Phytochemical Screenings, Thrombolytic Activity, Membrane Stabilizing Activity
and Cytotoxic Properties of Polygonum hydropiper
Md. Aminul Haque,
Polygonum hydropiper of the family Polygonaceae has been considered
as an important plant in the traditional Ayurvedic and in the indigenous medical
system in Bangladesh. As membrane stabilizing activity, thrombolytic activity
and cytotoxic activity of Polygonum hydropiper were not studied earlier,
our aim is to find out those activities of Polygonum hydropiper. In the
present study the phytochemical screening, thrombolytic activity, membrane stabilizing
activity and cytotoxic activity of Polygonum hydropiper was evaluated.
For phytochemical screening, some common standard tests those are available
for phytochemical screening was done. Cytotoxicity test was done by Brine shrimp
lethality bioassay. The thrombolytic and membrane stabilizing activities were
assessed by using human erythrocyte and the results were compared with standard
streptokinase (SK) and standard anti-inflammatory drug, Acetyl Salicylic Acid
(ASA) or aspirin, respectively. Carbohydrates, steroids, flavonoids and saponins
were present in different solvent extracts. The extracts demonstrated significant
toxicity to A. salina with LC50 values ranging from 1.59
to 3.39 μg mL-1 and LC90 values ranging from 32.36
to 85.11 μg mL-1 as compared to standard Vincristine sulphate
(VS, LC50 value 0.927 μg mL-1 and LC90 value
6.310 μg mL-1). The ethanol soluble fraction of ethanol extract
of Polygonum hydropiper revealed highest thrombolytic activity 43.08%.
For hypotonic solution induced haemolysis, at a concentration of 1.0 mg mL-1,
the Methanolic Extract (ME) inhibited 85.06% haemolysis of RBCs. Our study demonstrates
that Polygonum hydropiper possesses significant thrombolytic and membrane
stabilizing activity and shows moderate cytotoxic activity.
to cite this article:
Shahjabeen Sharif, Tanjida Ahmed, Md. Aminul Haque, M.A. Bhuiyan and Mohammad Shahriar, 2014. Phytochemical Screenings, Thrombolytic Activity, Membrane Stabilizing Activity
and Cytotoxic Properties of Polygonum hydropiper. Research Journal of Medicinal Plants, 8: 92-98.
Received: April 15, 2013;
Accepted: January 17, 2014;
Published: March 01, 2014
For decades, the utilization of herbal plants has drawn avalanche of interest
as they could accommodate therapeutic response and are promising candidate to
be developed as pharmaceutical products. Presently, complication has arises
in severity and extent in combating bacterial and fungal infections in behalf
of the development of bacteria and fungi resistant to many current antibiotics
(Aderogba et al., 2005; Rabaud
et al., 1997). Free radicals are responsible for initiating many
serious diseases (Malorni et al., 1998; Robert
and Meunier, 1998; Pauli et al., 2005; Shah,
2005; Rios and Recio, 2005). These free radicals
drive oxidative stress and transform the pathophysiological condition of the
patient by acting on immune system. It has been known that phenolic and flavonoid
compounds of the plant extracts are responsible for antioxidant and antibacterial
effects (Da-Silva et al., 2006; Majhenic
et al., 2007; Pereira et al., 2007).
Polygonum hydropiper (Fam. Polygonaceae) locally known as Bishkatali,
is an erect or ascending herb with stem decumbent at base, nodes below swollen,
linear-lanceolate leaves and small pink flowers in very slender erect or decurved
racemes, commonly found in wet places, particularly near the banks of canals
and ditches all over the country.
Aerial parts of P. hydropiper contain several flavonols and flavones
glycosides, including quercetin, quercitrin, kaempferol, rutin, persicarin and
its methyl ester, hyperoside, rhamnacin and its ester, a dialdehyde sesquiterpene,
tadeonal (polygodial) and its isomers, iso-tadeonal and confertifolin, iso-rhamnazin
(Yoshiyasu et al., 1983; Yoshiyasu,
1985; Haraguchi et al., 1996; Murai
et al., 1983). Acrid essential oil and dimethyl anthraquinones are
present in the leaves (Chopra et al., 1969). Tendines,
a glucoside, polygopiperin, an inactive alkaloid, tannins and a number of organic
acids are also found in this herb. Tannins, ellagic acid methyl ether, gallic
acid, anthraquinone and oxymethyl anthraquinone, quercetin glycosides and iso-coumarin
polygonolide are restrained in roots (Barnes and Loder,
1962; Furuta et al., 1986).
