INTRODUCTION:

Cancer is a disease due to decreased apoptotic activity and dysregulation of abnormal cell proliferation surrounding tissues, leading to death^1,2. Definition of a natural compound was a non-essential bioactive compound. They have various effects on human health, such as suspected chemo-preventive properties (anti-carcinogenic and antimutagenic) and interfere with tumor promotion and development³.

Natural biological material from plants was one source of natural medicinal ingredients for cancer treatment. Some of its derivatives include Vincristine, Vinblastin, Podophyllotoxin, Etoposide⁴.

The secondary metabolites of alkaloids, terpenoids, flavonoids, phenolics, and organic acids found in various plants have been shown to kill the growth of cancer cells⁵. Secondary metabolites formed in most living organisms, among other alkaloids, terpenoids, flavonoids, have potential anticancer activities^6–8. Most of the secondary metabolites have been reported to have anticancer effects, namely: alkaloid compounds have a cytotoxic effect on HCT-116 (colon cancer) and HeLa (cervical cancer) cells⁹. Terpenoids compounds and their derivatives: eugenol were active against the colorectal cell line HCT-116 and WiDr³^,¹⁰, and geraniol active on colon cancer cells⁷, Flavonoids group and their derivatives compounds such as quercetagenin, vitexin, artemetin, luteolin, vitetrifolin, curcumin were active on colon cancer, HeLa and Caco-2 cells line^8,^11,12.

Plant species that have reported cytotoxic activity on the human colon cancer cell line HT-29, including: Andrographis echioides, Saraca asoca bark, Mussaenda erythrophylla, and Alstonia scholaris L^13-16, on cell lines HCT-15 (Punica granatum L) species, and HCT-116 (Phaleria macrocarpa bark) and Pinus merkusii bark species^17–19. In addition, it was also reported that the plant species Cosmos caudatus, Majidea zanquebarica J. Krik, Eleutherine palmifolia L, Merr, and Cocos nucifera had a cytotoxic effect on HeLa cells of cervical cancer⁸^,20-22.

Some plant species from Verbenaceae were reported to have anticancer activity on HCT-15 cells, colon cancer, and HeLa cells such as family: Premna, Clerodendrum, Lippia, Verbena, and Vitex^23–28. P. canescens Jack was a plant with Family: Verbenaceae was a well-known Indonesian medicinal plant, which has been used as traditional medicine, especially the Dayak tribe of East Kalimantan, used as an anti-bloating, toothache, ringworm, and fever medicine⁶. Chemical compounds of P. canescens have been reported were β-sitosterol, phytol, β-amyrin, lantaden A, lantaden B, lanthanolic acid, lanthanic acid, and lantonin alkaloids²⁹, peronemin A1, A3, B2, B2, B3, C1, and D1³⁰. Secondary metabolites include flavonoids, terpenoids, steroids, alkaloids, phenolics, and saponins^31–33. They have cytotoxic potential mainly as anti-cancer^3,⁶.

Bioactivity studies that have been reported in South Sumatera and Lampung, the young leaves of P. canescens Jack used anti plasmodium and fever^29,34, antioxidant, immune-boosting, antipyretic^33,34, antibacteria³⁵, antidiabetic activities³⁶. In addition, research on the bioactivity of methanol, hexane, and ethyl acetate extracts has been reported using the animal model of the Brine Shrimp Lethality Test (BSLT) method. The results test showed that the three extracts had toxicity to animal model sea shrimp larvae³². From the description above, it is necessary to develop research on the cytotoxic effect (in vitro) of P. canescens leaf extract using an anticancer cell line. This study used HT-29 colon, and HeLa cervical cancer cells were used. This study used HT-29 colon cancer and HeLa cervical cancer cells. This study aimed to determine the cytotoxicity (IC₅₀) of P. canescens leaves extract of chloroform, ethyl acetate, and ethanol on HT-29 colon cancer and HeLa cervical cancer cells so that it can be developed as a source of alternative cancer treatment.

