In vitro cytotoxic and antimicrobial activities of Erythrina suberosa (Roxb) bark

ABSTRACT

Background:

The study was focused on evaluating cytotoxic and antimicrobial activities of Erythrina suberosa (Roxb.) bark through in vitro pharmacological screening.

Materials and Methods:

The bark was extracted using different solvents, for example, dichloromethane, ethyl acetate, methanol, and aqueous for obtaining the organic fractions. These organic fractions were then evaluated for their cytotoxic and antimicrobial activity compared with the standard. Cefixime was used as the standard for antibacterial assay, whereas clotrimazole was used as the standard for antifungal activities. Bacterial strains used were Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), whereas for antifungal activities Candida albicans, Candida parapsilosis, and Candida krusei strains were used.

Results:

The organic fractions obtained were evaluated for their cytotoxic and antimicrobial activities. In cytotoxic assay (Brine shrimp lethality assay), dichloromethane fraction was the most potent with LD₅₀ of 47.63, whereas aqueous, methanol, and ethyl acetate fractions showed LD₅₀ of 121.74, 422.2, and 201.96, respectively. Similarly, for antibacterial assay, dichloromethane fraction showed 32.2mm zone of inhibition against MRSA in comparison with standard cefixime (zone of inhibition, 30.5mm). A minimal zone of inhibition with crude saponins (13.1 and 12.2mm) was observed against C. albicans in comparison to standard (cefixime) with a zone of inhibition of 28.5mm. No prominent results were observed against C. parapsilosis and C. krusei strains.

Conclusion:

The study was based on the plant from Indo-Pak origin, and it has shown some prominent cytotoxic and antibacterial activities. Although the results of this study have provided a basic idea about the efficacy of plant extract, still more explanatory and high-scale studies can be beneficial for elaborating the cytotoxic and antimicrobial activities of this plant.

INTRODUCTION

Traditional systems of medicine have been a topic of global value for the past few years. In many developing countries, a sizeable ratio of the population depends on traditional medicinal systems to maintain primary health-care needs, which is shown in recent research studies.[1] Despite modern medicines present in the developed regions, herbal medicines have usually maintained fame for historical and cultural reasons.[2] Crude active extracts with medical properties are usually obtained from medicinal herbs, which are used in the production of all sorts of medical and chemical agents from simple purgative to the most developed form of antibiotics.[3] For instance, taxol, vincristine, and morphine are isolated from pacific yew, periwinkle (Catharanthus roseus), and poppy (Papaver somniferum), respectively.[4] About half a million plants exist and most of them are left uninvestigated, which can play a decisive role in controlling and treatment of various diseases.[5] According to the available literature, more than 9000 species of plants were documented to have some form of medical properties in traditional heritages, communities’ practices, traditional medicine, and in the modern scientific research.[6]

Erythrina suberosa (Roxb.), commonly known as Indian coral tree or corky coral tree, is a small- to medium-sized tree. The genus Erythrina belongs to the flowering plants of the pea family (Fabaceae). Alkaloids, isolated from the flowers of E. suberosa (Roxb.), were investigated for anxiolytic properties, and the results showed significant effects in Swiss male mice.[7] Erythraline is α-4, β-2-nicotinic receptors antagonist, and it has prominent anxiolytic effect.[8] The crude aqueous extract of flowers of E. suberosa (Roxb.) showed potential spasmolytic, bronchodilatory, and antioxidant effect.[9] From the alcoholic extract of stem bark of E. suberosa (Roxb.), certain flavonoids were isolated, which have shown apoptosis-inducing effects on human promyelocytic leukemia HL-60 cells.[10] The bark of E. suberosa (Roxb.) is locally used in India for diarrhea as a paste along with other plants.[11] Furthermore, bark has also shown some antitumor activities as well as some useful effects in treating liver disorders.[12,13] This study was focused on the cytotoxic and antimicrobial activities of the plant extract with different organic and aqueous solvents.