Traditionally juice of leaves is reported to be useful in pain, headache, toothache,
liver enlargement, gastric ulcer, dysentery, loss of appetite and dismenorrhoea.
Juice of paste is applied to wounds, skin diseases and painful carbuncles. Root
is used as stimulant. Preparation of the plant is used to cause premature abortion.
Leaf paste is also capable of stopping external haemorrhage (Ghani,
The aim of present study is to evaluate bioactivities including phytochemical
screening, thrombolytic activity, membrane stabilizing activity and cytotoxic
activity by using brine shrimp lethality assay of the plant extractives.
MATERIALS AND METHODS
Plant materials collection and identification: Whole plant sample of
Polygonum hydropiper was collected from Tongi, Gazipur, Dhaka, Bangladesh
in June 2012. Then the plant sample was submitted to The National Herbarium
of Bangladesh, Mirpur, Dhaka for its identification. One week later its voucher
specimen was collected after its identification (Accession No. 37756) which
was identified and authenticated by taxonomist of the National Herbarium of
Plant materials preparation: Whole plant of P. hydropiper was
separated in different parts and sun dried for 7 days. Then leaves were taken
and oven dried for 3 h at 40°C temperatures and then 800 g dried leaves
were grinded in coarse powder using high capacity grinding machine. About 715
g of grinded powders were sieved to get fine powder. Finally 630 g of fine powder
obtained which was then stored in air tight container with necessary marking
for identification and kept in cool, dark and dry place for further investigation.
Powdered leaves (15 g) were successively extracted in Soxhlet extractor at
elevated temperature (40-60°C) by using 300 mL of methanol (solvent). Then
by same process, ethanol, chloroform, petroleum ether and n-Hexane were extracted
too. All extracts were filtered individually by Watman filter paper. Then all
extracts were poured on petri dishes individually to evaporate liquid and proper
dry. After drying, crude extracts were stored in those petri dishes and kept
in refrigerator (0-4°C) for future investigation.
Preliminary phytochemical screening: Methanol extract (0.5 g) of
P. hydropiper was dissolved in 50 mL of methanol and was subjected to preliminary
phytochemical screenings for determining nature of phytoconstituents (Horbone,
1998; Kokate, 2001).
Thrombolytic activity: The thrombolytic activity of all extracts was
evaluated by the method developed by Prasad et al.
(2006) and using streptokinase (SK) as the standard.
Blood samples of volunteers: Blood (n = 6) was drawn from healthy human
volunteers without a history of oral contraceptive or anticoagulant therapy.
Streptokinase (SK): Commercially available lyophilized streptokinase
(15,00,000 I.U.) vial (Trade name: S-Kinase from Popular Pharmaceuticals Ltd.,
Tongi, Bangladesh) was used as standard in this test. On this vial 5 mL 0.9%
NaCl was added with powdered streptokinase and mixed properly. The concentration
the streptokinase became 30,000 I.U. which was used reference standard for in
Membrane stabilizing activity: The erythrocyte membrane resembles to
lysosomal membrane and as such, the effect of drugs on the stabilization of
erythrocyte could be extrapolated to the stabilization of lysosomal membrane
(Omale and Okafor, 2008). The membrane stabilizing activity
of the extractives was assessed by using hypotonic solution-induced and heat-induced
mice erythrocyte haemolysis (Shinde et al., 1999).
To prepare the erythrocyte suspension, 10 mL blood was obtained from healthy
human volunteers and was taken in syringes containing anticoagulant EDTA (3.1%
Na-citrate). The blood was centrifuged for 10 min at 3000 g and blood cells
were washed three times with solution (154 mM NaCl) in 10 mM sodium phosphate
buffer (pH 7.4) through centrifugation for 10 min at 3000 g.
