INTRODUCTION:

Medicinal ingredients derived from natural sources have attracted special interest and attention globally for reasons of safety, efficacy, and relatively lower side effects^1–3. This reason has led to a paradigm shift to preferring safer plant-based treatments⁴. Interestingly, therapeutic agents sourced from natural products have been consistently utilized since ancient times⁵.

Data shows that around 65-80% of the world's population in developing countries still depend on plants for their primary medicine due to lack of access to modern medicine^5,6.

Ethnomedicine-based plant screening is very important to be highlighted at this time to be able to manage and prove its potential as a medical therapy⁷. On the other hand, long-term use of antibiotics has undesirable effects such as losing their effectiveness against certain infectious diseases⁸, the emergence of resistant strains (cases of immunocompromised patients taking acyclovir)⁹, and accumulation of residues that are not friendly to environmental health^10,11.

Erythrina lithosperma Miq (Family: Fabaceae) is a potential traditional plant that is widely used in Ayurvedic, Siddha, and Usadha medicine systems. In Bali Province, Indonesia, this plant is commonly known as "Dadap Serep". The dried leaves of this plant are efficiently used as a treatment for typhoid fever by local indigenous people. Many studies have shown the bioactive properties contained in various Erythrina species as antibacterial^12,13, antidiabetic¹⁴, antiplasmodial¹⁵, gastroprotective¹⁶, hepatoprotective¹⁷, and anti-cancer^18,19.

However, information on the cytotoxicity of E. lithosperma leaves on normal human cells has not been widely reported. The use of cell lines such as Vero cells lines in pharmacology and medicine has been widely used to examine the cytotoxicity of herbal ingredients²⁰. In vitro cell culture experiments are often performed to investigate a biological process or reaction under specific circumstances²¹. However, the results of in vitro tests cannot be directly applied to real clinical circumstances, but these efforts are extremely therapeutically useful as a model for screening various characteristics and dangers offered by a drug²². The Vero cell lines were investigated for their potential utility in assessing the cytotoxicity of E. lithosperma.

The viability and reaction generated in Vero cell lines may change depending on the concentration of E. lithosperma leaf extract (ELLE) used. Viability testing is critical for better understanding the potency of ELLE on Vero cell lines. As a result, the purpose of this study was to determine the cytotoxicity of ELLE on the viability of Vero cell lines using the MTT test. The findings of this first test are critical in providing useful information about the future potential of ethnomedicinal plants.

MATERIALS AND METHODS:

EXTRACTION OF PLANT MATERIALS:

Plant material for ELLE was collected from the tropics, precisely in the plantation area of Negara Regency, Bali Province. The sample used in this study was the leaves of E. lithosperma. The leaf samples obtained were washed using clean water and dried using an oven at 40°C for 48 hours until the water content was reduced to obtain crude Simplicia. Furthermore, this crude Simplicia was weighed and then blended for 10 minutes, and then sieved using a 60 mesh sieve to produce Simplicia powder.

The Simplicia powder was then macerated by weighing 50 gr of extract and 1:10 (w/v) solvent. The sample was macerated using distilled water and then stored at room temperature for 2×24 hours and filtered. The results obtained were in the form of filtrate which was then concentrated using a rotary evaporator at a temperature of 40°C to produce a thick extract in the form of a paste.

PREPARATION OF VERO CELL LINES:

The cells used in this test are The normal African green monkey kidney epithelial cell line (Vero). Vero cells lines were cultured on an M199 (Sigma-Aldrich, US) medium with the addition of 10% FBS (Sigma-Aldrich, US) and the antibiotics Panstrep (Gibco, US) (2mL) and Fungison (Gibco, US) (1mL). The cells were cultured at 37°C in a CO₂ incubator until confluent. After incubation, the medium was replaced with a new medium, then cells were harvested by dripping with Trypsin. The harvested Vero cells were then re-cultured on a 96-well microplate with an initial density of 2×10⁴cells/100 mL/well. Vero cells lines were close to confluent at 24 hours of incubation. The addition of E. lithosperma extract was carried out 24 hours after culture on the microplate to prevent the Vero cell lines culture from being too dense²³.

