Chromatographic Analysis and Cytotoxic Effect of Extracts of Abrus precatorius a Medicinal Plant with Strong Anticancer Potential

Received: 03-Feb-2021 Accepted Date: May 22, 2021 ; Published: 28-May-2021

Introduction

Cancer is one of major cause of death in developed and developing countries [1]. Its treatment protocols depend on chemotherapy, radiotherapy and surgical intervention [2]. West Africa composed of mostly poor countries where cancer is an emergent disease. In 2008, men in this region of Africa had a doubly high rate of liver cancer while women had the highest cervical cancer cases worldwide [3]. The evolution of cancer in this part of Africa is very worrying, which is why WHO encourages countries in these regions to promote and integrate traditional medical practices into their health systems [4]. In the search for means of combating these diseases, man has used the medicinal properties of many cultivated and wild plants to combat these disturbing ailments. Among the many plant species exploited, Abrus precatorius is probably the best known of the 17 species of the genus Abrus. This climbing plant from the Fabaceae family is known mainly for its medicinal properties to cure various diseases [5]. The roots, leaves and seeds of this plant are used for different medicinal purpose [6]. En Inde, cesfeuillessontutilisées pour guérir la fièvre, les troubles d’estomac, l'asthmeet la bronchite [7]. In Nigeria leaf decocted is used in the treatment of diabetes [8]. In Côte d'Ivoire, the leaves of Abrus precatorius are used in the south of the country, among the Abbey and Krobou peoples of Agboville, to facilitate childbirth for women [9].The plant have been reported for activité antimicrobienne [10], Abortifacient effect, Antidiarrheal activity, Antifertility effect [11] and antidiabetic effect [12]. Several studies revealed that extracts of Abrus precatorius exhibit anticancer activity against human breast cancer cell line MDAMB-23 [13], against A-549 cancer cell lines [14], against four cancer cell lines (Colo-205, Y79, HepG2 and SupT1) [15 ]. The aqueous extract of A. precatorius leaves contains several chemical groups and possess in vitro anticancer effect against mastocytoma cancer cell line (P815) [16]. This present study, was carried out to study chromatographic aqueous extract and cytotoxic effect of ethanolic extract of Abrus precatorius leaves against the murinmastocytoma cancer cell line (P815).

Materials and Methods

Plant collection

The leaves of Abrus precatorius were collected in an urban area of Abidjan (Southern Ivory Coast) in month of October 2014. The plant had already been identified at the National Centre Floral of Abidjan (Ivory Coast) on the issue: Abrus precatorius (Fabaceae): Aboudé-Mandéké (Ivory Coast), 23 May 1990 N'Guessan Koffi 165 [9].

Preparation of extract

The aqueous extract of the leaves of Abrus precatorius was prepared by decoction according to the method of Lébriet al., [17]. 10 g of powder of leaves were introduced into a triple-neck round-bottom of 250 mL, 100 ml of distilled water were added. A round-bottom was topped with a cooler connected to a faucet opened by pipe. The round-bottom is put down into a warm balloon (ELECTROMANTLE) maintained in a constant temperature of heating during one hour. After cooling, the mixture is filtered with cotton wool three times and the obtained filtrate was moved in the stove (SELECTA) at 55°C during 24 h. The extract was dried and the aqueous extract (ETA) was obtained.

Chromatographiic anallysiis

Thiin Layer Chromatography

Thiin Layer Chromattographiic Anallysiis was performed wiitth tthree (3) sollventt miixiing systtems (Systtem 1:: Etthyll Acettatte // Hexane (1//9);; Systtem 2::
Etthyll Acettatte // Hexane (2//8) Systtem 3:: Etthyll acettatte // Hexane (3//7) One (1) mg of tthe samplle (aqueous exttractt,, etthanoll,, acettattiic,, hexane) was
diillutted iin a smallll quanttiitty of Diichllorometthane (spott solluttiion).. A samplle deposiitt iin smallll spotts (3 tto 4 ttiimes) iin tthe form of dotts usiing a capiillllary
was carriied outt..

