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The objective of this study was to evaluate the crude methanol extract of leaf of Grevillea robusta as well as its hexane, carbon tetrachloride, chloroform and aqueous soluble partitionates for cytotoxic, thrombolytic, membrane stabilizing and antimicrobial activities. In the brine shrimp lethality bioassay, the crude methanolic extract of G. robusta leaf revealed the highest cytotoxic activity with LC50 values as 1.50 ± 0.45 μg/ml as compared to 0.45 μg/ml for vincristine sulphate. Among the extractives of G. robusta, the carbon tetrachloride soluble fraction showed 69.95±0.11% clot lysis as compared to 70.77% clot lysis by standard streptokinase. At concentration of 1.0 mg/ml, the chloroform soluble fraction inhibited 40.31 ± 0.59% and 62.93 ± 0.73% of haemolysis of RBC induced by heat and hypotonic solution as compared to 42.12% and 71.90% by acetyl salicylic acid, respectively. The test samples also showed antimicrobial activity with zone of inhibition ranging from 7.0 to 17.0 mm in diameter. The chloroform soluble partitionate demonstrated the highest zone of inhibition (17.0 mm) against Salmonella Typhi.
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Bangladesh Pharmaceutical Journal 17(2): 135-137, 2014
Pharmacological Activities of Grevillea robusta, a Medicinal Plant of
Bangladesh
Md. Sharif Ullah1, Md. Al Amin Sikder2, Tasnuva Sharmin2 and Mohammad A. Rashid2
1Department of Pharmacy, State University of Bangladesh, Dhaka-1205, Bangladesh
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh
Received: May 01, 2014; Accepted: May 26, 2014; Published (Web): July 23, 2014
Abstract
The objective of this study was to evaluate the crude methanol extract of leaf of Grevillea robusta as well as its
hexane, carbon tetrachloride, chloroform and aqueous soluble partitionates for cytotoxic, thrombolytic, membrane
stabilizing and antimicrobial activities. In the brine shrimp lethality bioassay, the crude methanolic extract of G.
robusta leaf revealed the highest cytotoxic activity with LC50 values as 1.50 ± 0.45 μg/ml as compared to 0.45 μg/ml
for vincristine sulphate. Among the extractives of G. robusta, the carbon tetrachloride soluble fraction showed
69.95±0.11% clot lysis as compared to 70.77% clot lysis by standard streptokinase. At concentration of 1.0 mg/ml,
the chloroform soluble fraction inhibited 40.31 ± 0.59% and 62.93 ± 0.73% of haemolysis of RBC induced by heat
and hypotonic solution as compared to 42.12% and 71.90% by acetyl salicylic acid, respectively. The test samples
also showed antimicrobial activity with zone of inhibition ranging from 7.0 to 17.0 mm in diameter. The chloroform
soluble partitionate demonstrated the highest zone of inhibition (17.0 mm) against Salmonella Typhi.
Key words: Grevillea robusta, brine shrimp lethality, thrombolytic activity, membrane stabilizing activity, hypotonic
solution, zone of inhibition.
Introduction
Grevillea robusta A. Cunn. ex R.Br. (Synonyms:
Grevillea venusta A. Cunn. ex Meisn., Stylurus
robustus A. Cunn. O. Deg.; Bengali name: rupasi),
commonly known as the southern silky oak or Australian
silver oak, is a fast growing evergreen tree belonging to
the Proteaceae family. It is a native species of eastern
coastal Australia and in other riverine, subtropical and
rainforest environments that receive more than 1,000 mm
per year of average rainfall. The flowers and fruits of the
plant contain toxic hydrogen cyanide (Everist, 1974).
Tridecylresorcinol of G. robusta is responsible for contact
dermatitis (Menz et al., 2006). Previous phytochemical
investigation provided six new 5-alkyl resorcinols
(gravicycle, dehydrogravicycle, bisgravillol, dehydro-
bisgravillol, dehydrograviphane and methyldehydro-
graviphane) and eight known compounds (Chuang and
Wu, 2007). Leaves contain rutin (Philippine Medicinal
plants).
As part of our ongoing investigations on medicinal
plants of Bangladesh (Kaisar et al., 2011 and Sharmin
et al., 2012 and 2013), the crude methanol extract of leaf
of G. robusta growing in Bangladesh as well as its organic
and aqueous soluble fractions were studied for cytotoxic,
thrombolytic, membrane stabilizing and antimicrobial
activities for the first time and we, here in, report the
results of our preliminary investigations.
Materials and Methods
Collection of plant materials and extraction: The leaf
of G. robusta was collected from National Botanical
Garden of Bangladesh in June, 2012. A voucher specimen
(DACB-38206) for this plant has been deposited in
Bangladesh National Herbarium, Dhaka, Bangladesh for
future reference.
The sun dried and powdered leaves (500 g) were
macerated in 1.5 L of methanol for 7 days. The extract
was filtered through a fresh cotton bed and finally with
Whatman filter paper number 1. It was concentrated with
a rotary evaporator at reduced temperature and pressure.
