International Journal of Advanced Scientific Research and Publications ISSN-2454–9878

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Please cite this article as: Megha Vivek, Prashantha Karunakar, Evaluation of plants extracts for acetylcholinesterase inhibitory activity and GSK-3,

International Journal of Advanced Scientific Research and Publications, V2-3, page no 29 -34, (2016)

Evaluation of plants extracts for acetylcholinesterase inhibitory activity and GSK-3 Megha Vivek, Prashantha Karunakar Department of Biotechnology, PES Institute of Technology, BSK III Stage, Bangalore 560085, India

A b s t r a c t Acety lchol inesterase inhib i t ion is important fo r t reatment of neurodegenerat ive diseases l ike Alzheimer ’s , Parkinson ’s d isease.

Herbal medic inal p lants l ike Camel l ia s ines is , tul s i green tea, Z ingiber off i c inale, Ur t ica dioica, Lepidium meyeni i can be a source

of inhib itors . AchE activ ity was measured by color imetr ic methods in the presences of the extracts us ing Neost igma as the con trol .

For the treatment of neuro re lated diseases, an antiox idant assay ABT S and DPPH was performed and observed that Lepidium

meyeni i and Z ingiber off ic inale showed pos it ive resul t. From the MTT assay, the 3T3L1 adipocyte cel l l ine, tul s i green tea sh owed a

good effect of 93mg IC50 va lue, two which showed a h igh IC50 value they were Lepidium meyeni i and Urt ica diocia, as these

plants were experimental ly not much tested, Another sample cons idered was the combination of a l l the f ive plant extracts - A

Polyherbal formulations. The GSK-3T3L1 cel l l ine was sub-cultured, fur ther the glucose uptake assay was carr ied out and the

prol i ferations of the cel l s were observed.

Autho r In fo rmat ion Keywords L i cense

Email:

prashanthakarunakar@pes.edu

Alzheimer’s,

MTT-assay,

Ellman’s method

INTRODUCTION

Alzheimer‟s disease is the fourth cause of death worldwide which

affects an estimated 10 million people worldwide, which

demolishes the vital brain cells, memory loss and behavior

change, these brutal enough to affect work, lifelong hobbies,

and social life has become a threat to public health. A new

treatment strategies based on medicinal plants have become

the main focus (3). In a field of several theoretical options, several

kinds of AChEIs, such as donepezil, galantamine and rivastigmine

are available for the symptomatic treatment of patients with mild

to moderate AD (11).One of the most important approaches for

treatment of this disease involves the enhancement of

acetylcholine in neuron cells by using AChE inhibitors. Several

studies have reported acetylcholinesterase activity of the plant

extracts and drug (2).An acetylcholinesterase inhibitor (AChEI)

inhibits the acetylcholinesterase enzyme from breaking down

acetylcholine, hence will increase both the level and duration of

action of the neurotransmitter acetylcholine. Few inhibitors

increase the mechanism of acetylcholine by delaying or

reducing its degradation, some are used as nerve agents (Sarin)

or pesticides (organophosphates and the carbamates). Number

of research have been carried out on medicinal herbs showing

anti-inflammatory and antioxidant properties which may be

helpful in the treatment of Alzheimer‟s. Anti-inflammatory herbs

like turmeric, white willow bark may reduce the inflammation of

the brain tissue in Alzheimer‟s. The medicinal herbs that inhibit

Acetylcholinesterase contain natural COX-2 inhibitors that are

used for AD indication. It has been recently shown that a Chinese

herb, Yizhi Jiannao granules is effective in improving AD

symptoms (15).

The role of GSK-3 in regulating apoptosis is debatable however,

as some studies have shown that GSK-3β knockout mice are

sensitized to apoptosis and die in the embryonic stage, while

others have shown that over expression of GSK-3 can induce

apoptosis. The speed and efficacy of GSK-3 phosphorylation is

regulated by a number of factors. Due to its involvement in a

great number of signaling pathways, GSK-3 has been associated

with many high-profile diseases. GSK-3 inhibitors are currently

tested for medicinal effects in Alzheimer's disease, Type II

diabetes (Diabetes mellitus type 2), some forms of cancer,

and bipolar disorder(13).GSK-3 activity has been associated

with both pathological features of Alzheimer's disease,

namely Amyloid-β(Aβ) deposits and the formation of

neurofibrillary tangles. GSK-3 is believed to promote Aβ

production and to be tied to the process of the hyper-

phosphorylation of tau proteins, which leads to the tangles.