MATERIALS AND METHODS

Chemicals

All the standard drugs and chemicals used in this study were of analytical grade and were obtained from Mussa-G Chemicals (Abbottabad, Pakistan).

Plant collection

E. suberosa (Roxb.) bark was collected from the Government Postgraduate College, Haripur, Pakistan, and was identified by Prof. Dr. Manzoor Hussain (Head of Department), Botany Department, Hazara University, Mansehra, Pakistan, and a voucher specimen was kept in university herbarium. The bark collected was shade dried and powdered in grinding mill.

Plant extraction

Powder drug (950g) was extracted with 80% methanol (MeOH) for 21 days. The extract was filtered using four folds of muslin sheath followed by Whatman filter paper no. 42 (125mm). The process of extraction was repeated thrice. The major portion of the solvent was evaporated at 40°C using a water bath, whereas the remaining solvent was completely evaporated using a rotary evaporator under reduced pressure. A dark brown semisolid extract was obtained weighing 100g.

Fractions extracted with different organic solvents

The crude bark extract of E. suberosa (Roxb.) was subjected to fractionation with different organic solvents depending on the increasing order of polarity, for example, dichloromethane, ethyl acetate, and MeOH.

Separation of crude saponins

Crude methanolic extract of E. suberosa (Roxb.) bark was first dissolved in petroleum ether followed by extracting with an equal amount of MeOH, which was concentrated in rotary evaporator to get semisolid MeOH fraction, which was further extracted by adding n-butanol followed by the addition of an equal amount of distilled water gently until two layers got separated. The upper layer (n-butanol) was taken and dissolved in MeOH to form a concentrated solution. This solution was poured into precooled diethyl ether, resulting in yellow precipitates that settled at the bottom and were collected. The final calculated weight of crude saponins isolated was 5g.[14,15]

Toxicity study (brine shrimp lethality assay)

Brine shrimp lethality assay was carried out to find cytotoxicity of crude methanolic extract of E. suberosa (Roxb.) bark and its organic fractions. Sea salt weighing 3.8g was dissolved in 1L of distilled water to make 1L sea salt solution, which was filtered, and the pH was adjusted to 8.5. This solution was taken in a container having two portions separated by a perforated wall to allow the larvae movement. To the smaller portion, eggs were introduced and covered with aluminum foil so that no light could pass. The container was incubated at 28°C for 2 days.

Samples were made by dissolving crude methanolic extract of E. suberosa (Roxb.) bark and its organic fractions in 2mg/ mL MeOH, from which 1000, 100, and 10ppm dilutions were made in triplicates. After hatching, 10 shrimps were transferred to each vial. All vials were placed for 24h in the same environment as aforementioned, after which dead larvae were counted. LD₅₀ was calculated for the extract through Probit analysis software, Netanya, Israel.[16]

Probit software

Probit analysis is a type of analyzing software, which can correlate between stimulus (dose) and its response that may be all or nothing. Such programs are used for statistical analysis of data from bioassay studies, such as determining the effects of insecticides on insect mortality. These programs are written in the Mathematica language. With the help of these programs, different operations can be performed in no time such as to find out LC₅₀, LD₅₀, and dose-response curve.

Antimicrobial assays of Erythrina suberosa (Roxb.) bark

Antimicrobial assays were performed on crude bark extract of E. suberosa (Roxb.), organic fractions, crude saponins, and crude alkaloids. For these activities, following bacterial and fungal strains were used[17,18]:

• Staphylococcus aureus

• Methicillin-resistant S. aureus (MRSA)

• Candida albicans

• Candida paropsilosis

• Candida krusei

Preparing an enriched medium for antibacterial assay

Nutrient agar medium was prepared by adding 100 mL distilled water to 2.8g nutrient agar powder in a conical flask. Medium was dissolved completely by heating and sterilized at 121°C for 15min in an autoclave. Hot agar medium was distributed in culture discs on petri dishes inside laminar flow hood.[17]

Evaluation of antibacterial assay

Agar well diffusion method was used for this purpose, the culture plates were placed in an autoclave at 121°C for 15min. In every sterilized petri plate, 30 mL of agar medium was introduced inside the laminar flow hood. Strains of bacteria were spread gently on agar plates by using a sterile cotton swab.