An aliquot (5 g) of the concentrated methanol extract was
fractionated by the modified Kupchan (VanWagenen et
al., 1993) partitioning protocol and the resultant
partitionates were evaporated to dryness with a rotary
evaporator to yield hexane (HXSF, 1.5 g), carbon
tetrachloride (CTCSF, 1.5 g), chloroform (CSF, 1 g) and
*Correspondence to: Mohammad A. Rashid; Tel.: +880-2-9661900, Extn. - 8137; Fax: +880-2-9667222; E-mail: rashidma@du.ac.bd
136 Ullah et al. / Bangladesh Pharmaceutical Journal 17(2): 135-137, 2014
aqueous (AQSF, 0.5 g) soluble materials. The residues
were then stored in a refrigerator until further use.
Brine shrimp lethality bioassay: This technique was
applied for the determination of general toxic properties of
the dimethylsulfoxide (DMSO) solutions of plant
extractives against Artemia salina in a single day in vivo
assay (Meyer et al., 1982). Vincristine sulphate was used
as positive control.
Thrombolytic activity: The thombolytic activity was
evaluated by the method developed by Prasad et al. (2006)
by using streptokinase (SK) as positive control.
Membrane stabilizing activity: The membrane
stabilizing activity of the extractives was assessed by
evaluating their ability to inhibit heat- and hypotonic
solution- induced hemolysis of human erythrocytes
following the method developed by Omale et al. (2008).
Antimicrobial screening: Antimicrobial activity was
determined by disc diffusion method (Bauer et al., 1966).
Statistical analysis: For all bioassays, sample data
were compared with that of negative control by student’s
t-test
Results and Discussion
The present study was undertaken to evaluate the
cytotoxic, thrombolytic, membrane stabilizing and
antimicrobial activities of different organic and aqueous
soluble materials of the crude methanol extract of G.
robusta.
In brine shrimp lethality bioassay, all fractions
demonstrated cytotoxic potential against A. salina with
LC50 values ranging from 1.50 to 191.14 µg/ml. The crude
methanol extract and the carbon tetrachloride soluble
fraction revealed the presence of significant bioactive
principles with LC50 values 1.50 ± 0.45 μg/ml and 3.85 ±
0.22 μg/ml, respectively as compared to 0.45 μg/ml for
Vincristine sulphate (Table 1).
The extractives of G. robusta demonstrated mild to
moderate thrombolytic activity. The carbon tetrachloride
and aqueous soluble fractions displayed 69.95±0.11 % and
67.50±0.49% clot lysis, respectively as compared to
70.77% clot lysis revealed by standard streptokinase
(Table 1).
At concentration 1.0 mg/ml, the extractives of G.
robusta protected the hemolysis of RBCs induced by heat
and hypotonic solution as compared to the standard acetyl
salicylic acid (0.10 mg/ml). The chloroform soluble
fraction inhibited 40.31 ± 0.59% and 62.93 ± 0.73% of
hemolysis of RBCs induced by heat and hypotonic
solution as compared to 42.12 % and 71.90% by acetyl
salicylic acid, respectively (Table 1).
The antimicrobial activity of G. robusta test samples
was evaluated against five gram positive and eight gram
negative bacteria and three fungi and the results were
compared with a broad spectrum antimicrobial agent,
ciprofloxacin. Among the test samples of G. robusta, only
the carbon tetrachloride and chloroform soluble fractions
demonstrated antimicrobial activity with zone of
inhibition ranging from 7.0 to 17.0 mm. The highest zone
of inhibition (17.0 mm) was displayed by the chloroform
soluble fraction against Salmonella Typhi (Table 2).
Table 1. Cytotoxic, thrombolytic and membrane stabilizing activities of G. robusta.
Samples/ standard Brine shrimp lethality
bioassay LC50 (µg/ml)
% Clot lysis of RBCs % Inhibition of hemolysis
Heat- induced Hypotonic solution-induced
ME 1.50 ± 0.45 64.94 ± 0.56 39.39 ± 0.60 37.56 ± 0.42
HXSF 7.84 ± 0.74 29.76 ± 0.34 37.59 ± 0.17 26.91 ± 0.82
CTCSF 3.85 ± 0.22 69.95 ± 0.11 35.55 ± 0.14 11.89 ± 0.44
CSF 10.87 ± 0.70 47.67 ± 0.63 40.31 ± 0.59 62.93 ± 0.73
AQSF 191.14 ± 0.19 67.50 ± 0.49 10.82 ± 0.05 17.89 ± 0.52
VS 0.45 ± 0.04 - - -
Water - 3.791 ± 0.55 - -
SK - 70.77 ± 0.36 - -
ASA - - 42.12 ± 0.38 71.90 ± 0.78
Hypotonic medium - - - -
ME = Methanolic crude extract; HXSF = Hexane soluble fraction; CTCSF = Carbon tetrachloride soluble fraction; CSF = Chloroform
soluble fraction; AQSF = Aqueous soluble fraction; VS = Vincristine sulfate; SK = Streptokinase; ASA = Acetyl salicylic acid.
