ESTERIFICATION OF CINNAMIC ACID USING MENTHOL AND ITS ACTIVITY AS LOWERING GLUCOSE LEVELS USING ANTHRONE SULFATE

This study aimed to synthesize menthyl cinnamate and its activity as a glucose-lowering agent. Synthesis of menthyl cinnamate using Fischer esterification of cinnamic acid and menthol with sulfuric acid as a catalyst using reflux at a temperature of 60oC with a synthesis time variation of 4,5 and 6 hours. Identification of synthesis results using FTIR spectrophotometry and GC-MS. Glucose lowering activity of the synthesis product using anthrone sulfate. The synthesized compound was a yellow oil liquid with a sweet fruity aroma typical of cinnamic esters with a yield % of 95.83%, 96.38, and 91.79%, respectively, at 4, 5, and 6 hours. The synthesized product was soluble in nonpolar solvents. Analysis by FTIR showed several functional groups such as C=O, C=C, C-O, and C-H aliphatic. Analysis of the synthesis results with GC-MS showed a retention time of 18.38 minutes for menthyl cinnamate with m/z = 286. Test with anthrone sulfate gave an optimum concentration of 300 ppm with a % decrease in the glucose of 48.62%. Based on these results, it can be concluded that menthyl cinnamate can be synthesized with optimum yield in 5 hours and has potential as an antidiabetic agent.


INTRODUCTION
Diabetes mellitus (DM) is a metabolic disease caused by abnormalities in insulin secretion, insulin action, or both [1]. This disease is characterized by chronic hyperglycemia. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels [2].
People with diabetes in Indonesia showed a significant increase. For example, Riskesdas 2018 shows that DM's prevalence based on a doctor's diagnosis increased 2% compared to 2013 [3].
One class of compounds that can be used as antidiabetic agents is cinnamic acid derivatives in the form of esters. Cinnamic acid derivatives have low toxicity for humans, animals, and the environment, so these compounds are of great interest to researchers [4]. Cinnamic esters are cinnamic acid derivatives that replace the OH group in cinnamic acid with an alkyl group from alcohol. A cinnamic ester is a form of cinnamic acid derivative commonly found in nature. These compounds play an important role in various biological activities such as antidiabetic, anti-inflammatory [5], cytotoxicity, leishmanicidal activity [6], tyrosinase enzyme inhibitor [7], antioxidant [8], and antifungal [9].
In addition, cinnamic esters are important compounds in the flavor, perfume, and pharmaceutical industries [10].
The antidiabetic activity of cinnamate esters was reported by several researchers [11][12]. Pharmacokinetic studies have shown that cinnamic acid and its derivatives are easily absorbed from the small intestine through various mechanisms [11]. Ethyl cinnamate synthesized from cinnamon oil was able to inhibit the enzyme alphaglucosidase with IC50 at a concentration of 215.509 ppm [13]. One of the cinnamate ester compounds is menthyl cinnamate.
Menthyl cinnamate has activity as an antiinflammatory [14], and antifungal [15]. This compound can be synthesized using an acid catalyst by Fischer esterification of cinnamic acid with menthol [15].

Esterification of cinnamic acid using
Fischer esterification with a sulfuric acid catalyst under reflux can be carried out for several hours. Reaction time is one of the important aspects of secondary alcohol esterification [16]. Synthesized menthyl cinnamate through Fischer esterification using reflux for 3-4 hours and gave 85-92% yield [15]. Synthesized amyl cinnamate for 6 hours with % a yield of 80.73% [17]. This research synthesized menthyl cinnamate with variations in reaction time, namely 4,5 and 6 hours.
In vitro, a glucose reduction test can be done using anthrone sulfate. The anthrone sulfate method is one of the most commonly used techniques for determining carbohydrate content by the colorimetric method [18]. The anthrone test has advantages in terms of sensitivity and a simple test. In addition, a small number of carbohydrates can provide a color that is detected using a visible spectrophotometer [19].

Material and Instrument
The materials used in this synthesis were cinnamic acid from Sigma, menthol  regenerates the sulfuric acid catalyst and produces the menthyl cinnamate compound [25]. The esterification reaction is reversible, so the reaction equilibrium must be shifted to the ester side. One way is to make one of the reactants in excess so that in this study, the moles of cinnamic acid were made twice the moles of menthol. Another way is to remove the side-product water [26].    cm -1 is a C=C aromatic group [21], and at 1167 cm -1 is the C-O ester [29].

Test The Activity of Lowering Glucose Levels Using Anthrone Sulfate
The activity of reducing glucose levels by synthesizing compounds using the spectrophotometric method using anthrone sulfate reagent. This method is very simple, relatively fast, easy to do, and precise for determining glucose levels [18]. The principle of determining glucose levels using the anthrone sulfate method is the hydrolysis of carbohydrates into monosaccharides by sulfuric acid, which will then be hydrated to furfural. Furfural compounds react with anthrone to form a greenish-blue complex whose absorbance can be measured at the maximum wavelength [22]. The maximum wavelength used in this study was 626 nm, which was the same as the literature (623.80 nm) [30].
Using 1% anthrone in this test resulted in the highest absorbance. The anthrone solution of more than 1% showed a decrease in absorbance [18]. The synthesized compound binds to glucose. The remaining glucose, which is not bound by menthol cinnamate, is dehydrated by sulfuric acid to form 5hydroxymethylfurfural. This dehydration results in double bonds and ring formation [22]. The compound 5-hydroxymethyl furfural reacts with anthrone to form a compound (anthracene-9yloxy) (5-(hydroxymethyl) furan-2-yl) methanol which is blue-green, which can be measured through its absorption at a wavelength of 626 nm [30]. The anthrone sulfate test reaction can be seen in Figure 8.