PhotoAerative alterations of Antioxidant, Antibacterial and Antiplasmodial Activity of Hyptis suaveo

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Photo-Aerative alterations of Antioxidant,
Antibacterial and Antiplasmodial Activity of
Hyptis suaveolens Petroleum ether Leaf Extract.

• Iwalokun et al
• Biochemistry and Nutrition Division
• NIMR, Yaba Lagos
• Nigeria

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A

• Infectious diseases account for approximately 14
  million deaths world wide every year (WHO,
  2007).More than 80 of these diseases occur in
  developing countries with sub-Saharan Africa
  worst affected in terms of morbidity, GDP/DALYs
  loss and mortality. Malaria due to P.
  falciparum,diarhoea/gastroenteritis/food
  poisoning elicited by E. coli, Shigella sp.,
  Salmonella sp, and Staphylococci are major agents
  of infectious diseases in developing countries.
• These diseases evoked a myriad of clinical
  manisfestations that are initiated, propagated
  and sustained by oxidative stress.

B
Global distribution of Malaria (A) and Diarrhoea
(B), WHO, 2005.
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Control strategy Antimicrobial Antioxidant
agents
Malaria
Gastroenteritis
Oxidative Stress
Food poisoning
Diarrhea illnesses
ROS RNS Antioxidant Consumption
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• Chemotherapy and chemoprophylaxis, the mainstay
  of global control of infectious diseases are
  increasingly losing efficacy, promise and hope in
  developing countries where their impacts would
  have been more felt.
• Attributable factors include
• Emergence and spread of multidrug resistant
  pathogens (WHO, 2004)
• Lack of availability, affordability and
  accessibility of essential medicines (WHO, 2008,
  MOH, 2006)
• Complex ecology and biology of pathogens (E.g.
  Zoonotic infections)
• Counterfeit drugs in circulation
• Polypharmacy and inappropriate drug prescription
  (MOH, 2005).
• Over 80 of sub-Saharan Africa populations rely
  on TM for their health care needs.

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Plants have been used for medicines about 60,000
years ago, Paleolithic period (Solecki and
Shanidda, 1975). They remain the sources of novel
drugs, new leads and structural models for new
semi synthetic analogues (Farnsworth et al, 1985)
P H A R M A C O P E I A
Ethnobotany
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250,000 species
Veerporte, 2000
Pharmacology
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The Plant Hyptis suaveolens

• A species of Lamiaceae (mint) family
• Found along bush paths and grown as a weed in
  south-Western Part of Nigeria.

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The Plant Hyptis suaveolens contd..
The Nigerian Bush tea
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Reasons for studying this species

• Widely used in traditional medicine in
  south-Western Nigeria as a multiple remedy
  against diseases and vectors

• Infusion
• Decoction
• Poultice

Fever
Skin diseases
Gastrointestinal disorders
Pain Relief
Poor lactation
Repellant/Biocidal Anopheline/Culicine mosquitoes
Pesticide
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Reasons for studying this species

• Despite the numerous scientifically proven
  pharmacological activities of Hyptis suaveolens
  including its efficacy as, a repellant and
  cidal agent against Anopheles gambiae there are
  no in vivo data on its potential as
  anti-plasmodial agent.

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Proportion of reports of different therapeutic
effects of Hyptis spp.
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Reasons for studying this species
Nigerian cultivars of Hyptis suaveolens is unique
chemotypically Essential oil contains higher
level of Bergamotene but moderate level of
caryophylene compared to cultivars from Brazil,
Asia and Europe. Consumed extensively as a
vegetable, which has been scientifically
justified (Iwalokun et al, 2009). Hyptis
suaveolens is relatively non-toxic LD50 gt 5kg/kg
bw in mice and rats (Farnworth et al, 2002
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Reasons for studying this species
Nigerian cultivars of Hyptis suaveolens has also
been found to elicit antibacterial activity and
pharmacological efficacy against a wide spectrum
of pathogens and diseases but results have not
been consistent.
Why ?????
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How Hyptis suaveolens is prepared

• Most people prepared dried leaves using decoction
  in boiling water and consumed at a dose
  equivalent to 15 mg of freeze dried extract/kg
  body weight.

