This Presentation revolves round about the isolation and the characterization of the active molecule megastimene which has a power full activity as anti hyperglycemic ..and they have furthur processed it for formulation

1. Bioassay-guided identification of the
Antihyperglycaemic constituents of walnut
(Juglans regia) leaves.
By
Ch.Srija(NP02)
Debanjan Chatterjee(NP03)
Ashwini Armarkar(NP01)
(Dept. of Natural Products)
1

2. 2
Article
information
ELSEVIER
Journal of
functional
foods
3.14
29th
August
2016
Published on
Impact factor
Journal

3. Introduction
Materials and methods
in-vitro tests and
their results
Conclusion
Limitations
Characterisation of
isolated compounds

4. 1

5. Rationale
 The decoction of the leaves is consumed as tea since long back,
because of its beneficial effect as anti hyperglyceamic activity.
 The plant Juglans regia is well reported for the potent anti
hyperglyceamic activity at various levels i.e., in vitro, in vivo, and
even at human level.
 Most of the chemical structures of the compounds present in the plant
leaf extract were well established.
 For the first time, they have identified the main compounds from the
decoction which are responsible for the anti hyperglyceamic activity.
5
2

6. What is diabetes?
 Diabetes is a chronic, metabolic disease characterized by elevated levels
of blood glucose (or blood sugar), which leads over time to serious
damage to the heart, blood vessels, eyes, kidneys, and nerves.
 It is of mainly 2 types-
 The most common is type 2 diabetes, usually in adults, which occurs
when the body becomes resistant to insulin or doesn't make enough
insulin.
 Type 1 diabetes, once known as juvenile diabetes or insulin-dependent
diabetes, is a chronic condition in which the pancreas produces little
or no insulin by itself.
6
3

7. Current status
7
4

8. Why natural compounds are preferred ?
Though Allopathic medicines are widely used ,the main problems associated are-
 Nephrotoxicity, stomach upset, weight gain.
 As Diabetes is a life time disease and treatment involved is very costly.
5

9. Walnut tree
 Belonging to the genus Juglans (Juglandaceae).
 The fruits, have high nutritional value, with beneficial
effects including decrease of cardiovascular risks and
prevention of cancer and neurodegenerative diseases.
 The plant was reported to improve metabolic
parameters in patients affected by type 2 diabetes.
 Such health benefits have been attributed mainly to ω-3
fatty acids, dietary fibres, minerals, vitamins and
polyphenols, among others.
 The main activity is ANTI DIABETIC ACTIVITY.
 Besides the fruits, the leaves are also used for the
preparation of tea, which is being used not only as
beverage but also in traditional medicine mainly as
astringent and antiseptic or to promote bile flow. 9
6

10. Previous studies and traditional folk’s usage
of the walnut leaves…
 In Mediterranean and Asiatic countries, air-dried leaves of Juglans regia have been
employed to treat diabetic symptoms.
 The methanolic extract obtained from walnut leaves has been proven to enhance
glucose uptake in myocytes by inhibition of PTP1B receptor.
 A human trial was designed to determine the hypoglycaemic effects of J.regia leaf
aqueous extract in type II diabetic patients has been recently conducted.
 The study concluded that the leaf extracts lowered glycaemic levels without exerting
any significant adverse effects on both kidney and hepatic functions.
7

11. Which compound is responsible for antidiabetic action ?
 Since some flavonoids and anthocyanins are known to exert positive effects on diabetes.
 A number of secondary metabolites present in walnut leaf extract,including :
 chlorogenic acid,
 3-p-coumaroyl quinic acid,
 Tri hydroxyl naphthalene-hexoside,and
 Some kaempferol and quercetin glycosides, have been proposed as possible agents
responsible for the activity of the leaves.
11
8

12. 12
Materials
MATERIALS PROCURED
J.Regia leaves Avellion area, Italy in late october
HepG2, HB- 8065 and colon
adenocarcinoma cells ,Caco-2
American Type Culture Collection
Solvents for bioassay guided
fractionation
9

13. Instruments
13
10
NMR Spectometer Varian Unity Inova 700 spec-
trometer equipped with a 13C
Enhanced HCN Cold Probe
NMR Tubes [5mm] Shigemi
LTQ Orbitrap XL hybrid Fourier
Transform MS (FTMS) in- strument
Agilent 1100
Flash Chromatography COMBI Flash , Lumen
HPLC [Semi-Preparative , Analytical ] Agilent Technologies
RPMI 1640 Chemical Co., St. Louis, MO
Cell counter Cellometer Auto T4, Nexcelom
Bioscience, Lawrence, MA, USA). All
experiments

14. Extraction & Separation of Plant Material
14

15. Plant material collected &
dried in room temperature
150
mg
Powdered leaves
extracted twice
4.0L MeOH :water
=8:2
The resulating solvent
partitioned by 3 times
against
EtOAc (2.5L) = 1.1 gm
Butanol(2.0L)= 2.3 gm
Water (1L)=0.8 gm
EXTRACTION PROCESS-
Decoction
Method
Bioactivity checked
in HepG2 cell line
15
12

16. 16
What is Bioassay guided
fractionation ?

