|
Ethnobotanical
Leaflets 13: 338-342. 2009. Phytochemistry and Ethanopharmacological
Studies on Rubia cordifolia Linn. (Rubiaceae) M.Kannan1, A.J.A.Ranjit
Singh2 and M.Narayanan3 1Department of
Microbiology, V.H.N.S.N.College, Virudhunagar 2Department of Advanced
Zoology and Bio-technology, 3Aquatic Issued
Abstract Ethanolic
extracts of the leaves of Rubia cordifolia L. (Rubiaceae)
(ERC) were investigated for their phytocompounds,
in vitro and in vivo immunopharmacological
properties. Immunomodulatory effects of this plant
extract were proved in Cyclophosphamide (CP)
-induced immunosuppressed animal models. Albino
rats were treated with the aqueous extract of ERC and Cyclophosphamide.
The Total count (TC) and Differential count (DC) of microphages and blood
clotting assay were affected due to the suppressive effect of Cyclophosphamide. Administration of plant extract to CP-
exposed animals resulted in enhanced immune responses. Keywords: mmucopharmacology, Rubia cordifolia L., cyclophosphamide
and blood clotting assay. The family Rubiaceae
comprises about 450 genera and 6500 species and includes trees, shrubs and
infrequently herbs. Rubia cordifolia
L. is a perennial, herbaceous climbing plant, with very long roots,
cylindrical, flexuous, with a thin red bark. Stems often have a long, rough,
grooved, woody base. Plants belonging to this family are known to contain
substantial amounts of anthraquinones, especially
in the roots. The traditional therapeutic use of the plant has been for skin
disorders and for anticancer activity. Furthermore, the anthraquinones
of the Rubiaceae family exhibit some interesting in
vivo biological activities, such as antimicrobial, antifungal, hypotensive, analgesic, antimalarial,
antioxidant, antileukemic
and mutagenic functions. Apart from its medicinal value, this plant has also
been used as natural food colourants and as natural
hair dyes. The interest in the isolation of natural dyes and colouring matters is increasing due to their applications
in food, drugs and other human consumptions (Chopra et. al., 1992). Rubia cordifolia L. was extensively used in
traditional medicinal systems of Methodology Collection
of plant materials Healthy plant leaves were collected from South-Western Ghats of Tirunelveli range,
Tamil Nadu, Preparation
of plant extract Plant extracts were prepared by the method of Tiwari et al. (2004), with slight modification of the
method used to purify the ethanolic extract.
Briefly, the extract was filtered through Whatman
filter paper no. 1 (WHATMAN, ENGLAND) to remove all unextractable
matter, including cellular materials and other constituents that are
insoluble in the extraction solvent. To obtain a concentrated crude ethanolic extract, the crude extracts were evaporated at
45oC. The quantity was determined by weighing. Phytochemical
screening Standard phytochemical
screening tests were carried out for various constituents of the ERC
according to the methods of Trease and Evans
(1983). Observations were carried out for colour change,
precipitation or formation of an emulsion. The presence or absence of saponins tannins, alkaloids, glycosides, flavonoids, essential oils were observed. Experimental animals Swiss albino rats weighing 100 – 125 g of either sex
were used to study the immunopharmacological
activity. Rats were kept in 12 h light/12 h dark cycles under standard
conditions of temperature (28oC) and relative humidity (RH: 60%) with free
access to food and water. All protocols performed in this study were
conducted in accordance with internationally accepted principles for use and
care of laboratory animals. Experimental
design In each experiment, the animals were randomly divided
into four groups and each group consisted of six animals. The animals in
Group I served as a test control. Animals in Groups II & Hematological
changes Group II and Assay
of blood clotting time A modified method of Lee and White, as
reported by Wintrobe (1967) was used for the assay
of blood clotting time. Glass test tubes containing 0.1–1 ml each of ERC and
makeup with PBS, it was equilibrated in a water bath at 370C. Blood was
collected from healthy adult volunteers by venipuncture
into sterile plastic disposable syringes. The blood donors had been screened
for the study by a medical practitioner to ensure that they had not taken any
medications for at least 1 week before the blood was collected. Starting a
stopwatch, 1ml of this blood was immediately transferred into each of the
equilibrated test tubes by carefully allowing the blood to run down the side
of the tube. At intervals of 30 seconds, the tubes, still in the water bath,
were gently tilted to an angle of 450C to check for blood clot formation.