With the help of sterile cork borer, a well of 6-mm diameter was made. Sample solutions were made in 1 mL (1% dimethyl sulfoxide [DMSO]) of crude methanolic bark extract and its organic fractions. The assay plates were incubated at 37°C ± 2°C for 24h. Cefixime was considered as positive control. The zone of inhibitions was measured in millimeter.[16,17]

Antifungal assay

Potato agar medium was used as a growth media for evaluation of study, with the addition of 39g of potato dextrose agar powder to 1L of distilled water. Powdered solution was boiled to mix and completely dissolve the solid particles, which was distributed over the dish plates and autoclaved at 121°C for 15min. Samples were dissolved in 1 mL DMSO. The positive control used was clotrimazole. Media plates were incubated for 24h at 37°C.[16,17]

Distinct zones of bacterial growth inhibition were observed and measured for each of the experimental and positive/negative control cups. For the measurement of zones against fungal strains, incubation time was 48h at 28°C.

RESULTS AND DISCUSSION

Cytotoxic activity of crude extract of Erythrina suberosa (Roxb.) bark

Cytotoxic assay was performed on brine shrimps larvae at different doses. After 24h, the total number of dead larvae was counted and its LD₅₀ was calculated for the respective extract/fraction. As Dicholoromethane fraction showed LD₅₀ = 47.6ppm, this fraction was selected for further investigation. After obtaining significant results, we performed the isolation of crude alkaloids. The results of this assay are presented in Table 1. From DCM fraction, alkaloids were isolated in crude form. Crude alkaloids were also subjected to the cytotoxic assay. The results of this assay are mentioned in Table 2.

Table 1.

Cytotoxic assay of crude bark extract and organic fractions of Erythrina suberosa (Roxb.) bark

No.	Groups	No. of dead shrimps			LD50% (ppm)
		1000 ppm	100 ppm	10 ppm
1	Crude aqueous: methanol extract	23	17	4	121.7492
2	DCM fraction	24	22	19	47.6356
3	Ethyl acetate fraction	24	20	8	201.9651
4	MeOH fraction	13	12	9	422.6151

MeOH = methanol, DCM = Dichloromethane

Table 2.

Cytotoxic assay of crude alkaloids

No.	Groups	No. of deaths in total of 30 larvae			LD50% (ppm)
		500 ppm	250 ppm	125 ppm
1	Crude alkaloids	22	18	7	119.1384

Some phytoconstituents such as alkaloids and saponins of E. suberosa (Roxb.) have shown promising cytotoxic activity and are used to treat different types of tumors. In our study, the methanolic extracts have shown promising cytotoxic effects. Accordingly, a study based on the flavonoids of E. suberosa (Roxb.) has shown promising apoptotic and anticancer effects in HL₆₀ cells.[19] Crude alkaloidal portion was subjected to cytotoxicity assay resulting in LD₅₀ (119.13). Similarly, other members of the family have also been found with promising cytotoxic effects, for example, flowers of Erythrina lysistemonare are found to be toxic to brine shrimps.[20] Through human promyelocytic leukemia HL₆₀ cells, it has been found that stem bark of E. suberosa has apoptosis-inducing effects.[21] From aqueous leaf extracts of E. suberosa, biosynthesized silver nanoparticles were obtained, which were found with potentially effective anticancer activities.[18] Most of these studies were focused on leaf and flower extract, whereas our study was focused on the bark, and hence the results were nonetheless compatible with previous studies.

Antibacterial assay of crude methanolic extract of Erythrina suberosa (Roxb.) bark and its organic fractions

Antibacterial assay of crude (aqueous/MeOH) as well as various organic fractions of E. suberosa (Roxb.) bark was performed for antibacterial study against MRSA (C10 and C11 strains), and S. aureus as shown in Figure 1. By checking for appearance and disappearance of inhibition zones, microbial strains were assessed, which was determined by measuring its diameter with the help of scale.