The addition of ELLE begins with pouring the cell culture medium and replaces it with a new medium that has been added with ELLE with a final concentration of 10, 000; 8, 000; 4, 000; 2, 000; 1, 000; 500; 250; 125; 62.5; and 31.25µg/ml. Control cells are cell cultures without the addition of materials.

VIABILITY OF VERO CELL LINES ON ELLE:

The Vero cell line viability was determined by methyl-thiazole-tetrazolium assay (MTT)^7,24,25. A total of 10mL of M199 culture medium was added with 1 ml of MTT, mixed evenly. After the MTT medium mixture was prepared, the Vero cell line culture on the microplate was poured so that the remaining medium and dead cells (not attached to the plate) were removed. Then each well was filled with 100µl/well MTT mixture and incubated for 4 hours in a CO₂ incubator at 37°C. Stop solution was added to each well at a dose of 100µl/well then the plate was stored at room temperature for 12 hours. Cell growth was detected with a microplate reader at a wavelength of 550nm. The growth rate of cells exposed to ELLE was compared with the negative control, which was expressed in percent (%)²⁶.

STATISTICAL ANALYSIS:

Data are shown as mean and standard deviation. Statistical analysis was performed using SPSS software version 23.0 (SPSS Inc., Chicago, IL, USA). All data were tested for normality and a One Way ANOVA test was used to compare the control treatment and various concentrations of the ELLE group²¹.

RESULTS AND DISCUSSIONS:

Standardized traditional medicine must be active against certain infectious diseases and without causing significant toxicity to normal cell viability. A cytotoxicity test was carried out on Vero cells lines to confirm the safety of the aqueous ELLE.

Figure 1. Viability of cells seen with an inverted microscope: Control cells (A), concentration 31.25µg/ml (B), 62.5µg/ml (C), 125µg/ml (D), 250µg/ml (E), 500µg/ml (F), 1,000µg/ml (G), 2,000µg/ml (H), 4,000µg/ml (I), 8,000µg/ml (J), 10,000µg/ml (K).

The selection of Vero cells lines is ideal as a step-by-step model for in vitro studies due to their good sensitivity to toxicity, easy of culture technique, and availability in our various testing facilities. On the other hand, Vero cells lines are recommended as a model for detecting basal cytotoxicity^27,28. The viable cells viewed under an inverted microscope are presented in Figure 1. The mean and standard deviation (SD) values for the control group and the ELLE are shown in Table 1. Vero cell lines growth rates for various concentrations of ELLE are shown. in Figure 2.

MTT test is a colorimetric test that has sensitivity, is quantitative, and is reliable for measuring the viability of a cell. This assay is based on the capacity of the cellular mitochondrial dehydrogenase enzyme in living cells which is used to reduce the water-soluble yellow substrate 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) to dark blue/formazan products. purple which is insoluble in water. The number of cells in the cell line range has an amount that is directly proportional to the amount of formazan produced in this stage²⁹. Figure 1 clearly shows the effect of various concentrations of ELLE on Vero cells lines. The number of cells that experienced death increased along with the increase in the concentration of the extract treatment in this study. At concentrations of 8,000µg/ml and 10,000µg/ml (Figures 1J and 1K), the cells were more spherical in shape, small in size, and showed signs of detachment from the surface which could be suspected as cell death.

The previous findings showed a higher number of cell death along with an increase in the concentration of Elaeis guineensis extract in the cancer cell line type (MCF-7) when compared with the high after exposure to using Elaeis guineensis extract compared to Vero cells lines. Although overall, the number of cell death in both types of cells was still high after the concentration of E. guineensis extract increased³⁰. Cytotoxicity of various extract fractions of Aspilia pluriseta Schweinf. has been reported. The findings stated that all solvent extract fractions except the methanol solvent fraction had a cytotoxic concentration value that was able to kill 50% of Vero cell lines greater than 20µg/ml and with a selectivity index greater than 1.0. The methanol extract of Adenanthera pavonina seeds and leaves was shown to exhibit bone cancer cell lines, supporting the potential of this traditional plant to be explored further for anticancer medicines³¹. The chlorophyllin chemical isolated from the medicinal plant Phyllanthus emblica revealed its potential efficacy in suppressing the activity of breast cancer cells and exhibited no adverse impact on the Vero cell line by MTT test³².

