Journal of Applied Pharmaceutical Science Vol. 2 (10), pp. 050-053, October, 2012
Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2012.21010
ISSN 2231-3354
Phytochemical screening, cytotoxicity and anti-inflammatory
activities of the Zimbabwean endemic plant Phyllanthus serpentinicola
Radcl.-Sm. (Phyllanthaceae)
C. Zimudzi, L.F.Gwenhure, N.Kunonga, S. Kativu and J. Jere
Department of biological Sciences, University of Zimbabwe, P.O. Box MP 167, Mt Pleasant, Harare, Zimbabwe.
ARTICLE INFO
ABSTRACT
Article history:
Received on: 10/10/2012
Accepted on: 21/10/2012
Stem methanolic extract of Phyllanthus serpentinicola was screened for phytochemicals, and tested
for cytotoxicity and anti-inflammatory activities. Tests indicated presence of alkaloids, tannins,
terpenoids, reducing sugars and flavonoids. Saponins, anthraquinones, and cardiac glycosides were
absent. Cytotoxic effects of the plant were assessed through Brine shrimp lethality (BSL) bioassay.
An LC50 value of 1.3 µg/ml was found, thus indicating that the stem extract was strongly cytotoxic.
Anti-inflammatory activity was assessed by the egg albumen induced hind paw oedema test on
Swiss albino mice. The extract significantly (p<0.05) reduced the oedema, thus indicating that P.
serpentinicola has anti-inflammatory properties.
Available online:
Key words:
Phyllanthus serpentinicola,
phytochemicals, cytotoxicity,
BSL bioassay, antiinflammation
INTRODUCTION
Genus Phyllanthus belongs to family Phyllanthaceae, a
recent segregate of Euphorbiaceae sensu lato. The genus is widely
represented in tropical and subtropical regions of the world.
Thirty nine species have been recorded in Zimbabwe, with one,
Phyllanthus serpentinicola, being endemic to the country
(Radcliff-Smith, 1996).
The species is restricted to the serpentine soils of the
Great Dyke area of the country. P. serpentinicola is a suffrutex,
up to 30 cm high, with spirally arranged, simple, obovate leaves
which bear inconspicuous flowers in leaf axils. Male flowers are
solitary and yellowish green, whilst female flowers are dull green,
often pinkish tinged, and form a disk with broad rounded lobes.
Phytochemical and pharmacological
* Corresponding Author
C. Zimudzi, Department of biological Sciences,University of Zimbabwe,
P.O. Box MP 167, Mt Pleasant, Harare, Zimbabwe.
Telephone: +263-4-772669975, Fax: +263-4-333407
evaluation of several Phyllanthus species has identified a number
of useful biologically active compounds. Phyllanthimide, an
alkaloid used as a model for the synthesis of cystostatic agents in
treatment of cancer is one of several useful compounds derived
from Phyllanthus (Calixto et al., 1998). Phyllanthus species
reportedly have antipyretic, analgesic, anti-inflammatory,
antihepatotoxic and antiviral activities (Unander et al., 1990).
Phytochemical and pharmacological profiles of Phyllanthus show
variability according to species and environmental conditions
(Calixto et al., 1998).
In southern Africa, Phyllanthus species are used to treat a
variety of ailments including arthritis, bronchitis, earache, sore eyes,
swollen gums, burns and wounds (Bosch, 2008). P. serpentinicola
has never been phytochemically evaluated nor tested for its
cytotoxicity or anti-inflammatory activities. The present study
evaluates the phytochemical composition of the species and at the
same time tests its anti-inflammation and cytotoxic activities.
051
Zimudzi et al. / Journal of Applied Pharmaceutical Science 2 (10); 2012: 050-053
MATERIAL AND METHODS
Collection of plant material
Fresh stems of P. serpentinicola were collected from
Mutorashanga Pass along the Great Dyke of Zimbabwe in January
2012. The plant was identified in situ. Voucher specimens are
deposited at the University of Zimbabwe teaching herbarium.
Freshly collected stems were washed in tap water and air-dried for
two weeks.
Crude extraction
Dried stems were chopped into pieces and milled into a
fine powder by pounding manually with a clean pestle and mortar.
Some 2g of ground material was extracted with 100 ml analytical
grade methanol as solvent at room temperature for 48 h using the
cold maceration method.
