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Journal of Microbiology Research 2012, 2(6): 176-182
DOI: 10.5923/j.microbiology.20120206.04
Cytotoxic Effects and Safety Profiles of Extracts of Active
Medicinal Plants from South Africa
Morobe I. C.1,*, Mthethwa N. S.1, Bisi-Johnson M. A.1, Vasaikar S. D.1, Obi C. L.1,2, Oyedeji A. O.3,
Kambizi L.4, Eloff J. N.5, Hattori T.6
1Department of Microbiology, Walter Sisulu University, 5117, Mthatha, Eastern Cape, South Africa
2Academic and Research Division, Walter Sisulu University, 5117, Mthatha, Eastern Cape, South Africa
3Department of Chemistry and Chemical Technology, Walter Sisulu University, 5117, M thatha, Eastern Cape, South Africa
4Department of Botany, Walter Sisulu University, 5117, Mthatha, Eastern Cape, South Africa
5Department of Phyt omedicine, Faculty of Veterinary Medicine, University of Pretoria, Pretoria, South Africa
6Laboratory Emerging Infectious Diseases, Internal Medicine, Graduate School of Medicine, Tohoku University Sendai, Japan
Abstract Plant derived antimicrobial co mpounds that have no or minima l to xicity to host cells are considered candidates
for developing new antimicrobial drugs . Safety is therefore critical in the formulation of antimicrobia ls. The aim of this study
was to investigate the cytotoxic effects of some South African med icinal plant e xtracts. The methanolic and aqueous extracts
of nine South African medicinal plants were screened for cytotoxic activ ities against MAGI CC5+ cells us ing MTT assay.The
nine plant e xtracts us ed in the MTT assayrevealed Herb 2 (Cyanthula inculata) as the most potent extract identified with
activity of (1.4 Cc50values of 25.6 mg/ ml) and induced over 50% of cell deaths, followed by herb 3 (Croton grattismus) and
Herb 4 (Cassine trasvaalensis) with activity of (0.2 Cc50 values of3.7 mg/ml) each. The herbs that induced the least cell death,
were herbs 5 (Capris tomentosa) and 7 (Hypoxis hemerocallidea), with the activity of (0.05 Cc50 values of 0.9 mg/ml) each.
Of the nine p lant e xtracts, Croton grattisimus and Lycium inerme 2(22%), exh ibited minimal to xicity on MAGI cells a nd
7(77.8%) exh ibited 50% toxicity. In a similar study 2(22%) of the methanolic ext racts exhibited anti-HIV1 IIIB activit ies and
agains t Mycobacterium tuberculosis (TB) only one medicina l plant e xtract (Lysium inerme) e xhibited 29% act ivity. In this
study, a systematic eva luation of cytotoxic activities of methanolic extracts made fro m tested medicinal plants s howed
minimal to xicity on cell lines . Therefore, such plants could serve as sources for natural antimicrobial therapeutic agents .
Keywords South Africa, Med icinal Plants , Cytotoxic Activity, Antibacterial Activity, Herbs, Antimicrobial Compounds,
Respiratory Tract Infections
1. Introduction
The increas ing glob al preva lence of mult i res istan t
respiratory tract pathogens is beco ming a se rious pub lic
health and infect ion control p roblems worldwide (1). For
instance H. influenzaeis trans mitted by respiratory droplets
an d may ente r the bloo dst re a m, wh ere it ca n c ause
b ac terae mia a nd diss emin at e t o dis tan t s it es caus ing
d is o rd e rs s u ch as ot itis med ia, s in us itis , ep ig lott itis ,
bronchopneumonia, men ingitis, septic arthritis and cellulitis
resulting fro m invas ion of the bloodstrea m, fo llow ed by
localization of H. influenzae in these and other areas of the
body (1,2). S. pneumoniaeis found in the nasopharynx of 5 to
10% of the healthy adults and 20% of healthy children. The
organ is m att aches to the nasoph aryngea l cells through
interact ion o f bact erial surface adhes ions . This normal
* Corresponding author:
isaacmorob e@yahoo.com (Isaac Morobe Morobe)
Published online at http://journal.sapub.org/microbiology
Copyright © 2012 Scientific & Academic Publ ishing. All Rights Reserved
colonizat ion can become infectious in the Eustachian tube or
nas al sinuses where it caus es otitis med ia, sinus itis. The
organism spreads to the bloodstream, initiating bacteraemia
and is carried to the meninges, joint spaces, bones and
peritoneal cavity and may result in men ingitis, brain abscess ,
septic arthritis or osteomyelitis(2). The risk of pneu mococcal
infection is much increased in persons with impaired Ig G
specify the subset (e.g. IgG1,2,3, or4) and it poss ible
mo lecular phys iology activities in the manage ment of
pnemococcal synthesis, p hagocytosis or defective clearance
of pneumococci. In part icular, the abs ence of functional
spleen, through congenital asplenia, splenectomy, or
sickle-cell d iseas e predisposes one to a more s evere course
of infection (3). Bovine Tuberculosis and HIVare major
diseases , which have also impacted on the economies of
several countries. The morbidit ies and mortalities due to TB
bacilli and HIV are well docu mented (3).