Due to these roles of GSK-3 in promoting Alzheimer's disease,

GSK-3 inhibitors may have positive beneficial effects on

Alzheimer's patients and are currently in the early stages of

testing (5).

Green tea(Camellia sensis) contains a unique set of

catechins that possess biological activity in antioxidant, anti-

angiogenesis, and anti-proliferative assays potentially

relevant to the prevention and treatment of various forms of

cancer(14).The chemical components of green tea chiefly

include Flavonoids, polyphenols, caffeine and amino acids.

Tulsi Green Tea is a unique combination of RamaTulsi, Krishna

Tulsi, VanaTulsi& Green Tea, helps in weight reduction by

boosting metabolism and help burn fat. Both Tulsi and Green

tea are rich in antioxidants, which eliminates free radicals

from the body and have a vast array of remarkable health

benefits.Tulsi tea is a great stress buster and develops

resistance against anxiety, tension and stress.

India is now the largest producer of Ginger

(Zingiberofficinale). The characteristic odor and flavor of

ginger is caused by a mixture of zingerone, shogaols, and

gingerols, volatile oils that compose 1-3% of the weight of

fresh ginger. It was found to be more effective than placebo

for treating nausea caused by seasickness, morning sickness

and chemotherapy.Ginger and compounds isolated

therefrom include immuno-modulatory, anti-tumorigenic,

anti-inflammatory, anti-apoptotic, anti-hyperglycemic, anti-

lipidemic and anti-emetic actions. Ginger is a strong anti-

oxidant substance and may either mitigate or prevent

generation of free radicals. It is considered a safe herbal

medicine with only few and insignificant adverse/side effects.

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and Publications (IJASRP) www.stringsjournal.com

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Urticadioica, traditionally used as a nutritive and “blood

cleanser” or alterative agent, a substantial pharmacological and

clinical literature supports its use for arthritic and allergic

conditions (leaf/herb) and improving urological symptoms of

benign prostatic hyperplasia (root). It contains biologically active

compounds that reduce inflammation. It contains the mineral

boron that is reported to enhance the levels of estrogen, which is

a hormone in the body, which can be beneficial in short-term

memory. Stinging nettle has also been shown to elevate the

mood in some Alzheimer‟s patients (1).

The energetic power of Maca (Lepidium meyenii) comes from

the way its phyto chemicals work in the endocrine glands.

Glucosinolates, terpenoides, saponines, alkaloids, fat acids,

essential amino acids, are responsible of the tremendous energy

power of Maca, so it is a major non caloric energy source (4). It is

also helpful in stress management, dietary supplements and an

aid for menstrual disorder, menopausal symptoms and

osteoporosis. Maca shows beneficial improvement in memory

and learning. As black maca improves experimental memory

impairment, induced by ovariectomy, due in part, to its

antioxidant and AChE inhibitory activities (1).

MATERIALS AND METHODS

Methanol, DMSO, Dulbecco‟s modified eagle‟s medium

(DMEM), Fetal bovine serum (FBS), Phosphate buffer saline (PBS),

penicillin, Antimycotic and streptomycin antibiotic solution, 3-(4,5-

Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

reagent, DL-Glyceraldehyde, glucose, lithium sulfate, 2-

mercaptoethanol, NADPH, quercetin, Sucrose, Disodium

hydrogen phosphate, Sodium dihydrogen phosphate , Glucose

reagent kit, Insulin, dexamethasone ,isobutyl-methyl-xanthine

(IBMX) , Triton X-100,distilled water

Collection of Plant Extract

The samples collected were fresh and collected from Lal Bagh,

Bangalore and Lepidium meyenii was collected from the north

Indian market. Communication with the sellers were done to

ensure the sample collected were proper and accurate. The

sample were washed with slow running tap water and let to dry

for 1-2 weeks under the sun, then crushed into powder using a

pestle and mortar and finally made into a fine powdered state.

