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* Corresponding author: Sheila M.Maregesi E-mail address: smaregesi@hotmail.com.

Available online atwww.ijrpp.com Print ISSN: 2278 – 2648

Online ISSN: 2278 - 2656 IJRPP | Volume 2 | Issue 3 | 2013 Research article

In vitro antimicrobial activity and determination of essential metal and ash

value contents of Trichodesma zeylanicum

*Sheila M.Maregesi, Nyamwisenda T.Nyamwisenda, Denis Mwangomo, Abdul Kidukuli

Pharmacognosy Department - School of Pharmacy. Muhimbili University College of Health

and Allied Sciences. P.O. Box 65013 Dar Es Salaam, Tanzania.

ABSTRACT

Infectious diseases are responsible for morbidity and mortality of millions each year especially in developing

countries. Microbial drug resistance is a serious problem for current effective antimicrobial agents necessitating

the search for new antimicrobial agents from natural sources including plants. Trichodesma zeylanicum plants is

used for medicinal and nutrition purposes. The objective of this study was to confirm the antimicrobial activity

of T.zeylanicum leaves against selected pathogenic microbes, determine essential metal and ash content and

detect the presence of nitrate and calcium oxalate. The crude aqueous methanolic extract was prepared from the

dry powdered leaves of T.zeylanicum by maceration at room temperature. Eight bacterial and five fungi were

used in the antimicrobial testing employing broth micro dilution method. Atomic Absorption Spectrophotometer

was used for metal analysis. The crude extract of T.zeylanicum leaves exhibited activity against S.aureus,

P.aeruginosa, S typhi and the dermatophyte T.mentagrophyte with MIC value of 1.25 mg/ml. The content of

four essential minerals obtained are; iron (58.53 mg/kg), manganese (47.51 mg/kg), magnesium (3.43 mg/kg),

copper (3.28 mg/kg) and ash value of 23.46%. Microscopic and qualitative chemical analysis studies revealed

the presence abundant calcium oxalate crystal and nitrate respectively. Both antifungal and antibacterial activity

support the traditional use of T.zeylanicum leaves against scalp fungal infection which is normally accompanied

with secondary infection due to bacteria. Presence of essential elements justifies their nutritional potential but

safety issues need to be addressed. This is the first study on the plant T.zeylanicum with regard to antifungal

activity and metal analysis. Detail study is recommended to isolate bioactive principles and establishment of the

complete nutritional and mineral profiles and safety.

KEYWORDS: Antimicrobial activity, Trichodesma zeylanicum, Metal content, Ash value, Nitrate, Calcium

oxalate.

INTRODUCTION

Trichodesma zeylanicum (Burm.F.) R.Br. belongs

to the family Boraginaceae. It has various common

names like Camel bush, cattle bush, northern blue

bell (English), Herbe cipaye, bourrache sauvage,

herbe tourterelle (French). In Tanzania it bears

several names depending to the local languages

such as, Msasa mlanda or Mwagewage (Swahili)[1]

,

Nyabhugimbi (Jita)[2]

. It is a short-lived perennial,

less often annual herb, up to (1.5–2) m tall, much-

branched. Leaves are simple with lower leaves

opposite, upper leaves alternate; stipules absent;

petiole up to 1 cm long; blade oblong to oblong-

lanceolate, up to 16 cm×5 cm, base rounded to

slightly cordate narrowing towards apex. The plant

is widespread in the tropics and subtropics of the

Old World and in Australia. It occurs throughout

tropical Africa[3]

. (See photograph in the Figure:1)

International Journal of Research in Pharmacology & Pharmacotherapeutics

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Figure 1: Trichodesma zeylanicum plant

The plant has various medicinal uses; green leaves

and roots of Trichodesma zeylanicum are chewed

and applied as a poultice to wounds, boils and

snakebites. Roots are chewed or pounded and

soaked in water, and the infusion is used as a

remedy for tuberculosis, stomach-ache, poisoning

and rheumatism[1]

. In Nigeria, leaves are used to

treat fever, scorpion bite and as analgesic[4]

.