Currently, there is a continuous spread of multi -resistant
pathogens which have become a serious threat to public
health and infection control practices worldwide (3). This
problem has necessitated a search for new antimicrobial
177 Journal of Microbiology Research 2012, 2(6): 176-182
compounds from other sources including plants (4). It is
expected that plant e xtracts s howing target sites other than
those used by antibiotics will be act ive against drug resistant
pathogens as plant derived mater ials have provided the
models for about 50% o f western drugs (5,6). The enor mous
benefits of plants in the management of microbial infect ions
are huge and knowledge of herbal formations made fro m
roots, flowers, barks and their e xt racts are we ll docu mented
(7). The great civilization of the ancient Chinese, Indians and
North Africans provided written evidence of our ingenuity in
utilizing plants for the treatment of a wide variety of diseases,
including bacterial, viral, fungal and paras itic infections, as
well as immunological disorders (8). It was reported (9,10),
that about 25% o f prescribed drugs in the wo rld o riginated
from plants and a bout 80% of the population in developing
countries, including South Africa, rely on traditional
plants for their p rimary hea lth care needs . It was also reported
(10), that Bangladesh had a rich and prestigious heritage of
herbal medicines among the south As ian countries. More
than 500 s pecies of medicinal p lants were estimated to be
grown in Banglades h and about 250 s pecies of them were
used in the preparation of traditional medicines and
treatment of various diseases such as pneumonia, as thma,
bronchitis, epiglottitis, s epticae mia, meningitis, ce llulitis,
sep s i s, art h r i t is , d i ab et es, c an c er , i nfl a m m ati on a nd
diarrhoea
diseases (11). Medicinal plants may be used for totally
different ailments in different ethnic groups (12). It was
reported (12), that about 70% of South Africans consult
traditional healers and that 1% ofnurses are traditional
healers.
Antiviral s ubstances have also been isolated from higher
plants such as algae and lichens. The screening of plant
extracts for antiretroviral activ ity was important becaus e
plant-derived anti-HIV co mpounds could inhibit the
replication of the virus by interfering with one or more of the
ten steps of the HIV replication cycle (13).
Inspire of the therapeutic properties of plants derived
products, their usefulness and efficacies will be severely
compromised if they elicit adverse reactions on
administration. Th is potent that their safety profiles mus t be
guaranteed or that the side effects will be tolerab le and not
toxic to host cells. Therefore a determination of the
cytotoxicity level of any medic inal plant will reveal its safety
as a potential therapeutic agent. Consequently this study
determined the cytotoxic activ ities of aqueous and
methano lic extracts of n ine South African medicinal plants
in order to gauge their usefulnes s as potential candidates for
eventual drug development.
