Mutagenicity, cytotoxic and antioxidant activities of Ricinus communis different parts
Ricinus communis (castor plant) is a potent medicinal plant, which is commonly used in the treatment of various ailments. The present study was conducted to appraise the cytotoxicity and mutagenicity of R. communis along with antioxidant and antimicrobial activities. Cytotoxicity was evaluated by hemolytic and brine shrimp assays, whereas Ames test (TA98 and TA100) was used for mutagenicity evaluation. Plant different parts were extracted in methanol by shaking, sonication and Soxhlet extraction methods. The R. communis methanolic extracts showed promising antioxidant activity evaluated as through total phenolic contents (TPC), total flavonoid content (TFC), DPPH free radical inhibition, reducing power and inhibition of linoleic acid oxidation. R. communis seeds, stem, leaves, fruit and root methanolic extracts showed mild to moderate cytotoxicity against red blood cells (RBCs) of human and bovine. Brine shrimp lethality also revealed the cytotoxic nature of extracts with LC50 in the range of 0.22–3.70 (µg/mL) (shaking), 1.59–60.92 (µg/mL) (sonication) and 0.72–33.60 (µg/mL) (Soxhlet), whereas LC90 values were in the range of 345.42–1695.81, 660.50–14,794.40 and 641.62–15,047.80 µg/mL for shaking, sonication and Soxhlet extraction methods, respectively. R. communis methanolic extracts revealed mild mutagenicity against TA98 (range 1975 ± 67 to 2628 ± 79 revertant colonies) and TA100 (range 2773 ± 92 to 3461 ± 147 revertant colonies) strains and these values were 3267 ± 278 and 4720 ± 346 revertant colonies in case of TA98 and TA100 positive controls, respectively. R. communis methanolic extracts prevented the H2O2 and UV to Plasmid pBR322 DNA oxidative damage. Results revealed that R. communis is a potential source of bioactive compounds and in future studies the bioactive compounds will be identified by advanced spectroscopic techniques.
Introduction<!>Plant material<!>Sample preparation and extraction<!>Total phenolic contents (TPC)<!>Total flavonoid contents (TFC)<!>DPPH Radical scavenging assay<!>Antioxidant activity in linoleic acid system<!>Reducing power determination<!>Hemolytic assay<!>Brine shrimp lethality assay<!>Ames test<!><!>DNA protection<!>Conclusions
<p>Medicinal plants are commonly used to treat various ailments in most of the developing communities. Besides, these are a potent source of food, fodder and fuel, etc. Ethnopharmacology involves the investigation of those plants used by traditional communities without understanding the pharmacological basis of medicinal plants [1–3]. Ricinus communis (family Euphorbiaceae) is a soft wood small tree, located in tropical and warm temperate regions of the world and bioactivity has been studied well of this plant [4, 5]. R. communis plant is used for the treatment of hepatitis, skin and breast cancer [6]. Naturally, plants synthesize phytochemicals as a part of their defense system under variable and harsh environmental conditions, which provide defense for plants against microorganism, pests and insects [7–13]. In developing country, plant derived herbal medicine are used commonly due to easy access and affordable, which are also regarded as safe versus synthetic drugs [14–17]. Moreover, it is believed that plant based bioactive compounds have no side effects as compared to synthetic drugs and has wide range of therapeutic applications [18, 19]. However, plant extracts may contain toxic compounds [20], which can harm the living organisms. R. communis seeds, leaves, fruit, stem and bark are used in different traditional therapeutic practices by local practitioner (Hakeem) [21]. Therefore, the toxicity profiling (using bioassays) of such important plants is very helpful to appraise the safety [22–33]. In this regard, the bioassays (hemolytic and brine shrimp) are the standard tests to evaluate the cytotoxicity, whereas TA98 and TA100 (based on salmonella mutant strains) are the reference tests for mutagenicity evaluation. The shrimp lethality assay was developed by Michael [34], later Vanhaecke [35], and Sleet and Brendel [36]. In this assay, Artemia nauplii are exposed to test compound and lethality is used to estimate cytotoxicity. This has been used as a useful tool for preliminary assessment of toxicity [37] i.e., fungal [38], extract [39, 40], metals [41], toxins  [42], pesticides [43], wastewater [44–48], fumonisins [49] and dental materials [50]. Various authors also utilized this hemolytic test for cytotoxicity evaluation of different systems [51–56]. The Ames test was proposed by Ames and coworker [57–59] and have been used for mutagenicity evaluation of tobacco smoke [60], wastewater [61], treated wastewater [44, 45], herbal extracts [62] and toxic chemicals [63].