The chromattographiic pllatte was pllaced iin tthe satturatted ttank of tthe miixiing systtem.. The devellopmentt of tthe chromattogram was follllowed unttiill tthe
arriivall of tthe sollventt att tthe upper frontt.. The revellattiion of tthe consttiittuentts on tthe pllatte was carriied outt under a UV llamp:: Ullttra viiollett (λ = 254 nm
and 365 nm) and by iiodiine vapor.. For each consttiittuentt,, tthe fronttall rattiio or tthe rettenttiion facttor (Rf) was callcullatted accordiing tto tthe follllowiing
formulla::

Collumn chromatography

Collumn chromattography was carriied outt iin an attttemptt tto obttaiin diifferentt fracttiions of tthe ttottall aqueous exttractt of tthe lleaves of Abrus precattoriius iin
order tto make ttheiir iidenttiifiicattiions according to the method of Alljjerf [18]..The siilliica deposiitt was prepared from (one) 1 g of exttractt miixed wiitth
siilliica,, tthe wholle ttaken up iin a quanttiitty of hexane and tthe conttentts were concenttratted by evaporattiion iin a rottary evaporattor (HEIDOLPH WB 2000)..
The miixtture iis carriied outt att tthe ttop of a gllass collumn (55 x 2) fiilllled wiitth 60 Å siilliica gell iin hexane.. The fracttiionattiion was carriied outt by successiive
applliicattiions of an etthyll acettatte // hexane miixtture (1:: 9;; 2:: 8;; 3:: 7).. 10 mll fracttiions were collllectted and tthe sollventt was removed by rottary evaporattiion
(HEIDOLPH WB 2000) att a ttemperatture of 65°C

In vitro anticancer effect of aqueous extract of A. precatorius

In vitro anticancer effect of ethanolic extract of A. precatorius leaves was conducted in the Laboratory of biological engineering, Faculty of Sciences and Techniques, Sultan MoulaySlimane University of Beni-Mellal (Maroc).

Tumor cell line and culture

The mastocytoma tumor cell line, were grown in RPMI1640 (Sigama-Aldrich) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Sigama-Aldrich), 1% penicillin-streptomycin, and 0.2% sodium bicarbonate (Sigama-Aldrich), under a fully humidified atmosphere of 95% air and 5% CO2 at 37ºC.

Cytotoxicity assay

Cellular cytotoxicity was determined by the MTT reduction assay. This Colorimetric assay is based on the capacity of mitochondria succinate dehydrogenase enzymes in living cells to reduce the yellow water soluble substrate 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) into an insoluble, colored formazan product which is measured spectrophotometrically [19]. Growing concentrations of the tested extract (solubilised in Dimethyl sulfoxide (DMSO): 3, 12; 6, 25; 12,5; 25; 50; 100 and 200 μg/mL) were applied to the wells of a 96-well plate containing the confluent cell monolayer (106 cells per well) in duplicate. Methotrexate as positive control drug was added in the same concentrations and conditions. After 48 h of incubation, 20 μL of the MTT solution [5 mg/mL in Phosphate buffered saline (PBS)] was added. After incubation in the same conditions for 4 h, the plates were treated with a mixture of HCl /Isopropanol (24:1) to dissolve the blue intracellular formazan product. One hour later, the plates were read on a Micro ELISA reader using two wavelengths (540 and 630 nm). DMSO was used as negative control. The median inhibitory concentration (IC50) was calculated as the concentration of the sample that leads to 50% of cell lysis comparatively to the negative (positive) control.

The relative inhibition of cell proliferation was calculated by the formula:

% inhibition =100 x (1- A / A₀),

where A0 and A, are the absorbencies of negative control and ethanolic extract or methotrexate treated cells, respectively.

