Accepted Manuscript
Anti-aggregant effect of butanolic extract of Rubia tinctorum L on platelets in vitro and ex vivo
Fatima Zahra Marhoume, Mehdi Ait Laaradia, Youness zaid, Jawad Laadraoui, Sara Oufkir, Rachida Aboufatima, Abderrahmane Chait, Abdallah Bagri
PII: S0378-8741(19)30747-0
DOI: https://doi.org/10.1016/j.jep.2019.111971 Article Number: 111971
Reference: JEP 111971
To appear in: Journal of Ethnopharmacology
Received Date: 21 February 2019 Revised Date: 10 May 2019 Accepted Date: 22 May 2019
Please cite this article as: Marhoume, F.Z., Laaradia, M.A., Youness zaid, , Laadraoui, J., Oufkir, S., Aboufatima, R., Chait, A., Bagri, A., Anti-aggregant effect of butanolic extract of Rubia tinctorum L on platelets in vitro and ex vivo, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/
j.jep.2019.111971.
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Authors Affiliation
Fatima Zahra Marhoume Laboratory of Biochemistry & Neuroscience, Applied Biochemistry and Toxicology Team, Faculty of Sciences and Technology, Hassan First University, Settat, Morocco.
Mehdi Ait Laaradia Laboratory of Neurobiology, Pharmacology and Behavior, Faculty of Sciences Semlalia, Cadi Ayyad University, Marrakech. Morocco.
Youness zaid Laboratory of Thrombosis and Hemostasis, Research Center of Abulcasis University of Health Sciences, Rabat, Jawad Laadraoui Laboratory of Neurobiology, Pharmacology and Behavior,
Faculty of Sciences Semlalia, Cadi Ayyad University, Marrakech. Morocco.
Sara Oufkir Laboratory of Neurobiology, Pharmacology and Behavior, Faculty of Sciences Semlalia, Cadi Ayyad University, Marrakech. Morocco.
Rachida Aboufatima Laboratory of Génie Biologique, Sultan Moulay Slimane University, Faculty of Sciences and Techniques, Béni Mellal, Morocco.
Abderrahmane Chait
Laboratory of Neurobiology, Pharmacology and Behavior, Faculty of Sciences Semlalia, Cadi Ayyad University, Marrakech, Morocco. chait@uca.ac.ma
Abdallah Bagri
Laboratory of Biochemistry & Neuroscience, Applied Biochemistry and Toxicology Team, Faculty of Sciences and Technology, Hassan First University, Settat, Morocco.
abagri511@gmail.com
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1. Introduction
Platelets play pivotal role in primary hemostasis, inflammation and many other biological processes (Mancuso et al., 2017, Manne et al., 2017). Several cardiovascular diseases (CVD) including atherothrombotic disorders are associated with an increase in blood platelets aggregation (Ueno et al., 2011). Antiplatelets therapy is one of the most effective therapies for treatments of atherothrombotic disorders (Olas et al., 2005). In this regard, several antiplatelets drugs have been used clinically to treat and prevent coronary syndromes and stroke such as aspirin, clopidogrel and glycoprotein IIb-IIIa antagonists (Park et al., 2012;
Smart, 2007). However, their actions are restricted (Michelson, 2010), and they are may be responsible of adverse effects such as hemorrhage, gastric ulcers, thrombocytopenia, increased risk for recurrent cardiovascular events and therapeutic resistance (Angiolillo, 2009;
Barrett et al., 2008; Matetzky et al., 2004; Ridker et al., 2005).
Medicinal plants remain an important alternative source of new drugs by possessing many compounds that work in a synergistic manner with minimal side effects and are available to a large population. Interestingly, some natural compounds in the diet may inhibit platelets activation. In previous studies (Rahman and Billington, 2000) and (Dutta-Roy et al., 2001) were reported that garlic (Allium sativum) and tomato (Lycopersicum esculentum) may be beneficial in protecting against cardiovascular diseases as a result of inhibiting platelet aggregation.
