Potential of Usnic Acid Compound from Lichen Genus Usnea sp. as Antidiabetic Agents

12 ） , antiproliferative 13 ） , antioxidant 13 ） , and antiinflammatory 14 ） activity showed only five types of lichen, Ramalina Abstract: Lichen Usnea sp. is potential as a new natural medicine. This study report isolation of secondary metabolites from lichen Usnea sp. and α -glucosidase inhibitory, which is potential as an antidiabetic agent. Lichen powder was macerated using methanol, separated using column chromatography gravity and thin-layer chromatography. The crystalline was isolated and purified by the recrystallization process for obtaining pure compound. The isolated compound was determined using FTIR and NMR spectroscopy ( 1 H and 13 C). The results showed that the isolated compound was yellow needle crystals. Based on the spectra data interpretation, it was obtained usnic acid compound with the molecular formula of C 18 H 16 O 7 . The α glucosidase inhibitory activity test showed that the usnic acid had activity in inhibiting the α -glucosidase enzyme with an IC 50 value of 106.78 µg/mL. The usnic acid from Usnea sp. has a very good impact on the source of natural compounds as an antidiabetic drug in the future.

There are still few reports of secondary metabolites from lichens Usnea sp. as an antidiabetic agent. Based on data from the International Diabetes Federation 15 , the prevalence of global diabetes is expected to increase by 552 million by 2030. Data from the World Health Organization WHO 16 also reports that diabetes mellitus is the seventhlargest cause of death. One of the essential therapeutic methods for reducing plasma glucose levels is to inhibit α-glucosidase activity 17 19 . Several previous studies have reported the potential of lichen exploration as a good antidiabetic agent.

Extraction of lichen Usnea sp.
Lichen was cleaned of impurities and smoothed. The lichen weighed 1,000 grams was extracted by the maceration process. Lichen was immersed in methanol solvent for 3 24 hours, and then a filtering process was carried out to separate the filtrate and residue every 24 hours. The residue is macerated again with new methanol as solvent. The filtrate was combined and then concentrated using a rotary evaporator vacuum to obtain a methanol crude extract lichen Usnea sp.

Isolation and puri cation of lichen Usnea sp.
The initial separation stage uses the liquid-liquid extraction LLE technique. Methanol crude extract of lichen Usnea sp. is partitioned using a separating funnel with an increased polarity from nonpolar solvents to highly polar solvents n-hexane, ethyl acetate, butanol, and water . This stage aims to separate compounds based on their level of polarity. The partition results were tested using a thin layer chromatography TLC method for searching and determining eluents with a specific ratio that could separate compounds well.
Furthermore, the subsequent separation was using gravity column chromatography GCC on the ethyl acetate fraction. The ethyl acetate fraction is put into the column; the separation process uses a mixed solvent whose polarity is different from polar to nonpolar solvent. The solvent used starts with 1 L n-hexane 100 , then a mixture of nhexane:ethyl acetate and ethyl acetate:methanol in a gradient v:v . All eluate results from GCC were tested on the TLC plate using nonpolar solvent and polar solvent in a gradient until there was specific color and calculating the Rf value under ultraviolet irradiation at 365 and 254 nm. The isolates were re-crystallized using n-hexane, ethyl acetate, and acetone as solvents. The isolated compound was analyzed using Fourier-transform infrared spectroscopy FTIR Nicolet FT-IR iS10, USA was used to observe a typical absorption band at the wavenumber and confirmed using nuclear magnetic resonance NMR 1 H and 13 C JNM ECA 500 JEOL . We interpreted the data by comparing literature reviews and referring to the data obtained.

Glucosidase inhibitory activity
The antidiabetic test was carried out by inhibiting the α-glucosidase enzyme, according to Dewi et al. 2012 34 . Bioactivity testing was carried out using methanol crude extract, ethyl acetate fraction, n-hexane fraction, acetone fraction, and pure isolate compounds. Each test sample was made into four concentration series. The test sample was reacted with p-nitrophenyl-α-D-glucopyranoside and phosphate buffer solution pH 7.0, then incubated at 37 for 5 minutes in a water bath. Each test sample was added with α-glucosidase enzyme solution and incubated at 37 for 15 minutes in a water bath. The reaction was stopped by adding 1,000 μL of 0.2 M sodium carbonate solution. The amount of p-nitrophenyl released was then measured by UV-Vis spectrophotometry at 400 nm. Inhibitory activity is calculated by the equation as follows: The phytochemical screening of the lichen extract was used to establish the classification of chemical compounds in lichen Usnea sp. Maceration against 1,000 grams of lichen Usnea sp. crude powder obtained 53 grams of methanol extract. Secondary metabolites identified positively in lichen Usnea sp. are alkaloid, flavonoid, and tannin. Ethyl acetate fraction was isolated by GCC to produce 90 sub-fractions. The stationary phase used silica gel, and the mobile phase was a mixed eluent n-hexane: ethyl acetate and ethyl acetate:methanol v:v . Further purification using TLC showed specific isolates. TLC was carried out using a solvent mixture of n-hexane:ethyl acetate 6:4 v:v . TLC plate observations were carried out using UV lamps of 254 nm and 366 nm Fig. 1 . The single stain that appears on the TLC plate shows that isolate contains only one compound.

