1

Chapter One

Introduction and literature review

1.1 Medicinal plants

Infection diseases are one of the main reasons which cause the death, killing almost

50.000 people everyday [1]. Medicinal plants are used by 80% of the world

population as the only available medicines especially in developing countries [2],

the use of medicinal plants is very wide spread in many parts of the world because it

is commonly considered that herbal drugs are cheaper and safer as compared to

synthetic drugs and may be used without or minimum side effects.

Plants used for traditional medicine contain a wide range of substances that can be

used to treat chronic as well as infectious diseases. Clinical microbiologists have

great interest in screening of medicinal plants for new therapeutics [3]. The active

principles of many drugs found in plants are secondary metabolites. The

antimicrobial activities of plant extracts may reside in a variety of different

components, including aldehyde and phenolic compounds[4].

Resistance to antimicrobial agents is emerging in a wide variety of pathogens and

multiple drug, resistance is becoming common in divers organisms such as

Staphylococcus sp and Salmonella sp[5][6][7].

The appearance of resistant paved the way to the occurrence of infections that

are only treated by a limited number of antimicrobial agents the gradual rise in

resistance of bacterial and fungal pathogens for antibiotics and antifungals

highlights the need to find alternative sources from medicinal plants[8] .

2

In this study three medicinal plants were screened for their phytochemical contents

and were tested for their antibacterial activity against some types of standard

bacteria.

1.2 Trigonella Fenoum-Graeceum

Fenugreek, Trigonella foenum-graecum L. is an annual crop from the family

Leguminosae Fig(1), which have health potential with the ability to lower blood

glucose and cholesterol levels, and hence in the prevention and treatment of diabetes

and coronary heart diseases[9]. Due to its strong flavor and aroma, fenugreek in one

of such plants whose leaves and seeds are widely consumed in countries as a spice

in food preparations, and as an ingredient in traditional medicine. It is rich source of

calcium, iron, carotene and other vitamins. Fenugreek seeds have been found to

contain protein, vitamin C, niacin, potassium, and diosgenin (which is a compound

that has properties similar to estrogen). Other active constituents in fenugreek are

alkaloids, lysine and L-tryptophan, as well as steroidal saponins (diosgenin,

yamogenin, tigogenin, and neotigogenin).[10]

Fig.(1):Trigonella Fenoum-graeceum seeds

1.3 Artemisia herba- alba

3

Artemisia herba- alba (Asteraceae) [Fig. 2], commonly known as sheeh, is herb or

shrub which distributed in north Africa (Libya), and most of Europe [11], is a

chamaeophyte that grows to 20–40 cm (8–16 in). Leaves are strongly aromatic and

covered with fine glandular hairs that reflect sunlight giving a grayish aspect to the

shrub. The leaves of sterile shoots are grey. This plant has been used in traditional

medicine as antidiabetic (12) .

Fig. (2): Artemisia herba – alba leaves

1.4 Solenostemma argel

Solenostemma argel (Argel) [Fig. 3], known locally by the name (Hargel) and

scientifically known as Solenostemma argel is a plant in the "Apocynaceae" family.

It is indigenous to Africa. The parts used are the leaves and stems, the leaves

contain high carbohydrates and protein as well as crude oil, ash, calcium and

magnesium [13]. The leaves are used in herbal medicine for the treatment of some

diseases such as of liver and kidney and allergies.

It treats gastro-intestinal cramps, stomach-aches and urinary tract infections. Argel

tea is used for lessening the pains of childbirth and for treating eating disorders

since it increases appetite.

Fig. (3:) Solenostemma argel leaves

4

Chapter Two

Materials and Methods

2.1 Plants material

Specimens of three plants were collected in a form of seeds (Trigonella foenum-

graecum) and leaves (Artemisia herba-alba , Solenostemma argel) from different

retail markets of Khartoum in September, 2012. The spices were botanically

identified at the Department of Botany –University of Khartoum. The collected

plants were dried under shade at room temperature and ground with a grinder into a

powder.