In Morocco, as in many countries, medicinal plants are widely used in the traditional medicine systems to cure various ailments. Among these plants, Rubia tinctorum L (Madder root) belongs to Rubiaceae family, locally named (El foua or Tarûbya), one of the several plants, whose dried roots are used for treatment of cardiovascular disease including hypertension arterial (Bellakhder et al., 1991; Jouad et al., 2001) liver pain, anemia and diarrhea (Eddouks et al., 2002; El Haouari et al., 2016). The plant has been extensively studied for its biological activities and therapeutic potentials such as anti-inflammatory, antioxidants and antibacterial actions (Shipla et al., 2012; Kalyoncu et al., 2006).
However, there is no scientific research data about the anti-aggregant effect of Rubia tinctorum L on platelets activity. Thus, the aim of the present study was to evaluate in vitro and ex vivo the effects of butanolic extract of Rubia tinctorum L on platelets aggregation and to indentify polyphenols compounds that could be responsible these effects.
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2. Materials and methods 2.1 Chemicals and reagents
All chemical used in this study were of analytical grade. Collagen and collagen diluents were purchased from SD Medical (Lot: 44201-1), France. Aspirin was purchased from Bayer, Morocco. Butanol (lot: SZBB184SV). Quercitine (≥ 95%) (Lot: SLBM7736V), romarinci acid (≥ 98%), Cinnamic acid (≥ 99%) and sodium phosphate dibasic (98-100.5%) (Lot:
SZBF048AV) were purchase from Sigma-Aldrich (Germany). Vanillin (99.8 %,) (Lot:
080417CE) From SOLVACHIM (Casablanca, Maroc). Acetonitrile (≥ 99.9%) (Lot:
STBF9897V) methanol (≥ 99%) (Lot: STBG0885V) both HPLC grade from Sigma Aldrich (Germany).
2.2 Experimental animals and care
Male and female Sprague dawley rats weighting (200-260 g) were housed in the animal facility of the Faculty of Science Semlalia, Marrakech, Morocco. All rats were acclimated for 3 days in their cage before the experiments started. Management and care of animals were conducted in conformity with approved institutional protocols and in accordance with the provisions for animal care and use described in the scientific procedures on living animals ACT 1986 (European Council Directive: 86/609/EEC).
2.3 Collection of plant material and preparation of extracts
Rubia tinctorum L (RT) was collected in June 2017 from the province of Azilal, Ait M’hamed village, geographic coordinates (31°51’00”N, 6°30’48”W), Morocco. The botanical identity of the plant specimen was confirmed by Professor Ouhammou Ahmed Mohamed. A voucher specimen (reference 9825) was deposited in the herbarium of Semlalia Science Faculty, Cadi Ayyad University. The dried roots of RT were roughly powdered, then 303 g powder was placed into soxhlet column and an extraction with 70% v/v ethanol in water at 75-79°C for 15 hours. The obtained extract was evaporated at 45°C. The ethanol extract (12.75%) was successively separated with hexane, ethyl acetate, Butanol and distilled water according to the method of (Shaheen et al., 2000). The fraction obtained was concentrated with rotaevaporatory to obtain the yield following proportion: (4.13%) of hexanic extract, (1.3%) of ethyl acetate extract, (2 %) of Butanolic extract (B-RT) and (5.28 %) of aqueous extract.
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2.4 Acute toxicity study
Acute toxicity study was performed as per Organization for Economic Co-operation and Development guidelines 425 (2008). Swiss albino mice (10 mice per group, 5 females and 5 males), weighing (21 – 27g) received oral administration of Rubia tinctorum extract at doses (0.5, 1, 2, 3.5, 5 g/kg) by 14 days. The animals were observed daily for signs symptoms of toxicity, body weight changes and mortality. Kidney and liver organs were subjected to histopathological analysis.
2.5 Platelets anti-aggregant assay in vitro and ex vivo 2.5.1 Preparation of washed platelets (in vitro)
Rats (180-230g) were anaesthetized with Chloral hydrate (10mg/kg) and blood samples were collected from rat’s jugular vein, into tubes containing an anticoagulant solution (9 volumes of blood with 1 volume of trisodium citrate 170 mM, citric acid 130 mM, and dextrose 4%).
The platelets were prepared as reported previously (Zhou et al., 2005). Concisely, the platelets were obtained by series of differential centrifugations and adjusted to a final concentration of 225× 106/ ml using an automate cell counter. The platelets were allowed to stand a 37°C for 30 min before further experiments.