Statistical Analysis

Data are reported as means±SEM of 3 experiments Statistical differences were assessed by analysis of standard deviation using the Student’s t test , with the level of significance set at p<0.05

Results

Chromatographic study of extracts from the leaves of A. precatorius

Thin Layer Chromatography (TLC)

The phytochemical analysis of the aqueous extract of the leaves of Abrus precatorius revealed the presence of several chemical groups (alkaloids, tannins, flavonoids (flavones), saponins, quinone compounds (coumarins), sterols and triterpenes and reducing compounds) [12]. Analysis of the dry extract (aqueous) by thin-layer chromatography after UV detection and iodine vapor reveals the presence of 4; 5 and 6 different Rf spots with the systems (Ethyl acetate / Hexane: 1/9; 2/8 and 3/7) (Table 1).The number of different Rf spots was also revealed on the TLC plates with the dry extracts obtained from different organic solvents. The ethanolic extract exhibits 5 spots with the systems (Ethyl Acetate / Hexane: 1/9; 2/8 and 3/7). The acetate extract respectively 5 and 4 spots with the systems (Ethyl acetate / Hexane: 1/9; 2/8 and 3/7) and the hexane extract respectively 4; 6 and 5 with (Ethyl acetate / Hexane: 1/9; 2/8 and 3/7) (Table 1).

Table 1: Number of spots observed on the TLC plate of different extracts from the leaves of Abrus precatorius.

Column chromatography

The chromatographic study on a column using the elution solvent of increasing polarity (Ethyl acetate / Hexane: 1/9; 2/8 and 3/7) made it possible to obtain two fractions called LA1 and LA2 of appearance oily, of respective mass 100 mg and 300 mg per 1 g of dry extract (ETAAP).On TLC plate, the LA1 fraction presents a single spot and the LA2 fraction presents two spots (Table 2).

Table 2: Numbers of spots revealed in fractions LA1 and LA2 on TLC plate.

Invitro anticancer effect

The Invitro anticancer activity of ethanolic extract of Abrus precatorius was evaluated at 3.12; 6.25; 12.5; 25; 50 ; 100; 200 and 400 μg/mL.) against P815 tumor cell. The result is summarized in Figure 1. The results show that ethanolic extract has a best inhibitory effect on the growth of tumor cells compared to the aqueous extract which are less 20% at a concentration 200 μg/mL (Table 3). The ethanolic extract has aninhibitory effect which increases with concentration, this effect is similar to that of Methotrexate used as a reference (Figure 3). It was also observed, that the maximum effect of the ethanolic extract at the highest concentrations tested (400μg/mL) was 72,20 % (± 4,60) and (200 μg/mL) was 63.54% (±4,28) of lysis. Further, on lower doses of 3.12; 6.25; 12.15; 25 and 50 μg/mL percent growth inhibition observed by the extract was between 6,25% (±1,70) and 43.94 % (±3,30) (Table 3). The IC50 of the ethanol extract is 43.94 μg/mL (Figure 1), this IC50 is lower than that of the aqueous extract which are 200 μg/mL (Figure 2). The IC50 of ethanolic extract is more higher compared with the Methotrexate (2.5μg/mL) used as positive control (Figure 3).

Table 3: Percentage of lysis of cancer cell line according to the concentration of etanol extract.

Figure 1: in vitro anticancer effect of ethanolic extract of A. precatorius leaves on P815 cancer cell line Dose - response curve of ethanolic extract of A. precatorius leaves on P815 cancer cell line.

Figure 2: In vitro anticancer effect of aqueous extract (ETA) of A. precatorius leaves on P815 cancer line [16] Dose-response curve of aqueous extract of A. precatorius on P815 cancer cell line.

Figure 3: in vitro anticancer effect of methotrexate on P815 cancer line Dose-response curve of methotrexate on P815 cancer cell line.