Ullah et al. / Bangladesh Pharmaceutical Journal 17(2): 135-137, 2014 137
Table 2. Antimicrobial activity of carbon tetrachloride and chloroform soluble materials of G. robusta.
Test microorganisms Diameter of zone of inhibition (mm)
CTCSF CSF Ciprofloxacin
Bacillus cereus 9.0 ± 0.93 10.0 ± 0.22 45.0 ± 2.01
B. megaterium 8.0 ± 1.12 9.0 ± 0.61 42.0 ± 1.17
B. subtilis 8.0 ± 0.83 15.0 ± 0.26 42.0 ± 0.73
Staphylococcus aureus 9.0 ± 1.05 12.0 ± 0.64 42.0 ± 0.23
Sarcina lutea 9.0 ± 0.31 13.0 ± 0.57 42.0 ± 0.56
Escherichia coli 7.0 ± 0.66 13.0 ± 0.65 42.0 ± 0.43
Pseudomonas aeruginosa 10.0 ±0.39 9.0 ± 0.33 42.0 ± 1.11
Salmonella Typhi 11.0 ± 0.58 17.0 ± 0.42 45.0 ± 0.73
S. Paratyphi 8.0 ± 0.71 9.0 ± 0.58 47.0 ± 2.33
Shigella boydii 7.0 ± 0.56 11.0 ± 0.44 34.0 ± 0.58
Sh. dysenteriae 7.0 ± 0.47 11.0 ± 0.68 42.0 ± 0.22
Vibrio mimicus 9.0 ± 0.22 10.0 ± 0.31 40.0 ± 0.45
V. parahaemolyticus 8.0 ± 0.32
8.0 ± 0.17
10.0 ± 0.06 35.0 ± 0.44
Sacharomyces cerevisiae 10.0 ± 0.83 38.0 ± 0.49
Candida albicans 7.0 ± 0.47 8.0 ± 0.90 37.0 ± 0.33
Aspergillus niger 9.0 ± 0.66 12.0 ± 0.24 38.0 ± 0.11
CTCSF = Carbon tetrachloride soluble fraction; CSF = Chloroform soluble fraction
Conclusion
It is clearly evident from the above findings that G.
robusta possesses significant cytotoxic, thrombolytic and
membrane stabilizing activities. The plant is a good
candidate for further systematic, chemical and biological
studies to isolate the active principles.
Acknowledgement
The authors wish to thank Bangladesh National
Herbarium for the identification of the plant species
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Grevillea‘Robyn Gordon’is an Australian hybrid plant which may cause allergic contact dermatitis. Over the past 8 years, in bag been planted extensively in Australia and is now being exported to the United Slates mid other Western countries. Recently, fin explosion in incidence of contact dermatitis due to this plant has been seen. 24 patients who were suspected of being sensitive to it were patch tested to fresh plant and extracts with confirmation of sensitivity.A cross sensitivity to Toxicodendron was demonstrated clinically and on patch testing. The allergen in Toxicodendron, 3 pentadecylcatechol, is closely linked in chemical structure to 5-penladecylresorcinol. present in Grevillea banksii and tridecylresorcinol in Grevillea robusta. Patch letting in patients sensitive to Grevillea‘Robyn Gordon’with tridecylresorcinol produced positive reactions, thus implicating the sensitizing role of long chain alkyl resorcinols in Grevillea‘Robyn Gordon’allergy. There is a likelihood that (his sensitizing potential extends to other plants of the Grevillea genus, e.g. Grevillea robusta.With the spread of Grevillea‘Robyn Gordon’to other markets, this problem will become increasingly common.
Article
Grevillea 'Robyn Gordon' is an Australian hybrid plant which may cause allergic contact dermatitis. Over the past 8 years, it has been planted extensively in Australia and is now being exported to the United States and other Western countries. Recently, an explosion in incidence of contact dermatitis due to this plant has been seen. 24 patients who were suspected of being sensitive to it were patch tested to fresh plant and extracts with confirmation of sensitivity. A cross sensitivity to Toxicodendron was demonstrated clinically and on patch testing. The allergen in Toxicodendron, 3 pentadecylcatechol, is closely linked in chemical structure to 5-pentadecylresorcinol, present in Grevillea banksii and tridecylresorcinol in Grevillea robusta. Patch testing in patients sensitive to Grevillea 'Robyn Gordon' with tridecylresorcinol produced positive reactions, thus implicating the sensitizing rôle of long chain alkyl resorcinols in Grevillea 'Robyn Gordon' allergy. There is a likelihood that this sensitizing potential extends to other plants of the Grevillea genus, e.g., Grevillea robusta. With the spread of Grevillea 'Robyn Gordon' to other markets, this problem will become increasingly common.