2.5.2 Platelets aggregation assay
The aggregation of washed platelets was measured using an 8 channel aggregometer (SD Medical Innovation) (Théorêt et al., 2001; Caron et al., 2002). Washed platelets (270µl) were pre-incubated with (15µl) of the B-RT extract for 5 min at 37°C. Three doses of B-RT extract (250µg/ml, 500µg/ml, and 1000µg/ml) were tested in this experiment. Aggregation was triggered by adding (15µl) of collagen (2µ g/ml under continuous agitation (1000 rpm) at 37°C. The platelets aggregation was measured by monitoring the change in light absorbance (950 nm). As a control, a free B-RT extract tube was used.
2.5.3 Tail-bleeding time and platelets count (ex vivo)
Male and female Sprague-dawley rats were randomly divided into six groups (n=5) and treated by injection into jugular vein of anesthetized rats as follows: Control group received vehicle alone (0.9% w/v NaCl), second group received only high dose of collagen (2µg/ml), the third group received (100 mg/kg) of B-RT extract, the fourth group received (50mg/kg) of aspirin, the fifth group received aspirin (50 mg/kg) and high dose of collagen (2µl/ml) and the last group received 100 mg/kg of B-RT extract and high dose of collagen (2µl/ml). Collagen
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at high dose (2.5 µl/g of animal weight) was injected after 5 min of pre-incubation with (100 mg/kg) of B-RT extract and aspirin (50 mg/ml) into jugular vein of anesthetized rats. Rat tail bleeding times were determined by removing 1 cm of distal rat tail and immediately immersing the tail in 37°C isotonic saline. The bleeding time was defined as the time recorded for cessation of blood flow. Blood was collected and platelets count was determined with counter (Sysmex Kx-21).
2.6 Phytochemicals analysis
2.6.1 Determination of total polyphenols content in B-RT extract
The total polyphenolics content of Butanolic extract were determined using the Folin Ciocalteu method described by (Slinkard and Singleton, 1977). Briefly 100µl of test sample was mixed with 3.9 mL of water and 100µl of Folin-Ciocalteu’s phenol reagent. After 8 min, 1 mL of 20% (w/v) sodium carbonate was added to mixture. The reaction was kept in the dark for 40 min and after centrifugation; the absorbance was measured at 760 nm. The phenolic content was calculated as Gallic acid (1 – 0.062 mg/ml, Y= 0.981x+0.003, R2= 0.9999).
2.6.2 Determination of total flavonoids content in B-RT extract.
The flavonoids content was determined by the aluminum trichloride (AlCl3) method (Zhishen et al., 1999). (200µl) of diluted extract was mixed with 800 µl of distilled water and 60µl of 5% sodium nitrite (NaNO2). After 5 min, 40 µl of 10% (m/v) aluminum trichloride was added to the mixture. After 6 minutes of incubation at room temperature, 400 µl of sodium carbonate (1M) and 500 µl distilled water were added to the reaction medium. The absorbance of the reaction mixture was determined at 510 nm. A calibration curve was performed in parallel under the same operating conditions using Catechine as standard.
2.6.3 Determination of polyphenols compounds in B-RT extracts by HPLC analysis B-RT was standardized for its content of marker compound, flavonoids and polyphenols, by HPLC method. Chromatography separations were performed on a Reversed-Phase (RP-18) Columns, Agilent Technologies (250 mm × 4.6 mm, 5.0 µm), protected by Agilent technologies RP-18 (10 mm × 4.6 mm) pre-column. Both columns were placed in a column oven set at 25°C. The HPLC system consisted of Shimadzu SCL-10A pumping system, SIL- 10AD automatic injector, and the Shimadzu SPD 10A UV/Vis detector [wavelength scanning range 200 ~ 700 nm]. Data collection and analysis were performed using SHIAMDU
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LabSolutions software. Two solvents were used with a constant flow rate of 1 ml/min gradient program (table 1) and the sample volume injection was 10µl. Solvent A consisted of 5%
acetonitrile and 95% water. Solvent B is a phosphate buffer dissolved in water (pH 2.6). All the solvent used were of HPLC grad. HPLC analysis was first performed with the standards then followed by the B-RT extracts and finally spiking the samples with the standards.
