PHYTOCHEMICAL AND BIOLOGICAL SCREENING OF CRUDE EXTRACTS OF SELECTED PLANTS USED IN THE TREATMENT OF DIARRHOEA AND RELATED STOMACH AILMENTS

1
PHYTOCHEMICAL AND BIOLOGICAL SCREENING OF CRUDE
EXTRACTS OF SELECTED PLANTS USED IN THE TREATMENT
OF DIARRHOEA AND RELATED STOMACH
AILMENTS AUTHORS Bisi-Johnson MA, Obi CL,
Kambizi L, Eloff JN, Samuel B, Hattori T,
Vasaikar S and Baba K PRESENTER ADEJUMOKE
BISI-JOHNSON AT INTERNATIONAL SYMPOSIUM ON AIDS
TUBERCULOSIS (ISAT 2010) JANUARY 13-14, 2010,
GONRYO KAIKAN, AOBAKU, SENDAI, JAPAN
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OUTLINE
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BACKGROUND AND INTRODUCTION

• According to WHO reports (2003, 2005a),
  diarrhoea, HIV/AIDS, water and
• food-borne diseases account for a high
  percentage of morbidity and
• mortality in different age groups but
  mostly in children 0-5 years.
• In developing countries- 700 to 1000 million
  episodes of diarrhoea each
• year.
• Mortality 2.4 to 3.3 million deaths per year.
• Everyone has at least an episode or more of
  diarrhoea in a lifetime.
• Children may have up to 10 episodes before
  their 5th birthday.
• A child dies every 15 seconds from diarrhoea,
  caused largely by unsafe
• water and inadequate sanitation (WHO,
  2005b).

• WHO 2003. Emerging issues in water and Infectious
  Disease. Geneva World Health Organization.
• WHO. 2005a. WHO estimates of the causes of death
  in children. Geneva World Health Organization.
• WHO 2005b. Health and the Millennium Development
  Goals. MDGs, Health
• And Development Policy World Health Organization
  Publications of the
• World Health Organization can be obtained from
  WHO Press,
• World Health Organization, 20 Avenue Appia, 1211
  Geneva 27, Switzerland.

4

• Different organisms e.g. bacterial, viruses
  and protozoa are recognized as causative agents
  of acute gastroenteritis.
• Disease mild and self-limiting but symptoms
  may be severe in elderly and young children
  (Smith and Cheasty, 1998), as well as in HIV/AIDS
  patients.
• Basic objective of management include
• -prevention of dehydration
• -correction of dehydration
• -maintenance or improvement of nutrition
• -treatment of causative agents

Smith, H. R. Cheasty, T. 1998 Diarrhoeal
Disease Due to Escherichia coli and Aeromonas.
In Microbiology and Microbial Infections (ed.
Hausler, W. J. Sussman, M.), pp. 513529.
Oxford University Press, New York.
5

• 1. Use of Oral Rehydration Therapy (particularly
  in children)
• Based on facts that
• -Na and glucose transport in the small
  intestine are coupled and represent active
  transport while water follow passively
• -Sodium/glucose co-transport mechanism and
  other absorptive mechanism are maintained even
  in the face of considerable
• intestinal damage
• 2. Intravenous fluid treatment
• 3. Treatment through oral feeding
• Prepared drinks eg pedialyte, Resol
  (electrolytes)/ white grape juice
• Bland food e.g banana, apple sauce, toast may
  follow after 6-24 h clear fluid diet
• Additional food introduced slowly avoiding raw
  fruits , vegetables, beans, fatty and spicy foods
• Reduction of milk and other milk product intake
  for the first few days (reduce irrritation)
• 4. Use of Antibiotics
• Consider if stool is bloody or with high fever

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• The emerging global problem of multidrug
  resistant pathogens and the need for the
  discovery of lasting and sustainable therapy to
  combat
• diarrhoea has led to a paradigm shift to natural
  herbal product for
• succor.
• In most African countries, traditional healers
  and remedies made from plants play an important
  role in the health of millions of people.
• 80 of the worlds rural populations estimated to
  depend on
• plants for their primary health care, since
  western
• pharmaceuticals are often expensive or
  inaccessible (WHO, 1978).
• In South Africa (SA), different communities use a
  wide variety of plants to treat gastrointestinal
  disorders such as diarrhoea and infection by
  intestinal parasites, which are particularly
  prevalent in rural areas (McGaw et al., 2000).
• Among plants used in diarrhoea treatments are the
  following which were screened for antibacterial
  activities.