Table 1. Mean and standard deviation of control and treatment groups

Concentration (µg/ml)	Cell Viability (Mean±SD)
Control	100±0.000^a
31.25	86.0927±0.002^g
62.5	86.0927±0.00173^g
125	81.1258±0.00264^fg
250	85.4304±0.00208^fg
500	80.1324±0.00346^f
1, 000	69.5364±0.00230^e
2, 000	64.9006±0.00057^e
4, 000	39.7350±0.00057^d
8, 000	21.5231±0.0000^b
10, 000	28.4768±0.0000^c

Remarks: replication (n = 3 for each concentration). Columns with different notations showed significant differences between treatments based on Duncan's test (p<0.05).

Table 2. Significance between different concentration groups of ELLE and control

Concentrations

(µg/ml)

Concentrations (µg/ml)

10, 000

8, 000

4, 000

2, 000

1, 000

500

250

125

10, 000

0.007*

-0.011*

-0.036*

-0.041*

-0.052*

-0.057*

-0.058*

-0.072*

8, 000

0.00700*

-0.018*

-0.043*

-0.048*

-0.059*

-0.064*

-0.060*

-0.065*

-0.079*

4, 000

-0.011*

-0.018*

-0.0253*

-0.0300*

-0.040*

-0.046*

-0.041*

-0.046*

-0.060*

2, 000

-0.036*

-0.043*

-0.025*

-0.0046^ns

-0.004*

-0.020*

-0.0163*

-0.021*

-0.035*

1, 000

-0.041*

-0.048*

-0.030*

-0.004*

-0.010*

-0.016*

-0.011*

-0.016*

-0.030*

500

-0.052*

-0.059*

-0.040*

-0.015*

-0.010*

-0.005^ns

-0.001^ns

-0.006*

-0.020*

250

-0.057*

-0.064*

-0.046*

-0.020*

-0.016*

-0.005^ns

0.004^ns

-0.0006^ns

-0.014*

125

-0.053*

-0.060*

-0.041*

-0.016*

-0.011*

-0.001^ns

0.004^ns

-0.005^ns

-0.019*

-0.058*

-0.065*

-0.046*

-0.021*

-0.016*

-0.006*

-0.0006^ns

-0.005^ns

0.000^ns

-0.014*

-0.058*

-0.065*

-0.021*

-0.016*

-0.006*

-0.0006^ns

-0.005^ns

0.000^ns

-0.014*

-0.072*

-0.079*

-0.060*

-0.035*

-0.030*

-0.020*

-0.014*

-0.019*

-0.014*

Remarks: * : significant difference (P<0.001), ns: no significant different

The ELLE was found to be toxic at a concentration of 10,000µg/ml; 8,000µg/ml; and 4,000µg/ml is 28.4768%, 21.5231%, and 39.7350%, respectively. Significantly different when compared to the treatment at the lowest concentration of 31.25µg/ml, which was 86.0927%. However, our findings are still significantly lower when compared to the control group which has the highest percentage of viability (Table 1). According to ISO 109993-5, a substance is hazardous if the proportion of cell death is less than 30%³³, therefore E. lithosperma leaf extract at lower doses is considered as safe because it has a live cell viability percentage of more than 70%. The results of the MTT test at different concentrations of ELLE showed insignificant differences between 500, 250, 125, 63, and 31µg/ml. Furthermore, the data that have significant differences can be seen in Table 2.

