Comparative Study of Potential Thrombolytic and Anti-arthritic Activities of Pterospermum acerifolium and Sonneratia caseolaris Leaves
Sudipta Chowdhury1, Md. Irfan Amin Chowdury1, Mohammad Nazmul Alam1, Abu Hena Mustafa Kamal1, S. M. Arif Bin Alam1, Sharmin Sultana2, Tamanna Jahan2
1Department of Pharmacy, Faculty of Science and Engineering, International Islamic University, Chittagong, Bangladesh
2Department of Pharmaceutical Sciences, North South University, Dhaka, Bangladesh
To cite this article:
Sudipta Chowdhury, Md. Irfan Amin Chowdury, Mohammad Nazmul Alam, Abu Hena Mustafa Kamal, S. M. Arif Bin Alam, Sharmin Sultana, Tamanna Jahan. Comparative Study of Potential Thrombolytic and Anti-arthritic Activities of Pterospermum acerifolium and Sonneratia caseolaris Leaves.American Journal of Clinical and Experimental Medicine.Vol.3, No. 5, 2015, pp. 228-232. doi: 10.11648/j.ajcem.20150305.15
Abstract: This comparative study was performed for the evaluation of the thrombolytic and anti-arthritic effects of methanolic leaf extracts of P. acerifolium and S. caseolaris. The thrombolytic activity was evaluated by using the in vitro clot lysis model and anti-denaturation method was performed by using bovine serum albumin (BSA) to evaluate the anti-arthritic potential. Here, the thrombolytic activity of P. acerifolium leaves showed (35.15 ± 1.77)% whereas S. caseolaris leaves exhibited (26.05 ± 0.92)% and standard streptokinase demonstrated (63.54 ± 2.61)%. In the case of anti-arthritic study, P. acerifolium showed (35.48 ± 0.98)% at lower concentration and (76.64 ± 1.29)% at higher concentration and S. caseolaris exhibited (27.42 ± 0.98)% and (59.68 ± 1.07)% at lower and higher concentration respectively whereas standard diclofenac sodium showed (52.31 ± 0.56)% at 31.25 µg/ml and (86.67 ± 0.92)% at 1000 µg/ml. The results of these experiments suggest that methanolic leaf extract of P. acerifolium showed higher thrombolytic and anti-arthritic activities than S. caseolaris.
Keywords: P. acerifolium, S. caseolaris, Thrombolytic, Streptokinase, Anti-arthritic, Protein Denaturation
Pterospermum acerifolium (Sterculiaceae) is a tall evergreen tree. Locally it is known as Moochkunda, Mushkand, Madhurabura. Leaves are variable in size and shape, 20-35 cm long, orbicular or oblong, with a cordate base, entire or variously lobed, or remotely toothed, sometimes peltate. Flowers are tonic, laxative, anthelmintic; used in leucorrhoea, gastralgia, blood troubles, ulcers, tumours, leprosy, and inflammations. It is also used as an insecticide. Bark is used for impotence in Khagrachari . It is extensively found in forests of Chittagong, Chittagong Hill Tracts, Cox’s Bazar and Sylhet. Sonneratia caseolaris is a small, glabrous, evergreen tree, reaching 4.5 m high; Leaves subfleshy, 5-9.5 cm long, elliptic-oblong or obovate. Flowers solitary at the end of branchlets, about 5 cm across. Fruits are used as a poultice in sprains and swellings. Fermented juice of the fruits is useful in arresting haemorrhage.Leaves are rich in carbohydrates, lipids and proteins . It is widely distributed in tidal forest of Barisal, Chittagong, Sunderbans and Chakaria Sundarbans.
2. Materials and Methods
2.1. Plant Collection and Identification
Leaves of Pterospermum acerifolium and Sonneratia caseolaris were collected from different parts of the Chittagong region, Bangladesh. The plants were identified and authenticated by Dr. Shaikh Bokhtear Uddin, Taxonomist and Associate Professor, Department of Botany, University of Chittagong, Bangladesh.
2.2. Preparation of Extracts
Leaves of P. acerifolium and S. caseolaris were dried and ground into powder (40-80 mesh, 500 gm) through Moulinex Blender AK-241. Then the powder was soaked in 2 L of methanol at room temperature (23.0 ± 0.5)°C for a week. By using Whatman filter paper No. 1 and cheesecloth, filtrate was obtained. Filtrated solution was concentrated under reduced pressure by using a rotary evaporator. Less than 50°C was maintained for this filtration process. Glass Petri dishes were used to keep the extract. Each of the extracts (100 mg) was suspended in 10 mL distilled water. By using a vortex mixer, the suspension was shaken vigorously. Suspension of extract and distilled water was kept overnight and gradually poured through a 0.22 μm syringe filter for the filtration. In this way, soluble supernatant was removed. Then 100 μL of this filtrated aqueous preparation was added to microcentrifuge tubes which contained the clots to check the ex-vivo thrombolytic activity. The same concentration (10 mg/mL) of both plant extracts was prepared for the ex-vivo screening of anti-arthritic effects. In this process the leaves were dried and ground. The ground leaves (300g) were soaked in sufficient amount of methanol for one week at room temperature with occasional shaking and stirring then filtered through a cotton plug followed by Whitman filter paper No. 1. The solvent was evaporated under vacuum at room temperature to yield semisolid. The extract was then stored in a refrigerator till further use.
