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In vitro cytotoxicity effect of leaves methanol extract of Rumex crispus L. growing in Abha region, Saudi Arabia against
different human cancer cell lines
Mohammed Al-Shehri
Biology Department, Faculty of Science, King Khalid University
Keywords: Rumex crispus L., anticancer, methanol, leaves
1- INTRODUCTION
The Kingdom of Saudi Arabia is rich in weeds flora, particularly in the Asir region, which has about 70 out of a total of 2253 species, some of which usually used as part of various infection treatment. Rumex spp (family Polygonaceae) usually are spreading on a wide range of altitudes and common on road sides and in over-grazed regions. Rumex spp. appeared to prefer poor and quite drained soils. The plant is an erect herb, typically from 40 to 120 cm tall, but under favorable growing conditions plants over 150 cm can be found. It is a stationary perennial, which by means of a fleshy taproot can live for several years, up to 4 cm in width, more or less branched and to a depth of 150 cm or more in soil require deep root penetration [1]. This group includes many grasses and rosettes, since buds are located on the surface of the soil and protected by leaf and stem bases [2]. Rumex crispus L. is a tap-rooted, perennial forb and one of the most widely distributed non-cultivated plants in the world's flora [3]. It has spread to every continent as a follower of man, and is regarded in many countries as a dangerous weed of agriculture [4]. The species is possibly European and African in origin. Among native plant populations, it does
not occur in big community, but is clearly induced and spread by human activities [5]. The species occurs in a wide variety of habitats, but particularly in farmland, road sides, shingle beaches, disturbed areas, temporary grasslands, and arable land. It is present on virtually all types of soil except the most acidic one [4]. It is a perennial herb, with a fleshy taproot that can last for several years.
The root could be up to a width of 4 cm, reaching a depth of 150 cm or greater in the soil. The leaves are hairless, it has long inflorescence or stalk in a cluster which bears seeds.
Leaves, root, flower and stem from this genius usually shown to have biological activities. For example, R.
nervosus roots powder used as cutting edge powder to treat a low, rough, benign skin growth called Wart [6]. R.
nepalensis could be edible and its extracts and metabolites exhibited pharmacological activity including antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, insecticide, antipyretic, analgesic, antialgal, genotoxic, depressant central nervous system, wound healing and relaxing activity of the skeletal muscle [7]. Stems, leaves, roots, and flowers of R. nervosus plants methanol and n- hexane extracts demonstrated varied degree of antimicrobial inhibition activity [8]. R dentatus methanol (RM) and chloroform (RC) extracts inhibited cell proliferation, arrests the cycle of cells and induces Received: 15/01/2020 / Revised: 15/02/2020 / Accepted: 29/02/2020 / Published: 25/09/2020
Abstract: This study aimed to study the anticancer effects of leaves methanol extract of Rumex crispus using various human cancer cell cultures namely MCF-7, HEPG-2 and HCT116, in vitro using sulforhodamine B (SRB) dye assay.
Seven different concentrations of an extract from leaves of Rumex crispus were freshly prepared and used for cytotoxic activity. The leave extracts exhibited significant anti-proliferative activities against all human tumor cell lines whereas the ceased effect of almost RCLME was greater for the human colon cancer cells line (HCT116) than the breast cancer cells (MCF-7) and human liver cancer cells (HepG2). The numbers of dead cells increased from 7.490% for (HCT116) cell line, from 1.797% for (HepG-2) and from 2.80 % for (MCF-7) at concentration (0.01 ug/ml) of RCLME to be 93.94%, 94.81 % and 88.43% respectively at conc. (1000 ug/ml). The results showed also that the decline in the viable cells from conc. at (0.1 to 100 ug/ml) not so sever while the sudden increase in the alive cells found in the conc. (1000 ug/ml) of RCLME. The LC–MS/MS determination was performed directly from the leaves methanol extract of Rumex crispus without derivatization. Betaine, 2-Phenethylamine, Adenine, Apophedrin pyridoxine, Aceclidine, Adenosine, Tolycaine, Jasmolone, Desmosterol and psi-Tectorigenin were identified. In conclusion: Rumex crispus leaves methanol extract could be used as a natural product against various types of cancers. It is recommended to use the RCLME as it, due to chemicals synergetic effect against various types of tumor.
apoptosis in MDA-MB-231 cells by suppressing the NF- kB pathway [9].