Decoction of the leaves is credited with, demulcent

and diuretic properties and the flowers’ decoction

is sudorific and diuretic. Powdered root is analgesic

when applied to wounds and skin infections[5,6]

.

Infusion of leaves and roots used for intestinal

worms, coughing, chest complaints, itching and

throat pains. Root scrapings used for wound

dressing and against bacteria [3,7]

. In the Mascarene

Islands a decoction of the plant is used against

fever, dysentery and as a diuretic. In South Africa,

Zimbabwe and Comoros powder of dried leaves is

applied to infected wounds for healing and as

analgesic. The leaves are also used to prevent

stillbirth while ash of burnt plants enters in

prescriptions for cough and scabies[3]

. The seed oil

is used for its emollient properties in Tanzania,

India and Pakistan. The young leaves and shoots

are cooked and eaten as a vegetable in Tanzania,

Malawi and Madagascar. With the exception of

camels, other livestock avoid eating Trichodesma

zeylanicum[3,8]

. Root decoction is drunk and

douched for treatment of vaginitis. Leaf infusion is

used as eye drop for treatment of cataract [2]

. T.

zeylanicum leaves in combination with the rhizome

of Glycyrrhiza glabra, stem of Canna indica and

stem bark of Punica granatum are mixed and

ground into a paste is applied topically on affected

places for wound healing in India[9]

. Compounds

isolated from T.zeylanicum seed are ricinoleic acid

and cyclopropene acid, the pyrrolizidine alkaloids,

with low toxic alkaloid supinine as the principal

component[10,11]

. Pyrrolizidine alkaloids can cause

fatal liver conditions and several Trichodesma

species had been implicated for livestock poisoning

and not recommended for medicinal use[3,12]

.

Plants are the main source of minerals to the animal

kingdom. Human body needs many minerals

collectively called essential metals in quantities of

only a few milligrams or micrograms per day in

certain balance of levels in every organ, tissue and

cell of the human body for maintaining a healthy

existence. Major minerals (macronutrients) are

calcium, phosphorus, potassium, sulfur, sodium,

chlorine, and magnesium. Trace minerals

(micronutrients) are iron, zinc, iodine, selenium,

copper, manganese, fluoride, chromium and

molybdenum. Other trace nutrients known to be

essential in tiny amounts include nickel, silicon,

vanadium and cobalt[13]

. Iron is vital for almost

every living organisms involved in a wide variety

of metabolic processes including oxygen transport,

DNA, RNA and protein synthesis, electron

transport etc. It amounts to about 35 and 45 mg/kg

of body weight of women and men respectively. It

an essential component of haemoglobin found in

red blood cells that carry oxygen in the body

required for energy metabolism, myoglobin that

carries oxygen to the muscles, many enzyme

reactions and the cytochromes. There are several

disorders of iron metabolism encompassing a

broad spectrum of diseases with diverse clinical

manifestation ranging from iron deficiency i.e.

anemia to iron overload causing constipation,

diarrhea, nausea, vomiting, and gastrointestinal

irritation as well as neurodegenerative diseases [14]

Copper is essential to normal red blood

cell formation and connective tissue formation.

Acts as a catalyst to store and release iron for

hemoglobin formation and contributes to central

nervous system functioning. The deficiency in

copper can cause many hematological

manifestations, such as anemia, myelodyspla

sia, leucopenia and neutropenia. Another common

symptom of copper deficiency is peripheral

neuropathy which is numbness or tingling that can

start in the extremities and can sometimes progress

radially inward towards the torso[15]

.

Manganese is an essential trace nutrient in all

forms of life. It is a key component of enzyme

systems, including oxygen-handling enzymes [16]

.