MATERIAL AND METHOD:

Materials:

The P. canescens Jack was obtained in Tanah Merah Sub-District, Samarinda, East Kalimantan, in May 2019. It was determined at the Forest Ecology and Dendrology Laboratory of the Faculty of Forestry, Universita Mulawarman, Samarinda. The specimen voucher (33/H17.4.1.08/LL/VI/.2011). In this study, several chemicals were used, including methanol p.a, chloroform p.a, ethyl acetate p.a, ethanol pa (Merck, Indonesia), Bismuth nitrate, Potassium iodide, Magnesium powder, Sulfuric acid, and Iron (III) chloride (Merck, Indonesia), silica gel G60 and PF₂₅₄ (Merck®, Darmstadt, USA). Standard reagents and other chemicals were purchased from the Sigma Chemical Company (St. USA). Dulbecco's Modified Eagle Medium (DMEM) and Fetal Bovine Serum (FBS) (Biosera, South America Origin), Media Roswell Park Memorial Institute (RPMI) (Gibco BRL, Life Technologies, USA), Trypsin-EDTA solution, and fetal bovine serum from Sigma Chemical Company (St Louis, MO, USA). PBS (Biomatic, Canada. The USA). Dimethyl sulfoxide (DMSO) (Biomatic, Canada, USA). 1% Streptomycin and Penicillin (Meiji, Indonesia). MTT: Reagents Stopper Sodium Dodecyl Sulfate (SDS) 10% (Merck®, Darmstadt, Germany), Yellow tips, and Blue tips (Neptune, USA), Tissue Culture Flask 25 cm (Biologix Group Limited). Moreover, used tools, BioSafety Cabinet (ESCO: West Heidelberg, Australia), Waterbath (Elma: Singen, Germany), CO₂ Incubator (N-Biotek: Korea), Centrifuge (Sigma, Sartorius, Germany), Microscope Inverted (Axiovert 40 CLF, Zienhen- Germany), and ELISA (Model Accu Reader, Taiwan).

Methods:

Plant extraction:

Previous research used Macerator for the sample extraction process³⁷. The leaf of P. canescens was cleaned, dried, and pulverized was macerated with methanol for 2 x 24 h. Residue and extract solutions were separated using the Buchner funnel. The extracts solution was filtered using Whatman filter paper no. 1 was evaporated to obtain a concentrated extract under vacuum pressure.

Fractionation:

According to a previous study, the fractionation method was carried out using a separatory funnel^38,39, with some modification. Fractionation was carried out by suspending extract in 150mL water separately partitioned organic solvents (chloroform, ethyl acetate, and ethanol) according to a polarity gradient by using a separating funnel. The extract was stored at room temperature and ready for use.

Phytochemical screening:

Phytochemical screening was carried out using a qualitative method with slight modifications as described by^37,38. The following phytochemicals were tested for; alkaloids, terpenoids, steroids, phenolics, and saponins using specific reagents.

Cell preparation:

Culture HT-29 colon and HeLa cervical cancer cells were obtained from the Pathology Anatomy Laboratory of the Faculty of Medicine, University of Indonesia. The test cells were maintained in RPMI and DMEM 1640 supplemented with 10% FBS, added penicillin and streptomycin 100µg/mL, incubated at 37^oC with 5% CO₂. Both test cells were exposed to various extract concentrations for 24 hours³.

Cytotoxicity test (MTT assay):

Suspension of HT-29 and HeLa cancer cells (100μL) with a density of 2 x 10⁴cells/100μL were inserted media into the 96-well disc and incubated in 5% CO₂ at 37^oC for 24 hours. To the well was added 100μL of the test solution with a series of concentrations: 1.5, 3.123, 6.25, 12.5, 25.0, 50.0, 100.0, 200.0µg/mL. Next, MTT reagent (0.5mg/mL in PBS) was added 20μL of to each well incubated in 5% CO₂ for 4 hours at 37^oC. The negative control used DMSO. The condition of the cells was examined with an inverted microscope. If formazan was formed after incubated for 4 hours, the supernatant was removed by adding 150 μL DMSO to dissolve the formazan crystals in the cell, mixed in the orbitals for 2-3 minutes, incubated in a dark place at room temperature for 24 hours. Live cells react with MTT to form a purple color. ELISA read the test results at 90 - 590 nm^39–42. The inhibition percentage was calculated to get the IC₅₀ value using by probit analysis (Product Solutions and Service Statistics (IBM SPSS) 22.0 for Windows)

Absorption of treatment group

% inhibitory value = 1 ---------------------------------------------- x 100%

Absorption of a control group

RESULTS:

Profile of secondary metabolites:

The results of the identification of crude and extract fraction extract of P. canescens leaf were using chemical reagents of alkaloids, terpenoid-steroid, flavonoid, phenolic, and saponin groups, namely Dragendorf and Wagner (alkaloids), Lieberman-Bouchard (terpenoids-steroids), Magnesium powder plus HCl (flavonoid), Iron (III) chloride (phenolic and polyphenol groups), and Forth method (saponin). The profile of secondary metabolites was presented in (Table 1).