Isolation of plant extract

About 10g of the each plant extract (in powdered form) was

taken and was dissolved in 50 ml of methanol + water in 500 ml

beaker, which was covered with an aluminum foil. The beaker

was kept on hot water bath at 50º C for 4 hours with intermittent

shaking. After the incubation period of 4 hours the extract was

filtered using a Whatmann filter paper and the filtrate was

collected in 50 ml beaker. The residue present over the filter

paper was discarded and filtrate was taken for further use. Next

the filtrate was kept at 80 ºC for few more hours until the extract

gets semi dried up and turn into semisolid form.

Three different concentration stocks were prepared- 120 mg/ml

of extract dissolved in 1.2ml of distilled water which was used to

test the Phytochemical activities.32 mg/ml of extract dissolved in

1ml of DMSO and used in Antioxidant assay.100 mg/ml of extract

dissolved in 1 ml of Methanol used for Acetylcholinesterase assay.

ABTS RADICAL SCAVENGING ASSAY

Antioxidant effect of the 5 plant extracts was estimated using the

ABTS radical cations method, produced by reacting ABTS and

APS on incubating the mixture at room temperature in dark for 16

hours. The solution thus obtained is further diluted with PBS to give

an absorbance of 1.000.Stock solution of the extracts were

prepared as 100mg/ml in methanol. Different concentrations of

the test sample and the reference standard were tabulated, and

about 950 l of ABTS working solution was added to give a final

volume of 1ml (made up by adding PBS). The absorbance is

recorded immediately at 734nm. The percent inhibition is

calculated at different concentrations and the IC50 values are

calculated by Non-linear regression analysis.

DPPH ASSAY

Antioxidant potential of the 5 plant extract was analyzed in

the 96 micro-well plate .Stock solution of the extracts were prepared as 100mg/ml in methanol .About 90 l of DPPH

solution was treated with 180 micro liter of various

concentrations of test solution & standard. The different

concentrations tested for reference standard are 0.5, 1.0, 1.5,

2.0, 2.5 mcg/ml. The reaction mixture is mixed and incubated

at 25°C for 15 minutes. The absorbance is measured at 510

nm using Plate reader.

Determination of % Inhibition:

IC50 values for DPPH radical scavenging activity of test

compounds is derived from a nonlinear regression analysis

based on sigmoidal dose response curve and computed

using Graph Pad Prism 5

ACETYL CHOLINESTERACE ASSAY

The plant extract were further tested for the breakdown of

acetylcholine activity using Acetylcholinesterase inhibition

assay which is carried out as per the method of Ellaman.

(1961)(9). Acetylcholinesterase hydrolyses acetylthiocholine

to give thiocholine and acetate. The reaction between

thiocholine and DTNB (Dithiobisnitrobenzoate) gives 2-nitro-5-

mercaptobenzoate, a yellow compound which can be

measured at 412 nm.2985 µL of phosphate buffer along with

test samples / reference standard of various concentrations;

100 µL of DTNB; 15 µL of enzyme are pre-incubated at 25°C

for 5 minutes. Add 20 µL of substrate solution and incubate at

25°C for 5 minutes. The reaction mixture is arrested by adding

100 µL of Neostigmine. The percentage inhibition of acetyl

cholinesterase is calculated as follows:

HAEMOLYSIS -Isolation of erythrocytes

Five ml of blood was collected from healthy volunteers in the

tubes containing 5.4 mg of EDTA to prevent coagulation and

centrifuged at 1000 rpm for 10 min at 40C. Plasma was

removed carefully and the white buffy layer was completely

removed by aspiration with a pipette with utmost care. The

erythrocytes were then washed for additional three times

with 1X PBS, pH 7.4. Washed erythrocytes were stored at 4oC

and used within 6 h for the haemolysis assay.

Sample testing

The 5 plant extract were taken about 50 µl of 10 dilution (100

µl Erythrocytes suspension : 1000 µl 1XPBS) of erythrocytes

suspension was taken into 2 ml of new eppendoff tube and

add 100 µl of test samples (plant extracts), 100 µl of 1XPBS as

negative control, 100 µl of 1% Triton X-100 and 100 µl of 1%

SDS as positive controls. Reaction mixture is incubating at

370C water bath for 60 min. Adjust the volume of reaction

mixture to 1 ml by adding 850 µl of 1XPBS. Finally centrifuge at

300 rpm for 3 min and the resulting supernatant was

measured at 540 nm by spectrophotometer to determine the

concentration of hemoglobin.