</p><p>In view of importance of R. communis as a medicinal plant, nevertheless, researcher focused on cytotoxicity and mutagenicity using standard assays. Therefore, the principal objectives of the present study were to investigate the cytotoxicity and mutagenicity of different parts of R. communis parts along with bioactivity profiling. Hydrogen peroxide induced DNA damage protective efficiency was also evaluated of the extracts.</p><!><p>Ricinus communis plant was collected from the Botanical Garden, University of Agriculture, Faisalabad, Pakistan and seeds were purchased from local market, Faisalabad. The plants and seeds specimens were identified by Botanist, Dr. Mansoor Hameed, Department of Botany University of Agriculture Faisalabad, Pakistan.</p><!><p>The collected leaves, stem, fruit, roots and seeds of R. communis were washed with distilled water and shade dried. Dried plant parts were ground and passed through 80 mm mesh size. Different parts (20 g) were extracted in methanol (100 mL) using shaking, Soxhlet and sonication extraction methods. In case of shaking, extraction was performed for 6 h at room temperature (Shaker Gallenkamp, UK). For sonication, ultrasonic treatment (42 kHz, 135 W; Branson ultrasonic corporation, USA) was applied for 30 min. For Soxhlet, extraction was performed in Soxhlet extractor for 3 h. After extraction, methanol was evaporated and concentrated extracts were stored at − 4 °C.</p><!><p>The TPC was assessed using Folin–Ciocalteu reagent following reported method elsewhere [64]. The TPC was calculated using a calibration curve (gallic acid, 10–100 ppm) and data was expressed as GAE of dry plant matter.</p><!><p>Extract (0.1 g/mL) was placed in 10 mL volumetric flask and 5 mL distilled water was added. Then, 0.3 mL of 5% NaNO2 was added and after 5 min, 0.6 mL of 10% AlCl3 was added. After another 5 min, 2 mL of 1 M NaOH was added, mixed well and absorbance was measured at 510 nm. TFC amount was evaluated as catechin equivalents (g/100 g of DM) [65].</p><!><p>For DPPH activity measurement, extract (0.1 mg/mL) were mixed with 1 mL of 90 µM DPPH solution and then, final volume was made to 4 mL by adding 95% methanol. After 1 h of incubation at room temperature, the absorbance was recorded at 515 nm and response was calculated as in Eq. 1 [66].</p><p>1\documentclass[12pt]{minimal}
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				egin{document}$$Inhibition \, ( \%) = \left[ {rac{{A_{0} }}{{A_{s} - A_{0} }}} 
ight]*100$$\end{document}Inhibition(%)=A0As-A0∗100where, Ao is the absorbance of the control and As is the absorbance of the extract (sample).</p><!><p>The percent inhibition of peroxidation of linoleic acid system [67]. Extract (5 mg) and linoleic acid (0.13 mL), 99.8% ethanol (10 mL) and 10 mL of 0.2 M sodium phosphate buffer (PH 7.0) were mixed thoroughly. Then, 25 mL with distilled water was added and incubated at 40 °C. The degree of oxidation was measured following thiocyanate method and percent inhibition of linoleic acid was calculated using Eq. 2.</p><p>2\documentclass[12pt]{minimal}
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				egin{document}$$Inhibition \, ( \%) = 100 - \left[ {rac{{A_{{s,175\, {	ext{h}}}} }}{{A_{{0, 175 \,{	ext{h}}}} }}} 
ight]*100$$\end{document}Inhibition(%)=100-As,175hA0,175h∗100where, As,175 h and A0,175 h are the absorbance values at 175 h of sample and control, respectively.</p><!><p>The reducing power was determined as described elsewhere [68]. Sodium phosphate buffer (5.0 mL, 0.2 M, pH 6.6), and potassium ferricyanide (5.0 mL, 1.0%) and R. communis extract was mixed and incubated at 50 °C for 20 min. Then, 5 mL of trichloroacetic acid (10%) was added and centrifuged at 980×g for 10 min at 5 °C. The supernatant (5.0 mL) was collected and diluted with distilled water (5.0 mL) along with ferric chloride (1.0 mL, 0.1%) addition and absorbance was recorded at 700 nm (Hitachi U-2001, Tokyo, Japan).</p><!><p>Powell [69] method was adopted for hemolytic test. Blood sample (human and bovine, collected in heparinized tubes) was centrifuged for 5 min at 850×g for three to five times using chilled (4 °C) sterile isotonic phosphate buffer saline (PBS) having pH 7.4 and RBCs were separated. The separated RBCs were suspended in the PBS. Erythrocytes were counted using hemocytometer, which were 7.068 × 108 cells/mL. Then, 20 µL of plant extract was mixed with 180 µL blood cell suspension and samples were incubated with agitation for 30 min at 37 °C. The tubes were placed on ice for 5 min and contents were centrifuged for 5 min at 1310×g. A 100 µL supernatant was taken and 900 µL chilled PBS was added and eppendorfs were placed on ice for 5 min and absorbance was noted at 576 nm (BioTek, Winooski, VT, USA). The RBCs lysis (%) was calculated using relation shown in Eq. 3.</p><p>3\documentclass[12pt]{minimal}
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				egin{document}$$RBC_{lysis(\% )} = \left[ {\left( {{{A_{s} } \mathord{\left/ {phantom {{A_{s} } {A_{tx - 100} }}} 
ight. \kern-0pt} {A_{tx - 100} }}} 
ight) 	imes 100} 