Manganese deficiency causes skeletal deformation

in animals and inhibits the production

of collagen in wound [17]

among other medical

problems. Magnesium is found in bones, needed

for making protein, muscle contraction, nerve

transmission and immune system. It activates over

100 enzymes and helps to maintain the integrity of

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cell membranes and stabilizes the cell electrically

as well as proper heart functioning [18]. Symptoms

of magnesium deficiency include: hyper-

excitability, dizziness, muscle cramps, muscle

weakness and fatigue. Severe magnesium

deficiency can cause hypocalcemia, low serum

potassium levels (hypokalemia), retention of

sodium, low circulating levels of parathyroid

hormone (PTH), neurological and muscular

symptoms such as muscle spasms, loss of appetite,

nausea, vomiting, personality changes and death

from heart failure [19]

. Trichodesma zeylanicum is

among many documented Tanzanian medicinal

plants with little scientific proof of the

ethnomedical claims, established nutritional profile

and safety. This study dealt with leaves as an effort

to reconfirm the antibacterial activity and

determine the antifungal potential to avail the plant

for future work of isolating bioactive compounds.

Along with, metal content and ash value was

determined to probe on its nutritional value.

Preliminary microscopic study and the detection of

nitrate were as well conducted.

MATERIALS AND METHODS

Plant material collection, preparation and

identification

Leaves of Trichodesma zeylanicum were collected

from Kibubwa village of Mara region, Tanzania in

August 2012. The plant material was dried under

the shade and later powdered. Authentication using

the herbarium specimen was done by Mr. Haji

Selemani at Botany Department - University of Dar

Es Salaam.

Chemicals, Reagents and Media

Analytical grade Dilute Nitric acid and

Concentrated Hydrochloric acid, Standard solutions

of Iron, Magnesium, Manganese and Copper were

purchased from Scharlau Company (South Africa).

Dimethyl sulfoxide was purchased from SIGMA.

Poole, Dorset, England, Iodonitrotetrazolium

chloride was bought from SIGMA (Sigma-Aldrich)

St. Louis, USA. Magnesium carbonate was

manufactured by May and Baker (Dagenhan,

England), Sodium hydroxide was bought from

LOBA CHEMIE PVT. Ltd. (Mumbai 400005

India). Analytical grade methanol was purchased

from Lab Equip Ltd. (Dar Es Salaam, Tanzania),

distilled and deionized water. The media used

were Tryptone Soya Agar (TSA), Tryptone Soya

Broth (TSB) purchased from Himedia Labaratory

PVT. Ltd. (Mumbai, India), Saboraud’s Dextrose

Agar (SDA) and Saboraud’s Dextrose Broth (SDB)

bought from Biotech Laboratory Ltd. Ipswich,

United Kingdom. Reference drugs included

gentamicin injection bought from INTAS

Pharmaceuticals Ltd. (Ahmedabad, India).

Fluconazole was purchased from CADILA

Pharmaceutical Ltd. (Dholka, India). Clotrimazole

was purchased from Ceasar & Lorets GmbH. (D-

40721 Hilden, Germany). Polystyrene,

Nonpyrogenic Tissue Culture Plate, 96 well, U-

bottomed with Low evaporation Lid polystyrene

plates were purchased from Becton Dickinson

Labware Europe. (38800 Le Pont De Claix,

France).

Preparation of the plant crude extract

48 gm of powdered leaves was exhaustively

extracted by maceration with occasional shaking at

room temperature using 80% methanol (4 x700 ml)

for the period of 8 days. The filtered macerate was

concentrated under reduced vacuum pressure at

40oC using a rotary evaporator. This extract was

then stored in the refrigerator to avoid

decomposition and later used for antimicrobial

testing.