MTT- ASSAY

The 3T3L1 cell line was sub-cultured and trypsinized and

checked for the confluency of the cell line (70-80%

confluent) and then the cells were centrifuged. Seeding

procedure of the cell line (50000 cells / well of PC-3 in a 96

well plate) and incubate for 24 hours at 37oC, 5 % CO2

incubator. Compounds to be tested from 0-320 µg/ml [2 fold

variations] in RPMI media without FBS, are to be incubated for

24 hr. After incubation with compounds, the media is

removed from the wells and add 100l/well (50 µg /well) of

International Journal of Advanced Scientific Research and Publications

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the MTT (5 mg/10ml of MTT in 1X PBS, the solution is filtered

through a 0.2μM filter and stored at 2–8 °C for frequent use or

frozen for extended periods) working solution is added and

incubate for 3 to 4 hours. After incubation with MTT reagent, the

media is removed from the wells and 100 µlof DMSO was added

to rapidly solubilize the formazan. Measure the Absorbance at

590 nm.

Cell culture

In this study the selected 3T3l1 cell was isolated from adipose

tissue obtained from Skanda life Pvt Ltd. 3T3l1cells were grown in

Dulbecco‟s Modified Eagle‟s Medium (DMEM) (Gibco) media

supplemented with 10% heat inactivated fetal bovine serum

(FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were

incubated at 37°C in a 5% CO2 humidified incubator

Glucose uptake assay using 3T3 L1

Mouse 3T3-L1 pre-adipocytes are seeded in 10-cm dishes at

density of about 1x106. Upon reaching confluence, the cells are

maintained in M-1 for 1 DMEM for 2 days. The medium is replaced

by insulin and dexamethasone, and this is defined as day 0 of

differentiation induction. Cells are cultured for 2 days (day 0 to

day 2). The medium is replaced by another fresh media and

addition of insulin and dexamethasone on Day 2. Cells were

cultured for 5-10 days, with fresh medium fed every 2 days. For

lipolysis assays, on day 7-14 differentiation, split the cells into a 48-

well plate (one 10-cm plate to one 48-well plate) in the same

media, and incubate for 24 hour before performing assays. After

9-10 days there were formations of globules as shown in the figure

below (the normal 3T3L1 and the differentiated 3T3L1 cell line).

1. 3T3L1 ADIPOCYTE CONTROL2.3T3L1 ADIPOCYTE DIFFERENTIATION (GLOBULES)

PHF- Poly-herbal formulation

The methanol extracts were tested for inhibition for GSK-3 beta

and enhancement of glucose uptake by GSK-3 mechanism in

3T3L1 adipocytes. A poly herbal formulation of all the five plants

in a combined 1:1:1:1:1 ratio. About 40 micro litres were taken

from each plant extract to make a poly-herbal formulation and

was tested further

RESULTS Conduction of phytochemical analysis

Phytochemical analysis was conducted for the five plant extracts

– Camellia sinensis, Tulsi green tea, Zingerber officinale, Urtica

dioica and Lepidium meyenii. The results were tabulated and was

seen that two components carbohydrates and steroids were

common for all the plant extracts. And amino acids were absent

for all the extracts.

TABLE 1: Phytochemical analysis for the five plant extracts.

TEST Camellia

Sinensis

Tulsi

green

tea

Zingeber

Officinale

Urtica

Dioica

Lepidium

Meyenii

TANNINS + + - - -

SAPONIN + - + - -

PHOLBATANIN - - + - -

FALVANOID + + + + -

TERPENOID - - - + +

GLYCOSIDE + - - - -

STEROIDS + + + + +

CARBOHYDRATES + + + + +

AMINO ACIDS - - - - -

ALKALOIDS + + - - +

Flavonoid was shown absent in Lepidium meyenii, and

present in the other four plant extracts. Glycoside was

present in only Camellia sinensis and absent in the rest of the

plant extracts considered. Zingberofficinale shows presence

of pholbatanin which is absent in the rest.