2. Materials and Methods
2.1. Collection of Plant Material
From June 2010 to June 2011, the leaves, bark, stem, roots
and twigs of nine selected medicinal plants were collected
from different sources in the Eastern Cape and Limpopo
Provinces , South Africa, bas ed on their ethnomedical
application in the treatment of respiratory tract infect ions,
guided by the infor mation fro m trad itional healers on the
bas is of their various uses. The plants (Table 1.) we re
identified and authenticated by Taxonomist (Immelman K.L.)
in the Depart ment of Botany, W alter Sisulu Un iversity,
South Africa. Voucher specimens MI 001, MI 002, MI 003,
MI 004, MI 005, MI 006, M I 007, MI 008 and MI 009 were
deposited at the University herbarium.
All the nine plants in Table 1 are us ed by traditional
healers in South Africa, to treat diseas es such as pneumonia,
bronchitis, epiglottitis, asthma, septicaemia, cellulitis, sepsis,
men ingitis,and arthritis caus ed by respiratory tract
opportunistic pathogens such as Haemophilusinfluenzae and
Streptococcus pneumoniae.
Table 1. List of nine selected South African medicinal plants with their tradit ional uses
Botanical name (Family
Vernacular name
Part Used
Traditional Uses
Vangueriainfuesta (Rubiaceae)
Umviyo
Root
cough, heart beat
Cyanthulainculata
(Leguminosae)
Isinama
Leaf
cough, chest pains, T B
Croton grattismus
(Euphorbiaceae)
Umhlabakufeni
Leaf
cough, bronchitis, headache
Cassinetransvaalensis(Celastraceae)
Ingwavuma
Stem
cough, diarrhoea, fever
Capris tomentosa(Capparaceae)
Iquaningi
Bark
Sore throat, fever and cold
Plumbozafingerleaf(Plumbaginaceae)
Umpenduli
Stem
cough, sores, TB, chest pain
Hypoxishemarocallidea(Hyoxidaceae)
Ulabatheka
Corm
bronchitis, sore throat, rush
Lysiuminerme(Solanaceae)
Umvuthwamini
Leaf
cough, skin rush, sore throat
Dichrostachyscinerea(Leguminosae)
Umnukelambiba
Leaf
cough, tooth ache, fontanel
Morobe I. C. et al.: Cytotoxic Effects and Safety Profiles of Extracts of Active Medicinal Plants from South Africa 178
2.2. Preparation of Plant Extracts
Plant materials were washed with s terile distilled water,
dried in an oven for 2h and cut into small pieces us ing a sharp
knife and then air dried at room temperature for 7 days as
previously described (14 ).The dried material (50g) was
ground into a coars e powder using Macs alab mill (Model
200 LAB), Eriez, Bramley as previously des cribed (14).The
ground plant material was reduced to fine powder us ing an
electric blender and the physiochemical co mponents soaked
in methanol (500ml) for 72h with frequent shakings , as
previously described (14,15).The s amples were s uction
filtered through Whatman No1 filter paper. The filtrate was
evaporated to dryness under reduced pressure using a rotary
evaporator, collected in 10ml o f the solvent, placed in the
tube and allowed to dry at roo m temperature.A stock solution
of 0.2g/ml in dimethyl sulfoxide (DMSO) was made for each
extract.All the extracts were kept at 4°C in the dark until they
were further used.
2.3. Cytotoxic Screening of Nine Medicinal Plants
Extracts
2.3.1. Cell Culture
MAGI CCR5+ cells were us ed for cytotoxic screening of
the nine medicina l plant extracts. All ce ll lines were
purchased fro m AT CC, Manassas, VA 20108, USA. Cell
lines were cu ltured in Advanced Modified Eag le’s Medium
(DMEM) with 10% 5M m L-g lutamine (Gibco BRL) and
grown at 37°C in a 5% CO2 humidified incubator (Thermo
Fisher Scientific, Wakenyaku Co. Ltd, Japan). Cells we re
subcultured every 2 days after the confluent growth was
observed.