Table .1: gradient time table
Time [min]
A [%]
B [%]
Flow [mL/min]
0 95 5 1,000
10,00 87 13 1,000
13,00 82 18 1,000
20,00 82 18 1,000
23,00 75 25 1,000
35,00 75 25 1,000
40,00 70 30 1,000
41,00 15 85 1,000
50,00 10 90 1,000
52,00 5 95 1,000
53,00 5 95 1,000
54,00 0 100 1,000
59,00 0 100 1,000
60,00 95 5 1,000
2.7 Statistical analysis
Data are presented as mean ± standard error (SEM); P- values low than 0.05 were considered to be statistically significant. Statistical analyses were performed using the computer software Sigma Plot 12.5 for windows. Comparisons between different groups were performed using one-way (ANOVA) analysis of variance. Significant differences between control and experimental groups were assessed by tukey’s test.
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3- Results
3.1 Acute toxicity
Mice tolerated all doses (0.5, 1, 2, 3.5, 5 g/kg) of Rubia tinctorum ethanolic extract, no mortality was recorded and no external symptom of toxicity was observed within the period of treatment. Histopathological examination of liver and kidney sections, from all groups showed normal cells structures and no pathological lesions were observed (Figure 5 and 6 available as Supplementary Data).
3.2 Effect of butanolic extract of Rubia tinctorum L on human platelets aggregation In order to check the physiological status of platelets, a control aggregation (free from plant extract) induced by collagen was systematically achieved at the beginning of each experiment.
Washed platelets were separately pre-incubated with B-RT extract for 5 min at 37°C and then the aggregation was induced. Figure 1 (available as Supplementary Data) displays an original tracing of collagen induced platelets aggregation with and without (B-RT) extract. Our results show statistically significant differences between the effect of the different B-RT doses and the control on platelets aggregation (P<0.001, table 2). Moreover, the mean % of aggregation depended on the dose of B-RT extract.
Table.2: inhibitory effect of Rubia tinctorum butanolic extract on platelet aggregation induced by collagen
Collagen 2µg/ml (Control)
B-RT 250µg/ml B-RT 500µg/ml B-RT 1000µg/ml
% of Aggregation
69,85 ± 3,23 n=3
9,44 ± 0,68***
n=3
4,30 ± 3,15***
n=3
1,48 ± 1,32***
n=3 Mean value ± SD are presented; n=3
***P<0.001 significantly different compared to control
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3.3 Effect of B-RT extract on collagen induced change in platelets function ex vivo 3.3.1 Bleeding time
B-RT proved to be efficient in inhibiting platelets aggregation. Therefore, we checked whether this extract could also change functional parameters of platelets, ex vivo. In the bleeding time test, transaction bleeding time was found to be significantly (P<0.001) longer under pretreatment with B-RT compared to the vehicle control. Similar data was found with the group treated with aspirin. Administration of collagen at a high dose significantly reduced bleeding time. However, pretreatment with B-RT extract at the dose of (100 mg/kg) significantly (P<0.01) prevented platelets aggregation induced by the high dose of collagen as compared to collagen high dose group. This effect was of the same efficiency as aspirin (Figure 2).
Figure 2: The histogram represents the bleeding time of inhibitory effect of B-RT extract on platelets coagulation induced by high dose of collagen.
The Data shown in bar graphs expressed as the means ± SD (n= 5),
***P<0.001 versus vehicle Control;
# #
P<0.01, # # #P<0.001 versus Collagen high dose
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3.3.2 Platelets count
Pretreatment with B-RT or Aspirin did not change platelets count (PC) whereas pretreatment with collagen at high dose significantly (P<0.001) reduced PC. Aspirin and B-RT prevented significantly (P<0.001) the collagen induced PC reduction. The reversal effect of B-RT reached a level which did not differs statistically from control (Figure 3).
Figure 3: The histogram represents the platelets count of B-RT inhibitory effect on platelets aggregation induced by high dose of collagen.
The data shown in bar graphs expressed as the means ± SD (n = 5),
***P<0.001, *P<0.05 versus Vehicle Control;
# # #
P<0.001 versus Collagen high dose
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3.4 Phytochemicals analysis
3.4.1 Determination of total polyphenolics and total flavonoids contents
Mean total polyphenols content, calculated as Gallic acid equivalent (GAE), in B-RT extract was 1.1 ± 0.041 mg GAE/ 100g DM. Mean total flavonoids content, calculated in Catechin equivalent (CAE), of the B-RT extract was 0.7 ± 0.0055mg CAE /100 g DM.