MTT assay:

The MTT assay measures cell proliferation and viability and acts on macrophage-mediated living mitochondria cells⁴⁰. The test results showed a cytotoxic effect of the two test cells. From the graphic (Figures 1A and 1B) showed that the concentration of the extract to 200.0 µg/mL cell percentage viability in both cell lines, namely: chloroform (8.80%), ethyl acetate (45.00%), and ethanol extract (17.30%) on HT-29 cells, respectively. Whereas in HeLa cells (9.20%, 9.30%, and 54.00%), successively. The complete data can be seen in (Table 2).

Figure 4. Cytotoxicity (IC₅₀) value of P. canescens leaf extracts on HT-29 colon and HeLa cervical cancer cells after treatment 24 hours.

DISCUSSION:

The results of the secondary metabolite test with Dragendorf reagent showed a positive reaction for orange alkaloids and a brown precipitate with Wagner test⁴⁴. Dragendorf reagent forms an orange precipitate formed by bismuth (Bi₃⁺) complex reactions with potassium iodide (KI). Furthermore, the nitrogen in the alkaloid compound binds to a coordination covalent complex with K⁺ ion^44,45. However, the reactions of the negative alkaloid can occur if the bismuth nitrate (Bi(NO₃)₃ salt undergoes hydrolysis. The addition of acid can prevent the hydrolysis reaction. It will shift to the left so that the Bi₃⁺ ion reacts with KI to form BiI_3, which dissolves to form potassium tetraiodobismuthate [K(BiI₄)]^52,53.

The reaction of color formation and brown precipitate in Wagner reagent due to ligand replacement reaction, the nitrogen atom in the alkaloid has a lone pair of electrons, complex bonds (potassium-alkaloid) with K⁺ ions potassium tetraiodobismuthate by coordinate covalent bonds. Meanwhile, in the Wagner test, the complex reaction occurred between the alkaloids and K⁺ ions from potassium tetraiodobismuthate to produce a brown precipitate⁴⁵.

In the phenolic test with the Iron (III) chloride (FeCl₃) reagent, a positive reaction was indicated by blue-black solution⁴⁴. The black color was formed due to the complex response of the aromatic (OH^-) group reacting with FeCl₃. The iron (III) Hexa phenolate formed undergoes a bathochromic shift towards a larger wavelength^45,46. The results of the terpenoid test with Lieberman-Bouchard formed a brown ring on the tube, whereas steroid reaction green color solution. The difference in the test results was due to sulfate ionization (SO₂^-) in the C-4 atomic group for terpenoid compounds and the hydroxyl group (OH^-) in steroid compounds⁴⁶. The positive reaction of the red solution flavonoid test after the addition of magnesium powder (Wilstater method), due to the response of the Mg²⁺ complex with ortho-dihydroxy and hydroxy ketones on the flavonoid compound group, there was a more significant wavelength shift (red bathochromic)⁴⁷.

In the saponin test (Forth method), a stable foam was formed with the addition of HCl. Saponin compounds are surfactants because they have hydrophilic and hydrophobic groups to reduce surface tension⁴³. When shaken, compounds with surface-active (surfactant) properties will form micelles⁴⁸.

Terpenoids, alkaloids, and flavonoids have been reported as a group of compounds that have cytotoxic activity on HCT-116 and the WiDr colon cancer, and HeLa cervical cancer cells^3,8–10. MTT method to test the cytotoxic activity of a chemical compound. MTT was a pale yellow substrate that divided live cells to produce a dark blue formazan product. This process requires active mitochondria. This test was used and described proliferation or complement-mediated cytotoxicity assays⁴⁹. The principle of the reaction was through the reactions of succinate dehydrogenase; the tetrazolium salt is converted to purple formazan and then measured by spectrophotometry⁵⁰. This study aimed to determine the cytotoxicity (IC₅₀) of P. canescens leaf of the chloroform, ethyl acetate, and ethanol extract on HT-29 colon cancer and HeLa cervical cancer cells. Cytotoxic activity was determined based on the IC₅₀ value, and smaller values indicate stronger cytotoxic. The measurement of the IC₅₀ value was based on the percentage inhibition value of the test extract. Based on the study results (Table 2), it can be seen that the increase in the concentration of the cell viability test decreases; the suspect may be associated with DNA damage³⁹. It was supported by observations of the morphology of the two cells (Figure 2), cell condensation, nuclear shrinkage, and cytosol granulation might occur, cells die during exposure, cell membranes harden and rupture. Morphological changes are evident in line with the increase in test concentration. In comparison, the IC₅₀ value of each extract showed a relationship between the concentration of P. canescens leaf extract treatment with the percent (%) inhibition of the second cell (Figure 3).

