Screening of selected South African medicinal plants for anti-inflammatory properties

1. Screening of selected South African medicinal plants for anti-inflammatory properties Salmon Adebayo Dept: Biomedical Sciences Phytomedicine programme

2. Why screen medicinal plants for anti-inflammatory properties? • Background • Inflammation is a complex biological defence and repair mechanism; • Causes- infections, trauma, heat, chemicals, allergy; • Four classical signs- redness, heat, swollen and pain; • Types- Acute (common cold), Chronic (auto-immune diseases e.g RA, SLE) • Treatment- Blockage of molecular targets e.g. enzymes (COX-2/LOX), Interleukins (TNF-α) • NSAIDs (PGE₂) and DMARDs and biological agents (TNF-α) are effective inhibitors; • Extracts and compounds from medicinal plants could be as effective as many NSAIDs (e.g. aspirin) and DMARDs (e.g. Methotrexate); • Also, extracts and compounds from medicinal plants could serve as cheaper template for drug development.

3. Overview of inflammatory response Normal cells and tissues Inflammation caused by infections, stress, injury, disease e.t.c No Inflammation Activation of pro-inflammatory mediators such as Vascular cell adhesionmolecules, neutrophils, mast cells, lymphocytes, macrophages, platelets (histamine and serotonin) Up-regulation of anti-inflammatory cytokines from Th-2 cells e.g. IL-3, 4, 5, 10 and 13. Constitutive NOs and COX-1 Neutrophils, macrophages (cell phospholipids) Cellular metabolism *Inhibitors of iNOs synthetase, COX-2,15-LOX, NFкβ, STAT-1, and free radical scavengers are thought to prevent inflammation at this stage. Flavonoids, terpenoids, curcumins, essential oils and other phytochemicals from plant are effective inhibitors ROS iNO Arachidonic acid AO *NFкβ, STAT-1 *iNOs synthetase with O-free radicals *COX-2, *15-LOX DMARDs NSAIDs Pro-inflammatory cytokines from innate and Th 1 cells (IL-1β, IL-6, IL-8, TNF-α, IFN-γ, GM-CSF) and enzymes Peroxinitrite Prostaglandins PGE₂ Leukotrienes LDL • Sketch diagram to illustrate the interplay between inflammatory mediators and their inhibitors. COX (cyclo-oxygenase), NO (nitric oxide), O (oxygen), IL (interleukins), NFкβ (nuclear factor kappa β), STAT (Signal transducers and activators of transcription), TGF (tissue growth factor), PDGF (platelet derived growth factor), iNOs (inducible nitric oxide), Th (T-helper cells), VCAM ( cellular adhesion molecules), IFN-γ (interferon-gamma), GM-CSF (Granulocyte-Monocytecolony stimulating factor), ROS (reactive oxygen species), LDL (low density lipoprotein), 5-LOX (5-lipoxygenase), AO (anti-oxidants). Inflammatory-pain reaction associated with many diseases, arthritis, artherosclerosis, IDDM, inflammatory bowel disease, cancer etc.

4. What do we know? • Literature Review • Rheumatoid arthritis, an autoimmunedisorder affects 1 % of the adult population worldwide (Alarcon, 2001); • Globally, approx. US$7 Billion is spent on NSAIDs and DMARDs annually; • Most NSAIDs and DMARDs are toxic, expensive and with adverse side effects (e.g anorexia, vomiting, diarrhoea); • Estimates of 25 % of prescribed drugs globally are directly or indirectly derived from plant sources (Rates, 2001); • About 80 % of World population depend on medicinal plants for the management and treatment of various ailments (Farnswoth et al, 1985); • Bartogenic acid (Barringtoniaracemosa), Kolaviron (Garcinia kola), Shikonin (Lithospennumerythrorhizo), Falcarindinol (Aegopodiumpodagraria) esp. is a good 12, 15-LOX inhibitor; • A wide spectrum of bio-active phyto-compounds has been identified, but most have not been properly characterized, and the mode of actions of most compounds are poorly understood; • Can medicinal plants provide cheaper, effective and less toxic sources for the development of potent anti-inflammatory drugs comparable to current NSAIDs and DMARDs?

5. What can we achieve? • Objectives • To establish the presence of bio-active (anti-inflammatory) compounds in selected medicinal plants of South Africa; • To assess the immuno-modulatory and anti-inflammatory effects of extracts using known immunological biomarkers in in vitro bioassays; • To identify and characterize potential bio-active phyto-compound(s) that could exert anti-inflammatory effects on molecular targets from the selected plant species; • To develop a cheaper, non-toxic and effective plant based product that could be used to alleviate pain and inflammation (long term) .

6. Methodology • Selection of medicinal plants • Ethno-botanical use for inflammatory disorders; • Availability and sustainability; • Leaves portion of the selected plants were collected and dried. • Preparation of extracts • Grounded leaf material (3 g) was dissolved in 30 ml 70 % acetone shaken vigorously for 3 hr; • Filtered with Whatman no. 1 into pre-weighed honey jars (3 xce) ; • Solvent was dried completely and % yield determined; • For biological assay, the dried extract was re-constituted at 10 mg/ml in DMSO (Eloff, 1998);

7. Methodology (Contd) • Determination of total phenolics and flavonoids Total phenolics (Makkar, 1999 as adapted); • 25 ul crude extract oxidized with 250 ml Folin-Ciocaiteau’s reagent for 5 min ; • Reaction stopped with 750 ul 20 % anhydrous Na₂CO₃; • Volume made up to 5 ml with D/W and incubated in the dark for 2 hr at RT; • Phenolic content was calculated from the equation off the standard curve of Gallic acid (y = 0.019x + 0.205); • Standard curve (200 mg GA dissolve in DMSO and serially diluted); • Results expressed as mg/g GAE.