3.4.3 HPLC analysis of B-RT
Identification and quantification of phenolics compounds in B-RT extract were performed by using the HPLC analysis. On the basis of retention time (min) of standard compounds, the polyphenols identified in B-RT were Vanillin (Rt= 28.8), Rosmarinic Acid (Rt=29.4), Cinnamic Acid (Rt=41.8), Quercitine (Rt=45.1) (Figure 4). Vanillin is the most represented compound in B-RT extract. The phenols amounts found in the studied extracts ranged from 0.25 to 1.5 mg /EGA/100g DM. Vanillin presented the highest concentration in B-RT extract (22.61 mg/EGA/100g DM), followed by Rosmarinic acid (13.21 mg /EGA/100g DM), and then Cinnamic acid and Quercitine with concentrations of 13.04 mg /EGA/100 g DM and 12.39 mg /EGA/ 100g DM respectively.
HO O O va nillin
O
OH HO
O
O H OH OH O
Ro sm arinic a cid
O OH
cinna mic acid
O H O OH
HO O
O H O H
Q u erc eti n
Figure.4: HPLC chromatograms of the Butanolic extract of Rubia tinctorum: Vanillin (Rt =28.8), Rosmarinic Acid (Rt=29,4), Cinnamic Acid (Rt=41,8), Quercitine (Rt=45,1).
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4. Discussion
Traditional medicine is gaining popularity as a source of complementary and alternative therapies, in contrast to conventional pharmaceuticals, it generally presumed as safe and without side effects (Gilard et al., 2008). Many medicinal plants are antiplatelet agents and have been used in the therapy of cardiovascular disorders (Nor et al., 2016). Rubia tinctorum’s (Rubiaceae), one of several plants widely used in traditional medicine to cure different ailments among which kidney stones, rheumatic disorders and cardiovascular diseases (Agrawal et al., 2015; Adams et al., 2008; Bellakhder et al., 1991). According to literature surveys, no studies have been done about antithrombotic effects of Rubia tinctorum L extract.
In this study, we presented butanolic extract of Rubia tinctorum root as potent extract that exhibited an inhibition on platelets aggregation. Platelet aggregation is a complex process influenced by many elements. Collagen (a strong thrombogenic component) stimulates platelet aggregation and induces the activations of various intracellular mediators (ADP, thromboxane A2, Calcium) through receptors glycoproteins (GP IaIIa, GP VI) (Bross et al., 2011). Furthermore, platelet aggregation can be blocked by inhibiting the thrombin, ADP, phosphodiesterase and thromboxane pathways activation platelets (Sikka and Bindra, 2010).
In this study B-RT extract inhibits collagen induced platelets aggregation in dose dependent manner, indicating that the antiaggregant action of RT might be introduced by inhibiting intra cellular mediators or holding back fibrinogen binding to its platelet membrane receptor (glycoprotein (GP) IIb-IIIa), which is a final and common pathway of platelet aggregation.
Indeed, Endale et al., (2012) has demonstrated that ginsensoside- Rp1 isolated from Panax ginensis inhibit platelet activation induced by collagen via impaired glycoprotein VI signaling pathway.
In the ex vivo experiments a significant prolongation of bleeding time (BT) was obtained in pretreated groups with B-RT and collagen high dose as compared to collagen high dose group. This prolongation was also obtained in pretreated group with aspirin (good pharmaceutical antiplatelet drug). In practice, bleeding time is a useful test to detect abnormal platelets function and studying the in vivo effects of compounds interfering with platelets adhesion and aggregation (Praga et al., 1972). Furthermore the data of BT correlate well with platelets count which was significantly not affected by thrombocytopenia in pretreated groups with extract and control group pretreated with aspirin (aspirin was used as positive control to verify that washed platelets may be completely inhibited) when collagen was injected.
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According to the previous similar reports, the inhibition of platelet aggregation can be associated to a prolongation of bleeding time (Seo et al., 2013). These findings are similar to a previously data which have found that other plants such as Allium cepa (onion) (Moon et al., 2000), Allium sativum (garlic) (Rahman and Billington, 2000), Lycopersicum esculentum (tomato) (Dutta-roy et al., 2001), argan oil (Mekhfi et al., 2008), Petroselinum crispum (parsely) (Gadi et al., 2012), Vigna radiate (green gram) (Akbar basha et al., 2017) and Juglans regia root (bark) (Amirou et al., 2018) exhibited similar effects on platelet aggregation.