Tannins show positive result for Camellia sinensis and tulsi

green tea. Saponins show positive result for Camellis

sinensisand Zingeber officicnale. Urticadiocia and Lepidium

meyenii showed presences of Terpenoids.

The important component Alkaloids showed a positive result

for Camellia sinensis, Tulsi green tea and Lepidium meyenii.

ACETYLCHOLINE ACTIVITY Table 2: Acetylcholine activity forUrtica diocia,Lepidium

meyenii and Zingiber officinale

Plants Name Concentration

(µg/ml)

Absorbance

590nm

%

Inhibition IC50 (µg/ml)

Control 0.0 0.501 0.00

1.123 Standard

(Neostigmine)

0.1 0.478 4.59

0.2 0.453 9.58

0.4 0.387 22.75

0.8 0.314 37.33

1.6 0.169 66.27

3.2 0.126 74.85

Urtica dioica

0.0 0.501 0.00

59.06

3.1 0.432 13.77

6.3 0.387 22.75

12.5 0.328 34.60

25.0 0.275 45.11

50.0 0.229 54.29

100.0 0.097 80.64

Lepidium meyenii

0.0 0.501 0.00

85.70

3.1 0.445 11.18

6.3 0.410 18.16

12.5 0.339 32.34

25.0 0.289 42.32

50.0 0.232 53.69

100.0 0.078 84.43

Zingiber officinale

0.0 0.501 0.00

34.72

3.1 0.443 11.58

6.3 0.400 20.16

12.5 0.341 31.94

25.0 0.278 44.51

50.0 0.209 58.28

100.0 0.119 76.25 Acetylcholine activity showed good results for three plant extracts –Urtica diocia, Lepidium meyenii and Zingeber

officinale. Neostigmine was kept as the standard having an IC 50 value of 1.123. The results were tabulated for the three plant extracts shown above, where Zingeber officinale showing an IC50

value of 34.72 shows a better inhibitory effect compared to Urtica diocia(59.06) and Lepidium meyeni(85.70).

International Journal of Advanced Scientific Research and Publications ISSN-2454–9878

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ANTIOXIDANT ASSAY- DPPH assay and ABTS assay

Table 3: The antioxidant assay were conducted on Urtica

diocia,Lepidium meyeniiand Zingeber officinale

DPPH assay, an antioxidant assay was carried out for all the three plant extracts-- Urtica diocia, Lepidium meyeniiand Zingeber officinale. Two plant extracts Lepidium meyenii and Zingeber officinale showed a positive result. Quercetin is taken

as the standard for the samples .The IC50 activity of Lepidium meyenii and better than Zingeber offinale from the table. ABTS assay, an antioxidant assay was carried out for all the three plant extracts- Urtica diocia, Lepidium meyenii and

Zingeber officinale. Two plant extracts Lepidium meyenii and Zingeber officinale showed a positive result. Standard Quercetin sample shows an IC50 value of 17.67. The IC50 activity of Lepidium meyenii is better than Zingeber offinale from the table.

Hence from both the antioxidant assays Lepidium meyenii shows a good antioxidant property. MTT-ASSAY

MTT-Assay was carried out to check the % stimulation in 3T3L1 cell line. Zingeber officinale did not show the proliferation of 3T3L1, hence the term NA. The other four plant extracts - Tulsi green tea, Camillia sinensis, Urtica diociaand Lepidium meyenii

shows the stimulation of the cell line and hence does not destroy the cell line. Tulsi green tea showed a better EC value compared to the other three extracts. [Table 4]

HEMOLYSIS Haemolysis assay was tested for the four plant extracts- tulsi green tea(TG), Camellia sinensis(GT),Urtica diocia (N) and Lepidium meyenii(M). SDS was taken as the standard

which shows a hemolytic activity of 80%. From the tabular representation of the plant extracts is shown using different concentration, showing a positive result. Hence will not affect the erythrocytes in the body and can be used drug

development in future. [Table 5] 3T3L1 DIFFERETIATION OF THE PLANT EXTRACTS As compared to positive standards Insulin & Lithium

chloride, PHF exhibits significant activity in Glucose uptake studies compared to Lepidium meyenii, Urtica dioica, Tulsi green tea. Lepidium meyenii & Tulsi green tea exhibits 2 fold stimulations at 150 µg/ml, similar to PHF. Data is shown in

table 7 as well as Graph.

