Phytochemical screening, antibacterial, antioxidant, and anticancer activity of Coffee parasite acetone extract ( Loranthus ferrugineus Roxb)

antibacterial

wounds, and fever (Lohézic et al. 2002). L. ferrugineus is used to treat high blood pressure and gastrointestinal complaints (Ameer et al. 2015). It is also used to treat stomach and back pain and can treat cancer (Kwanda et al. 2012). L. parasiticus is used for the treatment of brain diseases. The genus Loranthus is used to treat diabetes, inflammation, and cancer (Noman et al. 2019).
The use of parasites in traditional medicine is popular. Therefore it attracted researchers to research it. Several studies published regarding parasite plant activity of the genus Loranthus, including L. micranthus, have found antihypertensive, antimicrobial, hypolipidemic immunomodulatory activity, antidiabetic antioxidant, and antidiarrheal (Onunogbo et al. 2012;Moghadamtousi et al. 2013). On the other hand, L. acaciae Zucc has antidiabetic, antiinflammatory and antioxidant activity (Noman et al. 2019).

Plants extract preparation
The research was carried out from April -November 2020; antibacterial testing and phytochemical screening were carried out at the Universitas Negeri Medan laboratory, while antioxidant and anticancer testing was carried out at the Central Lab of Universitas Padjadjaran Bandung. A 200 g of L. ferrugineus Roxb sample was macerated using acetone as a solvent to obtain a crude extract. This process is carried out for 3 × 24 hours at room temperature. The extract was filtered with Whatman filter paper, then evaporated at low pressure using a rotary evaporator at a temperature of 50 °C (Juwitaningsih et al. 2020a).

Phytochemical screening
Phytochemical screening was carried out, referring to the method that was used by Harborne (1987) using specific reagents. These tests include the alkaloid flavonoids, triterpenoids, steroids, saponins, and tannins test.

Antibacterial activity test
Antibacterial activity test was carried out by using the paper disc diffusion method (Juwitaningsih et al. 2020a) against Bacillus cereus ATCC 1178, Salmonella enterica ATCC 14028, Propionibacterium acnes ATCC 27853 and Streptococcus mutans ATCVC 35668 bacteria. The test solution was prepared at a concentration of 1% (10.000 μg/mL) in 10% Dimethyl sulfoxide (DMSO).

Antioxidant activity test
The antioxidant test used the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method (Molyneux 2004;Soltanzadeh et al. 2018). The concentrations of each of the test solutions were 100, 80, 60, 40, and 20 ppm. The test was replicated twice. Antioxidant activity was determined by the inhibition concentration (IC 50 )value that was calculated using the equation obtained from the linear regression curve y = a+bx, where x is the sample concentration, a is the regression coefficient, b is the intercept, and y is the % inhibition value. The IC 50 value is determined by the formula:

Anticancer activity test
The anticancer activity test was carried out as in previous studies, namely the MTT method (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Assay (Juwitaningsih et al. 2020b). The anticancer activity test was carried out on HeLa and A259 cells. The absorbance measurement used a Multimode Reader at a wavelength of 595 nm. Based on the absorbance value obtained, the percentage of cell viability is determined using the formula: The IC 50 value obtained from the linear regression equation y = a + bx. The graph was made with the concentration of the test sample (ppm) as abscissa (x-axis) against the percent Viability as the ordinate (y-axis) -data processed with Excel version 2016.

Phytochemical screening
Based on the phytochemical screening result against L. ferrugineus Roxb, the results are summarized in Table 1. It shows that the results of the phytochemical test of L. ferrugineus Roxb acetone extract attached to the coffee tree contain secondary metabolites of the alkaloid, triterpenoid, flavonoid, and tannin.
The results obtained were similar to L. ferrugineus Roxb from Bener Meriah Regency Aceh, which was extracted with ethanol (Yulian and Safrijal 2019). There is a similarity in results even though using different extracting solvents. This is because the polarity of acetone and methanol is almost the same, so the compounds that are extracted are the same.

