Avicularin: The major secondary metabolite isolated from Saurauia vulcani as a potential anti-colorectal cancer agent

Saurauia vulcani flora develops certainly in North Sumatra Province, Indonesia. It is normally consumed as an antidiabetic remedy and a treatment related to digestion diseases. This research aims to isolate the major compound from S. vulcani plants and assess the bioactivity of the S. vulcani plant for its anti-colorectal cancer potential. Subsequently, the ethyl acetate fraction was analyzed with a solvent system used as the eluent ethyl acetate: methanol: Aquadest = 4.6:0.2:0.2. Utilizing spectroscopy, we determined the chemical structure of the remote substance. Utilizing the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide technique in vitro , the anti-colorectal cancer activity was demonstrated against WiDr cancer cells. This study revealed that the major compound was identified as avicularin. The avicularin compound is a flavonoid group known to have anticancer activity. The cytotoxicity of avicularin compound was assessed against the WiDr cell line, with a 50% inhibition concentration value of 521.14 μg/m l. The bioactivity of the ethyl acetate fraction is stronger than that of the isolated part (avicularin compound).


INTRODUCTION
The Saurauia vulcani plant was used as traditional medicine in North Sumatra.This plant is called a pirdot, which grows wild on the forest edge.This plant is generally used as an antidiabetic medication.This plant has been planted on critical lands such as Merek, Karo Regency (Pasaribu et al., 2020).
Some previous studies reported that S. vulcani leaf extract has anticancer activities against WiDr and HCT 116 cell lines.The WiDr cell line exhibits cytotoxicity when exposed to fractions of n-hexane, ethyl acetate, and methanol; the 50% inhibition concentration (IC 50 ) values for these fractions are 456.19, 97.41, and 191.92 ppm, respectively.Our previous research showed that the leaf extract's cytotoxicity activity against the HCT cell lines is achieved at an IC 50 of 777.35, 568.53, and 529.39 ppm, respectively, with fractions of n-hexane, ethyl acetate, and methanol (Pasaribu et al., 2021).
Anticancer research using natural ingredients for cancer drugs will continuously be investigated worldwide due to the rising number of cancer patients, particularly those with colorectal cancer.Today, colorectal cancer is the third leading     cause of death, while ovarian and breast cancers are in the first and second places, respectively (Granados-Romero et al., 2017).
Colorectal cancer is very commonly found in people worldwide.The main causes of colorectal cancer are bad habits, a poor diet, smoking, colitis, inherited risk factors, and aging (Benarba and Pandiella, 2018;Hashim et al., 2016).
Conventional cancer treatment with chemotherapy sometimes gives unexpected side effects that cause drug resistance (Kuipers et al., 2015).The adverse effects of chemotherapy for colorectal cancer have been reduced in several ways, including renewable natural substances like plants and other living things.
Herbal medicines can be developed by finding active compounds from the plant extracts produced.This research aims to isolate an active compound from ethyl acetate fractions in WiDr cancer cells and determine the activities of the isolated compound as a colorectal anticancer.

Materials
The plant materials were collected from the Indonesian Ministry of Environment and Forestry research forest in the Sipiso-piso area, Karo Regency.For authentication, samples were tested at Herbarium Bogoriensis, Research Center for Biology LIPI, Indonesia.Meanwhile, WiDr cells made up the cancer cell components.

Fractionation
The S. vulcani leaf was extracted in the previous studies (Pasaribu et al., 2021).In the next step, the fractionation was carried out based on the level of polarity.The test results show that the ethyl acetate fraction gives the best bioactivity.Thus, in the next stage, the ethyl acetate fraction becomes the material to isolate and test its bioactivity.

Isolation
The ethyl acetate fraction was separated using vacuum liquid chromatography and purified using radial chromatography (RC) with a solvent system used as the eluent ethyl acetate: methanol: Aquadest = 4.6:0.2:0.2 to yield 50 mg of the isolated compound.To improve the separation process, the stationary phase is compacted by lowering the mobile phase and the column walls are tapped slowly.After that, the concentrated extract is added gradually with a dropper pipette through the column wall, then eluted with the eluent determined previously by TLC.The fractions were accommodated in test tubes, and each tube was identified by TLC.Next, the fractions with the same spots are combined into one fraction, and the eluent is evaporated using a rotary evaporator.Afterward, RC and Merck Kieselgel 60 PF254 round glass plates were used to perform the isolation.All of the prior profiles were detected using fragments from aluminum Merck TLC silica gel 60 F254 sheets measuring 20 × 20 cm and having a thickness of 0.25 mm.The profiles were then checked at UV light (254 nm) or using an H 2 SO 4 spray heating after the reaction.

