In vitro antimicrobial activity and determination of essential metal and ash value contents of Trichodesma zeylanicum

1. ~ 417 ~
_______________________________________
* 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
40o
C 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 0
C 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 80o
C 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 5000
C 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 100o
C per 1 hour up to
500o
C 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
500o
C 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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