• McGaw LJ, Jager AK and van Steden J. (2000).
  Antibacterial, antihelmintic and
  anti-amoebic activity in South African medicinal
  plants. J. Ethnopharmacol. 72(1-2) 247-63.
• WHO (World Health Organization). (1978). The
  Promotion and Development of Traditional
  Medicine. Technical Report No. 622. Geneva.

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BACKGROUND ON SELECTED MEDICINAL PLANTS

• Acacia mearnsii leguminous tree up to 15 m
  native to Australia invasive species in SA
• Bark grey-brown to blackish, smooth or rough on
  very old trunks.
• Common names Black Wattle
• Local name ublakweni, udywabasi

• A. mearnsii has some known medical applications,
  such as its use as a styptic or astringent..

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• Bulbine latifolia identified by its aloe-like
  growth, but with an absence of marginal teeth on
  the leaves.
• Forms solitary rosettes up to 20 cm high
• Local name intelezi
• It is utilized for a variety of folk-medicinal
  purposes quells vomiting and diarrhoea.

Bulbine natalensis
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• Eucomis autumnalis (Family Hyacinthaceae) are
  deciduousgeophytes
• Local name Isthibabala
• Common name Pineapple lily derived from the
  pineapple-like flower
• Eucomis have a rosette of large, broad,
  soft-textured, fleshy, wavy-edged leaves
  densely packed flower spikes.
• The bulbs are greatly valued in traditional
  medicine for the treatment of a variety of
  ailments. Decoctions are used for urinary
  diseases, stomach ache, fevers, colic,
  flatulence, hangovers and syphilis

Eucomis autumnalis
10

• Hydnora is a genus of unusual
• bizarre-looking plant parasites mainly
• African distribution.
• H. africana parasitize species of the genus
  Euphorbia commonly E. auretanica and E.
  tirucalli.

• Most species reside underground and all lack
  stomata and leaves.
• Similar to fungi, distinguishable from fungi when
  the flower has opened.
• The fruit of H. africana is said to be a
  traditional Khoi food (no recorded details to
  confirm this).

Other uses Diarrhoea, dysentery, kidney and
bladder complaints are all treated with infusions
and decoctions of Hydnora africana. Infusions
used as a face wash also treat acne. Local
name umafumbuka
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• Hypoxis spp, a well-known genus of the family
  Hypoxidaceae,
• Easily recognizable by its bright yellow
  star-shaped flowers and strap-like leaves.
• Local name ilabatheka

Hypoxis spp.
Hypoxis has a long history of medicinal use on
the African continent, currently being used in
South Africa in primary health care as an immune
booster for patients with HIV/AIDS.
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• Pelargonium sidoides forms a rosette-like plant
  with crowded leaves.
• Local name Umsongelo
• Easily distinguished by its blackish, rather than
  pink petals. The long-stalked leaves are mildly
  aromatic, heart-shaped and velvety.
• It is utilized for a variety of folk-medicinal
  purposes resulting in the colloguial name
  'Rabassam'.

Pelargonium sidoides
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Psidium guajava Family Myrtaceae Shrubs or
small trees. Bark gray, smooth. Branchlets
pubescent. Leaves opposite, petiolate leaf
blade pinnately veined. Common names Guava,
lemon guava, yellow guava. Local name ugwava
Used traditionally in African folk medicine to
manage, control and/or treat a plethora of human
ailments, including diarrhoea.
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RESEARCH PROBLEM

• Diarrhoea treatment failure particularly with the
  emerging multidrug resistant pathogens is a big
  challenge, resistance to extended-spectrum
  cephalosporin among the Enterobacteriaceae has
  become a growing problem (Bradford, 2001).
• Pharmaceutical industries produced a number of
  new antibiotics in the last three decades yet
  resistance to antibiotics by microorganisms has
  increased.
• Need to find lasting solution to the emerging
  infectious diseases
• necessitated the exploration of natural products
  to uncover new grounds in drug production.