Tested microorganisms

The microorganisms used in this study were

obtained from the Department of Microbiology and

Immunology - School of Medicine, Muhimbili

University of Health and Allied Sciences. These

were; four gram positive bacteria were

Staphylococcus aureus (ATCC25923),

Streptococcus pyogenes (clinical isolate), Bacillus

anthracis (NCTC 10073), Bacillus cereus and four

Gram negative bacteria Escherichia coli (ATCC

25922), Pseudomonas aureginosa (ATCC 29953),

Salmonella typhi and Klebsiella pneumonia (ATCC

700603) and five fungi; Trichophyton rubrum

(clinical isolate) Trichophyton mentagrophyte

(clinical isolate), Candida albicans (ATCC 90028),

Aspergillus niger (clinical isolate), Cryptococcus

neoformans (clinical isolate). All bacteria were

maintained on Tryptone Soya Agar while fungi

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were maintained on sabouraud’s dextrose agar

slants in universal bottles.

Antibacterial and antifungal testing

Both antibacterial and antifugal activities were

determined by the broth micro dilution technique

using sterile U-bottomed 96 well polystyrene

microtitre plates as per the method previously

described[20]

. In brief, microbial suspensions

equivalent to 0.5 McFarland concentrations were

prepared by suspending microbes’s inocula in

sterile distilled water in a universal bottle and

adjusting to get the right turbidity. Test extract was

prepared by dissolving 20mg in 0.1 ml of DMSO

and diluted with 0.9ml of sterile distilled water to

make a stock solution with a concentration of 20

mg/ml. The stock solution (50 µl) was added into a

first well of plates pre-loaded with 50 µl of broths

followed by serial dilution by transferring the test

sample from the first row well to well of the next

rows, down to the last rows. The 50 µl from the last

row were discarded. Then, 50 µl of suspension

containing the test micro organisms (0.5 McFarland

dilutions) was added to each of the wells. Wells in

last two columns were used as growth controls i.e.

no drug was added and the adjacent two other

column were used as positive controls. For bacteria

gentamycin was used as a positive control while

fluconazole and clotrimazole were used as positive

control for fungi. Two other columns were used as

negative/solvent controls in which 10%

DMSO/water was added to the first well and serial

diluted downwards. Tests for each sample were

done in duplicate. Microtitre plates were then

incubated at 37 0C for 24 hours except for T.

rubrum, T. mentagrophyte and A. niger that were

incubated for 72 hours.

After the incubation period, 20 µl of a 2% para

iodonitrotetrazolium chloride (INT) was added

followed by incubation for 30 minutes. The

evaluation for the inhibition of bacterial growth

was based on microbial growth indicated by change

of INT colour to pink, while absence of growth was

indicated by absence of colour change. The

minimum inhibitory concentration (MIC) was

determined as the lowest concentration at which

there was no observable microbial growth.

Determination of ash value

Air dried leaves were re-dried at 80oC overnight

then powdered with an electric blender. Three

samples of about 10 g of the powdered sample

were weighed in the pre-weighed crucibles then

subjected at 5000C in a furnace for 5 hours. The

samples were heated until the color changed to off

white/grey i.e. ash formation and constant weight

recorded. Ash value was obtained as an average of

ash contents using the formulas below;

( ) ( )

and,

Ash value = ∑

Determination of metal content

Equipment:

Atomic absorption spectrophotometer - Model:

AA-6300 Shimadzu Company AAS Graphite

Furnace from Japan.

Method:

To free metal atoms ashing by drying method for

food analysis as described by Shimadzu [21]

was

employed. Metal analysis was done by using

atomic absorption spectrophotometer.

Plant sample preparation:

Powdered 5 g of the sample was air dried and

placed in a quartz beaker then heated gently and

continuously on a hot plate until enough water is

driven off for partial carbonization to occur. The

beaker was placed in an electrical furnace and the

heat rate was increased at 100oC per 1 hour up to

500oC where it was heated for three hours to

conduct ashing. The sample was wetted with 2-5

ml of nitric acid, dried and heating continued at

500oC to attain complete ashing. 2 ml to 4 ml of

water were added to the ash, then dried followed by

addition of 5 ml of hydrochloric acid to dissolve

the salts. The volume was brought to 50 ml with

distilled water ready to inject in the AAS. Analysis

for each metal was determined in triplicate.