This was continued until the tubes could be inverted without blood flowing;
the stopwatch was immediately stopped and the blood clotting time was
recorded. Statistical
analysis The results are presented as mean ±
standard deviation (S.D.). Statistical significance between the groups was
analyzed by the Student’s t-test and P<0.01 was considered to be
statistically significant. Results Phytochemical screening Phytochemical
studies showed positive for alkaloids, cardiac glycosides, tannins, flavonoids and phenols, and negative for saponins, volatile oils, anthraquinones
and cyanogenic glycosides, and very trace for
steroids. Hematological
changes Total white blood cells and red blood
cells in the control and plant extracts treated rats for 21 days are given in
table 1. In the control rat the mean total WBC count was 13.08 ± 4.62 x 10 3/
µl. In R. cordifolia
L. treated rats showed 13.98 ± 2.68 x 103/µl respectively. When the rats were
given immune suppressive drugs cyclophosphamide
treated rats total WBC count was much reduced (4.8 ± 1.3 x 10 3/ µl). If
plant extract were given along with immune suppressant drugs the decrement in
total WBC was less when compared to the treatment in which immunosuppressant cyclophosphamide alone was given. The results indicate
that the active compounds in the extract of the plants had enhanced haematopoiesis. Assay
of blood clotting time The blood coagulation time of 4 adult
volunteers varied between 4 and 8 min, with a mean of 6.83±1.95 min. In the
presence of 1ml of ERC, the mean of clotting time was significantly (P <
0.01) reduced to 2.83±1.01 min with a range of 2–4 min. The use of 1ml PBS
instead of ERC, as a control, gave a mean clotting time of 7.29 ±1.98 min
with a range of 5–9 min. In the tubes containing anticoagulant, the blood
sample did not clot, as expected; this was a control used to ensure the
integrity of the blood samples. Discussion The preliminary chemical constituents
of selected plant Rubia cordifolia
L. was analysed and the results showed the bioactive
properties of medicinal plants are perhaps due to the presence of various
secondary metabolites such as alkaloids, cardiac glycosides, tannins, flavonoids and phenols.
Alkaloids are the lead molecules of therapeutic importance from the
selected plants. These are heterocyclic Indole
compounds which have proved to be have pharmacological properties such as
Antifungal activity (Ali et al., 2001), antiprotozoal,
antimicrobial and antimalarial activities (Frederich et al., 2002).Literature review on the phytochemical constituents of these plants revealed that
alkaloids are the major components of Rubia cordifolia L. The
presence of alkaloids in Rubia cordifolia L. (Toshihiro et al., 1992) had been
reported. In the present study, the immuno-pharmacological activity of Rubia
cordifolia L., an important plant in indigenous
medicinal practice, was explored. Administration of Rubia cordifolia L. increased total
counts of WBC and Blood clotting process is very complex,
involving many factors found in the plasma and tissues. It involves both the
intrinsic and extrinsic pathways (Brown, 1988; Jandl,
1996). Inhibitors (anticoagulants) and activators (procoagulant)
of blood coagulation may affect any of the factors. The blood clotting test
is used for distinguishing between the effects of ERC on the extrinsic and
intrinsic pathways. Bio-active substances from the selected plant Rubia cordifolia
L., which affect the clotting factors, are thought to act on the extrinsic
pathway factors: factors V, Some of the chemical constituents
isolated from Rubia cordifolia
L. include alkaloids, cardiac glycosides, tannins, flavonoids
and phenols (Gupta et al., 1995 and Ranjit et al.,
2008). Many of these constituents isolated from Rubia cordifolia L. plant sources possess immunomodulatory activity (Ranjit
singh et al., 2004). Most of the studies concerning
the evaluation of ethanopharmacological activities
of the plants have been undertaken utilizing the crude extracts. Table 1. Effect
of ethanolic extract of R.cordifolia
on peripheral blood count.
P<0.05 when compared with control. References Ali.Y., Munir.
O ., Vahit B., 2001,
Antioxidant activity of leaves Cydonia vulgaris. J Agric Food Chem. 29; 52(26):7884-90. Brown, B.A., 1988. Haematology: Principles and Procedures, 5th ed. Lea and Febiger, Chopra RN. Chopra IC, Handa KL, Kapur ID. 1992. Indigenous Drugs of Frederich M, Jacquier MJ, Thepenier P, De
Mol P, Tits M, Philippe G, Delaude C, Angenot L and Zaches Hanrot M,2002. Antiplasmodial
Activity of Alkaloids from Various Strychnos Species
J. Nat. Prod., 65, 1381. Gupta, A.K., Chaugule, M.A., Pakdalkar,
R.K., 1995. NMR structural characterization of gallactomannan
from Cassia occidentalis.
Indian Journal of Chemistry 34, 169–170. Jain, S.K., DeFillips, R.A., 1991. Medicinal Plants of Jandl, J.H., 1996. Blood:
Textbook of Haematology, 2nd ed. Little, Brown
& Ranjith M.S., Ranjith singh A.J.A. and Gokulshanker, S., 2008. Enhanced phagocytosis
and antibody production by Tinospora cardifolia – A new dimension in immunomodulation.
African Jr. of. Biotechnology, 7 (2), 081 – 085. Ranjith singh
A.J.A., Dhasarathan, P., Sujatha
N. and Jeypal. C., 2004, Antibacterial activities
of Cassytha capillaries, Asian Jr. of microbial. Biotech. Env. Sc, 6 (4), 609 – 612. Tiwari U, Rastogi
B, Thakur S, Jain S, Jain NK, Saraf
DK (2004). Studies on the Immunomodulatory effects
of Cleome viscose. Toshihiro,
A., Gebreyesus, T., Hiroshi, H., Toshitake, T., 1992. Cooccurrence
of C-24 epimeric 24-ethylsterols possessing and
lacking a _25 bond in Kalanchoe petitiana.
Phytochemistry 31, 163–166. Trease, G.E., Evans, M.S., 1983. Textbook of Pharmacognosy, 12. Bailliere Tindall, Varier, P.S., 1995. Indian Medicinal Plants,
A Compendium of 500 Species, vol. 4. Orient Longman, Wintrobe, M.W., 1967. Clinical Haematology, 6th ed. Lea and Febiger,
|