• Bradford PA. (2001). Extended-spectrum
  ß-lactamases in the 21st century
  Characterization, epidemiology, and
• detection of this important resistance threat.
  Clin. Microbiol. Rev, 14 933951.

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• Hence, the increase in the search for bioactive
  compounds from plants for pharmaceutical purposes
  (Hostettmann et al 2001).
• Against these backdrop, an ethnobotanical survey
  was conducted to
• identify indigenous herbal remedies for diarrhoea
  and associated
• stomach ailments in rural areas of O.R. Tambo
  district municipality in
• the Eastern Cape Province (ECP) of South Africa
  (RSA).

Hostettmann K, Wolfender J, Terreaux C.(2001).
Modern Screening Techniques for Plant Extracts.
Pharmaceutical Biology, 39, 1 (1)18 32.
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OBJECTIVES OF THE STUDY

• To screen the crude extracts of selected
  medicinal plants used in the treatment of
  diarrhoea and associated stomach ailments in
  Eastern Cape, RSA against enteric organisms
  isolated from diarrheic patients.
• To elucidate the active ingredients of
  potentially valuable selected medicinal plants
  for the development of new pharmaceuticals

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MATERIALS AND METHODS
STUDY AREA O.R. Tambo District Municipality
(ORTDM) Location falls within the latitudes
30 00' and 34 15' South and longitudes 22 45'
and 30 15' East. in the east of the Eastern
Cape Province along the Indian Ocean coastline of
South Africa. It is situated in the former
Transkei homeland area of the province
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SOURCES OF PLANT MATERIALS FOR EXTRACTS

• From the wild with the assistance of traditional
  healers.
• Selection based on frequent or consistent usage
  in the treatment of diarrhoea in Eastern Cape
  province.
• 12 herbs used in treatment of diarrhoea and
  stomach ailments were collected and air-dried.

• Plant materials include
• stem bark of Acacia spp., leaves of Bulbine
  natalensis, B. latifolia, Eucomis autumnalis, E.
  comosa, Hermbstaedtia odorata, root of Hydnora
  africana, Hypoxis latifolia, Pelargonium
  sidoides, spp. and Psidium guajava, Scilla
  nervosa (Burch.) Jessop, Scadoxus puniceus.

• Samples were deposited at the Kei Herbarium,
• at Walter Sisulu University (WSU).

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PREPARATION OF PLANT MATERIAL
PLANT MATERIAL AIR-DRIED
GROUND
STORED IN THE DARK IN AIR-TIGHT BOTTLES
20 g DISOLVED IN 200 ml ACETONE
SUCTION FILTER (Whatman No.1 FP)
EVAPORATION OF FILTERATE AT 40 0 C
STOCK SOLUTION ( 10 mg/ml in acetone)
SONICATE
ASSAY OF EXTRACTS
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PHYTOCHEMICAL ANALYSIS

• By thin layer chromatography (TLC) using
  silica-coated plates (Merck, Kieselgel 60 F254).

10 µl of the resuspended solution of extract was
loaded on the TLC plate
TLC PLATES IN MOBILE PHASE 3- Benzene-Ethanol-Amm
onium Hydroxide (BEA) (3640.4),
Chloroform-Ethyl Acetate-Formic acid (CEF)
(20164) Ethyl Acetate-Methanol-Water (EMW)
(405.44)
(Eloff, 1998)
PLATES VISUALIZED FOR CHROMATOGRAMS under the
short and long UV light
PLATES SPRAYED vanillin-sulphuric acid spray
reagent
PLATES OBSERVED FOR BAND COLOUR
(vanillin spray reagent 0.1 g vanillin, 28 ml
methanol, 1 ml sulphuric acid)
Eloff JN 1998. Which extractant should be used
for the screening and isolation of antimicrobial
components from plants? Journal of
Ethnopharmacology 60, 18.
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ANTIMICROBIAL ASSAY OF EXTRACTS
Determination of Minimum Inhibitory Concentration
(MIC)
MIC total activity determined by a serial
dilution microplate technique

• The bacteria species employed typed cultures of
  Enterococcus feacalis (ATCC 29212), Escherichia
  coli (ATCC 27853), Enterococcus faecalis,
  Pseudomonas aeruginosa (ATCC 25922), Salmonella
  enterica serovar Typhimurium (ESBL ), S.
  enterica serovar Typhimurium (ESBL-), Shigella
  flexneri type 2a, Sh. Sonnei and Staphylococcus
  aureus (ATCC 29213).