2.3. Chemicals and Drugs
Lyophilized streptokinase vial (1 500 000 IU) which was imported from Durakinase, Dongkook Phama. Co. Ltd, South Korea and sterile distilled water (5 mL) were added and mixed properly. Mixture of distilled water and lyophilized streptokinase was used as a stock. From this mixture, 100 μL (30 000 IU) was used for determination of thrombolytic activity. The chemicals used were Bovine serum albumin (BSA), Diclofenac Sodium, Sodium Dihydrogen phosphate, Disodium hydrogen phosphate, Sodium Chloride, Dextrose, sodium citrate, citric acid were purchased from Sigma-Aldrich. All chemicals in this experiment were of analytical reagent grade.
2.4. Thrombolytic Activity
2.4.1. Blood Sample
About 2 mL of blood was drawn from three healthy humans. They had no history of taking any types of contraceptive or anticoagulant therapy. A total of 500 μL of blood was transferred to each of the three previously weighed microcentrifuge tubes to form clots.
2.4.2. Clot Lysis
At first, three different sterile microcentrifuge tubes (0.5 mL/tube) were taken and weighed. Then 2 mL venous blood from human volunteers was added in pre-weighed sterile microcentrifuge tubes. The tubes were incubated at 37°C for 45 min. In this process, serum was totally eliminated after the formation of clots without disturbing the clots. Each tube containing clot was again weighed to know the weight of clot. For the determination of clot weight, weight of tube alone were excluded from the weight of clot and tube. For each microcentrifuge tube containing pre-weighed clot, 100μL of methanol extracts of both plants (Pterospermum acerifolium and Sonneratia caseolaris) were added separately. About 100μL of streptokinase was used as a positive control and 100 μL of distilled water was used as a negative control. At last, all the tubes were incubated at 37°C for 90 min. In this way, clot lysis was observed. After incubation, released fluid was removed and tubes were again weighed to observe the difference in weight after clot disruption. Difference in weight before and after clot lysis was expressed as percentage of clot lysis . Evaluation of thrombolytic effects of both methanolic extracts were performed by the formula below:
Percentage of clot lysis = (Weight of released clot/clot weight) ×100
The experiment was repeated three times with the blood samples from the three healthy volunteers.
2.5. In Vitro Anti-arthritic Activity
For the evaluation in vitro anti-arthritic activity of P. acerifolium and S. caseolaris, the method used was "inhibition of protein denaturation" with diclofenac sodium as a standard [4-7]. The test solution (0.5 ml) consists of 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of test solution (methanolic leaf extract of P. acerifolium and S. caseolaris). The test control solution (0.5 ml) consists of 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of distilled water. Product control (0.5 ml) consists of 0.45 ml of distilled water and 0.05 ml of test solution. Standard solution (0.5 ml) consists of 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of diclofenac sodium. Various concentrations (31.25, 62.5, 125, 250, 500, 1000 μg/ml) of methanolic leaf extract of sodium (standard) were taken respectively. All the solutions were adjusted to pH 6.3 using 1 N HCl. The samples were incubated at 37 °C for 20 min and the temperature was increased to keep the samples at 57 °C for 3 min. After cooling, 2.5 ml of phosphate buffer was added to the previous solutions. The absorbance was measured using UV-Visible spectrophotometer at 416 nm. The control represents 100% protein denaturation. The results were compared with diclofenac sodium. The percentage inhibition of protein denaturation of different concentrations is tabulated in table 2. The percentage inhibition of protein denaturation can be calculated as:
Percentage of inhibition = [100 − (OD of test solution − OD of product control)]×100
Where OD = optical density.
Control represents almost 100% protein denaturation. The results were compared with diclofenac sodium .
2.6. Statistical Analysis
In the case of thrombolytic study, statistical significance between percentage of clot lysis by streptokinase and both of the plant extracts were evaluated by paired t-test analysis. Expression of data was expressed as mean ± SD. The mean difference between positive and negative controls was considered significant at P<0.05. In the case of anti-arthritic study, the results were expressed as mean of the three repetitions and standard deviations were calculated. Statistical comparisons were made using the Independent t-test and P<0.01 was considered as significant. The tests were performed by using the software SPSS version 20.0 (SPSS for Windows, IBM Corporation, New York, USA).
