Isolation, structure elucidation, and antidiabetic test of vicanicin compound from lichen Teloschistes flavicans

Maulidiyah Maulidiyah1*, Akhmad Darmawan2, Asriani Hasan1, Dwiprayogo Wibowo3, La Ode Agus Salim1, Ansharullah Ansharullah4, Faizal Mustapa5, Ishmah Farah Adiba Nurdin6, Muhammad Nurdin1 1Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Halu Oleo, Kendari, Indonesia. 2Research Center for Chemistry, Indonesian Institute of Sciences, Tangerang, Indonesia. 3Department of Pharmacy, Faculty of Sciences and Technology, Institut Teknologi dan Kesehatan Avicenna, Kendari, Southeast Sulawesi, Indonesia. 4Department of Food Science Technology, Faculty of Agriculture, Universitas Halu Oleo, Kendari, Indonesia. 5Department of Aquaqulture, Faculty of Sciences and Technology, Institut Teknologi dan Kesehatan Avicenna, Kendari, Indonesia. 6Faculty of Medicine, Universitas Halu Oleo, Kendari, Indonesia.


INTRODUCTION
A recent study on natural phytopharmacy has attracted researchers to explore new medicines based on various types of medicinal plants, such as their uniqueness, benefits, and local wisdom (habitat and endemic location) (Pit'ay et al., 2019;Rohman et al., 2019). One of the types, local wisdom, has a unique appeal and exploration to observe the potential of medicinal plants from various countries (Rahardi, 2020). Indonesia is a mega-biodiverse country with a diversity of plants and organisms that have a high potential for exploration of natural products as medicinal ingredients (Sosilawaty, 2020;Yuniati et al., 2019). What is more, Indonesia is a tropical country that easily grows a variety of plants due to high sun exposure for high photosynthesis.
At present, the exploration of secondary metabolites from the plant has been studied based on the classification of algae and fungi because it produces the same secondary metabolite product (O'Neill, 2020;Selvakumar et al., 2020). It was identified for studying several active compounds from medicinal plants and will be developed for the basis of producing synthetic drugs (Yanuar et al., 2011). One of the unique organisms of lichen has attracted attention because it contains various types of secondary metabolites that are medicinal (Huneck and Yoshimura, 1996). Particularly, its symbiotic mutualism with algae (cyanobacteria) and fungi is very essential for exploring natural materials (Lutzoni et al., 2001;Maulidiyah et al., 2011).
The urgency and novelty of this study is the importance of exploring secondary metabolite compounds from mosses, such as Teloschistes flavicans, as a natural medicine to treat several human diseases, especially for their antidiabetic activity. We present the exploration of a bioactive compound (vicanicin) and observe the antidiabetic activity potential from lichen T. flavicans. It contains the chemical structures of chlorodepsidone, fatty acids, and polysaccharides present in apo-symbiotically cultured myco-and photobionts in the intact thallus and potential for the antidiabetic agent through the inhibitory activity of carbohydrate hydrolysis enzymes, namely α-amylase and α-glucosidase (Bate et al., 2018;Reis et al., 2005;Ruthes et al., 2008;Umeno et al., 2016).

Extraction of lichen T. flavicans
A sample of lichen T. flavicans was collected from the pine forest in Latimojong Village, Pasui District, Enrekang Regency, South Sulawesi, Indonesia. It was cleaned, dried, and mashed to eliminate the impurities. Then, it was weighed (560 g) and extracted by using acetone solvent (Merck, Germany) for 3 × 24 hourss (maceration process). Every 24 hours, the lichen extract was filtered to separate the filtrate and residue. This technique was repeated three times and the aim of the high-extraction process was to get the extracted lichen compound. The acetone extract was separated by using a rotary vacuum evaporator to produce high concentrates.

Separation and purification
The lichen extract was tested by using the thin layer chromatography (TLC) method to identify and determine variations in eluent ratios that can be used in gravity column chromatography (GCC) ( Table 1), with a column size of 50 ml and a diameter of 2 cm. The preparation of GCC was carried out by weighing 160 g of silica gel mixed with n-hexane solvent (Merck, Germany) and put in a GCC tube. On the other hand, the lichen extract was impregnated by weighing as much as 6 g containing silica gel in a ratio of 1: 1 w/w. The sample was eluted in a GCC tube in a solvent gradient as shown in Table 1. Every 250 ml of the lichen extract was put into a vial glass and evaporated to observe the crystal product. After that, 27 eluents were collected and tested on a TLC plate to review specific color spot by calculating the Rf value under ultraviolet (UV) irradiation at 254 and 365 nm, to determine if it has produced the specific color spot that provides a description of secondary metabolites found.