• 100 µl of 10 mg/ml of plant extracts in acetone
  were serially diluted two-folds with S/D/water
  and freshly prepared cultures of the test
  organisms.

• Triplicate experiment performed twice

• Controls
• Negative control Acetone
• Positive control Gentamycin (50 mg/ml)

Figure 1. Microtitre plates of MIC of extracts
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Determination of MIC Contd

• Plates were incubated at 37 0 C for 18 h to 24 h.
  
• 40 µl of 0.2 mg/ml iodonitrotetrazolium violet
  (INT, Sigma) was added to each well and incubated
  further.
• Plates were observed at 30 min, 60 min, 120 min
  and 24 h.
• Bacterial growth was indicated by the development
  of a red formazan colour.
• The MIC- the lowest concentration of the extract
  that inhibited bacterial growth.

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BIOAUTOGRAPHY

• Chromatograms of the different fractions of plant
  extracts were kept in stream of air for a day to
  remove all traces of the solvents from plates
  used for the TLC.
• The chromatograms were then sprayed with a high
  density of 48 h culture of the 5 test organisms
  mentioned above and incubated at
• 37 0 C under 100 relative humidity.
• After overnight incubation, plates were sprayed
  with a 2 mg/ml aqueous solution of
  p-iodonitrotetrazolium violet (INT, Sigma) as
  described by Masoko Eloff (2005).
• The clear zones against a pink background
  indicated inhibition of bacterial growth by
  bioactive compounds in the extract.

Masoko, P and Eloff, J.N. 2005 The diversity of
antifungal compounds in six South African
Terminalia species Combretaceae) determined by
bioautography. African Journal of Biotechnology.
Vol. 14(12) 1425-1431.
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RESULTS AND DISCUSSION
Plant extraction

• Acetone extracts of the plants yielded varying
  chromatograms. According to a previous
  comparative analysis of different solvents,
  acetone was the best solvent because of its
  volatility, ability to elute compounds over a
  wide range of polarities, and low toxicity in
  bioassays (Eloff, 1998).
• The TLC analysis of the extracts of the 12 plants
  revealed varying phytochemical constituents
  separated into bands based on polarities (figures
  1, 2 3).
• TLC detection agent was vanillin sulphuric acid
  (figures 1).
• Of the 3 solvent systems used CEF was the best
  followed by BEA and EMW

Eloff JN 1998. Which extractant should be used
for the screening and isolation of antimicrobial
components from plants? Journal of
Ethnopharmacology 60, 18.
25
Figure 1 Phytochemical screening
CEF
AC BB BS HY IQ MB MQ PE PS SC E1 E2 E3
Eluent CEF, Detection- Vanillin sulphuric acid
spray
AC ACACIA SPP
BB BULBINE LATIFOLIA
BS BULBINE NATALENSIS
HY HYPOXIS LATIFOLIA
IQ ALEPIDEA AMATYMBICA
MB HERMBSTAEDTIA ODORATA
MQ SCILLA NERVOSA
PE PELARGONIUM SIDOIDES
PS PSIDIUM GUAJAVA
SC SCADOXUS PUNICEUS
E1 EUCOMIS AUTUMNALIS
E2 EUCOMIS COMOSA
26
Figure 2 Phytochemical screening TLC plate
under UV Light
CEF
AC BB BS HY IQ MB MQ PE PS SC E1 E2 E3
AC ACACIA SPP
BB BULBINE LATIFOLIA
BS BULBINE NATALENSIS
HY HYPOXIS LATIFOLIA
IQ ALEPIDEA AMATYMBICA
MB HERMBSTAEDTIA ODORATA
MQ SCILLA NERVOSA
PE PELARGONIUM SIDOIDES
PS PSIDIUM GUAJAVA
SC SCADOXUS PUNICEUS
E1 EUCOMIS AUTUMNALIS
E2 EUCOMIS COMOSA
27
Figure 3 Phytochemical screening TLC plate
under UV Light
CEF
AC BB BS HY IQ MB MQ PE PS SC E1 E2 E3
AC ACACIA SPP
BB BULBINE LATIFOLIA
BS BULBINE NATALENSIS
HY HYPOXIS LATIFOLIA
IQ ALEPIDEA AMATYMBICA
MB HERMBSTAEDTIA ODORATA
MQ SCILLA NERVOSA
PE PELARGONIUM SIDOIDES
PS PSIDIUM GUAJAVA
SC SCADOXUS PUNICEUS
E1 EUCOMIS AUTUMNALIS
E2 EUCOMIS COMOSA
28
RESULT AND DISCUSSION CONTD
Percentage yield of plant extracts is as shown in
figure 4 Quantity extracted mg/g mass of
air-dried extract per gram of plant material
processed for extraction From the MIC values
taken at different times, total activity (TA,
ml/g) was calculated as the total mass extracted
from 1 g of plant material divided by MIC value
(figure 6) i.e. TA mass extracted (mg/g)/MIC
(mg/ml) Total activity is an indication of the
extent to which the bioactive compound present in
1 g of the dried plant material can be diluted
and still inhibit growth of the test organism
(Eloff, 1999). Total activity is largely
dependent on the quantity of material extracted
from the dried plant material and the higher the
total activity the more effective the plant
(Eloff, 2000).

