Transcript Slide 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
OUTLINE
BACKGROUND AND INTRODUCTION
RESEARCH PROBLEM
OBJECTIVES OF STUDY
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
ACKNOWLEDGEMENTS
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.
 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. 513–529. Oxford University Press, New York.
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
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 world’s 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
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..
 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 folkmedicinal purposes; quells
vomiting and diarrhoea.
Bulbine natalensis
 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, wavyedged 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
•
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

Hypoxis spp,

Easily recognizable by its bright
yellow star-shaped flowers and
strap-like leaves.

Local name: ilabatheka
a well-known
genus of the family Hypoxidaceae,
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.
 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 folkmedicinal purposes resulting in
the colloguial name 'Rabassam'.
Pelargonium sidoides
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.
RESEARCH PROBLEM
Diarrhoea treatment failure particularly with the emerging multidrug
resistant pathogens is a big challenge, resistance to extendedspectrum 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 β-lactamases in the 21st century: Characterization, epidemiology, and
detection of this important resistance threat. Clin. Microbiol. Rev, 14: 933–951.
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.
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
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
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).
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
 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-Ammonium Hydroxide (BEA) (36:4:0.4),
Chloroform-Ethyl Acetate-Formic acid (CEF) (20:16:4)
Ethyl Acetate-Methanol-Water (EMW) (40:5.4:4) (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, 1–8.
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
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.
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.
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, 1–8.
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
IQ
ALEPIDEA AMATYMBICA
PS
PSIDIUM GUAJAVA
BB
BULBINE LATIFOLIA
MB
HERMBSTAEDTIA ODORATA
SC
SCADOXUS PUNICEUS
BS
BULBINE NATALENSIS
MQ
SCILLA NERVOSA
E1
EUCOMIS AUTUMNALIS
HY
HYPOXIS LATIFOLIA
PE
PELARGONIUM SIDOIDES
E2
EUCOMIS COMOSA
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
IQ
ALEPIDEA AMATYMBICA
PS
PSIDIUM GUAJAVA
BB
BULBINE LATIFOLIA
MB
HERMBSTAEDTIA ODORATA
SC
SCADOXUS PUNICEUS
BS
BULBINE NATALENSIS
MQ
SCILLA NERVOSA
E1
EUCOMIS AUTUMNALIS
HY
HYPOXIS LATIFOLIA
PE
PELARGONIUM SIDOIDES
E2
EUCOMIS COMOSA
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
IQ
ALEPIDEA AMATYMBICA
PS
PSIDIUM GUAJAVA
BB
BULBINE LATIFOLIA
MB
HERMBSTAEDTIA ODORATA
SC
SCADOXUS PUNICEUS
BS
BULBINE NATALENSIS
MQ
SCILLA NERVOSA
E1
EUCOMIS AUTUMNALIS
HY
HYPOXIS LATIFOLIA
PE
PELARGONIUM SIDOIDES
E2
EUCOMIS COMOSA
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).
12. Eloff, J.N. 1999. The antibacterial activity of 27 southern African members of
the Combretaceae. South African Journal of Science 95, 148-152.
13. 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.
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
Figure 5: Total activity of plant extracts at 30 min, 1 h, 2 h & 24 h.
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.
Figure 6: Average MIC per plant at 2 h and 24 h
AC
ACACIA SPP
IQ
ALEPIDEA AMATYMBICA
PS
PSIDIUM GUAJAVA
BB
BULBINE LATIFOLIA
MB
HERMBSTAEDTIA ODORATA
SC
SCADOXUS PUNICEUS
BS
BULBINE NATALENSIS
MQ
SCILLA NERVOSA
E1
EUCOMIS AUTUMNALIS
HY
HYPOXIS LATIFOLIA
PE
PELARGONIUM SIDOIDES
E2
EUCOMIS COMOSA
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
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
organism by active compounds.
growth of test
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).
14. Begue WJ and Kline RM, 1972. The use of tetrazolium salts in bioautographic procedures. Journal of
Chromatography 64, 182 – 184.).
Figure 7: Bioautography of chromatograms
STE-
AC
BB
BS
HY
IQ
MB
MQ
PE
PS
SC
E1
CEF
E2
E3
Eluent CEF, detection- sprayed with S.enterica ser Typhimurium & INT
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.
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
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 104:14