17. Decoction (MeOH:Water)
Butanol extract Ethyl acetate extractHere both the extracts were
tested for bioactivity on
HepG2 cells.
Bioactive
fraction 2
Bioactive
fraction 1
Fractionated by flash
Bioactive
fraction 3
Bioactive
fraction 2
Bioactive
fraction 1
Fractionated by flash
Here both the fractions were
tested for bioactivity on
HepG2 and Caco 2 cells.
Separation by preparative HPLC
Identification by analytical HPLC 17
11

18. Extract (2.3gm)
Gradient
Propanol/water
4:6(v/v) for 80 min
Flow rate
10ml/min
20 fractions
Bioactive fraction
after (35 min)
Bioactive fraction
after (39 min)
Luna 10 μm column
Water/MeOH =6:4(v/v) flow
F.R-2.0mL/min
Bioactive fractions
were collected after
12 mins
1st Extract - Butanol
18
13

19. Bioactive
Fractions
Cont…
HPLC (5μm)
Water/MeOH(4:6)
0.8ml/min
Fraction collected after 2.5 min Fraction collected after 3.4 min
(3S,5R,6R,7E,9S)-3,5,6,9-
Tetrahydroxymegastigman-7-ene
(5.5mg)
(3S,6R,9S)
3,6,9-Trihydroxymegastigman-7-ene
(1.8mg)
19
14

20. Silica 5 μm column
N-hexene/EtoAc3:7(v/v)
flow rate,0.8mL/min
2nd Extract
Flow rate
10ml/min
45 fractions
Fractions collected after
Purified
EtOAc Extract (1.1gm)
Gradient
N-hexene/EtoAC
9:1, for 60 minBioactive fraction
after (14 min)
Bioactive fraction
after (25 min)
Bioactive fractions
were collected after
2.3 mins
20
15

21. Cont…. Bioactive
fraction 1
Bioactive
fraction 2
Identification by analytical HPLC
Fractioned sample are purified by prep HPLC
Esculentic acid Asiatic acid
Di-hydroquercetin / Taxifolin
21
16

22. 22

23. The mass of 3,5,6,9-Tetrahydroxymegastigman-7-ene in HRESIMS was obtained at m/z 267.1565
[M+Na]+
Where as the calculated mass for C13H24NaO4, is 267.1567.
The optical rotational value was found to be [α]D
25 - 20.1
1H-NMR spectrum of the decoction sample registered in deuterium oxide (blue) and 1H-NMR
spectrum of 1 registered in the same deuterated solvent (red)
17

24. 1H-NMR
(chemical
shift in PPM)
Intensity
of peaks
Splitting
Pattern
13C NMR
(chemical
shift in PPM)
1.43(j=2.1 Hz) 1H Dd C1=40.5
1.63(j=12.2Hz) 1H Triplet C2=46.3
4.04 1H Multiplet C3=65.1
1.75 2H Multiplet C4=45.5
6.05(j=15.9Hz) 1H Doublet C5=77.6
5.78(j=2.1 Hz), 1H Dd C6=78.6
4.33(j=2.1Hz) 1H Quardret C7=131.0
1.26(j=6.9Hz) 3H Doublet C8=135.9
1.18 3H singlet C9=694
0.83 3H singlet C10=24.0
1.13 3H singlet C11=26.0
4.49
5.09
2H
2H
doublet
doublet
C12=27.3
C13=27.0
3,5,6,9-
Tetrahydroxymegastigman-7-ene
For 1H-NMR
Solvent – CD3OD;
700 MHz
For 13C-NMR
Solvent – CD3OD;
700 MHz
18

25. 6.05
ppm
doublet
5.78
ppm
dd
4.78 ppm
for solvent
4.33ppm
quardret
4.04 ppm
multiplet
3.5 ppm
For solvent
1.75 ppm
multiplet 1.63ppm
triplet
1.43 ppm dd
19

26. 26
in-vitro assays and their results

27. Human hepatocellular
liver carcinoma cells
(HepG20, HB-8065)
were obtained from
American Type Culture
Collection.
Monolayers of cells were
grown in T75 flasks and
supplemented with high
concentration of glucose,
L-glutamine, foetal
bovine serum (FBS),
penicillin, streptomycin.
Cells were incubated at
37°C in a humidified
atmosphere of 5% CO2
in air.
CELL CULTURE OF HepG2 CELLS
27
20

28. Human colon adenocarcinoma cells
(Caco-2) were taken.They were
grown in h-glucose MEM.
Cells are supplemented with 10%
de-complemented foetal bovine
serum (FBS), penicillin,
streptomycin, L-glutamine and
sodium pyruvate.
Cells were incubated at 37°C in a
humidified atmosphere of 5% CO2
in air.
CELL CULTURE OF Caco-2 CELLS
28
21