DPPH ASSAY ABTS ASSAY

Plants Name Concentr ation

(µg/ml)

Absorb ance

590nm

% Inhibiti

on

IC 50

( µg/ml)

Absorb ance

590nm

% Inhibi tion

IC 50

( µg/ ml)

Control 0.0 0.545 0.00

13.13

0.424 0.00

17.6 7 Standard

(Quercitin)

0.3 0.524 3.85 0.405 4.48

0.6 0.511 6.24 0.386 8.96

1.3 0.479 12.11 0.359 15.33

2.5 0.407 25.32 0.304 28.30

5.0 0.244 55.23 0.232 45.28

10.0 0.109 80.00 0.105 75.24

Lepidiummey enii

0.0 0.545 0.00

76.33

0.424 0.00

41.8 2

3.1 0.510 6.42 0.401 5.42

6.3 0.465 14 .68 0.351 17.22

12.5 0.412 24.40 0.301 29.01

25.0 0.378 30.64 0.278 34.43

50.0 0.234 57.06 0.192 54.72

100.0 0.129 76.33 0.121 71.46

Zingiber officinale

0.0 0.545 0.00

121.8

0.424 0.00

89.0 6

3.1 0.512 6.06 0.401 5.42

6.3 0.478 12.2 9 0.356 16.04

12.5 0.432 20.73 0.321 24.29

25.0 0.367 32.66 0.278 34.43

50.0 0.256 53.03 0.209 50.71

100.0 0.098 82.02 0.096 77.36

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TABLE 4: MTT-Assay for the five plant extracts.

Plant name Conc. µg/ml OD at 540 nm % stimulation EC50

(µg/ml)

Control 0.00 0.5107 0.00

159.8 Tulsi Green Tea

10 0.5469 7.09

20 0.5923 15.98

40 0.6215 21.70

80 0.7257 42.10

160 0.8522 66.87

320 0.9418 84.41

Camellia sinesis

(green tea)