Leaves boiled with water
decoction
infusion
15 alcohol
40 alcohol
water
Hot water on the leaves
Leaves in cold water or alcohol
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A need to standardize natural product processing
for pharmacological validation
Sun drying
Oven drying
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Photoradiation
Extract in transparent bottles
Extract in brown bottles
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Study Rationale
Different extraction solvent produce different
extracts of different qualities and
phamacological activities. E.g. Hyptis suaveolens
Decreasing Polarity
Water
Methanol
High antibacterial activity but prone to lipid
peroxidation and product degradation
Ethanol
Oily extract
Chloroform
Petroleum ether
Extract defattening
Hexane
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Study Rationale Contd.

• The impact Photo-aerative exposure of H.
  suaveolens would have on its various
  pharmacological activities is not known.
• Scanty data on photo-aeration also exist for few
  medicinal plants.

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Aims of study

• To evaluate and analyse the pharmacological
  effects (antiplasmodial, antibacterial and
  antioxidant activities) of H. suaveolens
  petroleum ether leaf extract.

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• To determine whether the duration of
  photo-aeration influences the pharmacological
  activities of Hyptis suaveolens.

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• To determine the antiplasmodial activity of H.
  suaveolens in
• P. berghei challenged mice fed ascorbic acid
  supplemented diets.

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Methodology

• Study Types
• Animal study
• Antiplasmodial activity (Basal diet)
• Antiplasmodial activity (Ascorbic acid
  supplemented diet (50 mg, 100 mg per 100 g diet)
• Parasite Plasmodium berghei NK65
• Mice, 22.5 23 g (bw), acclimatized, ad libitum
  feeding and drinking, Ascorbic acid
  supplementation
• Challenged dose 107 parasitized erythrocytes in
  100 uL of vehicle Citrate-phosphate-glucose
  buffer (pH 6.9)
• Intervention tools
• Hyptis suaveolens leaf extract (62.5 500 mg/kg
  of body weight)
• Dihydroartemisin Cotecxin, (15 mg/kg of body
  weight)
• Vehicle (CPG, pH 6.9)

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Methodology contd.

• Antiplasmodial Activity determined using Peters
  4-day suppression test
• Indicators
• PCV (day 4 and 10)
• Day 4 Parasitaemia in extract and control drug
  groups.
• No of survivors

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Methodology Contd.

• Extract Preparation
• Plant collection Authentication
• Marceration in Petroleum ether (60-800C) (110,
  w/v) with stirring (150 rpm for 4 h) at 500C
• Evaporation of solvent in vacuo and yield
  determination
• Two storage conditions
• Airtight Brown bottle 40C
• Uncapped Plane bottle Photo-irradiation
• Four Serial dilutions of dried extract in CPG
  (62.5 500 mg/kg body weight)

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Methodology ContdIn vitro methods

• Antibacterial activity
• Well diffusion method ---- Zones of inhibition
• Agar dilution method - --- Minimum inhibitory
  concentration (MIC).
• Antioxidant Activity
• 1,1- Diphenyl-2-picrylhydrazyl radical (DPPH)
• inhibition assay
• (Ascorbic acid as a standard antioxidant)
• Read absorbance of colored solution at 517 nm.

