to have antidiabetic and antihyperuricemic activities [15]. One of such is Acalypha indica (AI) [15,16]. Various experimental studies had shown AI potential as antidiabetic and antihyperuricemic [17-21]. This study aimed to discover the effect of Acalypha indica Linn.’s root ethanol extract to the blood glucose and uric acid level of Sprague-Dawley rats which had been induced by high fructose high cholesterol (HFHC) diet to represent metabolic syndrome.
MATERIALS AND METHODS
Male Sprague-Dawley rats were assigned to seven groups (n =7) and given intervention for 4 weeks. One group received normal feed throughout the study period, one induced group were treated, the rest of the groups were still given HFHC diet and treated with 250 mg/kgBW AI extract, 30 mg/kgBW allopurinol, 100 mg/kgBW metformin, combination between 250 mg/kgBW AI extract + 30 mg/kgBW allopurinol, and combination between 250 mg/kgBW AI extract + 100 mg/kgBW metformin, respectively.
High cholesterol diet was a normal diet enriched with quail’s yolk composing 10% of its weight which dose was 10 g of fodder per day. Fructose was given in the form of 1.5 ml of fructose liquid 55% diluted in 0.5 ml of distilled water which dose was 2.0 ml per feeding. In this study, dried AI root was powdered with grinder, put in percolator tube and macerated with ethanol 70% for 3 x 24 hours. Extract was then condensed through rotavapor.
The end product was concentrated ethanol extract of AI roots with 68.75% purity (5.2% yield with 31.25% water content).
At the end of treatment period, rat bloods were put in EDTA tube and centrifugated in 3000 rpm speed for 15 minutes using Universal 320R Hettick Zentrifugen. Supernatant was then kept in cooler with -22°C temperature.
Glucose and uric acid level measurement was done by spectrophotometry with Randox GL364 and enzymatic colorimetric with Randox UA230, respectively. Glucose level was analyzed with Kruskal-Wallis test and post hoc Mann-Whitney test. Uric acid level was analyzed with One Way ANOVA test and post hoc Mann-Whitney test. Results were considered statistically significant if p value less than 0.05.
RESULTS AND DISCUSSION Blood Glucose Level
Statistical analysis was done to compare the glucose level before and after therapy. Glucose level difference for each group, excluding untreated and combination group, are distributed normally with p value 0,387; 0,039;
0.591; -; and 0,128, consecutively. Normality test for metformin+AI group can not be done due to too few subjects alive at the end of treatment period, hence, for the analysis purposes it is regarded not to be normal. Hypothesis test for normal, AI, and metformin group with paired T-test showed no significant difference in glucose level before and after therapy, with p=0,964; 0,831; and 0,056, consecutively. Wilcoxon test in untreated and combination group also showed no significant difference, p=0.138, and 0,655, respectively. Kruskal Wallis test also showed no significant difference of posttherapy glucose level between each group, p = 0.246.
FIGURE 1. Average Post Intervention Glucose Level (mg/dL)
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Blood uric acid level
Statistical analysis was done to compare the difference between uric acid level before and after therapy. Data distribution for each group are normal, p-value is 0,724; 0,552; 0.614; 0.937, and 0.724, consecutively. All groups in this study experienced increase in posttherapy uric acid level, this rise is significant in normal, negative, and positive control. The highest increment was observed in group receiving both allopurinol and AI. However, this rise is only significant in normal, untreated, and AI treated group. We also observed lowering of uric acid level in rat from HFHC and allopurinol group each -0,237 mg/dl and -0.183 mg/dl, respectively. Uric acid level differences between treatment group were not statistically significant, p-value 0.331.
FIGURE 2. Average Increase in Uric Acid Level (mg/dL)
Glucose and Uric Acid Levels Mice Induced with HFHC diet
It showed all mice group experienced an increased level of both blood uric acid and glucose level. Different to glucose, fructose has been metabolized into fructose-1-phosphate by ketohexokinase (KHK), an enzyme with no inhibition mechanism. Thus, high fructose diet may lower intracellular ATP level while inducing adenosine monophosphate (AMP) degeneration into inosine monophosphate, the end product of which is uric acid [22]. In hepatocytes, high level of uric acid induces oxidative stress and suppresses aconitase-2 activity, resulting in accumulation of citric acid in cytoplasm and lipogenesis or liver steatosis [5]. Additionally, high uric acid level inhibits endothelial vasodilation in response to insulin, thus inducing RAAS and hypertension [6].Therefore, high fructose diet may cause insulin resistance by escalating endogenous uric acid production, which is part of fructose metabolism cascade in hepatocytes.
Besides fructose, high cholesterol diet may also cause liver steatosis by reducing CPT1 enzyme expression which plays role in fatty acid oxidation promoting CYP7A1 enzyme expression in hepatic cholesterol excretion [23]. High fructose intake concomitantly with cholesterol may worsen liver steatosis. This is because fructose-1- phosphate is also a substrate in fatty acid synthesis [22]. In research by Hakim et al., [24] induced mice with HFHC diet for 4 weeks cause hypercholesterolemia, hypertriglyceridemia, increasing LDL and lowering HDL level, and liver steatosis.