R. crispus infusion or decoction is widely used in folk medicines by South African natives for the treatment of helminths, wounds, internal bleeding and vascular diseases especially in the Eastern Cape countryside [10].
Therefore, this study illustrate the impact of leaves methanol extract gained from R. crispus grown in Abha region (Saudi Arabia) and evaluation their anticancer properties against breast, liver and colon cancers cell lines.
This article aimed also to investigate phytocomopnents found in the leaves of R. crispus methanol extract using LC–MS/MStechnique.
II. MATERIALSANDMETHODS
Methods
R. crispus leaves methanol extract (RCLME) preparation R. crispus leaves were collected from the abandoned area, Abha area, Aseer Province Kingdom of Saudi Arabia (KSA) in October 2019. A voucher number was allocated to the plant and deposited in the Herbarium of Biology, Faculty of Science, King Khalid University, KSA. The leaves were air-dried for a week, using a mortar and pestle, before being ground into a powder. 3 grams of the powdered sample was placed into a Schott bottle containing 100 ml of methanol. The sample was incubated with agitation at 24 °C for 7 days, using a shaker [11]. It then filtered the sample using Whitman paper to eliminate any solid materials. The filtrate was dried with a freezer/lyophilizer of R. crispus leaves methanol extract as reported previously by Najmuddin and Romli, [12]. Seven different concentrations of RCLME were tested by dissolving the extract in de-ionized water to have the following concentrations, (0.00 ug/ml, 0.01 ug/ml, 0.1 ug/ml, 1.0 ug/ml, 10 ug/ml, 100 ug/ml and 1000 ug/ml).
Cell Culture
From the American Type Culture Collection (ATCC), human breast carcinoma cells (MCF-7), human liver cancer cells (HepG2), and human colon cancer cells (HCT116) had been collected. The cells were grown in media from the Roswell Park Medical Institute (RPMI). In a humidifier, RPMI media contains 100 units/ml of penicillin, 100μg/mL of streptomycin, and heat-inactivated bovine fetal serum (10%). The cells have been holding at 37 oC in 5 percent (v/v) of CO2 atmosphere. Cells were subcultured when it reach 80 % confluency [13].
Cytotoxicity Assays test Cytotoxicity
The cytotoxicity effect using seven various concentration of RCLME was examined for the three cell line of human breast carcinoma cells (MCF-7), human liver cancer cells (HEPG-2), and human colon cancer cells (HCT116) using sulforhodamine B (SRB) dye. The cells were collected using a 0.25% Trypsin-EDTA solution and placed at 2,000 cells/well on 96-well plates. For 72 hours, the cells were exposed to RCLME and then TCA (10) was used at 4°C as a fixative for 60 minutes. The cells were exposed for 10
minutes after washing to 0.4%of the SRB solution in a dark room. All the cells were then washed away with glacial acetic acid (%). After drying the stained SRB cells were liquefied with TRIS-HCl and the cytotoxic efficacy was assessed at 540 nm.
Cell Viability
SRB staining was used to distinguish between the viable and the dead cells. Use of a microplate reader (Anthos Zenyth-200RT, Cambridge, UK), as Tolba et al. [14], the viable or dead cells were calculated.
Data analysis
The dose-response curve of the compounds was analyzed using Excel program version 2016 and SPSS using one way ANOVA where P values at ≤ 0.01 and ≤ 0.05 considered highly and statistically significant respectively.
Chemicals analysis of leaves methanol extract
Study of LC-MS / MS was conducted on a 1260 Infinity (AgilentTechnologies, USA) HPLC equipped with a Zorbax Eclipse XDB C18 column (150 mm/4.6 mm, 5 um), coupled with a 4000 QTRAP ® System (Sciex, Canada) source of electrospray ionization (ESI). Fresh leaves methanol extract of R. crispus was analyzed according to Alexandra et al. [15].