2.2 Preparation of extracts

Extracts from the three tested plants were obtained by using cold extraction method

(14), 50g of each air-dried powder was taken in a 250 ml conical flask. A hundred

ml of (80%ethanol: 20% water) was added. The flask was plugged with cotton wool

and then kept on a rotary shaker at 190-220 rpm for 24 h. After 24 hours, the

supernatant was collected and the solvent was evaporated to make the final volume

one fourth of the original volume then stored at 4 °C in airtight pottles [Fig., 4].

Figure (4): ethanol extracts of the three tested plants

5

3 Method of identification

3.1 Alkaloids [Mayer’s test]

1.36 mg of mercuric chloride dissolved in 60 ml and 5 mg of potassium iodide were

dissolved in 10 ml of distilled water respectively. These two solvents were mixed

and diluted to 100 ml using distilled water. To one ml of acidic aqueous solution of

samples few drops of reagent was added. Formation of white or pale precipitate

showed the presence of alkaloids.

3.2 Flavonoids

In a test tube containing 0.5 ml of alcoholic extract of the samples, 5 to 10 drops of

diluted HCl and small amount of Zn or Mg were added and the solution was boiled

for few minutes. Appearance of reddish pink or dirty brown colour indicated the

presence of flavonoids.

3.3 Glycosides

A small amount of alcoholic extract of samples was dissolved in 1ml water and then

aqueous sodium hydroxide was added. Formation of a yellow colour indicated the

presence of glycosides.

3.4 Cardiac glycosides [Keller killiani’s test]

About 100 mg of extract was dissolved in 1ml of glacial acetic acid containing one

drop of ferric chloride solution and 1ml of concentrated sulphuric acid was added. A

brown ring obtained at the interface indicated the presence of a de oxy sugar

characteristic of cardenolides.

6

3.5 Steroids [Salkowski’s test]

About 100 mg of dried extract was dissolved in 2 ml of chloroform. Sulphuric acid

was carefully added to form a lower layer. A reddish brown colour at the interface

was an indicative of the presence of steroidal ring.

3.6 Saponins

A drop of sodium bicarbonate was added in a test tube containing about 50 ml of an

aqueous extract of sample. The mixture was shaken vigorously and kept for 3 min.

A honey comb like froth was formed and it showed the presence of saponins.

3.8 Phenols [Ferric Chloride Test]

To one ml of alcoholic solution of sample, 2 ml of distilled water followed by a few

drops of 10% aqueous ferric chloride solution were added. Formation of blue or

green color indicated the presence of phenols.

3.9 Tannin [Lead acetate test]

In a test tube containing about 5ml of an aqueous extract, a few drops of 1%

solution of lead acetate was added. Formation of a yellow or red precipitate

indicated the presence of tanine.

4 Antibacterial Assay

4.1Culture media

4.1.1 Nutrient broth

This medium contained ( peptic digest of animal tissue 5.00g, yeast extract 1.50g,

beef extract 1.50g and sodium chloride 5.00g [HiMedia Laboratories Pvt.Ltd-

Mumbai-400 086, India]. It was prepared by dissolving 13 grams of the medium

in one liter of distilled water. The pH of the medium was adjusted to 7.4 and

7

the medium was then distributed into screw capped bottles, 5 ml each and

sterilized by autoclaving at 121°C for 15 minutes.

4.1.2 Nutrient agar (oxoid, England)

The medium contained lab- lemco powder (1.0 g), yeast extract (2.0 g), peptone (5.0

g) and agar no.3 (15.0 g)-( HiMedia Laboratories Pvt.Ltd-Mumbai-400 086, India).

Twenty eight grams of dehydrated medium were dissolved in one liter of

distilled water, pH was adjusted to 7.4. The dissolved medium was sterilized

by autoclaving at 121°C for 15 minutes.