1. Eloff, J.N. 1999. The antibacterial activity of
   27 southern African members of the Combretaceae.
   South African Journal of Science 95, 148-152.
2. Eloff, J.N., 2000. A proposal on expressing the
   antibacterial activity of plant extracts-a small
   first step in applying scientific knowledge to
   rural primary health care in South Africa. South
   African Journal of Science. 96 116-118.

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Figure 4 Percentage yield of plant extract mg/g
AC ACACIA SPP
BB BULBINE LATIFOLIA
BS BULBINE NATALENSIS
E1 EUCOMIS AUTUMNALIS
E2 EUCOMIS COMOSA
HY HYPOXIS LATIFOLIA
IQ ALEPIDEA AMATYMBICA
MB HERMBSTAEDTIA ODORATA
MQ SCILLA NERVOSA
PE PELARGONIUM SIDOIDES
PS PSIDIUM GUAJAVA
SC SCADOXUS PUNICEUS
30
Figure 5 Total activity of plant extracts at 30
min, 1 h, 2 h 24 h.
31
MIC values of plants extracts

• Crude extracts of B.aspheloides, B. natalensis,
  E. autumnalis and P. guajava compared very well
  with Gentamicin in terms of the MIC against most
  pathogens (Figure 6).
• These plants also possess high total activity as
  seen in figure 5.
• Of striking importance is the MIC of B.
  natalensis which was even lower compared to
  Gentamicin against ESBL positive S. enterica
  serovar Typhimurium (Table 1).
• The MIC values of plants extracts ranged from
  0.078 mg/ml to 2.5 mg/ml within 30 min to 2 h
  incubation and from 0.018 mg/ml to 2.5 mg/ml
  after 24 h of incubation (Table 1).
• The average MIC values vary for the different
  bacterial species.