Identification of the bioactive compound
The isolated compound was analyzed using liquid chromatography-mass spectroscopy (LC-MS) (LCMS 8060 System) to review the molecular weight and provide the structural identity of each component with high molecular specificity and detection sensitivity. Also, Fourier-transform infrared spectroscopy (FTIR) (Shimadzu IR Affity-1S system) was used to observe a typical absorption band at the wavenumber and confirmed using 1D-nuclear magnetic resonance [(NMR) (1H and 13C)] (JEOL JNM ECA 500). Referring to these data, we interpret by comparison with the literature reviews.

Bioactivity test
Toxicity test for A. salina leach shrimp larvae Initial testing to overview the extracted toxicity compound was applied by using the Meyer method under A. salina Leach (shrimp larvae) (Meyer et al., 1982). The LC 50 value is calculated based on the equation: y = ax + b, where y states that the shrimp larvae have died 50% after 24 hours of incubation. Meanwhile, a and b values are data regressions based on a slope with concentration variations as the standard method. Then, we can calculate the value of x concentration to inhibit the larvae based on 50% mortality. The substance is active/toxic when the LC 50 value is ≤ 1,000 μg/ml (Meyer et al., 1982).

Antidiabetic activity test
Antidiabetic activity test was conducted by inhibiting the enzyme α-glucosidase that has been previously reported by Dewi et al. (2012) and Kim et al. (2008). A positive control using 1 mg of quercetin dissolved in 100 μl dimethyl sulfoxide (DMSO) was diluted with four variations of concentration to obtain a standard curve. The positive control was tested with the addition of or without enzyme solution. Meanwhile, the negative controls were prepared without the addition of 5 μl DMSO. Finally, we can calculate this with the following equation: Inhibitory activity (%) = , (1) where K is the absorbance of the negative control solution, S is the absorbance of test solution or positive control solution, and [S = S 1 (absorbance with the addition of enzymes) -S 0 (absorbance without adding enzymes)].

Isolation of lichen T. flavicans
The concentrated extract was identified by phytochemical screening to determine the classification of chemical compounds in T. flavicans, which were confirmed as alkaloids, flavonoids, saponins, tannins, and terpenoids, presented in Table 2.
Through the elution process, we obtained 136 eluates with various eluent systems ( Table 1). They were tested by using TLC to view patterns of a color spot under UV irradiation at 254 and 365 nm. Several organic compounds containing heterocyclicelectron are conjugated when exposed to UV light. So, we can determine the Rf value on the TLC plate and deduce a single spot.