4.2 Test of the extracts for antimicrobial activity

The cup-plate agar diffusion method was adopted to assess the antibacterial

activity of the prepared extracts(15). Two milliliters of a standardized bacterial

stock suspension (108-10

9cfu\ml) were thoroughly mixed with 200 ml of a sterile

molten nutrient agar. Twenty ml aliquots of the inoculated nutrient agar were

distributed in sterile Petri dishes. The agar was left to set and four cups (10 mm

in diameter) were cut using a sterile cork Borer (no.4) in each plate and the agar

discs were removed using a sterile wire loop. Each cup was filled with 100µ of

one of four extract concentration using microtiter pipette and the extract was

allowed to diffuse at room temperature for 2 h . Two replicates were carried out

for each extract against each of the bacterial organisms. The plates were then

incubated at 37°C for 18 h and simultaneously, in separate Petri dishes a cup was

made for each organism. After incubation, the diameters of the growth

inhibition zones were measured and the average values were tabulated.

8

Chapter Three

Results and Discussion

4.1 Moisture and ash contents

The moisture contents and ash contents of the tested were measured and the results

were varied slightly from previous studies (13). The amount of moisture in all

samples was in the range of 4.2 – 6.0%. The highest level of moisture content was

detected in Trigonella foneum seeds. Artemisia herba- alba and Solenostemma

argel leaves showed high amount of ash content as compared with Trigonella

foneum seeds (Table 1).

Table(1) the moisture content% and ash content% of three tested plants

Plants Moisture content% Ash content%

Trigonella foneum 6.0 4.1

9

4.2 Phytochemical screening

Aerial parts of S. argel, A.herba-alba and seedS of T. fengoum- graceum plants

were successively extracted with ethanol 80%. The crude extracts were

photochemical tested and the three plants showed some variation in their

composition. Very few amounts of alkaloids were found in each plant whereas the

other phytoconstituents were greater [Table 2].

The phytochemical screening revealed the presence of flavonids, glycocides, cardic

glycosides, phenols, tannins and terpenids in S. argel and this result agreed with

other studies (14). In case of T. fenoum graceum the phytochemical screening

showed highest content of phenolic ,flavinoids and revealed the presence of other

constituents, earlier reports have shown that ethanol extract had the same

phytochemical contents (15). The A. herba alba showed the same results with the

other two tested plants (16) [Table 2].

Table(2) : The comparison of the phytochemicals composition for the three

tested plants: Trigonella foneum, Solenostemma argel and Artemisia herba-alba

Artemisia herba- alba 4.5 7.6

Solenostemma argel 4.2 7.4

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Phytochemical

tests

Trigonella fenum

gream

Solenostemma

argel

Artesimia herba-

alba

Alkaloids

[Mayer,s test]

+

_

+

Flavonoids

+++

++

++

Glycosides

+++

+

+++

Cardic glycosides

[Killiani,s test]

++

+

+++

Steroids

[Salkowski,s test]

. +++

++

+++

Phenols [Ferric

Chloride test ]

+++

+++

+++

Tannin,s[Lead

acetate test]

+++

++

+++

Terpenoid

++

+

+++

4.3 Antibacterial Activity

The ethanolic extract of T. foneum graceum was adjusted for it,s antibacterial

activity against Escherichia coli and Pseudomonas aevregoma as agram negative

bacteria and the obtained zone of inhibition was found 2 mm with the Escherichia

coli and it exhibited no action with Pseudomonas aevregoma bacteria.

11

On the hand, the antibacterial activity of T. foneum showed a considerable effect on

gram positive bacteria Staphylococcus aureus, Bacillius subtilis which obtained 5-6

mm and 5 mm zone of inhibition, respectively [Table 3 and Fig. 5].

Table(2):T he measurement of inhibition zone diameter (mm) of the ethanolic extract of

Trigonella foenum- graceum against tested bacteria.

Concentration

mg/ml-1

E.c P.a B.s S.t

5 1 _ 6 5

10 1 _ 5 5

15 2 _ 6 5

*E.C = Escherichia coli P.s = Pseudomonas aevregoma

B.s = Bacillus subtilis S.t = Staphylococcus aureus

Other papers carried the same study but they have used another solvent and other

methods of extraction showed some difference in results (8)(9)(10).