Although the contribution of traditional plants has been highlighted to lead the development of promising therapeutic drugs for a long time until now, the facts show that plant extracts are not always safe when consumed in certain amounts and doses^30,34. However, traditional plants have been widely accepted as one of the promising products for the development of modern therapeutic drugs. Bioactive secondary metabolites derived from plants are very interesting to be used in an effort to prevent various health-related problems because they cause minimal side effects³⁵. The ELLE was chosen in this study because of its use as a wound-healing agent and reducing typhoid fever by native Indonesians and possibly as a therapeutic agent in other parts of the world. Previous studies have also confirmed the role of a large number of bioactive ingredients contained in this plant such as flavonoids, alkaloids, tannins, steroids, and terpenoids which show a modulating role in various biological activities. Therefore, several traditional herbal ingredients have great potential as new candidates for medicinal ingredients due to their safety, toxicity that can be tolerated at certain concentrations, and their relatively easy acceptance among the local community^36–38.

In our study, the significant decrease in cell viability at the highest concentration was probably due to apoptotic activity. Apoptosis is a mechanism of action of programmed cell death and is involved in the homeostatic management of the cell population, without showing an inflammatory reaction³⁹. Cell death by apoptosis can be identified from the form of cell death by observing the morphological and biochemical characteristics shown by dying cells. Blebbing and changes in the chemical composition of the plasma membrane, condensation, and fragmentation of chromosomal DNA, to the loss of mitochondrial membrane potential, are features that can be seen in cells undergoing rapid apoptosis^40,41.

The ELLE used in this study had a CC₅₀ (cytotoxic concentration) value of 390.59µg/ml or was included in the category of “low toxicity”. In previous studies, the potential cytotoxicity of Bungasia auriculata extracts against HeLa, Vero CRFK, and HEL cells which had cytotoxicity in the range of 100g/ml which was included in the moderate category for viral infections⁴². Other Indonesian plants, such as Abelmoschus Manihot from Palu, Central Sulawesi, inhibited breast cancer cells at a concentration of 185.06µg/ml while showing no effect on the development of Vero cells at a concentration of 200µg/ml⁴³.

The results of this study provide clear initial information that the ELLE tested for cytotoxicity on Vero cells lines is still safe for consumption at low concentrations and can be further developed as a traditional medicine product. Further research is still needed to evaluate other types of solvents on the cytotoxicity of this extract. Likewise, the ability of extracts against various types of cancer cells, bacteria, viruses still needs to be done in future research. In addition to cell culture tests, a molecular docking method is highly essential in rational drug design and discovery⁴⁴. Docking's principal role is to search for various virtual-based information and based on particular chemical substances, both current and novel, against certain target proteins, such as cancer proteins, viruses, bacteria, or other degenerative illnesses^45–47.

Figure 2. Vero cell line growth rate exposed to various concentrations of ELLE in this study.

CONCLUSION:

The overall results showed that ELLE which was tested for cytotoxicity levels on Vero cells lines was still safe for consumption by humans at lower concentrations and could be used as a promising alternative for standardized traditional medicinal ingredients in the future. Further research is still needed to evaluate the effect of various solvents on ELLE on its cytotoxicity. In vitro studies with various types of cancer cells, infectious diseases, and experimental animals (in vivo) are urgently needed to test the great potential of this extract in the future.

ACKNOWLEDGEMENT:

The authors would like to thank the Directorate General of Higher Education, Ministry of Education, Culture, Research, and Technology (KEMDIKBUDRISTEK) of the Republic of Indonesia and Universitas Dhyana Pura (Undhira Bali) for supporting this research.

AUTHORS’ CONTRIBUTIONS:

AAAPP and PAW: Supervision, Conceptualization, Methodology, Formal Analysis, Writing-Review, Editing, and Project Administration. IGW: Methodology, Formal Analysis, Writing-Review, and Editing. NW, NMV, I MFY: Investigation, Resources, Original Draft Preparation, Software, Validation, and Data Curation. THS: Methodology, Resources, and Data Curation. All authors declare that they contributed to the critical review of intellectual content and approval of the final version to be published.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 19.09.2021 Modified on 10.01.2022

Research J. Pharm. and Tech 2023; 16(1):153-158.

DOI: 10.52711/0974-360X.2023.00028