Hypotonic solution-induced haemolysis: The test sample consisted of
stock erythrocyte (RBC) suspension (500 μL) mixed with 5 mL of hypotonic
solution (50 mM NaCl) in 10 mM sodium phosphate buffered saline (pH 7.4) containing
all kinds of extracts (1.0 mg mL-1) and standard acetyl salicylic
acid (0.1 mg mL-1). The control sample consisted of 500 μL of
RBCs mixed with hypotonic-buffered saline alone. The mixture was incubated for
10 min at room temperature, centrifuged for 10 min at 3000 g and the absorbance
of the supernatant was measured at 540 nm. The percentage inhibition of either
haemolysis or membrane stabilization was calculated using the following equation:
Inhibition of haemolysis% = 100x(OD1-OD2/OD1)
where, OD1 is optical density of hypotonic-buffered saline solution
alone (control) and OD2 is optical density of test sample in hypotonic
Heat-induced haemolysis: Fresh human blood (10 mL) was collected and
transferred to the centrifuged tubes containing anticoagulant EDTA (1 mL of
3.1% Na-citrate). The tubes were centrifuged at 3000 rpm for 10 min and were
washed three times with equal volume of normal saline (0.9% NaCl). The volume
of the blood was measured and reconstituted as 10% v/v suspension with normal
The reaction mixture (2 mL) consisted of 1 mL of all kinds of extracts (1.0
mg mL-1) or test samples and 1 mL of 10% RBCs suspension, instead
of drug only saline was added to the control test tube. Aspirin or acetyl salicylic
acid (0.1 mg mL-1) was taken as a standard drug. All the centrifuge
tubes containing reaction mixture were incubated in a water bath at 56°C
for 30 min. At the end of the incubation, the tubes were cooled under running
tap water. The reaction mixture was centrifuged at 2500 rpm for 5 min and the
absorbance of the supernatants was taken at 560 nm. The experiment was performed
in triplicates. The percentage inhibition or acceleration of hemolysis in tests
and was calculated according to the equation:
Inhibition of hemolysis% = 100x [1- (OD2-OD1/
where, OD1 is optical density of unheated test sample, OD2
is optical density of heated test sample and OD3 is optical density
of heated control sample.
Brine shrimp lethality bioassay: Brine shrimp lethality bioassay (Meyer
et al., 1982; McLaughlin et al., 1998)
technique was applied for the determination of general toxic properties of the
plant extractives. DMSO solutions of the samples were applied against Artemia
salina Leach in a 1-day in vivo assay For the experiment.
Statistical analysis: Data were statistically analyzed with the help
of Microsoft office excel-2010.
RESULTS AND DISCUSSION
Preliminary phytochemical screening: Previously, the presence of triterpenes
and/or steroids, coumarins, flavonoids, polyphenols, tannins and saponins were
evaluated in different extracts of P. hydropiperoides in different research
studies (Jacome et al., 2004). Here, Phytochemical
screening of the crude extracts of the leaves of Polygonum hydropiper
(L.) revealed the presence of different kinds of chemical groups summarized
in the Table 1.
Thrombolytic activity: As a part of discovery of cardio-protective drugs
from natural sources of leaf extractives of P. hydropiper were assessed
for thrombolytic activity and the results are presented in Table
2. Addition of 100 μL SK, a positive control (30,000 I.U.), to the
clots and subsequent incubation for 90 min at 37°C, showed 59.20% lysis
|| Result of chemical group test of leaf extracts of Polygonum
|+++: Presence in high concentration, ++: Presence in moderate
concentration, +: Presence in low concentration, -: Absence
|| Clot lysis % by different extracts of Polygonum hydropiper
|| Effect of extractives of Polygonum hydropiper on hypotonic
solution (A) and heat induced (B) of erythrocyte membrane
At the same time, distilled water was treated as negative control which exhibited
negligible lysis of clot (3.215%). In this study, after treatment of clots with
100 μL methanolic, ethanolic, chloroform, pet ether, n-Hexane extract of,
clot lysis 24.43, 43.08, 36.48, 40.60 and 20.15% was obtained respectively.
In this study, the ethanol soluble fraction of ethanol extract of Polygonum
hydropiper revealed highest thrombolytic activity 43.08%, whereas methanolic,
chloroform, pet ether, n-Hexane extract of Polygonum hydropiper (24.43,
36.48, 40.60 and 20.15%) displayed moderate thrombolytic activities.
On the basis of the result obtained in this present study we can say that the
Polygonum hydropiper leaves extract have mild thrombolytic activity compared
to standard. So, in comparison with standard, Polygonum hydropiper can
be further use as a mild thrombolytic agent. In previous, the thrombolytic activity
of this plant was not carried out.