Some studies demonstrate wide spectrum antibacterial activity against gram

negative and gram positive bacteria, for both aqueous and ethanol extracts from

fenugreek seed Trigonella foenum-graecum (16).

Seed purchased from Pakistan was extracted into either water or ethanol, and then

used to make antibiotic discs, which prevented bacterial growth in zones

surrounding each disc.

Zones cleared of bacterial growth ranged in size from 12 to 21 mm, and exhibited

a direct dose response relationship when different concentrations of the extracts

were used. However, ethanol extracts from fenugreek seed purchased from India

12

did not inhibit growth of either bacteria or yeast (18). In our experiments, we

examined Local fenugreek seed, none of concentrations that we examined

possessed this activity. However, other study have shown that chemical

composition can vary among different fenugreek originating from different

countries of the world. This variation could be attributed to genetics and

environmental response of plants to production of phytochemicals (19).

The antibacterial activity which was obtained from the ethanolic extract of A. herba-

alba showed activity against both gram negative and gram positive bacteria [Table 4

and Fig. 6].

Table(4): T he measurement of inhibition zone diameter (mm) of the ethanolic extract of

Artemisia herba –alba against tested bacteria

*E.C = Escherichia coli P.s= Pseudomonas aevregom

B.s = Bacillus subtilis S.t= Staphylococcus aureus

The activity of the extract against gram positive bacteria was high as compared with

the activity against the gram negative one. This results agreed with other study

which had shown that only the essential oil was active against some gram negative

and gram positive bacteria,

Concentration

mg/ml-1

E.c

P.s

B.s

S.t

5

1.5

3

8

5

10

1

3

7

5

15

1

4

7

5

13

in this study the activity was perfumed by using the crude extraction which may

cause a decrease in the activity because the active principle component can be found

in one fraction (20).

The activity of crude ethanolic extraction of S .argel exhibit a weak zone of

inhibition against Bacillus subtilis ,Staphylococcus aureus and no action against

E.coli, P. aevregoma [Table(5) and Fig(7)].

Table(5) T he measurement of inhibition zone diameter (mm) of the ethanolic extract of

Solenostemma argel against tested bacteria

Concentration

mg/ml-1

E.c P.a B.s S.t

5

_

2

4

_

10

_

2

3

_

15

_

2

4

_

E.C = Escherichia coli P.s= Pseudomonas aevregoma

B.s = Bacillus subtilis S.t= Staphylococcus aureus

This results may attributed to different constituents found in the crude extract and

the type of solvent used , and this results agreed with other report (21).Earlier study

had shown that only one fraction had exhibited antibacterial activity against both

types of bacteria.

.

14

15

Fig(5) The zones of inhibition of the Trigonella foneum -graceum extract

against the four type of bacteria

16

17

Fig(6) The zones of inhibition of the Artemisia herba-alba extract against the four type ofbacteria

18

19

Fig(4) The zones of inhibition of the S. olenostemma argel extract against the

four type of bacteria

References

[1] Anonymous, Centre of Disease control and prevention. Preventing emerging

infectious diseases. Astrategy for the 21st century,2000, pp.1-3.

20

[2]Hashim H, Kamali EL, Mohammed Y, 2010. Antibacterial activity and

phytochemical screening of ethanolic extracts obtained from selected Sudanese

medicinal plants. Current Research Journal of Biological Science,

[3] Periyasamy A, Rajkumar, Mahalingam K, 2010. Phytochemical screening and

antimicrobial activity from five Indian medicinal plants against human pathogens.

Middle-East Journal of Scientific Research, 5(6): 477-482.

[4]. Lai PK, Roy J, 2004. Antimicrobial and chemo preventive properties of herbs

and spices. Current Medicinal Chemistry, 11: 1451-1460.