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Figure 6 Average MIC per plant at 2 h and 24 h
AC ACACIA SPP
BB BULBINE LATIFOLIA
BS BULBINE NATALENSIS
HY HYPOXIS LATIFOLIA
IQ ALEPIDEA AMATYMBICA
MB HERMBSTAEDTIA ODORATA
MQ SCILLA NERVOSA
PE PELARGONIUM SIDOIDES
PS PSIDIUM GUAJAVA
SC SCADOXUS PUNICEUS
E1 EUCOMIS AUTUMNALIS
E2 EUCOMIS COMOSA
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Table 1. MIC values (mg/ml) of plant extracts per
organism compared with Gentamycin
AC BB BS E1 E2 HY IQ MB MQ PE PS SC GENT
EC 2H 1.25 2.5 2.5 0.625 2.5 2.5 2.5 2.5 2.5 1.25 0.156 2.5 0.039
EC 24H 1.25 0.625 0.625 0.312 0.625 2.5 1.25 2.5 2.5 1.25 0.312 1.25 0.078
EF 2H 0.625 1.25 2.5 0.312 2.5 2.5 1.25 2.5 2.5 0.312 0.078 2.5 0.625
EF 24H 0.625 0.039 0.039 0.156 0.078 2.5 0.625 2.5 0.312 0.625 0.156 0.312 0.625
PA 2H 0.312 1.25 1.25 0.312 0.312 2.5 0.312 1.25 0.078 0.078 0.039 0.078 2.5
PA 24H 1.25 0.312 0.625 0.312 0.625 1.25 1.25 2.5 0.078 1.25 0.312 1.25 2.5
SA 2H 0.312 2.5 2.5 0.156 0.312 1.25 0.625 2.5 2.5 0.156 0.078 2.5 0.078
SA 24H 0.312 0.018 0.018 0.312 0.156 0.312 0.156 0.078 0.156 0.312 0.156 0.078 0.078
STE- 2H 1.25 2.5 2.5 0.156 0.625 2.5 1.25 2.5 1.25 1.25 0.312 1.25 2.5
STE- 24H 1.25 0.078 0.078 0.156 0.625 1.25 1.25 2.5 0.625 1.25 0.312 0.625 2.5
STE 2H 1.25 1.25 2.5 0.156 0.312 2.5 0.625 1.25 1.25 1.25 0.078 1.25 0.078
STE 24H 1.25 0.078 0.312 0.312 0.625 2.5 1.25 2.5 1.25 1.25 0.312 0.625 0.156
SHF 2H 0.312 0.078 0.156 0.625 0.625 0.312 0.625 1.25 1.25 0.625 0.078 1.25 0.078
SHF 24H 0.625 0.018 0.078 0.078 0.312 1.25 1.25 2.5 0.625 0.625 0.312 0.312 0.078
SHS 2H 0.625 0.156 0.312 0.312 1.25 2.5 0.625 1.25 2.5 1.25 0.156 2.5 0.156
SHS 24H 0.625 0.039 0.039 0.039 0.312 1.25 0.312 1.25 0.625 0.625 0.312 0.312 0.156
34
BIOAUTOGRAPHY

• The bioautograms revealed fractions of the
  extracts responsible for antibacterial activity.
• Active antibacterial compounds - clear zones
  against pinkish background.
• E. autumnalis showed bands of bioactive compounds
  (figure 7).
• Clear spots indicate location of inhibition of
  the growth of test organism by active compounds.
• It is the non-cleavage of the tetrazolium salt to
  yield the pinkish or purplish formazan product
  seen in the background (Begue and Kline, 1972).

1. Begue WJ and Kline RM, 1972. The use of
   tetrazolium salts in bioautographic procedures.
   Journal of Chromatography 64, 182 184.).

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Figure 7 Bioautography of chromatograms
STE- CEF
AC BB BS HY IQ MB MQ PE PS SC E1 E2 E3
Eluent CEF, detection- sprayed with S.enterica
ser Typhimurium INT
36
CONCLUSION

• If crude extract could compete very well with the
  tested antibiotic, the likelihood of the pure
  compound having higher activity is inferred.
  These plants may therefore be a succor to
  multidrug resistance among Salmonella spp.
• Further study to isolate and characterize pure
  compounds from bioactive plants is on-going with
  the bid to identify compounds with promising
  therapeutic usage.
• Cytotoxicity tests will be required to ascertain
  safety of extracts.

37
ACKNOWLEDGEMENTS

• I acknowledge
• Walter Sisulu University for the IRG support
• Prof. CL Obi and all the co-promoters for their
  contributions.
• Prof. JN. Eloff of the Phytomedicine Unit,
  University of Pretoria and his research team for
  guidance and technical support.
• The traditional healers and knowledgeable
  indigenes.
• Prof. Lamla (Anthropology Department), Mr.
  Wopula, Mpomelelo Nkomo, Tuli Jaca, Dr. Immelman
  and Ms. Cloete, of Botany Department, WSU for
  assistance in plant collection, interpretation
  and plant identification.
• Most importantly, GOD ALMIGHTY

38
He causeth the grass to grow for the cattle, and
herb for the service of man that he may bring
forth food out of the earth - Ps 10414
THANKS FOR LISTENING !!!