Antibacterial activity
In this study, the acetone extract of L. Ferrugineus Rox was tested against three gram-positive bacteria, namely P. acnes ATCC 27853, S. mutans ATCV 35668, B. cereus ATCC 1178, and one gram-negative bacterium, S. enterica ATCC 14028, so chloramphenicol was used as control positive which is able to inhibit the growth of bacteria with a broad spectrum. The acetone extract of L. ferrugineus Roxb has antibacterial activity against all tested bacteria, with a clear zone diameter between 6.2-11.1 mm. The results of the observations are summarized in Table 2.
Based on the results of observations on the in vitro antibacterial activity test, the acetone extract solution of L. ferrugineus Roxb showed strong activity against Streptococcus mutans, and other bacteria showed moderate activity. This is based on the following criteria: an extract has very strong antibacterial activity if the inhibition zone diameter is ≥ 20 mm, strong if the inhibition zone diameter is 10-20 mm, and moderate if the inhibition zone is 5-10 mm (Davis et al. 971).
Previous studies of the genus Loranthus showed antibacterial activity, such as L. micranthus, which was extracted with methanol, ethanol, chloroform, and petroleum ether solvent, which showed antibacterial activity against the test bacteria of Bacillus subtilis, Escherichia coli, and Klebsiella pneumonia (Osadebe and Akabogu 2006). Likewise, L. micranthus, grown to Periplaneta americana, provided stronger antibacterial activity against Pseudomonas aeruginosa than amoxicillin, while L. micranthus extracts that are attached to Azadirachta indica, Hydrangea macrophylla, and Irvingia gabonensis showed antibacterial activity against S. typhi and B. subtilis (Osadebe and Ukwueze 2004).
Based on these observations, the antibacterial activity is related to the content of secondary metabolites of L. ferrugineus Roxb, namely alkaloids, terpenoids, flavonoids, and tannins. These secondary metabolites have an antibacterial effect with different mechanisms of action. The alkaloid action mechanism is by disrupting the peptidoglycan component of bacterial (Darsana et al. 2012). The mechanism of flavonoids' action as antimicrobials can be through 3 ways, inhibiting nucleic acid synthesis, inhibiting cell membrane function, and inhibiting energy metabolism (Cushnie et al. 2005). Mechanism of terpenoids' action by disrupting the function of cell membranes (Saleem et al. 2010). Moreover, the mechanism of tannins' action as antibacterials by causing cells to become lysed. This happens because tannins have a target on the polypeptide wall of the cell wall, so the cell wall formation becomes less than perfect. This causes bacterial cells to become lysed due to osmotic and physical pressure so that the bacterial cells will die (Zega et al. 2021;Silaban et al. 2022).

Antioxidant activity
The test parameter for antioxidant activity is Inhibition Concentration (IC 50 ), which is the concentration of an antioxidant substance that causes 50% DPPH to lose its  radical character or its concentration antioxidant substance, which gives an inhibitory percentage of 50% (Nursid et al. 2013). The amount of antioxidant activity is inversely proportional to the IC 50 value, meaning that the greater the antioxidant activity, the smaller the IC 50 value is obtained. The acetone extract of L. ferrugineus Roxb has antioxidant activity with an IC 50 value of 48.7122 (µg/ml), which was the mean value of two repetitions. The results of IC 50 calculations can be seen in Table 3. The IC 50 value was determined from the linear regression graph in Fig. 1.
If the IC 50 value is < 50 µg/ml, the antioxidant activity is categorized as very strong. If the value is 50-100 µg/ml is classified as strong, and if the IC 50 value is 101-250 µg/ ml, it is categorized as moderate (Jun et al. 2003). Based on these criteria, the acetone extract of L. ferrugineus Roxb has strong antioxidant activity. Antioxidant activity may be related to secondary metabolites of phenolics (for example, flavonoids, tannins, phenolic acids, etc.) which can donate hydrogen atoms or electrons to free radicals (Sharififar et al. 2009). The results of previous studies of the genus Loranthus showed antioxidant activity, such as the L. acaciae Zucc chloroform fraction showed high inhibitory activity against DPPH radicals of 88.3% (Noman et al. 2019). Eleven compounds consisting of terpenoids and flavonoids isolated from the methanol extract of L. micranthus leaf twigs showed antioxidant activity in the range of IC 50 = 23.8-50.1 µM (Moghadamtousi et al. 2013).