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HSPE-0 has a comparable antioxidant activity as
Ascorbic acid in vitro. Photo-aeration reduces
the potency of HSPE as an antioxidant agent
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Table 1. Plasmodium berghei parasitaemia
suppression activity of Hyptis suaveolens
petroleum ether leaf extracts in mice.
Parasitaemia, rbc suppression, ()

Experimental groups
Controls Dose HSPE-0 HSPE-12 HSPE-24
HSPE-48 DHA (ve) DMSO-CPG (-ve) 15.0 -
- - 0.0 100 - Vehicle -
- - - - 25.11.7
0 a-500 8.60.7 65.8 11.90.8
52.7 12.20.9 51.6 12.40.8 50.8 -
- b-250 14.90.740.9 15.40.7 38.9 16.20.2
35.4 17.50.5 30.2 -
- c-125 15.91.836.6 16.51.5 34.2 17.60.7
29.8 18.90.9 24.8 -
- d-62.5 16.10.435.8 17.00.2 32.2
18.10.3 27.9 19.70.4 21.5 -
- F-statistics 28.1 174.6
141.5 101.7 P 0.03 0.006
0.007 0.0009 ED50, mg/mL 315.84.6a
402.74.3b 417.95.6c
482.76.0d. Parasitaemia data are expressed as
meanstandard deviation of infected mice per
group. Figures in parenthesis represent
percentage suppression of parasitaemia calculated
as 100 (Parasitaemia due to extract or DHA /
Parasitaemia due to vehicle). Changes in
parasitaemia suppression () with dose of extract
were evaluated using linear regression analysis.
P lt 0.05 was considered significant. ED50 is
defined as the effective dose of extract required
to suppress parasitaemia by 50 relative to the
vehicle treated-mice. ED50 values with different
superscripts are significant. DHA,
Dihidroartemisinin HSPE Hyptis suaveolens
petroleum extract, -0 (untreated), -12 (12 h post
Photo-aeration), -24 (24 h post Photo-aeration),
- 48 (48 h post-Photo-aeration). DMSO-CPG,
Dimethylsulfoxide (1)-citrate-phosphate glucose
buffer (pH 6.9)
Photo-aeration improves the antibacterial potency
of HSPE
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Table 2. Effects of photo-aeration on
antibacterial activity of Hyptis suaveolens
petroleum ether leaf extract
Activity Index (AI) Organism HSPE-0 HSPE-
12 HSPE-24 HSPE-48 Ofl (10 mg/disc)_at_ Gram
negatives E. coli ATCC 25922 0.38 0.41 0.44 0.54a
26.5 mm E. coli NG010 0.34 0.34 0.35 0.37 27.5
mm S. flexneri NG017 0.25 0.26 0.27 0.31a 23.0
mm P. aeruginosa NG025 0.09 0.10 0.11 0.12 23.0
mm Gram positives S. aureus ATCC25923
0.40 0.40 0.43 0.55a 24.0 mm S. Saprophyticus
0.37 0.39 0.41 0.51a 25.0 mm NG018 S. aureus
NG011 0.31 0.31 0.33 0.34 24.0 mm AI ,
Activity index was calculated as the ratio of GIZ
diameter due to HSPE (3000 mg/well) to GIZ
diameter due to ofloxacin _at_ Figures represent
mean growth inhibitory zone (GIZ) diameters in mm
due to ofloxacin (10 mg/disc) against the tested
isolates by diffusion method. Ofl, Ofloxacin
HSPE Hyptis suaveolens petroleum extract, -0
(untreated), -12 (12 h post Photo-aeration), -24
(24 h post Photo-aeration), - 48 (48 h
post-Photo-aeration). P lt 0.05 was considered to
be significant aPlt0.05 (Compared to HSPE-0)
Students t-test
Photo-aeration improves the antibacterial potency
of HSPE
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Ascorbic acid failed to improve antiplasmodial
potency of HSPE in Pberghei challenged mice
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HSPE normalizes PCV slowly as an antiplasmodial
agent but more slowly when photo-aerated
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Conclusion
Our results suggest that photoaeration has
potentials to modify the pharmacological
activities of Hyptis suaveolens.
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Future Plans

• Identify the active H. suaveolens compound(s).
• Understand the mechanisms of pharmacological
  activities of Hyptis suaveolens.
• Develop high throughput assays for screening for
  bioctivity of the plant

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Acknowledgements

• LOC
• Technologists CPHL, Microbiology Dept (NIMR).