2.3.2. MTT8 ASSAY 3-(Dimethylthiozole-2-yl-2,
5-diphenyltetrazolium bro mide)
MAGI cells were seeded into two 96 well p lates with 104
cells/well who was this determined?in 100µl of DM EM
supplemented with 10% foetus bovine serum (FBS). 11µl of
herbs was added into 2 wells of row B, with final
concentration of 1/20. Another 11µl of mixture was removed
from B to C and then to D, E, F, and 10µl was discarded from
F. 100µl of mediu m was added into each well fro m B to G.
After 48 h, cells were obs erved and 150µl o f supernatant
from each well was discarded and then 10µl o f MTT was
added into each well. The plates were incubated at 37°C for
4h. 100µl of s top s olution was added into each we ll and
OD570 was checked and then CC50 were determined, as
previously reported (14, 15).
2.3.3. Detection of Anti-HIV-1 III B Activity of Nine Herbs
Using MAGI As s ay
MAGI CCR5+ cells we re seeded into two 96 well plates
with 104 cells per well in 100µl of Dulbecco’s Modified
Eagle’s Medium (DM EM) supplemented with 10% foetal
calf seru m. Herbs in 50mg/ml DMS O, were serially diluted
with co mplete med iu m, by adding 11µl of herbs into 2 we lls
of row B, with a fina l concentration of 1/20. 11µl of mixture
was re moved fro m B to C and then to D, E, F and 10µl
discarded fro m F in flat bottomed microculture plates. The
diluted virus was added 30 minutes later. HIV-infected
(10ng of p24) or moc k-infected MA GI CCR+cells were
cultured with the e xtracts continuously present and no cell
washing was performed throughout the culture. Ce lls we re
stained with chlorophenol red β-D-ga lactopyranoside
(CPRG) as previously described (15). After 3 days of culture,
the medium was removed and the cells were lys ed with
100µl of phos phate buffered saline (PBS) containing 1%
Triton X-100 for 30 minutes at roo m te mperature. The cells
were then incubated at 37℃ for 1h with 100µl o f staining
solution containing 0.01 M KH2PO4, 0.1 M K2PO4, 2mM
MgCl2 and 10 mM CPRG. After 1h the results were observed
under the micros cope (14,15).
2.4. Virus Dilution
IIIB virus (1/250) was us ed for s creening of nine
med icina l plant extracts in a 96 well microtitre plate, in
which 100µl of the virus was added into each well. Cells
were fixed with 1% formaldehyde and 0.2 % gluteraldehyde
in Phosphate buffered saline (PBS) solution for 5 minutes at
room te mperature and then was hed 3 times with Phos phate
buffered saline. Cells we re incubated in 50µl of 4mM
potassium ferrocyanide, 4mM potassium ferricyanide, 2mM
MgCl2 and 0.4mg/ ml 5-bro mo-4-ch loro- 3-indolyl-2-D-gala
ctopyranoside (X-gal) at 37℃ for 1h. The reaction was
stopped by removing the staining solution and washing the
cells twice with Phos phate buffered saline in each well. Blue
cells were counted under a light micros cope at X100
magnificat ion (14,15).
2.5. Detection of Mycobacterium Tuberculosis Rv0679c
Protein Activity of Nine Herbs Using Enzyme
Linked Immunosorbent Assay (ELISA)
The antigen (Rv0679c) was diluted with carbonate coating
buffer (2µg/ml) and 50µl of the mixture was added into a 96
well ELISA microplate. The plate was then covered and
incubated at room te mperature for 24h. A fter 24h, the plate
was washed once with phosphate buffered saline (PBS). The
plate was then blocked with 250µl/well of the blocking
buffer ( PBS and 1% BSA), s ealed and incubated at room
temperature for 2h. The plate was washed 5 times with PBS
and then different herb extracts were added and incubated at
room temperature for 1h. After 1h the plate was was hed once
and the 1/20000 detection antibody (mAb5D4C2 conjugate)
was added and the plate incubated at room te mperature for
2h. After 2h the plate was washed and 1 drop of Vector Elite
ABC reagent was added and the plate incubated at room
temperature for 30 minutes . The plate was then was hed and
100µl/well substrate (H2O2 and TMB reagent) was added and
incubated in the dark roo m for 30 minutes. After 30 minutes
100µl of s top solution (2NH2SO4) was added and ODS was
read immediately at 45nm. Inhibit ion control of the detection
antibody (mAb8G10H2) and c rude Human s erum from TB
179 Journal of Microbiology Research 2012, 2(6): 176-182
pos itive patients was used (Cifuenteset al., 2010).