8. Methodology (contd) Total flavonoids (Zhishen et al, 1999; Yadav and Agarwala, 2011 as adapted) Crude extract (100 ul) dissolved in 300 ul methanol; 20 ul 10 % AlCl₃ was added; A further 20 ul Sodium Acetate was added to the solution; Resultant solution was made up to 1 ml with D/W; The mixture was incubated at RT for 30 min; After incubation, the solution was carefully transferred into a microplate and read at 450 nm; Quercetin (10 mM) was used as the standard; Flavonoid content was calculated from the equation off quercetin standard curve (y = 2.984x + 0.187) and expressed as mg/g QE

9. Methodology (Contd) Inhibition of 15-Lipoxygenase (LOX) enzyme; 15-LOX (Sigma) was made to a working solution of 200 units/ml and kept on ice; 487.5 ul 15-LOX was added to 12.5 ul extract, and incubated at RT for 5 min in a 96-well microplate in triplicates; After incubation, 500 ul substrate (10 ulLinoleic acid dissolved in 30 ul absolute Ethanol, made up to 120 ml with 2 M Borate buffer) was added; After 5 min of incubation at RT, the absorbance was read at 234 nm (SpectraMax 190, Molecular Devices); Quercetin (10 mM) was used for positive control, DMSO was used for negative control (100 % enzyme activity); % enzyme (15-LOX ) activity = X 100 %

10. Results and Discussion Percentage extract yield for the selected plants species (mean of triplicate experiments) from 3 g of dried leaf materials

11. Results and Discussion (contd)

12. Results and Discussion Relationship between total phenolic and flavonoid content. • Generally, there was a poor correlation between total phenolic and flavonoid contents (R=0.05) i.e the ratio of phenolic/flavonoid contents; • Terminaliaphanerophebiahad the highest flavonoid content (0.597 mgQE) while Dichrostachys cinerea yielded the highest amounts of total phenolic content (53.81 mgGAE); • Tulbaghiavioleceayielded the lowest amounts of flavonoids (0.1 mg QE) and total phenolics (9.47 mgGAE) .

13. Extracts of Acacia sieberiana(48.98 %), Erythrophleumlasianthum(49.29 %), and Tecomariacapensis(62.54 %), hadgood biological activities in the inhibition of 15-LOX respectively, compared with quercetin positive control (57.15 %), where 100 % enzyme activity represents lack of enzymatic inhibition. Results and Discussion Percentage 15-LOX activity when inhibited with plant extracts

14. Results and Discussion Correlation between flavonoid content and % 15-LOX activity

15. h Discussion • The three plants with the highest extract yield did not have good 15-LOX inhibitory properties; • Generally, there was a poor correlation between flavonoid content and 15-LOX inhibitory properties (R²= 0.064), indicating that other phyto-compounds could be involved e.g. Terpenoids, phytosterols, alkaloids e.t.c; • Flavonoids are best known for their free radical scavenging (AO) properties, hence observed 15-LOX-inhibitory effects could be due to the presence of other phyto-compounds in the extracts; • Results also indicated that some of the extracts possibly enhanced the activities of 15-LOX (pro-inflammatory); • Three of the selected plant species had good 15-LOX inhibitory activities, comparable to quercetin, a known inhibitor; • Identification and characterization of compound/s with potent anti-inflammatory activity could serve as a cheaper template for the development of cheaper and less toxic NSAIDs and DMARDs. • Only one pathway of inflammation was explored, hence extracts with poor activity via 15-LOX pathway could work better in other pathways of inflammation e.g. anti-oxidants or anti- NF-кβ

16. Conclusion and further work • Our result suggests that extracts from three of the selected plant species contained bio-active compounds with 15-LOX inhibitory properties; • This evidence supports the ethno-botanical use of the three plant species for inflammation related conditions; • The inhibitory activities was comparable to quercetin, a flavonoid and known inhibitor of LOX enzymes; • Further studies would be done on fractions of the extracts, separated by solvent/solvent fractionation; • Compound/s responsible for the observed 15-LOX inhibitory effects would be isolated and characterized; • Effects of the compound/s on the activation and release of pro-inflammatory biomarkers such as, NO, IL-1β, TNF-α, IL-6, IL-8, NF-кβ, PGE₂e.t.c would be assessed in future studies; • Possible cytotoxic effects of the compounds/s would be determined using standard methods e.g. MTT assays; • An in vivo assessment of the compounds/s would be conducted using a model organism in animal studies;

17. Acknowledgements • Research and Innovation Directorate, TUT ( scholarship) • National Research Foundation (funding) • Phytomedicineprogramme, UP (technical support) • Prof K Eloff & Dr J Shai (supervisors).

18. Thank You! • Mo dupe • Ke a leboga • Dankie • Yabonga • Merci