Fabrication Of Conjugated Linolenic

acid (CLnA) Nanoemulsion

Colloidal Delivery System : Its

Mitigative Role Against Alloxan

Induced Oxidative Stressed Diabetes

Mellitus RatsDebjyoti Paul

University of Calcutta

GRAPHICAL ABSTRACT

About bitter gourd seed oil….• Bitter gourd (Momordica charantia) seed oil

(BGO) is a unique oil which contains 9cis, 11trans, 13trans-conjugated linolenic acid (9c,11t,13t-CLN) commonly known as α-eleostearic acid (ESA) at a high level of >50%.

• Fatty Acid Composition (w/w %) of Bitter Gourd Seed Oil as obtained by Gas-Liquid Chromatography:

C14:0 C16:0 C18:0 C18:1 C18:2 C18:3

BITTER GOURD

SEED OIL- 2.4 % 31.3 % 8.0 % 7.9 % 50.4 %

Conjugated linolenic acidConjugated linolenic acid (CLnA) refers to a group of octadecatrienoic acid isomers that have three double bonds in conjugation.

Attributed with numerous health benefits.

Antioxidative Anti-Cancer

HypoglycemicHypolipidemic

Cytotoxic Apoptosis•Downregulation

bcl-2 gene •Upregulation of bax gene

• Upregulation of Peroxisome proliferatorActivated receptor proteinPPARs

• Down regulation of ApoB100 • Increase in Serum TG • Increase fatty acid mobilisation• Enhanced activity of mitochondrial peroxisomal β-oxidation

• Scavenging action against lipid peroxidation• Genoprotective role against oxidative stress

• Lipid peroxidation of membrane lipids• Alteration in membrane fluidity

• Antiangiogenesis Down regulation of VEGF,IL-4, Up-regulates MIF

Conjugated linolenic acid

OBJECTIVES OF THE STUDY

To prepare CLnA from seeds of Momordica charantia

(bitter gourd)

Develop colloidal delivery system in the form of nanoemulsion of the CLnA rich bitter gourd oil (BGO) to

increase bioavailability in in vivo system

Study the effects of the formulated system on alloxan induced diabetic rats

Why the trouble of formulation…

Here’s why…..• Reports suggest that o/w nanoemulsions (d <

200 nm) confers a distinct advantage over other delivery systems due to:

enhanced long term stability of the particles in the delivery system;

high optical clarity;

increased bioavailability

MATERIALS & METHODS

Extraction & refining of Bitter gourd seed oil

1. C r u s h e d2. S o l v e n t ex t ra c ti o n u s i n g n - h exa n e3. M i s c e l l a r e fi n i n g ( 2 : 1 )

Bitter Gourd Seed

Refined BGO

BGO Nanoemulsion(20-200 nm)

Preparation of bitter gourd seed oilnanoemulsion

High pressure homogenization

BGO nanoemulsion characterization for droplet size after preparation.Result represented as DelsaTM Nano (Beckman Coulter, Inc., USA) software generated data.

BGO nanoemulsion characterization for droplet size after 12 weeks.Result represented as DelsaTM Nano (Beckman Coulter, Inc., USA) software generated

data.

Animal experiment

ALLOXAN INJECTION (i.p.)

TYPE II DIABETES INDUCED

DIABETIC RATS FORCE FED WITH THE FORMULATED BGO-NE & BGO-CE AT

DIFFERENT DOSES (1% & 0.5%)

AMELIORATION IN CONDITION OF DISEASED STATE DETERMINED ON

THE BASIS OF PHYSIOLOGICAL,

BIOCHEMICAL AND HISTOPATHOLOGICAL

PARAMETERSHEALTHY RAT

Results & Discussion

Effect on blood glucose

0 5 10 15 20 25 30

40

60

80

100

120

140

160

BLO

OD

GLU

CO

SE

LE

VE

L(m

g/d

L)

NUMBER OF DAYS

NORMAL CONTROL ALLOXAN 1 % BGO NE 0.5 % BGO NE 1 % BGO CE 0.5 % BGO CE

Effect on biochemical parameters of liver homogenate:

Effect on biochemical parameters of Plasma homogenate:

Lipid peroxidation studies of Plasma, Erythrocyte membrane & tissue lipids

GROUP FATDIET

PLASMA LIPID PEROXIDATION

(nmole of MDA/mL)

ERYTHROCYTE MEMBRANE LIPID

PEROXIDATION(nmole of MDA/mL)

LIVER TISSUE LIPID PEROXIDATION

(nmole of MDA/mL)

CONTROL(NON-DIABETIC) SO 10.71 ± 0.414# 6.64 ± 0.44# 1.62 ± 0.34#

DIABETICGROUPS

SO27.21 ± 0.35 15.23 ± 0.26 3.4 ± 0.62

SO+0.5 % ESA NE a 5.34 ± 0.25* 3.26 ± 0.17* 1.42 ± 0.81*

SO+0.25 % ESA NE b 4.8 ± 0.33* 2.83 ± 0.91* 1.28 ± 0.33*

SO+0.5% ESA CE c 5.2±0.32* 4.23±0.42* 1.73±0.63*

SO+0.25 % ESA CE d 6.12±0.27* 4.4±0.31* 2.5±0.24*

[MDA] COMPARISONAMONG DIET GROUPS

ALX(SO)>CON(SO)>0.25 % ESA CE>0.5 % ESA NE>0.5 % ESA CE>0.25% ESA NE

HISTOPATHOLOGICALSTUDIES

Histopathology of pancreas

Histopathology of liver

CONCLUSIONBASED

ON RESULTS…….

THUS,THE FORMULATED BGO NE: A POTENT NUTRACEUTICAL

AGAINST TYPE II DIABETES

THE FORMULATED BGO-NE

DIABETIC PATIENT PATIENT RECOVERS

THE ATTEMPT OF FABRICATING A STABLE BGO-NE COLLOIDAL DELIVERY SYSTEM SUCCESSFULLY

INCREASED THE EFFICACY OF ESA MULTITUDINALLY WHEN ADMINISTERD IN ALLOXAN INDUCED

OXIDATIVELY STRESSED DIABETES MELLITUS CHARLES FOSTER STRAIN RATS (IN VIVO SYSTEM)