Phytochemical and biological activity studies of

Galenia africana

PHYTOPHARM 2015

15 July 2015

Lawrence Awa Ticha, PhD

Division of Clinical Pharmacology

Faculty of Medicine and Health Sciences

Stellenbosch University

E-mail: 19155611@sun.ac.za

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South Africa

Content:

Aim

Introduction

Methods

Results and Discussion

Conclusion

Acknowledgment

References

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Aim

This study focused on an 80% ethanol : water

extract of Galenia africana in which the

antimicrobial activity of isolated flovanoids

against S. aureus and methicillin-resistant

Staphylococcus aureus (MRSA) was investigated.

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Galenia africana

Belongs to the family Aizoaceae

It is an erect aromatic green to yellow-green soft woody shrub between 0.5 to 1.5 m high

Its distribution ranges from the dry flatlands and lower slopes of the Northern Cape

The plant was chewed by the indigenous Khoi-San to relieve toothache and has also been implicated in the treatment of venereal diseases, wounds, skin diseases and to relieve inflammation of the eye

Extracts of G. africana have demonstrated substantial activity against Mycobacterium tuberculosis (1)

Introduction…

A number of studies have demonstrated the beneficial effects of flavonoids in a variety of ailments including (2):

cancer

Coronary heart diseases

Bone mass loss

Age-related diseases

Mycobacterium tuberculosis

Antifungal agents

Antioxidants

Introduction…

Flavonoids are polyphenolic compounds with a major characteristic being the presence of a 1,3-diarylpropane skeleton capable of assuming different cyclic and alicyclic arrangements, depending on the various levels of oxidation.

Chalcones, are open chain flavonoids characterized by the presence of two aromatic or phenolic rings joined by an α,β-unsaturated carbonyl chain viz., 1,3-diphenyl-2-propen-1-one.

Methods

Fractionation and purification

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80% EtOH : H2O ExtractParceval (Pty) Ltd Pharmaceutical (South Africa) (2 L)

EtOAc/n-Hexane(5 – 80%)

A (1.72g) B (4.77g) C (2.62g) D (2.32g) E (1.03g)

(20.15g) Crude extract

Compounds 1- 9

Phytochemical screening

Methods…

Antimicrobial assay

An in vitro antimicrobial screening against S. aureus and

MRSA was carried out with vancomycin as standard. Kirby-

Bauer agar disc diffusion assays were used with slight

modifications (3)

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Results and Discussion…Phytochemical Screening

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Phytochemical constituents G. africana

Alkaloids - -

Anthraquinone - -

Coumarins - -

Flavonoids + + +

Leucoanthocyanins - -

Simple phenolics +

Steroids - -

Saponins +

Tannins +

Terpenoid +

Cardiac glycosides +

Results and Discussion…Nine flavanoids isolated by a meticulous chromatographic separation

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Results and Discussion…

(E)-2',4'-Dihydroxychalcone (1)

Yellow crystals, m.p. 145 – 146°C (EtOAc:hexane)

HRMS: found: 240.0786; calculated for C15H12O3: 240.0789; 1H NMR (200 MHz, CDCl3) δ: 5.98 (1H, single peak, 4'-OH), 6.44 (2H, m, H-3' and H-5'), 7.74 (3H, m, H-3, H-4 and H-5), 7.57 (1H, d, J = 15.7 Hz, H-α), 7.63 (2H, m, H-2 and H-6), 7.82 (1H, d, J = 8.8 Hz, H-6'), 7.87 (1H, d, J = 15.7 Hz, H-β) and 12.1 (1H, single peak, 2'-OH); 13C NMR (50 MHz, CDCl3) δ: 103.9 (C3'), 107.9 (C5'), 114.5(C1'), 120.4 (Cα), 128.7 (C3/5), 129.1 (C2/6), 130.8 (C4), 132.1 (C6'), 134.9 (C1), 144.7 (Cβ), 162.8 (C2'), 166.6 (C4') and 192.1 (C=O).

