OUTLINE

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

2. OUTLINE

3. 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. 513–529. 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

6. 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 of Traditional Medicine. Technical Report No. 622. Geneva.

7. 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..

8. 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.

9. 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

10. Hydnorais 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

11. 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 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.

12. Pelargonium sidoidesforms 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'.

13. 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.

14. 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 β-lactamases in the 21st century: Characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev, 14: 933–951.

15. 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.

16. OBJECTIVES OF THE STUDY • To screen the crude extractsofselected 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 valuableselected medicinal plants for the development of new pharmaceuticals

17. 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

18. 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).

19. 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

20. 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-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.

21. 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

22. 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.

23. 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.

24. 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.

25. Figure 1: Phytochemical screening CEF Eluent CEF, Detection- Vanillin sulphuric acid spray

26. Figure 2: Phytochemical screening: TLC plate under UV Light CEF

27. Figure 3: Phytochemical screening: TLC plate under UV Light CEF

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). • Eloff, J.N. 1999. The antibacterial activity of 27 southern African members of the Combretaceae. South African Journal of Science 95, 148-152. • 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.

29. Figure 4: Percentage yield of plant extract mg/g

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.

32. Figure 6: Average MIC per plant at 2 h and 24 h

33. Table 1. MIC values (mg/ml) of plant extracts per organism compared with Gentamycin

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

35. Figure 7: Bioautography of chromatograms STE- CEF 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 104:14 THANKS FOR LISTENING !!!