3. Results
3.1. Cytotoxic Effects of Nine Medicinal Plant Extracts
(Herbs)
The results on the Cytotoxic effects of the medic inal plant
extracts tested are presented below.
Figure 1. 1-Vangueria infausta (Umviyo), 2- Cyanthulainculata (Isinama), 3-Croton grattismus (Umhlabakufeni), 4-Cassine t ransvaalensis (Ingwavuma),
5-Caparis tom entosa (Iquaningi), 6-Plumboza fingerleaf (Umpenduli), 7-Hypoxis hemerocallidea (Ilabatheka), 8-Lysium inerme (Umvuthwamini),
9-Dichrostachys cinetia (Umnukelambiba)
The nine p lant ext racts were used in the MTT ass ay and the results of this study revealed Herb 2 ( Cyanthulainculata) as the
mos t to xic extract identified with activity of (1.4 Cc50 values of 25.6 mg/ml (Figure 1) and induced over 50% of cell death,
followed by herb 3 (Croton grattismus) and Herb 4 (Cassinetrasvaalensis) with activity of (0.2 Cc50 values of 3.7 mg/ml)
each. The herbs that induced the least cell death, were herbs 5 (Capris tomentosa) and 7 (Hypoxishemerocallidea), with the
activity of (0.05 Cc50 values of 0.9 mg/ml) each.
Figure 2. Anti-HIV-1 IIIB activity of nine medicinal plant extracts using MAGI cells
MTTassayresult
Morobe I. C. et al.: Cytotoxic Effects and Safety Profiles of Extracts of Active Medicinal Plants from South Africa 180
Figure 3. 1.Vangueriain faust a (Umviyo), 2. Cyanthulainculat a (Isinama), 3.Croton grattismus (Umhlabakufeni), 4.Cassinetransvaalensis (Ingwavuma),
5.Caparistom ent osa (Iquaningi), 6.Plumbozafingerleaf (Umpenduli), 7.Hypoxishemerocallidea (Ilabat heka), 8.Lysiumin erme (Umvut hwamini), and 9.
Dichrostachyscinetia (Umnukelambiba)
Only extracts 3 (Croton grattismus and 4
(Cassinetransvaalensis) s howed anti-HIV-1 IIIB act ivity
(figure 2). Herb 3 was not included in the list or the
experiment carried out previously by Shenewei on
anti-HIV-1 IIIB activity. Herb 4 has never being deter mined
before us ing MT4 cells (MTT Method). However, the results
revealed its anti-HIV-1 activity of 0.1µg/ ml and 0.2µg/ml.
For Herb 8 (Lysiuminerme), the plant part used in the
Shenwei experiment was a bark and in the current study, the
stem was used. Herb 5 (Caparistomentosa) showed the least
anti- HI V-1 activ ity.
3.2. Detection of Mycobacterium Tuberculosis Rv0679c
Protein Activity of Nine Herbs Using Enzyme
Linked Immunosorbent Assay (ELISA)
Inhibition control of the detection antibody (mAb8G10H2)
and crude Human serum fro m T B positive patients showed
an inhibition and binding activity of 52% (figure 3) wh ile
1mg/ml Herb-8 (Lysiuminerme, Umvuthwamini) showed an
inhibition and binding activity of 29%. Other herbs showed
no inhibition and s pecific binding agains t Mycobacterium
tuberculos is Rv0679c antigen.
4. Discussion
Microbial pathogens and diseases caused are enor mous
and are capable of impacting on several facets of education,
economy and health care systems. Concerted and pers istent
efforts are therefore warranted in gauging the efficac ies of
plant derived antimicrobials as well as critical evaluations
for their s afety profiles.A safety profile is e xemplified,
among other factors and considerations , by cytotoxicity
levels, although in-vitro results may not always simulate in
–vivo conditions . However in-vitro cytoto xicity determinati
ons could serve as one of the adjuncts in profiling
justifications for clinical trials.
