Phytochemical and antimicrobial screening

of Galium tricorne
Sikandar and Abdul Khaliq Jan

Sheringal High Altitude Research Centre (SHARC)

University of Malakand, NWFP, Pakistan

Corresponding author Email: uomians@yahoo.com

Phone +92(944) 885479, Cell 0300-5856017, Fax 0944-
885805
 Introduction
• The word Galium has been derived from a Greek term
”Gala” meaning milk, because the species of Galium
coagulate the milk.For this reason, it is known as “Yogurt
herb” in Turkish. while locally it is known as jeshay at
district Dir of Pakistan.

• Galium species are distributed in Pakistan from plains to

mountains and are used in folklore remedies.

• In recent times, a number of traditional healers claiming

the efficacy of various Galium species for a variety of
purposes, mainly as diuretic, in treatment of gout,
epilepsy and some stomach diseases.
• Cleavers are believed to have mild diuretic effects, which
may reduce stone formation by increasing urinary flow
and volume. Iridoid in Galium have anti-inflammatory
and cardiovascular activities.

• Due to little differences in characters, it is difficult to

distinguish species of the genus Galium L.

• The medicinal plant, Galium tricorne have been chosen

for the present study, which will be helpful in
chemotexonomic identification and will also help in full
plant utilization for a variety of medicinal purposes.
 Materials and Methods
Plant materials

• Whole plant of Galium tricorne was collected in village

Bunr of District Dir (L), NWFP.

• The Plant materials were identified by Ali Hazrat, plant

taxonomist at Sheringal Research Centre.

• A voucher specimen (SHPC-AKJ-40) has been

deposited at the Herbarium of Sheringal High Altitude
Research Centre, University of Malakand.
 Extraction
• The shade dried plant material (300g) was extracted
three times with 80% ethanol (3 liters) at room
temperature for fourteen days.

• The extract was concentrated to dryness under reduced

pressure to yield a gummy residue. The extract obtained
was concentrated in vacuo at 40 °C to yield crude
ethanol extracts.

• It was suspended in water, and then fractionated

successively with equal volumes of chloroform, ethyl
acetate and n-BuOH, leaving residual water-soluble
fraction.
 The fractions were then evaporated in vacuo to yield the
residues of relative solvents. After this all the fractions
were transferred to glass vials.
 Microorganisms
• Escherichia coli, Bacillus subtilis, Shigella flexenari,
Staphylococcus aureus, Pseudomonas aeruginosa,
Salmonella typhi.

• Candida albicans, Aspergillus flavus, Microsporum canis,

Fusarium solani and Candida glabrata.

• The bacteria were cultured on nutrient agar, while

Sabouraud's dextrose agar (SDA) was used for the
growth of fungus.
 Screening for antimicrobial activity
• Antimicrobial activity was tested by the agar-well diffusion
method (Mukherjee et al., 1995).

• Different concentrations of the extracts (0.075–15 mg/ml)

were prepared by reconstituting the extracts in suitable
solvents.

• The test microorganisms were seeded into respective

medium by gently mixing 0.5 ml (containing 106 cells/ml)
of the 24 h old cultures (for bacteria) and 48 h old
cultures (for fungi) with 20 ml sterile melted agar in sterile
Petri plates.
• After hardening, four 6 mm diameter wells were made
using a sterile borer.

• The wells were filled with 100 μl of the sample extracts

and standard drug. Imepenem, Miconazole and
Amphotericin B were used as standard antibiotics for
comparison with extracts and fractions.

• The plates were then incubated at 37 °C till the

appearance of visible results.

• The diameter of the zones of inhibition around each well

(diameter of well included) was taken as measure of the
antimicrobial activity.
• An inhibition zone of 16 mm or greater (including
diameter of the well) was considered as significant
antibacterial activity, while inhibition zone of 70 mm or
above was considered as significant antifungal activity.
 Phytochemical tests
• After concentrating the ethanolic extract to dryness, the
crude extract and the methanolic solution of the crude
extract were subjected to qualitative chemical screening
for the identification of various classes of compounds,
using classical methods described by Harbone et al.
 Results and Discussion
Table 1: Antibacterial activity (Zone of inhibition in mm) of the
extracts and fractions of Galium tricorne
E.Coli B.subtilis S.flexenari S.aureus P.aerugno S.typhi
Std 25 26 24 17 17 21

Cra 8 11 4 9 14 7

Ha 7 5 -- 7 9 --

Cla 14 17 7 15 17 12

Ea 9 13 6 11 6 --

Ba 6 8 2 6 -- --

Wa -- 5 -- -- -- --

Crf 6 8 -- 6 -- 7

Hf -- -- -- -- -- --

Clf 11 13 -- 2 -- --

Wf 4 9 -- 8 -- 8

-- no activity, 7-9 mm non significant, 17-18 mm (or above) significant,.A aerial part,F
fruit,Cr.crude fr.,H.hexane fr.,Cl.chloroform fr.,E.ethyl acetate fr.,B.butanol fr.,W.water fr
 Table 2: Antifungal activity (Zone of inhibition in mm) of the extracts
and fractions of Galium tricorne

C. albicans A. flavus M.canis F.solani C.glabrata T.longifusus

Std. 100M 90A 100M 100M 100M 100M

Cra 30 60 20 50 50 30

Ha -- -- 30 -- -- 20

Cla -- 20 10 30 -- 40

Ea 40 70 -- 70 10 10

Ba 10 10 -- 10 50 --

Wa -- 20 -- -- 40 --

Crf -- 20 10 -- 10 --

Hf -- -- -- -- -- --

Clf -- -- -- -- -- --

Wf -- 30 20 -- 20 --

M=Miconazole, A=Amphotericin B,-- no activity A.aerial part,F fruit,Cr.crude fr.,H.hexane

fr.,Cl.chloroform fr.,E.ethyl acetate fr.,B.butanol fr.,W.water fr.
Table 3: Phytochemical test for classes of compounds

Classes of Compounds Results

Saponins +
Alkaloids +
Flavonoids +
Tannins +
Steroids +
Coumarins +
Anthraquinones --
Glycosides +

+ Present, --Absent
 Summary
 Due to significant uses of medicinal plants of the genus
Galium (Rubiaceae) for a variety of purposes,
phytochemical tests of Galium tricorne were carried out
for various classes of compounds such as alkaloid,
steroid, Saponins, Flavonoids, tannin, Anthraquinones
and phenols etc.

• As the emergence of resistance to conventional

antimicrobial agents poses a serious problem for
physicians, therefore antimicrobial activities were also
elucidated. This will help ongoing development of new
antimicrobial agents that can inhibit the growth or kill
resistant microorganisms.
Thank You