1H NMR (200 MHz, acetone-d6) δ: 6.39 (1H, d, J = 2.2 Hz, H-3'), 6.48 (1H, J = 8.6 and 2.2 Hz, H-5'), 7.46 (3H, m, H-3, H-4 and H-5), 7.88 (4H, m, H-α, H-β, H-2 and H-6), 8.10 (1H, single peak, 4'-OH), 8.16 ( 1H, d, J = 8.6 Hz, H-6') and 12.20 (1H, single peak, 2'-OH); 13C NMR ( 50 MHz, acetone-d6) δ: 103.8 (C3'), 108.9 (C5'), 114.5 (C1'), 121.8 (Cα), 129.6 (C3/5), 129.8 (C2/6), 131.4 (C4), 133.5 (C6'), 135.9 (C1), 144.7 (Cβ), 165.9 (C2'), 167.7 (C4') and 192.8 (C=O).

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MIC for compounds P1, P2, P3, and P5 against S. aureus

MIC for compounds P1, P2, P3, and P5 against MRSA

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Concentration

(mg/ml)

Zone of Inhibition (mm)

P1 P2 P3 P5

2.000 14 0 11 0

1.000 12 0 0 0

0.500 11 0 0 0

0.250 0 0 0 0

0.125 0 0 0 0

0.063 0 0 0 0

0.031 0 0 0 0

0.016 0 0 0 0

0.008 0 0 0 0

0.004 0 0 0 0

0.002 0 0 0 0

Concentratio

n (mg/ml)

Zone of Inhibition (mm)

P1 P2 P3 P5

2.000 11 0 10 0

1.000 9 0 0 0

0.500 7 0 0 0

0.250 0 0 0 0

0.125 0 0 0 0

0.063 0 0 0 0

0.031 0 0 0 0

0.016 0 0 0 0

0.008 0 0 0 0

0.004 0 0 0 0

0.002 0 0 0 0

Methicillin/vancomycin susceptibility testing of S. aureus and MRSA

Antibiotic (10μg) Zone of Inhibition(mm)

S. aureus MRSA

Methicillin 15 0

Vancomycin 16 16

Results and Discussion…

Results and Discussion…

Compounds 2, 3 and 5 are being reported for the first time in this plant.

The observed antimicrobial dominance of chalcones over flavanones can be attributed to the α, β unsaturated keto group’s ability to undergo a Michael-type addition by a nucleophilic group such as a thiol of an essential amino acid as part of a protein (4).

As a result of the unsaturated carbonyl link between rings A and B, this favorsthe delocalization of π-electrons resulting in a lost of part of its individual character and partial integration into the adjacent olefinic group. This particular characteristic generates transference of electronic deficiency from the carbonyl carbon atom to Cβ, which is subsequently increased in 2'(OH)-chalcones.

Though we did not investigate the chemical reactivity, acid and basic properties of flavonoids play a crucial role in their reactivity. Pharmacologically, carbonyl compounds usually exert their effects in most cases by a direct interaction with some enzymes through hydrogen bonding.

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Conclusion

This study has shown that there is a good

possibility for these flavanoids being developed as

virulence therapies owing to their ability in

interfering with bacterial virulence factors such as

enzymes, toxins and signal receptors. However,

further studies are required to ascertain the

effects and toxicity of this compound in

experimental animals.

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Acknowledgments

Stellenbosch University

University of the Western Cape

Notational Research Foundation

Profs. IR Green and J Klaasen

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References

1. van Wyk B-E, van Oudtshoorn, B, Gericke N. (1997) Medicinal Plants of

South Africa. Briza Publications, Pretoria, 1st Ed. 118-119.

2. Wu, P., et al., Investigation of in vitro and in vivo antioxidant activities of

flavonoids rich extract from the berries of Rhodomyrtus tomentosa(Ait.) Hassk.

Food Chemistry, 2015. 173: p. 194-202.

3. Rajendra, P.N., Anandi, C., Balasubramanian, S., & Pugalendi, K.V.,

Antidermatophytic activity of extracts from Psoralea corylifolia (Fabaceae) correlated

with the presence of a flavonoid compound. Journal of Ethnopharmacology, 2004.

91(1): p. 21-24.

4. Zdzisława, N., A review of anti-infective and anti-inflammatory chalcones. European

Journal of Medicinal Chemistry, 2007. 42(2): p. 125-137.

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Thank you

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