Preparation of standard metal solutions:

Standard metal solutions were prepared by dilution

method; for Iron solutions of 0.4, 1.2 and 2.0

mg/L, for Copper solutions of 0.5, 1.0 and 2.0

mg/L, for Manganese solutions of 1.25, 2.5, and

5.0 mg/L, for Magnesium solutions of 0.1, 0.2 and

0.4mg/L. These were injected in ASS and the

calibration curve for each metal determined (see

Figure 2).

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Qualitative determination of nitrate

The method for preparation of the nitrate

containing extract followed the procedure

described by Cataldo et al. [21]

with slight

modifications as outlined here. To 100 mg of dried

plant material in the tube 10 ml hot deionized water

(90-95ºC) was added. Mixed thoroughly and more

10 ml hot deionized water was added. This was put

in a closed tube and placed in a water bath at 80ºC

for 30 minutes with occasional shaking. After

cooling the sample the open tube was centrifuged

at high speed (4500 rpm - 2649 g) in a table

centrifuge. The decanted supernatant was treated

with 1 gram of magnesium carbonate then

centrifuge at high speed to remove the chlorophyll.

The supernatant (containing nitrate) was treated

with 1.6 ml of reagent A (1.25 g of salicylic acid

dissolved in 25 ml of 96% Sulphuric acid - freshly

prepared in darkness) well mixed then left at room

temperature for 20 minutes. Slowly 38 ml of

reagent B (10 g of NaOH dissolved in 125 ml of

deionized water) was added, well mixed and left to

cool at room temperature to obtain the yellow

colour indicating the presence of nitrate.

Detection of Calcium oxalate

The powdered leaf was cleared by heating in

chloral hydrate solutions, mounted in glycerin then

viewed under microscope at 10 x and 40 x

magnifications.

RESULTS AND DISCUSSION

Antimicrobial activity

Table 1: Antibacterial and Antifungal activity of Trichodesma zeylanicum leaves

Microorganisms Minimum Inhibition Concentration (mg/ml)

Crude extract Gentamycin

Staphylococcus aureus 1.25 0.00033

Streptococcus pyogenes * Not determined

Bacillus anthracis * 0.00033

Bacillus cereus * 0.00125

Pseudomonas aeruginosa 1.25 0.00125

Salmonera typhi 1.25 0.00125

Klebsiella pneumonia * 0.00033

Escherichia coli * 0.00063

Clotrimazole

Trichophyton mentagrophyte 1.25 < 0.002

Trichophyton rubrum * < 0.002

Aspergillus niger * < 0.002

Fluconazole

Candida albicans * 0.00625

Cryptococcus neoformans * 0.00313

* = Not active at 5.00 mg/ml

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The aqueous methanolic of T.zeylanicum leaves

exhibited activity against three bacteria S. aureus,

P.aeruginosa, S.typhi and the dermatophyte T.

mentagrophyte. Activity against gram negative

bacteria is of interest particularly P.aeruginosa due

its resistance against various antibiotics. These

results support the traditional use of T.zeylanicum

against scalp fungal infection normally

accompanied by secondary bacterial infections. The

MIC value of 1.25mg/ml is regarded as weak

activity for bacteria and moderate for fungi

according to the rating of Cos and Algiannis teams

respectively[23, 24]

. In part, this could be contributed

by the presence of inactive compounds including

the mucilage. Previous study in agreement with our

results is that conducted in India using methanol

and ethyl acetate and diethyl ether extracts that

showed activity against Staphylococcus aureus and

Bacillus cereus respectively [25]

. At the genus level,

Trichodesma amplexicaule (root) extracts and

isolated compounds exhibited antimicrobial

activity against E.coli, S.aureus, Aspergillus flavus

and Penicillium chrysogenum etc. The benzene

extract was more potent against S.aureus and

R.phaseoli and among the isolated compounds,

hexacosane was more active against the gram

negative bacteria E. coli and hexacosanoic acid had

greater activity against the fungi A.flavus[26]

.