10 0.5186 1.55

274.2

20 0.5311 3.99

40 0.5519 8.07

80 0.6124 19.91

160 0.6927 35.64

320 0.8821 72.72

Urtica dioica

10 0.5329 4.35

239.9

20 0.5567 9.01

40 0.5924 16.00

80 0.6211 21.62

160 0.7519 47.23

320 0.8143 59.45

Zingiber officinale

10 0.0429 -91.60

NA

20 0.0871 -82.94

40 0.1058 -79.28

80 0.1174 -77.01

160 0.1365 -73.27

320 0.1863 -63.52

Lepidium meyenii

10 0.5237 2.55

204.7

20 0.5362 4.99

40 0.5615 9.95

80 0.6091 19.27

160 0.6526 27.79

320 0.8519 66.81

TABLE 5: Hemolysis assay activity for Camellia Sinensis, Tulsi

green tea, Urtica diocia and Lepidium meyenii

% Hemolysis

PBS 0

1%

SDS

80

TG

Concentration(µg) OD at

540nm

% Hemolysis

0.00 1.11 0.00

40.00 1.0918 1.64

80.00 1.1701 -5.41

160.00 1.1454 -3.19

320.00 1.0843 2.32

GT

40.00 1.141 -2.79

80.00 1.1605 -4.55

160.00 1.2025 -8.33

320.00 1.1737 -5.74

N

40.00 1.1683 -5.25

80.00 1.0894 1.86

160.00 1.1575 -4.28

320.00 1.116 -0.54

M

40.00 1.139 -2.61

80.00 1.3541 -21.99

160.00 1.2318 -10.97

320.00 1.2615 -13.65

TABLE 6: GLUCOSE UPTAKE ASSAY 3T3L1 ADIPOCYTES

Glucose uptake assay using 3T3 L1 Adipocytes

Test material mean CPM ± SEM Fold stimulation Remarks

Control 7033 ± 309 1

Insulin 100nM 22331 ± 1256 3.17 Positive control

Lithium chloride 50mM 23358 ± 1388 3.32 Positive control

PHF ----- 75µg/ml 12078 ± 1233 1.71

150µg/ml 19011 ± 1433 2.7

Maca ---- 75µg/ml 9088 ± 1088 1.43

150µg/ml 13786± 1188 2.24

Nettle ---- 75µg/ml 9003 ± 488 1.28

150µg/ml 11044 ± 988 1.57

Tulsi Green Tea ---- 75µg/ml 11088 ± 1099 1.57

150µg/ml 14089 ± 1099 2

TABLE 7: Graphical representation of the glucose uptake using 3T3L1 adipocyte

DISCUSSION

Research advances have been made in unravelling the

neuropathology and molecular mechanisms of AD there are

only few treatments option currently available. There is a

severe lack of effective therapies with respect to the

dramatic increase in AD or other related diseases in the

coming decades which calls for the demand of new drugs

.Hence a large number of direct and indirect activities of

traditional plants and its activity is studies that can influence

the therapeutic targets of AD. In the present study five plant

extracts were considered and checked for their

acetylcholinesterase activity which is the main true enzyme

which hydrolyses acetylcholine in the body , due to utilization

of acetylcholine by enzymes causes dementia or other

neurodegenerative disorders which indicates the messages is

not delivered properly from one neuron to another neuron.

Hence inhibitors of these enzymes may prevent the

hydrolases of acetylcholine substrate preferably natural

herbal plant extracts. There is research where oxidative stress

leading to AD and other neurological disorders (10,11).

Therefore antioxidant is the other key component to find a

cure or inhibition of this disease. Hemolytic activity

considered as one of the main biological effects of Saponins,

it have the ability to hemolysis human erythrocytes by form

pores in cell membrane based on affinity of a glycon moiety

for the membrane sterols particularly cholesterols which

leading to form insoluble complexes. The assay principle of

this experiment is that hydrogen peroxide, which crosses the

red blood cell (RBC) membrane, and acts on the intracellular

moiety, forms ferryl radical or hydroxyl radical by interacting

International Journal of Advanced Scientific Research and Publications ISSN-2454–9878

34[16]

with hemoglobin and initiates a series of reactions, resulting in

RBC lysis(7, 9). Since saponins have an ability to cause hemolysis

and may be present in chosen plants of herbal formulation.

Measuring hemolytic activity is important as it is an indicator for

cytotoxicity. Mechanical stability of erythrocytes membrane is

good indicator of in-vitro cytotoxicity. Performing hemolytic

activity is important to determine whether a drug possessing

antioxidant and other bioactivities can be used in

pharmacological applications.GSK-3 an enzyme involved in the

control of glycogen metabolism, it has been implicated in

Alzheimer‟s disease pathogen and alpha beta neurotoxicity(12).

Inhibition of GSK-3 leads to neuro-protective effects decrease the

beta amyloid production and reduction in tau hyper-

phosphorylation which have been associated in Alzheimer‟s

disease(15). From the results obtained it is seen from the glucose

uptake assay using 3T3L1 adipocyte the PHF(1:1:1:1:1 ratio of the

five plant extracts used) is showing a good effect at 150micro liter

concentration , about 2 fold stimulation whereas the control used

insulin shows about 3 fold. Lepidium meyenii and Tulsi green tea

also shows 2 fold stimulation similar to PHF. Therefore these three

extracts show a good level of possible inhibitory activity, further

analysis should be done on animals to see the effects. CONCLUSION

In the present work, five plant extracts were considered and

have seen that three plants - Urtica diocia, Lepidium meyenii and

Zigeber officinale show acetylcholine activity and can be tested

on animals to further study the different pathway and

mechanism to inhibit or control Alzheimer‟s disease or other

neurodegenerative disorders from moving forward and causing

neural cell death. Poly herbal formulations of natural herbal

extracts of different concentration can be tested for cytotoxicity

and will have lesser or no side effects as compared to the drugs

present today. Different concentrations can also be

experimented on the 3T3l1 cell line or similar adipocyte cell line to

see the proliferation or inhibition of the cell, which mimics the

similar mechanism of GSK-3 as studied in the human or animal

brain cells.

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