Based on the calculation results (Figure 4), The IC₅₀ values of chloroform extract in HT-29 and HeLa cells were obtained (10.353 and 38.913µg/mL), respectively, which showed high to moderate anticancer activity in both test cells. Ethyl acetate extract had a medium effect on both test cells (IC₅₀value48.635 and 28.186µg/mL) successively. The ethanol extract was moderately active on HT-29 colon cancer cells. Meanwhile, the ethanol extract had a weakly anticancer effect on HeLa cells (IC₅₀ value 253.190µg/mL). Based on the guidelines of the American National Cancer Institute and Geran et al., there are four categories of extract cytotoxic activity, namely; very active (IC₅₀ ≤ 20μg/mL), moderately active (IC₅₀ > 20–100μg/mL), weakly active (IC₅₀ > 100–1000μg/mL) and inactive (IC₅₀ > 1000μg/mL) after 24 h exposure⁵¹.

The results above showed that the cytotoxic activity of the chloroform extract was very active on human cells HT-29 colon cancer and moderately active on HeLa cervical cancer, and shows that the secondary metabolite content of P. canescens leaf extract can be anticancer activity.

The results showed that the cytotoxic activity of the chloroform extract had a strong effect on HT-29 human colon cancer cells, and the ethyl acetate extract had a moderate impact on HeLa cervical cancer cells. It was indicated that the secondary metabolites of P. canescens leaf extract have anticancer activity.

CONCLUSIONS:

The chloroform of P. canescens leaf extract, rich in chemical compounds, contributes to a potent cytotoxic effect on HT-29 colon cancer and a moderate impact on HeLa cervical cancer cells. These results could help the development of anticancer drugs for colon cancer and cervical cancer cells.

ACKNOWLEDGMENT:

We would like to thank for Faculty of Pharmacy, the University of Mulawarman, for the financial support in 2019 in our research project, Head of Research and Development Laboratory of Tropical Farmaka, the assistance permits, facilities to support research, as well as the laboratory for their assistance and cooperation.

CONFLICTS OF INTEREST:

The authors declared no conflicts of interest.

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Received on 25.09.2021 Modified on 11.12.2021

Research J. Pharm. and Tech 2022; 15(10):4739-4745.

DOI: 10.52711/0974-360X.2022.00796

S. No	Compound group	Extracts				Reactions	Reagents
S. No	Compound group	MeOH	CHCl₃	EtOAc	EtOH	Reactions	Reagents
1	Alkaloids	+	+	-	-	orange precipitate	Dragendorf
1	Alkaloids	+	+	-	-	brown precipitate	Wagner
2	Terpenoids	+	+	-	-	brown ring	Lieberman-Bouchard
3	Steroids	+	+	-	-	green solution	Lieberman-Bouchard
4	Flavanoids	+	+	+	+	red solution	Wilstater
5	Phenolic	+	+	+	+	blue-black solution	Iron (III) chloride
6	Saponin	+	-	-	+	foam	Forth

HT-29 cells				HeLa cells
Concentration (μg/mL)	Viability cells (%)			Concentration (μg/mL)	Viability cells (%)
Concentration (μg/mL)	CHCl₃	EtOAc	EtOH	Concentration (μg/mL)	CHCl₃	EtOAc	EtOH
3.125	66.20	91.80	121.60	3.125	68.80	61.90	83.50
6.25	68.00	98.40	96.30	6.25	70.70	63.50	81.00
12.5	66.90	68.70	90.70	12.5	69.60	70.40	73.20
25.0	52.30	74.60	49.20	25.0	54.40	55.00	65.30
50.0	42.90	54.10	38.20	50.0	44.70	39.20	69.00
100.0	23.00	46.50	30.50	100.0	23.90	41.70	66.80
200.0	8.80	45.00	17.30	200.0	9.20	9.30	54.00