Figure 1.

Graph of antibacterial assay of crude aqueous bark extract of Erythrina suberosa (Roxb.) bark and its fractions

The organic fractions, as shown in Figures 1 and 2, showed very promising results on the inhibition of MRSA and S. aureus. Comparable to cefixime, the ethyl acetate fraction showed better effects on the inhibition in the media. Similar results were found in a study conducted on leaf extract, which showed effective antimicrobial activity.[18] The antibacterial results showed that the majority of fractions were active but keeping in mind (minimum inhibitory concentrations), the zone of inhibition of MeOH fraction with 22.5 ± 0.76 (C10 strain) and 21.3 ± 1.63mm (C11 strain) and DCM fraction with 32.2 ± 0.67 (C10 strain) and 32.2 ± 0.67mm (C11 strain) was found to have significant activity against S. aureus and MRSA, whereas crude saponins showed very significant activity against MRSA (strain C11). Similar results were shown by a study on another species Erythrina cristagalli L.; when extracted with MeOH, it showed prominent results against gram-negative and gram-positive bacteria.[22] In accordance with our study results, Erythrina latissima flavonoids have been found to have effective antimicrobial activities.[23] In this study, focus was on the bark of the plants, and the effectiveness of alkaloids and saponins has been evaluated.

Figure 2.

Methicillin-resistant Staphylococcus aureus (C10 and C11) and S. aureus strains of aqueous and organic fractions. 1 = Ethyl acetate fraction, 2 = Dichloromethane fraction, 3 = methanol fraction, 4 = dimethyl sulfoxide, and S = standard (cefixime)

Antifungal assay of crude alkaloids and crude saponins

Antifungal assays of crude alkaloids and crude saponins of E. suberosa (Roxb.) bark were performed on fungal strains of C. albicans, C. krusei, and C. parapsilosis. Results were assessed by the presence or absence of zone of inhibition, which was determined by measuring its diameter with the help of scale. The results are shown in Figure 3.

Figure 3.

Graph of antifungal assay of crude alkaloids and crude saponins

In the previous few studies, it has been observed that the stem bark of another species from the same family Erythrina cristagalli L. has effective antifungal effects (C. krusei) with minimum inhibitory concentrations between 12.5 and 31.25 µg/mL.[22] In this study, the bark of E. suberosa has shown some insignificant effects against C. albicans, which is comparatively very less than standard. Furthermore, this study has not found any effect on any of the pool of C. krusei or C. parapsilosis Dalcol II.

CONCLUSION

The plant is native to the Indo-Pak region and is available abundantly. Analyzing and evaluating the cytotoxic and antimicrobial activities of this plant can be helpful in formulating cost-effective treatments. Furthermore, large-scale studies are of high importance so that they can be clinically evaluated in animal and human subjects.

Limitations

Although this research study was focused to discover the potential cytotoxic and antimicrobial activities of E. suberosa (Roxb.), but due to many reasons, for example, financial support, shortage of time, and space, we were not able to find the isolated pure compounds, which can be associated with these activities. Also because of the said reasons, sample sizes were kept smaller, which would have otherwise more significant statistical results.

Future implications

The cytotoxic assays of crude bark extract of E. suberosa (Roxb.) and its organic fractions suggest that this plant, especially its dichloromethane fraction may have potential anticancer compounds, which need to be further studied. For this purpose, activity-based isolation of natural compounds from this plant can be a significant addition to natural compounds research. Microbial resistance is a dilemma for medicinal experts because of which new and potent antimicrobials are the need of the hour. Antimicrobial analysis of this plant suggests that it has good antimicrobial activity, and further studies can unearth the potent future antibiotic compounds. Therefore, further research is necessary to elaborate our findings.

Acknowledgement

We acknowledge the Universiti Sains Malaysia (USM) fellowship for their support and all the members of the research team associated with the conduct of this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.