[5] Ahmed I and Beg AZ, Antimicrobial and phytochemical studies on 45 indian

medicinal plants againt multi-drug resistant human pathogens. J Ethnopharmacol,

2001, 74,113-123.

[6] Threlfall EJ.Frost JA and Ward LR, Increasing spectrum of resistance in multi-

resistant Samonella typhimurium, Lancet, 1996, 343, 1052-1054.

[7] Cowan MM, Plant products as antimicrobial agents, Clin Microbial Rev, 1999,

12,564-582.

[8]. Erdogrul OT, 2002. Antibacterial activities of some plant extracts used in folk

medicine. Pharmaceutical Biology, 40: 269-273.

[9] Karim ,A,M. Nouman, S, Munir Sattar,2011. Pharmacology and phytochemistry

of Pakistani herbs and herbal drugs used for treatement of diabetes.Int.J,

Pharmacol.,7:419-439.

[10]Tarka, P ,Antimicrobial studies of methanol and ethanol extracts (Soxhlet

extraction) of Fenugreek (Trigonella Foenum)SLC,S College of pharmacy JNTUH,

Hyderabad,500076.

21

[11]Jafri. S and Ei –Gadi . A . (1983) Asteraceae . In :Flora of Libya 107 . Al- Fateh

University , Tripoli –Libya .

[12] 2-Al –Shamaony , L., Al – Khazraji , S.M. and Twaji , H .A .A (1994).

Hypoglycemic effect of Artemisia herba alba ._ . Effect of a valuable extract on

some blood parameter in diabetic animals . Journal of ethanopharmacology 43:167-

171 .

[13] Murwan, K. El-Kheir, S. (2010). Chemical Composition, Minerals, Protein

Fractionation, and Anti-nutrition Factors in Leaf of Hargel Plant (Solenostemma

argel) Euro. Journals Publishing, Inc., 43, pp 430-434.

[14]Yogesh Mahida and JSS Mohan*

2007 Screening of plants for their potential

antibacterial activity against Staphylococcus and Salmonella spp.v6(4).pp301-305.

[15] Kavanagh, F. (1972). Analytical Microbiology. Vol.II. F.Kavanagh (Ed), Academic

Press, New York and London. PP.11.

[16] Dr. Jamal A. ElturbiG,Najib M. SufyaG,Aisha M. EllafiG 2007

(Phytochemical Investigation of Artemisia Herba Alba Asteraceae)

[17]Bhatti, M. A., Khan, M. T. J., Ahmed, B. and Jamshaid, M. 1996.

Antimicrobial activity of Trigonellafoenum-graecum seeds. Fitoterapia 67: 372-374.

[18]De, M., De, A. K. and Banerjee, A. B. 1999. Antimicrobial screening of some

Indian spices. Phytother. Res. 13: 616-618.

(19)Taylor, W. G., Zulyniak, H. J., Richards, K. W., Acharya, S. N., Bittman, S. and

Elder, J. L. 2002. Variation in diosgenin levels among 10 accessions of fenugreek

seeds produced in western Canada. J. Agric. Food Chern. 50: 5994-5997.

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[(20]) J. Yashphe, I. Feuerstein, S. Barel and R. Segal The Antibacterial and

Antispasmodic Activity of Artemisia herba alba Asso. II. Examination of Essential

Oils from Various Chemotypes

1987, Vol. 25, No. 2 , Pages 89-96 (doi:10.3109/13880208709088133)

[21] FATEN K. ABDO ELHADY* ,A.G.HEGAZI

**, NAGWA ATA

** AND

MONA L.ENBAAWY***

(Studied for determining antimicrobial activity of

solenostemma argel (DEL) HAYNE 2- extraction with chloroform, methanol in

different proportions .1994.14(c):pp143-146.

[22]Tharib,S.M., S. EL-Migirab and G.B.Veitch, 1986. Apreliminary investigation

of the potential antimicrobial activity of Solenostemma argel.Int.J.Crude Drug

Res.,24(2):101-104.

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