III- RESULTSANDDISCUSSIONS
Properties of RCLME against MCF-7, HEPG-2 and HCT116
Figure 1 shows result of RCLME's anti-proliferative effect on prostate, breast cancer, and colon cell lines. The extracts displayed strong anti-proliferative activity against various human tumor cell lines. For almost concentrations tested the ceased effect of RCLME was greater for the human colon cancer cells line (HCT116) than the breast cancer cells (MCF-7) followed by human liver cancer cells (HEPG-2). For colon cancer cells line (HCT116), the numbers of dead cells increased from 7.490% at concentration of RCLME (0.01 ug/ml) to be 93.94% at conc. (1000 ug/ml). For liver cell lines (HEPG-2) the numbers of dead cells increased from 1.797% at concentration of RCLME (0.01 ug/ml) to be 94.81 % at conc. (1000 ug/ml). While for human breast carcinoma (MCF-7), the numbers of dead cells increased from 2.80 % at concentration of RCLME (0.01 ug/ml) to be 88.43% at conc. (1000 ug/ml). It was noted that the rate of decreasing in the viable cells not so strong at the conc. from (0.01 ug/ml) to (0.100 ug/ml), as the rate for HEPG-2 (89.94%), HCT116 (84.53%) and for MCF-7 (82.60%). Numbers of declined cells in HCT116 found to be very small between (0.1 ug/ml) and (10 ug/ml) in between 9.07% to 9.73% and for HEPG-2 between (0.1 ug/ml to 1.0 ug/ml) declined from 3.041% to 3.55%. For MCF-7, the dead cell found to be increased in almost in 2 folds in every conc. from (0.01 ug/ml to 100 ug/ml) whereas it recorded an increment from 2.80% to 17.40%. The sudden increase in the alive cells found in the conc. (1000 ug/ml) of RCLME as for HCT116 (6.05%), HEPG-2 (5.18%) and for MCF-7 (11.57%).
Fig. 1. Cytotoxicity of methanol extract of R. crispus leaves against MCF-7, HEPG-2 and HCT116 cancer cell line using Sulforhodamine B (SRB) Assay. Data presented as mean ± SD (n=3), *p<0.05, **p<0.001, as compared with control.
As the cancer spreads worldwide and the percentage of deaths from this fatal disease is in alarming, especially in developing countries. So why scientists and researchers are now paying more attention to herbal medicine to treat more complex diseases such as cancer because the treatment of cancer patients with drug therapy has severe side effects.
Recently herbal medicinal products have come to play a more vital role in cancer treatment and its prevention and development. Assessment of plant extracts anti-cancer activity is important for safe care. This allows for the detection of the plant's inherent toxicity and the symptoms of acute overdose [16-17]. The results of the present research in agree with many studies for methanol extract of plant against human cancers cell lines. For example, MTT assay was carried out to assess the growth stimulatory effects of extractH. benghalensis on cell viability of three lines of cancer cellsnamely HeLa, MCF-7, and IMR-32 cells, which is based on the reduction of MTT at different concentrations. Methanol extract of Hiptage benghalensis was found to have a greater inhibitory effect on all tumor cells after 48 h of treatment, with varying degrees of efficiencies and selectivities [18]. Al-Shehri1 and Moustafa [11] studied the effect of methanol extract gained from extract Aerva javanica to determine its anticancerous effect on breast cancer cell lines and prostate cancer cell using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay found it had various levels of cytotoxicity toward both cancer cell lines. Recent studies have also shown that F carica's leave methanol extract had a higher anticancer activity compared to its fruit extracts tested by MTT (3-(4,5-dimethylthiazol-2yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium) assay on Huh7it liver cancer cells [19]. As the cancer is distinguished by an uncontrolled increase in cell proliferation and/or cell apoptosis reduction. Inhibition of growth and apoptosis activation in cancer cells were seen
as approaches for the treatment of cancer [20-21]. Since the cytotoxicity of RCLME was greater, therefore, there is an urgent need to reveal the mode of action for its extract cyto- toxicity.
Characterization of the active chemicals found in the fresh leaves methanol extracts of R. crispus are presented in the (Table. 1). LC–MS/MS analysis showed the presence of twelve various phytochemicals compounds. Chemical present in the methanol leaves extract namely, Betaine, 2- Phenethylamine, Adenine, Apophedrin pyridoxine, Aceclidine, Adenosine, Tolycaine, Jasmolone, Desmosterol and psi-Tectorigenin. Almost of those chemicals showed various biological activities, for example, very interesting results found that betaine alone efficiently inhibit tumour growth either in vitro or in vivio in rats [22]. Betaine (B) is another essential methyl donor [23] that preserves normal patterns of DNA methylation [23]. Naturally it can be found in a variety of food sources including sugar beet, wheat bran, spinach, shrimp and many others [25]. Betaine is actively engaged in methionine synthesis, which exists to serve as a major provider of cellular cysteine through a transsulfuration pathway for the synthesis of reduced glutathione which helps protect the cell against reactive oxygen species [26].