Identi cation of isolate structure
Pure isolate compounds isolated from the ethyl acetate fraction were identified for their functional groups using FTIR. The spectrum shows a typical absorption band in several regions of the wavenumber Fig. 2 . The absorption at wavenumber 3,091 cm 1 is identified as a bond C-H sp 2 supported by the absorption at wavenumber 1,630 cm 1 which is characteristic C C aromatic. The wavenumber 2,933 cm 1 is identified as a bond -C-H sp 3 . Typical absorption at 1,687 cm 1 indicates the presence of a carbonyl group C O aromatic ketone stretch. This absorption is slightly smaller than the typical carbonyl wave number because the carbonyl group C O forms hydrogen bonds with the hydroxy group OH , and there are bonds conjugation of a carbonyl group C O with a bond -C C-in the aromatic ring. The wavenumber 1,130 cm 1 is thought to signal the ether group C-O-C bond stretching. The absorption of the hydroxy group OH did not appear suspected because the formation of hydrogen bonds between the carbonyl group C O and the hydroxy group OH formed cyclic, which was considered more stable.
The determination number of carbon, hydrogen atoms and their positions was carried out using 1D NMR 1 H-NMR, 13 C-NMR, and DEPT and 2D NMR HMQC and HMBC .
The spectrum shows the similarity of data from usnic acid compounds 8,35 . Based on a 1 H-NMR spectrum Fig. 3 , the isolate has two methyl groups with the multiplication of each singlet -CH 3 at δ H 1.76 and 2.11 ppm. The shifts of 2.66 and 2.68 ppm indicate two methyl groups attached to the carboxylate group. The spectrum at the chemical shift δ H 5.98 ppm was identified as olefinic protons. The two hydroxy groups forming a hydrogen bond with the carbonyl group are shown by the chemical shifts δ H 13.32 and 11.04 ppm, respectively. At the H-16 position, the value of the methyl proton shift at the H-16 position is greater downfield due to the group position is directly attached to the phenolic group Ar-OH and the position that is also close to the hydroxy group -OH in position C-8 .
The 13 C-NMR spectrum Fig. 4 shows that the isolate has    Table 1 shows data regarding the correlation between protons and their neighboring carbon atoms, which are 2 to 3 bonds apart. The resonance of aromatic protons at δ H 5.98 ppm H-4 is correlated with δ C 59.   Usnic Acid Compound from Lichen Genus Usnea sp. as Antidiabetic C-9 , and 157.7 ppm C-11 .
In the final stage, we compare results with the appropriate literature by Huneck and Yoshimura 1996 35 and Maulidiyah et al. 2021 8 , it is assumed that the isolated compound from the ethyl acetate fraction is a usnic acid C 18 H 16 O 7 Table 2 . Usnic acid is one of the main contents of lichen Usnea sp. which has many benefits in the pharmacological field 36 . The molecular structure of the usnic acid compound is shown in Fig. 5.

Effect of lichen metabolites on α-glucosidase inhibition
The inhibition of α-glucosidase by quercetin, lichen Usnea sp. extract, and usnic acid compounds have been evaluated Table 3 . Quercetin acts as a positive control having a high inhibitory power compared to lichen Usnea sp. extract and usnic acid with IC 50 values of 5.69, 88.02, and 106.78 μg/mL, respectively. The usnic acid compound has a moderate performance in its activity to inhibit the α-glucosidase enzyme.   35) Lichen Usnea sp. extract shows excellent stability because it still contains various chemical compounds that work simultaneously to inhibit the α-glucosidase enzyme 37 39 , whereas pure usnic acid compound is a single compound and doesn t contain many hydroxy groups, which play an active role in inhibiting the α-glucosidase enzyme. Although it shows moderate performance in inhibiting the α-glucosidase enzyme, it has a potential positive impact as an antidiabetic 28,40,41 . In addition, the antidiabetic activity of usnic acid compounds from lichens Usnea sp. is not much reported about the inhibition of the α-glucosidase enzyme Table 4 .

Conclusion
The secondary metabolite compounds isolated from lichen Usnea sp. were usnic acid compounds with the molecular formula C 18 H 16 O 7 . The test of α-glucosidase inhibitory activity from lichen Usnea sp. extract has an IC 50 value of 88.02 μg/mL strong activity inhibiting the activity of the α-glucosidase enzyme. Meanwhile, usnic acid compounds have moderate activity in inhibiting the α-glucosidase enzyme with an IC 50 value of 106.78 μg/mL.   Table 4 Reported lichen and its compounds as antidiabetic agents α-glucosidase.