The research into cellular and molecular mechanisms that could produce the antiaggregant platelets effect of B-RT extract has led us to determinate the total polyphenols and total flavonoids contents, identify and quantify their polyphenolic composition. The Phytochemical studies performed on the extract revealed the presence of high amounts of total polyphenols and flavonoids, These results are consistent with other studies that have shown that flavonoids inhibit platelets adherence, aggregation and secretion (Middleton et al., 2000).
HPLC analysis of polyphenols compounds showed that vanillin was found in highest concentration followed by Rosmarinic acid, Cinnamic acid and Quercetin. The antiaggregant activity of B-RT extract it could be attributed to the presence of vanillin. As with flavonoids several reports have shown that some molecules like flavones, chrysin, apigenin and phloretin inhibited significantly platelet cyclo-oxygenase (CO) activity and decreased the cAMP response to prostacyclin (Middleton et al., 2000). This enzyme and lipoxygenase (12-LO) are implicated in the synthesis of platelet activators like thromboxane A2 (Kundu et al., 2006).
Furthermore it was previously reported that antiplatelets effect of vanillin is possibly due to cyclo-oxygenase inhibition (Lin et al., 2003). Other studies have shown that Rosmarinic acid has an inhibition of platelets aggregation that could be modulated by reducing Ca2+
mobilization (Chapado et al., 2010). Recently Du et al., (2016) reported that Quercitine and Rutin are the most flavonoids frequently studied for their cardiovascular effects. Quercitine was an active inhibitor of lipoxygenase (12-LO) activity in human platelets and its action has been associated to the inhibition of Arachidonic acid metabolism by CO (Middleton et al., 2000).
5. Conclusion
In conclusion, the present investigation has shown that B-RT extract has an anti-aggregant effect in which flavonoids could be involved, in particular vanillin. Further studies are needed to confirm the mechanism of action an efficacy of vanillin in platelets aggregation.
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Acknowledgements
This study was supported by Faculty of Sciences and Technology, University Hassan First, Settat and Faculty of Sciences Semlalia, Cadi Ayyad University Marrakech, Morocco.
Authors are extremely grateful to Mr Abderrazzak Regragi expert in animal laboratory handling for his assistance in this study.
Conflict of interest
The authors confirm that there are no Known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Author’s contributions
- Conception and design of study: A. Bagri, A. Chait, R. Aboufatima, F. Marhoume.
- Acquisition of data: F. Marhoume, S. Oufquir, J. Laadraoui.
- Analysis and interpretation of data: F. Marhoume, M. Ait Laaradia, A. Chait, R.
Aboufatima, Younes Zaid, A. Bagri
- Drafting the manuscript: F. Marhoume, A. Bagri
- Revising the manuscript critically for important intellectual content: A. Bagri, A. Chait - Approval of the version of the manuscript to be published: F. Marhoume, M. Ait Laaradia, S. Oufquir, J. Laadraoui, R. Aboufatima, Y. Zaid, A. Chait, A. Bagri.
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Supplementary data:
Figure.1. Original tracing showing the effect of Rubia tinctorum Butanolic extract (B-RT) on platelets aggregation inhibition induced by collagen. Platelets were incubated with different doses of B-RT extract (250 µg/ml, 500µg/ml and 1000µg/ml) for 5 min at 37°C and aggregation was induced by collagen (2µg/ml). The control response was platelets aggregation induced by collagen without B-RT extract. Aggregation curves are representative of 3 independent experiments.
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Figure 5: Microscopic images of hematoxylin and eosin stained kidney sections observed under light microscope (50×) from mice of: vehicle control (A), treated with E-RT at 0.5g/kg (B), treated with E-RT at 1 g/kg (C) and 2 g/kg (D), treated with E-RT at 3.5g/kg (E) and 5g/kg (F).
Figure 6: Microscopic images of hematoxylin and eosin stained liver sections observed under light microscope (50×) from mice of: vehicle control (A), treated with E-RT at 0.5g/kg (B), treated with E-RT at 1 g/kg (C) and 2 g/kg (D), treated with E-RT at 3.5g/kg (E) and 5g/kg (F).