It plays a key role in one-carbon metabolism mediated by choline, structural stability of the cell membrane and signalling functions, and synthesis of neurotransmitters.
Betaine looks safe at 9–15 g daily intake [26].
Phenethylamine, Aceclidine, asmolone and Tolycaine bioactivities have not been fully investigated. Adenine could inhibit dose and time dependent growth of Bel-7402 and Hela cells [27]. Studies have shown that adenosine nucleoside plays significant roles in cell growth and differentiation control, cardiac function, inflammation, renal function, and neurotransmission [28-30]. Mice who had a diets with 1, 7, 14 or 36 mg / kg of pyridoxine suppressed colon tumorigenesis for 22 weeks by reducing cell proliferation [31]. A3 adenosine receptor agonists had differential effects on cancer cells [32]. A variety of in vitro studies have explored aspects of the effects of desmosterol on inflammation and cell proliferation reported by
* ** ** **
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** **
** **
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** ** **
** **
**
0 20 40 60 80 100 120
0 0.01 0.1 1 10 100 1000
V iab il ity %
Conc. ug/ml
HCT116 HepG-2 MCF-7
McGrath et al. [33]. Psi-tectorigenin, an isoflavonoid, was isolated from actinomycete culture filtrate as an inhibitor of the phosphatidylinositol turnover in cultured A431 cells triggered by epidermal growth factor. With an IC50 of
about 1 microgram/ml it inhibited phosphatidylinositol turnover; its inhibitory activity estimated as 6 times stronger than that of genistein or orobol [34].
TABLE I: COMPOUNDS IDENTIFIED IN LEAVES METHANOL EXTRACT OF R. CRISPUS NAME USING LC-MS/MS
Library score % Proposed compound
MS/MS Fragment Formula
Measured m/z RT
(min) Peak
100 Betaine
115.0851, 116.0442, 116.0676, 117.0710, 118.0833, 118.2969, 119.0876, 120.0415, 121.0252, 122.0187, 123.0404
C5H11NO2
118.0832 1.16
25 1
99.8 2-Phenethylamine
123.086, 125.2, 125.6 C8H11N
122.0933 2.89
34 2
98.4 Adenine
126.3, 127.6 C5H5N5
136.0580 2.13
41 3
92.4 Apophedrin
128.6,129 C8H11NO
138.0878 1.75
45 4
84 pyridoxine
132.6, 133.5 C8H11NO3
170.0773 1.94
130 5
50.4 Aceclidine
135.6, 134.2 C9H15NO2
170.1136 1.28
131 6
93.2 Adenosine
136.4, 137.6 C10H13N5O4
268.0981 2.42
546 7
60.5 Tolycaine
137.9 C15H22N2O3
279.1732 2.92
612 8
75.6 Jasmolone
181.1234 C11H16O2
181.1222 2.90
632 9
77.3 Desmosterol
109.0649, 97.0648, 81.0704 C27H44O
385.3476 2.93
670 10
52.3 psi-Tectorigenin
286.0478, 258.0526 C16H12O6
301.0707 2.94
678 11
26.3 Cirsiliol
109.1011, 95.0655, 81.0697 C17H14O7
331.0813 2.96
680 12
V- CONCLUSION
In summary, methanol extract from the leaves of R. crispus were showed increased percentage inhibition associated with concentration up to (1000 ug/ml) for the human colon cancer cells line (HCT116), the breast cancer cells (MCF- 7) and for human liver cancer cells (HEPG-2) cells using Sulforhodamine B (SRB) Assay in Cell Culture. LC–
MS/MS analysis showed twelve various phytochemicals compounds that needed further works as isolation to elucidate the mode of action against various tumors types.
As many chemicals found in leaves of R. crispus had antitumor effect, therefore, it might worth to use the plant methanol extract as it due to the chemicals synergetic effect.
ACKNOWLEDGMENT
Author is thankful to the King Khalid University Abha, Saudi Arabia for the financial support of this research article.
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