The extract was filtered using Whatman filter paper (No:
1), concentrated and evaporated to dryness in vacuo at 40°C using
a rotary evaporator. The powder extract was re-dissolved in
distilled water and stored in a refrigerator at 4°C.
Phytochemical screening
Standard qualitative methods as described by Sowofora
(1993) and Tiwari et. al (2011) were adopted for phytochemical
screening. The crude extract was tested for phytochemical
constituents using the following tests and reagents: reducing
sugars with Fehlings test, anthraquinones with Borntragor’s test,
terpenoids with Salkowski test, flavonoids with ammonia and
suphuric acid, saponins with foam test, tannins with Ferric
Chloride test, alkaloids with Mayer’s and Draggendorff’s tests and
cardiac glycosides with Keller- Killian’s test.
Brine shrimp lethality test
The cytotoxicity activity of the crude extract was
assessed on brine shrimp nauplii (Artemia salina) according to
brine shrimp lethality bioassay (Meyer et al., 1982). Artificial sea
water was prepared by dissolving 12 g sodium chloride in 1 L of
distilled water and adjusting the pH to 8.5 using 40 % sodium
hydroxide. Some 2 g of brine shrimp eggs were hatched in 1L of
sterile sea water in a flask. The cysts were kept under bright light,
and were continuously agitated and aerated using an aquarium
pump. The nauplii hatched within 48 h at room temperature.
The methanol crude extract was dissolved in 1% aqueous
dimethyl sulfoxide (DMSO) in artificial sea water to obtain
concentrations of extract varying from 100 mg/ml to 0.25 mg/ml.
Some 50 µl of each solution was transferred, to clean sterile vials
containing 5 ml of aerated seawater. Ten shrimp nauplii were
transferred to each vial. The experiments were performed in
triplicate
for
each
extract
concentration.
Potassium
dichromate (5mg/ml) and 1% DMSO in seawater
were used as positive and negative controls, respectively. After
24h, the vials were examined against a lighted
background using a hand held magnifying glass, and
the number of nauplii that survived in each vial were counted.
Evaluation of anti-inflammatory activity
Animals
Adult Swiss albino mice of both sexes were used for
these experiments. They were obtained from the Animal House at
Central Veterinary Laboratories in Harare, Zimbabwe. The
animals were housed in cages, and were allowed free access to
standard pellets and water. All procedures used in the study
comply with guidelines on animal care provided by the University
of Zimbabwe Ethics Committee on the use of animals.
Anti-Inflammatory Test
The test was carried out using a phlogistic agent –
induced mouse hind paw oedema as a model of acute
inflammation (Winter et al, 1962). Fresh egg albumen was used as
the phlogistic agent. Adult Swiss albino mice of either sex were
fasted for 14 h and then divided into 5 groups of six mice each.
Each group received a different treatment. The extract (100,200
and 300 mg/kg) was administered intra-peritoneally using a
syringe 1h before inducing inflammation. The negative control
group received 10 ml/kg distilled water and the positive control
100 mg/kg of Acetic salicylic acid (ASA). Inflammation was
induced by injection of 0.1ml of fresh egg albumen into the
subplantar surface of the right hind paw of the mice. Paw oedema
was measured by wrapping a piece of cotton thread round the paw
and measuring the circumference with a ruler (Okokon et al.,
2012). The measures were determined at 1, 2 and 3 h following
egg albumen injection.
Data analysis
Results were analyzed using Microsoft Excel statistical
package (2007). Cytoxicity data were analysed using linear
regression analysis. All values were expressed as mean ± SEM. A
one-way ANOVA was applied to determine the significance of the
difference between the control groups and mice treated with the
extracts. A Student’s t-test was used to compare the treatments and
controls, and results were considered significant when p < 0.05.
RESULTS AND DISCUSSION
Phytochemical Screening
Table 1 shows the results of the phytochemical
screening. The extract tested positive for alkaloids, tannins,
terpenoids, reducing sugars and flavonoids, and negative for
saponins, anthraquinones, and cardiac glycosides. The observed
phytochemical profile of P. serpentinicola is similar to that of
other Phyllanthus species (Calixto et al., 1998).
Screening for phytochemicals in plants is important as a
first step in elucidating the pharmacological properties of a plant
species. The phytochemicals identified in P. serpentinicola belong
to large diverse groups with varied pharmacological activities.