Membrane stabilizing activity: The whole plant extractives of P.
hydropiper at concentration of 1.0 mg mL-1, were tested against
lysis of human erythrocyte membrane induced by hypotonic solution as well as
heat and compared with the standard Acetyl Salicylic Acid (ASA) (0.10 mg mL-1)
(Table 3). For hypotonic solution induced haemolysis, at a
concentration of 1.0 mg mL-1, the methanolic extract (ME) inhibited
85.06% haemolysis of RBCs as compared to 92.13% produced by acetyl salicylic
acid (0.10 mg mL-1). The ethanol and chloroform soluble extractives
also revealed significant inhibition of haemolysis of RBCs. To confirm the membrane
stabilizing activity of Polygonum hydropiper observed in the above mentioned
model (Omale and Okafor, 2008), experiments were performed
on the erythrocyte membrane. A possible explanation for the stabilizing activity
of the extractives due to an increase in the surface area/volume ratio of the
cells which could be brought about by an expansion of membrane or shrinkage
of the cell and an interaction with membrane proteins. The present investigation
suggests that the membrane stabilizing activity of Polygonum hydropiper
may be playing a significant role in its anti-inflammatory activity. Membrane
stabilizing activity of Polygonum hydropiper was not tested previously.
|| LC50 and LC90 values of the five extracts
of Polygonum Hydropiper and standard
Brine shrimp lethality bioassay: In the brine shrimp lethality bioassay,
the LC50 values of ME, EE, CE, PEE and n-HE of P. hydropiper
were found to be 3.09, 2.089, 3.311, 3.389 and 1.585 μg mL-1,
respectively and LC90 values of ME, EE, CE, PEE and n-HE of P.
hydropiper were found to be 85.113, 60.256, 32.359, 77.625 and 48.977 (Table
4). LC50 and LC90 were calculated by plotting graph
in Microsoft office excel-2010 grade sheet.
The phytochemical screening revealed the presence of carbohydrates, flavonoids,
glycosides, phenols, saponins, steroids and tannins. In percent mortality of
Brine Shrimp nauplii produced by the extracts of Polygonum hydropiper indicates
the presence of cytotoxic principles in these extracts. After thrombolytic and
membrane stabilizing activity significant results are shown in the present study.
1: Aderogba, M.A., E.K. Okoh and T.O. Idowu, 2005. Evaluation of the antioxidant activity of the secondary metabolites from Piliostigma reticulatum (DC.) hochst. J. Biol. Sci., 5: 239-242.
CrossRef | Direct Link |
2: Barnes, C.S. and J.W. Loder, 1962. The structure of polygodial: A new sesquiterpene dialdehyde from Polygonum hydropiper L. Aust. J. Chem., 15: 322-327.
CrossRef | Direct Link |
3: Chopra, R.N., L.C. Chopra and B.S. Varma, 1969. Supplement to Glossary of India Medicinal Plants. Publication and Information Division CSIR, New Delhi, India, Pages: 119
4: Prasad, S., R.S. Kashyap, J.Y. Deopujari, H.J. Purohit, G.M. Taori and H.F. Daginawala, 2006. Development of an in vitro model to study clot lysis activity of thrombolytic drugs. Thrombosis J., Vol. 4.
CrossRef | Direct Link |
5: Da-Silva, J.F.M., M.C. De-Souza, S.R. Matta, M.R. De-Andrade and F.V.N. Vidal, 2006. Correlation analysis between phenolic levels of brazilian propolis extracts and their antimicrobial and antioxidant activities. Food Chem., 99: 431-435.
6: Furuta, M., H. Yamagata, K. Tanaka, Z. Kasai and S. Fuji, 1986. Cell-free synthesis of the rice glutelin precursor. Plant Cell Physiol., 27: 1201-1204.
Direct Link |
7: Ghani, A., 2003. Medicinal Plants of Bangladesh-Chemical Constituents and Uses. 2nd Edn., The Asiatic Society of Bangladesh, Dhaka, Bangladesh, pp: 1-2, 55-57, 336, 402, 500
8: Haraguchi, H., I. Ohmi, S. Sakai and A. Fukuda, 1996. Effect of Polygonum hydropiper sulfated flavonoids on lens aldose reductase and related enzymes. J. Nat. Prod., 59: 443-445.