Anticancer activity
In Indonesia, through the Global Burden of Cancer Study (Globocan) report from the World Health Organization (WHO), the number of cancer patients reached 396,914 cases of cancer in 2020, and 54% of cases occurred in women. Cervical cancer ranks second with 36,633 cases, and lung cancer with 34,783 cases out of total cases. The anticancer activity test was carried out in vitro using the MTT assay method. This test aims to determine the toxicity of a compound. The IC 50 value of L. ferrugineus Roxb acetone extract against Hela cells was 47.62 (µg/mL), and the A549 cell was 192.83 (µg/mL). These values were obtained based on the linear regression equation (Figs 2, 3). NCI (National Cancer Institute) has established criteria for anticancer activity, namely an extract is declared active to have anticancer activity if it has a value of IC 50 < 30 μg/mL, moderate active if it has a value of IC 50 ≥ 30 μg/ mL and IC 50 < 100 μg/mL, it is said to be inactive if the value of IC 50 > 100 μg/mL (Suffness et al. 1990). Based on these provisions, the acetone extract of L. ferrugineus Roxb has strong anticancer activity against cervical cancer cells with an IC 50 value of 47.62 µg/mL. It is the result of the observations of HeLa cells and A549 cell morphology. L. ferrugineus Roxb. Acetone extract at the concentration of 62 µg/mL could kill all HeLa cells and against A549 cells at a concentration of 500 g/mL (Figs 4,5).
In the cell morphology (Figs 4, 5), there is a difference between the positive control (media + cells) and the acetone extract sample treatment with various concentrations and cisplatin as a negative control. Changes in cell morphology that may result from exposure to certain active compounds are a reflection of biochemical conditions, namely the activation of various endonucleases and proteases so that DNA is broken into fragments of different lengths that can lead to cell death either by necrosis or apoptosis. Apoptosis is an active process that requires specific gene transcription and translation and also requires the use of intracellular energy sources, whereas necrosis is cell death with a pathological condition. The difference in the fundamental mechanism of the two types of cell death is reflected in the morphology of cells that    experience death (Nursid et al. 2013). In cells that undergo apoptosis, their cytoplasm volume decreases, the cell nucleus shrinks, and the membrane and organelles remain united. On the contrary, in cells that undergo necrosis, the cell looks bulging or has swelled, the cell nucleus undergoes lysis, the plasma membrane and nuclear membrane are damaged, and cell organelles undergo disintegration (Nursid et al. 2013). In Fig. 4, normal living HeLa cells are polygonal with a clearly visible nucleus. At a sample concentration of 62 µg/mL, there was a change in cell morphology. The cells became rounder, and the cell density was lower than the control and could kill all HeLa cells. The cells experienced shrinkage and lost contact with surrounding cells (Hutomo et al. 2016). At a sample concentration of 62 µg/mL, all HeLa cells died by apoptosis. Although L. ferrugineus Roxb acetone extract did not show activity against A549 lung cells, L. ferrugineus Roxb acetone extract has potential as a lung anticancer. This is based on figure 5 of cell morphology; at a concentration of 250 µg/ mL, some of the A549 cells have died, the cell density was already reduced, and the cell morphology was similar to the positive control, namely cisplatin. At a concentration of 500 µg/mL, all A549 cells died by apoptosis.
In many studies carried out, phenolic, terpenoids, and alkaloids group compounds are known to have activities that can inhibit growth and kill cancer cells. Flavonoids such as Epicatechin, quercetin, and catechin are also isolated from L. micranthus (Moghadamtousi et al. 2013). Quercetin is a flavonoid derivative that has antioxidant, antiinflammatory, and anticancer activity (Alexander et al. 2014). The mechanism of flavonoid's action as an anticancer is by inhibiting the activity of DNA topoisomerase I/II, decreasing the expression of Bcl-2 and Bcl-xl genes, as well as activating endonuclease (Ren et al. 2003). The mechanism of the alkaloid action is by inhibiting cell growth in the G 1 phase through the exposure of p53 (Murti et al. 2007). The terpenoids by blocking the cell cycle in the M phase (mitosis), namely when cancer cells interact with terpenoid compounds, it will cause the mitotic stage to be inhibited, which follows there will be inhibition of cell proliferation and triggers apoptosis.

Conclusions
The acetone extract of parasitic L. ferrugineus Roxb in coffee trees contains flavonoids, alkaloids, triterpenoids, and tannins, and exhibits antibacterial, antioxidant, and anticancer activity. Acetone extract of L. ferrugineus Roxb is the potential as a source of antibiotic, antioxidant, and cervical anticancer compounds.