3.1. Thrombolytic Activity
At first, 100 μL of streptokinase (positive control) was added to the clots and kept in incubator at 37 °C for 90 min, which showed significant lysis of clot (63.54 ± 2.61)%. When distilled water (negative control) was used to treat clots, it showed negligible clot lysis (4.21 ± 0.73)%. A significant value (probability value of P<0.05) was obtained by calculating the mean difference in the clot lysis (%) between streptokinase (positive control) and distilled water (negative control). When methanolic leaf extracts of P. acerifolium and S. caseolaris were used to treat clots, the lysis of clot was (35.15±1.77)% and (26.05±0.92)% respectively. A statistically significant value (probability value of P<0.05) was obtained from the mean percentage of clot lysis of P. acerifolium and S. caseolaris. P. acerifolium exhibited a relatively higher percentage of clot lysis than S. caseolaris. However, both plant extracts showed statistically significant values when compared with streptokinase (positive control) and distilled water (negative control) (probability value of P<0.05).
|Treatment||% of clot lysis (Mean ± S. D.)|
|Streptokinase(Positive Control)||63.54 ± 2.61**|
|Distillled water (Negative Control)||4.21 ± 0.73**|
|PA||35.15 ± 1.77**|
|SC||26.05 ± 0.92**|
Statistical representation of the effective clot lysis percentage by herbal preparations, positive thrombolytic control (Streptokinase) and negative control (sterile distilled water) done by paired t-test analysis; clot lysis percentage is represented as mean ± S.D. **P<0.05 compared to control.
3.2. Anti-arthritic Study
Different concentrations of methanolic extract of P. acerifolium, S. caseolaris and diclofenac sodium was tested for anti-arthritic activity and found significant percentage inhibition in protein denaturation. Here, in lower concentration (31.25 μg/ml) the extract of P. acerifolium and S. caseolaris showed 35.48±0.98% (P<0.01) and 27.42±0.98% (P<0.01), where the standard drug diclofenac sodium showed 52.31±0.56% of inhibition. In the case of higher concentration (1000 μg/ml), methanolic extract of P. acerifolium and S. caseolaris exhibited the 76.64±1.29% (P<0.01) and 59.68±1.07% (P<0.01) of inhibition, whereas the diclofenac sodium showed 86.67±0.92% of inhibition of protein denaturation.
|Concentration(µg/ml)||Percent Inhibition of protein denaturation|
|PA(Test Solution)||SC(Test Solution)||Diclofenac sodium|
Values are expressed as mean ± SEM of three replicate (n=3), **P<0.01
In the case of thrombolytic study, methanolic leaf extracts of P. acerifolium, S. caseolaris and positive control (streptokinase) truly demonstrated the effect on clot lysis. By comparing the clot lysis percentage obtained through streptokinase and distilled water, a promisingly significant (P<0.05) thrombolytic effect was observed after the clots were treated with both extracts. It is established from the previous experiment that there are some bacterial pollutants of plants that have plasminogen receptors which are specific for plasminogen. Certain plasminogen on cell surface is rapidly activated to plasmin that could lead to fibrinolysis . Bacterial plasminogen activator which also acts as cofactor molecules, such as staphylokinase, and streptokinase, can cause formation of exosite and increase the substrate activity towards the enzyme. Staphylokinase activates plasminogen to be in a position to break down clots, and also damages the extracellular molecules secreted by cells and fibrin particles that keep cells organized [10-12]. From the above experiment, it would be interesting to examine both of the mechanisms correlated to clot lytic effects showed by P. acerifolium and S. caseolaris extracts. However, these activities might be due to the presence of biologically active or inhibitory compounds or synergism by the existence of some compounds. A different type of constituents, such as saponins, polyphenols, alkaloids and flavonoids, may be present in the extracts, so further vast investigations are required to determine the specific thrombolytic effects of the leaf extracts. In this comparative study, P. acerifolium demonstrated higher thrombolytic activity than S. caseolaris. Additional investigations are required to be performed because phytochemicals derived from these plant could be incorporated as a thrombolytic agent for the improvement of the patients suffering from cardiac diseases. Arthritis is a form of joint disorder that involves inflammation of one or more joints, responsible for painful swelling, stiffness, loss of function in the joint. Denaturation of protein is one of the causes of arthritis as documented. Production of autoantigen in certain arthritic disease may be due to denaturation of protein. The mechanism of denaturation probably involves alteration I electrostatic hydrogen, hydrophobic and di-sulphide bonding . In this comparative study, methanolic leaf extract of P. acerifolium showed higher anti-arthritic activity than S. caseolaris in both lower and higher concentration. According to the result of this comparative, it can be stated that methanolic leaf extract P. acerifolium has the capability of controlling the production of autoantigen to inhibit the denaturation of protein significantly.
Authors wish to thank the botanist Dr. Shaikh Bokhtear Uddin, Associate Professor, Department of Botany, University of Chittagong, Bangladesh, who helped to identify these plants. Authors are also grateful to the Department of Pharmacy, International Islamic University Chittagong, Chittagong, Bangladesh, for providing support and research facilities.