Liver steatosis, hyperuricemia, insulin resistance, hypercholesterolemia, and hypertension are all risk factors for metabolic syndrome [7, 25]. In this research, insulin resistance can be observed indirectly through increased blood glucose level in mice group given HFHC diet. Therefore, induction with HFHC supposed to create metabolic syndrome model in rats. However, this escalation was not significant statistically, with p-value > 0.05.
Antihyperuricemic Activity of Acalypha indica
In this research, therapy with AI extract showed less reduction in uric acid level when compared to a positive control (allopurinol). Interestingly, the statistically insignificant rise in uric acid level in this research (p=0.331) is different with previous researches where AI showed superior antihyperuricemic activity compared with allopurinol. In Azizahwati et al., AI antihyperuricemic activity was tested with 2.7 g/200 g BW of water extract of AI roots or about 13.5000 mg/kg BW, meanwhile the dose used in this research was 250 mg/kg BW [17].In Munthe’s experiment, AI antihyperuricemic activity was observed using ethanol extract of AI’s stem and leave with dose up to 200 mg/kg BW. Using this dose, AI extract results in decline of uric acid level similar with those
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treated with allopurinol. They used 10 mg/kg BW of allopurinol, meanwhile in this study used dosage up to 30 mg/kg BW [21]. Both Azizahwati and Munthe’s experiments used caffeine as induction method [17,21].
Antihyperglycemic Activity of Acalypha indica
In mice groups induced with HFHC, mean posttherapy glucose level in metformin group was higher than in AI group. Thus, AI antihyperglycemic activity exhibited tendency better than metformin one. However, this result was not statistically significant, with p value of 0.246.
Acalypha indica as An Add-On Theraphy
In this research, combination of AI extract with first line therapy for hyperuricemia and hyperglycemia gave different results. Combination of AI and allopurinol resulted in the biggest escalation compared to other groups, while combination of AI and metformin resulted in lowest post-therapy glucose level compared to other groups.
Therefore, the antihyperuricemic of AI and allopurinol has no synergy affect each other, while the antihyperglycemic activity of AI and metformin has. These however was not statistically significant, with p value 0.331 and 0.246 consecutively.
Endogenous uric acid production is affected by xanthine oxidase (XO) activity. The XO enzyme consists of a catalytic site surrounded by amino acids, creating a hydrophobic space [26].Beside direct inhibition through this site, uric acid production may also be inhibited indirectly via interaction of inhibitor with amino acids in the hydrophobic space [27]. In a molecular docking analysis by Lin et al., flavonoid compound is seen creating hydrogen bonds with amino acids in the hydrophobic space, while allopurinol directly interacts with XO’s catalytic site [26]. Thus, when given simultaneously, AI’s flavonoid content can inhibit the interaction between allopurinol and XO’s catalytic site. Free allopurinol may accumulate around the hydrophobic space and interact with free flavonoids. These interactions potentially destabilize both AI’s flavonoid and allopurinol, making way for xanthine to interact with XO. This can be observed by higher mean uric acid level in mice given both AI and allopurinol. However, this result was not statistically significant, with p=0.331.
The activation of 5’AMP- activated kinase (AMPK) enzyme can lower insulin resistance by decreasing lipogenesis, decreasing carboxylase acetyl-KoA activity, and decreasing gluconeogenesis in hepatocytes; and by increasing glucose uptake by muscles [28]. AMPK can be activated directly by molecular binding to subunit c
AMPK, or indirectly by inhibiting any step in respiration chain. Intake of metformin via organic cation transport- 1 (OCT1) in hepatocyte will inhibit the respiration chain, thus causing AMP build up which activates AMPK [29].
Similar to metformin, a derivate of flavonoid called quercetin is an AMPK activator as well [29,30]. AI has flavonoid content of 49,5 mg/g quercetin equivalent [31]. Therefore, the antihyperglycemic activity of AI and metformin was synergistic. It was proved by survived mice in group given both AI and metformin has lowest blood glucose level compared with other groups. This result, however, was not statistically significant, with p=0.246.
Acalypha indica as Preventive Therapy
In this research, HFHC diet was still concomitant with therapy, considering in human, change of dietary style during therapy is not easy to do. Additionally, this design enable an observation to the potential of Acalypha indica Linn.’s ethanol extract as part of preventive strategy against metabolic syndrome.
The average increased of blood uric acid and glucose level posttherapy in mice groups induced by HFHC given therapy with AI was higher than induced mice groups given no effect therapy at all. It can be deduced that AI extract is not effective in preventing rise in either blood uric acid nor glucose level. Yet this result was again not statistically significant, with p value 0.331 and 0.264 consecutively. This was probably due to short induction period during HFHC diet to rise blood glucose and uric acid level. Therefore, this treatment need to be optimised.
CONCLUSION
In this research, the antihyperuricemic and antihyperglycemic activity of AI extract have been observed albeit no statistically significant. The combination of Acalypha indica ethanol extract and allopurinol has antagonistic effect while Acalypha indica and metformin has synergistic efefct. For further research, longer induction period using HFHC supposed to get significant increase of uric acid and glucose level, before therapy period commences.
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ACKNOWLEDGEMENTS
The authors declare that they have no conflicts of interest. This research is funded by National Innovation System Research Incentive program from the Indonesian Ministry of Research and Technology. We also would like to express our gratitude for the financial aid from PITTA Grant, with contract number: nkb- 0532/un2.r3.1/hkp.05.00/2019
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