Test of anti-colorectal cancer activities
The stages of in vitro anti-colorectal cancer tests followed the procedures and were conducted in previous studies (Pasaribu et al., 2021).The avicularin compound's cytotoxicity was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT) assay.The method depended on the breakdown of the yellow tetrazolium salt, MTT, into a soluble blue formazan product by mitochondrial enzymes.The amount of formazan was proportional to the number of live cells present during the MTT exposure.Human colon adenocarcinoma WiDr cells were used in the test.The cells were cultivated in DMEM, 10% fetal bovine serum, and 1% antibiotic (Penicillin Streptomycin) containing media in a suitable environment (37°C, 5% CO 2 , 90% humidity).In total, 5,000 cells/well of the subcultured cells were placed into a 96-well tissue culture plate to adhere for 18-20 hours in a growth medium.Then triplicate tests on each concentration were conducted.The media was removed from the wells the next day and replaced with an avicularin compound in a growth medium.To make the sparingly soluble avicularin compound homogenous, dimethyl sulfoxide (DMSO) was added; the final concentration of DMSO was 0.5% in each well.The avicularin compound was then added to the plates at varied concentrations for 48 hours: 400, 200, 100, 50, 25, and 12.5 mg/l.Each well in the plates received a dose of MTT (10 l of 5 mg/ml in PBS).The plates underwent an additional 4 hours of incubation.After that, the medium was taken out, and 100 l of 1 N HCl in isopropanol was added.Finally, a microplate reader was used to read the plates' absorbance at 562 nm.The absorbance results were calibrated using 1 N HCl in isopropanol as a blank, and the absorbance of the cells exposed to the corresponding medium was interpreted as 100% cell viability (taken as negative control).Based on the relationship between percent inhibitions and concentrations, the IC 50 of the avicularin compound was calculated for each cell.

Isolation major compound
This study has discovered that the obtained compound is a yellow-pale solid, and the melting point is 215°C-217°C.The ultraviolet (UV) spectrum of compound 1 shows maximum absorption at λmax (MeOH) (log ε) 255-358 nm, which experiences a bathochromic shift with the addition of NaOH.The UV spectral pattern indicates that this compound has a phenol chromophore with an extended conjugation system.
Furthermore, it is also necessary to know the amount of carbon from this compound.The 13 C NMR spectrum (Fig. 3 Based on COSY spectrum, the H-6 and H-8 protons can be explained (Table 1).Although both protons have a doublet signal, the COSY spectrum does not indicate that they are correlated or adjacent (Fig. 5).This is because the value of J is 2.1 Hz for each proton suggests that they are interacting as a meta-coupling in the aromatic system.On the other hand, H-5′ and H-6′ are adjacent and show an ortho coupling in the aromatic system, with J = 8.5 Hz each.H-6′ has a dd multiplicity, and the second J = 2.2 Hz is meta coupled with H-2′.Finally, the remaining five protons H-1ʺ-H-5ʺ are part of the arabinofuranoside units, as indicated by this spectrum's correlation.
The heteronuclear multiple bond correlation (HMBC) spectrum is utilized to establish the relationship between structural units and confirm that the aglycone of the compound is quercetin (C6-C3-C6).The correlation between H-6 and C-5, C-8, C-9, and C-10 (Fig. 6) establishes the presence of 2,4-dihydroxyphenyl (ring A) and carbonyl at C-4 (ring B).Moreover, the correlation between H-2' and carbons C-2, C-3', C-4′, and C-6′ confirm the presence of 3,4-dihydroxyphenyl (ring C) (Fig. 8).Finally, the last HMBC correlation confirms that the arabinofuranoside group is bound to C-2 (Ugheighele et al., 2022) The mass spectrum (MS) data displays an [M-H]ion with a value of m/z 433.0475 (Fig. 7), suggesting that the molecular formula C20H17O11 (calculated value for C20H18O11 is 434.0475).The double bond equivalent (DBE) value is determined to be 12, obtained from the sum of 8 DBEs of the double bond (sp2) and 4 DBEs of the ring.Based on the analysis of the whole spectroscopy data and comparisons with existing literature, it can be inferred that this compound is similar to avicularin (Mohammed et al., 2022(Mohammed et al., , 2021;;Moharram et al., 2018).

Anticolorectal cancer activities
The cytotoxicity exerted by the avicularin compound in WiDr cells is 521.14 μg/ml.This material is derived from ethyl acetate of leaf fraction that has good cytotoxic activity against cancer cells.An extract or chemical compound is declared to provide very strong cytotoxic activity if the IC50 value is below 10 µg/ml.Meanwhile, the cytotoxic activity is classified as strong if the IC 50 value is 10-100 µg/ml.The level of toxicity to cells is considered to be moderate if the concentration at which 50% of cells are killed is between 100 and 500 µg/ml.This study has discovered that the IC50 value has leaf extract at the lowest possible dose to stop the growth of cancer cells; therefore, the growth of cell capacity is decreased or inhibited up to 50% (Weerapreeyakul et al., 2012).
The differences in the morphological changes in the control cells and the variations in concentrations were carried out (Fig. 9).Healthy cells are characterized by round cells and nuclei, protected by clear cell walls, and shine under a microscope (Kusuma et al., 2010).Meanwhile, dead cells appear dark with damaged cell nuclei.The morphological changes were pronounced at high isolate concentrations.In the control cells, the cells appeared in clusters, while at various concentrations, the growth of WiDr cancer cells was inhibited.

CONCLUSION
Avicularin was successfully isolated from the ethyl acetate fraction of S. vulcani extract originating from North

Figure 9 .
Figure 9.The appearance of WiDr cancer cells as observed under a microscope following therapy at the isolated compound; control cell (a); concentration 100 ppm (b); 200 ppm (c); 400 ppm (d).