Alkaloids are known to exhibit anti-inflammatory, cytotoxic
antibacterial, analgesic and anti cancer activities (Roberts and
Wink, 1998). Flavonoids and tannins are phenolic compounds that
are known to have antimicrobial, anti-inflammatory, cytotoxic,
Zimudzi et al. / Journal of Applied Pharmaceutical Science 2 (10); 2012: 050-053
antiallergic, analgesic and antioxidant properties (Cowan, 1999).
Terpenoids are reported to have anti-inflammatory, anti cancer,
antimalarial and antibacterial properties (Govindappa et al., 2011).
Table 1. Results of phytochemical screening tests on methanol extract of P.
serpentinicola
Test
Observation
Alkaloids
+
Cardiac Glycosides
Tannins
+
Anthraquinones
_
Terpenoids
+
Saponins
Reducing sugars
+
Flavonoids
+
Key – = absent and + = present
Table 2. Effect of stem methanolic extract of P.serpentinicola on fresh egg
albumen–induced inflammation in mice (means ± SD, n =6, *p<0.05).
Treatment
Dose(m
Paw Oedema (cm)
g/kg)
1h
2h
3h
Distilled
10ml/kg
1.47±0.12
1.58±0.08
1.73±0.15
water
PSE
100
1.90±0.13*
1.81±0.1*
1.77±0.10
PSE
200
1.87±0.08*
1.76±0.09*
1.68±0.073
PSE
300
1.85±1.17*
1.72±0.14*
1.62±0.13
ASA
100
1.95±0.04*
1.86±0.07*
1.79±0.06
Key: PSE-Phyllanthus serpentinicola extract; ASA-Acetosalicylic acid
Brine shrimp lethality test
The brine shrimp mortality was 100% in the Potassium
dichromate standard and in all extract concentrations above 8 µg
/ml. These data have been excluded from Figure 1 which shows
the mean percent mortality of the brine shrimp nauplii against the
logarithm of the sample concentrations. The mortality of brine
shrimps increased with increasing concentration of extract. The
regression equation on Figure 1 was used to calculate an LC50
value of 1.3 µg/ml.
052
for P. serpentinicola is comparable to that for Phyllanthus engleri
(0.47 µg/ml) where cytotoxicity has been attributed to presence of
triterpenoid phyllanthol (Bosch, 2008). P. engleri is a poison plant
commonly encountered in suicide cases in Zimbabwe (Bosch,
2008). Thus, care should be exercised in using P. serpentinicola as
a medicinal plant.
Anti-inflammation activity
Egg albumen induced oedema is a widely accepted
model for the evaluation of anti-inflammatory effect of plant
extracts. Table 2 shows the activity of P. serpentinicola extract
against egg albumen induced paw oedema in mice. Control mice
produced a local hind paw oedema that increased progressively
throughout the three hour
period. Pretreatment by P.
serpentinicola extract significantly (p<0.05) reduced the oedema
in the first two hours, but no significant differences between the
treatments and control were observed after three hours (p>0.05).
The standard showed a similar pattern to the treatments. Egg
albumen inflammation is biphasic, and the high anti-inflammatory
activity of the extract may be a response to the initial acute phase
of inflammation. The first phase occurs within two hours of
administering the phlogistic agent, and is due to the release of
histamine or serotonin, and the second phase of oedema is due to
the release of prostaglandin (Oyekunle and Idowu, 2012). Results
of the present study indicate that the methanol extracts of P.
serpentinicola significantly reduced paw oedema in mice. The
anti-inflammatory mechanism of action may be due to blocking of
the release of these chemical mediators.
The anti-inflammatory activity P. serpentinicola is
attributed to the phytochemical constituents of the plant extract
which include terpenoids, tannins, flavonoids and alkaloids. Plants
containing these chemical classes of compounds have been
reported to possess potent anti-inflammatory properties that act
through inhibiting prostaglandin pathways (Oyekunle and Idowu,
2012).
CONCLUSIONS
Fig. 1: Mortality of brine shrimp nauplii in different P. serpentinicola stem
extract concentrations.
Standard brine shrimp lethality bioassay stipulates that
LC50 values less than 1000 µg /ml are considered bioactive in
toxicity evaluation of plant extracts (Meyer et al., 1982). Based on
this benchmark, the P. serpentinicola extract is strongly cytotoxic
since the LC50 value was less than 1000 µg/ml. Cytotoxicity of the
extract is attributed to presence of alkaloids, tannins, flavonoids
and terpenoids ( Sandhar et al., 2011). The LC50 value of 1.3 µg/ml
The present study has demonstrated that stem methanolic
extract of P. serpentinicola exhibits anti-inflammatory and
cytotoxicity activities. Caution should; however, be exercised in
the use of this species for medicinal purposes because of its high
toxicity as evidenced by the LC50 values of 1.3 µg/ml. The results
also suggest that the plant contains bioactive constituents that are
responsible for the observed activities. The results from this study
suggest that P. serpentinicola like other Phyllanthus species can be
useful for the management of pain.