Discussion

The qualitative phytochemical study of the total aqueous extract of the leaves of Abrus precatorius showed that the plant contained alkaloids, tannins, flavonoids (flavones), saponins, quinone compounds (coumarins), sterols, triterpenes and reducing compounds [17]. The thin-layer chromatographic study of the different extracts (aqueous, ethanolic, acetate and hexane) made it possible to demonstrate the presence of several spots of different frontal ratio. Moreover, the chromatographic study on a column of the aqueous dry extract allowed to obtain two fractions, one containing a stain and the other two stains of different RF. The results of chromatographic studies confirm the richness of the extracts in active chemical compounds which could explain the traditional use of Abrus precatorius to treat many diseases. Work on the plant has isolated several groups of secondary compounds including alkaloids, steroids and triterpenoids, isoflavanoquinones, anthocyanins, tannins, flavonoids and phenolic compounds [20]. The root, leaf and stem containing (L +) abrin, glucosides (abralin, hemagglutinin), (N-methyltryptophan and urease) are traditionally used in the treatment of cough [21]. Studies have showed that compounds isolated from the Abrus precatorius plant (tannins, alkaloids, sterols, triterpenes and flavonoids) have anti-tuberculosis and antiplasmodial properties [22]. Hence, this study evaluates the potential in vitro anticancer activity of ethanolic extract of leaves Abrus precatorius against murinmastocytoma cells (P815). In this study, the cytotoxicity of extract was sought by determining the percentage of lysis of cancer cells and the inhibitory concentration 50% (IC50) by the MTT assay.

The results showed that the ethanolic extract of the leaves of Abrus precatorius exerts a dose-dependent inhibitory effect on the growth of cancer cells. The concentration of extract leading to 50% lysis is 43.94 μg/mL. The effect of the extract on the cancer cell line has been compared with that of methotrexate, which is a molecule used in the treatment of cancer. The control product showed a dose-dependent inhibitory effect with an IC50 (IC50=2.5 μg/mL) much lower than the extract. However, the inhibitory concentration 50 of the ethanolic extract (IC50 = 43.94 μg / mL) is lower than that of the aqueous extract of the leaves of A. precatorius ((IC50=200 μg /mL). The dose-dependent inhibitory effect of the ethanolic extract on the growth of cancer cells may be due to the action of the active ingredients contained in the extract. The results obtained are close to Tilaoui et al.,[23]. These authors have shown that the essential oil rich in monoterpenes extracted from the plant Artemisia herba-alba has a dose-dependent cytotoxic effect on the acute lymphoblastic leukemia (EMF) blood cancer cell line in humans (IC50=3 μg / mL).Several studies carried out using extracts of the A precatorius plant on different lines of cancer cells are in agreement with the results obtained with the ethanolic extract of the leaves of Abrusprecatorius. Sivakumar et al.,[14]. have shown that the crude insoluble methanolic fraction extracted from the seeds of Abrus precatorius exerts an effect on the cancer cell line A-549 (IC50 = 175 mg/ml). Mohammed Shafi Sofi et al.,[13]. Showed that the aqueous extract of the leaves of Abrus precatorius possesses anti-cancer activity on the MDAMB-231 female breast cancer cell line (IC50 = 98 μg/mL). Low cytoxic activity of the aqueous extract of the leaves of Abrus precatorius has also been described by Mir Z Gul et al.,[15]. Their work showed non-significant cytotoxic activity of the aqueous extract on four human tumor cell lines (Colo-205, Y79, HepG2 and SupT1) and unlike acetate and ethanolic extracts. Lebri et al., [16]. Also showed a weak cytotoxic activity in the aqueous extract of the leaves of Abrus precatorius on a murine mastocytoma P815 tumor cell line. These results show that the cytotoxic activity obtained with the ethanolic extract of the leaves of Abrus precatorius is better than that obtained with the aqueous extract. The results clearly demonstrate that the cytotoxic activity of the extracts of Abrus precatorius depends not only on the nature of the extract and its chemical composition, but also on the target tumor cells.

Conclusion

This present study confirmed that A. precatorius leaves could be potentially useful for the development of therapeutic agents against cancer. The ethanolic extract of Abrus precatorius showed the best cytotoxic effect. Furthermore, the study, the in vivo anticancer activity and the molecular mechanisms involved in such activity are needed to facilitate integration of Abrus precatorius as an anticancer herbal medicine.

Acknowledgements

We express gratitude to the UFR Biosciences, University Felix Houphouet-Boigny of Abidjan (Côte d’Ivoire), Ecology Research Center, Nangui Abrogoua University (Côte d’Ivoire) and to the Sultan Moulay Slimane University of Beni Mellal, Faculty of Technical and Sciences (Morocco) for providing the facilities for conducting this research

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