Anti-Platelet Effect of Bryophyllum Pinnatum Aqueous Extract in Human Blood
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ORIGINAL ARTICLE Anti-platelet effect of Bryophyllum Pinnatum Aqueous extract in human blood FARWA NAQVI1, NADEEM YAQOOB2, MAHEEN RANA3, SHOAIB AHMED4, SULTAN SIKANDAR5, BILAL HABIB6 1,4Assistant professor Pharmacology, Sargodha Medical College, Sargodha 2Assistant professor Pharmacology, Niazi Medical College Sargodha 3Senior Demonstrator Pathology, Rashid Latif Medical College Lahore 5Demonstrator Biochemistry Department, Sargodha Medical College, Sargodha 6Assistant Professor Physiology, Rai Medical College, Sargodha. Correspondence to Dr Shoaib Ahmed, Email: [email protected], Cell: 03216038797 ABSTRACT Background: Bryophyllum pinnatum has demonstrated anti-inflammatory effect through arachidonic acid pathway. Blockage of arachidonic acid pathway causes reduced production of thromboxane A2, which may be responsible for its possible anti-platelet effect. Aim: To compare the effect of Bryophyllum pinnatum aqueous extract on platelet aggregation in vitro, by using arachidonic acid, thrombin and ADP as agonists. Study design: Experimental Study Setting: This study was conducted in Hematology Laboratory of Children Hospital, Lahore and Pharmacology Department of Post Graduate Medical Institute, Lahore. Method: Twenty healthy volunteers aged between 24-35 years from both genders, were selected from patient’s attendants and staff of Children Hospital. Their blood samples were evaluated for platelet count. Platelet rich plasma was prepared and platelet aggregation was checked with three different concentrations of Bryophyllum pinnatum by using ADP, arachidonic acid and thrombin. The data collected was processed by using SPSS 20, using one way ANOVA followed by post hoc Tukey’s test. A p value of ≤ 0.05 was considered significant. Results: This study demonstrated in vitro anti-platelet effect of Bryophyllum pinnatum , which was not dose dependent. Inhibition of platelet aggregation was marked with arachidonic acid, minimal with thrombin and negligible with ADP. Conclusion: The possible mechanism of anti-platelet effect of Bryophullum pinnatum is mainly through arachidonic acid pathway and partially through thrombin inhibition. Key words: Bryophullum pinnatum, Platelet aggregation, Arachidonic acid, Thrombin, ADP INTRODUCTION meter. The thick leaves are 5 to 6 cm wide with curved margin6. Normal hemostasis of blood depends on formation of Anti-inflammatory effects of Bryophyllum pinnatum primary platelet plug at site of endothelial injury. Collagen is are known for centuries. It is used traditionally, exposed at sites of endothelial injury, which initiates ashemostatic, topical preparation for migraines and platelet plug formation, leading to formation of fibrin- headaches, and given orally for urethritis, fever, arthritis, containing thrombi. In certain pathological conditions, blood internal bruises, fractures, and respiratory inflammations.7 vessels are occluded by this platelet plug leading to In a recent study, ear edema was induced in Swiss mice by disorders like cerebro-vascular accidents, cardiac ischemia using different irritants like croton oil, capsaicin, arachidonic 1 and peripheral vascular diseases. In view of this sequence acid and phenol. The results showed that Bryophyllum of events, the incidence of acute ischemic cardiovascular pinnatum leaf extract effectively reduced edema, epidermal accidents can be prevented by inhibition of platelet hyperplasia, vasodilatation and inflammatory cell infiltration 2 aggregation . For this purpose, different agents are being possibly by inhibition of arachidonic acid pathway.4 Non- used, of which aspirin is the most important and frequently steroidal anti-inflammatory drugs act by same mechanism used. Other agents like Cangrelor, clopidogrel, prasugrel, and inhibit platelet aggregation. So, it was postulated that ticagrelor, and vorapaxar have been compared with aspirin Bryophyllum pinnatum may also inhibit platelet aggregation to check benefits versus side effects of these agents in and to check this hypothesis, effect of Bryophyllum 3 patients with coronary atherosclerosis . Even after pinnatum leaf extract was studied on platelet aggregation, discovery of multiple anti-platelet agents there is always using light transmission aggregometer (LTA). room for newer, safer and more efficient agents. In this background, herb called Bryophyllum pinnatum was MATERIALS AND METHODS evaluated for its effect on platelet aggregation. Bryophyllum pinnatum is a small erect plant that Leaves of Bryophyllum pinnatum were collected and belongs to family Crassulaceae.4 Its other common names identified by the Botany Department of University of are Kalanchoe pinnata, “pathar chat” in Urdu, air plant and Punjab, Lahore. This