ACKNOWLEDGEMENTS
The authors acknowledge the Department of Biological
Sciences, University of Zimbabwe, for providing its laboratory
facilities for this study. The Chief technician, Gerald Ashley, and
his technical staff provided invaluable technical assistance.
053
Zimudzi et al. / Journal of Applied Pharmaceutical Science 2 (10); 2012: 050-053
REFERENCES
Bosch C.H. (2008). Phyllanthus engleri. In G.H. Schmelzer &
A. Gurib-Fakim (Ed.). Plant Resources of Tropical Africa 11(1).
Medininal Plants 1. Leiden: Backhuys Publishers.
Calixto J.B., Santos A.R., Cechinel V. F., Yunes R.A. A review
of the plants of the genus Phyllanthus: their chemistry, pharmacology, and
therapeutic potential. Med. Res. Rev. 1998; 18(4): 225–258.
Cowan M.M. Plant products as antimicrobial agents. Clin.
Microbiol. Rev.1999; 12: 564-582.
Govindappa M., Naga S.S., Poojashri M. N., Sadananda T. S.
Chandrappa C. P. Antimicrobial, antioxidant and in vitro antiinflammatory activity of ethanol extract and active phytochemical
screening of Wedelia trilobata (L.) Hitchc. Journal of Pharmacognosy and
Phytotherapy. 2011; 3(3): 43-51.
Meyer B.N., Ferrigni N.R., Putnam J.E., Jacobsen L.B., Nichols
D.E., Mc Lauglin J.L. Brine shrimp: a convenient general bioassay for
active plant constituents. Planta Med. 1982; 45: 31-34.
Okokon J. E. Antia B. S. Ita B.N. Anti-Inflammatory and Antinociceptive Effects of Ethanolic Extract of Setaria megaphylla Leaves in
Rodents. Afric. J. Biomed. Res. 2006; 9: 229–233.
Oyekunle A.O., Idowu A.O. Anti-inflammatory activity of
methanolic extract of Cnidoscolous aconitifolius leaves in laboratory
rodents. Journal of Chemical, Biological and Physical Sciences.2012; 2(2):
770-774.
Radcliff-Smith A. Euphorbiaceae. Flora Zambesiaca.1996; 9(4):
59-60.
Roberts M.F., Wink M. Alkaloids: Biochemistry, ecology and
medicinal applications. ; Plenum, New York, 1998.
Sandhar H.K., Kumar B., Prasher S., Tiwari P., Salhan M.,
Sharma P. A Review of Phytochemistry and Pharmacology of Flavonoids.
Internationale Pharmaceutica Sciencia. 2011; 1(1):25-41.
Sofowora E.A. Phytochemical Assays in “Medicinal Plants and
Traditional Medicine in Africa”. Third edition. Spectrum Books Limited,
Abuja. (1993) 150-153.
Tiwari P., Kumar B., Kaur M., Kaur G., Kaur H.
Phytochemical screening and Extraction: A Review. Internationale
Pharmaceutica Sciencia. 2011; 1 (1): 98-106.
Unander D.W., Webster D.W., Blumberg B.S. Record of usage
or assays in Phyllanthus (Euphorbiaceae) I. Subgenera Isocladus,
Kirganelia, Cicca and Emblica. J. Ethnopharmacol. 1993; 30: 233-264.
Winter C.A., Risley E.A. Nuss G.W. Carragenin induced
oedema in hind paw of the rats as an assay of anti-inflammatory drugs.
Proceedings of the Society for Experimental Biology and Medicine. 1962;
111: 544-547.
How to cite this article:
C. Zimudzi, L.F.Gwenhure, N.Kunonga, S. Kativu and J. Jere.
Phytochemical screening, cytotoxicity and anti-inflammatory
activities of the Zimbabwean endemic plant Phyllanthus
serpentinicola Radcl.-Sm. (Phyllanthaceae). J App Pharm Sci.
2012; 2 (10): 050-053.