Interesting bactericidal and fungicidal activity was

also obtained from the ethanolic extract and

isolated compounds of Trichodesma indicum

(roots) against Bacillus subtilis, Staphylococcus

aureus, Staphylococcus epidermidis, Pseudomonas

aeruginosa, Klebsiella pneumonia, Escherichia

coli, Aspergillus niger, Aspergillus flavus, and

Candida albicans[27]

. T.zeylanicum leaves contain

nitrate as in the case of Trichodesma africanum[7]

.

Both nitrates and nitrites have been reported to

possess human health benefits. Acidified nitrite

exhibited growth inhibition against aerobic

bacterial such Escherichia coli O157, Shigella

sonnei, Yersinia enterocolitica, and Salmonella

Enteritidis [28]

. In vitro tests showed the bactricidal

of Helicobacter pylori in the acidified nitrite

conditions, speculating the protection of the

stomach from colonization of H.pylori associated

with ulcers and gastric cancers when the food

containing high nitrate content is ingested [29]

.

Besides gastrointestinal pathogens, acidified nitrite

had shown the ability to kill common

dermatological pathogens such as Trichophyton

mentgrophytes, Staphylococcus aureus, Strepto-

coccus pyogenes, and Propionibacterium

acnes[30]

.The therapeutical effect resulting from

oral and or topical administration with the

decoction, infusion, paste or ash for various

disease/conditions could be due to nitrate/nitrites in

addition to the organic biomolecules. On the other

hand, oral consumption of high amounts of nitrate

is known to have adverse effect on thyroid

function. Nitrite is more toxic than nitrate and is

produced in the human body during microbial

reactions. When nitrite is absorbed into the blood,

it reacts with haemoglobin and causes

methaemoglobin[31]

. Other human illnesses such as

non-Hodgkin's Lymphoma, cancer in the digestive

tract, diabetes, some birth defects and miscarriages

are also associated with high nitrate levels.

Recently, some studies have linked high nitrate to

bladder cancer, especially in women[32]

.

Preliminary microscopic study of powdered

T.zeylanicum revealed abundant calcium oxalate

crystals. These are quite sharp and abrasive

structures, ingestion of plants containing them can

cause abrasive and irritation injuries. Intake of

large amounts can cause kidney and liver damage

and even death in serious cases [33]

.

Metal content and Ash value

The present work afforded quantification of iron,

copper, manganese and magnesium and ash value

determination of T.zeylanicum leaves. The results

are presented in Table-2 together with the

Recommended Daily Intake values. Iron and

manganese contents are relatively higher compared

to copper and magnesium. Since the daily intake of

plant material does not exceed 100 g of the dry

weight, the four metals so far analyzed fall within

safe limits.

Table 2: Metal contents and Ash value of T. zeylanicum leaves Metal content Ash value *Recommended

Daily Intake (RDI)

Iron 58.53 mg/kg 18 mg

Manganese 47.51 mg/kg 2.0 mg

Copper 3.28 mg/kg 2mg Magnesium 3.43 mg/kg 400mg

Ash Value 11.97% -

*Values obtained from Council for Responsible Nutrition

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CONCLUSION

The results obtained from this work has

reconfirmed the activity of Trichodesma

zeylanicum leaves against Staphylococcus aureus

and reports for the first time the antimicrobial

activity against Pseudomonas aureginosa,

Salmonera typhii, and T. mentagrophyte as well as

the contents of iron, copper manganese and

magnesium metals. The observed antibacterial and

antifungal activity justify the reported use of leaf

powder and paste against scalp fungal infections

when applied topically. Recommendation on

suitability of this plant for medicinal /nutritional

use will depend on the safety established after

isolation the active fraction/compounds and

complete nutritional and mineral profiles.

ACKNOWLEDGEMENT

We are grateful to Ms. Kakomo Marwa for sharing

with us information of Trichodesma zeylanicum

medicinal use and Tanzania Food and Drug

Authority (TFDA) for their technical support.

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