In the present study, we do not have data on each of this
plant extracts and their activities, no significance level
determined, where are the data on binding of this compounds
to the cells? The fact that the compounds bound to the cells
does not s ignify their ability to kill the cells (cytotoxic ity)
leading to opsonisation; we should have the initial number of
cells and the number of cells after the plant extract have been
added to the wells. This assay would be best determinedwith
a Flo w cytometery, I EAs or by us e of florocho mes and not
direct ELISA. medicinal plant ext racts were screened for
cytotoxic effects on MA GI ce ll lines. Of the nine plant
extracts 2(22%), Croton grattismus and Lysiuminerme
exhibited minimal toxicity on MA GI cells and 7(77.8%)
exhibited 50% to xic ity. Therefore (Crotongrattismus and
Lysiuminerme) may be suitable for the treat ment of
infections caus ed by designated pathogens and this is
consistent with a previous finding ( 15 ). In a similar s tudy
2(22%) of the methanolic e xtracts exhibited anti-HIV1 IIIB
activities. Human immunodeficiency virus (HIV) is
currently one of the most s erious infectious pathogens
caus ing acquired immune deficiency s yndrome (AIDS) and
the observed anti-HIV activity as well as minimal to xicity of
the plants offer pro mise for che motherapy. Elucidation of the
structures of the active compounds will be another focus of
study. In a similar study methanolic e xtracts were screened
agains t Mycobacterium tuberculosis (TB) and only one
med icina l plant (Lysiuminerme) exh ibited 29% to xicity.
Tuberculosis is a mong the top three leading caus es of death
worldwide and it is aggravated by the increas ed
0
0.5
1
1.5
2
2.5
3
3.5
4
1mg/ml
.1mg/ml
.01mg/ml
.001mg/ml
ND
3*
10*
30*
181 Journal of Microbiology Research 2012, 2(6): 176-182
susceptibility to the human immunodeficiency virus (HIV).
The results obtained in this study had a s lightly lower
toxicities than the previous findings (1, 20)The results in this
study show that the active ingredients of the plant parts are
better ext racted with methanol than other solvents, as were
reported for Croton grattismus, Capris tomentosa, Cassine
transvaalensis, Lysium inerme and Cyantula inculata
(16,17,18,19).
Both the methods and result sections are inadequate in
content. They are not informative enough to bring out the
phytochemical activities of the plant e xtracts .
5. Conclusions
In this s tudy, a s ystematic eva luation of cytotoxic
activities of methanolic e xtractsmadefrom tested med icinal
plants showed minimal toxic ity of 22% on cell lines , therefo
re medicinal plantscould be used as natural antimic robial
therapeutic agents for the treatment ofdisorders caused by
thedesignated pathogens.
ACKNOWLEDGEMENTS
The authors wish to thank Walter Sisulu Un iversity
(WSU), the National Research Foundation (NRF) of South
Africa and the Medical Res earch Council (M RC) of South
Africa for financial assistance. We are indebted to the
technical s taff of the Department of Medica l Microbiology,
Walter Sisulu Un iversity (WSU) for the technical ass istance
they provided during this research work. Special thanks go to
the management and technical s taff of the National Health
Laboratory Services , Ne lson Mandela AcademicHospital,
Mthatha and The Laboratory for Emerging and Infectious
Diseas es , Tohoku University, Japan for the outs tanding
technical ass istance provided during this research work.
ABBREVIATIONS
BSA –Bovine serum Albu min
CPRG – β –D - galactopyranoside
DMEM – Dulbecco’s Modified Eagles’s Medium
DMSO – Dimethyl sulfoxide
ELIS A – Enzyme Lin ked Immunos orbant Assay
FBS – Foetus bovine serum
IgG – Immunoglobulin G
MTT–3- (4,5-Dimethy lthiozol– 2-yl)-2,5-d iphen yltetrazol
ium bromide
TB – Mycobacterium tuberculos is
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