Identification of the bioactive compound
White crystal needles were characterized by using the LC-MS/MS chromatogram (Fig. 2), showing a peak at m/z 383. We can deduce the molecular weight of the isolated compound to be 383 g mol −1 . Some chemical compounds of the lichen identified in m/z 383 are a depsidone group which enriches the -OH and O groups. LC-MS analysis only identifies the molecular weight of a compound to inform the total atoms in the isolated compound.
To predict the chemical groups in the isolated compound, we used FTIR (Fig. 3), which shows that there was a typical absorption band (wavenumber) at 3,511 cm −1 from the -OH (hydroxyl) group. Then, it showed a C-O stretch of the C-O-C ether group under wavenumbers 2,963 and 1,096 cm −1 (fingerprint  Lichen T. flavicans extract (acetone extract) + + + + + area). The strong intensity at wavenumber 2,744 cm −1 indicates the carbonyl group C=O. The presence of an aromatic ring at wavenumber 1,595 cm −1 and buckling bond is confirmed by the cyclic form at wavenumbers 1,437 and 1,359 cm −1 (methylene and methyl groups, respectively). On the other hand, we also discover two halogen groups (C-Cl) as hydrogen bridges with the -OH group at wavenumbers of 843 and 729 cm −1 . The presence of two chlorine atoms is strengthened by the fragment peaks in LC-MS with m/z 383 and 385 with the lower left to right intensity ratio which is characteristic of two chlorine atoms (Kadivar et al., 2011). Based on the FTIR spectrum analysis, the isolated compound had similar absorption data as the vicanicin compound (Huneck and Yoshimura, 1996).
In addition, 1D-NMR was applied to confirm the chemical structure of the isolated compound. The determination of the atomic structure is presented at 1 H and 13 C based on the   position of the H and C atoms. According to Figure 4, 1 H-NMR analysis shows that the isolated compound contains 16 hydrogen atoms with the appearance of a single signal. The chemical shift of δH = 6.95 ppm indicates the -OH group and δH = 3.86 ppm indicates the presence of methoxy group (-OCH 3 ) which are attached to the aromatic ring. Then, the chemical shift at 2.55, 2.49, 2.422, and 2.421 ppm shows four methyl groups attached to the aromatics. Furthermore, an analysis of 13 C-NMR (Fig. 5) shows that the isolated compound contained 18 carbon atoms at a chemical shift (δC) of 60.84 ppm, indicating one carbon atom in the methoxy group. The methyl group was confirmed at a chemical shift of 18. 59, 15.25, 11.27, and 11.26 ppm and the carbonyl group was presented at δC = 162.96 ppm, which also appears in the IR spectrum of 1,744 cm −1 . Moreover, signals from aromatic groups (C=C atom) were confirmed at chemical shifts of 139.00 and 144.50 ppm. Based on the interpretation of data compared with several other literature studies (Huneck and Yoshimura, 1996;Sargent et al., 1976), we conclude that the isolated compound was vicanicin with the molecular formula of C 18 H 16 Cl 2 O 5 .
The number of double bonds and rings in the isolated compound has been determined by the hydrogen deficiency index formula (Wang et al., 2012). The calculation of double bonds or ring number was obtained with an F value of 10 derived from 1 carbonyl group (C=O) and 6 double bonds of C=C in two aromatic rings. Three rings have been identified wherein two are aromatic rings and one is a nonaromatic ring of the carbonyl group.
Based on the comparison of physical properties and spectrum data of the isolated compound, they have the same data and similarity properties as the vicanicin compound. It is a depsidone group that previously has been isolated from lichen Ramalina javanica Nyl (Pranadita andYuliani, 2019;Sargent et al., 1976). The structural formula of vicanicin is shown in Figure 6.

Bioactivity test
Information of the bioactivity test inhibiting A. salina Leach and α-glucosidase enzyme is shown in Tables 5 and 6. Based on Table 5, the vicanicin compound has high toxicity against A. salina Leach because it contains cyclic groups, carbonyl groups, and oxygen bridges. Suryani et al. (2019) have reported that the role of the cyclic structure and carbonyl groups can inhibit high toxicity to A. salina Leach. Table 6 shows the test results of α-glucosidase enzyme activity by comparing the performances of quercetin, T. flavicans extract, and vicanicin compound. The quercetin as the positive control has a high inhibition compared to the T. flavicans extract and vicanicin with an IC 50 value of 4. 05, 54.05, and 197.04 μg/ ml, respectively. However, vicanicin has a lower activity because it does not contain many hydroxyl groups or an active role in inhibiting the α-glucosidase enzyme. The T. flavicans extract has shown good stability because it still contains a variety of chemical      compounds that synergize to inhibit the α-glucosidase enzyme (Li et al., 2005), while vicanicin is a single compound although it is weak in inhibiting the α-glucosidase enzyme, making an impact as a potential for antidiabetic activity (Wang et al., 2012).
With the chemical structure approach, vicanicin also does not have oxidative properties to inhibit α-glucosidase activity which is characterized by presenting oxygen atoms (Stojanovic et al., 2012). According to Gong et al. (2017), secondary metabolites with natural antioxidant properties have the ability to inhibit α-glucosidase as a type 2 diabetes treatment.

CONCLUSION
The secondary metabolite compound vicanicin was isolated from lichen T. flavicans, with the molecular formula C 18 H 16 Cl 2 O 5 . Current information about toxicity of A. salina Leach larvae also shows that the antidiabetic potential of T. flavicans extract has toxicity to A. salina with an LC 50 value < 1,000 μg/ ml, which is 9.38 μg/ml. Meanwhile, vicanicin compounds also showed toxicity, with an LC 50 value of 13.07 μg/mL. The antidiabetic test showed that the T. flavicans extract gave the highest inhibition compared to the vicanicin compound, with IC 50 values of 54.05 and 197.04 μg/ml, respectively. The hydroxyl group contains reactive oxygen compounds in T. flavicans extract which play an important role in inhibiting the larvae of A. salina Leach to lyse cell membranes and decide the chemical bonds in the α-glucosidase enzyme. This study provides a perspective on the natural products of lichen T. flavicans extract, which have the potential as antidiabetic drugs.