study was approved by the Ethical life plant in English5. This plant attains a height of 1- 1.5 Committee of the Institution. Leaves of Bryophyllum ------------------------------------------------------------------------------- pinnatum were washed with tape water then dried under Received on 26-06-2018 shade. They were crushed manually and immersed in Accepted on 06-11-2018 distilled water in 1:10 (w/v) for 24 hours8. Whatman filter paper no.1was used to filter supernatant, which was dried P J M H S Vol. 13, NO. 1, JAN – MAR 2019 87 Anti-platelet effect of Bryophyllum Pinnatum Aqueous extract in human blood in air to powder form. The dry extract was weighed and 28.85± 3.29 years with range of 24 to 35 years. Mean ± SD kept at 4°C. Different concentration of Bryophyllum haemoglobin level was 13.29±2.0 g/dl and mean ± SD pinnatum i.e. 30mg/ml, 100 mg/ml and 300 mg/ml were platelet count was 263.2±45.18 x 103/ µl. prepared daily using normal saline9. Figure 1 shows effect of different concentrations of B. Twenty normal healthy volunteers of both genders pinnatum on platelet aggregation by using different were selected from volunteer blood donors at blood bank of agonists. One way ANOVA revealed significant difference Children Hospital, Lahore. Individuals of age group 18-35 with arachidonic acid and thrombin with p value of years, with normal platelet count (150-400x103/µL were 0.001and 0.01 respectively, while it was insignificant with included in study after taking informed consent. Subjects ADPas agonist with p value of 0.061. Difference among were asked to refrain from coffee, cafinated beveregs and various concentrations of B. pinnatum was insignificant on exercise at least two hours before blood sampling. post hoc analysis. Exclusion criteria were history of bleeding disorder, Inhibition of platelet aggregation was calculated by pregnancy, any existing illness; and intake of any drug subtracting value of platelet aggregation with each affecting platelet aggregation in past two weeks. treatment from control value. Figure 2 Shows bar chart of After a loose tourniquet, 10 ml of blood was drawn inhibition of platelet aggregation. By applying one way from the antecubital vein. Two ml was put into EDTA ANOVA, difference among different concentrations of B. vacutainer and rest was taken in four 3.2% sodium citrate pinnatum was not significant with any agonist, while it was vials. All blood samples were processed within two hours of significant between different agonists with Pvalue <0.001. collection. Complete blood count of sample in EDTA Multiple comparison revealed that B. pinnatum extract vacutainer was performed by using automated hematology inhibited platelet aggregation induced by arachidonic acid analyser. Samples with haemoglobin greater than 10 g/dl more as compared to that induced by thrombin and ADP (p and normal platelet count were selected for further value ˂0.001), while inhibition of thrombin induced platelet processing. Clotted or hemolysed samples were discarded. aggregation was more as compared to that by ADP (p Platelet rich plasma (PRP) was prepared centrifuging value ˂0.01). Effect of different concentrations of citrated whole blood at 1500 rpm for 5 minutes at room Bryophyllum pinnatum on platelet aggregation (mean ± SD) temperature. Aim was to concentrate platelets from 150- by using different agonists (n=20). 400x109 /L. Platelet count was confirmed by automated hematology analyser. 90 Light transmission aggregometer (LTA) was used to % Platelet Aggregation check platelet aggregation in vitro by usingarachidonic acid, thrombin and ADP as agonists. The concentration of 80 arachidonic acid, thrombin and ADP were 0.5 mM, 1unit/ml and 10 μM respectively.10 Four microcuvettes were taken and were labelled as C, BL, BM and BH for Control, 70 C Bryophyllum pinnatum low, medium and high concentration respectively and 245 µl PRP was then taken in all the BL microcuvettes. Normal saline 2.5 µl was added in control, 60 * BM whereas 2.5 µl of different concentrations of Bryophyllum * * pinnatum were added in BL, BM and BH11 All * * BH o 12 * microcuvettes were incubated for 30 minutes at 37 C . 50 Aggregation was induced by adding arachidonic acid 2.5 µl, making final concentration 0.5mM (per chrono log USA arachidonic acid kit leaflet). Reading was then taken for 3 40 minutes and resulting percentage aggregation was * recorded on graph papers.13 This process was repeated * using 2.5µl of thrombin making final concentration 1unit/ml 30percentage aggregation * and 2.5 µl of ADP for a final concentration of 10 µM. Statistical analysis: The data collected was processed by * using SPSS 20. It was checked for normal distribution by 20 * * using Shapiro-wilk test of normality. Platelet aggregation * * and inhibition was presented as mean ± standard deviation