9: Horbone, J.B., 1998. Phytochemical Methods. 1st Edn., Chapman and Hall, London, pp: 60-66
10: Jacome, R.L.R.P., D.E.S. Lopes, R.A. Recio, J.F. Macedo and A.B. Oliveira, 2004. Pharmacognostic characterization of Polygonum hydropiperoides Michaux and P. spectabile (Mart.) (Polygonaceae). Rev. Bras. Farmacog., 14: 21-27.
11: Kokate, C.K., 2001. Practical Pharmacognosy. Vallabh Prakashan, New Delhi, India, Pages: 218
12: McLaughlin, J.L., L.L. Rogers and J.E. Anderson, 1998. The use of biological assays to evaluate botanicals. Drug Inform. J., 32: 513-524.
CrossRef | Direct Link |
13: Majhenic, L., M. Skerget and Z. Knez, 2007. Antioxidant and antimicrobial activity of guarana seed extracts. Food Chem., 104: 1258-1268.
14: Malorni, W., R. Rivabene, B.M. Lucia, R. Ferrara, A.M. Mazzone, R. Cauda and R. Paganelli, 1998. The role of oxidative imbalance in progression to AIDS: Effect of the thiol supplier N-Acetylcysteine. AIDS Res. Hum. Retroviruses, 14: 1589-1596.
PubMed | Direct Link |
15: Meyer, B.N., N.R. Ferrigni, J.E. Putnam, L.B. Jacobsen, D.E. Nichols and J.L. McLaughlin, 1982. Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med., 45: 31-34.
CrossRef | PubMed | Direct Link |
16: Murai, N., J.D. Kemp, D.W. Sutton, M.G. Murray and J.L. Slightom et al., 1983. Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors. Science, 222: 476-482.
17: Omale, J. and P.N. Okafor, 2008. Comparative antioxidant capacity, membrane stabilization, polyphenol composition and cytotoxicity of the leaf and stem of Cissus multistriata. Afr. J. Biotechnol., 7: 3129-3133.
Direct Link |
18: Pauli, G.F., R.J. Case, T. Inui, Y. Wang, S. Cho, H.H. Fischer and S.G. Franzblau, 2005. New perspectives on natural products in TB drug research. Life Sci., 78: 485-494.
CrossRef | PubMed |
19: Pereira, J.A., I. Oliveira, A. Sousa, P. Valentao and P.B. Andrade et al., 2007. Walnut (Juglans regia L.) leaves: Phenolic compounds, antibacterial activity and antioxidant potential of different cultivars. Food Chem. Toxicol., 45: 2287-2295.
CrossRef | Direct Link |
20: Rabaud, C., H. Tronel, S. Fremont, T. May, P. Canton and J.P. Nicolas, 1997. Free radicals and HIV infection. Ann. Biol. Clin., 55: 565-571.
PubMed | Direct Link |
21: Rios, J.L. and M.C. Recio, 2005. Medicinal plants and antimicrobial activity. J. Ethnopharmacol., 100: 80-84.
CrossRef | PubMed | Direct Link |
22: Robert, A. and B. Meunier, 1998. Is alkylation the main mechanism of action of the antimalarial drug artemisinin? Chem. Soc. Rev., 27: 273-274.
23: Shah, P.M., 2005. The need for new therapeutic agents: What is the pipeline? Clin. Microbiol. Infect., 11: 36-42.
PubMed | Direct Link |
24: Shinde, U.A., A.S. Phadke, A.M. Nair, A.A. Mungantiwar, V.J. Dikshit and M.N. Saraf, 1999. Membrane stabilizing activity-a possible mechanism of action for the anti-inflammatory activity of Cedrus deodara wood oil. Fitoterapia, 70: 251-257.
25: Yoshiyasu, H., S. Murata, S. Yamagata, Y. Wada and Y. Shibuya, 1983. Experimental study of electrode vapour flow in a free-burning arc. J. Phys. D,
26: Yoshiyasu, Y., 1985. A systematic study of the Nymphulinae and the Musotiminae of Japan (Lepidoptera: Pyralidae). Scientific Report No. 37, Kyoto Prefectural University Japan, pp: 1-162