ight]$$\end{document}RBClysis(%)=As/Atx-100×100where As is absorbance of the sample and Atx−100 is the absorbance of Triton X-100. Triton X-100 (0.1%) was used as a positive control and PBS was used as negative control.</p><!><p>Brine shrimp (Artemia sp.) eggs were hatched in a culture flask (15 × 15 × 15 cm) filled with sterile, artificial seawater (prepared using sea salt 38 g/L, the pH was adjusted to 8.5 with 1 M NaOH) under constant aeration (aquarium air pump) and illumination for 48 h at 25 °C. After 48 h the shrimp-larvae were collected and exposed extract under investigation. The brine shrimp lethality assay was performed following reported method [39, 70]. Plant extracts were diluted to concentrations of 10, 100, 1000 and 3000 µg/mL for cytotoxicity testing. Twenty brine shrimp larvae were placed in vials containing extract using a plastic pipette with a 2 mm diameter tip. The larvae survival was counted under the stereomicroscope after 24 h and percent death rate at each dose and control were calculated. Salt-water and cyclophosphamide were used as negative and positive controls, respectively, and LC50 and LC90 values were estimated.</p><!><p>Two S. typhimurium strains TA98 and TA100 were used [71]. The extract was considered mutagenic, if the number of revertant colonies on the plates containing test compounds was twice the number of revertant colonies in control plates (background) (extract/control revertant colonies ≥ 2.0) [72]. All the experiments were performed in triplicates and data, thus obtained was expressed as mean ± SD.</p><!><p>Antioxidant profile of extracts of Ricinus communis different parts, extracted by different extraction methods</p><p>The values are the mean ± SD of triplicate experiments. Capital letters in superscripts are representing significant different among extraction methods (P < 0.05) and small letter in superscripts are representing significance difference (P < 0.05) in activity within plant parts for individual extraction methods</p><p>Human and bovine red blood cell lysis (RBCs) assays of Ricinus communis different parts, extracted by different extraction methods</p><p>Explanations as given in Table 1</p><p>Brine shrimp lethality assay of Ricinus communis different parts, extracted by different extraction methods</p><p>Mutagenicity of Ricinus communis different parts extracts, extracted by different extraction methods, tested by TA98 and TA100 strains of Salmonella typhimurium</p><p>Negative (solvent) control; DMSO dimethyl sulphoxide, PC positive control: for TA98, TN (0.25 µg/plate); for TA100, NQNO (0.5 µg/plate); Values are insignificant (P < 0.05) among plant parts and extraction methods, and significantly different (P < 0.05) form positive control</p><p>Toxicities/activities reported for Ricinus communis plant</p><p>DNA damage/protection effect of methanol extract of Ricinus communis exposed to H2O2 and UV induced oxidative damage on pBR322. Lane 1 = 1 Kb DNA ladder: lane 2 = Plasmid pBR322 DNA without treatment (super coiled); lane 3 = Plasmid pBR322 DNA treated with H2O2 (open circular or damaged), lane 4 = Plasmid pBR322 DNA; treated with H2O2 + UV (open circular or damaged); lane 5 = Plasmid pBR322 DNA treated seed extract by shaking method + H2O2; lane 6 = Plasmid pBR322 DNA treated with stem extract by shaking method + H2O2; lane 7 = Plasmid pBR322 DNA treated with leaves extract by shaking method + H2O2; lane 8 = Plasmid pBR322 DNA treated with fruits extract by shaking method + H2O2; lane 9 = Plasmid pBR322 DNA treated with seeds extract by shaking method + H2O2 + UV light); lane 10 = Plasmid pBR322 DNA treated with stems extract by shaking method + H2O2 + UV); lane 11 = Plasmid pBR322 DNA treated with leaves extract by shaking method + H2O2 + UV); lane 12 = Plasmid pBR322 DNA treated with fruits extract by shaking method + H2O2 + UV)</p><!><p>4\documentclass[12pt]{minimal}
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				egin{document}$${	ext{H}}_{2} {	ext{O}}_{2} {	ext{--}}^{ \wedge } {	ext{-}}^{ \wedge } {	ext{--}}{>} 2{	ext{OH}} \cdot$$\end{document}H2O2--∧-∧-->2OH·</p><p>5\documentclass[12pt]{minimal}
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				egin{document}$${	ext{RH}} + {	ext{OH}} \cdot 	o {	ext{H}}_{ 2} {	ext{O}}/{	ext{N}} - {	ext{bases }} + {	ext{R}} \cdot 	o {	ext{Oxidative by-products}}$$\end{document}RH+OH·→H2O/N-bases+R·→Oxidative by-products</p><!><p>Cytotoxicity, mutagenicity, antioxidant as well as DNA protective efficiency of R. communis (seeds, stem, leaves, fruit and root) methanolic extracts were evaluated. Extracts showed variable antioxidant activity among plant parts and extraction methods. The R. communis also protected Plasmid pBR322 DNA from H2O2 and UV damage. Bioassays (Hemolytic, brine shrimp and Ames test) revealed that the R. communis methanolic extracts have compounds responsible for mild to moderate to moderate toxicity. R. communis may be a potential source of compounds for the development of new medicine and future studies will be focused on the identification of compounds responsible for bioactivity.</p>