29. Cells were washed
twice with 2 ml of
HEPES buffer
solution A in both
upper and lower
compartments
Cells were then
incubated for 30 min
at 37 º C, the
electrical resistance
was measured, and
buffer from the
upper and lower
compartments was
removed.
Glucose uptake
was stopped by
washing each
membrane twice
with ice-cold
PBS.
After that NaOH
solution was added
to lyse the cells,and
aliquots were
removed for
scintillation counting
and protein
measurement.
0.5 ml of the
different test
solutions were mixed
and analysed by
scintillation counting
using Liquid
Scintillation Analyzer.
22Assay for glucose uptake
0.05μg /μl of all extracts were
treated with the cells for 24 hrs

30. 30
Fig. 2 – (a) Effect of walnut leaf extract on glucose uptake in HepG2 cell lines. (b) Effect of
compounds 1 and 2 on glucose uptake in HepG2 cell lines. The cells were treated with and without
the purified compounds 1 and 2 for 24 h (0.05 g/L).The glucose was evaluated in the medium of the
cells.
23

31. 31
Fig. 3 – (a) Effect of 1 and 2 on the glucose uptake in Caco-2 cells.
24

32. PROLIFERATION ASSAY
The HepG2 cells and the
Caco-2 cells were
incubated for 24 h with 0
(control) and 0.05 g/L of
all extracts.
After that the cells were
trypsinized and counted using
a cell counter. All
experiments were performed
in triplicate.
The effects of these
compounds on the cell
growth were expressed as
percentage of proliferation
with respect to the
untreated cells (control).
32
25

33. 33
Fig.1 - Effect of 1 and 2 on the proliferation of Caco-2 cells.Cells were
treated for 24 h with 0.005 g/L.
26

34. The TBAR’s assay
was performed on
membranes
extracted from cells
using an ice cold
lysis buffer
supplemented with
protease inhibitors.
Aliquots (10 mL) of
the membrane
preparation were
added to TBA–
trichloroacetic acid
(TCA) solution at
100°C for 30 min.
The reaction was
stopped by cooling
the sample in cold
water, and then
centrifuged at
15000 ×g for 10 min
The homogenate
was centrifuged at
1200 ×g for 10 min
in order to separate
cytosol
(supernatants) from
membranes (pellet).
The pellet was
dissolved in
Tris,NaCl and EDTA,
and the protein
content of the
samples was
determined by Bio-
Rad assay
The chromogen
(TBARs) was
quantified by
spectrophotometry
at a wavelength of
532 nm.
The amount of
TBARS was
expressed as
mM/mM/protein
LIPID PEROXIDATION ASSAY-
34
27

35. NO PRODUCTION
100 µL of sample was mixed with 400 µl milli Q water and 500 µL
of Griess reagent (0.5% sulphanilamide,2.5% H3PO4, and 0.05%
naphthylethylene diamine in H2O) and incubated for 10 min at
room temperature.
The absorbance was measured at 550 nm and
compared with a standard curve obtained by using
sodium nitrite.
35
28

36. Results for lipid peroxidation and NO
production
 Compound 1 and 2 did not induce oxidative stress when tested by
TBARS assay and Griess assay.
 They concluded that the inhibition of proliferation could be
attributable to the reduced glucose uptake due to direct interaction
of the two compounds with the glucose transporters.
36
29

37. The leaves were pulverized,
submerged in water, brought to a boil
and simmered for about 20 minutes.
The final volume of the filtered
decoction was 600 mL.
A sample (5 mL) of the decoction was
evaporated to dryness and solubilized
in 500 µL of deuterium oxide for
NMR analysis.
37
Quantification of antidiabetic molecule in
walnut leaf decoction 30

38. 38
31

39. Conclusion-
 They have performed the research experiment based on Bioassay guided fractionation.
 In total five compounds were isolated in butanol and EtoAc fractions, and from butanol extract the
isolated two molecules were stereochemically identified by NMR as-
1. (3S,5R,6R,7E,9S)-3,5,6,9-Tetrahydroxymegastigman-7-ene
2. 3,6,9-Trihydroxymegastigman-7-ene,both with higher hydrophilicity.
 Taxifolin and Asiatic acid along with its stereoisomer esculentic acid were isolated from EtoAc, but
with lower degree of hydrophilicity.
 The isolated two compounds from butanol showed the reduced cellular uptake of glucose in HepG2
and Caco 2 cell line.
 On performing quantitative NMR ,the estimated total quantity of compound 1 was found to be
150mg/600ml.
39
32

40. Critics
• The structural elucidation of the isolated compounds was not discussed in
detail.
• They could have used LC-MS instead of FTMS.
• The data regarding lipid peroxidation and NO production was missing.
40
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41. Future Prospects-
• Many of the herbal plants remained at extract level with good potential activity, but the
compounds responsible for activity remaines unidentified.
• Not only qualitative analysis, the isolated compounds can also be quantified with the
advanced analytical tools available.
• This kind of experimental research helps in bridging the gap between the isolation and
formulation.
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42. Acknowledment-
We are sincerely thankful to Dr.Abhijit kate, Dr.Vinod Tiwari and Dr.Dinesh
kumar for constant support and valuable suggestions to understand this
article.
We are also thankful to Ph.D seniors, our seniors and our pals.
And finally we would like to thank Dr. Rakesh Tekade and Dr.Vinod Tiwari
for providing us this platform to present this article.
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